Determination of the human PTC transcriptomic landscape
Cells were isolated respectively from three patients with nodular goiter, four male patients with PTC, and seven female patients with PTC, followed by single-cell transcriptomic sequencing based on BD Rhapsody (Fig. 1A). All cell clusters were differentiated according to the three disease statuses (nodular, male PTC, and female PTC), which showed that the dominant clusters were different between male and female patients (Fig. 1B). The proportion of endothelial cells, fibroblasts, and immune cells was greatly different in patients with PTC and nodular goiter (Fig. 1C). Next, we focused on malignant epithelial cells, fibroblasts, endothelial cells, T cells and B cells by analyzing the differences in cell differentiation and gene expression profiles, as well as their interactions according to gender (Fig. 1D).
Differences in cell differentiation and gene profiles of malignant epithelial cells between male and female patients with PTC
CNV is the premise of normal cell cancerization. Epithelial cells from nodular goiter tissues were used as references, and the CNVs of epithelial cells in PTC samples from male and female patients were detected, respectively. The results showed the presence of significant differences in mutation regions and frequencies in both groups (Fig. 2A). Basing on the CNV of epithelial cells, the cell clusters of malignant epithelial cells could be determined (Fig. 2B). Moreover, the distribution of malignant epithelial cells in male and female PTCs showed two different subsets (Fig. 2C). The PCA revealed that the malignant epithelial cells in male and female patients with PTC could be classified into two subsets with obvious differences (Fig. 2D). Furthermore, the gene profiles of malignant epithelial cells were analyzed and characterized in male and female patients. The 50 genes with the highest expression levels were distinctly different between the two groups (Fig. S1). As shown in Figure S1, multiple members of the HLA gene family, including HLA-DRA, HLA-DPB1, and HLA-C, were significantly upregulated in the malignant epithelial cells of female patients with PTC. Additionally, the expression of many genes (such as RPS4Y1, S100A4, and SPOCK2) in male patients with PTC were significantly up-regulated compared with female patients. These results were also highlighted by a ridgeline plot (Fig. S2). The 100 genes with the highest expression were selected for KEGG analysis in malignant epithelial cells, which revealed that the top 10 signaling pathways with the most abundant genes were obviously different between male and female patients. Among them, four pathways (focal adhesion, proteoglycans in cancer, adherens junction, and pathways in cancer) held a dominant position in male patients, whereas an additional three pathways (herpes simplex infection, antigen processing and presentation, and HTLV-1 infection) were outstanding in female patients (Fig. 2E). Concomitantly, the selected genes were analyzed regarding molecular networks. The four most advantaged pathways were significantly different and had almost no intersection between male and female patients (Fig. 2F). Subsequently, the advantaged pathways were subjected to gene set enrichment analysis (GSEA), which showed that the advantaged pathways that played strong positive roles in female patients did not in male patients. Similarly, the advantaged pathways that played strong positive roles in male patients had a weak positive effect in female patients (Fig. 2G).
We performed a pseudotime analysis of all clusters of epithelial cells, and defined five differentiation states. Subsequently, we found an obvious aggregation of the malignant epithelial cells in male and female patients with PTC in state 3 (Fig. 3A). Cells in state 1 and state 2 were considered to be in the early stage of differentiation and could differentiate into malignant cells (state 3) and other cell subsets (state 4). The genes related to signal transduction, proliferation, extracellular matrix, and protein synthesis were significantly up-regulated in malignant tumor cells (Fig. 3B). Further analysis showed that the enrichment of malignant tumor cells was greater in male compared with female patients in states 2 and 7, whereas the distribution was uniform in other states according to gender (Fig. 3C). These results were further confirmed by cluster analyses of gene expression profiles and by a pathway enrichment analysis (Fig. 3D-3G and Fig. S3).
In summary, the characteristics of malignant epithelial cells, including gene profiles, cell clusters and cell differentiation, were significantly distinct according to the gender.
Interactions of malignant epithelial cells with fibroblasts and endothelial cells
Several other types of proximal cells, including fibroblasts and endothelial cells, interacted closely with malignant tumor cells and affected their growth, migration, and invasion. Fibroblasts and endothelial cells were less abundant in PTC tissue than the nodule tissue (Fig. 4A). In tumor tissues, malignant tumor cells, ordinary epithelial cells, endothelial cells and fibroblasts were used as the objects of analysis. A cell interaction analysis network was constructed. Obviously, the interactions of malignant epithelial cells with fibroblasts and endothelial cells were strong in both groups (Fig. 4B). Based on ligand-receptor pairs, there were significant differences in the interactions between cells from female and male patients with PTC (Fig. 4C and 4D).
Taken together, these results suggest that the interactions of malignant epithelial cells with fibroblasts and endothelial cells were different between male and female patients with PTC.
B lymphocyte infiltration in PTC tissues from male and female patients
The infiltration of immune cells into tumor tissues plays an important role in the development and treatment of cancers. B lymphocytes were the focus of our uniform manifold approximation and projection (UMAP) analysis. The count of B lymphocytes in PTC from male patients were significantly less than this in other two groups (Fig. 5A). All B lymphocytes in the three groups were clustered into five groups. Among them, it was particularly noteworthy that the proportion of exhausted B cells and plasmablasts was increased significantly in female patients compared with male patients (Fig. 5B and 5C). The representative genes of the exhausted B cell and plasmablast clusters were analyzed using tSNE and are shown in Figure S4. The differentiation of B lymphocytes formed two key nodes. Among them, there was almost no differentiation between state 2 and state 4 in male patients, which was obviously different from female patients with PTC and those with nodular goiter (Fig. 5D and 5E). The typical genes in the differentiation of state 2 and state 4 were screened and displayed in a trajectory distribution of pseudotime, with some overlap with the data in Figure 5D (Fig. 5F). Based on ligand-receptor pairs, malignant tumor cells (clusters 13 and 31 shown in Fig.1 A), exhausted B cells and plasmablasts were used as the objects of analysis. The results indicated that the interaction involving PDCD1, CD47, and CD94 between cluster 13 and exhausted B cells in male patients was stronger than that observed in female patients with PTC (Fig. 5G and 5H). In summary, the immune infiltration of B lymphocytes, including cell number, cell differentiation, and the interaction with malignant cells, was significantly different between male and female patients.
T lymphocyte infiltration in tissues from male and female patients with PTC
T lymphocytes were assessed by UMAP analysis according to the three experimental groups (Fig. 6A). PCA showed some differences in the differentiation of T lymphocytes between PTC from female and male patients (Fig. 6B). T lymphocytes were clustered in nine subsets (Fig. 6C). Among them, the proportion of three subsets (vd2_gd_T, NK, and CD8+T) was obviously different between female and male patients (Fig. 6D). It was also confirmed by the expression of the representative genes of the three subsets (Fig. 6E). The differentiation of T lymphocytes formed two key nodes, without significant differences between male and female patients (Fig. 6F).