CCL2 level is elevated in chemoresistant lung adenocarcinoma cells
To confirm that A549/Cis cells are the chemoresistant lung adenocarcinoma cells, we first compared the expression of chemoresistance-related genes in A549 (cisplatin-sensitive) and A549/Cis (cisplatin-resistant) cells by qPCR. Expression of these genes was higher in A549/Cis cells than in A549 cells (P < 0.05, Figure 1A). To determine the dominant factor involved in chemoresistance, the expression levels of several chemokines and cytokines in the supernatants of A549 and A549/Cis cells were detected by multiplex assay. We found that the CCL2 level was significantly higher in the supernatants of A549/Cis cells than in those of A549 cells (Figure 1B). The other cytokines and chemokines did not show significant differences. To confirm this result, we further detected the mRNA and protein expression of CCL2 in the A549 and A549/Cis cells by qPCR, and in their supernatants by ELISA. Similarly, CCL2 mRNA expression was markedly higher in A549/Cis cells than in A549 cells (P < 0.01, Figure 1C), and the CCL2 protein level in the A549/Cis cell supernatants was significantly higher than in the A549 cell supernatants (P < 0.001, Figure 1D). Therefore, these results suggest that the level of CCL2 is elevated in chemoresistant lung adenocarcinoma cells.
High CCL2 levels are closely associated with chemoresistance and poor survival in lung adenocarcinoma patients
To further investigate the association between CCL2 and chemoresistance in lung adenocarcinoma patients, CCL2 expression in chemosensitive and chemoresistant patients was assessed by immunohistochemical analysis. The level of CCL2 was significantly higher in chemoresistant patients than in chemosensitive patients (P < 0.01, Figure 2A). The association between CCL2 expression and clinicopathologic characteristics is shown in Table 1. In addition, chemosensitive patients with low CCL2 levels had a good overall survival (P = 0.038, Figure 2B). Therefore, we assumed that CCL2 was the key factor promoting chemoresistance in lung adenocarcinoma patients. To verify this further, CCL2 levels in tissues and serum from lung adenocarcinoma patients with chemosensitization and chemoresistance were measured. CCL2 mRNA expression was significantly lower in chemosensitive than in chemoresistant patients (P < 0.01, Figure 2C). CCL2 levels were significantly higher in serum from chemoresistant patients than in that from chemosensitive patients (P < 0.001, Figure 2D). We analyzed the relationship between CCL2 expression and the survival of lung adenocarcinoma patients. Patients with high CCL2 levels had a significantly worse survival rate (P = 0.023, Figure 2E, 2F). Together, these results indicate that CCL2 is a prognostic indicator in lung adenocarcinoma patients, and that it contributes to cisplatin-based chemotherapy resistance.
CCL2 enhances lung adenocarcinoma cell resistance to cisplatin in vitro
Next, we evaluated the effect of CCL2 on the chemoresistance of lung adenocarcinoma cells in vitro. The CCL2 stable knockdown A549/Cis cell line was successfully constructed, and was verified by qPCR analysis of CCL2 mRNA expression. CCL2 mRNA expression was significantly lower in shCCL2 A549/Cis cells than in controls (P < 0.001, Figure 3A). The expression of resistance-related genes was lower in shCCL2 A549/Cis cells than in controls (P < 0.05, Figure 3B). The proliferative ability of shCCL2 A549/Cis cells was significantly reduced compared to controls (P < 0.05, Figure 3C). After cisplatin treatment, shCCL2 A549/Cis cell viability was further reduced (P < 0.01, Figure 3C). Moreover, we found the apoptosis of shCCL2 A549/Cis cells was significantly higher than in the controls (P < 0.05, Figure 3D). The level of apoptosis in shCCL2 A549/Cis cells treated with cisplatin was higher than in those that were not treated with cisplatin (P < 0.01, Figure 3D).
Cancer stem cells are one of the key characteristics of chemoresistance in tumor cells. We therefore investigated the stemness of A549/Cis cells before and after CCL2 knockdown. Sphere-forming efficiency was significantly reduced after CCL2 knockdown in A549/Cis cells (P < 0.001, Figure 3E). Further, CSC-related gene expression was significantly lower in shCCL2 A549/Cis cells than in A549/Cis cells (Figure 3F). These findings demonstrate that CCL2 can enhance the chemoresistance of lung adenocarcinoma cells to cisplatin.
Chemoresistant lung adenocarcinoma cell-derived CCL2 promotes macrophage recruitment and polarization, which further mediates chemoresistance
CCL2 can promote monocyte recruitment to the tumor site, and mediate monocyte differentiation into M2 macrophages [16,17]. We therefore investigated the effect of lung adenocarcinoma cell-derived CCL2 on monocyte recruitment and macrophages polarization. THP-1 cells migrated faster in A549/Cis cell supernatants than in A549 cell supernatants (P < 0.01, Figure 4A). After CCL2-inhibitor treatment, the migration rates of THP-1 cells in the supernatants of both A549/Cis and A549 cells were significantly reduced (P < 0.05, Figure 4A). Thereafter, THP-1 cells were co-cultured with A549/Cis or A549 cells, and the proportions of CD163+CD14+ macrophages (mainly as M2 macrophages) before and after treatment with the CCL2 inhibitor were analyzed by flow cytometry. CD163+CD14+ macrophage frequency was significantly higher following co-culture with A549/Cis cells than following co-culture with A549 cells (P < 0.05, Figure 4B). After CCL2-inhibitor treatment, CD163+CD14+ macrophage significantly reduced relative to its frequency before CCL2-inhibitor treatment (P < 0.05, Figure 4B). The expression of anti-inflammatory cytokines IL-10 and TGF-b was significantly higher in macrophages co-cultured with A549/Cis cells than in those co-cultured with A549 cells (P < 0.05, Figure 4C). However, the expression of pro-inflammatory factors IFN-g and TNF-a in macrophages co-cultured with A549/Cis cells was lower than in macrophages co-cultured with A549 cells (P < 0.05, Figure 4C). The expression of IL-10 and TGF-b was reduced, and that of IFN-g and TNF-a was increased, after CCL2-inhibitor treatment (P < 0.05, Figure 4C). These results suggest that chemoresistant lung adenocarcinoma cell-derived CCL2 promotes recruitment of monocytes and polarization of M2 macrophages.
To further evaluate whether or not M2 macrophages affect lung adenocarcinoma chemoresistance, we investigated the effect of M2 macrophages on tumor cell proliferation, migration, and angiogenesis. A549 cells co-cultured with the supernatants of M2 macrophages polarized by A549/Cis cells had higher cell proliferative ability (Figure 4D), faster migration (Figure 4E), and higher angiogenesis (Figure 4F) than those co-cultured with the supernatants of M2 macrophages polarized by A549 cells and medium alone as a control group. Further, the expression of CCL2 and CD163 in chemoresistant lung adenocarcinoma tissues, assessed by immunohistochemistry, was significantly higher than that in tissues from chemosensitive patients (Figure 4G). Therefore, this indicates that chemoresistant lung adenocarcinoma cell-derived CCL2 promotes monocyte recruitment and polarization into M2 macrophages, which further mediates lung adenocarcinoma chemoresistance.
c-Fos is required for CCL2-enhanced chemoresistance of lung adenocarcinoma
To understand the underlying mechanism of CCL2-mediated chemoresistance, we analyzed the transcription factors in lung adenocarcinoma cells by RT2 profiler PCR array. The c-Fos expression level in A549/Cis cells was elevated compared to that in A549 cells (Figure 5A). To verify this, c-Fos expression in A549/Cis and A549 cells was analyzed by qPCR. The mRNA expression of c-Fos was higher in A549/Cis cells than in A549 cells (P < 0.001, Figure 5B). To further validate the high c-Fos level in resistant lung adenocarcinoma cells, we induced A549 cells to resistant cells with cisplatin treatment. Ninety days after treatment, c-Fos expression was significantly higher in these cisplatin-induced resistant A549 cells than in the controls (P < 0.05, Figure 5C).
Next, to evaluate whether CCL2-enhanced chemoresistance in lung adenocarcinoma cells is mediated by c-Fos, we further analyzed the association between CCL2 and c-Fos in lung adenocarcinoma data obtained from The Cancer Genome Atlas (TCGA) dataset. CCL2 was closely associated with c-Fos (P = 0.015, Figure 5D). A549/Cis cells were then infected with si-c-Fos to inhibit c-Fos expression (Figure 5E), to evaluate the change in CCL2 expression in A549/Cis cells. As expected, after downregulation of c-Fos expression in A549/Cis cells, CCL2 expression was significantly reduced compared to that in the controls (P < 0.001, Figure 5F). Further, A549/Cis cell proliferative ability was attenuated after treatment with si-c-Fos (P < 0.001, Figure 5G). To investigate whether c-Fos physically binds to the promoter region of CCL2, a qCHIP assay was performed using the A549/Cis and A549 cells. The CCL2 promoter region had greater c-Fos enrichment in A549/Cis cells than in the controls (P < 0.05), revealing that c-Fos is indeed located in the CCL2 promoter region in A549/Cis cells (Figure 5H-5J). These findings indicate that the involvement of c-Fos might promote chemoresistance during CCL2 expression in lung adenocarcinoma cells.
CCL2 blockade suppresses tumor progression and restores cisplatin sensitivity in lung adenocarcinoma
To evaluate the in vivo function of CCL2, the shCCL2, and the shRNA controls A549/Cis cells were injected subcutaneously into nude mice, and tumor growth was monitored twice per week. When tumor volumes reached 250 mm3, cisplatin was administrated (3 mg/kg, i.p.) once per week. After two weeks of cisplatin treatment, the mice were sacrificed. Three days before the mice were sacrificed, human CD14+ cells (5 × 106 cells) were transplanted into them by the caudal vein (Figure 6A). We found that tumor volume was dramatically reduced in the shCCL2 group compared to the control group (P < 0.05, Figure 6B). A significant reduction in tumor growth was detected in shCCL2 cell-derived xenografts following cisplatin administration, relative to the control groups, suggesting that shCCL2 A549/Cis cells enhanced chemosensitivity (P < 0.05, Figure 6B). However, after monocyte transfer, the tumor volume of the shCCL2 group treated with cisplatin was unchanged compared to the group without monocyte transfer (Figure 6B). Meanwhile, the final tumor volume results were measured (Figure 6C). Tumor weights were examined after the mice were sacrificed; the results were similar to those for tumor volume (Figure 6D). Last, to evaluate the effect of CCL2 depletion on monocyte recruitment to the tumor site, we investigated GFP-labeled monocyte infiltration in the xenograft tumor tissues, using flow cytometry. CCL2-knockdown significantly reduced macrophage infiltration in xenograft tissues (P < 0.05, Figure 6E). Accordingly, these results imply that CCL2 blockade of suppresses tumor progression and restores cisplatin sensitivity in lung adenocarcinoma.