OSBPL3 promotes colorectal cancer progression through modulation of TGF-β signaling and immune cell infiltration

Introduction

Colorectal cancer (CRC), as the third most common cancer, has gained increasing attention all over the world during the past decades (1). The development of CRC is closely related to unhealthy lifestyle habits such as a high-fat, low-fiber diet, lack of physical activity, obesity, smoking, and alcohol consumption. Additionally, about 10% to 15% of CRC patients have a genetic predisposition (2). The most common hereditary syndromes include hereditary nonpolyposis CRC (HNPCC), also known as Lynch syndrome (3), and familial adenomatous polyposis (FAP) (4,5). The 5-year survival rate for CRC varies significantly depending on the stage at diagnosis. In high-income countries where early detection is more common, 5-year survival rates range between 60% and 65% (6). In contrast, in low-income countries, where CRC is often diagnosed at a later stage, survival rates are much lower, typically below 40%. The incidence of CRC remains generally higher in men than in women, with men being more susceptible to lifestyle-related risk factors (such as alcohol consumption, smoking, and high-fat, low-fiber diets) (1,7).

Previous studies have shown that CRC patients with microsatellite instability-high (MSI-H) have better prognoses and respond well to immune checkpoint inhibitors and other immunotherapies (8,9). In recent years, an increasing number of CRC treatment strategies have been developed to offer personalized therapies specifically for MSI-H patient groups.

Additionally, KRAS, NRAS, and BRAF mutations are common molecular features in CRC (10,11). In recent years, testing for these mutations has increased, providing more guidance for targeted therapies. RAS mutations are often associated with resistance to anti-EGFR therapies, while BRAF mutations (especially BRAF V600E) typically indicate a poorer prognosis (12,13).

Oxysterol-binding protein-like 3 (OSBPL3) is a protein that belongs to the oxysterol-binding protein (OSBP) family, which is involved in regulating intracellular lipid transport, metabolism, and signaling (14). OSBPL3 plays an important role in lipid exchange between cell membranes and organelles, especially in the transport and metabolism of cholesterol and phospholipids (15). Emerging evidence suggests OSBPL3 exerts context-dependent functions in oncology. Studies indicate a tumor-suppressive role in specific malignancies, including colon cancer (16), lymphoma (17) and bladder cancer (18). Conversely, OSBPL3 overexpression is associated with invasiveness and disease progression in hepatic and gastric cancers (19,20). In patients with lung and gastric malignancies, elevated OSBPL3 expression correlates with metastasis, suggesting its potential utility as a prognostic marker (20,21). Despite these associations, the specific role of OSBPL3 in CRC remains relatively underexplored, and further research is needed to better understand its function in this context.

Given to these, we conduct a comprehensive study to explore the expression of OSBPL3 in patients with CRC through bioinformatic analysis and cellular experiments, detecting the role of OSBPL3 as well as related signaling pathway, especially the TGF-β signaling pathway, in CRC progression and development, providing potential therapeutic targets for clinical treatment. 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-2464/rc).

Methods

Cell culture

CRC cell lines HT29 and SW480 and normal colon epithelial cell line NCM460 were purchased from the Cell Bank of the Chinese Academy of Sciences. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Beijing Zhongshan Jinqiao, Beijing, China) containing 10% fetal bovine serum (FBS, Hangzhou Sijiqing, Hangzhou, China) and 1% penicillin-streptomycin solution (Shanghai Yuanye Biotechnology, Shanghai, China) in a constant temperature incubator at 37 °C and 5% CO₂.

To study the function of OSBPL3, we used lentiviral vectors to construct stable knockdown (sh-OSBPL3-1, sh-OSBPL3-2) and overexpression of OSBPL3 (oe-OSBPL3) cell lines, and the negative controls were sh-NC and empty vector, respectively. The design and synthesis of lentiviral vectors were completed by Guangzhou Jisai Biotechnology, and cell infection was carried out according to the manufacturer’s instructions. Puromycin (Shanghai Kaichuang Biotechnology, Shanghai, China) was used to screen positive clones 72 hours after infection. The primers used in this study were designed based on sequences obtained from the NCBI database and synthesized commercially. The primer sequences for quantitative reverse transcription polymerase chain reaction (qRT-PCR) are provided in Table S1.

Immunohistochemistry (IHC) analysis

Human CRC tissue samples used for IHC were collected from patients at Jiangmen Central Hospital after written informed consent was obtained from all patients. The human study was approved by the Institutional Research Ethics Committee of Jiangmen Central Hospital (decision No. JXY2023110) and was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

IHC was performed according to our previous publication (18). Sections were baked at 60 °C for 1 hour, followed by xylene dewaxing and gradient ethanol hydration. Antigen retrieval was performed using citrate buffer (pH 6.0) heated in a microwave oven and treated with 3% hydrogen peroxide for 15 minutes at room temperature to block endogenous peroxidase activity.

Sections were incubated with OSBPL3 primary antibody (1:200, Thermo Fisher, Waltham, USA) overnight, and incubated with biotin-labeled secondary antibody (Abcam, Cambridge, UK) for 1 hour the next day. DAB reagent (Abcam) was used for color development, and images were observed and taken under a microscope at high and low magnification fields.

Western blot (WB) analysis

Total protein was extracted from cells using Racial and Identity Profiling Act (RIPA) lysis buffer (Solarbio, Beijing, China), and protein concentration was quantified using a bicinchoninic acid (BCA) kit (Solarbio). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Solarbio). The membranes were blocked in 5% skim milk powder for 1 hour at room temperature and then incubated overnight with OSBPL3 (1:1,000, Thermo Fisher Scientific, Inc.) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3,000, Thermo Fisher Scientific, Inc.) antibodies. After incubation with secondary antibodies for 1 hour, the membranes were developed using electrochemiluminescence (ECL) luminescent solution (Thermo Fisher Scientific, Inc.), and the grayscale values of the bands were analyzed using ImageJ software. The original uncropped WB images are provided in Figure S1.

Real-time quantitative PCR (RT-qPCR)

Total RNA was extracted from cells using Trizol reagent (Solarbio), and cDNA was synthesized using a reverse transcription kit (Solarbio). qPCR was detected on a Bio-Rad CFX96 system using SYBR Green Master Mix (Qingdao Patel, Qingdao, China). The relative mRNA expression level of OSBPL3 was calculated by the ΔΔC^t method, and GAPDH was used as an internal reference gene.

Cell Counting Kit-8 (CCK-8) proliferation assay

Cells were seeded in 96-well plates (2,000 cells per well), and CCK-8 reagent (Solarbio) was used on the 1st, 2nd, 3rd, 4th, and 5th days after culture. The absorbance [optical density (OD) value] was measured at a wavelength of 450 nm after 2 hours of incubation. The experiment was repeated three times, with three replicates in each group.

Ethynyl deoxyuridine (EdU) incorporation assay

Using the EdU kit (Guangzhou Cell Probe, Guangzhou, China), cells were seeded in 24-well plates, and EdU solution was added for incubation for 2 hours, followed by fixation and fluorescent staining. Fluorescence microscopy (Nikon, Shanghai, China) was used to detect red fluorescently labeled EdUpositive cells to assess the DNA synthesis activity of cells.

Flow cytometry for apoptosis

Cells were labeled using Annexin V-FITC/PI double staining kit (Nanjing Keygen Biotechnology, Nanjing, China) and analyzed by flow cytometry (BD Biosciences, Franklin Lakes, USA). At least 10,000 events were detected for each group, and data were analyzed using FlowJo software.

Wound healing assay

Cells were seeded in 6-well plates. When the cell confluence reached more than 90%, a scratch was made on the cell monolayer with a sterile pipette tip. After 24 hours of culture, images of the scratch area were taken using a microscope (Olympus, Shanghai, China), and the wound closure rate was measured using ImageJ to evaluate cell migration ability.

Transwell invasion assay

The invasion assay used a 24-well Transwell chamber (8.0 µm pore size, Corning, Shanghai, China); 100 µL of Matrigel matrix gel (BD Biosciences) was added to the chamber and incubated for 30 minutes to solidify. The treated cells were seeded on the upper layer of the chamber and cultured in medium containing 10% FBS for 24 hours. The cells that passed through the membrane in the lower layer were fixed with methanol, stained with 0.1% crystal violet, photographed and counted under a microscope.

Gene set enrichment analysis (GSEA)

In order to explore the molecular mechanism of OSBPL3 in CRC, we downloaded the RNA-seq expression matrix and corresponding clinical data from The Cancer Genome Atlas (TCGA) CRC projects, including COAD and READ, using the TCGAbiolinks package in the R programming environment, focusing on the relationship between OSBPL3 and signaling pathways. According to the high and low expression groups of OSBPL3, the gene expression profile was analyzed to determine the relevant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially the association with the TGF-β signaling pathway. All bioinformatic analyses were completed in the R language environment, and the ClusterProfiler package was used for pathway enrichment analysis and visualization of the results.

Bioinformatic analysis

CRC and pan-cancer RNA sequencing data were obtained from the TCGA (https://www.cancer.gov/tcga) and Genotype-Tissue Expression (GTEx) database (https://gtexportal.org/), and the “limma” package was used for differentially expressed genes (DEGs) screening. The screening conditions were |log₂ fold change (FC)| >1 and p.adj value <0.05 to determine the significantly DEGs between high and low expression of OSBPL3. The R package corrplot was used to perform inter-gene co-expression network analysis to further explore the potential regulatory mechanism of OSBPL3. Immune infiltration analysis was performed using the CIBERSORTxR package. Protein interaction networks were completed using the STRING website (https://string-db.org/).

Nude mouse xenograft model

All animal experiments complied with the 2020 AVMA Guidelines for the Euthanasia of Animals and were approved by the Animal Ethics Committee of Guangdong Medical University (approval No. GDY2302537), in compliance with the institutional guidelines for the care and use of animals. Female BALB/c nude mice (4–6 weeks; Beijing Weitong Lihua, Beijing, China) were housed in a specific pathogen-free facility at the Laboratory Animal Center of Guangdong Medical University in individually ventilated cages (3–5 mice per cage) with corncob bedding, sterilized chow and water ad libitum, a 12-h light/12-h dark cycle, ambient temperature 22±2 °C, and relative humidity 45–65%; environmental enrichment (nesting material, shelters, and gnawing blocks) was provided, and mice were socially housed unless welfare or scientific considerations required temporary single housing. Animals were acclimatized for at least 7 days and checked at least once daily by trained staff. Mice were randomized to groups, and tumor measurements were performed by investigators blinded to allocation. For inoculation, control or OSBPL3-knockdown HT29 or SW480 cells (2×10⁶ per mouse) were injected subcutaneously under light isoflurane anesthesia (2–3% in oxygen via nose cone). Tumor length and width were measured at least 2–3 times per week with a caliper, and volume was calculated as V = 0.5 × length × width². At week 4 or upon reaching predefined humane endpoints, mice were euthanized by isoflurane overdose (≥5% in oxygen in an induction chamber); exposure was maintained for at least 1 min after cessation of respiration, followed by cervical dislocation to ensure death. Death was confirmed by the absence of a heartbeat and corneal reflexes before tissue collection. Ether, chloroform, and chloral hydrate were not used at any time.

Statistical analysis

All experiments were repeated at least three times. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were conducted using Student’s t-test, and comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). A P value <0.05 was considered statistically significant.

Results

Expression and clinical significance of OSBPL3 in CRC

We first examined the expression of OSBPL3 in tumors. Pan-cancer analysis of unmatched samples based on the TCGA database revealed that OSBPL3 was differentially expressed across various cancers, with significant differences observed in COAD (Figure 1A). Analysis of matched samples further confirmed that OSBPL3 expression levels were markedly different in COAD and READ (Figure 1B), and survival analysis indicated that high OSBPL3 expression was significantly associated with poor prognosis in COAD patients (Figure 1C). Similar protein expression patterns were observed in COAD in the unmatched and matched analyses (Figure 1D,1E). Additionally, receiver operating characteristic (ROC) curve analysis demonstrated that when OSBPL3 was used as a diagnostic marker, the area under the curve (AUC) reached 0.979 (Figure 1F), highlighting its excellent diagnostic accuracy. This suggests that OSBPL3 could effectively distinguish CRC patients from healthy individuals and holds potential as a biomarker for early diagnosis and monitoring of CRC. To further evaluate the clinical relevance of OSBPL3, we conducted analyses based on clinicopathological parameters (Figure 1G-1N). The results showed that OSBPL3 expression varied significantly (P<0.050) across different pathological N stages (N0 vs. N1&N2), indicating a potential association with lymph node metastasis (Figure 1K). Additionally, OSBPL3 expression differences between pathological M stages (M0 vs. M1) approached statistical significance (P=0.06), suggesting a possible role in metastatic CRC (Figure 1L). The remaining parameters, including age, body mass index, gender, pathologic T stage, histologic type, and carcinoembryonic antigen (CEA) level, did not show significant differences (Figure 1G-1J,1M,1N). In addition, by dividing the patients into high and low expression groups according to the expression level of OSBPL3, subgroup analysis of clinical indicators showed that there were statistical differences in N stage (P=0.05) and pathologic 214 stage (P=0.04) among different expression groups (Table 1).

Figure 1 Expression profile and clinical significance of OSBPL3 in CRC. (A,B) Pan-cancer analysis of unmatched TCGA samples showing the differential expression of OSBPL3 across multiple cancer types, with significant differences in COAD and READ. (C,D) Matched-sample analysis further confirming the differential expression of OSBPL3 in COAD. (E) Survival analysis showing that high OSBPL3 expression is associated with poorer prognosis in patients with COAD. (F) ROC curve showing the diagnostic performance of OSBPL3 for CRC, with an AUC of 0.979. (G-N) Clinicopathological analysis showing a significant difference in OSBPL3 expression between pathological N stages (N0 vs. N1/N2) and a borderline difference between pathological M stages (M0 vs. M1), while no significant differences were observed for the other clinicopathological parameters. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. AUC, area under the curve; BMI, body mass index; CI, confidence interval; CRC, colorectal cancer; FPR, false positive rate; HR, hazard ratio; M, metastasis; N, node; ROC, receiver operating characteristic; T, tumor; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TPR, true positive rate.

Comprehensive analysis of DEGs in CRC and its clinical significance

To elucidate the comprehensive differences in gene expression in CRC, we analyzed data from the TCGA database and identified significantly upregulated and downregulated DEGs, including a total of 4,694 upregulated and 1,032 downregulated genes (logFC >1, p.adj <0.05) (Figure 2A). Given that tumor progression often involves complex regulatory networks, we performed a co-expression analysis of OSBPL3 with these DEGs (Figure 2B-2F). The results revealed a strong positive correlation between OSBPL3 and ZBTB20, and a strong negative correlation with SNHG25. Gene Ontology (GO) analysis indicated that these genes are mainly involved in biological processes (BPs) such as chemical stimulus detection, oxygen transport, and cellular oxide detoxification (BP, Figure 2G), and are enriched in cellular components (CCs) like hemoglobin complexes and DNA packaging complexes (CC, Figure 2H). Molecular function (MF) analysis demonstrated associations with oxygen binding, antioxidant activity, and related functions (Figure 2I). KEGG pathway analysis revealed significant enrichment of these genes in pathways such as taste transduction, spliceosome, and systemic lupus erythematosus (Figure 2J).

Figure 2 Comprehensive analysis of DEGs and their coexpression with OSBPL3 in CRC. (A) Volcano plot showing significantly upregulated and downregulated DEGs identified from the TCGA database. (B-F) Co-expression analysis illustrating correlations between OSBPL3 and other DEGs, with notable positive and negative associations. (G-J) GO and KEGG pathway analysis, highlighting enrichment of DEGs in BPs, CCs, MFs, and pathways. (K-N) Co-expression subgroup analysis showing associations of OSBPL3 with DEGs across various T, N, and M stages, as well as survival status. (O,P) Heatmaps depicting the expression patterns of OSBPL3-associated DEGs clustered with OS and TNM staging, revealing significant correlations with patient prognosis and disease progression. *, P<0.05. BP, biological process; CC, cellular component; CRC, colorectal cancer; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; M, metastasis; MF, molecular function; N, node; OS, overall survival; T, tumor; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

To further explore the expression patterns of these genes across different clinicopathological characteristics, we conducted a co-expression subgroup analysis. The results showed that OSBPL3 was closely associated with multiple DEGs in the T2&3, N0, and M0 stages (Figure 2K-2M). Additionally, OSBPL3 exhibited stronger associations with differential genes in the survival group compared to the non-survival group (Figure 2N). Clustering analysis of gene expression patterns with overall survival (OS) and tumor-node-metastasis (TNM) staging indicated that key genes associated with OSBPL3 were significantly correlated with patient survival status and TNM pathological stages (Figure 2O,2P). These findings suggest that these genes may play critical roles in CRC staging and progression.

Correlation analysis between OSBPL3 expression and immune cell infiltration in tumor microenvironment (TME)

The TME is widely recognized as an important contributor to tumor progression. To examine the relationship between OSBPL3 expression and immune infiltration, we performed an in silico pan-cancer analysis based on CIBERSORT-derived immune cell estimates. This analysis suggested that OSBPL3 expression is associated with the inferred infiltration levels of multiple immune cell subsets across cancers (Figure 3A). In CRC, Spearman correlation analysis identified statistically significant associations between OSBPL3 expression and several immune cell types, including central memory T cells (Tcm) (r=0.478, P<0.001), Th2 cells (r=0.140, P=0.002), and Treg cells (r=−0.196, P<0.001) (Figure 3B). Notably, while the correlations with Th2 and Treg cells were statistically significant, their effect sizes were weak, indicating limited biological magnitude and warranting cautious interpretation.

Figure 3 Relationship between OSBPL3 expression and immune cell infiltration in TME. (A) Pan-cancer in silico analysis (CIBERSORT-based) showing associations between OSBPL3 expression and estimated infiltration levels of multiple immune cell subsets. (B) Spearman correlation analysis in CRC showing statistically significant associations between OSBPL3 expression and Tcm, Th2, and Treg cells (effect sizes for Th2 and Treg are weak). (C,D) Comparison of estimated immune cell infiltration between high and low OSBPL3 expression groups, highlighting differences in several immune subsets, including Treg, Th17, and Tcm cells. (E,F) Co-expression analysis between OSBPL3-related DEGs and immune cell subsets, showing predominantly negative associations with inferred immune infiltration patterns in the TME; these results are correlative and do not demonstrate functional immune regulation. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. CRC, colorectal cancer; DEGs, differentially expressed genes; NK, natural killer; TME, tumor microenvironment; TPM, transcripts per million.

To further characterize these associations, CRC samples were stratified into high and low OSBPL3 expression groups. Differences in estimated infiltration were observed for several immune subsets, including Treg cells, Th17 cells, and Tcm cells (Figure 3C,3D), supporting that OSBPL3 expression is linked to variations in the immune infiltration landscape. To explore potential molecular features related to these patterns, we assessed the co-expression relationships between OSBPL3-associated DEGs and immune cell subsets. Overall, OSBPL3 expression tended to show negative associations with multiple inferred immune populations, consistent with immune-excluded or immunosuppressive TME features observed in some CRC contexts (Figure 3E,3F). We also observed positive associations with Th cells (predominantly Th2), macrophages, and Tcm cells. Importantly, these findings are correlative and computationally inferred; they do not establish functional immune regulation and should be interpreted as hypotheses that require experimental validation.

Interaction and correlation analysis of OSBPL3 with key proteins in CRC

STRING database analysis revealed that OSBPL3 interacts with multiple key proteins in CRC, including NFE2L2, VAPA, GAK, and HNRNPA2B1, suggesting that OSBPL3 may participate in various BPs through these protein interactions (Figure 4A). To further clarify the association between OSBPL3 and these related proteins, we performed Spearman correlation analysis on the expression levels of OSBPL3 and several related proteins. The results showed that OSBPL3 expression was significantly correlated with the expression of multiple proteins, such as VAPB (r=0.377, P<0.001, Figure 4B), VAPA (r=0.356, P<0.001, Figure 4C), RRAS (r=−0.090, P=0.054, Figure 4D), NFE2L3 (r=0.334, P<0.001, Figure 4E), NFE2L2 (r=0.481, P<0.001, Figure 4F), HNRNPA3 (r=0.417, P<0.001, Figure 4G), and HNRNPA2B1 (r=0.518, P<0.001, Figure 4H). These correlations suggest that OSBPL3 may influence the biological behavior of tumor cells by regulating the expression of these genes. However, the analysis also found that certain genes, such as GAK (r=0.046, P=0.33, Figure 4I) and PDE11A (r=−0.090, P=0.05, Figure 4J), did not show significant correlation with OSBPL3 expression, suggesting that the role of OSBPL3 in CRC may not be mediated through these specific molecular mechanisms.

Figure 4 Interaction and expression correlation analysis of OSBPL3 with key proteins in colorectal cancer. (A) STRING database analysis showing OSBPL3’s interactions with multiple key proteins, including NFE2L2, VAPA, and HNRNPA2B1. (B-J) Spearman correlation analysis illustrating the relationship between OSBPL3 expression and various related proteins. TPM, transcripts per million.

In summary, the significant positive correlations between OSBPL3 and several key proteins in CRC indicate that OSBPL3 may have a crucial regulatory role in the biological behavior of tumor cells. However, its mechanisms may rely on specific molecular pathways, warranting further investigation.

OSBPL3 is highly expressed in CRC

To investigate the expression of OSBPL3 in CRC, we performed IHC and WB analyses. Using data from the Human Protein Atlas (HPA) database (https://www.proteinatlas.org/about/licence), the IHC results revealed low OSBPL3 expression in normal colon tissues but significantly elevated expression in CRC tissues. Under both high-magnification (10×10) and low-magnification (4×10) views, CRC samples exhibited markedly stronger positive staining for OSBPL3, confirming its high expression in these tissues (Figure 5A). Additionally, WB and PCR analyses of various CRC cell lines demonstrated that OSBPL3 protein levels (Figure 5B) and mRNA (Figure 5C) were significantly higher in CRC cell lines (SW620, SW480, HT29, HT115) compared to the normal colon cell line (NCM460).

Figure 5 Analysis of OSBPL3 expression in colorectal cancer. (A) IHC staining from HPA database (https://www.proteinatlas.org/about/licence) showing low OSBPL3 expression in normal colon tissues and significantly higher expression in colorectal cancer tissues at both high (10×10) and low (4×10) magnifications. (B) WB analysis of OSBPL3 protein levels, indicating higher expression in CRC lines (SW620, SW480, HT29, HT115) compared to the normal colon cell line (NCM460). (C) Relative mRNA expression of OSBPL3 in CRC cell lines, showing significant upregulation compared to normal colon cells. **, P<0.01; ***, P<0.001. CRC, colorectal cancer; HPA, Human Protein Atlas; IHC, immunohistochemistry; WB, Western blot.

OSBPL3 promotes proliferation, migration and invasion and inhibits apoptosis in CRC cells

To comprehensively assess the function of OSBPL3 in CRC cells, we conducted knockdown and overexpression experiments to examine its effects on cell proliferation, apoptosis, migration, and invasion (Figures 6,7). CCK-8 assays showed that OSBPL3 knockdown significantly inhibited the proliferation of HT29 and SW480 cells (Figure 6A, P<0.001), whereas OSBPL3 overexpression markedly enhanced their proliferation (Figure 7A, P<0.001). These findings were further supported by EdU incorporation assays, which demonstrated a significant reduction in the proportion of EdU-positive cells following OSBPL3 knockdown (Figure 6B, P<0.001) and a significant increase after OSBPL3 overexpression (Figure 7B, P<0.001). Flow cytometry analysis using Annexin V/PI staining showed that OSBPL3 knockdown was associated with an increased apoptotic fraction in HT29 and SW480 cells (Figure 6C), while OSBPL3 overexpression showed the opposite trend (Figure 7C). The bar graphs report the total apoptosis rate, defined as the sum of early apoptosis (Annexin V⁺/PI⁻) and late apoptosis (Annexin V⁺/PI⁺), with the corresponding quadrants and experimental conditions clearly labeled in the revised figures. Although the differences reached statistical significance, the absolute change was modest (≤~5%); therefore, the biological relevance of this effect should be interpreted cautiously and may warrant further validation. Wound healing assays demonstrated that OSBPL3 knockdown significantly impaired the migration ability of HT29 and SW480 cells (Figure 6D, P<0.001), whereas OSBPL3 overexpression significantly enhanced their migration (Figure 7D, P<0.001). Similarly, Transwell invasion assays showed that the invasive capacity of HT29 and SW480 cells was significantly diminished after OSBPL3 knockdown (Figure 6E, P<0.001), whereas OSBPL3 overexpression led to a marked increase in invasion (Figure 7E, P<0.001). These results suggest that OSBPL3 plays a crucial positive regulatory role in CRC cell proliferation, migration, and invasion, and may facilitate tumor cell survival by inhibiting apoptosis.

Figure 6 Effects of OSBPL3 knockdown on CRC cell behavior. (A) CCK-8 assays showing reduced proliferation of HT29 and SW480 cells after OSBPL3 knockdown. (B) The EdU incorporation assay showed decreased DNA synthesis in OSBPL3-knockdown cells. Cells were fixed and fluorescently stained with a 10× magnification before imaging. (C) Flow cytometry analysis of apoptosis using Annexin V-FITC/PI staining, showing increased apoptotic rates after OSBPL3 knockdown. (D) Wound healing assay showing impaired migration of HT29 and SW480 cells after OSBPL3 knockdown; images were acquired by bright-field microscopy at 10× magnification without additional staining. (E) Transwell invasion assay showing decreased invasive ability after OSBPL3 knockdown; invaded cells were fixed with methanol, stained with 0.1% crystal violet, and imaged under a microscope at 10× magnification. ns, not significant; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; CRC, colorectal cancer; EdU, 5-ethynyl-2'-deoxyuridine; PI, propidium iodide.

Figure 7 Effects of OSBPL3 overexpression on CRC cell behavior. (A) CCK-8 assays showing enhanced proliferation of HT29 and SW480 cells after OSBPL3 overexpression. (B) EdU incorporation assay showing increased DNA synthesis in OSBPL3-overexpressing cells; cells were fixed and fluorescently stained before imaging. (C) Flow cytometry analysis of apoptosis using Annexin V-FITC/PI staining, showing reduced apoptotic rates after OSBPL3 overexpression. (D) Wound healing assay showing enhanced migration of HT29 and SW480 cells after OSBPL3 overexpression; images were acquired by bright-field microscopy without additional staining. (E) Transwell invasion assay showing increased invasive ability after OSBPL3 overexpression; invaded cells were fixed with methanol, stained with 0.1% crystal violet, and imaged under a microscope. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; CRC, colorectal cancer; EdU, 5-ethynyl-2'-deoxyuridine; PI, propidium iodide.

OSBPL3 is associated with CRC cell behavior and correlates with TGF-β/SMAD signaling and epithelial-mesenchymal transition (EMT)-related changes

To explore the relationship between OSBPL3 and CRC cell behavior, we performed in vitro knockdown and overexpression experiments. Stable cell lines with reduced or elevated OSBPL3 expression were generated and confirmed by PCR and WB analyses (Figure 8A,8B). GSEA suggested that OSBPL3 expression is enriched in gene signatures related to the TGF-β signaling pathway, indicating a potential association with TGF-β-related downstream signaling in CRC (Figure 8C). Consistent with this association, WB analysis showed that OSBPL3 knockdown was accompanied by decreased levels of TGF-β, TGF-βR1, and p-SMAD2, together with increased E-cadherin and reduced N-cadherin expression. In contrast, OSBPL3 overexpression was accompanied by increased expression of TGF-β pathway components and corresponding changes in EMT markers (Figure 8D). Taken together, these results support a close relationship between OSBPL3 expression and TGF-β/SMAD pathway activity, as well as EMT-associated molecular alterations, in CRC cells.

Figure 8 Functional analysis of OSBPL3 in CRC cells. (A,B) In vitro experiments demonstrating stable knockdown and overexpression of OSBPL3 in CRC cell lines, confirmed by Western blot analyses. (C) GSEA suggests an association between OSBPL3 and the TGF-β signaling pathway. (D) WB analysis showing the effects of OSBPL3 knockdown and overexpression on TGF-β related proteins and EMT marker genes. ns, not significant; ***, P<0.001. CRC, colorectal cancer; EMT, epithelial-mesenchymal transition; GSEA, gene set enrichment analysis; WB, Western blot.

OSBPL3 knockdown inhibits tumor growth of CRC cells in vivo

To evaluate the effect of OSBPL3 on CRC tumor growth, we performed in vivo experiments in a nude mouse model. OSBPL3 knockdown CRC cells and untreated control CRC cells were injected into nude mice respectively, and the tumor volume was monitored.

The experimental results showed that knocking down OSBPL3 significantly inhibited the tumor growth of CRC cells compared with the control group (Figure 9A). Statistical analysis of tumor volume further showed that the tumor volume of the sh-OSBPL3 group was significantly smaller than that of the control group (P<0.001) (Figure 9B). These results indicate that OSBPL3 plays an important role in promoting the growth of CRC tumors. As summarized in Figure 10, our data suggest that OSBPL3 is linked to TGF-β/SMAD signaling activity and EMT-related molecular changes, which may contribute to CRC invasion and metastasis.

Figure 9 Effects of OSBPL3 knockdown on tumor growth in a CRC xenograft model. (A) Representative images of tumors from mice injected with control CRC cells and OSBPL3knockdown cells. (B) Tumor volume comparison showing a significant reduction in tumor size in the sh-OSBPL3 group compared to the control group. ***, P<0.001. CRC, colorectal cancer.

Figure 10 A schematic representation showed the effect of OSBPL3 on EMT in tumor cells. TGF-β binds to a tetrameric complex of TGF-β RI on cell membrane, which consequently phosphorylates Smad2 proteins and activates expression of EMT inducing transcription factors. OSBPL3 strongly enhances TGF-β induced phosphorylation of Smad2 and thereby enhances the downstream EMT program. EMT, epithelial-mesenchymal transition.

Discussion

OSBPL3, a member of the OSBP family, plays a crucial role in regulating intracellular lipid transport, metabolism, and signaling. Previous studies have indicated that OSBPL3 expression is closely associated with the invasiveness and progression of certain cancer types, though the specific mechanisms may vary across different cancers.

Evidence suggests that OSBPL3 exerts distinct, and sometimes opposing, effects across tumor types. For example, it has been reported to function as a tumor suppressor in lymphoma and bladder cancer, whereas in gastric and liver cancers it may facilitate tumor development. These discrepancies may reflect differences in tumor type and stage, molecular background, detection platforms, cohort composition, and microenvironmental influences, including context-specific shifts in TGF-β signaling. Notably, OSBPL3 is upregulated in gastric cancer and is associated with poor prognosis (20). In contrast, OSBPL3 is expressed at low levels in hepatocellular carcinoma and pancreatic cancer, and reduced expression has been linked to altered cholesterol and lipid metabolism that may influence hepatocellular carcinoma growth (19,22,23). These findings suggest that OSBPL3 exhibits diverse functions and regulatory mechanisms across different types of cancer. In this study, we performed pan-cancer analyses to explore OSBPL3 expression in various tumor cells and adjacent normal tissues. We found that OSBPL3 expression was significantly elevated in CRC patients, suggesting that its overexpression may be closely linked to the onset and progression of CRC. To further investigate the role of OSBPL3 in CRC, we conducted subgroup analyses using data from the TCGA database, which revealed that OSBPL3 expression was associated with the pathological N stage in CRC patients. Additionally, our analysis showed a correlation between OSBPL3 expression and tumor lymph node metastasis, as well as more advanced tumor stages.

Then we conducted the co-expression analysis in patients with CRC to explore relative DEGs. Database results showed that OSBPL3 was positively related to ZBTB20 significantly and negatively associated with SNHG25 on the opposite. ZBTB20 (zinc finger and BTB domain containing 20) is a transcription factor belonging to the zinc finger protein family, containing the BTB/POZ domain, regulating gluconeogenesis and insulin signaling pathways, maintaining normal blood glucose levels (24,25). The dysfunction of ZBTB20 is associated with metabolic diseases such as diabetes (26). Researchers have found that it can act as either an oncogene or a tumor suppressor, depending on the type of tumor and the environment (27). ZBTB20 modulates key cancer-related signaling pathways, such as the PI3K/AKT, Wnt/β-catenin, and TGF-β/SMAD pathways, thereby regulating the growth, differentiation, and metastasis of tumor cells (28). Both ZBTB20 and OSBPL3 are associated with the PI3K/AKT pathway, a critical oncogenic signaling pathway in various cancers. ZBTB20 activates this pathway to promote tumor cell proliferation and survival, while OSBPL3 may indirectly influence it by regulating cell migration and lipid metabolism. Consequently, these two proteins may overlap functionally within the PI3K/AKT pathway, collaboratively promoting tumor progression. In addition, SNHG25 (small nucleolar RNA host gene 25), a long non-coding RNA (lncRNA), is abnormally expressed in various tumors and may play a significant role in tumor development and progression (19,29). In hepatocellular carcinoma, elevated SNHG25 expression is associated with enhanced tumor proliferation, migration, and invasion (30). SNHG25 promotes cancer cell growth by acting as a “sponge” to sequester tumor-suppressive miRNAs, such as miR-497 (31,32). However, there is currently no direct research or clear evidence supporting a specific interaction between SNHG25 and OSBPL3, and further investigation is needed.

Immune infiltration is an important component of the TME and is closely linked to tumor progression (33). Based on CIBERSORT-derived estimates, Spearman correlation analysis suggested that OSBPL3 expression was positively associated with Tcm and Th2 cells, and negatively associated with Th17 cells and regulatory T cells (Tregs). Importantly, although the correlations with Th2 (r=0.140, P=0.002) and Treg cells (r=−0.196, P<0.001) reached statistical significance, their effect sizes were small, indicating that OSBPL3 is unlikely to be a dominant determinant of these immune subsets on its own. Instead, the biological relevance may lie in the directionality of these shifts and their consistency with broader immune-context features of CRC: even modest but reproducible changes in Th2- or Treg-associated signals can reflect a tendency toward immune polarization or immunoregulatory tone when integrated with other TME components and tumor-intrinsic programs. Tcm represent a long-lived T-cell subset that can contribute to anti-tumor immunity by sustaining immune memory and supporting rapid responses to tumor antigens (29,30). In CRC, the MSI-H subtype is often characterized by increased T-cell infiltration and has been associated with favorable responses to immunotherapy, and Tcm enrichment has been reported in this context (31). However, immune-suppressive features within the TME, including the presence of Tregs, may attenuate effective anti-tumor activity even when memory T-cell subsets are abundant. Th2 cells secrete cytokines such as IL-4, IL-5, and IL-13 and can shape immune polarization and macrophage function; in several tumor settings, Th2-skewed responses have been linked to immunosuppressive phenotypes and immune evasion (32). Th17 cells produce pro-inflammatory cytokines such as IL-17, and their roles in cancer are context dependent, with evidence supporting both anti-tumor and pro-tumor effects through inflammation and angiogenesis (34-36). For example, Liu et al. reported that Th17-related signaling may promote EMT and enhance migration and invasion in CRC through cytokine-mediated interactions involving Fn14 (37). Tregs are an immunosuppressive T-cell subset that can limit anti-tumor immunity via inhibitory cytokines including IL-10 and TGF-β (38,39), and elevated Treg infiltration has been associated with immune evasion and unfavorable outcomes in multiple cancers (40,41). Overall, these results support an association between OSBPL3 expression and variability in the inferred immune infiltration profile in CRC, with the strongest relationship observed for Tcm. The weaker Th2 and Treg associations should be viewed as subtle trends that may contribute to, rather than define, the immune milieu, and they are best interpreted alongside other immune populations and tumor-intrinsic pathways. Since these estimates were obtained through computational deconvolution and the analyses are correlational, they provide descriptive evidence of immune-context differences rather than direct proof of functional immune regulation.

Furthermore, we verified the role of OSBPL3 in CRC through cellular experiments and animal model. IHC staining showed the expression of OSBPL3 in tumor samples was significantly higher than that in normal tissues. In vitro knockdown of OSBPL3 in HT29 and SW480 cell lines resulted in a significant reduction in proliferation. Flow cytometry analysis revealed that both early and late apoptosis ratios in the experimental group were significantly higher compared to the control group. The wound healing assay demonstrated that the experimental group showed a marked reduction in cell healing ability after 24 hours, and the Transwell assay indicated a decrease in cell invasion ability in the OSBPL3 knockdown group compared to the control group. These findings suggest that in vitro knockdown of OSBPL3 in CRC cells can inhibit cell proliferation, invasion, and migration, while promoting tumor cell apoptosis. Further studies using a tumor-bearing animal model showed that after OSBPL3 knockdown, the subcutaneous tumor volume in nude mice was significantly reduced, confirming the results of the cell experiments. In vitro overexpression of OSBPL3 in CRC cells exhibited the opposite results, promoting cell proliferation, invasion, and migration, while reducing tumor cell apoptosis.

KEGG pathway analysis suggested that OSBPL3 is enriched in the TGF-β signaling pathway, and our WB results showed that changes in OSBPL3 expression were accompanied by altered levels of TGF-β pathway-related proteins and EMT markers. TGF-β signaling has well-recognized stage-dependent roles in cancer. In early tumorigenesis, TGF-β can restrain tumor development by inhibiting cell-cycle progression, promoting apoptosis, and limiting inflammatory responses. In contrast, at later stages, TGF-β signaling is frequently associated with tumor progression, invasion, and metastasis, partly through EMT induction and immunosuppressive effects (42,43). This functional shift reflects a transition of TGF-β signaling from predominantly tumor-suppressive to tumor-promoting activity in advanced disease (44). In addition, TGF-β can suppress anti-tumor immunity by inhibiting T-cell proliferation and activation and by promoting Treg differentiation (45). EMT facilitates tumor dissemination by enabling cancer cells to breach the basement membrane, invade surrounding tissues, and enter blood or lymphatic vessels, thereby promoting metastasis to distant organs. EMT is also linked to increased resistance to apoptosis and chemotherapy, complicating treatment (46), and may endow tumor cells with stem cell-like properties that enhance self-renewal and drug resistance (47). Mechanistically, TGF-β is a key inducer of EMT and can regulate EMT-related genes via both SMAD-dependent and SMAD-independent pathways (11,43,46). Other pathways, such as Wnt/β-catenin signaling, also contribute to EMT programs by promoting β-catenin nuclear translocation and inducing EMT-related gene expression (48). Collectively, these observations support the clinical relevance of EMT as a process associated with metastasis and therapy resistance; accordingly, EMT inhibition and EMT-related markers have potential value for therapeutic development and prognostic assessment.

In summary, OSBPL3, a pivotal regulator of lipid transport and a contributor to cancer progression, is increasingly recognized for its role in CRC. Elevated OSBPL3 expression is linked to heightened tumor aggressiveness, enhanced metastatic potential, and advanced disease stages. It positively correlates with the oncogene ZBTB20 and negatively with lncRNA SNHG25, thereby modulating the PI3K/AKT pathway and influencing tumor progression. OSBPL3 also interacts with immune cells, including Tcm, Th2, Th17, and Tregs, shaping the TME and regulating EMT. Functional assays reveal that OSBPL3 knockdown reduces CRC cell proliferation, migration, and invasion, while simultaneously inducing apoptosis in vitro and in vivo; conversely, its overexpression accelerates tumor growth. Furthermore, OSBPL3 is associated with the TGF-β signaling pathway and EMT—pathways integral to metastasis and contributors to drug resistance. Nonetheless, the study is constrained by limitations such as a small sample size, bioinformatic biases, and the necessity of further validating OSBPL3’s clinical implications and therapeutic potential.

There also remains several limitations about or study. First, because this was a single-center study with a relatively small sample size, the generalizability of our findings may be limited; validation in larger, multi-center cohorts is therefore needed to confirm the clinical relevance of OSBPL3 in CRC. Second, much of our analysis relied on public databases and bioinformatic pipelines, and cross-dataset heterogeneity—such as differences in platforms, batch effects, and cohort composition—may introduce systematic bias. Third, although we observed associations between OSBPL3 expression and TGF-β/SMAD signaling activity as well as EMT-related molecular changes, the evidence remains primarily correlative and does not establish a definitive mechanistic link. Finally, immune infiltration patterns were computationally inferred (e.g., using CIBERSORT-based deconvolution) and were not validated by experimental methods such as IHC, flow cytometry, cytokine profiling, or functional immune assays. In addition, the potential relationship between OSBPL3 and drug sensitivity, as well as its value for prognostic stratification, requires further confirmation in well-designed translational studies.

Conclusions

In summary, OSBPL3 is highly expressed in CRC and contributes to tumor cell proliferation, migration and invasion. Its expression is closely associated with activation of the TGF-β/SMAD signaling pathway, EMT-related molecular changes, and modulation of the tumor immune microenvironment. These findings highlight OSBPL3 as a potential biomarker for CRC aggressiveness and a promising therapeutic target. Future studies in larger, multi-center cohorts and functional investigations are warranted to further validate its clinical significance and therapeutic potential.

Acknowledgments

All authors would like to express their sincere thanks to the participants who provided colorectal tissue samples for this study and ensuring the successful completion of the study.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2464/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2464/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2464/prf

Funding: This work was supported by grants from Guangdong Administration of Traditional Chinese Medicine Research Foundation (Nos. 20221433, 20231378, and 20241364), Guangdong Basic and Applied Basic Research Foundation (No. 2023A1515220014), Guangdong Medical Science and Technology Research Foundation (No. A2023305), Jiangmen Planned Project of Science and Technology (No. 2320002000781), Technology Project of Traditional Chinese Medicine and Combined Chinese and Western Medicine (No. 20242A011003), Guangdong Provincial Medical Science and Technology Research Foundation (No. B2025584), and Jiangmen Central Hospital Foundation for Distinguished Young Scholars (No. J202103).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2464/coif). The authors have no conflicts of interest to declare.

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. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Research Ethics Committee of Jiangmen Central Hospital (decision No. JXY2023110) and informed consent was obtained from all patients. Animal experiments were performed under a project license (No. GDY2302537) granted by the Animal Ethics Committee of Guangdong Medical University, in compliance with the institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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