Effects of an Adipose Mesenchymal Stem Cell-Derived Conditioned medium and TGF-β1 on Human Keratinocytes In Vitro

Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro

Erythropoietin receptor regulates tumor mitochondrial biogenesis through iNOS and pAKT

Erythropoietin receptor (EPOR) is widely expressed in healthy and malignant tissues. In certain malignancies, EPOR stimulates tumor growth. In healthy tissues, EPOR controls processes other than erythropoiesis, including mitochondrial metabolism. We hypothesized that EPOR also controls the mitochondrial metabolism in cancer cells. To test this hypothesis, we generated EPOR-knockdown cancer cells to grow tumor xenografts in mice and analyzed tumor cellular respiration via high-resolution respirometry. Furthermore, we analyzed cellular respiratory control, mitochondrial content, and regulators of mitochondrial biogenesis in vivo and in vitro in different cancer cell lines. Our results show that EPOR controls tumor growth and mitochondrial biogenesis in tumors by controlling the levels of both, pAKT and inducible NO synthase (iNOS). Furthermore, we observed that the expression of EPOR is associated with the expression of the mitochondrial marker VDAC1 in tissue arrays of lung cancer patients, suggesting that EPOR indeed helps to regulate mitochondrial biogenesis in tumors of cancer patients. Thus, our data imply that EPOR not only stimulates tumor growth but also regulates tumor metabolism and is a target for direct intervention against progression

Iron- and erythropoietin-resistant anemia in a spontaneous breast cancer mouse model

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, where functional iron deficiency and iron-restricted erythropoiesis is induced by increased hepcidin levels in response to IL-6 elevation. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high IL-6 levels and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could neither be prevented by iron nor erythropoietin (EPO) treatment. Trp53floxWapCre mice are the first mouse model where EPO-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels that do not respond to EPO

Targeting neovascularization and respiration of tumor grafts grown on chick embryo chorioallantoic membranes

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin$^{®}$, AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research

Targeting neovascularization and respiration of tumor grafts grown on chick embryo chorioallantoic membranes.

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin®, AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research

Voluntary exercise does not always suppress lung cancer progression

Summary: Physical exercise can lower lung cancer incidence. However, its effect on lung cancer progression is less understood. Studies on exercising mice have shown decreased ectopic lung cancer growth through the secretion of interleukin-6 from muscles and the recruitment of natural killer (NK) cells to tumors. We asked if exercise suppresses lung cancer in an orthotopic model also. Single-housed C57Bl/6 male mice in cages with running wheels were tail vein-injected with LLC1.1 lung cancer cells, and lung tumor nodules were analyzed. Exercise did not affect lung cancer. Therefore, we also tested the effect of exercise on a subcutaneous LLC1 tumor and a tail vein-injected B16F10 melanoma model. Except for one case of excessive exercise, tumor progression was not influenced. Moderately exercising mice did not increase IL-6 or recruit NK cells to the tumor. Our data suggest that the exercise dose may dictate how efficiently the immune system is stimulated and controls tumor progression

Erythropoietin receptor regulates tumor mitochondrial biogenesis through iNOS and pAKT

Erythropoietin receptor (EPOR) is widely expressed in healthy and malignant tissues. In certain malignancies, EPOR stimulates tumor growth. In healthy tissues, EPOR controls processes other than erythropoiesis, including mitochondrial metabolism. We hypothesized that EPOR also controls the mitochondrial metabolism in cancer cells. To test this hypothesis, we generated EPOR-knockdown cancer cells to grow tumor xenografts in mice and analyzed tumor cellular respiration via high-resolution respirometry. Furthermore, we analyzed cellular respiratory control, mitochondrial content, and regulators of mitochondrial biogenesis in vivo and in vitro in different cancer cell lines. Our results show that EPOR controls tumor growth and mitochondrial biogenesis in tumors by controlling the levels of both, pAKT and inducible NO synthase (iNOS). Furthermore, we observed that the expression of EPOR is associated with the expression of the mitochondrial marker VDAC1 in tissue arrays of lung cancer patients, suggesting that EPOR indeed helps to regulate mitochondrial biogenesis in tumors of cancer patients. Thus, our data imply that EPOR not only stimulates tumor growth but also regulates tumor metabolism and is a target for direct intervention against progression