Abstract 2360: Digital spatial profiling of RNA and protein in the tumor microenvironment of invasive lobular breast cancer
Authors: Bennett Lynda B. , Sahoo Sunati , Lewis Cheryl M. , Raman Indu , Frerich Candace , Chen Guanchun , Xu Min , Conzen Suzanne D. 
Published: April 2023

Abstract
Invasive lobular breast cancer (ILC) is an understudied breast cancer subtype with late recurrence, metastasis to serosal surfaces including the peritoneum, and dismal long-term outcome. The interaction between a tumor and its microenvironment leads to phenotypic changes in stromal cells and the ECM that promote proliferation and invasion of malignant cells. ILC is histologically distinct from invasive ductal carcinoma, characterized by discohesive E-cadherin-negative tumor cells that grow in single file. We expect the TME to be unique in ILC. We hypothesized that differing levels of nuclear receptor expression in tumor cells would impact stromal cell composition, presumably through paracrine signaling. There is a range of glucocorticoid receptor (GR) expression in ILC. We sought to determine how crosstalk between GR+ or GR- ILC cells and their respective TME differentially impact stromal cell gene and protein expression, as well as the immune cell milieu. To profile the tumor and stroma separately, we performed nanoString GeoMx digital spatial profiling (DSP) of RNA and protein expression in GR-positive (+) and GR-negative (-) primary ILC. We performed RNA DSP for 12 primary ILCs: 5 strongly positive for GR, 5 GR- and 2 tumors with mixed GR expression. We also completed DSP of proteins on adjacent tissue sections for a total of 87 proteins. To profile tumor and TME independently we segmented regions into PanCK+ tumor and PanCK- stroma. RNA expression analyses revealed striking differences between GR+ and GR- ILC in both tumor cells and TME. Pathway analyses showed enrichment of genes encoding eukaryotic translation initiation and control factors and ribosomal proteins in GR+ tumor cells, as well as downregulation of immune response and antigen presentation pathways. In the TME of the GR- tumors, we saw significantly higher expression of collagen biosynthesis and extracellular matrix genes. Using a spatial deconvolution tool, we observed gene expression indicating markedly higher abundance of macrophages and other myeloid subsets, endothelial cells and fibroblasts in GR+ ILC stroma. There was also evidence for more regulatory T-cells, suggestive of an immune suppressive microenvironment in GR+ ILC. Protein DSP revealed upregulation of proteins involved in DNA repair and cell survival, and reduction of some immune checkpoint proteins in the GR+ tumor cells. In the TME of GR+ ILC, we observed more Ki-67 and ER-alpha as well as proteins expressed on T-regs, as well as downregulation of cytotoxic proteins such as granzyme A. We conclude that in-depth examination of molecular profiles in both the tumor cells and the TME of ILCs will give insight into how tumor GR expression/activation influences the crosstalk between ILC cells and their microenvironment. As expected, we observed heterogeneity between patients, but DSP will allow in-depth characterization of individual tumors at the molecular level.
Citation Format: Lynda B. Bennett, Sunati Sahoo, Cheryl M. Lewis, Indu Raman, Candace Frerich, Guanchun Chen, Min Xu, Suzanne D. Conzen. Digital spatial profiling of RNA and protein in the tumor microenvironment of invasive lobular breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2360.