Figure 6.

TAMs-assisted cancer cell invasion via MIP-1β is dependent on upregulation of MYO3A gene with in cancer cells. (A, B and C) Quantitative RT-PCR-based validation of selected genes (through c-DNA-based gene-expression analysis) in mono-cultured [C] and co-cultured (with macrophages) cancer cells [C+M]. Bars represent relative fold change in expression levels ±SE (*p < 0.05). (D, E and F) MYO3A gene exhibited a characteristic MIP-1β-responsive mRNA exprerssion profile. MIP-1β-neutralizing antibody (MIP-1β NA) treated cells showed decrease in MYO3A expression compared to cells that were co-cultured with macrophages [C+M] and/or IgG antibody control [C + M + IgG]. MIP-1β-purified cytokine (MIP-1β) enhanced the expression of MYO3A gene in monocultured cancer cells compared to cells that were not treated with MIP-1β-purified cytokine (C). Bars represent Quantitative RT-PCR relative fold change expression ±SE (*p <0.05.). All the experiments were done in triplicates and repeated at least thrice. (G and H) Blockade of MIP-1β with MIP-1β-neutralizing antibody (MIP-1β NA, Fig. 6G) and its cognate receptor CCR4 and CCR5 silencing (CCR4-siRNA and CCR5-siRNA, Fig. 6H)) downregulated expression levels of MYO3A in breast cancer cells (MDA-MB-231, MDA-MB-468 and MCF-7). MIP-1β-purified cytokine (MIP-1β) enhanced the expression of MYO3A gene in monocultured cancer cells compared to cells that were not treated with MIP-1β-purified cytokine (C) Fig. 6G. All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; C + M: Respective cancer cells co-cultured with macrophages; C+M(IgG): Respective cancer cells co-cultured with macrophages treated with isotype antibody control IgG; C + M + MIP-1β NA: Respective cancer cells co-cultured with macrophages treated with MIP-1β-neutralizing antibody; MIP-1β: Respective cancer cells treated with MIP-1β-purified cytokine.