Recent progress in aptamer-based microfluidics for the detection of circulating tumor cells and extracellular vesicles / 药物分析学报

Liquid biopsy is a technology that exhibits potential to detect cancer early,monitor therapies,and predict cancer prognosis due to its unique characteristics,including noninvasive sampling and real-time analysis.Circulating tumor cells(CTCs)and extracellular vesicles(EVs)are two important components of circu-lating targets,carrying substantial disease-related molecular information and playing a key role in liquid biopsy.Aptamers are single-stranded oligonucleotides with superior affinity and specificity,and they can bind to targets by folding into unique tertiary structures.Aptamer-based microfluidic platforms offer new ways to enhance the purity and capture efficiency of CTCs and EVs by combining the advantages of microfluidic chips as isolation platforms and aptamers as recognition tools.In this review,we first briefly introduce some new strategies for aptamer discovery based on traditional and aptamer-based micro-fluidic approaches.Then,we subsequently summarize the progress of aptamer-based microfluidics for CTC and EV detection.Finally,we offer an outlook on the future directional challenges of aptamer-based microfluidics for circulating targets in clinical applications.

Establishment and validation of a model for predicting infiltration of pulmonary subsolid nodules by circulating tumor cells combined with imaging features / 中国胸心血管外科临床杂志

@#Objective     To evaluate the clinical radiological features combined with circulating tumor cells (CTCs) in the diagnosis of invasiveness evaluation of subsolid nodules in lung cancers. Methods     Clinical data of 296 patients from the First Hospital of Lanzhou University between February 2019 and February 2021 were retrospectively included. There were 130 males and 166 females with a median age of 62.00 years. Patients were randomly divided into a training set and an internal validation set with a ratio of 3 : 1 by random number table method. The patients were divided into two groups: a preinvasive lesion group (atypical adenomatoid hyperplasia and adenocarcinoma in situ) and an invasive lesion group (microinvasive adenocarcinoma and invasive adenocarcinoma). Independent risk factors were selected by regression analysis of training set and a Nomogram prediction model was constructed. The accuracy and consistency of the model were verified by the receiver operating characteristic curve and calibration curve respectively. Subgroup analysis was conducted on nodules with different diameters to further verify the performance of the model. Specific performance metrics, including sensitivity, specificity, positive predictive value, negative predictive value and accuracy at the threshold were calculated. Results     Independent risk factors selected by regression analysis for subsolid nodules were age, CTCs level, nodular nature, lobulation and spiculation. The Nomogram prediction mode provided an area under the curve (AUC) of 0.914 (0.872, 0.956), outperforming clinical radiological features model AUC [0.856 (0.794, 0.917), P=0.003] and CTCs AUC [0.750 (0.675, 0.825), P=0.001] in training set. C-index was 0.914, 0.894 and corrected C-index was 0.902, 0.843 in training set and internal validation set, respectively. The AUC of the prediction model in training set was 0.902 (0.848, 0.955), 0.913 (0.860, 0.966) and 0.873 (0.730, 1.000) for nodule diameter of 5-20 mm, 10-20 mm and 21-30 mm, respectively. Conclusion     The prediction model in this study has better diagnostic value, and is more effective in clinical diagnosis of diseases.

Prognostic significance of circulating tumor cell measurement in the peripheral blood of patients with nasopharyngeal carcinoma

Abstract Objective: Nasopharyngeal Carcinoma (NPC) is lethal cancer. Typically, relapse and metastasis are the outcomes of most patients. Against this backdrop, this study aimed to investigate the correlation between Circulating Tumor Cell (CTC) profiles and clinicopathological features in patients with NPC. Patients and methods: A total of 119 blood samples from 79 patients were collected from patients with NPC during treatment. CanPatrol™ CTC enrichment and RNA In Situ Hybridization (RNA-ISH) were used to characterize CTCs, including epithelial, Mesenchymal (MCTCs), and epithelial/mesenchymal mixed types according to their surface markers. Results: The number of CTCs and MCTCs in the pre-treatment group was significantly higher than that in the post-treatment group (p < 0.05). The total number of CTCs and MCTCs cell numbers was significant correlation with Tumor-Node-Metastasis (TNM) staging (p < 0.05), Progression-Free Survival (PFS), and Overall Survival (OS). The PFS of patients with > 7 CTCs or > 5 MCTCs per 5 mL blood was significantly shorter PFS than those patients with ≤ 7 CTCs or ≤ 5 MCTCs (p < 0.05). Patients treated with targeted therapy combined with chemoradiother-apy had poorer PFS and OS rates than those treated with chemoradiotherapy (p < 0.05). The Kaplan-Meier survival analysis also demonstrated that patients with changes in CTC > 4 were strongly associated with PFS and OS rates (p < 0.05). Conclusion: CTC and MCTC number detection in patients with NPC is a useful biomarker for predicting patient progress. Patients with more than 7 CTCs or 5 MCTCs in 5 mL of blood had shorter PFS and OS rates. CTC and MCTC count changes were also significantly associated with the patient's therapy.

Research progress on liquid biopsy and its combination of radiology in diagnosis of pulmonary nodules / 中国胸心血管外科临床杂志

@#Lung cancer is a malignant tumor with the highest mortality worldwide, and its early diagnosis and evaluation have a crucial impact on the comprehensive treatment of patients. Early preoperative diagnosis of lung cancer depends on a variety of imaging and tumor marker indicators, but it cannot be accurately assessed due to its high false positive rate. Liquid biopsy biomarkers can detect circulating tumor cells and DNA in peripheral blood by non-invasive methods and are gradually becoming a powerful diagnostic tool in the field of precision medicine for tumors. This article reviews the research progress of liquid biopsy biomarkers and their combination with clinical imaging features in the early diagnosis of lung cancer.

Establishment and Application of in vitro and in vivo Model of Disseminated Tumor Cells with Dormancy / 肿瘤防治研究

Postoperative asymptomatic patients with early cancer (lung cancer) have dormant disseminated tumor cells (DTCs) in their metastatic target organs, and the proliferation of these DTCs is the key link leading to clinical metastasis. The development of therapeutic agents to maintain DTCs dormant or eradicate dormant DTCs will prevent tumor metastasis and break through the bottleneck of improving the overall efficacy of treating malignant tumors. This paper reviews the methods of establishing in vitro and in vivo research models of DTCs with dormant characteristics to promote the understanding of dormant DTCs and improve the research and development efficiency of anti-tumor metastasis drugs.

Research progress on function and molecular mechanism of transcription factor ELK1 of Ets / 中国生物制品学杂志

@#Ets transcription factor ELK 1,a member of the ternary complex factor(TCF) subfamily in the Ets family,is directly downstream signal of MAPK/ERK pathway and is activated by phosphorylation to execute the function of ERK signal.ELK1,which is highly expressed and phosphorylated in stem cells and tumor cells,plays a role in promoting proliferation,inhibiting apoptosis and differentiation in stem cells.In tumor cells,ELK1 has shown to promote proliferation,migration,and inhibit apoptosis.In neural cells,ELK1 is involved in long-term memory,learning,addiction and other functions.In this paper,the research progress on the main structure and basic biological characteristics of ELK1,the function and mechanism of ELK 1 in tumor cells,stem cells and nerve cells are reviewed in order to provide new ideas for the follow-up research.

Preliminary application evaluation of polydopamine nano-carriers labeled with multiple radionuclides / 中华核医学与分子影像杂志

Objective:To prepare nanoprobes by using polydopamine (PDA) as a carrier which is modified with the sonosensitizer protoporphyrin Ⅸ (PpⅨ) and labeled with 131I, 99Tc m or 177Lu, and to explore the value of these new nanoprobes in diagnosis and combination therapy of breast cancer. Methods:PDA particles were synthesized by aqueous oxidation, and a layer of polyethylene glycol (PEG) and PpⅨ were modified on the surface to product PDA-PEG-PpⅨ. Then the nuclides 131I, 99Tc m and 177Lu were labeled on PDA, respectively, and the labeling yield and stability were determined. The cytotoxicity test was conducted by comparing the viabilities of 4T1 tumor cells in free 131I group and 131I-PDA-PEG-PpⅨ group. The 4T1 cells were divided into 7 groups according to different treatment methods: PDA-PEG-PpⅨ group, PDA-PEG-PpⅨ+ photothermal therapy (PTT) group, PDA-PEG-PpⅨ+ sonodynamic therapy (SDT) group, 131I-PDA-PEG-PpⅨ+ PTT group, 131I-PDA-PEG-PpⅨ+ SDT group, 131I-PDA-PEG-PpⅨ+ PTT+ SDT group (100 μg/ml PDA-PEG-PpⅨ, 925 kBq/ml 131I), and the control group (DMEM culture medium). The cell viabilities of those groups were compared to evaluate the therapeutic effect. 4T1 tumor bearing mouse models were established, then 99Tc m-PDA-PEG-PpⅨ was injected through the tail vein (29.6 MBq) or intratumorally (14.8 MBq) to perform gamma imaging. The independent-sample t test and one-way analysis of variance were used for data analysis. Results:The PDA particles were uniform in size, with a particle size of (160.0±1.5) nm. They had a good photothermal conversion effect. A characteristic peak consistent with PpⅨ (400 nm) appeared in the UV-Vis absorption spectrum of PDA-PEG-PpIX. In the cytotoxicity test, when the radioactivity was 1.850 or 3.700 or 7.400 MBq/ml, the cell viabilities of free 131I group and 131I-PDA-PEG-PpⅨ group were significantly different ((72.18±6.57)% vs (86.07±5.17)%, (59.31±9.06)% vs (80.85±4.21)%, (42.90±1.30)% vs (72.99±5.73)%; t values: 3.71, 4.82, 11.46, P values: 0.006, 0.001, <0.001). The 131I-PDA-PEG-PpⅨ+ PTT+ SDT combination therapy had a better killing effect on 4T1 tumor cells than the combination of 131I-PDA-PEG-PpⅨ+ PTT and 131I-PDA-PEG-PpⅨ+ SDT (cell viabilities: (10.09±2.50)% vs (16.04±2.63)%, (28.65±4.72)%; F=351.66, P<0.001). In vivo imaging showed that 99Tc m-PDA-PEG-PpⅨ was stable in mouse models and could be effectively enriched in tumors. Conclusions:A multifunctional nanoprobe based on PDA is successfully prepared. The radionuclide labeling method is simple and effective, with a good stability. 131I-PDA-PEG-PpⅨ can kill 4T1 cells efficiently. 99Tc m-PDA-PEG-PpⅨ has an obvious tumor concentration effect in mouse models.

ImmunoPET studies of 64Cu-labeled CD30 monoclonal antibody 64Cu-NOTA-CD30 in lymphoma models / 中华核医学与分子影像杂志

Objective:To develop the anti-CD30 monoclonal antibody 64Cu-1, 4, 7-trizacyclononane-1, 4, 7-triacetic acid (NOTA)-CD30 and visualize CD30 expression in lymphoma non-invasively. Methods:The CD30 expression levels of 5 cell lines (Karpas299, Raji, Daudi, Ramos, and U266) were assessed by Western blot. Cell lines with high and low CD30 expression were selected for flow cytometry to evaluate the specific binding affinity of anti-CD30 monoclonal antibody. Thirteen NSG mice were used to established CD30 positive and negative subcutaneous xenograft models. 64Cu-NOTA-CD30 was obtained and 64Cu-NOTA-immunoglobulin (Ig)G was used as the control. ImmunoPET imaging was performed 2, 24, and 48 h after the injection of 64Cu-NOTA-CD30 or 64Cu-NOTA-IgG. Finally, the biodistribution studies were conducted. Repeated-measures analysis of variance and Bonferroni test were conducted for comparison. Results:Karpas299 showed the highest CD30 expression, while Raji showed the lowest. Flow cytometry showed specific binding affinity of the anti-CD30 monoclonal antibody to the Karpas299 cell line. The radiochemical purities of the probes were both higher than 95%. In microPET, the 64Cu-NOTA-CD30 uptake of Karpas299 xenograft tumors increased over time, with (11.46±0.58), (17.60±1.16) and (19.46±0.99) percentage activity of injection dose per gram of tissue (%ID/g) at 2, 24 and 48 h respectively. The contrast to normal tissue was good at 48 h, with the tumor/heart (blood) ratio of 2.20±0.22. The uptake of 64Cu-NOTA-CD30 in Karpas299 tumor at 48 h after injection was significantly higher than that in Raji tumor ((6.10±1.03) %ID/g) and 64Cu-NOTA-IgG in Karpas299 tumor ((5.12±0.89) %ID/g; F=290.99, t values: 19.65 and 22.25, all P<0.001). The uptake of 64Cu-NOTA-CD30 and the control probe in the heart and liver decreased over time in all groups. Ex vivo biodistribution at 48 h was mainly consistent with the results of microPET in vivo. Conclusions:64Cu-NOTA-CD30 is able to visualize the expression level and distribution of CD30 non-invasively. It is promising to be applied for screening the beneficial groups and evaluating efficacy for CD30-targeted immunotherapy.

Circulating Tumor Cells – A Review

Context: Circulating tumor cells (CTCs) are those cells that separate from the primary tumor to enter circulation. This is facili- tated by Epithelial -Mesenchymal Transition (EMT) or through Non EMT based modalities by passive entry into circulation. CTCs are responsible for causing distant metastasis. Objectives: This review article briefly describes few of the mechanisms of CTC production and survival and few methods that are used to detect CTCs Materials and Methods: Data was collected and analyzed from published literature and electronic database searches of PubMed and Google Scholar. Result: CTCs acquire genetic alteration that differentiates them from the primary tumor. Majority of CTCs do not survive in the circulation but the few that do, do so by adapting various survival mechanisms. Conclusion: Detection of CTCs help in the early diagnosis of cancer, providing patient tailored therapy and for monitoring of cancer.

Células tumorales circulantes en una paciente con sospecha clínica de mieloma múltiple / Circulating tumor cells in a patient with clinical suspicious of multiple myeloma

Introducción: La circulación de células tumorales en la sangre periférica, conocido como carcinocitemia, es un fenómeno raro y muy poco comunicado en la literatura científica y su diagnóstico diferencial puede constituir un desafío en la práctica clínica. Objetivos: Describir las causas más frecuentes de carcinocitemia, los retos diagnósticos que representa y contribuir a elevar el índice de sospecha de esta entidad. Presentación del caso: Paciente femenina de 71 años de edad que acude por dolores óseos y palidez cutánea. En el examen de sangre periférica se observa células de gran tamaño que recordaron células plasmáticas. El inmunofenotipo por citometría de flujo fue sugestivo de mieloma múltiple isotipo IgM. El ultrasonido de mamas y la tomografía de tórax mostraron lesión tumoral en la mama izquierda. El estudio inmunohistoquímico de la biopsia de médula ósea fue compatible con adenocarcinoma de mamas. La paciente falleció sin haber comenzado tratamiento específico. Conclusiones: Se presenta paciente con células circulantes tumorales secundaria a adenocarcinoma de la mama donde la inmunohistoquímica de la biopsia de médula ósea descartó el diagnóstico de mieloma múltiple sospechado clínica, radiológicamente, por la morfología celular y el inmunofenotipo(AU)

Introduction: The circulating tumor cells in peripheral blood, known as carcinocythemia is a rare and poorly documented phenomenon, that can be a challenge diagnosis. Objectives: To describe the most frequents causes of carcinocythemia, the diagnosis challenges that it represents and to contribute raising awareness of this entity. Case presentation: Female patient, 71-year-old who complained bone pain and skin pale. The peripheral blood smear showed big size cells mimicking plasma cells. The immunophenotype by flow cytometry suggested IgM isotype multiple myeloma. Breast ultrasound and thorax tomography showed a tumor in the left breast. The bone marrow biopsy immunohistochemical was compatible with adenocarcinoma of breast. The patient died short after before receive specific treatment. Conclusions: We present a patient with circulating tumor cells secondary to breast adenocarcinoma where the bone marrow biopsy immunohistochemical ruled out multiple myeloma diagnosis suspected by clinical, image studies, cell morphology and immunophenotype(AU)

Effects of miR-5011-5p on apoptosis and migration of bladder cancer cell line J82 and the underlying mechanism / 中国基层医药

Objective:To investigate the effects of miR-5011-5p on apoptosis and migration of bladder cancer cell line J82 and the underlying mechanism.Methods:J82 cells were transfected with random sequence molecules (NC group) and miR-5011-5p sequence molecules (miR-5011-5p group). Flow cytometry and scratch experiment were performed to analyze the effects of miR-5011-5p on apoptosis and migration of J82 cells. The target gene of miR-5011-5p was predicted by bioinformatics. Real-time fluorescent quantitative polymerase chain reaction and western blot assay were performed to investigate the effects of miR-5011-5p on target gene expression.Results:The relative expression of miR-5011-5p in J82 cells in the miR-5011-5p group was significantly higher than that in the NC group (10.73 ± 1.67 vs. 1.04 ± 0.16, t = 5.81, P < 0.01). There was significant difference in the apoptosis rate of J82 cells between NC and miR-5011-5p groups [(8.83 ± 1.67)% vs. (34.96 ± 3.80)%, t = 6.30, P < 0.01]. The migration rate of J82 cells differed significantly between NC and miR-5011-5p groups [(71.31 ± 7.69)% vs. (37.43 ± 5.01)%, t = 3.69, P < 0.05]. The target gene of miR-5011-5p may be Yes-related protein 1 (YAP1). Compared with the NC group, miR-5011-5p exhibited an obvious inhibitory effect on the YAP1 expression in J82 cells ( P < 0.01). Conclusion:miR-5011-5p may promote the apoptosis of J82 cells and inhibit their migration in bladder cancer through targeted inhibition of YAP1 gene expression.

Clinicopathological Significance of Poorly Differentiated Clusters in Liver Metastatic Lesions of Colorectal Carcinoma / 肿瘤防治研究

Objective To investigate the clinicopathological significance of PDC in liver metastases and analyze the correlation of PDC between liver metastases and primary lesions. Methods Retrospective analysis of 72 matched cases of colorectal cancer with liver metastases was performed. The PDC in primary tumor and liver metastatic lesion was interpreted synchronously, and then the relationship between PDC in liver metastasis and clinicopathological parameters was analyzed based on the correlation of PDC between primary and metastatic lesions. In addition, PDC were interpreted in accordance with Uenos' standard. Results Among the 72 cases of liver metastasis of colorectal cancer, the number of G1, G2, and G3 graded by PDC was 28, 24, and 20, respectively. The PDC in liver metastatic lesion was correlated with tumor budding in liver metastatic lesion and PDC grade of primary lesion. No significant correlation with the size and number of liver metastatic lesion, the site, WHO grade, depth of invasion, lymph node metastasis, vascular invasion or tumor budding of the primary lesion was observed. Conclusion A positive correlation is found between liver metastasis of colorectal adenocarcinoma and PDC grade of primary tumor. Evaluating the PDC grade of primary tumor may provide a reference for the risk of liver metastasis.

Dormancy and Survival Mechanism of Disseminated Tumor Cell and Its Role in Prevention and Treatment of Solid Tumor Metastasis / 肿瘤防治研究

The prevention and treatment of tumor metastasis can significantly improve the survival of patients with solid tumors. However, there is still a lack of effective drugs for the prevention and treatment of metastasis. The main reason is that the existing intervention and therapeutic drugs are difficult to achieve precise prevention and treatment of metastasis. Due to disseminated tumor cells (DTCs) already exist in the metastatic target organs of early postoperative patients, they are difficult to be detected with existing imaging techniques, and there is a lack of effective intervention drugs and efficacy evaluation systems. When DTCs grow to be detectable by imaging, the patient is already in the advanced stage of cancer, which has become a bottleneck restricting the breakthroughs in metastasis prevention and treatment. This paper reviews the dormancy and survival mechanism of DTCs in metastatic target organs and its intervention strategies, in order to promote the curative effect of metastasis prevention and treatment.

Citotoxicidad in vitro del polipéptido antimicrobiano nisina sobre células tumorales sanguíneas / In vitro cytotoxicity of the antimicrobial polypeptide nisin on blood tumor cells

RESUMEN: Las bacteriocinas son péptidos antimicrobianos de síntesis ribosomal secretadas por bacterias. Dentro de estas destaca nisina que posee potenciales usos en terapias antibióticas, como biopreservante de alimentos y probióticos. También se ha descrito que nisina posee citotoxicidad sobre líneas celulares neoplásicas, pero existe poca información de su efecto sobre células tumorales sanguíneas. Debido al potencial uso que presenta nisina, es relevante determinar la toxicidad que presenta sobre líneas celulares tumorales del tipo sanguíneo. Para esto, se realizaron ensayos de actividad hemolítica sobre eritrocitos humanos y de toxicidad sobre células mononucleares de sangre periférica humanas, determinándose que nisina no posee efecto citotóxico sobre este tipo de células normales humanas sanguíneas. Se realizaron también, ensayos de citotoxicidad con líneas celulares tumorales (K562 y U937), con el fin de determinar dosis, tiempo de exposición y selectividad en el efecto tóxico de nisina sobre las células tumorales humanas. Estos ensayos muestran que nisina presenta actividad citotóxica sobre líneas celulares K562 y U937 a las 72 h de exposición, a una concentración de 40 µg/mL, que corresponde a 100 veces la concentración mínima inhibitoria (MIC) usada para su acción sobre bacterias. Al comparar el efecto de nisina sobre células mononucleares de sangre periférica humanas con las líneas tumorales linfoides y mieloides (K562 y U937 respectivamente), se observa un efecto selectivo de nisina sobre las células tumorales sanguíneas.

SUMMARY: Bacteriocins are antimicrobial peptides of ribosomal synthesis secreted by bacteria. Among these, nisin stands out, which has potential uses in antibiotic therapies, as a food bio preservative and probiotics. Nisin has also been reported to have cytotoxicity on neoplastic cell lines, but there is little information on its effect on blood tumor cells. Due to the potential use that nisin presents, it is relevant to determine the toxicity it presents on tumor cell lines of the blood type. For this, hemolytic activity tests were carried out on human erythrocytes and toxicity on human peripheral blood mononuclear cells, determining that nisin does not have a toxic effect on this type of normal human blood cells. Cytotoxicity tests were also carried out with tumor cell lines (K562 and U937), to determine dose, exposure time and selectivity in the toxic effect of nisin on human tumor cells. These tests show that nisin shows cytotoxic activity on K562 and U937 cell lines at 72 h of exposure, at a concentration of 40 µg / mL, which corresponds to 100 times the minimum inhibitory concentration (MIC) used for its action on bacteria. When comparing the effect of nisin on human peripheral blood mononuclear cells with lymphoid and myeloid tumor lines (K562 and U937 respectively), a selective effect of nisin on blood tumor cells is observed.

Análise de marcadores sanguíneos em pacientes com osteossarcoma, sarcomas de partes moles e tumor desmóide / Analysis of blood markers in patients with soft tissue sarcoma and desmoid tumor

Introdução: O tumor desmoide (TD) é uma neoplasia rara com altas taxas de recorrência local, composto por células fibroblásticas que se caracterizam pela expressão de moléculas-chave, incluindo o filamento intermediário vimentina, ciclooxigenase-2 (COX-2) e ß-catenina nuclear. Células tumorais circulantes (CTCs) isoladas do sangue periférico de pacientes com sarcomas e outras neoplasias podem ser utilizadas como biomarcadores precoces de invasão e disseminação tumoral. A família dos Receptores do Fator de Crescimento Epidérmico (Epidermal Growth Factor Receptor, EGFR) também podem influenciar no processo de invasão das CTCs, na formação de metástases e na recolonização de seus tumores de origem por meio de um processo de "auto-semeadura do tumor". Objetivo: Nosso objetivo foi identificar CTCs no sangue periférico de pacientes com TD ou sarcomas e avaliar a expressão das proteínas ß-catenina, TGF-ßRI (do Inglês, Transforming Growth Factor-ß Receptor I), COX-2 (Cyclooxygenase2), vimentina, GLUT-1 (Glucose Transporter 1), LGR5 (G-Protein Coupled Receptor 5) e EGFR, e sua correlação com sobrevidas global (SG) e livre de progressão (SLP). Materiais e Métodos: Foi realizado um estudo prospectivo de pacientes com diagnóstico inicial ou TD recidivado com doença mensurável. Para sarcomas, utilizamos amostras coletadas de forma prospectiva e retrospectiva. As amostras de sangue de cada paciente foram processadas e filtradas pelo ISET® (Rarecells, França) para isolamento e quantificação de CTCs. A expressão das proteínas foi analisada por imunocitoquímica (ICC). Para análise molecular das CTCs provenientes de pacientes com TD foi padronizado o método de PCR digital. Resultados: Foram incluídos 18 pacientes com TD, todos com CTCs detectáveis, com níveis que variaram entre 0,5­13 CTCs/mL. Encontramos uma concordância da expressão de ß-catenina em CTCs e tumores primários de 42,8% (6/14) dos casos usando ICC e imunohistoquímica, respectivamente. Nos nossos testes prévios de PCR digital, encontramos cópias mutadas de S45Pro em 4 pacientes (40%) e de S45Phe em apenas um paciente (10%). Em contraste, não foram encontradas mutações Th41Ala. Nas amostras de sarcomas, analisamos 30 amostras e encontramos CTCs em 93% dos pacientes e os níveis variaram de 0-11,25 CTCs/mL. Observamos também que a SG dos pacientes positivos para EGFR (p=0,027) eram inferiores às sobrevidas dos pacientes negativos para as mesmas proteínas. Conclusões: Nosso estudo identificou alta prevalência de CTCs em pacientes com TD e sarcomas. A concordânciada expressão de ß-catenina entre tumor primário e CTCs traz novas perspectivas para avaliar a dinâmica das CTCs no compartimento sanguíneo, abrindo novos caminhos para o estudo da biologia e comportamento do TD. Este é o primeiro estudo a demonstrar a expressão da proteína LGR5 em CTCs de pacientes com diferentes tipos de sarcomas, o que pode abrir novas oportunidades para futuras investigações. O próximo passo é caracterizar CTCs em uma coorte maior de pacientes para entender melhor o papel do LGR5 e das demais proteínas no processo de metástases tumorais em sarcomas. Além disso, esses resultados abrem a possibilidade de usar CTCs para prever a dinâmica do TD no momento da progressão da doença e tratamento. Mais estudos com tamanhos de amostra maiores são necessários para validar nossos achados tanto em TD como em sarcomas

Introduction: Desmoid tumor (DT) is a rare neoplasm with high rates of local recurrence, composed of fibroblast cells that are characterized by the expression of key molecules, including the intermediate filament vimentin, cyclooxygenase-2 (COX-2) and ß-catenin. Circulating tumor cells (CTCs) isolated from the peripheral blood of patients with sarcomas and other neoplasms can be used as early biomarkers of tumor invasion and dissemination. The Epidermal Growth Factor Receptor (EGFR) family can also influence the process of CTC invasion, metastasis formation and recolonization of their tumors of origin through a process of "tumor selfseeding". Objective: Our objective was to identify CTCs in the peripheral blood of patients with TD or sarcomas and to evaluate the expression of ßcatenin proteins, transforming growth factor receptor beta I (TGF-ßRI), COX-2 (cyclooxygenase-2), vimentin, GLUT-1 (Glucose transporter 1), LGR5 (Gprotein coupled receptor 5) and EGFR and their relation with progression free (PFS) and overall suvival (OS). Methods: We performed a prospective study of patients with initial diagnosis or relapsed TD with measurable disease. For sarcomas, we used samples collected prospectively and retrospectively. Blood samples from each patient were processed and filtered by ISET® (Rarecells, France) for isolation and quantification of CTCs. Protein expression was analyzed by immunocytochemistry (ICC). For the molecular analysis of CTCs from patients with TD, the digital PCR method was standardized. Results: Eighteen TD patients were included, all with detectable CTCs, with levels ranging from 0.5­13 CTCs/mL. We found a concordance ofß-catenin expression in CTCs and primary tumors of 42.8% (6/14) of cases using ICC and immunohistochemistry, respectively. In our previous digital PCR tests, we found mutated copies of S45Pro in 4 patients (40%) and of S45Phe in only one patient (10%). In contrast, no Th41Ala mutations were found. In the sarcoma samples, we analyzed 30 samples and found CTCs in 93% of the patients and the levels ranged from 0-11.25 CTCs/mL. We also observed that the OS of EGFR positive patients (p=0.027) were lower than the survival of negative patients for the same proteins. Conclusions: Our study identified a high prevalence of CTCs in patients with TD and sarcomas. The agreement of ß-catenin expression between primary tumor and CTCs brings new perspectives to evaluate the dynamics of CTCs in the blood compartment, opening newavenues for the study of the biology and behavior of TD. This is the first study to demonstrate the expression of LGR5 protein in CTCs from patients with different types of sarcomas, which may open new opportunities for future investigations. The next step is to characterize CTCs in a larger cohort of patients to better understand the role of LGR5 and other proteins in the process of tumor metastases in sarcomas. Furthermore, these results open up the possibility of using CTCs to predict the dynamics of TD at the time of disease progression and treatment. More studies with larger sample sizes areneeded to validate our findings in both TD and sarcomas

Study on the prognostic value of circulating tumor cells in colorectal cancer patients / 中华普通外科杂志

Objective:To explore the prognostic value of circulating tumor cell (CTC) for colorectal cancer.Method:We analyze the correlation between CTC and clinicopathological data, survival curve and overall survival.Results:The positive rates of preoperative and postoperative CTC in 181 colorectal cancer patients were 66.3% and 65.7% respectively ( χ2=0.012, P=0.912). The postoperative CTC positive rates for recurrence and non-recurrence of stage Ⅱ colorectal cancer were 29.2% and 8.0%, respectively ( χ2=4.303, P=0.038). The progress free survuval of CTC-positive and CTC-negative in postoperative stage Ⅱ colorectal cancer patients were 28.7 months and 34.0 months, respectively ( χ2=4.096, P=0.043). Conclusion:Postoperative CTC detection has predictive prognostic value for patients with stage Ⅱ colorectal cancer.

Role of hsa_circ_0008039 and miR-484 in oxygen-glucose deprivation/reoxygenation injury in SK-N-SH cells: relationship with Fis1 / 中华麻醉学杂志

Objective:To evaluate the role of has_circ_0008039 and miR-484 in oxygen-glucose deprivation/reoxygenation (OGD/R) injury in SK-N-SH cells and the relationship with Fis1.Methods:SK-N-SH cells were cultured in vitro to logarithmic growth stage and divided into 5 groups ( n=25 each) according to the random number table method: control group (group C), OGD/R group, has_circ_0008039 siRNA group (group S), hsa_circ_0008039 over-expression group (group E) and has_circ_0008039 siRNA plus miR-484 inhibitor group (group S+ I). Cells were cultured in normal condition in group C. In S, E and S+ I groups, after the cells were transfected with hsa_circ_0008039 siRNA, has_circ_0008039 over-expression vector, hsa_circ_0008039 siRNA and miR-484 inhibitor, the cells were subjected to oxygen-glucose deprivation for 12 h followed by 24 h restoration of O 2-glucose supply to develop the OGD/R model.At 24 h of restoration of O 2-glucose supply, the cell viability and amount of lactic dehydrogenase (LDH) released were measured using CCK-8 assay, the expression of hsa_circ_0008039, miR-484 and Fis1 mRNA was detected using real-time polymerase chain reaction, and the expression of Fis1 was detected by Western blot.A dual-fluorescein experimental report was used to verify the targeting relationship between hsa_circ_0008039 and miR-484. Results:Compared with group C, the cell viability was significantly decreased, and the amount of LDH released was increased in the other 4 groups, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in OGD/R and E groups, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group OGD/R, the cell viability was significantly decreased, and the amount of LDH released was increased in E and S+ I groups, the cell viability was significantly increased, and the amount of LDH released was decreased in group S, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in group E, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and the expression of miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group S, the cell viability was significantly decreased, the amount of LDH released was increased, the expression of miR-484 was down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.01). The dual-fluorescein experimental report verified that miR-484 was the target of hsa_circ_0008039 which binded to miR-484 specifically. Conclusions:has_circ_0008039 is involved in OGD/R injury in SK-N-SH cells by targetedly binding to miR-484, which is associated with up-regulation of Fis1 expression.

A hybrid bacterium with tumor-associated macrophage polarization for enhanced photothermal-immunotherapy

Remodeling the tumor microenvironment through reprogramming tumor-associated macrophages (TAMs) and increasing the immunogenicity of tumors via immunogenic cell death (ICD) have been emerging as promising anticancer immunotherapy strategies. However, the heterogeneous distribution of TAMs in tumor tissues and the heterogeneity of the tumor cells make the immune activation challenging. To overcome these dilemmas, a hybrid bacterium with tumor targeting and penetration, TAM polarization, and photothermal conversion capabilities is developed for improving antitumor immunotherapy in vivo. The hybrid bacteria (B.b@QDs) are prepared by loading Ag2S quantum dots (QDs) on the Bifidobacterium bifidum (B.b) through electrostatic interactions. The hybrid bacteria with hypoxia targeting ability can effectively accumulate and penetrate the tumor tissues, enabling the B.b to fully contact with the TAMs and mediate their polarization toward M1 phenotype to reverse the immunosuppressive tumor microenvironment. It also enables to overcome the intratumoral heterogeneity and obtain abundant tumor-associated antigens by coupling tumor penetration of the B.b with photothermal effect of the QDs, resulting in an enhanced immune effect. This strategy that combines B.b-triggered TAM polarization and QD-induced ICD achieved a remarkable inhibition of tumor growth in orthotopic breast cancer.

Site-specific labelling and biological evaluation of PD-L1 targeted single-domain antibody / 中华核医学与分子影像杂志

Objective:To synthesize a novel site-specifically labelled probe 68Ga-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)-Cys-Asp-Val (CDV)-Nb109 and explore its potential for detection of the programmed cell death ligand 1 (PD-L1) expression level in different tumors. Methods:Firstly, CDV was inserted into the tail of the sequence of Nb109 by genetic engineering. Then the precursor DOTA-CDV-Nb109 was prepared by mixing the maleimide-DOTA and the single-domain antibody CDV-Nb109 (amount of substance ratio 1∶1) via the maleimide-cysteine site-specific coupling strategy. Subsequently, the DOTA-CDV-Nb109 was labeled with 68Ga and purified by PD-10 column. Human melanoma A375, human PD-L1 transfected melanoma A375-hPD-L1 and human glioma U87 tumor-bearing mice models were established, and the diagnostic value of 68Ga-DOTA-CDV-Nb109 was evaluated by stability assay, cellular uptake, and microPET imaging. One-way analysis of variance and the least significant difference t test were used to analyze the data. Results:The probe 68Ga-DOTA-CDV-Nb109 was obtained with the radiochemical yield of (69.79±4.69)%, radiochemical purity more than 97%, and molar activity of (12.85±1.51) GBq/μmol. 68Ga-DOTA-CDV-Nb109 had strong binding affinity for A375-hPD-L1 with the dissociation constant ( Kd) of (66.43±17.89) nmol/L. The uptake of 68Ga-DOTA-CDV-Nb109 in A375-hPD-L1 and U87 cells were (3.17±0.15) percentage of the added radioactivity dose (%AD) and (2.08±0.03) %AD respectively, which were significantly higher than that in A375 cells ((1.21±0.14) %AD; F=82.87, t values: 15.23, 9.98, P values: <0.001, 0.003). The tumor uptake of the probe in A375-hPD-L1 ((5.21±0.35) percentage of injected dose per ml (%ID/ml)) and U87 tumor-bearing mice ((3.44±0.69) %ID/ml) were significantly higher than that in A375 tumor-bearing mice ((2.17±0.36) %ID/ml; F=249.72, t values: 35.70, 3.43, both P<0.001). Conclusion:The site-specifically labelled probe 68Ga-DOTA-CDV-Nb109, which can non-invasively and dynamically monitor the change of PD-L1 expression level in different tumors and help screen patients who can benefit from PD-L1 immune checkpoint blocking therapy, is successfully synthesized with high radiochemical purity.

Preparation and microPET imaging of extradomain-B fibronectin specific probe 18F-AlF-NOTA-PEG 4-ZD2 / 中华核医学与分子影像杂志

Objective:To prepare specific molecular probe 18F-AlF-1, 4, 7-triazacylononane-1, 4, 7-triacetic acid-(polyethylene glycol) 4-ZD2 ( 18F-AlF-NOTA-PEG 4-ZD2) for targeting extradomain-B fibronectin (EDB-FN), and evaluate its properties in vitro and in vivo. Methods:18F-AlF-NOTA-PEG 4-ZD2 was prepared by one-step chelation labeling with Al 18F. The radiochemical purity and in vitro stability were determined by high performance liquid chromatography (HPLC). The partition coefficient (logP) of 18F-AlF-NOTA-PEG 4-ZD2 was evaluated, and the cell uptake experiment was carried out (triple-negative breast cancer (MDA-MB-231) cells (1×10 6/tube) were divided into 3 groups ( n=3 per group); positive group, inhibition group, control group). MicroPET imaging was performed on MDA-MB-231 bearing nude mice ( n=3) after 18F-AlF-NOTA-PEG 4-ZD2 injection (30, 60, 90, 120 min) and compared with blocking group ( n=3, NOTA-PEG 4-ZQ 2 was preinjected at 0.5 h before 18F-AIF-NOTA-PEG a-ZD2 injection). Independent-sample t test was used to analyze the data. Results:18F-AlF-NOTA-PEG 4-ZD2 was successfully prepared. The optimized radiochemical yield was (33.8±2.1)% (undecay corrected, n=8) and the radiochemical purity was >96%. After incubating 120 min at 37 ℃, the radiochemical purity of 18F-AlF-NOTA-PEG 4-ZD2 in human serum and PBS was >93%, indicating its good stability in vitro. The specific activity was (11.1±3.2) GBq/μmol, and logP was -1.43±0.05. The uptake value of tumor cells was (1.77±0.28) percentage applied activity (%AR)/10 6 cells at 120 min post-injection in positive group, and the total uptake value of the inhibition group was (0.76±0.07) %AR/10 6 cells ( t=4.30, P=0.032). MicroPET imaging in tumor bearing nude mice showed that 18F-AlF-NOTA-PEG 4-ZD2 was mainly metabolized by the liver and kidneys. The tumor uptake value was (1.94±0.21) percentage activity of injection dose per gram of tissue (%ID/g) at 60 min post-injection and the tumor/muscle ratio was 3.80±0.25 at 90 min post-injection in the experimental group, while the tumor uptake value of tumor bearing nude mice in the blocking group was (0.43±0.09) %ID/g at 60 min post-injection ( t=3.18, P=0.006). Conclusions:18F-AlF-NOTA-PEG 4-ZD2 can be prepared simply with high labeling rate and good stability in vitro, with high tumor uptake and tumor/muscle ratio in microPET imaging, and good specificity and long tumor residence time. The probe has good application prospect in breast cancer with high expression of fibronectin subtype EDB-FN.