Intratumoral heterogeneity in cancers arises from genomic instability and epigenomic plasticity and is associated with resistance to cytotoxic and targeted therapies. We show here that cell-state heterogeneity, defined by differentiation-state marker expression, is high in triple-negative and basal-like breast cancer subtypes, and that drug tolerant persister (DTP) cell populations with altered marker expression emerge during treatment with a wide range of pathway-targeted therapeutic compounds. We show that MEK and PI3K/mTOR inhibitor-driven DTP states arise through distinct cell-state transitions rather than by Darwinian selection of preexisting subpopulations, and that these transitions involve dynamic remodeling of open chromatin architecture. Increased activity of many chromatin modifier enzymes, including BRD4, is observed in DTP cells. Co-treatment with the PI3K/mTOR inhibitor BEZ235 and the BET inhibitor JQ1 prevents changes to the open chromatin architecture, inhibits the acquisition of a DTP state, and results in robust cell death in vitro and xenograft regression in vivo.

SUMMARY The tumor microenvironment plays a critical role in tumor growth, progression, and therapeutic resistance, but interrogating the role of specific tumor-stromal interactions on tumorigenic phenotypes is challenging within in vivo tissues. Here, we tested whether three-dimensional (3D) bioprinting could improve in vitro models by incorporating multiple cell types into scaffold-free tumor tissues with defined architecture. We generated tumor tissues from distinct subtypes of breast or pancreatic cancer in relevant microenvironments and demonstrate that this technique can model patient-specific tumors by using primary patient tissue. We assess intrinsic, extrinsic, and spatial tumorigenic phenotypes in bioprinted tissues and find that cellular proliferation, extracellular matrix deposition, and cellular migration are altered in response to extrinsic signals or therapies. Together, this work demonstrates that multi-cell-type bioprinted tissues can recapitulate aspects of in vivo neoplastic tissues and provide a manipulable system for the interrogation of multiple tumorigenic endpoints in the context of distinct tumor microenvironments.

Pancreatic ductal adenocarcinoma (PDAC) develops a pronounced stromal response reflecting an aberrant wound-healing process. This stromal reaction features trans-differentiation of tissue-resident pancreatic stellate cells (PSCs) into activated cancer-associated fibroblasts (CAFs), a process induced by PDAC cells but of unclear significance for PDAC progression. Here we show that PSCs undergo a dramatic lipid metabolic shift during differentiation in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated, fibroblastic state. Specifically, stroma-derived lysophosphatidylcholines support PDAC cell synthesis of phosphatidylcholines, key components of cell membranes, and also facilitate production of the potent wound-healing mediator lysophosphatidic acid (LPA) by the extracellular enzyme autotaxin, which is overexpressed in PDAC. The autotaxin-LPA axis promotes PDAC cell proliferation, migration and AKT activation, and genetic or pharmacologic autotaxin inhibition suppresses PDAC growth in vivo. Our work demonstrates how PDAC cells exploit the local production of wound healing mediators to stimulate their own growth and migration.

Intratumoral phenotypic heterogeneity has been described in many tumor types, where it can contribute to drug resistance and disease recurrence. We analyzed ductal and neuroendocrine markers in pancreatic ductal adenocarcinoma, revealing heterogeneous expression of the neuroendocrine marker Synaptophysin within ductal lesions. Higher percentages of Cytokeratin-Synaptophysin dual positive tumor cells correlate with shortened disease-free survival. We observe similar lineage marker heterogeneity in mouse models of pancreatic ductal adenocarcinoma, where lineage tracing indicates that Cytokeratin-Synaptophysin dual positive cells arise from the exocrine compartment. Mechanistically, MYC binding is enriched at neuroendocrine genes in mouse tumor cells and loss of MYC reduces ductal-neuroendocrine lineage heterogeneity, while deregulated MYC expression in KRAS mutant mice increases this phenotype. Neuroendocrine marker expression is associated with chemoresistance and reducing MYC levels decreases gemcitabine-induced neuroendocrine marker expression and increases chemosensitivity. Altogether, we demonstrate that MYC facilitates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma, contributing to poor survival and chemoresistance.

During normal tumor growth and in response to some therapies, tumor cells experience acute or chronic deprivation of nutrients and oxygen and induce tumor vascularization. While this occurs predominately through sprouting angiogenesis, tumor cells have also been shown to directly contribute to vessel formation through vascular mimicry (VM) and/or endothelial transdifferentiation. The extrinsic and intrinsic mechanisms underlying tumor cell adoption of endothelial phenotypes, however, are not well understood. Here we show that serum withdrawal induces mesenchymal breast cancer cells to undergo VM and that knockdown of the epithelial-to-mesenchymal transition (EMT) regulator, Zinc finger E-box binding homeobox 1 (ZEB1), or overexpression of the ZEB1-repressed microRNAs (miRNAs), miR-200c, miR-183, miR-96 and miR-182 inhibits this process. We find that secreted proteins Fibronectin 1 (FN1) and serine protease inhibitor (serpin) family E member 2 (SERPINE2) are essential for VM in this system. These secreted factors are upregulated in mesenchymal cells in response to serum withdrawal, and overexpression of VM-inhibiting miRNAs abrogates this upregulation. Intriguingly, the receptors for these secreted proteins, low-density lipoprotein receptor-related protein 1 (LRP1) and Integrin beta 1 (ITGB1), are also targets of the VM-inhibiting miRNAs, suggesting that autocrine signaling stimulating VM is regulated by ZEB1-repressed miRNA clusters. Together, these data provide mechanistic insight into the regulation of VM and suggest that miRNAs repressed during EMT, in addition to suppressing migratory and stem-like properties of tumor cells, also inhibit endothelial phenotypes of breast cancer cells adopted in response to a nutrient-deficient microenvironment.