| Background:Angiogenesis is a key process in the progression of cancer from an in situ lesion to an invasive and metastatic disease, providing the basis for the development of anti-angiogenic therapies. Vascular endothelial growth factor A(VEGF-A) is a major player in angiogenesis among the VEGF family that promotes angiogenesis through binding and activating the tyrosine kinase receptors, VEGFR1 and VEGFR2. CD47(also known as integrin-associated protein; IAP) is a transmembrane protein of the immunoglobulin superfamily that serves as a receptor for thrombospondin-1(TSP1). Interaction of TSP1 with CD47 inhibits nitric oxide(NO)-signaling in endothelial cells as well as vascular smooth muscle cells and platelets, mainly by inhibiting VEGFR2. TSP1 was also reported to suppress angiogenesis. It has been shown that TSP1 inhibits angiogenic responses by directly binding to VEGF. Moreover, TSP1 was found to inhibit VEGFR2 signaling by disrupting the association of CD47 with VEGFR2, suggesting that CD47 is involved in TSP1-mediated inhibition of angiogenesis.CD47 also serves as the counter-receptor for the inhibitory receptor, signal-regulatory protein-?(SIRP?), expressed on macrophages and dendritic cells. Binding of CD47 with SIRP? on macrophages and dendritic cells(DCs) conveys a “don't eat me” signal. CD47 expression is elevated in varying cancer cells, and injection of blocking antibodies against CD47 to block CD47-SIRP? signaling has been shown to induce antitumor immune responses. However, it is remains unclear as to whether and how administration of anti-CD47 antibody may affect tumor progression via its potential role in regulation of angiogenesis. Objective:To investigated the role of CD47 expression in non-tumor stromal cells in tumorigenesis by comparing tumor angiogenesis and progression in wild-type(WT) and CD47-deficient(CD47-/-) mice after injection of syngeneic cancer cells. Discuss the mechanism when lacking of CD47 in tumor stromal cells promotes angiogenesis and enhances vascular integrity and stability, leading to accelerated tumor progression. The therapy possibility of targeting CD47 by anti-CD47 antibody lead to promotion of angiogenesis through blocking TSP1-CD47 signaling. The similar phenomeno was shown in the mice liver after partial hepatectomy, which could play a role to indicate the conclusions. Methods:RM1 cells(prostate cancer RM1 cell line) were inoculated in the groin of WT or CD47-/- B6 mice. Tumor progression was determined by measuring tumor volume and tumor weight when mice were sacrificed at the indicated times. Briefly, the bilateral median lobe and left lateral lobe of mice were ligated and removed. Mice were sacrificed to collect the remaining lobes and blood samples at each indicated time point before and after partial hepatectomy. For histological analysis, tumor and liver tissue were fixed and then sectioned for hematoxylin and eosin(H&E) and immunohistochemistry(IHC) staining. Part of the tumor and liver were frozen in liquid nitrogen and stored for RNA analyses. Macrophage migration assay were acquired using flow cytometer and analyzed. Results:RM1 tumor grew significantly more aggressive in CD47-/- than in WT mice. We surgically removed the tumors 11 days after inoculation and found the weight and volume of the tumors from CD47-/- mice were significantly greater than those from WT mice. Necrotic lesions were readily detected in the tumors from WT mice, but were almost undetectable in those from CD47-/- mice. Tumors in CD47-/- mice exhibited an increased number of vessels compared to those in WT mice. Consistently, there was significantly increased expression of CD31 m RNA in tumors from CD47-/- than in those from WT mice. The levels of HIF-1A in the tumors of CD47-/- mice were significantly lower than in those from WT mice.We found that tumors grown in CD47-/- mice had significantly increased VE-cadherin expression compared to those grown in WT mice, implying that CD47 on endothelial cells may inhibit VE-cadherin expression. IHC analysis revealed that tumors from CD47-/- mice had significantly increased expression of VEGF-A and VEGFR2 than those from WT mice. In consistent with the protein levels, real time q PCR analysis showed a significantly increased expression of VEGF-A and VEGFR2 m RNA in the tumors from CD47-deficiet mice compared to those from WT mice.Then, we measured TSP1 expression in the tumors. Tumors from CD47-/- mice showed a significantly reduced TSP1 production, as measured by both IHC and real time q PCR, compared to those from WT mice. The number of F4/80+ tumor-infiltrating macrophages was significantly lower in CD47-/- than WT mice.In vitro, we performed a macrophage migration assay using Transwell plates, in which TSP1 showed a dose-dependent effect on macrophage recruitment. Then we confirmed that the level of TSP1 in the tumor from WT mice was significantly higher than that in tumor from CD47-deficietn mice. The culture supernatant of the tumor cells from WT mice was significantly more effective in recruiting macrophages than that from CD47-/- mice. However, addition of TSP1 into the tumor cell culture supernatant from CD47-/- mice significantly enhanced macrophage recruitment. Moreover, we found that although macrophages from CD47-/- and WT mice exhibited relatively lower migration than WT macrophages towards tumor cell culture supernatants from WT mice, CD47-/- macrophages showed significantly greater recruitment by the culture supernatant of tumor cells from WT mice. Thus, the lower number of tumor-infiltrating macrophages in CD47-/- mice was primarily due to reduced TSP1 production in the tumor, rather than defects of CD47-/-macrophages.In the model of mice partial hepatectomy, we found that newborn liver in CD47-/- mice had significantly increased CD31 and CD34 m RNA expression compared to those grown in WT mice. Then we confirmed that the level of VEGF and VEGFR2 m RNA in the newborn liver from WT mice was significantly lower than that from CD47-deficietn mice. IHC analysis revealed that newborn liver from CD47-/- mice had reduced recruitment macrophages than those from WT mice. Conclusions:(1) Enhanced tumor growth without apparent necrosis in CD47-/- mice;(2) Tumors from CD47-/- mice exhibited improved angiogenesis and vascular integrity than those from WT mice;(3) Increased VEGF and VEGFR2 expression in tumor from CD47-/- mice;(4) Reduced TSP1 expression and macrophage recruitment in tumor from CD47-/- mice;(5) The poor macrophage infiltration was attributed to reduced TSP1 production in the tumors in CD47-/- mice;(6) Liver from CD47-/- mice exhibited improved angiogenesis and reduced macrophage recruitment than those from WT mice after partial hepatectomy.Our data point out a potentially overlooked effect of cancer immunotherapy using anti-CD47 antibodies. Although blocking the interaction between CD47 on tumor cells and SIRP? on the host macrophages and DCs induces antitumor immune responses, simultaneous interruption of the TSP1-CD47 signaling in tumor stromal cells, such as endothelial cells, may potentially facilitate tumor progression. Our data also establish a fundamental role for CD47 as an important modulator of tissue angiogenesis as well as a regulatory role of TSP1 via macrophage. |
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