Expression Of PD-L1 In Tumor Microenvironment Of Triple Negative Breast Cancer And Its Regulatory Mechanism By Thymosin ?1(zadaxin)

Objective:Triple negative breast cancer(TNBC)is the most aggressive type of breast cancer,accounting for 10%-20%of all breast cancer.Due to the lack of corresponding targets,so far,the main treatment methods are surgery,radiotherapy and chemotherapy,so new treatment methods are urgently needed.Immunotherapy is a new method to treat tumor by activating specific T cells,targeted attacking and clearing tumor cells,activating anti-tumor immune system response in vivo.Among them,dendritic cells(DC),as a full-time antigen presenting cell(APC)in vivo,are important immune active cells in anti-tumor immunity.They detect peripheral tissues,recognize,ingest and process pathogens.In the presence of major histocompatibility complex(MHC),they present antigen peptides to the initial T lymphocytes in lymphoid tissues,causing T lymphocytes to become active,mature and respond to immune response.DC can not only initiate immune response,but also participate in immune response.In physiological state,immunostimulatory signals(such as PD-1/PD-L1)play an important role in maintaining autoimmune tolerance and preventing autoimmune diseases.PD-L1 is an immunosuppressive molecule,which can cause antigen-specific T lymphocyte apoptosis and promote tumor growth.It is one of the mechanisms of tumor cell immune escape.Therefore,immunotherapy for PD-1/PD-L1 negative regulatory pathway is widely used in clinical,but the therapeutic effect in breast cancer is not the same.It is mainly suitable for triple negative breast cancer,which may be similar to triple negative breast cancer The expression of PD-L1 in tumor cells increased.As PD-1/PD-L1 is one of the important immune checkpoints in immunotherapy,there are a lot of studies on the relationship between PD-L1 expression and prognosis in triple negative breast cancer patients,but the results are different.Some studies have proved that patients with high expression of PD-L1 have a good prognosis,and there are also opposite conclusions,which are worthy of further discussion and research.Moreover,there are few studies on the correlation between DC and PD-L1 expression,especially the relationship between tumor infiltrating dendritic cells(TIDCs)and PD-L1 in tumor microenvironment(TME).TIDCs are also a kind of antigen-presenting cells,which can activate primary T lymphocytes and initiate immune response.At present,most studies focus on tumor infiltrating lymphocytes(TILs)in TME,but the study of TIDCs in TME is more helpful to understand the anti-tumor immune function and tumor immune escape mechanism,and provides a new way to explore DC mediated tumor immunotherapy.Whether the invasion density of TIDCs can be used as an independent factor of tumor prognosis has always been a research hotspot.There are many reports that the number of CD1a~+TIDCs is positively correlated with the prognosis of breast cancer.There are also reports that the prognosis of breast cancer is related to the number of CD83~+TIDCs,but not to CD1a~+TIDCs.The conclusions are different and need to be further confirmed by large sample study.Based on the above research progress,the impact of PD-L1 and TIDCs on the prognosis of triple negative breast cancer and the relationship between them are still unclear.Clarifying these issues will improve our understanding of the unique biological characteristics of TIDCs and the relationship between TIDCs and PD-L1.Many clinical trials have shown that both anti-PD-1 and anti-PD-L1 antibodies are effective in the treatment of PD-L1 positive triple negative breast cancer,suggesting that the efficacy of anti-PD-1/PD-L1 immune checkpoint inhibitors in triple negative breast cancer is closely related to the expression of PD-L1.However,patients with malignant tumor are generally in the state of immunosuppression.Immunopotentiation is often used in clinic in order to achieve better therapeutic effect.One of the most commonly used immunopotentiators is thymosin?1(Zadaxin).There is no literature on whether immune enhancement can affect the expression of PD-L1 molecules,thus affecting the effect of immunotherapy.Everything has its two sides.We speculate that Zadaxin may affect the expression of PD-L1 on the surface of tumor cells,which is very worthy of our discussion and research,so as to provide a theoretical basis for the clinical application of Zadaxin,especially before the application of PD-1/PD-L1 immune checkpoint inhibitors.Methods:1.The tissue samples of 159 triple negative breast cancer patients were studied retrospectively.The tissue microarray array was constructed.The expression of PD-L1,CD1a and CD83 in primary tumor tissues was detected by immunofluorescence staining of tissue paraffin section.2.According to the interquartile distance,the expression of PD-L1,CD1a and CD83 were divided into low expression,medium expression and high expression.The relationship between the expression of PD-L1 and pathological features,TIDCs and the expression of TIDCs in primary breast cancer were studied.3.The relationship between the expression levels of PD-L1,CD1a and CD83 and the overall survival of triple negative breast cancer patients was further analyzed.4.Multiple stepwise logistic regression was used to analyze the determinants of overall survival in triple negative breast cancer patients.5.MDA-MB-231 cells were treated with thymosin?1(0?mol/L,40?mol/L,70?mol/L,100?mol/L,130?mol/L and 160?mol/L,respectively,for 12h,24h,48h and 96h),and each treatment concentration contained 3 wells.The protein and RNA were extracted and the expression of PD-L1 was detected by Western Blot and q RT-PCR.6.Western Blot and q RT-PCR were used to detect whether the effect of 160?mol/L thymosin?1 treatment for 96h was consistent with the previous experimental results.7.According to the results of 96h treatment with 160?mol/L thymosin?1,NC-1,-3,-4 and 160?mol/L-1,-2,-4 were selected for RNA-seq library construction and sequencing,and go and KEGG enrichment analysis tools were used for functional classification of differentially expressed genes based on KOBAS 2.0 software.8.According to the results of data analysis,q RT-PCR was used to verify the expression of 16 differentially expressed genes.9.The PD-L1 m RNA level was transiently transfected by lipo2000.The expression of PD-L1 was detected by q RT-PCR after 96h treatment with 160?mol/L thymosin?1.10.Knockdown of MMP1 by lipo2000 transient transfection,Western Blot and QRT PCR were used to detect the expression of PD-L1after treatment with 160?mol/L thymosin?1 for 96 h.11.Transwell method was used to detect the effect of thymosin?1 on the migration and invasion of triple negative breast cancer cells.Results:1.The median age was 51 years(ranging from 26 to 83 years).The median tumor diameter was 2cm(IQR1.5,2.5).According to postoperative pathology,149(93.7%)women had invasive ductal carcinoma.59(37.1%)women had ipsilateral axillary lymph node metastasis,and 14(8.8%)women had stage III breast cancer.2.Immunofluorescence analysis showed that the median of PD-L1 in breast cancer tissues was 882.6(IQR 184.54136.2),and the low,medium and high expression were 25.16%,49.05%and 25.79%,respectively;the median of CD1a in breast cancer tissues was 30.2(IQR184.54136.2)The median expression of CD83 in breast cancer tissues was 1073.9(IQR374.32110.9),and the low expression,medium expression and high expression were25.79%,47.79%and 26.42%respectively.3.Pearson correlation analysis showed that PD-L1 was significantly correlated with CD1a(r=0.30409,P<0.001)and CD83(r=0.6146,P<0.001).CD1a was significantly correlated with CD83(r=0.38244,P<0.001).4.The distribution of PD-L1 expression was different in different tumor sizes.PD-L1 levels in patients with larger tumors were significantly lower than those in patients with smaller tumors(P=0.020).The level of PD-L1 in T2 patients was also significantly lower than that in T1 patients(P=0.009);there was no significant difference in the expression of PD-L1 among different status of axillary lymph nodes,different TNM stages and different types of invasion(P=0.705,P=0.067 and P=0.229);5.In patients with high expression of PD-L1,the tumor was relatively small,only 17.07%of patients had tumor more than 2.5cm,24.39%of patients had tumor less than 1.5cm(P=0.011).The expression of PD-L1 was different in different stages.In the PD-L1 high expression group,56.10%of patients were in stage IA,and 2.44%were in stage III.In the low expression group of PD-L1,only 17.5%patients were in stage IA,and 82.5%patients were in stage IIA-?(P=0.018).There was no significant difference in the distribution of PD-L1 expression in different lymph node states(N0,N1 or N2).(P=0.471).6.The median OS was 83 months.The 3-year OS rate was 94.97%(95%CI:91.57-98.37%),and the 5-year OS rate was 86.79%(95%CI:81.53-92.06).In addition,the 5-year OS rate of patients with high median CD1a(75.6%)was significantly lower than that of patients with low median CD1a(93.5%,P=0.038).7.There was no significant difference in survival curve among different PD-L1,CD1a and CD83,but higher PD-L1,CD1a or CD83 levels predicted shorter survival trend at 5 years follow-up.8.Multivariate stepwise logistic regression analysis showed that T stage was an important determinant of OS.9.Thymosin?1 can inhibit the expression of PD-L1 in triple negative breast cancer.10.Thymosin?1(160?mol/L)inhibited the expression of PD-L1in triple negative breast cancer cells for 96h.11.Thymosin?1 also inhibited the expression of PD-L1 in PD-L1 overexpressed triple negative breast cancer cells.12.After the transcriptome sequencing of thymosin?1-treated triple negative breast cancer cells,GO/KEGG pathway enrichment analysis showed that there were 9 up-regulated differentially expressed genes,which were enriched in the immune pathway,and 16down regulated differentially expressed genes,which were enriched in the cancer pathway.There were 12 genes with significant difference in both expression level and alternative splicing level.13.15 differentially expressed genes were verified by q RT-PCR,and the results were basically consistent with RNA-seq.14.Knockdown of MMP1gene,The expression of PD-L1 in MMP1 knockdown group was higher than that in non knockdown group;the expression of PD-L1 in MMP1 knockdown and Thymosin treatment group was higher than that in non knockdown and and Thymosin treatment group.15.Compared with the control group,thymosin?1 treatment for 96h significantly inhibited cell migration,but had no significant effect on cell invasion.Conclusion:1.The expressions of PD-L1,CD1a and CD83 were different in triple negative breast cancer tissues,but they were mainly moderately expressed,and the expression of PD-L1 was correlated with the levels of CD1a and CD83 in breast cancer tissues.2.Higher levels of PD-L1,CD1a or CD83 tend to be negative prognostic indicators of OS.CD1a is highly expressed in triple negative breast cancer tissues,and the 5-year survival rate is lower.3.Thymosin?1 can inhibit the expression of PD-L1 in triple negative breast cancer.4.MMP1 may be the key gene regulating PD-L1expression.5.Thymosin?1 inhibits the migration of triple negative breast cancer cells,but has no significant effect on cell invasion,which may be related to the up regulation of MMP1 expression by thymosin?1.