Single Cell Sequencing of Pancreatic Ductal Adenocarcinoma Reveals a Paradoxical Immunosuppressive Microenvironment Following Stereotactic Body Radiation Therapy

Abstract

<h3>Purpose/Objective(s)</h3> Patients with locally advanced pancreatic ductal adenocarcinoma (PDAC) have limited therapeutic options with their disease being defined as neither metastatic nor amenable to surgical resection. With stereotactic body radiation therapy (SBRT) emerging as a viable treatment option in this context, disease progression remains far too common in PDAC. Understanding the interaction between radiation, malignant cells, and the tumor microenvironment (TME) may lead to an understanding of novel biological mechanisms that can be exploited. We hypothesized that a detailed itemization of the tumor components through single cell RNA (scRNA) sequencing may allow us to understand emerging immunosuppressive pathways following SBRT than can function as potential nodes of therapeutic actionability. <h3>Materials/Methods</h3> To do this we leveraged an institutional IRB approved protocol. Tumor biopsies were obtained under a phase I/II dose escalation trial of SBRT with a radiomodulating agent, GC4419, undertaken in patients with locally advanced PDAC. Core biopsies were obtained following neoadjuvant chemotherapy, but before and after SBRT with 50-60Gy in 5 fractions. A total of 16 biopsies from patients on trial were suitable for single cell sequencing with 7 patients with matched samples before and after radiation. ScRNA sequencing was performed using the 10x-genomics Chromium droplet-based platform. <h3>Results</h3> A total of ∼27,000 cells across 16 biopsy samples were analyzed. Subtypes of functionally distinct subtypes of cancer-associated fibroblasts (CAFs) were detected including myofibroblastic (myCAF), inflammatory CAFs (iCAF), and antigen presenting CAFs (apCAF). Specifically, following SBRT, there was an enrichment of apCAFs, which are involved in antigen presentation to CD4 T cells, but lack the costimulatory molecules to activate immune response, thus possibly leading to an immunosuppressive environment through anergy. Within the immune microenvironment, there was a shift to M1 macrophage (proinflammatory) polarization, following SBRT. Inversely, there was also an enrichment of regulatory T cells following SBRT, with no significant differences in populations of cytotoxic or memory T cells. <h3>Conclusion</h3> We have shown how single cell RNA sequencing can be used to profile the kinetics of cell populations following radiation therapy. This has revealed a paradoxical immunosuppressive effect following SBRT, whereby immunostimulatory signals are negated by an immunosuppressive microenvironment, providing potential therapeutic targets to be used concurrently with radiation therapy.