Graphical Abstract
INTRODUCTION
Liver cancer, particularly hepatocellular carcinoma (HCC) which accounts for about 90% of cases, is a significant global health challenge with a high fatality rate. Despite significant advancements in locoregional and systemic therapies, a substantial proportion of patients fail to respond adequately and ultimately succumb to the disease [1]. Therefore, the development of more effective treatment options remains imperative. Transmembrane 4 L six family member 1 (TM4SF1), known as tumor-associated antigen L6, has the capacity to engage in interactions with integrins, receptor tyrosine kinases, collagens and other proteins, facilitating the formation of tetraspanin-enriched microdomains. These interactions enable TM4SF1 to drive tumor angiogenesis, cell proliferation, and migration, underscoring its critical role in tumor progression [2,3]. TM4SF1 is significantly upregulated across multiple cancer types, including HCC [4]. Although extensively studied in epithelial malignancies, its precise role and underlying mechanisms in HCC have yet to be thoroughly elucidated.
Cellular senescence represents a state of proliferation arrest in response to endogenous or exogenous stressors. This cessation of cell cycle is predominantly facilitated by p16INK4a (encoded by CDKN1A) and p21CIP1 (encoded by CDKN2A) [5]. In addition, senescent cells often exhibit upregulation of p19ARF, p27, p53,and the loss of lamin B1. A subset of senescent cells, known as the senescence-associated secretory phenotype (SASP), actively secretes growth factors, cytokines, chemokines and proteases. Senescence in tumor cells is a double-edged sword. It halts tumor proliferation and SASP-associated cytokines, such as tumor necrosis factor-alpha (TNF-α), can induce reactive oxygen species-dependent apoptosis in tumor cells [6]. Moreover, senescent tumor cells can induce senescence in neighboring cells through SASPs and direct cell-to-cell interactions [7,8]. However, certain SASP-factors including IL-6, IL-8, CXCL5 and MMP7 contribute to tumorigenesis, invasion, migration and immune evasion [9-11]. In HCC, senescence in tumor cells has been shown to suppress proliferation and is modulated by various factors like ammonia metabolism, methionine metabolism and histone melthyltransferase [12-14]. Conversely, senescence in hepatic stellate cells and hepatocytes contributed to HCC promotion by SASP factors [15,16]. These studies suggest the ambiguous role of senescence in HCC, highlighting the need for further investigation to elucidate its context-dependent contributions to tumor progression.
Given the complicated intrinsic features of senescent tumor cells, they simultaneously influence tumor behavior and reshape the immune microenvironment. Through SASP, senescence can drive tumors toward either progression or elimination. However, the impact of non-secretory phenotypic senescence in tumor microenvironment remains poorly understood. In clinical practice, immunotherapies such as anti-programmed death 1 (anti-PD-1) have been widely utilized. The impact of senescence on immunotherapy efficacy remains debatable. Some studies revealed that the efficacy of immunotherapy was significantly affected by senescent tumor cells in a tissue-specific manner [17,18]. Notably, senescence in tumor cells has been reported to enhance immune surveillance and the efficacy of immunotherapy in HCC [19]. However, the interplay between immunotherapy and non-secretory phenotypic senescence, particularly in the context of HCC, has rarely been explored. In this study, we demonstrate the progressive role of TM4SF1 in HCC from the perspective of tumor senescence and immunotherapy for the first time. TM4SF1 inhibits non-secretory phenotypic senescence in liver cancer by recruiting PDPK1 and AKT1 to interact with each other, inducing upregulation of programmed cell death ligand 1 (PD-L1), downregulation of major histocompatibility complex class I (MHC I) in tumors, and promoting immune escape in liver cancer. Targeting TM4SF1 inhibits tumor progression and alleviates the suppressive immune microenvironment in HCC, enhancing immunotherapy efficacy.
MATERIALS AND METHODS
Public datasets and patient samples
GSE14520, GSE10143, GSE76297 and GSE32879 were obtained from the Gene Expression Omnibus. Non-HCC tumor samples were excluded. ERP117672 was obtained from European Nucleotide Archive. RNA-seq and clinical data from The Cancer Genome Atlas (TCGA) were obtained from Genome Data Commons data portal. All samples were collected postoperatively from patients with a confirmed pathological diagnosis of primary HCC who had not undergone any preoperative treatment. The samples were obtained from the Hepatic Surgery Center of Tongji Hospital. The first cohort (Tongji Cohort) consists of 121 clinical patients between 2012 and 2016, in which cancer and adjacent tissues were used to create tissue microarrays for immunohistochemical (IHC) analysis. All these patients had complete follow-up data. The second cohort (prospective cohort 1) includes 50 postoperative samples from HCC patients between 2019 and 2023. Tumor and adjacent tissue were used for IHC analysis and flow cytometry. The third cohort (prospective cohort 2) includes 14 postoperative samples from HCC patients between 2023 and 2024. Tumors and adjacent tissues were used for IHC analysis. Clinical data and magnetic resonance imaging (MRI)/computed tomography (CT) images were acquired from medical record system and classified. Clinicopathological characteristics of patients in the Tongji and prospective cohorts are provided in Supplementary Tables 1–3. Sample collection, utilization, storage, and destruction processes were supervised by the Medical Ethics Committee of Tongji Hospital (TJ-IRB20221136).
Mice and bioluminescent imaging
Male BALB/c nude mice and C57BL/6 mice (both 6–8 weeks old) were purchased from GemPharmatech Co., Ltd (Jiangsu, China). For subcutaneous model, nude mice were injected with 1×106 MHCC97H-luc or HepG2 cells. For orthotopic model, nude mice were injected with 1×106 MHCC97H-luc cells in the left lobe of liver. Subcutaneous tumors were measured every 3 days. Nude mice for subcutaneous were sacrificed on day 21. Nude mice orthotopically model were sacrificed on day 30. C57BL/6 mice were used to establish hydrodynamic tail vein injection (HTVi) HCC model. Plasmids were diluted in 2 mL warmed sterile PBS and rapidly injected into tail veins. For HTVi model, mice were sacrificed at day 30 [20]. In treatment experiment, self-complementary adeno-associated virus (scAAV) were injected intravenously. Anti-PD-1 or IgG were administered intraperitoneally (BE0146 and BE0089; BioXcell, West Lebanon, NH, USA). Before bioluminescent imaging, mice were anesthetized with isoflurane and injected peritoneally with D-Luciferin. Bioluminescence was imaged by IVIS Spectrum (PerkinElmer, Waltham, MA, USA) or LAGO system (Spectral Instruments Imaging, Tucson, AZ, USA). All animal experiments are approved by the Committee on the Ethics of Animal Experiments of Tongji Hospital (TJH-202304029).
Statistical analysis
All data analysis and figure plotting were performed using GraphPad Prism (version 8.0.2; GraphPad Software Inc., San Diego, CA, USA) or R software (version 4.1.0; R Foundation for Statistical Computing, Vienna, Austria). Values are represented as the mean±standard deviation. The log-rank test was used to assess the prognostic significance of the Kaplan–Meier curves. Significance in bar chart was determined using the Student’s t-test. Correlations were assessed by Pearson correlation analysis.
RESULTS
TM4SF1 promotes the progression of HCC
To explore the role of TM4SF1 in HCC, we conducted analysis in public databases and found a significant upregulation of TM4SF1 expression in tumor tissues across multiple HCC datasets (Fig. 1A, Supplementary Fig. 1A). Additionally, in the GES14520 dataset, patients with high TM4SF1 expression had worse overall survival (OS) and disease-free survival (DFS) (Fig. 1B), and similar results were confirmed in TCGA-LIHC dataset (Supplementary Fig. 1B). Immunohistochemistry (IHC) analysis of the HCC microarray from the Tongji cohort revealed that tumor tissues exhibited higher TM4SF1 expression than adjacent non-tumor tissues, with its localization predominantly on the cell membrane (Fig. 1C1). Meanwhile, in prospective cohort 1 from Tongji hospital, flow cytometry analysis of resected surgical samples confirmed the high expression of TM4SF1 in HCC tissues (Fig. 1C2). In addition, patients with high TM4SF1 expression indicated poorer OS and DFS (Fig. 1D). These different detection methods consistently demonstrated the high expression of TM4SF1 in HCC cells. To further understand the role of TM4SF1 in HCC, we examined several cell lines including MHCC97H, HepG2, Huh7 and HLF, which exhibit higher malignancy in HCC [21,22]. We confirmed elevated TM4SF1 expression in these cell lines relative to normal liver cells (Fig. 1E). Based on varying expression of TM4SF1, we selected MHCC97H and HLF for further investigation. A lentivirus was used to overexpress TM4SF1, and successful overexpression was confirmed by flow cytometry (Supplementary Fig. 1C). The effects of TM4SF1 on HCC were investigated. Overexpression of TM4SF1 significantly enhanced tumor cell proliferation, as shown by CCK-8 assay, colony formation assay, EdU assay and cell cycle analysis (Supplementary Fig. 1D–G). Additionally, TM4SF1 enhanced the anti-apoptotic ability of tumor cells (Supplementary Fig. 1H). This proliferative effect was further validated in a nude mouse subcutaneous tumor model and an orthotopic HCC model injected with luciferase-expressing MHCC97H cells (Supplementary Fig. 1I1, 1I2). In contrast, TM4SF1 knockdown markedly inhibited HCC cell growth in vitro and in vivo (Supplementary Fig. 2A–F). Overall, these findings indicate that TM4SF1 is overexpressed in HCC tissues and plays a key role in driving tumor progression.
TM4SF1 is associated with tumor cell senescence
To investigate the mechanism of TM4SF1-driven HCC progression, we performed transcriptome sequencing on TM4SF1 knockdown cell lines. GESA revealed significant enrichment in the Fridman senescence pathway in TM4SF1 knockdown groups. CDKN1A (encoding p21) and CDKN2A (encoding p16), markers of cellular senescence, were significantly upregulated following TM4SF1 knockdown (Fig. 2A). TM4SF1 overexpression decreased the proportion of cells in the G0/G1 phase (Supplementary Fig. 1G), whereas TM4SF1 knockdown induced G0/G1 phase arrest, a hallmark of senescence (Supplementary Fig. 2F). To confirm senescence, senescence-associated beta-galactosidase (SA-β-gal) staining was performed, showing an increase in number of senescent cells in TM4SF1 knockdown HCC cells (Fig. 2B, Supplementary Fig. 2G). However, SASP factors, including IL-6, IL-8, CCL-2, IL-1β, and IL-1α showed no significant changes following TM4SF1 knockdown (Supplementary Fig. 2H). We performed IHC analysis on tissues from the orthotopic HCC model. Consistent with the in vitro results, we observed increased protein levels of p21 and p16 following TM4SF1 knockdown, along with a decreased Ki-67 index (Fig. 2C). Additionally, SA-β-gal staining confirmed an increase in senescent tumor cells after TM4SF1 knockdown (Fig. 2C). Further analysis of TM4SF1 and tumor cell senescence in HCC tissue microarrays from the Tongji cohort revealed a negative correlation between TM4SF1 expression and senescence, as evidenced by p21 and p16 IHC staining (Fig. 2D, E). These findings suggest that reduced TM4SF1 expression induces tumor cell senescence, leading to slowed proliferation. However, further studies are required to elucidate the mechanisms by which TM4SF1 mediates this effect.
TM4SF1 mediates the phosphorylation of AKT through its interaction with AKT1 and PDPK1
To investigate how TM4SF1 affects HCC progression and cellular senescence, we performed immunoprecipitation mass spectrometry (IP-MS) to identify TM4SF1-interacting proteins. Silver staining confirmed high TM4SF1 expression, and IP-MS revealed AKT1 and PDPK1 as the top interactors (Supplementary Fig. 3A). Given the crucial roles of the AKT pathway and the molecular chaperone PDPK1 in cellular proliferation and senescence [23-25], we hypothesized that TM4SF1 potentially regulated the AKT pathway and influenced tumor cell senescence. First, we validated the interaction between exogenous TM4SF1 and AKT1, as well as PDPK1, by bimolecular fluorescence complementation (BiFC) assays and co-immunoprecipitation (co-IP) (Fig. 3A, Supplementary Fig. 3B). Immunofluorescence (IF) revealed the co-localization of TM4SF1 with AKT1 and PDPK1 in HEK293T cells transfected with exogenous plasmids (Supplementary Fig. 3C1). To detect endogenous interactions, we used cell lines overexpressing Flag-tagged TM4SF1. IF analysis confirmed the interactions between TM4SF1, AKT1, and PDPK1 (Supplementary Fig. 3C2).
The phosphorylation of AKT1 at S473 residue, facilitated by its binding to PDPK1, is crucial in the proliferation signaling pathway [26]. The interaction between TM4SF1, AKT1 and PDPK1 implies a link to AKT1 phosphorylation. Knockdown of TM4SF1 in MHCC97H and HLF cells led to a significant decrease in p-AKT-S473, and a significant upregulation of the senescence-related markers p21 and p16 (Fig. 3B1). These findings were further confirmed in the orthotopic tumor sections (Fig. 3B2, Supplementary Fig. 3D). Meanwhile, we assessed the phosphorylation levels of p38, p65, and STAT3, which are key modulators in SASP factors secretion [27]. However, no significant changes were observed in these pathways (Supplementary Fig. 3E). Further investigation of the regulatory relationship between PDPK1 and TM4SF1 in AKT1 phosphorylation revealed that PDPK1 did not affect TM4SF1 expression (Supplementary Fig. 3F). Knockdown of TM4SF1, even with overexpressed PDPK1, inhibited AKT1 phosphorylation and induced cellular senescence (Supplementary Fig. 3G1, 3H). Conversely, overexpression of TM4SF1 in cells with PDPK1 knockdown did not lead to changes in AKT1 phosphorylation or senescence (Supplementary Fig. 3G1, 3H). In addition, CCK-8 assay revealed impaired proliferation following TM4SF1 or PDPK1 knockdown (Supplementary Fig. 3G2). These findings suggest that the regulation of AKT1 phosphorylation and HCC cell senescence by TM4SF1 requires the involvement of PDPK1.
Further examination of the interaction domains between TM4SF1 and AKT1, as well as PDPK1, revealed that AKT1 interacted with the N-terminus of TM4SF1, whereas PDPK1 interacted with the C-terminus (Supplementary Fig. 4A–4C). Based on this, we hypothesized that TM4SF1 might mediate the interaction between AKT1 and PDPK1 through different regions. TM4SF1 was overexpressed exogenously in HEK293T cells. The interaction between AKT1 and PDPK1, as assayed by co-IP, confirmed the positive role of TM4SF1 (Fig. 3C1). Endogenous co-IP detection further confirmed that the overexpression or knockdown of TM4SF1 enhanced or inhibited the binding of AKT1 to PDPK1, respectively (Fig. 3C2, Supplementary Fig. 4D). To further validate the regulation of p-AKT-S473 by TM4SF1, we utilized patient-derived organoids (PDO). Expression of Glypican-3 (GPC3) and TM4SF1 was examined in PDO (Fig. 3D). Organoids were infected with lentivirus to knock down TM4SF1. Decreased Ki-67 index and organoid area indicated impaired proliferation (Fig. 3E1). Using multiplex immunohistochemistry (mIHC), we detected lower levels of pAKT-S473 and higher levels of p21 and p16 (Fig. 3E2). Taken together, these results indicate that TM4SF1 mediates tumor proliferation and senescence through the PDPK1/AKT/p21-p16 pathway.
This hypothesis was validated by treating TM4SF1-overexpressing cell lines with MK2206, an AKT1 inhibitor. The results showed that inhibition of the AKT pathway reversed the characteristics of TM4SF1 promoting HCC progression and inducing cellular senescence, in both control and TM4SF1-overexpressing HCC cells (Supplementary Fig. 5A–5C). Conversely, the restoration of AKT phosphorylation by exogenous AKT1 supplementation completely reversed the impaired proliferation and senescence induced by TM4SF1 knockdown (Supplementary Fig. 5D–5F). These findings were also validated in the orthotopic tumor model (Fig. 3F1, 3F2, Supplementary Fig. 5G). In conclusion, our data demonstrates that TM4SF1 recruits PDPK1 and AKT1 to promote tumor cell proliferation while inhibiting senescence.
TM4SF1 suppresses anti-tumor immunity
In tumor progression, the immune microenvironment plays a pivotal role. Given the complex regulatory role of tumor cell senescence in the immune microenvironment, we further investigated TM4SF1 in immunocompetent mouse. First, we found that Tm4sf1 expression was elevated in tumor tissues compared to that in normal liver tissues across multiple mouse HCC models, with the highest expression level detected in HTVi-induced model by injection of oncogenes (MYC+CCND1) (Supplementary Fig. 6A). Based on HCC model of this combination, we designed a strategy using three shRNA constructs to target and knock down Tm4sf1 (Fig. 4A). Bioluminescence intensity and gross tumor images indicated a reduced tumor burden in the Tm4sf1 knockdown group (Fig. 4B). mRNA analysis showed that the suppression of Tm4sf1 triggered an increase in Cdkn1a and Cdkn2a expression (Supplementary Fig. 6B). IHC and SA-β-gal staining further confirmed that silencing TM4SF1 induced senescence in the tumors and slowed tumor progression, consistent with previous findings (Fig. 4C, Supplementary Fig. 6C).
Since the role of tumor-derived TM4SF1 within the tumor microenvironment remains unclear, we performed flow cytometry analysis of tumor-infiltrating immune cells. Our results revealed that TM4SF1 knockdown increased the proportion of T cells among the total immune cells (Fig. 4D1). The proportions of major immune cell subgroups, including myeloid-derived suppressor cells (MDSCs), macrophages and conventional dendritic cells (cDCs), were partially increased. However, the proportions of B cells and plasmacytoid dendritic cells (pDCs) showed no significant changes between the control and knockdown groups (Fig. 4D1). Analysis of T cell subgroups revealed no significant changes in the ratio of CD8+ or CD4+ T cells. However, CD8+ T cells in the knockdown group exhibited higher proportions of Granzyme B+ and IFN-γ+ T cells, along with fewer PD-1+ T cells, indicating enhanced cytotoxic function and reduced exhaustion (Fig. 4D2). In TM4SF1 knockdown group, CD8+ T cells transitioned from naive state to effectors (Fig. 4D2). The immunosuppressive Foxp3+CD4+ subgroup did not show significant changes (Fig. 4D2). This implies that suppression of TM4SF1 may hinder tumor progression by increasing the number of CD8+ T cells while preserving their cytotoxic function.
Previous studies have reported that elevated AKT phosphorylation promotes PD-L1 expression while downregulating MHC I [28,29]. Both are closely related to immune cell recognition and exhaustion, particularly in CD8+ T cells. Therefore, we further investigated whether AKT1 phosphorylation, which is regulated by TM4SF1, affected the expression of PD-L1 and MHC I. In MHCC97H and HLF cell lines, TM4SF1 knockdown reduced PD-L1 expression and increased MHC I expression, whereas TM4SF1 overexpression had the opposite effect (Fig. 4E, Supplementary Fig. 6D). In TM4SF1 knockdown HCC cells, the exogenous AKT1 supplementation reversed the effect on PD-L1 and MHC I expression (Fig. 4F, Supplementary Fig. 6E). These findings suggest that TM4SF1 not only inhibits tumor senescence and promotes tumor proliferation but also influences the tumor microenvironment by modulating the expression of PD-L1 and MHC I in tumor cells through the AKT-senescence pathway.
TM4SF1 suppresses anti-tumor immunity by directly inducing CD8+ T cell exhaustion
The expression of PD-L1 and MHC I in tumor cells directly affects CD8+ T cell function. Therefore, we assessed the impact of tumor-derived TM4SF1 on CD8+ T cells in vitro. Human CD8+ T cells were isolated and co-cultured with HCC cells (Fig. 5A). The results demonstrated that, compared to the control group, the survival rate of TM4SF1-overexpressed HCC cells was significantly improved, while the reduction in survival was found in TM4SF1-knockdown HCC cells (Fig. 5B). These findings suggested that tumor cells with high TM4SF1 expression evaded CD8+ T cell-mediated cytotoxicity. The overexpression of TM4SF1 in tumor cells significantly impaired CD8+ T cell cytotoxicity, evidenced by reduced GZMB, TNF-α, and IFN-γ, and induced exhaustion, while inducing exhaustion, marked by increased PD-1 and TIM-3 expression (Fig. 5C). Additionally, it notably inhibited the proliferation of CD8+ T cells (Fig. 5C, Supplementary Fig. 7A, 7B). These findings demonstrate that TM4SF1 promotes tumor progression by directly impairing the function of CD8+ T cells. Finally, HCC tissues from prospective cohort 1 (n=50) were analyzed by IHC. Patients with low TM4SF1 expression (P-092 and P-081) exhibited higher levels of p16, p21, and MHC I, but decreased p-AKT-S473 and PD-L1 expression. In contrast, patients with high TM4SF1 expression (P-067 and P-096) exhibited the opposite pattern (Fig. 5D). Furthermore, TM4SF1 expression was positively correlated with proliferation (Fig. 5E), while p-AKT-S473 level was negatively correlated with p21, p16, and MHC I expression and positively correlated with PD-L1 expression (Fig. 5F). These findings corroborate our previous results. Therefore, we propose that TM4SF1 compromises the anti-tumor immune response of CD8+ T cells through the PDPK1/AKT1/PD-L1-MHC I pathway.
Targeting TM4SF1 inhibits HCC progression in vivo
Previous results elucidated the critical role of TM4SF1 in HCC. To further explore its therapeutic potential, we employed both immunodeficient and immunocompetent mouse models. Firstly, an orthotopic HCC model was established in nude mice. The scAAV-DJ serotype was designed to decrease the TM4SF1 expression in tumors (Fig. 6A). The results showed that TM4SF1 was successfully knocked down in vivo (Supplementary Fig. 8A1). The tumor burden in AAV-treated mice was significantly reduced compared to that in control mice (Fig. 6B). IHC staining of tumor tissue showed decreased TM4SF1 expression, along with reductions in p-AKT-S473, Ki-67 and PD-L1 levels. Meanwhile, senescence markers p21 and p16 were upregulated and SA-β-gal staining further confirmed the induction of tumor senescence in the treatment group (Fig. 6C, Supplementary Fig. 8A2). These findings in the nude mouse model validated the feasibility of AAV-based treatment. Next, we utilized the HTVi-induced HCC model in immunocompetent mice. Two weeks after injecting oncogenes (MYC+CCND1), we administered scAAV-8 to knock down TM4SF1 and combined it with anti-PD-1 treatment (Fig. 6D). Analysis of bioluminescence images (Fig. 6E1) and tumor burden (Fig. 6E2) revealed that both anti-PD-1 and AAV treatment alone could suppress tumor progression, with the combined treatment group demonstrating the most potent anti-tumor effect. Serum alanine aminotransferase and aspartate aminotransferase levels indicated a reduction in tumor burden following treatment, with the combination therapy group showing the greatest decrease (Supplementary Fig. 8B). Successful AAV infection was confirmed by zsGreen staining (Fig. 6F), and Tm4sf1 expression was effectively knocked down at the mRNA level. All treatment groups demonstrated increased Cdkn1a and Cdkn2a expression, with the combination therapy group showing the most pronounced increase (Supplementary Fig. 8C). AKT phosphorylation level and Ki-67 index decreased across all treatment groups, with the most pronounced reduction in the combined group. p21 and p16 levels were markedly higher in the combination treatment group than in the monotherapy groups (Fig. 6F, Supplementary Fig. 8D). Notably, tumor senescence was observed exclusively in AAV treatment group, and not in immunotherapy alone (Fig. 6F). These results indicate that targeting TM4SF1 effectively induces senescence to inhibit HCC progression, while the combined therapy with anti-PD-1 enhances this effect synergistically.
Targeting TM4SF1 sensitizes anti-PD-1 immunotherapy in HCC
To further investigate the synergistic effect of targeting TM4SF1 and anti-PD-1, our analysis focused on T cells, based on previous results that TM4SF1 accelerated CD8+ T cell exhaustion and impaired cytotoxic function. The results revealed that the treatment group had a significantly higher proportion of tumor-infiltrating T cells compared to the control group, with the combined therapy showing a more pronounced increase than either monotherapy (Fig. 7A1). In other subgroups, the proportions of B cells, macrophages, and cDCs increased following AAV monotherapy, whereas the proportion of pDCs remained unchanged across all treatment groups. Only MDSCs demonstrated a proportional increase following various treatments (Supplementary Fig. 8E). No significant changes were observed in the proportion of CD8+ or CD4+ T cells across various treatments, which is consistent with previous findings (Supplementary Fig. 8F). Besides, there was a notable reduction in naïve CD8+ T cells, with a trend of differentiation towards effector T cells in treatment groups, and the most pronounced shift was observed in the combined therapy group (Fig. 7A1). Moreover, the combination therapy group exhibited enhanced cytotoxic function and a reduced exhaustion status compared to monotherapy groups (Fig. 7A2). mIHC staining revealed that the combination therapy group had the highest proportion of GZMB+CD8+ cells and the lowest proportion of PD-1+CD8+ cells (Fig. 7B, Supplementary Fig. 9A). Collectively, these results demonstrate that the combined treatment enhances anti-PD-1 immunotherapy by increasing the total infiltrating CD8+ T cells and preserving their cytotoxic function.
To translate these findings into clinical practice, we examined prospective cohort 2 (n=14), consisting of HCC patients who underwent radical resection followed by anti-PD-1 immunotherapy. Based on relapse, patients were categorized into responsive and non-responsive groups. Follow-up MRI or CT images were collected (Fig. 7C, Supplementary Fig. 9B). mIHC revealed that tumors in the responsive group had a higher density of CD8+ and GZMB+CD8+ T cells, coupled with a lower presence of PD-1+CD8+ T cells (Fig. 7D, Supplementary Fig. 9C). The expression of TM4SF1 and its downstream pathways was assessed in both responsive and non-responsive patient groups using IHC. The results showed that responsive patients had lower expression levels of TM4SF1, p-AKT-S473, and Ki-67 (as reported pathologically), while p21 and p16 expression elevated (Fig. 7E, Supplementary Fig. 8D). Additionally, PD-L1, which is closely associated with immunotherapy efficacy, was overexpressed in non-responsive patients, whereas MHC I levels were lower (Fig. 7E). These findings confirm that TM4SF1 plays a critical role in modulating the response to immunotherapy. Furthermore, the ERP117672 data confirmed that non-responsive patients exhibited higher TM4SF1 expression (Supplementary Fig. 9E). In conclusion, TM4SF1 impairs anti-tumor immunity by modulating CD8+ T cell function; targeting TM4SF1 may offer a novel strategy to sensitize tumors to anti-PD-1 immunotherapy.
DISCUSSION
TM4SF1 has been extensively investigated in epithelial tumors such as colorectal cancer, breast cancer, and pancreatic cancer. TM4SF1, in conjunction with DDR1, reactivated metastasis in breast cancer by a non-canonical pathway [30]. TM4SF1 activated the JAK2-STAT3 signaling, enhancing cell stemness in colorectal cancer [4]. Tumor self-seeded cells with high TM4SF1 expression showed enhanced ability of invasion, migration and recolonization [31]. Unfortunately, due to the difference between HCC and other tumors, similar roles of TM4SF1 have not been well investigated in HCC. In this study, we confirmed elevated TM4SF1 expression in HCC tissues across multiple datasets. Through interactions involving AKT1 and PDPK1, TM4SF1 activated the classical AKT1-PDPK1 pathway and promoted AKT phosphorylation, which subsequently promoted tumor progression. Targeting TM4SF1 induced non-secretory phenotypic senescence and enhanced anti-tumor immunity. Although we successfully demonstrated TM4SF1 as a therapeutic target for HCC, this study has certain limitations. First, while we elucidated the mechanisms through which TM4SF1 promotes HCC progression, the upstream regulatory mechanisms of TM4SF1, such as transcriptional regulation and post-translational modifications, require further exploration, which we plan to address in future studies. Second, regarding model selection, we employed subcutaneous tumors, orthotopic tumors, and a hydrodynamic model with high TM4SF1 expression. Utilizing diverse combinations of hydrodynamic models may provide a more comprehensive understanding of TM4SF1’s functional roles. Third, the impact of tumor-derived TM4SF1 on other components of the tumor microenvironment, beyond CD8+ T cells, requires further investigation.
Senescence has emerged as a novel therapeutic target in cancer treatment. Senescent cells suppress tumor proliferation directly and influence tumor microenvironment by various means including paracrine, autocrine, and cell to cell interaction. AKT hyperactivation-induced senescence by cysteine import and trans-sulfuration protected gastric cancer cells from ROS-induced cell death [32]. In colorectal cancer, senescent cells at invasive margins promoted epithelial-mesenchymal transition (EMT) and metastasis [9]. Micro-RNA family prevented EMT-associated senescence in gastric cancer [33]. Similarly, chemotherapy-induced senescence in breast cancer facilitated immune evasion and subsequent tumor relapse by upregulating PD-L1 and CD80, while doxorubicin-induced senescence in brain metastases recruited exhausted T cells [17,34]. These findings underscore the varied impacts of senescence on tumor progression across cancer types. In HCC, senescent tumor cells upregulated IFN-γ receptor, remodeled tumor microenvironment and became hypersensitized to IFN-γ [20]. Other studies have demonstrated similar findings: glutaryl-CoA dehydrogenase induced HCC senescence by regulating the pentose phosphate pathway and glycolysis, benefiting immunosurveillance through SASP [35]. The decreased phosphorylation of mitochondrial protein in HCC cells induced senescence and impaired tumor proliferation as well as EMT process [36]. Interestingly, IL-17-induced AKT dependent IL-6 secretion has been reported to promote HCC progression without inducing senescence [37]. p21-activated secretory phenotype induced chemokine CXCL14 secretion, which attracted and polarized macrophages into M1, eliminating preneoplastic cells [38]. Pre-malignant senescent hepatocytes escaped from immunosurveillance by recruiting immunosuppressive myeloid cells on the early onset of HCC, underscoring context-dependent outcomes of senescence [39,40]. While similarities exist, our findings uniquely highlight non-secretory phenotypic senescence in HCC mediated by TM4SF1/AKT/p21-p16, which enhanced anti-PD-1 efficacy. In contrast to studies linking senescence to EMT or metastasis, our research found that senescent HCC cells induced by TM4SF1 knockdown demonstrated no discernible changes in SASP factors, including IL-6, IL-8, CCL-2, IL-1β, and IL-1α, which are implicated in tumor invasion, immune evasion and angiogenesis [27]. Likewise, the phosphorylation levels of pivotal modulators of SASP secretion, including p38, p65, and STAT3, remained unaltered. Within the tumor microenvironment, senescent HCC cells strengthened the cytotoxic function of CD8+ T cells by regulating PD-L1 and MHC I via the AKT pathway. The unaltered phosphorylation levels of p65 and STAT3 suggest a novel AKT-dependent pathway for the regulation of MHC I and PD-L1, which we plan to explore further in future studies. For other components in the tumor microenvironment, increased MDSCs following treatments require further exploration. We observed an increased proportion of CD4+ Tregs following anti-PD-1 monotherapy compared to that in the control group, consistent with other studies [41,42]. However, there was no significant change in the Treg subgroup after TM4SF1-targeted AAV treatment, and combination therapy further reduced Tregs compared to anti-PD-1 monotherapy. This phenomenon partially explained why anti-TM4SF1 therapy sensitized immunotherapy, but its underlying mechanism required further investigation (Supplementary Fig. 8F).
Our findings indicate that TM4SF1 may serve as a therapeutic target in HCC. In studies of other malignancies, TM4SF1-specific antibodies or antibody-drug conjugates have been used to suppress tumor stemness, growth, and vascularization [4,43]. TM4SF1-chimeric antigen receptor-T cell therapy exhibited anti-tumor capacity in vivo [44]. These systematic targeting strategies may also benefit HCC treatment. In this study, we employed systemic administration of AAV to knock down TM4SF1 in tumors transcriptionally. It can bypass post-translational modification of the target, which potentially compromises the therapeutic effect. However, AAV carries potential risks. In animal studies, it has been reported that intravenous administration of AAV induces hepatotoxicity, endothelial vessel injury, and microvascular thrombosis [45]. Neurotoxicity and insertional mutagenesis by AAV are also mentioned. In some cases, the immune system recognizes AAV, reduces its efficiency [45]. AAV also carries the potential risk of off-target effects, which may affect normal cells. These limitations could partly explain the restricted use of AAV in solid tumors, as evidenced by the limited number of clinical trials involving AAV. In our study, we utilized highly efficient and less immunogenic scAAV serotypes to target liver, incorporating a zsGreen tag to monitor infection efficiency. To mitigate these concerns, the precise administration, modification and optimization of AAV to enhance its specificity could help reduce adverse effects and off-target risks [46].
Importantly, targeting TM4SF1 in HCC not only suppresses tumor progression by inducing tumor cell senescence but also enhances the efficacy of immunotherapy. In clinical practice, assessing TM4SF1 expression in biopsy or surgical specimens could have a significant positive impact on subsequent treatment strategies. TM4SF1 may serve as a marker for predicting immunotherapy response. Patients with low TM4SF1 expression indicate a better immunotherapy response. For patients with high TM4SF1 expression, various approaches to downregulate its levels could be employed to simultaneously inhibit tumor progression and enhance sensitivity to immunotherapy. HCC treatment could benefit from systemic drug delivery as well as locoregional approaches such as transarterial chemoembolization or hepatic arterial infusion chemotherapy. With optimized AAV, it is feasible to utilize TM4SF1-targeted AAV-loaded beads in transarterial chemoembolization and integrate AAV into hepatic arterial infusion chemotherapy to downstage advanced HCC. These strategies provide opportunities to implement AAV-based targeting of TM4SF1 in HCC with greater precision and versatility. Recent advances in multiple cancers have led to the development of therapies targeting senescent cells, which aim to suppress tumors by enhancing the clearance of senescent cells [47]. We envision that combining TM4SF1 inhibition with therapies that eliminate senescent tumor cells, alongside immunotherapy, may provide a more effective strategy for HCC treatment.
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