2.1 TR-FRET Screening Assay for CD147–MCT1 Interaction Inhibitors

Since the interaction between CD147 and MCT1 plays pivotal roles in metabolic regulation and the microenvironment, we utilized the TR-FRET technique, which is sensitive, stable, and efficient, to screen specific inhibitors (Figure 1A). CD147 and MCT1 were overexpressed in 293T cells (Figure 1B), and TR-FRET signals were detected to evaluate their interaction. We used diluted protein at various concentrations to determine the optimal protein concentration for subsequent screening experiments (Figure 1C). We subsequently evaluated 276 selected compounds from the Food and Drug Administration (FDA)-approved drug library using the TR-FRET screening system to identify inhibitors capable of effectively disrupting CD147–MCT1 complex formation (Figure 1D). This rigorous screening process led to the identification of two promising candidate compounds, crizotinib (Figure 1E) and axitinib, both of which significantly reduced the TR-FRET signal, indicating their potential as inhibitors of the CD147–MCT1 interaction. To further validate the inhibitory effects of both compounds, coimmunoprecipitation was performed to assess the CD147–MCT1 interaction in the presence of crizotinib or axitinib. Notably, crizotinib exhibited greater efficacy than did axitinib, substantially blocking the formation of the CD147–MCT1 complex (Figure S1A). To determine whether crizotinib contributes directly to the inhibition of the CD147–MCT1 interaction, pulldown assays were employed, and the interaction of crizotinib with overexpressed CD147 and MCT1 is depicted in Figure 1F,G, confirming its potential as a CD147–MCT1 inhibitor. Quantitative analysis of the pulldown results is shown in Figures 1H,I. Consistent with the results obtained in 293T cells, crizotinib interacted directly with endogenous CD147 and MCT1 in melanoma cells (Figure 1J). Moreover, by performing Cell Counting Kit-8 (CCK-8) assays to assess the impact of crizotinib on melanoma cell proliferation, we found that crizotinib significantly inhibited melanoma cell growth (Figure 1K–N). In addition, crizotinib had a significantly lower inhibitory effect on normal cells (Figure S1B), further underscoring its potential as a therapeutic agent. Together, these results indicate that crizotinib is a novel PPI that blocks the CD147–MCT1 interaction.

2.2 Crizotinib Inhibits the Invasion and Metastasis of Melanoma and Disrupts the Membrane Localization of MCT1

Melanoma is a highly aggressive and lethal form of skin cancer, and its initiation and progression are closely associated with invasion and metastasis. To evaluate the antimetastatic potential of crizotinib, both wound healing and Transwell assays were performed. As a result, untreated control cells exhibited significant wound closure after 48 h, whereas crizotinib-treated cells demonstrated dose-dependent inhibition of migration (Figure 2A,B). Consistent with these findings, Transwell assays revealed a marked reduction in the number of cells that penetrated the Matrigel-coated membranes following crizotinib treatment (Figure 2C,D).

Considering the importance of lactate metabolism in tumor progression, we hypothesized that crizotinib could obstruct the CD147–MCT1 interaction and influence MCT1 localization on the cell membrane, thereby affecting lactate transport in melanoma cells. To further confirm our hypothesis, cells were exposed to varying doses of crizotinib, and membrane proteins were isolated and visualized via western blotting, which revealed that MCT1 expression on the cell membrane decreased with increasing crizotinib concentration (Figure 2E,F). Consequently, crizotinib treatment led to a decrease in extracellular lactate levels, which was consistent with the downregulation of MCT1 expression (Figure 2G). In addition, we knocked down CD147 and MCT1 in the cells. This resulted in reduced expression of both proteins on the membrane (Figure S1C,D) and decreased the extracellular concentration of lactate (Figure S1E), which is consistent with the effects noted following crizotinib treatment. These findings demonstrate that crizotinib effectively impairs melanoma cell invasion and metastasis, which may account for the blockade of lactate transport via disruption of MCT1 membrane localization.

2.3 Crizotinib Improves the Therapeutic Efficacy of PD-1 Blockade by Modulating Macrophage Polarization and Promoting CD8⁺ T-Cell Responses

Lactate, a key metabolic by-product, is known to impair antitumor immunity [24]. To evaluate the impact of crizotinib on immunotherapy efficacy, we administered an anti-PD-1 mAb to immune-competent mice bearing B16-F10 melanoma tumors. Our experiments demonstrated significant inhibition of tumor growth with monotherapy via either crizotinib or PD-1 immune checkpoint blockade (Figure 3A–C). Notably, the combined treatment further decreased the tumor volume and weight, indicating a potential synergistic effect between the two modalities. To explore the downstream mechanism by which crizotinib contributes to immunotherapy, flow cytometry analysis was performed. Interestingly, combined treatment with crizotinib and the anti-PD-1 mAb resulted in no significant changes in the overall macrophage population (Figure 3D), whereas substantial alterations in macrophage polarizations were observed, in which the proportion of anti-inflammatory and antitumor M1 macrophages notably increased (Figure 3E), whereas the subpopulation of M2 macrophages associated with immunosuppression and tumor promotion decreased (Figure 3F,H). Moreover, the proportion of IFN-γ⁺ CD8⁺ T cells, indicative of enhanced cytotoxic activity, was significantly elevated in the combination group (Figure 3G). This immunomodulatory effect of crizotinib was further validated in the YUMM1.7 melanoma model, where combination treatment similarly reduced M2 macrophage populations and enhanced IFN-γ⁺ CD8⁺ T-cell responses (Figure S2). These findings indicate that crizotinib improved the therapeutic efficacy of PD-1 blockade by modulating macrophage polarization and promoting CD8⁺ T-cell effector function.

2.4 Crizotinib Inhibits Macrophage Lactate Uptake and Promotes M2 Polarization

Although crizotinib affects lactate secretion in melanoma cells, several studies have suggested that MCT1 mediates lactate transport in a concentration gradient-dependent manner. Given the potential differences in lactate transport direction between macrophages and melanoma cells, we investigated the effects of crizotinib on lactate uptake and polarization in macrophages. Pull-down assays confirmed the interaction of crizotinib with CD147 and MCT1 in macrophages (Figure 4A). To assess the effect of crizotinib on MCT1 expression, membrane proteins were extracted from THP-1 cells treated with varying concentrations of crizotinib (0–1000 nM). Immunoblotting of the membrane fractions revealed a dose-dependent decrease in MCT1 expression (Figure 4B). We subsequently treated THP-1 cells with a medium containing 10 mM lactate and assessed changes in the lactate concentration to evaluate the influence of crizotinib on lactate transport. The results revealed that the lactate concentration in the control group was approximately 6.93 mM, whereas the lactate concentration in the crizotinib-treated macrophages was significantly higher, at approximately 8.60 mM (Figure 4C), suggesting that lactate uptake by crizotinib-treated THP-1 cells was inhibited. There was no significant change in the intracellular lactate concentration (Figure 4D), possibly because lactate is absorbed and subsequently used for synthesis or metabolism, thus maintaining intracellular homeostasis and preventing excessive accumulation of lactate.

Extensive research has demonstrated that lactate facilitates the M2 polarization of macrophages [6, 9, 10]. Therefore, we investigated the impact of lactate and crizotinib on macrophage polarization. Consistent with previous findings, lactate treatment significantly increased the expression of arginase 1 (ARG1) and CD206, whereas cotreatment with crizotinib markedly reduced the expression of M2 polarization markers (Figure 4E). Similar trends were observed when macrophages were treated with conditioned media (CM) collected from tumor cells, which contain higher lactate concentrations (Figure 4F–H). Additionally, we observed comparable results in mouse macrophage cell lines (Figure S3). Together, these results demonstrated that crizotinib inhibited M2 polarization of macrophages by hindering lactate uptake.

2.5 Lactate Promotes M2 Polarization of Macrophages and Increases CXCL13 Secretion

To investigate the influence of macrophages on melanoma, THP-1 cells were polarized into M2-like macrophages under previously described conditions, and the CM was collected (Figure 5A). We then assessed the effect of macrophage CM on melanoma cells. The results revealed that CM derived from lactate-treated macrophages did not significantly affect tumor cell proliferation (Figure 5B) but significantly increased the invasiveness of melanoma cells (Figure 5C,D). This proinvasive effect was attenuated when macrophages were cotreated with crizotinib, indicating that lactate-induced M2-like macrophages can promote melanoma cell invasion. Given that crizotinib could enhance the effectiveness of PD-1 treatment in mice with tumors, we hypothesized that changes in macrophage polarization status might affect the capacity of tumor-killing immune cells. By coculturing peripheral blood mononuclear cells (PBMCs) with tumor cells in CM from different macrophages, we observed that lactate-treated macrophage CM inhibited the PBMC-mediated killing of melanoma cells (Figure 5E).

To investigate the underlying mechanism by which lactate-induced macrophages affect immune cell-mediated killing of tumor cells, transcriptomic sequencing was conducted, which revealed that lactate induced widespread changes in gene expression in macrophages (Figure 5F, Figure S4A,B). We selected genes whose expression was upregulated in the lactate-treated group but whose expression was not significantly changed in the lactate- and crizotinib-treated groups for further study. These genes represent lactate-induced alterations that crizotinib could effectively intervene in. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the differentially expressed genes (DEGs) revealed that lactate-induced gene changes were involved mainly in cytokine‒cytokine receptor interactions, chemokine signaling, and cytokine activity pathways, with gene set enrichment analysis (GSEA) confirming these findings (Figure 5G,H, Figure S4C,D). Among these DEGs, significant changes were observed in the C-X-C motif chemokine ligand (CXCL) family, with CXCL13 showing the greatest fold change following crizotinib treatment (Figure S3E). Further validation through RT‒PCR confirmed that CXCL13 transcription in macrophages was upregulated under lactate induction and downregulated following crizotinib treatment, which was consistent with the transcriptomic sequencing results (Figure 5F, Figure S4F–H). Additionally, we observed a lactate concentration-dependent change in CXCL13 expression (Figure S4I). These findings suggest that lactate induces macrophage polarization toward the M2 phenotype and may promote tumor progression and increase CXCL13 secretion.

2.6 CXCL13 Promotes the Invasion and Metastasis of Melanoma Cells and Enhances Their Immune Evasion

Given the cancer-promoting role of M2 macrophages, we investigated the impact of CXCL13 on melanoma cells. CXCL13 treatment had no effect on melanoma cell proliferation (Figure 6A), but it markedly enhanced the invasion and metastatic potential of melanoma cells in a concentration-dependent manner (Figure 6B–E). Furthermore, in vitro cytotoxicity assays revealed that the inhibitory effect of CXCL13 on melanoma cells in PBMCs could be reversed, with even 50 ng/mL CXCL13 completely reversing the PBMC-mediated killing of melanoma cells (Figure 6F,G). To further understand how CXCL13 enhances the invasiveness and immune evasion capabilities of melanoma cells, we examined the protein levels in melanoma cells after CXCL13 treatment and observed a significant upregulation of matrix metallopeptidase 9 (MMP9) and programmed death-ligand 1 (PD-L1) (Figure 6H). These findings suggest that CXCL13 facilitates both the invasion and metastasis of melanoma cells, as well as their ability to evade immune surveillance, through the regulation of MMP9 and PD-L1 expression.

2.7 Lactate Promotes CXCL13 Expression in Macrophages via H3K18la-Mediated Histone Lactylation

Recent studies have revealed that lactate induces histone lactylation, an important epigenetic modification that influences chromatin structure, transcriptional regulation, and gene expression [11]. Based on these findings, we investigated whether histone lactylation was the mechanism underlying CXCL13 secretion by lactate-treated macrophages. Immunoblots revealed a significant increase in histone lactylation in macrophages after lactate treatment, which was reversed after crizotinib treatment (Figure 7A,B). Histone lactylation occurs at multiple sites, of which histone H3 lysine 18 (H3K18) is the most widely studied, and is commonly associated with the regulation of gene expression. Thus, we focused on changes in the expression levels at H3K18. Consistent with the overall level of histone lactylation, lactate treatment upregulated H3K18 lactylation (H3K18la), which was also attenuated by crizotinib (Figure 7A,C).

To further investigate the impact of histone lactylation on CXCL13 expression, we utilized the GSE156675 dataset to predict potential histone binding sites within the CXCL13 promoter region and designed primers based on peak locations (Figure 7D,E). Chromatin immunoprecipitation (ChIP) and DNA gel electrophoresis confirmed that lactate significantly enhanced histone binding to the CXCL13 promoter in macrophages (Figure 7F, Figure S5). Together, these results suggest that lactate promotes CXCL13 expression in macrophages via histone lactylation of H3K18la.

5.1 Cell Lines and Culture Conditions

SK-MEL-5, SK-MEL-28, THP-1, and RAW264.7 cells were obtained from our laboratory. For culturing, SK-MEL-5, SK-MEL-28, and RAW264.7 cells were cultured in complete Dulbecco's modified Eagle medium (DMEM) (Gibco, C11995500BT, USA), while THP-1 cells were grown in complete Roswell Park Memorial Institute (RPMI) 1640 (Gibco, C11875500BT, USA), with both media supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (ExCell Bio, 12J262, China). The cells were incubated at 37°C in a humidified environment with 5% CO₂.

5.2 TR-FRET Assay

pcDNA3.2/V5-DEST (Invitrogen, 12489019, USA) was used to construct Venus-Flag-CD147 and NLuc-HA-MCT1. HindIII and NheI restriction endonucleases were utilized to cleave the DNA strands, followed by ligation of the corresponding tag sequences. To express the CD147 and MCT1 proteins, 293T cells were transfected with Venus-Flag-CD147 and NLuc-HA-MCT1, followed by 48 h of culture. Subsequently, cell lysis was performed at 4°C using 0.5% NP-40 lysis buffer (Beyotime, China). After high-speed centrifugation, the supernatant was transferred to sterile ependorf tubes (EP tubes). Next, 384-well plates (Corning, 3573, USA) containing anti-Flag-Tb (Cisbio, 61FG2TLF, France) and anti-HA-d2 (Cisbio, 610HADAF, France) TR-FRET antibodies were prepared. The TR-FRET antibody mixture was prepared at 2X concentration using a TR-FRET buffer containing 50 mM Tris-HCl, 20 mM NaCl, and 0.01% NP-40, adjusted to pH 7.0. The library compounds (DMSO as a negative control), cell lysate, and 2X TR-FRET antibody mixture (in a 1:1 volume ratio) were sequentially added to the 384-well plate, followed by low-speed centrifugation (1000 rpm, 2 min) and overnight incubation at 4°C. TR-FRET signals were measured using a Cytation1 (BioTek, USA), and signal calculations were performed using the following formula: F_{665 nm}/F_{615 nm} × 10⁴.

5.3 Pull-Down Assay

Beads (Cytiva, 17043001, USA) were prepared and suspended in 1 mM HCl. The suspension was incubated in a rotary mixer for 5 min, followed by careful removal of the supernatant. This process was repeated three times. Finally, the supernatant was discarded. Crizotinib was prepared in a coupling buffer composed of 0.5 M NaCl and 0.1 M NaHCO₃, adjusted to pH 8.3. Excess ligand was removed by washing with coupling buffer. The coupling solution containing the ligand was added to the beads and mixed thoroughly at 4°C overnight. The beads were transferred alternately into a solution of 1 M acetic acid (pH 4.0) and a second solution containing 0.5 M NaCl and 0.1 M Tris-HCl (pH 8.0) and incubated in a rotary mixer for 5 min, and this process was repeated three times. Finally, the beads were rinsed in phosphate buffered saline (PBS), and after the supernatant was discarded, the beads were stored at 4°C in PBS with 0.01% sodium azide.

5.4 Histone Extraction and Western Blotting

Histones were prepared using a Histone Extraction Kit (Active Motif, 40028, USA). The protein samples were subsequently separated via 12% SDS-PAGE. The membranes were blocked with 5% milk and incubated overnight at 4°C with either pan-Kla (PTM BIO, PTM-1401RM, China) or anti-H3K18la (PTM BIO PTM-1427RM, China). Immunoreactive signals were detected using the ChemiDoc XRS+ System (Bio-Rad, USA).

5.5 Cell Proliferation Assays

The cells were seeded at a density of 2000 cells per well in 96-well plates and treated with crizotinib or the control. Then 10 µL of CCK-8 reagent (Selleck, B34302, USA) was added, followed by incubation at 37°C for 2 h. Absorbance was measured using a Cytation1 instrument (BioTek, USA), and the data were analyzed using Prism 8.

5.6 Wound-healing and Transwell Assays

For the wound-healing assay, cells were seeded into six-well plates. A wound was generated with a sterile 10 µL pipette tip. The cells were washed twice with PBS before treatment under the indicated conditions. The cells were then subjected to the indicated conditions, and images of the wounded areas were captured at 0, 24, and 48 h.

For the Transwell assay, a 24-well Transwell system (Corning, #3422, USA) was used. The cells were resuspended at 5 × 10⁵ cells/mL in serum-free DMEM, and 100 µL of this suspension was added to each Matrigel-coated insert. The lower chamber was filled with complete DMEM, and the plates were incubated at 37°C with 5% CO₂. After incubation, noninvasive cells were removed, fixed with 4% formaldehyde, and stained with crystal violet. Image analysis was performed using ImageJ, and data and statistical analyses were conducted using Prism 8.

5.7 Mouse Models

Female C57BL/6 mice (6–8 weeks, Shanghai Laboratory Animals Center [SLAC], China) were used to establish the model. Approximately 5 × 10⁵ B16-F10 cells were subcutaneously implanted into the right flanks of the mice. A YUMM1.7 melanoma model was established under the same conditions. When the tumor volume reached 50–100 mm³, the mice were randomly assigned to different groups. The mice were treated with crizotinib (25 mg/kg orally, MCE, HY-50878A, USA) or/and an anti-PD-1 mAb (200 µg/mouse, Bio X Cell, BE0146, USA). Crizotinib was administered every 2 days, and PD-1 was administered two times per week. The subcutaneous tumor size and animal weights were measured, and tumor volumes were calculated using the following formula: (length × width × height) × 0.5. The mice were euthanized, and the tumor tissue was harvested for analysis. This study was approved by the Ethics Committee of Xiangya Hospital (Central South University, China).

5.8 Flow Cytometry

Flow cytometry analysis was conducted on murine tumor tissues. Single-cell suspensions were obtained by filtering through a 70-µm cell strainer (Falcon, 352350, USA). For surface staining, the cells were incubated with fluorochrome-conjugated antibodies specific to the target surface markers for approximately 30 min at 4°C in the dark. For intracellular staining, the cells were first restimulated for 30 min using the eBioscience Intracellular Fixation & Permeabilization Buffer Set (Invitrogen, USA), followed by incubation with antibodies against intracellular targets under the same conditions (4°C, in the dark, for 30 min). Analysis was performed using a FACS LSRFortessa instrument (BD Biosciences, USA).

5.9 Lactate Measurement

The lactate concentration was measured using a lactate colorimetric assay kit (Nanjing Jiancheng Bioengineering Institute, A019-2, China). Lactate was converted to a chromogenic product and quantified spectrophotometrically at 530 nm.

5.10 RNA Sequencing and Data Processing

Total RNA was extracted and quality-checked before being submitted to BGI Genomics (Shenzhen, China) for transcriptome sequencing. Library preparation and sequencing were performed using the DNBSEQ platform according to the manufacturer's protocols. Clean reads were then aligned to the human reference genome (Homo sapiens, GCF_000001405.39_GRCh38.p13, downloaded from NCBI) using HISAT2. The RNA-seq data generated in this study have been deposited in the CNCB-NGDC under accession number HRA011699.

5.11 Coculture of CXCL13 and PBMCs

PBMCs were isolated from healthy human blood samples and cocultured with tumor cells at a ratio of 5:1, with or without the addition of CXCL13. At the specified time points, the remaining cell populations were assessed to observe the impact of CXCL13 on the cytotoxic function of PBMCs.

5.12 ChIP Assays

ChIP assays were performed following the manufacturer's protocol (CST, 9003, USA). Cells were cross-linked using 1% formaldehyde and subjected to sonication three times (20 s on, followed by 30 s of incubation on wet ice between each sonication). The sonicated chromatin was subsequently clarified via centrifugation. The cross-linked chromatin was then diluted with 1X ChIP buffer for each reaction, and H3K18la antibodies (PTM BIO, PTM-1427RM, China) were added to the supernatant. The control groups were treated with histone H3 (CST, 4620, USA) or rabbit IgG (CST, 2729, USA). Protein G magnetic beads were resuspended and added to each IP, followed by a 2-h incubation at 4°C. The magnetic beads were separated, and the supernatant was removed. Chromatin was eluted through gentle vortexing (1200 rpm) for 30 min at 65°C. The cross-links were reversed by treatment with proteinase K followed by incubation at 65°C for 2 h. The DNA was subsequently purified with spin columns. The PCR mixture was prepared following the manufacturer's guidelines and using standard parameters. The primer sequences used for ChIP-qPCR are listed in Table S1.

5.13 Statistical Analysis

All in vitro experiments were repeated at least three times. Statistical significance was assessed via an unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA), with GraphPad Prism 8 software. The error bars represent standard deviations, with statistical significance considered at *p < 0.05.