Hongxia Wang’s research while affiliated with Fudan University and other places

Cancer stem cells (CSC) play a crucial role in pancreatic ductal adenocarcinoma (PDAC) progression and therapeutic resistance. However, the underlying mechanisms and potential targeted treatment strategies remain poorly understood. In this study, we employed single-cell RNA sequencing and exosomal profiling, identifying TSPAN8-enriched exosomes secreted by CSC, which are associated with poor survival rates in PDAC patients. They enhanced stemness in the surrounding non-stem cancer cells (NSCC) by activating the Sonic Hedgehog (Hh) signalling pathway. This exosomal TSPAN8-Hh signalling axis significantly increases the clonogenic ability, invasiveness, and chemoresistance of PDAC cells. Furthermore, TSPAN8-enriched exosomes promoted a higher stem cell frequency, tumourigenicity, and tumour growth rate in vivo, confirming their critical roles in PDAC malignant progression. Our findings underscore the importance of TSPAN8-enriched exosomes for CSC-NSCC communication during PDAC progression.

Background Recurrent/metastatic head and neck squamous cell carcinoma (R/M-HNSCC) is a severe, frequently lethal condition. Oncogene addiction to epidermal growth factor receptor (EGFR) is a hallmark of HNSCC, but the clinical efficacy of EGFR-targeted therapies remains low. Understanding molecular networks governing EGFR-driven progression is paramount to the exploration of (co)-treatment targets and predictive markers. Methods We performed function-based mapping of differentially expressed genes in EGFR-mediated local invasion (fDEGs) using photoconvertible tracers and RNA-sequencing (RNA-seq) in a cellular 3D-model. Results Upon alignment with public single-cell RNA-seq (scRNA-seq) datasets and HNSCC-specific regulons, a gene regulatory network of local invasion (invGRN) was inferred from gene expression data, which was overrepresented in budding tumors. InvGRN comprises the central hubs inhibin subunit beta alpha (INHBA) and snail family transcriptional repressor 2 (SNAI2), and druggable fDEGs integrin subunit beta 4 (ITGB4), laminin 5 (LAMB3/LAMC2), and sphingosine kinase 1 (SPHK1). Blockade of INHBA repressed local invasion and was reverted by activin A, laminin 5, and sphingosine-1-phosphate, demonstrating a functional interconnectivity of the invGRN. Epithelial-to-mesenchymal transition (EMT) of malignant cells and the invGRN are induced by newly defined EGFR-activity subtypes with prognostic value that are promoted by amphiregulin (AREG) and epiregulin (EREG). Importantly, co-inhibition of SPHK1 showed synthetic effects on Cetuximab-mediated invasion blockade and high expression of selected fDEGs was associated with response to Cetuximab in patient-derived xenotransplantation (PDX) and R/M-HNSCC patients. Conclusions We describe an actionable network of EGFR-mediated local invasion and define druggable effectors with predictive potential regarding the response of R/M-HNSCC to Cetuximab.

Cancer‐associated fibroblasts (CAFs) play a crucial role in the progression of pancreatic ductal adenocarcinoma (PDAC). Here, integrated single‐cell RNA sequencing analysis is utilized to comprehensively map CAFs in the human PDAC tumor microenvironment (TME). Normal fibroblasts (NFs) and nine distinct CAF subtypes are identified including newly identified CAF subtypes, CDCP1⁺FTL⁺ CAFs, transitional CAFs (tCAFs), interferon simulated genes (ISG)⁺ myofibroblastic CAFs (myCAFs), and proliferative CAFs (pCAFs). CDCP1⁺FTL⁺ CAFs, pCAFs, and ISG⁺ myCAFs are associated with unfavorable clinical outcomes. CDCP1⁺FTL⁺ CAFs exhibit enhanced glycolysis and iron metabolism, resisting ferroptosis. The antigen‐presenting CAFs (apCAFs) show high heterogeneity, consisting of multiple subtypes expressing distinct immune cell signatures. The CAF subtypes display differentiation plasticity, transitioning from early normal‐like CAFs (nCAFs) to inflammatory CAFs (iCAFs) and myCAFs, ultimately leading to more invasive pCAFs. AP‐1 family members FOS and JUN regulate the malignant phenotype conversion of NFs to nCAFs, while transforming growth factor‐β (TGFβ) and interferon‐γ (IFNγ) signals trigger the interconversion between classic myCAFs and iCAFs, respectively. A close interaction between CAFs and myeloid cells (especially neutrophils) is further observed in PDAC‐TME, mainly mediated by CXCR4‐CXCL12 chemotaxis. This work depicts a detailed CAF map and its dynamic interconvertible shift, providing important insights for combined targeted CAFs therapy.

Tumor malignancy highly depends on the stiffness of tumor matrix, which mainly consists of collagen. Despite the destruction of tumor matrix is conducive to tumor therapy, it causes the risk of tumor metastasis. Here, metal‐anesthetic network‐coated dormant collagenase‐producing Clostridium is constructed to simultaneously destruct tumor matrix and inhibit tumor metastasis. By metal‐phenolic complexation and π–π stacking interactions, a Fe³⁺‐propofol network is formed on bacterial surface. Coated dormant Clostridium can selectively germinate and rapidly proliferate in tumor sites due to the ability of carried Fe³⁺ ions to promote bacterial multiplication. Intratumoral colonization of Clostridium produces sufficient collagenases to degrade tumor collagen mesh and the loaded propofol restrains tumor metastasis by inhibiting tumor cell migration and invasion. Meanwhile, the delivered Fe³⁺ ions are reduced to the Fe²⁺ form by intracellular glutathione, thereby inducing potent Fenton reaction to trigger lipid peroxidation and ultimate ferroptosis of tumor cells. In addition to a satisfactory safety, a single intratumoral injection of coated dormant Clostridium not only effectively retards the growth of established large primary tumors, but also significantly suppresses distal lung metastasis in two different orthotopic tumor models. This work proposes a strategy to develop advanced therapeutics for malignant tumor treatment and metastasis prevention.

Analysis of extracellular vesicles (EV) is a promising noninvasive liquid biopsy approach for breast cancer detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of breast cancer–specific EVs from human samples is required to realize the potential of this strategy. In this study, we applied a mass spectrometry–based, data-independent acquisition proteomic approach to characterize human serum EVs derived from patients with breast cancer (n = 126) and healthy donors (n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled the construction of specific EV protein classifiers for diagnosing breast cancer and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of breast cancer. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating breast cancer invasion and metastasis. Finally, high-throughput molecular docking and virtual screening of a library consisting of 271,380 small molecules identified a potent TALDO1 allosteric inhibitor, AO-022, which could inhibit breast cancer migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of breast cancer patients to guide the development of improved diagnosis, monitoring, and treatment strategies. Significance: Characterization of the proteomic composition of circulating extracellar vesicles in breast cancer patients identifies signatures for diagnosing primary and metastatic tumors and reveals tumor-promoting cargo that can be targeted to improve outcomes.

e16324 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the few cancer types that have essentially no clinical benefit from immune checkpoint inhibitors therapy, combined immunotherapy is expected to solve this dilemma. We conducted a randomized open-label, placebo-controlled phase II trial evaluating the efficacy of camrelizumab combined with apatinib or capecitabine in the treatment of patients with previously treated metastatic PDAC (ChiCTR1900024095). Methods: Eligible patients (n = 30) were randomized 1:1:1 to receive camrelizumab (200 mg q2w) combined with apatinib (250mg qd, d1-28, q4w) or capecitabine (1000 mg/m2, bid d1-14, q3w), or placebo plus capecitabine (1000 mg/m2, bid d1-14, q3w) until disease progression or unacceptable toxicity. Tumor response was assessed every 8 weeks according to RECISTv1.1. Primary endpoints were investigator-assessed 6-month overall survival (OS) rate. Secondary endpoints were progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), duration of response (DoR) and safety. Data cutoff date for the prespecified interim OS and final PFS analysis was Oct 30, 2022. Results: In the primary endpoint analysis, camrelizumab combined treatment showed improved 6-month OS rates, whether in the camrelizumab-apatinib arm (83.3% versus 33.3%, p = 0.1189) or in the camrelizumab-capecitabine arm (100% versus 33.3%, p = 0.0098), compared placebo-capecitabine, respectively. Median PFS was 5.4 months, HR 0.58, [95% confidence interval (CI), 0.21–1.59] in the camrelizumab-apatinib arm , 3.1 months HR 0.98, [95%CI, 0.35–2.77] in the camrelizumab-capecitabine arm , and 2.6 months in the placebo-capecitabine, respectively. The ORR and DCR were 0% and 66.7% in the camrelizumab-apatinib arm, 40% and 50% in the camrelizumab-capecitabine arm, 0% and 44.4% in the placebo-capecitabine, respectively. PR was observed in two patients receiving camrelizumab-capecitabine treatment. The median DoR were 5.3 months in the camrelizumab-apatinib arm, 4.3 months in the camrelizumab-capecitabine arm, and 2.1 months in the placebo-capecitabine arm. Treatment-related adverse event rates were slightly increased in camrelizumab combined arms. Incidences of grade ≥3 treatment-related adverse events were comparable between the three groups (20%、 10% vs 0%), with reactive cutaneous capillary endothelial proliferation (10%) as the most common one in Camrelizumab combinations. Conclusions: Camrelizumab combined treatment showed improved 6-month OS vs conventional capecitabine monotherapy, with a manageable safety profile. These analyses revealed combined immunotherapy could be used as a new option for posterior line treatment in previously treated metastatic PDAC. Clinical trial information: ChiCTR1900024095.

e14541 Background: Circulating tumor cell (CTCs) has the potential utility for screening, prognostic and predictive assessment, and offers the potential for personalized management. This trial aimed to evaluate the prognostic and predictive impact of programmed cell death ligand 1 (PD-L1) positive CTCs in patients with pancreatic ductal adenocarcinoma (PDAC). Methods: Twenty-three heavily treated advanced PDAC patients were randomized to receive camrelizumab combined with apatinib or capecitabine, or placebo plus capecitabine until disease progression or unacceptable toxicity, and peripheral blood CTCs samples were collected at baseline and after treatment. Forty-nine CTCs samples from 23 recruited advanced PDAC patients were detected by subtraction enrichment and immunostaining-FISH (SE-iFISH). PD-L1 expression level also was evaluated in tumor tissue samples by Immunohistochemistry (IHC). Investigator-assessed 6-month overall survival (OS) rate and 3-month progression-free survival (PFS) rate was evaluated. Results: The positive detection rate of CTCs in 23 PDAC patients was 95.7%. The cell sub-types of sizes of CTC analysis showed that 58.6% CTCs was the small CTCs (Diameter < 5μm). CTCs karyotypes analysis exhibited that triploid small CTCs were the main karyotype (41.2%) in small CTC, and pentaploid large CTCs was the major karyotype (74.7%) in large CTCs. PD-L1 ⁺ CTCs were detected in 43.5% of PDAC patients. Tissue PD-L1 positive detection rate was 50% (CPS > 1%) in available tissue samples(n=10). The presence of PD-L1 ⁺ CTCs cohort exhibited a significant increase in 6-month OS rate (80% versus 38.5%, P < 0.05) and 3-month PFS rate (70% versus 23.1%, P < 0.05) compared to PD-L1 ⁻ CTCs cohort. Compared with the PD-L1 ⁻ CTCs patients, the combination therapy camrelizumab markedly improved the 6-month OS rates (75% versus 50%) and 3-month PFS rate (62.5% versus 25%) in PD-L1 ⁺ CTC patients. Conclusions: PD-L1 ⁺ CTCs could be considered as a favorable prognostic marker in advanced PDAC. These results suggest that PD-L1 ⁺ CTCs might be used as a predictor of the efficacy of cancer immunotherapy in advanced PDAC, and provide a new monitoring method for the precise treatment of advanced PDAC. The expanding sample size test is now ongoing. Clinical trial information: ChiCTR1900024095 .

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8) ⁺ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8 ⁺ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)–related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8 ⁺ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser ⁷⁷² by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1 , which caused TSPAN8 ⁺ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8 ⁺ SIRT6 low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8 ⁺ myCAFs is a promising approach to circumvent chemoresistance.

BACKGROUND Analyses of immune cell subsets and immune indices are gaining in importance. By combining molecular probe and immunohistochemical (IHC) analysis with laser ablation, the imaging mass cytometry (IMC) offers spatially high-dimensional in situ measurements in tissue slides with a cell resolution of 1µm. The most critical challenge is how to implement stringent quality control strategies during application of IMC to facilitate reproducible data analysis. METHODS We discussed the multi-step experimental processing procedures of IMC, including specimen preparation, panel design, antibody selection, lanthanide metal labeling and data pre-processing workflows by IHC and fluorescent multiplex immunohistochemistry (mIHC) analysis. Based on IMC, we developed a standard operation and well-established agreement for 29 BC patients to identify a structured tumor immune microenvironment. RESULTS Metal labeling does not affect the antibodies target specificity (p > 0.05). The 3 µm-thick formalin-fixed and paraffin-embedded (FFPE) and fresh-frozen slides exhibited the least blur (p < 0.01) and the lowest nonspecific adsorption of antibodies (p < 0.01). Detailed analysis of human BC tissues revealed a wide range of differences in the composition and frequency of immune cells within the tumor microenvironment. CONCLUSIONS This study offered optimized proposals on procedures for IMC analysis as well as a standard quality control strategy.