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Tumor-associated macrophage-derived chemokine ligand 5 regulates the Hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway to promote breast cancer invasion and migration
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Received: ,
Accepted: ,
How to cite this article: Zhu Y, Zhang M, Zhu Z. Tumor-associated macrophage-derived chemokine ligand 5 regulates the Hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway to promote breast cancer invasion and migration. CytoJournal. 2026;23:26. doi: 10.25259/Cytojournal_6_2025
Abstract
Objective:
Tumor-associated macrophages (TAMs) are important components of the breast cancer (BC) microenvironment that contributes to tumor progression by secreting cytokines. Chemokine ligand 5 (CCL5) expression is upregulated in BC, but the specific mechanism of TAM-derived CCL5 in BC microenvironment remains elucidated. This article aims to explore the role of TAM-derived CCL5 in the progression of BC.
Material and Methods:
M2-type TAMs were induced from Tohoku Hospital Pediatrics-1 (THP-1) monocytes. CCL5 expression in TAM cells was evaluated. The biological effects of recombinant or TAM-derived CCL5 on ZR-75-30 cells were examined using proliferation, migration, invasion assays, and epithelial-mesenchymal transition (EMT) markers. CCL5 was silenced in TAMs and hypoxia-inducible factor-1α (HIF-1α) was overexpressed to explore the downstream signaling pathway.
Results:
CCL5 expression was significantly increased in TAMs compared with that in BC cells (P < 0.001). Recombinant CCL5 and TAM-conditioned medium promoted ZR-75-30 cell proliferation, migration, and invasion and EMT (P < 0.01), whereas CCL5 knockdown in TAMs markedly reversed these effects (P < 0.001). EMT-related changes, including decreased E-cadherin expression and increased Vimentin, matrix metalloproteinase (MMP)2, and MMP9 expression levels, were reversed by CCL5 silencing (P < 0.01). Inhibition of CCL5 significantly reduced HIF-1α and vascular endothelial growth factor (VEGF) expression levels (P < 0.001), and these reductions were rescued by HIF-1α overexpression (P < 0.01). Functional rescue experiments confirmed that HIF-1α overexpression restored cell proliferation and invasion and EMT suppressed by CCL5 knockdown. Angiogenesis was suppressed by CCL5 silencing and subsequently restored by HIF-1α overexpression (P < 0.001).
Conclusion:
TAM-derived CCL5 plays an oncogenic role in BC by regulating HIF-1α/VEGF pathway-mediated malignancy and angiogenesis.
Keywords
Breast cancer
Chemokine ligand 5
Hypoxia-inducible factor-1α/vascular endothelial growth factor pathway
Transfer
Tumor-associated macrophages
INTRODUCTION
In China and around the world, the incidence and mortality of breast cancer (BC) are in the forefront of common malignant tumors.[1,2] Although the current clinical managements for BC have been developing and the 5-year survival rate of patients is improved, most patients still suffer from tumor metastasis and recurrence.[3] Tumor metastasis is a multi-step and multi-factor dynamic process involving various interaction networks between cell components and cytokines.[4-6] The formation of tumor microenvironment (TME) can indirectly affect tumor progression due to their interaction and crosstalk.[7] Hence, revealing the specific mechanism of TME in BC is of great value to develop novel treatments and improve patient prognosis.
TME includes various non-malignant stromal cells that play key roles in tumor progression and metastasis.[8] Among them, tumor-associated macrophage (TAM) is the most migratory hematopoietic cell type and the major contributor to tumor metastasis.[9,10] Evidence from clinical and epidemiological studies suggests that the presence of a large number of TAM is strongly associated with poor outcomes in patients with BC.[11,12] Recent studies have revealed that TAM, as an important component of BC microenvironment, can mediate the interaction between TAM and tumor cells through the secretion of cytokines, thereby promoting the proliferation, metastasis, immune response evasion, and drug resistance of BC cells.[13,14] For instance, chemokine ligand 2 (CCL2) secreted by TAM induces tamoxifen resistance in BC cell.[15] TAM promotes epithelialmesenchymal transformation (EMT) and tumor stem cell properties in triple-negative BC through the CCL2/protein kinase B (AKT)/β-catenin pathway[16] These findings suggest that inhibiting BC metastasis by targeting matrix TAMs is a prospective approach.
Chemokines are key secretions derived from TAM that mediate cancer progression and metastasis.[17] The C-C chemokine ligand 5 (CCL5) belongs to the CC-class chemokine family. Current studies have revealed that CCL5 can substantially facilitate tumor growth, metastasis, angiogenesis, and immune escape.[18-20] Certain studies have investigated the corresponding inhibitors against CCL5 or its receptor CCR5, but they have not been applied in clinical practice. Previous studies found that overexpression of CCL5 affected the differentiation of co-cultured cell line THP-1-M2, and CCL5 secreted by BC cells induced polarization of M2 macrophages through activation of the mitogen-activated protein kinase (MEK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway by CCR5. Other studies showed that TAM participates in the immunosuppressive microenvironment and malignant progression of tumors by secreting CCL5.[19,21] However, at present, reports on the progress of TAM-derived CCL5 in BC transfer are few, and the complex functions of TAM-derived CCL5 in BC have not been fully revealed, may be due to previous research techniques.
Hypoxia-inducible factor-1α (HIF-1α) is a transcriptional regulator closely related to angiogenesis, and it plays a crucial role in the hypoxia response of rapidly growing tumors. Previous studies have confirmed that HIF-1α overexpression is associated with BC metastasis and poor clinical prognosis.[22] Studies have shown that the HIF-1α/vascular endothelial growth factor (VEGF) signaling axis participates in the malignant phenotype of BC cells by inducing the angiogenesis pathway.[23] TAM-derived CCL5 promoted hepatocellular carcinoma metastasis by upregulating HIF-1α expression.[24] However, whether TAM-derived CCL5 regulates HIF-1α/VEGF signaling pathway-mediated vascular regeneration and participates in BC metastasis have not been reported yet. Therefore, this study focused on the association of TAM-derived CCL5-mediated-HIF-1α/VEGF signaling in the pathogenesis of BC cell transfer phenotypes and its potential molecular mechanisms.
This study hypothesized that TAM-derived CCL5 may be involved in the transfer phenotype of BC cells through the regulation of HIF-1α/VEGF signaling axis mediating angiogenesis. The present study aimed to deeply explore the specific mechanism of TAM secretion of CCL5 on BC cells through in vitro cell experiments and identify the receptor or signaling pathway mediating TAM-derived CCL5 effect.
MATERIAL AND METHODS
Bioinformatics analysis
The gene expression profiling interactive analysis database (http://gepia.cancer-pku.cn/), which integrates data from the cancer genome atlas and genotype-tissue expression projects, was utilized to evaluate the expression and clinical relevance of CCL5 in BC. The CCL5 expression levels in breast invasive carcinoma (BRCA) and normal breast tissues were analyzed using the “Expression DIY” module. Kaplan–Meier survival analysis for overall survival was performed via the “Survival Analysis” module, using the median expression as the cutoff. The association between CCL5 expression and tumor-node-metastasis (TNM) stage was assessed through the “Stage Plot” module.
Cell culture
Human BC cells ZR-75-30 (IM-H253, Xiamen Immocell Biotechnology Co., Ltd., Fujian, China), normal breast epithelial cells Michigan Cancer Foundation-10A (MCF-10) (IM-H315, Xiamen Immocell Biotechnology Co., Ltd., Fujian, China), THP-1 cells (IM-H260, Xiamen Immocell Biotechnology Co., Ltd.), and human umbilical vein endothelial cells (HUVECs; IM-H205, Xiamen Immocell Biotechnology Co., Ltd., Fujian, China) were inoculated with high-glucose Dulbecco’s modified eagle medium (DMEM) (31053036, Gibco, China) containing 10% fetal bovine serum (FBS) (A5670701, Gibco, China) and 1% double antibody (C0222 penicillin-streptomycin solution, Beyotime, Shanghai, China). They were routinely cultured in a 5% CO2 cell incubator at 37°C. The ZR-75-30 cells at logarithmic growth stage were taken and divided into three groups: control, CCL5 (20 ng/mL), and CCL5 (40 ng/mL). The concentrations of CCL5 (HY-P70450, MedChemExpress, New Jersey, USA) were selected on the basis of the authors’ previous reports.[19,21] Cell line authentication was performed via STR profiling, and mycoplasma contamination was not detected based on routine testing.
Induction and establishment of M2-type macrophages
THP-1 cells at logarithmic growth stage were selected and collected by centrifugation. First, these cells were stimulated with 160 μg/L of phorbol 12-myristate 13-acetate (HY-18739, MedChemExpress, New Jersey, USA) for 24 h to differentiate into M0 macrophages.[25] The cells were then treated with interleukin-4 (IL-4) (20 ng/mL; HYP70445, MedChemExpress, New Jersey, USA) for 24 h to induce polarization towards M2 macrophages.[26] Then, the expression levels of CD14 and CD206 were identified by flow cytometry. For surface marker analysis, the harvested cells were blocked with Human TruStain FcX for 10 min at 4°C to prevent non-specific binding, followed by staining with anti-human CD14 (ab221678, Abcam, Cambridge, UK) and CD206 (ab270647, Abcam, Cambridge, UK) antibodies for 30 min in the dark. The samples were analyzed within 1 h using a BD FACSCanto II flow cytometer equipped with 488 nm fluorescein isothiocyanate/phycoerythrin (FITC/PE) and 633 nm allophycocyanin (APC) excitation lasers. Fluorescence signals were collected through 530/30 (FITC), 585/42 (PE), and 660/20 nm (APC) filters. Compensation matrices were established using single-stained controls, and a minimum of 10,000 events per sample was acquired. FlowJo software (version 10.8.1, BD Biosciences) was used to quantify apoptotic populations (Annexin V+/PI- for early apoptosis; Annexin V+/PI+ for late apoptosis) and surface marker expression levels. Gating strategies excluded debris on the basis of forward/side scatter profiles, and fluorescence-minus-one controls defined positive populations for CD14 and CD206.
Establishment and grouping of co-culture system of M2 macrophages and ZE-75-30 cells
The experiment was divided into seven groups: Control group (no treatment), TAM-CM group (co-culture system treatment), SH-NC-TAM-CM group (sh-NC vector virus transfected M2 macrophages with ZR-75-30 co-culture), SHCCL5-TAM-CM group (sh-CCL5 vector virus transfected M2 macrophages co-cultured with ZR-75-30 co-culture), OE-NC-TAM-CM group (OE-NC vector virus transfected M2 macrophages co-cultured with ZR-75-30), OE-HIF-1α-TAM-CM group (OE-HIF-1α vector virus transfected M2 macrophages co-cultured with ZR-75-30), and sh-CCL5 + OE-HIF-1α-TAM-CM group (sh-CCL5 and OE-HIF-1α vector viruses co-transfected M2 macrophages co-cultured with ZR-75-30).
Three CCL5-specific shRNA sequences were designed on the basis of the human CCL5 coding sequence and cloned into the pLKO.1 lentiviral vector ([Table S1]; Suzhou GenePharma Co., Ltd., China). For HIF-1α overexpression, the full-length coding sequence synthesized by Sangon Biotech (Shanghai, China) was inserted into the pLV-eGFP-N-Puro vector (Suzhou GenePharma Co., Ltd., China). Lentiviral particles were packaged using the ViraPower Packaging Mix (K497500, Thermo Fisher Scientific, Waltham, MA, USA), with sequence fidelity confirmed by restriction digestion (AgeI/EcoRI for pLKO.1; BamHI/XbaI for pLV-eGFP-NPuro) and Sanger sequencing. For cellular transfection, THP-1-derived TAMs and ZR-75-30 cells (1 × 106 cells/well in six-well plates) were transduced at 80% confluence using lentiviral particles complexed with Lipofectamine 3000 following the manufacturer’s protocol (L3000015, Thermo Fisher Scientific, Waltham, MA, USA). After being incubated for 15 min at room temperature, the cells were added with the mixture and incubated for 48 h at 37°C with 5% CO2. The transduced TAMs received either pLKO.1-CCL5 shRNA (shRNA-1/2/3) or control shNC, whereas the ZR-75-30 cells were transfected with pLV-eGFP-N-Puro-OE-HIF-1α.
The ZR-75-30 cells (1 × 106) were inoculated into the lower chamber of a six-well Transwell plate with a 0.4 μm pore size insert to establish the indirect co-culture system. After the cells adhered to the well, 1 × 106 M2 macrophages were seeded into the upper chamber for 48 h. The 0.4 μm pore size was specifically chosen to allow soluble factors (such as cytokines and chemokines) to diffuse between compartments while preventing direct cell-to-cell contact, thereby ensuring that the observed effects were mediated exclusively by paracrine signaling rather than direct interaction. Following 48 h of incubation, the ZR-75-30 cells from the lower chamber were collected for subsequent experiments.
Enzyme-linked immunosorbent assay (ELISA)
After each group of cells was treated, the cell culture supernatant was collected, and the CCL5 content was detected using a CCL5 ELISA kit (HB2330-Hu, Shanghai Hengyuan Biological Co., Ltd., Shanghai, China). In brief, samples were added to pre-coated wells along with standards and incubated to allow binding. After the samples were washed, they were added with a biotinylated antibody and incubated, followed by the introduction of horseradish peroxidase (HRP)-conjugated streptavidin. Additional washing steps were performed before tetramethylbenzidine substrate was added, and the color reaction was stopped with a stop solution. Absorbance at 450 nm was measured, and the concentration of the target protein in the samples was determined by comparing their absorbance values to a standard curve.
Cell counting kit-8 (CCK-8) experiment
The cells were inoculated into 96-well plates at a density of 2000 cells per well, with six replicate wells per group. The plates were incubated at 37°C in a 5% CO2 atmosphere for 4–8 h to allow cell attachment. After the cells adhered, they were treated with 20 or 40 ng/mL of CCL5 for 24 h or treated with TAM-conditioned medium (CM) in accordance with the experimental design. After 24 h of incubation, 10 μL of CCK-8 reagent (BA00208, Bioss, Beijing, China) was added to each well 2 h before the incubation ended. The absorbance at 450 nm was measured using a microplate reader (RT-6000, Rayto, Shenzhen, China).
Wound healing assay
Cells were seeded into six-well plates at a density of 1 × 106 cells per well. When the cell monolayer reached about 90% confluence, a linear scratch was introduced across each well using the tip of a sterile pipette, aligned perpendicular to a pre-marked reference line. Images of the wound area were captured at 0 and 24 h using an inverted microscope (BZX810, KEYENCE, Osaka, Japan). The percentage of wound closure was calculated as: [(scratch area at 0 h - scratch area at 24 h)/scratch area at 0 h] × 100%.
Transwell experiment
Cell invasion capacity was measured by Transwell assay in plates coated with Matrigel (8 μm, 3422, CORNING, Maine, USA). ZR-75-30 cells (2 × 105) suspended in serum-free medium were inoculated in the upper chamber. The medium supplemented with 10% FBS was placed in the lower chamber. After incubation was performed for 12–24 h, the invaded cells were fixed, stained, and counted using an inverted microscope (BZ-X810, KEYENCE, Osaka, Japan).
Real-time quantitative PCR (RT-qPCR) analysis
Total RNA was extracted from cells using the TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions. The concentration and purity of the RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Reverse transcription was performed using the PrimeScript RT Reagent Kit (Takara, Japan) to synthesize complementary DNA. RT-qPCR was conducted using the SYBR Green PCR Master Mix (Takara, Japan) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems, USA). The thermal cycling conditions were set as follows: Initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, and relative gene expression was calculated using the 2^−ΔΔCt method. The primer sequences were designed by Sangon Biotech (Shanghai, China) in accordance with previous reports, as shown in Table S2.[27]
Western blot (WB) analysis
Cell proteins were collected and extracted with radio immunoprecipitation assay lysis buffer (C5029, Bioss, Beijing, China). Protein concentration was determined by BCA kits (P0009, Beyotime Biotechnology, Shanghai, China). Total protein samples of 50 μg were taken for each group, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, wet transferred to nitrate cellulose filter membrane, sealed with 5% skim milk for 2 h, washed with team-based simulation training (TBST, ST671-500 mL, Beyotime, Shanghai, China), added with mono-antibody diluent, and incubated at 4°C overnight. The second antibody was added after TBST washing the next day and incubated at room temperature for 2 h. After TBST film washing for 3 times and enhanced chemiluminescence color rendering, image information was collected in a gel imaging system (1658000 Mini-PROTEAN Tetra Handcast Systems, Bio-rad, California, USA), and the gray value of the strip was measured. The following primary antibodies were used: rabbit anti-e-cadherin antibody (1:1000, abs130068, absin, Shanghai, China), mouse anti-vimentin monoclonal antibody (1:1000, abs149750, absin, Shanghai, China), matrix metalloproteinase (MMP)2 polyclonal antibody (1:1000, 10373-2-AP, Proteintech, Hubei, China), MMP9 (N-terminal) polyclonal antibody (1:1000, 10375-2-AP, Proteintech, Hubei, China), HIF-1 alpha polyclonal antibody (1:1000, 20960-1-AP, Proteintech, Hubei, China), polyclonal antibody for VEGFA (1:1000, 19003-1-AP, Proteintech, Hubei, China), and CCL5 polyclonal antibody for CCL5 (1:1000, 12000-1-AP, Proteintech, Hubei, China). Then, the following secondary antibodies were incubated to the membranes: HRP-labeled goat anti-rabbit IgG (H + L; 1:1000, A0208, Beyotime, Shanghai, China) and HRP-labeled goat anti-Mouse IgG (H + L; 1:1000, A0216, Beyotime, Shanghai, China). GAPDH was used as the internal reference protein to normalize the expression levels of target proteins across all WB analyses. The primary antibody against GAPDH was purchased from Proteintech (Cat No. 60004-1-Ig, 1:1000 dilution, Proteintech, Hubei, China). Protein bands were analyzed with ImageJ software (version 1.53k; National Institutes of Health, USA).
HUVEC tube formation detection
The treated ZR-75-30 cells were inoculated with 5 × 105 cells/well in the upper chamber of a 12-well Transwell plate (0.4 μm model), whereas HUVECs were digested and inoculated with 5 × 105/well in the lower chamber for 48 h. Subsequently, the HUVECs were collected and incubated on extracellular matrix gel for 4 h, and the blood vessel formation was observed and photographed with an inverted microscope (BZ-X810, KEYENCE, Osaka, Japan). Tube formation was quantified using ImageJ software (version 1.53, NIH, USA) with the Angiogenesis Analyzer plugin. The number of nodes, the junctions, and total tube length were measured from three randomly selected fields per well.
Data analysis
Data were analyzed and plotted using GraphPad Prism 9 (version 9.4.0), and the diagram was collated. All data were expressed as means ± standard deviation. Unpaired Student’s t-test was used for comparisons between two groups. For multiple group comparisons, one-way analysis of variance followed by Tukey’s post hoc test was applied to determine statistical significance. P < 0.05 was considered as significant.
RESULTS
Abnormally high expression of CCL5 in BC
The expression of CCL5 in BC was analyzed through the bioinformatics website to clarify the expression pattern of CCL5 in BC. As exhibited in Figure 1a-c, CCL5 was abnormally high expressed in BRCA (P < 0.05), and patients with high levels of CCL5 had worse long-term survival, but CCL5 was not associated with the TNM stage of patients. Subsequently, a higher expression of CCL5 in human BC cell line (ZR-75-30) than in normal breast epithelial cells (MCF-10) was detected (P < 0.001, [Figure 1d]). These data suggest that CCL5 may be a cancer-promoting factor in BC.
- Analysis of CCL5 expression and clinical relevance in breast cancer. (a) CCL5 expression in breast invasive carcinoma versus normal breast tissues analyzed through gene expression profiling interactive analysis database (http://gepia.cancer-pku.cn/index.html); n (T) = 1085, n (N) = 291. (b) Kaplan–Meier overall survival curves comparing patients with BC with high and low CCL5 expression; n (high) = 535, n (low) = 535. (c) Violin plot showing CCL5 expression across different TNM stages of BC. (d) ELISA-based quantification of CCL5 secretion in MCF-10 and ZR-75-30 cells; n = 3. ✶P < 0.05, ✶✶✶P < 0.001. CCL5: Chemokine ligand 5; BC: Breast cancer, ELISA: Enzyme-linked immunosorbent assay.
CCL5 promotion of EMT and metastasis of ZR-75-30 cells
ZR-75-30 cells were treated with 0 (control), 20, and 40 ng/mL of recombinant CCL5 to further elucidate the biological function of CCL5 in BC cells. The CCK-8 assay demonstrated that 20 and 40 ng/mL of CCL5 significantly enhanced cell viability compared with control (P < 0.001), with a further increase observed at 40 ng/mL (P < 0.001 vs. 20 ng/mL, [Figure 2a]). In the wound healing assay, the relative migration rate was significantly increased in the CCL5-treated groups (P < 0.01, [Figure 2b]). Similarly, the Transwell invasion assays revealed a dose-dependent increase in invasive capacity (P < 0.05 for 20 ng/mL; P < 0.001 for 40 ng/mL, [Figure 2c]). Compared with 20 ng/mL of CCL5, 40 ng/mL of CCL5 pronouncedly enhanced the viability, migration, and invasion of ZR-75-30 cells (P < 0.001).
- Effects of 20 or 40 ng/mL CCL5 on proliferation, migration, invasion, and EMT-related protein expression in ZR-75-30 cells. (a) Cell viability evaluated by CCK-8 assay after treatment with different concentrations of CCL5. (b) Wound healing assay for assessing cell migration at 0 and 24 h (Scale bar: 50 μm, ×20). (c) Transwell assay for assessing cell invasion under varying CCL5 concentrations (Scale bar: 50 μm, ×20). (d-h) Western blot analysis and quantification of EMT markers (E-cadherin and vimentin) and metastasis-associated proteins (MMP2 and MMP9). ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001; n = 3. CCL5: Chemokine ligand 5; EMT: Epithelial-mesenchymal transition, MMP2: Matrix metalloproteinase-2, MMP9: Matrix metalloproteinase-9.
At the molecular level, CCL5 treatment downregulated the epithelial marker E-cadherin (P < 0.05) and upregulated the mesenchymal marker vimentin (P < 0.05 and P < 0.001 for 20 ng/mL; both P < 0.001 for 40 ng/mL, [Figure 2d-f]), indicating enhanced EMT. The expression levels of MMP2 and MMP9, two critical regulators of tumor cell invasion, significantly increased after CCL5 stimulation (both P < 0.001 for 20 ng/mL; both P < 0.001 for 40 ng/mL, [Figure 2g and 2h]). Moreover, 40 ng/mL of CCL5 exhibited significant effects compared with 20 ng/mL of CCL5 in regulating these proteins (P < 0.01 for E-cadherin, P < 0.01 for vimentin, P < 0.05 for MMP2, and P < 0.001 for MMP9).
These findings demonstrate that CCL5 promotes ZR-75-30 cell proliferation, migration, and invasion and EMT in a dose-dependent and statistically significant manner.
TAM-derived CCL5 promotion of metastasis and EMT process of ZR-75-30 cells
Previous studies have shown that TAM can induce tumorigenesis by secreting CCL5 to regulate TME.[28] In the subsequent experiments, M2-type TAMs were generated through IL-4-induced polarization of THP-1-derived macrophages. The successful induction of M2 phenotype was confirmed by the increased expression levels of M2 markers CD14 and CD206, as assessed by flow cytometry [Figure S1], ensuring the reliability of downstream co-culture experiments involving TAMs.
The CCL5 secretion in normal breast epithelial cells (MCF-10), BC cells (ZR-75-30), M0 macrophages, and M2-polarized TAMs was assessed using ELISA to investigate whether TAM-derived CCL5 contributes to the malignant progression of BC. As expected, the CCL5 level in BC cells markedly increased (P < 0.001, [Figure 3a]). The CCL5 levels in M0 and M2 macrophages significantly increased compared with that in ZR-75-30 cells, with M2 macrophages exhibiting the highest CCL5 concentration (all P < 0.001, [Figure 3a]), suggesting a potential role of TAM-derived CCL5 in promoting BC aggressiveness.
- Effects of TAM-derived CCL5 on ZR-75-30 cell proliferation, migration, and invasion. (a) CCL5 secretion in MCF-10, ZR-75-30, M0, and M2-polarized macrophages assessed by ELISA. (b and c) Confirmation of CCL5 knockdown efficiency in TAMs by Western blot and RT-qPCR. (d) Cell viability of ZR-75-30 cells following co-culture with different TAM-conditioned media, as assessed by CCK-8 assay. (e) Cell migration evaluated by wound healing assay (Scale bar: 50 μm, ×20). (f) Invasive capacity analyzed using Transwell assay (Scale bar: 50 μm, ×20). ✶P < 0.05, ✶✶P < 0.01, and ✶✶✶P < 0.001; n = 3. CCL5: Chemokine ligand 5; TAM: Tumor-associated macrophage, ELISA: Enzyme-linked immunosorbent assay.
M2-polarized TAMs were transduced with either control (sh-NC) or CCL5-silencing (sh-CCL5) lentiviral vectors and co-cultured with ZR-75-30 cells using an indirect Transwell system. Efficient knockdown of CCL5 was confirmed by WB and RT-qPCR analyses (P < 0.001, [Figure 3b and c]). The ZR-75-30 cells co-cultured with TAM-CM showed significantly increased cell viability (P < 0.01), migratory capacity (P < 0.05), and invasive potential (P < 0.01, [Figure 3d-f]), compared with the control cells. However, these effects were markedly reversed when TAMs were silenced for CCL5 expression (P < 0.001 for viability, migration, and invasion, [Figure 3d-f]), indicating that the pro-metastatic influence of TAMs is largely dependent on CCL5.
Compared with control, the expression of E-cadherin in ZR-75-30 cells co-cultured with TAM-CM significantly decreased, whereas that of vimentin significantly increased (P < 0.01, [Figure 4a-c]). The expression levels of MMP2 and MMP9 in the TAM-CM and sh-NC-TAM-CM groups markedly increased compared with that in the control group (P < 0.001). These effects were significantly attenuated after co-culturing BC cells with CCL5-silencing TAMs (P < 0.001 for E-cadherin, P < 0.01 for vimentin, P < 0.001 for MMP2, and P < 0.001 for MMP9; [Figure 4a-e]).
- Effect of TAM-derived CCL5 on EMT- and invasion-related protein expression in ZR-75-30 cells. (a) Representative Western blot images of E-cadherin, vimentin, MMP2, and MMP9 following treatment with different TAM-conditioned media. (b-e) Quantification of protein expression levels normalized to GAPDH. ✶P < 0.05, ✶✶P < 0.01, and ✶✶✶P < 0.001; n = 3. CCL5: Chemokine ligand 5; TAM: Tumor-associated macrophage, EMT: Epithelial-mesenchymal transition.
These results confirm that CCL5 is a key functional chemokine secreted by TAMs, mediating their ability to promote the proliferation, EMT, and invasive behavior of BC cells. Silencing CCL5 in TAMs effectively attenuates these malignant phenotypes in ZR-75-30 cells.
TAM-derived CCL5 promotion of metastasis, EMT process, and angiogenesis of ZR-75-30 cells by regulating HIF-1α/VEGF pathway
Given the existing evidence that CCL5 may influence tumor angiogenesis through hypoxia-related pathways, this work hypothesized that TAM-derived CCL5 may regulate the HIF-1α/VEGF signaling axis. A series of overexpression and knockdown experiments was performed. As shown in Figure 5a and b, the transfection of OE-HIF-1α into TAMs significantly increased the HIF-1α mRNA expression (P < 0.001), confirming successful overexpression. Conversely, CCL5 knockdown through sh-CCL5 markedly suppressed the HIF-1α mRNA levels (P < 0.001), an effect that was partially reversed by co-transfection with OE-HIF-1α (P < 0.05). This result indicates that CCL5 likely functions upstream of HIF-1α. Notably, manipulation of HIF-1α expression had no significant effect on CCL5 mRNA levels, supporting the unidirectional regulatory relationship.
- Effects of TAM-derived CCL5 and HIF-1α on proliferation, migration, and invasion of ZR-75-30 cells. (a and b) mRNA expression levels of HIF-1α and CCL5 in ZR-75-30 cells co-cultured with TAMs transduced with sh-CCL5 or OE-HIF-1α, as assessed by RT-qPCR. (c-e) Western blot and quantification of HIF-1α and VEGF protein levels. (f) Cell viability measured by CCK-8 assay. (g) Migration assessed by wound healing assay (Scale bar: 50 μm, ×20). (h) Cell invasion evaluated using Transwell assay (Scale bar: 50 μm, ×20). ✶P < 0.05, ✶✶P < 0.01, and ✶✶✶P < 0.001; n = 3. TAM: Tumor-associated macrophage, CCL5: Chemokine ligand 5, HIF-1α: Hypoxia-inducible factor-1α, VEGF: Vascular endothelial growth factor.
WB analysis further confirmed that silencing CCL5 in TAMs reduced the protein levels of HIF-1α and VEGF (P < 0.001, [Figure 5c-e), and OE-HIF-1α restored the expression despite CCL5 knockdown (P < 0.001 for HIF-1α; P < 0.01 for VEGF, [Figure 5c and e]). These results suggest that TAM-derived CCL5 promotes BC malignancy, at least in part, through activation of the HIF-1α/VEGF signaling pathway.
A rescue experiment was performed using exogenous HIF-1α overexpression to further validate the functional relevance of this pathway. As expected, OE-HIF-1α significantly enhanced ZR-75-30 cell proliferation, migration, and invasion (all P < 0.001), and this effect was significantly ameliorated by sh-CCL5 co-transfection (P < 0.01, [Figure 5f-h]). OE-HIF-1α abolished the positive effects of CCL5 knockdown on the malignant phenotypes, including EMT reversal, as evidenced by increased vimentin and MMP2/9 expression levels and decreased E-cadherin expression in the sh-CCL5 + OE-HIF-1α group (all P < 0.001, [Figure 6a-e]). The angiogenesis assessed by HUVEC tube formation was significantly suppressed by CCL5 knockdown and subsequently rescued by HIF-1α overexpression [Figure 6f], providing strong evidence that CCL5 promotes angiogenesis through HIF-1α signaling.
- Regulation of EMT and angiogenesis in ZR-75-30 cells by TAM-derived CCL5 via HIF-1α/VEGF pathway. (a) Western blot analysis of EMT-related (E-cadherin and vimentin) and metastasis-related (MMP2 and MMP9) protein levels in ZR-75-30 cells co-cultured with differentially modified TAMs. (b-e) Quantification of protein expression normalized to GAPDH. (f) Angiogenic activity assessed by HUVEC tube formation assay (Scale bar: 50 μm, ×20). ✶P < 0.05, ✶✶P < 0.01, and ✶✶✶P < 0.001; n = 3. TAM: Tumor-associated macrophage, CCL5: Chemokine ligand 5, EMT: Epithelial-mesenchymal transition, HUVEC: Human umbilical vein endothelial cells.
These findings demonstrate that TAM-derived CCL5 contributes to BC cell EMT, metastasis, and angiogenesis by activating the HIF-1α/VEGF axis, and targeting this pathway may offer therapeutic potential.
DISCUSSION
Studies have revealed that the TME in solid tumors is composed of malignant tumor cells and some non-malignant mesenchymal cells.[8] Among these non-malignant cells, macrophages play a crucial role in promoting tumor migration, invasion, and neovascularization.[9,10] On the one hand, infiltrating TAM in TME can lead to tumorigenic inflammation, which plays an important role in tumorigenesis. On the other hand, TAM can express chemokines and cytokines, thus promoting the formation of immunosuppressive TME. A notable detail that current studies have confirmed that TAM infiltrates BC tissues and is related to poor prognosis in patients.[11,12] Recent studies found that TAM can promote the progression of certain solid tumors by secreting CCL5.[19,21] However, the specific mechanism of action of TAM-derived CCL5 in BC TME has not been fully expounded. Here, the antitumor function of TAM-derived CCL5 in BC was investigated primarily by inhibiting cancer progression and angiogenesis. For the 1st time, the importance of TAM-derived CCL5 in regulating HIF-1α/VEGF signaling axle-mediated angiogenesis to regulate BC development was demonstrated.
Chemokines have long been associated with cancer, and they play a key role in coordinating the recruitment and localization of white blood cells.[29] Among the many chemokines, the present study focused on CCL5 due to its increased expression in BC and its association with poor prognosis in patients.[30] The results confirmed that CCL5 can promote the proliferation, migration, and invasion of BC cells. Increasing evidence suggests that researchers should understand not only the regulatory mechanisms of malignant phenotypes, such as tumor cell proliferation, migration, and invasion but also the complex interactions between tumor cells and their microenvironment.[31,32] In the process of tumor development, tumor cells can be transformed into mesenchymal cells through EMT, making tumor cells obtain increased invasion and migration ability.
E-cadherin is the main molecule of intercellular adhesion, and it plays an important role in maintaining cell polarity and tissue integrity. Vimentin is an intermediate filament protein that is mainly found in mesenchymal cells. Decreased E-cadherin-mediated intercellular adhesion and increased vimentin-mediated plasticity of tumor cells led to tumor cells being able to cross the basement membrane, making the cells highly invasive and able to migrate.[33,34] Therefore, the downregulation of E-cadherin and the upregulation of vimentin are considered to be the signature features of EMT. Here, CCL5 or TAM intervention downregulated E-cadherin and upregulated vimentin, suggesting that CCL5 is a participant in EMT and metastatic processes in BC cells. TAM in BC is currently believed to have the characteristics of M2-type macrophages, manifested as promoting the growth, invasion, and metastasis of tumor cells.[13] CCL5 is produced by various stromal cells, including TAM, in TME under various stimuli, which may affect the aggressiveness and metastasis potential of cancer cells.[19,21] To further clarify the anti-tumor function of TAM-derived CCL5 in BC, here we first induced TAM and observed its effect on the biological function of BC cells by inhibiting the secretion of CCL5 in TAM. The inhibition of CCL5 secretion in TAM inhibited the transfer phenotype and EMT process of ZR-75-30 cells. These results indicate that TAM is a participant in the EMT and metastasis process in BC cells, and CCL5 is a key chemokine secreted by TAM.
In the past decade, angiogenesis has been associated with benign and malignant transformation of tumors.[35] Rapid growth of tumor tissue leads to local hypoxia, which activates HIF-1α expression. The activation of HIF-1α prompts tumor cells to secrete various angiogenic factors, which can stimulate new angiogenesis and provide the tumor with sufficient oxygen and nutrients, thereby maintaining tumor growth and survival.[36,37] In this process, HIF-1α is most closely related to tumor angiogenesis. Therefore, by targeting the HIF-1α/VEGF signaling axis, tumor cells can escape the restriction of hypoxia and obtain more nutrient supply by promoting angiogenesis, thus promoting further tumor growth and spread. TAM-derived CCL5 can upregulate HIF-1α expression and participate in the progression of other malignancies.[38] The present study found that TAM-derived CCL5 can play an active role by activating the HIF-1α/VEGF pathway. TAM-derived CCL5 can also promote the malignant phenotype of BC cells by regulating HIF-1α/VEGF pathway-mediated angiogenesis. This finding suggests that TAM-derived CCL5 can promote the formation of blood vessels by regulating the HIF-1α/VEGF pathway, thus providing a prerequisite for tumor cell invasion and metastasis. Beyond this pathway, previous studies suggest that CCL5 may interact with other key oncogenic signaling cascades, such as NF-κB[39] and MEK/STAT3,[40] which have been implicated in tumor progression and immune modulation.[41] Notably, NF-κB activation can lead to increased HIF-1α transcription, potentially linking CCL5 to hypoxia-independent regulation of angiogenesis.[42] Given the complexity of TME, CCL5 possibly cooperates with multiple pathways to create a pro-tumorigenic niche. Future studies should focus on elucidating whether CCL5 functions synergistically with other inflammatory cytokines to enhance HIF-1α stability and VEGF-mediated angiogenesis. In vivo models could further validate the therapeutic potential of targeting CCL5 to inhibit tumor angiogenesis and metastasis.
This study has some limitations. First, it was conducted in BC cell lines, which need to be validated at the animal level. In addition, TAM’s involvement in tumor metastasis is a complex physiological process involving various pathological mechanisms, including tumor immune escape and energy metabolism. TAM-derived CCL5 regulates the HIF-1α/VEGF-mediated angiogenesis signaling pathway, which is only one of the pathways and cannot fully cover all pathological mechanisms.
SUMMARY
The results reveal that CCL5 is a TAM-induced BC metastasis cytokine, and its mechanism may be related to the HIF-1α/VEGF-mediated angiogenesis pathway. This research provides a theoretical foundation for further research on the clinical application of TAM targeted therapy for BC metastasis.
ACKNOWLEDGMENT
The authors express their appreciation to all the staff involved in this study.
AVAILABILITY OF DATA AND MATERIALS
The datasets used and analyzed during the present study are available from the corresponding author on reasonable request.
ABBREVIATIONS
BC: Breast cancer
CCK-8: Cell Counting Kit-8
CCL5: Chemokine ligand 5
ELISA: Enzyme-linked immunosorbent assay
EMT: Epithelial-mesenchymal transition
FBS: Fetal bovine serum
GEPIA: Gene Expression Profiling Interactive Analysis
GTEx: Genotype-Tissue Expression
HIF-1α: Hypoxia-inducible factor 1 alpha
HUVECs: Human umbilical vein endothelial cells
IL-4: Interleukin-4
MMP2: Matrix metalloproteinase-2
MMP9: Matrix metalloproteinase-9
RT-qPCR: Real-time quantitative polymerase chain reaction
STAT3: Signal transducer and activator of transcription 3
TAMs: Tumor-associated macrophages
TCGA: The Cancer Genome Atlas
TME: Tumor microenvironment
TNM: Tumor-node-metastasis
VEGF: Vascular endothelial growth factor
WB: Western blot
AUTHOR CONTRIBUTIONS
YYZ and ZZZ: Concepted and designed the research. MXZ: Acquired the data. YYZ and ZZZ: Analyzed and interpreted data. YYZ, MXZ, and MXZ: Drafted the manuscript. ZZZ: Revised manuscript for important intellectual content. All authors contributed to this present work. All authors read and approved the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors gave final approval of the version to be published. All authors meet ICMJE authorship requirements.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
This research did not involve human or animal studies, and therefore, ethics committee approval and consent to participate are not required.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
EDITORIAL/PEER REVIEW
To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double-blind model (authors are blinded for reviewers and vice versa) through an automatic online system.
Supplementary material available at
FUNDING: The present study is supported by the Scientific Research Program of Shanghai Pudong New Area Health Commission (the General Program) (PW2023A-18) and the Academic Leaders Training Program of Shanghai Pudong New Area Health Commission (PWRd2023-10) from Yongyun Zhu.
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