Abstract PO-083: Utilizing spatial transcriptomics to elucidate tertiary lymphoid structure heterogeneity in human cancer

Blockade of the inhibitory PD1 pathway on CD8+ and CD4+ tumor infiltrating lymphocytes (TILs) has revolutionized standard of care for cancer patients. However, these approaches only benefit approximately 20% of cancer patients, thus a more comprehensive understanding of the immune response is paramount for the development of new therapeutic approaches and enhancement of the anti-tumor immune response. The presence of tertiary lymphoid structures (TLS) correlates with enhanced anti-tumor immunity and improved prognosis in several solid tumors. These ectopic lymphoid aggregates can exhibit features like secondary lymphoid organs (SLOs), including high endothelial venules, a T cell zone with mature dendritic cells (DCs), and a germinal center (GC) with follicular DCs and B cells, that facilitate the induction of immune responses in situ. Furthermore, the presence of TLS correlate with superior responses to immunotherapy and mature, GC-containing TLS correlate with increased T cell function in human tumors. However, current immunotherapies do not target TLS despite their predominance in the tumor microenvironment (TME) and key role in the adaptive immune response. Developing therapies that adequately induce TLS is limited by a lack of objective, bioinformatic analyses to elucidate the cellular and locational heterogeneity of TLS. TLS differences in composition are thought to define maturation levels i.e. early TLS have B and T cell aggregates but lack follicular DC and GC appear as the TLS mature. However, cellular composition is just one aspect of TLS heterogeneity. To successfully increase mature TLS in cancer patients for maximal humoral immunity, we must first understand the complete transcriptomic and proteomic profile of TLS in cancer patients. We have utilized the innovative Nanostring GeoMx platform to spatially interrogate the transcriptomics and proteomics associated with TLS. By pairing this technology with high level multispectral immunofluorescence via the Vectra Polaris system, we can link TLS heterogeneity to TLS position within the tumor. Further, we can analyze the complete activation profile of B and T cells to understand how TLS heterogeneity affects lymphocyte function. Our data have indicated that TLS can be linked to increased CD8+ T cell infiltrate and function. In fact, when mature TLS are not present, CD8+ T cell and Treg interactions increase, ultimately creating a more immunosuppressive TME. Lastly, utilizing a novel, robust bioinformatic pipeline, we have identified new genes that will aid in an objective definition of TLS. Specifically, CD27 is increased within TLS that are proximal to the tumor compared to those in the normal adjacent tissue. These studies will revolutionize the way in which TLS are defined within the TME, ultimately offering comprehensive gene and protein analysis of TLS that includes spatial geography of the structures and an unbiased, objective definition of TLS for future clinical utility and immunotherapeutic targeting. Citation Format: Dongyan Liu, Xiang Li, Rajesh Acharya, Ernest M. Meyer, Shelley Reynolds, Ayana Ruffin, Robert L. Ferris, Dario A.A. Vignali, Riyue Bao, Tullia C. Bruno. Utilizing spatial transcriptomics to elucidate tertiary lymphoid structure heterogeneity in human cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-083.