CD47 blockade improves the therapeutic effect of osimertinib in non-small cell lung cancer / 医学前沿

The third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib (OSI) has been approved as the first-line treatment for EGFR-mutant non-small cell lung cancer (NSCLC). This study aims to explore a rational combination strategy for enhancing the OSI efficacy. In this study, OSI induced higher CD47 expression, an important anti-phagocytic immune checkpoint, via the NF-κB pathway in EGFR-mutant NSCLC HCC827 and NCI-H1975 cells. The combination treatment of OSI and the anti-CD47 antibody exhibited dramatically increasing phagocytosis in HCC827 and NCI-H1975 cells, which highly relied on the antibody-dependent cellular phagocytosis effect. Consistently, the enhanced phagocytosis index from combination treatment was reversed in CD47 knockout HCC827 cells. Meanwhile, combining the anti-CD47 antibody significantly augmented the anticancer effect of OSI in HCC827 xenograft mice model. Notably, OSI induced the surface exposure of "eat me" signal calreticulin and reduced the expression of immune-inhibitory receptor PD-L1 in cancer cells, which might contribute to the increased phagocytosis on cancer cells pretreated with OSI. In summary, these findings suggest the multidimensional regulation by OSI and encourage the further exploration of combining anti-CD47 antibody with OSI as a new strategy to enhance the anticancer efficacy in EGFR-mutant NSCLC with CD47 activation induced by OSI.

Radix Tetrastigme Polysaccharide Promotes Antitumor Immune Response in Lewis Lung Cancer Mice / 中国肺癌杂志

BACKGROUND@#Lung cancer has a high incidence and mortality rate, but the treatment of lung cancer still lacks low toxicity and efficient anti-tumor drugs. Polysaccharide from radix tetrastigme has development value in anti-tumor treatment methods. This study was to observe the effect of polysaccharide from radix tetrastigme on immune response of Lewis lung cancer mice and explore its molecular mechanism.@*METHODS@#Lewis lung cancer mouse models were established and randomly grouped. The spleen polypeptide group was intragastric with 50 mg/kg spleen polypeptide, and the radix tetrastigme polysaccharide low, medium and high dose groups were intragastric with 62.5, 125 and 250 mg/kg radix tetrastigme polysaccharide, respectively, and the model group and the control group were intragastric with equivolume normal saline. Tumor formation and metastasis were compared. Haematoxylin-eosin (HE) staining was used to observe the pathological changes of tumor cells. Macrophage phagocytosis, apoptosis, M1/M2 polarization, T cell subsets and cytokine levels in peripheral blood were detected by flow cytometry. The proliferation activity of macrophages was detected by methyl thiazolyldiphenyl tetrazolium (MTT) assay. Dendritic cell (DC) antigen presenting function was detected by chlorophenol red-β-D-galactopyranoside (CPRG) method. Tumor tissue differentiation antigen cluster 47 (CD47) mRNA and protein expression and macrophage signal regulatory protein α (SIRRP α) expression were detected by real time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB).@*RESULTS@#The tumor inhibition rates and anti-metastasis rates in the 3-dose radix tetrastigme polysaccharide group and the spleen polypeptide group were higher than those in the model group, and the pathological injury of tumor tissue were severer, and the positive rate of phagocytosis of ink by macrophages and the efficiency of phagocytosis of tumor cells were increased; the apoptosis rate of macrophages was decreased; the proliferation activity of macrophages, polarization ratio of macrophages to M1 type, DC antigen presenting ability, CD4+, CD4+/CD8+ levels were increased; the level of serum tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and the expression of tumor tissue CD47, macrophage SH2-containing protein tyrosine phosphatase 1 (SHP-1), SH2-containing protein tyrosine phosphatase 2 (SHP-2), and phosphorylation signal regulatory protein α (p-SIRPα) were decreased, and the differences were statistically significant (P<0.05). There were no significant differences in the above indexes between low-dose radix tetrastigme polysaccharide group and spleen polypeptide group (P>0.05), and the effects of radix tetrastigme polysaccharide were dose-dependent.@*CONCLUSIONS@#Radix tetrastigme polysaccharide can inhibit tumor growth, metastasis and immune response in Lewis lung cancer mice, and its mechanism may be related to inhibiting SIRP/CD47 signaling pathway.

CD47 expression is decreased in hematopoietic progenitor cells in patients with myelofibrosis

Myelofibrosis (MF) is characterized by increased circulating hematopoietic progenitor cells (HPCs), abnormal cytokine levels, and the survival advantage of neoplastic progenitors over their normal counterparts, which leads to progressive disappearance of polyclonal hematopoiesis. CD47 is a surface glycoprotein with many functions, such as acting as a phagocytosis inhibitor of the expressing cell, that is increased in normal hematopoietic stem and progenitor cells mobilized into the blood and several human cancer-initiating cells, such as in acute myeloid leukemia. We compared CD47 expression in hematopoietic stem and progenitor cells of patients with MF and controls and found it to be decreased in progenitors of MF. Exposure of control HPCs to the cytokines transforming growth factor β and stromal-derived factor 1, which are important regulators of hematopoietic stem cell cycling and are overexpressed in patients with MF, did not modulate CD47 expression.

Expressions of CD44,CD47,and c-met in Ovarian Clear Cell Carcinoma and Their Clinical Significance / 中国医学科学院学报

Objective To investigate the expressions of CD44,CD47,and c-met in ovarian clear cell carcinoma (OCCC) tissue and their correlations with clinical variables and prognosis. Methods Immunohistochemical method was used to investigate the expressions of CD44,CD47,and c-met in tissues from 86 OCCC patients and the relationships of their expressions with the clinicopathological factors of OCCC were analyzed. Results The expressions of CD44,CD47,and c-met were significantly high in OCCC tissues (90.7%,91.9%,and 94.2%,respectively). The strong positive expressions of CD44 and CD47 were significantly correlated with advanced International Federation of Gynecology and Obstetrics stages,chemotherapeutic resistance,and poor prognosis (all P<0.05),the strong positive expression of c-met was significantly correlated with chemotherapeutic resistance and poor prognosis (all P<0.05),whereas there was no correlation between the strong positive expressions of CD44,CD47,and c-met and the lymphatic node metastasis. COX survival analysis revealed that advanced International Federation of Gynecology and Obstetrics stages and high expressions of CD44,CD47 and c-met were independent risk factors for poor prognosis (P<0.05). There was a positive correlation between CD44 (or CD47) and c-met and between CD44 and CD47 (the Spearman correlation coefficient rwas 0.783,0.776,and 0.835,respectively,all P<0.01). Conclusions The expressions of CD44,CD47,and c-met increase in OCCC tissues and are correlated with each other. High expressions of CD44,CD47,and c-met are independent factors for poor prognosis.

Inflammatory cytokines and oxidative stress markers in the inhibition of osteoarthritis by curcumin / 浙江大学学报·医学版

To observe the influence of matrix metalloproteinases-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), CD47, L-selectin and advanced oxidation proteinproducts (AOPP) in osteoarthritis and the intervention of curcumin.A total of 20 male C57BL/6 mice (10.05-15.00 g) were randomly divided into control group, OA group, Cur25 group and Cur50 group (intraperitoneal injected 25 μmol/L or 50 μmol/L of curcumin everyday after modeling). After 4 weeks treatment, we observed the morphological changes of the gross specimen by immunohistochemical method, and observed the ultrastructure of cartilage tissue under electron microscope. The expression of MMP-2, MCP-1 and CD47 were detected by western blotting, and L-selectin and AOPP were detected by ELISA and spectrophotometer, respectively.In the cartilage tissue morphology, the chondrocytes of OA group showed obvious change, while Cur25 and Cur50 groups maintained the good cartilage cell membrane intact. Compared with control group, the expressions of MMP-2, MCP-1, L-selectin and AOPP in OA group, Cur25 group and Cur50 group were increased (all<0.05), while CD47 levels were decreased (all<0.05). Compared with OA group, the expressions of MMP-2, MCP-1, L-selectin and AOPP in Cur25 group and Cur50 group were decreased (all<0.05), while CD47 levels were increased (all<0.05), and such changes were more significant in Cur50 group (all<0.05).The MMP-2, MCP-1, CD47, L-selectin and AOPP are closely associated with the pathology course of OA. Curcumin has protection effect on cartilage, which can relieve joint cartilage degeneration, reduce cartilage inflammation and increase the metabolic activity of chondrocytes.

Effectiveness of anti-CD47 antibody and Ara-C combination targeting therapy NOD/SCID mouse of myeloid leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the prognostic significance of CD47 in a NOD/SCID mouse model of acute myeloid leukemia (AML) and the best strategy for targeted therapy for this disease.</p><p><b>METHODS</b>CD34(+)CD38(-) leukemia stem cells (LSCs) were separated and transplanted into NOD/SCID mice to establish a mouse model of acute monocytic leukemia (AMoL). Anti-human CD47 antibody, alone or combined with cytosine arabinoside (Ara-C), was used to treat the mice with AMoL for 7-14 days, and therapeutic efficacy was assessed. LSCs were cultured together with mouse macrophages in culture medium containing anti-CD47 or anti-CD45 monoclonal antibody for 2 hours, to observe the phagocytic ability of macrophages to LSCs.</p><p><b>RESULTS</b>CD34(+)CD38(-) LSCs existed among THP-1 cells, with a content of about (0.12 ± 0.06)%, and a mouse model of AML was successfully established after the purified CD34(+)CD38(-) LSCs (97.0% ± 1.7%) were transplanted into NOD/SCID mice. The in vivo experiment showed that mice with AMoL had the most significant decrease in CD33(+)CD45(+) leukemia cells in peripheral blood and bone marrow and survived the longest after being treated with Ara-C (7 days) plus anti-CD47 monoclonal antibody (14 days) (P < 0.01). After 2 hours of in vitro culture, the phagocytic index in the culture medium containing anti-CD47 monoclonal antibody was significantly higher than in the culture medium containing anti-CD45 monoclonal antibody (76.9% ± 12.2% vs 7.60% ± 2.4%; P < 0.05).</p><p><b>CONCLUSIONS</b>High expression of CD47 is an adverse prognostic factor in AML. Combination therapy with anti-CD47 monoclonal antibody and Ara-C can effectively eliminate leukemia cells and LSCs, demonstrating great clinical significance in curing AML.</p>

Study on the expression levels of CXCR4, CXCL12, CD44, and CD147 and their potential correlation with invasive behaviors of pituitary adenomas / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate the factors of CXCR4, CXCL12, CD44, and CD147 as early potential diagnostic biomarkers by determining their expression levels in invasive and non-invasive pituitary adenomas.</p><p><b>METHODS</b>Fresh pituitary adenoma specimens were collected from 35 pituitary adenoma (21 invasive and 14 non-invasive) patients who underwent surgical treatment in our Neurosurgery Department between January and April of 2009. The expression levels of CXCR4, CXCL12, CD44, and CD147 were evaluated firstly by flow cytometry, fluorescence microscopy in single cell suspensions, and then by immunohistochemical staining of paraffin tissue sections.</p><p><b>RESULTS</b>Flow cytometric analyses showed that the percentage of CXCR4- and CXCL12-positive cells from invasive pituitary adenomas (IPA) was significantly higher in the single cell suspensions than that from non-invasive pituitary adenomas (nIPA) (P<0.05). Immunohistochemical staining revealed that CXCR4 and CXCL12 staining index scores of the invasive pituitary adenomas were significantly higher than those of the non-invasive pituitary adenomas (P<0.05). In contrast, neither flow cytometry nor immunohistochemical staining demonstrated significant difference between CD44 and CD147 expression levels, respectively.</p><p><b>CONCLUSION</b>Expression levels of CXCR4 and CXCL12 are correlated with the invasiveness of pituitary adenomas. Therefore, rather than CD44 and CD147, CXCR4 and CXCL12 may potentially serve as biomarkers for early detection of pituitary adenomas.</p>

Role of CD47 in hematologic malignancies / 中国实验血液学杂志

CD47 is a ubiquitously expressed transmembrane glycoprotein on surface of many cells. Through its interaction with integrin, signal regulatory protein alpha (SIRPα) and thrombin sensitive protein-1 (TSP-1), it plays important roles in various immunological processes including inflammatory response, immune response and tumor immunity. Recently, it has been found that CD47 interacts with SIRPα expressed on phagocytic cells, which transfers a negative signal when being activated. By the mechanisms described above, CD47-SIRPα signal complex is involved in the pathogenesis of hematological diseases and might provide some informations for the therapy of patients. This review focuses on the structure and immunoregulatory functions of CD47, the mechanism of CD47 in tumor therapy, the CD47 and hematologic malignancies including acute leukemia, B-cell lymphoma and multiple myeloma, as well as CD47 and hematopoietic stem cell transplantation.

CD47 and leukemia stem cells / 中国实验血液学杂志

CD47, also known as integrin-associated protein (IAP), is an immunoglobulin-like protein. It can inhibit the phagocytosis of macrophages through binding with signal-regulatory protein alpha chain of inhibitory receptor on macrophage (SIRPα). The expression of CD47 on normal hematopoietic stem cells (HSCs) is useful for maintaining the stability of HSCs in body, but the high expression of CD47 existed on leukemia stem cell (LSCs) of AML patients which can reduce the macrophage-induced phagocytosis to LSCs and decrease the clearance of innate immune system of organism to LSCs. In this article, the expression and function of CD47 on HSCs and LSCs as well as the role of CD47 in the prognosis and target therapy of AML are reviewed.

Anti-CD47 monoclonal antibody (B6H12) impairs the maturation and function of human dendritic cells / 中国实验血液学杂志

To investigate the influence and mechanisms of CD47 engagement by its soluble mAb B6H12 on the maturation and function of cultured dendritic cells (DCs), monocyte-derived DCs were propagated in granulocyte-macrophage colony stimulating factor (GM-CSF) combined with lipopolysaccharide (LPS) and interleukin (IL)-4, in the presence or absence of soluble anti-CD47 monoclonal antibodies (anti-CD47 mAbs, B6H12). The characteristic morphology of DCs was identified by using the transmission electron microscopy. Flow cytometry was used to detect the cell surface phenotypes. The concentration of IL-12 P70 in supernatant was measured by ELISA. The antigen-presenting functions of DCs were determined in one-way mixed leukocyte reaction by BrdU-ELISA. Electrophoretic mobility shift assay (EMSA) was applied to examine the activity of NF-kappaB. The results indicated that the anti-CD47 mAbs markedly suppressed the expression of CD80, CD86, CD83, CD1a and HLA-DR on the surface of DCs (P < 0.05). The data of the mixed leukocyte reaction and IL-12 P70 production were consistent with the results by flow cytometry (P < 0.01). Pre-exposure to B6H12 mAb during the development of DCs resulted in a dramatic depletion of the DNA binding activity of NF-kappaB toward nucleus protein. Moreover, such an inhibition effect seemed to be dose dependent. In conclusion, the soluble mAb B6H12 inhibits the expression of the costimulatory molecules and MHCII molecules on the DCs. The antigen-presenting function of DCs was also impaired by B6H12. And these modulations are closely related with the depletive DNA binding activity of NF-kappaB. It is suggested that the soluble B6H12 exerts a negative effect on the maturation and function of in vitro cultured DCs due to inhibition of NF-kappaB binding activity.

Effect of soluble anti-CD47 monoclonal antibody on the differentiation and function of human dendritic cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the influences of anti-CD47 monoclonal antibody (mAb) B6H12 on the differentiation and function of cultured dendritic cells (DCs).</p><p><b>METHODS</b>Human peripheral monocyte derived DCs were propagated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) plus interleukin-4 (IL-4) in the presence or absence of soluble B6H12. Flow cytometry was used to analyze the immunophenotypes of cells, semi-quantitative RT-PCR and ELISA methods to analyse the mRNA and protein expression levels of interleukin-12 (IL-12). The antigen-presenting function of DCs was determined in one-way mixed leukocyte reaction by Brdu-ELISA technique.</p><p><b>RESULTS</b>There was a high expression rate (94% approximately 98%) of CD47 molecules in DCs. The cell immunophenotypes in B6H12 mAb treated and untreated DC groups were as follows: CD80(+) (68.14 +/- 7.41)% vs (89.17 +/- 8.59)%; CD86(+) (67.33 +/- 4.71)% vs (87.27 +/- 3.56)%; CD83 (40.08 +/- 14.80)% vs (72.77 +/- 8.68)%; CD1a(+) (66.45 +/- 4.06)% vs (95.93 +/- 3.03)%; and HLA-DR (40.67 +/- 13.48) vs (98.97 +/- 1.01)%, respectively. The expression levels of mRNA and protein of IL-12 were strongly inhibited in B6H12 mAb treated DC (P < 0.01). The quantity of Brdu was also lower in B6H12 mAb treated DC than that in untreated DCs (P < 0.01).</p><p><b>CONCLUSION</b>The anti-CD47 McAb exerts a negative effect on the maturation and functions of cultured DCs.</p>

Research on physiological function of CD47 / 中国实验血液学杂志

CD47, also known as integrin-associated protein (IAP), is an ubiquitously expressed 50 kD cell membrane glycoprotein. This article reviews that as an integrin-associated molecule, CD47 regulates a series of integrin-dependent cell functions; CD47 regulates cell adhesiveness, migration and activation and platelet aggregation through binding to thrombospondins; CD7 is the extracellular ligand for human signal-regulatory proteins, thus it causes negative regulatory effect; CD47 induces costimulatory signals on activation of T cell, T cell apoptosis and T cell anergy and enhances the efficiency of TCR signaling; CD47 is a member of Rh antigen complex. CD47 is involved in stroma-supported erythropoiesis. This review also discussed that CD47 as a marker on red blood cells, associated with haemolysis, which provides a new way to recognize, diagnose, and treat diseases.