Suppression Of Phenotype Research Articles

LINC00514 facilitates cell proliferation, migration, invasion, and epithelial-mesenchymal transition in non-small cell lung cancer by acting on the Wnt/β-catenin signaling pathway

ABSTRACT The long non-coding RNA (lncRNA) LINC00514 was identified to play an essential oncogenic function in different human cancers, but its effects in non-small cell lung cancer (NSCLC) are yet to be elucidated. In this study, we evaluated the function of LINC00514 in NSCLC. LINC00514 expression and prognosis in NSCLC were analyzed using qRT-PCR and online bioinformatic tools. The bioeffects of LINC0514 in NSCLC cells were examined using cell counting kit-8, colony formation, and transwell assays. Western blotting was used to measure the expression of the target proteins. The LINC00514 regulation of the Wnt/β-catenin signaling pathway was assessed using a specific agonist (LiCl) and luciferase reporter assay. We found that LINC00514 expression was elevated in NSCLC cells and clinical samples and that increased LINC00514 expression predicted poorer patient prognosis. Silencing LINC00514 suppresses proliferation, migration, and invasion of NSCLC cells. Downregulation of LINC00514 inhibited Wnt/β-catenin signaling and epithelial-mesenchymal transition (EMT). Moreover, suppression of the biological phenotypes of NSCLC cells induced by LINC00514 gene silencing was restored after LiCl treatment. Finally, we found that silencing LINC00514 attenuated the growth of xenograft tumors in vivo. Altogether, this study provides the latest convincing evidence that LINC00514 facilitates the malignant biological behavior of NSCLC cells through activation of the Wnt/β-catenin pathway, which might offer a beneficial approach for the treatment of NSCLC.

Altered T Regulatory Cell Phenotype due to Chronic Hypoxia Exposure Contributes to the Pathogenesis of Pulmonary Hypertension

Hypoxic pulmonary hypertension (PH) can be caused by chronic obstructive lung diseases, acute lung injury, sleep apnea, and high altitude. Hallmarks for PH include recruitment of immune cells to the perivascular region of the lungs, thickening of the pulmonary arterial walls, elevated pulmonary vascular resistance and pressure, and right ventricular remodeling, ultimately causing right heart failure and death.We previously reported that chronic hypoxia (CH) leads to an upregulation of pro‐inflammatory Th17 cells but does not alter T regulatory (Treg) cell numbers within the lung. This change in Th17‐Treg ratio can upset the pro‐inflammatory/anti‐inflammatory balance required to maintain homeostasis leading to disease pathogenesis. We also demonstrated that these Th17 cells react to the cryptic self‐antigen collagen V (col V), which is upregulated following CH. Nasally instilling mice with col V attenuated CH‐induced PH, presumably by inducing mucosal tolerance to col V and restoring Treg‐Th17 balance. Tregs can lose their suppressive phenotype and transition into Th17 cells (deemed exTreg Th17 cells) in certain autoimmune conditions. However, it is unknown whether CH triggers this mechanism. We hypothesized that CH disrupts the Th17‐Treg cell balance due to the pathogenic conversion of Tregs into Th17 cells, contributing to PH development. Using a mouse model (Foxp3tm9(EGFP/cre/ERT2)Ayr/J x Ai14‐tdTomato) that allows the study of lineage stability and genetic mapping of Tregs, together with single‐cell RNA sequencing, we discovered that CH resulted in a gene expression profile consistent with exTregs or Tregs with less suppressive activity, such as downregulation of Il2ra and upregulation of Tnfrsf4 within CD4 memory T cell cluster (adjusted p‐value 0.03). The single‐cell sequencing data were processed with the conventional pipeline. Seurat (v4.0.5), R (v4.1.0), single R (v1.6.1) were used for downstream analysis and cell type prediction against the mouse Immunologic Genome Project. Differential gene expression was determined using the R package DESEq2, and significant genes were defined by adjusted p‐value ≤ 0.05. Our future studies include using multicolor flow cytometry to characterize these changes further and determine if and how CH decreases Treg suppressive activity, contributing to the phenotypic switch to Th17 cells. In conclusion, our data suggest that CH alters Treg cell phenotype, which aids in the pathogenesis of PH.

A Novel Connection Between [PSI+] Prion Formation and RNA Splicing in Saccharomyces cerevisiae

Relatively new studies on prions in Saccharomyces cerevisiae show that some protein prion conformations may be beneficial to cell survival in response to environmental stress. Sup35, a translation termination release factor, forms the prion [PSI+]. When [PSI+] forms, translation termination efficiency decreases, which has been shown to result in the alteration of the abundance of cellular proteins, including splicing factor abundance. Therefore, we are testing the hypothesis that [PSI+] prion formation alters splicing efficiency in yeast. First, we employed quantitative RT‐PCR and revealed that splicing efficiency improves upon [PSI+] prion formation. The proposed mechanisms for the effect of [PSI+] on protein abundance include nonsense suppression and non‐stop decay. Non‐stop decay would lead to degradation of the mRNA encoding a splicing protein and therefore would reduce splicing factor protein levels. Non‐sense suppression, a result of translation readthrough, generates a C‐terminally extended protein, which could alter the function of splicing factors, or alter protein levels. We are initiating RT‐PCR and Western Blot analyses to test the contribution of the two mechanisms on splicing factor abundance. To further test the relationship between RNA splicing and [PSI+] prion formation, we used knockout PCR to create splicing factor deletion mutations in a [psi‐] strain, as well as a weak [PSI+]SC37 strain or a strong [PSI+]Str strain. We found that the [PSI+]SC37 conformation can suppress the growth defect in a yeast strain with a deletion of the gene encoding SNU66, a component of the tri‐snRNP. Similarly, the [PSI+]SC37conformation can suppress the growth defect in a yeast strain with a deletion of the MUD2gene, which encodes a gene important for commitment complex formation. In addition, we found that [PSI+]SC37can rescue the splicing defect observed in a mud2Δstrain, consistent with the suppressive growth phenotype. Together, the results show that [PSI+] formation impacts RNA splicing in S. cerevisiae and our future work will focus on determining the mechanisms that underlie this effect.

Role of the extracellular ATP/adenosine pathway in neutrophil-mediated T cell suppression in ovarian cancer microenvironment

Abstract The extracellular ATP (eATP)/adenosine (ADO) pathway is a key modulator of innate and adaptive immunity and is a target for immunotherapy for cancer. In the tumor microenvironment (TME) and conditions of chronic injury, ATP is released by dying cells and is hydrolyzed by the ecto-nucleotidase CD39 to AMP, and further to ADO by CD73. We previously observed that normal neutrophils (PMN) acquire a T cell suppressor phenotype mimicking PMN-MDSCs when exposed to the TME. Using a coculture system consisting of ascites supernatants (ASC) from patients with ovarian cancer (OC) as an authentic component of the TME and concentrations of healthy donor PMN and T cells that mimic those in OC ascites, we found that ASC-primed PMNs inhibit stimulated T cell proliferation, activation and metabolic responses. This suppressor activity was dependent on several PMN pathways, including complement signaling and NADPH oxidase. Since PMNs regulate and respond to signaling from the eATP/ADO pathway, we asked whether eATP/ADO mediates PMN suppressor function. PMNs had high constitutive expression of CD39 that was unaffected by ASC exposure, and inhibition of CD39 by POM-1 abrogated PMN suppressor function. CD73 inhibitor Adenosine 5′-(α,β-methylene) diphosphate also abrogated PMN suppressor function while using a different inhibitor (AB-680) had no effect. Neither ADO receptor inhibitors nor the addition of adenosine deaminase to deplete ADO affected PMN suppressor function. Together, these studies point to eATP as a modulator of PMN suppressor function in the TME and the potential to abrogate suppression by targeting CD39. Future studies will address the specific signaling functions of eATP on PMN in the TME that drive suppressor function. Supported by grant from NIH (5R01CA188900)

Tim-3 deletion on Treg increases virus-specific T cell response and reduces viral burden in chronic LCMV infection

Abstract Tim-3, a surface protein, is upregulated on Treg during chronic viral infections. During HCV infection in humans there is an increase in frequency of Tim-3+ Treg, which have increased proliferation, survival, and expression of immunosuppressive cytokines like IL-10. Similarly, it’s been recently reported that there is an increase in Tim-3+ Treg in people with HIV (PWH). However, the role of regulatory T cells (Treg) during chronic viral infection has not been fully defined. We hypothesize that Tim-3 promotes an effector phenotype on Treg during chronic viral infection, which limits the virus-specific T cell response and impairs viral clearance. Using an inducible Treg-specific Tim-3 loss-of-function (Tim-3 Treg KO) model, we found a significant decrease in morbidity during LCMV chronic infection. Tim-3 Treg KO mice mounted a higher virus-specific T cell response and had lower viral burden in blood, liver, and kidney at 30dpi. Lack of Tim-3 expression on Treg limited their ability to upregulate IL-10, TGFb, CD39, PD-1, CD25, CTLA-4 and CD44. Similarly, in PWH Tim-3+ Treg have increased expression of IL-10 compared to HIV(−). In addition, Tim-3+ Treg from PWH show significant upregulation of Ki67+, CD73+, Gzmb+ and TGFb+ compared to their Tim-3− Treg counterpart. Our findings demonstrate that modulation of a surface protein in Treg can lead to a reduction in viral burden and enhance LCMV-specific T cell response. We found that Tim-3+ Treg have an increased suppressive phenotype, compared with Tim-3− Treg in PWH, regardless of viral suppression. Elucidation of the mechanism of Tim-3 function in Treg may provide insights into the biology of chronic infections and for developing novel potential strategies to achieve HIV remission off ART. Supported by grants from NIH (T32GM008208, R01AI138504, U01AI131285)

Thrombin cleavage of osteopontin initiates osteopontin’s tumor‐promoting activity

BackgroundOsteopontin (OPN) is a multifunctional proinflammatory matricellular protein overexpressed in multiple human cancers and associated with tumor progression and metastases. Thrombin cleavage of OPN reveals a cryptic binding site for α4β1 and α9β1 integrins. MethodsThrombin cleavage‐resistant OPNR153A knock‐in (OPN‐KI) mice were generated and compared to OPN deficient mice (OPN‐KO) and wild type (WT) mice in their ability to support growth of melanoma cells. Flow cytometry was used to analyze tumor infiltrating leukocytes. ResultsOPN‐KI mice engineered with a thrombin cleavage‐resistant OPN had reduced B16 melanoma growth and fewer pulmonary metastases than WT mice. The tumor suppression phenotype of the OPN‐KI mouse was identical to that observed in OPN‐KO mice and was replicated in WT mice by pharmacologic inhibition of thrombin with dabigatran. Tumors isolated from OPN‐KI mice had increased tumor‐associated macrophages with an altered activation phenotype. Immunodeficient OPN‐KI mice (NOG‐OPN‐KI) or macrophage‐depleted OPN‐KI mice did not exhibit the tumor suppression phenotype. As B16 cells do not express OPN, thrombin‐cleaved fragments of host OPN suppress host antitumor immune response by functionally modulating the tumor‐associated macrophages. YUMM3.1 cells, which express OPN, showed less tumor suppression in the OPN‐KI and OPN‐KO mice than B16 cells, but its growth was suppressed by dabigatran similar to B16 cells. ConclusionsThrombin cleavage of OPN, derived from the host and the tumor, initiates OPN’s tumor‐promoting activity in vivo.

TIM-3+ T regulatory cells alter the lymphoid compartment in cold and Hot solid tumor Model.

Abstract There is a progressive TIM-3+ T reg increase in solid tumors as the tumor progress. W have found that knocking out the ability of T reg to induce TIM-3 lead to delayed progression of tumors in the TIM-3 conditional knockout mice upon both hot (MC38) and cold (B16F10) syngeneic tumor challenges. We are employing our conditional T reg specific TIM-3 Knockout mouse to study if presence or absence of TIM-3 on T reg cells within these tumor models. Preliminary data has shown that our conditional Tim3 knockout T reg cells are found at a much lower frequency, and have an altered suppressive phenotype within the microenvironment of these tumors. We are currently investigating the other changes in the different subsets of lymphoid compartment in tumors. These data suggest that altering the expression of TIM-3+ on T reg has the potential to act as an effective target to alter tumor microenvironment. Targeting TIM-3+ T reg may therefore be attractive model to study, along with other combination immunotherapy to increase the success in immunotherapy on patients who develop resistance to primary immunotherapy.

Molecular Mechanism by which Coumestrol Exerts its Anticancer Activity in Triple‐Negative Inflammatory Breast Cancer

Coumestrol (Cou) is a phytoestrogen present in soy and clover sprouts which is structurally similar to estrogen (E2) and has a cytotoxic effect in breast cancer cell lines. Nevertheless, the molecular mechanism by which Cou can exert its effect in the most aggressive breast cancer subtype, triple‐negative inflammatory breast cancer (TN‐IBC), is still unknown. Several studies show that TN‐IBC cell lines are unresponsive to hormonal therapies, but E2 can activate signaling pathways involved in pro‐oncogenic phenotypes such as motility and invasion. Therefore, the objective of this study was to determine if Cou has an anti‐cancer effect in TN‐IBC cell lines and the molecular mechanism that Cou exerts for its anti‐cancer activity using 2D cell culture models. For this, a dose‐response curve of Cou in 2D models of TN‐IBC cell lines was generated to determine the half‐maximal inhibitory concentration (IC50). Also, relative cell viability was performed in ER‐positive, triple‐negative, TN‐IBC, and HER2‐amplified IBC cell lines. Functional assays were performed in TN‐IBC cell lines to determine the effect of E2 alone, Cou alone, and in a combination of treatments on cell viability, migration, and invasion. Also, the effect on phosphorylation of downstream kinases was analyzed by a proteome profile human phospho‐kinase array. An RNA‐seq was performed in TN‐IBC cell lines to identify transcriptome changes after Cou treatment. Preliminary observations demonstrated that Cou treatment using the IC50 (13µM) caused a decrease in cell viability in TN‐IBC cell lines. Similarly, Cou treatment reduces triple‐negative non‐IBC viability. Also, in comparison with E2, Cou decreases migration and invasion in TN‐IBC cell lines. Finally, Cou treatment reduces the phosphorylation of kinases that promote pro‐oncogenic phenotypes and upregulate genes involved in breast cancer phenotype suppression. In summary, the data gathered in this study will guide us in elucidating a signaling pathway affected by Cou’s anti‐cancer activity. More importantly, it will open the opportunities to design more effective targeted therapeutic strategies to improve the prognosis and survival rates of IBC patients.

Multiplexed single-cell analysis reveals prognostic and nonprognostic T cell types in human colorectal cancer.

Clinical outcomes in colorectal cancer (CRC) correlate with T cell infiltrates, but the specific contributions of heterogenous T cell types remain unclear. To investigate the diverse function of T cells in CRC, we profiled 37,931 T cells from tumors and adjacent normal colon of 16 patients with CRC with respect to transcriptome, TCR sequence, and cell surface markers. Our analysis identified phenotypically and functionally distinguishable effector T cell types. We employed single-cell gene signatures from these T cell subsets to query the TCGA database to assess their prognostic significance. We found 2 distinct cytotoxic T cell types. GZMK+KLRG1+ cytotoxic T cells were enriched in CRC patients with good outcomes. GNLY+CD103+ cytotoxic T cells with a dysfunctional phenotype were not associated with good outcomes, despite coexpression of CD39 and CD103, markers that denote tumor reactivity. We found 2 distinct Treg subtypes associated with opposite outcomes. While total Tregs were associated with good outcomes, CD38+ Tregs were associated with bad outcomes independently of stage and possessed a highly suppressive phenotype, suggesting that they inhibit antitumor immunity in CRC. These findings highlight the potential utility of these subpopulations in predicting outcomes and support the potential for novel therapies directed at CD38+ Tregs or CD8+CD103+ T cells.

Euphorbia factor L3 ameliorates rheumatoid arthritis by suppressing the inflammatory response by targeting Rac family small GTPase 1

ABSTRACT Euphorbia factor L3 (EFL3) is extracted from Euphorbia lathyris and is known for its anti-inflammatory properties. This study focused on the potential anti-inflammatory and therapeutic effects of EFL3 on rheumatoid arthritis (RA) using fibroblast-like synoviocytes (FLSs) and arthritis animal models. Functional analysis showed that EFL3 could ameliorate the inflammatory phenotype of FLSs derived from RA patients, as evidenced by the decreases in cell viability, migration, invasion and cytokine production. Luciferase activity, Western blotting and immunofluorescence assays demonstrated that EFL3 inhibited the nuclear translocation of the p65 subunit and the subsequent activation of the nuclear factor kappa-Β (NF-κB) pathway. Furthermore, the therapeutic effects of EFL3 against arthritic progression were evidenced by decreases in joint swelling, arthritis scores, inflammatory factor production, synovial hyperplasia, and bone destruction in collagen-induced arthritis (CIA) and tumor necrosis factor-α (TNF-α) transgenic (TNF-tg) mouse models. Molecular analysis identified Rac family small GTPase 1 (Rac1) as the potential target that was required for EFL3-mediated suppression of the inflammatory RA FLS phenotype. In summary, this study uncovered the therapeutic potential of EFL3 in RA, which suggests its future clinical use.

Agonists Specific for κ-Opioid Receptor Induces Apoptosis of HCC Cells Through Enhanced Endoplasmic Reticulum Stress.

Cancer pain is an important factor affecting life quality of patients especially in the advanced stage and relieving pain is one of fundamental strategies for cancer treatment. Opioids such as morphine are the most widely used in clinics. However, they have been reported to be associated with the occurrence and development of several types of cancer. Thus, search for an opioid that has analgesic effect and can retard cancer progress simultaneously is critical for cancer management. In this study, we first examined the expression of μ and κ (MOR and KOR) in cell lines and tumor tissues of hepatocellular carcinoma (HCC), a malignant tumor with high mortality, and then compared the effects of opioid receptors-specific agonists on malignant phenotypes of HCC cells in vitro and tumor growth in an HCC xenograft mouse model. KOR and MOR were found to be highly expressed in HCC cell lines and HCC tissues. The KOR-specific agonist U50488h, oxycodone (agonist for both KOR and MOR) and the MOR-specific agonist morphine inhibited HCC cell proliferation, while only U50488h and oxycodone suppressed colony formation and migration of HCC cells. U50488h and oxycodone, but not morphine, induced HCC apoptosis. Further detection of PERK, GRP78 and CHOP revealed that PERK signaling was upregulated by treatment with U50488h, while treatment with the PERK inhibitor GSK2656157 partially reversed the promotion of apoptosis and inhibition of cell proliferation by U50488h, indicating that endoplasmic reticulum stress is associated with its suppressing effect on HCC malignant phenotypes. Similar to the in vitro results, HCC growth was significantly reduced by administration of U50488h and oxycodone, but not by morphine, in the HCC xenograft mouse model. PERK and caspase-3 in the HCC tissues were up-regulated by U50488h treatment as detected by immunohistochemistry and western blotting. Taken together, our results revealed that activation of KOR by U50488h inhibited malignant phenotypes of HCC both in vitro and in vivo, while activation of MOR by morphine did not have such effect. Because of their dual roles in the relief of pain and in the suppression of malignant phenotypes, opioids such as U50488h that act on KOR should be considered as the first choice for HCC management.

A Comprehensive Analysis of the Glutathione Peroxidase 8 (GPX8) in Human Cancer.

ObjectiveNowadays, cancer is still a leading public health problem all over the world. Several studies have reported the GPX8 could be correlated with the poor prognostic of Gastric Cancer and Breast Cancer. However, the prognostic potential of GPX8 in pan-cancer remains unclear. In this work, we aimed to explore the prognostic and immunological role of GPX8 in human cancer and confirm the oncogenic value in GBM.MethodsThe data of TCGA, CPTAC and GEO databases were adopted for the survival analysis. Based on the RNAseq and Methylation450 data of TCGA, the R language and package “ggplot2” were used to analyze the DNA methylation at the region of the promoter of GPX8 in tumors. The genetic alteration of GPX8 from TCGA cancers was investigated in cBioPortal. The R package “GSVA” and “ssGSEA” were employed to evaluate the correlation of GPX8 expression with the immune infiltration. The KEGG website was used for pathway analysis. The STRING website and GEPIA were performed to predict GPX8-binding proteins. The R package “ggplot2” and “clusterprofile” were used to analyze and visualize the GO and KEGG analysis. A normal human astrocyte cell line and three GBM cell lines were cultured under suitable conditions. The shRNA was transferred to cells by Lipofectamine 3000. The qRT-PCR and WB were adopted to detect the expression of GPX8. The wound-healing assay and transwell assay were taken to analyze the invasive and metastatic abilities. The tumor tissues and paracancerous ones were collected from patients with GBM. WB assay was employed to analyze the expression of GPX8 protein.ResultsGPX8 was a valuable diagnostic biomarker in multiple cancers, including GBM/LGG (glioblastoma multiforme/Brain lower grade glioma), KIRC (kidney renal clear cell carcinoma), KIRP (kidney renal papillary cell carcinoma) and STAD (stomach adenocarcinoma). Moreover, we observed a correlation between the expression of GPX8 and the reduced DNA methylation at the promoter region in several tumors, such as GBM/LGG. Our results indicated a positive correlation between the GPX8 expression and immune infiltration. In addition, the enrichment analysis demonstrated that antioxidant activity was mainly involved in the functional mechanism of GPX8. In particular, we first confirmed the up-regulated of GPX8 in GBM cells and observed the suppression of migrative and invasive phenotypes by knockdown of GPX8. Furthermore, we confirmed the expression of GPX8 was higher in GBM tumor tissues than paracancerous ones.ConclusionOur study showed a correlation of GPX8 expression with clinical prognosis, DNA methylation and immune infiltrates. Furthermore, we first confirmed GPX8 was highly expressed in GBM cells and contributed to migration and invasion. These results provided a predictive biomarker and an inclusive understanding of the GPX8 expression in multiple tumors types, especially in GBM.

LINC00982 Inhibits the Proliferation, Migration, and Invasion of Breast Cancer Cells Through the miR-765/DPF3 Axis.

Breast cancer (BC) is one of the most frequently occurring malignant tumors in female adults. The long intergenic nonprotein coding RNA 00982 (LINC00982) has been regarded as a cancer suppressor in several human cancers. However, the function and the underlying mechanisms of LINC00982 have not been studied in BC. The present study found that LINC00982 was significantly downregulated in BC tumor tissues, and the low LINC00982 level predicts a poor prognosis of BC. Through the overexpression and suppression of LINC00982 in two BC cell lines, we found that LINC00982 could inhibit cell proliferation, migration, and invasion by suppressing the activity of the signal transducer and activator of transcription 3 (STAT3)/nuclear factor kappa B (NF-κB) signal pathway. Furthermore, luciferase reporter assay has been used to verify that LINC00982 functions as a molecular sponge for miR-765, which could target DPF3. The relative expression of miR-765 decreased with LINC00982 overexpressing, and DPF3 increased at the same time. In addition, the suppression of cell malignant phenotype caused by overexpression of LINC00982 can be reversed by inhibition of DPF3. To verify the function of LINC00982 in vivo, the BC cells were implanted in nude mice and the results suggested the tumor growth and malignant phenotype were suppressed by LINC00982. In this study, we prove that LINC00982 regulates the growth and development of BC through STAT3/NF-κB signal pathway, mediated by the miR-765/DPF3 axis. LINC00982 may function as a target molecule to take part in the prognosis and therapy of BC.

Xenogeneic Sertoli cells modulate immune response in an evolutionary distant mouse model through the production of interleukin-10 and PD-1 ligands expression.

Immunomodulatory mechanisms of Sertoli cells (SCs) during phylogeny have not been described previously. This study attempted to reveal mechanisms of SC immune modulation in an evolutionary distant host. The interaction of the SC cell line derived from Xenopus tropicalis (XtSC) with murine immune cells was studied in vivo and in vitro. The changes in the cytokine production, the intracellular and surface molecules expression on murine immune cells were evaluated after co-culturing with XtSCs. Migration of XtSCs in mouse recipients after intravenous application and subsequent changes in spleen and the testicular immune environment were determined by flow cytometry. The in vitro co-culture model was established, allowing the study of XtSCs interaction with murine immune cells. Intracellular staining of interleukin (IL-)10 revealed a significant increase in its expression in macrophages and B cells co-cultured with XtSCs, compared to both unstimulated cells and xenogeneic control. On the contrary, a significant decrease in Th lymphocytes expressing interferon-gamma was observed. The expression of both PD-1 ligands (PD-L1 and PD-L2) was upregulated on the macrophage surfaces after co-culture with XtSCs, but not with the controls. XtSCs migrated specifically to testes when administered intravenously and modulated systemic and local testicular microenvironment; this was detected by the expression of molecules associated with suppressive phenotype by CD45+ cells in both spleen and testes. We have demonstrated for the first time that SCs can migrate and modulate immune response in a phylogenetically distant host. It was further observed that SCs induce expression of molecules associated with immunosuppression, such as IL-10 and PD-1 ligands.

Inhibition of Anti-Inflammatory Macrophage Phenotype Reduces Tumour Growth in Mouse Models of Brain Metastasis.

Breast cancer brain metastasis is a significant clinical problem and carries a poor prognosis. Although it is well-established that macrophages are a primary component of the brain metastasis microenvironment, the role of blood-derived macrophages (BDM) and brain-resident microglia in the progression of brain metastases remains uncertain. The aim of this study, therefore, was to determine the role, specifically, of pro- and anti-inflammatory BDM and microglial phenotypes on metastasis progression. Initial in vitro studies demonstrated decreased migration of EO771 metastatic breast cancer cells in the presence of pro-inflammatory, but not anti-inflammatory, stimulated RAW 264.7 macrophages. In vivo, suppression of the anti-inflammatory BDM phenotype, specifically, via myeloid knock out of Krüppel-like Factor 4 (KLF4) significantly reduced EO771 tumour growth in the brains of C57BL/6 mice. Further, pharmacological inhibition of the anti-inflammatory BDM and/or microglial phenotypes, via either Colony Stimulating Factor 1 Receptor (CSF-1R) or STAT6 pathways, significantly decreased tumour burden in two different syngeneic mouse models of breast cancer brain metastasis. These findings suggest that switching BDM and microglia towards a more pro-inflammatory phenotype may be an effective therapeutic strategy in brain metastasis.

Poor clinical outcomes and immunoevasive contexture in SIRPα+ tumor-associated macrophages enriched muscle-invasive bladder cancer patients.

In tumor immune microenvironment, the functions of tumor-associated macrophages (TAMs), including phagocytosis and immunomodulatory, have attracted increasing attention recently. With the discovery of CD47-signal regulatory protein-α (SIRPα) as "don't eat me" signaling pathway, the role of novel subpopulation of TAMs expressing SIRPα has not been fully elucidated in a wide spectrum of solid tumors including bladder cancer. In this study, we investigated the prognostic and predictive implication of SIRPα+ TAMs regarding clinical outcomes and adjuvant chemotherapeutic benefit in muscle-invasive bladder cancer (MIBC), and preliminarily characterized the phenotypic features of SIRPα+ TAMs and its relationship with immune contexture. A total of 141 histochemical MIBC samples from Zhongshan Hospital (ZS), 45 fresh tissue samples, and 391 MIBC patients from TCGA database were enrolled in this study. SIRPα+ TAMs was evaluated by immunohistochemical staining of CD68 and SIRPα, and flow cytometry fluorescence staining. Our results illustrated that SIRPα+ TAMs were enriched in MIBC specimens. Patients with high SIRPα+ TAMs infiltration suffered significant poor overall survival and recurrence-free survival (P = 0.0030 and P = 0.0282). SIRPα+ TAMs infiltration was an independent prognosticator in multivariate Cox model. Moreover, adjuvant chemotherapy (ACT) application showed significantly survival benefit in patients with low SIRPα+ TAMs infiltration (P = 0.0135). SIRPα+ TAMs with suppressive phenotype exhibited a positive correlation with immune tolerance and dysfunctional CD8+ T cells in MIBC. SIRPα+ TAMs infiltration indicated poor prognosis and ACT resistance in MIBC. Immunosuppressive SIRPα+ TAMs is closely related to immune evasion with exhausted T cells states, suggesting the prospect of SIRPα+ TAMs as a potential therapeutic target in MIBC.

Candida auris Bloodstream Infection Induces Upregulation of the PD-1/PD-L1 Immune Checkpoint Pathway in an Immunocompetent Mouse Model.

ABSTRACTCandida auris is a globally spreading yeast pathogen causing bloodstream infections with high mortality in critically ill patients. The inherent antifungal drug resistance of most C. auris isolates and threat of multidrug-resistant strains create a need for adjunct immunotherapeutic strategies. While C. albicans candidemia was shown to induce immune paralysis and activation of inhibitory immune checkpoints, in vivo data on host responses to C. auris bloodstream infection are lacking as is an immunocompetent murine infection model to study the immunopathology and immunotherapy of C. auris sepsis. Therefore, herein, we developed an immunocompetent C. auris sepsis model by intravenously infecting C57BL/6 mice with 1.5 × 108 to 8 × 108 yeast cells of aggregate-forming (AR-0384) and nonaggregative (AR-0381) C. auris reference isolates. Both isolates caused reproducible, inoculum-dependent increasing morbidity, mortality, and fungal burden in kidney tissue. Notably, morbidity and mortality outcomes were partially decoupled from fungal burden, suggesting a role of additional modulators of disease severity such as host immune responses. Flow cytometric analyses of splenic immune cells revealed significant upregulation of the programmed cell death protein 1 (PD-1) on T cells and its ligand PD-L1 on macrophages from mice infected with C. auris AR-0384 compared to uninfected mice. PD-L1 expression on macrophages from AR-0384-infected mice strongly correlated with fungal tissue burden (Spearman’s rank correlation coefficient [ρ] = 0.95). Altogether, our findings suggest that C. auris sepsis promotes a suppressive immune phenotype through PD-1/PD-L1 induction, supporting further exploration of PD-1/PD-L1 blockade as an immunotherapeutic strategy to mitigate C. auris candidiasis.IMPORTANCE Health authorities consider Candida auris to be one of the most serious emerging nosocomial pathogens due to its transmissibility, resistance to disinfection procedures, and frequent antifungal drug resistance. The frequency of multidrug-resistant C. auris isolates necessitates the development of novel therapeutic platforms, including immunotherapy. However, in vivo data on host interactions with C. auris are scarce, compounded by the lack of reliable immunocompetent mammalian models of C. auris candidemia. Herein, we describe a C. auris sepsis model in immunocompetent C57BL/6 mice and demonstrate reproducible and inoculum-dependent acute infection with both aggregate-forming and nonaggregative reference isolates from different clades. Furthermore, we show that C. auris sepsis induces upregulation of the PD-1/PD-L1 immune checkpoint pathway in infected mice, raising the potential of a therapeutic benefit of immune checkpoint blockade. Our immunocompetent model of C. auris sepsis could provide a facile preclinical platform to thoroughly investigate immune checkpoint blockade and combination therapy with antifungals.

PERK is a critical metabolic hub for immunosuppressive function in macrophages.

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

Abstract P3-10-06: High levels of miR-18a is associated with increased proliferation but suppression of EMT phenotype in ER negative breast cancer

Abstract Introduction: miRNA-based regulation has been implicated in tumor evolution and progression. miR-18a belonging to the miR-17-92 polycistronic cluster has also been reported to have oncogenic effects across multiple cancer types including breast cancer. We have previously demonstrated the epigenetic regulation of ER by miR-18a and its high levels as a poor prognostic marker in ER-positive breast tumors (Nair et al, Cancer Med. 2016). Here, we have examined the effects of high expression of miR-18a in the ER-negative subtype of breast cancer. Methods: 275 surgically excised specimens of primary breast cancers were analyzed. Samples were segregated into ER-positive and ER-negative tumors based on ER positivity as determined by Immunohistochemistry. Relative abundance of hsa-miR-18a-5p in these samples was assessed using a TaqMan qRT-PCR and used to correlate with a probability distribution of proliferation that was derived by fitting a binomial logistic regression model using 4 genes - FOXM1, UBE2C, BIRC5 & ANLN. miR-18a was inhibited using synthetic inhibitors in ER-negative breast cancer cell lines - MDA-MB-468 AND MDA-MB-231. Migratory ability was assessed using wound-healing assays. The expression of miR-18a was further analyzed in ER-negative breast cancer samples from the TCGA (n= 116) and the METABRIC cohort (n=107). ER-negative tumors with higher than the third quartile and lower than the first quartile expression of miR-18a were segregated into tumors with high and low expression, respectively. Functional enrichment of differentially expressed genes (DEGs) between these groups was performed using the G: profiler to identify the deregulated pathways. Result: Evaluation of the levels of miR-18a in 275 breast tumor samples showed that the microRNA was highly expressed (p<0.0001) in the ER-negative tumors (n=105) when compared to ER-positive samples (n=170). The ER-negative tumors were further stratified based on the 3rd quartile expression of miR-18a into high (n=27) and low miR-18a (n=78) expressing tumors. The high miR-18a expressing tumors were associated with a higher proliferation score (p=0.005). Inhibition of miR-18a levels in ER-negative breast cancer increased migration ability up to 33 % in MDA-MB-468 (p=0.009) and up to 26 % in MDA-MB-231 (p=0.0003). Analysis of ER-negative tumors of TCGA and METABRIC cohort revealed that the high miR-18a expressing tumors expressed low levels of EMT master regulators-ZEB1 and ZEB2 and low levels of Matrix metalloproteinases -MMP2, MMP3, MMP10, MMP11, MMP13, and MMP17 (p<0.01). Functional enrichment of differentially expressed genes (DEGs) demonstrated suppression of pathways related to cell motility and migration, ECM activation, Wnt signaling, and Focal adhesion-PI3K-Akt signaling pathways (p<0.01). Conclusion: In ER-positive tumors, high levels of miR-18a are associated with activated EMT, increased migratory ability, and poor prognosis. On the contrary, in ER-negative breast tumors, high levels of miR-18a is associated with a higher rate of proliferation but lower migratory ability and suppression of signaling pathways necessary for an epithelial to mesenchymal transition. Citation Format: Madhumathy G Nair, Chandrakala M, Apoorva D, Snijesh VP, Jyothi S Prabhu, Savitha Rajarajan, Aruna Korlimarla, Rakesh S Ramesh, Srinath BS, Sridhar TS. High levels of miR-18a is associated with increased proliferation but suppression of EMT phenotype in ER negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-10-06.

VSSP abrogates murine ovarian tumor-associated myeloid cell-driven immune suppression and induces M1 polarization in tumor-associated macrophages from ovarian cancer patients.

The ovarian tumor microenvironment (TME) is characterized by the accumulation of immunosuppressive tumor-associated macrophages (TAMs) and granulocytic cells. Very small size particles (VSSP), comprised of the ganglioside NAcGM3 and Neisseria meningitidis derived outer membrane vesicles, is being developed as a nanoparticulated modulator of innate immunity. Prior studies have shown that VSSP enhanced antigen-specific cytotoxic T cell responses and reduced the suppressive phenotype of splenic granulocytic cells in tumor-bearing mice. Here, we hypothesized that intraperitoneal VSSP would modify myeloid cell accumulation and phenotypes in the ovarian TME and abrogate suppressor function of TAMs and tumor-associated granulocytic cells. In the ID8 syngeneic model of epithelial ovarian cancer, VSSP reduced peritoneal TAMs and induced M1-like polarization in TAMs. In addition, VSSP stimulated peritoneal inflammation characterized by increased granulocytes and monocytes, including inflammatory monocytic cells. VSSP treatment resulted in peritoneal TAMs and granulocytic cells being less suppressive of ex vivo stimulated CD8+ T cell responses. VSSP alone and combined with anti-PD-1 modestly but significantly prolonged survival in tumor-bearing mice. In addition, ex vivo treatment with VSSP induced M1-like polarization in TAMs from patients with metastatic ovarian cancer and variably abrogated their suppressor phenotype. VSSP treatment also partially abrogated the induction of suppressor function in healthy donor neutrophils exposed to ascites supernatants from patients with ovarian cancer. Together, these results point to VSSP reprogramming myeloid responses resulting in abrogation of suppressive pathways and raise the potential for administration of VSSP into the TME to enhance anti-tumor immunity.