Comprehensive Explorations of CCL28 in Lung Adenocarcinoma Immunotherapy and Experimental Validation

BACKGROUND: Chemokines have been reported to play an important role in cancer immunotherapy. This study aimed to explore the chemokines involved in lung cancer immunotherapy. METHODS: All the public data were downloaded from The Cancer Genome Atlas Program database. Quantitative real time-PCR was used to detect the mRNA level of specific molecules and Western blot was used for the protein level. Other experiments used include luciferase reporter experiments, flow cytometric analysis, Chromatin immunoprecipitation assay, ELISA and co-cultured system. RESULTS: We found that the CCL7, CCL11, CCL14, CCL24, CCL25, CCL26, CCL28 had a higher level, while the CCL17, CCL23 had a lower level in immunotherapy non-responders. Also, we found that immunotherapy non-responders had a higher level of CD56dim NK cells, NK cells, Th1 cells, Th2 cells and Treg, yet a lower level of iDC and Th17 cells. Biological enrichment analysis indicated that in the patients with high Treg infiltration, the pathways of pancreas beta cells, KRAS signaling, coagulation, WNT BETA catenin signaling, bile acid metabolism, interferon alpha response, hedgehog signaling, PI3K/AKT/mTOR signaling, apical surface, myogenesis were significantly enriched in. CCL7, CCL11, CCL26 and CCL28 were selected for further analysis. Compared with the patients with high CCL7, CCL11, CCL26 and CCL28 expression, the patients with low CCL7, CCL11, CCL26 and CCL28 expression had a better performance of immunotherapy response and this effect might partly be due to Treg cells. Furthermore, biological exploration and clinical correlation of CCL7, CCL11, CCL26 and CCL28 were conducted, Finally, CCL28 was selected for validation. Experiments showed that under the hypoxia condition, HIF-1α was upregulated, which can directly bind to the promoter region of CCL28 and lead to its higher level. Also, CCL28 secreted by lung cancer cells could induce Tregs infiltration. CONCLUSION: Our study provides a novel insight focused on the chemokines in lung cancer immunotherapy. Also, CCL28 was identified as an underlying biomarker for lung cancer immunotherapy.