耦联CD133/ABCG2抗体的荧光磁性纳米微粒的制备与靶向性实验

目的制备耦联抗CD133及ABCG2抗体的荧光Fe3O4纳米微粒,用于肝癌的早期诊断和癌细胞转移后的定位。方法采用共沉淀法制备Fe3O4磁性纳米粒子,在其外面包裹羧基化葡聚糖,然后与带有荧光基团的CD133及ABCG2抗体耦联,制备成一种可以检测CD133和ABCG2双阳性细胞的免疫磁性纳米微粒,继而检测其表征和抗性;体外检测制备的磁性纳米微粒对人SP细胞的靶向性;肝癌SP细胞裸鼠皮下种植制作肝癌模型,尾静脉注射经筛选的纳米微粒,用荧光显微镜观察磁性纳米微粒的体内靶向作用。结果成功制备得到了具有磁性、粒度均匀的磁性纳米微粒,且在体内外均有荧光和对肿瘤干细胞的靶向性。结论成功制备得到了可用于检测CD133及ABCG2双阳性肝癌干细胞的免疫荧光磁性纳米微粒。     

Combining CD47 blockade with trastuzumab eliminates HER2-positive breast cancer cells and overcomes trastuzumab tolerance

Trastuzumab, a targeted anti-human epidermal-growth-factor receptor-2 (HER2) monoclonal antibody, represents a mainstay in the treatment of HER2-positive (HER2⁺) breast cancer. Although trastuzumab treatment is highly efficacious for early-stage HER2⁺ breast cancer, the majority of advanced-stage HER2⁺ breast cancer patients who initially respond to trastuzumab acquire resistance to treatment and relapse, despite persistence of HER2 gene amplification/overexpression. Here, we sought to leverage HER2 overexpression to engage antibody-dependent cellular phagocytosis (ADCP) through a combination of trastuzumab and anti-CD47 macrophage checkpoint immunotherapy. We have previously shown that blockade of CD47, a surface protein expressed by many malignancies (including HER2⁺ breast cancer), is an effective anticancer therapy. CD47 functions as a “don’t eat me” signal through its interaction with signal regulatory protein-α (SIRPα) on macrophages to inhibit phagocytosis. Hu5F9-G4 (magrolimab), a humanized monoclonal antibody against CD47, blocks CD47’s “don’t eat me” signal, thereby facilitating macrophage-mediated phagocytosis. Preclinical studies have shown that combining Hu5F9-G4 with tumor-targeting antibodies, such as rituximab, further enhances Hu5F9-G4’s anticancer effects via ADCP. Clinical trials have additionally demonstrated that Hu5F9-G4, in combination with rituximab, produced objective responses in patients whose diffuse large B cell lymphomas had developed resistance to rituximab and chemotherapy. These studies led us to hypothesize that combining Hu5F9-G4 with trastuzumab would produce an anticancer effect in antibody-dependent cellular cytotoxicity (ADCC)-tolerant HER2⁺ breast cancer. This combination significantly suppressed the growth of ADCC-tolerant HER2⁺ breast cancers via Fc-dependent ADCP. Our study demonstrates that combining trastuzumab and Hu5F9-G4 represents a potential new treatment option for HER2⁺ breast cancer patients, even for patients whose tumors have progressed after trastuzumab.

Overexpression of human epidermal-growth-factor receptor-2 (HER2) occurs in ∼16% of breast cancers in the United States (1–3) and has been associated with a number of adverse prognostic factors (summarized in ref. 4). Prior to the advent of HER2-targeted therapeutics, HER2 overexpression was associated with increased risk of recurrence and poor survival rates (1, 2). Trastuzumab is a humanized monoclonal antibody that selectively binds HER2. Clinical use of trastuzumab has dramatically improved the outcomes of patients with HER2⁺ breast cancer and remains the foundational component of modern standard of care treatment regimens for HER2⁺ breast cancer in the neoadjuvant, adjuvant, and metastatic settings (5, 6). Early studies of trastuzumab’s mechanism of action focused on trastuzumab’s inhibition of protumor growth HER2 signaling pathways (7–9). Subsequent research revealed that trastuzumab also coopts a patient’s immune system to promote an antitumor response (7–11). This later body of research initially elucidated trastuzumab’s ability to engage Fc-receptors on natural killer cells (NKs) to promote antibody-dependent cellular cytotoxicity (ADCC) (12, 13). Recent reports have further illuminated trastuzumab’s ability to engage Fc-γ receptors (FcγR) on macrophages and promote antibody-dependent cellular phagocytosis (ADCP) (14).Administering trastuzumab to early-stage HER2⁺ breast cancer patients significantly increases disease-free survival and overall survival rates (15–17). Treating advanced-stage HER2⁺ breast cancer patients with the most efficacious trastuzumab-based regimens, however, produces less hopeful outcomes. For example, the Food and Drug Administration (FDA)-approved regimen studied in the CLEOPATRA clinical trial for HER2⁺ metastatic breast cancer in the first line of treatment utilized trastuzumab in combination with docetaxel and pertuzumab; this regimen resulted in a median progression-free survival of 18.7 mo (18, 19). In the same study, 19.8% of patients did not achieve an objective clinical response to trastuzumab-based treatment (20). And, of the advanced-stage HER2⁺ breast cancer patients who initially responded to trastuzumab, pertuzumab, and docetaxel, the median duration of response was 20.2 mo; thereafter, the majority of patients experienced objective disease-progression, defining acquired clinical resistance to trastuzumab-based therapy (18).A myriad of potential mechanisms of trastuzumab resistance have been reported, such as: 1) perturbation of HER family receptors or binding of therapeutic antibodies to HER2 (e.g., shedding of the HER2 extracellular domain, expression of the Δ16HER2 splice isoform, overexpression of MUC4/MUC1 resulting in steric hindrance to trastuzumab binding to the HER2 extracellular domain, and increased phosphorylation of HER3); 2) parallel receptor pathway activation (e.g., overexpression of other HER family members, up-regulation of IGF1 receptor, erythropoietin receptor, AXL receptor, or MET receptor); and 3) activation of downstream signaling events distal to HER2 receptor (e.g., hyperactivation of the PI3 kinase/Akt pathway by loss of PTEN or PIK3CA mutational activation, cyclin E amplification/overexpression, up-regulation of miR-21, and expression of the estrogen receptor) (21). Impairments in trastuzumab-mediated ADCC may also lead to relative resistance to trastuzumab (22, 23). Interestingly, it has been shown that even after HER2⁺ breast cancers relapse or progress after trastuzumab, resistant cells most often still overexpress HER2 (24). Given the many ways in which trastuzumab resistance develops, there is an urgent clinical need for novel treatment approaches that provide HER2 specificity without eliciting the same mechanisms of trastuzumab resistance that are currently seen in the clinic (25). This led us to hypothesize that engagement of macrophages and activation of ADCP could still be effective even in the face of various resistance mechanisms—as long as the HER2 ectodomain target epitope is present.Our laboratory has previously shown that many different cancers overexpress the CD47 surface protein to convey a “don’t eat me” signal to macrophages (26–29) and to counteract “eat me” signals (30–32), thereby resulting in immune evasion through inhibition of macrophage phagocytosis. This led to the development of a new type of immunotherapy based on macrophage checkpoint inhibition through blockade of CD47. Hu5F9-G4 (magrolimab) is a humanized monoclonal antibody against CD47 that blocks CD47''s interaction with signal regulatory protein-α (SIRPα), thereby diminishing the inhibition of macrophages by cancer cells (33). As a monotherapy, Hu5F9-G4’s anticancer activity works by blocking CD47’s antiphagocytic signaling. The combination of Hu5F9-G4 and tumor-targeting antibodies, moreover, promotes ADCP by increasing the amount of “eat me” signals provided by the interaction of cancer-targeting antibody Fc domains and macrophage Fc-receptors (33–36). We have previously demonstrated this principle of ADCP enhancement in the context of rituximab-resistant CD20-expressing diffuse large B cell non-Hodgkin’s lymphoma. These studies showed that combining Hu5F9-G4 with rituximab (an anti-CD20 tumor-targeting antibody) in human xenograft models produced an anticancer ADCP response (34). In the clinical trials that followed, about half of the patients who are relapsed and refractory to rituximab plus or minus chemotherapy nevertheless responded to magrolimab plus rituximab (35). This clinical study suggested that combining Hu5F9-G4 with rituximab may resensitize refractory lymphoma cells to rituximab via an ADCP mechanism (35, 37).Based on these previous studies, we hypothesized that administering a combination of trastuzumab and Hu5F9-G4 to ADCC-tolerant HER2⁺ breast cancer cells would resensitize these cells to trastuzumab. We found that this combination was more efficacious than either treatment alone. We also found that this combinatorial treatment augments ADCP via Fc-receptor-mediated phagocytosis. Taken together, our study suggests that combining Hu5F9-G4 and trastuzumab may represent an alternative or complementary approach to the current standard of care for advanced HER2⁺ breast cancer that expands trastuzumab’s efficacy through engaging ADCP while preserving and utilizing trastuzumab’s HER2-targeting capabilities.     

血清铁蛋白与透明质酸和肝硬化的相关性

目的 探讨肝硬化患者血清铁蛋白(SF)与透明质酸(HA)间的相关性.方法 70例肝硬化患者,其中女性16例,男性54例,平均(46.93±13.34)岁,Child-Pugh A级15例,B级19例,C级36例,采用化学发光法检测SF及HA,对SF与HA水平进行统计并分析二者之间的关系.结果 SF在肝硬化中明显增高,随着肝硬化分级的加重,患者的SF也增高,在C级处于最高水平,不同分级间比较差异均有统计学意义(P＜0.01).HA在肝硬化中明显增高,随着肝硬化分级的加重,患者的HA也增高,不同分级之间差异有统计学意义(P＜0.01).不同分级患者的SF与HA呈显著正相关(r=0.828,P＜0.001).结论 肝硬化患者中SF与HA有显著相关性,在反映肝硬化程度上SF与HA同等重要,是反映肝纤维化程度的敏感指标.     

A close relationship between Helicobacter pylori infection and the serum level of CD47 in adults

Abstract

Objectives: Although Helicobacter pylori (H. pylori) infection is one of the most important risk factors for gastric cancer, the molecular mechanisms underlying the progression of H. pylori-induced gastric cancer remain unclear. Previous studies have demonstrated that Integrin-associated protein (CD47) plays an important role in the development of gastric cancer. Hence, the aim of this study was to preliminarily explore the relationship between CD47 and H. pylori infection.

Methods: A total of 417 adults who underwent health checkups at a hospital were recruited in 2018. Serum levels of CD47 and tumor necrosis factor-α (TNF-α) were determined using an enzyme-linked immunosorbent assay. ¹³C urea breath test (¹³C-UBT) was carried out to diagnose H. pylori infection in all participants.

Results: Compared with H. pylori-negative participants, H. pylori-positive participants have higher levels of serum CD47 and TNF-α. H. pylori infection, the levels of serum TNF-α and low density lipoprotein (LDL) are the independent predictors of serum level of CD47 in adults. In addition, a potential diagnostic value of serum CD47 level for H. pylori infection has been demonstrated in our study.

Conclusion: H. pylori infection is closely associated with the serum level of CD47 in adults, suggesting that H. pylori may promote gastric cancer progression by activating CD47-mediated oncogenic pathways.     

受试者工作特征曲线分析血小板计数与透明质酸对肝硬化的诊断价值

目的 应用受试者工作特征(ROC)曲线分析血小板( PLT)计数与透明质酸(HA)对肝硬化的诊断价值.方法 收集377例慢性HBV感染者,分为慢性乙型肝炎组233例,乙型肝炎肝硬化组144例.化学发光法检测HA,全自动血细胞分析仪检测外周血的PLT计数,进行ROC曲线分析.结果 (1 )PLT计数诊断肝硬化的ROC曲线下面积(AUC)为0.888±0.0165,截断值为91×109/L,以PLT计数≤91×109/L来预测肝硬化灵敏度为84.03％,特异度为82.83％.HA的AUC为0.920±0.0138,截断值为308nmol· ml -1·h-1),以HA ＞308 nmol·ml-1 ·h-1来预测肝硬化灵敏度为84.72％,特异度为88.84％.PLT与HA的AUC之间的差异无统计学意义(P=0.089),二指标均为诊断肝硬化的优良指标.(2)以公式log10( PLT/HA)判断两个指标联合对肝硬化的诊断价值,所得AUC为0.945±0.0113,截断值为-0.595,以log10(PLT/HA)≤-0.595来预测肝硬化灵敏度为88.19％,特异度为89.7％.AUC两两比较显示log10( PLT/HA)与PLT之间的差异有统计学意义(P=0.0008);同样log10( PLT/HA)与HA之间的差异也有统计学意义(P＜0.0001).可见PLT与HA联合检测对肝硬化诊断的灵敏度和特异度更高.结论 在慢性HBV感染者缺乏肝组织病理学检查时,联合检测PLT计数与HA对肝硬化的诊断比单—检测有更高灵敏度和特异度,临床价值更高.     

Ligation of CD47 mediates phosphatidylserine expression on erythrocytes and a concomitant loss of viability in vitro

CD47 ligation has been shown to induce phosphatidylserine (PS) expression as part of a death pathway in nucleated blood cells. Using Annexin V binding assays we showed that ligation of CD47 with the specific CD47-binding peptide 41NK, anti-CD47 monoclonal antibody, and its natural ligand thrombospondin-1 also induced PS expression on enucleated erythrocytes. PS expression was associated with a concomitant loss of erythrocyte viability in vitro. Further characterisation of the CD47-PS signalling pathway on erythrocytes may help develop clinical strategies to further preserve the life of blood donations and improve our understanding of certain types of haemolytic anaemias.     

Hemodynamic patterns of phosphatidylcholine hydroperoxide and hyaluronic acid during hepatic ischemia-reperfusion

We investigated the hemodynamic pattern of serum hyaluronic acid (HA) and compared it with that of plasma phosphatidylcholine hydroperoxide (PCOOH) in terms of a convenient parameter of reperfusion injury. Using pig models, we designed two continuous ischemia groups, prepared by blockage of the blood flow at the hepatic hilum for 10 or 30 min. A discontinuous ischemia model was prepared by repeating the 10-min ischemia procedure, followed by 10 min of reperfusion, to a total ischemia period of 30 min. The PCOOH level started to increase just after reperfusion and reached the peak at 90 min, followed by a gradual decline after 6 h. The HA level increased rapidly in the continuous ischemia groups, starting immediately after ischemia onset until immediately before reperfusion, followed by a gradual decrease during up to 6 h of reperfusion. The HA levels in the three groups were almost normalized after 90 min of reperfusion, when the PCOOH level reached the peak. These results indicated that the plasma PCOOH level is a useful parameter for predicting the onset and progress of reperfusion injury in its initial stages.     

From the Cover: Overexpression of CD47 is associated with brain overgrowth and 16p11.2 deletion syndrome

Copy number variation (CNV) at the 16p11.2 locus is associated with neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia. CNVs of the 16p gene can manifest in opposing head sizes. Carriers of 16p11.2 deletion tend to have macrocephaly (or brain enlargement), while those with 16p11.2 duplication frequently have microcephaly. Increases in both gray and white matter volume have been observed in brain imaging studies in 16p11.2 deletion carriers with macrocephaly. Here, we use human induced pluripotent stem cells (hiPSCs) derived from controls and subjects with 16p11.2 deletion and 16p11.2 duplication to understand the underlying mechanisms regulating brain overgrowth. To model both gray and white matter, we differentiated patient-derived iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). In both NPCs and OPCs, we show that CD47 (a “don’t eat me” signal) is overexpressed in the 16p11.2 deletion carriers contributing to reduced phagocytosis both in vitro and in vivo. Furthermore, 16p11.2 deletion NPCs and OPCs up-regulate cell surface expression of calreticulin (a prophagocytic “eat me” signal) and its binding sites, indicating that these cells should be phagocytosed but fail to be eliminated due to elevations in CD47. Treatment of 16p11.2 deletion NPCs and OPCs with an anti-CD47 antibody to block CD47 restores phagocytosis to control levels. While the CD47 pathway is commonly implicated in cancer progression, we document a role for CD47 in psychiatric disorders associated with brain overgrowth.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction and communication. Copy number variation (CNV) at the 16p11.2 locus is associated with ASD (1–8). People who have 16p11.2 deletion syndrome tend to have larger head circumferences (macrocephaly), with disproportionate enlargement in both gray and white matter volume (8–13). Individuals with ASD and macrocephaly have more severe behavioral and cognitive problems and are less responsive to standard medical and therapeutic interventions than those with ASD and normal head circumferences (14). In addition, prior work has documented a very strong cross-sectional and temporal association between macrocephaly and ASD symptoms (8, 9, 11, 12, 14–17). These findings suggest that understanding the underlying mechanisms regulating macrocephaly could provide a window of opportunity for intervention or mitigation of symptoms.Here, we used patient-derived human induced pluripotent stem cells (hiPSCs) to interrogate the underlying mechanisms contributing to gray and white matter enlargement. We focused on individuals with intellectual disability (IQ < 70) or ASD associated with brain overgrowth in 16p11.2 deletion carriers. We differentiated the iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs) and investigate the hypothesis that brain enlargement in 16p11.2 deletion carriers may be due to improper cellular elimination. Under normal conditions, classic “eat me” and “don’t eat me” signaling mechanisms associated with phagocytosis maintain cellular homeostasis across diverse tissue types (18, 19). CD47 (a “don’t eat me” signal) protects normal cells from getting cleared (18), but can become overexpressed in many types of cancer cells, preventing tumorigenic cells from getting engulfed or phagocytosed (20–22). In fact, CD47 plays an important role in many pathological disorders associated with an overproduction of cells and cell removal, including cancer (20–22), atherosclerosis (23), and fibrotic diseases (24). NPCs derived from iPSCs of autistic individuals with macrocephaly have increased proliferation relative to controls (25, 26). Therefore, we hypothesized that CD47 may be involved in these disorders.We find that CD47 is overexpressed in NPCs and OPCs derived from 16p11.2 deletion carriers, leading to reduced phagocytosis by macrophages and microglia. Furthermore, the 16p11.2 deletion NPCs and OPCs have increased cell surface expression of calreticulin (CRT, a prophagocytic “eat me” signal), indicating that these cells should be eliminated but are not due to high levels of CD47 (27). Importantly, treatment with a CD47 blocking antibody restores phagocytosis of 16p11.2 deletion NPCs and OPCs to control levels, particularly in 16p_del NPCs and OPCs that have increased cell surface expression of CRT, indicating that the changes in phagocytosis are mediated by cell surface expression of CD47. We thus identify a role for CD47 in 16p11.2 deletion syndrome and highlight the potential importance of blocking CD47 to promote clearance of unhealthy NPCs and OPCs in 16p11.2 deletion with brain overgrowth.