Tumor cell-derived conditioned medium induced pro-tumoral phenotypes in macrophages through calcium-nuclear factor κB interaction
Authors: Zhang Yuexin , Zhang Ziqi , Chen Lei , Zhang Xiuyue 
Published: December 2022

Abstract
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 κB (NF-κB) is the most crucial event. The regulation of NF-κB 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-κB signal. BAPTA-AM was applied to sequestrate cytosolic calcium to further investigate the relationship between calcium and NF-κB signal. The polarization, calcium alteration, and NF-κB 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-$$\mathrm{\alpha }$$
α
(TNF-$$\mathrm{\alpha }$$
α
) were secreted. RNA-seq indicated that LL-2-CM activated both canonical and non-canonical NF-κB signal in BMDMs. Western blot showed that canonical NF-κB was temporarily elicited and attenuated at 24 h, while non-canonical NF-κB 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-κB 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-κB and expression of M1-like marker induced by LPS, which further confirmed the relationship between cytosolic calcium and canonical NF-κB 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-κB 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-κB and M1-like polarization in macrophages by tumor cells.