| Tumor, a big threat to human life, brought about a big economic burden for sufferers and the whole society. Solid tumors cause numerous incidence and mortality, while no perfect therapy is available up to now. It is necessary to explore the tumor genesis and development mechanism for the sake of producing better tumor control strategies.Tumor angiogenesis and anti-tumor immune response are the most important determinants of tumor growth. Neoplasm, within 2 or 3 cubic micrometers, can slowly expand by diffusions of nutritients in cell neighborhood. But when the tumor size gets bigger and bigger, the growth requires tumor neovascularization to supply the nutrition. On the other hand, immune responses can be stimulated by tumor antigen, leading to cellular and humoral responses against tumor cells.The latest tumor control strategy emphasizes the angiogenesis inhibition because neovascularization was thought indispensable to tumor growth. Lab and clinic practice have proved that this strategy is effective in some solid tumors. But drug resistance and lower efficacy has been observed also. As a result, the understanding of tumor angiogenesis mechanism is the premise of vascular network formation inhibition.Notch signal, a classical and conserved pathway associated with development regulation, is consisted of ligands and receptors on cell membrane, transcription factors in cytoplasm, downstream targets and regulators. Notch intracellular domain can be cleaved by different proteases after ligand and receptor binding, then the cleaved domain translocates into nuclear and interact with the transcription factor, RBP-J. After that, the RBP-J-containing co-repressor is switched into co-activator and triggers target transcription factors to initiate downstream genes expression. These activated genes then participate in developmental events.Notch signal is an important player in vascular network formation in embryos and adults. Notch pathway was reported to be involved with the choice of artery or vein fate, commitment of tip cell or stalk cell, endothelial proliferation, filopodia formation, migration and stability maintenance. Thus Notch pathway has been hought as a potential target in tumor angiogenesis inhibition. When Notch signal is interrupted by Dll4 intervention, some reports have observed retarded tumor growth while tumor angiogenesis augmented. In those new formed vascular, poor perfusion of blood was recorded. But the paradox is that after Notch signal activation, some solid tumor growth is slowed down.Furthermore, Notch signal is related to immune cell development. Bulk of evidence has showed that T cell, B cell, dendritic cell and myloid cell commitments require Notch singal. Consquently, when tumor angiogenesis inhibition is central for therapy, the immunological changes should be evaluated since anti-tumor responses contribute to prognosis.Because of functional redundancy in Notch pathway result from varied receptors and ligands, the completely interruption of this signal can be fulfilled by RBP-J inhibition. Actually, RBP-J is the key player in Notch pathway. Thus in the current study conditional RBP-J gene knockout mice were employeed to analyze solide tumor growth. In the same mice, immune cells involved in tumor growth were evaluated also. These following results provided some important informations for Notch singal intervention in solid tumor therapy.1. Conditional RBP-J gene knockout mice were obtained and some solid tumor cell lines were selected for direct inoculation.Mating RBP-Jflox/wr and Mx-Cre mice got Mx-Cre-RBP-Jflox/wr and RBP-Jflox/flox mice. Mating ROSA and Mx-Cre got Mx-Cre-ROSA mice. Ear from Mx-Cre-ROSA mice were stained by X-gal after peritoneal injection with 500μg polyI:C. The obvious blue color indicated that Cre recombinants began is expressed in vascular cells. RT-PCR analysis showed that Notch4,Dll4 transcripts presented in S180 and H22 cell line, while Notch1 was expressed in B16 cell line and Notch2 presented in LLC cell line.2. In the RBP-J gene knockout mice, different tumor growth was characterized. Hepatocarcinoma could metastasize to the liver in the RBP-J knockout mice.Compared to the tumors in the control mice, the S180, H22 and LLC tumor grew slower in the RBP-J gene knockout mice. Melanoma grew faster in the RBP-J gene knockout mice. Furthermore, liver metastasis was observed in RBP-J gene knockout mice after H22 cell inoculation. H.E staining showed maglinant cell invasion in the metastasized livers. The control mice suvived the metastasized mice after H22 cell inoculation. These results implied that completely interruption of Notch signal mighg lead to different growth for different tumors.3. Tumor angiogenesis, resulted from completely Notch signal interruption, did not necessarily bring about retarded growth; Tumor hypoxia and immune cell infiltration were not the reasons of different tumor growth after RBP-J gene knockout.Tissue section from S180, H22, LLC and B16 tumor were stained with anti-CD31 antibody to show the micro-vessel density. All the micro-vessel density in the tissue from different tumors growing in RBP-J gene knockout mice increased. Anti-HIF-1αstaining indicated that all the hypoxia area in the tissue from different tumors growing in RBP-J gene knockout mice increased. FACS analysis showed that T cell, B cell and DC infiltration in the tissue from different tumors growing in RBP-J gene knockout mice did not changed, while Gr-1+/Mac-1+ cell infiltration increased in the tissue from different tumors growing in RBP-J gene knockout mice. Air sac analysis based on H22 cells showed more typical tumor-like vessels in RBP-J gene knockout mice. These data suggested that tumor angiogenesis result from Notch signal interruption dissociated with melanoma growth. Hypoxia and immune cell infiltration were not the cause of different tumor growth after RBP-J gene knockout. H22 hepatocarcinoma with higher metastasis in RBP-J gene knockout mice might be linkedwith more typical tumor-like vessels.4. The process of Gr-1+ cell developing into DC was retarded after Notch signal blocking; More dendritic cells located in the vascular network when RBP-J gene was knockout, which meant poor antigen process ability.Culture analysis in vitro showed that the Gr-1+ cell from bone marrow of RBP-J gene knockout mice slowed down the process of commitment into DC. Wild type Gr-1+ cells were marked by Dio and tranfered into RBP-J gene knockout mice proved that the commitment of DC was blocked. What's more, after splenic CD11c+ cells were sorted and marked by Dio, co-location staining result implied that the merge signal based on CD11c+ and agglutinin staining increased in RBP-J gene knockout group. These results indicated that RBP-J dependent Notch pathway played a role in the commitment DC from Gr-1+ cells. Dendritic cells located more in the vascular network when RBP-J gene was knockout, which meant poor antigen process ability and possible retarded tumor growth.5. ConclusionThe current study showed that in the RBP-J gene knockout mice, different tumor growth was characterized. At least hepatocarcinoma can metastasize to the liver in the Notch signal interruption mice. Tumor angiogenesis, resulted from completely Notch signal interruption, did not necessarily bring about retarded growth; Hypoxia and immune cell infiltration were not the reason of different tumor growth after RBP-J gene knockout. But the function of DCs and myeloid cells after Notch sigal intervention need further analysis to ascertain the possible effects on tumor growth.Tumor angiogenesis supports tumor growth while DCs process tumor antigen to inhibit tumor growth. Notch signal is an important player in tumor angiogenesis and immune cell commitment. Thus intervention of Notch signal, whether up or down regulated, can retard tumor growth. The therapy result depends on the plolarization after Notch signal is changed. So it is important to take the different tumor types into account for the sake of therapy targeting on Notch singal.
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