Hypoxia as a potential inducer of immune tolerance, tumor plasticity and a driver of tumor mutational burden: Impact on cancer immunotherapy.

In cancer patients, immune cells are often functionally compromised due to the immunosuppressive features of the tumor microenvironment (TME) which contribute to the failures in cancer therapies. Clinical and experimental evidence indicates that developing tumors adapt to the immunological environment and create a local microenvironment that impairs immune function by inducing immune tolerance and invasion. In this context, microenvironmental hypoxia, which is an established hallmark of solid tumors, significantly contributes to tumor aggressiveness and therapy resistance through the induction of tumor plasticity/heterogeneity and, more importantly, through the differentiation and expansion of immune-suppressive stromal cells. We and others have provided evidence indicating that hypoxia also drives genomic instability in cancer cells and interferes with DNA damage response and repair suggesting that hypoxia could be a potential driver of tumor mutational burden. Here, we reviewed the current knowledge on how hypoxic stress in the TME impacts tumor angiogenesis, heterogeneity, plasticity, and immune resistance, with a special interest in tumor immunogenicity and hypoxia targeting. An integrated understanding of the complexity of the effect of hypoxia on the immune and microenvironmental components could lead to the identification of better adapted and more effective combinational strategies in cancer immunotherapy. Clearly, the discovery and validation of therapeutic targets derived from the hypoxic tumor microenvironment is of major importance and the identification of critical hypoxia-associated pathways could generate targets that are undeniably attractive for combined cancer immunotherapy approaches.

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.

Endometriosis development in relation to hypoxia: a murine model study.

BACKGROUND: Endometriosis, due to its ambiguous symptoms, still remains one of the most difficult female diseases to treat, with an average diagnosis time of 7-9 years. The changing level of hypoxia plays an important role in a healthy endometrium during menstruation and an elevated expression of the hypoxia-inducible factor 1-alpha (HIF-1α) has been demonstrated in ectopic endometria. HIF-1α mediates the induction of proangiogenic factors and the development of angiogenesis is a critical step in the establishment and pathogenesis of endometriosis. Although the inhibition of angiogenesis has been proposed as one of the actionable therapeutic modalities, vascular normalization and re-oxygenation may become a possible new approach for therapeutic intervention. METHODS: Our goal was to investigate whether a selected murine model of endometriosis would be suitable for future studies on new methods for treating endometriosis. Non-invasive, high-resolution ultrasound-monitored observation was selected as the preclinical approach to obtain imaging of the presence and volume of the endometriotic-like lesions. The EF5 (2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide) compound that selectively binds to reduced proteins in hypoxic cells was used for hypoxia detection. The expression of Pten and other crucial genes linking endometriosis and hypoxia were also assessed. RESULTS: Using EF5, a pentafluorinated derivative of the 2-nitroimidazole that is metabolically reduced by oxygen-inhibitable nitroreductase, we confirmed that hypoxia did develop in the selected model and was detected in uterine and ectopic endometriotic lesions. Moreover, the changes in oxygen tension also influenced the expression level of significant genes related to endometriosis, like Pten, Trp53, Hif1a, Epas1, and Vegfa. Their strong modulation evidenced here is indicative of model reliability. Using high-resolution ultrasound-based imaging, we present a non-invasive method of visualization that enables the detection and observation of lesion evolution throughout the duration of the experiment, which is fundamental for further preclinical studies and treatment evaluation. CONCLUSIONS: The selected model and method of visualization appear to be suitable for the study of new treatment strategies based on hypoxia alleviation and blood flow restoration.

Molecular tracking of interactions between progenitor and endothelial cells via Raman and FTIR spectroscopy imaging: a proof of concept of a new analytical strategy for in vitro research.

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24 h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions.

Polyploidy formation in cancer cells: How a Trojan horse is born.

Ploidy increase has been shown to occur in different type of tumors and participate in tumor initiation and resistance to the treatment. Polyploid giant cancer cells (PGCCs) are cells with multiple nuclei or a single giant nucleus containing multiple complete sets of chromosomes. The mechanism leading to formation of PGCCs may depend on: endoreplication, mitotic slippage, cytokinesis failure, cell fusion or cell cannibalism. Polyploidy formation might be triggered in response to various genotoxic stresses including: chemotherapeutics, radiation, hypoxia, oxidative stress or environmental factors like: air pollution, UV light or hyperthermia. A fundamental feature of polyploid cancer cells is the generation of progeny during the reversal of the polyploid state (depolyploidization) that may show high aggressiveness resulting in the formation of resistant disease and tumor recurrence. Therefore, we propose that modern anti-cancer therapies should be designed taking under consideration polyploidization/ depolyploidization processes, which confer the polyploidization a hidden potential similar to a Trojan horse delayed aggressiveness. Various mechanisms and stress factors leading to polyploidy formation in cancer cells are discussed in this review.

Tumour suppressor PTEN activity is differentially inducible by myo-inositol phosphates.

Tumour evolution and efficacy of treatments are controlled by the microenvironment, the composition of which is primarily dependent on the angiogenic reaction to hypoxic stress. Tumour angiogenesis normalization is a challenge for adjuvant therapy strategies to chemo-, radio- and immunotherapeutics. Myo-inositol trispyrophosphate (ITPP) appears to provide the means to alleviate hypoxia in the tumour site by a double molecular mechanism. First, it modifies the properties of red blood cells (RBC) to release oxygen (O2 ) in the hypoxic sites more easily, leading to a rapid and stable increase in the partial pressure of oxygen (pO2 ). And second, it activates the endothelial phosphatase and tensin homologue deleted on Chromosome 10 (PTEN). The hypothesis that stable normalization of the vascular system is due to the PTEN, a tumour suppressor and phosphatase which controls the proper angiogenic reaction was ascertained. Here, by direct biochemical measurements of PTEN competitive activity in relation to PIP2 production, we show that the kinetics are complex in terms of the activation/inhibition effects of ITPP with an inverted consequence towards the kinase PI3K. The use of the surface plasmon resonance (SPR) technique allowed us to demonstrate that PTEN binds inositol derivatives differently but weakly. This method permitted us to reveal that PTEN is highly sensitive to the local concentration conditions, especially that ITPP increases the PTEN activity towards PIP3, and importantly, that PTEN affinity for ITPP is considerably increased by the presence of PIP3, as occurs in vivo. Our approach demonstrates the validity of using ITPP to activate PTEN for stable vessel normalization strategies.

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.

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.

Heme Oxygenase-1 Has a Greater Effect on Melanoma Stem Cell Properties Than the Expression of Melanoma-Initiating Cell Markers.

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.

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.

The GTPase-activating protein-related domain of neurofibromin interacts with MC1R and regulates pigmentation-mediated signaling in human melanocytes.

The melanocortin 1 receptor (MC1R) is a G-protein coupled receptor (GPCR) which plays a major role in controlling melanogenesis. A large body of evidence indicates that GPCRs are part of large protein complexes that are critical for their signal transduction properties. Among proteins which may affect MC1R signaling, neurofibromin (Nf1), a GTPase activating protein (GAP) for Ras, is of special interest as it regulates adenylyl cyclase activity and ERK signaling, two pathways involved in MC1R signaling. Moreover, mutations in this gene encoding Nf1 are responsible for neurofibromatosis type I, a disease inducing hyperpigmented flat skin lesions. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer experiments we demonstrated a physical interaction of Nf1 with MC1R. In particular, the GAP domain of Nf1 directly and constitutively interacts with MC1R in melanocytes. Pharmacologic and genetic approaches revealed that the GAP activity of Nf1 is important to regulate intracellular signaling pathways involved in melanogenesis and, consequently, melanogenic enzyme expression and melanin production. These finding shed new light on the understanding and cure of skin pigmentation disorders.

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.

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.

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.

Initial experience with novel Embocure Plus microspheres for transarterial chemoembolization (TACE) of liver metastatic colorectal cancer tumours - a clinical and in vitro study.

PURPOSE: Transarterial chemoembolization (TACE) is currently recommended for unresectable intrahepatic tumours with no vascular invasion or metastasis to other organs. It is based on drug-eluting microspheres pre-loaded with chemotherapeutics, which are injected selectively into vessels supplying the tumour, to embolize them inducing ischaemia, and elute the drug, to induce tumour response. We present our initial experience with novel irinotecan- loaded Embocure Plus microspheres in patients with metastatic colorectal cancer tumours in the liver, and their effect on HCT-116 cell cultures in vitro. MATERIAL AND METHODS: Three consecutive male patients (median age 62 [50-76] years) with liver metastatic colorectal cancer tumours were selected. All patients had a pre-procedure contrast-enhanced computed tomography, confirming multiple metastatic liver tumours (mean tumour diameter = 42 mm; range: 14-77 mm) and periprocedural dyna-CT scans for rapid treatment results assessment. In vitro: Human colon HCT116 cancer cell line was cultured, irinotecan loaded Embocure Plus microspheres were added. Cultures were assessed after 24 hours and 72 hours of incubation in normoxia or hypoxia. RESULTS: All embolizations were technically successful, and no complications were observed. Stabilization of the targeted metastatic liver tumours in all patients was noted. In vitro: Significant decrease of the growth of HTC 116 cell lines were observed in controls compared to cells treated with Embocure Plus loaded with irinotecan in normoxia and hypoxia after 48 and 72 hours. We observed a tendency for less inhibited cell proliferation in low-oxygen conditions. CONCLUSIONS: TACE therapy of liver metastatic tumours shows satisfactory results and a low complication rate. Embocure Plus microspheres are safe and technically feasible for superselective chemoembolization of metastatic colorectal cancer liver tumour. Dyna-CT can be used for assessment of treatment results during repeated TACE procedures.

Treatment of hypoxia-dependent cardiovascular diseases by myo-inositol trispyrophosphate (ITPP)-enhancement of oxygen delivery by red blood cells.

Heart failure is a consequence of progression hypoxia-dependent tissue damages. Therapeutic approaches to restore and/or protect the healthy cardiac tissue have largely failed and remain a major challenge of regenerative medicine. The myo-inositol trispyrophosphate (ITPP) is a modifier of haemoglobin which enters the red blood cells and modifies the haemoglobin properties, allowing for easier and better delivery of oxygen by the blood. Here, we show that this treatment approach in an in vivo model of myocardial infarction (MI) results in an efficient protection from heart failure, and we demonstrate the recovery effect on post-MI left ventricular remodelling in the rat model. Cultured cardiomyocytes used to study the molecular mechanism of action of ITPP in vitro displayed the fast stimulation of HIF-1 upon hypoxic conditions. HIF-1 overexpression was prevented by ITPP when incorporated into red blood cells applied in a model of blood-perfused cardiomyocytes coupling the dynamic shear stress effect to the enhanced O2 supply by modification of haemoglobin ability to release O2 in hypoxia. ITPP treatment appears a breakthrough strategy for the efficient and safe treatment of hypoxia- or ischaemia-induced injury of cardiac tissue.

A Role of NKR-P1A (CD161) and Lectin-like Transcript 1 in Natural Cytotoxicity against Human Articular Chondrocytes.

Normal cartilage cells are susceptible to lysis by NK cells. This phenomenon may play a role in immune cartilage destruction; however, the mechanisms of chondrocyte recognition by NK cells remain poorly understood. Therefore, the aim of this study was to reveal a possible role of NKR-P1A/lectin-like transcript 1 (LLT1) interaction in NK cell-mediated cytotoxicity against normal human articular chondrocytes. Chondrocytes were isolated from articular cartilage obtained during talonavicular joint surgery. PBMC or polyclonal NK cells isolated from normal donors served as effector cells. Cell-mediated cytotoxicity against chondrocytes was evaluated by means of 18-h 51Cr-release assay. Specific mRNA expression was evaluated by classical and quantitative RT-PCR, and proteins were detected by Western blot analysis. We found that lysis of articular chondrocytes by PBMC or polyclonal NK cells was potentiated by stimulation with IL-2. Stimulation of effector cells with IL-2 downregulated mRNA expression of inhibitory NKR-P1A NK cell receptor, and blocking of NKR-P1A with specific mAbs resulted in increased chondrocyte killing. Chondrocytes constitutively expressed LLT1, a ligand of NKR-P1A. LLT1 expression by chondrocytes could be upregulated by IL-1α and TNF. Chondrocyte treatment with IL-1α resulted in their increased resistance to killing by natural cytotoxic cells. This could be reversed by blocking of NKR-P1A. These results show that susceptibility of normal articular chondrocytes to lysis by NK cells is modulated by NKR-P1A/LLT1 interactions. Thus, NKR-P1A/LLT1 interaction might provide some novel target for therapeutic interventions in the course of pathological cartilage injury.

Activity of the human immortalized endothelial progenitor cell line HEPC-CB.1 supporting in vitro angiogenesis.

The human HEPC-CB.1 cell line with many characteristics of endothelial progenitor cells (EPC) was tested for its proangiogenic properties as a potentially therapeutic compound. HEPC-CB.1 cells' potential to differentiate into endothelial cells was revealed after treating the cells with a mixture of ATRA, cAMP and VEGF, as shown by the reduced expression levels of CD133, CD271 and CD90 antigens, augmentation of CD146 and CD31, and a decrease in cell clonogenicity. The cooperation of HEPC-CB.1 with the endothelial cell line HSkMEC.2 resulted in the formation of a common network. Tube formation was significantly more effective when resulting from HEPC-CB.1 and HSkMEC.2 cell co-culture as compared to a monoculture of each cell line. The exocrine mechanism of HEPC-CB.1 and HSkMEC.2 cross talk by secreted factors was evidenced using the HEPC-CB.1 supernatant to increase the efficacy of HSkMEC.2 tube formation. The proangiogenic factors produced by HEPC-CB.1 were identified using cytokine antibody array. Out of 120 examined factors, the HEPC-CB.1 cell line produced 63, some with known angiogenic activity. As in vivo the angiogenic process occurs at low oxygen tension, it was observed that in hypoxia, the production of defined factors was augmented. The presented results demonstrate that HEPC-CB.1 cells are able to both cooperate and integrate in a newly formed network and produce factors that help the network formation. The results suggest that HEPC-CB.1 cells are indeed endothelial progenitors and may prove to be an effective tool in regenerative medicine.

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.