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Previous studies show that LDH-A knockdown reduces orthotopic 4T1 breast tumor lactate and delays tumor growth and the development of metastases in nude mice. Here, we report significant changes in the tumor microenvironment (TME) and a more robust anti-tumor response in immune competent BALB/c mice. 4T1 murine breast cancer cells were transfected with shRNA plasmids directed against LDH-A (KD) or a scrambled control plasmid (NC). Cells were also transduced with dual luciferase-based reporter systems to monitor HIF-1 activity and the development of metastases by bioluminescence imaging, using HRE-sensitive and constitutive promoters, respectively. The growth and metastatic profile of orthotopic 4T1 tumors developed from these cell lines were compared and a primary tumor resection model was studied to simulate the clinical management of breast cancer. Primary tumor growth, metastasis formation and TME phenotype were significantly different in LDH-A KD tumors compared with controls. In LDH-A KD cells, HIF-1 activity, hexokinase 1 and 2 expression and VEGF secretion were reduced. Differences in the TME included lower HIF-1α expression that correlated with lower vascularity and pimonidazole staining, higher infiltration of CD3+ and CD4+ T cells and less infiltration of TAMs. These changes resulted in a greater delay in metastases formation and 40% long-term survivors (>20 weeks) in the LDH-A KD cohort following surgical resection of the primary tumor. We show for the first time that LDH-depletion inhibits the formation of metastases and prolongs survival of mice through changes in tumor microenvironment that modulate the immune response. We attribute these effects to diminished HIF-1 activity, vascularization, necrosis formation and immune suppression in immune competent animals. Gene-expression analyses from four human breast cancer datasets are consistent with these results, and further demonstrate the link between glycolysis and immune suppression in breast cancer.

Chimeric antigen receptor (CAR) T cell therapy in hematologic malignancies has shown remarkable responses, but the same level of success has not been observed in solid tumors. A new prostate cancer model (Myc-CaP:PSMA(+)) and a second-generation anti-hPSMA human CAR T cells expressing a Click Beetle Red luciferase reporter) were used to study hPSMA targeting and assess CAR T cell trafficking and persistence by bioluminescence imaging (BLI). We investigated the antitumor efficacy of human CAR T cells targeting human prostate-specific membrane antigen (hPSMA), in the presence and absence of the target antigen; first alone and then combined with a monoclonal antibody targeting the human programmed death receptor 1 (anti-hPD1 mAb). PDL-1 expression was detected in Myc-CaP murine prostate tumors growing in immune competent FVB/N and immune-deficient SCID mice. Endogenous CD3+ T cells were restricted from the centers of Myc-CaP tumor nodules growing in FVB/N mice. Following anti-programmed cell death protein 1 (PD-1) treatment, the restriction of CD3+ T cells was reversed, and a tumor-treatment response was observed. Adoptive hPSMA-CAR T cell immunotherapy was enhanced when combined with PD-1 blockade, but the treatment response was of comparatively short duration, suggesting other immune modulation mechanisms exist and restrict CAR T cell targeting, function, and persistence in hPSMA expressing Myc-CaP tumors. Interestingly, an “inverse pattern” of CAR T cell BLI intensity was observed in control and test tumors, which suggests CAR T cells undergo changes leading to a loss of signal and/or number following hPSMA-specific activation. The lower BLI signal intensity in the hPSMA test tumors (compared with controls) is due in part to a decrease in T cell mitochondrial function following T cell activation, which may limit the intensity of the ATP-dependent Luciferin-luciferase bioluminescence signal.

Purpose Liver cancers are among the deadliest malignancies due to a limited efficacy of early diagnostics, the lack of appropriate biomarkers and insufficient discrimination of different types of tumors by classic and molecular methods. In this study, we searched for novel long non-coding RNA (lncRNA) as well as validated several known candidates suitable as probable biomarkers for primary liver tumors of various etiology. Methods We described a novel lncRNA HELIS (aka “HEalthy LIver Specific”) and estimated its expression by RT-qPCR in 82 paired tissue samples from patients with hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), combined HCC-CCA, pediatric hepatoblastoma (HBL) and non-malignant hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH). Additionally, we examined expression of cancer-associated lncRNAs HULC, MALAT1, UCA1, CYTOR, LINC01093 and H19, which were previously studied mainly in HCC. Results We demonstrated that down-regulation of HELIS strongly correlates with carcinogenesis; whereas in tumors with non-hepatocyte origin (HBL, CCA) or in a number of poorly differentiated HCC, this lncRNA is not expressed. We showed that recently discovered LINC01093 is dramatically down-regulated in all malignant liver cancers; while in benign tumors LINC01093 expression is just twice decreased in comparison to adjacent samples. Conclusion Our study revealed that among all measured biomarkers only down-regulated HELIS and LINC01093, up-regulated CYTOR and dysregulated HULC are perspective for differential diagnostics of liver cancers; whereas others demonstrated discordant results and cannot be considered as potential universal biomarkers for this purpose.

Hepatocellular carcinoma (HCC) is the most common and aggressive type of malignant liver tumor. HCC progression depends significantly on its vascularization and formation of new blood vessels. Vascular endothelial growth factor A (VEGFA) is a crucial regulator of tumor vascularization and components of VEGF-induced cell signaling pathways are important targets of therapeutical drugs that demonstrated the highest efficiency in case of advanced HCC (sorafenib and regorafenib). VEGFA is expressed as a set of isoforms with different functional properties, thus VEGFA isoform expression pattern may affect tumor sensitivity to anti-angiogenic drugs. However, information about VEGFA isoforms expression in HCC is still incomplete and contradictory. The present study aims to quantitatively investigate VEGFA isoform expression aberrations in HCC tissue. A total of 50 pairs of HCC and non-tumor tissue samples were used to evaluate the VEGFA isoform spectrum using RT-PCR and quantitatively estimate changes in isoform expression using RT-qPCR. Correlations between these changes and tumor clinicopathological characteristics were analyzed. We identified VEGFA-189, VEGFA-165, and VEGFA-121 as predominant isoforms in liver tissue. Anti-angiogenic VEGFA-xxxb variants constituted no more than 5% of all mature VEGFA transcripts detected and their expression was not changed significantly in HCC tissue. We demonstrated for the first time that the least active variant VEGFA-189 is frequently repressed in HCC ( < 0.001), while no uniform changes were detected for potent angiogenesis stimulators VEGFA-165 and VEGFA-121. Isoform balance in HCC shifts from VEGFA-189 towards VEGFA-165 or VEGFA-121 in the majority of cases ( < 0.001). Changes in fractions, but not expression levels, of VEGFA-189 (decrease) and VEGFA-121 (increase) correlated with advanced Tumor-Node-Metastasis (TNM) and Barcelona Clinic Liver Cancer (BCLC) tumor stages ( < 0.05), VEGFA-189 fraction reduction was also associated with poor tumor differentiation ( < 0.05). A distinct shift in VEGFA isoform balance towards more pro-angiogenic variants occurs in HCC tissue and may modulate overall impact of VEGFA signaling. We suppose that the ratio between VEGFA isoforms is an important parameter governing HCC angiogenesis that may affect HCC progression and be used for optimizing the strategy of HCC therapy by predicting the response to anti-angiogenic drugs.

HIF-1alpha is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1alpha chimeric reporter systems, HIF-1alpha/FLuc and HIF-1alpha(DeltaODDD)/FLuc, to investigate the tightly controlled level of HIF-1alpha protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1alpha in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1alpha/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1alpha in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1alpha was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1alpha/FLuc protein degradation and quantify the half-life of HIF-1alpha fusion proteins. The rapid clearance component (t(1/2) approximately 4-6 min) was abolished by the hypoxia-mimetic CoCl2 MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t(1/2) approximately 200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1alpha/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1alpha.

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α.

Influenza vaccination is recommended for vulnerable individuals, including active drug users, to prevent influenza complications and decrease influenza spread. Recent studies suggest that opioids negatively regulate immune responses in experimental models, but the extent to which opioid use will affect the humoral responses to influenza vaccine in humans is unknown. This information is critical in maximizing vaccination efforts. To determine whether there is a difference in antibody response after influenza vaccination in heroin or methadone users compared to control subjects. We studied active heroin users, subjects on methadone maintenance treatment (MMT) and subjects that did not use any drugs before and 1 and 4 weeks after vaccination with trivalent influenza vaccine (TIV). We measured hemagglutination inhibition and microneutralization titers, and we compared geometric mean titers (GMT), and rates of seroprotection and seroconversion for each of the vaccine strains among the 3 groups of subjects. Heroin users, subjects on MMT and non-user controls mount a similarly robust serologic response to TIV. GMT and rates of seroprotection and seroconversion were not significantly different among groups. Our results suggest that opioid use do not significantly alter antibody responses to influenza vaccine supporting the vaccination effort in these populations.

HIF-1[alpha] is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1[alpha] chimeric reporter systems, HIF-1[alpha]/FLuc and HIF-1[alpha]([DELTA]ODDD)/FLuc, to investigate the tightly controlled level of HIF-1[alpha] protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1[alpha] in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1[alpha]/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1[alpha] in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1[alpha] was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl.sub.2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1[alpha]/FLuc protein degradation and quantify the half-life of HIF-1[alpha] fusion proteins. The rapid clearance component (t.sub.1/2 ~4-6 min) was abolished by the hypoxia-mimetic CoCl.sub.2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t.sub.1/2 ~200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1[alpha]/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1[alpha].

Cationic antimicrobial peptides (CAMPs) are considered as next-generation antibiotics with a lower probability of developing bacterial resistance. In view of potential clinical use, studies on CAMP biocompatibility are important. This work aimed to evaluate the behavior of synthetic short CAMPs (designed using bioinformatic analysis of the medicinal leech genome and microbiome) in direct contact with blood cells and plasma. Eight CAMPs were included in the study. Hemolysis and lactate dehydrogenase assays showed that the potency to disrupt erythrocyte, neutrophil and mononuclear cell membranes descended in the order pept_1 > pept_3 ~ pept_5 > pept_2 ~ pept_4. Pept_3 caused both cell lysis and aggregation. Blood plasma and albumin inhibited the CAMP-induced hemolysis. The chemiluminescence method allowed the detection of pept_3-mediated neutrophil activation. In plasma coagulation assays, pept_3 prolonged the activated partial thromboplastin time (APTT) and prothrombin time (at 50 μM by 75% and 320%, respectively). Pept_3 was also capable of causing fibrinogen aggregation. Pept_6 prolonged APTT (at 50 μM by 115%). Pept_2 was found to combine higher bactericidal activity with lower effects on cells and coagulation. Our data emphasize the necessity of investigating CAMP interaction with plasma.

The unique ability of oncolytic viruses (OVs) to replicate in and destroy malignant cells while leaving healthy cells intact and activating the host immune response makes them powerful targeted anti-cancer therapeutic agents. Vesicular stomatitis virus (VSV) only causes mild and asymptomatic infection, lacks pre-existing immunity, can be genetically engineered for enhanced efficiency and improved safety, and has a broad cell tropism. VSV can facilitate targeted delivery of immunostimulatory cytokines for an enhanced immune response against cancer cells, thus decreasing the possible toxicity frequently observed as a result of systemic delivery. In this study, the oncolytic potency of the two rVSV versions, rVSV-dM51-GFP, delivering green fluorescent protein (GFP), and rVSV-dM51-mIL12-mGMCSF, delivering mouse interleukin-12 (mIL-12) and granulocyte-macrophage colony-stimulating factor (mGMCSF), was compared on the four murine cancer cell lines of different origin and healthy mesenchymal stem cells (MSCs) at 24 h post-infection by flow cytometry. Lewis lung carcinoma (LL/2) cells were demonstrated to be more susceptible to the lytic effects of both rVSV versions compared to melanoma (B16-F10) cells. Detection of expression levels of antiviral and pro-apoptotic genes in response to the rVSV-dM51-GFP infection by quantitative PCR (qPCR) showed lower levels of IFIT, RIG-I, and N-cadherin and higher levels of IFNβ and p53 in LL/2 cells. Subsequently, C57BL/6 mice, infused subcutaneously with the LL/2 cells, were injected intratumorally with the rVSV-dM51-mIL12-mGMCSF 7 days later to assess the synergistic effect of rVSV and immunostimulatory factors. The in vivo study demonstrated that treatment with two rVSV-dM51-mIL12-mGMCSF doses 3 days apart resulted in a tumor growth inhibition index (TGII) of over 50%.