PMID- 36536301 OWN - NLM STAT- MEDLINE DCOM- 20221221 LR - 20230321 IS - 1471-2407 (Electronic) IS - 1471-2407 (Linking) VI - 22 IP - 1 DP - 2022 Dec 19 TI - Tumor cells-derived conditioned medium induced pro-tumoral phenotypes in macrophages through calcium-nuclear factor kappaB interaction. PG - 1327 LID - 10.1186/s12885-022-10431-8 [doi] LID - 1327 AB - BACKGROUND: The malignant behaviors of lung cancers are affected by not only cancer cells but also many kinds of stromal cells in tumor microenvironment (TME), including macrophages. Macrophages have been proven to extensively influence tumor progression through several mechanisms, among which switching of macrophages from pro-inflammatory phenotypes (M1-like) to anti-inflammatory phenotypes (M2-like) mediated by transcription factors such as nuclear factor kappaB (NF-kappaB) is the most crucial event. The regulation of NF-kappaB has been well studied, however some details remain fuzzy. METHODS: Mouse primary bone marrow-derived macrophages (BMDMs) were cultured in Lewis lung carcinoma cell line LL-2-derived conditioned medium (LL-2-CM). Proliferation, migration, and polarization of BMDMs were tested by CCK8, scratch test, transwell, and flow cytometry. Secretion of several cytokines were detected by ELISA or cytometric bead array. To further explore the underlying mechanisms, BMDMs cultured in LL-2-CM were harvested for RNA-seq. Cytosolic calcium was detected by calcium probe Fluo-4-AM. Western blot was applied to exam the activation of NF-kappaB signal. BAPTA-AM was applied to sequestrate cytosolic calcium to further investigate the relationship between calcium and NF-kappaB signal. The polarization, calcium alteration, and NF-kappaB signal activation were further validated in BMDMs treated by CMT-64-derived conditioned medium (CMT-64-CM). RESULTS: LL-2-CM promoted proliferation, migration, and M2-like polarization of BMDMs and inhibited M1-like polarization of BMDMs. However two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]) were secreted. RNA-seq indicated that LL-2-CM activated both canonical and non-canonical NF-kappaB signal in BMDMs. Western blot showed that canonical NF-kappaB was temporarily elicited and attenuated at 24 h, while non-canonical NF-kappaB was consistently activated. At the same time, expression of genes that regulate cytosolic calcium ion concentration were down regulated, which caused diminution of cytosolic calcium in BMDMs treated with LL-2-CM. The decreased cytosolic calcium, M2-like polarization, and NF-kappaB activation was also observed in CMT-64-CM treated BMDMs. On the contrary, elevated cytosolic calcium was observed during M1-like polarization of BMDMs elicited by lipopolysaccharide (LPS). Interestingly, administration of calcium chelator, BAPTA-AM, impeded activation of canonical NF-kappaB and expression of M1-like marker induced by LPS, which further confirmed the relationship between cytosolic calcium and canonical NF-kappaB signal. CONCLUSIONS: In summary, lung cancer cell-derived conditioned medium promoted migration, proliferation, and M2-like polarization of BMDMs. The suppressed M1-like polarization was achieved through mitigating canonical NF-kappaB pathway via diminishing cytosolic calcium concentration. As far as we know, our work firstly revealed that cytosolic calcium is the key during inhibition of canonical NF-kappaB and M1-like polarization in macrophages by tumor cells. CI - (c) 2022. The Author(s). FAU - Zhang, Yuexin AU - Zhang Y AD - Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610064, China. AD - Gastric and Colorectal Surgery Division, Department of General Surgery, Daping Hospital, Army Medical University, No. 10, Changjiangzhilu, Daping, Yuzhong District, Chongqing, 400042, China. FAU - Zhang, Ziqi AU - Zhang Z AD - Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu, Sichuan, 610041, People's Republic of China. FAU - Chen, Lei AU - Chen L AD - Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610064, China. FAU - Zhang, Xiuyue AU - Zhang X AD - Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610064, China. zhangxiuyue@scu.edu.cn. LA - eng GR - 32070529/National Natural Science Foundation of China/ PT - Journal Article DEP - 20221219 PL - England TA - BMC Cancer JT - BMC cancer JID - 100967800 RN - 0 (NF-kappa B) RN - SY7Q814VUP (Calcium) RN - 0 (Culture Media, Conditioned) RN - 0 (Lipopolysaccharides) RN - 139890-68-9 (1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester) RN - 0 (Cytokines) SB - IM MH - Mice MH - Animals MH - *NF-kappa B/metabolism MH - Calcium/metabolism MH - Culture Media, Conditioned/metabolism MH - Lipopolysaccharides MH - Macrophages/metabolism MH - Cytokines/metabolism MH - *Neoplasms/metabolism MH - Phenotype MH - Tumor Microenvironment PMC - PMC9762082 OTO - NOTNLM OT - CMT-64 macrophages OT - Calcium OT - LL-2 OT - NF-kappaB OT - RNA-seq COIS- The authors declare that they have no conflict of interest. EDAT- 2022/12/20 06:00 MHDA- 2022/12/22 06:00 PMCR- 2022/12/19 CRDT- 2022/12/19 23:45 PHST- 2022/07/26 00:00 [received] PHST- 2022/12/09 00:00 [accepted] PHST- 2022/12/19 23:45 [entrez] PHST- 2022/12/20 06:00 [pubmed] PHST- 2022/12/22 06:00 [medline] PHST- 2022/12/19 00:00 [pmc-release] AID - 10.1186/s12885-022-10431-8 [pii] AID - 10431 [pii] AID - 10.1186/s12885-022-10431-8 [doi] PST - epublish SO - BMC Cancer. 2022 Dec 19;22(1):1327. doi: 10.1186/s12885-022-10431-8.