Downregulating PD-L1 Expression Research Articles

Atorvastatin Enhances the Efficacy of Immune Checkpoint Therapy and Suppresses the Cellular and Extracellular Vesicle PD-L1.

According to clinical studies, statins improve the efficacy of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) blockade therapy for breast cancer; however, the underlying mechanisms are unclear. Herein, we showed that atorvastatin (ATO) decreased the content of PD-L1 in extracellular vesicles (EVs) by reducing cellular PD-L1 expression and inhibiting EV secretion in breast cancer cells, thereby enhancing the efficacy of anti-PD-L1 therapy. ATO reduced EV secretion by regulating the Rab proteins involved in EV biogenesis and secretion. ATO-mediated inhibition of the Ras-activated MAPK signaling pathway downregulated PD-L1 expression. In addition, ATO strongly promoted antitumor efficacy by inducing T cell-mediated tumor destruction when combined with an anti-PD-L1 antibody. Moreover, suppression of EV PD-L1 by ATO improved the reactivity of anti-PD-L1 therapy by enhancing T-cell activity in draining lymph nodes of EMT6-bearing immunocompetent mice. Therefore, ATO is a potential therapeutic drug that improves antitumor immunity by inhibiting EV PD-L1, particularly in response to immune escape during cancer.

Multicomponent siRNA/miRNA-loaded modified mesoporous silica nanoparticles targeted bladder cancer for a highly effective combination therapy.

Bladder cancer is one of the concerning urological malignant diseases in the world, which has a clinical need for effective targeted therapy. The development of nanotechnology-based gene delivery to bladder tumor sites is an effective strategy for targeted cancer therapy with low/no toxicity. With this view, in the present work, the mesoporous silica nanoparticles (MSNs) modified with c(RGDfK)-PLGA-PEG [c(RGDfK)-MSN NPs] were constructed for co-delivery of miR-34a and siPD-L1 within bladder cancer cells and tissues. Our findings showed that miR-34a is downregulated while PD-L1 is up-regulated in cell lines and animal studies. This nano-carrier is biocompatible in the serum environment and effectively protects miR-34a and siPD-L1 against serum degradation. However, we showed that c(RGDfK)-MSN NPs could simultaneously downregulate PD-L1 expression and up-regulate miR-34a in the T24 cells and T24 mice model and enhance anti-tumor effects both in vivo and in vitro. In conclusion, these findings presented new suggestions for improving targeted therapeutic strategies with specified molecular objectives for bladder cancer treatment.

Osimertinib Improves the Immune Microenvironment of Lung Cancer by Downregulating PD-L1 Expression of Vascular Endothelial Cells and Enhances the Antitumor Effect of Bevacizumab.

Objective To investigate the effect and mechanism of osimertinib combined with bevacizumab on lung cancer through cell and transplanted tumor animal experiments and to provide theoretical basis for further clinical trials. Methods Immunohistochemistry was used to detect the expression of PD-L1 in tumor vessels of nonmetastatic lung adenocarcinoma and metastatic lung adenocarcinoma. At the same time, the expression of CD8 and FoxP3 in tumor tissue was detected. qRT-PCR was used to detect the effect of osimertinib on PD-L1 expression in HUVECs. The expression levels of p-Akt and p-ERK in HUVECs treated with osimertinib were analyzed by Western blot. AKT was blocked by AKT specific inhibitor Ly294002 to analyze the expression of PD-L1 in HUVECs. An animal model of transplanted tumor was constructed to analyze whether osimertinib could enhance the antitumor effect of bevacizumab. Results PD-L1 was highly expressed in vascular endothelial cells of metastatic lung cancer. FoxP3 was highly expressed in metastatic lung adenocarcinoma, while CD8 expression was low. Osimertinib inhibits PD-L1 expression in endothelial cells. Mechanism studies have shown that osimertinib inhibits PD-L1 expression in endothelial cells through the AKT/ERK pathway. Osimertinib inhibited endothelial cell PD-L1 expression, increased CD8+T cell infiltration, inhibited tumor growth, and enhanced the tumor effect of bevacizumab. Conclusion Osimertinib can significantly increase the killing ability of bevacizumab against tumor. Osimertinib can improve the tumor microenvironment and enhance the antitumor effect of bevacizumab by reducing the expression of PD-L1 in tumor blood vessels.

Over-Expression of GUSB Leads to Primary Resistance of Anti-PD1 Therapy in Hepatocellular Carcinoma.

Immunotherapy treatments, particularly immune checkpoint blockade, can result in benefits in clinical settings. But many pre-clinical and clinical studies have shown that resistance to anti-PD1 therapy frequently occurs, leading to tumor recurrence and treatment failure, including in patients with hepatocellular carcinoma (HCC). In this study, 10 patients with HCC were remedied with anti-PD1, and pre-treatment biopsy samples were sequenced for 289 nanostring panel RNA to compare responsive and non-responsive tumors to identify possible pretreatment biomarkers or targets of anti-PD1 therapeutic responses. Fortunately, the expression of β-Glucuronidase (GUSB) in the non-responding tumors was found to be remarkably higher than that in responding tumors. Results of the cell counting kit 8 (CCK8), 5-ethynyl-2’-deoxyuridine (EdU), transwell, wound healing test, and flow cytometry showed that GUSB facilitated proliferation, invasion, as well as migration of human HCC cells and downregulated PD-L1 expression by promoting miR-513a-5p. Additionally, as a GUSB inhibitor, amoxapine can reduce the progression of human HCC cells, and was an effective treatment for HCC and improved the sensitivity of anti-PD1 therapy. In summary, this study reveals that increased GUSB downregulates PD-L1 expression by promoting miR-513a-5p, leading to primary resistance to anti-PD1 treatment in HCC, and amoxapine enhances the sensitivity of anti-PD1 therapy by inhibiting GUSB, providing a new strategy and method for improving the efficacy of anti-PD1 therapy and bringing new prospects for therapy of HCC.

Combination treatment with 17β-estradiol and anti-PD-L1 suppresses MC38 tumor growth by reducing PD-L1 expression and enhancing M1 macrophage population in MC38 colon tumor model

17β-estradiol (E2) is known to have a protective effect in colorectal cancer (CRC); thus, E2 may be effective for cancer immunotherapy in CRC. The aim of this study is to evaluate the effect of combination therapy with E2 and anti-programmed cell death receptor-1 ligand (PD-L1) antibodies, and the effects of sex and estrogen on colon tumor growth, PD-L1 expression, and tumor-associated cell populations in an MC38 colon tumor model. Male mice showed increased MC38 colon tumor growth and PD-L1 expression in tumor sections as well as higher proportion of cancer-associated fibroblasts (CD45−CD31−CD140a+), PD-L1-expressing tumor cells (CD45−CD274+) and tumor-associated macrophages (TAMs) (CD11b+F4/80+CD274+) compared to female mice. E2 treatment prior to MC38 injection significantly reduced these phenomena in male mice. Furthermore, co-treatment with E2 and anti-PD-L1 antibodies significantly inhibited MC38 tumor growth and reduced PD-L1-expressing cells in male mice compared to treatment with either E2 or anti-PD-L1 antibodies alone. Combination treatment with E2 and anti-PD-L1 decreased TAM population (CD11b+F4/80+) in the tumor mass while increasing M1 TMAs (CD11b+F4/80+CD86+). These results suggest that estrogen inhibits MC38 tumor growth by downregulating PD-L1 expression and regulating tumor-associated cell populations. Furthermore, estrogen boosted the effect of anti-PD-L1 antibody in the MC38 tumor model.

Dual drugs decorated bacteria irradiate deep hypoxic tumor and arouse strong immune responses

Intratumoral environment as a hypoxic, non-inflamed “cold” state is difficult for many agents to accumulate and activate the immune system. Intrinsically, facultative anaerobic Salmonella VNP20009 target the tumor hypoxic areas, invade into tumor cells and exhibit an immune effect. Here we engineer the bacteria by decorating their surface with newly synthesized heptamethine cyanine dyes NHS–N782 and JQ-1 derivatives to obtain the biohybrid agent N–V-J, leading to the deep tumor targeted photothermal therapy and magnified immunotherapy. Due to the mitochondrial targeting capacity of NHS–N782, N–V-J becomes susceptive to the temperature rise when reaching tumors. This synergistic strategy promotes the systemic immunity by creating an inflamed “hot” tumor state from three different dimensions, which include the inherent immunogenicity of bacteria, the near-infrared laser triggered tumor antigens and the downregulation of PD-L1 expression. All these approaches result in effective and long-lasting T cell immune responses to prevent local and distant tumors for extended time. Leveraging the attenuated bacteria to transport dual drugs to the tumor tissues for self-synthetic vaccines provides a novel paradigm to enhance the bacteria-mediated cancer immunotherapy.

Effects of Cardamonin on PD‐1/PD‐L1 Checkpoint in Triple‐Negative Breast Cancer

Breast cancer is the most common type of cancer in women and the second leading cause of death. Trible‐negative breast cancer (TNBC) accounts for about 10% of all breast cancer. Unlike other forms of breast cancer, TNBC does not express estrogen, progesterone, or HER2 receptors. These cancer cells instead employ mechanisms to escape the immune system. One such mechanism involves the upregulation of the expression of PD‐L1, a ligand encoded by the CD274 gene. Because cancer cells tend to produce higher levels of PD‐L1 compared to normal cells, therapies that inhibit PD‐L1 may be helpful. Flavonoids, polyphenols found abundantly in fruits and vegetables, have already been shown to be effective antioxidants, anti‐inflammatories, and anticancer. Cardamonin, an aromatic enone within the flavonoid family, has been shown to display an array of pharmacological activities. The objective of this work was to investigate the ability of Cardamonin in decreasing the expression of PD‐L1 in MDA‐MB‐231 (Caucasian) and MDA‐MB‐468 (African American) TNBC cell lines which are genetically distinct. The methodology included cytotoxicity assays, human B7H1/PD‐L1 ELISA, and Real Time‐Polymerase Chain Reaction (RT‐PCR) assays. The results obtained showed that cardamonin treatment caused a dose‐dependent decrease in cell viability in both cell lines in concentrations ranging from 3.12 µM to 200µM. ELISA data showed low levels of PD‐L1 expression in both cell lines. Furthermore, RT‐PCR assays demonstrated that cardamonin downregulated PD‐L1 expression in MDA‐MB‐231cells in the presence or absence of IFN‐γ, showing a more effective effect after the cells were stimulated. In MDA‐MB‐468 cells, cardamonin had an opposite effect, upregulating the expression of PD‐L1. The data show that cardamonin may alter the production of PD‐L1 at the transcription level in MDA‐MB‐231 TNBC cells, but not in MDA‐MB‐468 cells, corroborating with literature showing differences in the tumor microenvironments between African Americans and non‐African Americans. In conclusion, cardamonin may have the therapeutic potential to decrease the levels of PD‐L1 in the tumor microenvironment combating the cancer cells’ effectiveness of evading the immune system.

Histone Methyltransferase SETDB1 Promotes Immune Evasion in Colorectal Cancer via FOSB-Mediated Downregulation of MicroRNA-22 through BATF3/PD-L1 Pathway.

Tumors may develop a variety of immune evasion mechanisms during the progression of colorectal cancer (CRC). Here, we intended to explore the mechanism of histone methyltransferase SETDB1 in immune evasion in CRC. The expression of SETDB1, microRNA-22 (miR-22), BATF3, PD-L1, and FOSB in CRC tissues and cells was determined with their interactions analyzed also. Gain-of-function and loss-of-function approaches were employed to evaluate the effects of the SETDB1/FOSB/miR-22/BATF3/PD-L1 axis on T cell function, immune cell infiltration, and tumorigenesis. Aberrant high SETDB1 expression in CRC was positively associated with PD-L1 expression. SETDB1 negatively regulated miR-22 expression by downregulating FOSB expression, while miR-22 downregulated PD-L1 expression via targeting BATF3. Furthermore, SETDB1 silencing promoted the T cell-mediated cytotoxicity to tumor cells via the FOSB/miR-22/BATF3/PD-L1 axis and hindered CRC tumor growth in mice while leading to decreased immune cell infiltration. Taken together, SETDB1 could activate the BATF3/PD-L1 axis by inhibiting FOSB-mediated miR-22 and promote immune evasion in CRC, which provides a better understanding of the mechanisms underlying immune evasion in CRC.

Engineered a dual-targeting biomimetic nanomedicine for pancreatic cancer chemoimmunotherapy

The therapeutic effect of chemotherapeutics such as gemcitabine against pancreatic cancer is considerably attenuated by immune-suppressive tumor microenvironment. Improvement of chemotherapeutic efficacy by targeting tumor-associated macrophage and reprograming tumor microenvironment to enhance their efficacy may become a promising strategy. To this end, we developed a biomimetic dual-targeting nanomedicine (PG@KMCM) where gemcitabine-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles are coated with a layer of bioengineered cancer cell membrane that stably expresses peptides targeting M2-like macrophages (M2pep) while reserving tumor-associated antigens (TAAs). The PG@KMCM nanomedicine enables the simultaneous targeted delivery of gemcitabine to pancreatic tumor sites and TAMs to potentiate its therapeutic effects. Furthermore, the combination of an immune checkpoint inhibitor (PD-L1 antibody) with PG@KMCM synergistically enhanced the anti-tumoral effect by reprogramming the immune-suppressive tumor microenvironment, including the elimination of PD-L1-positive macrophages and the downregulation of PD-L1 expression. Our study proved dual-targeting PG@KMCM nanomedicine in combination with PD-L1 immune checkpoint inhibitor therapy is able to effectively reprogram the tumor microenvironment and kill pancreatic cancer cells to enhance overall therapeutic potential.Graphical

Combination of Tipifarnib and Sunitinib Overcomes Renal Cell Carcinoma Resistance to Tyrosine Kinase Inhibitors via Tumor-Derived Exosome and T Cell Modulation

Simple SummaryMetastatic renal cell carcinoma continues to have a poor prognosis. Chemotherapies and immuno-oncologic therapies have garnered increasing importance in cancer therapy, with improvements in patient care and survival. However, a large proportion of patients present with tumors resistant to these treatments. Exosomes are small extracellular vesicles secreted by all nucleated cells that have proven to be key actors in this resistance. Exosomes carry bioactive oncogenic cargos that reprogram target cells to promote tumor growth, migration, metastasis, immune evasion, and chemotherapy resistance. Tipifarnib, in combination with standard therapy, decreased tumor growth in the setting of chemotherapeutic resistance through an exosome-mediated mechanism. After using a qNANO IZON system to compare tumor-derived exosomes collected from untreated and tipifarnib-treated cells, all cancerous cell lines displayed a reduction of vesicle concentration. Tipifarnib also directly inhibited PD-L1 protein expression in chemo-sensitive cell lines and resistant cell lines.Background: Tyrosine kinase inhibitors (TKI) were initially demonstrated as an efficacious treatment for renal cell carcinoma (RCC). However, after a median treatment length of 14 months, a vast majority of patients develop resistance. This study analyzed a combination therapy of tipifarnib (Tipi) + sunitinib that targeted exosome-conferred drug resistance. Methods: 786-O, 786-O-SR (sunitinib resistant), A498, A498-SR, Caki-2, Caki-2-SR, and 293T cells were cultured. Exosomes were collected using differential ultracentrifugation. Cell proliferation, Jurkat T cell immune assay, and immunoblot analysis were used for downstream analysis. Results: SR exosomes treatment displayed a cytotoxic effect on immune cells. This cytotoxic effect was associated with increased expression of PD-L1 on SR exosomes when compared to sunitinib-sensitive (SS) exosomes. Additionally, Tipi treatment downregulated PD-L1 expression on exosomes derived from SR cell lines. Tipi’s ability to downregulate PD-L1 in exosomes has a significant application within patients. Exosomes collected from patients with RCC showed increased PD-L1 expression over subjects without RCC. Next, exosome concentrations were then compared after Tipi treatment, with all SS cell lines displaying an even greater reduction. On immunoblot assay, 293T cells showed a dose-dependent increase in Alix with no change in either nSMase or Rab27a. Conversely, all the SS and SR cell lines displayed a decrease in all three markers. After a cell proliferation employed a 48-h treatment on all SS and SR cell lines, the drug combination displayed synergistic ability to decrease tumor growth. Conclusions: Tipifarnib attenuates both the exosome endosomal sorting complex required for endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways, thereby blocking exosome biogenesis and secretion as well as downregulating PD-L1 on SS and SR cells.

Enhancing the anti-leukemia immunity of acute lymphocytic leukemia-derived exosome-based vaccine by downregulation of PD-L1 expression.

Cell-released nanovesicles can induce anti-leukemia immunity. Leukemia cell-derived exosomes (LEXs) are promising anti-tumor vaccine components for cancer immunotherapy. Nonetheless, LEX-based vaccines show modest potency in vivo, likely due to the presence of immunosuppressive PD-L1 proteins in the exosomes. We hypothesized that targeting exosomal PD-L1 could optimize LEX-based vaccines. To test this hypothesis, we compared the capacity of exosomes derived from PD-L1-silenced acute lymphocytic leukemia-derived leukemia cells (LEXPD-L1si) and non-modified exosomes to induce anti-leukemia immunity. Lentivirus-mediated PD-L1 shRNA was used to downregulate PD-L1 expression in parental leukemia cells and LEXs. LEXPD-L1si were characterized by electron microscopy, Western blotting, nanoparticle tracking analysis and flow cytometry, and their anti-leukemia immune effects were tested on immune cells and in animal models. In the present study, lentivirus-mediated PD-L1 shRNA successfully downregulated PD-L1 expression in parental leukemia cells and in LEXs. LEXPD-L1si induced better DC maturation and subsequently enhanced T cell activation, as compared with non-modified LEXs. Consistently, immunization with LEXPD-L1si induced greater T cell proliferation and Th1 cytokine release. LEXPD-L1si was a more potent inducer of antigen-specific cytotoxic lymphocyte (CTL) response. Finally, we vaccinated DBA/2 mice with exosome formulations to test their ability to induce both protective and therapeutic anti-tumor CTL responses in vivo. Vaccination with LEXPD-L1si strongly inhibited tumor growth and prolonged survival of immunized mice. Downregulation of exosomal PD-L1 expression in LEXs effectively induces more potent anti-leukemia immunity. Therefore, our strategy for optimizing LEX-based vaccine has a potential application in leukemia immunotherapy.

INCREASE IN MK2 ACTIVITY IN CROHN’S DISEASE: ROLE IN THE INFLAMMATORY ACTIVATION OF FIBROBLASTS

Abstract INTRODUCTION AND HYPOTHESIS Mesenchymal cells known as myo-/fibroblasts (MFs) are critical immunosuppressors under gut mucosal homeostasis. Expression of immune checkpoint PD-L1 by MFs plays a key role in the control of T cell inflammatory responses. In Crohn’s disease (CD), MFs switch their activity from immunosuppressive to pro-inflammatory, where they are also known as Inflammatory Fibroblasts. However, the mechanisms responsible for these pathological changes in MF activity are unknown. Map-kinase-activated protein kinase 2 (MK2) is a major regulator of inflammation in the gut. MK2 is downstream of p38 signaling, it evokes a sub-pathway that directly regulates the production of key inflammatory cytokines implicated in CD (such as TNF-α, IL-1, and IL-6). Thus, we hypothesized that activation of MK2 signaling is critical to the pathological changes in MFs during the immunopathogenesis of CD. METHODS Human normal and CD tissues and derived MFs, as well as animal models relevant to CD, were used in this study. MF signaling/activity was analyzed using RNAseq, qRT-PCR, western blot (WB), cytokine/chemokine multiplex arrays, and confocal microscopy. RESULTS In situ analysis demonstrated an increase in MK2 activity within the inflamed compared to the non-inflamed CD and healthy control intestinal tissues, which was confirmed by WB and multiplex signaling array analysis. In situ increase in MK2 activity in CD intestinal mucosa was greatly associated with mesenchymal stromal cells that bear a “myofibroblast” phenotype (positive for α-SMA expression). An increase in MK2 activity was also observed in primary MF cultures isolated from CD (CD-MFs) when compared to normal (N-) MFs. MK2 activity within CD-MFs was also associated with a significant decrease in the expression of the immunosuppressive checkpoint PD-L1 and an increase in the expression of inflammatory CCL2 and IL-6. Inhibition of MK2 activity within CD-MFs through using the MK2-specific inhibitor PF-3644022 (10 μM) reversed the inflammatory activity of MFs. Remarkably, we observed a differential role of p38 and MK2 in the regulation of PD-L1 expression in MFs: while p38 was required for basal expression of PD-L1, activation of MK2 downregulates PD-L1 expression. These data indicate a unique role of MK2 activation in pathological reprogramming of MFs in CD. Use of MK2 inhibitor in a therapeutic modality in chronic DSS and IL-10 KO murine models of CD also significantly reduce MF-linked inflammatory responses in vivo. CONCLUSION Our data suggest that an increase in MK2 activity in CD is critical to the reprogramming of the MF from immunosuppressive toward pathological Inflammatory fibroblasts. Targeting MK2 activity within MFs could be a desirable strategy for improving the efficacy of current IBD therapeutic approaches.

Metformin Downregulates PD-L1 Expression in Esophageal Squamous Cell Catrcinoma by Inhibiting IL-6 Signaling Pathway.

PurposeTo characterize the mechanism by which metformin inhibits PD-L1 expression in esophageal squamous cell carcinoma (ESCC) and to evaluate the effect of metformin on the antitumor immune response.MethodsThe Cancer Genome Atlas (TCGA) database was used to analyze the correlations between IL-6 and prognosis and between IL-6 and PD-L1 gene expression in esophageal cancer. Reverse transcription-quantitative polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence were used to study the mechanism by which metformin affects PD-L1 expression. Additionally, T cell function was assessed in a coculture system containing ESCC cells and peripheral blood mononuclear cells (PBMCs) treated with metformin or IL-6. In an in vivo assay, we used a model established with NPIdKO™ mice, which have a reconstituted immune system generated by transplanting PBMCs through intravenous injection, to evaluate the effect of metformin on tumors.ResultsThe TCGA esophageal cancer data showed that IL-6 expression was positively correlated with PD-L1 expression and that patients with high IL-6 expression had a significantly lower overall survival rate than patients with low IL-6 expression. PD-L1 expression in ESCC cell lines was significantly inhibited by metformin via the IL-6/JAK2/STAT3 signaling pathway but was not correlated with the canonical AMPK pathway. In the coculture system, the metformin pretreatment group showed higher T cell activation and better T cell killing function than the control group. Animal experiments confirmed that metformin downregulated PD-L1 expression and that combination treatment with metformin and PD-1 inhibitors synergistically enhanced the antitumor response.ConclusionsMetformin downregulated PD-L1 expression by blocking the IL-6/JAK2/STAT3 signaling pathway in ESCC, which enhanced the antitumor immune response.

Colorectal Cancer Progression Is Potently Reduced by a Glucose-Free, High-Protein Diet: Comparison to Anti-EGFR Therapy.

Simple SummaryTo study the interplay between nutrition and intestinal metabolism in the context of colitis-driven colorectal carcinoma (CRC), we here investigated a nutritional therapy strategy in the presence or absence of EGFR-directed antibody therapy in mice to treat established colitis-driven CRCs in vivo. After CRC development, mice were fed a control diet or an isoenergetic glucose-free high-protein (GFHP) diet in the presence or absence of EGFR-directed antibody therapy. The GFHP diet was accompanied by a metabolic shift of the mice towards lower glycolysis activity. Both, GFHP diet or anti-EGFR antibody treatment, improved tumor differentiation and anti-tumor immune response, resulting in an efficient reduction of colonic tumor burden.To enable rapid proliferation, colorectal tumor cells up-regulate epidermal growth factor receptor (EGFR) signaling and aerobic glycolysis, resulting in substantial lactate release into the tumor microenvironment and impaired anti-tumor immune responses. We hypothesized that a nutritional intervention designed to reduce aerobic glycolysis may boost the EGFR-directed antibody (Ab)-based therapy of pre-existing colitis-driven colorectal carcinoma (CRC). CRC development was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) administration to C57BL/6 mice. AOM/DSS-treated mice were fed a glucose-free, high-protein diet (GFHPD) or an isoenergetic control diet (CD) in the presence or absence of an i.p. injection of an anti-EGFR mIgG2a or respective controls. AOM/DSS-treated mice on a GFHPD displayed a reduced systemic glucose metabolism associated with reduced oxidative phosphorylation (OXPHOS) complex IV expression and diminished tumor loads. Comparable but not additive to an anti-EGFR-Ab therapy, the GFHPD was accompanied by enhanced tumoral goblet cell differentiation and decreased colonic PD-L1 and splenic CD3ε, as well as PD-1 immune checkpoint expression. In vitro, glucose-free, high-amino acid culture conditions reduced proliferation but improved goblet cell differentiation of murine and human CRC cell lines MC-38 and HT29-MTX in combination with down-regulation of PD-L1 expression. We here found GFHPD to systemically dampen glycolysis activity, thereby reducing CRC progression with a similar efficacy to EGFR-directed antibody therapy.

Combination of epigenetic regulation with gene therapy-mediated immune checkpoint blockade induces anti-tumour effects and immune response in vivo

Immunotherapy has become a powerful cancer treatment, but only a small fraction of patients have achieved durable benefits due to the immune escape mechanism. In this study, epigenetic regulation is combined with gene therapy-mediated immune checkpoint blockade to relieve this immune escape mechanism. PPD (i.e., mPEG-b-PLG/PEI-RT3/DNA) is developed to mediate plasmid-encoding shPD-L1 delivery by introducing multiple interactions (i.e., electrostatic, hydrogen bonding, and hydrophobic interactions) and polyproline II (PPII)-helix conformation, which downregulates PD-L1 expression on tumour cells to relieve the immunosuppression of T cells. Zebularine (abbreviated as Zeb), a DNA methyltransferase inhibitor (DNMTi), is used for the epigenetic regulation of the tumour immune microenvironment, thus inducing DC maturation and MHC I molecule expression to enhance antigen presentation. PPD plus Zeb combination therapy initiates a systemic anti-tumour immune response and effectively prevents tumour relapse and metastasis by generating durable immune memory. This strategy provides a scheme for tumour treatment and the inhibition of relapse and metastasis.

Distinct roles but cooperative effect of TLR3/9 agonists and PD-1 blockade in converting the immunotolerant microenvironment of irreversible electroporation-ablated tumors.

Irreversible electroporation (IRE) is a new cancer ablation technology, but methods to improve IRE-induced therapeutic immunity are only beginning to be investigated. We developed a mouse model bearing large primary (300 mm3) and medium distant (100 mm3) EG7 lymphomas engineered to express ovalbumin (OVA) as a nominal tumor antigen. We established experimental protocols including IRE alone and IRE combined with Toll-like receptor (TLR)3/9 agonists (poly I:C/CpG) (IRE + pIC/CpG), PD-1 blockade (IRE + PD-1 blockade), or both (IRE + Combo) to investigate therapeutic effects on primary and distant EG7 tumors and conversion-promoting effects on the immunotolerant tumor microenvironment (TME). We demonstrated that IRE alone simulated very weak OVA-specific CD8+ T cell responses and did not inhibit primary tumor growth. IRE + pIC/CpG synergistically stimulated more efficient OVA-specific CD8+ T cell responses and primary tumor growth inhibition than IRE + PD-1 blockade. IRE + pIC/CpG played a major role in the modulation of immune cell profiles but a minor role in the downregulation of PD-L1 expression in the TME and vice versa for IRE + PD-1 blockade. IRE + Combo cooperatively induced potent OVA-specific CD8+ T cell immunity and rescued exhausted intratumoral CD8+ T cells, leading to eradication of not only primary tumors but also untreated concomitant distant tumors and lung metastases. IRE + Combo efficiently modulated immune cell profiles, as evidenced by reductions in immunotolerant type-2 (M2) macrophages, myeloid-derived suppressor-cells, plasmacytoid dendritic cells, and regulatory T cells and by increases in immunogenic M1 macrophages, CD169+ macrophages, type-1 conventional dendritic cells, and CD8+ T cells, leading to conversion of immunotolerance in not only primary TMEs but also untreated distant TMEs. IRE + Combo also showed effective therapeutic effects in two breast cancer models. Therefore, our results suggest that IRE + Combo is a promising strategy to improve IRE ablation therapy in cancer.

Nano-Coated si-SNHG14 Regulated PD-L1 Expression and Decreased Epithelial-Mesenchymal Transition in Nasopharyngeal Carcinoma Cells.

To investigate the expression characteristics of long non-coding RNA SNHG14 in nasopharyngeal carcinoma (NPC) and its effects on epithelial-mesenchymal transition and development of nano-coated si-SNHG14 as an anti-tumor agent. The SNHG14 expression in cancerous and adjacent non-cancerous tissues was monitored using reverse transcriptionpolymerase chain reaction (RT-PCR). Gain- and loss-of-function experiments tested the regulation of SNHG14, miR- 5590-3p, and ZEB1 on PD-L1. The binding association between the above three factors was verified using bioinformatics analysis. EMT-related E-cadherin, N-cadherin, and Vimentin were tested using Western blot. Animal experiments in nude mice verified the function of SNHG14 in the EMT of NPC in vivo. The nano-coated si-SNHG14 was developed as an anti-tumor agent and was verified NPC cell in vitro. SNHG14 was upregulated in NPC tissues. Knocking down SNHG14 markedly inhibited the EMT of NPC. Additionally, the expression of ZEB1 was positively related to that of the SNHG14, while it was inversely correlated with that of miR-5590-3p. Moreover, ZEB1 transcription upregulated PD-L1 and promoted the EMT, while SNHG14 could accelerate the EMT of NPC in vivo by regulating the PD-1 and PD-L1. SNHG14-miR-5590- 3p-ZEB1 positively regulated PD-L1 and facilitate the EMT of NPC. Nano-coated si-SNHG14 significantly downregulated PD-L1 expression and decreased EMT.

Engineering Endogenous Tumor-Associated Macrophage-Targeted Biomimetic Nano-RBC to Reprogram Tumor Immunosuppressive Microenvironment for Enhanced Chemo-Immunotherapy.

Immunotherapy has shown encouraging results in various cancers, but the response rates are relatively low due to the complex tumor immunosuppressive microenvironment (TIME). The presence of tumor-associated macrophages (TAMs) and tumor hypoxia correlates significantly with potent immunosuppressive activity. Here, a hemoglobin-poly(ε-caprolactone) (Hb-PCL) conjugate self-assembled biomimetic nano red blood cell (nano-RBC) system (V(Hb)) is engineered to deliver chemotherapeutic doxorubicin (DOX) and oxygen for reprogramming TIME. The Hb moiety of V(Hb)@DOX can bind to endogenous plasma haptoglobin (Hp) and specifically target the M2-type TAMs via the CD163 surface receptor, and effectively kill the cells. In addition, the O2 released by the Hb alleviates tumor hypoxia, which further augments the antitumor immune response by recruiting fewer M2-type macrophages. TAM-targeting depletion and hypoxia alleviation synergistically reprogram the TIME, which concurrently downregulate PD-L1 expression of tumor cells, decrease the levels of immunosuppressive cytokines such as IL-10 and TGF-β, elevate the immunostimulatory IFN-γ, enhance cytotoxic T lymphocyte (CTL) response, and boost a strong memory response. The ensuing TAM-targeted chemo-immunotherapeutic effects markedly inhibit tumor metastasis and recurrence. Taken together, the engineered endogenous TAM-targeted biomimetic nano-RBC system is a highly promising tool to reprogram TIME for cancer chemo-immunotherapy.

Histone deacetylase 2 knockout suppresses immune escape of triple-negative breast cancer cells via downregulating PD-L1 expression

The PD-L1 overexpression is an important event of immune escape and metastasis in triple-negative breast cancer (TNBC), but the molecular mechanism remains to be determined. Interferon gamma (IFNγ) represents a major driving force behind PD-L1 expression in tumor microenvironment, and histone deacetylase 2 (HDAC2) is required for IFN signaling. Here, we investigated the regulation of HDAC2 on the IFNγ-induced PD-L1 expression in TNBC cells. We found the HDAC2 and PD-L1 expression in TNBC was significantly higher than that in non-TNBC, and HDAC2 was positively correlated with PD-L1 expression. HDAC2 promoted PD-L1 induction by upregulating the phosphorylation of JAK1, JAK2, and STAT1, as well as the translocation of STAT1 to the nucleus and the recruitment of STAT1 to the PD-L1 promoter. Meanwhile, HDAC2 was recruited to the PD-L1 promoter by STAT1, and HDAC2 knockout compromised IFNγ-induced upregulation of H3K27, H3K9 acetylation, and the BRD4 recruitment in PD-L1 promoter. In addition, significant inhibition of proliferation, colony formation, migration, and cell cycle of TNBC cells were observed following knockout of HDAC2 in vitro. Furthermore, HDAC2 knockout reduced IFNγ-induced PD-L1 expression, lymphocyte infiltration, and retarded tumor growth and metastasis in the breast cancer mouse models. This study may provide evidence that HDAC2 promotes IFNγ-induced PD-L1 expression, suggesting a way for enhanced antitumor immunity when targeting the HDAC2 in TNBC.

MM-129 as a Novel Inhibitor Targeting PI3K/AKT/mTOR and PD-L1 in Colorectal Cancer.

Simple SummaryMM-129 (1,2,4-triazine derivative) is a novel promising drug candidate against colon cancer. It has the ability to inhibit intracellular pathways promoting tumorigenesis with a simultaneous reduction of PD-L1 expression, a key element of the cancer immune escape axis. MM-129 may also act as a chemosensitizer, overcoming chemoresistance against 5-FU, the first-line agent in the chemother-apy of colon cancer. Our results significantly expand knowledge and help better understand the process of tumorigenesis, the intracellular pathways involved, and the mutual interactions of in-dividual proteins, and create the possibility of their pharmacological blockade. There is a real chance that the obtained results and the conclusions drawn on their basis will help in the development of a new, effective therapy, which could be an attractive alternative to the already existing methods of colon cancer treatment.Background and aims: The purpose of the present study was to examine the pharmacodynamics features of MM-129 (1,2,4-triazine derivative) as a novel promising drug candidate against colon cancer. Methods: MM-129 was assessed for antitumor activity through an in vivo study on Cby.Cg-Foxn1nu/cmdb mice. The mechanistic studies investigated cellular affinity of a new 1,2,4-triazine derivative by measuring levels of intracellular/extracellular signal molecules participating in tumorigenesis. Results: The results revealed that MM-129 significantly reduced tumor growth in mice challenged with DLD-1 and HT-29 cells. It exerted the ability to inhibit intracellular molecules promoting tumorigenesis and inducing cell cycle arrest, like Akt, mTOR, and CDK2. Simultaneously, it was able to downregulate PD-L1 expression, which involves immunological self-tolerance. Combined administration of MM-129 and 5-fluorouracil (5-FU) additionally amplified these effects, which were manifest as an increase population of cells in the G0/G1 phase. Conclusions: A novel 1,2,4-triazine derivative with a dual mechanism of antitumor activity—MM-129, may act as a chemosensitizer, overcoming chemoresistance against 5-FU, the first-line agent in the chemotherapy of colon cancer.