Abstract 2635: Role of LRRC15, a candidate immunotherapy target, in cell adhesion, migration and spheroid formation in osteosarcoma cells

Background: Leucine-rich repeat containing 15 (LRRC15), a transmembrane protein, has been found to be highly expressed in stromal cells in multiple solid tumors especially tumors of mesenchymal origin (e.g. sarcoma, glioma, 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. In addition, LRRC15 has been found to be significantly upregulated during osteogenic induction [2] and TGFβ induction in mesenchymal stem cells [3]. The present study explores the functional role of LRRC15 in osteosarcoma (OS) cells. Methods: To delineate the role of LRRC15 in OS cells, we generated inducible shRNA knockdown derivatives of high LRRC15 expressing OS cells (SAOS2/HuO9) and induced LRRC15 in low expressing OS cells (HOS & U2OS). We explored various cell-cell and cell-ECM interactions using invasion, migration, adhesion, 3D colony formation (Matrigel), spheroid formation and spheroid migration assays. Results: LRRC15 expression and knockdown in different OS cells were confirmed by transcriptional, flow cytometric and western blot analysis We found that expression of LRRC15 varies significantly among osteosarcoma (OS) cell-lines. Here we report that inducible knockdown of LRRC15 in high expressing OS cells significantly reduces collagen type 1 based adhesion (using extracellular matrix screening array), migration, invasion and 3D colony formation using Matrigel. Enlarged colonies were observed in TGFβ treated HOS cells in Matrigel. Spheroids were formed using ultra low attachment plates with SaOS2/HuO9/HOS & U2OS cells. Knockdown of LRRC15 significantly affects the spheroid compaction in high LRRC15 expressing cells (SaOS2 & HuO9). A spheroid-based migration assay on collagen type 1 coated plates revealed significantly reduced migratory phenotype in LRRC15-knockdown SAOS2 cells. Conclusion: Taken together, these results suggest that LRRC15 plays a key role in cell-cell and cell-ECM interactions of OS cells providing insights relevant to biological function of LRRC15 in osteosarcoma cells. 1. Satoh, K., M. Hata, and H. Yokota, A novel member of the leucine-rich repeat superfamily induced in rat astrocytes by beta-amyloid. Biochem Biophys Res Commun, 2002. 290(2): p. 756-62. 2. Wang, Y., et al., LRRC15 promotes osteogenic differentiation of mesenchymal stem cells by modulating p65 cytoplasmic/nuclear translocation. Stem Cell Res Ther, 2018. 9(1): p. 65. 3. Purcell, J.W., et al., LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates. Cancer Res, 2018. 78(14): p. 4059-4072. Citation Format: Sanjit Mukherjee, Konrad Huppi, Robert L. Walker, Jack Zhu, Marbin Pineda, James W. Purcell, Lee J. Helman, Paul S. Meltzer. Role of LRRC15, a candidate immunotherapy target, in cell adhesion, migration and spheroid formation in osteosarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2635.