Abstract 5328: Dual role of Kupffer cell secreted CXCL5 in hepatocellular carcinoma
Authors: Tu Taojian , Hong Handan , He Lina , Alba Mario , Qi Tang , Okamoto Curtis T. , Stiles Bangyan L. 
Published: March 2024

Abstract
Hepatocellular carcinoma (HCC) is hallmarked by inflammatory cell infiltration. Chemokines secreted during inflammation are crucial at directing immune cell infiltration during cancer development. We screened patient dataset from different etiologies including HBV, HCV, alcohol and non-alcoholic steatohepatitis (NASH) and identified CXCL5 as the only chemokine consistently upregulated in HCC. Immunohistochemistry (IHC) of patient liver tumor tissues further showed the production of CXCL5 by tumor surrounding non parenchymal cells. Using a mouse model (Pte loxP/loxP; Alb-Cre+, Pten null) which recapitulates human liver disease progression from non-alcoholic fatty liver disease (NAFLD) to NASH to liver cancer, we found that CXCL5 was expression was gradually increased during disease progression. Further analysis using IHC and flow cytometry analysis showed that CXCL5 is predominantly expressed by Kupffer cells in Pten null livers bearing tumors. Furthermore, we found that LPS stimulated the expression and secretion of CXCL5 in Kupffer cells, but not other macrophages including peritoneal macrophage and macrophage cell line Raw264.7 cells. We hypothesized that the Kupffer cell-secreted CXCL5 in response to LPS promotes tumor progression by promoting HCC cell proliferation and regulating HCC associated immune cell infiltration. To investigate whether CXCL5 increases tumor cell proliferation, we treated wild type and Pten null hepatocytes with various concentrations of CXCL5. Our results showed that CXCL5 increased hepatocytes viability in a dose dependent manner. BrdU incorporation assay further confirmed that CXCL5 treatments induces HCC cell proliferation through its receptor CXCR2. We further demonstrated that CXCR2 knockdown led to reduced xenograft tumor growth using HepG2 cells. Additionally, we injected Pten null mice with CXCR2 inhibitor to block CXCL5 signal and observed partial response. We analyzed the immune cell profile of the responder mice vs. non-responder mice and showed that tumor inhibition is correlated with T cell infiltration. To test whether Kupffer cells regulate T cells through CXCL5, we isolated primary Kupffer cells from murine livers. We performed transwell migration assay using immune cells from murine spleens. Our results indicate that LPS stimulation in Kupffer cells reduced the ratio of migrated CD4+ T cells, while blocking CXCL5 using a CXCL5 antibody significantly increased CD4+ T cell recruitment to the LPS-stimulated Kupffer cells and moderately decreased neutrophil recruitment. Consistently, we found that recombinant CXCL5 protein reduces CD4+ T cell migration independent of neutrophils. In summary, our study suggests that Kupffer cell secreted CXCL5 reduces liver CD4+ T cell recruitment and increases liver cancer cell proliferation.
Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Tang Qi, Curtis T. Okamoto, Bangyan L. Stiles. Dual role of Kupffer cell secreted CXCL5 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5328.