High expression of USP15 affects tumor progression and immune infiltration in hepatocellular carcinoma

Abstract

Background and Aim

Ubiquitin-specific protease 15 (USP15) is closely associated with the occurrence and progression of hepatocellular carcinoma (HCC). However, its role in shaping the immune landscape of HCC remains unclear.

Materials and Methods

The expression levels, proportions, and spatial distributions of USP15 and specific immune cell subsets in HCC tissues were evaluated using multiplex immunohistochemistry (mIHC).

Results

In the tumor parenchyma of HCC tissues, the infiltration of immune cells, particularly natural killer (NK) cells, was significantly reduced (p<0.05). Further analyses revealed that USP15 expression levels were significantly associated with clinical stage and other clinicopathological parameters (p<0.05). In particular, NK cell infiltration was significantly correlated with N stage, M stage, and overall tumor–node–metastasis (TNM) stage (p<0.05).

Conclusion

USP15 contributes to the establishment of an immunosuppressive tumor microenvironment in HCC by inhibiting T cell and NK cell infiltration, facilitating programmed death-ligand 1 (PD-L1)-mediated immune evasion, and enhancing macrophage recruitment. These findings indicate that USP15 may serve as a potential therapeutic target for HCC.

Highlights & Insights

• Scientific Gap: The role of USP15 in shaping the tumor immune microenvironment and immunotherapy response in hepatocellular carcinoma remains unclear.

• Key Finding: High USP15 expression is associated with advanced stage, reduced T-cell and NK-cell infiltration, and increased PD-L1 expression and macrophage presence, indicating an immunosuppressive milieu.

• Clinical Impact: USP15 may serve as a prognostic biomarker and its inhibition, especially with PD-1/PD-L1 blockade, may enhance immunotherapy response.

Introduction

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is characterized by abnormal cell proliferation and dysregulation of the immune system. Consequently, HCC is often diagnosed at an advanced stage, which limits treatment options and contributes to a persistently high mortality rate.^[1,2] Although various therapeutic strategies, including surgical resection, locoregional ablation, and systemic therapies, are available, the overall prognosis of patients with HCC remains poor. Because the efficacy of programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-based immunotherapies critically depends on the tumor microenvironment (TME), it is essential to elucidate the key factors that regulate the TME to optimize therapeutic responses, improve prognostic accuracy, and develop novel treatment strategies.

The liver is an immune-rich organ that contains numerous immune-active cells and defense mechanisms against pathogens. However, its ability to combat cancer is often compromised by tumor cell-mediated evasion mechanisms and the establishment of an immunosuppressive TME.^[3] The composition of the TME is highly complex and heterogeneous, consisting of various cellular and acellular components, as well as the molecules they produce. These components can modulate immune function, ultimately suppressing effective antitumor immune responses while creating a microenvironment that favors tumor survival.

The dynamic interaction between the immune microenvironment and tumor cells drives tumorigenesis, progression, metastasis, and treatment response.^[4] Immune cell populations within the immune microenvironment play crucial roles in the development of HCC by regulating antitumor immune responses. When the antitumor activity of immunocytes in HCC is impaired, the TME promotes cancer cell growth and metastasis while limiting the immune system’s ability to eliminate tumor cells.^[5] Therefore, exploring changes in the TME during HCC progression, particularly alterations in the aforementioned immune cell populations, is essential for guiding HCC treatment strategies.

Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinases (DUBs) and play pivotal roles in regulating tumor progression, immune cell function, immune responses, and the establishment of an immunosuppressive TME.^[6,7] Ubiquitin-specific protease 15 (USP15) has been reported to be overexpressed in various malignancies, including glioblastoma, ovarian cancer, myeloma, and pancreatic cancer.^[8] Our previous research demonstrated that USP15 expression is relatively low in normal liver tissue but markedly elevated in metastatic HCC. In addition, increased USP15 in HCC tissues is strongly associated with unfavorable clinical outcomes and negatively correlated with patient survival time.^[9] These findings suggest that USP15 expression may be closely linked to inflammatory and immune-mediated pathways that promote HCC metastatic spread, highlighting the pivotal role of USP15 in driving HCC progression and disease deterioration. Furthermore, some studies have suggested that ubiquitin-specific peptidase 6 (USP6) in pancreatic ductal adenocarcinoma is strongly associated with various immune cell infiltrations.^[10] Therefore, we hypothesize that members of the USP family may influence the immune response in the TME of HCC. Exploring the relationship between USP15 and the TME holds considerable clinical significance and may provide deeper insights into the critical roles of USP15 in tumor development and immune regulation in patients with HCC.

This study aims to explore USP15 expression in HCC and analyze its impact on the TME, particularly on the infiltration of immune cell subpopulations such as T cells and natural killer (NK) cells. Additionally, we investigated the association between USP15 expression levels and the clinical characteristics of HCC patients to evaluate its potential as a therapeutic target.

Materials and Methods

Clinical Samples

Tumor tissue microarrays (TMAs; HLivH060CD03) were obtained from Shanghai Outdo Biotechnology. The array consists of 60 tissue cores, each with a diameter of 2.0 mm. The cohort includes two normal liver tissue samples, six cirrhotic specimens, 17 primary HCC lesions, and 15 metastatic HCC samples. For the majority of HCC cases, paired sampling was performed between the central region of the primary tumor and the adjacent peritumoral liver tissue. In addition, a subset of advanced-stage cases included distant hepatic tissue specimens. This study was conducted in accordance with the Declaration of Helsinki. All patients provided informed consent, and the study was approved by the Shanghai Outdo Ethics Committee (No: SHYJS-CP-1601006) on January 4, 2016. USP15 expression levels across the HCC cohort were ranked in ascending order, and the median value (50^th percentile) was used as the stratification threshold. The cohort was subsequently divided into two subgroups: those with above-median USP15 expression (high-expression group) and those with below-median expression (low-expression group).

Antibody Information

The antibodies used in multiplex immunohistochemistry (mIHC) were as follows: PD-L1 antibody (AD80167) from Abcepta; USP15 antibody (14354-1-AP) from Proteintech; cluster of differentiation 56 (CD56) antibody (ZM0057) from Beijing Zhong Shan Golden Bridge Biological Technology Co., Ltd.; cluster of differentiation 4 (CD4) antibody (PA285); cluster of differentiation 68 (CD68) antibody (PA014); cluster of differentiation 8 (CD8) antibody (PA577); forkhead box P3 (FoxP3) antibody (PA448); and cytokeratin (CK) antibody (PA125) from Suzhou Abcarta Medtech Co., Ltd.

Multiplex Immunohistochemistry

The experimental protocols were verified by the EACRI IHC Core (Providence Cancer Institute, Portland, OR) following established guidelines.^[11] Briefly, tissue microarray (TMA) sections underwent sequential processing: initial baking at 63°C for one hour, followed by dewaxing in xylene and rehydration through graded ethanol. Antigen unmasking was performed using an automated staining platform (Leica ST5020; Leica Biosystems). To neutralize endogenous peroxidase activity, slides were treated with 3% hydrogen peroxide for 10 minutes. Subsequent blocking with 5% bovine serum albumin (BSA) preceded a 60-minute incubation with primary antibodies targeting PD-L1 (ready to use), USP15 (1:200 dilution), CD56 (ready to use), CD4 (ready to use), CD68 (ready to use), CD8 (ready to use), FoxP3 (ready to use), and cytokeratin (ready to use). Following three washes with Tris-buffered saline with Tween 20 (TBST), a secondary antibody (SM802; Dako; ready to use) was applied for 10 minutes. Fluorescent labeling was performed using Opal 7-color dye (PerkinElmer; NEL801001KT) diluted 1:100, with a 10-minute incubation at room temperature. Antibody stripping between markers was achieved via microwave treatment in the citrate buffer. Sequential staining cycles were repeated for all targets, followed by nuclear counterstaining with DAPI for 5 minutes and mounting with antifade medium. Two independent antibody panels were processed.

Whole-slide imaging was conducted using a TissueFAXS Spectra system (TissueGnostics GmbH, Austria). Acquired multispectral data were analyzed using StrataQuest software (v7.1.129; TissueGnostics), employing spectral unmixing to resolve individual fluorescence channels. Nuclear segmentation was performed using DAPI signals, and cytoplasmic or membrane markers were quantified within a user-defined radius from the nuclei. Threshold parameters were dynamically adjusted for each marker to classify positive cells based on fluorescence intensity and spatial distribution. Multiplex positivity was determined through co-localization analysis of two or more markers, with cell counts and signal intensities recorded for statistical evaluation.

Statistical Analysis

USP15 expression and the levels of TME cells in HCC tumor tissues and paracancerous tissues were compared using the Wilcoxon test. Comparisons among multiple groups were performed using the Kruskal–Wallis test. The relationship between USP15 expression and immunohistochemical factors was examined using the Spearman rank correlation coefficient. All statistical analyses were performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and p<0.05 was considered statistically significant.

Results

USP15 Expression Level Correlates with Clinical Characteristics and Progression in Patients with HCC

To investigate the regulatory role of USP15 within the TME of HCC, mIHC was used to detect USP15 expression and specific immune markers, including CD4 (a marker of CD4⁺ T cells), CD8 (a marker of CD8⁺ T cells), CD56 (a marker of NK cells), and CD68 (a marker of macrophages), in HCC tissues. The infiltration of various immune cells in HCC tissues with high USP15 expression was analyzed (Fig. 1A-D), and their correlation with USP15 expression levels was assessed.

Figure 1.

(a, c) Statistical demonstration of overall expression levels of various cell types in TMA in the two mIHC panels respectively. (b, d) Statistical demonstration of the expression levels of various cell types in each clinical sample in the two mIHC panels respectively. (e). Representative multiplex immunofluorescence image of a HCC tissue core selected for analysis of USP15-high peritumoral regions. Markers: USP15 (red), CD8 (T cells, green), CD4 (T cells, yellow), CK (tumor epithelial cells, cyan), FOX P3 (purple). Scale bar =200 µm. (f) The difference of USP15 infiltration between different progression of HCC.

According to the mIHC results, USP15 staining was predominantly observed on the tumor cell membrane and in the cytoplasm, with a red positive signal (Fig. 1E). In contrast, USP15 staining varied among paracancerous tissues, with some samples showing strong staining and others weak staining. These findings indicate that USP15 may play a specific role in tumor cells, and its expression is upregulated during the progression of HCC (Fig. 1F), consistent with our previous research.

Our previous studies have revealed that USP15 expression is positively correlated with HCC recurrence.^[9] To further investigate whether differential USP15 expression in HCC tissues is associated with clinical characteristics, we analyzed the clinical data of all patients with HCC. Our analysis revealed a strong association between high USP15 expression and advanced disease stages, showing a marked trend with M stage and a statistically significant association with tumor–node–metastasis (TNM) stage (p<0.05) (Table 1). These results are consistent with our previous findings and suggest that USP15 expression may serve as a therapeutic target in HCC.

Table 1.

Expression of USP15 in HCC of patients with different clinical characteristics

Character	Group	Low	High	Total	p value	r value
Age(year)	≤52	1	16	17	0.319	-0.213
	>52	3	12	15
Tumor size	≤6.25 cm	2	6	8	1	0.16
	>6.25 cm	1	7	8
T stage	I-II	3	6	9	0.585	0.218
	III	1	6	7
N stage	N0	0	16	16	0.066	0.149
	N1	4	11	15
M stage	M0	0	1	1	0.038	0.402
	M1	4	11	15
Pathological grade	I	0	17	17	1	-0.203
	II-III	0	2	2
TNM stage	I-III	2	8	10	0.028	0.429
	IV	4	10	14

USP15: Ubiquitin-specific protease 15; HCC: Hepatocellular carcinoma; TNM: Tumor node metastasis.

USP15 as a Negative Regulatory Factor for T-Cell Infiltration

The activation and function of T cells are tightly regulated by various negative regulators to ensure that the immune system effectively combats foreign pathogens while preventing abnormal immune responses and autoimmune diseases.^[12] Our results indicated significant differences in the staining of CD4⁺ T cells, CD8⁺ T cells, and regulatory T (Treg) cells between HCC tissues and paracancerous tissues (p<0.05) (Fig. 2A-C). The observed low densities of CD4⁺ T cells and CD8⁺ T cells suggest limited infiltration into the tumor parenchyma. These findings imply that USP15 may function as a negative regulator of T cells in HCC, with high USP15 expression correlating with reduced T-cell activation and infiltration.

Figure 2.

(a–c) Differential analysis of the percentage of CD4⁺ T cells, CD8⁺ T cells and Treg cells between cancer tissues and adjacent normal tissues. (d) Heat map of correlation analysis between indicators in panel 1. (e) Correlation analysis of Tumor epithelial cells (CK⁺) and CD4 ⁺ T cell expression, p<0.001, R=-0.627. (f) Correlation analysis of Tumor epithelial cells (CK⁺) and USP15⁺ Tumor epithelial cells expression, p<0.001, R=0.621.

Furthermore, we observed a strong association between tumor epithelial cells (cytokeratin-positive, CK⁺) and both CD4⁺ T-cell expression and USP15⁺ CK⁺ cells. Specifically, CD4⁺ T-cell expression showed a significant negative correlation (p<0.001) (Fig. 2D-E), whereas USP15⁺ CK⁺ cells showed a significant positive correlation (p<0.001) (Fig. 2F). However, although a negative correlation was observed between USP15⁺ CK⁺ cells and CD4⁺ T cells, it was not statistically significant (p>0.05). Therefore, we speculate that USP15 may influence CD4⁺ T cells indirectly through modulation of the tumor microenvironment rather than through direct interaction.

The Degree of NK Cell Infiltration Is Correlated with USP15 Expression and the Clinical Features of Patients with HCC

To better evaluate NK-cell infiltration, we selected tumor peripheral regions exhibiting high USP15 expression for detailed analysis (Fig. 3A). The mIHC data revealed a significant difference in NK-cell infiltration between HCC tissues and paracancerous tissues (p<0.001) (Fig. 3B). The low density of CD56⁺ cells indicates minimal NK-cell infiltration in the tumor parenchyma (Fig. 3C). In addition, mIHC analysis revealed a significant negative association between the level of NK-cell infiltration and USP15⁺ cells (p<0.05) (Fig. 3D-E). Similarly, the expression level of CD56⁺ cells was significantly negatively correlated with USP15⁺ tumor epithelial cells (p<0.05) (Fig. 3F). To investigate potential associations between NK-cell infiltration in HCC tissues and clinical characteristics, we analyzed the clinicopathological data of all patients with HCC. The results showed that NK-cell infiltration was significantly associated with the N stage, M stage, and TNM stage (p<0.05) (Table 2). Therefore, these findings suggest that high USP15 expression may inhibit NK-cell infiltration, activation, and cytotoxic function within the TME.

Figure 3.

(a) Representative immunohistochemical staining of peritumoral regions in HCC tissue microarray. Markers: USP15 (red), CD68 (macrophages, yellow), CD56 (NK cells, green), CK (tumor epithelial cells, cyan), PD-L1 (purple). Scale bar =200 µm. (b, c) Differential analysis of the percentage of NK cells and PD-L1⁺ cells between HCC tissues and adjacent normal tissues respectively. (d) Heat map of correlation analysis between indicators in panel 1. (e) Correlation analysis of USP15⁺ cells and NK cells expression, p<0.05, R=-0.397. (f) Correlation analysis of NK cells and USP15⁺ Tumor epithelial cells expression, p<0.05, R=-0.368.

Table 2.

Expression of NK cells in hepatocellular carcinoma tissues of patients with different clinical characteristics

Character	Group	Low	High	Total	p value	r value
Age(year)	≤52	8	9	17	0.735	0.071
	>52	6	9	15
Tumor size	≤6.25cm	6	2	8	0.608	0.258
	>6.25cm	4	4	8
T stage	I-II	6	3	9	1	0.098
	III	4	3	7
N stage	N0	4	12	16	0.031	0.333
	N1	10	5	15
M stage	M0	0	1	1	0.031	0.434
	M1	10	5	15
Pathological grade	I	4	13	17	1	-0.112
	II-III	1	1	2
TNM stage	I-III	7	3	10	0.011	0.492
	IV	10	4	14

NK: Natural killer; TNM: Tumor node metastasis.

USP15 Exhibits a Positive Correlation with PD-L1⁺ Cells and Tumor-Associated Macrophages

PD-L1, an important target for tumor immunotherapy, is expressed not only on tumor cells but also on tumor-associated macrophages (TAMs). Our mIHC analysis revealed a modest correlation between USP15 expression and PD-L1⁺ cells (Fig. 4A). Although the proportion of PD-L1⁺ cells showed no significant difference between HCC tissues and adjacent normal tissues (Fig. 3C), we observed a significant negative correlation between PD-L1⁺ cell infiltration and tumor epithelial cell expression levels (p<0.05) (Fig. 4B). Concurrently, a significant positive correlation was observed with the expression of PD-L1⁺ tumor epithelial cells (p<0.05) (Fig. 4C). These findings suggest that USP15 may be involved in regulating PD-L1⁺ cells, particularly PD-L1⁺ tumor epithelial cells.

Figure 4.

(a) Correlation analysis of USP15⁺ cells and PD-L1⁺ cells expression, p<0.05, R=0.356. (b) Correlation analysis of tumor cells and PD-L1⁺ cells expression, p<0.005, R=-0.524. (c) Correlation analysis of PD-L1⁺ cells and PD-L1⁺ tumor epithelial cells expression, p<0.05, R=0.804. (d) Correlation analysis of PD-L1⁺ tumor epithelial cells and macrophages expression, p<0.05, R=0.385. (e, f) Complex Heatmap based on the expression of different cells in different samples and the corresponding clinical information of each sample of two mIHC panels.

Notably, our data demonstrated a modest positive correlation between PD-L1⁺ tumor epithelial cells and CD68⁺ cell infiltration (p<0.05) (Fig. 4D). Therefore, the sequential chain of correlations from USP15⁺ cells to CD68⁺ cells may suggest that USP15⁺ tumor cells influence macrophage infiltration indirectly via PD-L1 co-expression.

Discussion

In recent years, the TME has gradually emerged as a major focus of cancer research. In this study, we explored the relationship between USP15 and the TME by performing mIHC staining to analyze the expression of USP15 and various immune cell subsets associated with the TME in tissues from patients with HCC (Fig. 4E-F). Our findings revealed that USP15 significantly influences tumor cell expression and shows negative correlations with the infiltration of most immune cell types. Furthermore, our study demonstrated that USP15 is overexpressed in HCC tissues and is associated with adverse clinicopathological parameters. These findings suggest that USP15 may function as an important immunomodulatory regulator in HCC and highlight its potential as a therapeutic target in HCC.

The therapeutic value of combining USP inhibitors with tumor immunotherapy has been increasingly recognized. For example, combination therapy strategies that synergistically enhance the efficacy of anti-PD-1/PD-L1 immunotherapy by inhibiting deubiquitinating enzymes can reshape the inflammatory TME. In addition, inhibition of USP22 expression has been shown to enhance the sensitivity of patients with pancreatic ductal adenocarcinoma to immunotherapy.^[13,14] Therefore, our study provides a theoretical basis for the development of novel USP inhibitors.

The observed correlations between USP15 and immune cell infiltration provide important insights into the potential role of USPs as therapeutic targets for HCC. Numerous mechanisms involved in tumorigenesis and tumor progression, including regulation of the TME, are closely associated with ubiquitination processes. Previous studies have reported that USP15 functions as a negative regulator of T-cell activation, and targeting USP15 may induce tumor cell apoptosis while enhancing the antitumor T-cell response.^[15] These findings are consistent with our mIHC results. High USP15 expression was associated with reduced T-cell infiltration and increased metastasis in HCC. This suggests that USP15 may promote tumorigenesis and disease progression through both intrinsic tumor-cell mechanisms and suppression of host antitumor immune defense.

Natural killer cells, as essential innate immune effector cells, play a critical role in defense against infections and tumor progression.^[16] However, the functional activity of NK cells within the TME can be inhibited by certain cytokines, such as interleukin-6 (IL-6), IL-10, and indoleamine 2,3-dioxygenase (IDO), which suppress NK-cell activity by promoting immunosuppressive cells and cytokines that antagonize NK-cell activation.^[16,17] To examine the relationship between immune infiltration and clinicopathological features of HCC, we performed a comprehensive clinical correlation analysis using patient-derived clinical data. Our results demonstrated significant associations between NK-cell infiltration and multiple clinicopathological features in patients with HCC. Therefore, we believe that the immunosuppressive TME represents one of the main limiting factors preventing NK cells from exerting effective immune responses. Future studies will further investigate the intrinsic mechanisms by which USP15 regulates NK-cell activity, with the aim of optimizing NK cell-based immunotherapy and identifying new therapeutic strategies for tumor immunotherapy.

Currently, TAMs are important targets for cancer therapy. Their two main subtypes, M1 and M2, can transform into each other in response to changes in the microenvironment. Among them, M2 macrophages promote the formation of an immunosuppressive TME by secreting various immunosuppressive factors, including transforming growth factor-β (TGF-β) and IL-10, thereby exacerbating the immunosuppressive state of the TME and leading to HCC metastasis and resistance to immunotherapy.^[18-20] Previous studies have shown that downregulation of USP7 expression can induce phenotypic transformation of TAMs from the M2 to the M1 subtype through the p38 mitogen-activated protein kinase (MAPK) pathway, while also upregulating PD-L1 expression in the TME.^[21] Both USP7 and USP15 belong to the USP family and play similar roles in regulating intracellular ubiquitination processes. Therefore, USP15 could be a new therapeutic target and prognostic factor. In this study, we initially attempted to determine the relationship between USP15 and macrophages; however, no significant changes in macrophage infiltration were observed. We suspect that macrophages may instead undergo polarization changes, which provides a direction for our subsequent research. Furthermore, in both the tumor parenchyma and stroma of HCC, the co-expression of USP15 and PD-L1, as well as the co-expression of macrophages and PD-L1, was significant, indicating a close relationship among these three factors. Therefore, we speculate that macrophages may alter their polarization state due to the combined effects of USP15 and PD-L1, thereby promoting tumor growth. Over the past decade, PD-L1/PD-1 monoclonal antibodies have been widely used in cancer treatment; however, anti-PD-L1/PD-1 monotherapy still has unavoidable limitations. It has been reported that the immunotherapeutic effect of PD-1/PD-L1 inhibitors can be enhanced by inhibiting macrophage infiltration in the TME.^[22] Our findings suggest that combining USP15-targeted inhibition with PD-1/PD-L1 blockade may represent a novel therapeutic strategy for the treatment of hepatocellular carcinoma.

Our findings demonstrate that USP15 functions as a crucial immune regulatory factor in hepatocellular carcinoma, underscoring its potential as a therapeutic target. However, it is necessary to note that this study has several limitations. First, the cohort size is limited and the group distribution is imbalanced. For example, the M0 group contains only one patient, rendering statistical comparisons between M0 and M1 highly unstable and unreliable. Similarly, regarding lymph node stage, all N0 cases exhibited high USP15 expression, while 11 of 15 N1 cases showed high expression (p=0.066). This indicates that the observed associations with M and N stages—although biologically suggestive—require validation in larger and more balanced independent cohorts. Second, although TMA technology enables efficient high-throughput analysis, its limited sampling range may fail to fully capture intratumoral heterogeneity. To address this limitation, we conducted paired sampling of “peritumoral liver tissue - primary tumor site” for most HCC cases. For some advanced cases, we further included “distal liver tissue - peritumoral liver tissue - primary tumor site” for three-point sampling. However, this technical compensation still cannot fully overcome the challenges posed by intratumoral heterogeneity. Finally, although the present data support the potential of USP15 as a therapeutic target, the limited sample size, absence of survival analysis, and lack of functional validation preclude definitive clinical recommendations at this stage. In future studies, we will expand the cohort size, integrate comprehensive survival data, and perform in-depth mechanistic investigations to further elucidate the role of USP15 and robustly validate its potential as a viable therapeutic target in HCC. In summary, this study provides novel evidence supporting the role of USP15 in modulating the immune microenvironment of HCC. However, the aforementioned limitations underscore the need for larger and more diverse cohorts in future investigations for further validation.

Conclusion

In summary, our study investigated the role and regulatory effects of USP15 within the TME and characterized the tumor microenvironment of primary and metastatic liver tumors under different USP15 expression levels. We elucidated the critical role of USP15 in shaping the TME and driving the progression of HCC. Our findings demonstrate that high USP15 expression in HCC tissues is strongly associated with advanced clinical stages, including M stage and TNM stage, and correlates with reduced infiltration of immune cells, particularly T cells and NK cells. Furthermore, USP15 shows a positive association with PD-L1⁺ cells and TAMs, suggesting its dual role in promoting immune evasion and fostering an immunosuppressive TME. These results underscore USP15 as a pivotal regulator of tumor-immune interactions in HCC, highlighting its potential as a therapeutic target.

Ethics Committee Approval

All patients gave their consent, and the Shanghai outdo Ethical Committee authorized this study (No: SHYJS-CP-1601006), on January 4, 2016. Informed Consent: Written informed consent was obtained from participants.

Informed Consent

All patients gave their consent.

Conflict of Interest

The authors declare that they have no competing interests.

Financial Disclosure

This project received funding from Jilin Provincial Department of Science and Technology (No. YDZJ202201ZYTS203).

Use of AI for Writing Assistance

No AI-assisted technologies were used in the preparation of this manuscript.

Author Contributions

Concept: Z-LL; Design: Z-LL; Supervision: Z-LL; Analysis and/or Interpretation: Z-LL; Writing: S-PW; Critical Reviews: X-YZ, H-DC, F-ZJ, NJ, J-XZ.

Peer-review

Externally peer-reviewed.