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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., Competing Interests: The authors declare no competing interest.

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., (© 2023. The Author(s).)

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., (© 2022. The Society for In Vitro Biology.)

Tumour microenvironment determines the fate of treatments. Reconstitution of tumour conditions is mandatory for alternative in vitro methods devoted to cancer development and the selection of therapeutic strategies. This work describes a 3D model of melanoma growth in its environment. Introducing means to mimic tumour angiogenesis, which turns on tumour progression, the model shows that melanoma tumour spheroids allow reconstitution of solid tumours with stromal cells. Angiogenesis evidenced the differential recruitment of endothelial cells (EC) from early progenitors (EEPCs) to mature ECs. Hypoxia was the key parameter that selected and stabilized melanoma cancer stem like cells (CSCs) phenotype based on aldehyde dehydrogenase expression as the best criterion. The 3D-tumour-model demonstrated the distinct reactivity of ECs toward tumour cells in terms of cellular cross-talk and humoral response. Intra-spheroid cell-to-cell membrane dye exchanges, mediated by intercellular interactions, uncovered the melanoma-to-EEPC cooperation. The resulting changes in tumour milieu were evidenced by the chemokinic composition and hypoxia-related variations in microRNA expression assessed in each cellular component of the spheroids. This method brings new tools to decipher the molecular mechanism of tumour-mediated cell recruitment and for in vitro assessment of therapeutic approaches., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.

Cellular interactions in the tumor stroma play a major role in cancer progression but can also induce tumor rejection. To explore the role of endothelial cells in these interactions, we used an in vitro three-dimensional collagen matrix model containing a cytotoxic T lymphocyte CTL clone (M4.48), autologous tumor cells (M4T), and an endothelial cell (M4E) line that are all derived from the same tumor. We demonstrate in this study that specific killing of the endothelial cells by the CTL clone required the autologous tumor cells and involved Ag cross-presentation. The formation of gap junctions between endothelial and tumor cells is required for antigenic peptide transfer to endothelial cells that are then recognized and eliminated by CTL. Our results indicate that gap junctions facilitate an effective CTL-mediated destruction of endothelial cells from the tumor microenvironment that may contribute to the control of tumor progression.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: Heme oxygenase-1 (HO-1) is a cytoprotective, proangiogenic and anti-inflammatory enzyme that is often upregulated in tumors. Overexpression of HO-1 in melanoma cells leads to enhanced tumor growth, augmented angiogenesis and resistance to anticancer treatment. The effect of HO-1 in host cells on tumor development is, however, hardly known. Methods and results: To clarify the effect of HO-1 expression in host cells on melanoma progression, C57BL/6xFvB mice of different HO-1 genotypes, HO-1+/+, HO-1+/−, and HO-1−/−, were injected with the syngeneic wild-type murine melanoma B16(F10) cell line. Lack of HO-1 in host cells did not significantly influence the host survival. Nevertheless, in comparison to the wild-type counterparts, the HO-1+/− and HO-1−/− males formed bigger tumors, and more numerous lung nodules; in addition, more of them had liver and spleen micrometastases. Females of all genotypes developed at least 10 times smaller tumors than males. Of importance, the growth of primary and secondary tumors was completely blocked in HO-1+/+ females. This was related to the increased infiltration of leukocytes (mainly lymphocytes T) in primary tumors. Conclusions: Although HO-1 overexpression in melanoma cells can enhance tumor progression in mice, its presence in host cells, including immune cells, can reduce growth and metastasis of melanoma. [ABSTRACT FROM AUTHOR]