Supplemental Figure S3. Body weight changes in ApcÎ"14/+ mice. Body weights of mice in each experimental group were recorded weekly throughout the study; each point represents average body weight {plus minus} SEM. At the end of the study, weights of mice in groups 2 (MDS pair fed) and 3 (MDD) were reduced by approximately 45%. Mice in group 4 (MDD:MDS) initially failed to gain weight and at the time of transfer to MDS diet (15 weeks of age) weighed approximately 33% less than group 1 (MDS ad lib.) mice. However, following methyl donor repletion with the MDS diet, group 4 mice rapidly gained weight and had returned to a normal body weight by the end of the study. Error bars indicate means {plus minus} SEM.

Supplemental Figure S4. (A) Representative images of liver H&E staining from the four experimental groups. Mice maintained on MDD diet for 18 weeks develop hepatic steatosis, as evidenced by the presence of vacuoles formed by fat accumulation. These vacuoles are not present in the liver of MDD:MDS mice, indicating that hepatic steatosis is reversed after 7 weeks of methyl donor repletion. (B) Representative images of spleen H&E staining; MDD mice show a loss of white pulp (stained purple) and follicles, indicating a reduction in splenic lymphocyte populations. As before, spleens from MDD:MDS mice do not show evidence of lymphocytic depletion, indicating that this effect of the MDD diet is reversible by methyl donor repletion. (C) Chronic choline deficiency has been reported to cause renal fibrosis, particularly around glomeruli. Using Masson's Trichrome stain to visualize collagen deposition (blue), we did not find evidence of renal fibrosis in MDD mice. Scale bars, 200 and 800 µm.

Comparison of tumor burden metrics

Immunohistochemistry and quantification of ki-67+ cells within colonic crypts

Supplemental Table S1. Nutritional composition of the methyl donor sufficient (MDS; TD.99366, Harlan Laboratories, Madison WI) and methyl donor deficient (MDD; TD.00605, Harlan Laboratories, Madison WI).

Figure S2. Immunohistochemistry for cleaved caspase-3 (CC3) in normal and tumor tissue from the small intestine. (A) Representative images of CC3 staining in tumors of the small intestine, with quantification. Caloric restriction (MDS pair fed) and MDD caused 2-fold (P<0.01) and 3.1-fold (P<0.01) increases in apoptotic index, respectively. Apoptotic cells were scattered throughout the tumor with no obvious localization. (B) Representative images of PHH3 staining in tumors, with quantification. Tumor mitotic index was calculated by averaging the number of PHH3+ cells in 10 40x microscope fields; MDD caused a large reduction (81%, P<0.01) in tumor mitotic index. Mitotic index was also reduced in tumors from MDD:MDS mice (31%, P<0.05), indicating that mitotic inhibition in tumors persists, to a lesser degree, at least 7 weeks beyond methyl donor repletion. Bars indicate means {plus minus} SEM. Statistically significant differences (P<0.05) between groups, measured by ANOVA with Bonferroni's post-test, are indicated within bar graphs by differences in the letter placed above each group. Scale bars, 120 µm.

Supplemental Figure S1. Immunohistochemistry for Ki-67 in normal crypts and tumors of the small intestine. (A) Representative images of Ki-67 staining in normal crypts, with quantification. MDD caused a small reduction (12%, P<0.01) in the average percentage of Ki-67+ cells per crypt. MDD:MDS mice exhibited a small (6%, P<0.01), but significant, reduction in the average percentage of Ki-67+ cells per crypt, possibly indicating that MDD-induced inhibition of proliferation persists during methyl donor repletion in normal crypts. (B) Representative images of Ki-67 staining in tumors, with quantification. Tumor proliferative index was determined by calculating the average percentage of Ki-67+ cells in 5 40x fields per mouse (minimum 2000 cells counted). MDD induced a modest reduction (17%, P<0.01) in proliferative index in tumors of the small intestine. There was no reduction in proliferative index in tumors from MDD:MDS mice, indicating that MDD-induced reductions in proliferation are reversed by repletion with MDS diet. Bars indicate means {plus minus} SEM. Statistically significant differences.(P<0.05) between groups, measured by ANOVA with Bonferroni's post-test, are indicated within bar graphs by differences in the letter placed above each group. Scale bars, 80 and 200 µm.

Intestinal health relies on the immunosuppressive activity of CD4 + regulatory T (T reg ) cells 1 . Expression of the transcription factor Foxp3 defines this lineage, and can be induced extrathymically by dietary or commensal-derived antigens in a process assisted by a Foxp3 enhancer known as conserved non-coding sequence 1 (CNS1) 2 – 4 . Products of microbial fermentation including butyrate facilitate the generation of peripherally induced T reg (pT reg ) cells 5 – 7 , indicating that metabolites shape the composition of the colonic immune cell population. In addition to dietary components, bacteria modify host-derived molecules, generating a number of biologically active substances. This is epitomized by the bacterial transformation of bile acids, which creates a complex pool of steroids 8 with a range of physiological functions 9 . Here we screened the major species of deconjugated bile acids for their ability to potentiate the differentiation of pT reg cells. We found that the secondary bile acid 3β-hydroxydeoxycholic acid (isoDCA) increased Foxp3 induction by acting on dendritic cells (DCs) to diminish their immunostimulatory properties. Ablating one receptor, the farnesoid X receptor, in DCs enhanced the generation of T reg cells and imposed a transcriptional profile similar to that induced by isoDCA, suggesting an interaction between this bile acid and nuclear receptor. To investigate isoDCA in vivo, we took a synthetic biology approach and designed minimal microbial consortia containing engineered Bacteroides strains. IsoDCA-producing consortia increased the number of colonic RORγt-expressing T reg cells in a CNS1-dependent manner, suggesting enhanced extrathymic differentiation.

Abstract Our understanding of the role of folate one-carbon metabolism in colon carcinogenesis remains incomplete. Previous studies indicate that a methyl donor–deficient (MDD) diet lacking folic acid, choline, methionine, and vitamin B12 is associated with long-lasting changes to the intestinal epithelium and sustained tumor protection in Apc-mutant mice. However, the metabolic pathways by which the MDD diet affects these changes are unknown. Colon samples harvested from ApcΔ14/+ mice fed the MDD diet for 18 weeks were profiled using a GC-MS and LC-MS/MS metabolomics platform. Random forest and pathway analyses were used to identify altered metabolic pathways, and associated gene expression changes were analyzed by RT-PCR. Approximately 100 metabolites affected by the MDD diet were identified. As expected, metabolites within the methionine cycle, including methionine (−2.9-fold, P < 0.001) and betaine (−3.3-fold, P < 0.001), were reduced. Elevated homocysteine (110-fold, P < 0.001) was associated with increased flux through the transsulfuration pathway. Unexpectedly, levels of deoxycholic acid (−4.5-fold, P < 0.05) and several other secondary bile acids were reduced. There were also unexpected reductions in the levels of carnitine (−2.0-fold, P < 0.01) and a panel of acylcarnitines involved in fatty acid β-oxidation. Finally, metabolites involved in redox balance, including ascorbate and hypotaurine, were found to be persistently elevated. These findings provide clues to the molecular changes underlying MDD-mediated tumor protection and identify regulatable metabolic pathways that may provide new targets for colon cancer prevention and treatment. Implications: Metabolomic profiling reveals molecular changes underlying MDD-induced tumor protection and may provide new targets for colorectal cancer prevention and treatment.

Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.

3 Background: Expression of inhibitory immune checkpoints (ICPs) within tumors has emerged as an important barrier for effective anti-tumor immunity. Antibody-mediated blockade of ICPs can lead to durable responses in patients. Interestingly, only a small subset of tumor infiltrating lymphocytes (TILs) express these checkpoints and there is a need to better understand the characteristics of this subset. We undertook this study to understand characteristics of TILs within melanoma. Methods: We used single cell mass cytometry, gene expression profiling of purified T cell subsets, T cell receptor (TCR) sequencing as well as functional studies to understand the characteristics of TILs in melanoma patients (n=50). We also performed exome sequencing of tumor cells in some patients. Results: We find that TILs are functionally and phenotypically distinct from circulating T cells. They express higher levels of inhibitory ICPs (PD-1, TIM-3) and secrete less IL2, IFNg and TNFa than T cells in circulation. Expression of vascular endothelial growth factor within tumors correlated with reduced T cell infiltration. Expression of ICPs (PD-1, TIM-3, PD-L1) were enriched in T cells with a phenotype and expression profile of tissue resident memory T (TRM) cells with most cells expressing multiple checkpoints. Within the myeloid compartment, ICPs were predominantly expressed on CD14+CD16+ subset. TCR sequencing revealed that individual melanoma metastases revealed that the top clones within each of the lesions have distinct TCRs. Concurrent TCR and tumor exome sequencing of individual metastases in the same patient revealed that inter-lesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. Conclusions: Our findings suggest that TRM cells and CD16+ myeloid cells may be the major target of ICP blockade within tumors. The ability to activate, and retain TRM cells may be an important determinant of the T cell content of the tumor microenvironment and should be a goal for future vaccines. Importantly, our study illustrates inter-lesional diversity of TCRs within individual metastases which may differentially impact the outcome of immune therapy at each site.

SUMMARYKnowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We created an immune map of breast cancer using single-cell RNA-seq data from 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph node. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous tumor-specific phenotypic expansions driven by environmental cues. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer, with important implications for characterizing tumor-infiltrating immune cells.

Heterogeneity of tumor cells and their microenvironment can affect outcome in cancer. Blockade of immune checkpoints (ICPs) expressed only on a subset of immune cells leads to durable responses in advanced melanoma. Tissue-resident memory T (TRM) cells have recently emerged as a distinct subset of memory T cells in nonlymphoid tissues. Here, we show that functional properties and expression of ICPs within tumor-infiltrating lymphocytes (TILs) differ from those of blood T cells. TILs secrete less IL-2, IFN-γ, and TNF-α compared with circulating counterparts, and expression of VEGF correlated with reduced TIL infiltration. Within tumors, ICPs are particularly enriched within T cells with phenotype and genomic features of TRM cells and the CD16+ subset of myeloid cells. Concurrent T cell receptor (TCR) and tumor exome sequencing of individual metastases in the same patient revealed that interlesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. These findings suggest that the TRM subset of TILs may be the major target of ICP blockade and illustrate interlesional diversity of tissue-resident TCRs within individual metastases, which did not equilibrate between metastases and may differentially affect the outcome of immune therapy at each site.

AbstractThe role of folate one-carbon metabolism in colorectal cancer development is controversial, with nutritional intervention studies producing conflicting results. It has been reported that ApcMin/+ mice maintained on a diet deficient in the methyl donors folic acid, methionine, choline, and vitamin B12, and supplemented with homocysteine, show a greater than 95% reduction in intestinal tumor development. The present study extends these findings and shows that tumor protection afforded by dietary methyl donor deficiency (MDD) is long-lasting. After 11 weeks of MDD, tumor protection persisted for at least an additional 7 weeks of methyl donor repletion (22.2 ± 3.5 vs. 70.2 ± 4.6 tumors per mouse; P < 0.01). Sustained tumor protection was associated with a reduction in intestinal crypt length (26%, P < 0.01), crypt cell division and crypt fission, and an increase in apoptosis of both normal crypts and tumors (4.9- and 3.2-fold, respectively, P < 0.01). MDD also caused a significant reduction in the number of Dclk1-positive cells in the intestine (62%, P < 0.01), a long-lived crypt cell with cancer stem cell potential. Several undesirable effects associated with methyl donor restriction (e.g., reduced body weight gain) were shown to be transient and readily reversible following methyl donor repletion. Taken together, these results indicate that even temporary dietary methyl donor restriction in adenoma-prone mice can induce persistent changes to the intestinal epithelium and provide long-lasting tumor protection. These data also suggest that transient reductions in dietary methyl donor consumption should be considered when studying the impact of folate on colon cancer risk in humans. Cancer Prev Res; 9(10); 812–20. ©2016 AACR.

Meeting abstracts Blockade of inhibitory immune checkpoints (ICPs) improves survival in melanoma patients. Interestingly, the expression of these ICPs in most tumor tissues including melanoma is restricted to only a subset of tumor-infiltrating immune cells (TIICs). Here, we utilize single cell

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in both men and women. In humans genetic changes in intestinal epithelia commencing with inactivation of the APC tumor suppressor gene along with mucosal inflammation, facilitate the development of intestinal adenomas. The role of host immune system to detect and eliminate these altered cells is debatable based on its failure to prevent tumor growth. A physiological approach to prevent CRC formation is to use dietary compounds, which can inhibit inflammation and induce apoptosis to transformed cells. Capsaicin (CP), the principal pungent ingredient of red chili peppers is highly consumed spice throughout the world. It exhibits a wide range of immunological and pharmacological properties. We have previously reported that orally administered CP enhances a discreet population of CD11b+/F4/80+ macrophages and a CD4+ T cells population in the gut associated lymphoid tissue (GALT) expressing anti-inflammatory factors such as IL-10 and PD-L1. Here we demonstrate that orally administered CP can suppress spontaneous intestinal polyposis in the Apc(Min/+) mouse model, a widely used spontaneous colorectal cancer model for human familial adenomatous polyposis (FAP) in a time and dose dependent manner. We further demonstrate the role of different immune cells (myeloid and lymphoid) in CP mediated prevention of intestinal tumorigenesis. The use of dietary CP will be a novel approach for chemoprevention of human CRC.

Abstract The goal of the present study is to determine the effects of folate deficiency on intestinal tumorigenesis in ApcMin/+ mice, a widely used animal model for human familial adenomatous polyposis (FAP). Since folate plays an important role in maintaining methyl donor status, dietary folate levels were reduced in combination with a panel of methyl donors, including choline, methionine and vitamin B12. Although the impact of folate on colorectal cancer (CRC) has been studied extensively, its role in cancer chemoprevention remains controversial. For example, folate supplementation has been widely discussed as a strategy for lowering cancer incidence. However, excess folate has also been associated with increased cancer risk. The following studies were designed to evaluate the effects of folate deficiency on intestinal tumorigenesis in ApcMin/+ mice. In Study 1, methyl donor sufficient diet (MDS) and methyl donor deficient diet (MDD) were fed to mice, beginning at either 5 or 10 weeks of age. Intestinal tumors were evaluated at 16 weeks of age. MDD diet suppressed intestinal tumor formation in ApcMin/+ mice by ∼80% when started at 5 weeks of age. Importantly, the protective effect was lost when the MDD diet was started at 10 weeks of age, indicating an important time-dependency on cancer suppression. Gene expression profiling of normal colonic mucosa after 11 weeks on MDD diet identified the most significantly down-regulated genes related to immune response and inflammation, data that are consistent with a recently published human colon expression profiling study (Protiva et al., 2011). The cancer suppression, however, was associated with reduced food intake and lowered body weight gain (up to 40%) throughout the experimental period. To control for this effect, Study 2 was performed in which ApcMin/+ mice were pair-fed with either methyl donor sufficient (MDS) or methyl donor deficient (MDD) diet, beginning at 5 weeks of age for a total of 11 weeks. A third group received MDS ad libitum. At 16 weeks of age, intestinal tumor formation was significantly reduced (54%, p<0.001) in the caloric-restricted MDS group. In the MDD group, however, tumor suppression was almost complete (∼96%, p<0.001). Associated with tumor protection were increased numbers of apoptotic cells within the villous epithelium of the small intestine, with a concomitant reduction in cell proliferation assessed by Ki-67 and Phospho-histone H3 (pHH3). While cancer protection by methyl donor deficiency is partially dependent on reduced caloric intake, additional mechanisms dependent on altered cell turnover must be invoked to explain these results. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-182. doi:1538-7445.AM2012-LB-182

Abstract Despite recent population data, the influence of dietary folate supplementation on colon cancer risk remains controversial. This study examines the effects of folate deficiency, in combination with choline, methionine, and vitamin B12 depletion, on intestinal tumorigenesis in ApcMin/+ mice. Methyl donor sufficient (MDS) and deficient (MDD) diets were started at five or 10 weeks of age and tumors evaluated at 16 weeks. MDD suppressed intestinal tumor formation in ApcMin/+ mice (∼80%) when started at five weeks of age. The protective effect was lost when MDD was initiated at 10 weeks of age, indicating an important time dependency on cancer suppression. Concomitant with cancer protection, MDD restricted body weight gain. Therefore, a second study was conducted in which MDS was given ad libitum or pair-fed with MDD. Although small intestinal tumors were reduced 54% in pair-fed MDS mice, MDD caused a further reduction (96%). In colon, although MDD did not affect tumor numbers, tumor size was reduced. Gene expression profiling of normal-appearing colonic mucosa after 11 weeks on MDD identified a total of 493 significantly downregulated genes relative to the MDS group. Pathway analysis placed many of these genes within general categories of inflammatory signaling and cell-cycle regulation, consistent with recently published human data obtained during folate depletion. Further studies are warranted to investigate the complex interplay of methyl donor status and cancer protection in high-risk populations. Cancer Prev Res; 5(7); 911–20. ©2012 AACR.