Khazaie, Khashayarsha

Abstract Many tumors exhibit increased incorporation of sialic acids into cell-surface glycans, which impact the tumor microenvironment. Sialic acid immunoglobulin-like lectins (Siglec) are receptors that recognize sialic acids and modulate immune responses, including responses to tumors. However, the roles of individual sialyltransferases in tumorigenesis and tumor growth are not well understood. Here, we examined the sialyltransferase ST8Sia6, which generated α2,8-linked disialic acids that bind to murine Siglec-E and human Siglec-7 and -9. Increased ST8Sia6 expression was found on many human tumors and associated with decreased survival in several cancers, including colon cancer. Because of this, we engineered MC38 and B16-F10 tumor lines to express ST8Sia6. ST8Sia6-expressing MC38 and B16-F10 tumors exhibited faster growth and led to decreased survival, which required host Siglec-E. ST8Sia6 expression on tumors also altered macrophage polarization toward M2, including upregulation of the immune modulator arginase, which also required Siglec-E. ST8Sia6 also accelerated tumorigenesis in a genetically engineered, spontaneous murine model of colon cancer, decreasing survival from approximately 6 months to 67 days. Thus, ST8Sia6 expression on tumors inhibits antitumor immune responses to accelerate tumor growth.

Abstract TCF1 is essential for the development and function of T regulatory cells (Tregs). However, how TCF1 regulates Treg function in homeostasis or under pathogenic conditions is poorly understood. Here, we ablated TCF1 in Tregs to elucidate its role in Treg specification in healthy mice and mice with colon cancer. RNAseq revealed that TCF1-deficient Tregs maintain their core transcriptional signature, but promote T-cell receptor, Tgfβ receptor, TH17, and Wnt/β-catenin signaling pathways. Single-cell RNAseq identified central-memory-Tregs with low Klf2 or high Mif expression, which upon downregulation of TCF1 gained TH17 characteristics and matured into Maf expressing effector Tregs. Tcf1-deficient Tregs exhibited enhanced suppression of T-cell proliferation and cytotoxicity but were compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Tcf1-deficient Tregs promoted inflammation and tumor growth. Thus, TCF1 differentially regulates Treg control of TH17 inflammation and T-cell cytotoxicity, and its action in Tregs determines colorectal cancer outcome.

Abstract The molecular and functional diversity of regulatory T-cells (Tregs) in health and in disease remains unclear. We previously described in colorectal cancer (CRC) patients a subpopulation of RORγt+ Tregs with elevated expression of β-catenin and pro-inflammatory properties. Here we observed progressive expansion of RORγt+ Tregs in inflammatory bowel disease (IBD) patients during inflammation and early dysplasia. Activating Wnt/β-catenin signaling in human and murine Tregs was sufficient to recapitulate the disease-associated increase in frequencies of RORγt+ Tregs expressing IL-17, IFN-γ, and TNFa. We found that binding of the β-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt/β-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Tregs. Activation of ꞵ-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

Abstract Purpose: Intratumorally injected Clostridium novyi-NT (nontoxic; lacking the alpha toxin), an attenuated strain of C. novyi, replicates within hypoxic tumor regions resulting in tumor-confined cell lysis and inflammatory response in animals, which warrants clinical investigation. Patients and Methods: This first-in-human study (NCT01924689) enrolled patients with injectable, treatment-refractory solid tumors to receive a single intratumoral injection of C. novyi-NT across 6 dose cohorts (1 × 104 to 3 × 106 spores, 3+3 dose-escalation design) to determine dose-limiting toxicities (DLT), and the maximum tolerated dose. Results: Among 24 patients, a single intratumoral injection of C. novyi-NT led to bacterial spores germination and the resultant lysis of injected tumor masses in 10 patients (42%) across all doses. The cohort 5 dose (1 × 106 spores) was defined as the maximum tolerated dose; DLTs were grade 4 sepsis (n = 2) and grade 4 gas gangrene (n = 1), all occurring in three patients with injected tumors >8 cm. Other treatment-related grade ≥3 toxicities included pathologic fracture (n = 1), limb abscess (n = 1), soft-tissue infection (n = 1), respiratory insufficiency (n = 1), and rash (n = 1), which occurred across four patients. Of 22 evaluable patients, nine (41%) had a decrease in size of the injected tumor and 19 (86%) had stable disease as the best overall response in injected and noninjected lesions combined. C. novyi-NT injection elicited a transient systemic cytokine response and enhanced systemic tumor-specific T-cell responses. Conclusions: Single intratumoral injection of C. novyi-NT is feasible. Toxicities can be significant but manageable. Signals of antitumor activity and the host immune response support additional studies of C. novyi-NT in humans.

e16062 Background: Lynch syndrome (Ly) but not Sporadic (Sp) CRC patients are responsive to immunotherapies. We have shown micorbiota from CRC patients induces precancerous colonic lesions in germ-free mice. The aim was to investigate microbiota linked compromised anti tumor immune response. Methods: We compared 1) Whole metagenomic fecal microbiotas of CRC patients [Sp, n = 53+Ly, n = 19] to controls (n = 90) or being first degree relatives of Ly patients (n = 14), respectively; 2) Tumoral to normal mucosa-adherent microbiota (16sRNA sequences); 3) Normal to tumoral tissues’ mRNA RT-PCRs of IL1b, IL4, IL6, IL8, IL17, TNFa, TGFb, FasL, Granzyme A&B, Thy1; 4) CD3, CD4, CD8, Granzym, KL1, FoxP3, RORgT, MastCell, cells in tumor tissues quantified by immunohistochemistry (IHC), between subgroups. Associations between bacteria, tissue immune cell infiltrates and clinical data (3-yrs follow up or death) were established by using unsupervised Systems Biology and Bioinformatics analyses (Institut Pasteur Shaman platform and Qlucor program). Anti tumoral immune response links with bacteria were based on differences between Ly and Sp CRC patients. Tumor tolerance links with bacteria were identified using germ-free mice (n = 185) who orally received microbiota (9 Sp CRC patients vs 9 controls) with colonic mucosa examined 3 months after human fecal transfer (whole exome sequencing, methylchips, IHC for CD3, CD11b, FoxP3, RORgT, ILC3). Results: Enrichment in Peptostreptococcus, Thermodesulfovibrio and diminution in Ruminococcus, Eubacterium, Coprococcus were linked with FoxP3 and IL-17 over expressions in tumor tissues. Enrichment in Escherichia, Enterobacter, Thioalkalimicrobium, and diminution in Eubacterium, Faecalibacterium and Butyrovibrio were associated with higher FoxP3, FasL, Granzyms A&B, Perforin, Thy1 mRNAs/mRNACD3. Diminution of Eubacterium, Dorea, Faecalibacterium genera and enrichment in Fusobacterium, E. Coli/Shigella were associated with higher CD3, FoxP3, GranzymA and B and FOXP3-RORgT cells in tumor tissues. Higher fecal enrichments in oral bacteria (parvimonas micra) and lack of Thermodesulfovibrio enrichment were associated with higher FoxP3-RORgt cells and short survival; in mice ILC3 and FoxP3-RORgT cells were linked with Sp CRC dysbiosis. Species linked with immunotolerance are identified. Conclusions: Fecal and tissue microbiota can impact failure of anti tumoral immune response in sporadic CRC patients by enhancing overexpression of IL-17 and reducing cytotoxic T-cell infiltration in tumors. Clinical trial information: NCT01270360 .

Abstract The BALB/c-neuT mouse has been a faithful model for investigating metastatic breast cancer. Spontaneous breast tumors from BALB/c-neuT mice are readily transplantable orthotopically into syngeneic or rat neuT negative BALB/c WT hosts facilitating controlled studies of developing breast cancers. When introduced into the breast ducts of BALB/c WT hosts, lung metastases form similarly to how they appear in mice with spontaneous tumors. Tumor grafts grow out with comparable kinetics and induce similar systemic immune abnormalities in the spleens and lymph nodes of both lines of mice as seen in mice with spontaneous tumors. Factors inducing these abnormal immune cell configurations are not yet known. Immunization of BALB/c WT mice against the rat Her2 antigen can induce both T cell and antibody mediated rejection of the transplanted tumors, but these approaches have not been equally successful in syngeneic, rat neuT+ positive hosts, indicating tolerance to rat Her2 is a significant barrier to the induction of immunity against these breast tumors. The MHC encodes class I and class II antigen presenting molecules which are known to be highly polymorphic in both structure and function. We introduced the H-2b MHC haplotype into the MMTV-NeuT model to assess the influence of a different set of antigen presenting molecules on tumor biology. In BALB.B-neuT mice spontaneous tumors appeared with frequencies and in numbers similar to those seen in the BALB/c model. In anything, lung metastases are more frequent and more aggressive in BALB.B-neuT mice. Again, the tumors were readily transplantable into the breast fat pads of syngeneic BALB.B-neuT hosts which developed systemic immune abnormalities very similar to those observed in BALB/c -neuT or BALB/c WT mice. However, BALB.B WT hosts initially accepted the grafts, but subsequently rejected them after they were established. Mice that were in the process of rejecting the tumors retained normal cellularity within their spleens and lymph nodes indicating the importance of an ongoing immune response and/or tumor rejection in shaping the microenvironment in secondary lymphoid organs. Adoptive transfer of spleen cells from BALB.B WT mice which had rejected established tumor conferred resistance to the tumor grafts as long as B cells were included (P<0.01), indicating active humoral immunity was important to tumor rejection. A mechanistic understanding or how antibody-mediated immunotherapy works in patients is incomplete. This new model provides an opportunity to address this question. The Her2 ecto-domain is known to be shed from tumor cells. We propose a model in which the ecto-domain of rat neuT is shed, but is insufficiently immunogenic in BALB/c (H-2d) hosts, but highly immunogenic in BALB.B (H-2b) mice. Consistent with this model is the prediction using the Immune Epitope and Data Base Resource of the presence of a potentially immunogenic IAb-restricted class II epitope within the ecto-domain of rat Her2 neuT oncogene, but no such epitope restricted by IAd or IEd was identified. This provides a compelling hypothesis explaining why grafted spontaneous tumors grow in BALB/c, but not in BALB.B WT mice. An important prediction from this model is that linkage of a potent class II epitope to the Erbb2-encoded ecto-domain may provide a basis for a potent vaccine against Her2 positive breast tumors which should be a strategy adaptable to any MHC genotype. It also will be of interest to determine whether the presence of an antibody reactive against established tumor restores the normal cellular composition of secondary lymphoid organs which have been perturbed by established breast tumors. Citation Format: Sarah Anne Castro, Alvaro Pena, Khashayarsha Khazaie, Larry R. Pease. An immunogenic mouse model using BALB.B-MMTV-neuT breast cancers reveals spontaneous protective humoral immunity [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-15.

Sporadic colorectal cancer (CRC) is a result of complex interactions between the host and its environment. Environmental stressors act by causing host cell DNA alterations implicated in the onset of cancer. Here we investigate the stressor ability of CRC-associated gut dysbiosis as causal agent of host DNA alterations. The epigenetic nature of these alterations was investigated in humans and in mice. Germ-free mice receiving fecal samples from subjects with normal colonoscopy or from CRC patients were monitored for 7 or 14 wk. Aberrant crypt foci, luminal microbiota, and DNA alterations (colonic exome sequencing and methylation patterns) were monitored following human feces transfer. CRC-associated microbiota induced higher numbers of hypermethylated genes in murine colonic mucosa (vs. healthy controls’ microbiota recipients). Several gene promoters including SFRP1,2,3, PENK, NPY, ALX4, SEPT9, and WIF1 promoters were found hypermethylated in CRC but not in normal tissues or effluents from fecal donors. In a pilot study ( n = 266), the blood methylation levels of 3 genes ( Wif1 , PENK , and NPY ) were shown closely associated with CRC dysbiosis. In a validation study ( n = 1,000), the cumulative methylation index (CMI) of these genes was significantly higher in CRCs than in controls. Further, CMI appeared as an independent risk factor for CRC diagnosis as shown by multivariate analysis that included fecal immunochemical blood test. Consequently, fecal bacterial species in individuals with higher CMI in blood were identified by whole metagenomic analysis. Thus, CRC-related dysbiosis induces methylation of host genes, and corresponding CMIs together with associated bacteria are potential biomarkers for CRC.