Abstract B57: Studying the role of OPN-CD44 interaction in colorectal cancer antitumor immunity suppression using scRNA-seq data


 Colorectal cancer (CRC) remains one of the deadliest malignancies worldwide despite recent progress in screening and treatment strategies. Osteopontin (OPN) is a multifunctional protein expressed by multiple cells in the tumor microenvironment (TME) and is associated with immune suppression and poor prognosis in CRC. Tumor-associated macrophages (TAM), are also associated with immune suppression and poor prognosis in CRC, and may express OPN. While both TAM and OPN overlap in the TME, the role of OPN and OPN expressing TAM is poorly understood. In this study, we show the critical interaction and strategy for targeting this axis to overcome therapeutic resistance in CRC. Five publicly available CRC scRNA-seq data (571,818 cells total) from both untreated and treated patients were analyzed to determine the OPN expression and OPN-CD44 interaction in primary CRC and liver metastases using CellChat ligand-receptor interaction analysis. For experimental analyses, patient tissue was collected from the University of Missouri (MU) and the Harry S. Truman VA (HSTVA) on an approved IRB. OPN-positive TAMs were determined by immunofluorescence (IFC) and flow cytometry (FC). Analysis of FC data was completed using analyzed (FlowJo). Wilcoxon rank sum test was performed to find differentially expressed genes between the samples with and without OPN-CD44 interaction in each dataset. Data from scRNA-seq, IFC, and FC all demonstrated that TAMs were the predominant cells expressing OPN in the TME and the majority of TAM are OPN+. Most interestingly, in scRNA-seq data, we found minimal OPN+TAM in primary tumors from treated patients. These patients had a good response to therapy. Conversely, in FC data from our own patients, we found OPN+ TAM were preserved in primary tumors from patients’ post-therapy that had no response. Gene set enrichment analysis (GSEA) in treated patients with minimal OPN+TAM showed decreased exhaustion markers (logFC>0.5, FDR adjusted p-value<0.05). We then found that the strongest OPN-CD44 interactions were predicted between OPN+TAM and other TAM as well as T cells. Separating patients into high and low predicted OPN-CD44 interactors, GSEA demonstrated enrichment of cell death regulation and apoptosis pathways in CD8+ T cells in high interactors. To determine potential targets for modulation of OPN+TAM, we undertook trajectory analysis demonstrating that OPN+TAM were an intermediate phase, suggesting they are malleable. We then found that these OPN+TAM highly express TLR8 in both scRNAseq and FC, suggesting a potential target to modulate OPN+TAM. In this study, we demonstrate that OPN+TAM are the largest proportion of TAM in CRC tumors. Additionally, the presence of OPN+ TAM in treated samples is associated with response to systemic therapy. OPN+TAM appear to represent an intermediate phase of TAMs differentiation and highly express TLR8 suggesting a mechanism for modulation to improve patient response. Further ex vivo studies targeting these cells and their role in therapeutic response are critical.
 Citation Format: Yulia I Nussbaum, Yariswamy Manjunath, Elizabeth Shumway, Kaifi T Jussuf, Wesley C Warren, Jonathan B Mitchem. Studying the role of OPN-CD44 interaction in colorectal cancer antitumor immunity suppression using scRNA-seq data [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B57.