Gastroenteropancreatic neuroendocrine tumors (GEP-NET) account for roughly 60% of all neuroendocrine tumors. Low/intermediate grade human GEP-NETs have relatively low proliferation rates that animal models and cell lines fail to recapitulate. Short-term patient-derived cancer organoids (PDCOs) are a 3D model system that holds great promise for recapitulating well-differentiated human GEP-NETs. However, traditional measurements of drug response (i.e., growth, proliferation) are not effective in GEP-NET PDCOs due to the small volume of tissue and low proliferation rates that are characteristic of the disease. Here, we test a label-free, non-destructive optical metabolic imaging (OMI) method to measure drug response in live GEP-NET PDCOs. OMI captures the fluorescence lifetime and intensity of endogenous metabolic cofactors NAD(P)H and FAD. OMI has previously provided accurate predictions of drug response on a single cell level in other cancer types, but this is the first study to apply OMI to GEP-NETs. OMI tested the response to novel drug combination on GEP-NET PDCOs, specifically ABT263 (navitoclax), a Bcl-2 family inhibitor, and everolimus, a standard GEP-NET treatment that inhibits mTOR. Treatment response to ABT263, everolimus, and the combination were tested in GEP-NET PDCO lines derived from seven patients, using two-photon OMI. OMI measured a response to the combination treatment in 5 PDCO lines, at 72 h post-treatment. In one of the non-responsive PDCO lines, heterogeneous response was identified with two distinct subpopulations of cell metabolism. Overall, this work shows that OMI provides single-cell metabolic measurements of drug response in PDCOs to guide drug development for GEP-NET patients.

The gut microbiota has a significant impact on the development and function of intestinal epithelial cells (IECs) by modifying bile acid (BA) metabolites. Recently, specific gut microbiome-derived BAs, such as 7-oxo-deoxycholic acid (7-oxo-DCA) and isodeoxycholic acid (isoDCA), have been identified to be shifted inversely in colitis and hepatic liver diseases. Although the responsible gut microbes have been identified, metabolites’ effects on IECs remain largely unclear. We found that although high-fat diet treatment in mice elevated both 7-oxo-DCA and isoDCA levels, during intestinal tumorigenesis, 7-oxo-DCA levels rise while isoDCA levels decrease. Interestingly, 7-oxo-DCA promotes cancer cell growth, while isoDCA suppresses it. Moreover, 7-oxo-DCA promotes whereas isoDCA inhibits the proliferation of intestinal stem cells in organoids derived from WT and APC ^Min/+ mice, as well as in patient-derived colon cancer organoids. The APC ^Min/+ mice administered with 7-oxo-DCA heightened gut permeability and increased tumor burden, whereas isoDCA protected gut barrier and reduced tumor loads. Both BAs reshape the BA pool and shifted gut microbiome. Mechanistically, we identified 7-oxo-DCA as a natural antagonist of Farnesoid X Receptor (FXR) to downregulate FXR signaling, as opposed to isoDCA, which is a potent FXR agonist to upregulate FXR signaling. In conclusion, we unveiled the opposing roles of 7-oxo-DCA and isoDCA to promote or inhibit intestinal tumorigenesis, respectively. Manipulating the BA–FXR axis during tumor initiation and progression holds great promise for developing innovative diagnostic and therapeutic approaches for the treatment of colorectal cancer.

Human colorectal cancers often possess multiple mutations, including three to six driver mutations per tumor. The timing of when these mutations occur during tumor development and progression continues to be debated. More advanced lesions carry a greater number of driver mutations, indicating that colon tumors might progress from adenomas to carcinomas through the stepwise accumulation of mutations following tumor initiation. However, mutations that have been implicated in tumor progression have been identified in normal-appearing epithelial cells of the colon, leaving the possibility that these mutations might be present before the initiation of tumorigenesis. We utilized mouse models of colon cancer to investigate whether tumorigenesis still occurs through the adenoma-to-carcinoma sequence when multiple mutations are present at the time of tumor initiation. To create a model in which tumors could concomitantly possess mutations in Apc, Kras, and Pik3ca, we developed a novel minimally invasive technique to administer an adenovirus expressing Cre recombinase to a focal region of the colon. Here, we demonstrate that the presence of these additional driver mutations at the time of tumor initiation results in increased tumor multiplicity and an increased rate of progression to invasive adenocarcinomas. These cancers can even metastasize to retroperitoneal lymph nodes or the liver. However, despite having as many as three concomitant driver mutations at the time of initiation, these tumors still proceed through the adenoma-to-carcinoma sequence. Cancer Prev Res; 8(10); 952–61. ©2015 AACR.

In colorectal cancer (CRC), attempts to identify cancer cell-specific markers to guide antibody-mediated therapeutics have failed to uncover markers that are both exclusive to cancer tissues and abundant across CRCs. Alternatively, cancer-associated fibroblasts (CAFs), which are abundant in the tumor microenvironment and upregulate unique surface markers, are not found in healthy tissues. Here, we evaluated the expression patterns of CAF-associated proteins α-smooth muscle actin (αSMA), fibroblast activation protein (FAP), podoplanin (PDPN), matrix metalloproteinase-2 (MMP2), transgelin (TAGLN), and THY1. While αSMA and THY1 were abundant in cancer tissues, high abundance in normal tissues limited their targeting potential. FAP was present in 94.5% of primary and metastatic CRC tissues and absent in 93.7% of adjacent normal colon and liver tissues assessed. These results indicate that FAP is a promising target for antibody conjugates with potential for broad application in CRC. Co-expression analyses showed that CRCs simultaneously expressing high levels of PDPN, MMP2, and THY1 were enriched for immune-related signatures, indicating potential for antibody-mediated immune engagers. Overall, this work highlights the potential of CAF proteins to act as therapeutic targets for novel anticancer agents and become important therapeutic biomarkers.

Bile acids (BA) not only influence the gut microbiome composition but are also metabolized by gut bacteria to form various microbial BAs. Among these, 3-oxo-lithocholic acid (3-oxo-LCA) and isoallo-LCA have been reported to modulate host immunity, suppress intestinal pathogens, and provide antiaging benefits, suggesting that they could also affect intestinal epithelial cells and colorectal cancer progression. To investigate the impact of 3-oxo-LCA on intestinal tumorigenesis, we evaluated its activity in vitro on mouse and human colorectal cancer cell lines, as well as primary mouse intestinal organoids and patient-derived colorectal cancer organoids, and in vivo using a genetically engineered mouse model, cell line–derived syngeneic and xenograft tumors, and patient-derived xenografts. 3-Oxo-LCA functioned as a potent FXR agonist that restored FXR signaling both in vitro and in vivo. Activation of FXR signaling reduced the growth of colorectal cancer cell lines and suppressed the proliferation of intestinal stem cells in both mouse organoids and patient-derived colorectal cancer organoids. In the APCMin/+ genetically engineered mouse model, 3-oxo-LCA reduced BA levels, enhanced gut barrier function, decreased tumor burden, and suppressed tumor initiation. Furthermore, 3-oxo-LCA significantly inhibited tumor progression in syngeneic and xenograft mouse models and promoted apoptosis within the tumors. Together, these results underscore the function of 3-oxo-LCA as an FXR agonist with the ability to inhibit colorectal cancer tumorigenesis and progression by modulating epithelial cell growth and death. Significance: The microbial bile acid 3-oxo-LCA activates FXR signaling in intestinal epithelial cells that inhibits cancer stem cell proliferation and induces apoptosis, highlighting the potential of 3-oxo-LCA for treating intestinal tumorigenesis.

Targeting PIK3CA-mutant colorectal cancers with precision medicine strategies is of great clinical interest. However, resistance to single-agent PI3K pathway inhibitors has been observed across multiple clinical trials, necessitating the identification of combination therapies that overcome or prevent resistance to precision medicine strategies. Previously, our group identified that inhibition of mTORC1/2 is necessary to induce a response in PIK3CA-mutant colorectal cancers. The PI3K/mTORC1/2 inhibitor copanlisib has demonstrated some clinical activity in PIK3CA-mutant solid tumors as part of the NCI-MATCH trial. In this study, we evaluate potential combination therapies that could enhance the efficacy of copanlisib and other similar inhibitors in PIK3CA-mutant colorectal cancers. Using a novel high-throughput drug screen method in Apc- and Pik3ca-mutant mouse-derived cancer organoids, we identify navitoclax, a BCL-2 family inhibitor, as a drug that could potentially enhance the response to copanlisib. Across multiple in vitro and in vivo colorectal cancer models, navitoclax enhanced PI3K/mTOR inhibition (copanlisib, sapanisertib, and dactolisib) and induced apoptosis. Furthermore, we examine these combination therapies across a panel of patient-derived cancer organoids with a range of mutation profiles. These studies indicate that KRAS mutations could confer resistance. Furthermore, we identify BCL-xL as the major BCL-2 family target important for the response to this combination in this setting. This provides a strong rationale for mTORC1/2 and BCL-2 family inhibition as a potential treatment strategy for PIK3CA-mutant colorectal cancers.