High-throughput CRISPR-mediated 3D enrichment platform for functional interrogation of chemotherapeutic resistance.

Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is a need for rapid, high-throughput, unbiased in vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identify mutations that confer chemotherapeutic drug resistance. Taking the example of the CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a three-dimensional (3D) setting. Gene-edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug-resistant phenotypes, detectable by next-gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug-resistant cell within a large, drug-responsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Furthermore, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring noncell autonomous interactions, selection pressures, and clonal competition.

Functional profiling of microtumors to identify cancer associated fibroblast-derived drug targets.

Recent advances in chemotherapeutics highlight the importance of molecularly-targeted perturbagens. Although these therapies typically address dysregulated cancer cell proteins, there are increasing therapeutic modalities that take into consideration cancer cell-extrinsic factors. Targeting components of tumor stroma such as vascular or immune cells has been shown to represent an efficacious approach in cancer treatment. Cancer-associated fibroblasts (CAFs) exemplify an important stromal component that can be exploited in targeted therapeutics, though their employment in drug discovery campaigns has been relatively minimal due to technical logistics in assaying for CAF-tumor interactions. Here we report a 3-dimensional multi-culture tumor:CAF spheroid phenotypic screening platform that can be applied to high-content drug discovery initiatives. Using a functional genomics approach we systematically profiled 1,024 candidate genes for CAF-intrinsic anti-spheroid activity; identifying several CAF genes important for development and maintenance of tumor:CAF co-culture spheroids. Along with previously reported genes such as WNT, we identify CAF-derived targets such as ARAF and COL3A1 upon which the tumor compartment depends for spheroid development. Specifically, we highlight the G-protein-coupled receptor OGR1 as a unique CAF-specific protein that may represent an attractive drug target for treating colorectal cancer. In vivo, murine colon tumor implants in OGR1 knockout mice displayed delayed tumor growth compared to tumors implanted in wild type littermate controls. These findings demonstrate a robust microphysiological screening approach for identifying new CAF targets that may be applied to drug discovery efforts.

PAR2 regulates regeneration, transdifferentiation, and death.

Understanding the mechanisms by which cells sense and respond to injury is central to developing therapies to enhance tissue regeneration. Previously, we showed that pancreatic injury consisting of acinar cell damage+ß-cell ablation led to islet cell transdifferentiation. Here, we report that the molecular mechanism for this requires activating protease-activated receptor-2 (PAR2), a G-protein-coupled receptor. PAR2 modulation was sufficient to induce islet cell transdifferentiation in the absence of ß-cells. Its expression was modulated in an islet cell type-specific manner in murine and human type 1 diabetes (T1D). In addition to transdifferentiation, PAR2 regulated ß-cell apoptosis in pancreatitis. PAR2's role in regeneration is broad, as mice lacking PAR2 had marked phenotypes in response to injury in the liver and in digit regeneration following amputation. These studies provide a pharmacologically relevant target to induce tissue regeneration in a number of diseases, including T1D.

RAGE, carboxylated glycans and S100A8/A9 play essential roles in colitis-associated carcinogenesis.

Patients with inflammatory bowel diseases are at increased risk for colorectal cancer, but the molecular mechanisms linking inflammation and cancer are not well defined. We earlier showed that carboxylated N-glycans expressed on receptor for advanced glycation end products (RAGE) and other glycoproteins mediate colitis through activation of nuclear factor kappa B (NF-kappaB). Because NF-kappaB signaling plays a critical role in the molecular pathogenesis of colitis-associated cancer (CAC), we reasoned that carboxylated glycans, RAGE and its ligands might promote CAC. Carboxylated glycans are expressed on a subpopulation of RAGE on colon cancer cells and mediate S100A8/A9 binding to RAGE. Colon tumor cells express binding sites for S100A8/A9 and binding leads to activation of NF-kappaB and tumor cell proliferation. Binding, downstream signaling and tumor cell proliferation are blocked by mAbGB3.1, an anti-carboxylate glycan antibody, and by anti-RAGE. In human colon tumor tissues and in a mouse model of CAC, we found that myeloid progenitors expressing S100A8 and S100A9 infiltrate regions of dysplasia and adenoma. mAbGB3.1 administration markedly reduces chronic inflammation and tumorigenesis in the mouse model of CAC and RAGE-deficient mice are resistant to the onset of CAC. These findings show that RAGE, carboxylated glycans and S100A8/A9 play essential roles in tumor-stromal interactions, leading to inflammation-associated colon carcinogenesis.

Carboxylated glycans mediate colitis through activation of NF-kappa B.

The role of carbohydrate modifications of glycoproteins in leukocyte trafficking is well established, but less is known concerning how glycans influence pathogenesis of inflammation. We previously identified a carboxylate modification of N-linked glycans that is recognized by S100A8, S100A9, and S100A12. The glycans are expressed on macrophages and dendritic cells of normal colonic lamina propria, and in inflammatory infiltrates in colon tissues from Crohn's disease patients. We assessed the contribution of these glycans to the development of colitis induced by CD4(+)CD45RB(high) T cell transfer to Rag1(-/-) mice. Administration of an anti-carboxylate glycan Ab markedly reduced clinical and histological disease in preventive and early therapeutic protocols. Ab treatment reduced accumulation of CD4(+) T cells in colon. This was accompanied by reduction in inflammatory cells, reduced expression of proinflammatory cytokines and of S100A8, S100A9, and receptor for advanced glycation end products. In vitro, the Ab inhibited expression of LPS-elicited cytokines and induced apoptosis of activated macrophages. It specifically blocked activation of NF-kappaB p65 in lamina propria cells of colitic mice and in activated macrophages. These results indicate that carboxylate-glycan-dependent pathways contribute to the early onset of colitis.