Jing Zhang

Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin (Epo)-independent growth.

BACKGROUND: Therapeutic antibodies targeting programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis induce potent and durable anti-tumor responses in multiple types of cancers. However, only a subset of patients benefits from anti-PD-1/PD-L1 therapies. As a negative regulator of anti-tumor immunity, TGF-β impairs the efficacy of anti-PD-1/PD-L1 and induces drug resistance. Developing a novel treatment strategy to simultaneously block PD-1/PD-L1 and TGF-β would be valuable to enhance the effect of anti-PD-1/PD-L1 and relieve drug resistance. METHODS: Based on the Check-BODY™ technology platform, we developed an anti-TGF-β/PD-L1 bispecific antibody YM101. The bioactivity of the anti-TGF-β moiety was determined by Smad-luciferase reporter assay, transwell assay, western blotting, CCK-8, and flow cytometry. The bioactivity of the anti-PD-L1 moiety was measured by T cell activation assays. EMT-6, CT26, and 3LL tumor models were used to investigate the anti-tumor activity of YM101 in vivo. RNA-seq, immunohistochemical staining, and flow cytometry were utilized to analyze the effect of YM101 on the tumor microenvironment. RESULTS: YM101 could bind to TGF-β and PD-L1 specifically. In vitro experiments showed that YM101 effectively counteracted the biological effects of TGF-β and PD-1/PD-L1 pathway, including activating Smad signaling, inducing epithelial-mesenchymal transition, and immunosuppression. Besides, in vivo experiments indicated the anti-tumor activity of YM101 was superior to anti-TGF-β and anti-PD-L1 monotherapies. Mechanistically, YM101 promoted the formation of 'hot tumor': increasing the numbers of tumor infiltrating lymphocytes and dendritic cells, elevating the ratio of M1/M2, and enhancing cytokine production in T cells. This normalized tumor immune microenvironment and enhanced anti-tumor immune response might contribute to the robust anti-tumor effect of YM101. CONCLUSION: Our results demonstrated that YM101 could simultaneously block TGF-β and PD-L1 pathways and had a superior anti-tumor effect compared to the monotherapies.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an important active protein for the induction of angiogenesis and improvement in cardiac function after myocardial ischemia; however, the lack of a delivery system targeted to the injured myocardium reduces the local therapeutic efficacy of VEGF and increases its possible adverse effects. METHODS AND RESULTS: We produced a fusion protein (CBD-VEGF) consisting of VEGF and a collagen-binding domain (CBD). The fusion protein specifically bound to type I collagen in vitro. In addition, CBD-VEGF promoted human umbilical vein endothelial cell proliferation after binding to collagen, which indicates that it retained both growth factor activity and collagen-binding ability. When implanted subcutaneously in rats, collagen membranes loaded with CBD-VEGF were significantly vascularized. After it was injected into rats with acute myocardial infarction, CBD-VEGF was largely retained in the cardiac extracellular matrix, in which collagen I was rich. Four weeks after VEGF or CBD-VEGF was injected into the infarct border zone, cardiac function detected by echocardiography and hemodynamics was preserved in the CBD-VEGF group. Administration of CBD-VEGF also induced reduction of scar size, whereas native VEGF did not have these effects. In addition, a significant increase in the number of capillary vessels in infarcted hearts was found in the CBD-VEGF group. CONCLUSIONS: The injection of CBD-VEGF improved cardiac function in rats with induced acute myocardial infarction. This could potentially provide a new treatment option for myocardial infarction.