Our understanding of glioma biology has relied heavily on the use of cell lines and xenograft animal models. However, the recent development of transgenic mouse models offers a unique opportunity to examine the pathophysiology of these tumors in immunocompetent models in vivo. Transgenic models are highly informative for a number of reasons. First, the resulting tumors are genetically and histologically similar to human gliomas. Second, transgenic models allow the study of causality of genetic/pathway alterations reminiscent of human gliomas. Third, new therapies can be tested in established tumors to truly evaluate their potential efficacy. This review describes the available technologies involved in transgenic and knockout mouse modeling, including the generation of cell-type-specific genetic alterations. Finally, genetics are discussed with a focus on how transgenic murine gliomas recapitulate alterations found in human counterparts.
Background: Triple-negative breast cancer (TNBC) is a uniquely aggressive cancer with high rates of relapse due to resistance to chemotherapy, the current major option for treatment. TNBC expresses higher levels of programmed cell death-ligand 1 (PD-L1) compared to other breast cancers, providing the rationale for the recently approved immunotherapy with anti-PD-L1 monoclonal antibodies (mAbs). A huge effort is dedicated to identify actionable biomarkers that may allow for novel combination therapies with immune-checkpoint blockade in TNBC. Platelet-derived growth factor receptor β (PDGFRβ) is highly expressed in mesenchymal invasive TNBC, both on tumor cells and tumor microenvironment (TME). We recently proved that tumor growth and lung metastases are impaired in mouse models of human TNBC by a high efficacious PDGFRβ aptamer. Hence, we aimed at investigating the effectiveness of a novel combination treatment with the PDGFRβ aptamer and anti-PD-L1 mAbs in TNBC.Methods: The targeting ability of the anti-human PDGFRβ aptamer toward the murine receptor was verified by streptavidin-biotin assays and confocal microscopy, and its inhibitory function by transwell migration assays on PDGFRβ-positive cells. The anti-proliferative effects of the PDGFRβ aptamer/anti-PD-L1 mAbs combination was assessed in human MDA-MB-231 and murine 4T1 TNBC cells, both grown as monolayer or co-cultured with lymphocytes. Tumor cell lysis and cytokines secretion by lymphocytes were analyzed by LDH quantification and ELISA, respectively. Orthotopic 4T1 xenografts in syngeneic mice were used for dissecting the effect of aptamer/mAbs combination on tumor growth, metastasis and lymphocytes infiltration. Ex vivo analyses through immunohistochemistry, RT-qPCR and immunoblotting were performed. Results: We show that the PDGFRβ aptamer potentiates the anti-proliferative activity of anti-PD-L1 mAbs on both human and murine TNBC cells, according to its human/mouse cross-reactivity. Further, by binding to activated human and mouse lymphocytes, the aptamer enhances the anti-PD-L1 mAbs-induced cytotoxicity of lymphocytes against tumor cells. Importantly, the aptamer heightens the antibody efficacy in inhibiting tumor growth and lung metastases in a syngeneic mouse model by acting on both TME and cancer cells. Conclusion: Co-treatment of PDGFRβ aptamer with anti-PD-L1 mAbs is a viable strategy, thus providing for the first an evidence of the efficacy of PDGFRβ/PD-L1 co-targeting combination therapy in TNBC.
Background: Triple-negative breast cancer (TNBC) is a uniquely aggressive cancer with high rates of relapse due to resistance to chemotherapy. TNBC expresses higher levels of programmed cell death-ligand 1 (PD-L1) compared to other breast cancers, providing the rationale for the recently approved immunotherapy with anti-PD-L1 monoclonal antibodies (mAbs). A huge effort is dedicated to identify actionable biomarkers allowing for combination therapies with immune-checkpoint blockade. Platelet-derived growth factor receptor β (PDGFRβ) is highly expressed in invasive TNBC, both on tumor cells and tumor microenvironment. We recently proved that tumor growth and lung metastases are impaired in mouse models of human TNBC by a high efficacious PDGFRβ aptamer. Hence, we aimed at investigating the effectiveness of a novel combination treatment with the PDGFRβ aptamer and anti-PD-L1 mAbs in TNBC.Methods: The targeting ability of the anti-human PDGFRβ aptamer toward the murine receptor was verified by streptavidin-biotin assays and confocal microscopy, and its inhibitory function by transwell migration assays. The anti-proliferative effects of the PDGFRβ aptamer/anti-PD-L1 mAbs combination was assessed in human MDA-MB-231 and murine 4T1 TNBC cells, both grown as monolayer or co-cultured with lymphocytes. Tumor cell lysis and cytokines secretion by lymphocytes were analyzed by LDH quantification and ELISA, respectively. Orthotopic 4T1 xenografts in syngeneic mice were used for dissecting the effect of aptamer/mAb combination on tumor growth, metastasis and lymphocytes infiltration. Ex vivo analyses through immunohistochemistry, RT-qPCR and immunoblotting were performed. Results: We show that the PDGFRβ aptamer potentiates the anti-proliferative activity of anti-PD-L1 mAbs on both human and murine TNBC cells, according to its human/mouse cross-reactivity. Further, by binding to activated human and mouse lymphocytes, the aptamer enhances the anti-PD-L1 mAb-induced cytotoxicity of lymphocytes against tumor cells. Importantly, the aptamer heightens the antibody efficacy in inhibiting tumor growth and lung metastases in mice. It acts on both tumor cells, inhibiting Akt and ERK1/2 signaling pathways, and immune populations, increasing intratumoral CD8+T cells and reducing FOXP3+Treg cells. Conclusion: Co-treatment of PDGFRβ aptamer with anti-PD-L1 mAbs is a viable strategy, thus providing for the first an evidence of the efficacy of PDGFRβ/PD-L1 co-targeting combination therapy in TNBC.
Introduction. Autophagy, a catabolic process of protein and organelle recycling by transferring defective cytoplasm and organelles into double-membraned vesicles to degrade and regenerate materials, plays a critical role in maintaining energy homeostasis. Autophagy also protects against stress and infection, participates at the development of autoimmune disease. In recent years, the existence of alternative blood circulation system in tumors, vasculogenic mimicry (VM), which can partially compensate the lack of nutrients and oxygen under the hypoxic conditions, has been described. Objective. To elucidate the relationship between autophagy and VM. Materials and methods. In this study we used 2D- and 3D-culturing of melanoma cells derived from surgical species of patients with disseminated melanoma, electrophoresis and western blot, knockdown of the genes by using small interfering RNA (siRNA), flow cytometry, fluorescence microscopy. Results. We detected the basal level autophagy by examining the expression of autophagy-specific protein (LC-3B) by flow cytometry and cellular immunofluorescence staining by monodancylcadaverine. Both assays are the markers of autophagy late stage. Here we show that the level of autophagy in melanoma cells mel P, participated in capillary-like structures (CLS) formation in matrigel, was considerably higher than in mel Me cells which do not involve in VM. To explore the function of autophagy in the ability of melanoma cells to form CLS 3-methyladenine (3-MA) or chloroquine - inhibitors of initiation and terminal stage of autophagy - were used. Both inhibitors reduced the ability of melanoma cells to engage in VM. The data obtained were confirmed by siRNA-mediated gene silencing of BECN1 involved in the initiation of autophagy and ATG5 gene which is considered to be a marker of late stage of autophagy. Knockdown of BECN1 or ATG5 in mel P melanoma cells reduced the level of protein Beclin-1 and Atg5 about 70-75 %, and suppressed CLS formation in matrigel. Melanoma cells with the ATG5 gene knockdown changed the shape but maintained the ability to migrate and recognize each other, the formation of CLS was not observed. Low molecular weight VM inhibitor LCS-1269, significantly reduced the basic level of autophagy. Conclusion. Our data indicate that autophagy participates in CLS formation, and inhibition of autophagy suppresses CLS formation. We suggest that autophagy plays a dual role in the survival and development of tumors: autophagy helps cancer cells against environment stress and provides a temporary survival pathway by promoting energy regeneration, autophagy also promotes VM formation which supplies nutrients and oxygen to less vascularized area of tumor.
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