R-Spondin 3 as an independent biomarker and potential therapeutic target for colon cancer immunotherapy.

e15557 Background: R-spondin 3 (RSPO3) is a potentiator of the Wnt/β-catenin pathway and its dysregulation has been implicated in cancer development. However, the clinicopathologic features of RSPO3 expression and targeting RSPO3 therapy in colorectal cancer (CRC) remain largely unknown. Methods: Serum RSPO3 level was measures by ELISA in CRC patients, individuals with colon polyps, and healthy controls, respectively. RSPO3 expression in CRC tissues were assessed through immunohistochemistry (IHC) on tissue arrays from 100 pairs of CRC and adjacent normal tissues. Transcription levels of RSPO3 were analyzed using Real-Time PCR in 30 pairs of CRC and non-tumor tissues. The prognostic significance of RSPO3 was evaluated in a cohort of 279 CRC patients. The correlation between RSPO3 expression and immune cell infiltration were analyzed in human CRC tissues. The RSPO3-overexpressing CRC cells lines were established and the effects of RSPO3 on CRC tumor growth was explored using subcutaneous tumor mouse models. Results: The RSPO3 levels in serum was significantly lower in CRC patients than in colon polyp patients (p < 0.01) and healthy individuals (p < 0.001). The mRNA expression of RSPO3 was significantly decreased in CRC tissues compared with the paired adjacent tissues in 73% (22/30) of CRC cases. Accordingly, the protein expression of RSPO3 was significantly lower in CRC tissues than in adjacent normal tissues in 70% (70/100) of CRC patients. Interestedly, the low expression of RSPO3 indicated less infiltration of CD11C+ dendritic cells (DCs), NK cells and CD8+ T cells in CRC tissues (R = 0.38, p < 0.05). Kaplan-Meier survival analysis revealed that low expression of RSPO3 was correlated with poor prognosis in CRC (p < 0.01). The restoration expression of RSPO3 in human (RKO and HCT116) and mouse (MC38) CRC cells did not affect the cell proliferation, cell cycle and apoptosis, but significantly inhibited tumor growth in both immunocompetent and immunocompromised mice, implicating that RSPO3 inhibits tumor growth through activation of immune response in tumor microenvironment (TME). Importantly, recombinant RSPO3 (rRSPO3) treatment significantly inhibited CRC tumor growth in xenograft and allograft mouse models. rRSPO3 and anti-PD-1 combination exhibited greater inhibitory effects than either treatment alone (p < 0.05). FACS analysis confirmed that rRSPO3 treatment notably increased the infiltration of various immune cells, particularly DCs, in the TME (p < 0.05). Conclusions: RSPO3 is lowly expressed in CRC and its low expression was associated with less infiltration of immune cells and poor prognosis in CRC patients. The restoration of RSPO3 expression inhibits CRC tumor growth in vivo . Our results for the first time demonstrate that RSPO3 function as a tumor suppressor that activates in vivo immune response and has a potential implication for CRC immunotherapy.