Tumor cell-derived CCL2 promotes chemoresistance via c-Fos in lung adenocarcinoma

Background The tumor microenvironment plays an important role in promoting chemoresistance in solid cancers. This study aimed to evaluate the underlying molecular mechanisms in the tumor microenvironment potentially involved in chemoresistance in lung adenocarcinoma patients. Methods A multiplex assay was used to determine the transcription factor levels in chemoresistant lung adenocarcinoma cells. The role of chemokine (C-C motif) ligand 2 (CCL2) in chemoresistance of lung adenocarcinoma was evaluated both in vitro and in vivo. A ChIP assay was performed to determine the molecular mechanism by which c-Fos regulates “CCL2”. CCL2 in controlling chemoresistance induced by c-Fos in lung adenocarcinoma. The c-Fos-CCL2 axis is a potential molecular target for lung adenocarcinoma therapy.


Introduction
One of the main causes of treatment failure in cancer patients is resistance to chemotherapy [1,2]. Although there are many studies on the mechanisms underlying anticancer drug resistance, these mechanisms are not fully understood. In recent years, there has been increasing evidence that the tumor microenvironment (TME) plays a key role in the occurrence of chemotherapy resistance in solid tumors [3,4].
Various factors can lead to resistance in the immunosuppressive TME. In metastatic renal cell carcinoma, the higher in ltration of CD4 + FOXP3 + regulatory T cells may be closely related to resistance to antiangiogenic therapy [5]. Tumor-associated macrophage-derived inducible nitric oxide synthase protected tumor cells from chemotherapeutic drug-induced apoptosis [6]. In addition, chemokines, including CXCL12 (C-X-C motif chemokine 12), play an important role in supporting cancer cell survival and promoting drug resistance [7][8][9]. Anti-apoptotic molecules, including IL-6, IL-10, and TNF-α, were involved in promoting the chemoresistance of non-Hodgkin's lymphoma, breast cancer, and glioma [10][11][12][13]. We previously reported the importance of CXCR7 in controlling IL-6-induced cancer-cell proliferation and chemoresistance in esophageal squamous cell carcinoma. Therefore, the underlying molecular mechanisms whereby the TME regulates drug resistance provide potential targets to overcome resistance.
Lung adenocarcinoma is the most common type of lung cancer in non-small-cell lung cancer. Current research suggests that genetic factors, second-hand smoke, and exposure to sputum in the home play key roles in the development of lung cancer, but the speci c reasons remain unclear. Most commonly, treatment for patients with advanced lung adenocarcinoma begins with either cisplatin or carboplatin combined with another medication. It is necessary to reverse drug resistance in order to improve the clinical prognosis of these patients.
Here, we found that the expression levels of chemokine (C-C motif) ligand 2 (CCL2) in cisplatin-resistant A549/Cis cells and tumor tissues of patients with cisplatin resistance were signi cantly higher than in controls. CCL2 expression was closely associated with poor prognosis in lung adenocarcinoma patients, and may therefore serve as a potential biomarker for the prognosis of lung adenocarcinoma. CCL2 enhanced cisplatin-resistance in A549 cells via c-Fos. Moreover, A549/Cis cell-derived CCL2 promoted the recruitment and polarization of monocytes into M2 macrophages, which further induced chemoresistance. This study demonstrates the importance of CCL2 in controlling chemoresistance induced by c-Fos in lung adenocarcinoma. The c-Fos-CCL2 axis is a potential molecular target for lung adenocarcinoma therapy.

Materials And Methods
Patients and tumor samples Eighty-one lung adenocarcinoma tissue samples were obtained from The First A liated Hospital of Zhengzhou University and were used for immunohistochemical analysis. All patient diagnoses were con rmed by pathological analysis and then staged according to the TNM system. The study was approved by the Ethics Committee of our hospital, and all patients signed a written informed-consent form.

Multiplex assay
To identify which factors play key roles in determining and maintaining chemoresistance in lung adenocarcinoma cells, a multiplex assay was used. Cytokine and chemokine levels (for 13 human cytokines and 13 human chemokines) in the supernatants of A549 and A549/Cis cells were analyzed using a Multianalyte Flow Assay Kit (BioLegend, USA), according to the manufacturer's instructions.

RNA extraction and qPCR
Total RNA was extracted from cells and tissues. RNA concentration and purity were then measured using a NanoDrop 2000 (Thermo Scienti c, USA). Total RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, Japan). qPCR was performed using SYBR Premix Ex Taq II (TaKaRa, Japan) in the Agilent Mx3005P system. GAPDH was used as an endogenous control for normalization.

ELISA
The concentration of CCL2 in the supernatants of A549 and A549/Cis cells, and in the supernatants of tissue samples from lung adenocarcinoma patients, was measured by ELISA (R&D Systems Inc., USA), as previously described [14].

Immunohistochemistry
The expression of CCL2 in para n-embedded tissue samples from 81 lung adenocarcinoma patients was examined. The primary antibody (anti-CCL2, 1:200; Abcam, USA) was incubated overnight at 4 °C.
The secondary antibody was incubated at 37 °C for 1 h. The incubated samples were then counterstained with hematoxylin and observed under a microscope (Olympus, Japan). Each sample was selected using three elds of view.

Lentiviral generation and cell sorting
The gene sequence of interest was stably transfected into A549/Cis cells using a lentiviral vector, and the expression of CCL2 was con rmed by qPCR and western blotting. All inserted sequences were con rmed by DNA sequencing. After 48 h, cells expressing green uorescent protein (GFP) were sorted by ow cytometry (MoFlo XDP, Beckman Coulter, USA).

Cell lines and cell culture conditions
Human non-small cell lung cancer cell line A549, cisplatin resistant cell line A549/Cis and HUVEC were purchased from the Institute of basic medicine, Chinese Academy of Medical Sciences. The cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS, Hyclone) at 37 ℃ and 5% CO2. The growth was observed regularly, and the medium was changed and digested for passage. All cells were tested in logarithmic growth phase.

Cell viability assay
Cell proliferation rate was determined by CCK8 assay (Dojindo, Japan) according to the manufacturer's protocol. Cells were seeded in ve replicates in a 96-well plate at a density of 5,000 cells per well. The cells were cultured with 100 μL Dulbecco's Modi ed Eagle Medium (DMEM) containing 10 % FBS. Cells were incubated with 10 μL of CCK-8 for 4 h at 37 °C. Viable cells were counted every day by reading the absorbance at 450 nm with a plate reader (Multiskan MK3, Thermo Scienti c, USA).

Flow cytometrical evaluation of apoptosis
After treatment with cisplatin (also known as DDP), A549/Cis and shCCL2 cells were harvested. Cell concentration was then adjusted to 10 6 cells/mL. Cells were incubated with Alexa Fluor 647 Annexin V (BioLegend, USA) for 15 min in the dark, and samples were analyzed by ow cytometry (FACSCanto II, BD Biosciences, USA) immediately after addition of PI (Sigma, USA).
Migration assay A 0.4 μm diameter chamber (Corning, USA) was used in a transwell assay, whereby 1 × 10 5 THP-1 cells were seeded in the upper chamber, and 600 μL of A549 or A549/Cis cell supernatants were co-cultured in the bottom chamber. CCL2 inhibitor (Selleck, China) was included in these cells. The cells were incubated at 5 % CO 2 and 37 °C for 48 h. The change in the percentage of CD14 + CD163 + cells, and the expression of in ammatory cytokine genes, was measured.
In another migration assay, 5 × 10 4 A549 or A549/Cis cells were seeded in the upper chamber with 600 μL of M2 macrophage supernatant using an 8 μm diameter chamber (Corning, USA). After 24 h incubation, the migrated cells stained with 0.1 % crystal violet were counted. All experiments were repeated independently three times.

Tube-formation assay
Subcon uent human umbilical vein endothelial cells (HUVECs) were harvested resuspended in the A549 or A549/Cis co-cultured with M2 macrophages supernatants. This suspension was seeded (3 × 10 4 cells/well) in BD Matrigel (Becton Dickinson, USA) in a 96-well plate, and incubated for 3 h at 5 % CO 2 and 37 °C. Tube formation was examined under an inverted microscope.

RT 2 pro ler PCR array
Total RNA was isolated from A549 and A549/Cis cells according to the manufacturer's instructions (QIAGEN, Germany). Detailed RT 2 pro ler PCR array experiments were performed as previously described [15].
ChIP and qChIP assay A ChIP assay was performed with 2.5 µg anti-CCL2 and goat anti-rabbit IgG (Cell Signaling Technology, USA) as the negative controls, using a Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, USA) according to the manufacturer's protocol. The following c-Fos primers were used: forward, 5′-AACCTAACGAAAGCTGGGTTG-3′; reverse, 5′-AATGGCATTGTTGAGTGCAT-3′. Detailed ChIP and qChIP assay experiments were performed as previously described [15].

Animal model
Twenty-four 6-week-old female NOD SCID mice (Beijing Vital River Experimental Animal Science and Technology Co., Ltd.) were randomly divided into six groups (four mice/group). The two groups were injected subcutaneously with shRNA control or shCCL2 A549/Cis cells (5 × 10 6 cells resuspended in 100 μL PBS). Tumor growth was assessed by measuring the length and width of the tumor mass with a Vernier caliper. When the tumor volume reached 250 mm 3 , treatment with cisplatin (3 mg/kg once a week, i.p.) was started. Human CD14 + cells (5 × 10 6 cells) were transplanted through the tail vein 3 d before the mice were sacri ced. Three days after transplantation of human CD14 + cells into tail vein, mice were sacri ced by intraperitoneal injection of pentobarbital sodium 150mg / kg. All animal experiments were approved by the Institutional Animal Care and Use Committee of the First A liated Hospital of Zhengzhou University.

Statistical analysis
Data were analyzed using Student's t-tests or chi-square tests, and are reported as means ± SD. The overall survival curve was plotted using the Kaplan-Meier method. Statistical analysis was performed using GraphPad Prism 7 (La Jolla, CA, USA). Differences were considered statistically signi cant at P < 0.05.

CCL2 level is elevated in chemoresistant lung adenocarcinoma cells
To con rm that A549/Cis cells are the chemoresistant lung adenocarcinoma cells, we rst compared the expression of chemoresistance-related genes in A549 (cisplatin-sensitive) and A549/Cis (cisplatinresistant) 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 signi cantly higher in the supernatants of A549/Cis cells than in those of A549 cells ( Figure 1B). The other cytokines and chemokines did not show signi cant differences. To con rm 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 signi cantly 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 signi cantly 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 signi cantly lower in chemosensitive than in chemoresistant patients (P < 0.01, Figure 2C). CCL2 levels were signi cantly 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 signi cantly 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 veri ed by qPCR analysis of CCL2 mRNA expression. CCL2 mRNA expression was signi cantly 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 signi cantly 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 signi cantly 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 e ciency was signi cantly reduced after CCL2 knockdown in A549/Cis cells (P < 0.001, Figure 3E). Further, CSCrelated gene expression was signi cantly lower in shCCL2 A549/Cis cells than in A549/Cis cells ( Figure  3F). These ndings 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 signi cantly 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 ow cytometry. CD163 + CD14 + macrophage frequency was signi cantly 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 signi cantly reduced relative to its frequency before CCL2-inhibitor treatment (P < 0.05, Figure 4B). The expression of antiin ammatory cytokines IL-10 and TGF-b was signi cantly 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-in ammatory 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 signi cantly 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 RT 2 pro ler 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 signi cantly 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 signi cantly 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 ndings 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 mm 3 , cisplatin was administrated (3 mg/kg, i.p.) once per week. After two weeks of cisplatin treatment, the mice were sacri ced. Three days before the mice were sacri ced, human CD14 + cells (5 × 10 6 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 signi cant 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 nal tumor volume results were measured ( Figure 6C). Tumor weights were examined after the mice were sacri ced; 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 in ltration in the xenograft tumor tissues, using ow cytometry. CCL2-knockdown signi cantly reduced macrophage in ltration 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.

Discussion
Non-small-cell lung cancer, of which lung adenocarcinoma is a subtype, accounts for about 80 % of lung cancer cases [18]. The currently used chemotherapeutic or targeted therapeutic drugs inevitably develop drug resistance after a period of application, presenting an obstacle to lung adenocarcinoma therapy.
Cisplatin has been used for decades as rst-line chemotherapy for lung adenocarcinoma. Cisplatin's main drawback is that, following treatment, those cancer cells that evade apoptosis become resistant, leading to poor prognosis [19]. Cancer cells develop cisplatin resistance via various mechanisms, such as increasing cell growth and development, repairing DNA damage, and stimulating endocytosis, although many other factors in the immunosuppressive TME can contribute to chemoresistance. The aim of this study was to explore the molecular mechanisms whereby the TME regulates lung adenocarcinoma chemoresistance, to identify potential targets for overcoming resistance. Our ndings highlight the importance of the role of CCL2 in controlling c-Fos-induced chemoresistance in lung adenocarcinoma. Accordingly, the c-Fos-CCL2 axis might provide a potential molecular target for lung adenocarcinoma therapy.
Chemokines are important in the migration of leukocytes [20,21]. The upregulation of chemokines is closely related to tumor progression, angiogenesis, tumor invasion and metastasis [22][23][24], and there is much experimental evidence that chemokines contribute cancer chemoresistance. B lymphocytes, which can be recruited by CXCL13 into the tumor site, produce lymphotoxins, which promote castration-resistant prostate cancer by activating the IKKα-Bmi1 module in prostate cancer stem cells [25,26]. Steinberg et al. found that myeloid-derived suppressor cells promote BRAF kinase inhibitor (BRAFi) resistance in melanoma cells, by activating the MAPK signaling pathway to induce downstream CCL2 production.
MDSC depletion/blocking (via anti-Gr-1 and CCR2 antagonists) can inhibit the growth of BRAFi-resistant tumors [27]. In addition, Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy in colorectal cancer (CRC). CX3CR1 is essential for the migration of Ly6Clo monocytes to mouse CRC tumors [28]. Similarly, we identi ed a close association between CCL2 and drug resistance: CCL2 enhanced cisplatin-resistance in lung adenocarcinoma cells. Moreover, lung adenocarcinoma cell-derived CCL2 promoted the recruitment and polarization of monocytes into M2 macrophages, which further induced resistance.
Binding of CCL2 to CCR2 promotes the recruitment of monocytes to primary tumors and metastases [29].
In primary sclerosing cholangitis, senescent and BV6-treated human cholangiocytes released monocyte chemoattractant CCL2, which promoted monocyte recruitment to the tissue site [30]. Interestingly, high levels of CCL2 were detected within the follicular lymphoma-cell niche, and CCL2 is overexpressed by follicular lymphoma-mesenchymal stromal cells; this is consistent with the capacity of these cells to recruit monocytes more e ciently than healthy donor-mesenchymal stromal cells [31]. Moreover, in liver disease, the chemokine pathways that involve CCR2 or CCL2 modulate monocyte recruitment or macrophage polarization and differentiation [16]. CCL2 stimulates macrophage proliferation and polarization in multiple myelomas [17,32]. Our ndings are consistent with this: we found that CCL2 promoted monocyte recruitment into tumor sites, and polarization of monocytes into M2 macrophages.
Our results showed that c-Fos might be required in order for CCL2 expression to enhance chemoresistance of lung adenocarcinoma. The association between c-Fos and drug resistance has previously been reported. Overexpression of c-Fos in human ovarian cancer cell lines can lead to cisplatin resistance. In addition, the expression of the c-Fos gene may affect cisplatin sensitivity, and c-Fos antisense oligonucleotide alone, or in combination with cisplatin, can effectively kill parental and cisplatin-resistant ovarian cancer cells [33]. c-Fos increased the expression of P-gp and mdr1 in HEp-2/chemotherapeutic vincristine cells, and enhanced the e ux function of these cells, thereby contributing to the development of multidrug resistance [34]. c-Fos in platinum-resistant cells was up-regulated early in the transcriptional process after 5-FdU-ECyd treatment [35]. For a wide range of leukemias, Kesarwani et al. [36] reported that growth-factor-induced c-Fos expression conferred intrinsic resistance to tyrosinekinase-inhibitor treatment, because the c-Fos expression levels determined the threshold for tyrosine kinase inhibitor e cacy.
Targeting the c-Fos-CCL2 axis in tumors may provide a novel potential therapeutic strategy for controlling lung adenocarcinoma. Li et al. [37] found that CCL2 was overexpressed in human liver cancers and was prognostic for patients with hepatocellular carcinoma. Blockade of CCL2/CCR2 signalling via CCR2 knockout, or using a CCR2 antagonist, inhibited malignant growth and metastasis, reduced postsurgical recurrence, and enhanced survival, suggesting the translational potential of CCL2/CCR2 blockade for treating hepatocellular carcinoma [37]. Moreover, anti-CCL2 antibody is effective in treating mouse hepatocellular carcinoma by blocking the CCL2-CCR2 axis [38]. In a mouse model, DNA vaccines targeting Fos-associated antigen 1 can induce CD8 + T cell-mediated speci c immunity, eradicating spontaneous and experimental lung cancer metastasis; these vaccines are equally effective in breast cancer metastasis models [39]. In the current study, we investigated tumor growth in vitro using si-c-Fos cells and in vivo using shCCL2 cells, and we found that the involvement of c-Fos in CCL2 expression in lung adenocarcinoma cells might promote chemoresistance. Further, we found that CCL2 blockade suppressed tumor progression and restored cisplatin sensitivity in lung adenocarcinoma, suggesting that the c-Fos-CCL2 axis may be a potential therapeutic target for lung adenocarcinoma.

Conclusions
In summary, high levels of CCL2 mediated chemoresistance in lung adenocarcinoma via the action of c-Fos. CCL2 blockade suppressed tumor progression and restored cisplatin sensitivity in lung adenocarcinoma. Therefore, therapeutic strategies that target c-Fos/CCL2 axis could represent an effective approach in lung adenocarcinoma treatment.

Declarations
Ethics approval and consent to participate The research protocol was reviewed and approved by the Ethics Committee of The First A liated Hospital of Zhengzhou University, and written informed consent was obtained from each patient included in the study. All procedures involving animals were performed according to the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care International.

Consent for publication
No con ict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.

Availability of data and materials
The datasets supporting the ndings of this study are included within the article.
Competing interests: The authors con rm that there are no con icts of interest.   Figure 1 The level of CCL2 is elevated in lung adenocarcinoma cells with chemoresistance. A. Resistance-related gene expression levels in A549 and A549/Cis cells were analyzed by qPCR. B. Heatmap showing the concentration of 13 chemokines (pg/mL) in supernatants obtained from A549 and A549/Cis cells measured by multiplex assay. C. CCL2 relative expression in A549 and A549/Cis cells was analyzed by qPCR. D. CCL2 concentration (pg/mL) in supernatants obtained from A549 and A549/Cis cells was measured by ELISA. Results are expressed as means ± SD. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.  treatment was analyzed by CCK8 assay. D. Cell apoptosis of shCCL2 and scramble shRNA A549/Cis cells before and after treatment with cisplatin was evaluated by ow cytometry. A representative analysis from one experiment is shown. E. shCCL2 and scramble shRNA A549/Cis cells were cultured for sphereformation assay. One representative micrograph is shown (200 ×). F. Heatmap showing the expression of stemness-related genes in shCCL2 and scramble shRNA A549/Cis cells. Results are expressed as means ± SD. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; ns = non-signi cant.

Figure 4
Page 21/24 CCL2 derived from A549/Cis cells promotes macrophage recruitment and polarization, which further mediates chemoresistance. A. Cell migration of THP-1 cells co-cultured with the supernatants of A549 or A549/Cis cells before and after CCL2-inhibitor treatment was analyzed by transwell assay. B. The percentage of CD163+CD14+ cells in THP-1 cells co-cultured with the supernatants of A549 or A549/Cis cells before and after CCL2-inhibitor treatment was analyzed by ow cytometry. C. The relative expression of IL-10, TGF-, IFN-, and TNF-in THP-1 cells co-cultured with the supernatants of A549 or A549/Cis cells before and after CCL2-inhibitor treatment was analyzed by qPCR. D. The viability of A549 cells cocultured with the supernatants of M2 macrophages induced by A549 or A549/Cis cells was analyzed by CCK8 assay. E. Cell migration of A549 cells co-cultured with the supernatants of M2 macrophages induced by A549 or A549/Cis cells was analyzed by transwell assay (200 ×). The results are presented as a histogram. F. HUVEC cells co-cultured with the supernatants of M2 macrophages induced by A549 or A549/Cis cells were analyzed by tube-formation assay (200 ×). The results are presented as a histogram.