Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.
Central to anti-tumor immunity are dendritic cells (DCs), which stimulate long-lived protective T cell responses. Recent studies have demonstrated that DCs can achieve a state of hyperactivation, which is associated with inflammasome activities within living cells. Herein, we report that hyperactive DCs have an enhanced ability to migrate to draining lymph nodes and stimulate potent cytotoxic T lymphocyte (CTL) responses. This enhanced migratory activity is dependent on the chemokine receptor CCR7 and is associated with a unique transcriptional program that is not observed in conventionally activated or pyroptotic DCs. We show that hyperactivating stimuli are uniquely capable of inducing durable CTL-mediated anti-tumor immunity against tumors that are sensitive or resistant to PD-1 inhibition. These protective responses are intrinsic to the cDC1 subset of DCs, depend on the inflammasome-dependent cytokine IL-1β, and enable tumor lysates to serve as immunogens. If these activities are verified in humans, hyperactive DCs may impact immunotherapy.
Prostate cancer is the second most common malignancy in men worldwide and consists of a mixture of tumor and non-tumor cell types. To characterize the prostate cancer tumor microenvironment, we performed single-cell RNA-sequencing on prostate biopsies, prostatectomy specimens, and patient-derived organoids from localized prostate cancer patients. We identify a population of tumor-associated club cells that may act as progenitor cells and uncover heterogeneous cellular states in prostate epithelial cells marked by high androgen signaling states that are enriched in prostate cancer. ERG- tumor cells, compared to ERG+ cells, demonstrate shared heterogeneity with surrounding luminal epithelial cells and appear to give rise to common tumor microenvironment responses. Finally, we show that prostate epithelial organoids recapitulate tumor-associated epithelial cell states and are enriched with distinct cell types and states from their parent tissues. Our results provide diagnostically relevant insights and advance our understanding of the cellular states associated with prostate carcinogenesis.
Bulk transcriptomic studies have defined classical and basal-like gene expression subtypes in pancreatic ductal adenocarcinoma (PDAC) that correlate with survival and response to chemotherapy; however, the underlying mechanisms that govern these subtypes and their heterogeneity remain elusive. Here, we performed single-cell RNA-sequencing of 23 metastatic PDAC needle biopsies and matched organoid models to understand how tumor cell-intrinsic features and extrinsic factors in the tumor microenvironment (TME) shape PDAC cancer cell phenotypes. We identify a novel cancer cell state that co-expresses basal-like and classical signatures, demonstrates upregulation of developmental and KRAS-driven gene expression programs, and represents a transitional intermediate between the basal-like and classical poles. Further, we observe structure to the metastatic TME supporting a model whereby reciprocal intercellular signaling shapes the local microenvironment and influences cancer cell transcriptional subtypes. In organoid culture, we find that transcriptional phenotypes are plastic and strongly skew toward the classical expression state, irrespective of genotype. Moreover, we show that patient-relevant transcriptional heterogeneity can be rescued by supplementing organoid media with factors found in the TME in a subtype-specific manner. Collectively, our study demonstrates that distinct microenvironmental signals are critical regulators of clinically relevant PDAC transcriptional states and their plasticity, identifies the necessity for considering the TME in cancer modeling efforts, and provides a generalizable approach for delineating the cell-intrinsic versus -extrinsic factors that govern tumor cell phenotypes.
Crucial transitions in cancer—including tumor initiation, local expansion, metastasis, and therapeutic resistance—involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.
Whether cultured in vitro or part of a complex tissue in vivo, a cell’s phenotype and function are significantly influenced by dynamic interactions with its microenvironment. To explicitly examine how a cell’s spatiotemporal activity impacts its behavior, we developed and validated a strategy termed SPACECAT – Spatially PhotoActivatable Color Encoded Cell Address Tags – to annotate, track, and isolate specific cells in a non-destructive, viability-preserving manner. In SPACECAT, a biological sample is immersed in a photocaged fluorescent molecule, and cells within a location of interest are labeled for further study by uncaging that molecule with user-patterned near-UV light. SPACECAT offers high spatial precision and temporal stability across diverse cell and tissue types, and is compatible with common downstream assays, including flow cytometry and single-cell RNA-Seq. Illustratively, we leveraged this approach in patient-derived intestinal organoids, a spatially complex system less amenable to genetic manipulations, to select for crypt-like regions enriched in stem-like and actively mitotic cells. Moreover, we demonstrate its applicability and utility on ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model, uncovering spatially-biased gene expression patterns among immune cell subsets and identifying rare myeloid phenotypes enriched around tumor/healthy border regions. In sum, our method provides a minimally invasive and broadly applicable approach to link cellular spatiotemporal features and/or behavioral phenotypes with diverse downstream assays, enabling fundamental insights into the connections between tissue microenvironments and biological (dys)function.
Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) programme that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway.
The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor: macrophage ratio, was recapitulated in spleen by partial macrophage depletion and was overcome by multiple, highdose alkylating agents. Cyclophosphamide induced ER-stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGF-A, massive macrophage infiltration and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGF-A blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a “super-phagocytic” subset that expressed CD36/FcgRIV. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance.
Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.
Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.
We develop single-cell genomic approaches to comprehensively profile complex biological ensembles. To date, the majority of our work has focused on establishing, validating, and scaling single-cell transcriptomics, often through the development of microdevices to enable genome-wide identification of the cell types/states that comprise functional or dysfunctional biological samples.
Most recently, we have developed Seq-Well, a portable, low-cost platform for high-throughput single-cell RNA-Seq (scRNA-Seq). By providing open access to resources and protocols, we hope to democratize access to cutting-edge approaches in single-cell genomics.
To complement and inform the analysis of scRNA-Seq datasets, we create methods to simultaneously profile additional cellular characteristics of interest (e.g. genome, epigenome, or proteome), independently, or in combination with, scRNA-Seq. For a given technique or system, we ask what additional information would help us better interpret our scRNA-Seq results and develop methods to collect these data. These novel methods often map ancillary information into a DNA-based readout that can be coanalyzed with cellular mRNA or developing/applying microdevices. Recently, we have developed a method for integrated mRNA and protein detection that leverages proximity extension assays (Genshaft et al. 2016). To extract the information content from these novel datasets more effectively, we also formulate new computational methods and analyses.
We explore how the extracellular milieu impacts intracellular decision-making by experimentally controlling the cellular microenvironment or leveraging naturally occurring sources of variation within a tissue. Here, we employ solutions that include controlled culture conditions with cells (Shalek et al., 2014) or organoids, chemical or genetic perturbations (Kumar et al., 2014), and constant microfluidic perfusion. We are also developing in silico approaches that are powered by in-situ cellular tagging techniques. In each instance, we aim to understand the degree to which extracellular environments modulate, and can be used to rationally control, the responses of individual cells or the overall distribution thereof, with an eye toward engineering ensemble responses.
We use microdevices, coupled with functional signal readouts, to create and study defined cell-cell interactions. By explicitly enumerating cell type, number, and additional functional properties (e.g., cytokine secretion), we model ensemble behaviors, looking for synergies and antagonisms. These genetic signatures, along with those collected via our other platforms, provide a unique and essential reference for deconvolving behaviors in complex ensembles. We are also using genetic tracing strategies to examine differences between interacting and random cell pairs in vivo, and are developing computational methods (Tirosh et al., 2016) to identify putative interactions from scRNA-Seq data.
As the amount of data we have relating to cells, properties, surroundings, and interactions increases exponentially, we are motivated to develop pan-system measurements and analyses to paint comprehensive pictures of immune response in health and disease. Relying on massive transcriptomic datasets generated from complex tissues, like melanoma tumors, inflamed human gut, M. tuberculosis (MTB)-induced granulomas, and healthy or SHIV-infected monkey tissues, we have begun to construct social networks of integrated responses to physiological perturbations. The technologies outlined above uniquely enable us to generate foundational datasets (e.g., transcriptomes from interacting cell pairs) for deconvolving and interpreting the potential drivers of observed ensemble behaviors, as well as for identifying which properties we cannot explain, and thus need to study. To date, our lab has generated over 2 million single-cell transcriptomes across multiple tissues, individuals, and species; we are utilizing this data, paired with metadata and additional characteristics, to look for common cellular network motifs, such as division of labor, quorum sensing, persistence, or bet-hedging.
Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.
Seq-Well is a portable, low-cost platform for single-cell RNA sequencing designed to be compatible with low-input, clinical biopsies. We have recently published a manuscript detailing the development and validation of the Seq-Well plaftorm. Here, we provide an in-depth protocol and videos describing how to perform Seq-Well experiments.
We hope that you will use Seq-Well and apply it in your work.
CAD files for Seq-well devices and molds are provided here: CAD Files
Note: These files are made available under the “Attribution-NonCommercial-NoDerivatives 4.0 International” creative commons license.
Copyright 2017, Massachusetts Institute of Technology and the Broad Institute.
A diverse array of mechanisms—including genetic mutations, environmental triggers, and diet—can alter cell function and reduce tissue stability, ultimately leading to malignancy, autoimmunity, or immunodeficiency. By identifying which cells these factors affect and in what ways, we aim to develop targeted therapeutic interventions in areas such as cancer, allergy, and inflammatory bowel disease.
Motivating questions that drive our research include:
- How do the coordinated interactions between epithelial and immune populations inform barrier tissue function in the context of homeostasis, inflammation and malignancy?
- How can we leverage information across systems to derive a set of unifying principles of cellular ecology in health and disease?
Current projects aim to contrast the cellular microenvironments of healthy, inflamed, and malignant (Tirosh et al., 2016; Patel et al., 2014) tissues to examine inflammation-induced changes and the drivers of malignant transformation, as well as to identify which cells remember prior insult. We are similarly profiling aberrant immune behaviors in immune privileged tissues, such as the nervous systems. As in our host-microbial studies, our goal is to identify common features shared across different immune-related diseases that we can probe further in natural (tissues, models) and engineered (patterned cells and cellular structures, organoids) ensembles.
To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.
One major goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. While many studies have identified candidate driver genes based on recurrence of mutations in individual genes, subsets of genes with non-recurrent mutations may also be defined as putative drivers if they affect a single biological pathway. In this fashion, we previously identified Wnt signaling as significantly mutated through large-scale massively-parallel DNA sequencing of chronic lymphocytic leukemia (CLL). Here, we use a novel method of biomolecule delivery, vertical silicon nanowires, to efficiently introduce small interfering RNAs into CLL cells, and interrogate the effects of 8 of 15 mutated Wnt pathway members identified across 91 CLLs. In HEK293T cells, mutations in 2 genes did not generate functional changes, 3 led to dysregulated pathway activation, and 3 led to further activation or loss of repression of pathway activation. Silencing 4 of 8 mutated genes in CLL samples harboring the mutated alleles resulted in reduced viability compared to leukemia samples with wild-type alleles. We demonstrate that somatic mutations in CLL can generate dependence on this pathway for survival. These findings support the notion that non recurrent mutations at different nodes of the Wnt pathway can contribute to leukemogenesis.
Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes, and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used single-cell RNA sequencing (RNA-seq) to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response, and hypoxia.We also observed a continuum of stemness related expression states that enabled us to identify putative regulators of stemness in vivo. Finally,we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity.Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.
Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumorpropagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, and OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements and are sufficient to fully reprogram differentiated GBM cells to ‘‘induced’’ TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies candidate therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies.
Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs,
a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.
A circuit level understanding of immune cells and hematological cancers has been severely impeded by a lack of techniques that enable intracellular perturbation without significantly altering cell viability and function. Here, we demonstrate that vertical silicon nanowires (NWs) enable gene-specific manipulation of diverse murine and human immune cells with negligible toxicity. To illustrate the power of the technique, we then apply NW-mediated gene silencing to investigate the role of the Wnt signaling pathway in chronic lymphocytic leukemia (CLL). Remarkably, CLL-B cells from different patients exhibit tremendous heterogeneity in their response to the knockdown of a single gene, LEF1. This functional heterogeneity defines three distinct patient groups not discernible by conventional CLL cytogenetic markers and provides a prognostic indicator for patients’ time to first therapy. Analyses of gene expression signatures associated with these functional patient subgroups reveal unique insights into the underlying molecular basis for disease heterogeneity. Overall, our findings suggest a functional classification that can potentially guide the selection of patient-specific therapies in CLL and highlight the opportunities for nanotechnology to drive biological inquiry.