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Tumor aerobic glycolysis confers immune evasion through modulating sensitivity to T cell-mediated bystander killing via TNF-α
Abstract
Metabolic reprogramming toward glycolysis is a hallmark of cancer malignancy. The molecular mechanisms by which the tumor glycolysis pathway promotes immune evasion remain to be elucidated. Here, by performing genome-wide CRISPR screens in murine tumor cells co-cultured with cytotoxic T cells (CTLs), we identified that deficiency of two important glycolysis enzymes, Glut1 (glucose transporter 1) and Gpi1 (glucose-6-phosphate isomerase 1), resulted in enhanced killing of tumor cells by CTLs. Mechanistically, Glut1 inactivation causes metabolic rewiring toward oxidative phosphorylation, which generates an excessive amount of reactive oxygen species (ROS). Accumulated ROS potentiate tumor cell death mediated by tumor necrosis factor alpha (TNF-α) in a caspase-8- and Fadd-dependent manner. Genetic and pharmacological inactivation of Glut1 sensitizes tumors to anti-tumor immunity and synergizes with anti-PD-1 therapy through the TNF-α pathway. The mechanistic interplay between tumor-intrinsic glycolysis and TNF-α-induced killing provides new therapeutic strategies to enhance anti-tumor immunity.
... Under hypoxia, glycolysis is further enhanced in cancer cells to maintain energy supply and promote antioxidant defense, while the tricarboxylic acid (TCA) cycle is inhibited to reduce reactive oxygen species (ROS) generation [26,27]. When the glycolysis pathway is inhibited, the cancer cells shift the cellular metabolism to OXPHOS and TCA cycle due to their metabolic plasticity [28], which produces cellular ROS during mitochondrial respiration [29]. Although the metabolic flexibility of cancer cells could help them adapt to harsh environments and resist therapy, the specific metabolic fitness of hypoxic cancer cells under glycolysis inhibition might be a potential therapeutic target. ...
... EGCG is able to inhibit glycolysis in various types of cancer cells through inhibition of the glycolytic enzymes and glucose internalization [38]. The inhibition of glycolysis in hypoxic cancer cells would induce a shift in cellular metabolism towards OXPHOS, therefore enhancing ROS generation [29,50]. In addition, copper ions were demonstrated to amplify the amount and toxicity of cellular ROS via GSH consumption and Fenton-like reactions [30]. ...
... The selective and effective ROS generation in hypoxic cancer cells might be attributed to the synergistic effects of CuGI, including that (1) CuGI oxidizes the intracellular GSH to be GSSG (Fig. 5A), (2) inhibition of glycolysis forces cellular metabolic shift to OXPHOS to increase ROS production in hypoxic cancer cells (Figs. S28A-B) [29], and (3) the GSH metabolism was suppressed by CuGI and the antioxidant agents (L-ascorbate, NADH) were markedly decreased (Fig. 4D), significantly reducing the antioxidant capability of cancer cells, (4) the released Cu + decomposes H 2 O 2 to generate •OH via Fenton-like reactions, amplifying the oxidative stress (Fig. 1N). Conversely, in the noncancerous cells (HUVECs), EGCG did not increase the level of cellular ROS; instead, it reduced the ROS content under hypoxic conditions (Fig. S28C). ...
Hypoxia, a prevalent characteristic of solid tumors, substantially impairs the efficacy of cancer treatments. However, there are no feasible clinical approaches for treating hypoxic tumors. Here, we develop metal-phenolic networks (CuGI) utilizing the natural glycolysis inhibitor (epigallocatechin gallate) and the essential metal element in the human body (copper ions), specifically targeting and annihilating hypoxic cancer cells. CuGI redirects the metabolic pathway of hypoxic cancer cells from anaerobic glycolysis to oxidative phosphorylation, thereby enhancing reactive oxygen species production and promoting oligomerization of lipoylated proteins in the tricarboxylic acid cycle. Through targeted induction of oxidative and proteotoxic stresses, CuGI induces apoptosis and cuproptosis specifically in cancer cells under hypoxic conditions while sparing normal cells. Moreover, cancer cell membrane-coated CuGI (CuGI@CM) exhibits enhanced tumor penetration effect and demonstrates commendable biocompatibility, effectively suppressing colorectal tumor growth. Importantly, CuGI@CM, when combined with vascular disruptors or radiotherapy which aggravate tumor hypoxia, synergistically potentiates therapeutic efficacy. Thus, CuGI represents a specific and potent nanotherapeutic capable of selectively eliminating hypoxic tumors, offering promise in combination therapies to address tumor hypoxia.
... Currently, research on metabolic reprogramming and its interplay with tumor immunity following radiotherapy remains scarce. Inactivation of GLUT1 enhances the sensitivity of tumor cells to TNF-α-induced anti-tumor immunity by elevating ROS levels (22). The function of cytotoxic T cells, which expresses GLUT3 at high levels, is not significantly affected by inhibiting GLUT1 (23). ...
... The function of cytotoxic T cells, which expresses GLUT3 at high levels, is not significantly affected by inhibiting GLUT1 (23). Combining GLUT1 inhibitor BAY-876 with anti-PD-1 treatment leads to a significant increase in the number of infiltrated CD45+ immune cells (22). Li et al. develop a strategy that combines the GLUT1 inhibitor BAY-876 with the PD-L1 inhibitor BMS-1, delivered via an injectable thermogel formulation, fostering sustained antitumor immunity and improved survival outcomes in glioblastoma models (24). ...
The interplay between metabolic pathways and immune escape has emerged as a captivating research area in oncobiology. Among these, the Warburg effect stands out as a hallmark metabolic reprogramming in cancer, characterized by elevated glucose utilization and excessive lactic acid production through anaerobic glycolysis. Key glycolytic enzymes not only fulfill the bioenergetic demands of cancer cells but also exhibit moonlighting roles, including regulation of epigenetic modifications, protein kinase activity, and immune escape mechanisms, thereby reshaping the tumor microenvironment. Tumor-specific vascular architecture facilitates lactate accumulation, which drives tumor progression by impairing immune cell function and acting as a signaling molecule to recruit immunosuppressive cells and modulate immune checkpoint pathways. The PD-1/PD-L1 co-stimulatory pathway plays a crucial role in negatively modulating the activation, proliferation, and cytokine secretion by T-lymphocytes. This review primarily focuses on elucidating the regulation and mechanisms underlying PD-1/PD-L1 signaling axis during glycolysis in tumor cells as well as surrounding cells. In the era of precision medicine, there is a particular interest in leveraging ¹⁸F-FDG PET/CT imaging as a valuable tool to assess PD-L1 expression status for more targeted therapeutic interventions. Additionally, the development of natural compounds capable of modulating metabolism opens new avenues for metabolism-based immunotherapy, though further studies are required to validate their in vivo efficacy.
... T cells can impede tumor growth through direct cytotoxicity towards tumor cells and modulate immune cell activity by secreting diverse immune regulatory molecules [30][31][32][33]. During inflammation, infection, and particularly in the presence of tumors, MDSCs migrate to injury sites and around tumor cells through the bloodstream, where they impede the normal functions of innate and adaptive immune cells [34][35][36]. ...
Background
Myeloid-derived suppressor cells (MDSCs) are a subset of immature myeloid cells with immunosuppressive properties. Evidence suggests that abnormal immune system can lead to immune dysfunction and increase the risk of developing diffuse large B-cell lymphoma (DLBCL). This study investigated the abnormality of MDSCs in the peripheral blood of patients with DLBCL.
Methods
Expression, apoptosis, and proliferation of MDSCs was measured in the peripheral blood DLBCL patients and healthy donors (HDs) via flow cytometer. The co-culture groups included the MDSCs and DLBCL cells line and MDSCs and T cells. Using flow cytometry detected MDSCs and T cells proliferation, apoptosis, T cells activation and function in the co-culture groups. RNA transcriptome sequencing analysis was conducted on DLBCL-MDSCs and HDs-MDSCs. Combined with the clinicopathological data of DLBCL patients, the correlation between MDSCs and DLBCL progression was analyzed.
Results
The expression of MDSCs in patients newly diagnosed with DLBCL was elevated. DLBCL tumor cells could stimulate MDSCs growth. DLBCL-MDSCs showed stronger immunosuppressive ability to T cells proliferation, activation and secretion of cytokines and associated with several clinical indicators such as Ann Arbor stage, serum LDH level, and lymphoma IPI score.
Conclusion
This study investigated the abnormality of MDSCs and underscored the critical role of MDSCs in suppressing T cell function in DLBCL patients. It provides certain laboratory evidence for MDSCs as biomarkers of disease progression and treatment response in DLBCL.
... On the one hand, when the level of aerobic glycolysis in cancer cells decreases, mitochondrial oxidative phosphorylation significantly increases, leading to the production of a large amount of ROS. Increased ROS levels result in the downregulation of antiapoptotic proteins [80]. On the other hand, increased aerobic glycolysis directly affects the levels of antiapoptotic and proapoptotic proteins [81]. ...
Annexin A2 (ANXA2) is a multifunctional protein that binds to calcium and phospholipids and plays a critical role in various pathological conditions, including cancer and inflammation. Recently, there has been increasing recognition of the significant role of ANXA2 in inhibiting apoptosis and promoting immune evasion in tumour cells. Therefore, a deep understanding of the regulatory mechanisms of ANXA2 in tumour cell apoptosis and its relationship with immune evasion can provide new targets for cancer therapy. This review summarizes the role and mechanisms of ANXA2 in regulating apoptosis in tumour cells, the connection between apoptosis regulation and tumour immunity, and the potential role of ANXA2 in therapy resistance.
... Interaction of TNF with its receptor (TNFR) initiates cell death signals through the apoptotic/necrotic pathway in tumor cells. Glut1 is present mainly in tumors, while Glut3 is mostly represented in T cells, suggesting that Glut1 could be a promising candidate for cancer therapy (Wu et al. 2023). Cancer cells secrete GPI into the extracellular matrix, where it acts as an autocrine motility factor (AMF). ...
Cancer, an important global health problem, is defined by aberrant cell proliferation and continues to be the main cause of death globally. The tumor microenvironment (TME) plays an essential role in the development of cancer, resistance to therapy, and regulation of the immune response. Some immune cells in the TME, like T cells, B cells, macrophages, dendritic cells, and natural killer cells, can either stop or help tumor growth, depending on how metabolic and cytokine changes happen. Cytokines function as essential signaling molecules that modulate immune cell metabolism, altering their functionality. This review focuses on how cytokine-mediated metabolic reprogramming affects the activity of immune cells inside the TME, which can either make the immune response stronger or weaker. New ways of treating cancer that focus on metabolic pathways and cytokine signaling, such as using IL (Interleukin) - 15, IL- 10, and IL- 4, show promise in boosting immune cell activity and making cancer treatments more effective. Finding these pathways could lead to new ways to treat cancer with immunotherapy that focus on metabolic competition and immune resistance in the TME.
... NF-κB activation is pivotal to IR-AKI progression. During IR-AKI, NF-κB-driven inflammatory cytokines (e.g., TNFα) activate Caspase-8, triggering the mitochondrial apoptotic pathway via proapoptotic Bax upregulation [26,27]. Consequently, we further explored DKS26's anti-inflammatory and antiapoptotic effects. ...
Acute kidney injury (AKI) is a common critical clinical disease with high morbidity and mortality rates. Ischemia-reperfusion (IR) is the main cause of AKI, and there is no effective treatment or prevention. Therefore, it is critical to screen for effective therapeutic agents and to find therapeutic targets. DKS26 is a derivative of oleanolic acid (OA) optimized for bioavailability while retaining the anti-inflammatory, antioxidant, and anti-apoptotic properties of OA. This study aimed to investigate the therapeutic effects of DKS26 on AKI and its underlying molecular mechanisms. We established an AKI model in vivo and in vitro and observed that DKS26 had an ameliorative effect on IR or H/R-induced renal tubular epithelial cell injury and reduced oxidative stress, inflammation, and apoptosis. Meanwhile, Swiss TargetPrediction and AutoDock Vina analysis revealed that DKS26 may interact with vitamin D receptors (VDR) through hydrogen bonding, suggesting that DKS26 may exert effects through VDR. In this study, we found that DKS26 treatment enhanced the stability of the VDR protein, promoted the binding of VDR to p-NF-κB P65Ser311, reduced the entry of p-NF-κB P65Ser311 into the nucleus, and inhibited the transcription of downstream inflammatory genes as well as their own expression, thus exerting its protective effect. In summary, these findings suggest that DKS26 may be a promising preventive strategy and provide a theoretical and experimental basis for AKI treatment.
... Moreover, glycolytic enzyme inhibitors enhance memory T cell formation and tumor clearance [60]. A deficiency in glycolytic enzymes, such as Glut1 and Gpi1 (glucose-6-phosphate isomerase 1) in tumor cells, can enhance a T cell-mediated antitumor response [61]. Furthermore, Lactic acid, a toxic metabolite to activating T cells, supports Treg growth and function [51], whereas lactate catabolism decreases Treg induction, promotes antitumor immunity, and inhibits tumor growth in mice [62]. ...
Liver cancer is the sixth most common cancer worldwide and the third most common cause of cancer mortality. The development and progression of liver cancer and metastases is a multifaceted process involving numerous metabolic pathways. T cells have a protective role in the defense against cancer, and manipulating metabolic pathways in T cells can alter their antitumor activity. Furthermore, Liver cancer and T cell nutrition competition lead to T cell dysfunction through various molecular mechanisms. Some nanomaterials and drugs can improve T cell metabolism and promote the anti-liver cancer function of T cells. This review discusses the current literature regarding metabolic changes in liver cancer, the role of T cells in liver cancer, T cell metabolism in liver cancer, and targeted T cell metabolism therapy for liver cancer. The promise and challenges of studying target T cell metabolism for treating liver cancer are also addressed. Targeting T cell metabolism is a promising approach for treating liver cancer.
... PLGA, a hydrophobic and biodegradable polymer, hydrolyzes in the body to produce lactic and glycolic acids [44]. The presence of lactate may exacerbate the acidification of the TME, inhibiting the anti-tumor ability of immune cells within the TME [45]. This condition can even drive tumor angiogenesis [46] and facilitate immune evasion [47]. ...
Transarterial chemoembolization (TACE) serves as a locoregional therapy for hepatocellular carcinoma (HCC) patients. Nevertheless, the rapid dissociation of conventional TACE (cTACE) preparations, attributed to the instability of the emulsion, often leads to inadequate concentrations of chemotherapeutic agents within the tumor site. Consequently, there exists a pressing demand for an embolic agent that possesses facile injectability and the capacity to provide continuous delivery of chemotherapy drugs. Herein, we leveraged the inherent drug-loading capabilities and distinctive structural attributes of Spirulina platensis (SP) to formulate a novel microalgae embolic agent, doxorubicin loaded-Spirulina platensis (DOX-SP). The DOX-SP formulation exhibited a notable capacity for drug loading and demonstrated the ability to sustain drug release in response to acidic tumor microenvironments (TME). The spiral structure and micron-scale size of SP contributed to effective vascular embolization and continuous localized release of DOX. Furthermore, the biodegradability of SP as a natural biomaterial ensured good biosafety, with its degradation products potentially enhancing the pH of TME. In a rat model of in-situ hepatocellular carcinoma, DOX-SP effectively suppressed tumor growth and significantly reduced tumor size following intra-arterial injection, while exhibiting minimal adverse effects. Taken together, the high drug loading capacity, effective vascular embolization, pH sensitivity, TME pH modulation, and biodegradability of DOX-SP made it a promising embolic agent for hepatocellular carcinoma treatment.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12951-025-03290-5.
... TNF-α is mainly produced by activated macrophages and is a multi-functional pro-inflammatory cytokine that can activate T cells and stimulate their production of IFN-γ. The cytokines TNF-α and IFN-γ are recognized for a synergistic anti-tumor effect [18,19]. The elevated levels of these cytokines in the traditional grafting model may contribute to the more rapid decline of surviving tumor cells in the traditional model compared to our optimized model. ...
Brain metastasis is thought to be related to the high mortality and poor prognosis of lung cancer. Despite significant advances in the treatment of primary lung cancer, the unique microenvironment of the brain renders current therapeutic strategies largely ineffective against brain metastasis. The lack of effective drugs for brain metastasis treatment is primarily due to the incomplete understanding of the mechanisms underlying its initiation and progression. Currently, our understanding of brain metastasis remains limited, primarily due to the absence of appropriate models that can realistically simulate the entire process of tumor cell detachment from the primary site, circulation through the bloodstream, and eventual colonization of the brain. Therefore, there is a pressing need to develop more suitable lung cancer brain metastasis models that can effectively replicate these critical stages of metastasis. Here, based on the traditional carotid artery injection model, we established a novel orthotopic mouse model by using a light-controlled hydrogel to repair the puncture site on the carotid artery, with sustained cerebral blood circulation and the capability of multiple delivery cancer cell to mimic lung cancer brain metastasis. The optimized orthotopic mouse model significantly reduced cerebral ischemia and improved cerebral oxygenation by 60% compared to the traditional orthotopic mouse model, enhancing post-operative survival rates. It also showed a reduction in pro-inflammatory cytokines and featured less inflammatory and more resting states of microglial and astrocyte cells. Furthermore, the optimized orthotopic mouse model markedly increased the success rate and absolute number of the metastatic clones in the brain. Additionally, the multiple delivery model based on the optimized orthotopic mouse model substantially augmented the tumor clone number and formation rates compared to single injection in the optimized orthotopic mouse model. This model overcomes previous limitations by maintaining cerebral circulation, providing a more accurate simulation of the continuous entry of tumor cells into cerebral circulation. It offers a robust platform for studying the interactions of cancer cells with the brain microenvironment and testing new therapeutic approaches.
Graphical abstract
... A large-scale study on cancer survivors showed that systemic inflammatory markers (e.g., IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, and C-reactive protein) are closely related to physical, emotional, and cognitive CRF [25]. These cytokines influence cancer progression and fatigue severity through centralnervous-system and neuroimmune mechanisms [26,27]. Some studies target these cytokines with biological therapies to relieve CRF [28], focusing on reducing the pro-inflammatory state or inhibiting key inflammatory signaling pathways [29,30]. ...
Objective
To investigate the association between the systemic immunity‐inflammation index (SII) and fatigue, cancer, and cancer‐related fatigue (CRF) populations.
Methods
The National Health and Nutrition Examination Survey (NHANES) from 2005 to 2018 provided data for this retrospective cross‐sectional study. By dividing the platelet count by the neutrophil count and the lymphocyte count, SII was calculated. Participants were categorized into four groups: normal, fatigue, cancer, and cancer‐related fatigue (CRF), with the normal group serving as the reference. Binary logistic regression was applied to assess the correlations. The dose–response relationship between SII and outcomes in the four groups was evaluated using restricted cubic splines. Use threshold effect analysis to determine the optimal SII value for each of the three groups. Stratified and subgroup analyses were performed based on sociodemographic factors and confounders, with specific attention to fatigue severity levels (mild, moderate, severe) in the fatigue and CRF groups.
Results
Data analysis included a total of 32,491 participants, including 14,846 in the normal group, 14,581 in the fatigue group, 1520 in the cancer group, and 1544 in the CRF group. The results of binary logistic regression showed that SII was positively correlated with the fatigue group (1.43[1.33, 1.55]), cancer group (1.67 [1.43, 1.95]) and CRF group (1.93 [1.66, 2.25]). Restricted cubic spline analysis revealed a linear relationship between SII and outcomes. The threshold values (k) for each of these groups were identified as 464.78 × 10³ cells/μL, 448.97 × 10³ cells/μL, and 454.65 × 10³ cells/μL, respectively. Stratified analysis indicates that most groups exhibit significant differences. The subgroup analysis indicated that fatigue severity increased with higher SII levels, with the CRF group exhibiting the highest rate of severe fatigue (171% increase).
Conclusion
SII is positively correlated with fatigue, cancer, and CRF in a linear way. Higher SII values are associated with greater fatigue, particularly in the CRF population.
... Signature DRGs may be involved in the different immune landscape in two subtypes. Genetic and drug-induced inactivation of GLUT1 enhances tumor sensitivity to anti-tumor immunity and complements anti-PD-1 therapy through the TNF-α pathway [57]. Meanwhile, TAMs promote the formation of an immunosuppressive phenotype by increasing metabolic pathways such as glycolysis and fatty acid oxidation [58]. ...
Disulfidptosis, a novel form of disulfide stress-induced cell death involved in tumor progression, hasn’t be well defined the function in tumor progression. And the clinical impacts of disulfidptosis-related genes (DRGs) in pancreatic adenocarcinoma (PAAD) remain largely unclear. In this study, we identified two distinct disulfidptosis subtypes and found that multilayer DRG alterations were associated with prognosis and TME infiltration characteristics. A three-gene prognostic signature was constructed to predict prognosis, and its clinical significance was characterized in the TCGA-PAAD cohort. The disulfidptosis signature was significantly correlated with prognosis, molecular subtype, CD8 T-cell infiltration, response to immune checkpoint inhibitors and chemotherapeutic drug sensitivity, and its predictive capability in PAAD patients was validated in multiple cohorts. Meanwhile, two anti-PD-L1 immunotherapy cohorts confirmed that low-risk patients exhibited substantially enhanced clinical response and treatment advantages. Furthermore, the expression patterns of DRGs were validated by quantitative real-time PCR. The expression and prognostic predictive capability of GLUT1 were verified by 87 PAAD patients from our cohort. These findings may help us understand the roles of DRGs in PAAD and the molecular characterization of disulfidptosis subtypes. The disulfidptosis signature could be a promising biomarker for prognosis, molecular subtypes, TME infiltration characteristics and immunotherapy efficacy.
... However, it has been proposed that increased glycolytic activity impairs the function and development of memory CD8 + T cells, driving their conversion into effector T cell phenotypes and subsequent depletion, which ultimately weakens anti-tumor efficacy [138]. Inactivation of GLUT1 impairs glycolysis and promotes OXPHOS pathways, resulting in the accumulation of ROS, which enhances CTL-mediated bystander killing and promotes tumor cell death [139]. FAO, mediated by several signaling pathways, increases the lifespan of memory CD8 + T cells while dampening the effector activity of CD8 + T cells [15]. ...
Epigenomic modifications—such as DNA methylation, histone acetylation, and histone methylation—and their implications in tumorigenesis, progression, and treatment have emerged as a pivotal field in cancer research. Tumors undergo metabolic reprogramming to sustain proliferation and metastasis in nutrient-deficient conditions, while suppressing anti-tumor immunity in the tumor microenvironment (TME). Concurrently, immune cells within the immunosuppressive TME undergo metabolic adaptations, leading to alterations in their immune function. The complicated interplay between metabolites and epigenomic modulation has spotlighted the significance of epigenomic regulation in tumor immunometabolism. In this review, characteristics of the epigenomic modification associated with tumors are systematically summarized alongside with their regulatory roles in tumor metabolic reprogramming and immunometabolism. Classical and emerging approaches are delineated to broaden the boundaries of research on the crosstalk research on the crosstalk between tumor immunometabolism and epigenomics. Furthermore, we discuss potential therapeutic strategies that target tumor immunometabolism to modulate epigenomic modifications, highlighting the burgeoning synergy between metabolic therapies and immunotherapy as a promising avenue for cancer treatment.
... Of interest, the metabolic rewiring observed in Spry1 KO cells can render the tumor microenvironment less immunosuppressive [52]. Considering that glycolysis has been associated with decreased immuneinfiltration [53], and immune-evasion [54], the potential contribution of Spry1 on immune resistance mechanisms should be addressed in future studies. Although the mitochondrial control over HIF1α is still debated, it is known that mitochondrial dysfunction can reduce HIF1α stabilization along with the expression of its downstream targets [28], and several mitochondriaderived molecules were shown to modulate HIF1α stability and activity, including ROS [55]. ...
Background
About 50% of cutaneous melanoma (CM) harbors the activating BRAF V600 mutation which exerts most of the oncogenic effects through the MAPK signaling pathway. In the last years, a number of MAPK modulators have been identified, including Spry1. In this context, we have recently demonstrated that knockout of Spry1 (Spry1 KO ) in BRAF V600 -mutant CM led to cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and reduced tumor growth in vivo. Despite these findings, however, the precise molecular mechanism linking Spry1 to BRAF V600 -mutant CM remains to be elucidated.
Materials and methods
Immunoprecipitation coupled to mass spectrometry was employed to gain insight into Spry1 interactome. Spry1 gene was knocked-out using the CRISPR strategy in the BRAF-mutant cell lines. Transmission electron microscopy was used to assess the relationship between Spry1 expression and mitochondrial morphology. By using in vitro and in vivo models, the effects of Spry1 KO were investigated through RNA-sequencing, quantitative real-time PCR, Western blot, and immunofluorescence analyses. The Seahorse XF24 assay allowed real-time measurement of cellular metabolism in our model. Angiogenic potential was assessed through in vitro tube formation assays and in vivo CD31 staining.
Results
Spry1 was mainly located in mitochondria in BRAF V600 -mutant CM cells where it interacted with key molecules involved in mitochondrial homeostasis. Spry1 loss resulted in mitochondrial shape alterations and dysfunction, which associated with increased reactive oxygen species production. In agreement, we found that nuclear hypoxia-inducible factor-1 alpha (HIF1α) protein levels were reduced in Spry1 KO clones both in vitro and in vivo along with the expression of its glycolysis related genes. Accordingly, Ingenuity Pathway Analysis identified “HIF1α Signaling” as the most significant molecular and cellular function affected by Spry1 silencing, whereas the glycolytic function was significantly impaired in Spry1 depleted BRAF V600 -mutant CM cells. In addition, our results indicated that the expression of the vascular endothelial growth factor A was down-regulated following Spry1 KO , possibly as a result of mitochondrial dysfunction. Consistently, we observed a substantial impairment of angiogenesis, as assessed by the tube formation assay in vitro and the immunofluorescence staining of CD31 in vivo.
Conclusions
Altogether, these findings identify Spry1 as a potential regulator of mitochondrial homeostasis, and uncover a previously unrecognized role for Spry1 in regulating nuclear HIF1α expression and angiogenesis in BRAF V600 -mutant CM.
Significance
Spry1 KO profoundly impacts on mitochondria homeostasis, while concomitantly impairing HIF1α-dependent glycolysis and reducing angiogenesis in BRAF-mutant CM cells, thus providing a potential therapeutic target to improve BRAF V600 -mutant CM treatment.
... For host retinal cells, more photoreceptors were preserved in retinas with PD-L1 + hESC-RPE-transplants, companied by enhanced light-sensing and light transduction signals in photoreceptors (rod and cone), BC and Müller cells compared to CTL RPE, supporting the visual protection provided by PD-L1 + hESC-RPE. Additionally, oxidative phosphorylation was significantly downregulated in PD-L1 + hESC-RPE grafts, host photoreceptors and BC, potentially reducing ROS accumulation and cell apoptosis [61]. ...
Immune rejection is a major barrier to the successful human embryonic stem cell‐derived retinal pigment epithelial (hESC‐RPE) transplantation for age‐related macular degeneration (AMD). Traditional strategies to mitigate immune rejection involve ablating major histocompatibility complex (MHC) molecules on hESC‐RPE. An alternative approach is immune checkpoint overexpression, avoiding natural killer (NK) cell‐mediated destruction due to MHC‐I deficiency. Our study highlights the benefits of PD‐L1 overexpression without requiring MHC gene deletion, which preserved the immunosuppressive functions of hESC‐RPE on NK cells. In Vivo experiments in retinal degeneration models showed that PD‐L1‐expressing hESC‐RPE grafts exhibited significantly higher survival, reduced apoptosis and enhanced visual protection. Single‐cell transcriptomics revealed reduced immune activation and oxidative stress in PD‐L1‐overexpressing grafts. PD‐L1's protective role was further evidenced by improved light transduction in host photoreceptors. These findings support PD‐L1 overexpression as a promising strategy to improve the efficiency of hESC‐RPE‐based therapy for AMD.
... Notably, silencing GPR81 in mouse models revealed a suppression of Tregs production (74). Targeting glucose transporter protein 1 (GLUT1) has a promotive effect on the differentiation of CD8 + T cells into effector cells, and suppression of GLUT1 not only affects tumor metabolism but also induces the accumulation of reactive oxygen species (ROS), which mediates tumor cell apoptosis through activation of the TNF-a signaling pathway (75,76). The checkpoint blockade antibodies against CTLA-4, PD-1, and PD-L1, can restore glucose in tumor microenvironment, permitting T cell glycolysis and IFN-g production (77). ...
In the realm of oncology, the tumor microenvironment (TME)—comprising extracellular matrix components, immune cells, fibroblasts, and endothelial cells—plays a pivotal role in tumorigenesis, progression, and response to therapeutic interventions. Initially, the TME exhibits tumor-suppressive properties that can inhibit malignant transformation. However, as the tumor progresses, various factors induce immune tolerance, resulting in TME behaving in a state that promotes tumor growth and metastasis in later stages. This state of immunosuppression is crucial as it enables TME to change from a role of killing tumor cells to a role of promoting tumor progression. Gastric cancer is a common malignant tumor of the gastrointestinal tract with an alarmingly high mortality rate. While chemotherapy has historically been the cornerstone of treatment, its efficacy in prolonging survival remains limited. The emergence of immunotherapy has opened new therapeutic pathways, yet the challenge of immune tolerance driven by the gastric cancer microenvironment complicates these efforts. This review aims to elucidate the intricate role of the TME in mediating immune tolerance in gastric cancer and to spotlight innovative strategies and clinical trials designed to enhance the efficacy of immunotherapeutic approaches. By providing a comprehensive theoretical framework, this review seeks to advance the understanding and application of immunotherapy in the treatment of gastric cancer, ultimately contributing to improved patient outcomes.
... In addition, increased ROS levels also correlated with the TNF--mediated tumor-killing capacity in a caspase-8 and Fadd-dependent manner. [19] Furthermore, interleukin-2 (IL-2) [20] and IL-12, [21] which promote CD8 + T cell infiltration and expansion, were more than twice as high in the NP/OXA-ASP 2 -treated tumor compared to those of the controls, indicating robust activation of the T cell-mediated anticancer immune responses ( Figure 6E). Finally, the median survival time of mice in the NP/OXA-ASP 2 group was extended by 1.77-fold compared to the OXA plus ASP group ( Figure S48, Supporting Information). ...
Inappropriate glucose metabolism in cancer cells is associated with immunosuppressive tumor microenvironments (TMEs). Although glycolysis inhibition enhances T cell‐mediated immune responses, the integrated platforms combining glycolysis inhibition with immunotherapy remain underdeveloped. To address this gap, a glucose metabolism‐targeted poly(amino acid) nanoformulation of oxaliplatin(IV)‐aspirin prodrug (NP/OXA‐ASP2) is developed to improve chemo‐immunotherapy by suppressing tumor glycolysis. This poly(amino acid) nanoparticle exhibits selective release, discharging 90.0% of OXA‐ASP2 under reductive conditions within 36 h. Furthermore, over 80% of the prodrug converts to OXA and ASP within 12 h, promoting mitochondrial damage and glycolysis inhibition, which amplifies immunogenic cell death induced by OXA. In addition, suppressing glycolytic flux reduces lactate leakage, mitigating the immunosuppressive TMEs. Together, these mechanisms contribute to stronger chemo‐immunotherapy efficacy. Compared to the OXA plus ASP formulation, NP/OXA‐ASP2 demonstrates superior performances, reducing lactate levels at the tumor site by 25.4%, increasing the proportion of cytotoxic T lymphocytes by 1.53 times, decreasing the proportion of regulatory T cells by 2.20 times, and improving 1.39‐fold of the tumor inhibition rate. These findings underscore that NP/OXA‐ASP2 is a promising platform for integrating tumor metabolic regulation with immunomodulation and holds significant potential for advancing clinical chemo‐immunotherapy.
... Studies have found that metabolic competition in the immunosuppressive TME, such as lactate accumulation and glucose depletion, inhibits the function of effector T cells [213][214][215]. By using metabolic modulators, such as inhibitors of lactate dehydrogenase or glucose transporter protein, the metabolic state of the TME can be reprogrammed, restoring T-cell antitumor activity [216,217]. Furthermore, regulating mitochondrial function in tumor cells is also considered a potential approach to enhancing the efficacy of immunotherapy [218,219]. ...
Gastric cancer (GC) remains a leading cause of cancer-related mortality worldwide, with limited treatment options in advanced stages. Immunotherapy, particularly immune checkpoint inhibitors (ICIs) targeting PD1/PD-L1, has emerged as a promising therapeutic approach. However, a significant proportion of patients exhibit primary or acquired resistance, limiting the overall efficacy of immunotherapy. This review provides a comprehensive analysis of the mechanisms underlying immunotherapy resistance in GC, including the role of the tumor immune microenvironment, dynamic PD-L1 expression, compensatory activation of other immune checkpoints, and tumor genomic instability. Furthermore, the review explores GC-specific factors such as molecular subtypes, unique immune evasion mechanisms, and the impact of Helicobacter pylori infection. We also discuss emerging strategies to overcome resistance, including combination therapies, novel immunotherapeutic approaches, and personalized treatment strategies based on tumor genomics and the immune microenvironment. By highlighting these key areas, this review aims to inform future research directions and clinical practice, ultimately improving outcomes for GC patients undergoing immunotherapy.
... 46 The latest reports confirmed that inhibition of Glut1 genetically and pharmacologically sensitises tumours to antitumour immunity and synergises with anti-PD-1 therapy through the TNF-a pathway. 52 In our study, we also confirm the role of glucose metabolism restriction in reversing the increased cellsurface levels of PD-L1 and the resistance to PD-1/PD-L1 inhibitor therapy caused by EDEM3 overexpression. ...
Background
Immunotherapy is beneficial for some colorectal cancer (CRC) patients, but immunosuppressive networks limit its effectiveness. Cancer‐associatedfibroblasts (CAFs) are significant in immune escape and resistance toimmunotherapy, emphasizing the urgent need for new treatment strategies.
Methods
Flow cytometric, Western blotting, proteomics analysis, analysis of public database data, genetically modified cell line models, T cell coculture, crystal violetstaining, ELISA, metabonomic and clinical tumour samples were conducted to assess the role of EDEM3 in immune escape and itsmolecular mechanisms. We evaluated theeffects of FMD plus 2‐DG on antitumour immunity using multipleximmunofluorescence, flow cytometry, cytokine profiling, TUNEL assays, xenografttumours, and in vivo studies.
Results
We show thatCAFs upregulate PD‐L1 glycosylation and contribute to immune evasion byglycosyltransferase EDEM3. Additionally, EDEM3 plays a role in tumour immunityduring tumour progression. However, the EDEM3‐mediated upregulation of PD‐L1 expression underpins PD‐1/PD‐L1 blockade resistance in vivo. This finding contradictsthe previous trend that positive PD‐L1 expression indicates a strong responseto PD‐1/PD‐L1 blockade. Mechanistically, high‐EDEM3 expression facilitates M2‐like This finding contradictsthe previous trend that positive PD‐L1 expression indicates a strong responseto PD‐1/PD‐L1 blockade.Mechanistically, polarizationand chemotactic migration of macrophages, which are enriched in theperipheral region of tumours compared to thecore region, precluding access of CD8+ T cells to tumourfoci. Furthermore, we EDEM3 predominantly activates the recruited M2‐like macrophagesvia a glucose metabolism‐dependent mechanism. Manipulationof glucose utilization by a fasting‐mimicking diet(FMD) plus 2‐DG treatmentsynergistically with PD‐1 antibody elicits potent antitumour activity byeffectively decreasing tumour glycosylated PD‐L1 expression, augmenting the CD8+effector T cell infiltration and activation while concurrently reducing the infiltration.TheCAFs‐EDEM3‐M2‐like macrophage axis plays a critical role in promotingimmunotherapy resistance. infiltration.TheCAFs‐EDEM3‐M2‐like macrophage axis plays a critical role in promotingimmunotherapy resistance.
Conclusions
Our study suggests that blocking EDEM3‐induced M2‐like macro phage trafficking by FMD plus 2‐DG is a promising and effective strategy to overcomeresistance to checkpoint blockade therapy offeringhope for improved treatment outcomes.
Key points
Cancer‐associated fibroblasts (CAFs) can enhance PD‐L1 glycosylation through the glycosyltransferase EDEM3, contributing to immune evasion during tumour progression.
EDEM3 predominantly activates the recruit M2‐like macrophages via a glucose metabolism‐dependent mechanism.
Blocking glucose utilization antagonizes recruiting and polarizing M2‐like macrophages synergistically with PD‐1 antibody to improve anticancer immunity.
... [29] Tumor cells primarily obtain energy through aerobic glycolysis to meet the needs of proliferation and survival in adverse environments, ultimately leading to tumor progression. [30] Related studies have shown that MRTO4 promotes glycolysis in liver cancer cells by downregulating the expression of ALDOB, subsequently inducing the proliferative and invasive phenotype of liver cancer cells. [31] Therefore, it is speculated that the MRTO4 gene may play an important role in the progression of colorectal cancer. ...
Colorectal cancer refers to malignant tumors occurring in the walls of the colon or rectum. The roles of WD Repeat Domain 12 (WDR12) and mitochondrial ribosome-associated tumor suppressor 4 (MRTO4) genes in colorectal cancer remain unclear. The colorectal cancer dataset GSE113513 configuration file was downloaded from the gene expression omnibus database generated from GPL15207. Differentially expressed genes screening, functional enrichment analysis, gene set enrichment analysis, Weighted Gene Co-expression Network Analysis, construction and analysis of protein–protein interaction networks, survival analysis, and gene expression heatmap plotting were conducted. Comparative toxicogenomics database analysis was performed to find diseases most relevant to core genes. TargetScan was used to screen miRNAs regulating core genes. A total of 3106 differentially expressed genes were identified. According to gene ontology analysis, they mainly enriched in organic acid metabolic processes, condensed chromosome kinetochore, oxidoreductase activity, and cell cycle. In Kyoto encyclopedia of genes and genomes analysis, they primarily concentrated in the cell cycle, TGF-β signaling pathway, Jak-STAT signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, TNF signaling pathway, p53 signaling pathway, NF-kB signaling pathway, and WNT signaling pathway. Weighted Gene Co-expression Network Analysis with a soft thresholding power set to 12 generated 29 modules. The protein–protein interaction network identified 6 core genes (DDX27, NAT10, WDR12, DKC1, MRTO4, and NOP56). Survival analysis showed core genes (POSTN, MYH11, LUM, COL6A3, and COL4A1) as risk factors. Gene expression heatmap revealed high expression of core genes (WDR12 and MRTO4) in colorectal samples. Comparative toxicogenomics database analysis linked core genes (WDR12 and MRTO4) with local tumor infiltration, bowel obstruction, abdominal pain, and colorectal neoplasms. WDR12 and MRTO4 genes are highly expressed in colorectal cancer, potentially influencing its progression.
... At the molecular level, oxidative stress triggers several key pathways that contribute to these complications. ROS activate the NF-κB signaling pathway (Morgan and Liu 2011), leading to the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β (Wu et al. 2023). These cytokines exacerbate inflammation and apoptosis in placental and fetal cells. ...
Recurrent pregnancy loss (RPL) is the occurrence of two or more consecutive miscarriages before 20 weeks of gestation. Recent research has increasingly focused on the role of oxidative stress in RPL, providing insights into its underlying mechanisms and potential therapeutic targets. Oxidative stress arises from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, leading to cellular damage and inflammation. Oxidative stress has been implicated in disrupting placental blood flow, inducing apoptosis in fetal and placental cells, and exacerbating inflammatory responses, all of which can contribute to pregnancy loss. Elevated levels of ROS have been associated with compromised placental function, impaired fetal development, and increased risk of RPL. Additionally, oxidative stress can modulate maternal immune responses, potentially leading to immune-related pregnancy complications. This review synthesizes current evidence on the mechanisms by which oxidative stress contributes to RPL and highlights emerging research on potential interventions, including antioxidant therapies and lifestyle modifications. Understanding these mechanisms is crucial for developing effective preventive and therapeutic strategies to reduce the risk of RPL and improve pregnancy outcomes. Future research should focus on elucidating the specific pathways involved and exploring novel treatments aimed at mitigating oxidative damage during pregnancy.
... The interdependence of aerobic glycolysis and immune escape in cancer progression is underscored by the role of glycolytic metabolites in modulating key immune checkpoints, thereby directly influencing the immune system's ability to recognize and attack tumor cells (19). Immunotherapy for HCC represents a promising avenue for treatment, aiming to enhance the immune system's ability to recognize and eliminate cancer cells (20). ...
Introduction
Energy metabolism abnormity emerges as a crucial factor that facilitates tumorigenesis by accelerating aerobic glycolysis. However, the function of N⁶-methyladenosine (m⁶A) on hepatocellular carcinoma (HCC) aerobic glycolysis and immune escape is still unclear. Here, this investigation was intended to elucidate the regulation of m⁶A ‘reader’ IGF2BP1 involved in HCC aerobic glycolysis and immune escape.
Methods
The aerobic glycolysis was tested by glucose uptake, lactate, ATP generation and ECAR. The CD8⁺ T cell-mediated killing effect was tested by cytotoxicity, IFN-γ and granzyme B. The molecular interaction was confirmed by luciferase reporter assay, immunoprecipitation assay and chromatin immunoprecipitation (ChIP)-PCR.
Results
Elevated IGF2BP1 expression was associated with poor prognosis in HCC patients. Functionally, IGF2BP1 emerged as an oncogenic factor that accelerated HCC aerobic glycolysis (glucose uptake, lactate, ATP generation and ECAR) and oxaliplatin resistance. Meanwhile, IGF2BP1 repressed the activated CD8⁺ T cell-mediated killing effect (cytotoxicity, IFN-γ and granzyme B) and apoptosis of HCC cells, indicating a suppressed cytotoxic T-cell response. By recognizing and binding to the m⁶A-modified sites on c-Myc mRNA, IGF2BP1 enhanced the stability of c-Myc mRNA, consequently upregulating c-Myc expression. In addition, transcription factor c-Myc targeted the programmed death ligand 1 (PD-L1) promoter region to strengthen its transcription.
Discussion
Taken together, this study illustrates IGF2BP1 as a potential therapeutic target in HCC, aiming to disrupt the interplay between aberrant metabolism and immune escape.
... PDK1, a key regulator of MR, is elevated in liver metastasis of breast cancer patients and can promote tumor cell proliferation and migration by enhancing the Warburg effect (23). Glucose transporter 1 (GLUT1) inactivation leads to MR for oxidative phosphorylation, generating excess reactive oxygen species (ROS), and accumulated ROS enhances TNF-a-mediated tumor cell death (72). FASN promotes lymph node metastasis in cervical cancer via cholesterol reprogramming and lymphangiogenesis (73). ...
Background
Metabolic reprogramming (MR) in cancer (CA) has been a focus of intense research in the recent two decades. This phenomenon has attracted great interest because it offers potential targets for cancer therapy. To capture the intellectual landscape of this field, we conducted a bibliometric analysis to assess the scientific output, major contributors, and trends in the MR/CA research.
Methods
We performed a systematic search using the Web of Science to retrieve articles published on MR of cancer from 2006 until 2023. The bibliometric tools such as Biblioshiny, VOSviewer, and Microsoft Excel were used to identify the most prolific authors, institutions, citation patterns, and keywords. We also used co-citation analysis to map the conceptual structure of the field and identify influential publications. Furthermore, we examined the literature by analyzing publication years, citations, and research impact factors.
Results
A total of 4,465 publications about MR/CA were retrieved. Publications on MR/CA increased rapidly from 2006 to 2023. Frontiers in Oncology published the most papers, while Cell Metabolism had the most citations. Highly cited papers were mainly published in Cancer Cell, Nature, Cell, Science and Cell Metabolism. China and the United States led the way in publications and contributed the most to MR/CA research. The University of Texas System, Chinese Academy of Sciences, and Fudan University were the most productive institutions. The profitable authors were Deberardinis Ralph J and Chiarugi Paola. The current topics included MR in tumorigenesis and progression of CA, MR of tumor cells and tumor microenvironment, the effect of MR on the CA treatment, the underlying mechanisms of MR (such as gene regulation, epigenetics, extracellular vesicles, and gut microbiota), and the modulation of MR. Some topics such as tumor microenvironment, lipid MR, circular RNA, long noncoding RNA, exosome, prognostic model, and immunotherapy may be the focus of MR/CA research in the next few years.
Conclusion
This study evaluated the global scientific output in the field of MR/CA research, analyzing its quantitative characteristics. It identified some significant and distinguished papers and compiled information regarding the current status and evolving trends of MR/CA research.
... The enhancement of aerobic glycolysis is a significant feature of the cancer metabolic process (9). The process of glycolysis is the conversion of glucose to pyruvate, which eventually produces lactic acid (10). ...
Background
Lactate dehydrogenase A (LDHA) has been confirmed as a tumor promoter in various cancers, but its role in endometrial cancer remains unclear.
Methods
The Cancer Genome Atlas (TCGA), quantitative real-time polymerase chain reaction and the Human Protein Atlas were utilized to analyzed the LDHA expression in EC. The LDHA levels of patients with different clinical features were compared based on the TCGA cohort. The Genome Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis of LDHA-related genes were conducted by R language. The influence of LDHA knockdown on cell proliferation, apoptosis, migration and invasion was detected by in vitro experiment. The relationship between LDHA expression and immune infiltration was explored by Tumor Immune Estimation Resource 2.0 and Gene Expression Profiling Interactive Analysis. The association of LDHA level with N6-methyladenosine (m6A) modification and ferroptosis was investigated based on the TCGA-UCEC and the GEO cohort.
Results
The LDHA was overexpressed in EC tissues and EC cell lines, and had high predictive accuracy for the EC diagnosis. The LDHA level was associated with age, histological type, histologic grade, and radiation therapy. LDHA-related genes participated in multiple biological functions and signaling pathways. LDHA downregulation significantly promoted cell apoptosis and inhibited the proliferation, migration, and invasion of EC cells. LDHA expression was connected to multiple tumor-infiltrating lymphocytes (TILs), m6A-related genes, and ferroptosis-related genes.
Conclusion
LDHA has the potential to work as an EC biomarker associated with TILs, m6A modification, and ferroptosis in EC.
... In the 70 KEGG metabolic pathways, the MyD88 high expression group exhibited upregulation in 52 pathways, including the majority of pathways involved in amino acid metabolism, carbohydrate metabolism, lipid metabolism, and nucleic acid metabolism, which are well-recognized as oncogenic metabolic pathways (Fig. 4). However, within the MyD88 high expression group, the pathways of taurine and hypotaurine metabolism [13] and oxidative phosphorylation [14] were downregulated. These pathways are associated with the suppression of the tumor immune microenvironment. ...
... Cancer cells consume two-thirds of glucose while myeloid cells consume one-third in the tumor microenvironment (53)(54)(55). Glut1 was previously considered the primary transporter of the Rg3-lipo drug delivery system in cancer cells (24,25). Consistent with prior results, we found that tumor cells mostly expressed the Glut1-encoding gene Slc2a1, whereas myeloid cells predominantly expressed Slc2a3. ...
Liposomal drug delivery systems have revolutionized traditional cytotoxic drugs. However, the relative instability and toxicity of the existing liposomal drug delivery systems compromised their efficacy. Herein, we present Rg3-lipo, an innovative drug delivery system using a glycosyl moiety-enriched ginsenoside (Rg3). This system is distinguished by its glycosyl moieties exposed on the liposomal surface. These moieties imitate human cell membranes to stabilize and evade phagocytic clearance. The Rg3-lipo system loaded with paclitaxel (PTX-Rg3-lipo) demonstrated favorable bioavailability and safety in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. With its glycosyl moieties recognizing tumor cells via the glucose transporter Glut1, PTX-Rg3-lipo inhibited gastric, breast, and esophageal cancers in human cancer cell lines, tumor-bearing mice, and patient-derived xenograft models. These glycosyl moieties selectively targeted myeloid-derived suppressor cells (MDSCs) through the glucose transporter Glut3 to attenuate their immunosuppressive effect. The mechanism study revealed that Rg3-lipo suppressed glycolysis and downregulated the transcription factors c-Maf and Mafb overcoming the MDSC-mediated immunosuppressive microenvironment and enhancing PTX-Rg3-lipo's antitumor effect. Taken together, we supply substantial evidence for its advantageous bioavailability and safety in multiple animal models, including nonhuman primates, and Rg3-lipo's dual targeting of cancer cells and MDSCs. Further investigation regarding Rg3-lipo's druggability will be conducted in clinical trials.
... Metabolic reprogramming in both tumor cells and T cells plays a crucial role in tumor immunity, particularly in pancreatic ductal adenocarcinoma. Genetic screening is employed to explore fundamental metabolic pathways during pancreatic tumor progression and TME-induced dependencies, identifying potential therapeutic targets [164,165]. To pinpoint the metabolic pathways critical for T cell activation, a screening of a metabolite inhibitor library was conducted on sgRNA-modified Jurkat T cells and primary human T cells. ...
Adoptive T cell therapy is a pivotal strategy in cancer immunotherapy, demonstrating potent clinical efficacy. However, its limited durability often results in primary resistance. High-throughput screening technologies, which include both genetic and non-genetic approaches, facilitate the optimization of adoptive T cell therapies by enabling the selection of biologically significant targets or substances from extensive libraries. In this review, we examine advancements in high-throughput screening technologies and their applications in adoptive T cell therapies. We highlight the use of genetic screening for T cells, tumor cells, and other promising combination strategies, and elucidate the role of non-genetic screening in identifying small molecules and targeted delivery systems relevant to adoptive T cell therapies, providing guidance for future research and clinical applications.
... The glucose transporter type 1 (GLUT1) has been identified to be over-expressed on the membranes of colorectal cancer cells (CRC) [27,28], and relatively low expressed in the immune cells [29], which is a potential target for drug delivery for CRC. Thus, mannose ligands (the epimer of glucose that could specifically bind to GLUT1 [30]) were modified on the surface of CuPDA (CuPDA-PEGM) to endow it with targeting capability to enhance the drug delivery efficacy. ...
Background
Traditional chemotherapeutic agents suffer from a lack of selectivity, poor targeting ability, and drug resistance. Developing tumor-specific therapies is crucial for precisely eliminating tumors while circumventing toxicity to normal tissues. Disulfiram (DSF), an FDA-approved drug for treating alcohol dependence, exhibits antitumor effect by forming complexes with copper ions (Cu(DDC)2). Here, we developed a Cu-doped polydopamine-based nanosystem (DSF@CuPDA-PEGM) to achieve in situ generation of toxic Cu(DDC)2.
Results
In cancer cells with elevated H2O2 contents, CuPDA responsively degrades to release Cu ions and DSF, allowing on-site synthesis of Cu(DDC)2 with potent antitumor activity. DSF@CuPDA-PEGM exhibits excellent therapeutic efficacy against both drug-sensitive and drug-resistant cancer cells while minimizing toxicity to noncancerous cells. Moreover, DSF@CuPDA-PEGM promotes the immune response by inducing cancer cell immunogenic death, thereby augmenting anti-PD-1-based immune checkpoint blockade therapy.
Conclusion
A tumor-specifically degradable Cu-doped polydopamine-based nanosystem is developed to achieve in situ synthesis of antitumor compounds, providing a promising approach to precisely eliminate tumors and heighten chemo-immunotherapy.
Graphical Abstract
... IL-8 is known for its role in neutrophil chemotaxis and activation, contributing to neutrophil-mediated tissue damage [30]. TNF-α promotes the production of various cytokines involved in the body's defense mechanisms, with its levels rising significantly during early inflammation [31]. CRP, an acute-phase protein synthesized by the liver, reflects the severity of the body's stress response and serves as a major indicator of inflammation [32]. ...
Energy metabolism is indispensable for sustaining physiological functions in living organisms and assumes a pivotal role across physiological and pathological conditions. This review provides an extensive overview of advancements in energy metabolism research, elucidating critical pathways such as glycolysis, oxidative phosphorylation, fatty acid metabolism, and amino acid metabolism, along with their intricate regulatory mechanisms. The homeostatic balance of these processes is crucial; however, in pathological states such as neurodegenerative diseases, autoimmune disorders, and cancer, extensive metabolic reprogramming occurs, resulting in impaired glucose metabolism and mitochondrial dysfunction, which accelerate disease progression. Recent investigations into key regulatory pathways, including mechanistic target of rapamycin, sirtuins, and adenosine monophosphate-activated protein kinase, have considerably deepened our understanding of metabolic dysregulation and opened new avenues for therapeutic innovation. Emerging technologies, such as fluorescent probes, nano-biomaterials, and metabolomic analyses, promise substantial improvements in diagnostic precision. This review critically examines recent advancements and ongoing challenges in metabolism research, emphasizing its potential for precision diagnostics and personalized therapeutic interventions. Future studies should prioritize unraveling the regulatory mechanisms of energy metabolism and the dynamics of intercellular energy interactions. Integrating cutting-edge gene-editing technologies and multi-omics approaches, the development of multi-target pharmaceuticals in synergy with existing therapies such as immunotherapy and dietary interventions could enhance therapeutic efficacy. Personalized metabolic analysis is indispensable for crafting tailored treatment protocols, ultimately providing more accurate medical solutions for patients. This review aims to deepen the understanding and improve the application of energy metabolism to drive innovative diagnostic and therapeutic strategies.
The abnormal metabolism of tumor cells fulfills their high energy demands for rapid growth while simultaneously reshaping the tumor microenvironment (TME), which suppresses immune cell function and facilitates immune evasion. Herein, a peptide‐based nanocomplex (DCK@siGLUT1) that synergizes with photodynamic therapy (PDT) to disrupt tumor cell energy metabolism is developed. DCK@siGLUT1, utilizing a mitochondria‐targeting peptide (dKLA) selectively accumulates in mitochondria, where it impairs mitochondrial membrane integrity, disrupts energy metabolism, and induces apoptosis. Upon apoptosis, activated caspase‐3 (Casp3) cleaves DCK@siGLUT1, releasing siGLUT1 to silence glucose transporter 1 (GLUT1) expression, which further inhibits glucose uptake and intensifies metabolic collapse, thereby amplifying apoptotic effects. Moreover, Ce6, conjugated to dKLA, is co‐delivered to the mitochondria and, upon light activation, exacerbates mitochondrial damage and metabolic disruption. These combined mechanisms intensify oxidative stress and apoptosis, further activate Casp3, and promote DCK@siGLUT1 cleavage, thereby driving a self‐amplifying tumoricidal cascade. Furthermore, DCK@siGLUT1 effectively induces immunogenic cell death (ICD), triggers antitumor immune responses, and inhibits both primary and distant tumor growth and metastasis. This strategy offers a novel approach for targeting tumor energy metabolism in antitumor immunotherapy.
The glycolytic enzyme, fructose‐1,6‐bisphosphate aldolase B (ALDOB), is recognized for its key role in shaping tthe umor immune microenvironment. However, the precise ways in which it influences the CD8 ⁺ T cell immune response in colorectal cancer (CRC) are still largely unknown. This study is designed to elucidate the interplay between ALDOB and the immune system in CRC. We analyzed the high expression of ALDOB in CRC tissues and cells through bioinformatics, clinical samples and in vitro experiments, finding that it promoted tumor progression. Its high expression was negatively correlated with CD8 expression and positively correlated with PDL1 expression. Further cell experiments revealed that ALDOB overexpression enhanced the expression of WNT signaling pathway‐related proteins (β‐catenin and c‐myc), which in turn promoted PDL1 expression in CRC cells, inhibiting the proliferation and killing effect of CD8 ⁺ T cells in co‐culture systems. Our findings disclose how ALDOB influences CD8 ⁺ T cell recruitment and antitumor immune function, proposing it as a potential target for the treatment of CRC.
Higher and richer nutrient requirements are typical features that distinguish tumor cells from AU: cells, ensuring adequate substrates and energy sources for tumor cell proliferation and migration. Therefore, nutrient deprivation strategies based on targeted technologies can induce impaired cell viability in tumor cells, which are more sensitive than normal cells. In this review, nutrients that are required by tumor cells and related metabolic pathways are introduced, and anti‐tumor strategies developed to target nutrient deprivation are described. In addition to tumor cells, the nutritional and metabolic characteristics of other cells in the tumor microenvironment (including macrophages, neutrophils, natural killer cells, T cells, and cancer‐associated fibroblasts) and related new anti‐tumor strategies are also summarized. In conclusion, recent advances in anti‐tumor strategies targeting nutrient blockade are reviewed, and the challenges and prospects of these anti‐tumor strategies are discussed, which are of theoretical significance for optimizing the clinical application of tumor nutrition deprivation strategies.
GLUT1 is an essential glucose transporter, the expression of which increases in tumor cells, especially under conditions of hypoxia, and correlates with their active proliferation. This study aimed to investigate the relationship between GLUT1 expression and biological parameters and to evaluate the potential impact on survival in patients with radically treated non-small cell lung cancer (NSCLC). Forty-two patients who received radical treatment for NSCLC were involved in the study. Gender, age, smoking history, disease stage, and tumor histological type were considered when analyzing the data. GLUT1 antibodies were used to assess the degree of hypoxia. A semi-quantitative immunohistochemical score ranging from 0 to 12 was used for calculation. The chi2 and Student's t-test were used to compare categorical and parametric variables. The Cox proportional hazards model, the Kaplan-Meier method, and the Log-rank test were used to evaluate the effect of GLUT1 expression on survival. The results were considered statistically significant at p<0.05. A moderate correlation was found between GLUT1 expression and histological type of NSCLC (r=0.432, p<0.0001), sex (r=0.336, p<0.0009), and smoking (r=0.325, p<0.0009). GLUT1 overexpression was observed more in squamous cell carcinomas than in adenocarcinomas (p=0.0001). In patients with adenocarcinomas, the level of GLUT1 expression depended on age and T category. In patients with squamous cell carcinomas, GLUT1 expression was not associated with the studied clinicopathological characteristics. Patients with T1b-2a categories, without regional lymph node metastases, younger than 60, and non-smokers have better survival. Kaplan-Meier curves demonstrated no statistically significant differences in recurrence-free survival and overall survival between the patients with high and low GLUT1 (Log-rank p=0.3284 and Log-rank p=0.7161, respectively). In conclusion, GLUT1 overexpression is associated with squamous cell lung carcinomas. GLUT1 expression has no prognostic value and does not correlate with recurrence-free and overall survival in radically treated patients with NSCLC.
Mitochondria are essential double-membrane organelles with intricate structures and diverse functions within cells. Under normal physiological conditions, mitochondria regulate cellular metabolism and maintain energy homeostasis via the electron transport chain, mediate stem cell fate, and modulate reactive oxygen species production, playing a pivotal role in energy supply and lifespan extension. However, mitochondrial dysfunction can lead to various pathological changes, including cellular aging, necrosis, dysregulated tumor immunity, and the initiation and progression of cancer. Moreover, abnormal mitochondrial metabolism is closely associated with numerous diseases, such as neurodegenerative disorders, metabolic syndromes, and cancers. In recent years, targeting mitochondria has emerged as a promising anticancer strategy, aiming to modulate mitochondrial functions and metabolism for therapeutic benefits. Nonetheless, such approaches face limitations, including low delivery efficiency and insufficient specificity. This review systematically explores mitochondrial structure and function, their physiological and pathological roles, and the potential and challenges of mitochondria-targeted strategies in cancer therapy, providing insights for future research directions.
This study investigated the role of swimming exercise in regulating melanoma tumour growth and glycolysis in cancer cells, the specific mechanism involved was also studied. In our study, a murine melanoma tumour model was established to assess the impact of swimming on tumour growth. The mRNA and protein expressions were assessed using qRT-PCR, western blot, and IHC. The metabolic behavior of melanoma cells was examined through lactic acid level measurements and glucose consumption assessments. CCK-8 and colony formation assays were used to detect cell viability and proliferation. ELISA was employed to determine the levels of cytokines secreted by macrophages. The interaction between APOL3 and STAT3 was analyzed by dual luciferase reporter gene and ChIP assays. Our results demonstrated that swimming exercise suppressed melanoma growth in mice by suppressing glycolysis, which might be related to APOL3 upregulation. In addition, downregulation of APOL3 in melanoma was associated with poor prognosis, and APOL3 overexpression markedly suppressed melanoma cell proliferation by reducing glucose uptake and lactate production in vitro. Mechanistically, STAT3 directly down-regulated APOL3 transcription. Swimming upregulated APOL3 by inactivating the IL-6R-STAT3 signaling axis in melanoma cells by inhibiting the secretion of IL-6 by M2 macrophages. As expected, IL-6 secreted by M2 macrophages promoted glycolysis in melanoma cells by reducing APOL3 expression. In summary, swimming inactivated the IL-6R/STAT3 signaling axis in melanoma cells by inhibiting the secretion of IL-6 by M2 macrophages, which could suppress the growth of melanoma in the body by upregulating APOL3 to inhibit glycolysis.
Increased aerobic glycolysis is a metabolic hallmark of proinflammatory leukocytes including macrophages and T cells. To take up glucose from the environment and fuel glycolysis, activated leukocytes upregulate the glucose transporter GLUT1. The orally bioavailable selective GLUT1 inhibitor BAY-876 was developed primarily as an anti-tumor drug. Our study assessed its activity on activated macrophages and CD4+ T cells. BAY-876 significantly attenuated glucose uptake by cultured CD4+ T cells and macrophages by 41% and 15%, respectively. Extracellular flux analysis of activated CD4+ T cells in vitro showed that BAY-876 significantly decreases glycolytic proton flux rate and lactate production, effects that are accompanied by an increased oxidative phosphorylation-mediated ATP production rate, leaving intracellular ATP levels per cell unchanged. However, GLUT1 inhibition reduced CD4+ T cell proliferation without compromising cell viability and reduced IFN-γ secretion by 20%. Moreover, TNF secretion from macrophages was reduced by 27%. We conclude that GLUT1-specific inhibitors, like BAY-876, deserve further in vivo testing in a broad range of (auto-) inflammatory disease models.
Tumor escape mechanisms for immunotherapy include deficiencies in antigen presentation, diminishing adaptive CD8+ T cell antitumor activity. Although innate natural killer (NK) cells are triggered by loss of MHC class I, their response is often inadequate. To increase tumor susceptibility to both innate and adaptive immune elimination, we performed parallel genome-wide CRISPR-Cas9 knockout screens under NK and CD8+ T cell pressure. We identify all components, RNF31, RBCK1, and SHARPIN, of the linear ubiquitination chain assembly complex (LUBAC). Genetic and pharmacologic ablation of RNF31, an E3 ubiquitin ligase, strongly sensitizes cancer cells to NK and CD8+ T cell killing. This occurs in a tumor necrosis factor (TNF)-dependent manner, causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I. A small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF and greatly enhances bystander killing of MHC antigen-deficient tumor cells. These results merit exploration of RNF31 inhibition as a clinical pharmacological opportunity for immunotherapy-refractory cancers.
Pancreatic ductal adenocarcinoma (PDA) is an inherently immune cell deprived tumor, characterized by desmoplastic stroma and suppressive immune cells. Here we systematically dissect PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identify Vps4b and Rnf31 as essential factors required for escaping CD8+ T cell killing. For Vps4b we find that inactivation impairs autophagy, resulting in increased accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. For Rnf31 we demonstrate that it protects tumor cells from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction, a mechanism that is conserved in human PDA organoids. Orthotopic transplantation of Vps4b- or Rnf31 deficient pancreatic tumors into immune competent mice, moreover, reveals increased CD8+ T cell infiltration and effector function, and markedly reduced tumor growth. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system. Pancreatic cancer is characterized by an immunosuppressive microenvironment, leading to immune evasion. Here, based on in vitro and in vivo CRISPR screens, the authors identify Rnf31 and Vps4b as drivers of immune escape, showing that loss of their function leads to an increase in T cell-mediated killing and reduced tumor growth in preclinical pancreatic cancer models.
Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD+-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.
Background
Solid tumors such as pancreatic ductal adenocarcinoma (PDAC) comprise not just tumor cells but also a microenvironment with which the tumor cells constantly interact. Detailed characterization of the cellular composition of the tumor microenvironment is critical to the understanding of the disease and treatment of the patient. Single-cell transcriptomics has been used to study the cellular composition of different solid tumor types including PDAC. However, almost all of those studies used primary tumor tissues.
Methods
In this study, we employed a single-cell RNA sequencing technology to profile the transcriptomes of individual cells from dissociated primary tumors or metastatic biopsies obtained from patients with PDAC. Unsupervised clustering analysis as well as a new supervised classification algorithm, SuperCT, was used to identify the different cell types within the tumor tissues. The expression signatures of the different cell types were then compared between primary tumors and metastatic biopsies. The expressions of the cell type-specific signature genes were also correlated with patient survival using public datasets.
Results
Our single-cell RNA sequencing analysis revealed distinct cell types in primary and metastatic PDAC tissues including tumor cells, endothelial cells, cancer-associated fibroblasts (CAFs), and immune cells. The cancer cells showed high inter-patient heterogeneity, whereas the stromal cells were more homogenous across patients. Immune infiltration varies significantly from patient to patient with majority of the immune cells being macrophages and exhausted lymphocytes. We found that the tumor cellular composition was an important factor in defining the PDAC subtypes. Furthermore, the expression levels of cell type-specific markers for EMT ⁺ cancer cells, activated CAFs, and endothelial cells significantly associated with patient survival.
Conclusions
Taken together, our work identifies significant heterogeneity in cellular compositions of PDAC tumors and between primary tumors and metastatic lesions. Furthermore, the cellular composition was an important factor in defining PDAC subtypes and significantly correlated with patient outcome. These findings provide valuable insights on the PDAC microenvironment and could potentially inform the management of PDAC patients.
The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1–3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.
Acinar metaplasia is an initial step in a series of events that can lead to pancreatic cancer. Here we perform single-cell RNA-sequencing of mouse pancreas during the progression from preinvasive stages to tumor formation. Using a reporter gene, we identify metaplastic cells that originated from acinar cells and express two transcription factors, Onecut2 and Foxq1. Further analyses of metaplastic acinar cell heterogeneity define six acinar metaplastic cell types and states, including stomach-specific cell types. Localization of metaplastic cell types and mixture of different metaplastic cell types in the same pre-malignant lesion is shown. Finally, single-cell transcriptome analyses of tumor-associated stromal, immune, endothelial and fibroblast cells identify signals that may support tumor development, as well as the recruitment and education of immune cells. Our findings are consistent with the early, premalignant formation of an immunosuppressive environment mediated by interactions between acinar metaplastic cells and other cells in the microenvironment.
Metabolic reprogramming dictates the fate and function of stimulated T cells, yet these pathways can be suppressed in T cells in tumor microenvironments. We previously showed that glycolytic and mitochondrial adaptations directly contribute to reducing the effector function of renal cell carcinoma (RCC) CD8+ tumor-infiltrating lymphocytes (TILs). Here we define the role of these metabolic pathways in the activation and effector functions of CD8+ RCC TILs. CD28 costimulation plays a key role in augmenting T cell activation and metabolism, and is antagonized by the inhibitory and checkpoint immunotherapy receptors CTLA4 and PD-1. While RCC CD8+ TILs were activated at a low level when stimulated through the T cell receptor alone, addition of CD28 costimulation greatly enhanced activation, function, and proliferation. CD28 costimulation reprogrammed RCC CD8+ TIL metabolism with increased glycolysis and mitochondrial oxidative metabolism, possibly through upregulation of GLUT3. Mitochondria also fused to a greater degree, with higher membrane potential and overall mass. These phenotypes were dependent on glucose metabolism, as the glycolytic inhibitor 2-deoxyglucose both prevented changes to mitochondria and suppressed RCC CD8+ TIL activation and function. These data show that CD28 costimulation can restore RCC CD8+ TIL metabolism and function through rescue of T cell glycolysis that supports mitochondrial mass and activity.
The metabolic properties of tumor microenvironment (TME) are dynamically dysregulated to achieve immune escape and promote cancer cell survival. However, in vivo properties of glucose metabolism in cancer and immune cells are poorly understood and their clinical application to development of a biomarker reflecting immune functionality is still lacking.
Methods: We analyzed RNA-seq and fluorodeoxyglucose (FDG) positron emission tomography profiles of 63 lung squamous cell carcinoma (LUSC) specimens to correlate FDG uptake, expression of glucose transporters (GLUT) by RNA-seq and immune cell enrichment score (ImmuneScore). Single cell RNA-seq analysis in five lung cancer specimens was performed. We tested the GLUT3/GLUT1 ratio, the GLUT-ratio, as a surrogate representing immune metabolic functionality by investigating the association with immunotherapy response in two melanoma cohorts.
Results: ImmuneScore showed a negative correlation with GLUT1 (r = -0.70, p < 0.01) and a positive correlation with GLUT3 (r = 0.39, p < 0.01) in LUSC. Single-cell RNA-seq showed GLUT1 and GLUT3 were mostly expressed in cancer and immune cells, respectively. In immune-poor LUSC, FDG uptake was positively correlated with GLUT1 (r = 0.27, p = 0.04) and negatively correlated with ImmuneScore (r = -0.28, p = 0.04). In immune-rich LUSC, FDG uptake was positively correlated with both GLUT3 (r = 0.78, p = 0.01) and ImmuneScore (r = 0.58, p = 0.10). The GLUT-ratio was higher in anti-PD1 responders than nonresponders (p = 0.08 for baseline; p = 0.02 for on-treatment) and associated with a progression-free survival in melanoma patients who treated with anti-CTLA4 (p = 0.04).
Conclusions: Competitive uptake of glucose by cancer and immune cells in TME could be mediated by differential GLUT expression in these cells.
Ironing out a survival strategy
Metastatic cells display a notable ability to adapt to—and even thrive in—harsh microenvironments. One extreme example is leptomeningeal metastases (LM), cancer cells that enter a region of the central nervous system called the subarachnoid space, which is filled with cerebral spinal fluid (CSF). This anatomic site has a limited supply of micronutrients such as iron, and it harbors immune cells. Chi et al. used single-cell RNA sequencing to study CSF samples from cancer patients with LM (see the Perspective by Garzia and Taylor). They found that LM cells express and use components of a high-affinity iron-capturing system. Through this mechanism, the LM cells avoid the adverse effects of iron deprivation and potentially escape immune attack by limiting the supply of iron to macrophages.
Science this issue p. 276 ; see also p. 250
We developed Lisa (http://lisa.cistrome.org/) to predict the transcriptional regulators (TRs) of differentially expressed or co-expressed gene sets. Based on the input gene sets, Lisa first uses histone mark ChIP-seq and chromatin accessibility profiles to construct a chromatin model related to the regulation of these genes. Using TR ChIP-seq peaks or imputed TR binding sites, Lisa probes the chromatin models using in silico deletion to find the most relevant TRs. Applied to gene sets derived from targeted TF perturbation experiments, Lisa boosted the performance of imputed TR cistromes and outperformed alternative methods in identifying the perturbed TRs.
Single-cell RNA sequencing (scRNA-seq) enables the systematic identification of cell populations in a tissue, but characterizing their spatial organization remains challenging. We combine a microarray-based spatial transcriptomics method that reveals spatial patterns of gene expression using an array of spots, each capturing the transcriptomes of multiple adjacent cells, with scRNA-Seq generated from the same sample. To annotate the precise cellular composition of distinct tissue regions, we introduce a method for multimodal intersection analysis. Applying multimodal intersection analysis to primary pancreatic tumors, we find that subpopulations of ductal cells, macrophages, dendritic cells and cancer cells have spatially restricted enrichments, as well as distinct coenrichments with other cell types. Furthermore, we identify colocalization of inflammatory fibroblasts and cancer cells expressing a stress-response gene module. Our approach for mapping the architecture of scRNA-seq-defined subpopulations can be applied to reveal the interactions inherent to complex tissues. Combining single-cell RNA-seq data and microarray-based spatial transcriptomics maps the location of different cell types and cell states in pancreatic tumors.
Immune-checkpoint blockade (ICB) has demonstrated efficacy in many tumor types, but predictors of responsiveness to anti-PD1 ICB are incompletely characterized. In this study, we analyzed a clinically annotated cohort of patients with melanoma (n = 144) treated with anti-PD1 ICB, with whole-exome and whole-transcriptome sequencing of pre-treatment tumors. We found that tumor mutational burden as a predictor of response was confounded by melanoma subtype, whereas multiple novel genomic and transcriptomic features predicted selective response, including features associated with MHC-I and MHC-II antigen presentation. Furthermore, previous anti-CTLA4 ICB exposure was associated with different predictors of response compared to tumors that were naive to ICB, suggesting selective immune effects of previous exposure to anti-CTLA4 ICB. Finally, we developed parsimonious models integrating clinical, genomic and transcriptomic features to predict intrinsic resistance to anti-PD1 ICB in individual tumors, with validation in smaller independent cohorts limited by the availability of comprehensive data. Broadly, we present a framework to discover predictive features and build models of ICB therapeutic response.
Tumor‐infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA‐seq (scRNA‐seq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early‐stage non‐small cell lung cancer (NSCLC) patients. Our scRNA‐seq analyses identified 11 485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor‐mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte‐to‐M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (eg, MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (eg, CXCL2, IL1B) with significantly enriched functions (TNF‐α signaling via NF‐κB and inflammatory response). Our analysis further identified a co‐regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte‐to‐M2 differentiation, and activated ligand‐receptor interactions (eg, SFTPA1‐TLR2, ICAM1‐ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte‐to‐M2 differentiation. Overall, our study identified the prevalent monocyte‐to‐M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand‐receptor interactions.
Immune checkpoint inhibitors have been successful across several tumor types; however, their efficacy has been uncommon and unpredictable in glioblastomas (GBM), where <10% of patients show long-term responses. To understand the molecular determinants of immunotherapeutic response in GBM, we longitudinally profiled 66 patients, including 17 long-term responders, during standard therapy and after treatment with PD-1 inhibitors (nivolumab or pembrolizumab). Genomic and transcriptomic analysis revealed a significant enrichment of PTEN mutations associated with immunosuppressive expression signatures in non-responders, and an enrichment of MAPK pathway alterations (PTPN11, BRAF) in responders. Responsive tumors were also associated with branched patterns of evolution from the elimination of neoepitopes as well as with differences in T cell clonal diversity and tumor microenvironment profiles. Our study shows that clinical response to anti-PD-1 immunotherapy in GBM is associated with specific molecular alterations, immune expression signatures, and immune infiltration that reflect the tumor’s clonal evolution during treatment. © 2019, The Author(s), under exclusive licence to Springer Nature America, Inc.
Cancer treatment by immune checkpoint blockade (ICB) can bring long-lasting clinical benefits, but only a fraction of patients respond to treatment. To predict ICB response, we developed TIDE, a computational method to model two primary mechanisms of tumor immune evasion: the induction of T cell dysfunction in tumors with high infiltration of cytotoxic T lymphocytes (CTL) and the prevention of T cell infiltration in tumors with low CTL level. We identified signatures of T cell dysfunction from large tumor cohorts by testing how the expression of each gene in tumors interacts with the CTL infiltration level to influence patient survival. We also modeled factors that exclude T cell infiltration into tumors using expression signatures from immunosuppressive cells. Using this framework and pre-treatment RNA-Seq or NanoString tumor expression profiles, TIDE predicted the outcome of melanoma patients treated with first-line anti-PD1 or anti-CTLA4 more accurately than other biomarkers such as PD-L1 level and mutation load. TIDE also revealed new candidate ICB resistance regulators, such as SERPINB9, demonstrating utility for immunotherapy research.
Clinical studies support the efficacy of programmed cell death 1 (PD-1) targeted therapy in a subset of patients with metastatic gastric cancer (mGC). With the goal of identifying determinants of response, we performed molecular characterization of tissues and circulating tumor DNA (ctDNA) from 61 patients with mGC who were treated with pembrolizumab as salvage treatment in a prospective phase 2 clinical trial. In patients with microsatellite instability-high and Epstein-Barr virus-positive tumors, which are mutually exclusive, dramatic responses to pembrolizumab were observed (overall response rate (ORR) 85.7% in microsatellite instability-high mGC and ORR 100% in Epstein-Barr virus-positive mGC). For the 55 patients for whom programmed death-ligand 1 (PD-L1) combined positive score positivity was available (combined positive score cut-off value ≥1%), ORR was significantly higher in PD-L1(+) gastric cancer when compared to PD-L1(-) tumors (50.0% versus 0.0%, P value <0.001). Changes in ctDNA levels at six weeks post-treatment predicted response and progression-free survival, and decreased ctDNA was associated with improved outcomes. Our findings provide insight into the molecular features associated with response to pembrolizumab in patients with mGC and provide biomarkers potentially relevant for the selection of patients who may derive greater benefit from PD-1 inhibition.
Cancer cells are embedded in the tumor microenvironment (TME), a complex ecosystem of stromal cells. Here, we present a 52,698-cell catalog of the TME transcriptome in human lung tumors at single-cell resolution, validated in independent samples where 40,250 additional cells were sequenced. By comparing with matching non-malignant lung samples, we reveal a highly complex TME that profoundly molds stromal cells. We identify 52 stromal cell subtypes, including novel subpopulations in cell types hitherto considered to be homogeneous, as well as transcription factors underlying their heterogeneity. For instance, we discover fibroblasts expressing different collagen sets, endothelial cells downregulating immune cell homing and genes coregulated with established immune checkpoint transcripts and correlating with T-cell activity. By assessing marker genes for these cell subtypes in bulk RNA-sequencing data from 1,572 patients, we illustrate how these correlate with survival, while immunohistochemistry for selected markers validates them as separate cellular entities in an independent series of lung tumors. Hence, in providing a comprehensive catalog of stromal cells types and by characterizing their phenotype and co-optive behavior, this resource provides deeper insights into lung cancer biology that will be helpful in advancing lung cancer diagnosis and therapy.
We describe results from IMmotion150, a randomized phase 2 study of atezolizumab (anti-PD-L1) alone or combined with bevacizumab (anti-VEGF) versus sunitinib in 305 patients with treatment-naive metastatic renal cell carcinoma. Co-primary endpoints were progression-free survival (PFS) in intent-to-treat and PD-L1+ populations. Intent-to-treat PFS hazard ratios for atezolizumab + bevacizumab or atezolizumab monotherapy versus sunitinib were 1.0 (95% confidence interval (CI), 0.69-1.45) and 1.19 (95% CI, 0.82-1.71), respectively; PD-L1+ PFS hazard ratios were 0.64 (95% CI, 0.38-1.08) and 1.03 (95% CI, 0.63-1.67), respectively. Exploratory biomarker analyses indicated that tumor mutation and neoantigen burden were not associated with PFS. Angiogenesis, T-effector/IFN-γ response, and myeloid inflammatory gene expression signatures were strongly and differentially associated with PFS within and across the treatments. These molecular profiles suggest that prediction of outcomes with anti-VEGF and immunotherapy may be possible and offer mechanistic insights into how blocking VEGF may overcome resistance to immune checkpoint blockade.
Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)–based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8⁺ T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8⁺ T cell–mediated killing and blunted antitumor immune responses in vivo. Deletion of a number of genes in the TNF pathway also emerged as the key mechanism of immune evasion from primary NK cells. Our screens also identified that the metabolic protein 2-aminoethanethiol dioxygenase (Ado) modulates sensitivity to TNF-mediated killing by cytotoxic lymphocytes and is required for optimal control of tumors in vivo. Remarkably, we found that tumors delete the same genes when exposed to perforin-deficient CD8⁺ T cells, demonstrating that the dominant immune evasion strategy used by tumor cells is acquired resistance to T cell–derived cytokine-mediated antitumor effects. We demonstrate that TNF-mediated bystander killing is a potent T cell effector mechanism capable of killing antigen-negative tumor cells. In addition to highlighting the importance of TNF in CD8⁺ T cell– and NK cell–mediated killing of tumor cells, our study also provides a comprehensive picture of the roles of the TNF, IFN, and antigen presentation pathways in immune-mediated tumor surveillance.
Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor β (TGFβ) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFβ-blocking and anti-PD-L1 antibodies reduced TGFβ signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFβ shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.
Tumor infiltrating leukocytes (TILs) are an integral component of the tumor microenvironment and have been found to correlate with prognosis and response to therapy. Methods to enumerate immune subsets such as immunohistochemistry or flow cytometry suffer from limitations in phenotypic markers and can be challenging to practically implement and standardize. An alternative approach is to acquire aggregative high dimensional data from cellular mixtures and to subsequently infer the cellular components computationally. We recently described CIBERSORT, a versatile computational method for quantifying cell fractions from bulk tissue gene expression profiles (GEPs). Combining support vector regression with prior knowledge of expression profiles from purified leukocyte subsets, CIBERSORT can accurately estimate the immune composition of a tumor biopsy. In this chapter, we provide a primer on the CIBERSORT method and illustrate its use for characterizing TILs in tumor samples profiled by microarray or RNA-Seq.
SNF'ing out antitumor immunity
Immune checkpoint inhibitors induce durable tumor regressions in some, but not all, cancer patients. Understanding the mechanisms that determine tumor sensitivity to these drugs could potentially expand the number of patients who benefit (see the Perspective by Ghorani and Quezada). Pan et al. discovered that tumor cells in which a specific SWI/SNF chromatin remodeling complex had been experimentally inactivated were more sensitive to T cell–mediated killing. The cells were more responsive to interferon-γ, leading to increased secretion of cytokines that promote antitumor immunity. Miao et al. examined the genomic features of tumors from patients with metastatic renal cell carcinoma who had been treated with immune checkpoint inhibitors. Tumors harboring inactivating mutations in PBRM1 , which encodes a subunit of the same SWI/SNF complex, were more likely to respond to the drugs.
Science , this issue p. 770 , p. 801 ; see also p. 745
Tissues are complex milieus consisting of numerous cell types. Several recent methods have attempted to enumerate cell subsets from transcriptomes. However, the available methods have used limited sources for training and give only a partial portrayal of the full cellular landscape. Here we present xCell, a novel gene signature-based method, and use it to infer 64 immune and stromal cell types. We harmonized 1822 pure human cell type transcriptomes from various sources and employed a curve fitting approach for linear comparison of cell types and introduced a novel spillover compensation technique for separating them. Using extensive in silico analyses and comparison to cytometry immunophenotyping, we show that xCell outperforms other methods. xCell is available at http://xCell.ucsf.edu/.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-017-1349-1) contains supplementary material, which is available to authorized users.
ELife digest
Malignant tumors do not only contain cancer cells. Normal cells from the body also infiltrate tumors. These often include a variety of immune cells that can help detect and kill cancer cells. Many evidences suggest that the proportion of different immune cell types in a tumor can affect tumor growth and which treatments are effective.
Researchers often study tumors by measuring the expression of genes, i.e., which genes are active in tumors. However, the proportion of different cell types in the tumor is often not measured for tumors studied at the gene expression level.
Racle et al. have now demonstrated that a new computer-based tool can accurately detect all the main cell types in a tumor directly from the expression of genes in this tumor. The tool is called “Estimating the Proportion of Immune and Cancer cells” – or EPIC for short. It compares the level of expression of genes in a tumor with a library of the gene expression profiles from specific cell types that can be found in tumors and uses this information to predict how many of each type of cell are present. Experimental measurements of several human tumors confirmed that EPIC’s predictions are accurate.
EPIC is freely available online. Since the active genes in tumors from many patients have already been documented together with clinical data, researchers could use EPIC to investigate whether the cell types in a tumor affect how harmful it is or how well a particular treatment works on it. In the future, this information could help to identify the best treatment for a particular patient and may reveal new genes that cause malignant tumors to develop and grow.
Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.
Single-cell transcriptome profiling of tumour tissue isolates allows the characterization of heterogeneous tumour cells along with neighbouring stromal and immune cells. Here we adopt this powerful approach to breast cancer and analyse 515 cells from 11 patients. Inferred copy number variations from the single-cell RNA-seq data separate carcinoma cells from non-cancer cells. At a single-cell resolution, carcinoma cells display common signatures within the tumour as well as intratumoral heterogeneity regarding breast cancer subtype and crucial cancer-related pathways. Most of the non-cancer cells are immune cells, with three distinct clusters of T lymphocytes, B lymphocytes and macrophages. T lymphocytes and macrophages both display immunosuppressive characteristics: T cells with a regulatory or an exhausted phenotype and macrophages with an M2 phenotype. These results illustrate that the breast cancer transcriptome has a wide range of intratumoral heterogeneity, which is shaped by the tumour cells and immune cells in the surrounding microenvironment.
High expression of PD-L1 in tumor cells contributes to tumor immune evasion. However, whether PD-L1 expression in tumor cells is regulated by the availability of nutrients is unknown. Here, we show that in human glioblastoma cells, high glucose promotes hexokinase (HK) 2 dissociation from mitochondria and its subsequent binding and phosphorylation of IκBα at T291. This leads to increased interaction between IκBα and μ-calpain protease and subsequent μ-calpain-mediated IκBα degradation and NF-κB activation-dependent transcriptional upregulation of PD-L1 expression. Expression of IκBα T291A in glioblastoma cells blocked high glucose-induced PD-L1 expression and promoted CD8⁺ T cell activation and infiltration into the tumor tissue, reducing brain tumor growth. Combined treatment with an HK inhibitor and an anti-PD-1 antibody eliminates tumor immune evasion and remarkably enhances the anti-tumor effect of immune checkpoint blockade. These findings elucidate a novel mechanism underlying the upregulation of PD-L1 expression mediated by aerobic glycolysis and underscore the roles of HK2 as a glucose sensor and a protein kinase in regulation of tumor immune evasion.
The tumor microenvironment (TME) is a unique metabolic niche that can inhibit T cell metabolism and cytotoxicity. To dissect the metabolic interplay between tumors and T cells, we establish an in vitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as an inhibitor of CD8⁺ T cell cytotoxicity, revealing an unexpected metabolic shunt in the TCA cycle. Metabolically fit cytotoxic T cells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1). Cytotoxic T cells are reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates. By contrast, lactate reduces PC-mediated anaplerosis. The inhibition of pyruvate dehydrogenase (PDH) is sufficient to restore PC activity, succinate secretion, and the activation of SUCNR1. These studies identify PDH as a potential drug target to allow CD8⁺ T cells to retain cytotoxicity and overcome a lactate-enriched TME.
Interferon-gamma (IFN-γ) has pleiotropic effects on cancer immune checkpoint blockade (ICB), including roles in ICB resistance. We analyzed gene expression in ICB-sensitive versus ICB-resistant tumor cells and identified a strong association between interferon-mediated resistance and expression of Ripk1, a regulator of tumor necrosis factor (TNF) superfamily receptors. Genetic interaction screening revealed that in cancer cells, RIPK1 diverted TNF signaling through NF-κB and away from its role in cell death. This promoted an immunosuppressive chemokine program by cancer cells, enhanced cancer cell survival, and decreased infiltration of T and NK cells expressing TNF superfamily ligands. Deletion of RIPK1 in cancer cells compromised chemokine secretion, decreased ARG1⁺ suppressive myeloid cells linked to ICB failure in mice and humans, and improved ICB response driven by CASP8-killing and dependent on T and NK cells. RIPK1-mediated resistance required its ubiquitin scaffolding but not kinase function. Thus, cancer cells co-opt RIPK1 to promote cell-intrinsic and cell-extrinsic resistance to immunotherapy.
In brief Hochrein et al. report that inflammatory T cells express high levels of GLUT3. Ablation of GLUT3 curtailed Th17-cell-mediated immune responses and protected mice from autoimmune colitis and encephalomyelitis. GLUT3-dependent glucose metabolism controls the generation of nucleo-cytosolic acetyl-CoA and the epigenetic regulation of cytokine responses through histone acetylation. SUMMARY Metabolic reprogramming is a hallmark of activated T cells. The switch from oxidative phosphorylation to aer-obic glycolysis provides energy and intermediary metabolites for the biosynthesis of macromolecules to support clonal expansion and effector function. Here, we show that glycolytic reprogramming additionally controls inflammatory gene expression via epigenetic remodeling. We found that the glucose transporter GLUT3 is essential for the effector functions of Th17 cells in models of autoimmune colitis and encephalomyelitis. At the molecular level, we show that GLUT3-dependent glucose uptake controls a metabolic-transcriptional circuit that regulates the pathogenicity of Th17 cells. Metabolomic, epigenetic, and transcriptomic analyses linked GLUT3 to mitochondrial glucose oxidation and ACLY-dependent acetyl-CoA generation as a rate-limiting step in the epigenetic regulation of inflammatory gene expression. Our findings are also important from a translational perspective because inhibiting GLUT3-dependent acetyl-CoA generation is a promising metabolic checkpoint to mitigate Th17-cell-mediated inflammatory diseases.
The hallmarks of cancer conceptualization is a heuristic tool for distilling the vast complexity of cancer phenotypes and genotypes into a provisional set of underlying principles. As knowledge of cancer mechanisms has progressed, other facets of the disease have emerged as potential refinements. Herein, the prospect is raised that phenotypic plasticity and disrupted differentiation is a discrete hallmark capability, and that nonmutational epigenetic reprogramming and polymorphic microbiomes both constitute distinctive enabling characteristics that facilitate the acquisition of hallmark capabilities. Additionally, senescent cells, of varying origins, may be added to the roster of functionally important cell types in the tumor microenvironment.
Significance
Cancer is daunting in the breadth and scope of its diversity, spanning genetics, cell and tissue biology, pathology, and response to therapy. Ever more powerful experimental and computational tools and technologies are providing an avalanche of “big data” about the myriad manifestations of the diseases that cancer encompasses. The integrative concept embodied in the hallmarks of cancer is helping to distill this complexity into an increasingly logical science, and the provisional new dimensions presented in this perspective may add value to that endeavor, to more fully understand mechanisms of cancer development and malignant progression, and apply that knowledge to cancer medicine.
One hundred years have passed since Warburg discovered alterations in cancer metabolism, more than 70 years since Sidney Farber introduced anti-folates that transformed the treatment of childhood leukaemia, and 20 years since metabolism was linked to oncogenes. However, progress in targeting cancer metabolism therapeutically in the past decade has been limited. Only a few metabolism-based drugs for cancer have been successfully developed, some of which are in — or en route to — clinical trials. Strategies for targeting the intrinsic metabolism of cancer cells often did not account for the metabolism of non-cancer stromal and immune cells, which have pivotal roles in tumour progression and maintenance. By considering immune cell metabolism and the clinical manifestations of inborn errors of metabolism, it may be possible to isolate undesirable off-tumour, on-target effects of metabolic drugs during their development. Hence, the conceptual framework for drug design must consider the metabolic vulnerabilities of non-cancer cells in the tumour immune microenvironment, as well as those of cancer cells. In this Review, we cover the recent developments, notable milestones and setbacks in targeting cancer metabolism, and discuss the way forward for the field. Despite the link between metabolism and oncogenes, very few metabolism-based drugs for cancer have been successfully developed. This Review covers the setbacks and recent developments in targeting cancer metabolism, and discusses the path forward for the field.
Precision oncology has made significant advances, mainly by targeting actionable mutations in cancer driver genes. Aiming to expand treatment opportunities, recent studies have begun to explore the utility of tumor transcriptome to guide patient treatment. Here, we introduce SELECT (synthetic lethality and rescue-mediated precision oncology via the transcriptome), a precision oncology framework harnessing genetic interactions to predict patient response to cancer therapy from the tumor transcriptome. SELECT is tested on a broad collection of 35 published targeted and immunotherapy clinical trials from 10 different cancer types. It is predictive of patients’ response in 80% of these clinical trials and in the recent multi-arm WINTHER trial. The predictive signatures and the code are made publicly available for academic use, laying a basis for future prospective clinical studies.
Although T cell checkpoint inhibitors have transformed the treatment of cancer, the molecular determinants of tumor cell sensitivity to T cell-mediated killing need further elucidation. Here, we describe a mouse genome-scale CRISPR knockout screen that identifies tumor cell TNFα signaling as an important component of T cell-induced apoptosis, with NF-κB signaling and autophagy as major protective mechanisms. Knockout of individual autophagy genes sensitized tumor cells to killing by T cells that were activated via specific TCR or by a CD3 bispecific antibody. Conversely, inhibition of mTOR signaling, which results in increased autophagic activity, protected tumor cells from T cell killing. Autophagy functions at a relatively early step in the TNFα signaling pathway, limiting FADD-dependent caspase-8 activation. Genetic inactivation of tumor cell autophagy enhanced the efficacy of immune checkpoint blockade in mouse tumor models. Thus, targeting the protective autophagy pathway might sensitize tumors to T cell-engaging immunotherapies in the clinic.
Purpose:
Pembrolizumab improved survival in recurrent or metastatic head and neck squamous-cell carcinoma (HNSCC) patients. The aims of this study were to determine if pembrolizumab would be safe, result in pathologic tumor response (pTR), and lower the relapse rate in patients with resectable human papillomavirus (HPV)-unrelated HNSCC.
Methods:
Neoadjuvant pembrolizumab (200 mg) was administered and followed 2-3 weeks later by surgical tumor ablation. Post-operative (chemo) radiation was planned. High-risk pathology patients (positive margins and/or extranodal extension) received adjuvant pembrolizumab. pTR was quantified as the proportion of the resection bed with tumor necrosis, keratinous debris, and giant cells/histiocytes: pTR-0 (<10%), pTR-1 (10-49%), and pTR-2 (≥50%). Co-primary endpoints were pTR-2 among all patients and one-year relapse rate in patients with high-risk pathology (historical: 35%). Correlations of baseline PD-L1 and T-cell infiltration with pTR were assessed. Tumor clonal dynamics were evaluated (ClinicalTrials.gov NCT02296684).
Results:
Thirty-six patients enrolled. After neoadjuvant pembrolizumab, serious (grades 3-4) adverse events and unexpected surgical delays/complications did not occur. pTR-2 occurred in eight patients (22%), and pTR-1 in eight other patients (22%). One-year relapse rate among eighteen patients with high-risk pathology was 16.7% (95%CI: 3.6-41.4%). pTR≥10% correlated with baseline tumor PD-L1, immune infiltrate, and IFN-γ activity. Matched samples showed upregulation of inhibitory checkpoints in patients with pTR-0, and confirmed clonal loss in some patients.
Conclusions:
Among patients with locally advanced, HPV-unrelated HNSCC, pembrolizumab was safe, and any pathologic response was observed in 44% of patients with 0% pathologic complete responses. The one-year relapse rate in patients with high-risk-pathology was lower than historical.
Single-cell transcriptomics has transformed our ability to characterize cell states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to measure distinct cellular modalities, a key analytical challenge is to integrate these datasets to better understand cellular identity and function. Here, we develop a strategy to "anchor" diverse datasets together, enabling us to integrate single-cell measurements not only across scRNA-seq technologies, but also across different modalities. After demonstrating improvement over existing methods for integrating scRNA-seq data, we anchor scRNA-seq experiments with scATAC-seq to explore chromatin differences in closely related interneuron subsets and project protein expression measurements onto a bone marrow atlas to characterize lymphocyte populations. Lastly, we harmonize in situ gene expression and scRNA-seq datasets, allowing transcriptome-wide imputation of spatial gene expression patterns. Our work presents a strategy for the assembly of harmonized references and transfer of information across datasets.
Cancer immunotherapies provide survival benefits in responding patients, but many patients fail to respond. Identifying the biology of treatment response and resistance are a priority to optimize drug selection and improve patient outcomes. We performed transcriptomic and immune profiling on 158 tumor biopsies from melanoma patients treated with anti-PD-1 monotherapy (n = 63) or combined anti-PD-1 and anti-CTLA-4 (n = 57). These data identified activated T cell signatures and T cell populations in responders to both treatments. Further mass cytometry analysis identified an EOMES+CD69+CD45RO+ effector memory T cell phenotype that was significantly more abundant in responders to combined immunotherapy compared with non-responders (n = 18). The gene expression profile of this population was associated with longer progression-free survival in patients treated with single agent and greater tumor shrinkage in both treatments.
‘Immune checkpoint blockade’ for cancer describes the use of therapeutic antibodies that disrupt negative immune regulatory checkpoints and unleash pre-existing antitumour immune responses. Antibodies targeting the checkpoint molecules cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death 1 (PD1) and PD1 ligand 1 (PD-L1) have had early success in the clinic, which has led to approval by the US Food and Drug Administration of multiple agents in several cancer types. Yet, clinicians still have very limited tools to discriminate a priori patients who will and will not respond to treatment. This has fuelled a wave of research into the molecular mechanisms of tumour-intrinsic resistance to immune checkpoint blockade, leading to the rediscovery of biological processes critical to antitumour immunity, namely interferon signalling and antigen presentation. Other efforts have shed light on the immunological implications of canonical cancer signalling pathways, such as WNT–β-catenin signalling, cell cycle regulatory signalling, mitogen-activated protein kinase signalling and pathways activated by loss of the tumour suppressor phosphoinositide phosphatase PTEN. Here we review each of these molecular mechanisms of resistance and explore ongoing approaches to overcome resistance to immune checkpoint blockade and expand the spectrum of patients who can benefit from immune checkpoint blockade. Understanding why some patients and not others respond to immune checkpoint blockade for cancer is crucial for extending benefit from this therapy. Here the authors describe how tumour cells can resist immune checkpoint blockade, for example, by resistance to interferon signalling and through immune-evasive oncogenic signalling pathways.
Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.
Tumor-infiltrating myeloid cells (TIMs)comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq)to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients’ blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets.
Adoptive T cell therapy (ACT) produces durable responses in some cancer patients; however, most tumors are refractory to ACT and the molecular mechanisms underlying resistance are unclear. Using two independent approaches, we identified tumor glycolysis as a pathway associated with immune resistance in melanoma. Glycolysis-related genes were upregulated in melanoma and lung cancer patient samples poorly infiltrated by T cells. Overexpression of glycolysis-related molecules impaired T cell killing of tumor cells, whereas inhibition of glycolysis enhanced T cell-mediated antitumor immunity in vitro and in vivo. Moreover, glycolysis-related gene expression was higher in melanoma tissues from ACT-refractory patients, and tumor cells derived from these patients exhibited higher glycolytic activity. We identified reduced levels of IRF1 and CXCL10 immunostimulatory molecules in highly glycolytic melanoma cells. Our findings demonstrate that tumor glycolysis is associated with the efficacy of ACT and identify the glycolysis pathway as a candidate target for combinatorial therapeutic intervention.
Chemokines are mostly known for their chemotactic properties, and less for their ability to direct the biological function of target cells, including T cells. The current review focuses on a key chemokine named CXCL10 and its role in directing the migratory propertied and biological function of CD4+ and CD8+ T cells in the context of cancer and inflammatory autoimmunity.
CXCR3 is a chemokine receptor that is abundant on CD4+ T cells, CD8+ T cells and NK cells. It has three known ligands: CXCL9, CXCL10 and CXCL11. Different studies, including those coming form our laboratory, indicated that aside of attracting CD8+ and CD4+ effector T cells to tumor sites and sites of inflammation CXCL10 directs the polarization and potentiates the biological function of these cells. This makes CXCL10 a “key driver chemokine” and a valid target for therapy of autoimmune diseases such as Inflammatory Bowl’s Disease, Multiple Sclerosis, Rheumatoid arthritis and others. As for cancer this motivated different groups, including our group to develop CXCL10 based therapies for cancer due to its ability to enhance T-dependent anti cancer immunity. The current review summarizes these findings and their potential translational implication.
SNF'ing out antitumor immunity
Immune checkpoint inhibitors induce durable tumor regressions in some, but not all, cancer patients. Understanding the mechanisms that determine tumor sensitivity to these drugs could potentially expand the number of patients who benefit (see the Perspective by Ghorani and Quezada). Pan et al. discovered that tumor cells in which a specific SWI/SNF chromatin remodeling complex had been experimentally inactivated were more sensitive to T cell–mediated killing. The cells were more responsive to interferon-γ, leading to increased secretion of cytokines that promote antitumor immunity. Miao et al. examined the genomic features of tumors from patients with metastatic renal cell carcinoma who had been treated with immune checkpoint inhibitors. Tumors harboring inactivating mutations in PBRM1 , which encodes a subunit of the same SWI/SNF complex, were more likely to respond to the drugs.
Science , this issue p. 770 , p. 801 ; see also p. 745
Tumor necrosis factor (TNF) is a proinflammatory cytokine that coordinates tissue homeostasis by regulating cytokine production, cell survival, and cell death. However, how life and death decisions are made in response to TNF is poorly understood. Many inflammatory pathologies are now recognized to be driven by aberrant TNF-induced cell death, which, in most circumstances, depends on the kinase Receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Recent advances have identified ubiquitin (Ub)-mediated phosphorylation of RIPK1 as belonging to crucial checkpoints for cell fate in inflammation and infection. A better understanding of these checkpoints might lead to new approaches for the treatment of chronic inflammatory diseases fueled by aberrant RIPK1-induced cell death, and/or reveal novel strategies for anticancer immunotherapies, harnessing the ability of RIPK1 to trigger immunogenic cell death.
The mechanisms by which immune checkpoint blockade modulates tumor evolution during therapy are unclear. We assessed genomic changes in tumors from 68 patients with advanced melanoma, who progressed on ipilimumab or were ipilimumab-naive, before and after nivolumab initiation (CA209-038 study). Tumors were analyzed by whole-exome, transcriptome, and/or T cell receptor (TCR) sequencing. In responding patients, mutation and neoantigen load were reduced from baseline, and analysis of intratumoral heterogeneity during therapy demonstrated differential clonal evolution within tumors and putative selection against neoantigenic mutations on-therapy. Transcriptome analyses before and during nivolumab therapy revealed increases in distinct immune cell subsets, activation of specific transcriptional networks, and upregulation of immune checkpoint genes that were more pronounced in patients with response. Temporal changes in intratumoral TCR repertoire revealed expansion of T cell clones in the setting of neoantigen loss. Comprehensive genomic profiling data in this study provide insight into nivolumab's mechanism of action.
Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR–Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.
Choices have consequences. Immune cells survey and migrate throughout the body and sometimes take residence in niche environments with distinct communities of cells, extracellular matrix, and nutrients that may differ from those in which they matured. Imbedded in immune cell physiology are metabolic pathways and metabolites that not only provide energy and substrates for growth and survival, but also instruct effector functions, differentiation, and gene expression. This review of immunometabolism will reference the most recent literature to cover the choices that environments impose on the metabolism and function of immune cells and highlight their consequences during homeostasis and disease.