Lrrc15, A Candidate Immunotherapy Target, Regulates Cell-Cell Interaction, Migration And Spheroid Formation In Osteosarcoma Cells.

Abstract Background: Leucine-rich repeat containing 15 (LRRC15), a transmembrane protein, whose expression can be modulated by TGFβ, has been found to be highly expressed in multiple solid tumors (e.g. sarcoma, glioma and melanoma) (1). LRRC15 has been nominated as a candidate target for immunotherapy with antibody drug conjugates in cancers, notably osteosarcomas that strongly express this surface marker (2). The present study explores the functional role of LRRC15 in osteosarcoma (OS) cells. Methods: We obtained the gene expression and ChiPseq profiles at the LRRC15 locus including H3K4me3, H3K27Ac, H3K27me3 and RNA pol II for both high (SAOS2/HuO9) and low (HOS/U2OS) LRRC15 expressing OS cells. We generated inducible shRNA knockdown (KD) derivatives to explore the cell-cell & cell-ECM interactions using cell adhesion to ECM array, migration (scratch assay), 3D colony and spheroid formation assays. Gap junction mediated intercellular communication (GJIC) was determined by the ‘Parachute assay' using Dil & Calcein Blue, AM as marker dyes. LRRC15 localization in spheroids was obtained using high resolution 3D imaging of entire spheroids using light-sheet fluorescence microscopy, as well as immunohistochemical localization in cross-sections of spheroids. Results: We found a strong enrichment of activation marks around the LRRC15 locus in high LRRC15 expressing cells (SaOS2/HuO9). In contrast, the LRRC15 locus in low expressing HOS and U2OS cells was enriched in H3K27Me3, suggesting epigenetic control of LRRC15 expression in this OS cell lines. We observed that LRRC15 expression can be induced with TGFβ in low expressing HOS & U2OS cells establishing the gene is still intact and poised for expression. KD of LRRC15 in high expressing OS (SaOS2 & HuO9) cells significantly reduces migration, 3D colony formation, and adhesion to Type 1 collagen. We observed reduced spheroid compaction after LRRC15 KD as well as loss of peripheral cell adherence in high LRRC15 expressing cells. Lateral transfer of Calcein Blue, AM via gap junctions was reduced in LRRC15 KD cells when compared to non-target control cells. Spheroid imaging and IHC revealed a significant reduction in LRRC15 on the cell surface and ECM in disintegrating LRRC15 KD spheroids. Conclusion: Taken together, these results suggest that LRRC15 plays a key role in the cell-cell and cell-ECM interactions of OS cells suggesting that LRRC15 plays a critical role in osteosarcoma cell biology in vitro with likely relevance to osteosarcoma tumors. Reference:1.Purcell JW, Tanlimco SG, Hickson J, Fox M, Sho M, Durkin L, et al. LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates. Cancer Res 2018;78:4059-4072.2.Slemmons KK, Mukherjee S, Meltzer P, Purcell JW, Helman LJ. LRRC15 antibody-drug conjugates show promise as osteosarcoma therapeutics in preclinical studies. Pediatr Blood Cancer 2020:e28771. Citation Format: Sanjit Mukherjee, Konrad Huppi, Robert L. Walker, Jack Zhu, Marbin Pineda, Fan Yang, James Purcell, Lee J. Helman, Paul S. Meltzer. LRRC15, a candidate immunotherapy target, regulates cell-cell interaction, migration and spheroid formation in osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3119.