Biology subjects

Izar, B.

Publications and source records attributed to Izar, B..

25 records · Page 2Linked to original sources

bioRxiv2022

Multi-modal single-cell and whole-genome sequencing of minute, frozen specimens to propel clinical applications

Single-cell genomics are enabling technologies, but their broad clinical application remains challenging. We report an easily adaptable approach for single-cell transcriptome and T cell receptor (TCR)-sequencing, and matched whole-genome sequencing from tiny, frozen clinical specimens. We achieve similar quality and biological outputs while reducing artifactual signals compared to data from matched fresh tissue samples. Profiling sequentially collected melanoma samples from the KEYNOTE-001 trial, we resolve cellular, genomic, and clonotype dynamics that encapsulate molecular patterns of tumor evolution during anti-PD-1 therapy. To demonstrate applicability to banked biospecimens of rare diseases, we generate a large uveal melanoma liver metastasis single-cell and matched WGS atlas, which revealed niche-specific impairment of clonal T cell expansion. This study provides a foundational framework for propelling single-cell genomics to the clinical arena.

genomics↗

bioRxiv2021

Microenvironmental correlates of immune checkpoint inhibitor response in human melanoma brain metastases revealed by T cell receptor and single-cell RNA sequencing

Melanoma-derived brain metastases (MBM) represent an unmet clinical need due to central nervous system (CNS) progression as a frequent, end-stage site of disease. Immune checkpoint inhibition (ICI) represents a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique MBM-TME elements and correlates of MBM-ICI response, we collected 32 fresh MBM and performed single cell RNA sequencing of the MBM-TME and T cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity, most notably multiple distinct neutrophil states including an IL-8 expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. Additionally, we observe significant relationships between intracranial T cell phenotypes and the distribution of T cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data demonstrate unique features of the MBM-TME, which may represent potential targets to improve clinical outcomes for patients with MBM.

cancer biology↗

bioRxiv2020

Multi-modal pooled Perturb-CITE-Seq screens in patient models define novel mechanisms of cancer immune evasion

Resistance to immune checkpoint inhibitors (ICI) that activate T cell mediated anti-tumor immunity is a key challenge in cancer therapy, yet the underlying mechanisms remain poorly understood. To further elucidate those, we developed a new approach, Perturb-CITE-seq, for pooled CRISPR perturbation screens with multi-modal RNA and protein single-cell profiling readout and applied it to screen patient-derived autologous melanoma and tumor infiltrating lymphocyte (TIL) co-cultures. We profiled RNA and 20 surface proteins in over 218,000 cells under ~750 perturbations, chosen by their membership in an immune evasion program that is associated with immunotherapy resistance in patients. Our screen recovered clinically-relevant resistance mechanisms concordantly reflected in RNA, protein and perturbation effects on susceptibility to T cell mediated killing. These were organized in eight co-functional modules whose perturbation distinctly affect four co-regulated programs associated with immune evasion. Among these were defects in the IFN{gamma}-JAK/STAT pathway and in antigen presentation, and several novel mechanisms, including loss or downregulation of CD58, a surface protein without known mouse homolog. Leveraging the rich profiles in our screen, we found that loss of CD58 did not compromise MHC protein expression and that CD58 was not transcriptionally induced by the IFN{gamma} pathway, allowing us to distinguish it as a novel mechanism of immune resistance. We further show that loss of CD58 on cancer cells conferred immune evasion across multiple T cell and Natural Killer cell patient co-culture models. Notably, CD58 is downregulated in tumors with resistance to immunotherapy in melanoma patients. Our work identifies novel mechanisms at the nexus of immune evasion and drug resistance and provides a general framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal singlecell profiles, including in systems consisting of multiple cell types.

genomics↗

bioRxiv2020

Transcriptional mediators of treatment resistance in lethal prostate cancer

Metastatic castration resistant prostate cancer (mCRPC) is primarily treated with therapies that prevent transcriptional activity of the androgen receptor (AR), cause DNA damage, or prevent cell division. Clinical resistance to these therapies, including second-generation androgen-targeting compounds such as enzalutamide and abiraterone, is nearly universal. Other treatment modalities, including immune checkpoint inhibitors, have provided minimal benefit except in rare subsets of patients1,2. Both tumour intrinsic and extrinsic cellular programs contributing to therapeutic resistance remain areas of active investigation. Here we use full-length single-cell RNA-sequencing (scRNA-seq) to identify the transcriptional states of cancer and immune cells in the mCRPC microenvironment. Within cancer cells, we identified transcriptional patterns that mediate a significant proportion of inherited risk for prostate cancer, extensive heterogeneity in AR splicing within and between tumours, and vastly divergent regulatory programs between adenocarcinoma and small cell carcinoma. Moreover, upregulation of TGF-{beta} signalling and epithelial-mesenchymal transition (EMT) were both associated with resistance to enzalutamide. We found that some lymph node metastases, but no bone metastases, were heavily infiltrated by dysfunctional CD8+ T cells, including cells undergoing dramatic clonal expansion during enzalutamide treatment. Our findings suggest avenues for rational therapeutic approaches targeting both tumour-intrinsic and immunological pathways to combat resistance to current treatment options.

genomics↗

bioRxiv2019

A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors

Single cell genomics is essential to chart the complex tumor ecosystem. While single cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumor tissues, single nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each strategy requires modifications to fit the unique characteristics of different tissue and tumor types, posing a barrier to adoption. Here, we developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We tested eight tumor types of varying tissue and sample characteristics (resection, biopsy, ascites, and orthotopic patient-derived xenograft): lung cancer, metastatic breast cancer, ovarian cancer, melanoma, neuroblastoma, pediatric sarcoma, glioblastoma, pediatric high-grade glioma, and chronic lymphocytic leukemia. Analyzing 212,498 cells and nuclei from 39 clinical samples, we evaluated protocols by cell quality, recovery rate, and cellular composition. We optimized protocols for fresh tissue dissociation for different tumor types using a decision tree to account for the technical and biological variation between clinical samples. We established methods for nucleus isolation from OCT embedded and fresh-frozen tissues, with an optimization matrix varying mechanical force, buffer, and detergent. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types and intrinsic expression profiles, but at different proportions. Our work provides direct guidance across a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

genomics↗

bioRxiv2019

Opposing immune and genetic forces shape oncogenic programs in synovial sarcoma

Synovial sarcoma is an aggressive mesenchymal neoplasm, driven by the SS18-SSX fusion, and characterized by immunogenic antigens expression and exceptionally low T cell infiltration levels. To study the cancer-immune interplay in this disease, we profiled 16,872 cells from 12 human synovial sarcoma tumors using single-cell RNA-sequencing (scRNA-Seq). Synovial sarcoma manifests antitumor immunity, high cellular plasticity and a core oncogenic program, which is predictive of low immune levels and poor clinical outcomes. Using genetic and pharmacological perturbations, we demonstrate that the program is controlled by the SS18-SSX driver and repressed by cytokines secreted by macrophages and T cells in the tumor microenvironment. Network modeling predicted that SS18-SSX promotes the program through HDAC1 and CDK6. Indeed, the combination of HDAC and CDK4/6 inhibitors represses the program, induces immunogenic cell states, and selectively targets synovial sarcoma cells. Our study demonstrates that immune evasion, cellular plasticity, and cell cycle are co-regulated and can be co-targeted in synovial sarcoma and potentially in other malignancies.

cancer biology↗

bioRxiv2017

A simple open-source method for highly multiplexed imaging of single cells in tissues and tumours

The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.

cancer biology↗