The tumor microenvironment shows a hierarchy of cell-cell interactions dominated by fibroblasts.

The tumor microenvironment (TME) is comprised of non-malignant cells that interact with each other and with cancer cells, critically impacting cancer biology. The TME is complex, and understanding it requires simplifying approaches. Here we provide an experimental-mathematical approach to decompose the TME into small circuits of interacting cell types. We find, using female breast cancer single-cell-RNA-sequencing data, a hierarchical network of interactions, with cancer-associated fibroblasts (CAFs) at the top secreting factors primarily to tumor-associated macrophages (TAMs). This network is composed of repeating circuit motifs. We isolate the strongest two-cell circuit motif by culturing fibroblasts and macrophages in-vitro, and analyze their dynamics and transcriptomes. This isolated circuit recapitulates the hierarchy of in-vivo interactions, and enables testing the effect of ligand-receptor interactions on cell dynamics and function, as we demonstrate by identifying a mediator of CAF-TAM interactions - RARRES2, and its receptor CMKLR1. Thus, the complexity of the TME may be simplified by identifying small circuits, facilitating the development of strategies to modulate the TME.

DNA methylation landscape of tumor-associated macrophages reveals pathways, transcription factors and prognostic value relevant to triple-negative breast cancer patients.

The accumulation of myeloid cells, particularly tumor-associated macrophages (TAMs), characterizes the tumor microenvironment (TME) of many solid cancers, including breast cancer. Compared to healthy tissue-resident macrophages, TAMs acquire distinct transcriptomes and tumor-promoting functions by largely unknown mechanisms. Here, we hypothesize the involvement of TME signaling and subsequent epigenetic reprogramming of TAMs. Using the 4T1 mouse model of triple-negative breast cancer, we demonstrate that the presence of cancer cells significantly alters the DNA methylation landscape of macrophages and, to a lesser extent, bone marrow-derived monocytes (BMDMs). TAM methylomes, dissected into BMDM-originating and TAM-specific epigenetic programs, implicated transcription factors (TFs) and signaling pathways involved in TAM reprogramming, correlated with cancer-specific gene expression patterns. Utilizing published single-cell gene expression data, we linked microenvironmentally-derived signals to the cancer-specific DNA methylation landscape of TAMs. These integrative analyses highlighted the role of altered cytokine production in the TME (eg, TGF-ß, IFN-γ and CSF1) on the induction of specific TFs (eg, FOSL2, STAT1 and RUNX3) responsible for the epigenetic reprogramming of TAMs. DNA methylation deconvolution identified a TAM-specific signature associated with the identified signaling pathways and TFs, corresponding with severe tumor grade and poor prognosis of breast cancer patients. Similarly, immunosuppressive TAM functions were identified, such as induction of the immune inhibitory receptor-ligand PD-L1 by DNA hypomethylation of Cd274. Collectively, these results provide strong evidence that the epigenetic landscapes of macrophages and monocytes are perturbed by the presence of breast cancer, pointing to molecular mechanisms of TAM reprogramming, impacting patient outcomes.

BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking clusterin expression in cancer associated fibroblasts with HSF1 signaling.

Tumors initiate by mutations in cancer cells, and progress through interactions of the cancer cells with non-malignant cells of the tumor microenvironment. Major players in the tumor microenvironment are cancer-associated fibroblasts (CAFs), which support tumor malignancy, and comprise up to 90% of the tumor mass in pancreatic cancer. CAFs are transcriptionally rewired by cancer cells. Whether this rewiring is differentially affected by different mutations in cancer cells is largely unknown. Here we address this question by dissecting the stromal landscape of BRCA-mutated and BRCA Wild-type pancreatic ductal adenocarcinoma. We comprehensively analyze pancreatic cancer samples from 42 patients, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA Wild-type tumors. In particular, we detect an increase in a subset of immune-regulatory clusterin-positive CAFs in BRCA-mutated tumors. Using cancer organoids and mouse models we show that this process is mediated through activation of heat-shock factor 1, the transcriptional regulator of clusterin. Our findings unravel a dimension of stromal heterogeneity influenced by germline mutations in cancer cells, with direct implications for clinical research.

Global DNA Methylation Analysis of Cancer-Associated Fibroblasts Reveals Extensive Epigenetic Rewiring Linked with RUNX1 Upregulation in Breast Cancer Stroma.

Cancer cells recruit and rewire normal fibroblasts in their microenvironment to become protumorigenic cancer-associated fibroblasts (CAF). These CAFs are genomically stable, yet their transcriptional programs are distinct from those of their normal counterparts. Transcriptional regulation plays a major role in this reprogramming, but the extent to which epigenetic modifications of DNA also contribute to the rewiring of CAF transcription is not clear. Here we address this question by dissecting the epigenetic landscape of breast CAFs. Applying tagmentation-based whole-genome bisulfite sequencing in a mouse model of breast cancer, we found that fibroblasts undergo massive DNA methylation changes as they transition into CAFs. Transcriptional and epigenetic analyses revealed RUNX1 as a potential mediator of this process and identified a RUNX1-dependent stromal gene signature. Coculture and mouse models showed that both RUNX1 and its stromal signature are induced as normal fibroblasts transition into CAFs. In breast cancer patients, RUNX1 was upregulated in CAFs, and expression of the RUNX1 signature was associated with poor disease outcome, highlighting the relevance of these findings to human disease. This work presents a comprehensive genome-wide map of DNA methylation in CAFs and reveals a previously unknown facet of the dynamic plasticity of the stroma. SIGNIFICANCE: The first genome-wide map of DNA methylation in breast cancer-associated fibroblasts unravels a previously unknown facet of the dynamic plasticity of the stroma, with far-reaching therapeutic implications.

Impact of TP53 Genomic Alterations in Large B-Cell Lymphoma Treated With CD19-Chimeric Antigen Receptor T-Cell Therapy.

PURPOSE: Tumor-intrinsic features may render large B-cell lymphoma (LBCL) insensitive to CD19-directed chimeric antigen receptor T cells (CAR-T). We hypothesized that TP53 genomic alterations are detrimental to response outcomes in LBCL treated with CD19-CAR-T. MATERIALS AND METHODS: Patients with LBCL treated with CD19-CAR-T were included. Targeted next-generation sequencing was performed on pre-CAR-T tumor samples in a subset of patients. Response and survival rates by histologic, cytogenetic, and molecular features were assessed. Within a cohort of newly diagnosed LBCL with genomic and transcriptomic profiling, we studied interactions between cellular pathways and TP53 status. RESULTS: We included 153 adults with relapsed or refractory LBCL treated with CD19-CAR-T (axicabtagene ciloleucel [50%], tisagenlecleucel [32%], and lisocabtagene maraleucel [18%]). Outcomes echoed pivotal trials: complete response (CR) rate 54%, median overall survival (OS) 21.1 months (95% CI, 14.8 to not reached), and progression-free survival 6 months (3.4 to 9.7). Histologic and cytogenetic LBCL features were not predictive of CR. In a subset of 82 patients with next-generation sequencing profiling, CR and OS rates were comparable with the unsequenced cohort. TP53 alterations (mutations and/or copy number alterations) were common (37%) and associated with inferior CR and OS rates in univariable and multivariable regression models; the 1-year OS in TP53-altered LBCL was 44% (95% CI, 29 to 67) versus 76% (65 to 89) in wild-type (P = .012). Transcriptomic profiling from a separate cohort of patients with newly diagnosed lymphoma (n = 562) demonstrated that TP53 alterations are associated with dysregulation of pathways related to CAR-T-cell cytotoxicity, including interferon and death receptor signaling pathway and reduced CD8 T-cell tumor infiltration. CONCLUSION: TP53 is a potent tumor-intrinsic biomarker that can inform risk stratification and clinical trial design in patients with LBCL treated with CD19-CAR-T. The role of TP53 should be further validated in independent cohorts.

Cancer-Associated Fibroblasts Promote Aggressive Gastric Cancer Phenotypes via Heat Shock Factor 1-Mediated Secretion of Extracellular Vesicles.

Gastric cancer is the third most lethal cancer worldwide, and evaluation of the genomic status of gastric cancer cells has not translated into effective prognostic or therapeutic strategies. We therefore hypothesize that outcomes may depend on the tumor microenvironment (TME), in particular, cancer-associated fibroblasts (CAF). However, very little is known about the role of CAFs in gastric cancer. To address this, we mapped the transcriptional landscape of human gastric cancer stroma by microdissection and RNA sequencing of CAFs from patients with gastric cancer. A stromal gene signature was associated with poor disease outcome, and the transcription factor heat shock factor 1 (HSF1) regulated the signature. HSF1 upregulated inhibin subunit beta A and thrombospondin 2, which were secreted in CAF-derived extracellular vesicles to the TME to promote cancer. Together, our work provides the first transcriptional map of human gastric cancer stroma and highlights HSF1 and its transcriptional targets as potential diagnostic and therapeutic targets in the genomically stable tumor microenvironment. SIGNIFICANCE: This study shows how HSF1 regulates a stromal transcriptional program associated with aggressive gastric cancer and identifies multiple proteins within this program as candidates for therapeutic intervention. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1639/F1.large.jpg.

Heat Shock Factor 1-dependent extracellular matrix remodeling mediates the transition from chronic intestinal inflammation to colon cancer.

In the colon, long-term exposure to chronic inflammation drives colitis-associated colon cancer (CAC) in patients with inflammatory bowel disease. While the causal and clinical links are well established, molecular understanding of how chronic inflammation leads to the development of colon cancer is lacking. Here we deconstruct the evolving microenvironment of CAC by measuring proteomic changes and extracellular matrix (ECM) organization over time in a mouse model of CAC. We detect early changes in ECM structure and composition, and report a crucial role for the transcriptional regulator heat shock factor 1 (HSF1) in orchestrating these events. Loss of HSF1 abrogates ECM assembly by colon fibroblasts in cell-culture, prevents inflammation-induced ECM remodeling in mice and inhibits progression to CAC. Establishing relevance to human disease, we find high activation of stromal HSF1 in CAC patients, and detect the HSF1-dependent proteomic ECM signature in human colorectal cancer. Thus, HSF1-dependent ECM remodeling plays a crucial role in mediating inflammation-driven colon cancer.

Stromal Expression of the Core Clock Gene Period 2 Is Essential for Tumor Initiation and Metastatic Colonization.

The circadian clock regulates diverse physiological processes by maintaining a 24-h gene expression pattern. Genetic and environmental cues that disrupt normal clock rhythms can lead to cancer, yet the extent to which this effect is controlled by the cancer cells versus non-malignant cells in the tumor microenvironment (TME) is not clear. Here we set out to address this question, by selective manipulation of circadian clock genes in the TME. In two different mouse models of cancer we find that expression of the core clock gene Per2 in the TME is crucial for tumor initiation and metastatic colonization, whereas another core gene, Per1, is dispensable. We further show that loss of Per2 in the TME leads to significant transcriptional changes in response to cancer cell introduction. These changes may contribute to a tumor-suppressive microenvironment. Thus, our work unravels an unexpected protumorigenic role for the core clock gene Per2 in the TME, with potential implications for therapeutic dosing strategies and treatment regimens.

Cancer-associated fibroblast compositions change with breast cancer progression linking the ratio of S100A4+ and PDPN+ CAFs to clinical outcome.

Tumors are supported by cancer-associated fibroblasts (CAFs). CAFs are heterogeneous and carry out distinct cancer-associated functions. Understanding the full repertoire of CAFs and their dynamic changes as tumors evolve could improve the precision of cancer treatment. Here we comprehensively analyze CAFs using index and transcriptional single-cell sorting at several time points along breast tumor progression in mice, uncovering distinct subpopulations. Notably, the transcriptional programs of these subpopulations change over time and in metastases, transitioning from an immunoregulatory program to wound-healing and antigen-presentation programs, indicating that CAFs and their functions are dynamic. Two main CAF subpopulations are also found in human breast tumors, where their ratio is associated with disease outcome across subtypes and is particularly correlated with BRCA mutations in triple-negative breast cancer. These findings indicate that the repertoire of CAF changes over time in breast cancer progression, with direct clinical implications.

A TIGIT-based chimeric co-stimulatory switch receptor improves T-cell anti-tumor function.

BACKGROUND: Tumors can employ different mechanisms to evade immune surveillance and function. Overexpression of co-inhibitory ligands that bind to checkpoint molecules on the surface of T-cells can greatly impair the function of latter. TIGIT (T cell immunoreceptor with Ig and ITIM domains) is such a co-inhibitory receptor expressed by T and NK cells which, upon binding to its ligand (e.g., CD155), can diminish cytokine production and effector function. Additionally, the absence of positive co-stimulation at the tumor site can further dampen T-cell response. METHODS: As T-cell genetic engineering has become clinically-relevant in the recent years, we devised herein a strategy aimed at enhancing T-cell anti-tumor function by diverting T-cell coinhibitory signals into positive ones using a chimeric costimulatory switch receptor (CSR) composed of the TIGIT exodomain fused to the signaling domain of CD28. RESULTS: After selecting an optimized TIGIT-28 CSR, we co-transduced it along with tumor-specific TCR or CAR into human T-cells. TIGIT-28-equipped T-cells exhibited enhanced cytokine secretion and upregulation of activation markers upon co-culture with tumor cells. TIGIT-28 enhancing capability was also demonstrated in an original in vitro model of T-cell of hypofunction induction upon repetitive antigen exposure. Finally, we tested the function of this molecule in the context of a xenograft model of established human melanoma tumors and showed that TIGIT-28-engineered human T-cells demonstrated superior anti-tumor function. CONCLUSION: Overall, we propose that TIGIT-based CSR can substantially enhance T-cell function and thus contribute to the improvement of engineered T cell-based immunotherapy.