Galanin receptor 2 (GALR2) functions as a tumor suppressor in experimental tumors of SCC9 cells

Galanin Receptor 2 (GALR2) is one of three known receptors for the neuropeptide galanin, and conflicting evidence indicates that GALR2 may be either a tumor suppressor or an oncogene in OSCC. This study evaluated the influence of GALR2 expression by neoplastic cells on tumor progression, innate immunity and response to a non-curative dose of cisplatin. We used a murine orthotopic xenograft model of OSCC. Experimental tumors were induced in 20 athymic nude Balb/c mice by injecting 5×105 SCC9 cells directly into their floor of the mouth. We used SCC9 cells stably-transfected with CMV-driven expression of human GALR2 coding region (pcDNA3.1 plasmid vector, GR2 group, n = 10 mice). As negative controls we used SCC9 cells stably-transfected with the empty vector (pcDNA group, n = 10 mice). Two weeks after injection, half of the mice were treated with a single non-curative dose of cisplatin (5 mg/Kg, i.p.) forming the groups GR2+CP (n = 5 mice) and pcDNA+CP (n = 5 mice). The other half of the mice received i.p. injections of the vehicle. A week after treatment, animals were euthanized and tumors were collected, processed and analyzed descriptively, by immunofluorescence (IF), RT-qPCR and flow cytometry. Volume and weight of experimental tumors were similar in GR2 and pcDNA groups, but the overall leukocyte infiltration (CD45+ cells) was increased in GR2 tumors. Cisplatin treatment reduced tumor volume and weight similarly in GR2+CP and pcDNA+CP groups. GR2 tumors were poorly differentiated in comparison with pcDNA tumors. Treatment with cisplatin increased infiltration of inflammatory cells in both groups and caused pulmonary metastasis in pcDNA tumors, which is associated with increased expression of vimentin. Flow cytometry analysis indicated that macrophage (F4/80+ cells) infiltration was similar in pcDNA and GR2 tumors, but cisplatin treatment increased macrophages only in pcDNA tumors (pcDNA+CP). Cisplatin treatment increased infiltration of total myeloid cells (CD45+CD11b+), PMNs (CD45+Ly6G+) and activated PMNs (CD45+Ly6G+CD80+) in both GR2 and pcDNA tumors. These results indicate that overexpression of GALR2 induces poorly differentiated tumors with increased infiltration of inflammatory cells. The increased pulmonary metastasis in pcDNA tumors indicates that for SCC9 cells GALR2 expression functions as a tumor suppressor. Moreover, GALR2 expression did not affect the response to cisplatin. Financial Support: FAPESP (2022/06041-0; 2020/00394-2).

Corresponding author: Ianny Brum Reis (e-mail: iannybrumreis@gmail.com).