Programmed Death-ligand 1 mRNA Research Articles

PD-L1 mRNA derived from tumor-educated platelets as a potential immunotherapy biomarker in non-small cell lung cancer.

To date, the role of programmed death ligand-1 (PD-L1) messenger RNA (mRNA) derived from tumor-educated platelets (TEPs) has not been well investigated in patients with advanced non-small cell lung cancer (NSCLC). A few reports have examined whether mRNA in TEPs can predict the clinical responses of patients with advanced NSCLC following immunotherapy. This study aimed to identify novel biomarkers to improve the clinical benefits and outcomes of NSCLC patients. Advanced NSCLC patients receiving a combination of immunotherapy and chemotherapy, or immunotherapy alone as a first- or second-line treatment at the Fudan University Shanghai Cancer Center were enrolled in this study. All the patients had wild-type epidermal growth factor receptor/anaplastic lymphoma kinase. The patients were enrolled in clinical trials for immune checkpoint inhibitors (ICIs), including nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, and camrelizumab. Tumoral PD-L1 expression was tested by immunohistochemistry (PD-L1 22C3 pharmDx kit, Agilent, Santa Clara, CA, USA) in archived tissue samples, when available, to calculate the tumor proportion scores (TPSs). RNA and exosomal RNA of blood were isolated before immunotherapy using the Yunying RNA extraction kit (Yunying Medicine, Shanghai, China). The concentration and quality of the RNA was determined using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). Finally, we analyzed the predictive value of TEP-derived PD-L1 mRNA expression and association with the level of the tumoral PD-L1 expression. In total, 72 patients were enrolled in this study. Most of the patients were male (n=54, 75.0%), had adenocarcinoma (n=49, 68.1%). We found there was no significant correlation between the TEP-derived mRNA of PD-L1 and tumoral PD-L1 expression based on the results of the Pearson Correlation test (r=-0.19, P=0.233). Based on the median of PD-L1 mRNA, 72 patients were divided into a high PD-L1 group and a low PD-L1 group. We found that 19 patients (44.4%) responded to immunotherapy [partial response or progression-free survival (PFS) >6 months] in the high PD-L1 group, but only five patients (13.9%) responded to immunotherapy in the low PD-L1 group (P<0.01). The median PFS of the low PD-L1 group was lower than that of the high PD-L1 group (2.8 vs. 8.3 months, P<0.001). For the patients who were treated with immunotherapy alone (n=64), a similar PFS advantage was observed in the high PD-L1 group (2.8 vs. 8.0 months, P=0.002). This article presented the first data on TEP-derived PD-L1 mRNA in advanced NSCLC patients following immunotherapy and showed the potential advantage of using it as the surrogate biomarker for predicting the PFS and overall survival of patients following immunotherapy.

PD-L1 mRNA and protein expression in canine mammary carcinomas: Correlation with histopathological grade and molecular markers.

Programmed death ligand 1 (PD-L1) is an immune checkpoint molecule that plays a crucial role in regulating antitumor immune responses. Canine mammary carcinomas (CMCs) are common tumors of dogs. Despite extensive studies on the heterogeneity of CMCs, there is still a lack of effective precision therapies for the treatment of CMCs. In this study, we aimed to investigate the correlation between PD-L1 mRNA and protein expression in CMCs and explore its association with histopathological grade and molecular markers, including the estrogen receptor, epidermal growth factor receptor 2, and cytokeratin 5/6 (CK5/6). Formalin-fixed paraffin-embedded samples were evaluated for PD-L1 mRNA expression using RNA in situ hybridization and PD-L1 protein expression using immunohistochemistry. We observed no substantial correlation between PD-L1 mRNA and protein expression in CMCs; however, PD-L1 mRNA levels were significantly higher in grade 3 than in grade 1 tumors (P = .001). In addition, we observed a positive correlation between PD-L1 protein expression and CK5/6 expression in CMCs (P = .032). These findings suggest that PD-L1 expression in CMCs is heterogeneous and may be regulated post-transcriptionally. Further studies are needed to explore the prognostic and therapeutic implications of PD-L1 expression in different molecular subtypes of CMCs and their potential as predictive biomarkers for immunotherapy.

Splicing factor proline- and glutamine-rich regulates cytotoxic T lymphocytes-mediated cytotoxicity on non-small cell lung cancer by directly binding to PD-L1 3'UTR.

Splicing factor proline- and glutamine-rich (SFPQ) can interact with RNAs to regulate gene expression. The function of SFPQ in the immunotherapy of non-small cell lung cancer (NSCLC) is investigated in this study. H1299 and A549 cells were transfected with shSFPQ plasmid. Cell counting kit-8 (CCK-8) and cell clone formation were utilized to detect survival and proliferation. Programmed death-ligand 1 (PD-L1) and SFPQ were detected in NSCLC patients treated with anti-PD-L1 antibody. Dual-luciferase assays, RNA immunoblotting, RNA pull-down, and mRNA stability assay were applied to verify the regulation of PD-L1 with SFPQ. Human peripheral blood mononuclear cells (PBMC)-derived dendritic cells were loaded with irradiated A549 and H1299 cells, which were cultured with autologous CD8+T cells and tumor cells to perform in vitro tumor-specific cytotoxic T lymphocytes (CTL) cytotoxicity analysis. SFPQ silencing inhibited the survival and proliferation of H1299 and A549 cells with down-regulated PD-L1 expression. PD-L1 and SFPQ expression were markedly higher in anti-PD-L1 antibody treatment responders compared to non-responders, which showed a positive Pearson correlation (R = 0.76, P < .001). SFPQ up-regulated the relative mRNA and protein expression of PD-L1 by binding to the PD-L1 3'UTR to slow the decay of PD-L1 mRNA. SFPQ silencing promoted the killing effect of CTL on A549 and H1299 cells. SFPQ up-regulates PD-L1 expression by binding with PD-L1 3'UTR to slow the decay of PD-L1 mRNA, and SFPQ silencing promotes CTL-mediated cytotoxicity on NSCLC cells.

Predicting PD-L1 Status in Solid Tumors Using Transcriptomic Data and Artificial Intelligence Algorithms

Programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) is routinely used to predict the clinical response to immune checkpoint inhibitors (ICIs); however, multiple assays and antibodies have been used. This study aimed to evaluate the potential of targeted transcriptome and artificial intelligence (AI) to determine PD-L1 RNA expression levels and predict the ICI response compared with traditional IHC. RNA from 396 solid tumors samples was sequenced using next-generation sequencing (NGS) with a targeted 1408-gene panel. RNA expression and PD-L1 IHC were assessed across a broad range of PD-L1 expression levels. AI was used to predict the PD-L1 status. PD-L1 RNA levels assessed by NGS demonstrated robust linearity across high and low expression ranges, and those assessed using NGS and IHC (tumor proportion score and tumor-infiltrating immune cells) had a similar pattern. RNA sequencing provided in-depth information on the tumor microenvironment and immune response, including CD19, CD22, CD8A, CTLA4, and PD-L2 expression status. Subanalyses showed a sustained correlation of mRNA expression with IHC (tumor proportion score and immune cells) across different solid tumor types. Machine learning showed high accuracy in predicting PD-L1 status, with the area under the curve varying between 0.83 and 0.91. Targeted transcriptome sequencing combined with AI is highly useful for predicting PD-L1 status. Measuring PD-L1 mRNA expression by NGS is comparable to measuring PD-L1 expression by IHC for predicting ICI response. RNA expression has the added advantages of being amenable to standardization and avoiding interpretation bias, along with an in-depth evaluation of the tumor microenvironment.

N6-methyladenosine reader protein IGF2BP1 suppresses CD8 + T cells-mediated tumor cytotoxicity and apoptosis in colon cancer.

Tumor immune escape is an important manner for colon cancer to escape effective killing by immune system. Currently, the immune checkpoint PD-1/PD-L1-targeted immunotherapy has emerged as a promising therapeutic strategy in colon cancer. Here, present work aims to investigate the biological function of N6-methyladenosine (m6A) reader insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in regulating colon cancer's immune escape and CD8 + T cells-mediated tumor cytotoxicity and apoptosis. Results illustrated that IGF2BP1 was closely correlated to the colon cancer patients' poor clinical outcome. Functionally, upregulation of IGF2BP1 suppressed the CD8+ T-cells mediated antitumor immunity through reducing their tumor cytotoxicity. Mechanistically, MeRIP-Seq revealed that programmed death ligand 1 (PD-L1) mRNA had a remarkable m6A modified site on 3'-UTR genomic. Moreover, PD-L1 acted as the target of IGF2BP1, which enhanced the stability of PD-L1 mRNA. Overall, these results indicated that IGF2BP1 targeted PD-L1 to accelerate the immune escape in colon cancer by reducing CD8 + T cells-mediated tumor cytotoxicity in m6A-dependent manner. The findings demonstrate the potential of m6A-targeted immune checkpoint blockade in colon cancer, providing a novel insight for colon cancer immune escape and antitumor immunity in further precise treatment.

Comprehensive Detection of PD-L1 Protein and mRNA in Tumor Cells and Extracellular Vesicles through a Real-Time qPCR Assay.

A growing number of studies have shown that tumor cells secrete extracellular vesicles (EVs) containing programmed death-ligand 1 (PD-L1) protein. These vesicles can travel to lymph nodes and remotely inactivate T cells, thereby evading immune system attack. Therefore, the simultaneous detection of PD-L1 protein expression in cells and EVs is of great significance in guiding immunotherapy. Herein, we developed a method based on qPCR for the simultaneous detection of PD-L1 protein and mRNA in EVs and their parental cells (PREC-qPCR assay). Lipid probes immobilized on magnetic beads were used to capture EVs directly from samples. For RNA assay, EVs were directly broken by heating and quantified with qPCR. As to protein assay, EVs were recognized and bound with specific probes (such as aptamers), which were used as templates in subsequent qPCR analysis. This method was used to analyze EVs of patient-derived tumor clusters (PTCs) and plasma samples from patients and healthy volunteers. The results revealed that the expression of exosomal PD-L1 in PTCs was correlated with tumor types and significantly higher in plasma-derived EVs from tumor patients than that of healthy individuals. When extended to cells and PD-L1 mRNAs, the results showed that the expression of PD-L1 protein was consistent with mRNA in cancer cell lines, while PTCs demonstrated significant heterogeneity. This comprehensive detection of PD-L1 at four levels (cell, EVs, protein, and mRNA) is believed to enhance our understanding of the relationship among PD-L1, tumors, and the immune system and to provide a promising tool for predicting the benefits of immunotherapy.

The tumor promoter cysteinyl leukotriene receptor 1 regulates PD-L1 expression in colon cancer cells via the Wnt/β-catenin signaling axis

Immunotherapy targeting programmed death-ligand 1 (PD-L1) or PD-1 in solid tumors has been shown to be clinically beneficial. However, in colorectal cancer (CRC), only a subset of patients benefit from PD-1/PD-L1 treatment. Previously, we showed that high cysteinyl leukotriene receptor 1 (CysLT1R) levels are associated with poor prognosis in CRC patients. Recently, we have revealed the role of the tumor promoter CysLT1R in drug resistance and stemness in colon cancer (CC) cells. Here, we show the role of the CysLT1R/Wnt/β-catenin signaling axis in the regulation of PD-L1 using both in vitro and in vivo preclinical model systems. Interestingly, we found that both endogenous and IFNγ-induced PD-L1 expression in CC cells is mediated through upregulation of CysLT1R, which enhances Wnt/β-catenin signaling. Therapeutic targeting of CysLT1R with its antagonist montelukast (Mo), as well as CRISPR/Cas9-mediated or doxycycline-inducible functional absence of CysLT1R, negatively regulated PD-L1 expression in CC cells. Interestingly, an anti-PD-L1 neutralizing antibody exhibited stronger effects together with the CysLT1R antagonist in cells (Apcmut or CTNNB1mut) with either endogenous or IFNγ-induced PD-L1 expression. Additionally, mice treated with Mo showed depletion of PD-L1 mRNA and protein. Moreover, in CC cells with combined treatment of a Wnt inhibitor and an anti-PD-L1 antibody was effective only in β-catenin-dependent (APCmut) context. Finally, analysis of public dataset showed positive correlations between the PD-L1 and CysLT1R mRNA levels. These results elucidate a previously underappreciated CysLT1R/Wnt/β-catenin signaling pathway in the context of PD-L1 inhibition in CC, which might be considered for improving the efficacy of anti-PD-L1 therapy in CC patients.7T-oMpUEusLw1sD21V8GVPVideo

The Immunosuppressive Roles of PD-L1 during Influenza A Virus Infection.

The clinical benefits of targeting programmed death-ligand 1 (PD-L1) in various cancers represent a strategy for the treatment of immunosuppressive diseases. Here, it was demonstrated that the expression levels of PD-L1 in cells were greatly upregulated in response to H1N1 influenza A virus (IAV) infection. Overexpression of PD-L1 promoted viral replication and downregulated type-I and type-III interferons and interferon-stimulated genes. Moreover, the association between PD-L1 and Src homology region-2, containing protein tyrosine phosphatase (SHP2), during IAV/H1N1 infection was analyzed by employing the SHP2 inhibitor (SHP099), siSHP2, and pNL-SHP2. The results showed that the expressions of PD-L1 mRNA and protein were decreased under SHP099 or siSHP2 treatment, whereas the cells overexpressing SHP2 exhibited the opposite effects. Additionally, the effects of PD-L1 on the expression of p-ERK and p-SHP2 were investigated in PD-L1-overexpressed cells following WSN or PR8 infection, determining that the PD-L1 overexpression led to the decreased expression of p-SHP2 and p-ERK induced by WSN or PR8 infection. Taken together, these data reveal that PD-L1 could play an important role in immunosuppression during IAV/H1N1 infection; thus, it may serve as a promising therapeutic target for development of novel anti-IAV drugs.

Examination of the Functional Relationship between PD-L1 DNA Methylation and mRNA Expression in Non-Small-Cell Lung Cancer

Immunotherapy targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) is a treatment option for patients with non-small-cell lung cancer (NSCLC). The expression of PD-L1 by the NSCLC cells determines treatment effectiveness, but the relationship between PD-L1 DNA methylation and expression has not been clearly described. We investigated PD-L1 DNA methylation, mRNA expression, and protein expression in NSCLC cell lines and tumor biopsies. We used clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) to modify PD-L1 genetic contexts and endonuclease deficient Cas9 (dCas9) fusions with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) to manipulate PD-L1 DNA methylation. In NSCLC cell lines, we identified specific PD-L1 CpG sites with methylation levels inversely correlated with PD-L1 mRNA expression. However, inducing PD-L1 mRNA expression with interferon-γ did not decrease the methylation level for these CpG sites, and using CRISPR-Cas9, we found that the CpG sites did not directly confer a negative regulation. dCas9-TET1 and dCas9-DNMT3A could induce PD-L1 hypo- and hyper-methylation, respectively, with the latter conferring a decrease in expression showing the functional impact of methylation. In NSCLC biopsies, the inverse correlation between the methylation and expression of PD-L1 was weak. We conclude that there is a regulatory link between PD-L1 DNA methylation and expression. However, since these measures are weakly associated, this study highlights the need for further research before PD-L1 DNA methylation can be implemented as a biomarker and drug target for measures to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC.

Classification of Tumor Immune Microenvironment According to Programmed Death-Ligand 1 Expression and Immune Infiltration Predicts Response to Immunotherapy Plus Chemotherapy in Advanced Patients With NSCLC

IntroductionAccording to mechanisms of adaptive immune resistance, tumor immune microenvironment (TIME) is classified into four types: (1) programmed death-ligand 1 (PD-L1)–negative and tumor-infiltrating lymphocyte (TIL)–negative (type I); (2) PD-L1–positive and TIL-positive (type II); (3) PD-L1–negative and TIL-positive (type III); and (4) PD-L1–positive and TIL-negative (type IV). However, the relationship between the TIME classification model and immunotherapy efficacy has not been validated by any large-scale randomized controlled clinical trial among patients with advanced NSCLC. MethodsOn the basis of RNA-sequencing and immunohistochemistry data from the ORIENT-11 study, we optimized the TIME classification model and evaluated its predictive value for the efficacy of immunotherapy plus chemotherapy. ResultsPD-L1 mRNA expression and immune score calculated by the ESTIMATE method were the strongest predictors for the efficacy of immunotherapy plus chemotherapy. Therefore, they were determined as the optimized definition of the TIME classification system. When compared between combination therapy and chemotherapy alone, only the type II subpopulation with high immune score and high PD-L1 mRNA expression was significantly associated with improved progression-free survival (PFS) (hazard ratio = 0.12, 95% confidence interval: 0.06–0.25, p < 0.001) and overall survival (hazard ratio = 0.27, 95% confidence interval: 0.13–0.55, p < 0.001). In the combination group, the type II subpopulation had a much longer survival time, not even reaching the median PFS or overall survival, but the other three subpopulations were susceptible to having similar PFS. In the chemotherapy group, there was no marked association between survival outcomes and TIME subtypes. ConclusionsOnly patients with both high PD-L1 expression and high immune infiltration could benefit from chemotherapy plus immunotherapy in first-line treatment of advanced NSCLC. For patients lacking either PD-L1 expression or immune infiltration, chemotherapy alone might be a better treatment option to avoid unnecessary toxicities and financial burdens.

Abstract P6-14-15: PD-L1 expression regulated by JAK/STAT signaling pathway contributes to cell migration and invasion in breast cancer

Abstract Background: Programmed death-ligand 1 (PD-L1) implicated in tumor immune evasion and predictive biomarker in immunotherapy is widely recognized, however, the role of PD-L1 in modulating tumor invasion remains largely unexplored. PD-L1 expression on tumor tissue is usually limited by the invasiveness, tumor heterogeneity as well as insufficient tumor tissue samples, while PD-L1 expression on circulating tumor cells (CTCs) might overcome the limitation. In the present study, we aimed to investigate the role and mechanism of PD-L1 in regulating the migration and invasion of breast cancer cells and to evaluate PD-L1 expression on CTCs in metastatic breast cancer patients. Methods: The expression level of PD-L1 in MCF-7 cells and MDA-MB-231 cells was assessed by quantitative real-time RT-PCR and Western Blot. Gain-of-function and loss-of-function study on cell migration and invasion abilities were carried out by overexpression of PD-L1 or silencing PD-L1. PD-L1 expression on CTCs in thirty-six metastatic breast cancer patients were detected. A novel staining procedure which included fluorescent glucose analog staining for CTC enumeration and immunostaining targeting CD45, vimentin and PD-L1 were analyzed. Survival curves were estimated by the Kaplan-Meier method and the log-rank test was used to compare between groups. All tests were two-sided, and p values were considered significant at the 0.05 level. Results: Compared with MCF-7 cells, PD-L1 mRNA and PD-L1 protein expression were significantly increased in MDA-MB-231 cells. Down-regulation of PD-L1 expression in MDA-MB-231 cells inhibited the migration and invasion of MDA-MB-231 cells, while overexpression of PD-L1 in MCF-7 cells increased the migration and invasion of MCF-7 cells. In addition, we found that PD-L1 expression was regulated by JAK/STAT signaling pathway. PD-L1 expression on CTCs in metastatic breast cancer patients was associated with triple negative breast cancers subtype (P=0.013). High expression of PD-L1 on CTCs in metastatic breast cancer patients was correlated to poor overall survival (HR=3.165, 95%CI: 1.121-8.938, P=0.029). Conclusion: Our results indicate that high expression of PD-L1 contributes to cell migration and invasion in breast cancer cell possibly partially through JAK/STAT signaling pathway. The PD-L1 expression on CTCs might serve as a promising non-invasive prognostic biomarker in patients with metastatic breast cancer. Citation Format: Junqing Chen. PD-L1 expression regulated by JAK/STAT signaling pathway contributes to cell migration and invasion in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-15.

The regulation of N6-methyladenosine modification in PD-L1-induced anti-tumor immunity.

There is growing evidence that programmed death ligand-1 (PD-L1) has exciting therapeutic efficacy in hematological malignancy and partial solid tumors. However, many patients still face failure with the treatment of immune checkpoint blockade because of PD-L1 expression regulation during transcription and post-transcription processes, including N6-methyladenosine (m6A). Similar to the epigenetic regulation in DNA and histones, recent research has revealed the essential regulation of m6A modification in RNA nuclear export, metabolism and translation. Recent studies have shown that m6A-induced PD-L1 expression emerges as one of the main reasons for the immunological alteration in this process and contributes to the failure of Tcell-induced anti-tumor immunity. The results of preclinical studies demonstrate the potential of m6A-targeted therapy in combination with immune checkpoint blockade. The comprehensive expression of m6A-related genes also provided the possibility to indicate the prognosis and to optimize the treatment for patients of various cancer types. In this review, we focus on the m6A modification in PD-L1 mRNA as well as the regulation of PD-L1 expression in cancer cells and summarize its clinical value in anti-PD-L1 cancer immune therapy.

Characterization of expression and prognostic implications of transforming growth factor beta, programmed death-ligand 1, and T regulatory cells in canine histiocytic sarcoma

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-β), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this study was to investigate the expression and prognostic significance of TGF-β, PD-L1, and FoxP3/CD25 in canine HS utilizing RNA in situ hybridization (RNAscope®). After validation was performed, RNAscope® on formalin-fixed paraffin-embedded (FFPE) patient HS tissue samples was performed for all targets and expression quantified with HALO® software image analysis. Cox proportional hazard model was conducted to investigate the association between survival time and each variable. Additionally, for categorical data, the Kaplan-Meier product-limit method was used to generate survival curves. TGF-β and PD-L1 mRNA expression was confirmed in the DH82 cell line by reverse transcription polymerase chain reaction (RT-PCR) and CD25 + FoxP3 + cells were detected by flow cytometry in peripheral blood. Once the RNAscope® method was validated, TGF-β H-score and dots/cell and FoxP3 dots/cell were assessed in HS samples and found to be significantly correlated with survival. Moderate positive correlations were found between FoxP3 and PD-L1 H-score, percent staining area, and dots/cell, and FoxP3 and TGF-β dots/cell. In summary, RNAscope® is a valid technique to detect TGF-β and PD-L1 expression and identify Tregs in canine HS FFPE tissues. Furthermore, canine HS expresses TGF-β and PD-L1. Increased TGF-β and FoxP3 correlated with worse prognosis. Prospective studies are warranted to further investigate TGF-β, PD-L1, and Tregs effect on prognosis.

Effect of MiR-424-5p on the Drug Resistance of Diffuse Large B-Cell Lymphoma Cells by Regulating PD-1/PD-L1 Signaling Pathway

To explore the effect of microRNA-424-5p (miR-424-5p) on the drug resistance of diffuse large B-cell lymphoma (DLBCL) cells by regulating the programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway. Human DLBCL cell line CRL2631 cells were induced to construct CRL2631-CHOP resistant cell line. RT-qPCR and Western blot were used to detect the expression levels of MiR-424-5p, PD-L1 mRNA and protein, and multidrug resistance gene-1 (MDR-1) protein in CRL2631 cells and CRL2631-CHOP cells, respectively. The target genes of MiR-424-5p was verified by dual luciferase reporter assay. The miRNA simulation/interference technology and thiazole blue (MTT) method were used to detect the resistance of CRL2631 cells and CRL2631-CHOP cells to CHOP. Compared with CRL2631 cells, the drug resistance of CRL2631-CHOP cells to CHOP and the levels of MDR-1 protein (P<0.05), PD-L1 mRNA and protein in the cells were significantly increased (both P<0.001), while the relative level of MiR-424-5p was significantly reduced (P<0.001). The result of the dual luciferase reporter assay showed that PD-L1 was the direct downstream target gene of MiR-424-5p (P<0.001). After transfection of MiR-424-5p inhibitor, the resistance of CRL2631 cells to CHOP drugs increased, and the expression level of MDR-1 protein (P<0.01), PD-L1 mRNA and protein also increased significantly (both P<0.01). After transfection of MiR-424-5p mimics, the resistance of CRL2631-CHOP cells to CHOP drugs decreased, and the expression level of MDR-1 protein (P<0.001), PD-L1 mRNA and protein also decreased significantly (both P<0.001). Overexpression of PD-L1 could reverse the inhibitory effect of upregulating MiR-424-5p on PD-L1 (P<0.001). Down-regulation of MiR-424-5p enhances the drug resistance of DLBCL cells by regulating the PD-1/PD-L1 signaling pathway.

&lt;i&gt;Dictyostelium&lt;/i&gt; Differentiation-inducing Factor Derivatives Reduce the Glycosylation of PD-L1 in MDA-MB-231 Human Breast Cancer Cells

Objectives Triple-negative breast cancer (TNBC) is a metastatic and intractable cancer with limited treatment options. Refractory cancer cells often express the immune checkpoint molecules programmed death-ligand 1 (PD-L1) and PD-L2, which inhibit the anticancer effects of T cells. Differentiation-inducing factors, originally found in Dictyostelium discoideum, and their derivatives possess strong antiproliferative activity, at least in part by reducing cyclin D1 expression in various cancer cells, but their effects on PD-L1/PD-L2 have not been examined. In this study, we investigate the effects of six DIF compounds (DIFs) on the expression of PD-L1/PD-L2 and cyclin D1/D3 in MDA-MB-231 cells, a model TNBC cell line.Methods MDA-MB-231 cells were incubated for 5 or 15 h with or without DIFs, and the mRNA expression of cyclin D1, PD-L1, and PD-L2 were assessed by quantitative polymerase chain reaction (qPCR). Whereas, MDA-MD-231 cells were incubated for 12 or 24 h with or without DIFs, and the protein expression of cyclins D1 and D3, PD-L1, and PD-L2 were assessed by Western blotting.Results As expected, some DIFs strongly reduced cyclin D1/D3 protein expression in MDA-MB-231 cells. Contrary to our expectation, DIFs had little effect on PD-L1 mRNA expression or increased it transiently. However, some DIFs partially reduced glycosylated PD-L1 and increased non-glycosylated PD-L1 in MDA-MB-231 cells. The level of PD-L2 was very low in these cells.Conclusion Since PD-L1 glycosylation plays an important role in preventing T cells from attacking cancer cells, such DIFs may promote T cell attack on cancer cells in vivo.

The Effect of Secreted IL-10 from Mesenchymal Stem Cell on Immune Checkpoint Molecules.

Immunosuppression in sepsis is hypothesized to result from the increased expression of the immune checkpoint molecules programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1). PD-1 and PD-L1 blockade therapies have been reported to increase survival in septic animals. Currently, the interleukin (IL)-10 within mesenchymal stem cell (MSC) secretome is known for its immunomodulatory capacity. To study the effect of IL-10 within MSC secretome on the expression of immune checkpoints in the rat model of sepsis. Methods: We used 48 male Rattus norvegicus rats in this research and divided them into four groups: sham (rats without sepsis induction and treatment), control (sepsis-induced rats without treatment), T1 (sepsis-induced rats treated with 150 μL of secreted IL-10 from MSC), and T2 (sepsis-induced rats treated with 300 μL of secreted IL-10 from MSC). Forty-eight hours after sepsis induction, we terminated the rats and collected the blood to examine the PD-1 and PD-L1 expression levels. We found a decrease in the relative expression of PD-1 in the septic rat group given 150 μL and 300 μL of secreted IL-10 from MSC compared to the control group, but the decrease was not significant. We also found a decrease in the relative expression of PD-L1 mRNA in the septic rat group given 150 μL and 300 μL of secreted IL-10 from MSC compared to the control group. Administering secreted IL-10 from MSC reduces the expression of PD-1 and PD-L1 in sepsis. These findings suggest that MSC secretome can improve the immunosuppression in sepsis.

Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer

Programmed Death Ligand 1 (PD-L1) is crucial in regulating the immunological tolerance in non-small cell lung cancer (NSCLC). Alveolar macrophage (AM)-derived PD-L1 binds to its receptor, PD-1, on surveilling lymphocytes, leading to lymphocyte exhaustion. Increased PD-L1 expression is associated with cigarette smoke (CS)-exposure. However, the PD-L1 role in CS-associated lung diseases associated with NSCLC, such as chronic obstructive pulmonary disease (COPD), is still unclear. In two different cohorts of ever smokers with COPD or NSCLC, and ever and never smoker controls, we evaluated PD-L1 expression: (1) via cutting-edge digital spatial proteomic and transcriptomic profiling (Geomx) of formalin-fixed paraffin-embedded (FFPE) lung tissue sections (n = 19); and (2) via triple immunofluorescence staining of bronchoalveolar lavage (BAL) AMs (n = 83). PD-L1 mRNA expression was also quantified in BAL AMs exposed to CS extract. PD-L1 expression was increased in the bronchiolar wall, parenchyma, and vascular wall from mild-moderate (GOLD 1–2) COPD patients compared to severe-very severe (GOLD 3–4) COPD patients and controls. Within all the COPD patients, PD-L1 protein expression was associated with upregulation of genes involved in tumor progression and downregulation of oncosuppressive genes, and strongly directly correlated with the FEV1% predicted, indicating higher PD-L1 expression in the milder vs. more severe COPD stages. In bronchioles, PD-L1 levels were strongly directly correlated with the number of functionally active AMs. In BAL, we confirmed that AMs from patients with both GOLD 1–2 COPD and NSCLC had the highest and similar, PD-L1 expression levels versus all the other groups, independently from active cigarette smoking. Intriguingly, AMs from patients with more severe COPD had reduced AM PD-L1 expression compared to patients with mild COPD. Acute CS extract stimulation increased PD-L1 mRNA expression only in never-and not in ever-smoker AMs. Lungs from patients with mild COPD and NSCLC are characterized by a similar strong PD-L1 expression signature in bronchioles and functionally active AMs compared to patients with severe COPD and controls. Active smoking does not affect PD-L1 levels. These observations represent a new resource in understanding the innate immune mechanisms underlying the link between COPD and lung cancer onset and progression and pave the way to future studies focused on the mechanisms by which CS promotes tumorigenesis and COPD.

Electric Field-Based Cancer Therapy Induces the Expression of HMGB1 and PD-L1 mRNA Genes on Breast Tumor of Female Rats

Electro Capacitive Cancer Therapy (ECCT) is an electric field-based cancer therapy method using intermediate frequency (150 kHz) and low intensity (18 Vpp). High Mobility Group Box 1 (HMGB1) is a cytokine related to damage-associated molecular patterns (DAMPs) secreted by dead cells. The expression of Programmed Death Ligand 1 (PD-L1) is ligand present on the surface of tumor cells and its expression is associated with the increase in the number CD8+ T lymphocytes. This study aims to examine the effect of ECCT exposure on the expression of HMGB1 and PD-L1 genes on the breast tumor, brain, and liver tissues of Rattus norvegicus (Berkenhout, 1769). The tissues were obtained from the previous studies stored in RNAlater (-20˚C). Female rat tissues of the previous study from four treatment groups, namely the control group (NINT), non-DMBA-induction with therapy (NIT), DMBA-induction with non-therapy (INT), and DMBA-induction with therapy (IT). Gene expression was analyzed using the RT-qPCR. Statistical t-test with a p&lt;0.05 significance level was performed using GraphPad Prism 9.4.0 software. The result shows HMGB1 and PD-L1 mRNA genes were both significantly expressed in breast tumor samples. The liver and brain samples of normal rats did not show any significant changes in the activity of these genes after exposure to the electric field. This study indicates that exposure to electric fields may trigger the expression of HMGB1 and PD-L on the rat’s breast tumor samples. This study also provides information related to the safety of ECCT in healthy organs of female rats, especially the brain and liver.Keywords: ECCT, breast tumor, HMGB1, PD-L1, IFN- γ.

PD-L1 on Circulating Tumor Cells Indicates Poor Prognosis in Breast Cancer

Background: In breast cancer, research concerning programmed death-ligand 1 (PD-L1) on circulating tumor cells (CTCs) is limited. Besides, the best cell type for determining the prognostic and predictive values of PD-L1 has not been established. This study investigated associations between PD-L1 on tumor cells (TCs), CTCs, and tumor-infiltrating immune cells (TIICs), respectively, and prognosis and clinicopathological features in breast cancer. Methods: Twenty patients with breast cancer were recruited; one was excluded for confirmed lymphoma. The PD-L1 on TCs and TIICs was determined via immunohistochemistry; PD-L1 mRNA expression on CTCs was analyzed. The chi-squared test and Kaplan-Meier and Cox proportional hazards model analyses were applied. Results: The median follow-up time was 60 months. Of the 19 patients, 14 had &gt;1 CTC/10 mL peripheral blood. Among these, each had ≥1 CTC showing PD-L1. At baseline, there was no difference between groups with or without metastasis regarding CTCs and PD-L1 expression. Patients with high PD-L1 levels on CTCs had poor overall survival (p = 0.034). In the multivariate analysis, PD-L1 levels on CTCs and T stage were independent prognostic factors (p = 0.029, 0.010, respectively). Conclusions: High levels of PD-L1 on CTCs may prognose shorter overall survival in breast cancer.

EP01.01-008 Expression of PD-L1 mRNA and Its Epigenetic Regulators: MicroRNA-17, -93 and -142 in Early Stages NSCLC Patients’ Plasma

One of the leading treatment options for advanced non-small cell lung cancer (NSCLC) patients is immunotherapy based on immune checkpoint inhibitors, including programmed death ligand 1 (PD-L1) inhibitors. PD-L1 molecule expressed on tumor cells causes PD-1+ tumor infiltrating lymphocytes (TILs) exhaustion and thus escape from immune surveillance. Blockade of the PD-1/PD-L1 pathway by monoclonal antibodies enables restoration of the anti-tumor response. PD-L1 expression is regulated by epigenetic factors, e.g.: microRNAs (miRNAs).