KIAA1199 Recruits Neutrophils To Promote Colorectal Cancer Liver Metastasis Via TGF-?/SMAD3 Signaling

Objectives:Liver metastasis is the main cause of treatment failure and death in patients with colorectal cancer(CRC).The establishment of tumor immunosuppressive microenvironment is closely related to successful metastasis.Immuneopanoramic analysis of hepatic metastases suggests that the liver inflammatory microenvironment with extensive infiltration of Polymorphonuclear Neutrophils(PMN)plays an important role in supporting the colonization and proliferation of metastatic CRC cells,but the driving factors and recruitment mechanisms have not been elucidated.The key gene KIAA1199,which promotes the distant metastasis of CRC cells,has been screened and identified by our group using high-throughput transcriptome sequencing.However,the mechanism of KIAA1199's tumor promotion has been mainly focused on the regulation of tumor cells'own invasive metastatic ability,while its role in modulating the local microenvironment of distal target organs is less clear.The aim of this study was to elucidate the role and mechanism of tumor-derived KIAA1199 in the formation of immunosuppressive microenvironment in CRC liver metastases,and to provide a new theoretical basis and therapeutic strategy for the prevention and treatment of CRC liver metastases.Methods:(1)Tissue specimens were collected from the primary tumor and liver metastases of CRC patients,and the differences in KIAA1199 expression between primary foci and paired hepatic metastases were analyzed by immunohistochemistry(IHC)and multiplex immunofluorescence(IF)staining;the correlation between KIAA1199 and the proportion of immune cell infiltration in tumor tissues was further analyzed via combining with The Cancer Genome Atlas(TCGA)CRC data.(2)KIAA1199 overexpression or knockdown MC38 cell lines were constructed and were inoculated in C57BL/6 mice to construct trans-splenial liver metastasis model and cecum in situ tumor spontaneous liver metastasis model.General view and Hematoxylin-eosin staining(HE)were used to observe liver metastasis directly.Flow cytometry and multiplexed tissue IF were applied to analyze the composition and functional status of immune cells in the primary tumor and liver metastases of the tumor-bearing mice.(3)Anti-Ly6G and anti-CD8 were used to remove PMN and CD8⁺T cells in the tumor-bearing mice to further verify that KIAA199 is mainly dependent on PMN to inhibit CD8⁺T cell infiltration and anti-tumor activity,and thus mediates immune evasion.(4)Human peripheral blood or mouse bone marrow-derived PMN were sorted by gradient centrifugation or immunomagnetic bead method,and the efficiency of PMN migration induced by different KIAA1199-expressing CRC cell conditioned medium(CM)was examined by transwell assay.(5)PMN were cultured in different KIAA1199-expressing CRC supernatants,and 12 hours later,total RNA was extracted for high-throughput transcriptome sequencing to compare the phenotypic differences of PMN between groups,and quantitative Real-time PCR(q RT-PCR)was used to further verify the expression of phenotype-related molecules.The above domesticated PMN were co-cultured with CD8⁺T cells,and the proliferation of T cells and secretion of IFN-?were detected after 72 hours.(6)q RT-PCR was applied to screen KIAA1199-induced chemokines and further confirmed by Enzyme linked immunosorbent assay(ELISA).(7)CXCR2 inhibitors were adopted in in vitro neutrophil chemotaxis assay and in vivo mouse trans-splenial model,respectively,aiming to further clarify CXCL/CXCR2 as the core factor of KIAA1199-mediated PMN recruitment.(8)Western Blotting(WB)and Co-immunoprecipitation(Co-IP)were performed to detect the correlation between KIAA1199 and TGF-?/SMAD3 signaling pathway.TGFBRI/II inhibitors were applied to clarify the TGF-?/SMAD3 signaling pathway as a key factor in KIAA1199-driven CXCL chemokine upregulation,PMN recruitment and phenotypic transformation,respectively.(9)Pirfenidone(PFD)was aimed to inhibit KIAA1199 expression,and to observe the efficacy of the combined effect with anti-PD-1 in multiple animal models.(10)Using the TCGA CRC data,we further validated the correlation between KIAA1199 m RNA expression levels and the efficacy of PD-1 blocking antibodies.Results:(1)IHC and IF analysis for primary and paired liver metastases from CRC patients showed that KIAA1199 gene expression was elevated in liver metastases and CD66B-positive PMN infiltration was increased compared with primary foci,while the proportion of CD8⁺T cells was decreased.Subsequently,the CIBERSORT method was applied to assess the proportion of immune cell infiltration from TCGA CRC data,which further verified that the KIAA1199 m RNA expression level in tumor tissues was positively correlated with the number of intratumoral PMN and negatively correlated with CD8⁺T cell infiltration.(2)KIAA1199 overexpression and knockdown MC38 cell lines were successfully constructed by genetic engineering technology and inoculated into mice to construct liver metastasis model and in situ tumor model.The results showed that KIAA1199 could promote liver metastasis formation in mice,and further flow analysis of liver metastasis and primary foci showed that KIAA1199 could induce PMN aggregation in tumor tissues,and conversely inhibited CD8⁺T cell infiltration and IFN-?secretion.(3)Blocking PMN accumulation by applying PMN clearance antibody in mice significantly counteracted the ability of KIAA1199 to promote CRC proliferation and liver metastasis.Quantitative analysis of immune cell ratios and functions in primary tumor and liver metastases showed that PMN clearance upregulated intra-tumoral CD8⁺T cell aggregation and enhanced killer T cell activity,thereby reversing KIAA1199-mediated immune tolerance.In addition,the difference in liver metastasis formation between the KIAA1199overexpression group and the control group was also attenuated after clearance of CD8⁺T cells in mice,although the number of liver metastases increased significantly between the 2groups,thus further suggesting that inhibition of T cell anti-tumor activity is the core downstream event of PMN recruitment by KIAA1199.(4)Compared with KIAA1199^lowCRC cells,supernatants of KIAA1199^high CRC cells significantly induced PMN migration and reshaped their phenotype toward pro-tumor-like polarization,allowing them to function as inhibitors of CD8⁺T cell proliferation and antitumor activity.(5)KIAA1199 has clear regulatory effects on the synthesis and secretion of CXCL1 and CXCL3 as verified by q RT-PCR screening and ELISA assay.(6)In vitro transewell assay revealed that the ability of KIAA1199 to recruit PMN could be blocked by CXCL1,CXCL3 neutralizing antibodies and CXCR2 inhibitors,respectively.In mouse liver metastasis model,the CXCR2inhibitors reversed KIAA1199-mediated immunosuppression and significantly attenuated KIAA1199-driven liver metastasis formation.(7)The results of enrichment analysis suggested that KIAA1199 was closely associated with the TGF-?/SMAD3 signaling pathway.Further immunoblotting and Co-IP assays suggested that KIAA1199 may act on the TGF-?receptor and subsequently activate SMAD3 phosphorylation.The results suggest that KIAA1199 mainly relies on the TGF-?/SMAD3 signaling pathway to upregulate CXCL1/3 expression,regulate PMN recruitment and reprogram its phenotype.We also observed that KIAA1199-mediated PMN accumulation and T-cell deficiency in liver metastases could be blocked by TGFBRI/II inhibitors in a trans-splenial model.(8)Knockdown or inhibition of KIAA1199 expression with pirfenidone induced massive CD8⁺T cell infiltration in tumor and enhanced sensitivity to PD-1 blocking antibodies.The combined use of pirfenidone and PD-1 monoclonal antibody significantly curbed the development of CRC liver metastases.(9)KIAA1199 expression was significantly lower in CRC patients with high microsatellite instability(MSI-H)than in patients with microsatellite stable(MSS)or low microsatellite instability(MSI-L).Conclusions:In summary,KIAA1199 activates TGF-?signaling and up-regulate CXCL1/3chemokines production via interacting with TGF-?receptor in tumor cells,which can recruit more CXCR2⁺PMN and therefore eventually promote liver metastasis.Pirfenidone is expected to block CRC cell proliferation and liver metastasis,and increase sensitivity to PD-1 monoclonal antibody by inhibiting KIAA1199 expression and breaking its mediated immune evasion.This project focuses on the impact of tumor-derived KIAA1199 on the shaping of metastatic immunosuppressive microenvironment,and intends to develop novel approaches to boost anti-tumor immunity by targeting KIAA1199-driven suppressive PMNs.The foundings of this study will generate new knowledge about mechanism for CRC liver metastases that will be important in prognostication and designing new therapeutic strategies.