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Colonoscopic images over time, whole mount image and H&E-stained section of a tumor from a Apcfl/fl Pik3cap110* mouse inoculated with Adeno-Cre that spontaneously decreased in size.

Supplementary Figure S1 presents the viability of SW48 and SW48PK cells after treatment with BEZ235 and TAK-228 at the 100-400nM concentrations.

PDF file - 30K, Relative gene expression is shown for three adenomas and three intramucosal carcinomas taken from the colons of DSS-treated F1 Min mice.

Supplementary Figure Legends

PDF file - 21K, Characteristics of F1 ApcMin/+ mice treated with 4% dextran sodium sulfate.

S1. AP spheroids were plated and allowed to mature for 48 hours. S2. To address the issue of potentially slower growth of the spheres with the largest initial sizes, a change-point analysis was performed. S3. AP mice were treated with control (A) or BEZ235 (B) for 14 days. S4. AP mice were treated with BEZ235 or control for 14 days. S5. AP spheroids were treated with NVP-BYL719, BEZ235, LY3023414, or control for 24 hours. S5. AP spheroids were treated with NVP-BYL719, BEZ235, LY3023414, or control for 24 hours. Supplementary Table S1. AP spheroids were treated with NVP-BYL719, GDC0941, BEZ235, LY3023414, or control for 48 hours. Supplemental Table S2. AP mice were treated with BEZ235, LY3023414 or control for 14 days.

PDF file - 54K, Panel A illustrates the colonoscopic surveillance protocol. Panel B shows the serial biopsy protocol.

PIK3CA mutations are common in clinical molecular profiling, yet an effective means to target these cancers has yet to be developed. MTORC1 inhibitors are often used off-label for patients with PIK3CA mutant cancers with only limited data to support this approach. Here we describe a cohort of patients treated with cancers possessing mutations activating the PI3K signaling cascade with minimal benefit to treatment with the MTORC1 inhibitor everolimus. Previously, we demonstrated that dual PI3K/mTOR inhibition could decrease proliferation, induce differentiation, and result in a treatment response in APC and PIK3CA mutant colorectal cancer. However, reactivation of AKT was identified, indicating that the majority of the benefit may be secondary to MTORC1/2 inhibition. TAK-228, an MTORC1/2 inhibitor, was compared with dual PI3K/mTOR inhibition using BEZ235 in murine colorectal cancer spheroids. A reduction in spheroid size was observed with TAK-228 and BEZ235 (−13% and −14%, respectively) compared with an increase of >200% in control (P < 0.001). These spheroids were resistant to MTORC1 inhibition. In transgenic mice possessing Pik3ca and Apc mutations, BEZ235 and TAK-228 resulted in a median reduction in colon tumor size of 19% and 20%, respectively, with control tumors having a median increase of 18% (P = 0.02 and 0.004, respectively). This response correlated with a decrease in the phosphorylation of 4EBP1 and RPS6. MTORC1/2 inhibition is sufficient to overcome resistance to everolimus and induce a treatment response in PIK3CA mutant colorectal cancers and deserves investigation in clinical trials and in future combination regimens.

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.

Characteristics of the mice treated with Adeno-Cre.

PDF file - 42K, Complete list of differentially expressed genes between intramucosal carcinomas and adenomas.

Colorectal cancer often arises from adenomatous colonic polyps. Polyps can grow and progress to cancer, but may also remain static in size, regress, or resolve. Predicting which polyps progress and which remain benign is difficult. We developed a novel long-lived murine model of colorectal cancer with tumors that can be followed by colonoscopy. Our aim was to assess whether these tumors have similar growth patterns and histologic fates to human colorectal polyps to identify features to aid in risk stratification of colonic tumors. Long-lived ApcMin/+ mice were treated with dextran sodium sulfate to promote colonic tumorigenesis. Tumor growth patterns were characterized by serial colonoscopy with biopsies obtained for immunohistochemistry and gene expression profiling. Tumors grew, remained static, regressed, or resolved over time with different relative frequencies. Newly developed tumors demonstrated higher rates of growth and resolution than more established tumors that tended to remain static in size. Colonic tumors were hyperplastic lesions (3%), adenomas (73%), intramucosal carcinomas (20%), or adenocarcinomas (3%). Interestingly, the level of β-catenin was higher in adenomas that became intratumoral carcinomas than those that failed to progress. In addition, differentially expressed genes between adenomas and intramucosal carcinomas were identified. This novel murine model of intestinal tumorigenesis develops colonic tumors that can be monitored by serial colonoscopy, mirror growth patterns seen in human colorectal polyps, and progress to colorectal cancer. Further characterization of cellular and molecular features is needed to determine which features can be used to risk-stratify polyps for progression to colorectal cancer and potentially guide prevention strategies. Cancer Prev Res; 7(1); 105–13. ©2013 AACR.

PDF file - 28K, Differentially Expressed Gene Functional Categories between Adenomas and Intramucosal Carcinomas

Dot plots corresponding to population distribution curves and graphs of individual replicates for bar graphs in Figure 4.

Purpose:Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed.Experimental Design:Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively.Results:We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer.Conclusions:These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.

Supplementary Methods

Success rates for growing human cancers in spheroid culture

WMP file - 834MB, The tumors in FC+ PIK3ca*+ mice exhibit high avidity for 124I-CLR1404. A mouse was injected with the imaging agent and then dual hybrid PET/CT colonography was performed. The agent was detected in all three tumors. This mouse is the same as shown in Figure 1, panels C and D. The agent was also detected in heart.

PDF file - 583K, Mice expressing activated PI3K were scanned to identify those bearing tumors. After sacrifice, the intestine was removed to characterize tissue morphology, cell signaling and cell proliferation within the epithelium and the associated neoplasms

Colorectal cancer (CRC) is a leading cause of cancer death in the United States. Standard treatment for advanced stage CRC for decades has included 5-fluorouracil based chemotherapy. More recently, targeted therapies for metastatic CRC are being used based on the individual cancer's molecular profile. In the past few years, several different molecular subtype schemes for human CRC have been developed. The molecular subtypes can be distinguished by gene expression signatures and have the potential to be used to guide treatment decisions. However, many subtyping classification methods were developed using mRNA expression levels of hundreds to thousands of genes, making them impractical for clinical use. In this study, we assessed whether an immunohistochemical approach could be used for molecular subtyping of colorectal cancers. We validated two previously published, independent sets of immunohistochemistry classifiers and modified the published methods to improve the accuracy of the scoring methods. In addition, we evaluated whether protein and genetic signatures identified originally in the mouse were linked to clinical outcomes of patients with CRC. We found that low DDAH1 or low GAL3ST2 protein levels in human CRCs correlate with poor patient outcome. The results of this study have the potential to impact methods for determining the prognosis and therapy selection for CRC patients.

Background Data are steadily accruing that demonstrate that intestinal tumors are frequently derived from multiple founding cells, resulting in tumors comprised of distinct ancestral clones that might cooperate or alternatively compete, thereby potentially impacting different phases of the disease process. Aim We sought to determine whether tumors with a multi-ancestral architecture involving at least two distinct clones show increased tumor number, growth, progression, or resistance to drug intervention. Methods Mice carrying the Min allele of Apc were generated that were mosaic with only a subset of cells in the intestinal epithelium expressing an activated form of PI3K, a key regulatory kinase affecting several important cellular processes. These cells were identifiable as they fluoresced green, whereas all other cells fluoresced red. Results Cell lineage tracing revealed that many intestinal tumors from our mouse model were derived from at least two founding cells, those expressing the activated PI3K (green) and those which did not (red). Heterotypic tumors with a multi-ancestral architecture as evidenced by a mixture of green and red cells exhibited increased tumor growth and invasiveness. Clonal architecture also had an impact on tumor response to low-dose aspirin. Aspirin treatment resulted in a greater reduction of heterotypic tumors derived from multiple founding cells as compared to tumors derived from a single founding cell. Conclusion These data indicate that genetically distinct tumor-founding cells can contribute to early intratumoral heterogeneity. The coevolution of the founding cells and their progeny enhances colon tumor progression and impacts the response to aspirin. These findings are important to a more complete understanding of tumorigenesis with consequences for several distinct models of tumor evolution. They also have practical implications to the clinic. Mouse models with heterogenous tumors are likely better for predicting drug efficacy as compared to models in which the tumors are highly homogeneous. Moreover, understanding how interactions among different populations in a single heterotypic tumor with a multi-ancestral architecture impact response to a single agent and combination therapies are necessary to fully develop personalized medicine.

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. These results were corroborated using ~ 1700 patient-derived cancer organoids (PDCOs) isolated from 19 patients. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies.

Tumor heterogeneity is predicted to confer inferior clinical outcomes, however modeling heterogeneity in a manner that still represents the tumor of origin remains a formidable challenge. Sequencing technologies are limited in their ability to identify rare subclonal populations and predict response to the multitude of available treatments for patients. Patient-derived organotypic cultures have significantly improved the modeling of cancer biology by faithfully representing the molecular features of primary malignant tissues. Patient-derived cancer organoid (PCO) cultures contain numerous individual organoids with the potential to recapitulate heterogeneity, though PCOs are most commonly studied in bulk ignoring any diversity in the molecular profile or treatment response. Here we demonstrate the advantage of evaluating individual PCOs in conjunction with cellular level optical metabolic imaging to characterize the largely ignored heterogeneity within these cultures to predict clinical therapeutic response, identify subclonal populations, and determine patient specific mechanisms of resistance.

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3,500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies using murine colorectal cancer organoids.

Simple Summary Gastroenteropancreatic neuroendocrine tumors (GEP-NET) account for roughly 60% of all neuroendocrine tumors, and low/intermediate grade human GEP-NETs have relatively slow growth rates that many laboratory culture methods fail to capture. Patient-derived cancer organoids (PDCOs) are an attractive model to address this need for relevant 3D cultures of GEP-NETs for laboratory drug testing. However, traditional measurements of drug response are not effective in GEP-NET PDCOs due to the small volume of tissue and slow growth 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 measured a response to the novel treatment combination of ABT-263 and everolimus in five out of seven PDCO lines, at 72 h post-treatment. Overall, this work shows that OMI provides single-cell metabolic measurements of drug response in PDCOs to guide drug development for GEP-NET patients. Abstract 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 normal colon epithelium is transformed into its neoplastic counterpart through a series of genetic alterations in driver genes including activating mutations in PIK3CA. Treatment often involves surgery followed by 5-fluorouracil (5-FU) based therapy, which has limited efficiency and serious side effects. We sought to determine whether fisetin, a dietary flavonoid, alone or in combination with 5-FU affected tumorigenesis in the mammalian intestine. We first determined the effect of fisetin, 5-FU or their combination on PIK3CA-mutant and PIK3CA wild-type colon cancer cells by assessing cell viability, colony formation, apoptosis and effects on PI3K/AKT/mTOR signaling. Treatment of PIK3CA-mutant cells with fisetin and 5-FU reduced the expression of PI3K, phosphorylation of AKT, mTOR, its target proteins, constituents of mTOR signaling complex and this treatment increased the phosphorylation of AMPKα. We then determined whether fisetin and 5-FU together or singly affected tumorigenesis in Apc(Min/+) mice that also express constitutively active PI3K in the distal small intestine and colon. Tumor incidence was markedly lower in fisetin-treated FC(1)3K(1)Apc(Min/+) mice that also express constitutively active PI3K in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5-FU also reduced the total number of intestinal tumors. Fisetin could be used as a preventive agent plus an adjuvant with 5-FU for the treatment of PIK3CA-mutant colorectal cancer.

Significance The premalignant colonic adenoma presents a long-lived target to prevent colon cancer. The levels of peptide biomarkers for four serum proteins—F5, ITIH4, LRG1, and VTN—are each elevated in sera in two adenoma-bearing animal models and in patients. The elevated levels of these serum proteins are correlated with total colonic adenoma number in the rat model. Longitudinal analysis of patients demonstrated their association with the subset of adenomas that continue to grow, but not with the total volume of the adenoma burden. This pattern of correlation with the number and growth, but not total adenoma volume, indicates that this class of marker can complement an emergent class of volume-dependent markers to detect the growing early colonic adenoma, minimizing overdiagnosis., A major challenge for the reduction of colon cancer is to detect patients carrying high-risk premalignant adenomas with minimally invasive testing. As one step, we have addressed the feasibility of detecting protein signals in the serum of patients carrying an adenoma as small as 6–9 mm in maximum linear dimension. Serum protein biomarkers, discovered in two animal models of early colonic adenomagenesis, were studied in patients using quantitative mass-spectrometric assays. One cohort included patients bearing adenomas known to be growing on the basis of longitudinal computed tomographic colonography. The other cohort, screened by optical colonoscopy, included both patients free of adenomas and patients bearing adenomas whose risk status was judged by histopathology. The markers F5, ITIH4, LRG1, and VTN were each elevated both in this patient study and in the studies of the Pirc rat model. The quantitative study in the Pirc rat model had demonstrated that the elevated level of each of these markers is correlated with the number of colonic adenomas. However, the levels of these markers in patients were not significantly correlated with the total adenoma volume. Postpolypectomy blood samples demonstrated that the elevated levels of these four conserved markers persisted after polypectomy. Two additional serum markers rapidly renormalized after polypectomy: growth-associated CRP levels were enhanced only with high-risk adenomas, while PI16 levels, not associated with growth, were reduced regardless of risk status. We discuss biological hypotheses to account for these observations, and ways for these signals to contribute to the prevention of colon cancer.

Background: Treating colorectal cancer (CRC) using targeted therapies based on each patient’s individual mutational profile continues to gain interest. Oncogenic mutations of the PIK3CA gene leads to tumor initiation and progression. Copanlisib inhibits the PI3K pathway and has proven to be an effective treatment strategy for various cancers characterized by overactivation of the PI3K signaling cascade. However, resistance mechanisms to targeted therapies such as copanlisib are frequently encountered, and can be overcome through combination with another molecule such as romidepsin, a histone deacetylase (HDAC) inhibitor. Here we aim to identify a novel therapeutic regimen for improved treatment of the molecular subtypes of CRC that can be targeted with PI3K inhibition. Methods: Organoids were derived from colorectal cancer tumor tissue from two different biopsy sites from one patient with PIK3CA mutant rectal cancer (RC46A and RC46B), as well as from a patient with APC and TP53 mutant CRC (MTB74) under approved IRB protocols. All organoids were plated and allowed to mature for 24 to 48 hours before treatment and baseline images were obtained. The organoids were imaged again 48 hours later, and response was determined by measuring the change in organoid diameter using ImageJ. Additionally, organoids were collected for immunoblotting to examine alterations in the PI3K pathway as well as histone acetylation, and proteins involved in apoptosis. Results: Control organoids demonstrated increased median relative changes in diameter of +53.38%, +43.04% and +46.2% for RC46A, RC46B and MTB74, respectively. In comparison, the combination treatment group exhibited median relative changes for RC46A, RC46B and MTB74 as -29.19%, -46.46% and -100% (p Conclusions: As molecular diagnostic technology continues to advance, treating each individual cancer with a targeted therapy based on molecular profiling is becoming more feasible. Dual inhibition of the PI3K pathway and HDAC is effective in reducing cell growth and proliferation of CRC organoids characterized by PIK3CA mutations, as well as other molecular subtypes such as cancers with APC and TP53 mutations. This combination strategy shows promise for improving treatment response and reducing resistance in patients across these molecular subtypes. Citation Format: Alyssa K. DeZeeuw, Rebecca A. DeStefanis, Susan N. Payne, Autumn M. Olson, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Combined inhibition of PI3K and HDAC1/2 as a novel treatment strategy for RAS/RAF wildtype colorectal cancer in a patient derived organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2673.

Background: Cancer Associated Fibroblasts (CAFs) are a significant component of tumor stroma, and have an important impact on immune infiltration in the tumor microenvironment (TME). Two major subtypes of CAFs have been previously identified by literature: myofibroblastic (myCAF) and inflammatory (iCAF). Our lab has identified subtype markers for each CAF phenotype and previously analyzed a sampling of 153 colorectal cancer (CRC) patients. Here, we validate these subtype markers and investigate CAF phenotypes in metastatic colorectal cancer patients. Methods: Dual immunofluorescence on formalin fixed paraffin embedded tissue sections was performed to analyze co-staining between combinations of myCAF markers, αSMA and TAGLN, and iCAF markers, PDPN and ICAM1. Slides were imaged using a fluorescent microscope. Also, tissue microarrays sampling 212 CRC patients spanning all stages of disease, 90 with matched metastatic cores, were stained via immunohistochemistry (IHC) for the CAF subtype markers then quantified on an intensity scale from 0-3+. iCAF and myCAF marker scores were averaged to get a composite score for each, then split into low (average score Results: Significant co-staining was observed between iCAF markers PDPN and ICAM1, as well as myCAF markers αSMA and TAGLN. Co-staining did not occur, or was minimal, between combinations of myCAF and iCAF markers. There is not significant different in abundance of iCAFs or myCAFs in primary site cores of patients with metastatic versus non-metastatic disease (p = 0.67 for iCAF, p = 0.57 for myCAF). Of matched primary and metastatic samples able to be scored, 43.3% of samples had a decrease in iCAF score from primary to metastatic site while only 18.8% increased. Overall, 34.4% of samples had a decrease in score of more than 1 and only 2.2% of samples had an increase of more than 1. However, the percentage of samples that had a decrease in myCAF score was 32.2% while 22.2% increased. In all primary cores of patients with metastatic disease, there was higher average CD8+ TILs in those with high iCAF scores compared to those with low iCAF scores (12.0 vs 5.5, p=0.03). There was not a significant difference in average CD8+ TILs in those with high myCAF scores compared to those with low myCAF scores (9.3 vs 7.1, p=0.7). Conclusions: Here, we validate the myCAF markers TAGLN and αSMA, as well as, iCAF markers ICAM1 and PDPN by demonstrating co-staining between CAFs of the same subtype and exclusion between different subtypes. These data indicate that that CAF phenotype correlates with CD8 T cell infiltration into the TME. iCAFs correlate with immune infiltration and myCAFs with immune exclusion. Citation Format: Anna L. Lippert, Katherine A. Johnson, Cheri A. Pasch, Sean G. Kraus, Philip B. Emmerich, Linda Clipson, Kristina A. Matkowskyj, Wei Zhang, Dustin A. Deming. Validation and analysis of cancer associated fibroblast subtype markers in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3198.

Background: A major roadblock to immune-therapy response in many solid malignancies is the lack of tumor-infiltrating lymphocytes (TILs) found within their immune micro-environments. These immunologically cold tumors have lower response rates to immune-based therapies and among all colorectal cancers (CRC), only about six percent of patients have an objective response to immune checkpoint blockade (ICB). Versican (VCAN) accumulation has been found to be negatively correlated with TILs and its proteolysis into versikine (Vkine) correlates with higher TILs. In this study we analyze the association between VCAN accumulation/proteolysis and TILs in CRC from patients with both resectable primary CRCs and matched liver metastases. Methods: VCAN, Vkine, and TIL abundance were assessed via immunohistochemistry, with VCAN and Vkine being measured using an intensity binning system ranging from 0-3+ and TILs counted at 40X magnification for each tumor. Tumors were then designated as high (2 or 3+) or low (0 or 1) for both VCAN and Vkine. Tumors that were designated VCAN low and Vkine high are considered VCAN proteolytic predominant (VPP) and all other combinations are considered VCAN proteolytic weak (VPW). Results: 53% of both primary and metastatic tumors were designated VCAN low and 47% VCAN high. 59% of metastases from patients with VCAN-high primary tumors were also designated VCAN high, whereas 43% of metastases from patients with VCAN low primary tumors were designated VCAN high. When assessing proteolysis, it was found that 37% of primary tumors were designated VPP but only 26% of metastases were found to be VPP. When comparing proteolysis in a pairwise fashion, it was found that only 37% of VPP primaries had a VPP metastasis. An inverse correlation was again found between VCAN accumulation and TILs within primary CRCs. Tumors containing high levels of VCAN had an average of three TILs/high powered field (HPF), whereas tumors with low levels of VCAN had an average of 16 TILs/HPF (p=0.007). VPW tumors had an average of three TILs/HPF and VPP cores had an average of 21 TILs/HPF (p=0.009). VPW metastatic tumors had an average of eight TILs/HPF, whereas those that were VPP had an average of 15 TILs/HPF (p=0.01). Conclusion: Overall, these data confirm the inverse correlations between VCAN accumulation and TILs in primary CRCs. Additionally, VCAN proteolysis correlates with TILs in primary tumors and metastases. The potential for different VCAN accumulation/proteolysis in the primary tumor and metastatic disease will be an important consideration when studying VCAN further as a potential biomarker of immunotherapy response. Citation Format: Sean G. Kraus, Kristina A. Matkowskyj, Philip B. Emmerich, Wei Zhang, Linda Clipson, Cheri A. Pasch, Dustin A. Deming. Versican proteolysis is a predictive biomarker of tumor infiltrating lymphocytes within primary and metastatic colorectal cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3842.

Background: Cancer-associated fibroblasts (CAFs) are major regulators of the immune microenvironment and therapeutic response in colorectal cancer (CRC). Neutralizing their role in modulating the immune landscape could be the key to enhancing immunotherapy success. Imatinib, dasatinib, and nilotinib are tyrosine kinase inhibitors with several kinase targets. Methods: Tissue microarrays spanning 153 patients were stained for αSMA, TAGLN, PDPN, ICAM1, and CD8. CD8 stains were quantified as number of tumor infiltrating lymphocytes per high powered field (TILs/HPF) in the epithelial compartment. All other stains were quantified by intensity on a 0-3+ scale. Scores for αSMA and TAGLN were combined into a myCAF score, and PDPN and ICAM1 into an iCAF score. myCAF gene expression signatures derived from a re-analysis of scRNA-seq data previously done by our lab were entered into the LINCS database to discover potential drugs to reverse the phenotype. Primary cancer associated fibroblasts were derived from patient tumor samples, then treated with clinically relevant concentrations of imatinib, dasatinib, or nilotinib for 96 hours. RNA was isolated and RT-qPCR was performed to quantify the myCAF genes ACTA2, COL11A1, and TAGLN, and the iCAF genes ICAM1, PDPN, IL1R1, CXCL1 and CXCL2. TGFB1 expression was also measured. Expression was normalized to untreated cells and GAPDH expression levels. Results: Cancers with high expression of myCAF markers but low expression of iCAF markers had the most CD8+ TILs (average 10.2; median 1.5; range 0-73), while cancers with low myCAF scores and high iCAF scores had the least (average 1.5; median 0; range 0-19; p < 0.01). Reversing the myCAF signature relative to iCAFs in the LINCs database revealed nilotinib as a top hit. Treatment with imatinib did not significantly alter the expression of myCAF genes (control vs. max dose: p = 0.06 for ACTA2, COL11A1 p =0.2, TAGLN p = 1), while treatment with dasatinib significantly increased these genes (ACTA2 1.4x higher, p < 0.001; COL11A1 2.6x higher, p < 0.01; TAGLN 1.5x higher, p < 0.001). Only treatment with nilotinib significantly decreased myCAF genes (ACTA2 2.2x lower, p Conclusions: myCAFs may be major actors in immune exclusion in the microenvironment, and the reversal of the myCAF phenotype may be a target for treatment with immunotherapy. Nilotinib, but not imatinib or dasatinib, is effective at decreasing expression of myCAF genes. Further research is warranted into the mechanisms of this drug on altering expression and whether these trends continue in vivo. Citation Format: Katherine Anne Johnson, Anna L. Lippert, Sean G. Kraus, Grace E. McGrath, Philip B. Emmerich, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Wei Zhang, Dustin A. Deming. Effects of tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib on cancer-associated fibroblast phenotypes in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3533.

Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related deaths. PIK3CA mutations are found in 18% of CRCs which lead to the constitutive activation of the PI3K/MTOR pathway and can promote tumorigenesis. Here, we examine the efficacy of the FDA approved inhibitors copanlisib, a PI3K/MTOR inhibitor, and romidepsin, a HDAC1/2 inhibitor, in CRC with a PIK3CA mutation. Methods: CRC mouse derived cancer organoids (MDCOs) were derived from Apc and Pik3ca mutant mice (Fc1Apcfl/+Pik3caH1047R/+). Brightfield images of the MDCOs were taken prior to treatment with copanlisib, romidepsin, or the combination and 48 hours post-treatment. The change in diameter of each organoid over the 48-hour treatment was determined. Additionally, immunoblotting was performed to confirm known targets of copanlisib and romidepsin were altered in response to drug treatment. MTORC1/2 and HDAC1/2 inhibition were also investigated in vivo. SW48 and SW48PIK3CA-H1047R (SW48PK) xenograft mice were treated with a vehicle control, copanlisib, romidepsin, or the combination therapy. Results: In the diameter analysis study, the combination therapy had the largest effect size compared to control (Glass’s Δ 2.82). Single agents copanlisib and romidepsin had smaller effect sizes (Glass’s Δ 1.75 and 2.04, respectively). Immunoblotting results indicated a decrease in phosphoAKT (Ser473) and pRPS6 (Ser235/236) in the copanlisib treated samples and an increase in H3K27 acetylation was seen in the romidepsin treated samples. There was also increased cleaved PARP, an indicator of apoptosis, in the romidepsin and combination therapy treatment groups. In vivo, SW48 xenografts showed a greater response in the combination therapy compared to either single agent therapy (median relative change in tumor volume: control=339%; combination therapy=107%(p-value Conclusion: MDCOs with Apc and Pik3ca mutations had an increased response to treatment with the combination therapy as compared to the single agents alone. Additionally, in vivo studies with human CRC xenografts showed enhanced inhibition of tumor growth with both MTORC1/2 and HDAC1/2 inhibition. These data demonstrate potential for this combination treatment strategy for the treatment of PIK3CA mutant CRC and this combination warrants further investigation in other models and clinically. Citation Format: Autumn M. Olson, Rebecca A. DeStefanis, Alyssa K. DeZeeuw, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. MTORC1/2 and HDAC1/2 inhibition as therapy for colorectal cancer with PIK3CA mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3721.

Background: The identification of treatment strategies targeting PIK3CA mutant colorectal cancer (CRC) are of great clinical interest. Previous work from our lab has identified MTORC1/2 and HDAC1/2 inhibition with copanlisib (cop; PI3K/MTOR) and romidepsin (romi; HDAC1/2) as a potential therapeutic strategy. We hypothesized that changes in c-MYC protein levels and c-MYC target gene (CTG) alterations might be a potential mechanism of action for this combination. Methods: Known CTGs were identified and their expression levels were examined in PIK3CA mutant vs WT CRCs using the cBioPortal Colorectal Adenocarcinoma (TCGA, PanCancer Atlas) dataset. Murine derived cancer organoids (MDCO) were generated from adenocarcinomas of Apc and Pik3ca mutant transgenic mice (F1 (FVB x B6) Apcfl/+ Pik3caH1047R/+). MDCO results were corroborated using the human 2D isogenic cell lines SW48 and SW48PIK3CA-H1047R (SW48PK) and RAS/RAF WT patient derived cancer organoids (PDCO) generated from CRC patient samples under approved IRB protocols. Immunoblots (IB) were used to assess c-MYC levels after treatment with cop, romi, and the combination across all models. RNA sequencing was conducted on PDCOs and SW48PK cells and changes in 16 CTGs were examined. An aggregate score was created for each treatment group where a statistically significantly altered CTG with log fold change ≥1.5 added one point and ≤-1.5 subtracted one point. All others were scored 0. Results: c-MYC target genes were assessed for differential expression in PIK3CA mutant CRC vs PIK3CA WT CRC with only 1/16 genes decreased in PIK3CA mutant CRC (GADD45A: log ratio -0.21, q=0.01). Next, MDCOs treated with the combination showed a decrease in total c-MYC levels in the combination therapy. These results were corroborated in two human 2D CRC cell lines, SW48 and SW48PK and a panel of PDCOs. Interestingly, c-MYC levels decreased in both romi alone and the combination in both the SW48 and SW48PK cell lines after 24 hours of treatment. However, the extent to which c-MYC levels were decreased was not as substantial in the panel of PDCOs. Across all in vitro models a decrease in PI3K signaling, as illustrated by decreased pRPS6 and p4EBP1 in response to cop treatment and an increase in H3K27 acetylation in response to romi, was observed in the single agent and combination therapies. RNA sequencing demonstrated that cop, romi, and combo had a CTG score of 0, -4, and -7 respectively in the PIK3CA mutant PDCO. Similar results were seen in an additional RAS/RAF WT PDCO (0, 1, and -4, respectively) and SW48PK cells (0, -3, and -7, respectively). Conclusion: A potential mechanism by which cop and romi treatment promote tumor response is through a decrease in c-MYC protein levels and expression of downstream c-MYC target genes. This regimen deserves further mechanistic investigations in vivo. Citation Format: Rebecca A. DeStefanis, Autumn M. Olson, Alyssa K. DeZeeuw, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. MTORC1/2 and HDAC1/2 inhibition promote tumor response through inhibition of MYC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1128.

Purpose. Estimating the cancer risk associated with interplanetary travel is complicated by the lack of data for human exposure to high atomic number, high-energy (HZE) radiation encountered in space. Therefore, human exposure data from low linear energy transfer (low-LET) γ-ray radiation is used in cancer risk models with the critical assumption that HZE and γ-ray radiation have comparable biological effects, including the induction of the same spectrum of tumors with similar lethality. Recently this assumption has been challenged by several reports indicating that HZE radiation might produce more aggressive tumors. The goal of this research is to test the hypothesis that high-LET HZE radiation induced tumors are more aggressive than those that arise spontaneously or from exposure to low-LET γ-rays. Materials & methods. The FVB/N-Tg(MMTV-PyMT) and (DBAxFVB)F1-Tg(MMTV-PyMT) murine models of mammary cancer and the C3H-Tg(Afp-mCherry) and C3B6F1-Tg(Afp-mCherry) liver cancer models were used to determine the impact of exposure to 0.2 Gy of HZE 300 MeV/n silicon ions in comparison to 3 Gy of γ-rays and no radiation. Various measures of tumor aggressiveness including latency, size, pathological grade, prognostic biomarkers, metastasis, and survival were assessed. Results. For both mammary cancer models, there was no significant change in the latency of mammary tumors or in the number or size of pulmonary metastasis in silicon-irradiated mice compared to their sham-irradiated controls. For the C3H-Tg and C3B6-Tg liver cancer models, we did observe an increase in the incidence of liver cancer in irradiated mice, but no differences in measures of aggressiveness, including tumor size, grade, metastasis, or overall survival. Conclusion. The principle finding of our study is that tumors in the HZE-irradiated mice were not more aggressive than those arising from exposure to low-LET γ-rays or spontaneously. Thus, we conclude that enhanced aggressiveness does not appear to be a uniform characteristic of all tumors in HZE-irradiated animals.

Though many advancements in personalized medicine have been made, better methods are still needed to predict treatment benefit for patients with colorectal cancer. Patient-derived cancer organoids (PDCOs) are a major advance towards true personalization of treatment strategies. A growing body of literature is demonstrating the feasibility of PDCOs as an accurate and high-throughput preclinical tool for patient treatment selection. Many studies demonstrate that these cultures are readily generated and represent the tumors they were derived from phenotypically and based on their mutation profile. This includes maintenance of the driver muatations giving the cancer cells a selective growth advantage, and also heterogeneity, including molecular and metabolic heterogeneity. Additionally, PDCOs are now being utilized to develop patient biospecimen repositories, perform high to moderate-throughput drug screening, and to potentially predict treatment response for individual patients that are undergoing anti-cancer treatments. In order to develop PDCOs as a true clinical tool, further studies are required to determine the reproducibility and accuracy of these models to predict patient response.

Background: The incidence of colorectal cancer (CRC) in those of pre-screening age continues to rise. A deeper understanding of the differences in tumor biology in early age onset (EAO) cancers is needed. Differences in the tumor microenvironment have been understudied in this setting. We previously have identified versican (VCAN), a large matrix proteoglycan, as an important factor in lymphocyte exclusion in CRC. Here we explore the impact of the mutation profile and VCAN status on the infiltration of CD8+ and CD4+ lymphocytes in EAO CRC. Methods: Cancer tissues from 153 patients with CRC were stained via immunohistochemistry for CD4, CD8, and VCAN. CD4 and CD8 stains were quantified by number of positive-staining tumor-infiltrating lymphocytes per high powered field (TILs/HPF). VCAN stains were quantified on an intensity scale from 0-3+. 121 of these patients had tissue that was sequenced using the Qiagen Comprehensive Cancer targeted sequencing panel, and variations were called using Strelka. Patients were split by age into early-onset (EAO; age at diagnosis Results: Mutations in BRAF and APC were significantly more common in LAO cancers (p Conclusions: The impacts of TP53 and PIK3CA mutations on immune infiltration in CRC differ in EAO compared to later onset cancers, indicating that the processes controlling immune infiltration vary between age cohorts. Such differences in immune infiltration may be linked to changes in microenvironmental factors such as VCAN. These data warrant further investigation into the relationship between tumor microenvironment and mutation profiles in EAO CRC. Citation Format: Katherine A. Johnson, Philip B. Emmerich, Anna L. Lippert, Cheri A. Pasch, Linda Clipson, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Impact of the mutation profile and versican status on lymphocyte infiltration in early age onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2734.

Impact of baseline culture condition on the growth and treatment response of mouse derived cancer organoidsAuthors: Autumn Olson, Rebecca A. DeStefanis, Alyssa DeZeeuw, Samantha Anderson, Gioia Sha, Jeremy Kratz, Cheri Pasch, Linda Clipson, Dustin A. DemingIntroduction: Colorectal cancer (CRC) is a leading cause of cancer related deaths and there has been an increased focus on developing therapies targeted to the mutational profile of individual patients. Mouse derived cancer organoids (MDCOs) are used as a model for therapeutic drug testing. How the culture conditions can impact the determination of treatment response from MDCOs has been understudied and is the focus of these investigations. Here, we examine how baseline size, passage number, plating density, location within the Matrigel droplet, and lineage impact MDCO growth and treatment response.Methods: CRC MDCOs were derived from multiple tumors across several Apc and Pik3ca mutant mice (Fc1 Apcfl/+ Pik3caH1047R/+) totaling 3,152 individual MDCOs. Untreated MDCO growth was analyzed across 902 MDCOs. Additionally, the PI3K pathway inhibitors, AZD2014, Everolimus, BEZ235, MLN0128, Palbociclib, and Copanlisib were analyzed across 2,250 MDCOs. In each study, baseline brightfield images of the MDCOs were taken and following 48 hours. Individual MDCOs were evaluated for the impact of baseline size, passage number (passage 1-15), plating density, location within the Matrigel droplet, and the generation of MDCOs across different mice on MDCO growth and treatment response. A change point analysis was used to assess the impact of baseline size. The percent relative change in diameter over 48 hours was compared to each other culture condition by calculating an R2 value.Results: A change in the organoid baseline growth rate was observed if the MDCOs were greater than 0.373 mm in baseline was identified. There were no significant differences in growth due to changes in the other culture conditions for the untreated MDCOs. The R2 values comparing the percent relative change in diameter over 48 hours to the culture conditions of passage number, plating density, location within the Matrigel droplet, and from which mouse the MDCOs were derived from were 0.0103, 0.0243, 0.0001, and 0.0295, respectively. There were no significant differences in the response to PI3K pathway inhibition of the MDCOs related to passage number, plating density, location within the Matrigel droplet, and from which mouse the MDCOs were derived from (R2 values: 0.1174, 0.0979, 0.0221, and 0.1267, respectively).Conclusions: Mouse derived cancer organoids are a useful tool in modeling drug response of cancers with specific mutational profiles. While the baseline size of MDCOs can change the growth rate, the growth rate and treatment response of MDCOs is not impacted by the passage number, plating density, location within the Matrigel, and from which mouse the MDCOs were derived. Citation Format: Autumn Olson, Rebecca A. DeStefanis, Alyssa DeZeeuw, Samantha Anderson, Gioia Sha, Jeremy Kratz, Cheri Pasch, Linda Clipson, Dustin A. Deming. Impact of baseline culture condition on the growth and treatment response of mouse derived cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2969.

Background: Incidences of early age onset colorectal cancer (CRC) (those diagnosed before age 50) have been increasing by 2% every year since the 1990s, with the rate expecting to double by 2030. Although there has been some research done on differences in mutational profiles between patients diagnosed before and after age 50, little research has been done to understand the immune and stromal environments of early age onset (EAO) CRC. As identified by previous literature, there are two distinct phenotypes of cancer associated fibroblasts (CAFs): myofibroblastic (myCAFs) and non-myofibroblastic (non-myCAFs). Here, we evaluate both CAF environments as well as immune infiltrating cells in context of EAO CRC. Methods: A total of 153 CRC patient samples were obtained with matching adjacent normal tissue. Of these, 60 patients had EAO CRC. Tissue slides were stained via immunohistochemistry (IHC) for the CAF subtype markers αSMA, FAP, PDPN, and MMP2, by Masson's Trichrome for collagen, and then quantified on an intensity scale from 0-3. MyCAF and non-myCAF scores were calculated by averaging the scores of αSMA and collagen, or FAP, PDPN, and MMP2, respectively. Once these scores were determined, they were split into low (average score Results: Cancers with a low myCAF and non-myCAF score display the highest average number of CD4+ (10.6) and CD8+ (10.3) TILs across both age groups. Furthermore, cancers with both myCAF and non-myCAF high scores had reduced average CD4+ and CD8+ TILs when compared to both CAF scores being low (p value: 0.018 for CD8+ TILs, Conclusions: Here we demonstrate that there are differences in the stromal and immune microenvironments between both age groups of CRC. We indicate that increased myCAF and non-myCAF scores are associated with T cell exclusion. However, the extent of T cell exclusion is different in EAO CRC compared to patients diagnosed after age 50. The reasoning for the difference in T-cell exclusion remains unknown and this implicates the importance for further research into the stromal and immune microenvironments of EAO CRC. Citation Format: Anna Lippert, Katherine A. Johnson, Philip B. Emmerich, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyi, Wei Zhang, Dustin A. Deming. Impact of cancer associated fibroblast phenotypes on the infiltration of t-lymphocytes in early age onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3166.

Background: Mutations in PIK3CA occur in 18% of colorectal cancers (CRC) and result in constitutive activation of the PI3K pathway. Previous work from our lab has determined that MTORC1/2 inhibition, with copanlisib (cop), a PI3K/MTOR inhibitor, is sufficient to induce a treatment response. We hypothesized that the BCL2 family inhibitor navitoclax (nav), a BCL2/BCLxL inhibitor, would enhance the therapeutic response of cop and increase the induction of apoptosis. Methods: Murine derived cancer organoids (MDCOs) were generated from adenocarcinomas arising in Apc and Pik3ca mutant transgenic mice ((FVB x B6) F1 Apcfl/+ Pik3caH1047R/+). MDCOs were plated and matured for 24 hours. MDCOs were treated for 48 hours. Change in diameter was used to assess response. Additionally, human 2D isogenic cell lines SW48 and SW48PIK3CA-H1047R (SW48PK) were plated and treated for 48 hours. WST-1 proliferation assay was used to determine response. In vivo response was assessed as median relative change (MRC) in tumor volume of SW48 and SW48PK xenografts in immunocompromised Rag2-Il2rg-/- (R2G2) mice. Induction of apoptosis was determined with immunoblotting (IB) for PARP cleavage. Results: MDCOs were treated with cop (200nM) or nav (250nM) alone and in combination. Single agent cop resulted in -16% MRC (p Conclusion: These data indicate that navitoclax, through BCLxL inhibition, enhances the efficacy of copanlisib with a greater induction of apoptosis in several models of PIK3CA mutant CRC. Citation Format: Rebecca Anna DeStefanis, Alyssa DeZeeuw, Gioia Sha, Autumn Olson, Samantha J. Anderson, Christopher P. Babiarz, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Navitoclax enhances the efficacy of copanlisib predominantly through inhibition of BCLxL in PIK3CA mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1944.