Comparative transcriptomic and genomic analysis of tumor cells in the marginal and center regions of tumor nests in human hepatocellular carcinoma

Abstract

Background: The tumor nests in solid tumors, including hepatocellular carcinoma (HCC), possess tumor-initiating potential, with the capacity to metastasize and form new metastatic lesions. However, the biological characteristics and heterogeneity of tumor cells at the central and marginal regions of these tumor nests remain poorly understood.

Method: Based on pathological tissue sections, data integration and dimensionality reduction, we defined the boundaries and centers of tumor nests and fibrous nodules within 19 spatial transcriptomics (ST) samples from 8 HCC patients. Differential gene expression was analyzed at the single-unit, sample, patient, and pseudobulk levels, followed by Gene Ontology (GO) enrichment analysis. Additionally, spatial copy number variation (CNV) was inferred using inferCNV, and comparisons were made at the single-unit, sample, patient, and pseudobulk levels.

Results: Ultimately, 24 tumor nests and 15 liver fibrosis nodules were analyzed. The spatial gene expression patterns of the tumor nests exhibited significant heterogeneity, with gene enrichment analysis revealing upregulation of immune-related pathways (e.g., humoral immune response mediated by circulating immunoglobulin; B cell receptor signaling pathway, etc.) at the tumor nest margins and growth/metabolism-related pathways (e.g., sulfur amino acid metabolic process; proteinogenic amino acid metabolic process, etc.) within the tumor nest center. Similar expression patterns were also observed in liver fibrous nodule samples. CNV and clustering analyses demonstrated transcriptional differences between tumor nests within individual patients, suggesting the evolutionary diversity, or heterogeneity, of tumor nests within the same tumor.

Conclusion: Tumor nests exhibit significant transcriptional differentiation along spatial axes: the central regions show high expression of metabolism-related genes, while the marginal regions are enriched in immune-regulatory genes. This pattern is also observed in liver fibrotic nodules. This center-margin functional division may inform rational design of therapeutics that simultaneously modulate metabolism and immune responses.

Keywords: fibrotic nodules; hepatocellular carcinoma; heterogeneity; spatial transcriptomic; tumor nest.

FIGURE 1

Dimensionality reduction clustering and sampling schematic of ST data. (A) Clustering results of ST data. A total of 19 samples (right-top) obtained from 8 HCC patients were included. Hepatocyte_BEC: Hepatocyte-Biliary Epithelial Cells; (B) Schematic representation of ST data sampling. f-c, Fibrotic nodule-center; f-m, Fibrotic nodule-margin; t-c, Tumor nest-center; t-m, Tumor nest-margin.

FIGURE 2

Analysis of differentially expressed genes (DEGs) across tumor nests in HCC patient samples. (A,B) Venn diagram showing upregulated DEGs in the marginal (A) or central (B) regions of three tumor nests from patient HCC-BT; (C,D) Upset plot displaying upregulated DEGs in the marginal (C) or central (D) regions of tumor nests, analyzed at the individual patient level. (E) Functional enrichment of genes upregulated in the marginal regions of the tumor nest; (F) Functional enrichment of genes upregulated in the central regions of the tumor nest. Enrichment categories include Biological Process (BP), Molecular Function (MF), and Cellular Component (CC).

FIGURE 3

Differential gene expression analysis of fibrotic nodules in individual patients. Shown are the results of the representative sample HCC-1N. See also Supplementary Figure 4. (A,B) Venn diagram showing upregulated DEGs in the marginal (A) or central (B) regions of three fibrotic nodules from patient HCC_1N; (C,D) Upset plot displaying upregulated DEGs in the marginal (C)or central (D) regions of fibrotic nodules, analyzed at the individual patient level. (E) Functional enrichment of upregulated genes in the marginal regions of fibrotic nodules; (F) Functional enrichment of upregulated genes in the central regions of fibrotic nodules. Enrichment analyses were categorized into Biological Process (BP), Molecular Function (MF), and Cellular Component (CC).

FIGURE 4

Functional enrichment analysis of differential genes in tumor nests. (A) Functional enrichment analysis of upregulated genes in the marginal region of tumor nests; (B) Functional enrichment analysis of upregulated genes in the central region of tumor nests. Enrichment analyses are categorized into Biological Process (BP), Molecular Function (MF), and Cellular Component (CC).

FIGURE 5

Comparison of CNV levels in tumor nests and fibrotic nodules. (A) Comparison of CNV levels between the central and marginal regions of tumor nests. c, center; m, margin; (B) Overall CNV levels across different tumor nests within the same sample. (C) Comparison of CNV levels between the central and marginal regions of fibrotic nodules. c, center; m, margin; (D) Overall CNV levels across different fibrotic nodules within the same sample. *, **, ***, **** indicate, p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

FIGURE 6

Comparison of transcriptomic and CNV profiles at the pseudobulk level. (A) Comparison of CNV at the pseudobulk level in tumor nests. c, center; m, margin; (B) Clustering of transcriptomic similarity at the pseudobulk level across tumor nests and analysis units. c, center; m, margin.