Targeting LRPPRC lactylation disrupts metabolic-immune crosstalk and restores antitumor immunity in hepatocellular carcinoma

Abstract

Background

The Warburg effect drives lactate accumulation in the tumor microenvironment (TME), where it functions as a signaling molecule. Lactate-derived lysine lactylation (Kla) is a novel post-translational modification (PTM) implicated in regulating immune cell function. Leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is overexpressed in hepatocellular carcinoma (HCC) and plays key roles in mitochondrial metabolism and immune evasion. However, whether and how LRPPRC is regulated by lactylation to coordinate metabolic-immune crosstalk in HCC remains unknown. This study aims to investigate the role and mechanism of LRPPRC lactylation in linking tumor glycolysis to macrophage polarization in HCC.

Methods

Bioinformatics analysis identified lactate metabolism-related genes and hub nodes in HCC datasets. LRPPRC lactylation was detected via immunoprecipitation and western blot using pan-Kla antibody. The specific lactylation site was mapped by prediction database and validated by site-directed mutagenesis (K326R). Functional impacts of LRPPRC-Kla³²⁶ on HCC cell proliferation, invasion, and glycolysis were assessed using Cell Counting Kit-8 (CCK-8), Transwell, wound-healing, and Seahorse assays. The role of LRPPRC-Kla³²⁶ in macrophage polarization was examined in bone marrow-derived macrophages (BMDMs) from LRPPRC^WT and LRPPRC^K326R knock-in mice using flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and cytokine measurement. An in vivo tumor admix model co-injecting Lewis lung carcinoma (LLC) cells with polarized BMDMs was used to evaluate tumor growth and immune cell infiltration.

Results

LRPPRC was identified as a hub gene among lactate metabolism-related genes in HCC and was upregulated in tumor tissues, correlating with poor prognosis. LRPPRC undergoes lactylation in a lactate-dependent manner, with K326 being the major modification site. The LRPPRC^K326R mutation impaired HCC cell proliferation, invasion, and glycolytic flux. In macrophages, lactylation at LRPPRCK326was required for lactate-induced M2 polarization and glycolytic reprogramming; the K326R mutation skewed polarization towards an M1 phenotype with reduced glycolysis. In the tumor admix model, co-injection of LRPPRC^K326R M2 macrophages significantly suppressed tumor growth compared to LRPPRC^WT M2 macrophages, which was associated with increased infiltration of activated IFN-γ⁺ CD8⁺ and CD4⁺ T cells.

Conclusions

Lactate-induced lactylation of LRPPRC at K326 serves as a critical metabolic-immune switch in HCC. It enhances tumor glycolysis and simultaneously drives M2-like macrophage polarization, fostering an immunosuppressive TME conducive to tumor progression. Targeting the LRPPRC-Kla³²⁶ axis may represent a promising therapeutic strategy to disrupt the metabolic symbiosis between tumor cells and immune cells in HCC.

Highlight box.

Key findings

• Lactate induces lactylation of the mitochondrial protein leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) at lysine 326 (Kla³²⁶) in hepatocellular carcinoma (HCC).

• LRPPRC-Kla³²⁶ promotes tumor cell glycolysis, proliferation, and invasion.

• In macrophages, LRPPRC-Kla³²⁶ drives M2-like tumor-associated macrophage polarization and glycolytic reprogramming, creating an immunosuppressive tumor microenvironment.

• Disrupting Kla³²⁶ (via K326R mutation) reverses these effects, inhibiting tumor growth and enhancing anti-tumor T-cell responses.

What is known and what is new?

• The Warburg effect increases lactate in tumors. Lactate drives histone lactylation (Kla) to regulate immunity. LRPPRC protein promotes cancer metabolism and immune evasion. M2-like macrophages support tumor progression.

• We discover lactate-induced lactylation of the mitochondrial protein LRPPRC at K326. This modification dually enhances tumor glycolysis and drives M2 macrophage polarization, coupling metabolism to immunosuppression. Targeting LRPPRC-Kla³²⁶ is a novel therapeutic strategy.

What is the implication, and what should change now?

• This study identifies LRPPRC-Kla³²⁶ as a crucial link coupling tumor glycolysis to immunosuppression, expanding the realm of lactylation biology and presenting a novel dual-target for cancer therapy.

• Future research and therapeutic development should shift towards targeting the specific LRPPRC-Kla³²⁶ axis to simultaneously disrupt tumor metabolism and immune evasion, potentially overcoming resistance in aggressive cancers like HCC.

Introduction

The Warburg effect, a hallmark of cancer metabolism, describes the preference of tumor cells to rely on glycolysis for adenosine triphosphate (ATP) production even under normoxic conditions, resulting in excessive lactate secretion into the tumor microenvironment (TME) (1-3). Compared to traditional oxidative phosphorylation (OXPHOS), glycolysis—though less efficient in ATP generation—rapidly supplies biosynthetic precursors [e.g., acetyl-coenzyme A, nicotinamide adenine dinucleotide phosphate (NADPH)] for tumor cells and fosters an acidic microenvironment (pH 6.0–6.5) (4,5). Lactate, the end product of glycolysis, not only maintains intracellular pH homeostasis via efflux through monocarboxylate transporters (MCT1/4) but also acts as a precursor for epigenetic modifications (6,7). Specifically, lactate drives lysine (Kla) lactylation, a novel post-translational modification (PTM) that regulates gene expression and modulates immune cell functionality (5,8,9).

Tumor-associated macrophages (TAMs) are a dynamic, heterogeneous, and abundant cell population within the TME (10,11). TAMs exhibit phenotypic and functional plasticity, broadly categorized into M1 and M2 subtypes (12). Anti-inflammatory, proangiogenic M2-polarized macrophages promote wound healing and immune resolution (13). However, in advanced cancers, this phenotype is co-opted by tumors to evade antitumor immunity (14). Conversely, while proinflammatory M1 macrophages enhance antitumor immune responses, they paradoxically fuel tumorigenesis by promoting chronic inflammation-associated DNA damage and mutations (15). Emerging evidence indicates that pro-tumorigenic TAMs display distinct molecular signatures in cancers such as hepatocellular carcinoma (HCC) and colorectal cancer (CRC) (16). Macrophage polarization is sensitive to TME lactate levels, with high lactate concentrations impairing TAM glucose metabolism to drive HCC progression (17,18). Lactate accumulation in the TME directly correlates with histone lactylation in macrophages, which influences their immunophenotype and effector functions (19).

The leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), a member of the pentatricopeptide repeat (PPR) family, plays a critical role in mitochondrial function, RNA metabolism, and cellular homeostasis (20,21). Studies demonstrate that LRPPRC coordinates mitochondrial OXPHOS by stabilizing mitochondrial mRNAs and enhancing the translation of OXPHOS complex subunits encoded by the mitochondrial genome, thereby promoting metabolic adaptation in cancer cells (22,23). LRPPRC is overexpressed in various cancers, including prostate, esophageal, lung, and breast cancers, where its upregulation correlates with advanced tumor stage, metastasis, and poor prognosis (24-28). In HCC, LRPPRC enhances programmed death-ligand 1 (PD-L1) expression by recruiting m6A readers (e.g., YTHDF1) to stabilize PD-L1 mRNA, thereby suppressing CD8⁺ and CD4⁺ T-cell infiltration and fostering an immunosuppressive TME. Given its central role in cancer metabolism and immune evasion, LRPPRC represents a promising therapeutic target for precision oncology.

Our study unveils LRPPRC-Kla³²⁶ as a metabolic-immune key point in HCC, where lactate-induced lactylation couples Warburg metabolism to TAM polarization, fostering an immunosuppressive TME. Targeting this axis may offer therapeutic strategies to disrupt metabolic symbiosis between tumors and immune cells. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2533/rc).

Methods

Data sources

Gene expression profiles and clinical data were obtained from the TCGA-LIHC cohort [The Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma (LIHC); https://portal.gdc.cancer.gov] and Gene Expression Omnibus (GEO) datasets (GSE263786 and GSE244826), comprising 125 normal and 314 tumor tissue samples. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Animal experiments

Male C57BL/6 mice (GemPharmatech Co., Ltd., Nanjing, China) were randomized into 3 groups (n=6/group) and housed under a 12-hour light/dark cycle with ad libitum access to food and water. For the tumor admix model (29), bone marrow-derived macrophages (BMDMs) from LRPPRC^K326R or LRPPRC^WT mice were polarized with interleukin (IL)-4 (20 ng/mL, PeproTech, Cranbury, USA) for 24 h, mixed with Lewis lung carcinoma (LLC) cells (CD45.2⁺) at a 1:2.5 ratio in Matrigel, and subcutaneously injected into syngeneic B6/SJL mice (CD45.1⁺). Tumors were measured every 3 days from day 7 using the formula: volume = (length × width²)/2. At endpoint (day 30), tumors were excised, weighed, and enzymatically digested (collagenase IV, hyaluronidase, and DNase I; 37 ℃, 30 min). Experiments were performed under a project license (No. 20240125032) granted by Animal Experiment Committee of An Hui Medical Early Diagnosis (AHMED), in compliance with Chinese guidelines for the care and use of animals.

Cell culture

HepG2 and Hep3B cells (Cell Bank of Type Culture Collection, Chinese Academy of Sciences, Shanghai, China) were maintained in DMEM (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX at 37 ℃/5% CO₂. Mycoplasma-negative cells were used for experiments.

LRPPRC stable knockdown

Lentiviral vectors encoding LRPPRC-specific shRNAs (Genechem Co., Ltd., Shanghai, China) were transfected into HepG2/Hep3B cells for 48 h, followed by puromycin selection (2 µg/mL) for 72 h.

Cell proliferation assay

Cells (5×10³/well) were seeded into 96-well plates. At indicated timepoints (12–72 h), 10 μL Cell Counting Kit8 (CCK-8) reagent (Dojindo, Rockville, USA) was added, incubated for 2 h, and absorbance measured at 450 nm (BioTek Synergy H1, Winooski, USA).

Transwell invasion assay

Cells (2×10⁵) in serum-free medium were seeded into 6.5 mm Transwell inserts (8 μm pores; Corning, Shanghai, China) with Matrigel-coated (invasion) membranes. After 24 h, migrated cells were fixed with 4% paraformaldehyde (PFA), stained with 0.1% crystal violet, and imaged (Nikon Eclipse Ti, Tokyo, Japan).

Glycolytic metabolite quantification

Glucose, lactate, ATP, and pyruvate levels were measured in cell lysates using commercial kits (Leagene Biotech, Beijing, China; Abbkine, Wuhan, China; Beyotime Biotechnology, Shanghai, China) per manufacturer protocols.

BMDM differentiation

Bone marrow cells from euthanized mice were flushed from femurs/tibiae and cultured in RPMI 1640 with 10% FBS and recombinant murine M-CSF (25 ng/mL; PeproTech) for 7 days. Differentiated BMDMs were polarized with IL-4 (20 ng/mL) or lipopolysaccharide (LPS; 100 ng/mL) + IFN-γ (20 ng/mL) for 48 h.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands), reverse-transcribed (HighCapacity cDNA Reverse Transcription Kit; Applied Biosystems, Shanghai, China), and quantified via TaqMan assays (7500 Fast Real-Time PCR System; Applied Biosystems). Relative mRNA expression was calculated using the 2−ΔΔCt method normalized to β-actin.

Western blotting

Proteins were extracted with RIPA lysis buffer (Beyotime Biotechnology), quantified (BCA Assay; Beyotime), separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Blots were probed with anti-LRPPRC (1:1,000; Abcam, Shanghai, China, EPR24052-38) and HRP-conjugated secondary antibodies (1:5,000; Cell Signaling Technology, Shanghai, China), visualized via ECL (Thermo Fisher, Grand Island, USA), and quantified (Image J, Maryland, USA).

Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR)

Cells (6×10⁴/well) were seeded into Seahorse XF96 plates (Agilent, Billerica, USA). ECAR/OCR was measured using the Seahorse XFe96 Analyzer (Agilent) following sequential injection of 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxyglucose. Data were analyzed using Wave Software (Agilent).

Statistical analysis

Normality of the data was tested using the Shapiro-Wilk normality test. Nonparametric data with multiple comparisons were analyzed by Kruskal-Wallis one-way analysis of variance (ANOVA) followed by Holm’s Stepdown Bonferroni procedure for adjusted P values. The Mann-Whitney t-test was used for comparison between two groups. Data with normal distribution were analyzed by one-way ANOVA with Dunnett’s post-test or Tukey’s correction for multiple comparisons as described in the figure legends. Statistical significance was set at P<0.05. Data were analyzed using Prism 8.0 software (GraphPad, La Jolla, CA, USA).

Results

Identification of biological characteristics of lactate metabolism-related genes (LMRGs) in HCC patients

Under the criteria of an adjusted P value <0.05 and |log₂ fold change (FC)| >1, a total of 5,410 differentially expressed genes (DEGs) were identified across the GSE263786 and GSE244826 datasets, comprising 2,989 upregulated and 2,421 downregulated genes (Figure 1A, available online: https://cdn.amegroups.cn/static/public/tcr-2025-aw-2533-1.xlsx). By overlapping the DEGs and LMRGs in HCC, we identified 56 LMRGs that were differentially expressed (Figure 1B). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were subsequently performed on these 56 LMRGs. Functional annotation demonstrated that the lactate-centric molecular subnetwork was predominantly enriched in biological processes such as proton transmembrane transport mitochondrial electron transport chain (ETC), and glycolytic processes, all of which are mechanistically linked to lactate biosynthesis and oncogenic progression (Figure 1C,1D). Further analysis of the heatmap depicting Pearson’s correlation coefficients and hub genes within the protein-protein interaction (PPI) network highlighted LRPPRC as a pivotal node (Figure 1E,1F).

Figure 1.

Differences in LMRGs expression in normal and HCC samples and its enrichment analysis. (A) Volcano plot of differentially expressed genes between normal and HCC tissues. Red represents genes highly expressed in tumors, and blue represents genes highly expressed in normal tissues. Gray represents no difference in expression. (B) 56 LMRGs associated with HCC. (C) GO enrichment analysis of differentially expressed genes between the normal and HCC subtypes; (D) KEGG pathway enrichment analysis of differentially expressed genes between the normal and HCC subtypes. (E) Spearman correlation analysis of the relationship with LMRGs associated with HCC. (F) The PPI network analysis among 11 differentially expressed LMRGs associated with HCC. DEG, differentially expressed gene; GO, Gene Ontology; HCC, hepatocellular carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; LMRG, lactate metabolism related gene; PPI, protein-protein interaction.

Identification of lactylation at the K326 site of LRPPRC

To identify key regulatory molecules driving HCC progression, we integrated DEG analysis with PPI network data, prioritizing LRPPRC as a candidate oncoprotein aberrantly expressed in HCC. Analysis of TCGA datasets confirmed LRPPRC upregulation in HCC patients, which correlated positively with advanced tumor stage and poor prognosis (Figure 2A,2B). This observation prompted our hypothesis that LRPPRC may undergo lactylation, a lactate-dependent PTM. Western blotting using a pan-Kla antibody revealed elevated lactylation levels of LRPPRC in Hep3B cells (Figure 2C,2D). Given the reliance of rapidly proliferating tumor cells on glycolysis and consequent lactate accumulation, we investigated whether lactate availability regulates LRPPRC lactation. Exogenous D-lactate supplementation significantly enhanced LRPPRC lactylation levels, whereas treatment with oxamate [an inhibitor of lactate dehydrogenase A (LDHA)] to suppress intracellular lactate production markedly reduced lactylation (Figure 2E). These findings demonstrate that LRPPRC lactylation is dynamically modulated by lactate levels in HCC cells.

Figure 2.

Identification of lactylation at the K326 site of LRPPRC. (A) LRPPRC level in HCC and control specimens across TCGA-LIHC patients. (B) Overall survival probability of patients with lowly or highly expressed LRPPRC. (C) Hep3B cell lysates were subjected to Co-IP with pan-lactate modified antibody, and Western blot analysis with anti-LRPPRC antibody. (D) Hep3B cell lysates were subjected to Co-IP, Western blot analysis with anti-Lactylation modified antibody. (E) Hep3B cell lines were treated with D-lactate (20 mM) or sodium oxamate (20 mM) for 24 h, and Western blot analysis of the level of LRPPRC lactylation. (F) LRPPRC of lactic acid modified site through the FSL-Kla database (http://kla.zbiolab.cn/). (G) Hep3B cell lysates were subjected to Co-IP, Western blot analysis with anti-lactylation modified antibody. HR, hazard ratio; IgG, immunoglobulin G; LIHC, liver hepatocellular carcinoma; LRPPRC, leucine-rich pentatricopeptide repeat-containing protein; TCGA, The Cancer Genome Atlas; WT, wild type.

To map the specific lactylation site(s) on LRPPRC, we utilized the FSL-Kla database for in silico prediction, identifying eight high-confidence lysine residues (ranked by prediction scores), with K326 as the top candidate (Figure 2F). In order to examine whether K326 serves as the major Kla site of LRPPRC, we constructed K326R mutant of LRPPRC and compared the dynamics of Kla levels in wild type (WT) and mutation LRPPRC in response to lactate treatments. Flag-tagged LRPPRC^WT and LRPPRC^K326R were overexpressed in HepG2 and Hep3B cells with treatment of D-lactate, followed by Flag-immunoprecipitation. As expected, compared with the LRPPRC^WT, LRPPRC^K326R mutation in HepG2 or Hep3B cells led to a notable decrease in Kla levels after treatment (Figure 2G), indicating that lactylation mainly occurs at Kla³²⁶ of LRPPRC in high-lactate environments.

LRPPRC-Kla326 contributes to energy disorder and HCC cell malignancy

For the assessment of LRPPRC function during HCC progression, its expression was effectively knockout through transfection of specific shRNAs and Flag-tagged LRPPRC^WT and LRPPRC^K326R were overexpressed in HepG2 and Hep3B cells. The LRPPRC^K326R mutation significantly impaired the proliferative capacity of HepG2 and Hep3B cells, through CCK-8 assays (Figure 3A-3D). Transwell invasion assays further demonstrated that the LRPPRC^K326R mutation suppressed the invasive potential of both HepG2 and Hep3B cells (Figure 3E,3F). In wound-healing assays, LRPPRC^K326R expression in HepG2 and Hep3B cells resulted in a marked reduction in migrated cell numbers compared to LRPPRC^WT (Figure 3G,3H). The ECAR difference between HCC cells was measured using the extracellular flux detector Seahorse XF. The results indicate that, K326R-mutated LRPPRC have a lower level of glycolysis level and glycolysis capacity compared with the WT LRPPRC (Figure 3I,3J). These findings demonstrate that LRPPRC lactylation enhances glycolysis level and attenuates oncogenic phenotypes—including proliferation, migration, and invasion—suggesting its role as a central regulatory node in HCC pathogenesis.

Figure 3.

LRPPRC K326R mutation significantly constrains tumorigenesis ability and glycolysis in hepatocellular carcinoma cell line. (A,C) IP validation of LRPPRC lactylation differences with LRPPRC^WT and LRPPRC^K326R mutation treatment in HepG2 and Hep3B cells. (B,D) The cell viabilities of HepG2 and Hep3B with LRPPRC^WT and LRPPRC^K326R mutation treatment. (E,F) Transwell invasion assays of HepG2 cells with LRPPRC^WT and LRPPRC^K326R mutation treatment. Cells were stained with crystal violet and imaged under bright-field microscopy. (G,H) Wound-healing assays of HepG2 cells with LRPPRC^WT and LRPPRC^K326R mutation treatment. The cell images were obtained using bright-field microscopy without any staining. (I) Lactate content assay, glucose content assay and ATP content assay of HepG2 cells with LRPPRC^WT and LRPPRC^K326R mutation treatment. (J) The ECAR analyses of HepG2 cells with LRPPRC^WT and LRPPRC^K326R mutation treatment. Data are presented as means ± SEM of 5 replicates. *, P≤0.05; **, P≤0.01; ***, P≤0.001. IP, immunoprecipitation; LRPPRC, leucine-rich pentatricopeptide repeat-containing protein; OD, optical density; SEM, standard error of the mean; WT, wild type.

Mice with LRPPRCK326R mutation revealed a profound dysregulation in M1/M2 macrophage polarization dynamics

Lactate-mediated lysine lactylation, a PTM, epigenetically regulates immune cell functionality, serving as a critical mechanistic link between tumor metabolic reprogramming and immunosuppression. Comparative analysis of immune microenvironment composition revealed that HCC patients exhibit significantly elevated infiltration of M0/M1/M2 macrophages, activated CD8⁺ T cells, dendritic cells (DCs), and monocytes compared to healthy controls (Figure 4A,4B). Warburg effect-driven lactate accumulation remodels the tumor immune landscape via lactylation-dependent pathways, with macrophage polarization emerging as a central regulatory node. Notably, we observed substantial enrichment of TAMs in HCC tissues, aligning with their dual roles in promoting immunosuppression through M2-like polarization and sustaining pro-tumorigenic inflammation via M1-like activation states.

Figure 4.

Mice with LRPPRC^K326R mutation revealed a profound dysregulation in M1/M2 macrophage polarization dynamics. (A,B) Immune cell infiltration analysis using CIBERSORT for the GSE263786 and GSE233421 cohorts. (C) Western blot analysis of lactylation of LRPPRC in LRPPRC^WT mice and LRPPRC^K326R mutation mice. (D) qPCR analysis of F4/80 and CD11d expression in Kupffer cells of LRPPRC^WT mice and LRPPRC^K326R mutation mice. (E,F) Flow cytometry analysis CD11c⁺ and CD206⁺ in F4/80⁺CD11b⁺ cells percentage. (G) qPCR analysis of Arg1 and Fizz1 in M2-typed macrophages. (H) qPCR analysis of CD11c and iNOS in M1-typed macrophages. Data are presented as means ± SEM of 6 replicates. *, P≤0.05; **, P≤0.01; ***, P≤0.001. HCC, hepatocellular carcinoma; iNOS, inducible nitric oxide synthase; LRPPRC, leucine-rich pentatricopeptide repeat-containing protein; NK, natural killer; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; WT, wild type.

To explore the precise role of LRPPRC-Kla³²⁶ lactylation in the macrophage polarization, we generated a mouse model in which LRPPRC-Kla³²⁶ was mutated to a nonacetylation alanine through a Cas9-mediated site mutagenesis. The knocking mutation did not affect expression of LRPPRC in BMDM of mice with LRPPRC^K326R mutation (Figure 4C). As expected, this mutation prevented lactylation of LRPPRC recognized by pan-Kla antibody in the knock-in mice (Figure 4C).

To verify the potential regulatory role of LRPPRC in hepatic macrophages, we performed qRT-PCR to examine the mRNA expression levels of macrophage-associated markers in liver tissues. The mRNA expression levels of macrophage markers F4/80 and CD11b in the livers of LRPPRC^K326R mice were significantly increased (Figure 4D). Additionally, M1-type macrophages (stained with CD11c) and M2-type macrophages (stained with CD206) were detected and quantified via flow cytometry. In LRPPRC^K326R mice, M2-type macrophages, but not M1-type macrophages, were markedly decreased (Figure 4E,4F). Hepatic macrophages isolated from the livers of both genotypes exhibited a mixed M1/M2 phenotype. Compared to LRPPRC^WT mice, the expression of M2-type Kupffer cell markers, including Arg1 and Fizz1, was significantly downregulated in LRPPRC^K326R mice. However, the expression of iNOS and CD11c in M1-type Kupffer cells was upregulated (Figure 4G,4H). Collectively, these findings suggest that M1/M2 macrophage polarization is regulated by lactylation at the K326 residue of LRPPRC.

Lactylation of LRPPRC regulates macrophage polarization by reprogramming glycolytic metabolism

The role of LRPPRC-Kla³²⁶ lactylation in macrophage characterization and glycolysis during direct M0→M1/M2-like TAM polarization and effector functions remains poorly understood. To investigate this, BMDMs were isolated from LRPPRC^WT and LRPPRC^K326R mice and polarized into M2-type macrophages with IL-4 or M1-type macrophages with LPS + IFN-γ. Exposure to D-lactate enhanced M2 polarization (increased CD206⁺) and suppressed M1 polarization (decreased CD11c⁺) in BMDMs from LRPPRC^WT mice, but this pro-M2/anti-M1 effect was abolished in BMDMs from LRPPRC^K326R mice (Figure 5A,5B). This phenotypic reprogramming was further corroborated by marked downregulation of M2-associated markers, including Arg1 in mice with LRPPRC^K326R mutation (Figure 5C). In contrast, the expression of iNOS in M1-type macrophages was markedly induced (Figure 5D). Furthermore, IL-10 secretion levels were decreased in M2-type macrophages, whereas TNF-α levels were elevated in M1-type macrophages (Figure 5E,5F). These findings suggest that loss of lactylation at K326 impairs M2 polarization while promoting M1 polarization.

Figure 5.

Lactylation of LRPPRC regulates macrophage polarization by reprogramming glycolytic metabolism. (A) Flow cytometry analysis CD206⁺ in F4/80⁺CD11b⁺ cells percentage in IL-4 induced M2-typed macrophages treat with D-lactate. (B) Flow cytometry analysis CD11c⁺ in F4/80⁺CD11b⁺ cells percentage in LPS + IFN-γ induced M1-typed macrophages treat with D-lactate. (C) qPCR analysis of Arg1, expression in IL-4 induced M2-typed macrophages treat with D-lactate. (D) qPCR analysis of iNOS expression in LPS + IFN-γ induced M1-typed macrophages treat with D-lactate. (E) Quantification of secreted cytokine TNF-α levels in M1typed macrophages treat with D-lactate. n=7 per group. (F) Quantification of secreted cytokine IL-10 levels in M2-typed macrophages treat with D-lactate. n=7 per group. (G) Lactate content assay, glucose content assay and ATP content assay of IL-4 induced M2-typed macrophages. (H) The ECAR analyses of IL-4 induced M2-typed macrophages. Data are presented as means ± SEM of 6 replicates. ***, P≤0.001; n.s., not significant. BMDM, bone marrow-derived macrophage; CTL, control without D-lactate; DL, D-lactate; ECAR, extracellular acidification rate; IFN-γ, interferon-gamma; IL, interleukin; KI, knock-in; LPS, lipopolysaccharide; LRPPRC, leucine-rich pentatricopeptide repeat-containing protein; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; TNF-α, tumor necrosis factor-alpha; WT, wild type.

To assess glycolytic activity, the Seahorse XF extracellular flux analyzer was employed to measure the ECAR of BMDMs. Macrophages from LRPPRC^K326R mice significantly reduced the ECAR (Figure 5G). Additionally, key glycolytic indicators including glucose consumption, intracellular lactate production, and ATP generation were substantially diminished in LRPPRC^K326R mice (Figure 5H). Collectively, these findings establish that lactylation at the K326 site of LRPPRC critically regulates macrophage polarization by reprogramming glycolytic metabolism, thereby orchestrating an immunosuppressive TME in HCC.

Lactylation of LRPPRC is necessary for immune-suppressive activity, and M2 TAM-dependent tumor progression

Given the established role of M2 macrophages in suppressing antitumor immunity to promote tumor progression, we employed an in vivo tumor admixture model to assess the functional requirement of LRPPRC lactylation in M2 macrophage-mediated oncogenesis. LLC cells were co-injected with either LRPPRC^WT or LRPPRC^K326R M2-polarized BMDMs (CD45.2⁺) into congenic B6.SJL mice (CD45.1⁺). Tumors co-injected with LRPPRC^WT M2-BMDMs exhibited accelerated tumor growth (Figure 6A,6B) and significantly higher endpoint tumor mass (Figure 6C) compared to LLC-only controls. In stark contrast, tumors co-injected with LRPPRC^K326R M2-BMDMs showed markedly attenuated tumor outgrowth and reduced tumor burden relative to LRPPRC^WT M2-BMDM cohorts.

Figure 6.

Lactylation of LRPPRC is required for M2 macrophage polarization and tumor progression. (A) Schematic work flow of in vivo tumor admixture model. (B,C) Growth and weight of tumors consisting of LLC cells only, LLC cells + M2-polarized LRPPRC^WT BMDMs, or LLC cells + M2-polarized LRPPRC^K326R mutation BMDMs. (D,E) IFN-γ production in CD8⁺ cytotoxic T cells and CD4⁺ helper T cells in the respective tumors. Data are presented as means ± SEM of 6 replicates. **, P≤0.01; ***, P≤0.001. BMDM, bone marrow-derived macrophage; IFN-γ, interferon-gamma; LLC, Lewis lung carcinoma; LRPPRC, leucine-rich pentatricopeptide repeat-containing protein; SEM, standard error of the mean; WT, wild type.

Flow cytometric profiling of tumor-infiltrating immune cells revealed a significant increase in IFN-γ⁺CD8⁺ cytotoxic T cells and IFN-γ⁺CD4⁺ helper T cells in tumors harboring LRPPRC^K326R M2-BMDMs compared to LRPPRC^WT M2-BMDM groups (Figure 6D,6E). These data demonstrate that LRPPRC lactylation is indispensable for the immunosuppressive activity of tumor-associated M2 macrophages. Abrogation of lactylation at K326 disrupts TAM-mediated immune suppression, thereby unleashing robust antitumor T cell responses and impeding HCC progression (Figure 7).

Figure 7.

LRPPRC-Kla326 glycolytic metabolism to M2-TAM polarization, driving immune evasion in HCC. GLUT, glucose transporter; HCC, hepatocellular carcinoma; Lactoyl-CoA, lactoyl-coenzyme A; LRPPRC, leucine-rich pentatricopeptide repeat-containing protein; MCT, monocarboxylate transporter; TAM, tumor-associated macrophage.

Discussion

The present study elucidates a pivotal role of LRPPRC lactylation at lysine 326 (Kla³²⁶) in orchestrating metabolic-immune crosstalk within the HCC microenvironment. By integrating multi-omics analyses with functional validation, we demonstrate that lactate-driven LRPPRC-Kla³²⁶ serves as a molecular switch coupling glycolytic reprogramming to M2-like macrophage polarization, thereby establishing an immunosuppressive niche conducive to tumor progression. These findings not only resolve a long-standing knowledge gap regarding how lactate metabolites directly modulate non-histone lactylation to regulate immune evasion but also position LRPPRC-Kla³²⁶ as a novel therapeutic target for disrupting metabolic symbiosis in HCC.

Our results establish that Warburg effect-derived lactate induces LRPPRC lactylation at K326, which functionally reprograms glycolytic metabolism in HCC cells while simultaneously skewing TAMs toward immunosuppressive M2 phenotypes. Mechanistically, LRPPRC-Kla³²⁶ enhances mitochondrial transcription stability and the coupling between OXPHOS and glycolysis as critical upstream events that orchestrate immunometabolic reprogramming in the TME. LRPPRC, canonically known for stabilizing mitochondrial mRNAs encoding OXPHOS complex subunits, presents a paradox in the glycolytic context of HCC. Our data suggest that lactylation at K326 may functionally repurpose this protein. Instead of solely bolstering OXPHOS, lactylated LRPPRC appears to instigate a metabolic flux diversion. We hypothesize that Kla326 alters LRPPRC’s interaction with its mitochondrial RNA targets or associated proteins, potentially leading to a compromised assembly of ETC complexes. This incomplete or dysfunctional OXPHOS would not only be consistent with the Warburg phenotype but also generate mitochondrial retrograde signals that activate cytosolic glycolytic enzymes, thereby coupling impaired mitochondrial respiration to enhanced glycolytic flux. This realigned metabolic flux, resulting in exaggerated lactate production, creates a self-amplifying feedforward loop: lactate lactylates more LRPPRC, which further skews metabolism towards glycolysis.

This metabolic reprogramming directly correlates with impaired CD8⁺/CD4⁺ T-cell infiltration and M2-TAM dominance, as evidenced by in vivo tumor admix models where LRPPRC^K326R mutation reversed immune suppression and attenuated tumor growth. Crucially, the Kla³²⁶ site specificity was validated through site-directed mutagenesis and lactylation profiling, confirming its indispensability in mediating metabolic-immune crosstalk.

Our findings align with emerging paradigms of lactate as an epigenetic modulator beyond its metabolic roles. While previous studies established histone lactylation as a regulator of macrophage polarization (19), we extend this concept by identifying LRPPRC as the first mitochondrial protein of which its lactylation directly governs both tumor metabolism and immune remodeling. This dual functionality contrasts with prior reports focusing solely on lactate’s cell-autonomous effects in cancer cells or its paracrine immunosuppression via pH modulation (5). The observed LRPPRC-mediated PD-L1 stabilization and M2-TAM polarization synergize with recent work demonstrating lactate’s role in enhancing immune checkpoint expression, suggesting a unified mechanism whereby lactylation coordinates metabolic adaptation and immune evasion. However, our data diverge from studies emphasizing lactate’s pro-inflammatory effects in early tumorigenesis, highlighting context-dependent roles of lactate signaling across cancer stages.

Conclusions

This study establishes LRPPRC lactylation as a linchpin connecting glycolytic metabolism to immune suppression in HCC. Therapeutically, our findings support developing LRPPRC-Kla³²⁶ inhibitors to simultaneously target tumor metabolism and immune evasion—a strategy potentially overcoming limitations of single-pathway inhibitors. Our work advances the emerging field of lactylation biology while offering tangible strategies to overcome immunotherapy resistance in metabolically aggressive cancers.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Experiments were performed under a project license (No. 20240125032) granted by Animal Experiment Committee of An Hui Medical Early Diagnosis (AHMED), in compliance with Chinese guidelines for the care and use of animals.

Data Sharing Statement

Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2533/dss

tcr-15-03-148-dss.pdf ^{(70KB, pdf)}

DOI: 10.21037/tcr-2025-aw-2533