Circulating tumor cells isolation:the "post-EpCAM era"

Circulating tumor cells （CTCs） represent a submicroscopic fraction detached from a primary tumor and in transit to a secondary site. The prognostic significance of CTCs in metastatic cancer patients was demonstrated for the first time more than ten years ago. To date, it seems clear enough that CTCs are highly heterogeneous and dynamically change their shape. Thus, the inadequacy of epithelial cell adhesion molecule （EpCAM） as universal marker for CTCs detection seems unquestionable and alternative methods able to recognize a broader spectrum of phenotypes are definitely needed. In this review the pleiotropic functions of EpCAM are discussed in detail and the role of the molecule in the biology of CTCs is critically dissected.

Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells

Background： Circulating tumor cells （CTCs） are often undetected through the immunomagnetic epithelial cell adhesion molecule （EpCAM）-based CellSearch~ System in breast and colorectal cancer （CRC） patients treated with bevacizumab （BEV）, where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. Methods： The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch, untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch. Results： Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins （CK）, while no evidence of epithelial to mesenchymal transition （EMT） in treated cells was observed. The recovery rate of cells through CellSearch was gradually reduced in course of treatment with BEV, being 84% , 70% and 40% at l, 2 and 3 months, respectively. Conclusions： We hypothesize that BEV may prevent CellSearch from capturing CTCs through altering EpCAM isoforms.

Epithelial-mesenchymal transition status of circulating tumor cells in breast cancer and its clinical relevance

Objective:Circulating tumor cells(CTCs)play a critical role in cancer metastasis,but their prevalence and significance remain unclear.This study attempted to track the epithelial-mesenchymal transition(EMT)status of CTCs in breast cancer patients and investigate their clinical relevance.Methods:In this study,the established negFACS-IF:E/M platform was applied to isolate rare CTCs and characterize their EMT status in breast cancer.A total of 89 breast cancer patients were recruited,including stage 0–III(n=60)and late stage(n=29)cases.Results:Using the negFACS-IF:E/M platform,it was found that in human epidermal growth factor receptor 2(HER2)+patients,mesenchymal CTCs usually exhibited a high percentage of HER2+cells.Stage IV breast cancer patients had considerably more CTCs than stage 0–III patients.Among stage 0–III breast cancers,the HER2 subtype included a significantly higher percentage of mesenchymal and biphenotypic(epithelial and mesenchymal)CTCs than the luminal A or B subtypes.Among stage IV patients,CTCs were predominantly epithelial in cases with local recurrence and were more mesenchymal in cases with distant metastasis.By applying a support vector machine(SVM)algorithm,the EMT status of CTCs could distinguish between breast cancer cases with metastasis/local recurrence and those without recurrence.Conclusions:The negFACS-IF:E/M platform provides a flexible and generally acceptable method for the highly sensitive and specific detection of CTCs and their EMT traits in breast cancer.This study demonstrated that the EMT status of CTCs had high clinical relevance in breast cancer,especially in predicting the distant metastasis or local recurrence of breast cancer.

Epidermal growth factor receptor-targeted immune magnetic liposomes capture circulating colorectal tumor cells efficiently

AIM To compare the capacity of newly developed epidermal growth factor receptor(EGFR)-targeted immune magnetic liposomes(EILs) vs epithelial cell adhesion molecule(Ep CAM) immunomagnetic beads to capture colorectal circulating tumor cells(CTCs).METHODS EILs were prepared using a two-step method, and the magnetic and surface characteristics were confirmed. The efficiency of capturing colorectal CTCs as well as the specificity were compared between EILs and Ep CAM magnetic beads. RESULTS The obtained EILs had a lipid nanoparticle structure similar to cell membrane. Improved binding with cancer cells was seen in EILs compared with the method of coupling nano/microspheres with antibody. The binding increased as the contact time extended. Compared with Ep CAM immunomagnetic beads, EILs captured more CTCs in peripheral blood from colorectal cancer patients. The captured cells showed consistency with clinical diagnosis and pathology. Mutation analysis showed same results between captured CTCs and cancer tissues. CONCLUSION EGFR antibody-coated magnetic liposomes show high efficiency and specificity in capturing colorectal CTCs.

Cytokine-Induced Cell Surface Expression of Adhesion Molecules in Vascular Endothelial Cells In vitro

Regulation of the adhesion molecules expression by cytokine in vascular endothelial cells was investigated. Human umbilical vein endothelial cells (HUVEC) were stimulated with cytokines, TNF α (1-250 U/ml) or IL 1β (0.1-50 U/ml) for 24 h. HUVEC were also cultured with cytokines, TNF α (100 U/ml) or IL 1β (10 U/ml), for 4-72 h, cell surface expression of adhesion molecules (ICAM 1 and VCAM 1) were detected and quantitated by immunocytochemical methods and computerized imaging analysis technique. Adhesion molecules expression were up regulated by TNF α, IL 1β in a concentration and time dependent manner. Some significant differences were observed between the effects of cytokines on the ICAM 1 and on VCAM 1 expression. Cytokines might directly induce the expression of ICAM 1 and VCAM 1 in vascular endothelial cells. Our observations indicate differential functions of the two adhesion molecules during the evolution of inflammatory responses in stroke.

Adhesion molecule and proinflammatory cytokine gene expression in hepatic sinusoidal endothelial cells following cecal ligation and puncture

INTRODUCTIONMultiple organ dysfunction syndrome (MODS) isthought to be a frequent consequence of sepsis[1-3].Despite substantial advances in our knowledge and understanding of the basic pathophysiologic mechanisms[4-7], in critically ill patients infections and sepsis are still associated with a high mortality[8,9].

Ubiquitin Reduces Expression of Intercellular Adhesion Molecules and Tumor Necrosis Factor-α in Lung Tissue of Experimental Acute Lung Injury

Background Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are two important cytokines in inflammatory response, which may induce rolling and adhesion of both leukocytes and lymphocytes, while modulating vascular permeability at the same time. These adhesion molecules usually serve as surrogate markers of activation and injury of vascular endothelial cells. Tumor necrosis factor-α (TNF-α) is a key factor to induce the expression and production of the above cell adhesion molecules. However, it remains to be elucidated whether exogenous ubiquitin exerts any effect on the cytokines in sepsis-induced ALI. Methods Sixty mice were devided randomly into five groups with twelve mice in each group, i.e. CLP group, SHAM group, UB1 group (10 mg/kg), UB2 group (5 mg/kg) and UB3 group(1 mg/kg). Mice of SHAM group underwent sham operation, and other four groups underwent CLP. Six hours after surgery, mice of three UB groups received ubiquitin by caudal vein injection while CLP and SHAM group received vehicle. Seven hours after surgery, blood and lungs of all mice were collected. ICAM-1, VCAM-1 and TNF-α level of 9% lung homogenate and serum TNF-α level were measured by ELISA. Results Pulmonary ICAM-1, VCAM-1 and TNF-α level of three UB groups were lower than CLP and SHAM group, and there were several comparisons with a statistically significant difference. Serum TNF-α level of three UB groups were slightly lower than CLP group, but far higher than SHAM group. Pulmonary ICAM-1 level, VCAM-1 level and serum TNF-α level of UB3 group were lower than UB1 and UB2 group, and there was a significant difference in VCAM-1 between UB3 and UB1 group. Pulmonary TNF-α level of UB3 group was slightly higher than UB1 and UB2 group.

Expression of platelet endothelial cell adhesion molecule-1 between pancreatic microcirculation and peripheral circulation in rats with acute edematous pancreatitis

OBJECTIVE: To study the ehanges of platelet endothelial cell adhesion molecule-1 (PECAM-1) expression on polymorphonuclear leukocytes (PMNs) in peripheral circulation anti pancreatic microcirculation in rats with acute edematous pancreatitis (AEP). METHODS: The model of AEP was established with 50 Wistar rats, and the changes of PECAM-1 expression on PMNs from the splenic vein and inferior vena cava were determined by flow cytometry. RESULTS: PECAM-I expression on PMNs showed no significant difference between pancreatic microcirculation and peripheral circulation at AEP2h and AEP4h time points. From the AEP4h to the AEP8h time point, PECAM-1 expression in peripheral circulation was up-regulated, but PECAM-1 expression in pancreatic microcirculation was down-regulated. PECAM-1 expression had a significant difference between pancreatic microcirculation and peripheral circulation at the AEP8h time point (P<0.05). CONCLUSION: PECAM-1 expression on PMNs is in a converse way between pancreatic microcirculation and peripheral circulation in AEP.

Re-expression of Cell Adhesion Molecule Inhibits Growth and Induces Apoptosis of Human Pancreatic Cancer Cell Line PANC-1

This study examined the expression of cell adhesion molecule 1 （CADM1） in pancreatic cancer and the possible mechanism. The expression of CADM 1 was detected by immunohistochemistry in tissues of pancreatic cancer, pancreatitis, and normal pancreas. The plasmid pcDNA3.1-Hy- gro（＋）/CADM1 was transfected into PANC-1 cells （a pancreatic cancer cell line）. The expression of CADM1 in the transfected cells was determined by RT-PCR and Western blotting. Cell growth was measured by the MTT method and cell apoptosis by flow cytometry. The results showed that CADM1 was weakly expressed in tissues of pancreatic cancer in contrast to its high expression in normal pancreatic and pancreatitis tissues. The expression level of CADM in pancreatic caner was intensely correlated with the differentiation degree, lymph node metastasis and TNM stages. The growth of CADMl-transfected PANC-1 cells was significantly suppressed in vitro by a G1 cell cycle arrest and apoptosis occurrence. It was concluded that re-expression of CADM1 inhibits the growth of pancreatic cancer cells and induces their apoptosis in vitro. As a tumor suppressor gene, CADM1 plays an important role in the occurrence, progression and metastasis of pancreatic cancer.

Early detection of circulating tumor DNA and successful treatment with osimertinib in thr790met-positive leptomeningeal metastatic lung cancer:A case report

BACKGROUND Patients diagnosed with non-small-cell lung cancer with activated epidermal growth factor receptor mutations are more likely to develop leptomeningeal(LM)metastasis than other types of lung cancers and have a poor prognosis.Early diagnosis and effective treatment of leptomeningeal carcinoma can improve the prognosis.CASE SUMMARY A 55-year-old female with a progressive headache and vomiting for one month was admitted to Peking University First Hospital.She was diagnosed with lung adenocarcinoma with osseous metastasis 10 months prior to admittance.epidermal growth factor receptor(EGFR)mutation was detected by genomic examination,so she was first treated with gefitinib for 10 months before acquiring resistance.Cell-free cerebrospinal fluid(CSF)circulating tumor DNA detection by next-generation sequencing was conducted and indicated the EGFR-Thr790Met mutation,while biopsy and cytology from the patient’s CSF and the first enhanced cranial magnetic resonance imaging(MRI)showed no positive findings.A month later,the enhanced MRI showed linear leptomeningeal enhancement,and the cytology and biochemical examination in CSF remained negative.Therefore,osimertinib(80 mg/d)was initiated as a second-line treatment,resulting in a good response within a month.CONCLUSION This report suggests clinical benefit of osimertinib in LM patients with positive detection of the EGFR-Thr790Met mutation in CSF and proposes that the positive findings of CSF circulating tumor DNA as a liquid biopsy technology based on the detection of cancer-associated gene mutations may appear earlier than the imaging and CSF findings and may thus be helpful for therapy.Moreover,the routine screening of chest CT with the novel coronavirus may provide unexpected benefits。

Effects of tanshinone Ⅱ-A sullfonate on adhesion molecule expression of endothelial cells and platelets in vitro

探讨丹参酮ⅡＡ磺酸钠（Ｔａｎ）对培养人脐静脉内皮细胞（ＨＵＶＥＣ）和人血小板表达粘附分子的影响．方法：用流动血细胞计数仪测定肿瘤坏死因子（ＴＮＦα）诱导人脐静脉内皮细胞ＩＣＡＭ１和凝血酶诱导人血小板Ｐ选择素的表达．结果：ＨＵＶＥＣ经ＴＮＦα处理后，明显增加细胞表面ＩＣＡＭ１的表达，增加ＨＬ６０细胞粘附到内皮细胞表面达加入细胞总数的３０％±６％（对照组为４６％±０７％）．在ＴＮＦα处理前，用Ｔａｎ（２５－２００μｍｏｌ·Ｌ－１）与ＨＵＶＥＣ共孵育，则Ｔａｎ剂量依赖性地抑制ＴＮＦα的作用．Ｔａｎ（２５－２００μｍｏｌ·Ｌ－１）与人血小板孵育后，可剂量依赖性地抑制凝血酶诱导人血小板表面Ｐｓｅｌｅｃｔｉｎ的表达．结论：Ｔａｎ可抑制内皮细胞和血小板表达粘附分子．

活化白细胞黏附分子在乳腺癌中的研究进展

乳腺癌是全球女性最常见的肿瘤,也是导致女性死亡最主要的疾病之一。肿瘤相关标志物及治疗靶点的研究已成为肿瘤领域的一个热点话题。研究发现活化白细胞黏附分子(Activated leukocyte cell adhesion molecule,ALCAM)在乳腺癌的发生发展过程中发挥重要的作用,并且与乳腺癌患者的预后具有相关性。本文针对ALCAM的分子结构特征、ALCAM与乳腺癌临床指征及预后的相关性,以及ALCAM作为乳腺癌临床诊断及治疗靶点的研究和应用进展进行综述。

重组人表皮生长因子联合纳米银敷料治疗烧伤的效果及对血清炎性因子的影响

目的探讨重组人表皮生长因子联合纳米银敷料治疗烧伤的效果及对血清炎性因子等的影响。方法选取2020年1月—2022年1月收治的烧伤100例为研究对象,按照不同的治疗方式将其分成2组,每组50例。对照组采用重组人表皮生长因子治疗,观察组在对照组治疗基础上增加纳米银辅料治疗。比较2组治疗前后血清细胞间黏附因子-1(ICAM-1)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)水平,统计2组烧伤分泌物细菌培养阳性率、炎性症状消失时间及创面愈合时间,使用疼痛数字评分法(NRS)对患者治疗前后创面疼痛情况进行评估,统计2组创面愈合率并使用温哥华瘢痕量表(VSS)对2组瘢痕情况予以评估,比较2组治疗期间不良反应发生情况。结果与治疗前相比,治疗7 d后2组血清ICAM-1、TNF-α均降低,血清IL-10均升高,且观察组变化更显著(P<0.01)。观察组治疗7和14 d后烧伤分泌物细菌培养阳性率均低于对照组(P<0.05)。观察组炎性症状消失时间、创面愈合50%所需时间、创面愈合80%所需时间、创面愈合100%所需时间均短于对照组,创面愈合率高于对照组(P<0.01,P<0.05)。与治疗前相比,2组治疗14 d后NRS评分和治疗6个月后VSS评分均降低,且观察组低于对照组(P<0.01)。结论重组人表皮生长因子联合纳米银敷料治疗烧伤可促进创面愈合,改善创面疼痛程度,降低血清炎性因子水平。

乳腺癌患者循环肿瘤细胞及其激素受体、人表皮生长因子受体2表达意义的研究进展

乳腺癌作为女性发病率最高的肿瘤,严重威胁女性生命健康,同时肿瘤具有高度异质性,也给检测及治疗带来困难。液体活检突破常规组织学活检壁垒,其无创、便捷、实时的特征在精准医疗中展现出巨大潜力。循环肿瘤细胞(CTC)检测作为液体活检中最具代表性的检查手段被广泛应用于临床。本文基于循环肿瘤细胞特点及特性以及对目前循环肿瘤细胞检测技术的介绍,结合乳腺癌经典分子标志物雌激素受体(ER)、孕激素受体(PR)、人表皮生长因子受体2(HER2)在肿瘤细胞上的表达发现,在乳腺癌疾病的进展及转移过程中ER、PR及HER2表达会发生动态改变。因此,通过检测CTC上ER、PR及HER2受体的表达情况,可以及时发现肿瘤细胞的动态变化,有助于实时监测复发及转移,及时调整治疗方案,从而从最大程度上改善乳腺癌患者的预后。

重症肺炎64例外周血微RNA-34a、沉默信息调节因子1的表达

目的研究重症肺炎病人外周血微RNA-34a(miR-34a)、沉默信息调节因子1(Sirt1)的表达变化及临床意义。方法选取2017年10月至2020年4月武警重庆总队医院收治的64例重症肺炎病人为重症肺炎组,同期收治的80例普通肺炎病人为普通肺炎组,进行健康体检的70例健康志愿者为对照组。采用实时荧光定量PCR法(qRT-PCR)检测外周血miR-34a表达水平,采用酶联免疫吸附法检测血清Sirt1、炎症细胞因子表达水平;Kaplan-Meier生存分析研究miR-34a与Sirt1表达水平与重症肺炎组病人预后的关系;Cox回归分析影响重症肺炎病人预后的因素。结果重症肺炎组miR-34a的表达水平高于普通肺炎组和对照组(1.65±0.28比1.32±0.33、1.00±0.25),Sirt1表达水平低于普通肺炎组和对照组[(6.83±1.59)μg/L比(8.94±1.62)μg/L、(12.11±1.77)μg/L](P<0.05);miR-34a表达水平与Sirt1表达水平存在明显负相关(P<0.05)。高危、中危病人miR-34a表达水平高于低危病人,Sirt1表达水平低于低危病人(P<0.05),高危病人miR-34a表达水平高于中危病人,Sirt1表达水平低于中危病人(P<0.05)。miR-34a高表达病人血清肿瘤坏死因子-α(TNF-α)、细胞间黏附分子-1(ICAM-1)、白细胞介素(IL)-6和高迁移率族蛋白B-1(HMGB1)水平高于miR-34a低表达病人,30 d累积生存率低于miR-34a低表达病人(P<0.05);Sirt1高表达病人血清TNF-α、ICAM-1、IL-6、HMGB1水平低于Sirt1低表达病人,30 d累积生存率高于Sirt1低表达病人(P<0.05)。ICAM-1、miR-34a及Sirt1均是影响重症肺炎病人预后的独立危险因素(P<0.05)。结论重症肺炎病人外周血中miR-34a表达增加与血清Sirt1表达降低具有相关性,且miR-34a、Sirt1的变化与病情加重、炎症反应激活、预后不良有关。

血清癌胚抗原相关细胞黏附分子1 脂质运载蛋白-2 恶性肿瘤特异生长因子联合检测鉴别诊断乳腺癌的价值

目的 探讨血清癌胚抗原相关细胞黏附分子1(CEACAM1)、脂质运载蛋白2(LCN-2)、恶性肿瘤特异生长因子(TSGF)联合检测鉴别诊断乳腺癌的价值。方法 本研究为回顾性分析,对我院2021年1月至2023年1月收治的40例良性乳腺肿瘤患者临床资料进行收集,作为对照组;并对同期医院收治的40例乳腺癌患者临床资料进行收集,作为观察组。设计基线资料填写表,详细对2组基线资料、血清检查指标进行填写、比较,重点分析血清CEACAM1、LCN-2、TSGF联合检测鉴别诊断乳腺癌的价值。结果 观察组血清CEACAM1、LCN-2、TSGF水平比对照组高,差异有统计学意义(P<0.05),组间年龄、体质指数(BMI)、肿瘤直径、绝经及患侧比较,差异无统计学意义(P>0.05);绘制受试者工作曲线(ROC)结果显示,血清CEACAM1、LCN-2、TSGF检测鉴别诊断乳腺癌的曲线下面积(AUC)均>0.70,且以联合检测(并联)价值最佳。结论 血清CEACAM1、LCN-2、TSGF联合检测鉴别诊断乳腺癌有较高的灵敏度、特异度,鉴别诊断价值满意。

Nrf2、LTBP2、PECAM1与NSCLC患者EGFR靶向治疗反应性关系及意义

目的:探讨血清核因子E2相关因子2(Nrf2)、转化生长因子结合蛋白2(LTBP2)、血小板内皮细胞黏附分子1(PECAM1)与非小细胞肺癌(NSCLC)患者表皮生长因子受体(EGFR)靶向治疗反应性关系及意义。方法:选取我院2021年1月—2023年1月收治的95例NSCLC患者为研究对象,根据EGFR靶向治疗3个月后的治疗反应性分为有效组(54例)、无效组(41例)。比较2组治疗前及治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平,分析治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平与NSCLC患者EGFR靶向治疗反应性相关性,偏回归分析治疗无效的影响因素,受试者工作特征曲线(ROC)分析治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平联合检测对NSCLC患者EGFR靶向治疗效果的预测价值。结果:治疗1个月、2个月后无效组血清Nrf2、LTBP2、PECAM1水平均高于有效组(P<0.05);相关性分析发现,治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平与治疗反应性均呈负相关(P<0.05);Logistic回归分析发现,治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平为NSCLC患者EGFR靶向治疗效果的影响因素(P<0.05);ROC曲线分析发现,治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平联合预测治疗无效的曲线下面积(AUC)分别为0.761、0.816,最佳预测敏感度、特异度分别为(85.37%、66.67%)、(92.68%、70.37%),高于单一指标预测(P<0.05)。结论:血清Nrf2、LTBP2、PECAM1水平与NSCLC患者EGFR靶向治疗反应性密切相关,并在预测治疗效果方面具有较高效能。

L-Tetrahydropalamatine Inhibits Tumor Necrosis Factor-α-Induced Monocyte-Endothelial Cell Adhesion through Downregulation of Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1 Involving Suppression of Nuclear Factor-κ B Signaling Pat

Objective： TO investigate whether I-tetrahydropalmatine （I-THP）, an alkaloid mainly present in Corydalis family, could ameliorate early vascular inflammatory responses in atheroscleroUc processes. Methods： Fluorescently labeled monocytes were co-incubated with human umbilical vein endothelial cells （HUVECs）, which were pretreated with I-THP and then simulated with tumor necrosis factor （TNF）-α in absence of I-THP to determine if I-THP could reduce thecytokine-induced adhesion of monocytes to HUVECs. Then I-THP were further studied the underlying mechanisms through observing the transcriptional and translational level of intercellular adhesion molecule-1 （ICAM-1） and vascular cell adhesion molecule-1 （VCAM-1） and the nuclear translocation of nuclear factor （NF）- K B in HUVECs. Results： L-THP could block TNF-α-induced adhesion of monocytes to HUVECs and could significantly inhibited the expression of ICAM-1 and VCAM-1 on cell surface by 31% and 36% at 30 μmol/L. L-THP pretreatment could also markedly reduce transcriptional and translational level of VCAM-1 as well as mildly reduce the total proteJn and mRNA expressJon levels of ICAM-1. Furthermore, I-THP attenuated TNF- α -stimulated NF- κ B nuclear translocation. Conclusion： These results provide evidences supporting that I-THP could be a promising compound in the prevention and treatment of the early vascular inflammatory reaction in atherosclerosis by inhibiting monocyte adhesion to vascular endothelial cell through down- regulating ICAM-1 and VCAM-1 in vascular endothelial cell based on suppressing NF- κ B.