Conserved serum protein biomarkers associated with growing early colorectal adenomas.

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.

The conserved protective cyclic AMP-phosphodiesterase function PDE4B is expressed in the adenoma and adjacent normal colonic epithelium of mammals and silenced in colorectal cancer.

Conservation over three mammalian genera-the mouse, rat, and human-has been found for a subset of the transcripts whose level differs between the adenoma and normal epithelium of the colon. Pde4b is one of the triply conserved transcripts whose level is enhanced both in the colonic adenoma and in the normal colonic epithelium, especially adjacent to adenomas. It encodes the phosphodiesterase PDE4B, specific for cAMP. Loss of PDE4B function in the ApcMin/+ mouse leads to a significant increase in the number of colonic adenomas. Similarly, Pde4b-deficient ApcMin/+ mice are hypersensitive to treatment by the inflammatory agent DSS, becoming moribund soon after treatment. These observations imply that the PDE4B function protects against ApcMin-induced adenomagenesis and inflammatory lethality. The paradoxical enhancement of the Pde4b transcript in the adenoma versus this inferred protective function of PDE4B can be rationalized by a feedback model in which PDE4B is first activated by early oncogenic stress involving cAMP and then, as reported for frank human colon cancer, inactivated by epigenetic silencing.

Human Colon Organoids and Other Laboratory Strategies to Enhance Patient Treatment Selection.

OPINION STATEMENT: 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.

Fisetin and 5-fluorouracil: Effective combination for PIK3CA-mutant colorectal cancer.

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 ApcMin/+ mice that also express constitutively active PI3K in the distal small intestine and colon. Tumor incidence was markedly lower in fisetin-treated FC1 3K1 ApcMin/+ 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.

Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer.

Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8+ T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8+ T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103+CD11chiMHCIIhi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow-derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents.

Metastatic Bulk Independently Predicts Outcomes for EGFR Precision Targeting in Colorectal Cancer.

Background: Molecular profiles guide the clinical management of metastatic colorectal cancer (mCRC), particularly related to the use of anti-epidermal growth factor receptor (EGFR) antibodies. Tumor sidedness has also been implicated in resistance to these therapies, but has largely been studied in the first-line setting. We examined the role of tumor sidedness and disease bulk in predicting clinical outcomes to anti-EGFR therapy in the treatment-refractory setting. Methods: We identified a retrospective cohort of 62 patients with KRAS wild-type mCRC who received anti-EGFR therapy in the late-line setting. Response was assessed per RECIST 1.1, with bulky disease defined as any single lesion >35 mm in longest cross-sectional diameter or nodal short axis. Primary sidedness was defined in relation to the splenic flexure. Results: Patients with right-sided primary tumors at time of late-line EGFR therapy presented with increased tumor bulk and worsened overall survival (OS) relative to left-sided primary tumors. Tumor bulk, defined as either a categorical or continuous variable, predicted worsened progression-free survival (PFS) and OS, which persisted when controlling for differences in the primary tumor location. Within the right-sided cohort, no objective responses were observed for bulky disease or during treatment with anti-EGFR monotherapy. The nonbulky cohort experienced clinical benefit with anti-EGFR monotherapy, showing similar PFS and an improved response rate compared with sequential chemotherapy. Conclusions: In an effort to expand understanding of the role of primary sidedness in clinical response to anti-EGFR therapy, we identified sidedness and tumor bulk as potential predictive biomarkers of clinical response in late-line mCRC. Future prospective studies of EGFR targeting should consider tumor bulk in addition to molecular profiling in the identification of populations most likely to achieve meaningful clinical benefit.

Subclonal diversity arises early even in small colorectal tumours and contributes to differential growth fates.

OBJECTIVE AND DESIGN: The goal of the study was to determine whether the mutational profile of early colorectal polyps correlated with growth behaviour. The growth of small polyps (6-9 mm) that were first identified during routine screening of patients was monitored over time by interval imaging with CT colonography. Mutations in these lesions with known growth rates were identified by targeted next-generation sequencing. The timing of mutational events was estimated using computer modelling and statistical inference considering several parameters including allele frequency and fitness. RESULTS: The mutational landscape of small polyps is varied both within individual polyps and among the group as a whole but no single alteration was correlated with growth behaviour. Polyps carried 0-3 pathogenic mutations with the most frequent being in APC, KRAS/NRAS, BRAF, FBXW7 and TP53. In polyps with two or more pathogenic mutations, allele frequencies were often variable, indicating the presence of multiple populations within a single tumour. Based on computer modelling, detectable mutations occurred at a mean polyp size of 30±35 crypts, well before the tumour is of a clinically detectable size. CONCLUSIONS: These data indicate that small colon polyps can have multiple pathogenic mutations in crucial driver genes that arise early in the existence of a tumour. Understanding the molecular pathway of tumourigenesis and clonal evolution in polyps that are at risk for progressing to invasive cancers will allow us to begin to better predict which polyps are more likely to progress into adenocarcinomas and which patients are at greater risk of developing advanced disease.

Sex disparity in colonic adenomagenesis involves promotion by male hormones, not protection by female hormones.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc(Pirc/+) (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc(Min/+) mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

Transformation of epithelial cells through recruitment leads to polyclonal intestinal tumors.

Intestinal tumors from mice and humans can have a polyclonal origin. Statistical analyses indicate that the best explanation for this source of intratumoral heterogeneity is the presence of interactions among multiple progenitors. We sought to better understand the nature of these interactions. An initial progenitor could recruit others by facilitating the transformation of one or more neighboring cells. Alternatively, two progenitors that are independently initiated could simply cooperate to form a single tumor. These possibilities were tested by analyzing tumors from aggregation chimeras that were generated by fusing together embryos with unequal predispositions to tumor development. Strikingly, numerous polyclonal tumors were observed even when one genetic component was highly, if not completely, resistant to spontaneous tumorigenesis in the intestine. Moreover, the observed number of polyclonal tumors could be explained by the facilitated transformation of a single neighbor within 144 µm of an initial progenitor. These findings strongly support recruitment instead of cooperation. Thus, it is conceivable that these interactions are necessary for tumors to thrive, so blocking them might be a highly effective method for preventing the formation of tumors in the intestine and other tissues.

Cholecalciferol or 25-hydroxycholecalciferol neither prevents nor treats adenomas in a rat model of familial colon cancer.

BACKGROUND: Epidemiologic studies in humans have shown associations between greater sunlight exposure, higher serum 25-hydroxycholecalciferol [25(OH)D3] concentrations, and reduced colon cancer risk. However, results from a limited number of vitamin D supplementation trials in humans have not shown a protective effect. OBJECTIVE: We sought to determine whether adding to the diet increasing amounts of either 25(OH)D3, the stable metabolite measured in serum and associated with cancer risk, or cholecalciferol (vitamin D3), the compound commonly used for supplementation in humans, could reduce emergent adenomas (chemoprevention) or decrease the growth of existing adenomas (treatment) in the colons of vitamin D-sufficient rats carrying a truncation mutation of adenomatous polyposis coli (Apc), a model of early intestinal cancer. METHODS: Apc(Pirc/+) rats were supplemented with either vitamin D3 over a range of 4 doses [6-1500 µg/(kg body weight · d)] or with 25(OH)D3 over a range of 6 doses [60-4500 µg/(kg body weight · d)] beginning after weaning. Rats underwent colonoscopy every other week to assess effects on adenoma number and size. At termination (140 d of age), the number of tumors in the small intestine and colon and the size of tumors in the colon were determined, and serum calcium and 25(OH)D3 measurements were obtained. RESULTS: At lower doses (those that did not affect body weight), neither of the vitamin D compounds reduced the number of existing or emergent colonic tumors (P-trend > 0.24). By contrast, supplementation at higher doses (those that caused a suppression in body weight gain) with either 25(OH)D3 or vitamin D3 caused a dose-dependent increase in colonic tumor number in both males and females (P-trend < 0.003). CONCLUSIONS: No evidence for protection against colon tumor development was seen with lower dose supplementation with either cholecalciferol or 25-hydroxycholecalciferol. Thus, the association between sunlight exposure and the incidence of colon cancer may involve factors other than vitamin D concentrations. Alternative hypotheses warrant investigation. Furthermore, this study provides preliminary evidence for the need for caution regarding vitamin D supplementation of humans at higher doses, especially in individuals with sufficient serum 25(OH)D3 concentrations.

Monoallelic silencing and haploinsufficiency in early murine intestinal neoplasms.

Studies of tumors from human familial adenomatous polyposis, sporadic colon cancer, and mouse and rat models of intestinal cancer indicate that the majority of early adenomas develop through loss of normal function of the Adenomatous polyposis coli (APC) gene. In murine models of familial adenomatous polyposis, specifically the multiple intestinal neoplasia mouse (Min) and the polyposis in the rat colon (Pirc) rat, most adenomas have lost their WT copy of the Apc gene through loss of heterozygosity by homologous somatic recombination. We report that large colonic adenomas in the Pirc rat have no detectable copy number losses or gains in genomic material and that most tumors lose heterozygosity only on the short arm of chromosome 18. Examination of early mouse and rat tumors indicates that a substantial subset of tumors shows maintenance of heterozygosity of Apc in genomic DNA, apparently violating Knudson's two-hit hypothesis. Sequencing of the Apc gene in a sampling of rat tumors failed to find secondary mutations in the majority of tumors that maintained heterozygosity of Apc in genomic DNA. Using quantitative allele-specific assays of Apc cDNA, we discovered two neoplastic pathways. One class of tumors maintains heterozygosity of Apc(Min/+) or Apc(Pirc/+) RNA expression and may involve haploinsufficiency for Apc function. Another class of tumors exhibits highly biased monoallelic expression of the mutant Apc allele, providing evidence for a stochastic or random process of monoallelic epigenetic silencing of the tumor suppressor gene Apc.

Impact of baseline culture conditions of cancer organoids when determining therapeutic response and tumor heterogeneity.

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.

Validation of genetic classifiers derived from mouse and human tumors to identify molecular subtypes of colorectal cancer.

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 CRCs. 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 outcomes. The results of this study have the potential to impact methods for determining the prognosis and therapy selection for patients with CRC.

Multi-ancestral origin of intestinal tumors: Impact on growth, progression, and drug efficacy.

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.

Supplementation by vitamin D compounds does not affect colonic tumor development in vitamin D sufficient murine models.

Epidemiological studies indicate that sunlight exposure and vitamin D are each associated with a lower risk of colon cancer. The few controlled supplementation trials testing vitamin D in humans reported to date show conflicting results. We have used two genetic models of familial colon cancer, the Apc(Pirc/+) (Pirc) rat and the Apc(Min/+) (Min) mouse, to investigate the effect of 25-hydroxyvitamin D(3) [25(OH)D(3)] and two analogs of vitamin D hormone on colonic tumors. Longitudinal endoscopic monitoring allowed us to test the efficacy of these compounds in preventing newly arising colonic tumors and in affecting established colonic tumors. 25(OH)D(3) and two analogs of vitamin D hormone each failed to reduce tumor multiplicities or alter the growth patterns of colonic tumors in the Pirc rat or the Min mouse.

Autofluorescence Imaging of Treatment Response in Neuroendocrine Tumor Organoids.

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.

Tumor aggressiveness is independent of radiation quality in murine hepatocellular carcinoma and mammary tumor models.

PURPOSE: Estimating cancer risk associated with interplanetary space travel is complicated. Human exposure data to high atomic number, high-energy (HZE) radiation is lacking, so data from low linear energy transfer (low-LET) γ-ray radiation is used in risk models, with the assumption that HZE and γ-ray radiation have comparable biological effects. This assumption has been challenged by reports indicating that HZE radiation might produce more aggressive tumors. The goal of this research is to test whether high-LET HZE radiation induced tumors are more aggressive. MATERIALS AND METHODS: Murine models of mammary and liver cancer were used to compare the impact of exposure to 0.2Gy of 300MeV/n silicon ions, 3 Gy of γ-rays or no radiation. Numerous measures of tumor aggressiveness were assessed. RESULTS: For the mammary cancer models, there was no significant change in the tumor latency or metastasis in silicon-irradiated mice compared to controls. For the liver cancer models, we observed an increase in tumor incidence but not tumor aggressiveness in irradiated mice. CONCLUSION: Tumors in the HZE-irradiated mice were not more aggressive than those arising from exposure to low-LET γ-rays or spontaneously. Thus, enhanced aggressiveness does not appear to be a uniform characteristic of all tumors in HZE-irradiated animals.

The pleiotropic phenotype of Apc mutations in the mouse: allele specificity and effects of the genetic background.

Familial adenomatous polyposis (FAP) is a human cancer syndrome characterized by the development of hundreds to thousands of colonic polyps and extracolonic lesions including desmoid fibromas, osteomas, epidermoid cysts, and congenital hypertrophy of the pigmented retinal epithelium. Afflicted individuals are heterozygous for mutations in the APC gene. Detailed investigations of mice heterozygous for mutations in the ortholog Apc have shown that other genetic factors strongly influence the phenotype. Here we report qualitative and quantitative modifications of the phenotype of Apc mutants as a function of three genetic variables: Apc allele, p53 allele, and genetic background. We have found major differences between the Apc alleles Min and 1638N in multiplicity and regionality of intestinal tumors, as well as in incidence of extracolonic lesions. By contrast, Min mice homozygous for either of two different knockout alleles of p53 show similar phenotypic effects. These studies illustrate the classic principle that functional genetics is enriched by assessing penetrance and expressivity with allelic series. The mouse permits study of an allelic gene series on multiple genetic backgrounds, thereby leading to a better understanding of gene action in a range of biological processes.

MTORC1/2 Inhibition as a Therapeutic Strategy for PIK3CA Mutant Cancers.

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.

Patient-Derived Cancer Organoid Cultures to Predict Sensitivity to Chemotherapy and Radiation.

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.