Extrinsic KRAS Signaling Shapes the Pancreatic Microenvironment Through Fibroblast Reprogramming.

BACKGROUND & AIMS: Oncogenic Kirsten Rat Sarcoma virus (KRAS) is the hallmark mutation of human pancreatic cancer and a driver of tumorigenesis in genetically engineered mouse models of the disease. Although the tumor cell-intrinsic effects of oncogenic Kras expression have been widely studied, its role in regulating the extensive pancreatic tumor microenvironment is less understood. METHODS: Using a genetically engineered mouse model of inducible and reversible oncogenic Kras expression and a combination of approaches that include mass cytometry and single-cell RNA sequencing we studied the effect of oncogenic KRAS in the tumor microenvironment. RESULTS: We have discovered that non-cell autonomous (ie, extrinsic) oncogenic KRAS signaling reprograms pancreatic fibroblasts, activating an inflammatory gene expression program. As a result, fibroblasts become a hub of extracellular signaling, and the main source of cytokines mediating the polarization of protumorigenic macrophages while also preventing tissue repair. CONCLUSIONS: Our study provides fundamental knowledge on the mechanisms underlying the formation of the fibroinflammatory stroma in pancreatic cancer and highlights stromal pathways with the potential to be exploited therapeutically.

Pancreatic cancer is marked by complement-high blood monocytes and tumor-associated macrophages.

Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3 Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8 In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.

Transcriptomic Analysis of Diffuse Intrinsic Pontine Glioma (DIPG) Identifies a Targetable ALDH-Positive Subset of Highly Tumorigenic Cancer Stem-like Cells.

Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH-, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence. IMPLICATIONS: Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.

Regulatory T-cell Depletion Alters the Tumor Microenvironment and Accelerates Pancreatic Carcinogenesis.

Regulatory T cells (Treg) are abundant in human and mouse pancreatic cancer. To understand the contribution to the immunosuppressive microenvironment, we depleted Tregs in a mouse model of pancreatic cancer. Contrary to our expectations, Treg depletion failed to relieve immunosuppression and led to accelerated tumor progression. We show that Tregs are a key source of TGFß ligands and, accordingly, their depletion reprogramed the fibroblast population, with loss of tumor-restraining, smooth muscle actin-expressing fibroblasts. Conversely, we observed an increase in chemokines Ccl3, Ccl6, and Ccl8 leading to increased myeloid cell recruitment, restoration of immune suppression, and promotion of carcinogenesis, an effect that was inhibited by blockade of the common CCL3/6/8 receptor CCR1. Further, Treg depletion unleashed pathologic CD4+ T-cell responses. Our data point to new mechanisms regulating fibroblast differentiation in pancreatic cancer and support the notion that fibroblasts are a heterogeneous population with different and opposing functions in pancreatic carcinogenesis. SIGNIFICANCE: Here, we describe an unexpected cross-talk between Tregs and fibroblasts in pancreatic cancer. Treg depletion resulted in differentiation of inflammatory fibroblast subsets, in turn driving infiltration of myeloid cells through CCR1, thus uncovering a potentially new therapeutic approach to relieve immunosuppression in pancreatic cancer.See related commentary by Aykut et al., p. 345.This article is highlighted in the In This Issue feature, p. 327.

Multimodal Mapping of the Tumor and Peripheral Blood Immune Landscape in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an immune-suppressive tumor microenvironment that renders it largely refractory to immunotherapy. We implemented a multimodal analysis approach to elucidate the immune landscape in PDA. Using a combination of CyTOF, single-cell RNA sequencing, and multiplex immunohistochemistry on patient tumors, matched blood, and non-malignant samples, we uncovered a complex network of immune-suppressive cellular interactions. These experiments revealed heterogeneous expression of immune checkpoint receptors in individual patient's T cells and increased markers of CD8+ T cell dysfunction in advanced disease stage. Tumor-infiltrating CD8+ T cells had an increased proportion of cells expressing an exhausted expression profile that included upregulation of the immune checkpoint TIGIT, a finding that we validated at the protein level. Our findings point to a profound alteration of the immune landscape of tumors, and to patient-specific immune changes that should be taken into account as combination immunotherapy becomes available for pancreatic cancer.

Degree of Tissue Differentiation Dictates Susceptibility to BRAF-Driven Colorectal Cancer.

Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers; however, the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2), stem cell activity is restored in BRAFV600E intestines, and the oncogenic capacity of BRAFV600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors that reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.