Comprehensive analysis the diagnosis, malignant progression and immune infiltrate of ANXA6 in prostate cancer

Annexins (ANXAs) play a crucial role in the development and progression of tumors. However, their specific involvement in prostate cancer (PCa) remains unclear. To investigate the function and clinical significance of key ANXAs in PCa. Multiple databases were used to analyze the expression levels, genetic variations, potential prognostic value and clinical significance of ANXAs in PCa. Then, the co-expressed genes of ANXA6 were identified, and the correlation between ANXA6 and immune cell infiltration was validated using the Tumor Immune Estimation Resource (TIMER) database. Additionally, in vitro assays such as Cell Counting Kit-8 (CCK-8), Colony Formation, Transwell and T-cell Chemotaxis assays were conducted to validate the functions of ANXA6. Moreover, multiple types of in vivo assays were performed to further validate the identified ANXA6 functions. The results demonstrated that ANXA2, ANXA6 and ANXA8 were significantly downregulated in PCa. ANXA6 upregulation was significantly associated with improved PCa patients’ overall survival. Enrichment analysis revealed that ANXA6 and its co-expressed genes were involved in tumor progression, and ANXA6 overexpression could effectively inhibit the proliferation, migration and invasion of PC-3 cells. In vivo studies also demonstrated that ANXA6 overexpression suppressed tumor growth. Importantly, ANXA6 was found to promote the chemotaxis of CD4+ T cells and CD8+ T cells towards PC-3 cells, and the overexpression of ANXA6 in PC-3 cells promoted the polarization of macrophages into M1 macrophages in the supernatant of PCa cells. ANXA6 demonstrated promising potential for consideration as a prognostic biomarker in PCa as it was found to play key roles in regulating immune cell infiltration and the malignant progression to PCa.


Introduction
Prostate cancer (PCa) is one of the most common urinary system tumors (Siegel et al. 2019). Currently, the primary approach to treating PCa involves targeting androgen receptor activity, for which androgen deprivation therapy (ADT) is the standard treatment modality for PCa patients Banggao Huang sunson33@126.com it was found that the human genome contains 13 Annexin subfamilies (Bode et al. 2019). In eukaryotes, Annexins have been reported to play a significant role in a range of calcium-dependent membrane-related biological activities, including the formation of biomembranes, establishment of ion channels, regulation of cell apoptosis, and secretion of membrane proteins (Fatimathas and Moss 2010). Moreover, Annexins have been found to be involved in regulating tumor cell growth during malignant progression. For instance, in nasopharyngeal carcinoma, Annexin A1 (ANXA1) was identified as a protein that binds to Epithelial cell kinase 2 (EphA2), thereby influencing the growth of nasopharyngeal carcinoma cells (Feng et al. 2020). In cervical cancer, ANXA2 has been recognized as a potential diagnostic marker ). In addition, some researchers also reported that ANXA2 could regulate the β-catenin pathway in ovarian cancer (Liu et al. 2017). Apart from their roles in cell proliferation and differentiation, abnormal expression of certain Annexins has been linked to chemoresistance in various types of tumors (Jin et al. 2020;Uchihara et al. 2020). However, the specific molecular mechanisms and functions of many Annexins remain unclear.
In the context of PCa, several Annexins have been found to be closely associated with malignant progression and diagnosis. For instance, high expression of ANXA2 has been linked to higher-grade and higher-stage prostate tumors (Tan et al. 2021), and ANXA1 has been shown to promote the nuclear localization of epidermal growth factor receptor (EGFR) in Pca (Mota et al. 2020). Therefore, exploring the diagnostic and prognostic value of Annexin family genes in PCa and studying the genes and pathways regulated by Annexins in PCa progression might hold significant importance in improving the diagnosis, prognosis and therapeutic strategies of patients with PCa.
In this study, the differential expression levels of Annexins between PCa and normal tissues were analyzed, and survival analysis of each Annexin family member in PCa was conducted. ANXA6 was found to be significantly related to the malignant progression of PCa. Furthermore, the co-expressed genes of ANXA6 were identified, their biological functions were analyzed, and the relationship between ANXA6 and immune-infiltrated cells in the tumor microenvironment was also assessed. Lastly, the function of ANXA6 was characterized through various assays. Collectively, this research aimed to determine the clinical significance of ANXA6 in predicting the diagnosis, prognosis and treatment options for PCa.

Analysis of differential expression genes with gene expression profiling interactive analysis (GEPIA)
The expression data of Annexins (ANXA1-11, 13) in PCa and normal tissues were downloaded and analyzed by GEPIA (http://gepia.cancer-pku.cn/) (Tang et al. 2019). A significant difference was determined based on a threshold of P < 0.05. GEPIA uses the UCSC Xena server to analyze gene expression levels in various tumor samples. The database comprises a total of 9,736 tumor samples and 8,587 normal samples (Tang et al. 2017) and provides comprehensive information on patient survival, prognosis and gene expression levels in tissues for multiple cancer types.

UALCAN
The differential expression level of ANXAs (ANXA1-11, 13) in PCa and normal tissues were analyzed using UAL-CAN (http://ualcan.path.uab.edu) (Chandrashekar et al. 2017). UALCAN is a comprehensive and user-friendly web resource designed to analyze cancer omics data. It utilizes PERL-CGI and incorporates high-quality graphics using javascript and cascading style sheets (CSS). The expression levels of ANXA6 were specifically compared between PCa and normal tissues, considering factors such as age, molecular signature, TP53 mutation status, Gleason score, and nodal metastasis score. Statistical significance was determined with a threshold of P < 0.05.

Clinical significance in PCa
The clinical significance of the 12 ANXAs on the overall survival (OS) of PCa patients was assessed using the Encyclopedia of RNA Interactomes (ENCORI) (http://starbase. sysu.edu.cn/) (Li et al. 2014) with log-rank p-value and hazard ratio (HR) with 95% confidence intervals (CIs). A log-rank test with a P-value < 0.05 indicated significant differences. ENCORI, previously known as starBase v2.0 (Li et al. 2014), is a publicly accessible platform widely utilized for studying the interactions between mRNAs and noncoding RNAs across 23 different species. The ENCORI database enables researchers to perform OS analysis of cancer patients, providing valuable insights into clinical outcomes.

Gene co-expression network and functional enrichment analysis
The co-expression genes of ANXA6 were investigated using LinkedOmics (http://www.linkedomics.org/login. php/) (Vasaikar et al. 2018) and The Cancer Genome Atlas (TCGA)-PRAD dataset. Genes with a false discovery rate (FDR) < 0.01 were considered co-expressed genes. Linke-dOmics is a publicly accessible portal integrating multiomics data from all 32 TCGA cancer types and 10 Clinical Proteomics Tumor Analysis Consortium (CPTAC) cancer cohorts. It is a valuable platform for biologists and clinicians to access, analyze and compare multi-omics data across different tumor types. For functional network analysis of the coexpressed genes, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, gene set enrichment analysis (GSEA) was performed. Enriched functions and pathways were considered significant at a threshold value of FDR < 0.05. GSEA is a computational method to assess whether a predefined set of genes exhibits statistically significant and concordant differences between two biological states. It provides insights into the functional implications and molecular pathways associated with the co-expressed genes.

Correlation analysis of gene expression level with immune cell infiltration and tumor titer
The correlation between ANXA6 and tumor-associated immune cells was investigated using TIMER (http://timer. cistrome.org/) (Liang et al. 2020). TIMER is a comprehensive resource for systematically analyzing immune infiltrates across diverse cancer types. This web server provides estimates of immune infiltrate abundances using multiple immune deconvolution methods and offers dynamic generation of high-quality figures, allowing users to comprehensively explore tumor immunological, clinical and genomic features.

Cell culture and transfection
Mouse RM-1 cells, human RWPE-1, 293T, and PC-3 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human peripheral blood mononuclear cells (PBMC) were purchased from BNCC (Beijing, China), and human monocytic cells (THP-1) were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). RWPE-1 cell lines were cultured in keratinocyte serum-free medium (K-SFM) supplemented with 50 µg/mL bovine pituitary extract and 5 ng/mL epidermal growth factor. PC-3 and 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA). PBMC, RM-1, and THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). To differentiate THP-1 monocytes into macrophages, they were treated with 10 ng/mL phorbol-12-myristate-13-acetate (PMA, P8139, Sigma, St. Louis, Missouri, USA) for 24 h. All cell cultures were maintained at 37 °C with 5% CO 2 . pcDNA-ANXA6 was synthesized by RiboBio (Guangzhou, China) and cloned into the pcDNA.3.1 vector, incorporating the full-length sequences of ANXA6. In vitro experiments, cell transfection was performed using Lipofectamine® 2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Cells were collected for subsequent studies approximately 48 h post-transfection. In vivo experiments, a lentiviral expression vector was constructed using the ViraPower™ II Lentiviral GatewayTM Expression System (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols. The stable cell lines were used for further experiments at least 72 h after transduction.

Co-culture experiments
Different groups of PC3 cells were seeded at a density of 1 × 10 6 cells/mL in 6-well plates, with each well containing 3 mL of culture medium. The supernatant of PC3 cells was collected, and the final concentration was adjusted to 25% of the total volume. PMA-induced THP-1 cells (macrophages) were then seeded at a density of 1 × 10 6 cells/mL in 6-well plates, with each well containing 3 mL of RPMI-1640 medium supplemented with the PC3 cell supernatant, which was maintained for 48 h.

Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted from the cell lines using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The collected cells were lysed with 1 mL of TRIzol. The purity of the extracted RNA was assessed using UV A260/ A280 spectrophotometry (Nanodrop ND2000; Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized using the PrimeScript RT kit (Takara, Shiga, Japan). Reverse transcription was performed using 0.5 µg of RNA, and the resulting cDNA was stored at -20 °C. RT-qPCR was conducted on an ABI7900 system (Applied Biosystems, Foster City, CA, USA) using SYBR Green Premix Ex Taq (Takara, Shiga, Japan). The reaction protocol consisted of an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of amplification at 95 °C for 1 min and 60 °C for 30 s. GAPDH was used as the reference gene for ANXA6, and RPS9 was used as the reference gene for IL-6, CXCL10, CD86, CD206, Fibronectin, and CCL22. The data were analyzed using the 2 −ΔΔCt method. The primer sequences used were as follows: ANXA6: Forward (F): 5'-ACGGTTGATTGTGGGCCTG-3', Reverse (R): 5'-TCAGCCTCCAGGTCCCGCTC-3'; the Transwell chamber, and a complete medium containing 600 µL and 10% fetal bovine serum (FBS) was added to the lower layer. The Transwell chamber was then incubated in the incubator for 36 h, following which cells that did not penetrate the membrane were carefully wiped off using cotton swabs. Cells that passed through the membrane were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet. Subsequently, the cells were counted under a light microscope at a magnification of ×100 (Olympus, Tokyo, Japan). Three replicates were performed for each sample.

T-Cell chemotaxis assay
CD8 + T cells were isolated from PBMC cells using the CytoSinctTM CD8 NanoBeads isolation kit (L00864, Gen-Script, Nanjing, China) and CD4 + T cells from PBMC cells using the MACS MicroBeads with negative selection and the CD4 + T-cell isolation kit (Bergisch Gladbach, Germany). Then, the supernatant from the cell culture medium was collected. In the migration assay, the lower chamber of the Transwell system was filled with PCa cell culture medium. T cells were added to the Transwell cells with either 5-or 3-µm pore size (Costar, Shanghai, China), and the cells were allowed to migrate for a determined period. After migration, the migrated cells were determined using a hemocytometer for cell counting. Three replicates were performed for each sample.

Cell counting kit-8 (CCK-8) assay
To assess the cell viability of prostate cancer (PCa), CCK-8 solution (Dojindo, Kumamoto, Japan) was utilized. Briefly, the cells were seeded into 96-well plates and cultured in a 5% CO2 incubator at 37 °C for 0, 24, 48, 72, and 96 h. After each time point, 10 µL of the CCK-8 reagent was added to each well and mixed gently. The plates were then incubated for 2 h in the incubator. The optical density (OD) at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Three replicates were performed for each sample to ensure reliable results.

Colony formation assay
PCa cells were cultured in 6-well plates at a density of 200 cells per well, and the culture medium was changed every 3 days. Once the cell colonies became visible to the naked eye, the medium was aspirated. The cells were then washed twice with 1x phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 15 min. After the formaldehyde was washed off, the cells were stained with 0.25% crystal violet (Beyotime, Shanghai, China) for 25 min. Subsequently, the cells were washed with sterile water, allowed to dry, and photographed. Three replicates were performed for each sample.

Transwell assay
An uncoated substrate (BD Biosciences, San Jose, CA, USA) with an upper lumen was used to assess cell migration and invasion. Briefly, the upper layer was used to identify migrating cells and the coated membrane to recognize invading cells. The cells were added to the upper layer of

Expression analysis of ANXAs in PCa
To explore the role of Annexins in PCa, we analyzed their expression using GEPIA. The results revealed no significant differences in the expression levels of ANXA1, ANXA3-5, ANXA7, ANXA9-11 and ANXA13 between PCa and normal tissues (P > 0.05), while a notable reduction in the expression of ANXA2, ANXA6 and ANXA8 in PCa tissues compared to normal tissues was observed (P < 0.05) (Fig. 1). In addition, the differential expression level of ANXAs in PCa and normal tissues were also illustrated using UAL-CAN, which revealed an upregulation in the expression levels of ANXA5 and ANXA10 in PCa tissue compared to normal tissue (P < 0.05), while the expression levels of other ANXAs were down-regulated in PCa tissue (P < 0.05) (Fig. 2). Collectively, ANXA2, ANXA6 and ANXA8 consistently exhibited the same expression patterns in both GEPIA and UALCAN databases, indicating that they could be closely associated with the progression of PCa.

ANXA6 played a significant prognostic role in patients with PCa
Next, we investigated the prognostic significance of Annexins with the OS of patients with PCa. The results indicated that, except for ANXA6, no significant associations were observed between the expression of other Annexins and OS in PCa patients (Fig. 3). However, ANXA6 expression was found to be significantly correlated with a favorable OS outcome (P = 0.041, Hazard Ratio = 0.15), suggesting that it might serve as a key gene associated with the malignant progression of PCa.

Correlation of ANXA6 expression and clinical significance of PCa
Here, the UALCAN database was used to assess the correlation between ANXA6 expression in PCa and clinical significance. We first analyzed the correlation between ANXA6 expression and the Gleason score of PCa patients, and the results showed that ANXA6 showed lower expression in patients with 6-9 Gleason score (P < 0.05) (Fig. 4a). Additionally, we found that the expression of most ANXA6 was related to the molecular signature (P < 0.05) (Fig. 4b). Similarly, the expression of ANXA6 was also lower in patients with N0 and N1 metastasis status (P < 0.05). However, there was no difference in ANXA6 expression between the N0 and N1 metastasis status group (P > 0.05) (Fig. 4c). Further, although the expression of ANXA6 was lower in samples of TP-53 mutation and non-mutation patients compared with euthanasia chamber, following the approved protocols and guidelines.

Immunohistochemistry
The paraffin-embedded xenograft tumor tissues were first fixed and then dewaxed, followed by rinsing with PBS. The embedded tissue sections were sliced into 5 μm sections for staining, incubated overnight with rabbit polyclonal antibodies specific to Ki67 and caspase-3 (Cell Signaling Technology, Boston, USA), and after the incubation with the primary antibodies, a secondary antibody (rabbit IgG; Cell Signaling Technology, Boston, USA) was applied for 1 h. The stained tissue sections were visualized and imaged using an inverted phase contrast microscope from Olympus (Tokyo, Japan).

Multi-color immunohistochemistry
After collecting tumor tissue from the mice, the samples were initially fixed in formalin for 48 h. Following the fixation period, routine methods were employed for dehydration and embedding the samples in paraffin. The embedded paraffin samples were sectioned and cut into about 5 mm sections. Then, the paraffin sections were baked and gradient dehydrated using ethanol. To process the antigen, a citric acid buffer was utilized. The primary antibodies, including CD8, CD4, Panck, ANXA6 and C86 (Abcam, Cambridge, UK), were incubated with the samples overnight at 4 °C. After washing the samples with PBS, the GTVisionTM II Detection System/Mo&Rb was employed for imaging and result processing.

Statistical analysis
All experiments, except the in vivo procedures, were conducted in triplicate to ensure reproducibility. Data obtained from these experiments were analyzed using Graph-Pad Prism software (version 8.0) and are presented as mean ± standard deviation (SD). For comparisons among multiple groups, one-way ANOVA followed by Tukey's post hoc test was performed. Student's t-test, on the other hand, was used for comparisons between two groups. To analyze the co-expression genes of ANXAs, the Pearson correlation coefficient was calculated, and a bilateral test was applied. Statistical significance was set at P < 0.05. Fig. 2 The expression of ANXAs in PCa using the UALCAN database. a-l Box plot of mRNA expression of ANXA1-11, 13 in PCa (red plot) and normal tissues (blue plot). *P < 0.05 Fig. 1 The expression of ANXAs in PCa (GEPIA). a-l Box plot of mRNA expression of ANXA1-11, 13 in PCa (red plot) and normal tissues (gray plot). *P < 0.05

Gene co-expressed analysis of ANXA6 in PCa
The results above demonstrate that ANXA6 exhibits lower expression in tumors and is closely associated with the clinical significance of PCa. To explore the co-expression network of PCa regulated by ANXA6, bioinformatic prediction on the website LinkedOmics was performed. The results normal tissue (P < 0.05), there was no significant difference in ANXA6 expression between the TP53-mutant and TP53-non-mutant groups (P > 0.05) (Fig. 4d). Moreover, the level of ANXA6 was not related to the age of PCa patients (P > 0.05) (Fig. 4e). Altogether, these results suggested that ANXA6 might play a tumor suppressor role in PCa. Fig. 4 Expression of the ANXAs in PCa with different clinical significance using the UALCAN database. a-e Expression of ANXA6 based on the Gleason score, molecular signature, metastasis status, TP53 mutation status and patients' age. *P < 0.05 Fig. 3 The correlation between ANXAs expression and the prognosis of PCa in OS using ENCORI. a-l OS of ANXA1-11, 13 in PCa.
trimer," "extracellular matrix," and "preribosome" (Fig. 6b). Furthermore, they exhibit molecular functions (MF) such as "extracellular matrix structural constituent," "collagen binding," and "tRNA binding" (Fig. 6c). Additionally, the KEGG pathway analysis revealed that these genes are involved in pathways such as "Focal adhesion," "ECMreceptor interaction," and "tRNA biosynthesis" (Fig. 6d). These findings highlight the close relationship between ANXA6 and various pathways that influence the development of PCa. Next, we aimed to investigate the regulatory effect of ANXA6 on the biological functions of PCa cells to validate these results.

ANXA6 inhibits multiple biological functions of PCa
Next, we explored whether ANXA6 could regulate the biological function of PCa cells. The results showed that the expression level of ANXA6 was lower in PC-3 cells (P < 0.05) (Fig. 7a). ANXA6 expression in PC-3 cells was higher in the oe-ANXA6 group (P < 0.05) (Fig. 7b). Further, the cell growth capacity of PCa cells with oe-ANXA6 was found to be lower compared with those of PCa cells with oe-NC (P < 0.05) (Fig. 7c, d). As shown in Fig. 7e, the Transwell assay revealed that overexpression of ANXA6 inhibited tumor migration and invasion of PCa cells (P < 0.05).
To evaluate the anti-tumor effect of ANXA6 in an in vivo setting, we generated a PC-3 mice model. The results demonstrated that treatment with oe-ANXA6 resulted in a smaller tumor volume compared to the control group (P < 0.05) (Fig. 8a, b). Additionally, there was a significant increase in ANXA6 expression in the oe-ANXA6 group compared to the control group (P < 0.05) (Fig. 8c). Furthermore, immunohistochemical analysis revealed that the oe-ANXA6 group exhibited a significant lower Ki67 positive ratio, indicating reduced proliferation, compared to the control group (P < 0.05) (Fig. 8d). Moreover, there was a significant enhancement in the caspase-3 positive ratio in the oe-ANXA6 group, suggesting increased apoptosis (P < 0.05).
revealed the prediction of 7,195 genes positively correlated with ANXA6 and 4,619 genes negatively correlated with ANXA6 (Fig. 5a). The top 50 genes are shown on the heatmaps in Fig. 5b, c. Notably, these top 50 genes are known to play significant roles in the initiation, malignant progression, and metastasis of PCa (Guo et al. 2022;Kamdar et al. 2019;Lee et al. 2015;Sun et al. 2016;Teng et al. 2021;Wlazlinski et al. 2007). Thus, these results indicated that the ANXA6-correlated gene might be associated with tumor progression in PCa.

Enrichment analysis of ANXA6 -correlated gene functional networks in PCa
The genes that are correlated with ANXA6 are involved in various biological processes (BPs), including "positive regulation of cell adhesion," "chemokine production," and "RNA metabolic process" (Fig. 6a). In terms of cellular component (CC), these genes are associated with "collagen

ANXA6 regulates the chemotaxis of T cells to PCa and macrophage polarization
To investigate the impact of ANXA6 on immune infiltration in PCa, we conducted chemotactic function experiments to validate its functionality. Our findings revealed that the overexpression of ANXA6 in PCa cells led to an increased migration rate of CD4 + and CD8 + T cells towards PCa cells (P < 0.05) (Fig. 9b, c). Additionally, we examined the expression levels of M1 and M2 macrophage markers in PCa and macrophage co-culture experiments and observed higher transcription levels of the M1 macrophage markers CXCL10 and CD86 in the oe-ANXA6 group (P < 0.05) (Fig. 9d). Conversely, the expression level of the M2 macrophage marker CCL22 was lower in the oe-ANXA6 group (P < 0.05) (Fig. 9e). In vivo experiments indicated that the expression levels of T cell markers (CD8 and CD4) and M1 macrophage marker (CD86) were higher in tumor tissues of oe-ANXA6 (Fig. 10a, b). Collectively, these results suggested that ANXA6 could regulate the immune escape of cancer cells.

Discussion
Abnormal expression of Annexin genes has been identified in various malignant tumors, contributing to their biological processes. However, the specific impact and regulatory mechanisms of certain Annexins in PCa remain unclear. This study aimed to investigate the expression levels of 12 Annexin genes in PCa. Additionally, OS analysis was performed to assess the prognostic significance of Annexins in PCa.
The lack of effective methods for early diagnosis and prognosis of PCa highlights the significance of discovering new biomarkers to enhance diagnostic approaches. Research has shown that ANXA2(+) tumors are associated with grade group 4-5 (Tan et al. 2021), while ANXA3 has been found to exhibit a significant correlation with tumor volumes in clinical settings (Jeun et al. 2017). Some researchers also found that PRDX6 and ANXA11 antibodies in the serum of PCa patients could be used as diagnostic biomarkers for PCa (Ummanni et al. 2015). Our study showed a significant reduction in ANXA2, ANXA6 and ANXA8 expression in PCa tissue compared to normal tissue. In addition, we also found that ANXA6 was correlated with OS for PCa patients. These findings suggest that certain ANXAs, particularly ANXA6, may be involved in the malignant progression of PCa.
The initial findings of our study suggest that ANXA6 may play a role in the tumorigenesis and progression of PCa. Previous reports have demonstrated the correlation

Correlation analysis between ANXA6 expression and 6 kinds of immune infiltrate
We utilized the TIMER database to analyze the relationship between ANXA6 expression and immune infiltrate. We observed a negative correlation between ANXA6 expression and tumor purity (Fig. 9a). Additionally, ANXA6 showed a significant positive correlation with various types of immune cells, including B cells (P = 1.49e-19, partial correlation = 0.426), CD8 + T cells (P = 2.54e-10, partial correlation = 0.304), CD4 + T cells (P = 2.25e-34, partial correlation = 0.554), macrophages (P = 5.93e-35, partial correlation = 0.555), neutrophils (P = 1.41e-20, partial correlation = 0.435) and dendritic cells (P = 6.01e-30, partial correlation = 0.519) (Fig. 9a). Altogether, these findings suggested that ANXA6 was closely related to the immune response in PCa tumor microenvironment by influencing immune cells. Quantitative data are presented as the mean ± SD of triplicate experiments. * P < 0.05. c The effect of overexpression of ANXA6 on the proliferation of PC-3 cells was measured by CCK-8 assay. Quantitative data are presented as the mean ± SD of triplicate experiments. * P < 0.05. d The effect of overexpression of ANXA6 on the proliferation of PC-3 cells was measured by colony formation assay. Cells were incubated in fresh media every 3 days. When cell colonies were visible to the naked eye, the medium was removed. Quantitative data are presented as the mean ± SD of triplicate experiments. * P < 0.05. e The impact of overexpression of ANXA6 on migration and invasive ability of PC-3 cells was assessed via Transwell assay. Quantitative data are presented as the mean ± SD of triplicate experiments. * P < 0.05 immune cell infiltration and ANXA6 expression, which revealed a significant positive correlation between ANXA6 expression and the infiltration of B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils and dendritic cells. To the best of our knowledge, there have been no previous reports on the relationship between ANXA6 and immune infiltration in PCa. However, in bladder cancer, ANXA6 was previously reported as an immune-related gene and a potential prognostic factor . Additional studies have highlighted the regulatory role of ANXA6 in the signaling and proliferation of T cells (Cornely et al. 2016). In the context of uveal melanoma, ANXA6 has been identified as an immune-related gene associated with CD8 + T cells (Sun et al. 2021). Our current study revealed that overexpression of ANXA6 in PCa cells enhanced the chemotaxis of CD4 + and CD8 + T cells towards PCa cells. In our coculture experiments, we observed a potential polarization of macrophages towards the M1 phenotype in the presence of of ANXA6 with prognosis and diagnosis in triple-negative breast cancer (Korolkova et al. 2020). ANXA6 has also been found to suppress tumorigenesis in cervical cancer through autophagy (Sun et al. 2020) and inhibit ubiquitination in triple-negative breast cancer (Li et al. 2021). However, limited reports have investigated the relationship between ANXA6 and PCa. In this present research, we identified co-expressed genes with ANXA6, analyzed their biological functions, and identified their involvement in tumor metastasis, cell adhesion, chemokine production and other functional pathways. Moreover, ANXA6 was found to inhibit PCa proliferation both in vitro and in vivo. Thus, ANXA6 might be a crucial Annexin influencing the malignant progression of PCa.
The immune-related mechanism is increasingly recognized as playing a crucial role in the progression of PCa (Rui et al. 2019;Schepisi et al. 2019). Consequently, immunotherapy has emerged as a potential clinical strategy for PCa treatment. Our study investigated the association between Tumor volumes were examined every 7 days and then calculated (0.5×length×width×width) for 5 weeks. * P < 0.05. c ANXA6 expres-sion was investigated by RT-qPCR. Quantitative data are presented as the mean ± SD of triplicate experiments. * P < 0.05. d Immunohistochemical analysis was used to detect the positive cell ratio of Ki67 and caspase-3 in tumor tissue In summary, our study investigated the diagnostic significance and tumor-immune interaction of ANXA6 in PCa. The findings suggest that ANXA6 holds promise as a new prognostic biomarker for PCa. Our results indicate that elevated ANXA6 expression inhibits PCa progression by suppressing proliferation, migration, and invasion. Furthermore, ANXA6 appears to regulate the immune evasion of supernatant from oe-ANXA6 PCa cells. Furthermore, tumor tissues with oe-ANXA6 exhibited elevated expression levels of T cell markers and the M1 macrophage marker. These findings emphasize the pivotal role of ANXA6 in regulating tumor-mediated T cells and macrophage polarization, highlighting its importance in the PCa microenvironment. PCa cells by influencing interactions with various immune cells. However, there are certain limitations to our research. Firstly, additional clinical information is required to analyze the correlations between our molecular findings and patients' clinical characteristics/prognosis, providing a more comprehensive understanding of our results. Secondly, differences among various databases, limited sample sizes, and a lack of relevant experimental studies represent further limitations that should be addressed in future investigations.
Author contributions Banggao Huang raised the concepts, design, definition of the study. Kewei Yang contribute to literature search, data acquisition, and analysis. Banggao Huang is responsible for manuscript editing and modification. All the authors contribute to and approved the manuscript.
Funding This work was supported by the Public Benefit Technology Research Program of Zhejiang Science and Technology Department (LBY21H050001).

Data Availability
The data are available from the corresponding author on reasonable request.
Code Availability not applicable.

Conflicts of interest/Competing interests Banggao Huang and Kewei
Yang declare that they have no conflict of interest.

Ethics approval
The animal experiment protocol has been reviewed and approved by laboratory animal management and ethics committee of Zhejiang Provincial People's Hospital (Approval No: IACUC-A2022079).
Consent for publication not applicable.