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.

Germline BRCA–associated pancreatic ductal adenocarcinoma (glBRCA PDAC) tumors are susceptible to platinum and PARP inhibition. The clinical outcomes of 125 patients with glBRCA PDAC were stratified based on the spectrum of response to platinum/PARP inhibition: (i) refractory [overall survival (OS) <6 months], (ii) durable response followed by acquired resistance (OS <36 months), and (iii) long-term responders (OS >36 months). Patient-derived xenografts (PDX) were generated from 25 patients with glBRCA PDAC at different clinical time points. Response to platinum/PARP inhibition in vivo and ex vivo culture (EVOC) correlated with clinical response. We deciphered the mechanisms of resistance in glBRCA PDAC and identified homologous recombination (HR) proficiency and secondary mutations restoring partial functionality as the most dominant resistant mechanism. Yet, a subset of HR-deficient (HRD) patients demonstrated clinical resistance. Their tumors displayed basal-like molecular subtype and were more aneuploid. Tumor mutational burden was high in HRD PDAC and significantly higher in tumors with secondary mutations. Anti–PD-1 attenuated tumor growth in a novel humanized glBRCA PDAC PDX model. This work demonstrates the utility of preclinical models, including EVOC, to predict the response of glBRCA PDAC to treatment, which has the potential to inform time-sensitive medical decisions. Significance: glBRCA PDAC has a favorable response to platinum/PARP inhibition. However, most patients develop resistance. Additional treatment options for this unique subpopulation are needed. We generated model systems in PDXs and an ex vivo system (EVOC) that faithfully recapitulate these specific clinical scenarios as a platform to investigate the mechanisms of resistance for further drug development.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is remarkably resistant to standard modalities, including radiotherapy (RT). The mechanisms of radiation resistance in general, and pancreatic cancer in particular, are poorly understood. We hypothesized that metabolic reprogramming may underlie this radioresistance, and moreover, that it would be possible to exploit these changes in metabolism for therapeutic intent. Experimental Design: We established multiple isogenic models of radioresistant PDAC cells. Metabolic profile was investigated using Nanostring technology, labeled-glucose tracing by liquid chromatography-mass spectrometry, Seahorse analysis and exposure to metabolic inhibitors. Patient-derived xenografts (PDXs) were established from patients treated with radiation and RNA sequencing performed. The PDXs were grouped according to clinical RECIST response to radiation (responsive/stable disease vs disease progression) and differential gene expression analysis was performed. Results: The radioresistant cells overexpressed pyruvate dehydrogenase kinase (PDK) and were radiosensitized by the PDK inhibitor dichloroacetate. In keeping with PDK overexpression, radioresistant cells displayed increased glycolysis and downregulated both the tricarboxylic acid cycle and oxidative phosphorylation. Metabolic flux through the pentose-phosphate pathway (PPP) was increased, as were levels of reduced glutathione; PPP inhibition dramatically potentiated radiation-induced cell death. Critically, the PPP was upregulated in PDXs derived from patients who demonstrated clinical resistance to radiotherapy. High transcription levels of 6PGD, the rate-limiting enzyme of the PPP, were associated with a poor radiological response to radiation therapy (p=0.0004) and a lower overall survival (p=0.004). Conclusions: We demonstrate that radioresistant PDAC cells divert the glycolytic flux from the tricarboxylic acid cycle and oxidative phosphorylation to the PPP, thereby increasing their antioxidant capacity and promoting nucleotide synthesis for DNA repair. Furthermore, we show that PDAC cells can be radiosensitized via PPP inhibition. Exploitation of metabolic vulnerabilities to radiosensitize tumors constitutes a novel approach to pancreatic cancer with a real potential to improve clinical outcomes. Citation Format: Ariel Shimoni-Sebag, Ifat Abramovich, Bella Agranovich, Yaarit Sirovsky, Chani Stossel, Dikla Atias, Maria Raitses-Gurevich, Yulia Glick-Gorman, Ofer Margalit, David Regev, Rotem Tal, Itay Tirosh, Talia Golan, Keren Yizhak, Eyal Gottlieb, Yaacov R. Lawrence. The pentose-phosphate pathway induces pancreatic cancer radioresistance, a preclinical study with clinical validation [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 2411.