Comparative analysis of the effect of hypoxia in two different tumor cell models shows the differential involvement of PTEN control of proangiogenic pathways.

Hypoxia, low, non-physiological oxygen tension is a key regulator of tumor microenvironment, determining the pathological tumor vascularization. Alleviation of hypoxia through vessel normalization may be a promising therapeutic approach. We aimed to assess the role of low oxygen tension in PTEN-related pathways and proangiogenic response, in vitro, in two different tumor cell lines, focusing on potential therapeutic targets for tumor vessel normalization. Downregulation of PTEN in hypoxia mediates the activation of distinct mechanisms: cytoplasmic pAKT activation in melanoma and pMDM2 modulation in kidney cancer. We show that hypoxia-induced proangiogenic potential was stronger in Renca cells than B16 F10-confirmed by a distinct secretory potential and different ability to affect endothelial cells functions. Therefore, the impact of hypoxia on PTEN-mediated regulation may determine the therapeutic targets and effectiveness of vessel normalization and intrinsic characteristics of cancer cell have to be taken into account when designing treatment.

Pten knockout affects drug resistance differently in melanoma and kidney cancer.

BACKGROUND: PTEN is a tumor suppressor that is often mutated and nonfunctional in many types of cancer. The high heterogeneity of PTEN function between tumor types makes new Pten knockout models necessary to assess its impact on cancer progression and/or treatment outcomes. METHODS: We aimed to show the effect of CRISPR/Cas9-mediated Pten knockout on murine melanoma (B16 F10) and kidney cancer (Renca) cells. We evaluated the effect of PTEN deregulation on tumor progression in vivo and in vitro, as well as on the effectiveness of drug treatment in vitro. In addition, we studied the molecular changes induced by Pten knockout. RESULTS: In both models, Pten mutation did not cause significant changes in cell proliferation in vitro or in vivo. Cells with Pten knockout differed in sensitivity to cisplatin treatment: in B16 F10 cells, the lack of PTEN induced sensitivity and, in Renca cells, resistance to drug treatment. Accumulation of pAKT was observed in both cell lines, but only Renca cells showed upregulation of the p53 level after Pten knockout. PTEN deregulation also varied in the way that it altered PAI-1 secretion in the tested models, showing a decrease in PAI-1 in B16 F10 Pten/KO and an increase in Renca Pten/KO cells. In kidney cancer cells, Pten knockout caused changes in epithelial to mesenchymal transition marker expression, with downregulation of E-cadherin and upregulation of Snail, Mmp9, and Acta2 (α-SMA). CONCLUSIONS: The results confirmed heterogenous cell responses to PTEN loss, which may lead to a better understanding of the role of PTEN in particular types of tumors and points to PTEN as a therapeutic target for personalized medicine.

Microenvironment commits breast tumor ECs to dedifferentiation by micro-RNA-200-b-3p regulation and extracellular matrix remodeling.

Introduction: Hypoxia shapes the tumor microenvironment, modulates distinct cell population activities, and activates pathological angiogenesis in cancer, where endothelial cells (ECs) are the most important players. This study aimed to evidence the influences of the tumor microenvironment on the global gene expression pattern characteristic for ECs and the distinct responses displayed by tumor-derived ECs in comparison to the healthy endothelium during endothelial to mesenchymal transition (EndMT) and its regulation by miR-200-b-3p. Methodology: Immortalized lines of ECs from the same patient with breast cancer, healthy breast tissue (HBH.MEC), and primary tumor (HBCa.MEC) were used. The experiments were performed in normoxia and hypoxia for 48 h. By using the wound healing test, we investigated the migration abilities of ECs. Global gene expression analysis with NGS was carried out to detect new pathways altered in pathological ECs and find the most changed miRNAs. The validation of NGS data from RNA and miRNA was estimated by qPCRs. Mimic miR-200b-3p was used in HBH.MEC, and the targets VEGF, Bcl2, ROCK2, and SP1 were checked. Results: Hypoxia influences EC migration properties in wound healing assays. In hypoxia, healthy ECs migrate slower than they do in normoxia, as opposed to HBCa.MEC, where no decreased migration ability is induced by hypoxia due to EndMT features. NGS data identified this process to be altered in cancer ECs through extracellular matrix (ECM) organization. The deregulated genes, validated by qPCR, included SPP1, ITGB6, COL4A4, ADAMST2, LAMA1, GAS6, PECAM1, ELN, FBLN2, COL6A3, and COL9A3. NGS also identified collagens, laminins, fibronectins, and integrins, as being deregulated in tumor-derived ECs. Moreover, the analysis of the 10 most intensively modified miRNAs, when breast tumor-derived ECs were compared to healthy ECs, shed light on miR-200b-3p, which is strongly upregulated in HBCa.MECs when compared to HBH.MECs. Discussion and conclusion: The pathological ECs differed significantly, both phenotypically and functionally, from the normal corresponding tissue, thus influencing their microenvironment cross-talk. The gene expression profile confirms the EndMT phenotype of tumor-derived ECs and migratory properties acquisition. Moreover, it indicates the role of miR-200b-3p, that is, regulating EndMT in pathological ECs and silencing several angiogenic growth factors and their receptors by directly targeting their mRNA transcripts.

Hypoxia, but Not Normoxia, Reduces Effects of Resveratrol on Cisplatin Treatment in A2780 Ovarian Cancer Cells: A Challenge for Resveratrol Use in Anticancer Adjuvant Cisplatin Therapy.

Natural compounds, such as resveratrol (Res), are currently used as adjuvants for anticancer therapies. To evaluate the effectiveness of Res for the treatment of ovarian cancer (OC), we screened the response of various OC cell lines to the combined treatment with cisplatin (CisPt) and Res. We identified A2780 cells as the most synergistically responding, thus optimal for further analysis. Because hypoxia is the hallmark of the solid tumor microenvironment, we compared the effects of Res alone and in combination with CisPt in hypoxia (pO2 = 1%) vs. normoxia (pO2 = 19%). Hypoxia caused an increase (43.2 vs. 5.0%) in apoptosis and necrosis (14.2 vs. 2.5%), reactive oxygen species production, pro-angiogenic HIF-1α (hypoxia-inducible factor-1α) and VEGF (vascular endothelial growth factor), cell migration, and downregulated the expression of ZO1 (zonula occludens-1) protein in comparison to normoxia. Res was not cytotoxic under hypoxia in contrast to normoxia. In normoxia, Res alone or CisPt+Res caused apoptosis via caspase-3 cleavage and BAX, while in hypoxia, it reduced the accumulation of A2780 cells in the G2/M phase. CisPt+Res increased levels of vimentin under normoxia and upregulated SNAI1 expression under hypoxia. Thus, various effects of Res or CisPt+Res on A2780 cells observed in normoxia are eliminated or diminished in hypoxia. These findings indicate the limitations in using Res as an adjuvant with CisPt therapy in OC.

"Endothelial Antibody Factory" at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases.

The failures of anti-ß-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-ß-amyloid and anti-TDP43 Fabs turning them into an "antibody fragment factory". When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect.

miRNA Pattern in Hypoxic Microenvironment of Kidney Cancer-Role of PTEN.

MicroRNAs are post-transcriptional regulators of gene expression, and disturbances of their expression are the basis of many pathological states, including cancers. The miRNA pattern in the context of tumor microenvironment explains mechanisms related to cancer progression and provides a potential target of modern therapies. Here we show the miRNA pattern in renal cancer focusing on hypoxia as a characteristic feature of the tumor microenvironment and dysregulation of PTEN, being a major tumor suppressor. Methods comprised the CRSPR/Cas9 mediated PTEN knockout in the Renca kidney cancer cell line and global miRNA expression analysis in both in vivo and in vitro (in normoxic and hypoxic conditions). The results were validated on human cancer models with distinct PTEN status. The increase in miR-210-3p in hypoxia was universal; however, the hypoxia-induced decrease in PTEN was associated with an increase in miR-221-3p, the loss of PTEN affected the response to hypoxia differently by decreasing miR-10b-5p and increasing miR-206-3p. In turn, the complete loss of PTEN induces miR-155-5p, miR-100-5p. Upregulation of miR-342-3p in knockout PTEN occurred in the context of the whole tumor microenvironment. Thus, effective identification of miRNA patterns in cancers must consider the specificity of the tumor microenvironment together with the mutations of key suppressors.

Spheroid culture models adequately imitate distinctive features of the renal cancer or melanoma microenvironment.

Tumor development studies should adapt to cancer cells' specific mechanisms in connection with their microenvironment. Standard two-dimensional cultures and gas composition are not relevant to the real cancer environment. Existing three-dimensional models are often requiring sophisticated conditions. Here, we propose and characterize, in two cancer models, melanoma (B16F10) and kidney cancer (RenCa), a three-dimensional culture method, reporting the presence of hypoxia-related genes/proteins and aggressiveness mechanisms (epithelial mesenchymal transition and cancer stem cells). We validate the designed three-dimensional method by comparing it with in vivo growing tumors. The developed method brings simplicity and data reproducibility. Melanoma spheroid-growing cells reached a cell cycle arrest at the G0/G1 phase and showed induction of hypoxia. Spheroid-recovered RenCa cells were enriched in proliferating cells and displayed delayed hypoxia. Moreover, the responses to hypoxia observed in spheroids were validated by in vivo tumor studies for both lines. Three-dimensional shapes induced cancer stem cells in renal cancer, whereas epithelial to mesenchymal transition occurred in the melanoma model. Such distinction in the use of different aggressiveness-leading pathways was observed in in vivo melanoma vs kidney tumors. Thus, this 3D culture model approach is adequate to uncover crucial molecular pathways using distinct mechanisms to reach aggressiveness; i.e., B16F10 cells perform epithelial to mesenchymal transition while RenCa cells dedifferentiate into cancer stem cells. Such three-dimensional models help mimic the in vivo tumor features, i.e., hypoxia and aggressiveness mechanisms as validated here by next-generation sequencing analysis, and are proposed for further alternative methods to in vivo studies.

The Effect of Hypoxia and Hypoxia-Associated Pathways in the Regulation of Antitumor Response: Friends or Foes?

Hypoxia is an environmental stressor that is instigated by low oxygen availability. It fuels the progression of solid tumors by driving tumor plasticity, heterogeneity, stemness and genomic instability. Hypoxia metabolically reprograms the tumor microenvironment (TME), adding insult to injury to the acidic, nutrient deprived and poorly vascularized conditions that act to dampen immune cell function. Through its impact on key cancer hallmarks and by creating a physical barrier conducive to tumor survival, hypoxia modulates tumor cell escape from the mounted immune response. The tumor cell-immune cell crosstalk in the context of a hypoxic TME tips the balance towards a cold and immunosuppressed microenvironment that is resistant to immune checkpoint inhibitors (ICI). Nonetheless, evidence is emerging that could make hypoxia an asset for improving response to ICI. Tackling the tumor immune contexture has taken on an in silico, digitalized approach with an increasing number of studies applying bioinformatics to deconvolute the cellular and non-cellular elements of the TME. Such approaches have additionally been combined with signature-based proxies of hypoxia to further dissect the turbulent hypoxia-immune relationship. In this review we will be highlighting the mechanisms by which hypoxia impacts immune cell functions and how that could translate to predicting response to immunotherapy in an era of machine learning and computational biology.

Spheroid Culture Differentially Affects Cancer Cell Sensitivity to Drugs in Melanoma and RCC Models.

2D culture as a model for drug testing often turns to be clinically futile. Therefore, 3D cultures (3Ds) show potential to better model responses to drugs observed in vivo. In preliminary studies, using melanoma (B16F10) and renal (RenCa) cancer, we confirmed that 3Ds better mimics the tumor microenvironment. Here, we evaluated how the proposed 3D mode of culture affects tumor cell susceptibility to anti-cancer drugs, which have distinct mechanisms of action (everolimus, doxorubicin, cisplatin). Melanoma spheroids showed higher resistance to all used drugs, as compared to 2D. In an RCC model, such modulation was only observed for doxorubicin treatment. As drug distribution was not affected by the 3D shape, we assessed the expression of MDR1 and mTor. Upregulation of MDR1 in RCC spheroids was observed, in contrast to melanoma. In both models, mTor expression was not affected by the 3D cultures. By NGS, 10 genes related with metabolism of xenobiotics by cytochrome p450 were deregulated in renal cancer spheroids; 9 of them were later confirmed in the melanoma model. The differences between 3D models and classical 2D cultures point to the potential to uncover new non-canonical mechanisms to explain drug resistance set by the tumor in its microenvironment.

Effectiveness of Hyperbaric Oxygen Therapy in SARS-CoV-2 Pneumonia: The Primary Results of a Randomised Clinical Trial.

Mortality in COVID-19 is mainly associated with respiratory failure, cytokine storm, and macrophage activation. Oxygenation and anti-inflammatory effects of Hyperbaric Oxygen Therapy (HBOT) suggest that it is a promising adjunct treatment for COVID-19. Repeated sessions of HBO with standard COVID-19 therapy were used to reduce the inflammation and increase oxygenation. We evaluated the safety and efficacy of HBOT in avoiding the replacement ventilation and/or ECMO and its effect on the inflammatory process. Twenty-eight moderate-to-severe COVID-19 patients were randomized into control or HBOT group. HBOT patients participated in 5 hyperbaric sessions (60 min). Before and after each session blood gas levels and vital parameters were monitored. Blood samples were collected for extended biochemical tests, blood morphology and immunological assays. There were 3 deaths in the control, no deaths in the HBOT group. No adverse events leading to discontinuation of HBOT were observed and patients receiving HBOT required lower oxygen delivery. We observed decrease in CRP, ferritin and LDH and increase in CD3 in HBOT group compared to control. This study confirmed the feasibility and safety of HBOT in patients with COVID-19 and indicated HBOT can lead to alleviation of inflammation and partial restoration of T cell responses.

Effect of Hyperbaric Oxygenation on Blood Cytokines and Arginine Derivatives; No Evidence for Induction of Inflammation or Endothelial Injury.

(1) Background: Hyperbaric oxygen therapy (HBOT) uses 100% oxygen delivered at 1.5-3 times the atmospheric pressure in a specialised chamber to achieve supraphysiological oxygen tension in blood and tissues. Besides its target, HBOT may affect inflammation, endothelial function or angiogenesis. This study analysed the effect of HBOT on blood concentrations of factors that may affect these processes in patients with necrotizing soft-tissue infections (NSTI), aseptic bone necrosis (ABN) and idiopathic sudden sensory neural hearing loss (ISSNHL). (2) Methods: Concentrations asymmetric dimethylarginine (ADMA) and other arginine derivatives were measured with liquid chromatography/mass spectrometry, whereas ELISA was used to quantitate vascular endothelial growth factor (VEGF) and cytokines (IL-1, IL-4, IL-6, IL-10, TGF-ß) before and after HBOT in 80 patients (NSTI n = 21, ISSNHL n = 53, ABN n = 6). (3) Results: While some differences were noted between patient groups in ADMA and other arginine derivatives as well as in cytokine concentrations, HBOT did not affect any of these parameters. (4) Conclusions: While cytokines and arginine derivatives concentrations were modified by underlying pathology, hyperbaric oxygenation did not immediately modify it suggesting that it is neutral for inflammation and is not inducing endothelial injury.

The impact of physical training on neutrophil extracellular traps in young male athletes - a pilot study.

Neutrophils are an important component of the innate immune response against various pathogens. However, there is a lack of research concerning the effects of short intensive training on neutrophil functions, especially neutrophil extracellular traps (NET) formation. The study aim was to determine the effects of a 19-day training cycle on innate immunity among young male athletes. Six male ice hockey players (< 20 years old) from the Polish national team were monitored across a five-day training camp and after a return to normal club training. The first blood collection took place before training (T1), the second after the training camp (T2) and the third 14 days later (T3). The counts/concentrations of blood biochemical, immune and endocrine markers were compared across each training period. Creatine kinase activity tended to increase at T2 (546 ± 216 U·L-1) when compared to T1 (191 ± 111 U·L-1; p=0.063). Neutrophil extracellular traps formation and neutrophil counts also differed between training periods (p=0.042 and p=0.042, respectively). Neutrophil counts tended to decrease, in contrast to NET formation which tended to rise, at T2 in comparison to T1 (2.51 ± 0.45 vs 3.04 ± 0.47 109·L-1; 24 ± 13 vs 8 ± 15%, respectively). No significant differences in other leucocyte counts were observed. A short period of intensive training was accompanied by some muscle damage and inflammation, as evidenced by CK and NET up-regulation, whilst neutrophil counts were diminished in the blood. Thus, neutrophils and NET could be involved in muscle damage and local inflammatory processes following intensive physical training in young male athletes.

Right ventricular myocardial oxygen tension is reduced in monocrotaline-induced pulmonary hypertension in the rat and restored by myo-inositol trispyrophosphate.

Pulmonary hypertension (PH) initially results in compensatory right ventricular (RV) hypertrophy, but eventually in RV failure. This transition is poorly understood, but may be triggered by hypoxia. Measurements of RV oxygen tension (pO2) in PH are lacking. We hypothesized that RV hypoxia occurs in monocrotaline-induced PH in rats and that myo-inositol trispyrophosphate (ITPP), facilitating oxygen dissociation from hemoglobin, can relieve it. Rats received monocrotaline (PH) or saline (control) and 24 days later echocardiograms, pressure-volume loops were obtained and myocardial pO2 was measured using a fluorescent probe. In PH mean pulmonary artery pressure more than doubled (35 ± 5 vs. 15 ± 2 in control), RV was hypertrophied, though its contractility was augmented. RV and LV pO2 was 32 ± 5 and 15 ± 8 mmHg, respectively, in control rats. In PH RV pO2 was reduced to 18 ± 9 mmHg, while LV pO2 was unchanged. RV pO2 correlated with RV diastolic wall stress (negatively) and LV systolic pressure (positively). Acute ITPP administration did not affect RV or LV pO2 in control animals, but increased RV pO2 to 26 ± 5 mmHg without affecting LV pO2 in PH. RV oxygen balance is impaired in PH and as such can be an important target for PH therapy. ITPP may be one of such potential therapies.

Distinctive Properties of Endothelial Cells from Tumor and Normal Tissue in Human Breast Cancer.

Tumor microenvironments shape aggressiveness and are largely maintained by the conditions of angiogenesis formation. Thus, endothelial cells' (ECs) biological reactions are crucial to understand and control the design of efficient therapies. In this work, we used models of ECs to represent a breast cancer tumor site as well as the same, healthy tissue. Cells characterization was performed at the transcriptome and protein expression levels, and the cells functional biological responses (angiogenesis and permeability) were assessed. We showed that the expression of proteins specific to ECs (ACE+, VWF+), their differentiation (CD31+, CD 133+, CD105+, CD34-), their adhesion properties (ICAM-1+, VCAM-1+, CD62-L+), and their barrier formation (ZO-1+) were all downregulated in tumor-derived ECs. NGS-based differential transcriptome analysis confirmed CD31-lowered expression and pointed to the increase of Ephrin-B2 and SNCAIP, indicative of dedifferentiation. Functional assays confirmed these differences; angiogenesis was impaired while permeability increased in tumor-derived ECs, as further validated by the distinctly enhanced VEGF production in response to hypoxia, reflecting the tumor conditions. This work showed that endothelial cells differed highly significantly, both phenotypically and functionally, in the tumor site as compared to the normal corresponding tissue, thus influencing the tumor microenvironment.

Calendula officinalis Triterpenoid Saponins Impact the Immune Recognition of Proteins in Parasitic Nematodes.

The influence of triterpenoid saponins on subcellular morphological changes in the cells of parasitic nematodes remains poorly understood. Our study examines the effect of oleanolic acid glucuronides from marigold (Calendula officinalis) on the possible modification of immunogenic proteins from infective Heligmosomoides polygyrus bakeri larvae (L3). Our findings indicate that the triterpenoid saponins alter the subcellular morphology of the larvae and prevent recognition of nematode-specific proteins by rabbit immune-IgG. TEM ultrastructure and HPLC analysis showed that microtubule and cytoskeleton fibres were fragmented by saponin treatment. MASCOT bioinformatic analysis revealed that in larvae exposed to saponins, the immune epitopes of their proteins altered. Several mitochondrial and cytoskeleton proteins involved in signalling and cellular processes were downregulated or degraded. As possible candidates, the following set of recognised proteins may play a key role in the immunogenicity of larvae: beta-tubulin isotype, alpha-tubulin, myosin, paramyosin isoform-1, actin, disorganized muscle protein-1, ATP-synthase, beta subunit, carboxyl transferase domain protein, glutamate dehydrogenase, enolase (phosphopyruvate hydratase), fructose-bisphosphate aldolase 2, tropomyosin, arginine kinase or putative chaperone protein DnaK, and galactoside-binding lectin. Data are available via ProteomeXchange with identifier PXD024205.

Tumour angiogenesis normalized by myo-inositol trispyrophosphate alleviates hypoxia in the microenvironment and promotes antitumor immune response.

Pathologic angiogenesis directly responds to tumour hypoxia and controls the molecular/cellular composition of the tumour microenvironment, increasing both immune tolerance and stromal cooperation with tumour growth. Myo-inositol-trispyrophosphate (ITPP) provides a means to achieve stable normalization of angiogenesis. ITPP increases intratumour oxygen tension (pO2 ) and stabilizes vessel normalization through activation of endothelial Phosphatase-and-Tensin-homologue (PTEN). Here, we show that the tumour reduction due to the ITPP-induced modification of the tumour microenvironment by elevating pO2 affects the phenotype and properties of the immune infiltrate. Our main observations are as follows: a relative change in the M1 and M2 macrophage-type proportions, increased proportions of NK and CD8+ T cells, and a reduction in Tregs and Th2 cells. We also found, in vivo and in vitro, that the impaired access of PD1+ NK cells to tumour cells is due to their adhesion to PD-L1+ /PD-L2+ endothelial cells in hypoxia. ITPP treatment strongly reduced PD-L1/PD-L2 expression on CD45+/CD31+ cells, and PD1+ cells were more numerous in the tumour mass. CTLA-4+ cell numbers were stable, but level of expression decreased. Similarly, CD47+ cells and expression were reduced. Consequently, angiogenesis normalization induced by ITPP is the mean to revert immunosuppression into an antitumor immune response. This brings a key adjuvant effect to improve the efficacy of chemo/radio/immunotherapeutic strategies for cancer treatment.

A new in vitro model applied 90Y microspheres to study the effects of low dose beta radiation on colorectal cancer cell line in various oxygenation conditions.

We propose a new in vitro model to assess the impact of 90Y-microspheres derived low-dose beta radiation on colorectal cancer cell line under various oxygenation conditions that mimic the tumor environment. Cancer cells (HCT116) proliferation was assessed using Alamar Blue (AB) assay after 48, 72, and 96 h. FLUKA code assessed changes in cancer cell populations relative to the absorbed dose. In normoxia, mitochondrial activity measured by Alamar Blue after 48-72 h was significantly correlated with the number of microspheres (48 h: r = 0.87 and 72 h: r = 0.89, p < 0.05) and absorbed dose (48 h: r = 0.87 and 72 h: r = 0.7, p < 0.05). In hypoxia, the coefficients were r = 0.43 for both the number of spheres and absorbed dose and r = 0.45, r = 0.47, respectively. Impediment of cancer cell proliferation depended on the absorbed dose. Doses below 70 Gy could reduce colorectal cancer cell proliferation in vitro. Hypoxia induced a higher resistance to radiation than that observed under normoxic conditions. Hypoxia and radiation induced senescence in cultured cells. The new in vitro model is useful for the assessment of 90Y radioembolization effects at the micro-scale.

Endothelial Cells as Tools to Model Tissue Microenvironment in Hypoxia-Dependent Pathologies.

Endothelial cells (ECs) lining the blood vessels are important players in many biological phenomena but are crucial in hypoxia-dependent diseases where their deregulation contributes to pathology. On the other hand, processes mediated by ECs, such as angiogenesis, vessel permeability, interactions with cells and factors circulating in the blood, maintain homeostasis of the organism. Understanding the diversity and heterogeneity of ECs in different tissues and during various biological processes is crucial in biomedical research to properly develop our knowledge on many diseases, including cancer. Here, we review the most important aspects related to ECs' heterogeneity and list the available in vitro tools to study different angiogenesis-related pathologies. We focus on the relationship between functions of ECs and their organo-specificity but also point to how the microenvironment, mainly hypoxia, shapes their activity. We believe that taking into account the specific features of ECs that are relevant to the object of the study (organ or disease state), especially in a simplified in vitro setting, is important to truly depict the biology of endothelium and its consequences. This is possible in many instances with the use of proper in vitro tools as alternative methods to animal testing.

Children were less frequently infected with SARS-CoV-2 than adults during 2020 COVID-19 pandemic in Warsaw, Poland.

Clinical data suggest that during the current COVID-19 pandemic, children are less prone than adults to SARS-CoV-2 infection. Our purpose was to determine the frequency of SARS-CoV-2 in children vs. adults during the 2020 pandemic in Warsaw, Poland, and to investigate whether RSV and/or influenza A/B infections were associated with SARS-CoV-2 infections. We present results of RT-PCR tests for SARS-CoV-2 performed in Warsaw, Poland. Some of the pediatric subjects were also PCR-tested for RSV, and A and B influenza. We compared the test results from the four groups of symptomatic and asymptomatic subjects: 459 symptomatic pediatric patients (children 0-18 years old), 1774 symptomatic adults, 445 asymptomatic children, and 239 asymptomatic adults. 3.26% (15/459) of symptomatic pediatric patients were positive for SARS-CoV-2 in contrast to 5.58% (99/1774) of symptomatic adults (p = 0.0448). There were no SARS-CoV-2 positive cases in the group of asymptomatic children (0/445) and two positive cases in the group of asymptomatic adults (2/239), i.e., 0.83%. In the group of symptomatic pediatric patients, 17.14% (6/35) (p = 0.0002) were positive for RSV, 8.16% (4/49) were positive for influenza A, and 2.04% (1/49), thus 10.20% (5/49) (p = 0.0176) for influenza A/B. Children were less prone to SARS-CoV-2 infection than the adults during the COVID-19 pandemic in Warsaw. Higher percentage of symptomatic children was infected with RSV or influenza A/B than with SARS-CoV-2. This suggests a necessity for the testing for all these viruses for an early identification and isolation of SARS-CoV-2-positive patients for an ensuing 2020 autumn return of COVID-19.

Hypoxia-Mediated Decrease of Ovarian Cancer Cells Reaction to Treatment: Significance for Chemo- and Immunotherapies.

Hypoxia, a common factor ruling the microenvironment composition, leads to tumor progression. In this hypoxic context, cytokines and cells cooperate to favor cancer development and metastasis. Tumor hypoxia is heterogeneously distributed. Oxygen gradients depend on the vicinity, functionality of blood vessels, and oxygen ability to diffuse into surrounding tissues. Thus, the vasculature state modulates the microenvironment of the tumor cells. Cells sense and react to small variations in oxygen tension, which explains the lack of tumor cells' unicity in their reaction to drugs. Ovarian cancers are highly hypoxia-dependent, ascites worsening the access to oxygen, in their reactions to both chemotherapy and new immunotherapy. Consequently, hypoxia affects the results of immunotherapy, and is thus, crucial for the design of treatments. Controlling key immunosuppressive factors and receptors, as well as immune checkpoint molecule expression on tumor, immune and stromal cells, hypoxia induces immunosuppression. Consequently, new approaches to alleviate hypoxia in the tumor microenvironment bring promises for ovarian cancer immunotherapeutic strategies. This review focuses on the effects of hypoxia in the microenvironment and its consequences on tumor treatments. This opens the way to innovative combined treatments to the advantage of immunotherapy outcome in ovarian cancers.