Mounting clinical and preclinical evidence demonstrates an important role for the intestinal microbiome in mediating efficacy of immune checkpoint inhibitor (ICI) antibody therapy across a number of tumor contexts. We sought to determine the effects of microbiome modulation on ICI therapy in a clinically recapitulative orthotopic murine lung cancer model. The microbiome of C57Bl/6NHsd mice was sterilized with antibiotic (ampicillin, streptomycin and colistin) for ten days, resulting in a 99.9% mean decrease in fecal aerobic and anaerobic bacterial load in comparison to naïve mice, handled under specific pathogen free (SPF) conditions. Luciferase-expressing murine Lewis lung carcinoma cells (LL/2-Red-FLuc) were surgically implanted into the left lung parenchyma of all animals. Animals were treated with locoregional radiotherapy (2x 9Gy fractions) targeted to the left lung. The gastrointestinal microbiome was reconstituted via oral gavage Q3D of ~1e09/dose commensal A. muciniphila (A. muc) and E. hirae (E. hir) over five doses or sterile saline as control and animals were randomized within commensal/saline treatment groups to equivalent mean tumor burden as measured by Lumina Series III In-Vivo Imaging System (IVIS). Animals were then administered anti-murine PD-1 or isotype control (0.25 mg) antibody (Ab) treatments Q3D over four doses via intraperitoneal injection. Tumor growth was monitored by IVIS over the course of the study, and ex-vivo IVIS was performed on lungs at endpoint (Day 12 post-tumor implantation). Tumor growth of microbiota non-reconstituted antibiotic-sterilized animals was only slightly inhibited by anti-PD-1 therapy. Animals administered commensal A. muc and E. hir and treatments also displayed slightly inhibited tumor growth kinetics, similar to those observed under saline/anti-PD-1 therapy. Fecal microbial sequencing and immunophenotypic analyses are ongoing. This study demonstrates the utility and ongoing development of a clinically recapitulative contextually accurate preclinical murine lung cancer model to assess the effects of specific microbiota in mediating the efficacy of anti-tumor immunotherapy. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Nicholas Rivelli, Alexandra Kury, Sallyann Vu, Gavin Gagnon, Veronica Ritchie, Kasey Reardon, Catarina Costa, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1499.

The intestinal microbiome has become increasingly appreciated as a significant mediator of systemic antitumor immunity/response in both naïve and treatment contexts. In naïve contexts, an intact intestinal microbiome has been demonstrated to enhance tumorigenesis, and its composition to mediate primary tumor growth kinetics. In the context of cancer treatment, antibiotic depletion of the intestinal microbiota has been reported to inhibit the efficacy of cyclophosphamide and that of the immune checkpoint inhibitor αCTLA4. Compositional modulation of the intestinal microbiota has been found to be sufficient to enhance the antitumor efficacy of αPD-L1. Here, we assessed the relative importance of the intestinal microbiota in mediating αPD-L1 antitumor efficacy in a B16.F10.SIY murine model of melanoma, by performing parallel efficacy studies in C57BL/6 germ-free (Taconic) or specific pathogen free (Taconic or Jackson) mice. We observed that αPD-L1 treatment provided significant antitumor efficacy of in Taconic mice carrying an intact microbiome; however, this efficacy was abolished in germ-free Taconic mice. Furthermore, we observed that tumors of Jackson mice carrying an intact but compositionally different microbiome did not respond to αPD-L1 treatment. Phenotyping of local tumor and systemic immune responses, as well as characterization of the intestinal microbiome in responder vs nonresponder animals provided mechanistic insights. Taken together, these observations suggest that rational modulation of the microbiome may enhance response to immune checkpoint inhibition, and indicate that the gastrointestinal microbiome and its composition are critical for the antitumor efficacy of αPD-L1. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Katie Pedrick, Alexandra Kury, Catarina Costa, Brett Van Dam, Jonathan Jung, Gregory D. Lyng, Stephen T. Sonis. The gastrointestinal microbiome and its composition are critical for antitumor efficacy of immune checkpoint inhibition by anti-PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2017-2683

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Accuracy of context is critical in efforts to improve the translational potential of preclinical oncology models. Pancreatic cancer has high mortality and current models are insufficient for advancing therapeutics. Here we note key observations in the ongoing development of a new pancreatic cancer mouse model designed to enable translational immuno-oncology and/or tumor microenvironment (TME) targeted therapeutics in a “triple-humanized” orthotopic context including: 1) tumor: bioluminescent PD-L1+ BxPC3-Red-FLuc human pancreatic ductal adenocarcinoma (PDAC) cells surgically implanted into the pancreas of aged NSG™ mice; 2) T cells: human peripheral blood mononuclear cells (PBMCs) derived from a healthy donor and shown to respond to PD-1 inhibition; and 3) tumor-tropic stem-like stromal cells: bone-marrow (BM) derived human mesenchymal stem/stromal cells (MSCs) derived from a healthy donor. MSCs are described to home to PDAC stroma where they react to TME and contribute to PDAC progression via pleiotropic therapeutic refractory effects including stromal remodeling favoring effector immunosuppression, angiogenesis and desmoplasia. To assess the role of MSCs in PDAC response to PD-1 blockade, we injected BxPC3-Red-FLuc cells into the pancreas of 14-week NSG™ mice, +/-MSCs; coinjected animals received weekly MSCs bolus injections to simulate continuous migration from BM. Some groups concurrently received PBMCs characterized for aPD-1 response. Ten days after injection with human cells, animals were randomized by tumor radiance into treatment groups administered +/- 150µg anti-human PD-1 mAb (clone: EH12.2H7) (aPD-1) 2x/week. Immunohistochemistry analysis of tumors was performed. Engraftment/characterization of PBMCs was assessed by flow cytometry of PB in-life and spleen at terminus. MSCs alone did not increase primary tumor growth, but PBMC engraftment was increased by administration of MSC. Tumor growth was decreased with PBMC but primary tumor growth was increased in animals receiving PBMC+MSC compared to PBMC alone. The antigen-experienced status of the T cells was not affected by MSC treatment. In contrast, the diversity of engrafting T cells was increased by MSC treatment. Strikingly, aPD-1 treatment completely suppressed PBMC engraftment, regardless of MSC presence. This work details ongoing development of a contextually improved “triple-humanized” mouse model of pancreatic cancer. Key findings agree with recent findings of the importance of timing in aPD-1 treatment in humanized models and clinical applications. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Alexandra Kury, Scott Anderson, Gregory D. Lyng, Stephen T. Sonis. Early PD-1 inhibition abrogates PBMC engraftment in a triple-humanized (tumor/PBMC/MSC) bioluminescent orthotopic model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1146.