Integrative Analysis of Bulk RNA-Seq and Single-Cell RNA-Seq Unveils the Characteristics of the Immune Microenvironment and Prognosis Signature in Prostate Cancer

The immune microenvironment is a culmination of the collaborative effort of immune cells and is important in cancer development. The underlying mechanisms of the tumor immune microenvironment in regulating prostate cancer (PRAD) are unclear. In the current study, 144 natural killer cell-related genes were identified using differential expression, single-sample gene set enrichment analysis, and weighted gene coexpression network analysis. Furthermore, VCL, ACTA2, MYL9, MYLK, MYH11, TPM1, ACTG2, TAGLN, and FLNC were selected as hub genes via the protein-protein interaction network. Based on the expression patterns of the hub genes, endothelial, epithelial, and tissue stem cells were identified as key cell subpopulations, which could regulate PRAD via immune response, extracellular signaling, and protein formation. Moreover, 27 genes were identified as prognostic signatures and used to construct the risk score model. Receiver operating characteristic curves revealed the good performance of the risk score model in both the training and testing datasets. Different chemotherapeutic responses were observed between the low- and high-risk groups. Additionally, a nomogram based on the risk score and other clinical features was established to predict the 1-, 3-, and 5-year progression-free interval of patients with PRAD. This study provides novel insights into the molecular mechanisms of the immune microenvironment and its role in the pathogenesis of PARD. The identification of key cell subpopulations has a potential therapeutic and prognostic use in PRAD.


Introduction
PRAD is a common malignancy in males [1,2], ranking first in incidence and second in mortality [3]. However, the aetiology of a disease as common as PRAD remains unclear [4]. Hormones regulate the growth and maintenance of the prostate gland, with androgens playing a key role in PRAD progression [1,5]. Additionally, several dietary factors, such as soy protein, animal fat, and fibers, have been considered etiologic factors that are vital for the progression of PRAD [6].
Current PRAD examinations, including digital rectal diagnosis, serum prostate-specific antigen (PSA), ultrasound detection, computed tomography (CT), and magnetic resonance imaging (MRI), present certain limitations, especially in patients suffering from biochemical recurrence and re-evaluation after treatment [7]. However, the current diagnoses cannot reveal PRAD's heterogeneity [8], which is vital in predicting drug treatment sensitivity or primary resistance [9]. erefore, exploring the pathogenesis of PRAD is highly significant as it can aid in identifying new targets for its diagnosis, thereby developing new drugs and improving patient prognosis.
It has been widely reported that tumorigenesis, tumor growth, and tumor metastasis are affected by the tumor immune microenvironment (TME), which includes immune cells, stromal cells, blood vessels, and the extracellular matrix [10]. Genetic alterations have been reported to alter the microenvironment in epithelial cells [11]. Furthermore, numerous studies report the complexity of TME, with the tumor-infiltrating immune cells (TIICs) playing a crucial role in TME [12]. TIICs in the TME are correlated with the prognosis and treatment response [10]. However, many modulatory interactions between the immune cells remain unknown because of TME complexity. Because of the heterogeneous and dynamic nature of cancer's microenvironment, the immune contexture of TME has been shown to affect the disease's outcome [13]. Hence, it is necessary to conduct an in-depth analysis of the PRAD TME at the single-cell level.
Single-cell sequencing is a technology that performs sequencing and analysis at the level of the genome, transcriptome, and epigenome in a single cell. Single-cell resolution is beneficial for analyzing intratumor heterogeneity [14]. Unlike bulk RNA-Seq data analysis, single-cell RNA-Seq (scRNA-seq) analysis allows for an in-depth profile of cell populations at the cellular level and aids in the discovery of rare subpopulation functions, thus eliminating the problem of deconvolution caused by bulk measurements. However, most of the present study uses bulk RNA-Seq data. RNA-Seq data from bulk samples assembles the variant allele frequency profiles of thousands to millions of cells, which cannot be obtained from scRNA-Seq data. e mechanism underlying the effects of immune cell infiltration on the prostate at the single-cell level remains unclear. erefore, considering the heterogeneity of PRAD cells, this study uses bioinformatic analyzes, including extensive genomic and transcriptomic characterization with both bulk RNA-Seq and scRNA-Seq to elucidate the mechanisms. Firstly, bulk RNA-Seq data are used to find key genes associated with PRAD TME. Secondly, the single-cell sequencing data of PRAD is used to analyze the expression patterns of key genes in different cell subsets.
us, the role and potential mechanisms of TME in PRAD could be explained at the single-cell level.

Data Source.
For bulk RNA-seq analysis, the R package "TCGAbiolinks" was used to obtain level 3 mRNA expression data of PRAD from the cancer genome atlas (TCGA) database. en, the data was further filtered by the TCGAanalyze_Preprocessing function using coefficient >0.6 among samples as the criterion, and the genes with an average expression level ranking in the top 75% were retained by the TCGAanalyze_Filtering function. After data processing, 498 PRAD and 52 adjacent control samples (normal tissue), namely the TCGA-PRAD cohort consisting of 13,125 genes, were used for downstream analysis. e normalized PRAD single-cell sequencing data, including 36,424 cells and 24,391 genes, which have been preprocessed in a previous study [15], were downloaded from the gene expression omnibus (GEO) GSE141445 dataset for downstream analysis. Additionally, the bulk RNA-seq data and survival information of patients with PRAD in GSE54460 (N � 106), GSE46602 (N � 36), GSE70768 (N � 111), and GSE70769 (N � 92) were used as testing sets. e pipeline of the entire process is shown in Figure S1.

Identification and Functional Analysis of Differentially
Expressed Genes (DEGs) in TCGA-PRAD. DEGs in PRAD and adjacent control samples were identified using the "DESeq2" R package, with a threshold of |log2FC| > 1 and adjusted p-value < 0.05. R package "ClusterProfiler" was employed to perform the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyzes of DEGs. A q-value <0.05 indicated significant enrichment.

Landscape of Immune Infiltration in TCGA-PRAD.
Single-sample gene set enrichment analysis (ssGSEA) algorithm in the R package "GSVA" was utilized to calculate the infiltration levels of 27 immune cell types in PRAD and adjacent control samples [16]. e difference in immune cell infiltration between PRAD and adjacent control samples was determined using the Wilcoxon test [17]. Immune cells with a p-value < 0.05 were considered differentially infiltrated immune cells (DIICs).

Identification of DIIC-Related DEGs in TCGA-PRAD.
Weighted correlation network analysis (WGCNA) was performed based on the gene expression profiles and DIICs. A sample clustering treemap was constructed to detect and eliminate outliers. Subsequently, WGCNA was performed based on the gene expression profiles and sample traits (differentially enriched immune cell types). e "pick Soft reshold" function of WGCNA was used to calculate β and select the best soft threshold. Based on the selected soft threshold, the adjacency matrix was converted to a topological overlap matrix for constructing the network. Moreover, the gene dendrogram and module color were established using a dissimilarity degree. e initial module was further divided using dynamic tree cutting, and similar modules were merged. Pearson's correlation coefficient between the module eigengenes and DIICs was calculated to identify the most relevant module associated with DIICs. Using module membership (MM) > 0.8 and gene significance (GS) > 0.5 as criteria [18], DEGs in the most relevant module associated with DIICs were screened for further analysis.
DEGs. Pearson's correlations among the candidate hub DIICs-DEGs were calculated and visualized using the R package "ggcorrplot." e function of candidate hub DIICs-DEGs was analyzed using the Metascape online tool (https:// metascape.org/). e expression patterns of the candidate hub DIICs-DEGs in PRAD and adjacent control samples in TCGA-PRAD were tested in an external dataset, GSE54450. e candidate hub DIICs-DEGs with consistent expression patterns in the TCGA-PRAD and GSE54460 datasets were identified as differentially infiltrated immune cell-related genes (DIICRGs).

Single-Cell Analysis.
e Seurat pipeline was used to analyze single-cell sequencing data and classify the cell groups. e cell types were identified using SingleR [19], and the cell differentiation trajectory was analyzed using Monocle [20].

Construction and Validation of the Risk Score Model.
After converting the gene symbol of the selected DEGs in TCGA to ENTREZ ID, the intersection of DEGs and marker genes of cell clusters was analyzed using univariate Cox regression to screen genes related to the progression-free interval (PFI) (HR ≠ 1, p-value < 0.0001 for stricter screening) [21]. e LASSO algorithm was employed to further screen for prognostic signatures under the optimal lambda with the smallest classification error using the R package "glmnet." After the proportional hazards (PH) assumption, prognostic signatures with a p-value >0.05 were used to construct the risk score (multivariate Cox regression) model in the training set. en, the risk score was determined using the following formula: n 1 ExpGenei * Coefi (ExpGenei, expression of the gene; Coefi, coefficient of the gene). To evaluate the performance of the risk score model, the receiver operating characteristics (ROC) curve was plotted using the R package "survi-valROC." Moreover, the risk score model was tested in external datasets, including GSE54460, GSE46602, GSE70768, and GSE70769. According to the expressions and coefficients of model genes, the risk score of each patient was calculated, and the patients with PRAD were divided into low-and high-risk score groups based on the median value of the risk score. e PFIs in the low-and high-risk groups were analyzed and compared using the Kaplan-Meier curves and the log-rank test.
e "pRRophetic" R package and Wilcoxon test were used to analyze and compare the chemotherapeutic response of patients in the low-and high-risk groups to 138 drugs [22].

Establishment of the Predictive Nomogram for PFI.
e risk score and clinical features of patients with PRAD (age and TNM stage) were analyzed using univariate Cox regression to detect independent prognostic factors (p-value <0.05). e screened independent prognostic factors were then analyzed using multivariate Cox regression to construct the nomogram for predicting the 1-, 3-, and 5-year PFI of patients with PRAD. e nomogram's efficiency was evaluated using ROC curves.

DEGs Identified in TCGA-PRAD Have Multiple
Functions. A total of 1,750 DEGs, including 714 upregulated (PRAD versus control) and 1,036 downregulated genes (PRAD versus control), were identified in the TCGA-PRAD cohort (Figure 1(a) and Table S1). e DEGs were significantly enriched in 473 biological processes, 65 cellular components, and 55 molecular functions (Table S2). Moreover, the top biological processes were mainly associated with the muscle system, urogenital system, circulatory system, and cell adhesion, such as muscle contraction, blood circulation regulation, and cell-cell adhesion via plasma-membrane adhesion molecules (Figure 1(b)). Additionally, 29 significantly enriched KEGG pathways were identified (Table S3). ese pathways were further found to be relevant to cell proliferation, cell adhesion, cardiomyopathy, and immunity, such as focal adhesion, extracellular matrix (ECM)-receptor interaction, cell cycle, complement and coagulation cascades, hypertrophic cardiomyopathy, pI3K-Akt signaling pathway, and calcium signaling pathway (Figure 1(c)). erefore, the complexity of PRAD aetiology could be attributed to multiple biological processes and signaling pathways that contribute to the development of PRAD.

TME Is Altered in PRAD.
A growing body of evidence reveals the important role of the TME in cancer [10,11,23]. erefore, the infiltration levels in PRAD and control samples were analyzed using ssGSEA. A total of 21 DIICs (immature B cells, memory B cells, activated B cells, central memory CD8 + T cells, central memory CD4 + T cells, activated CD8 + T cells, NK cells, effector memory CD8 + T cells, type 2T helper cells, effector memory CD4 + T cells, CD56dim, NK cells, type 17T helper cells, monocytes, NK T cells, immature dendritic cells, plasmacytoid dendritic cells, mast cells, T follicular helper cells, eosinophils, neutrophils, and type 1T helper cells), most of which showed higher infiltration levels in control samples than PRAD samples, were identified (Tables S4-S5 and Figure 2(a)). Further analysis showed that these DIICs had moderate to strong correlations with each other (Figure 2(b)).

Nine Hub DIICRGs Identified in PRAD.
To explore genes associated with DIICs, WGCNA analysis was performed. After sample clustering, outlier samples were excluded (data not shown). Using the "pick Soft reshold" function of WGCNA, the optimal soft threshold power was found to be 3, wherein R 2 was approximately 0.9 (Figure 3(a)). ree modules were identified from the coexpression network. According to the module-trait relationships shown in Figure 3(b), the turquoise module was selected as the most relevant module with NK cells (cor � 0.78, p-value <0.05). Moreover, using GS > 0.5 and MM > 0.8, 144 genes were obtained from the turquoise module for further analysis (Figure 3(c)). e PPI network of the genes was constructed  and visualized using Cytoscape (Figure 3(d)). Furthermore, the top ten genes with the highest interaction degrees, including, FLNA, VCL, ACTA2, MYL9, MYLK, MYH11, TPM1, ACTG2, TAGLN, and FLNC, were selected using the "cytohubba" plug-in (Figure 3(d)) and identified as candidate hub genes in PRAD.
Correlation analysis revealed that candidate hub genes had a moderate to a strong relationship with each other (Figure 4(a)). Metascape revealed that they were associated with the contraction of the smooth muscle, RHO GTpase activation of PAKs, regulation of the actin cytoskeleton, focal adhesion, tissue morphogenesis, striated muscle contraction pathway, and development of muscle structure and muscle cell (Figure 4(b)). ereafter, the expression patterns of the genes in the TCGA-PRAD and GSE54460 datasets were examined, revealing that the expressions of VCL, ACTA2, MYL9, MYLK, MYH11, TPM1, ACTG2, TAGLN, and FLNC were significantly different between PRAD and control samples in both cohorts (Figures 4(c)-4(d)).
us, these nine genes were identified as hub DIICRGs in PRAD.

Hub DIICRGs Are Mainly Expressed in ree Cell
Subpopulations. e top 4000 highly variable genes ( Figure 5(a)) were selected for downstream analysis. After performing the principal component analysis (PCA) on highly variable genes, 70 significant principal components (PCs) were identified ( Figure 5(b)). Using the tSNE method, PRAD cells were clustered into nine distinct cell subpopulations, including B cells, chondrocytes, common myeloid progenitor, endothelial and epithelial cells, induced pluripotent stem cells, monocytes, T cells, and tissue stem cells ( Figure 5(c)). e examination of the expression patterns of hub genes in those cell subpopulations revealed that they were highly expressed in endothelial, epithelial, and tissue stem cells (Figures 5(d)-5(e)), suggesting that these cell subpopulations could be crucial to PRAD aetiology.
To illustrate each cluster's role, marker genes in each cluster were identified (Figure 8(a)), and the functional analysis of the markers in each cluster was performed (all enrichment results can be found in Tables S6-S13 for cluster 0-7). e common and distinct functions among the clusters could contribute to the complexity of PRAD. e markers of clusters 0 and 4 were mainly enriched in immune-related biological processes, such as neutrophil activation (Figures 8(b) and 8(f )). Markers in clusters 6 and 7 were mainly associated with ECM processes, such as ECM organization and cell-substrate adhesion (Figures 8(h)-8(i). Furthermore, the functions of the markers in cluster 5 were similar to those in clusters 6 and 7, which were also relevant      to endothelial cell differentiation (Figure 8(g)). Notably, markers in clusters 1, 2, and 3 had important roles in the formation of proteins, such as PERK-mediated unfolded protein response, translation initiation, and plasma-membrane protein development (Figures 8(c)-8(e)).
Moreover, similar results were observed in four external datasets ( Figures S1-S4), demonstrating the reliability of the risk score model. Additionally, the chemotherapeutic responses of patients to 85 drugs were significantly different between the low-and high-risk groups ( Figures S5 and S6), indicating that the prognostic biomarkers may affect the chemotherapy response of patients with PRAD. Among them, the half-maximal inhibitory concentration (IC 50 ) of Bicalutamide, which is used in the treatment of advanced PRAD, in the high-risk group was significantly lower than that in the low-risk group. Cisplatin, another chemotherapy drug, presented significantly lower IC 50 values in the lowrisk group, suggesting its efficacy in treating localized PRAD or early-stage PRAD.

A Predictive Nomogram Is Established in PRAD.
Finally, the risk score as an independent prognostic factor in PRAD was investigated. Using the univariate Cox regression analysis, the risk score, T stage, and N stage were found to be significantly related to the survival of patients with PRAD ( Figure 10(a)). After the multivariate Cox regression analysis, the risk score remained significantly associated with the survival of patients with PRAD ( Figure 10(b)), indicating that the risk score was an independent prognostic factor in PRAD. us, a nomogram based on the risk score and other clinical features was established to predict the 1-, 3-, and 5year PFI of patients with PRAD (Figure 10(c)). e calibration curves showed that the predicted PFI was very close to the actual PFI (Figure 10(d)), suggesting the clinical application of the nomogram. Furthermore, the areas under the ROC curves were 0.79, 0.83, and 0.81 for the 1-, 3-, and 5year PFI, respectively (Figure 10(e)), further indicating the good performance of the nomogram.

Discussion
A total of 1,750 DEGs were identified in the TCGA-PRAD cohort. ssGSEA showed that the proportions of 21 immune cells were significantly different between PRAD and control samples. WGCNA identified 144NK cell-related genes and nine hub genes, including VCL, ACTA2, MYL9, MYLK, MYH11, TPM1, ACTG2, TAGLN, and FLNC, which were selected by the PPI network. Using Single R, nine cell subpopulations were detected in PRAD. Based on the expression patterns of hub genes, endothelial, epithelial, and tissue stem cells were identified as key cell subpopulations, which regulate PRAD via immune response, extracellular signaling, and protein formation. e functional analysis of DEGs in cancer and peritumoral samples was performed, revealing that the muscle tissue development and the blood circulatory system were mainly related to the DEGs. It has been reported that angiogenesis is closely related to the progression of cancer cells [24][25][26]. e increased local angiogenesis could be a marker for detecting PRAD [27]. Some drugs include Zoledronic acid [28], curcumin [29], and the EZH2 inhibitor GSK126 or EPZ6438 [26], which inhibit neoangiogenesis in patients with PRAD. To date, to the best of our knowledge, there is no direct evidence that muscle tissue development is associated with cancer progression. Hypoxia occurring within the muscle during exercise could stimulate angiogenesis [30][31][32]. Moreover, hypoxia is common in many cancers [33]. erefore, it is inferred that a high oxygen demand during cancer cell growth could lead to hypoxia, wherein hypoxia simulates the muscle near the prostate to generate a stimulus for angiogenesis, thereby leading to cancer progression.
Subsequently, the immune microenvironment between the tumor and peritumoral samples was compared, revealing that the TME of PRAD was significantly altered, which was reflected by the significant change in infiltration levels in all 21 types of immune cells. Similarly, consistent with the results of Gao et al. [34], the current study showed a similar trend in terms of the infiltration level of T follicular helper cells. An increase in neutrophils was also reported [10,35]. ese increased immune cells are speculated to be involved in the progression of PRAD [35,36]. Among these immune cells, NK cells, which were reduced in the tumor tissues, were found to be heavily involved in the anticancer activity as a type 12 Journal of Oncology  Journal of Oncology 13 of lymphocyte population [37,38]. Several studies indicate that a lower degree of NK cells is associated with an increased risk of PRAD [38][39][40]. e reduction of NK cell levels (Figure 2(a)) in PRAD, as reported by Li et al. [41], could be a favorable environment for neutrophils to promote metastasis. Conversely, a high level of NK cells indicates a good prognosis or lower risk of PRAD [42,43]. us, NK cells could be used as a potential therapeutic target. Although immunotherapy has been used to treat various solid tumors, Sipuleucel-T is the only approved PRAD immunotherapy for castration-resistant PRAD, which is the advanced stage of PRAD [44,45]. It is suggested that effective NK activation and tumor targeting/binding are essential mechanisms in NK cell-mediated cancer treatment. Highly effective NK cells are critical limiting factors in cancer immunotherapy efficacy [46,47]. Hence, based on the current study, the increasing activity of the NK cells via checkpoint inhibitors, NK cell engagers, and cytokines could improve PRAD immunotherapy efficacy at different stages. Next, nine hub genes, namely VCL, ACTA2, MYL9, MYLK, MYH11, TPM1, ACTG2, TAGLN, and FLNC, were obtained using WGCNA, PPI, and external gene set validation. VCL is a focal adhesion-related cytoskeletal protein that plays an essential role in cell adhesion and signal transduction. ese nine hub genes mainly are involved in the processes, including metastasis, progression, and survival. MYL9 encodes an actin-binding protein involved in cell motility, division, and adhesion. Previous studies have confirmed that the expression level of MYL9 is downregulated in the stroma of PRAD, indicating the poor prognosis of patients with PRAD [48,49]. MYL9 has the potential to become a molecular marker for diagnosing PRAD and predicting cancer progression and prognosis. TPM1 belongs to the tropomyosin family and plays a vital role in cytoskeletal functions, such as cell proliferation, migration, and apoptosis, thereby playing a key role in tumor growth and metastasis. MYH11 is a member of the Myosin family, which regulates functions, such as signal transduction, muscle contraction, and cell movement in the body, and the mutation of MYH11 has also been observed in prostate cancer, however, further exploration is needed [50,51]. Myosin light-chain kinase (MYLK) is a member of the immunoglobulin superfamily, an enzyme independent of calcium-/calmodulin that facilitates myosin interaction with actin filaments and produces contractile activity. ACTA2 are found in muscle tissues and are the significant constituents of the contractile apparatus. ACTG2 gene encodes actin, a gamma-enteric smooth muscle protein found in human enteric tissues. e PPI network (Figure 3(d)) indicates the interactions among the hub genes (MYH11, MYLK, ACTA2, and ACTG2). erefore, PRAD development was speculated to be correlated with muscle development (Figure 1(b)). Moreover, TAGLN and FLNC genes have functions in muscle tissues, indicating their involvement in PRAD progression. 14 Journal of Oncology Subsequently, using PRAD single-cell sequencing data, three cell subsets that could play an essential role in the occurrence and development of PRAD were screened. e tumorigenicity of epithelial cells was reported to be the major reason for PRAD development [52]. Epithelialmesenchymal transition (EMT) is a normal cellular physiological process that involves transforming epithelial cells into cells with a mesenchymal phenotype. EMT is a  TIMP3  A2M  ADGRF5  THY1  EDNRB  HSPG2  ESM1  PLPP3  COL4A1  COL4A2  SPARC  FLT1  RGCC  COL1A1  COL3A1  PPP1R14A  NOTCH3  IGFBP7  FRZB  COL1A2  HIGD1B  PDGFRB  BGN  CRIP1  MFGE8  MYH11  NDUFA4L2 LGALS1 C1orf64  CST1  RPS4Y1  TFF3  AGR2  SNHG19  HSD17B6  SFTPA2  FABP5  SLC38A11  CRISPLD1  LRRC75A  CRISP3  KRT7  SLPI  KRT19  MMP7  KRT15  TMEFF2  PCAT4  ADIRF  ACPP  WFDC2  KRT17  RLN1  NPY  PLA2G2A  VGLL3  CXCL8  BANK1  SERPINB11  RDH10  IGF1  KLK12  KLK11  OGN   0  1  2  3  4  5     necessary process that drives the metastasis of PRAD [53,54]. Various proteins regulate e-cadherin expression, including snail [55], epidermal growth factor, epidermal growth factor receptor [56], twist [57], and miRNAs [58,59]. e decreased expression, including the silencing of e-cadherin in the epithelial cells, leads to the sustained loss of normal polarity and adhesion [60] and subsequently to PRAD cell invasion and metastasis. In other words, the abnormal expression of genes in epithelial cells could promote PRAD development. However, further in-depth research is required to clarify the mechanisms and provide new targets and directions for the diagnosis, prognosis, and treatment of PRAD. Endothelial cells are strongly linked to metastasis. Endothelial cells surrounding tumors are the basis of angiogenesis [61]. Additionally, they boost autophagy and accelerate focal adhesion protein disassembly [62]. is type of cell is also associated with drug resistance in PRAD. Recent studies have shown that vascular endothelial cells can modulate the response of tumor cells to chemotherapy [63]. Akiyama et al. [64] found that endothelial cells can acquire drug resistance from TME.
Tissue stem cells are the dominant cell types in PRAD, suggesting that PRAD could be a stem cell disease. In addition to the stem cell characteristics of proliferation, selfrenewal, and differentiation potential [65], cancer stem cells (CSCs) have the ability to generate the heterogeneous lineages of cancer cells that develop into tumors [66,67]. Only a tiny portion of PRAD exhibit the phenotypical and functional characteristics of normal prostate stem cells and participate in tumorigenesis, metastasis, and drug resistance [68][69][70]. However, PRAD CSC markers remain undefined in clinical practice.
ere is increasing evidence that PRAD-CSC-specific markers (CD44, CD133, CD166, FAM65 B, MFI2, and LEF1) can predict the patients' overall survival, which suggests the clinical potential of PRAD CSC as biomarkers and therapeutic targets [71,72]. e combined use of CSC vaccines with immunomodulators, such as anti-PD-L1 antibodies, can significantly improve the antitumor efficiency of CSC-based vaccines and block the immunosuppressive effect of the TME [73,74]. us, immunotherapy targeting CSC could have clinical implications in treating PRAD.
To further reveal the function of the three cell subsets in PRAD, they were subdivided into eight cell clusters, for which cell trajectory and functional analyses were performed. e pseudotemporal cell trajectory analysis indicated that the epithelial cells were at the beginning of the entire cell tree. It has been suggested that endothelial cells are a type of epithelial cells, indicating that epithelial cells could transform into endothelial cells [75]. Chen et al. [15] claimed that this differentiation occurs in carcinoma cells that have undergone EMT, which contributes to tumor growth. Additionally, following the treatment of proangiogenic signals, which is a common phenomenon during tumors, epithelial cells can transition to endothelial cells in vivo [76]. ere is little robust evidence that epithelial cells could be differentiated into stem cells in PRAD. However, it is assumed that epithelial cells reacquiring self-renewal capabilities could lead to carcinogenesis [77,78]. Following EMT, tumor cells could gain stem cell-associated properties [79][80][81][82].
erefore, similar signals in vivo could be applied to epithelial cells to validate the transition to stem cells through EMT.
ese enrichment results match the cell subpopulations' functions (Figures 6(a) and 6(b)), indicating that cell clusters from the same source have similar functions but cell clusters from different sources have dissimilar functions. is ability to differentiate into different types of cells contributes to the         e pseudotime trajectory and pathway enrichment (functional analysis) analysis might provide speculations regarding the occurrence of PRAD. In the beginning, epithelial cells (clusters 0 and 4) were the main subpopulations of the cells, and functional analysis deciphers the activation of neutrophils as the primary function. Following activation, neutrophils, inflammation, and cancer can elevate DNA replication errors and release reactive oxygen species in epithelial cells [23]. us, activated neutrophils trigger the oncogenic transformation, and epithelial cells are subsequently driven to carcinogenesis and the abnormal expression of specific genes. Following this,  ALDH1A2 Reduction in ALDH1A2 protein is an early event in human prostate cancer Kim et al. [120] 22 Journal of Oncology clusters 1, 2, and 3, which were considered the main types, including epithelial and endothelial cells, were found to be involved in the pathways of PERK-mediated unfolded protein response (UPR) and translation initiation, which correlates with previous studies. PERK-mediated pathways introduce endoplasmic reticulum stress into cells and reduce the expression of e-cadherin [83,84]. us, epithelial cells could be differentiated into endothelial cells. UPR, to some extent, aids in the survival of tumor cells [83,85]. Furthermore, PERK-mediated processes assist the production of proangiogenic factors and the growth of blood vessels, offering tumor cells a route to metastasis [83,86]. Proteins that are relevant to angiogenesis are then produced by endothelial cells [87,88]. During angiogenesis, endothelial cells grow and promote the formation of new blood vessels through the vascular endothelial growth factor (VEGF). UPR also reduces cell-cell junction markers, promoting metastasis [89,90]. However, epithelial cells could also transition to CSCs or stem cells. Stem cells, including CSCs, induce the expression of endothelial markers [91,92], and VEGF also promotes endothelial cell differentiation [93]. roughout angiogenesis and EMT, ECM organization is dysregulated and cell-substrate adhesion is reduced [94], resulting in cancer cells acquiring an invasive and migratory phenotype.
To further explore the prognostic value of cell subset marker genes, 25 prognostic genes were identified through univariate, LASSO, and multivariate analyses. eir roles in PRAD are summarized in Table 1. Based on these 25 genes, a risk model was constructed, which divided patients into high-and low-risk groups. Interestingly, a significant difference was observed in chemosensitivity between the highand low-risk groups. Bicalutamide was found to be more effective for patients with PRAD in the high-risk group than in the low-risk group. Clinically, Bicalutamide is used to treat advanced PRAD [121,122], corroborating our results. Cisplatin is used to treat early-stage PRAD [123], which corroborates with the result that the patients in the low-risk group are more sensitive to Cisplatin than the high-risk group. Moreover, a significant difference was observed in the chemotherapy response to the other 83 drugs, although they are not yet approved for the treatment of PRAD. Despite the advances in PRAD treatment, therapeutic options for PRAD remain limited [124,125]. ese 83 drugs can be considered a pool when looking for novel therapies. For instance, GDC.0449 is a drug targeting the Hedgehog pathway, whose activation could indicate the potential effectiveness of GDC.0449 in PRAD [8,126]. Moreover, the SRC family kinase activity is observed in hormone-refractory PRAD [127], suggesting that medications targeting the SRC family, such as A.770041, AZD.0530, and WH.4.023, could be effective in PRAD treatment. e small sample size (13 tumors) limits the applicability of the study [15]. is study combined the analysis of highthroughput gene chip data and single-cell sequencing data, which avoided the insufficiency of a single research method and improved the credibility of the results. e occurrence and development of PRAD, a highly heterogeneous disease, involves complex mechanisms, and the DEGs obtained form only a small part of the mechanism. Furthermore, novel insights into the molecular mechanisms of TME in the pathogenesis of PARD are reported. Hence, precision medicine is crucial in PRAD treatment because of the disease's heterogenicity. Further immunological functional studies to clarify the biological functions of these genes and cell subsets in PRAD are essential, which could provide a solid foundation for improved clinical diagnosis and treatment.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Ruisong Wang finished the manuscript. Yaqian Xiao and Meisen Pan, together with Ruisong Wang, performed the R and statistical analyses. Zhongyuan Chen designed and supervised the study. Pinhong Yang provided the idea for the research and proofread the manuscript. Ruisong Wang and Yaqian Xiao have contributed equally to this work and share the first authorship.
curve analysis for 1-, 3-and 5-year PFI using the clinical information of patients of the GSE54460 validation dataset. Figure S3. Validation of the risk score model using the GSE46602 dataset. A. Patients with prostate cancer (PRAD) in the GSE46602 cohort are listed in ascending order of risk score. B. Progression-free interval (PFI) distribution versus the risk score of each patient in the GSE46602 cohort. C. Kaplan-Meier (KM) curves of patients with different risk levels in the GSE46602 validation dataset. D. Receiver Operating Characteristic (ROC) curve analysis for 1-, 3-and 5-year PFI using the clinical information of patients of the GSE46602 validation dataset. Figure S4. Validation of the risk score model using the GSE70768 dataset. A. Patients with prostate cancer (PRAD) in the GSE70768 cohort are listed in ascending order of risk score. B. Progression-free interval (PFI) distribution versus the risk score of each patient in the GSE70768 cohort. C. Kaplan-Meier (KM) curves of patients with different risk levels in the GSE70768 validation dataset. D. Receiver Operating Characteristic (ROC) curve analysis for 1-, 3-and 5-year PFI using the clinical information of patients of the GSE70768 validation dataset. Figure S5. Validation of the risk score model using the GSE70769 dataset. A. Patients with prostate cancer (PRAD) in the GSE70769 validation dataset are listed in ascending order of risk score. B. Progression-free interval (PFI) distribution versus the risk score of each patient in the GSE70769 cohort. C. Kaplan-Meier (KM) curves of patients with different risk levels in the GSE70769 validation set. D. Receiver Operating Characteristic (ROC) curve analysis for 1-, 3-and 5-year PFI using the clinical information of patients of the GSE70768 validation dataset. Figure S6. Drug response is estimated by the half-maximal inhibitory concentration (IC 50 ) value wherein the sensitivity of the highrisk patients is higher than that of the low-risk patients in the TCGA training cohort. Figure S7. Drug response is estimated by the half-maximal inhibitory concentration (IC 50 ) value wherein the sensitivity of the high-risk patients is lower than that of the low-risk patients in the TCGA training cohort. Table S1. DEGs identified in the TCGA-PRAD cohort. Table S2. DEGs enrichment by GO. Table S3. DEGs enriched in KEGG pathway. Table S4. Immune score in each sample of TCGA-PRAD cohort. Table S5. e altered immune microenvironment in prostate cancer (PRAD). Table  S6. GO enrichment for cluster 0.