The Role of PLIN3 in Prognosis and Tumor-Associated Macrophage Infiltration: A Pan-Cancer Analysis

Abstract

Background: Nucleolar and spindle-associated protein 1 (PLIN3), a member of the perilipin family, plays a critical role in lipid droplet dynamics and is implicated in promoting tumor progression across several cancers. However, its influence on the tumor immune microenvironment and its potential as a prognostic indicator regarding immunotherapy responses have yet to be systematically evaluated. This study leverages data retrieved from multiple databases to address these questions.

Methods: PLIN3 mRNA and protein expressions were analyzed across a diverse range of normal and cancerous tissues, utilizing data retrieved from multiple databases. The potential of PLIN3 as a diagnostic and prognostic biomarker in cancers was assessed. Advanced computational algorithms were employed to examine the impact of PLIN3 on immune cell infiltration. The association between PLIN3 expression and the presence of M2 macrophages was validated through analyses incorporating bulk and single-cell transcriptomics, spatial transcriptomics, and multicolor fluorescence staining techniques. Furthermore, the effects of PLIN3 on tumor malignancy and growth were investigated in vitro in lung adenocarcinoma (LUAD) cells. Potential compounds targeting PLIN3 were identified using the Connectivity Map (cMap) web tool, and their efficacy was further assessed through molecular docking.

Results: PLIN3 was predominantly upregulated in various cancers, correlating with adverse prognostic outcomes. A strong positive association was observed between PLIN3 levels and M2 macrophage infiltration in several cancer types, establishing it as a potential pan-cancer marker for M2 macrophage presence. This was confirmed by integrative multi-omics analysis and multiple fluorescence staining. Additionally, PLIN3 knockdown in LUAD cells diminished their malignant traits, resulting in decreased proliferation and migration. In LUAD, clofibrate was identified as a potential inhibitor of PLIN3's pro-oncogenic functions.

Conclusion: PLIN3 may serve as a potential biomarker and oncogene, particularly in LUAD. It plays a key role in mediating M2 macrophage infiltration in various cancers and presents a promising immunotherapeutic target.

Keywords: M2 macrophage; biomarker; immunotherapy; pan-cancer analysis; prognosis.

Figure 1

Study flowchart.

Figure 2

Differential expression of PLIN3 and its prognostic potential across various cancers. (A) Comparison of PLIN3 levels in tumor versus normal tissue samples from GTEx and TCGA databases. (B) PLIN3 mRNA expression in paired tumor and normal samples from TCGA. (C) PLIN3 expression profiles across various organs comparing tumor and normal tissues. (D) Diagnostic ROC curves evaluating PLIN3 as a biomarker across multiple cancers. Abbreviation list of tumor cohorts from TCGA is given in.Supplementary Table 1.

Figure 3

Differential analysis of PLIN3 protein levels. (A) Wilcoxon Rank Sum Tests to compare the statistical differences in expression levels between the tumor and normal groups from the CPTAC dataset. (B) IHC images of PLIN3 staining sourced from the Human Protein Atlas. Abbreviation list of tumor cohorts from TCGA is given in.Supplementary Table 1.

Figure 4

Survival analysis correlating PLIN3 expression with patient outcomes in pan-cancer. (A–D) Forest plots displaying the prognostic significance of PLIN3 for OS, DSS, DFI, and PFI via univariate Cox regression analysis. (E) Kaplan-Meier plots for OS, with the red and blue lines representing high and low PLIN3 expression groups, respectively. Abbreviation list of tumor cohorts from TCGA is given in.Supplementary Table 1.

Figure 5

Genetic alterations of PLIN3 across various cancers. (A) Overview of PLIN3 mutation types using data from the COSMIC database. (B) Mutation frequency of PLIN3 across different cancer types. (C) Detailed mapping of PLIN3 genetic alterations, including sites and numbers, sourced from cBioPortal. (D) Histogram depicting the frequency of somatic copy number alterations of PLIN3 in each cancer type. (E) Analysis of differential PLIN3 expression across various CNV types in pan-cancer. Abbreviation list of tumor cohorts from TCGA is given in.Supplementary Table 1.

Figure 6

Associations of PLIN3 with DNA repair, stemness, and epigenetic modifications. (A) Heatmap illustrating correlations between PLIN3 and five MMR-related genes. (B) Lollipop chart detailing the correlation of PLIN3 levels with DNA methylation-based stem scores. (C) Bar chart showing the correlation of PLIN3 levels with RNA methylation-based stem scores. (D) Heatmap of the relationships between PLIN3 levels and RNA modifications. (E) Heatmap displaying associations of PLIN3 with four methyltransferases. (F) Radar chart presenting the Spearman correlation between PLIN3 expression levels and TMB across pan-cancer. (G) Radar chart showing the Spearman correlation between PLIN3 expression levels and MSI across pan-cancer. (H) Heatmap displaying relationships between PLIN3 expression and ESTIMATE, Immune, and Stromal scores, with statistical significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviation list of tumor cohorts from TCGA is given in.Supplementary Table 1.

Figure 7

Functional analysis of PLIN3 in human cancers using GSEA. Bubble plot illustrating differential enrichment of hallmark gene sets between PLIN3-high and -low tumor patients. Circle size corresponds to the magnitude of the P-value, while color transitions from red to white to blue indicate the strength of NES. The red boxes highlight key pathways that are significantly enriched in PLIN3-high tumor patients, emphasizing their potential biological importance in cancer progression. Abbreviation list of tumor cohorts from TCGA is given in Supplementary Table 1.

Figure 8

Correlation analysis between PLIN3 expression and immune cell infiltration. (A) Heatmap showing correlations between PLIN3 mRNA expression and the expression of chemokines, chemokine receptors, immune-inhibitors, immune-stimulatory, and MHC genes. (B) Heatmaps displaying correlations between PLIN3 expression and infiltration levels of various immune cells. Abbreviation list of tumor cohorts from TCGA is given in.Supplementary Table 1.

Figure 9

PLIN3 as a Potential Marker of M2 Macrophage Infiltration. (A) Heatmap showing PLIN3 gene expression across various microdomains in pan-cancer spatial transcriptomic sections. Rows represent different datasets, each labeled in a color specific to the disease type. Columns represent different cell types, with the color intensity on the right scale indicating data values—darker red signifies higher values, and lighter colors indicate lower values. Gray indicates that the cell type is absent in the microregion. The red boxes highlight PLIN3’s widespread expression in macrophage and malignant cell types. (B) Spearman correlation analysis used to calculate the correlation between cell content across all spots and between cell content and gene expression levels. Red lines indicate positive correlations, green lines indicate negative correlations, and gray lines indicate non-significant correlations. Line thickness represents the magnitude of the correlation coefficient (C) Spatial transcriptomics exploring co-localization patterns of PLIN3 with CD68 and CD163, color-coded by expression levels. Each dot represents a microdomain (spot) with deeper red indicating higher gene expression. (D) Expression of PLIN3 in cancer-specific single-cell clusters analyzed using the TISCH database. The red boxes highlight PLIN3’s widespread expression in monocyte, macrophage and malignant cell types. (E-F) UMAP plots detailing cell type distributions and PLIN3 intensity in KIRC (E) and NSCLC (F).Abbreviation list of tumor cohorts from TCGA is given in.Supplementary Table 1.

Figure 10

Investigation of PLIN3’s role in regulating malignancy in LUAD tumor cells. (A) Fluorescent staining of tumor tissues showing CD68 (red) and PLIN3 (green), with DAPI (blue) for counterstaining. (B) Fluorescent images of tumor tissues with CD163 (red) and PLIN3 (green), counterstained with DAPI (blue). (C) PLIN3 mRNA expression levels in transfected cells. (D) Colony formation assay to evaluate the impact of PLIN3 on tumor cell proliferation. (E) Wound healing assay assessing the effect of PLIN3 knockdown on tumor cell migration.

Figure 11

Drug Sensitivity Analysis. (A) Bubble plot illustrating correlations between PLIN3 expressions and drug sensitivity across various databases. A p-value < 0.05 was considered statistically significant. (B) The heatmap illustrates potential compounds that target PLIN3, identified using CMap analysis across various cancers. (C) Identification of PLIN3-targeting compounds through CMap analysis for LUAD. (D) 3D molecular docking illustrations showing interactions between PLIN3 and compound clofibrate. Abbreviation list of tumor cohorts from TCGA is given in.Supplementary Table 1.