Characterization of obesity-related diseases and inflammation using single cell immunophenotyping in two different diet-induced obesity models.

BACKGROUND: Obesity is a growing problem worldwide and a major risk factor for many chronic diseases. The accumulation of adipose tissue leads to the release of significant amounts of pro-inflammatory cytokines and adipokines, resulting in a low-grade systemic inflammation. However, the mechanisms behind the development of obesity-related diseases are not fully understood. Therefore, our study aimed to investigate the pathological changes and inflammatory processes at systemic level and in individual organs in two different diet-induced mouse obesity models. METHODS: Male C57BL6/J mice were fed by high-fat diet (HFD), high-fat/high-fructose diet (HFD + FR) or normal chow for 21 weeks starting at 3 months of age (n = 15 animals/group). Insulin resistance was tested by oral glucose tolerance test. Pathological changes were investigated on hematoxylin-eosin-stained liver and brown adipose tissue sections. The gene expression levels of adipokines and cytokines were analyzed by qPCR in adipose tissues, whereas serum protein concentrations were determined by multiplex immunoassays. Immunophenotyping of isolated blood, bone marrow and spleen cells was performed by single-cell mass cytometry. RESULTS: Weight gain, glucose intolerance and hepatic steatosis were more severe in the HFD + FR group than in the control and HFD groups. This was accompanied by a higher level of systemic inflammation, as indicated by increased expression of pro-inflammatory genes in visceral white adipose tissue and by a higher serum TNFα level. In addition, immunophenotyping revealed the increase of the surface expressions of CD44 and CD69 on various cell types, such as CD8+ and CD4 + T-cells, B-cells and macrophages, in animals with obesity. CONCLUSIONS: The combination of HFD with fructose supplementation promotes more properly the symptoms of metabolic syndrome. Therefore, the combined high-fat/high-fructose nutrition can be a more suitable model of the Western diet. However, despite these differences, both models showed immunophenotypic changes that may be associated with increased risk of obesity-related cancer.

Dominant suppressor genes of p53-induced apoptosis in Drosophila melanogaster.

One of the major functions of programmed cell death (apoptosis) is the removal of cells that suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes that, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS promoters at both ends, which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, 7 genes were identified, which suppressed the p53-induced apoptosis. In 4 mutants, the suppression effect resulted from single genes activated by 1 UAS promoter (Pka-R2, Rga, crol, and Spt5). In the other 3 (Orct2, Polr2M, and stg), deleting either UAS promoter eliminated the suppression effect. In qPCR experiments, we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eukaryotic genomes, there are coexpressed gene clusters. Three of the DEP insertion mutants are included, and 2 are in close vicinity of separate coexpressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.

Lectin-Based Immunophenotyping and Whole Proteomic Profiling of CT-26 Colon Carcinoma Murine Model.

A murine colorectal carcinoma (CRC) model was established. CT26 colon carcinoma cells were injected into BALB/c mice's spleen to study the primary tumor and the mechanisms of cell spread of colon cancer to the liver. The CRC was verified by the immunohistochemistry of Pan Cytokeratin and Vimentin expression. Immunophenotyping of leukocytes isolated from CRC-bearing BALB/c mice or healthy controls, such as CD19+ B cells, CD11+ myeloid cells, and CD3+ T cells, was carried out using fluorochrome-labeled lectins. The binding of six lectins to white blood cells, such as galectin-1 (Gal1), siglec-1 (Sig1), Sambucus nigra lectin (SNA), Aleuria aurantia lectin (AAL), Phytolacca americana lectin (PWM), and galectin-3 (Gal3), was assayed. Flow cytometric analysis of the splenocytes revealed the increased binding of SNA, and AAL to CD3 + T cells and CD11b myeloid cells; and increased siglec-1 and AAL binding to CD19 B cells of the tumor-bearing mice. The whole proteomic analysis of the established CRC-bearing liver and spleen versus healthy tissues identified differentially expressed proteins, characteristic of the primary or secondary CRC tissues. KEGG Gene Ontology bioinformatic analysis delineated the established murine CRC characteristic protein interaction networks, biological pathways, and cellular processes involved in CRC. Galectin-1 and S100A4 were identified as upregulated proteins in the primary and secondary CT26 tumor tissues, and these were previously reported to contribute to the poor prognosis of CRC patients. Modelling the development of liver colonization of CRC by the injection of CT26 cells into the spleen may facilitate the understanding of carcinogenesis in human CRC and contribute to the development of novel therapeutic strategies.

Single-cell mass cytometric analysis of peripheral immunity and multiplex plasma marker profiling of non-small cell lung cancer patients receiving PD-1 targeting immune checkpoint inhibitors in comparison with platinum-based chemotherapy.

Introduction: The effect of platinum-based chemotherapy (Chem.) and second- or multiple- line immune checkpoint PD-1 blocking therapy by Nivolumab or Pembrolizumab (ICI) was assayed in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Methods: Flow cytometry was used to detect NSCLC-related antigen binding IgG antibodies. The Luminex MagPix multiplex bead-based cytokine/chemokine detecting system was used to quantitatively measure 17 soluble markers in the plasma samples. Single-cell mass cytometry was applied for the immunophenotyping of peripheral leukocytes. Results: The incubation of patient derived plasma with human NSCLC tumor cell lines, such as A549, H1975, and H1650, detected NSCLC-specific antibodies reaching a maximum of up to 32% reactive IgG-positive NSCLC cells. The following markers were detected in significantly higher concentration in the plasma of Chem. group versus healthy non-smoker and smoker controls: BTLA, CD27, CD28, CD40, CD80, CD86, GITRL, ICOS, LAG-3, PD-1, PD-L1, and TLR-2. The following markers were detected in significantly higher concentration in the plasma of ICI group versus healthy non-smoker and smoker controls: CD27, CD28, CD40, GITRL, LAG-3, PD-1, PD-L1, and TLR-2. We showed the induction of CD69 and IL-2R on CD4+ CD25+ T-cells upon chemotherapy; the exhaustion of one CD8+ T-cell population was detected by the loss of CD127 and a decrease in CD27. CD19+CD20+, CD79B+, or activated B-cell subtypes showed CD69 increase and downregulation of BTLA, CD27, and IL-2R in NSCLC patients following chemotherapy or ICI. Discussion: Peripheral immunophenotype caused by chemotherapy or PD-1 blocking was shown in the context of advanced NSCLC.

Introduction of an Ultraviolet C-Irradiated 4T1 Murine Breast Cancer Whole-Cell Vaccine Model.

The advent of immunotherapy has revolutionized cancer treatments. However, the application of immune checkpoint inhibitors may entail severe side effects, with the risk of therapeutic resistance. The generation of chimeric antigen receptor (CAR) T-cells or CAR-NK cells requires specialized molecular laboratories, is costly, and is difficult to adapt to the rapidly growing number of cancer patients. To provide a simpler but effective immune therapy, a whole-cell tumor vaccine protocol was established based on ultraviolet C (UCV)-irradiated 4T1 triple-negative breast cancer cells. The apoptosis of tumor cells after UVC irradiation was verified using resazurin and Annexin V/propidium iodide flow cytometric assays. Protective immunity was achieved in immunized BALB/c mice, showing partial remission. Adoptive transfer of splenocytes or plasma from the mice in remission showed a protective effect in the naive BALB/c mice that received a living 4T1 tumor cell injection. 4T1-specific IgG antibodies were recorded in the plasma of the mice following immunization with the whole-cell vaccine. Interleukin-2 (IL-2) and oligonucleotide 2006 (ODN2006) adjuvants were used for the transfer of splenocytes from C57BL/6 mice into cyclophosphamide-treated BALB/c mice, resulting in prolonged survival, reduced tumor growth, and remission in 33% of the cases, without the development of the graft-versus-host disease. Our approach offers a simple, cost-effective whole-cell vaccine protocol that can be administered to immunocompetent healthy organisms. The plasma or the adoptive transfer of HLA-matching immunized donor-derived leukocytes could be used as an immune cell therapy for cancer patients.

Tilorone increases glucose uptake in vivo and in skeletal muscle cells by enhancing Akt2/AS160 signaling and glucose transporter levels.

Skeletal muscle plays a major role in whole-body glucose metabolism. Insulin resistance in skeletal muscle is characterized by decreased insulin-stimulated glucose uptake resulting from impaired intracellular trafficking and decreased glucose transporter 4 (GLUT4) expression. In this study, we illustrated that tilorone, a low-molecular-weight antiviral agent, improves glucose uptake in vitro and in vivo. Tilorone increased bone morphogenetic protein (BMP) signaling in C2C12 myoblasts, the transcription of multiple BMPs (BMP2, BMP4, BMP7, and BMP14), Smad4 expression, and the phosphorylation of BMP-mediated Smad1/5/8. The activation of Akt2/AS160 (TBC1D4) signaling, the critical regulator of GLUT4 translocation, was also increased, as well as the levels of GLUT4 and GLUT1, leading to enhanced uptake of the radioactively labeled glucose analog 18 F-fluoro-2-deoxyglucose (18 FDG). However, this excess glucose content did not result in increased ATP formation by mitochondrial respiration; both basal and ATP-linked respiration were diminished, thereby contributing to the induction of AMPK. In differentiated myotubes, AS160 phosphorylation and 18 FDG uptake also increased. Moreover, tilorone administration further increased insulin-stimulated phosphorylation of Akt2 and glucose uptake of myotubes indicating an insulin-sensitizing effect. Importantly, during in vivo experiments, the systemic administration of tilorone resulted in increased 18 FDG uptake of skeletal muscle, liver, and adipose tissue in C57BL/6 mice. Our results provide new perspectives for the treatment of type 2 diabetes, which has a limited number of treatments that regulate protein expression or translocation.

Three-Component Reaction of 3-Formyl-6-Methylchromone, Primary Amines, and Secondary Phosphine Oxides: A Synthetic and Mechanistic Study.

A fast, mild, and efficient catalyst-free approach has been developed for the synthesis of chromonyl-substituted α-aminophosphine oxides by the three-component reaction of 3-formyl-6-methylchromone, primary amines, and secondary phosphine oxides at ambient temperature. Carrying out the reaction with aliphatic amines or aminoalcohols at a higher temperature (80 °C), phosphinoyl-functionalized 3-aminomethylene chromanones were formed instead of the corresponding chromonyl-substituted α-aminophosphine oxides. No reaction occurred when 3-formyl-6-methylchromone and secondary phosphine oxides were reacted with aromatic amines in the absence of any catalyst. Applying a basic catalyst, the formation of the phosphinoyl-functionalized 3-aminomethylene chromanones was observed; however, the reaction was not complete. Detailed experimental and quantum chemical studies were performed to study the transformation. Moreover, the in vitro cytotoxicity of phosphinoyl-functionalized 3-aminomethylene chromanones was also investigated in three different cell lines, such as human lung adenocarcinoma (A549), mouse fibroblast (NIH/3T3), and human promyelocytic leukemia (HL60) cells. Several derivatives showed modest activity against the human promyelocytic leukemia (HL60) cell line.

Single-cell immunophenotyping revealed the association of CD4+ central and CD4+ effector memory T cells linking exacerbating chronic obstructive pulmonary disease and NSCLC.

Introduction: Tobacco smoking generates airway inflammation in chronic obstructive pulmonary disease (COPD), and its involvement in the development of lung cancer is still among the leading causes of early death. Therefore, we aimed to have a better understanding of the disbalance in immunoregulation in chronic inflammatory conditions in smoker subjects with stable COPD (stCOPD), exacerbating COPD (exCOPD), or non-small cell lung cancer (NSCLC). Methods: Smoker controls without chronic illness were recruited as controls. Through extensive mapping of single cells, surface receptor quantification was achieved by single-cell mass cytometry (CyTOF) with 29 antibodies. The CyTOF characterized 14 main immune subsets such as CD4+, CD8+, CD4+/CD8+, CD4-/CD8-, and γ/δ T cells and other subsets such as CD4+ or CD8+ NKT cells, NK cells, B cells, plasmablasts, monocytes, CD11cdim, mDCs, and pDCs. The CD4+ central memory (CM) T cells (CD4+/CD45RA-/CD45RO+/CD197+) and CD4+ effector memory (EM) T cells (CD4+/CD45RA-/CD45RO+/CD197-) were FACS-sorted for RNA-Seq analysis. Plasma samples were assayed by Luminex MAGPIX® for the quantitative measurement of 17 soluble immuno-oncology mediators (BTLA, CD28, CD80, CD27, CD40, CD86, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TLR-2) in the four studied groups. Results: Our focus was on T-cell-dependent differences in COPD and NSCLC, where peripheral CD4+ central memory and CD4+ effector memory cells showed a significant reduction in exCOPD and CD4+ CM showed elevation in NSCLC. The transcriptome analysis delineated a perfect correlation of differentially expressed genes between exacerbating COPD and NSCLC-derived peripheral CD4+ CM or CD4+ EM cells. The measurement of 17 immuno-oncology soluble mediators revealed a disease-associated phenotype in the peripheral blood of stCOPD, exCOPD, and NSCLC patients. Discussion: The applied single-cell mass cytometry, the whole transcriptome profiling of peripheral CD4+ memory cells, and the quantification of 17 plasma mediators provided complex data that may contribute to the understanding of the disbalance in immune homeostasis generated or sustained by tobacco smoking in COPD and NSCLC.

Outcome Quality After Colorectal Cancer Resection in Certified Colorectal Cancer Centers­Patient-Reported and Short-Term Clinical Outcomes.

BACKGROUND: In this observational study, patient-reported outcomes and short-term clinical outcome parameters in patients with colorectal cancer were studied 12 months after the start of treatment. Outcomes were also compared across German Certified Colorectal Cancer Centres. METHODS: Data were collected from 4239 patients with colorectal cancer who had undergone elective tumor resection in one of 102 colorectal cancer centers and had responded to a quality-of-life questionnaire before treatment (EORTC QLQ-C30 and -CR29). 3142 (74.1%) of these patients completed a post-treatment questionnaire 12 months later. Correlation analyses were calculated and case-mix adjusted comparisons across centers were made for selected patient-reported outcomes, anastomotic insufficiency, and 30-day-mortality. RESULTS: At 12 months, mild improvements were seen in mean quality-of-life scores (66 vs. 62 points), constipation (16 vs. 19), and abdominal pain (15 vs. 17). Worsening was seen in physical function (75 vs. 82) and pain (22 vs. 19). Better patient-reported outcomes at 12 months were associated with better scores before treatment. Better results in at least three of the five scores were associated with male sex, higher educational level, higher age, and private health insurance. Major worsening of fecal incontinence was seen among patients with rectal cancer without a stoma. The largest differences across centers were found with respect to physical function. Anastomotic insufficiency was found in 4.3% of colon cancer patients and 8.2% of rectal cancer patients. 1.9% of patients died within 30 days after their resection. CONCLUSION: Clinicians can use these findings to identify patients at higher risk for poorer patient-reported outcomes. The differences among cancer centers that were found imply that measures for quality improvement would be desirable.

A cytotoxic survey on 2-amino-1H-imidazol based synthetic marine sponge alkaloid analogues.

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 µM on HL-60 cells, and 29: 3.1 µM on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 µM for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development.

Comparison of Homologous and Heterologous Booster SARS-CoV-2 Vaccination in Autoimmune Rheumatic and Musculoskeletal Patients.

Vaccination against SARS-CoV-2 to prevent COVID-19 is highly recommended for immunocompromised patients with autoimmune rheumatic and musculoskeletal diseases (aiRMDs). Little is known about the effect of booster vaccination or infection followed by previously completed two-dose vaccination in aiRMDs. We determined neutralizing anti-SARS-CoV-2 antibody levels and applied flow cytometric immunophenotyping to quantify the SARS-CoV-2 reactive B- and T-cell mediated immunity in aiRMDs receiving homologous or heterologous boosters or acquired infection following vaccination. Patients receiving a heterologous booster had a higher proportion of IgM+ SARS-CoV-2 S+ CD19+CD27+ peripheral memory B-cells in comparison to those who acquired infection. Biologic therapy decreased the number of S+CD19+; S+CD19+CD27+IgG+; and S+CD19+CD27+IgM+ B-cells. The response rate to a booster event in cellular immunity was the highest in the S-, M-, and N-reactive CD4+CD40L+ T-cell subset. Patients with a disease duration of more than 10 years had higher proportions of CD8+TNF-α+ and CD8+IFN-γ+ T-cells in comparison to patients who were diagnosed less than 10 years ago. We detected neutralizing antibodies, S+ reactive peripheral memory B-cells, and five S-, M-, and N-reactive T-cells subsets in our patient cohort showing the importance of booster events. Biologic therapy and <10 years disease duration may confound anti-SARS-CoV-2 specific immunity in aiRMDs.

A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver.

BACKGROUND: Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. RESULTS: We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. CONCLUSIONS: We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.

Sequential Combination of a Strong Interferon Inducer Viral Vector With Low Doses of Nivolumab Plus Ipilimumab Could Provide Functional Cure in Chronic Hepatitis B Virus infections: Technical Report Proposing a New Modality.

Based on the recommendation of the International Coalition to Eliminate hepatitis B virus (ICE-HBV), we intend to mimic the spontaneous resolution of HBV infection to achieve a functional cure of chronic hepatitis B virus (HBV) infection. To this end, we propose sequential targeting of the innate and adaptive host immune responses. Long-term suppression of HBV replication and hepatitis B surface antigen (HbsAg) production will be achieved first by inducing a strong innate immune response. The clinically validated viral superinfection therapy (SIT) will be administered, which employs an attenuated, non-lytic, double-stranded RNA (dsRNA) infectious bursal disease virus (IBDV) that provides an exceptionally strong interferon (IFN) response. Then, the exhausted HBV-specific T cell function will be restored by blocking the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1) receptors with immune checkpoint inhibitors (ICIs). In order to minimize any risk of toxicity, off-label low doses of nivolumab (0.5 mg/kg) plus ipilimumab (0.3 mg/kg) will be administered, the safety and efficacy of which has already been demonstrated in 131 unselected stage IV cancer patients. We predict that this combination therapy will provide sustained off-treatment virological and clinical responses during a relatively short treatment period.

Humoral and Cellular Immunogenicity and Safety of Five Different SARS-CoV-2 Vaccines in Patients With Autoimmune Rheumatic and Musculoskeletal Diseases in Remission or With Low Disease Activity and in Healthy Controls: A Single Center Study.

Background: Vaccine-induced immunity is essential for controlling the COVID-19 pandemic. Data on humoral and cellular immunogenicity and safety of different SARS-CoV-2 vaccines in patients with autoimmune rheumatic and musculoskeletal diseases (RMDs) are limited. Methods: A single center observational study evaluated the immunogenicity and safety of the two-dose regimen of the BBIBP-CorV inactivated, Gam-COVID-Vac and AZD1222 adenovirus-based, and BNT162b2 and mRNA-1273 mRNA-based vaccines in patients with RMDs (n = 89) compared with healthy controls (n = 74). Neutralizing anti-RBD (receptor binding domain) specific antibodies and SARS-CoV-2 specific T-cell response were measured one and four months after the second vaccine dose in parallel with vaccination efficacy and safety. Results: Disease-specific comparison showed that antibody response at four months was higher in spondylarthropathies compared to rheumatoid arthritis and autoimmune RMDs. Risk factors for reduced immunogenicity included longer disease duration, positive immunoserological profile and anti-CD20 therapy of patients. The rate of positive anti-RBD antibody response for healthy controls versus patients after 4 months post vaccination was 69% vs. 55% for the inactivated viral vaccine BBIBP-CorV, 97% vs. 53% for the pooled data of adenovirus vector-based vaccines Gam-COVID-Vac and AZD1222, or 100% vs. 81% for the pooled data of mRNA vaccines BNT162b2 and mRNA-1273, respectively. Patients who received the Gam-COVID-Vac or mRNA-1273 vaccines had a higher proportion of TNF-α producing CD4+ T-cells upon SARS-CoV-2 antigen stimulation compared to the inactivated viral vaccine. Conclusion: All five investigated vaccines were immunogenic in the majority of patients and healthy controls with variable antibody and T-cell response and an acceptable safety profile.

Identification of a Tumor Cell Associated Type I IFN Resistance Gene Expression Signature of Human Melanoma, the Components of Which Have a Predictive Potential for Immunotherapy.

We developed a human melanoma model using the HT168-M1 cell line to induce IFN-α2 resistance in vitro (HT168-M1res), which was proven to be maintained in vivo in SCID mice. Comparing the mRNA profile of in vitro cultured HT168-M1res cells to its sensitive counterpart, we found 79 differentially expressed genes (DEGs). We found that only a 13-gene core of the DEGs was stable in vitro and only a 4-gene core was stable in vivo. Using an in silico cohort of IFN-treated melanoma tissues, we validated a differentially expressed 9-gene core of the DEGs. Furthermore, using an in silico cohort of immune checkpoint inhibitor (ICI)-treated melanoma tissues, we tested the predictive power of the DEGs for the response rate. Analysis of the top four upregulated and top four downregulated genes of the DEGs identified WFDC1, EFNA3, DDX10, and PTBP1 as predictive genes, and analysis of the "stable" genes of DEGs for predictive potential of ICI response revealed another 13 genes, out of which CDCA4, SOX4, DEK, and HSPA1B were identified as IFN-regulated genes. Interestingly, the IFN treatment associated genes and the ICI-therapy predictive genes overlapped by three genes: WFDC1, BCAN, and MT2A, suggesting a connection between the two biological processes.

Patient-specific Boolean models of signalling networks guide personalised treatments.

Prostate cancer is the second most occurring cancer in men worldwide. To better understand the mechanisms of tumorigenesis and possible treatment responses, we developed a mathematical model of prostate cancer which considers the major signalling pathways known to be deregulated. We personalised this Boolean model to molecular data to reflect the heterogeneity and specific response to perturbations of cancer patients. A total of 488 prostate samples were used to build patient-specific models and compared to available clinical data. Additionally, eight prostate cell line-specific models were built to validate our approach with dose-response data of several drugs. The effects of single and combined drugs were tested in these models under different growth conditions. We identified 15 actionable points of interventions in one cell line-specific model whose inactivation hinders tumorigenesis. To validate these results, we tested nine small molecule inhibitors of five of those putative targets and found a dose-dependent effect on four of them, notably those targeting HSP90 and PI3K. These results highlight the predictive power of our personalised Boolean models and illustrate how they can be used for precision oncology.

Blocking the Increase of Intracellular Deuterium Concentration Prevents the Expression of Cancer-Related Genes, Tumor Development, and Tumor Recurrence in Cancer Patients.

The possible role of the naturally occurring deuterium in the regulation of cell division was first described in the 1990s. To investigate the mechanism of influence of deuterium (D) on cell growth, expression of 236 cancer-related and 536 kinase genes were tested in deuterium-depleted (40 and 80 ppm) and deuterium-enriched (300 ppm) media compared to natural D level (150 ppm). Among genes with expression changes exceeding 30% and copy numbers over 30 (124 and 135 genes, respectively) 97.3% of them was upregulated at 300 ppm D-concentration. In mice exposed to chemical carcinogen, one-year survival data showed that deuterium-depleted water (DDW) with 30 ppm D as drinking water prevented tumor development. One quarter of the treated male mice survived 344 days, the females 334 days, while one quarter of the control mice survived only 188 and 156 days, respectively. In our human retrospective study 204 previously treated cancer patients with disease in remission, who consumed DDW, were followed. Cumulative follow-up time was 1024 years, and average follow-up time per patient, 5 years (median: 3.6 years). One hundred and fifty-six patients out of 204 (77.9%) did not relapse during their 803 years cumulative follow-up time. Median survival time (MST) was not calculable due to the extremely low death rate (11 cancer-related deaths, 5.4% of the study population). Importantly, 8 out of 11 deaths occurred several years after stopping DDW consumption, confirming that regular consumption of DDW can prevent recurrence of cancer. These findings point to the likely mechanism in which consumption of DDW keeps D-concentration below natural levels, preventing the D/H ratio from increasing to the threshold required for cell division. This in turn can serve as a key to reduce the relapse rate of cancer patients and/or to reduce cancer incidence in healthy populations.

Deuterium Content of the Organic Compounds in Food Has an Impact on Tumor Growth in Mice.

Research with deuterium-depleted water (DDW) in the last two decades proved that the deuterium/hydrogen ratio has a key role in cell cycle regulation and cellular metabolism. The present study aimed to investigate the possible effect of deuterium-depleted yolk (DDyolk) alone and in combination with DDW on cancer growth in two in vivo mouse models. To produce DDyolk, the drinking water of laying hens was replaced with DDW (25 ppm) for 6 weeks, resulting in a 60 ppm D level in dried egg yolk that was used as a deuterium-depleted food additive. In one model, 4T1, a cell line with a high metastatic capacity to the lung was inoculated in the mice's mammary pad. After three weeks of treatment with DDW and/or DDyolk, the tumor volume in the lungs was smaller in all treated groups vs. controls with natural D levels. Tumor growth and survival in mice transplanted with an MCF-7 breast cancer cell line showed that the anticancer effect of DDW was enhanced by food containing the deuterium-depleted yolk. The study confirmed the importance of the D/H ratio in consumed water and in metabolic water produced by the mitochondria while oxidizing nutrient molecules. This is in line with the concept that the initiation of cell growth requires the cells to generate a higher D/H ratio, but DDW, DDyolk, or the naturally low-D lipids in a ketogenic diet, have a significant effect on tumor growth by preventing the cells from raising the D/H ratio to the threshold.

Crosstalk between the redox signalling and the detoxification: GSTs under redox control?

Reactive oxygen species (ROS), antioxidants and their reduction-oxidation (redox) states all contribute to the redox homeostasis, but glutathione is considered to be the master regulator of it. We aimed to understand the relationship between the redox potential and the diverse glutathione transferase (GST) enzyme family by comparing the stress responses of two tomato cultivars (Solanum lycopersicum 'Moneymaker' and 'Ailsa Craig'). Four-week-old plants were treated by two concentrations of mannitol, NaCl and salicylic acid. The lower H2O2 and malondialdehyde contents indicated higher stress tolerance of 'Moneymaker'. The redox status of roots was characterized by measuring the reduced and oxidized form of ascorbate and glutathione spectrophotometrically after 24 h. The redox potential of 'Ailsa Craig' was more oxidized compared to 'Moneymaker' even under control conditions and became more positive due to treatments. High-throughput quantitative real-time PCR revealed that besides overall higher expression levels, SlGSTs were activated more efficiently in 'Moneymaker' due to stresses, resulting in generally higher GST and glutathione peroxidase activities compared to 'Ailsa Craig'. The expression level of SlGSTs correlated differently, however Pearson's correlation analysis showed usually strong positive correlation between SlGST transcription and glutathione redox potential. The possible redox regulation of SlGST expressions was discussed.

Chronic Obstructive Pulmonary Disease: Epidemiology, Biomarkers, and Paving the Way to Lung Cancer.

Chronic obstructive pulmonary disease (COPD), the frequently fatal pathology of the respiratory tract, accounts for half a billion cases globally. COPD manifests via chronic inflammatory response to irritants, frequently to tobacco smoke. The progression of COPD from early onset to advanced disease leads to the loss of the alveolar wall, pulmonary hypertension, and fibrosis of the respiratory epithelium. Here, we focus on the epidemiology, progression, and biomarkers of COPD with a particular connection to lung cancer. Dissecting the cellular and molecular players in the progression of the disease, we aim to shed light on the role of smoking, which is responsible for the disease, or at least for the more severe symptoms and worse patient outcomes. We summarize the inflammatory conditions, as well as the role of EMT and fibroblasts in establishing a cancer-prone microenvironment, i.e., the soil for 'COPD-derived' lung cancer. We highlight that the major health problem of COPD can be alleviated via smoking cessation, early diagnosis, and abandonment of the usage of biomass fuels on a global basis.