Abstract 4753: Bone Morphogenetic Protein 7 is a candidate promoter of cancer immune evasion
Authors: Routh Eric D. , Chifman Julia , Pullikuth Ashok , Miller Lance D. 
Published: August 2018

Abstract
Tumors evade immune recognition and destruction by various established mechanisms, but less is known about how tumors short-circuit successful immune cell trafficking. To identify candidate proteins that inhibit immune trafficking, we developed a bioinformatics screen that utilizes an intratumoral cytotoxic T lymphocyte (CTL) gene signature that quantifies the relative abundance of tumor infiltrating CTLs. This gene signature was used as a stratification tool to examine differential gene expression between tumors of “low” versus “high” CTL status. We hypothesized that a subset of recurrently overexpressed genes in low-CTL tumors (i.e., immunologically “cold” tumors) may functionally act to limit lymphocyte infiltration into the tumor mass. A pan-TCGA analysis of 23 solid tumor types (n=8,875 tumors) revealed a number of recurrent genes highly significantly associated with low CTL infiltration. We found that 88.5% of tumor pairwise comparisons (among the 23 tumor types) showed significant overlap of top genes associated with low CTL status, consistent with the existence of conserved, tumor-agnostic mechanisms of immune evasion. From these analyses, the secreted TGFβ superfamily member, BMP7, emerged as a top-ranking candidate regulator of immune trafficking. To investigate the role of BMP7 in CTL tumor trafficking, an immunogenic variant of the 4T1 mouse breast cancer cell line, 4T1-S, was modified using CRISPR-Cas9 to inducibly express BMP7 from the ROSA26 locus. 4T1-S BMP7-inducible cells were orthotopically transplanted into Balb/C female mice and tumors were allowed to form with or without BMP7 induction. Mice were sacrificed at 2, 3, and 4 wks post-tumor inoculation, and tumors, tumor-draining LNs (TDLNs), and spleens were harvested. At both 2 wks and 3 wks, a BMP7-dependent reduction in tumor CTLs was observed by anti-CD8a IF microscopy (p=0.002 and p&lt;0.001, respectively). Flow cytometric analysis confirmed the significant reduction in tumor-infiltrating CD8+ CTLs in the BMP7-induced group at 3 wks (p=0.02). BMP7 expression did not alter tumor trafficking of natural killer (NK) cells. Flow analysis also showed CTL reduction in TDLNs at 2 wks (p=0.02), and NKs were reduced in the TDLNs at 3 wks (p=0.04). Spleen sizes were also significantly smaller at all time points, indicating a systemic immunomodulatory function of tumor-derived BMP7 (p=0.002, p&lt;0.001, and p=0.002 at 2, 3, and 4 wks, respectively). This study provides a framework for discovering yet unappreciated mechanisms by which tumors evade the immune system, and presents first evidence that BMP7 expression by breast tumors can function to limit CTL trafficking.
Citation Format: Eric D. Routh, Julia Chifman, Ashok Pullikuth, Lance D. Miller. Bone Morphogenetic Protein 7 is a candidate promoter of cancer immune evasion [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 4753.