A Preliminary Study of Tumor Microenvironment Interleukin-4 as Promoter in Immune Escape Event in Prostate Cancer

Introduction : This study aims to investigate the relationship between IL-4 expression with Apoptosis-associated gene receptors (PD-1, CTLA-4) and Programmed Death-1 Ligands (PD-L1, PD-L2) in the microenvironment of prostate cancer tissue. Methods : The samples were collected from single-center hospital in a period from 2014 to 2020. Deparanize formalin-xed paran-embedded and RNAs extraction by manufacturer’s protocol with slight modication was performed. The RNAs expressions were investigated by using quantitative real-time polymerase chain reaction. Then we categorize them into 4 groups. The ANOVA test is used to compare mean expression between groups and followed by a correlation test using Pearson test. Result : In the BPH group sample, CTLA-4 had the highest expression level, followed by the expression of IL-4, PD-L2, then PD-1 and PD-L1. The concentration of IL-4 in prostate cancer, both metastatic and non-metastatic, is higher than in BPH, with a p-value of 0.006. the correlation between IL-4 and PD-L1 is the strongest (r=0.919), between IL-4 and PD-L2 comes the second (0.832) and between PD-1 is comes the third (r=0.626). Conclusion : In this study, we nd that the expression of IL-4 and Apoptosis-Associated Gene Receptors (PD-1, CTLA-4) and Programmed Death-1 Ligands (PD-L1, PD-L2) in the prostate cancer tissue microenvironment have a signicant relationship. In conclusion, it is possible that IL-4 is a promoter of the Immune Escape mechanism in prostate cancer.


Introduction
Prostate cancer is currently the most commonly diagnosed cancer in 105 countries and the 2nd most common type of malignancy in male malignancies worldwide. 1 In 2018, there were 1.3 million new cases of prostate cancer and 359,000 related deaths worldwide.
Tumor cells express various cytokines, cytokine receptors, and proteins which then in situ helps tumor cells in tumor survival, tumor progression, tumor invasion, tumor metastasis, and apoptosis resistance to immune cell responses, this mechanism is called Immune Escape. 1,2 Programmed Death-1 (PD-1) or CD279 is an apoptosis-associated gene receptor for T cell activity that plays a role in inhibiting the immune response. 3,4 PD-1 has protein binding with Programmed Death-1-Ligand 1 (PD-L1) and Programmed Death-1-Ligand 2 (PD-L2). 5 PD-L1 and PD-L2 are expressed on immune cells, stromal cells, tumor cells, and antigen-presenting cells (APCs) from the Tumor Micro Environment (TME). [6][7][8][9] IL-4 is known to be expressed in malignancies such as melanoma, colorectal, non-Hodgkin's lymphoma, gastric, breast, skin, and bladder. The expression of IL-4 was increased under conditions of low androgen levels and increased the expression of Mir-21. 10,11 Mir-21 increases Androgen Receptor expression in the Androgen Receptor negative group and has a feedback loop mechanism. 12 Interleukin-4 is able to activate the Androgen Receptor (AR) by increasing the expression of CBP/p300 and the interaction of the AKT signal which results in the growth of tumors without androgen (androgen-independent). [13][14][15] The purpose of this study was to determine the relationship between IL-4 expression with Apoptosisassociated gene receptors (PD-1, CTLA-4) and Programmed Death-1 Ligands (PD-L1, PD-L2) in the microenvironment of prostate cancer tissue.

Method
Cluster Random Sampling was used as the sampling method. The sample is the FFPE of a patient's tissue who was diagnosed with prostate cancer histopathologically. FFPE samples were obtained from prostate tissue after prostate biopsy or transurethral prostatectomy. Exclusion criteria includes: no baseline Total PSA data, no baseline Total Testosterone data, FFPE age greater than 3 years, and invalid DNA integrity.
The Prostate biopsy and transurethral prostatectomy were carried out according to the guidelines and standard operating procedures in our center hospital. All experimental protocols were approved by ethics

RNA Extraction
The RNA genome was extracted from formalin-xed para n-embedded (FFPE) prostate tissue. In FFPE tissue specimens, a depara nization procedure was carried out using depara n liquid, RNA was extracted using the Hybrid-R miRNA kit.

RT-qPCR
The RNA extraction product was examined by RT-qPCR using the Bioner Accupower Greenstar RT-qPCR

Statistical Analysis
The descriptive and analytic data analysis methods were used. An analytical test was performed to ensure that the data was normal. If the sample size is greater than 50 using the Kolmogorov Smirnov Test and greater than 50 using the Shapiro Wilk Test, and the data distribution is normal, the statistical test employs a comparative parametric test Independent T-Test for two groups of categories and ANOVA Test for more than two groups of categories. If the data distribution is not normal, the statistical test employs the comparative non-parametric test, Mann Whitney U test for two groups of categories, and Kruskal Wallis Test for more than two groups of categories, followed by the Pos Hoc Test if the test is signi cant. The comparative test is preceded by the correlation test. The Pearson Test is used for parametric tests, while the Spearman Test is used for non-parametric tests.

Result
The samples taken were 40 FFPE preparations, including 12 samples diagnosed with Benign Prostate Hyperplasia (BPH), 8 samples of non-metastatic prostate cancer, and 20 samples of metastatic prostate cancer. The mean age of the patients is 68.5 years. In the BPH group sample, CTLA-4 had the highest expression level, followed by the expression of IL-4, PD-L2, then PD-1 and PD-L1. However, in the third column of prostate cancer, in non-metastatic prostate cancer and metastatic prostate cancer, PD-L2 had the highest expression level, followed by PD-L1, CTLA-4, IL-4, and PD-1 with a p-value 0.012.  Table 2 we can observe that the concentration of IL-4 in prostate cancer, both metastatic and nonmetastatic, is higher than in BPH, with a p-value of 0.006. There is also a signi cant difference in the average concentration of PD-L1 and PD-L1 between BPH and prostate cancer, with p-values of 0.004 and 0.012 respectively. In the case of PD-1 and CLTA-4, there was no signi cant difference in average concentration.  Serum IL-4 increases as prostate cancer progresses. Data from a study conducted by Goldstein et al showed that IL-4 increased as it developed into Castrase Resistant. This is also consistent with the evidence that after radical prostatectomy, the prognosis worsens when a signi cant increase in IL-4 is found. 21 As research conducted by Ueda et al, showed that serum IL-4 in hormone-refractory prostate cancer patients compared with pre-treatment prostate cancer patients was signi cantly signi cant. 22 However, serum IL-4 between healthy patients, patients with benign prostate enlargement, and patients with pretreatment prostate cancer was not signi cant. Furthermore, IL-4 levels were also not signi cantly different in pre-treatment patients in terms of clinical stage, histologic grade, and tumor Gleason score. A study on prostate cancer showed elevated IL-4 levels in patients with hormone-refractory cancer.
Moreover, recombinant IL-4 application also upregulated the expression of the two proteins -annexin A5 and syncytin that play important roles in several cell-cell fusion processes. IL-4 inhibition, indeed, lowers their expression and suppresses cell proliferation and fusion. In pancreatic tumors, IL-4 autocrine origin is essential in the control of normal macrophages transition into tumor-promoting macrophages. IL-4 expression was low in normal islets and increased by 4.5-fold at the hyperplastic stage. 24

Conclusion
In this study, we nd that the expression of IL-4 and Apoptosis-Associated Gene Receptors (PD-1, CTLA-4) and Programmed Death-1 Ligands (PD-L1, PD-L2) in the prostate cancer tissue microenvironment have a signi cant relationship. As a result, it is possible that IL-4 is a promoter of the Immune Escape mechanism in prostate cancer. This study is the rst study in Indonesia to look into the relationship between IL-4 expression in the tumor microenvironment of prostate cancer tissue. However, because it is still a local study, it is recommended that a larger sample size be used for future research in Indonesia using a multicenter approach.