PD-L1 In Cancer Cells Research Articles

Tissue Expression and Plasma Soluble PD-L1 Levels in Patients With Endometrial Cancer.

The prognostic role of tissue PD-L1 expression in endometrial cancer (EC) remains controversial. Moreover, its value in guiding anti-PD1/PD-L1 immunotherapy is questionable. The eventual role of soluble PD-L1 (sPD-L1), released by cancer tissue and circulating immune cells, is largely unexplored. In this pilot study, we investigated the expression of PD-L1 in cancer cells and tumor stroma infiltrating inflammatory cells (TIICs), in parallel with sPD-L1 levels in the plasma of 19 patients with early-stage endometrioid EC, before and after hysterectomy. Cancer cell membrane staining was noted in 5/19 (26.3%) cases (range=1-5%; median 1% of the cancer cell population). IICs showed positive reactivity in 14/19 (73.7%) cases. sPD-L1 plasma levels ranged from 53-408 pg/ml and 113-557 pg/ml, respectively, in the plasma of patients before and after hysterectomy (p=0.01). High PD-L1 expression by IIC was marginally associated with a high histology grade (p=0.08). High sPD-L1 levels before surgery were significantly associated with ICC PD-L1 expression (p=0.0007), high histology grade (p=0.04), and marginally with T2-stage (p=0.08). sPD-L1 is easily detectable in the plasma of EC patients and may be associated with aggressive clinical features and PD-L1-expressing immune cells infiltrating the tumor stroma. sPD-L1 plasma levels in EC patients undergoing immunotherapy can be further tested as a biomarker for therapeutic stratification.

IMPDH inhibitors upregulate PD-L1 in cancer cells without impairing immune checkpoint inhibitor efficacy.

Tumor cells are characterized by rapid proliferation. In order to provide purines for DNA and RNA synthesis, inosine 5'-monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo guanosine biosynthesis, is highly expressed in tumor cells. In this study we investigated whether IMPDH was involved in cancer immunoregulation. We revealed that the IMPDH inhibitors AVN944, MPA or ribavirin concentration-dependently upregulated PD-L1 expression in non-small cell lung cancer cell line NCI-H292. This effect was reproduced in other non-small cell lung cancer cell lines H460, H1299 and HCC827, colon cancer cell lines HT29, RKO and HCT116, as well as kidney cancer cell line Huh7. In NCI-H292 cells, we clarified that IMPDH inhibitors increased CD274 mRNA levels by enhancing CD274 mRNA stability. IMPDH inhibitors improved the affinity of the ARE-binding protein HuR for CD274 mRNA, thereby stabilizing CD274 mRNA. Guanosine supplementation abolished the IMPDH inhibitor-induced increase in PD-L1 expression. In CT26 and EMT6 tumor models used for ICIs based studies, we showed that despite its immunosuppressive properties, the IMPDH inhibitor mycophenolate mofetil did not reduce the clinical response of checkpoint inhibitors, representing an important clinical observation given that this class of drugs is approved for use in multiple diseases. We conclude that PD-L1 induction contributes to the immunosuppressive effect of IMPDH inhibitors. Furthermore, the IMPDH inhibitor mycophenolate mofetil does not antagonize immune checkpoint blockade.

Discovering New Metallo-Deubiquitinase CSN5 Inhibitors by a Non-Catalytic Activity Assay Platform.

COP9 signalosome catalytic subunit CSN5 plays a key role in tumorigenesis and tumor immunity, showing potential as an anticancer target. Currently, only a few CSN5 inhibitors have been reported, at least partially, due to the challenges in establishing assays for CSN5 deubiquitinase activity. Here, we present the establishment and validation of a simple and reliable non-catalytic activity assay platform for identifying CSN5 inhibitors utilizing a new fluorescent probe, CFP-1, that exhibits enhanced fluorescence and fluorescence polarization features upon binding to CSN5. By using this platform, we identified 2-aminothiazole-4-carboxylic acids as new CSN5 inhibitors, which inhibited CSN5 but slightly downregulated PD-L1 in cancer cells. Furthermore, through the integration of deep learning-enabled virtual screening, we discovered that shikonins are nanomolar CSN5 inhibitors, which can upregulate PD-L1 in HCT116 cells. The binding modes of these structurally distinct inhibitors with CSN5 were explored by using microsecond-scale molecular dynamics simulations and tryptophan quenching assays.

Abstract PO1-25-03: Programmed Death-Ligand 1 and Receptor Tyrosine Kinases in Breast Cancer

Abstract Programmed death-ligand 1 (PD-L1) is an immune checkpoint expressed in a wide range of malignancies leading to immune tolerance and cancer cell immune evasion. Receptor tyrosine kinases (RTKs) are key regulators of cancer cell proliferation, survival, and invasion. The Hepatocyte Growth Factor (HGF) receptor, MET, and the Epidermal Growth Factor Receptor (EGFR) are RTKs known for their tumorigenic potential in a variety of human malignancies. This study aimed to evaluate the effect of the small-molecule tyrosine kinase inhibitors (TKIs) on the expression of PD-L1 in breast cancer cells and the effect of their combination with inflammatory cytokines. The study also assessed the association of the expression of the tumoral PD-L1 mRNA with each of MET and EGFR mRNA expression and the tumor features and treatment outcomes in breast cancer patients using the METABRIC dataset publicly available from cBioPortal for Cancer Genomics. The TKIs crizotinib [a MET inhibitor] and gefitinib [an EGFR inhibitor] were used as a single treatment and in combination with the cytokines; tumor necrosis factor-α (TNF-α) or interferon-γ (INF-γ) in MCF7 and MDA-MB-231 breast cancer cells in vitro. The cells were also treated with the mitogens HGF and EGF. The viability of cells after the different treatments was determined using the MTT viability assay. The effects of the combination treatments were analyzed using combination index (CI) analysis. The expression level of PD-L1 in cancer cells was assessed using Western blotting. The combination treatment of crizotinib with TNF-α and INF-γ produced a synergistic growth inhibition of MCF7 and MDA-MB-231 cells with CI values of 0.31 and 0.55, respectively. Alternatively, combined crizotinib and TNF-α produced an antagonist effect on the viability of MCF7 cells (CI=2.49). The combination of gefitinib with TNF-α produced a synergistic growth inhibition in both MCF7 and MDA-MB-231 cells with CI values of 0.39 and 0.78, respectively. Alternatively, gefitinib resulted in an additive effect when combined with INF-γ (CI=0.99) and an antagonistic effect when combined with TNF-α (CI=2.68) in MCF7 and MDA-MB-231 cells, respectively. Treatment with HGF (50 and 100 ng/ml) increased the protein levels of PD-L1 while EGF (50 and 100 ng/ml) reduced its levels in MDA-MB-231 cells. Treatment with the TKIs crizotinib (0.1-4 µM) and gefitinib (1-40 µM) significantly reduced PD-L1 levels in MDA-MB-231 cells compared to vehicle-treated cells. The analysis of the METABRIC dataset revealed that the mRNA expression of PD-L1 was positively correlated with the mRNA expression of the EGFR gene (r= 0.081, p< 0.001) but not the MET gene. A double-high PD-L1/MET expression was significantly associated with younger age at diagnosis, high-grade carcinoma, greater tumor size, hormone receptor-negative status, HER2-positivity, and non-luminal disease compared to patients with a double-low PD-L1/MET expression. Similar findings were reported for patients with a double-high PD-L1/EGFR expression compared to patients with a double-low PD-L1/EGFR expression. Nevertheless, the mRNA expression of the PD-L1, MET, and EGFR genes as well as the co-expression of PD-L1/MET or PD-L1/EGFR genes did not affect the overall survival of breast cancer patients. Collectively, MET and EGFR TKIs could modulate the effects of inflammatory cytokines differently based on the type of cytokine and the molecular subtype of breast cancer cells. The expression of PD-L1 in breast cancer cells was upregulated by HGF while TKIs reduced its expression. The co-expression of the PD-L1 gene with MET and EGFR genes in breast cancer was associated with advanced disease presentation and worse prognosticators in patients. Together, TKIs that target MET or EGFR could be an appealing therapeutic target in breast cancer, particularly in younger patients with the non-luminal disease. Citation Format: Nehad Ayoub, Muhsen Al-Diabat, Moath Al-Shorman, Laith Al-Eitan. Programmed Death-Ligand 1 and Receptor Tyrosine Kinases in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-03.

GSK0660 enhances antitumor immunotherapy by reducing PD-L1 expression

Blockade of PD-1/PD-L1 immune checkpoint is wildly used for multiple types of cancer treatment, while the low response rate for patients is still completely unknown. As nuclear hormone receptor, PPARδ (peroxisome-proliferator-activated receptor) regulates cell proliferation, inflammation, and tumor progression, while the effect of PPARδ on tumor immune escape is still unclear. Here we found that PPARδ antagonist GSK0660 significantly reduced colon cancer cell PD-L1 protein and gene expression. Luciferase analysis showed that GSK0660 decreased PD-L1 gene transcription activity. Moreover, reduced PD-L1 expression in colon cancer cells led to increased T cell activity. Further analysis showed that GSK0660 decreased PD-L1 expression in a PPARδ dependent manner. Implanted tumor model analysis showed that GSK0660 inhibited tumor immune escape and the combined PD-1 antibody with GSK0660 effectively enhanced colorectal cancer immunotherapy. These findings suggest that GSK0660 treatment could be an effective strategy for cancer immunotherapy.

RCAd-LTH-shPD-L1, a double-gene recombinant oncolytic adenovirus with enhanced antitumor immunity, increases lymphocyte infiltration and reshapes the tumor microenvironment

BackgroundWith the successful development of modern immunotherapy, immune checkpoint inhibitors (ICIs) are currently considered potential therapeutic options for patients with cancer. However, the therapeutic potential of ICIs in human cancer...

Tumor-infiltrating lymphocytes, PD-L1, and MMR-deficiency combined characterization may identify subgroups of rectal cancer patients who would benefit from immunotherapy

IntroductionMismatch repair deficiency, immunological fertility, and PD-L1 expression status are key histopathological and molecular features defining tumor responsiveness to immunotherapy and, eventually, prognosis. These were investigated in a series of locally advanced rectal cancer patients treated with postoperative chemotherapy and radiotherapy. Materials and methodsTumor-infiltrating lymphocyte (TIL) density was assessed in hematoxylin-eosin tissue sections. PD-L1 expression and the expression of MMR proteins (MLH1, PSM2, MSH2, and MSH6) were assessed with immunohistochemistry. Their association with histopathological variables (node involvement and tumor budding) and prognosis was assessed. ResultsThe TIL-density was significantly higher in the invading tumor front and was inversely related to tumor budding and directly with better overall survival (OS) and distant metastasis-free survival (DMFS) (p = 0.02 and 0.02, respectively). Cancer cell PD-L1 expression was related to high TIL-density (p < 0.01) but not to prognosis, although its overexpression defined a trend for poorer OS in patients with high TIL-density. High PD-L1 expression by stroma infiltrating immune cells was linked with better OS and DMFS (p = 0.007 and 0.001, respectively. MMR deficiency was recorded in 26.2 % of cases, and this was linked with higher TIL-density, but not with prognosis. ConclusionsDense intratumoral lymphocytic infiltration relates to a better prognosis in rectal cancer, although it is also linked with PD-L1 expression that may adversely modulate the anti-tumor effects of TILs. This latter subgroup of patients (high TIL-density/high cancer cell PD-L1 expression) could be an additional target for anti-PD-1/PD-L1 immunotherapy, along with the established subgroup of MMR deficient patients.

Abrine, an IDO1 inhibitor, suppresses the immune escape and enhances the immunotherapy of anti-PD-1 antibody in hepatocellular carcinoma.

Indoleamine-2,3-dioxygenase 1 (IDO1) is responsible for tumor immune escape by regulating T cell-associated immune responses and promoting the activation of immunosuppressive. Given the vital role of IDO1 in immune response, further investigation on the regulation of IDO1 in tumors is needed. Herein, we used ELISA kit to detect the interferon-gamma (IFN-γ), Tryptophan (Trp), and kynurenic acid (Kyn) levels; western blot, Flow cytometry, and immunofluorescence assays detected the expression of the proteins; Molecular docking assay, SPR assay and Cellular Thermal Shift Assay (CETSA) were used to detect the interaction between IDO1 and Abrine; nano live label-free system was used to detect the phagocytosis activity; tumor xenografts animal experiments were used to explore the anti-tumor effect of Abrine; flow cytometry detected the immune cells changes. The important immune and inflammatory response cytokine interferon-gamma (IFN-γ) up-regulated the IDO1 expression in cancer cells through the methylation of 6-methyladenosine (m6A) m6A modification of RNA, metabolism of Trp into Kyn, and JAK1/STAT1 signaling pathway, which could be inhibited by IDO1 inhibitor Abrine. CD47 is IFN-γ-stimulated genes (ISGs) and prevents the phagocytosis of macrophages, leading to the cancer immune escape, and this effect could be inhibited by Abrine both in vivo and in vitro. The PD-1/PD-L1 axis is an important immune checkpoint in regulating immune response, overexpression of PD-1 or PD-L1 promotes immune suppression, while in this study Abrine could inhibit the expression of PD-L1 in cancer cells or tumor tissue. The combination treatment of Abrine and anti-PD-1 antibody has a synergistic effect on suppressing the tumor growth through up-regulating CD4+ or CD8+ T cells, down-regulating the Foxp3+ Treg cells, and inhibiting the expression of IDO1, CD47, and PD-L1. Overall, this study reveals that Abrine as an IDO1 inhibitor has an inhibition effect on immune escape and has a synergistic effect with the anti-PD-1 antibody on the treatment of HCC.

Abstract 4438: Bypassing immune evasion in colorectal cancer by integrin inhibition-mediated downregulation of PD-L1

Abstract Integrin receptors have long posed a potentially attractive target for disrupting cancer hallmarks. Promising preliminary findings with integrin inhibition as an adjuvant to chemotherapy have not translated to clinical success. However, the effect of integrin inhibition on tumor-immune cell interactions remains largely unexplored. Further investigation could shed light on a connection between integrin signaling and immune checkpoint expression, opening the path for using integrin inhibitors to sensitize otherwise resistant tumors to immunotherapy. Fluorescently labeled wild-type HCT-116 colorectal cancer cells and TALL-104 T-cells were co-cultured and treated with GLPG0187, a small molecule integrin inhibitor, at various doses. Co-cultures were observed at different time points using fluorescence microscopy and cell counts were quantified using ImageJ software. The co-culture included the cell viability marker ethidium homodimer so the number of viable cells of each type could be determined using colocalization. This assay revealed synergistic cancer cell killing, indicating that integrin inhibition may be sensitizing cancer cells to immune cells. The hypothesized mechanism involves TGF-beta-mediated PD-L1 expression in cancer cells. GLPG0187 has been shown to inhibit the ɑvβ6 integrin receptor, which is responsible for the activation of latent-TGFβ into the active form. By blocking this receptor, there would potentially be a reduction of TGFβ signaling and therefore a reduced expression of PD-L1 leading to an enhanced immune response. To investigate this mechanism, both WT and p53-/- HCT-116 cells were pre-treated with GLPG0187 and subsequently with latent-TGFβ. Western blot analysis demonstrated that the addition of latent-TGFβ increased the expression of PD-L1 in cancer cells. Additionally, a low dose of integrin inhibitor rescued these effects, returning PD-L1 expression back to control levels. However, the higher dose of the drug did not reduce PD-L1 expression. This could potentially be due to off-target effects conflicting with the proposed pathway, however, these findings are still under active investigation. Ongoing proteomic experiments include a larger range of both drug and latent-TGFβ doses. Probing for additional downstream markers of TGFβ and up-stream markers of PD-L1 will help to further elucidate the mechanism. Further co-culture experiments include anti-PD-L1 and anti-PD-1 therapy to investigate the viability of integrin inhibition as an adjuvant to immune checkpoint blockade. Citation Format: William J. MacDonald, Brooke Verschleiser, Lindsey Carlsen, Kelsey E. Huntington, Wafik S. El-Deiry. Bypassing immune evasion in colorectal cancer by integrin inhibition-mediated downregulation of PD-L1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4438.

Abstract 6330: Countering antidrug antibodies to programmed cell death-1 blockade in mouse Pten-null prostate cancer

Abstract Therapeutic biologics have the potential to elicit immune responses that results in the development anti-drug antibodies (ADAs). ADAs that develop after immune checkpoint blockade (ICB) limit their therapeutic efficacy and increase the risk of adverse events. Mitigating ADAs would improve the efficacy of antibody-based therapeutics and identifying reliable biomarkers would allow for rational, evidence-based criteria for tailoring treatments. However, detecting ADAs is challenging and data regarding their biology and means to counter this phenomenon are limited. Here, we examined the development of ADAs to anti-programmed cell death-1 (PD-L1) monoclonal antibodies in a mouse model Pten/Trp53-null (DKO) prostate cancer and countered their negative effect with systemic administration of a JAK1/2 inhibitor. Like human prostate cancer, adding PD-L1 ICB to androgen deprivation therapy has failed to produce a therapeutic effect in DKO cancer model. Our analysis showed that tumor-bearing DKO mice have increased levels of peripheral dendritic cells (pDC) of which &amp;gt;80% exhibited high levels of PD-L1. Given this finding, we developed a system to monitor the development of ADAs against two murine anti-PD-L1 (aPD-L1) monoclonal antibodies clone 80 and 10F9.G2. Pharmacodynamic studies showed that a single-dose administration of the blocking antibodies effectively neutralized PD-L1 in pDC that lasted for four days. Chronic (twice/week) dosing of the aPD-L1 antibodies strongly neutralized PD-L1 in pDCs, but the efficacy waned after 22 days. Using crossover approach, consisting of clone 10F9.G2 dosing for two weeks followed by clone 80, extended PD-L1 blockade in pDCs to 36 days, but did not improve antitumor activity. Blocking STAT3 signaling with the JAK1/2 inhibitor AZD1480 restored PD-L1 blockade in pDCs and was associated with improved reduction of tumor burden. Further analysis showed improved neutralization of PD-L1 in cancer cells and tumor infiltrating immune cells in mice treated with AZD1480. These effects were associated with greater T and NK cell reactivity in tumors. Our study shows that the presence of ADAs is associated with lack of antitumor responses to ant-PD-L1 blockade in a preclinical model of prostate cancer and provides additional data that will improve our understanding of ADAs against immune checkpoint inhibitors. This study also offers an approach to monitor ADA development and a platform to investigate novel approaches to target ADAs. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazutoshi Fujita, Mitsuhisa Nishimoto, Kazuko Sakai, Kazuhiro Yoshimura, Masahiro Nozawa, Scott A. Hammond, Simon J. Dovedi, Barry R. Davies, Kazuto Nishio, Hirotsugu Uemura. Countering antidrug antibodies to programmed cell death-1 blockade in mouse Pten-null prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6330.

A Potential Off-Target Effect of the Wnt/β-Catenin Inhibitor KYA1797K: PD-L1 Binding and Checkpoint Inhibition

Introduction: The quest for small molecule inhibitors of the PD-1/PD-L1 checkpoint continues in parallel to the extensive development of monoclonal antibodies directed against this immune checkpoint. Drug screening strategies are being set up to identify novel PD-L1 inhibitors. Methods: A virtual screening based on molecular docking with the PD-L1 protein dimer has been performed to identify a new binder. Binding of the identified ligand to PD-L1 has been validated experimentally using a microscale thermophoresis (MST) assay. The cellular effect of the compound was evidenced using a fluorescence resonance energy transfer (FRET) assay based on activation of tyrosine phosphatase SHP-2. Results: We have identified the potent Wnt/β-catenin inhibitor KYA1797K as a weak PD-L1 binder. Molecular docking suggested that the compound can bind to the interface of a PD-L1 dimer, with a geometry superimposable to that of the reference PD-L1 inhibitor BMS-202. The atypical 2-thioxo-4-thiazolidinone motif of KYA1797K, derived from the natural product rhodanine, plays a major role in the interaction with PD-L1. Binding of KYA1797K to recombinant hPD-L1 was validated experimentally, using MST. The drug was found to bind modestly but effectively to hPD-L1. The FRET assay confirmed the weak capacity of KYA1797K to interfere with the activation of SHP-2 upon its interaction with human PD-1. Discussion: Collectively, the data show that KYA1797K could function as a weak modulator of the PD-1/PD-L1 checkpoint. This effect may contribute, at least partially, to the reported capacity of the β-catenin inhibitor to downregulate PD-L1 in cancer cells. The work also underlines the interest to further consider the rhodanine moiety as a chemical motif for the design of new PD-L1 binders.

SPOP mutations promote tumor immune escape in endometrial cancer via the IRF1-PD-L1 axis.

Blockade of programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L1) has evolved into one of the most promising immunotherapy strategies for cancer patients. Tumor cells frequently overexpress PD-L1 to evade T cell-mediated immune surveillance. However, the specific genetic alterations that drive aberrant overexpression of PD-L1 in cancer cells remain poorly understood. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in endometrial cancer (EC). Here, we report that SPOP negatively regulates PD-L1 expression at the transcriptional level. Wild-type SPOP binds to IRF1, a primary transcription factor responsible for the inducible expression of PD-L1, and subsequently triggers its ubiquitin- proteasomal degradation to suppress IRF1-mediated transcriptional upregulation of PD-L1. In contrast, EC-associated SPOP mutants lose their capacity to degrade IRF1 but stabilize IRF1, and upregulate PD-L1 expression. EC-associated SPOP mutations accelerate xenograft tumor growth partially by increasing IRF1 and PD-L1 expression. Together, we identify SPOP as a negative regulator of the IRF1-PD-L1 axis and characterize the critical roles of IRF1 and PD-L1 in SPOP mutation-driven tumor immune evasion in EC.

CD47 blockade improves the therapeutic effect of osimertinib in non-small cell lung cancer.

The third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib (OSI) has been approved as the first-line treatment for EGFR-mutant non-small cell lung cancer (NSCLC). This study aims to explore a rational combination strategy for enhancing the OSI efficacy. In this study, OSI induced higher CD47 expression, an important anti-phagocytic immune checkpoint, via the NF-κB pathway in EGFR-mutant NSCLC HCC827 and NCI-H1975 cells. The combination treatment of OSI and the anti-CD47 antibody exhibited dramatically increasing phagocytosis in HCC827 and NCI-H1975 cells, which highly relied on the antibody-dependent cellular phagocytosis effect. Consistently, the enhanced phagocytosis index from combination treatment was reversed in CD47 knockout HCC827 cells. Meanwhile, combining the anti-CD47 antibody significantly augmented the anticancer effect of OSI in HCC827 xenograft mice model. Notably, OSI induced the surface exposure of "eat me" signal calreticulin and reduced the expression of immune-inhibitory receptor PD-L1 in cancer cells, which might contribute to the increased phagocytosis on cancer cells pretreated with OSI. In summary, these findings suggest the multidimensional regulation by OSI and encourage the further exploration of combining anti-CD47 antibody with OSI as a new strategy to enhance the anticancer efficacy in EGFR-mutant NSCLC with CD47 activation induced by OSI.

Docosahexaenoic acid reverses PD-L1-mediated immune suppression by accelerating its ubiquitin-proteasome degradation

PD-L1 interacts with its receptor PD-1 on T cells to negatively regulate T cell function, leading to cancer cell immune escape from the immune surveillance. Therefore, targeting PD-L1 is considered to be an attractive approach for cancer immunotherapy. In this study, we demonstrated for the first time that ω-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) reduced the expression of PD-L1 in cancer cells both in vitro and in vivo. Promotion of PD-L1 ubiquitin-proteasome degradation by DHA resulted in a decrease of PD-L1 expression, leading to reduction of PD-L1 and PD-1 interaction, and reversing PD-L1-mediated immune suppression, which in turn contributed to the inhibitory effect on tumor growth. Furtherly, DHA significantly reduced fatty acid synthase (FASN) expression in cancer cells, which inhibited the palmitoyltransferases DHHC5, promoting the CSN5-dependent PD-L1 degradation. Our present finding uncovered a novel mechanism involved in the anti-cancer activity of DHA, and implicated that DHA holds promising potential to be developed as a novel immune-enhancer for cancer treatment and prevention.

Computational design of PD-L1 small molecule inhibitors for cancer therapy.

Drug repurposing opens new avenues in cancer therapy. Drug repurposing, or finding new uses for existing drugs, can substantially reduce drug discovery time and costs. Cheminformatics, genetics, and systems biology advances enable repositioning drugs. Clinical usage of PD-1/PD-L1 blocking has been approved because of its efficacy in improving prognosis in select groups. The PD-1/PD-L1 axis was considered to represent a mechanism for tumour evasion of host tumour antigen-specific T-cell immunity in early preclinical research. The expression of PD-L1 in cancer cells causes T lymphocytes to become exhausted by transmitting a co-inhibitory signal. A better understanding of how PD-L1 is regulated in cancer cells could lead to new therapeutic options. In this view, the study was aimed to repurpose the existing FDA-approved drugs as a potential PD-L1 inhibitor through e-Pharmacophore modelling, molecular docking and dynamic simulation. e-Pharmacophore screening retrieved 324 FDA-approved medications with the fitness score ≥ 1. The top 10-docked FDA candidates were compared with IN-35 (Clinical trial candidate) for its interaction pattern with critical amino acid residues. Mirabegron and Indacaterol exhibited a greater affinity for PD-L1 with docking scores of - 9.213kcalmol-1 and - 8.023kcalmol-1, respectively. Mirabegron retain interactions at all three major hotspots in the PD-L1 dimer interface similar to IN-35. MM-GBSA analyses indicated that Mirabegron uses less energy to create a more stable complex and retains all of the inhibitor's positive interactions found in clinical trial ligand IN-35. Molecular dynamics simulation analysis of the Mirabegron complex showed a similar pattern of deviation in correlation with IN-35, and it retains the interaction with the active key amino acids throughout the simulation time. Our present study has shown Mirabegron as a powerful inhibitor of PD-L1 expression in cancer cells using a drug-repurposing screen.

Platelets Increase the Expression of PD-L1 in Ovarian Cancer.

Simple SummaryOne-third of patients with ovarian cancer have elevated platelet counts associated with a poor prognosis. We found that platelets increase the expression of immune checkpoint (PD-L1) in ovarian cancer in mice and patients. Reducing platelet counts or inhibiting platelet function reduced the expression of PD-L1 in tumors. We investigated the mechanism of platelet-induced PD-L1 and showed that platelets increase PD-L1 on cancer cells both directly (contact-dependent through NF-κB signaling) and indirectly (contact-independent via TFGβ released from platelets through TFGβR1/Smad signaling). Our results show that platelets dampen the antitumor immune response in the tumor microenvironment. Based on our preclinical results, we speculate that platelet counts might be a predictive biomarker for immunotherapy, i.e., patients with thrombocytosis respond better to anti-PD1/PDL-1 therapy. On the other hand, the use of aspirin or other antiplatelet reagents may impact the effectiveness of immunotherapy. These speculations need to be examined in clinical trials.The interactions between platelets and cancer cells activate platelets and enhance tumor growth. Platelets increase proliferation and epithelial–mesenchymal transition in cancer cells, inhibit anoikis, enhance the extravasation of cancer cells, and protect circulating tumor cells against natural killer cells. Here, we have identified another mechanism by which platelets dampen the immune attack on cancer cells. We found that platelets can blunt the antitumor immune response by increasing the expression of inhibitory immune checkpoint (PD-L1) on ovarian cancer cells in vitro and in vivo. Platelets increased PD-L1 in cancer cells via contact-dependent (through NF-κB signaling) and contact-independent (through TFGβR1/Smad signaling) pathways. Inhibition of NF-κB or TGFβR1 signaling in ovarian cancer cells abrogated platelet-induced PD-L1 expression. Reducing platelet counts or inhibiting platelet functions reduced the expression of PD-L1 in ovarian cancer. On the other hand, an increase in platelet counts increased the expression of PD-L1 in tumor-bearing mice.

SiRNA Nanoparticle Targeting PD-L1 Activates Tumor Immunity and Abrogates Pancreatic Cancer Growth in Humanized Preclinical Model.

Pancreatic cancer is characterized by late detection, frequent drug resistance, and a highly metastatic nature, leading to poor prognosis. Antibody-based immunotherapy showed limited success for pancreatic cancer, partly owing to the low delivery rate of the drug into the tumor. Herein, we describe a poly(lactic-co-glycolic acid;PLGA)-based siRNA nanoparticle targeting PD-L1 (siPD-L1@PLGA). The siPD-L1@PLGA exhibited efficient knockdown of PD-L1 in cancer cells, without affecting the cell viability up to 6 mg/mL. Further, 99.2% of PDAC cells uptake the nanoparticle and successfully blocked the IFN-gamma-mediated PD-L1 induction. Consistently, the siPD-L1@PLGA sensitized cancer cells to antigen-specific immune cells, as exemplified by Ovalbumin-targeting T cells. To evaluate its efficacy in vivo, we adopted a pancreatic PDX model in humanized mice, generated by grafting CD34+ hematopoeitic stem cells onto NSG mice. The siPD-L1@PLGA significantly suppressed pancreatic tumor growth in this model with upregulated IFN-gamma positive CD8 T cells, leading to more apoptotic tumor cells. Multiplex immunofluorescence analysis exhibited comparable immune cell compositions in control and siPD-L1@PLGA-treated tumors. However, we found higher Granzyme B expression in the siPD-L1@PLGA-treated tumors, suggesting higher activity of NK or cytotoxic T cells. Based on these results, we propose the application of siPD-L1@PLGA as an immunotherapeutic agent for pancreatic cancer.

Cancer-associated fibroblast senescence and its relation with tumour-infiltrating lymphocytes and PD-L1 expressions in intrahepatic cholangiocarcinoma.

Caveolin-1 (CAV1) in cancer-associated fibroblasts (CAFs) has pro- or anti-tumourigenic effect depending on the cancer type. However, its effect in intrahepatic carcinoma (ICC) remains unknown. Therefore, this study aimed to investigate the relationship between CAV1 in CAFs and tumour-infiltrating lymphocyte (TIL) numbers or PD-L1 levels in ICC patients. Consecutive ICC patients (n = 158) were enrolled in this study. The levels of CAV1 in CAFs, CD8 + TILs, Foxp3+ TILs and PD-L1 in cancer cells were analysed using immunohistochemistry. Their association with the clinicopathological factors and prognosis were evaluated. The correlation between these factors was evaluated. CAV1 upregulation in CAFs was associated with a poor overall survival (OS) (P < 0.001) and recurrence-free survival (P = 0.008). Clinicopathological factors were associated with high CA19-9 levels (P < 0.001), advanced tumour stage (P = 0.046) and lymph node metastasis (P = 0.004). CAV1 level was positively correlated with Foxp3+ TIL numbers (P = 0.01). There were no significant correlations between CAV1 levels and CD8 + TIL numbers (P = 0.80) and PD-L1 levels (P = 0.97). An increased CD8 + TIL number and decreased Foxp3+ TIL number were associated with an increased OS. In multivariate analysis, positive CAV1 expression in CAFs (P = 0.013) and decreased CD8 + TIL numbers (P = 0.021) were independent poor prognostic factors. Cellular senescence, represented by CAV1 levels, may be a marker of CAFs and a prognostic indicator of ICC through Foxp3+ TIL regulation. CAV1 expression in CAFs can be a therapeutic target for ICC.

Correction: THADA drives Golgi residency and upregulation of PD-L1 in cancer cells and provides promising target for immunotherapy.

Correction: THADA drives Golgi residency and upregulation of PD-L1 in cancer cells and provides promising target for immunotherapy.

Increased PD-1 and EGFR expression levels of T lymphocytes in patients with non-small cell lung cancer.

e21018 Background: Programmed cell death protein 1 (PD-1) and its ligand (PD-L1) are proteins that negatively regulate immune responses. Increased expression of PD-L1 in cancer cells leads to inhibition of the immune response against cancer, and causes cancer development and metastasis. Epidermal growth factor receptor (EGFR) activation upregulates PD-L1 expression in lung cancer cells and contribute to escape from immune response. In this study, we aimed to investigate the circulating T lymphocyte subsets and, PD-1 and EGFR expression levels of these cells in non-small cell lung cancer (NSCLC) progression. Methods: 40 NSCLC patients with 63.8 ± 8.0 mean age and 40 healthy controls with 60.9 ± 14.6 mean age were included in the study. In the peripheral blood samples of the patients taken at the time of diagnosis and controls, CD4+ T helper (Th) subsets (Th1, Th2, Th9, Th17, Th1Th17, CD4+ follicular T [Tfc] and peripheral T cells [Tpcs]), CD8+ cytotoxic T cell (CTL) subsets (CD8+ Tfcs and Tpcs), EGFR and PD-1 expression levels of T cells were determined by flow cytometric analysis. Results: Total lymphocyte ratio was decreased in patients with NSCLC compared to control (11.5 ± 5.2 in patients and 17.6 ± 6.1 in controls, p = 0.001), but there was no significant difference in CD3+ total T, Th and CTLs as well as their subsets (p &lt; 0.05). Increased PD-1 expression level (mean fluorescence intensity-MFI) on total lymphocyte (p = 0.001), CD3+ T (p = 0.001), CD4+ Th (p = 0.001), CTL (p = 0.016), CD4+ Tpc (p = 0.002) and CD8+ Tpc subsets (p = 0.017) were determined in the patients compared with controls. EGFR expressions were also increased on total lymphocyte, CD3+ T, Th and Th2 cells (p = 0.034, p = 0.020, p = 0.030, respectively). 19 NSCLC patients were metastatic, while 21 patients were non-metastatic. There was no significant difference in mean age between metastatic and non-metastatic NSCLC patients. Reduced total lymphocytes and Th cells, increased Th1Th17 cells were found in metastatic NSCLC patients (p = 0.003, p = 0.046 and p = 0.022, respectively). The percentage of EGFR in CD3+ T cell and Th17 cells decreased in metastatic patients. Conclusions: This study showed the increased PD-1 and EGFR expression levels of T lymphocytes in patients with NSCLC. Cancer cells may upregulate PD-1 expression by inducing EGFR activation on lymphocytes to suppress immune responses, leading to programmed cell death of lymphocytes and a decrease in the percentage of these cells in NSCLC. Therapeutic approaches focused on changing EGFR need to be considered for the distribution of T cell subsets and EGFR expressions.