A375 melanoma-derived lactate controls A375 melanoma phenotypes by inducing macrophage M2 polarization via TCA cycle and TGF-β signaling

Cell lines and cell cultures

The human melanoma cell line A375 (Cat#CL-0014; Procell, Miami, China) was kindly provided by Procell Life Science & Technology Co., Ltd. The human melanocyte cell line PIG1 was obtained from Amrican Type Culture Collection (ATCC). A375 and PIG1 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (HyClone, Logan, UT, USA) in an incubator with 37 °C, 5% CO₂. All cell lines were used within 10–50 passages.

PBMCs separation

Written informed consent was obtained from all subjects prior to their participation in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Peking University Third Hospital (IRB00006761–M2022199). PBMCs were separated by density gradient centrifugation using human peripheral blood lymphocyte isolation fluid (Cat#7111011; DAKEWE, Shenzhen, China). Fresh anticoagulated whole blood was diluted with an equal volume of phosphate-buffered saline (PBS). The separation liquid was added to the centrifuge tube. The volume of separation fluid, anticoagulated undiluted whole blood, and PBS is 1:1:1. The mixed blood was then centrifuged at 800 g in a horizontal rotor for 30 min at room temperature. A thin and dense white film, known as mononuclear cells (including lymphocytes and mononuclear cells), formed between the plasma layer and the separation fluid layer. The white film layer was carefully transferred to another centrifuge tube and diluted with PBS and mixed by inverting. The mixture was centrifuged at 250 g for 10 min. Subsequently, 80 ul buffer and 20 ul CD14 microbeads were added to the cell pellet. After incubation at 4 °C for 15 min, PBMCs were separated. The separated PBMCs were prepared for the following experiments.

Conditioned medium preparation

PIG1 and A375 cells were seeded into 6 cm dishes at a density of 2,000,000 cells per dish in 6 ml DMEM and cultured for 48 h. The conditioned medium was collected (namely PIG1-CM or A375-CM) and centrifuged at 800 g for 5 min to remove detached cells and cellular debris. Processed conditioned medium (PIG1-CM or A375-CM) was mixed with RIPM medium containing 10% FBS at a ratio of 1:1 to incubate PBMCs for 48 h, and old media were replaced with fresh culture media to incubate cells for another 48 h. The above conditioned medium (termed PIG1-M-CM or A375-M-CM) was collected and centrifuged to remove cellular debris. The acquired conditioned medium was stored at −80 °C for future experiments.

Optimal oxygen level determination

Firstly, 4,000 A375 cells per well were seeded into 96-well plates and cultured in a hypoxic incubator (HERAcell 150i; Thermo Fisher Scientific, Waltham, Massachusetts, US) with oxygen concentrations of 1%, 2%, 3%, 4%, 5%, and 21% for 48 h. After removing the old media, 100 µl of DMEM and 10 µl of CCK-8 (Cat# C0038, Beyotime, Shanghai and Nantong, China) were added to each well. The plates were then incubated for an additional 2 h at 37 °C. Cell proliferation was assessed by measuring the optical density (OD) at 450 nm using a spectrophotometric plate reader.

Lactate content determination

Lactate was determined with lactate assay kit (Cat#BC2235; Solarbio, Beijing, China). Briefly, 3,000 cells were seeded into 96-well plates with 100 μL of DMEM. After incubation for 36 h, the cellular supernatant was collected, and lactate levels in PIG1-CM and A375-CM were measured according to the specifications provided by the kit The absorbances at 570 nm were recorded on a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) to quantify lactate production.

Cell viability

Trypan blue dye (0.4% Trypan Blue solution; Solarbio, Beijing, China) was used to assess cell viability according to the manufacturer’s instructions. Briefly, A375 cells and PBMCs were seeded into six-well culture plates and cultured under varying O₂ levels (1%, 2%, 3%, 4%, 5%, and 21%) or with conditioned media containing 2-deoxyglucose (2-DG) or AA5 for 24 h, as per experimental requirements. After cell collection, viable and dead cells were counted under a microscope (Leica, Wetzlar, Hesse, Germany). Cell viability was calculated using the following formula: Viable cells/(Viable cells + Dead cells).

Western blot

Cells were lysed in RIPA lysis buffer (Solarbio, Beijing, China), which contained a phosphatase and protease inhibitor cocktail (Solarbio, Beijing, China). The cell lysates were centrifuged, and protein content was quantified using a bicinchoninic acid (BCA) Protein Assay kit (Beyotime, Shanghai and Nantong, China). Twenty-five micrograms of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes with 0.45 μm pores (Millipore, MA, USA). After blocking with 5% bovine serum albumin (BSA) for 1 h at room temperature, the membranes were incubated with primary antibodies (HIF1α,1:1,000, CST; β-actin,1:3,000, CST) overnight at 4 °C, followed by three washes. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, washed with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST). Protein bands were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Amersham, United Kingdom). Densitometric analysis was performed with ImageJ software, and relative protein levels were normalized to β-actin.

Flow cytometry

Flow cytometry was performed to evaluate M1/M2 marker expression. PBMCs were incubated in conditioned medium that contained AZD3965 (Cat#S7339; Selleck), lactate (Cat#E4473; Selleck, Washington, USA), 2-deoxy-D-glucose (2DG) (S4701; Selleck) or AA5 (Cat#ab144194; Abcam, Cambridge, USA) as experimental requirements. Cells were collected after centrifugation at 500 g for 5 min. Cell pellets were incubated with Human TruStain FcXTM (Fc Receptor Blocking Solution) (Cat#422301; Biolegend, San Diego, CA, USA) for 10 min at room temperature to block Fc receptors on human cells. To fix and permeabilize cells, they were incubated with Cyto-Fast™ Fix/Perm Solution (Cat#426803; Biolegend, San Diego, CA, USA) for 30 min at 4 °C. Cells were stained with Brilliant Violet 421™ anti-human CD206 (Cat#321126; Biolegend, San Diego, CA, USA), PE anti-human CD68 (Cat#333808; Biolegend, San Diego, CA, USA) and PerCP/Cyanine5.5 anti-human ARG1 (Cat #369710; Biolegend, San Diego, CA, USA) for 40 min at 4 °C in the dark. Fluorescence intensities of different antibody were determined with a flow cytometer (BD, Franklin Lakes, New Jersey, USA).

ECAR and OCR analysis

ECAR and OCR were analyzed using the XF96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA), following the protocol described in our previous study (Wang et al., 2021, 2023). Briefly, to begin with, PBMCs were treated with different levels of 2-Deoxy-D-glucose (2-DG) or Atpenin A5 (AA5). 4 × 10⁴ PIG1, A375 or PBMCs underwent with different treatments were seeded into a well of seahorse cell culture plate and cultured for 24 h. The Seahorse XF stress test assay medium was prepared in accordance with the protocol for glycolysis and mitochondrial stress tests. Cells were washed three times with the conditioned medium, and the final volume in each well was 175 μl before the seahorse assay. For ECAR determination, cells were treated with sequential injections of glucose (10 mM), oligomycin (2 μM) and 2DG (50 mM). While for OCR measurement, oligomycin (2 μM), FCCP (2 μM) and rotenone/antimycin A (1 μM) were loaded sequentially. ECAR and OCR are reported as mpH/min and pmol/min, respectively.

ELISA

TNF-α and TGF-β concentrations were detected by TNF-α ELISA kit (Cat#Z10020761; CUSABIO, Houston, USA) and TGF-β ELISA kit (Cat#Z09020760; CUSABIO, Houston, USA), respectively. Samples were prepared as experimental requirements. The measurement was performed according to user manual. Briefly, 100 μl standard or sample was added to each well and incubate 2 h. The liquid was removed. A total of 100 ul Biotin-antibody (1x) was added to each well and incubate 1 h. After aspiration and wash 3 times, 100 μl HRP-avidin (1x) was added to each well and incubate 1 h. 90 μl TMB substrate was added to each well and incubate 30 min at 37 °C in the light. Finally, 50 μl stop solution was added to each well and read at 450 nm within 5 min.

Proliferation assays

Proliferation was evaluated with Cell Counting Kit-8 (CCK8) (Cat#CK04, DOJINDO, Kumamoto, Japan). Briefly, 4 × 10³ cells were seeded into 96-well culture plates and cultured in conditioned medium, such as PIG1-M-CM and A375-M-CM, for 48 h. After removing cell supernatant with a pipette, 100 μl fresh DMEM stock solution and 10 μl CCK8 reagent were added to each well. After incubation at 37 °C for 30 min in the dark, the optical density (OD) values at 450 nm were recorded on a microplate reader. Three replicates of each assay were performed.

Clone formation of A375

Each well of six-well plates was seeded with 1,000 A375 cells and incubated with different conditioned media such as PIG1-M-CM, A375-M-CM etc for 2 weeks. The media were changed every 3 days and cell status was observed under microscope. Cells were washed twice with PBS and fixed with pure methanol for 15 min, followed by staining with 0.1% crystal violet (Cat#G1063; Solarbio, Beijing, China) for 15 min. The dyeing liquid was slowly washed away with running water and pictures were taken with a digital camera. The clone numbers were counted.

Migration and invasion assays of A375

Migration was evaluated using transwell chambers with 8.0 μM pores (Cat#3422; Corning, NY, USA). Briefly, A375 cells were cultured with different conditioned media. A total of 50,000 pretreated cells were seeded into the upper compartment with 200 μl DMEM, and the lower compartment was filled with 500 μl complete medium containing 10% FBS. Cells were cultured for 24 h. Migrated cells were fixed with 4% paraformaldehyde (Cat#1110; Solarbio, Beijing, China) for 15 min. Cells in the upper chambers were removed with cotton swabs. And then migrated cells were stained with 0.1% crystal violet for 15 min. Five random fields were chosen to count the number of migrated cells using a microscope (100 × magnification).

The invasion assay in our study differed slightly from the migration assay. Matrigel was diluted in DMEM at a volume ratio of 1:6. A total of 40 μl of diluted Matrigel (Cat#356234; BD) was added to the upper chamber. Subsequently, 50,000 pretreated cells were seeded into the upper chamber and cultured for 36 h. The remaining steps were performed identically to those performed in the migration assay.

Apoptosis assay

A375 apoptosis levels were detected with an Annexin V-PE/PI apoptosis kit (Cat#KGA1030; KeyGEN, Nanjing, China) following the manufacturer’s instructions. Briefly, 200,000 A375 cells were seeded into six-well plates and treated with different conditioned media for 48 h. Cells were collected using 0.25% trypsin without ethylenediaminetetraacetic acid (EDTA) and then resuspended in 50 μl binding buffer along with 5 μl propidium iodide (PI). The cell suspension was incubated in the dark for 15 min. Subsequently, 450 μl binding buffer and 1 μl Annexin V-PE were added to the suspension, which was then incubated for an additional 15 min in the dark. Finally, apoptosis levels were analyzed by flow cytometry (BD).

Statistical analysis

Statistical analyses of all data were performed with SPSS (version 26.0; SPSS Inc.). Data are shown as mean ± standard deviation. Student’s t-test was performed to analyze differences between two groups, and one-way ANOVA was used to analyze intergroup differences with a Bonferroni multiple comparison post-test. Experiment data represent at least three sample replicates. The level of statistical significance was p < 0.05.

A375-CM induces macrophage M2 polarization

To investigate whether melanoma influences M1/M2 polarization, PBMCs were cultured with PIG1-CM or A375-CM. Flow cytometry results showed that the expression of the M1 macrophage surface marker CD68 (Figs. 1A, 1B) was reduced in PBMCs treated with A375-CM compared to those incubated with PIG1-CM. In contrast, the levels of the M2 macrophage marker CD206 (Figs. 1C, 1D) and arginase 1 (ARG1) (Figs. 1E, 1F) were elevated in PBMCs co-cultured with A375-CM compared to those cultured with PIG1-CM or normal medium (control group). ELISA analysis revealed that the levels of TNF-α (indicative of M1 polarization) in A375-CM did not differ from those in the control and PIG1-CM groups (Fig. 1G). However, the secretion of TGF-β (indicative of M2 polarization) from PBMCs treated with A375-CM was significantly higher than that observed in the PIG1-CM group and the control group (Fig. 1H). These findings collectively suggest that A375-CM promotes M2 polarization.

Hypoxia enhances macrophage M2 polarization induced by A375-CM

A375 and PBMCs were cultured under various hypoxic conditions. Proliferation results (Fig. S1A) indicated that hypoxia (1%, 2%, and 3% O₂) promoted A375 proliferation. However, cell viability assessments (Figs. S1B, S1C) revealed that 1% and 2% O₂ levels significantly inhibited PBMC viability. Consequently, 3% O₂ was chosen as the optimal condition for further studies. Our results demonstrated that hypoxia suppressed the expression of the M1 marker CD68 (Figs. 2A, 2B) in PBMCs treated with either PIG1-CM or A375-CM, with a more pronounced effect observed in the A375-CM group. In contrast, expressions of M2 markers, including CD206 (Figs. 2C, 2D) and arginase 1 (ARG1) (Figs. 2E, 2F), were elevated in PBMCs co-cultured with A375-CM following hypoxia treatment. In addition, cytokine secretions are also modulated by hypoxia. In PBMCs co-cultured with A375-CM under hypoxic conditions, TNF-α levels (Fig. 2G) remained unchanged, while TGF-β levels (Fig. 2H) increased. Collectively, these findings suggest that hypoxia reinforces M2 polarization induced by A375-CM.

M2 polarization of macrophage induced by A375-CM is regulated by lactate

Firstly, optimal concentrations of lactate and AZD3965 were determined by evaluating CD206 expression in PBMCs (Figs. S2A–S2D). The lactate production from A375 was found to be higher than that from PIG1 (Fig. S2E). To explore whether lactate in A375-CM alters macrophage polarization, the MCT1 inhibitor AZD3965 was utilized to block lactate transport into cells. In this study, flow cytometric analysis revealed that CD68 expression in PBMCs co-cultured with control medium or PIG1-CM decreased upon the addition of lactate (Figs. 3A, 3B). In contrast, CD68 expression in PBMCs co-cultured with A375-CM increased following treatment with AZD3965 (Figs. 3A, 3B). Furthermore, expressions of CD206 (Figs. 3C, 3D) and ARG1 (Figs. 3E, 3F) in PBMCs exhibited opposite trends. Their expressions increased in the control or PIG1-CM groups after lactate treatment, but decreased when cells cultured with A375-CM were treated with AZD3965. While TNF-α secretion was unaffected by treatment with lactate or the MCT1 inhibitor (Fig. 3G), TGF-β secretion (Fig. 3H) from PBMCs increased upon the addition of lactate to PIG1-CM, whereas it decreased following the addition of AZD3965 to A375-CM.

TCA cycle instead of glycolysis is in involved in macrophage M2 polarization induced by lactate

Melanoma is characterized by the Warburg effect. The glycolysis and mitochondrial respiration of A375 cells were analyzed using a Seahorse analyzer. The results indicated that ECAR of A375 was higher than that of PIG1 (Fig. 4A), while OCR of A375 was lower compared to PIG1 (Fig. 4B). To assess the inhibitory effects on PBMCs, the glycolysis inhibitor 2-DG and the mitochondrial respiration inhibitor AA5 were utilized to evaluate their impacts on ECAR (Fig. 4C) and OCR (Fig. 4D), respectively. Concentrations of 1 mM for 2-DG and 1 μM for AA5 were employed for further studies. Additionally, we blocked glycolysis and the TCA cycle to analyze their roles in lactate-induced M2 polarization. Our findings showed that glycolysis inhibition did not affect the expression of CD68, CD206 and ARG1, nor did it alter TGF-β and TNF-α secretion. In brief, glycolysis did not influence macrophage polarization induced by A375-CM. Interestingly, blockage of TCA cycle enhanced the expression of M1 markers, such as CD68 (Figs. 4E, 4F), while inhibiting the expression of M2 markers (CD206 and ARG1) (Figs. 4G–4J). Furthermore, the results of cytokine TGF-β (Fig. 4L) supported these conclusions. Collectively, these results demonstrate that lactate induces M2 polarization via TCA cycle.

M2 macrophages promote melanoma proliferation, cell cycle progression, clone formation, invasion and inhibit apoptosis

To investigate the impact of polarized macrophages on A375 phenotypes, PIG1-M-CM and A375-M-CM were first prepared to culture A375 cells. CCK8 assays indicated that A375-M-CM significantly promoted the proliferation of A375 compared to PIG1-M-CM (Fig. 5A). Consistently, A375-M-CM markedly promoted A375 clone formation (Figs. 5B, 5C) and increased the cell ratio in the G2/M phase (Figs. 5D, 5E). These findings suggest that polarized macrophages induced by A375-CM promote A375 growth. Additionally, flow cytometry analysis demonstrated that the apoptotic level of A375 cells was inhibited following treatment with A375-M-CM (Figs. 5F, 5G). Transwell assays revealed that co-culturing A375 cells with A375-M-CM resulted in enhanced migration (Figs. 5H, 5I) and invasion (Figs. 5J, 5K).

Altered melanoma phenotypes by M2 macrophages are modulated by TCA and TGF-β secretion

Our findings indicate that polarized macrophages have an impact on various melanoma phenotypes. However, the specific mechanism underlying these alterations requires further investigation. Herein, we explored whether TCA cycle exerts a role in melanoma phenotype alterations induced by polarized macrophages. We observed that A375-M-TCA inhibitor-CM decreased A375 proliferation, as detected by CCK8 test (Fig. 6A). In addition, TCA cycle inhibitor of macrophages inhibited A375 clone formation (Figs. 6B, 6C) and cell cycle progression (Figs. 6D, 6E), and promoted apoptosis (Figs. 6F, 6G). Moreover, the migration (Figs. 6H, 6I) and invasion (Figs. 6J, 6K) of A375 were attenuated upon TCA cycle inhibition in polarized macrophages. Despite these observed alterations in melanoma phenotypes the specific mediators involved in the interaction between these two cell types remain unclear. Our above findings showed TCA cycle enhances macrophage M2 polarization induced by lactate and promoted TGF-β secretion. Therefore, we hypothesize that the interaction between polarized macrophages and melanoma cells is mediated through TGF-β. Notably, the elimination of TGF-β from A375-M-CM through the use of neutralizing antibodies resulted in reduced A375 proliferation (Fig. 6A). The similar results were observed in clone formation (Figs. 6B, 6C) and cell cycle progression (Figs. 6D, 6E), migration (Figs. 6H, 6I) and invasion (Figs. 6J, 6K). In contrast, the level of apoptosis in A375 cells increased following the removal of TGF-β from the conditioned medium.