Abstract 879: Chemokine engineering significantly enhanced migration and tumor infiltration of <i>ex vivo</i>-expanded natural killer cells in osteosarcoma

Abstract Metastatic and relapsed osteosarcoma (OS) has poor prognosis despite multiagent chemoradiotherapy. Novel therapies are urgently needed. Natural killer (NK) cells are highly cytotoxic to OS cells. Increased abundance of tumor infiltrating activated NK cells has been correlated with extended overall survival in OS. However, the OS tumor microenvironment (TME) impairs NK cell TME infiltration. We and others found that CXCR3, a receptor for CXCL9, -10, and -11, is highly expressed on ex vivo-expanded NK cells. We hypothesize that inducing OS cells to secrete CXCL9, -10, or -11 will enhance NK infiltration into the OS TME and improve outcomes. We expanded NK cells from PBMCs using K562-mbIL21-41BBL feeder cells in the presence of IL-2 (50 IU/mL) and confirmed high CXCR3 expression (98%) on expanded NK cells by flow cytometry. CXCL9, -10, or -11 cDNA was cloned into pMSCVneo to produce lentiviruses for infecting OS 143B cells. ELISA assay showed significantly increased secretion of CXCL9, -10, and -11 from CXCL9, -10, and -11 virus-infected 143B cells compared with wild-type (WT) cells (135.0 ± 51.3 vs. 1.7 ± 1.7 pg/mL, p &amp;lt; 0.05; 1165.4 ± 102.4 vs. 0.4 ± 0.4 pg/mL, p &amp;lt; 0.01; and 86.1 ± 10.5 vs. 0.0 ± 0.0 pg/mL, p &amp;lt; 0.01, respectively). Importantly, we observed significantly enhanced migration of expanded NK cells toward CXCL9, -10, or -11 conditioned media (CM) relative to WT CM (1.6 ± 0.1-fold, p &amp;lt; 0.001; 1.7 ± 0.1-fold, p &amp;lt; 0.05; and 1.5 ± 0.0-fold, p &amp;lt; 0.001, respectively) in transwell assays. Luciferase-based in vitro cytotoxicity assay showed that chemokine (CXCL9, -10, or -11)-secreting 143B cells were similarly if not more sensitive than WT cells to NK cells. To compare the migration of expanded NK cells towards WT and chemokine-secreting OS, we inoculated WT and chemokine-secreting 143B cells subcutaneously into separate flanks of the same NSG mice and administered luciferase-expressing human NK cells intraperitoneally after tumor establishment. IVIS imaging of the mice showed significantly enhanced NK migration toward CXCL9, -10, or -11-secreting tumors compared with WT tumors (2.0 ± 0.3-fold, p &amp;lt; 0.05; 2.0 ± 0.2-fold, p &amp;lt; 0.05; and 2.1 ± 0.1-fold, p &amp;lt; 0.01, respectively). To evaluate NK cell tumor infiltration, we injected human NK cells with an IL-15 agonist, NKTR-255, and harvested and dissociated tumors 5-7 days after NK injection for flow cytometry analysis. Intriguingly, we found significantly increased number of human CD45+ cells in CXCL9, -10, or -11-secreting tumors compared with WT tumors (2.4 ± 0.2-fold, p &amp;lt; 0.05; 11.3 ± 2.8-fold, p &amp;lt; 0.05; and 4.7 ± 1.2-fold, p &amp;lt; 0.05, respectively). Our in vitro and in vivo data demonstrated that secretion of CXCL9, -10, and -11 from OS significantly enhanced NK migration and NK infiltration into the OS TME. We are actively investigating the in vivo anti-tumor efficacy of expanded NK cells against CXCL9, -10, or -11-secreting disseminated OS tumors. Citation Format: Shiori Eguchi, Wen Luo, Hai M. Hoang, Hongwen Zhu, Mitchell S. Cairo. Chemokine engineering significantly enhanced migration and tumor infiltration of ex vivo-expanded natural killer cells in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 879.