No abstract

Cultured cell lines are the workhorse of cancer research, but it is unclear to what extent they recapitulate the cellular heterogeneity observed among malignant cells in tumors. To address this, we used multiplexed single cell RNA-seq to profile ~200 cancer cell lines from 22 cancer types. We uncovered 12 expression programs that are recurrently heterogeneous within many cancer cell lines. These programs are associated with diverse biological processes including cell cycle, senescence, stress and interferon responses, epithelial-mesenchymal transition, and protein maturation and degradation. Notably, most of these recurrent programs of heterogeneity recapitulate those recently observed within human tumors. The similarity to tumors allowed us to prioritize specific cell lines as model systems of cellular heterogeneity. We used two such models

Each tumor contains malignant cells that differ in genotype, phenotype, and in their interactions with the tumor micro-environment (TME). This results in distinct integrated cellular states that govern intra-tumor heterogeneity (ITH), a central challenge of cancer therapeutics. Dozens of recent studies have begun to describe ITH by single cell RNA-seq, but each study typically profiledonly a small number of tumors and provided a narrow view of transcriptional ITH. Here, we curate, annotate and integrate the data from 77 different studies to reveal the patterns of ITH across 1,163 tumor samples covering 24 tumor types. Focusing on the malignant cells, we find thousands of transcriptional ITH programs that can be described by 41 consensus meta-programs (MPs), each consisting of dozens of genes that are coordinately upregulated in subpopulations of cells within many different tumors. The MPs cover diverse cellular processes and differ in their cancer-type distribution. General MPs associated with processes such as cell cycle and stress vary within most tumors, while context-specific MPs reflect the unique biology of particular cancer types, often resembling developmental cell types and suggesting the co-existence of variable differentiation states within tumors. Some of the MPs are further associated with overall tumor proliferation or immune state, highlighting their potential clinical significance. Based on functional similarities among MPs, we propose a set of 11 hallmarks that together account for the majority of observed ITH programs. Given the breadth and scope of the investigated cohort, the MPs and hallmarks described here reflect the first comprehensive pan-cancer description of transcriptional ITH.

Cultured cell lines are the workhorse of cancer research, but it is unclear to what extent they recapitulate the cellular heterogeneity observed among malignant cells in tumors, given the absence of a native tumor microenvironment. Here, we used multiplexed single cell RNA-seq to profile ~200 cancer cell lines. We uncovered expression programs that are recurrently heterogeneous within many cancer cell lines and are largely independent of observed genetic diversity. These programs of heterogeneity are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-to-mesenchymal transition, and protein maturation and degradation. Notably, some of these recurrent programs recapitulate those seen in human tumors, suggesting a prominent role of intrinsic plasticity in generating intra-tumoral heterogeneity. Moreover, the data allowed us to prioritize specific cell lines as model systems of cellular plasticity. We used two such models to demonstrate the dynamics, regulation and drug sensitivities associated with a cancer senescence program also observed in human tumors. Our work describes the landscape of cellular heterogeneity in diverse cancer cell lines, and identifies recurrent patterns of expression heterogeneity that are shared between tumors and specific cell lines and can thus be further explored in follow up studies.

Insufficient erythropoiesis due to increased demand is usually met by hypoxia-driven up-regulation of erythropoietin (Epo). Here, we uncovered vascular endothelial growth factor (VEGF) as a novel inducer of Epo capable of increasing circulating Epo under normoxic, nonanemic conditions in a previously unrecognized reservoir of Epo-producing cells (EPCs), leading to expansion of the erythroid progenitor pool and robust splenic erythropoiesis. Epo induction by VEGF occurs in kidney, liver, and spleen in a population of Gli1+SMA+PDGFRβ+ cells, a signature shared with vascular smooth muscle cells (VSMCs) derived from mesenchymal stem cell–like progenitors. Surprisingly, inhibition of PDGFRβ signaling, but not VEGF signaling, abrogated VEGF-induced Epo synthesis. We thus introduce VEGF as a new player in Epo induction and perivascular Gli1+SMA+PDGFRβ+ cells as a previously unrecognized EPC reservoir that could be harnessed for augmenting Epo synthesis in circumstances such as chronic kidney disease where production by canonical EPCs is compromised.

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor-A (VEGF) biologics, termed VEGF “Sticky-traps,” with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky-traps could locally inhibit angiogenesis to at least the same extent as the original VEGF-trap that also gains whole-body access. Sticky-traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky-trap remained localized to various regions of the eye, such as the inner-limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky-trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre-maturity.

Communication between cancer cells and immune cells is a key determinant of the glioblastoma ecosystem and its response to therapies, but remains poorly understood. Here we leveraged single-cell RNA-sequencing (scRNA-seq) of human samples and mouse models, deconvolution analysis of bulk specimens from The Cancer Genome Atlas (TCGA) and functional approaches to dissect cellular states and cross-talk in glioblastoma. We demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal-like (MES-like) states. This effect is mediated, both in vitro and in vivo, by macrophage-derived Oncostatin M (OSM) and its cognate receptor OSMR on glioblastoma cells. We show that MES-like glioblastoma states are associated with increased T cells cytotoxicity and potentially with better clinical response to immunotherapies. Overall, our work dissects the cellular interactions within the glioblastoma microenvironment, with potential implications for therapies.

The lethality of glioblastoma and the failure of surgical treatment are in part attributable to the invasive properties of this cancer; however, our understanding of the cell-biological process is limited due to the complex representation of invasion phenotype in glioblastoma models. Here, to overcome this limitation, we developed an in vivo approach with human glioblastoma spheroid lines multiplexed for mouse intracranial xenografts. We validated this with 20 patient-derived models and identified, by 3’end single-cell RNA-sequencing (scRNA-seq) coupled with deconvolution analysis, in vivo models capable of infiltrating along the periphery of either neurons or blood vessels. We previously found that the diverse malignant cells in glioblastoma specimens exist in a limited set of four cellular states: neural progenitor-like (NPC-like), oligodendrocyte progenitor-like (OPC-like), astrocyte-like (AC-like), and mesenchymal-like (MES-like), with potential for state plasticity under the influence of tumor microenvironment. To determine whether or not the cell state heterogeneity is implicated in glioblastoma invasion, we profiled and characterized programs, states, and clonality of invading glioblastoma cells by comparing cells from the contralateral invasive edge to their counterparts residing at the original bulk tumor. This analysis showed evidence of a bi-directional state transitioning model for cells undergoing distinct environmental spaces. Exploration of cell state distribution at the primary tumors revealed that presence of an OPC-like state was significantly associated with invasion phenotype regardless of the infiltration routes. Our results suggest that cellular plasticity may be a prerequisite for the topographically diffusive nature of glioblastoma, urging further assessment by perturbing critical regulators of the cellular state programs. In conclusion, this work describes multiplexed in vivo models as an enabling platform for the rapid interrogation of patient-derived xenografts, coupling the molecularly measurable states with functional identity driving malignant phenotypes such as cancer invasion. Citation Format: Toshiro Hara, Rony Chanoch-Myers, Alissa Greenwald, Lillian Bussema, Jinan Behnan, Rossella Galli, Hiroaki Wakimoto, Itay Tirosh, Mario Suvà. Single-cell profiles of multiplexed in vivo models facilitate the characterization of phenotypic plasticity and invasion in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1246.