Supplemental material and figure legend

Role of CD40:CD40L and cell-to-cell contact in the activation of M-MDSC:MC suppressive axis.

The file contains the lists of MC and MDSC related genes

<p>Supplementary Materials and Methods. Description of additional methods and procedures used in the study.</p>

Characterization of BC cell lines established from spontaneous tumors, commercially available and BMMCs. (A) Western blot to compare the levels of ER and HER2 in cell lines established by us from spontaneous tumors developed in NeuT (N2C), PyMT B6 (PyMT41c) and PyMT Wsh (WPY25F) transgenic mice. (B) Purity of BMMCs employed in co-culture experiments was evaluated by flow cytometry analysis by excluding debris and doublets (FSC-A vs FSC-H) and evaluating the percentage of FceRIβ and cKit double positive cells. (C) WPY25F tumor cells were first gated to include only viable cells (7AA negative), then they were gated on CD45-CD31-Ter119- to exclude leukocytes, endothelial cells and erythrocytes. Gated cells were finally analyzed for the expression of CD49f and EpCAM to define the basal/luminal phenotype as shown in figure 2D. (D) Absolute levels of ESR1 evaluated by Real-Time PCR in both human and mouse cell lines are shown.

The file contains the list and information of all antibodies used in the study

Supplementary Figures S1-S5. Colon length in wild-type and KitW-sh mice during colitis progression (S1); Il22 and Il22bp relative expression in colon in wild-type and KitW-sh mice (S2); Polyps count in colon of WT and KitW-sh mice (S3); Colorectal cancer development in WT, KitW-sh and KitW-sh REC mice (S4); Summary of MC activity in intestinal mucosal healing and in transformation toward malignancy (S5).

Supplementary Figure 1. Histologic evaluation of adenocarcinoma and NE tumors from TRAMP mice; Supplementary Figure 2. MCs infiltration in prostates of TRAMP mice; Supplementary Figure 3. OPT 7714 cells originate NE tumors in vivo; Supplementary Figure 4. Murine B16 F10 cells express cKit and PDGFR-β; Supplementary Figure 5. Human PC3 cells express cKit but are insensitive to Imatinib treatment; Supplementary Figure 6. Murine normal seminal vesicles do not express PDGFR-β; Supplementary Figure 7. Human phyllodes breast tumors express PDGFR-β.

Supplementary Figure 1. Representative plots illustrating analysis of tumor-specific CD8+ T cell response in TRAMP and KitWsh-TRAMP mice. Supplementary Figure 2. CD8+T cells from PDLN and prostates of KitWsh-TRAMP mice are responsive. Supplementary Figure 3. Quantification of MCs or T cells after reconstitution of depletion, respectively. Supplementary Figure 4. Flow cytometry identification of T cells in spleen and prostate of TRAMP and KitWsh-TRAMP mice. Supplementary Figure 5. Flow cytometry identification of myeloid cells in spleen and prostate of TRAMP and KitWsh-TRAMP mice. Supplementary Figure 6. MCs and PMN-MDSC can interact via CD40L-CD40 in TRAMP mice. Supplementary Figure 7. Wild type and CD40L-/- BMMC are equally functional, but CD8+T cells from PDLN and prostates of KitWsh-TRAMP mice reconstituted with BMMC CD40L-/- are still responsive. Supplementary Figure 8. MCs, CD40L and CD33 co-localize in human tumor prostate cancer samples. Supplementary Figure 9. Correlation between MC-genes and MDSC-activity genes in human prostate cancer data sets. Supplementary Figure 10. MC/PMN-MDSC signatures outperform random signatures of the same length.

Supplementary Table S1. Genes differentially expressed (FDR < 0.05) between PyMT B6 and PyMT Wsh mice.

MDSC accumulation in CT-26 tumor bearing mice and migration in response to mast cells and to conditioned media from colon cancer cell line.

Abstract Tumor growth and development is determined by both cancer cell–autonomous and microenvironmental mechanisms, including the contribution of infiltrating immune cells. Because the role of mast cells (MC) in this process is poorly characterized and even controversial, we investigated their part in breast cancer. Crossing C57BL/6 MMTV-PyMT mice, which spontaneously develop mammary carcinomas, with MC-deficient C57BL/6-KitW-sh/W-sh (Wsh) mice, showed that MCs promote tumor growth and prevent the development of basal CK5-positive areas in favor of a luminal gene program. When cocultured with breast cancer cells in vitro, MCs hindered activation of cMET, a master regulator of the basal program, and simultaneously promoted expression and activation of estrogen receptor (ESR1/ER) and its target genes (PGR, KRT8/CK8, BCL2), which are all luminal markers. Moreover, MCs reduced ERBB2/HER2 levels, whose inhibition further increased ESR1 expression. In vivo and in silico analysis of patients with breast cancer revealed a direct correlation between MC density and ESR1 expression. In mice engrafted with HER2-positive breast cancer tumors, coinjection of MCs increased tumor engraftment and outgrowth, supporting the link between MCs and increased risk of relapse in patients with breast cancer. Together, our findings support the notion that MCs influence the phenotype of breast cancer cells by stimulating a luminal phenotype and ultimately modifying the outcome of the disease. Significance: Mast cells impact breast cancer outcome by directly affecting the phenotype of tumor cells through stimulation of the estrogen receptor pathway.

Benefits from biologic therapy for inflammatory bowel disease (IBD) come at a cost of side effects and antidrug antibodies.1Gareb B. Pharmaceutics. 2020; 12: E539Crossref PubMed Scopus (5) Google Scholar This necessitates novel anti-inflammatory therapies. Histone acetylation is an important epigenetic gene expression regulator. Acetylation of histone-3 lysine-27 (H3K27ac), H3K9ac, and trimethylation of H3K4 (H3K4me3) mark active enhancers. H3K27ac can differentiate active from poised enhancers and is linked to gene expression.2Creyghton M. PNAS. 2010; 107: 21931-21936Crossref PubMed Scopus (2088) Google Scholar Trimethylation of H3K27 leads to gene repression. IBD-associated single-nucleotide polymorphisms overlap with regulatory elements marked by H3K27ac.3Mokry M. Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar H3K27ac is reduced in dextran sulfate sodium (DSS)-induced colitis in mice and broad-acting histone deacetylases (HDAC) inhibitors (eg, SAHA and valproic acid [VPA]) restore H3K27ac levels and inhibit inflammatory cytokine production.4Guo Y. Carcinogenesis. 2016; 37: 616-624Crossref PubMed Scopus (28) Google Scholar,5Felice C. Aliment Pharmacol Ther. 2015; 41: 26-38Crossref PubMed Scopus (71) Google Scholar We extend findings from models to patients with IBD to evaluate the therapeutic potential of HDAC inhibitors. We assessed H3K27ac in patients with IBD and tested whether VPA could inhibit inflammatory cytokine production in IBD biopsies cultured ex vivo. VPA was selected because of its safety record in treating neurologic disorders. Methods and patient characteristics are given in the Supplementary Material. To validate our approach, we confirmed a reduction in the percentage of H3K27ac positive (H3K27ac+) cells in conjunction with the development of inflammation in a DSS model (Figure 1A–C).4Guo Y. Carcinogenesis. 2016; 37: 616-624Crossref PubMed Scopus (28) Google Scholar Moreover, after removal of DSS, H3K27ac+ cells significantly increased during resolution of inflammation (Figure 1C). No significant changes in HDAC mRNA were detected during the inflammatory phase, despite an increase in interleukin (IL) 6 mRNA levels (positive control for DSS-treatment) (Figure 1D). During the resolution of inflammation (Day 17), expression of 4 Hdacs differed from baseline (downregulated, Hdac1, 5, and 10; upregulated, Hdac2) (Figure 1D). A reduction in H3K27ac+ cells was confirmed in actively inflamed IBD biopsies relative to inactive IBD control subjects (Figure 2A and B) and was maintained in a paired subanalysis of the same samples between patient matched active and inactive segments (-16.25%; P = .0243; n = 11). This indicated that confounding factors, including disease duration and medications, could not account for the observed differences. A similar pattern of change was observed in patients with Crohn’s disease (CD) and ulcerative colitis (UC), although the reduction in H3K27ac+ cells in active biopsies was significant only in patients with UC (Figure 2B). As in the DSS model, active disease in patients with IBD was marked by increased IL6 mRNA but was not associated with changes in HDAC mRNA levels (Figure 2C). A subanalysis of patients with UC and CD (Supplementary Figure 1) found increased HDAC 9 in UC consistent with its inflammatory role.6De Zoeten E.F. Gastroenterology. 2010; 138: 583-594Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar Inhibiting HDAC activity in mucosal IBD biopsies cultured ex vivo with 5 mM VPA (Figure 2D) increased H3K27ac levels and reduced expression of IL6, IL10, IL1B, and IL23 mRNA significantly relative to patient-matched biopsies treated with control media (Figure 2E). Reduced production of IL6, IL10, and IL23 protein into culture media was confirmed (Figure 2F), and a reduction in tumor necrosis factor and interferon gamma was found (Figure 2F). Analysis of IL6 in an expanded cohort again indicated that VPA effects were greater in biopsies from areas with active disease, and in patients with UC (Figure 2G); namely, groups that had the most pronounced reductions in H3K27ac+ cells (Figure 2B). In biopsy cultures, we cannot discern the primary cellular source of IL6 or other inflammatory cytokines. VPA can suppress inflammation by inducing apoptosis of laminal propria mononuclear cells,7Glauben R. J Immunol. 2006; 176: 5015-5022Crossref PubMed Google Scholar and we observed a decrease in BLC3 mRNA and an increase in CASP9 expression in VPA-treated IBD biopsies (Supplementary Figure 2). Although not significant, there was an increase in the percentage of CASP+ cells in VPA-treated biopsies and the proportion of CASP+/CD3+ T cells (Supplementary Figure 2). Other potentially relevant mechanisms of action for VPA include modulation of acetylation at inflammatory gene promoters and regulation of microRNAs. For example, in a model of colitis-accelerated colon carcinogenesis, DSS treatment increased HDAC activity and decreased H3K27ac levels in the intestine. However, promoters of inflammatory genes, including the IL6 promoter, were hyperacetylated. Conversely, anti-inflammatory treatment with aspirin increased global acetylation levels but reduced H3K27ac levels at the promoter regions of proinflammatory genes.4Guo Y. Carcinogenesis. 2016; 37: 616-624Crossref PubMed Scopus (28) Google Scholar Others have reported that in T cells, HDAC inhibitors promote FOXP3 expression by acetylating its promoter and inducing microRNA signatures associated with regulatory T cells. Thus, reducing IL6/STAT3/IL17 signaling in naive CD4+ T cells and blocking the polarization of Th17 cells.8Glauben R. J Biol Chem. 2014; 289: 6142-6151Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar HDAC inhibitors also acetylate nonhistone proteins including STAT1, blocking their phosphorylation and inhibiting proinflammatory signalling.9Dahllöf M.S.J. Interferon Cytokine Res. 2015; 35: 63-70Crossref PubMed Scopus (8) Google Scholar Increasing histone acetylation may alter other histone modifications (eg, H3K27me3), which is altered by DSS treatment. VPA can have HDAC-independent effects, including regulating DNA methylation leading to gene repression and metabolic changes. Because chemically diverse HDAC inhibitors have similar effects, an HDAC-dependent mechanism seems likely. Indeed, SAHA (vorinostat), which is more potent than VPA in IBD models but currently only used for cutaneous T-cell lymphoma, is being trailed in patients with CD.10Available at: https://clinicalcenter.nih.gov/recruit/protocols/17_crohns.html Refer to study #17-I-0101. Accessed June 2020.Google Scholar Our data identify H3K27ac levels in the mucosa as a potential biomarker of response to HDAC therapy and establish ex vivo biopsy cultures as an alternative screen for HDAC inhibitors. Although further work is needed to elucidate VPA mechanism of action, we highlight the potential to repurpose VPA, a widely used and inexpensive drug, to treat inflammation in IBD. Animal experiments were approved by the Ethics Committee for Animal Experimentation of the Fondazione IRCCS Istituto Nazionale dei Tumori of Milan. C57BL/6 wild-type mice (4–6 weeks) were purchased from Charles River and housed in filter-top cages held under pathogen-free conditions. Mice were administered 1.5% DSS in drinking water for 10 days. Recovery was evaluated 10 days after DSS withdrawal. Mice were sacrificed at Day 0 (n = 3), Day 3 (n = 4), Day 10 (n = 4), Day 17 (n = 3), and Day 22 (n = 3). The colon was removed, washed in phosphate-buffered saline (PBS) and formalin fixed and paraffin embedded (FFPE). Other mice were sacrificed at Day 0 (n = 3), Day 10 (n = 3), and Day 17 (n = 3) and the colon homogenized in Trizol reagent (Invitrogen, Waltham, MA) before RNA extraction. Robarts histopathology index score of trichromatin-stained FFPE sections monitored disease activity.1Mosli M.H. Gut. 2017; 66: 50-58Crossref PubMed Scopus (126) Google Scholar The Robarts histopathology index score correlated well with previously published measures in these mice.2Creyghton M. PNAS. 2010; 107: 21931-21936Crossref PubMed Scopus (2088) Google Scholar,3Mokry M. Gastroenterology. 2014; 146: 1040-1047Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar H3K27ac+ nuclei were quantified using standard immunohistochemistry protocols. Antigen retrieval was performed in citrate buffer pH 6.0 for 20 minutes in a microwave. Slides were cooled in a 1x PBS solution for 2 minutes, before blocking in goat serum for 15 minutes (Dake, diluted 1:25). Primary anti-H3K27ac (ab4729, Abcam, Cambridge, MA; 1:1000) was added and incubated for 45 minutes at room temperature, followed by a PBS wash (5 minutes under agitation). The secondary antibody (goat antirabbit horseradish peroxidase secondary antibody, 1:250; Dako) was added and incubated for 45 minutes. Next, slides were washed in 1x PBS, before incubation with 3,3’-dianminobenzidine solution for 10 minutes, washed in distilled water and counterstained with hematoxylin. Finally, slides were dehydrated and mounted from xylene using distyrene plasticizer reagent. All slides were scanned using the NanoZoomer Digital Pathology (NDP.view2) system (Hamamatsu Corp, Bridgewater, NJ). For H3K27ac quantification 3 high-power fields of the mucosa were taken from each slide, processed on ImageJ, and each field counted to assess positively stained (brown) and total nuclei (brown and purple). H3K27ac+ cells were expressed as a percentage of the total number of cells. Between 600 and 1800 nuclei were analyzed per field. Surgically resected intestinal FFPE tissue sections from patients with IBD were obtained from the Royal London Hospital Pathology Archive. Specimens with dysplasia or established malignancy were excluded. IBD FFPE tissues analyzed (n = 28) included 11 inactive IBD samples (CD, n = 8; UC, n = 3) and 17 active IBD samples (CD, n = 9; UC, n = 8). The cohort included 11 inactive and active patient matched pairs. Disease activity for each block was assessed using pathology reports. Cohort characteristics are shown in Supplementary Table 1. For H3K27ac analysis antigen retrieval and primary antibody incubations were performed as described in the immunohistochemistry section. However, H3K27ac staining was visualized using a fluorescent secondary antibody (Ab150077, diluted 1:250 in PBS), and Vectashield HardSet (Vector Biosystems, Malvern, PA) mounting medium with DAPI was used for counterstaining. Imaging used ×40 magnification. H3K27ac+ nuclei were scored manually from 3 independent mucosal images. H3K27ac+ nuclei were expressed as a percentage of the total number of cells in that field. Typically, 200–400 nuclei were scored per field. Data were normally distributed and differences in the percentage of H3K27ac+ cells between active and inactive IBD tissue determined using a 2-tailed Student t test assuming equal variance. A subanalysis of the paired patient-matched inactive and active tissue was performed using a paired Student t test. IBD biopsies from the same area of the intestine were collected during routine endoscopies at the Royal London Hospital. For each patient the disease activity, age, date of diagnosis, extension and behavior of disease (Montreal classification), clinical activity of disease (Harvey-Bradshaw Index for CD and Mayo score for UC), comorbidities, and all previous and current medications were recorded. Exclusion criteria included a diagnosis of infective colitis and prior use of VPA. In total 29 patients with IBD were recruited, but not all patients had sufficient biopsies taken to be included in all analyses. The numbers for each analysis are listed individually under the appropriate methods sections. The cohort characteristics as a whole are in Supplementary Table 2. Biopsies were placed immediately into ice-cold RPMI 1640 medium and then washed in Hank’s balanced salt solution with dithiothreitol 0.01% to eliminate mucus and debris before culturing. Biopsies were culture with either control medium (RPMI 164 supplemented with fetal bovine serum [5%], L-glutamine [0.01%], penicillin/streptomycin [0.01%], and gentamycin [0.0025%]) or medium containing VPA (5 mM) at 37°C in a humidified incubator at 5% CO2 for 24 hours. The dose of VPA used corresponded to that used to reduce inflammatory cytokine production and treat in DSS-exposed mice.4Glauben R. J Immunol. 2006; 176: 5015-5022Crossref PubMed Scopus (226) Google Scholar VPA was diluted from a stock 50-mM solution dissolved in RPMI medium. Following culture, the supernatant was removed and stored at -80°C, and the biopsies stored either in RNALater at -80°C or FFPE embedded. As a positive control for treatment (n = 6 pairs), the percentage of H3K27ac cells was determined as in the immunofluorescence protocol. Difference between patient-matched biopsies treated with either VPA or a vehicle control (RPMI media) was determined using a paired Student t test. IBD biopsies stored in RNALater were homogenized in 700 μL of Qiazol reagent and RNA extracted using the miRNeasy Kit (Qiagen, Hilden, Germany) and RNA quality determined using a bioanalyzer. RNA was converted to cDNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Wilmington, DE). The cDNAs were diluted 1:10 before incubation with commercial Taqman gene expression probes and Universal Mastermix (Applied Biosystems) on a 7500 System RealTime PCR cycler (Applied Biosystems). Resultant cycle threshold (Ct) values were exported and normalized to control genes RPLPO using the 2-ΔCt method. All normalized data were log2 transformed before statistical analysis and normally distributed. IL6 and HDAC mRNA levels in the DSS mouse at Day 10 (n = 3) and Day 17 (n = 3) were compared with baseline at Day 0 (n = 3) using a 2-tailed Student t test assuming equal variance. Differences in IL6 mRNA and HDAC mRNA between inactive (n = 8) and active IBD biopsies (n = 11) were similarly analyzed. Supernatants from cultured IBD biopsies (n = 21 pairs) were assessed using a customized 6-plex Luminex panel according to manufacturer’s instructions (EMD Millipore, Billerica, MA). Changes in IL6 were further validated by enzyme-linked immunosorbent assays (DY206-05; n = 17 pairs). Data were not normally distributed and differences between VPA-treated biopsies and biopsies cultured with the vehicle control (RPMI media) were determined using a Wilcoxon matched-pairs signed rank test. CD, Crohn’s disease; CRP, C-reactive protein; FFPE, formalin fixed and paraffin embedded; UC, ulcerative colitis. CD, Crohn’s disease; CRP, C-reactive protein; UC, ulcerative colitis.

Abstract Immunotherapy, including the use of checkpoint inhibitors, is a potent therapeutic approach for some cancers, but has limited success with prostate tumors, in which immune suppression is instigated by the tumor. The immunosuppressive capacity of mast cells, which promote adenocarcinoma development in the prostate, prompted our investigation on whether mast cells promote tolerance to SV40 Large-T antigen, the transforming oncogene in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. The incidence of adenocarcinoma was reduced in the offspring of a cross between TRAMP mice and mast cell–deficient KitWsh mice. TRAMP mice are tolerant to the SV40 Large T antigen, which is otherwise immunogenic in normal syngeneic B6 mice. Genetic ablation of mast cells in TRAMP mice restored their ability to mount a tumor-specific cytotoxic T-cell response. In KitWsh-TRAMP mice, the restored T-cell immunity correlated with the reduced activity of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC), along with their reduced expression of Arg1, Nos2, and Stat3. Having found that CD40L-expressing mast cells can interact in vivo with CD40-expressing PMN-MDSC, we then determined that only KitWsh-TRAMP mice reconstituted with mast cells expressing CD40L could restore PMN-MDSCs suppressive functions, T-cell unresponsiveness and adenocarcinoma development. Thus, mast cells have an immunoregulatory effect on PMN-MDSCs activity through CD40L-CD40 interaction, favoring immunosuppression and tumor onset. In prostate cancer patients, in silico analyses correlated poor clinical outcomes with high expression of genes related to mast cells and PMN-MDSCs. Cancer Immunol Res; 6(5); 552–65. ©2018 AACR.

Basophils, eosinophils and mast cells were first recognized by Paul Ehrlich in the late 19th century. These cells have common, but non-redundant roles, in the pathogenesis of allergic diseases and in the protection against parasites. Nevertheless, in virtue of their shared-adeptness to produce a huge variety of immunological mediators and express membrane-bound receptors, they are able to interact with immune and non-immune components of the tissue microenvironment, contributing to the regulation of tissue homeostasis and immune response while participating to further deregulation of tissues transforming into neoplasia.

Background DUOX2 and DUOXA2 form the predominant H2O2-producing system in human colorectal mucosa. Inflammation, hypoxia, and 5-aminosalicylic acid increase H2O2 production, supporting innate defense and mucosal healing. Thiocyanate reacts with H2O2 in the presence of lactoperoxidase (LPO) to form hypothiocyanate (OSCN-), which acts as a biocide and H2O2 scavenging system to reduce damage during inflammation. We aimed to discover the organization of Duox2, Duoxa2, and Lpo expression in colonic crypts of Lieberkühn (intestinal glands) of mice and how distributions respond to dextran sodium sulfate (DSS)-induced colitis and subsequent mucosal regeneration. Methods We studied tissue from DSS-exposed mice and human biopsies using in situ hybridization, reverse transcription quantitative polymerase chain reaction, and cDNA microarray analysis. Results Duox2 mRNA expression was mostly in the upper crypt quintile while Duoxa2 was more apically focused. Most Lpo mRNA was in the basal quintile, where stem cells reside. Duox2 and Duoxa2 mRNA were increased during the induction and resolution of DSS colitis, while Lpo expression did not increase during the acute phase. Patterns of Lpo expression differed from Duox2 in normal, inflamed, and regenerative mouse crypts (P < 0.001). We found no evidence of LPO expression in the human gut. Conclusions The spatial and temporal separation of H2O2-consuming and -producing enzymes enables a thiocyanate- H2O2 “scavenging” system in murine intestinal crypts to protect the stem/proliferative zones from DNA damage, while still supporting higher H2O2 concentrations apically to aid mucosal healing. The absence of LPO expression in the human gut suggests an alternative mechanism or less protection from DNA damage during H2O2-driven mucosal healing.

Abstract Prostate cancer is a leading cause of cancer-related death in males worldwide. Indeed, advanced and metastatic disease characterized by androgen resistance and often associated with neuroendocrine (NE) differentiation remains incurable. Using the spontaneous prostate cancer TRAMP model, we have shown that mast cells (MCs) support in vivo the growth of prostate adenocarcinoma, whereas their genetic or pharmacologic targeting favors prostate NE cancer arousal. Aiming at simultaneously targeting prostate NE tumor cells and MCs, both expressing the cKit tyrosine kinase receptor, we have tested the therapeutic effect of imatinib in TRAMP mice. Imatinib-treated TRAMP mice experience a partial benefit against prostate adenocarcinoma, because of inhibition of supportive MCs. However, they show an unexpected outgrowth of prostate NE tumors, likely because of defective signaling pathway downstream of cKit receptor. Also unexpected but very effective was the inhibition of epithelial–stromal tumors of the seminal vesicles achieved by imatinib treatment. These tumors normally arise in the seminal vesicles of TRAMP mice, independently of the degree of prostatic glandular lesions, and resemble phyllodes tumors found in human prostate and seminal vesicles, and in breast. In both mice and in patients, these tumors are negative for cKit but express PDGFR-β, another tyrosine kinase receptor specifically inhibited by imatinib. Our results imply a possible detrimental effect of imatinib in prostate cancer patients but suggest a promising therapeutic application of imatinib in the treatment of recurrent or metastatic phyllodes tumors. Mol Cancer Ther; 16(2); 365–75. ©2016 AACR.

Abstract Mast cells (MC) are immune cells located next to the intestinal epithelium with regulatory function in maintaining the homeostasis of the mucosal barrier. We have investigated MC activities in colon inflammation and cancer in mice either wild-type (WT) or MC-deficient (KitW-sh) reconstituted or not with bone marrow-derived MCs. Colitis was chemically induced with dextran sodium sulfate (DSS). Tumors were induced by administering azoxymethane (AOM) intraperitoneally before DSS. Following DSS withdrawal, KitW-sh mice showed reduced weight gain and impaired tissue repair compared with their WT littermates or KitW-sh mice reconstituted with bone marrow-derived MCs. MCs were localized in areas of mucosal healing rather than damaged areas where they degraded IL33, an alarmin released by epithelial cells during tissue damage. KitW-sh mice reconstituted with MC deficient for mouse mast cell protease 4 did not restore normal mucosal healing or reduce efficiently inflammation after DSS withdrawal. In contrast with MCs recruited during inflammation-associated wound healing, MCs adjacent to transformed epithelial cells acquired a protumorigenic profile. In AOM- and DSS-treated WT mice, high MC density correlated with high-grade carcinomas. In similarly treated KitW-sh mice, tumors were less extended and displayed lower histologic grade. Our results indicate that the interaction of MCs with epithelial cells is dependent on the inflammatory stage, and on the activation of the tissue repair program. Selective targeting of MCs for prevention or treatment of inflammation-associated colon cancer should be timely pondered to allow tissue repair at premalignant stages or to reduce aggressiveness at the tumor stage. Cancer Res; 75(18); 3760–70. ©2015 AACR.

Abstract Treatments for hormone refractory and metastatic prostate cancer (PC) still remain palliative. Also tumor specific vaccinations when tested in the clinical setting showed results lower than expected. A major limitation to active immunotherapy relies on mechanisms of tolerance adopted by the tumor. Indeed, an immunosuppressive environment is established in PC patients, as well as in the TRAMP mouse model of PC, in which peripheral T cell tolerance to the tumor-associated antigen Tag is acquired early during neoplastic transformation, with mechanisms that still need to be fully clarified. Mast cells (MCs) have been described to mediate immunological tolerance in transplantation and in several disease models, including cancer. We have demonstrated their involvement in early stage of PC development. Therefore, we investigated whether immunosuppression is part of their supporting adenocarcinoma outgrowth in TRAMP mice. We crossed TRAMP mice with KitWsh mice, genetically lacking MCs, and followed tumor growth and Tag-specific CD8+ T cells response after immunization, measured as in vivo lytic activity or ex vivo cytokine production. MCs depletion restrained adenocarcinoma growth in about 70% of mice that at 30 weeks of age only showed evidences of intraepithelial neoplasia or local in situ well-differentiated adenocarcinoma. These lesions were much different from those raised in MCs-sufficient age-matched TRAMP counterparts that developed poor-differentiated multifocal invasive carcinoma. Notably, reduced tumor onset and aggressiveness correlated with improved immune surveillance. Indeed, 16 weeks old KitWsh-TRAMP mice matched for the size of prostatic lesions and for Tag expression, were able to mount a Tag-specific CTL response, whereas their TRAMP counterparts were tolerant. Reconstitution of KitWsh-TRAMP with bone marrow-derived MCs restored complete T cell tolerance. Interestingly, treatment of TRAMP mice with Cromolyn, a compound that inhibit MCs degranulation, had no effect on Tag-specific immune response while partially inhibited tumor growth. These results suggest that independent mechanisms may regulate local immunosuppression and tumor promotion by MCs. They also hint that surface receptors, rather than soluble mediators, are involved in MCs-mediated T cell tolerance. MCs contribute to the maintenance of an immunosuppressive environment, favoring the growth of well-differentiated prostate adenocarcinoma. This open the way for the identification of new therapeutic approaches in PC, aimed at coupling MCs targeting with other immunotherapeutic strategies. Citation Format: Elena Jachetti, Alice Rigoni, Lucia Bongiovanni, Claudio Tripodo, Mario P. Colombo. Mast cells contribute to T cell tolerance against prostate cancer- associated antigens favoring tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2015-4054