Wenting Zhao’s research while affiliated with Columbia University and other places

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Publications (29)


DDEL-17. INTRA-TUMORAL CONVECTION ENHANCED DELIVERY OF DEXAMETHASONE REDUCES INFLAMMATORY SIGNATURES AND EXTENDS SURVIVAL IN GBM MODEL
  • Article

November 2024

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37 Reads

Neuro-Oncology

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Damian Teasley

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Dexamethasone is the most widely utilized drug for glioblastoma (GBM). However, systemic delivery of dexamethasone in GBM patients is associated with significant side effects and increasing doses with worse survival. Convection-enhanced delivery (CED) can deliver high doses of immunotherapy directly into the tumor microenvironment (TME) while avoiding systemic toxicity. Here, we report that high doses of dexamethasone can be delivered locally by CED without side effects and increase survival in a GBM model along with reduced inflammatory myeloid signatures. We investigated CED of dexamethasone treatment on survival(n=40), adverse side effects, and the immunological TME through peripheral analyses, liquid chromatography–mass spectrometry, and histology in a preclinical syngeneic mouse model. Additionally, we assessed the transcriptional responses of human GBM slices and stem-cell-derived human microglia after steroid treatment through bulk and single-cell RNA sequencing. We found that 7-day treatment with CED of dexamethasone produced a significant survival advantage in glioma-bearing mice compared to non-treated control(p=0.03). Systemic dexamethasone achieved low levels of drug in the TME and caused dysregulated blood glucose, blood counts, and peripheral organ weights, while high CED doses avoided these side effects(p< 0.05 each). Steroid treatment of acute GBM slices and lipopolysaccharide-activated microglia in-vitro both reversed inflammatory myeloid signatures associated with poor survival and recurrence on RNA sequencing analyses. We demonstrate the preclinical efficacy of delivering high local doses of dexamethasone in a GBM model through CED and observed prolonged survival along with reduced adverse inflammatory myeloid signatures. No side effects were demonstrated after chronic CED treatment of dexamethasone while systemic delivery achieved poor tumor penetration and caused known hematologic and metabolic side effects supporting the potential to optimize the clinical use of this therapy. CED of dexamethasone provides us a new treatment paradigm to locally control inflammatory signatures in the glioma TME in a controlled fashion.


TMIC-78. CHEMOTHERAPY INDUCED IMMUNOGENIC RESPONSE IN GLIOMA AND IMPLICATIONS FOR THERAPEUTIC STRATEGIES

November 2024

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37 Reads

Neuro-Oncology

INTRODUCTION Many glioblastoma (GBM) treatments fail because they treat tumor cells in isolation and ignore the surrounding microenvironment. In our phase 1b clinical trial of delivery of Topotecan (TPT) via Convection Enhanced Delivery (CED) in glioma, we demonstrated effective tumor cell elimination accompanied by a robust inflammatory response. We hypothesized that TPT induces an immunogenic response resulting in an unfavorable inflammatory landscape. METHODS To characterize chemotherapy’s effect on myeloid cells, we analyzed pre- and post-treatment biopsies from our trial using bulk RNA sequencing to measure canonical myeloid markers. We validated survival benefit of CED in vivo in our tumor model and characterized post-treatment tissue using single cell RNA sequencing (scRNA seq) after 7 days of treatment. We treated slice cultures generated from surgical samples of GBM with TPT and performed scRNA seq and histologic analysis to assess microenvironment changes after 24 hours of treatment. We used several in vitro assays to characterize the direct and indirect mechanisms of myeloid activation. RESULTS Bulk RNA sequencing of post treatment biopsies demonstrates upregulation of markers associated with activated and exhausted immune cell populations such as IBA1, CD68, MSR1, MARCO and loss of markers associated with homeostatic microglia such as P2RY12 and TMEM119. Similar results are found within 7 days of treatment in vivo, however, are not found in human derived samples treated acutely. Treatment of in vitro mono and co-culture systems demonstrate a tumor cell death dependent activation of myeloid cell populations via immunogenic molecules. CONCLUSIONS Chemotherapies eliminate proliferating glioma cells but also induce inflammation. While many investigators have tried to leverage this activation, our results suggest that chronic inflammation contributes to poor prognosis and recurrence. The identification of molecules responsible for this activation point to new therapeutic targets aimed at controlling treatment-induced inflammation, as part of a combinatorial approach to treat GBM.


MAPK/ERK signaling in gliomas modulates interferon responses, T cell recruitment, enhances tumor-microglia crosstalk, and drives immune checkpoint blockade efficacy.
  • Preprint
  • File available

September 2024

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86 Reads

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1 Citation

Background Glioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. Method We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8 ⁺ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single cell RNA- sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAF V600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Results CRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8 ⁺ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAF V600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAF V600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAF V600E mutant GB. Conclusion Our data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.

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A cell state-specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma

August 2024

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69 Reads

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3 Citations

The EMBO Journal

Glioma cells hijack developmental programs to control cell state. Here, we uncover a glioma cell state-specific metabolic liability that can be therapeutically targeted. To model cell conditions at brain tumor inception, we generated genetically engineered murine gliomas, with deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling astrocyte differentiation during brain development. N1IC tumors harbored quiescent astrocyte-like transformed cell populations while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. Further, N1IC transformed cells exhibited increased mitochondrial lipid peroxidation, high ROS production and depletion of reduced glutathione. This altered mitochondrial phenotype rendered the astrocyte-like, quiescent populations more sensitive to pharmacologic or genetic inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Treatment of patient-derived early-passage cell lines and glioma slice cultures generated from surgical samples with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles. Collectively, these findings reveal a specific therapeutic vulnerability to ferroptosis linked to mitochondrial redox imbalance in a subpopulation of quiescent astrocyte-like glioma cells resistant to standard forms of treatment.


Multiplexed single-cell lineage tracing of mitotic kinesin inhibitor resistance in glioblastoma

April 2024

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7 Reads

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4 Citations

Cell Reports

Glioblastoma (GBM) is a deadly brain tumor, and the kinesin motor KIF11 is an attractive therapeutic target with roles in proliferation and invasion. Resistance to KIF11 inhibitors, which has mainly been studied in animal models, presents significant challenges. We use lineage-tracing barcodes and single-cell RNA sequencing to analyze resistance in patient-derived GBM neurospheres treated with ispinesib, a potent KIF11 inhibitor. Similar to GBM progression in patients, untreated cells lose their neural lineage identity and become mesenchymal, which is associated with poor prognosis. Conversely, cells subjected to long-term ispinesib treatment exhibit a proneural phenotype. We generate patient-derived xenografts and show that ispinesib-resistant cells form less aggressive tumors in vivo, even in the absence of drug. Moreover, treatment of human ex vivo GBM slices with ispinesib demonstrates phenotypic alignment with in vitro responses, underscoring the clinical relevance of our findings. Finally, using retrospective lineage tracing, we identify drugs that are synergistic with ispinesib.


A pharmacological toolkit for human microglia identifies Topoisomerase I inhibitors as immunomodulators for Alzheimer’s disease

February 2024

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124 Reads

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5 Citations

While efforts to identify microglial subtypes have recently accelerated, the relation of transcriptomically defined states to function has been largely limited to in silico annotations. Here, we characterize a set of pharmacological compounds that have been proposed to polarize human microglia towards two distinct states – one enriched for AD and MS genes and another characterized by increased expression of antigen presentation genes. Using different model systems including HMC3 cells, iPSC-derived microglia and cerebral organoids, we characterize the effect of these compounds in mimicking human microglial subtypes in vitro . We show that the Topoisomerase I inhibitor Camptothecin induces a CD74 high /MHC high microglial subtype which is specialized in amyloid beta phagocytosis. Camptothecin suppressed amyloid toxicity and restored microglia back to their homeostatic state in a zebrafish amyloid model. Our work provides avenues to recapitulate human microglial subtypes in vitro , enabling functional characterization and providing a foundation for modulating human microglia in vivo .


Abstract 102: Microanatomy of Intracranial Human Thrombi by Single Nuclei Transcriptomics

February 2024

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14 Reads

Stroke

Introduction: Microvascular dysfunction and inflammation are thought to underlie reperfusion failure following endovascular thrombectomy. Here we present the first study to produce a transcriptome-based cellular landscape of human intracranial thrombi highlighting the importance of cellular plasticity and intercellular communication at the site of occlusion. Methods: We performed single nuclei RNA-sequencing (snRNA-seq) on intracranial human thrombi harvested during revascularization. Unsupervised clustering, data visualization, differential expression and functional gene ontology/pathway analysis were utilized to deconvolve expression data into cell type-specific expression profiles. Thrombi snRNA-seq data were integrated with single cell RNA-seq data from symptomatic and asymptomatic human carotid plaques and healthy brain vasculature to contextualize thrombi in atherosclerotic disease, perform diffusion coefficient analysis, and identify ligand-receptor interactions. Results: Unbiased clustering identified six cellular populations in distinct effector states within the thrombus microenvironment. Functional gene ontology and pathway analysis revealed a monocyte subpopulation in a distinct endothelial-activation and neuroplasticity state, supporting the notion of cellular trans-differentiation and interconversion. Integration with carotid plaque data and diffusion coefficient analysis revealed shared transcriptional programs existing in a continuum, progressing from asymptomatic plaque to embolized thrombus. Ligand-receptor analysis identified macrophage mediated T-cell activation, pro-inflammatory response, immunological synapse stabilization, neuroplasticity, and monocyte trans-endothelial migration. Conclusion: Our novel application of snRNA-seq to thrombectomy specimens provides the highest resolution exploration of cellular differences and a best understanding of individual contextualized cell function to date. This detailed cellular landscape will facilitate mapping novel interventional targets with direct functional relevance and allow for greater personalization of post-reperfusion therapy.




Figure 4. Fold-changes in the frequencies of cells with high cell scores in drug-treated vs. vehicle control-treated slice cultures in the transformed glioma cell scHPF model for A) a proliferation factor; B) a metallothionein factor; C) an astrocyte/mesenchymal factor; and D) a mesenchymal factor. Here, each dot represents an individual patient (i.e. biological replicates). For each drug, ** indicates FDR<0.05 based on a linear mixed model.
Consensus scHPF Identifies Cell Type-Specific Drug Responses in Glioma by Integrating Large-Scale scRNA-seq

December 2023

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31 Reads

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1 Citation

Single-cell transcriptomic analyses now frequently involve elaborate study designs including samples from multiple individuals, experimental conditions, perturbations, and batches from complex tissues. Dimensionality reduction is required to facilitate integration, interpretation, and statistical analysis. However, these datasets often include subtly different cellular subpopulations or state transitions, which are poorly described by clustering. We previously reported a Bayesian matrix factorization algorithm called single-cell hierarchical Poisson factorization (scHPF) that identifies gene co-expression patterns directly from single-cell RNA-seq (scRNA-seq) count matrices while accounting for transcript drop-out and noise. Here, we describe consensus scHPF, which analyzes scHPF models from multiple random initializations to identify the most robust gene signatures and automatically determine the number of factors for a given dataset. Consensus scHPF facilitates integration of complex datasets with highly multi-modal posterior distributions, resulting in factors that can be uniformly analyzed across individuals and conditions. To demonstrate the utility of consensus scHPF, we performed a meta-analysis of a large-scale scRNA-seq dataset from drug-treated, human glioma slice cultures generated from surgical specimens across three major cell types, 19 patients, 10 drug treatment conditions, and 52 samples. In addition to recapitulating previously reported cell type-specific drug responses from smaller studies, consensus scHPF identified disparate effects of the topoisomerase poisons etoposide and topotecan that are highly consistent with the distinct roles and expression patterns of their respective protein targets.


Citations (13)


... Bulk-RNA sequencing analyses on these biopsies demonstrated a significant correlation with the histological analysis, and post-treatment biopsies revealed increases in several proinflammatory cytokines and immunoreactive markers [19]. In other emerging treatments for rGBM, histological analyses of CD68 alongside RNA sequencing also revealed an inflammatory response following treatment [59]. ...

Reference:

Unveiling the Inflammatory Landscape of Recurrent Glioblastoma through Histological-Based Assessments
A cell state-specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma
  • Citing Article
  • August 2024

The EMBO Journal

... Transcriptional cell states are informative for modeling and predicting treatment response and resistance on the single-cell level. 13,16,16,30,50,[57][58][59][60][61][62][63] Devising effective therapies requires identification of those states and transcriptional modules specifically, over cell types, are either the most vulnerable to drug treatment or the most essential to tumor integrity. This comprehensive characterization of GBM vulnerabilities across both scRNA-and stRNA-seq data highlights the importance of multimodal approaches to drug discovery. ...

Multiplexed single-cell lineage tracing of mitotic kinesin inhibitor resistance in glioblastoma
  • Citing Article
  • April 2024

Cell Reports

... Recent studies have focused on optimization of in vitro systems that recapitulate disease/lipidassociated microglia/macrophages. These models use iPSC-derived microglia, CNS-relevant substrates, or genetic manipulations of DLAM master transcription factors 19,20,67 . Although they can model human microglia under controlled conditions and offer insights into microglial biology, they also have several caveats. ...

A pharmacological toolkit for human microglia identifies Topoisomerase I inhibitors as immunomodulators for Alzheimer’s disease

... In order to assess whether the tissue imparted distinct gene signatures across lineages, we implemented consensus single cell hierarchical Poisson Factorization (scHPF) 11,35 , a Bayesian factorization algorithm that identifies the major co-expression patterns ("factors") in the data, rather than considering each gene in isolation (as we have done for DE). We applied scHPF to each major immune cell lineage in separate models with balanced cell input across tissues ( Supplementary Table 7 ), which allowed us to identify a variety of transcriptional programs. ...

Consensus scHPF Identifies Cell Type-Specific Drug Responses in Glioma by Integrating Large-Scale scRNA-seq

... Recent research has shown quiescent, astrocyte-like glioma cell populations have a unique metabolic vulnerability to GPX4 inhibition, which induces ferroptosis [133]. While GPX4 inhibition via RSL3 led to a non-significant survival benefit in a murine MG3 tumor model, a significant survival benefit was seen when GPX4 inhibition was combined with cysteine and methionine restriction, suggesting a future role for ferroptotic agents may exist in the treatment of malignant gliomas [134]. ...

A cell state specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma

... This promising profile places TPT as a strong candidate for further exploration in this field [10,28], especially considering its recent identification as a potential candidate for drug repurposing in GBM therapy, with tests performed in various cell lines [29]. TPT targets proliferating cells in the S-phase of the cell cycle, and since only a small percentage of glial cells undergo division and at a slower rate than cancer cells [30], it may be assumed that normal cells would be less effected than cancer cells. However, further comparative investigation applying normal-cancer cell pairs is required to support such a statement. ...

Chronic convection-enhanced delivery of topotecan for patients with recurrent glioblastoma: a first-in-patient, single-centre, single-arm, phase 1b trial
  • Citing Article
  • October 2022

The Lancet Oncology

... Additionally, local delivery methods, such convection-enhanced delivery (CED) as well as intranasal delivery, are being used to circumvent the BBB/BTBB and achieve the direct delivery of repurposed drugs into the (peri)tumoral area. Such examples include topotecan [257], which is delivered via CED against recurrent GBM, as well as perillyl alcohol via intranasal delivery [258]. Similarly, a novel introduced catheter systems for direct delivery of therapeutics to the brain (Neuroinfuse™) aims to improve chronic and acute implantable intra-parenchymal drug delivery [259]. ...

Treatment of Recurrent Glioblastoma by Chronic Convection-Enhanced Delivery of Topotecan

... 91 Phosphorylation of ERK is a hallmark of MAPK activation. 92 A considerable amount of research has demonstrated that the MAPK signaling pathway simultaneously serves as input and output pathways in the circadian clock. [93][94][95] ERK can directly interact with components of the circadian oscillator and phosphorylate them, including BMAL1, CLOCK, CRY1, and CRY2, playing a significant role in maintaining circadian rhythms. ...

ERK1/2 phosphorylation predicts survival following anti-PD-1 immunotherapy in recurrent glioblastoma

Nature Cancer

... Multi-omics approaches at the single-cell resolution have emerged as a promising method for studying tumor biology [10] [11]. The increasing availability of single-cell RNA sequencing data has uncovered significant features of GBM and its intricate immune microenvironment [12]. Single-cell transcriptomics affords insights into the genetic characteristics of individual cell subtypes, facilitating the identification of targeted treatment options. ...

Single-cell characterization of macrophages in glioblastoma reveals MARCO as a mesenchymal pro-tumor marker

Genome Medicine

... 36 scRNA-seq has uncovered diverse drug effects; for instance, etoposide influences proliferating tumour cells, whereas panobinostat impacts not only tumour cells but also the immune microenvironment in surprising ways. 37 In addition to the above-mentioned applications, the human microbiome which comprises a vast community of microorganisms, including bacteria, viruses, fungi, and other microbes, is another system that can significantly influence the efficacy of nutraceuticals. 38 For instance, certain bioactive compounds in nutraceuticals are metabolised by gut microbiota, leading to bioactive metabolites that may contribute to the health benefits associated with those compounds. ...

Deconvolution of cell type-specific drug responses in human tumor tissue with single-cell RNA-seq

Genome Medicine