Abstract 3511: Melanoma-shed, lymph-borne CSPG4 conditions the pre-metastatic lymph node niche

Abstract

Abstract Tissues are primed for metastasis prior to the arrival of tumor cells. Primary tumors drive this transformation by shedding tumor-derived factors (TDFs), including extracellular vesicles and secreted/shed proteins (TSPs) into tumor-associated blood and lymphatic vasculature. Despite being the most common site of metastasis across solid tumor types, we know very little about how the lymph node (LN) is primed for metastasis by TDFs. Here, we test the hypothesis that lymph-borne TDFs are sufficient to initiate the formation of the pre-metastatic LN niche. Using a murine melanoma model (YUMM1.7), we demonstrate primary melanomas initiate remodeling in their draining LNs that enhances metastatic outgrowth relative to contralateral, non-draining and naïve LN controls. Furthermore, we have demonstrated that TDFs isolated from YUMM1.7-conditioned media uniquely activate this pro-metastatic LN phenotype rather than factors derived from benign dermis. The focus of this field has been on the role tumor-derived extracellular vesicles play in establishing this pre-metastatic niche, however our data further demonstrates TSPs are also sufficient to activate this niche. To identify candidate TDFs in vivo that have the potential to initiate LN transformation, we adopted a novel chemical biology strategy to ubiquitously tag TDFs in vivo using non-canonical amino acids and engineered expression of mutant tRNA synthetases in YUMM1.7 melanoma cells. Using this approach, we identified several TDFs by mass spectrometry present in pre-metastatic LNs, and identified one candidate (CSPG4; Melanoma-Associated Chondroitin Sulfate Proteoglycan) as a potential mediator of pre-metastatic niche development. Shed CSPG4 is found within TSPs isolated from media conditioned by YUMM1.7, but not benign dermal, cells. We generated stable shRNA-expressing YUMM1.7 cell lines for efficient knockdown of CSPG4, and found that loss of CSPG4 confers loss of TSP-dependent pre-metastatic niche development in the draining LN. Furthermore, using an enzyme specific to chondroitins (the main glycan decorating CSPG4), we demonstrate that loss of chondroitin glycosylation of CSPG4 is sufficient to attenuate metastasis, even with the core protein present. We find that fibroblastic reticular cells in the draining LN respond to TSPs by becoming more myofibroblast like, resembling cancer-associated fibroblasts that provide direct trophic support to tumor cells. This phenotypic switch is partially rescued in the absence of CSPG4. Our results collectively indicate TSPs prime the LN for metastasis and we have identified melanoma-shed CSPG4 as a driver of pre-metastatic niche development, potentially through its influence on the LN-resident fibroblasts. Citation Format: Haley du Bois, Annie L. Chen, Katherine S. Ventre, Maria S. Steele, Triantafyllia Karakousi, Amanda W. Lund. Melanoma-shed, lymph-borne CSPG4 conditions the pre-metastatic lymph node niche [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 3511.