Lactate drives immune resistance via a pharmaceutically reversible H3K18la-KIF20A-c-Myc-PD-L1 axis in hepatocellular carcinoma

Clinical samples

Eighty-nine pairs of HCC tissues and matched adjacent non-tumor liver tissues were harvested from patients who underwent radical hepatectomy at the Tianjin Second People’s Hospital between 2019 and 2022 (Table S1). Additionally, 46 patients with advanced HCC received post-surgical anti-PD-1 monotherapy (Table S2). Ethical approval for this study was granted by the hospital’s ethics committee (approval No. LL-BG-032), and all enrolled patients provided signed written informed consent forms. Clinical therapeutic efficacy was determined in line with the Response Evaluation Criteria in Solid Tumors criteria, which categorize outcomes into partial response (PR), stable disease (SD), and progressive disease (PD).

Cell lines and culture

Human HCC cell lines, including Huh7, HCCLM3, Hep3B, MHCC97-H, and MHCC97-L, as well as the normal mouse hepatocyte line AML12, were obtained from the Cell Bank of the Chinese Academy of Sciences. All these cell lines underwent short tandem repeat authentication to ensure quality control. The mouse HCC cell line Hepa1-6 was procured from the American Type Culture Collection. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco, cat. No. C11995500BT) supplemented with 10% fetal bovine serum (Bioind, cat. No. 04-001-1ACS) and 1% penicillin-streptomycin (YEASEN, cat. No. 60162ES76). The cells were maintained in a humidified incubator at 37°C under a 5% carbon dioxide environment.

Plasmids, small interfering RNAs, and transfection

Huh7 and HCCLM3 cells were subjected to transfection with small interfering RNA (siRNA) targeting lactate dehydrogenase (LDH)A, LDHB, KIF20A, or c-Myc, as well as KIF20A overexpression plasmids (Santa Cruz), with Lipofectamine 2000 reagent (cat. 40802ES01, Yeasen). Transfection efficiency was confirmed 48 h after transfection. LDHA/LDHB double knockout (DKD) cell lines were established by transduction of lentiviral particles carrying LDHA and LDHB shRNAs, or transfection of pcDNA3.1 plasmids encoding KIF20A (Santa Cruz). Positive cell clones were screened and selected with puromycin (cat. P8230, Solarbio). Detailed primer sequences used in the experiments are listed in Table S3.

Chromatin immunoprecipitation (ChIP)-PCR

ChIP-PCR was performed by fixing HCC cells with formaldehyde, lysing them, sonicating the chromatin to 200–500 bp, and incubating the lysates with anti-H3K18la, anti-H3K18ac, anti-c-Myc, or IgG control overnight at 4°C. Immune complexes were captured with Protein A/G agarose beads, washed, eluted, and subjected to cross-link reversal and protein digestion. Quantitative PCR was conducted with specific primers (Table S4) on a Bio-Rad system, with promoter enrichment normalized to input controls. The primer sequences are provided in Table S3.

Immunohistochemistry (IHC)

For IHC, tissue sections were dewaxed, rehydrated, and processed for antigen retrieval. Non-specific binding sites were blocked with 3% hydrogen peroxide and goat serum before incubation with antibodies to Pan Kla, H3K18la, KIF20A, and Ki-67 at 4°C overnight. After incubation with the secondary antibody, diaminobenzidine was used for the chromogenic reaction, and hematoxylin counterstaining was subsequently performed. Images were captured with a microscope, and positive protein expression levels were quantified according to the average optical density and positivity rate in ImageJ. All scores were independently assessed by 2 pathologists in a double-blinded manner. Detailed antibody information is listed in Table S4.

Western blotting (WB)

For WB, cells or tissues were lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology) containing protease inhibitor cocktail (Roche) to extract total protein. Protein concentrations were determined with a bicinchoninic acid assay kit (Beyotime Biotechnology). Equal amounts of protein were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk, then incubated with primary antibodies at 4°C overnight, followed by a 1-h room-temperature incubation with secondary antibodies (Beyotime Biotechnology). Signals were detected with enhanced chemiluminescence reagent, and band gray values were quantified in ImageJ. Primary antibodies to the following were used: Pan Kla, H3K18la, histone H3, KIF20A, c-Myc, PD-L1, LDHA, LDHB, and β-actin (Table S4).

ChIP assays and ChIP-seq

ChIP assays were performed with a ChIP kit (Cell Signaling Technology) according to the manufacturer’s instructions. Briefly, cells were cross-linked with 1% formaldehyde, and chromatin was sheared by sonication, then incubated with anti-H3K18la or IgG control at 4°C overnight. After immunocomplex precipitation and washing, the cross-linking was reversed, and the DNA was purified and analyzed with PCR or sequencing. ChIP-seq was performed on Huh7 cells with anti-H3K18la, and sequencing data were analyzed to screen binding peaks.

Dual-luciferase reporter assays

The pGL3-KIF20A/pGL3-PD-L1 plasmid, pRL-TK internal reference plasmid, and specified plasmids/siRNAs were co-transfected into cells (Table S3). At 24 h post-treatment, dual-luciferase reporter assays (Promega) were performed to measure activity, with Renilla luciferase serving as an internal control for normalization of relative luciferase activity.

CD8⁺ T cell isolation, activation, and co-culture assays

CD8⁺ T cells were purified from mouse spleens with a Miltenyi Biotec CD8⁺ T cell isolation kit, then activated with 1 μg/mL anti-CD3/CD28 (BioLegend). HCC cells and activated CD8⁺ T cells were seeded into 96-well plates for 48-h co-culture. Flow cytometry was used to detect GzMB, IFN-γ, and TNF-α expression in CD8⁺ T cells. For colony formation assays, co-cultured cells were plated in 6-well plates and stained with crystal violet after 14 days of incubation, and colonies were counted.

Flow cytometry

TILs were isolated from mouse tumor tissues through mechanical grinding and collagenase digestion. Cells were stained with surface antibodies (to CD3, CD8, CD44, CD62L, and Ki-67) and intracellular antibodies (to GzMB, IFN-γ, and TNF-α) (BioLegend), then detected with a BD FACSCanto II flow cytometer. Antibody information is detailed in Table S4.

Animal experiments

Male C57BL/6J mice (6–8 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. For subcutaneous xenograft model establishment, 3 × 10⁵ Hepa1-6 cells (control or LDHA/B DKD) were inoculated into the right dorsum. In the combination therapy experiment, mice were randomly assigned to 4 groups (n = 6 per group): control, anti-PD-1 (50 μg/mouse, intraperitoneal injection every 3 days), oxamate (cat. HY-W013032A, MCE; 250 mg/kg/day, intraperitoneal injection), and combination therapy. Every 3 days, tumor volumes were measured, and the mice were sacrificed on day 21 post-inoculation. Survival curves were constructed with the Kaplan-Meier method. For CD8⁺ T cell depletion, the mice received intraperitoneal anti-CD8 injections (500 μg/mouse) at 3-day intervals. All animal experiments were approved by the Animal Management and Use Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (approval No. IRM/2-IACUC-2311-006).

Lactate detection

For in vitro experiments, Huh7 and HCCLM3 cells were treated with solvent control, 2-DG (cat. B1027, APExBIO), or oxamate (cat. HY-W013032A, MCE) for 24 h. Cell lysates were centrifuged, and supernatants were collected to measure intracellular lactate levels. Similarly, Huh7 and HCCLM3 cells transfected with siLDHA, siLDHB, or both were plated to 80%–90% confluency and incubated for 24 h, and lactate was quantified in lysate supernatants. For in vivo tumor tissue analysis, samples were mechanically homogenized in extraction buffer (1:10 w/v) and centrifuged for supernatant collection. For cell experiments, HCC cells were first lysed, and the supernatant was collected for measurement of intracellular lactate concentrations. Lactate levels were detected with a kit (Solarbio, cat. BC2235), and the results were normalized to total protein content or tissue mass.

Statistical analysis

Experimental data are presented as mean ± standard deviation (SD) and were analyzed in GraphPad Prism 9.0. Paired t-test was applied for paired samples, unpaired t-test was applied for 2 independent samples, and one-way/two-way analysis of variance (ANOVA) was applied for multiple groups. Pearson correlation coefficient was used for correlation analysis, and log-rank test was used for survival analysis. P < 0.05 was considered statistically significant.

Enhanced H3K18 lactylation correlates with inferior clinical prognosis and anti-PD-1 therapy resistance in HCC

To elucidate the clinical relevance of histone lactylation in HCC, we initially performed IHC to measure lactylation levels in 89 pairs of HCC tissues and adjacent liver tissues. TPan Kla levels were markedly higher in HCC tissues than adjacent tissues (Figure 1A, B). Notably, among patients with HCC undergoing anti-PD-1 therapy, a significant discrepancy in tumor tissue lactylation levels was observed between responders and non-responders (Figure 1C). Because the proportion of non-responders was considerably higher in the high lactylation group than the low lactylation group (Figure 1D), the lactylation level appeared to closely correlate with the efficacy of anti-PD-1 therapy.

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Figure 1

H3K18 lactylation correlates with unfavorable clinical prognosis and compromised anti-PD-1 immunotherapeutic efficacy in HCC. (A) Representative IHC staining images showing lactylation levels in HCC and paracancerous tissues (scale bar: 100 μm). (B) Statistical analysis of relative lactylation levels in 89 pairs of HCC and paracancerous tissues. (C) Representative tissue images of responders and non-responders after anti-PD-1 therapy. (D) Proportions of patients with HCC with PR or SD/PD in the low and high lactylation groups. (E) Relative lactylation levels of 7 common lysine residues on histones, analyzed with IHC (n = 10). (F) Representative WB images of total lactylation (Pan Kla), H3K18 lactylation (H3K18la), and internal reference protein H3 in paired samples. (G) Quantitative analysis results of WB data for Pan Kla and H3K18la. (H) IHC staining micrographs of Pan Kla and H3K18la in HCC and paracancerous tissues (scale bar: 100 μm). (I, J) Quantitative analysis of IHC results, presented as average optical density (I) and positivity rate (J), respectively. (K–N) Levels of Pan Kla and H3K18la, stratified by TNM stage. (O) WB analysis of Pan Kla, H3K18la, and H3 levels in the normal hepatocyte line (AML12) and multiple HCC cell lines. Data are represented as mean ± SD. (B, I, J) Paired t-test and (D, K-N) ANOVA were used for statistical analysis. All experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; PD, progressive disease; PR, partial response; SD, stable disease; WB, western blotting.

To screen key lactylation sites related to HCC, we used IHC to analyze the lactylation levels of 7 common lysine residues in histones (n = 10). H3K18la was among the most significantly differentially lactylated sites (Figure 1E). WB assays further confirmed significantly greater Pan Kla and H3K18la levels in paired HCC tissues than adjacent tissues (Figure 1F, G). Quantitative IHC analysis revealed notably higher Pan Kla and H3K18la levels in HCC tissues than adjacent tissues (Figure 1H–J). Furthermore, TNM stage-stratified analysis revealed that Pan Kla and H3K18la levels gradually increased with tumor progression from stage I to III (Figure 1K–N). At the cellular level, WB results indicated that Pan Kla and H3K18la were significantly upregulated in HCC cells (Figure 1O). These data suggested that H3K18 lactylation closely correlates with HCC malignant progression and poor anti-PD-1 therapy response.

The high lactate tumor microenvironment promotes KIF20A expression via H3K18 lactylation in the promoter region

We next sought to explore the downstream target genes of H3K18la. Previous studies have reported relatively high H3K18la enrichment in the KIF20A promoter in HCC tissues⁷. On this basis, we investigated the potential role of H3K18la modification and KIF20A in immune evasion in HCC.

The protein level of KIF20A was significantly higher in all HCC cell lines than in the normal hepatocyte line AML12 (Figure 2A, B). In clinical specimens, WB analysis verified notably greater KIF20A protein expression in HCC tissues than adjacent non-tumor tissues (Figure 2C, D). IHC staining combined with correlation analysis further revealed a significant positive correlation between KIF20A and H3K18la levels in HCC tissues (Figure 2E, F).

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Figure 2

High lactate TME promotes KIF20A expression via H3K18 lactylation in the promoter region. (A, B) WB analysis and quantitative statistics of KIF20A protein levels in a normal hepatocyte line (AML12) and HCC cell lines (β-actin as internal reference). (C, D) Detection and quantitative analysis of KIF20A protein expression in clinical HCC tissues and adjacent non-tumor tissues (n = 7). (E, F) Representative IHC images (E) and correlation analysis (F) of KIF20A expression and H3K18la levels in the HCC cohort (n = 89). (G) After treatment with lactate at various concentrations (10, 15, and 20 μM), ChIP-qPCR was performed to examine H3K18ac and H3K18la levels at the KIF20A promoter. (H, I) The pRL-TK plasmid was co-transfected into Huh7 and HCCLM3 cells with the PGL3-KIF20A plasmid or empty PGL3-basic vector, and luciferase activity was detected after 24 h with or without treatment with 20 mM lactate. (J) After 24-h treatment of HCC cells with 2-DG or oxamate, ChIP-PCR was used to examine H3K18la levels at the KIF20A promoter region. (K, L) mRNA expression levels of KIF20A in Huh7 (K) and HCCLM3 (L) cells after 24 h of treatment with 2-DG or oxamate. (M, N) Protein expression levels of KIF20A in Huh7 (M) and HCCLM3 (N) cells after 24 h of treatment with 2-DG or oxamate. Data are expressed as mean ± SD. (B, D, F) Student’s t-test, (J, M, N) one-way ANOVA, and (H, I) two-way ANOVA were used for analysis. All experiments were performed in triplicate. ***P < 0.001. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; TME, tumor microenvironment; WB, western blotting.

To investigate lactate’s specific regulatory effect on H3K18 modification at the KIF20A promoter, we established a normal control group and lactate gradient treatment groups at concentrations of 10, 15, and 20 μM. ChIP-qPCR was performed to detect the enrichment levels of IgG, H3K18ac, and H3K18la at the KIF20A promoter under the various lactate concentrations. H3K18la enrichment at the KIF20A promoter increased in a lactate concentration-dependent manner, whereas H3K18ac exhibited an opposite pattern (Figure 2G). Therefore, lactate specifically promoted H3K18la enrichment at the KIF20A promoter while inhibiting H3K18ac modification in this region.

Dual-luciferase reporter assays revealed that in Huh7 and HCCLM3 cells transfected with the PGL3-KIF20A plasmid, luciferase activity was significantly enhanced after 20 mM lactate treatment, whereas cells transfected with the empty PGL3-basic vector showed no such effect (Figure 2H, I). These findings suggested that lactate promotes KIF20A expression by regulating its promoter activity. To verify the dependence of this regulation on H3K18la, we treated HCC cells with the glycolysis inhibitors 2-DG or oxamate. ChIP-PCR results demonstrated that both inhibitors markedly decreased H3K18la in the KIF20A promoter region (Figure 2J). Moreover, RT-PCR and WB assays confirmed that 2-DG and oxamate downregulated the mRNA and protein levels of KIF20A in a dose-dependent manner in Huh7 and HCCLM3 cells (Figure 2K–N). These results indicated that the high lactate TME promotes KIF20A transcription and expression by increasing H3K18 lactylation levels in the KIF20A promoter region.

KIF20A regulates PD-L1 expression via c-Myc

To elucidate the regulatory role of KIF20A in c-Myc and PD-L1 expression, we transfected Huh7 and HCCLM3 cells with KIF20A siRNA or overexpression plasmid. Our prior study demonstrated that KIF20A interacts with and stabilizes the c-Myc protein. Targeting KIF20A promotes c-Myc ubiquitination, thereby inducing mismatch repair defects and enhancing the efficacy of PD-1 inhibitors²⁰. Herein, we further explored the downstream regulatory cascade linking KIF20A, c-Myc, and PD-L1. WB revealed that KIF20A knockdown significantly decreased c-Myc and PD-L1 protein levels in Huh7 and HCCLM3 cells (Figure 3A, C), whereas KIF20A overexpression markedly upregulated the protein expression of both molecules (Figure 3B, D). We therefore concluded that KIF20A exerts positive regulatory effects on c-Myc and PD-L1.

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Figure 3

KIF20A modulates PD-L1 expression through c-Myc. (A–D) Huh7 and HCCLM3 cells were transfected with KIF20A siRNA or KIF20A overexpression pcDNA3.1 plasmid, respectively. WB assays were used to detect the protein levels of KIF20A, c-Myc, and PD-L1 in Huh7 (A, B) and HCCLM3 (C, D) cells. (E) ChIP-PCR was used to assess c-Myc binding to the PD-L1 promoter region in Huh7 and HCCLM3 cells. (F) After transfection of Huh7 and HCCLM3 cells with c-Myc siRNA, RT-PCR was used to verify c-Myc knockdown efficiency. (G–H) Huh7 (G) and HCCLM3 (H) cells were co-transfected with KIF20A overexpression plasmid and c-Myc siRNA, and WB was used to determine the protein levels of KIF20A, PD-L1, and c-Myc. (I, J) Huh7 (I) and HCCLM3 (J) cells were co-transfected with pGL3-PD-L1 or pGL3-basic plasmid, KIF20A overexpression pcDNA3.1 plasmid, c-Myc siRNA, and pRL-TK plasmid, and the luciferase activity was measured. Data are represented as mean ± SD. (B, D, F) Student’s t-test, (E) ANOVA, and (I, J) two-way ANOVA were used for statistical analysis. Experiments were performed in triplicate. ***P < 0.001. OE, overexpression; WB, western blotting.

ChIP-PCR experiments further confirmed that c-Myc directly binds the promoter region of PD-L1. This binding ability was enhanced by KIF20A overexpression but weakened by KIF20A knockdown (Figure 3E). To verify whether the regulation of PD-L1 by KIF20A was dependent on c-Myc, we transfected Huh7 and HCCLM3 cells with c-Myc siRNA and confirmed favorable c-Myc knockdown efficiency with RT-PCR (Figure 3F). Co-transfection assays demonstrated that KIF20A overexpression markedly upregulated PD-L1 protein levels, whereas concurrent c-Myc knockdown abrogated this regulatory effect (Figure 3G, H). Dual-luciferase reporter gene assays further confirmed that KIF20A overexpression enhanced the luciferase activity of the pGL3-PD-L1 plasmid, whereas c-Myc silencing suppressed this activity (Figure 3I, J). Consequently, KIF20A mediates PD-L1 expression by regulating c-Myc activity.

KIF20A promotes HCC cell immune evasion by regulating c-Myc-mediated PD-L1 expression

To clarify KIF20A’s function in HCC immune evasion, we co-cultured treated HCC cells with CD8⁺ T cells, then evaluated CD8⁺ T cell activation and HCC cell colony formation. FC analysis revealed markedly greater GzMB and IFN-γ levels in CD8⁺ T cells co-cultured with KIF20A-knockdown Huh7 and HCCLM3 cells than controls. In contrast, KIF20A overexpression exerted an opposite regulatory effect on the expression of both molecules (Figure 4A–D). Collectively, these findings indicated that KIF20A suppresses the activation of CD8⁺ T cells.

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Figure 4

KIF20A facilitates immune evasion of HCC cells via regulating c-Myc-mediated PD-L1 expression. HCC cells from distinct groups were co-cultured with CD8⁺ T cells in a 1:3 ratio. (A, B) Representative FC dot plots and quantitative analysis of GzMB and IFN-γ expression in CD8⁺ T cells post co-culture with Huh7 cells. (C, D) Representative FC profiles and corresponding quantitative data of GzMB and IFN-γ levels in CD8⁺ T cells after co-culture with HCCLM3 cells. (E, F) Representative images and quantitative results of colony formation assays in Huh7 cells cultured alone or in the presence of CD8⁺ T cells. (G, H) Representative micrographs and quantitative analysis of colony formation for HCCLM3 cells under solitary culture or co-culture with CD8⁺ T cells. Data are presented as mean ± SD. Statistical analyses were performed with one-way ANOVA (B, D) or two-way ANOVA (F, H). Experiments were independently replicated 3 times. *P < 0.05; **P < 0.01; ***P < 0.001. HCC, hepatocellular carcinoma; OE, overexpression.

In colony formation assays, we observed minimal changes in the colony-forming ability of Huh7 and HCCLM3 cells after KIF20A knockdown or overexpression when CD8⁺ T cells were absent from the co-culture system. In contrast, after co-cultivation with CD8⁺ T cells, KIF20A silencing markedly impaired the colony-forming potential of HCC cells, whereas KIF20A overexpression promoted this phenotype (Figure 4E–H). These results indicated that KIF20A inhibits CD8⁺ T cell activation and consequently promotes HCC cell immune evasion by regulating c-Myc-mediated PD-L1 expression.

LDH mediates the HCC lactate-histone lactylation axis and regulates CD8⁺ T cell activation function

To clarify the role of LDH in lactate metabolism and histone lactylation of HCC cells, we transfected Huh7 and HCCLM3 cells with si-LDHA, si-LDHB, or LDHA/B DKD. The transfected cells were then cultured for 24 h in the presence or absence of exogenous lactate. WB indicated that LDHA/B DKD significantly decreased the protein levels of LDHA and LDHB in the cells, and simultaneously significantly downregulated the levels of Pan Kla and H3K18la; moreover, this effect was not affected by exogenous lactate supplementation (Figure 5A–D). ELISA indicated that LDHA/B DKD significantly decreased the lactate concentrations in the supernatants of Huh7 and HCCLM3 cells, and the effect of single knockdown was weaker than that of DKD (Figure 5E, F). These findings suggested that LDH is a key regulator of HCC lactate production and histone lactylation.

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Figure 5

LDH mediates the HCC lactate-histone lactylation axis and regulates CD8⁺ T cell activation function. HCC cells were transfected with si-LDHA, si-LDHB, or both, then cultured in the presence or absence of lactate for 24 h. (A–D) WB detected the expression of LDHA and LDHB in Huh7 (A, B) and HCCLM3 (C, D) cells under different treatments, and the levels of Pan-Kla and H3K18la. (E, F) Detection of lactate concentrations in the supernatants of Huh7 (E) and HCCLM3 (F) cells from different treatment groups. Huh7 and HCCLM3 cell lines co-transfected with si-LDHA and si-LDHB were co-cultured with CD8⁺ T cells in a 1:3 ratio under anti-CD3/CD28 stimulation. (G–I) Colony formation assays on HCC cells from different treatment groups under solitary culture or co-culture with CD8⁺ T cells (G), and quantitative analysis of colony numbers (H, I). (J, K) CCK-8 assays were used to assess the proliferative activity of Huh7 (J) and HCCLM3 (K) cells. (L, M) FC analysis was performed to quantify the levels of GzMB (L) and IFN-γ (M) in CD8⁺ T cells within the co-culture system. All data are presented as mean ± SD. Statistical analyses were conducted with one-way ANOVA for (C, D, E, F, H, I), two-way ANOVA for (J, K), or Student’s t-test for (L, M). Each experiment was independently repeated 3 times. *P < 0.05; **P < 0.01; ***P < 0.001. DKD, double knockout.

Under stimulation with anti-CD3/CD28, LDHA/B DKD HCC cells were co-cultured with CD8⁺ T cells at a 1:3 ratio. Colony formation assay results revealed notably lower colony-forming ability of HCC cells in the DKD group than the control group, and this inhibitory effect was further exacerbated after co-culture with CD8⁺ T cells (Figure 5G–I). In agreement with this finding, CCK-8 assays demonstrated that LDHA/B DKD significantly inhibited HCC cell proliferation, and this effect became more pronounced after co-cultivation with CD8⁺ T cells (Figure 5J, K). Flow cytometric analysis indicated that, relative to those in the control group, the expression levels of GzMB and IFN-γ in CD8⁺ T cells within the LDHA/B DKD group co-culture system were significantly upregulated (Figure 5L, M). Collectively, these findings suggested that LDH facilitates HCC cell proliferation and colony formation by mediating lactate production and histone lactylation, and consequently inhibiting the activation of CD8⁺ T cells.

In vivo experiments confirm that LDH is an effective target for reversing HCC lactate metabolism and immune evasion

To verify the regulatory role of LDH in vivo, we constructed the LDHA/B DKD Hepa1-6 cell line. WB confirmed that the protein levels of LDHA and LDHB were effectively knocked down (Figure 6A). Control cells and LDHA/B DKD cells were subcutaneously inoculated into C57BL/6J mice to establish xenograft models. Mice bearing LDHA/B DKD cells, compared with controls, displayed substantially smaller tumor volumes (Figure 6B, C) and lower tumor weights (Figure 6D), lower lactate concentrations in tumor tissues (Figure 6E), and longer overall survival (Figure 6F). IHC staining demonstrated markedly lower Pan Kla and H3K18la in tumor tissues from the LDHA/B DKD group than the control group (Figure 6G). FC analysis of TILs revealed that the LDHA/B DKD group had significantly higher proportions of CD3⁺CD8⁺ CTLs, CD44hiCD62Llo effector memory CTLs, and Ki-67⁺ proliferating CTLs in tumor tissues than observed in the control group (Figure 6H–J). Furthermore, the percentages of GzMB⁺, IFN-γ⁺, and TNF-α⁺ CTLs in tumors were significantly elevated in the LDHA/B DKD group. (Figure 6K–M). These data collectively suggested that LDH knockdown enhances the recruitment, activation, and proliferation of tumor-infiltrating CD8⁺ T cells.

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Figure 6

In vivo experiments confirm that LDH is an effective target for reversing abnormal HCC lactate metabolism and immune evasion. (A) WB analysis of LDHA, LDHB, and the internal reference protein β-actin in control and LDHA/B DKD Hepa1-6 cells. (B) Representative images of dissected tumor tissues from tumor-bearing mice (n = 6). (C–F) Tumor growth curves (C), tumor weights (D), lactate concentrations in tumor tissues (E), and K-M survival curves (F) of mice in the control and LDHA/B DKD groups (n = 6). (G) Representative micrographs of IHC staining, showing H3K18la and Pan Kla levels in tumor tissues (scale bar: 100 μm; n = 6). (H–M) FC analysis of tumor-infiltrating immune cells: TILs were isolated from tumor tissues of the control and LDHA/B DKD groups. Flow plots and quantitative statistics show the proportions of CD3⁺CD8⁺ (H), CD44hiCD62Llo effector memory (I), Ki-67⁺ proliferating (J), GzMB⁺ (K), IFN-γ⁺ (L), and TNF-α⁺ (M) CTLs in tumors (n = 6). (N, O) Tumor volume changes in mice treated with anti-CD8 to deplete CD8⁺ T cells or IgG (n = 6). (P) Body weight changes in mice at the end of the experiment (n = 6). Data are expressed as mean ± SD. (C–F, H–O) Student’s t-test and (P) two-way ANOVA were used for analysis. All experiments were independently replicated 3 times. *P < 0.05; **P < 0.01; ***P < 0.001. CTLs, cytotoxic T lymphocytes; DKD, double knockout HCC, hepatocellular carcinoma.

To verify that the tumor-suppressive effect of LDH knockdown was dependent on CD8⁺ T cells, we depleted CD8⁺ T cells with anti-CD8. After CD8⁺ T cell depletion, the difference in tumor volume between the LDHA/B DKD group and the control group disappeared (Figure 6N, O), thus confirming that the tumor-suppressive effect of LDH knockdown was mediated by CD8⁺ T cells. In addition, the absence of a significant difference in the body weight of mice in each group at the end of the experiment indicated that LDHA/B knockdown had no obvious toxicity (Figure 6P). These in vivo experimental results indicated that LDH is an effective target for reversing abnormal HCC lactate metabolism and immune evasion.

Combined glycolysis inhibitor and immune checkpoint inhibitor (ICI) therapy exhibits synergistic efficacy

To evaluate the combined therapeutic effects of glycolysis inhibitor and anti-PD-1 inhibitor, we verified the effects of glycolysis inhibitor treatment on HCC cell lactate levels and lactylation in vitro. Treatment with 2-DG and oxamate significantly decreased lactate levels in Huh7 and HCCLM3 cells (Figure 7A, B), and simultaneously significantly downregulated the levels of Pan Kla and H3K18la (Figure 7C–F), in agreement with the effects of LDH knockdown described above. In vivo combination therapy significantly suppressed tumor growth in mice, and the combination group showed markedly smaller tumor volumes than the monotherapy and control groups (Figure 7G–I). The substantially diminished lactate content in tumor tissues (Figure 7J) and significantly prolonged overall survival of mice (Figure 7K) suggested synergistic tumor-suppressive effects of the combined treatment. IHC staining revealed a notably lower proportion of Ki-67⁺ proliferating cells in tumor tissues in the combination therapy group than the monotherapy and control groups (Figure 7L, M). Moreover, WB revealed that combination therapy significantly decreased Pan Kla and H3K18la modification levels in tumor tissues, and also decreased the expression of KIF20A, c-Myc, and PD-L1 (Figure 7N). FC analysis demonstrated that the combination therapy group exhibited significantly higher proportions of Ki-67⁺ proliferating CTLs, and GzMB⁺, IFN-γ⁺, and TNF-α⁺ activated CTLs in tumors than the monotherapy and control groups (Figure 7O–R). Collectively, these findings indicated that combination treatment with a glycolysis inhibitor and an anti-PD-1 inhibitor synergistically enhanced the anti-tumor immune response and suppressed HCC progression by targeting the lactate-histone lactylation-KIF20A-c-Myc-PD-L1 axis. Figure 8 shows the mechanistic pathway.

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Figure 7

Evaluation of the synergistic efficacy of combined therapy with glycolysis inhibitors and immune checkpoint inhibitors. (A, B) Changes in lactate levels in Huh7 (A) and HCCLM3 (B) cells after treatment with 2-DG and oxamate. (C–F) WB analysis of Pan-Kla and H3K18la levels in Huh7 (C, D) and HCCLM3 (E, F) cells after inhibitor treatment. A total of 3 × 10⁵ Hepa1-6 cells in logarithmic growth phase were subcutaneously implanted into the right dorsum in C57BL/6J mice. After the tumor volumes reached approximately 100 mm³, the mice were randomly assigned to 4 groups (n = 6). (G–K) Representative images of tumor sizes (G), tumor volume curves (H), tumor weights (I), lactate content in tumor tissues (J), and survival analysis (K) for each group. (L, M) IHC staining results for Ki-67 in tumor tissues from the treatment groups (n = 3; scale bar: 100 μm). (N) WB analysis of Pan-Kla, H3K18la, KIF20A, c-Myc, and PD-L1 levels in tumor tissues (n = 5). (O–R) Proportions and representative scatter plots of Ki-67⁺ (O), GzMB⁺ (P), IFN-γ⁺ (Q), and TNF-α⁺ (R) CTLs in tumor tissues (n = 5). Data are presented as mean ± SD. Statistical analyses were performed with one-way ANOVA (A, B, D, F, H-J, O-R) and two-way ANOVA (K). Experiments were independently repeated 3 times. **P < 0.01; ***P < 0.001.

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Figure 8

Schematic illustration of lactic acid-mediated HCC immune tolerance through the pharmacologically reversible H3K18la-KIF20A-c-Myc-PD-L1 axis. Pathological state (left panel): Under the Warburg effect, lactic acid, a glycolytic product, substantially accumulates in the cytoplasm (1); after entering the nucleus, it promotes lactylation modification of H3K18la in the KIF20A promoter, thereby upregulating KIF20A transcription (2). Subsequently, KIF20A interacts with c-Myc and competitively inhibits FBXW7 E3 ubiquitin ligase-mediated ubiquitin-dependent degradation of c-Myc (3), and the c-Myc transcription factor further enhances PD-L1 expression (4) and ultimately leads to the presentation of PD-L1 on HCC cell surfaces and inhibition of the cytotoxic activity of CD8⁺ T cells (5), thereby forming an immunosuppressive TME. Immune reconstruction strategy (right panel): 2-DG inhibits the conversion of glucose to pyruvate through competitive binding to HK, while oxamate inhibits LDH (6); both agents synergistically block lactate production, H3K18 lactylation levels and KIF20A expression (7, 8), thus restoring c-Myc degradation (9), downregulating c-Myc-mediated PD-L1 transcription and presentation (10), and eventually reversing the immunosuppressive microenvironment (11). HCC, hepatocellular carcinoma; H3K18la, histone H3 lysine 18 lactylation; HK2, hexokinase 2; La, lactylation; PD-L1, programmed death ligand 1; TCR, T-cell receptor; TME, tumor microenvironment; Ub, ubiquitination; 2DG, 2-deoxy-D-glucose (created with https://www.citexs.com).