Correlation analysis between cancer cell stemness and clinical grade in prostate cancer
Based on OCLR's results and transcriptome data of PCa clinical specimen in TCGA, the correlation between the stemness scores and Gleason grade was analyzed. The results showed that the PCa cell stemness was closely related to Gleason grade and increased with the increased of Gleason classification (Gleason scores; Fig. 1a). In addition, by using WGCNA (Weighted Gene Co-expression Network Analysis) to analyze the PCa transcriptome data for cancer cell stemness-related gene transcripts, we got 30 transcript modules which related to PCa cell stemness (Fig. 1b, Supplementary Fig.1a and 1b), and many stemness-related genes were included in each transcript module. About 88.2% of the 2158 known cell stemness-related genes  were found in the results of our WGCNA analysis (Supplementary Materials S1; Supplementary Fig. 1c). From the results, we also found that MEmagenta module had the most positive correlation with the stemness of PCa cells (Fig. 1b), suggesting that the genes in this module (Supplementary Table S1) might be significant and play key roles in the development of PCa.
From the analysis results, we found that the expression of genes in the MEmagenta module in PCa cancer specimen was generally higher than that in normal prostate specimen; and expression of these stemness gene in PCa increased with Gleason grade, and the highest expression level was found at Gleason score 5 (Fig. 1c). Further, we analyzed the data from 33 sample of GEO database and found that most of the stemness gene expression levels in small cell prostate cancer (SCPC) were higher than those in prostate adenocarcinoma (PRAD) (Supplementary Fig. 1D). Subsequently, the transcriptome data of different prostate (normal or PRAD) cell lines and SCPC cell lines were analyzed and it's found that the expression of stemness gene in PCa cells (PRAD cell lines) was higher than that in normal cells. while the expression level in SCPC cell lines was slightly higher than that in PRAD cell lines (Supplementary Fig. 1e).
In the genes of MEmagenta module (Supplementary Table S1), we found that the known genes closely related to cancer stemness in non-prostate cancer, such as BRCA1 , EZH2 , FOXM1 , CDC20  and CDCA8 , were all clustered in this module, indicating these genes were also closely related to the stemness of prostate cancer cells. From supplementary table S1, we also found that EZH2 was the most significant gene in the correlation with stemness of PCa cells.
The CNVs of stemness-related genes were closely correlated to cell stemness and malignancy of prostate cancer
The segments containing all genes in the MEmagenta module were obtained from TCGA and then analyzed using GISTIC (Genome Identification of Significant Targets in Cancer) method. Analysis results showed that most of genes in MEmagenta module were found to have high variations, which were distinctly higher than those in normal prostate tissue, in prostate cancer, indicating that the stemness of PCa cells was influenced by the CNVs (Copy Number Variations) of these genes (Fig. 2a and Supplementary Fig. 2a). In two types of CNV (amplification and deletion, Supplementary materials S2-S3), the change of deletion CNVs was more than amplification CNVs of the genes in MEmagenta module of PCa samples; and both amplification and deletion CNVs in PCa samples were all much more than those in prostate normal samples (Fig. 2b and 2c, and Supplementary Fig. 2b and 2c). In addition, by combining analysis with Gleason classification of PCa, GISTIC results showed that CNVs of genes in MEmagenta module were also increased with the Gleason socre (Fig. 2d), suggesting that CNVs of genes in MEmagenta module were related to the malignancy of PCa.
In details of CNVs of some genes in MEmagenta module, it was found that the CNVs (amplification) and expression of SKA3 and RUVBL1, which promoted tumor metastasis and played a role in the development of stem cells [35-38], were all increased with the clinical Gleason grade (Supplementary Fig. 3a and 3b); and the CNVs (deletion) and expression of MCM6 and CENPH, which enhanced cancer cell proliferation, stemness and metastasis and promoted cancer development [39-43], were also increased with the increase of clinical Gleason grade (Supplementary Fig. 3c and Fig. 3d).
Transcription regulation of stemness genes was correlated to stemness of PCa cells
ATAC-seq data of the MEmagenta module genes in PCa samples from TCGA, and the sequence data of 2kb range of the transcription start site (TSS) of the genes were analyzed and displayed. Results showed that both normal and tumor prostate samples had the binding signals of transcription factors, and the binding signals of all PCa samples (including different stage of PCa and small cell PCa) were weaker than that of normal prostate samples (Fig. 3a); and the binding signals of transcription regulators in small cell PCa was the weakest one (Fig. 3a, Supplementary Fig. 4b). These results indicated that the transcriptional regulation manner and the types of transcription regulators of the gene of MEmagenta moduel in PCa might be different with those in normal prostate tissues.
After clustering transcriptome of 253 common transcription factors in PCa, it was found that FOXA1, HOXB13, ERG1, et al. were highly expressed and NANOG, FOXA3, SOX3, et al. were lowly expressed in all prostate cancer (Supplementary Fig. 4a). In the top results of principal component analysis (PCA) of 253 common transcription factors, it was found that stemness-related PUM1 [44, 45], CLOCK , SP1 and TCF12 played a major positive regulation role on PCa cell stemness, while IRF3  was a negative correlation with other 9 transcription factors and played negative regulation role on PCa cell stemness (Fig. 3b, Supplementary Fig. 4c). As we know, IRF3 (interferon regulatory factor 3) signaling played an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways , suggesting that the immune system might play a role in suppressing the stemness of PCa cells. Furthermore, out analysis results showed that AR, FOXA1, NFYA, CTCF and FOXO3 might enhance the stemness of PCa cells, where FOXF1 might be negatively correlated with these transcription factors (Fig. 3c).
In normal prostate samples, we found that the major transcription regulators were HOXB4, NFYB and TFE3 (Supplementary Fig. 5a and 5c), which were different from those in prostate cancer. In normal samples, the role of FOXA1, NFYA and FOXP1 in regulating stemness genes was changed, by comparing their results of PCA analysis in prostate cancer (Supplementary Fig. 5b and Fig. 3c). These results indicated that same transcription factors might play different roles in regulating the cell stemness in prostate normal and cancer samples.
Our results of transcriptional regulator analysis showed that the upstream of EZH2 gene, the most relevant gene to the stemness of PCa cells, could be significantly bound by NFY (Fig. 3d). As a transcription factor, NFY not only regulates the self-renewal of hematopoietic stem cells , but also promotes the self-renewal and expansion of prostate cancer cells and their stemness . Hence EZH2 might play a stemness roles in prostate cancer.
Immunological microenvironment negatively related to the stemness of PCa cells
By scoring the stemness and immunity of immune microenvironment of PCa in different Gleason stages of clinical samples, it was found there was a negative correlation between the PCa cell stemness and the immunity of microenvironment of PCa clinical samples in all stages; and the correlation coefficient of stage I-II was almost the same as that of stage III-V (Fig. 4a). By analyzing and scoring the stromal and immune cells of PCa microenvironment, we found that the scores of stromal and immune cells in PCa microenvironment were all inversely related to the stemness of PCa cells (Supplementary Fig. 6a); and immunity and immune cells of PCa microenvironment were improved and enhanced with PCa progression (Supplementary Fig. 6b). After clustering the genes of MEmangenta module based on cell stemness and immune scores of PCa, we found that PCa cell stemness was negatively related to immunity of PCa microenvironment in the clinical samples with high expression of genes of MEmagenta module (Supplementary Fig. 6d), indicating the immune microenvironment had an inhibitory effect on the cell stemness of PCa. Furthermore, by analyzing and scoring the immune infiltration of PCa clinical samples from TCGA, the number of 22 types of immune cells in prostate cancer with different clinical grades was obtained (Fig. 4b). The number of most types of immune cells in microenvironment of PCa was increased with the Gleason score increase; and CD8+ T cells  and macrophage M1  were the most significantly increased in all types of immune cells, while the plasma cells  were significantly reduced with the Gleason score increase (Fig. 4b), indicating that plasma cells (B cells) in microenvironment of PCa played an important role in anti-PCa immunity. By analyzing the prostate tumor microenvironment immune cell network, it was found that the correlation among immune cells in cluster-A was more complicated than that in cluster-B, cluster-C and cluster-D (Fig. 4c; Supplementary Materials S4). For examples, the activated NK cell was negatively correlated with resting NK cell, which was most significant in all correlation among immune cells (Supplementary Fig. 6c) and the number of activated NK cell increased with the Gleason score increase in PCa (Fig. 4b), indicating that activated NK cell might play a major inhibitory funciton on prostate cancer stemness; the most significant positive correlation was between the activated dendritic cell and the memory B cell, and the number of both activated dendritic cells and memory B cells were all increased with the Gleason score increase (Fig. 4c), which indicated that these two type of cells might play important roles in inhibiting the stemness of PCa cells.
Protein interaction network of MEmagenta module genes and relationship between expression of MEmagenta module genes and immune infiltration of PCa
By screening and analyzing human protein interaction data containing MEmagenta module genes from STRING database, we not only found the important protein-protein interaction network in the proteins of MEmagenta module genes, but also found that EZH2 interacted directly with 17 proteins. In the EZH2-related 17 protein-protein interactions, we also found that EZH2 could regulate entire protein interaction network of MEmagenta module stemness genes by mainly interacting with CENPA, BUB1B and PARP1 (Fig. 5a) [51-54]. Furthermore, function enrichment analysis of genes of MEmagenta module revealed that most function of these genes were concentrated in cell mitosis, and the most significant functional pathway was related cell cycle (Supplementary Fig. 7a and 7b), suggesting that these stemness genes might involve in the regulation of PCa stem cell mitosis.
From the analysis results of relationship between expression of MEmagenta module gene and immune infiltration of PCa, we found that different immune cells had different effects on the expression of stemness genes in PCa; and different type of immune cells could have effects on the same stemness gene expression, as well as one stemness gene expression could also reversely affect different type of immune cells (Supplementary materials S5). In correlation of expression of stemness genes and immune cells, we found that expression of most stemness genes were positively correlated to memory B cells and naive B cells and negatively correlated to plasma cells (Fig .5b); and further, the number of these B cells increased, while the number of plasma cells decreased, with the increase of PCa Gleason score (Fig. 4b). These results indicated that B cells might play the opposite effects on PCa cell stemness in different conditions (it's consistent with reference ). In addition, the number of activated NK cells and memory CD4+ T cells were all increased with the Gleason score; and the expression of stemness genes of MEmagenta module was positively correlated to resting NK cells and memory CD4+ T cells, while negatively correlated to activated NK cells (Fig. 4b and 5b).
Expression of EZH2, the most relevant gene to PCa cell stemness, was most positively correlated to the activated memory CD4+ T cells and most negatively correlated to the resting Mast cells (Fig. 5c and 5d). Most types of cells positively correlated to the expression of EZH2 gene were T cells and B cells (Fig. 5c), suggesting that T cells and B cells were the important immune cells in regulating PCa cell stemness by controlling the expression of EZH2 gene. Further, the number of resting mast cells decreased and number of activated mast cell increased with the increased PCa Gleason scores (Fig. 4b); and the resting mast cells were the most negative correlated to expression of EZH2 gene (Fig. 5c and 5d). These results indicated that the immunity and immune cells of the microenvironment of PCa played an important role in the tumorigenesis and development of prostate cancer.