Journal articles: 'ISET (Isolation by SizE of Tumor'

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Ma, Yu-Chao, Li Wang, and Feng-Lei Yu. "Recent Advances and Prospects in the Isolation by Size of Epithelial Tumor Cells (ISET) Methodology." Technology in Cancer Research & Treatment 12, no. 4 (August 2013): 295–309. http://dx.doi.org/10.7785/tcrt.2012.500328.

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Massard, Christophe, Marianne Oulhen, Sylvestre Le Moulec, Yohann Loriot, Nathalie Auger, Philippe Beuzeboc, Laurence Albiges, et al. "Molecular characterization of heterogeneity in circulating tumor cells (CTCs) and tumor samples of metastatic castration-resistant prostate cancer (mCRPC) patients: The multicenter PETRUS program." Journal of Clinical Oncology 33, no. 7_suppl (March 1, 2015): 165. http://dx.doi.org/10.1200/jco.2015.33.7_suppl.165.

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165 Background: In order to optimize molecular screening in advanced prostate cancer patients, we implemented a prospective molecular triage trial PETRUS (NCT01786031), based on molecular characterization of fresh tumor biopsies and circulating tumor cells (CTCs) in patients with mCRPC. The primary objective was to investigate the feasibility of prospectively performing molecular profiling of CRPC patients. Methods: A tumor biopsy from a metastatic and tumor primary site, along with blood samples specimen were collected from patients, and both frozen and FFPE samples were obtained. CTCs were detected using two methods, CellSearch and filtration (ISET, isolation by Size of Epithelial Tumor cells) combined to four-color immunofluorescent staining. Results: From December 2012 and March 2014, 51 CRPC patients with metastases accessible for biospy were included in 5 centres in France. Tumor biospy were successful (≥ 50% tumor cells) in 36/51 pts (72%). CTCs with different phenotypes were detected by CellSearch and ISET combined to four-color immunofluorescent staining in 28 (51%) pts. FISH AR and ERG was successful in only 54% and 32% of metastasis biopsies and primitive tumors respectively, most likely due to the absence of tumor cells or overfixation. The ISET platform was able to detect CTCs clusters, CTCs expressing epithelial and vimentin markers. AR amplification and ERG rearrangement analyses showed high level of heterogeneity with evidence of multiple CRPC iCTC subpopulation. A high level of heterogeneity between CTCs, metastasis biopsies and primitive tumors was observed for ERG rearrangement and AR amplification status. Conclusions: Our results demonstrate that CTCs could be an alternative source for surrogate molecular marker assessment in mCRPC, but neither approach could truly reflect the extreme heterogeneity observed in CTCs from mCRPC. Our preliminary results suggest also the operational feasibility of metastatic tumor biopsy: high interest for patients, significant yield of tumor biopsies and ad hoc molecular analysis. Clinical trial information: NCT01786031.

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Tamminga, Menno, Kiki C. Andree, T. Jeroen N. Hiltermann, Maximilien Jayat, Ed Schuuring, Hilda van den Bos, Diana C. J. Spierings, et al. "Detection of Circulating Tumor Cells in the Diagnostic Leukapheresis Product of Non-Small-Cell Lung Cancer Patients Comparing CellSearch® and ISET." Cancers 12, no. 4 (April 7, 2020): 896. http://dx.doi.org/10.3390/cancers12040896.

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Circulating tumor cells (CTCs) detected by CellSearch are prognostic in non-small-cell lung cancer (NSCLC), but rarely found. CTCs can be extracted from the blood together with mononuclear cell populations by diagnostic leukapheresis (DLA), therefore concentrating them. However, CellSearch can only process limited DLA volumes (≈2 mL). Therefore, we established a protocol to enumerate CTCs in DLA products with Isolation by SizE of Tumor cells (ISET), and compared CTC counts between CellSearch® and ISET. DLA was performed in NSCLC patients who started a new therapy. With an adapted protocol, ISET could process 10 mL of DLA. CellSearch detected CTCs in a volume equaling 2 × 108 leukocytes (mean 2 mL). CTC counts per mL were compared. Furthermore, the live cell protocol of ISET was tested in eight patients. ISET successfully processed all DLA products—16 with the fixed cell protocol and 8 with the live cell protocol. In total, 10–20 mL of DLA was processed. ISET detected CTCs in 88% (14/16), compared to 69% (11/16, p < 0.05) with CellSearch. ISET also detected higher number of CTCs (ISET median CTC/mL = 4, interquartile range [IQR] = 2–6, CellSearch median CTC/mL = 0.9, IQR = 0–1.8, p < 0.01). Cells positive for the epithelial cell adhesion molecule (EpCAM+) per mL were detected in similar counts by both methods. Eight patients were processed with the live cell protocol. All had EpCAM+, CD45−, CD235- cells isolated by fluorescence-activated cell sorting (FACS). Overall, ISET processed larger volumes and detected higher CTC counts compared to CellSearch. EpCAM+ CTCs were detected in comparable rates.

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Lelievre, L., P. Paterlini-Brechot, S. Camatte, E. Tartour, M. Aggerbeck, F. Vilde, and F. Lecuru. "Effect of laparoscopy versus laparotomy on circulating tumor cells using isolation by size of epithelial tumor cells." International Journal of Gynecologic Cancer 14, no. 2 (2004): 229–33. http://dx.doi.org/10.1136/ijgc-00009577-200403000-00008.

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AimTo assess the effect of laparoscopy on circulating tumor cell (CTC) detection in case of carcinosis.Material and methodsWe compared laparoscopy versus laparotomy on tumor cell blood release in an animal model of ovarian carcinosis obtained by intraperitoneal inoculation of IGR-OV1 cells in nude rats. Animals were randomly assigned to one of the following groups: CO2 laparoscopy (L), gasless laparoscopy (GL), midline laparotomy (ML), or general anesthesia as control (C). A 0.5 ml blood sample was taken in each case before and after experiment and tested with a novel assay, ISET (isolation by size of epithelial tumor cells), which isolates CTC by filtration on account of their size. Statistics were performed with the Fisher's and the Chi-square tests.ResultsTen rats were included in each group. We did not find any significant difference in CTC prevalence before and after surgery (2/14 versus 3/19, respectively, P = 1). Similarly, the three surgical accesses were equivalent with one postexperiment detection per group: 1/5 for L, 1/7 for ML, 1/7 for GL, and 1/6 for C (P = 0.9).ConclusionThis trial did not show any deleterious effect of laparoscopy on CTC when compared to laparotomy.

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Sun, Na, Xinpan Li, Zhili Wang, Yuzhi Li, and Renjun Pei. "High-purity capture of CTCs based on micro-beads enhanced isolation by size of epithelial tumor cells (ISET) method." Biosensors and Bioelectronics 102 (April 2018): 157–63. http://dx.doi.org/10.1016/j.bios.2017.11.026.

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Clarke, Noel W., Sarah E. Marley, Andrew M. Hughes, Anthony F. Nash, Michael D. Malone, Jim W. Growcott, Darren R. Hodgson, et al. "Assessment of the variability of and effect of hormone therapy on circulating tumor cell numbers and androgen receptor expression in patients with prostate cancer." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e15141-e15141. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e15141.

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e15141 Background: In prostate cancer (PC) CTC number and character may offer a means of assessing disease load and target engagement. However, capture platforms differ, presenting challenges to enumeration and molecular characterisation. We report the findings of a pilot study assessing the intra- and inter-patient variability of 2 different platforms, and the feasibility of measuring CTC-androgen receptor (AR) expression by immunohistochemistry (IHC) in a single platform. Methods: Following ethical approval, 4 PC cohorts (n = 12, 12, 10, and 6 respectively) were recruited; #1 localised, no hormonal therapy; #2 and #3: castrate-resistant, receiving LHRHa or LHRHa and bicalutamide, respectively; #4: newly diagnosed locally advanced, no hormonal therapy. Blood (2 x 10 mL, 2 visits #1-3; 2 x 10 mL,1 visit #4) was taken and CTCs isolated using either the Cell Search CTC Test (Veridex) or Isolation by Size of Epithelial Cells Technique (ISET) for enumeration, and ISET for AR expression (H-score [(% 1*1+) + (% 2*2+) + (% 3*3+)]). Results: There was no correlation between Veridex and ISET for detection of CTCs (Veridex enumeration:14%, 63%, 53% and 0% of samples in #1 - 4, respectively, vs ISET enumeration: 100% of samples across all cohorts) with the latter platform detecting a significantly higher number of CTCs/4mls from patients in cohort #4 vs #1 (GLS Means 119 vs 46, p=0.0135). For both platforms there was no evidence of a systematic change in the counts at two separate visits 2 weeks apart. AR was detectable in approximately 25-35% CTCs from all cohorts and there were no significant differences in the H-score between the cohorts, although the number of AR-positive cells/4mls was significantly higher in #4 vs #1, and #2 (GLS Means 25 vs 14, 15, p= 0.0197, 0.0398, respectively). Conclusions: Populations of CTCs detected by Veridex and ISET appear stable over short durations (2 weeks) and AR was detected in a proportion of CTCs by ISET using IHC. Further work is required to find alternative methodologies with greater specificity.

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Paterlini-Brechot, P., S. Camatte, E. Tartour, M. Aggerbeck, F. Vilde, F. Lecuru, and L. Lelievre. "EFFECT OF LAPAROSCOPY VERSUS LAPAROTOMY ON CIRCULATING TUMOR CELLS USING ISET (ISOLATION BY SIZE OF EPITHELIAL TUMOR CELLS), A NEW METHOD OF DETECTION." International Journal of Gynecologic Cancer 13, Suppl 1 (March 2003): 58.2–58. http://dx.doi.org/10.1136/ijgc-00009577-200303001-00210.

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Oliveira, Thiago Bueno, Alexcia Camila Braun, Ulisses Ribaldo Nicolau, Emne Ali Abdallah, Vanessa Silva Alves, Victor Hugo Fonseca Jesus, Vinicius Fernando Calsavara, Luiz Paulo Kowalski, and Ludmilla T. D. Chinen. "Prognostic impact of baseline circulating tumor cells (CTCs) detected by the isolation by size of epithelial tumor cells (ISET) in locally advanced head and neck squamous cell carcinoma (LAHNSCC): Results of a prospective study." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 6061. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.6061.

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6061 Background: The prognostic impact of CTCs in LAHNSCC is yet to be determined, with conflicting results in previous trials, the majority utilizing cytokeratin dependent techniques for identification and counting of CTCs. The primary objective of this study is to determine the detection rates using the ISET method, and the prognostic role of CTCs in LAHNSCC patients (pts) treated with a curative intent. Methods: In this prospective study, peripheral blood samples of pts with non-metastatic LAHNSCC, stages III/IV, were analyzed for CTCs using the ISET method, in two scenarios: curative surgical resection and adjuvant radiotherapy (CTCs before RT) and candidates for a non-surgical strategy (unresectable or organ preservation) either with upfront RT concurrent with chemotherapy (CT) or cetuximab (CTCs before RT), or preceded by induction CT (CTCs before ICT). Results: Eighty-three pts were included, the majority males (83%), with oropharynx primary (50%) and submitted to ICT (40%). The detection rate of baseline CTCs was 94% (78/83), and CTCs counts were significantly correlated with survival. For each increase of 1 CTC at baseline there was a relative increase of 18% in the risk of death (HR = 1.18; 95%CI: 1.06-1.31; p < 0.001), 16% in the risk of progression (HR = 1.16; 95%CI: 1.04-1.28; p = 0.004) and a reduction of 26% in the odds of complete response to treatment (non-surgical group only - OR = 0.74; 95%CI: 0.58-0.95; p = 0.022). Using the maximum of the standardized log-rank statistic proposed by Lausen and Schumacher we establish cut off points for overall survival (OS) and progression-free survival (PFS). Pts with CTCs < 6.5/ml had an estimated 2y OS of 85.6% versus 22.9% for CTCs ≥ 6.5/ml (HR = 0.18; 95%CI: 0.06-0.49; P < 0.0001) and pts with CTCs ≤ 3.8/ml had an estimated 2y PFS of 71.8% versus 37% for CTCs > 3.8/ml (HR = 0.32; 95%CI:0.15-0.67; p = 0.001). Conclusions: The detection rate of baseline CTCs using the ISET method was very high in LAHNSCC and the counts of CTCs were strongly correlated with survival and response to treatment.

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Ilie, Marius, Véronique Hofman, Sylvie Leroy, Charlotte Cohen, Simon Heeke, Florian Cattet, Coraline Bence, et al. "Use of circulating tumor cells in prospective clinical trials for NSCLC patients – standardization of the pre-analytical conditions." Clinical Chemistry and Laboratory Medicine (CCLM) 56, no. 6 (May 24, 2018): 980–89. http://dx.doi.org/10.1515/cclm-2017-0764.

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Abstract Background: Circulating tumor cells (CTCs) hold potential for noninvasive diagnosis, prognosis and prediction testing in non-small cell lung cancer (NSCLC) patients. Minimizing degradation or loss of CTCs is pivotal for detection and profiling of the low abundance and fragile CTCs, particularly in clinical trials. We prospectively investigated (NCT02372448) whether a new blood collection device performed better compared to commonly used K3EDTA tubes, when subjected to long-term sample storage. Methods: Blood samples were drawn into K3EDTA and blood collection tubes (BCT) (Streck), and filtered by the Isolation by SizE of Tumor/Trophoblastic Cells (ISET® system), for CTC detection in two study populations of NSCLC patients; the training set of 14 patients with stage II/IV NSCLC, and the validation set of 36 patients with stage IV NSCLC). MET expression was evaluated by immunocytochemistry (ICC) and anaplastic lymphoma kinase (ALK) gene rearrangement by break-apart fluorescence in situ hybridization (FISH) on ISET-enriched CTCs. Results: Blood processed after 24 h and 48 h in BCT tubes showed stable CTCs counts and integrity, whereas CTCs in K3EDTA tubes showed an altered morphology in all patients. CTCs recovered in BCT or K3EDTA tubes at 24 and 48 h were evaluable by ICC for MET expression and by FISH for ALK rearrangement. Conclusions: The BCT tubes gave a high yield and preserved the integrity of CTCs after 24 and 48 h of storage at room temperature, which facilitate their molecular characterization in NSCLC patients entering clinical trials.

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Ilie, Marius, Elodie Long, Catherine Butori, Veronique Hofman, Celine Coelle, Virginie Mauro, Katia Zahaf, et al. "EML4-ALK-gene rearrangement comparative analysis in circulating tumor cells and in tumor tissue of patients with lung adenocarcinoma." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 7591. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.7591.

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7591 Background: The implementation of new theranostic biomarkers in Oncology is leading to impressive therapeutic improvements. In patients with lung cancer, the possibility to use Circulating Tumor Cells (CTCs) as a non-invasive theranostic approach is a clinically appealing challenge. Adenocarcinomas with EML4-ALK rearrangement are a new molecular subgroup of lung tumors with very good response to Crizotinib, an ALK inhibitor. We have thus aimed at developing an informative assay characterizing the ALK-gene status in CTCs isolated from patients with lung cancer. Methods: CTCs were isolated preoperatively using Isolation by Size of Epithelial Tumor cells method (ISET) from 65 patients with lung adenocarcinoma and blindly screened for ALK-gene status. ALK break-apart fluorescence in situ hybridization (FISH) (LSI ALK dual colour probes set) and immunochemistry using an anti-ALK antibody (5A4 clone) were blindly performed on CTCs and corresponding tumor tissues and results were compared. Results: Two patients consistently showed ALK-gene rearrangement and strong ALK protein expression in CTCs and corresponding tumor samples. Negative results (both ALK FISH and ALK immunochemistry) were found in CTCs and corresponding tumor samples from the other 63 patients. Conclusion: We have developed an approach allowing to characterize ALK-gene status in CTCs from patients with lung cancer and shown consistent results in CTC and tumor tissues. These preliminary results encourage larger studies and open new avenues for non-invasive, real-time, theranostic monitoring of cancer patients.

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Oliveira, Thiago Bueno, Ulisses Ribaldo Nicolau, Daniel Garcia, Victor Hugo Fonseca Jesus, Vanessa Alves Silva, Juliana Valim Romero, Marcilei Eliza Cavicchioli Buim, et al. "Detection of circulating tumor cells (CTCs) in locally advanced head and neck squamous cell carcinoma using the isolation by size of epithelial tumor cell (ISET) method: Preliminary results of a prospective study." Journal of Clinical Oncology 33, no. 15_suppl (May 20, 2015): e17057-e17057. http://dx.doi.org/10.1200/jco.2015.33.15_suppl.e17057.

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Hofman, V., E. Long, M. Ilie, C. Bonnetaud, J. M. Vignaud, J. F. Fléjou, S. Lantuejoul, et al. "Morphological analysis of circulating tumour cells in patients undergoing surgery for non-small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method." Cytopathology 23, no. 1 (January 6, 2011): 30–38. http://dx.doi.org/10.1111/j.1365-2303.2010.00835.x.

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Castello, Angelo, Francesco Giuseppe Carbone, Sabrina Rossi, Simona Monterisi, Davide Federico, Luca Toschi, and Egesta Lopci. "Circulating Tumor Cells and Metabolic Parameters in NSCLC Patients Treated with Checkpoint Inhibitors." Cancers 12, no. 2 (February 19, 2020): 487. http://dx.doi.org/10.3390/cancers12020487.

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Circulating tumor cells (CTC) count and characterization have been associated with poor prognosis in recent studies. Our aim was to examine CTC count and its association with metabolic parameters and clinical outcomes in non-small cell lung carcinoma (NSCLC) patients treated with immune checkpoint inhibitors (ICI). For this prospective study, data from 35 patients (23 males, 12 females) were collected and analyzed. All patients underwent an 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) scan and CTC detection through Isolation by Size of Tumor/Trophoblastic Cells (ISET) from peripheral blood samples obtained at baseline and 8 weeks after ICI initiation. Association of CTC count with clinical and metabolic characteristics was studied. Progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan–Meier method and the log-rank test. Median follow-up was 13.2 months (range of 4.9–21.6). CTC were identified in 16 out of 35 patients (45.7%) at baseline and 10 out of 24 patients at 8 weeks (41.7%). Mean CTC numbers before and after 8 weeks were 15 ± 28 and 11 ± 19, respectively. Prior to ICI, the mean CTC number was significantly higher in treatment-naïve patients (34 ± 39 vs. 9 ± 21, p = 0.004). CTC count variation (ΔCTC) was significantly associated with tumor metabolic response set by European Organization for Research and Treatment of Cancer (EORTC) criteria (p = 0.033). At the first restaging, patients with a high tumor burden, that is, metabolic tumor volume (MTV) and total lesion glycolysis (TLG), had a higher CTC count (p = 0.009). The combination of mean CTC and median MTV at 8 weeks was associated with PFS (p < 0.001) and OS (p = 0.024). Multivariate analysis identified CTC count at 8 weeks as an independent predictor for PFS and OS, whereas ΔMTV and maximum standardized uptake value variation (ΔSUVmax) was predictive for PFS and OS, respectively. Our study confirmed that CTC number is modulated by previous treatments and correlates with metabolic response during ICI. Moreover, elevated CTC count, along with metabolic parameters, were found to be prognostic factors for PFS and OS.

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Kallergi, Galatea, Eleni Politaki, Saad Alkahtani, Christos Stournaras, and Vassilis Georgoulias. "Evaluation of Isolation Methods for Circulating Tumor Cells (CTCs)." Cellular Physiology and Biochemistry 40, no. 3-4 (2016): 411–19. http://dx.doi.org/10.1159/000452556.

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Background: Detection of CTCs is a poor prognostic factor for many cancer types; however, their very low frequency represents an obstacle for their detection. The objective of the current study was to compare the performance of commonly used methods for CTCs isolation. Methods: The evaluated methods using spiking experiments of MCF7, SKBR3 and MDA MB-231 breast cancer cell lines were (i) ficoll density gradient separation (DGS), (ii) red blood cell lysis (Erythrolysis) isolation, (iii) positive immunomagnetic selection (EpCAM Dynal beads), (iv) two different negative immunomagnetic separation systems (Dynal vs Miltenyi CD45 beads) as well as (v) the Cell Search platform and (vi) the ISET system. Results: The recovery rates of Erythrolysis and DGS were 39% and 24%, respectively. Magnetic isolations are ranked from the worse to the best recovery rate as follows:, Myltenyi-anti-CD45 microbeads (24%); Dynal-anti-EpCAM beads (75%); Dynabeads-anti-CD45 (97%). CTCs isolation from blood samples using the CellSearch and ISET systems revealed that the recovery rate for Cell Search and ISET was 52% and 95%, respectively. Conclusions: Dynal-anti-CD45 beads have the best recovery rate compared to other magnetic methods. Furthermore the recovery rate of ISET was higher compared to Cell Search, especially for the more aggressive MDA-MB 231 cell line.

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Vona, Giovanna, Abdelmajid Sabile, Malek Louha, Veronique Sitruk, Serge Romana, Karin Schütze, Frédérique Capron, et al. "Isolation by Size of Epithelial Tumor Cells." American Journal of Pathology 156, no. 1 (January 2000): 57–63. http://dx.doi.org/10.1016/s0002-9440(10)64706-2.

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Nogaibayeva, Kakharman, and Gulbarshin Sultanova. "Circulating Tumor Cells: Atomic Properties And Hostile To Malignant Growth Treatment Checking." American Journal of Medical Sciences and Pharmaceutical Research 2, no. 07 (July 7, 2020): 1–6. http://dx.doi.org/10.37547/tajmspr/volume02issue07-01.

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Presence of Сirculating tumor cells (CTCs) in blood of disease patients, affirms about a scattering of malignant growth cells to the fringe circulatory system, on the most punctual phases of malignancy improvement and all the time vouches for an unfavorable clinical ebb and flow, particularly it is associated with arrangement of metastases. Moreover, these phones can speak to the base lingering infection and quantitative observing of CTCs level in time of hostile to malignant growth treatment furnishes specialiCDC with important data. For now the job of epithelial-to-mesenchymal travel (EMT) and mesenchymal-to-epithelial travel (MET) in arrangement of different subpopulations CTCs, in development of malignancy forceful properties. In this article we need to demonstrate the reason and issues of our logical exploration - the cytological atomic investigation of flowing tumor cells have separated by the size ward ISET innovation

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Mohamed, Hisham, Megan Murray, James N. Turner, and Michele Caggana. "Isolation of tumor cells using size and deformation." Journal of Chromatography A 1216, no. 47 (November 2009): 8289–95. http://dx.doi.org/10.1016/j.chroma.2009.05.036.

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Silva, Virgílio Souza e., Emne Ali Abdallah, Angelo Borsarelli Carvalho de Brito, Alexcia Camila Braun, Milena Shizue Tariki, Celso Abdon Lopes de Mello, Vinicius Fernando Calsavara, Rachel Riechelmann, and Ludmilla Thomé Domingos Chinen. "Baseline and Kinetic Circulating Tumor Cell Counts Are Prognostic Factors in a Prospective Study of Metastatic Colorectal Cancer." Diagnostics 11, no. 3 (March 12, 2021): 502. http://dx.doi.org/10.3390/diagnostics11030502.

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The discovery of predictive biomarkers in metastatic colorectal cancer (mCRC) is essential to improve clinical outcomes. Recent data suggest a potential role of circulating tumor cells (CTCs) as prognostic indicators. We conducted a follow-on analysis from a prospective study of consecutive patients with mCRC. CTC analysis was conducted at two timepoints: baseline (CTC1; before starting chemotherapy), and two months after starting treatment (CTC2). CTC isolation/quantification were completed by ISET® (Rarecells, France). CTC expressions of drug resistance-associated proteins were evaluated. Progression-free survival (PFS) and overall survival (OS) were estimated by the Kaplan–Meier method. Seventy-five patients were enrolled from May 2012 to May 2014. A CTC1 cut-off of >1.5 CTCs/mL was associated with an inferior median OS compared to lower values. A difference of CTC2−CTC1 > 5.5 CTCs/mL was associated with a reduced median PFS. By multivariate analysis, CTC1 > 1.5 CTCs/mL was an independent prognostic factor for worse OS. Multi-drug resistance protein-1 (MRP-1) expression was associated with poor median OS. CTC baseline counts, kinetics, and MRP-1 expression were predictive of clinical outcomes. Larger studies are warranted to explore the potential clinical benefit of treating mCRC patients with targeted therapeutic regimens guided by CTC findings.

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Hofman, Véronique J., Marius I. Ilie, Christelle Bonnetaud, Eric Selva, Elodie Long, Thierry Molina, Jean Michel Vignaud, et al. "Cytopathologic Detection of Circulating Tumor Cells Using the Isolation by Size of Epithelial Tumor Cell Method." American Journal of Clinical Pathology 135, no. 1 (January 2011): 146–56. http://dx.doi.org/10.1309/ajcp9x8ozbeiqvvi.

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Lelievre, L., P. Paterlini-Brechot, S. Camatte, E. Tartour, M. Aggerbeck, F. Vilde, and F. Lecuru. "Effect of laparoscopy versus laparotomy on circulating tumor cells using isolation by size of epithelial tumor cells." International Journal of Gynecological Cancer 14, no. 2 (March 2004): 229–33. http://dx.doi.org/10.1111/j.1048-891x.2004.014205.x.

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Xu, Lei, Xueying Mao, Ahmet Imrali, Ferrial Syed, Katherine Mutsvangwa, Daniel Berney, Paul Cathcart, John Hines, Jonathan Shamash, and Yong-Jie Lu. "Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation System." PLOS ONE 10, no. 9 (September 23, 2015): e0138032. http://dx.doi.org/10.1371/journal.pone.0138032.

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Faugeroux, Vincent, Celine Lefebvre, Emma Pailler, Valerie Pierron, Fanny Billiot, Charles Marcaillou, Sebastien Tourlet, et al. "Whole-exome sequencing of single circulating tumor cells is a useful tool for studying the intrapatient genetic heterogeneity in metastatic prostate cancer." Journal of Clinical Oncology 35, no. 6_suppl (February 20, 2017): 148. http://dx.doi.org/10.1200/jco.2017.35.6_suppl.148.

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148 Background: Molecular characterization of metastatic castration resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTC) offers an attractive noninvasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in mCRPC patients. Methods: Blood samples were drawn from 11 enzalutamide or abiraterone pre-treated mCRPC patients enrolled in the clinical program MOSCATO (NCT02613962). CTC enrichment, immunofluorescent detection and single cell isolation were performed using three methods (ISET filtration, CellSearch and the VyCap puncher system and RosetteSep enrichment) to obtain pools of 1-10 CTCs with distinct epithelial or mesenchymal phenotypes. After Whole Genome Amplification (WGA), WES was performed on the Illumina HiSeq 2000 platform. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively. Results: 189 WGA of CTC pools were performed. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5-10 cells. 17/34 (50%) CTC samples had shared sSNV with the paired tumor sample (range 0.35%-68%) Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large non epithelial CTC pointing out a high level of genetic heterogeneity between CTC. Overall, 89 deleterious protein-coding mutations were found only in pools of CTC, including mutations affecting oncogenic drivers such as MAPK1, HSP90AB1 or KDM5B. Conclusions: We present single cell WES of CTCs harboring distinct phenotypes. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity.

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Matos, Ignacio, Rebeca Lozano, Emilio Fonseca, Jesus MariÂa Hernandez, Arantxa Amores, Lina Lopez, Carolina Lopez, Maria Angeles Hernandez, Juan Luis Hernandez, and Juan J. Cruz. "Isolation of circulating tumor cells in colon cancer patients by size and chromosomal abnormalities." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e22058-e22058. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e22058.

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e22058 Background: Determining the number of circulating tumor cells (CTCs) in patients with metastatic colorectal cancer (CRC) has shown to be a prognostic factor, recent studies have proven the existence of EpCAM and cytokeratin negative CTCs. These cells present morphological and genetic alterations. Our study is based on an algorithm determined by the selection of the size of the cells and the genetic alterations detected by FISH which enables the isolation of CTCs in CRC patients by an automated system of fluorescence. Methods: Patients with metastatic CRC with normal blood differential and healthy controls between June 2011 and June 2012 were chosen. The objective was to show the differences between the quantification of morphogenetically altered cells in peripheral blood obtained from control and patients. The selected cells were over 7 micra in size and presented two or more genetic alterations. A combination of four probe sets was used: centromeric probe for CEP3, CEP7 and CEP17 and LSI p16INK4A 9p21.3. Fluorescent signals in specimens were quantified using an automated fluorescent system (Metafer) that is capable of scanning and classifying hundreds of cells under fluorescent illumination and allows for detection of cells according to nuclear size > 7 um and FISH pattern based on the algorithm that we have developed. Results: We selected 28 healthy individuals and 45 patients with CRC. In all the cases, 1,000 cells were analyzed in each individual. Only 30 cells >7 micra being found in the controls out of the 28,000 cells analyzed (0.1%). In the case of the patients, 640 cells were found (1.42%), the difference becoming statistically relevant after the T-test (p=0.003). None of the controls presented cells with two or more genetic alterations and over 7 micra in size, whereas 38% of the patients presented these characteristics despite having received treatment. The most frequent chromosomal changes were trisomy 3 and 17 (26% and 18%, respectively). Conclusions: This method allows us to select genetically abnormal circulating cells in a simple and automatic way. This opens the door for future studies to analyze the prognostic factor and the response rate to treatment under this method.

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Bauer, Natalie, Jyoti Rai, Hairu Chen, Lillianne Harris, Lalita Shevde, Tim Moore, and Judy King. "Microparticles/Exosomes: Isolation and TEM Analysis." Microscopy Today 17, no. 2 (March 2009): 42–45. http://dx.doi.org/10.1017/s1551929500054493.

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Microparticles and exosomes are small vesicular fragments of cell membrane which are released from activated and apoptotic cells. Microparticles (MPs) range in size from 0.5-1.5 μm, and exosomes are 0.5 μm and under. For the purposes of this article we will refer to both categories as microparticles. They differ from apoptotic bodies based on their smaller size, intact structure, and lack of degraded nuclear material. MPs have been shown to be released from a variety of cell types including platelets, endothelium, vascular smooth muscle cells, dendritic cells, and tumor cells. Jimenez and others have shown that based on the stimulus and cell type the MPs released are both quantitatively and phenotypically distinct. More recent data have shown the proteomics of MPs released from human umbilical vein endothelial cells differ dependent on whether they are stimulated with PAI or TNF-α.

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Tian, Fei, Lili Cai, Jianqiao Chang, Shanshan Li, Chao Liu, Tiejun Li, and Jiashu Sun. "Label-free isolation of rare tumor cells from untreated whole blood by interfacial viscoelastic microfluidics." Lab on a Chip 18, no. 22 (2018): 3436–45. http://dx.doi.org/10.1039/c8lc00700d.

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Low, Wan Shi, and Wan Abu Bakar Wan Abas. "Benchtop Technologies for Circulating Tumor Cells Separation Based on Biophysical Properties." BioMed Research International 2015 (2015): 1–22. http://dx.doi.org/10.1155/2015/239362.

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Circulating tumor cells (CTCs) are tumor cells that have detached from primary tumor site and are transported via the circulation system. The importance of CTCs as prognostic biomarker is leveraged when multiple studies found that patient with cutoff of 5 CTCs per 7.5 mL blood has poor survival rate. Despite its clinical relevance, the isolation and characterization of CTCs can be quite challenging due to their large morphological variability and the rare presence of CTCs within the blood. Numerous methods have been employed and discussed in the literature for CTCs separation. In this paper, we will focus on label free CTCs isolation methods, in which the biophysical and biomechanical properties of cells (e.g., size, deformability, and electricity) are exploited for CTCs detection. To assess the present state of various isolation methods, key performance metrics such as capture efficiency, cell viability, and throughput will be reported. Finally, we discuss the challenges and future perspectives of CTC isolation technologies.

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Ludwig, Nils, Aparna Rao, Saigopalakrishna Saileelaprasad Yerneni, Nduka Amankulor, and Theresa Whiteside. "Isolation and characterization of exosomes from IDH mutant gliomas." Journal of Clinical Oncology 37, no. 8_suppl (March 10, 2019): 152. http://dx.doi.org/10.1200/jco.2019.37.8_suppl.152.

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152 Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are found in > 70% of intermediate grade gliomas. Gain-of-function mutations in IDH1 promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and in body fluids of patients. TEX serve as a communication system between tumor and normal cells. Circulating TEX carry a complex molecular and genetic cargo and interfere with functions of immune cells. This study characterizes TEX produced by IDH mutant glioma spheres and TEX from plasma of patients with IDH mutant gliomas. Methods: TEX produced by patient-derived IDH1(mut) or IDH1(wt) glioma spheres or TEX in patient’s plasma were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers and molecular profile were characterized and the crossing of the blood-brain barrier (BBB) and biodistribution was investigated by in vivo imaging of TEX. Interactions of TEX with endothelial cells, astrocytes and immune cells were demonstrated by confocal microscopy. Results: TEX isolated from supernatants of IDH1(mut) or IDH1(wt) glioma spheres by mini-SEC carried TSG101 and showed the typical size and vesicular morphology of exosomes. BCA protein assays and qNano analysis revealed an elevated exosome production by IDH mutant cells ( p< 0.01). TEX carried immunosuppressive proteins (FasL, CTLA-4 and TRAIL) and IDH mutant exosomes carried higher levels of these proteins and of adenosine pathway components CD39 and CD73. Labeled TEX injected in the white matter of mice crossed the BBB and were detectable in distant organs 24h after injection. TEX were rapidly internalized by endothelial cells and astrocytes, but not by T cells, which interacted with TEX by surface signaling. These results were confirmed by investigating TEX isolated from glioma patient’s plasma. Conclusions: The data suggest that TEX play an important role in IDH mutant gliomas and drive tumor progression in part by inducing suppression of immune cells. Future efforts will focus on characterizing immunosuppressive effects of TEX derived from IDH mutant cells vs gliomas without IDH mutations.

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Gottlin, Elizabeth B., Xiangrong Guan, Charles Pegram, Allen Cannedy, Michael J. Campa, and Edward F. Patz. "Isolation of Novel EGFR-Specific VHH Domains." Journal of Biomolecular Screening 14, no. 1 (November 21, 2008): 77–85. http://dx.doi.org/10.1177/1087057108327064.

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Epidermal growth factor receptor (EGFR) is overexpressed or mutated in a high percentage of tumors. EGFR has long been considered a promising target for cancer diagnostic and therapeutic applications. However, monoclonal antibodies and other large antibody constructs diffuse into tumors slowly, limiting their efficacy. To develop lower molecular weight probes for EGFR and other tumor cell receptors, the authors immunized a llama with the extracellular domains (ECDs) of EGFR and an oncogenic mutant receptor, EGFRvIII, and with extracts of tumor cell lines. From the immune repertoire of the llama, the authors constructed a heavy chain variable domain (VHH domain)—phage library. At ~16 kDa, the VHH domain is a tenth of the size of a monoclonal antibody and is the smallest antibody fragment that retains specificity. By affinity selection from this library, the authors isolated many VHH domains with specificity for EGFR. The VHH domains bind to whole cells expressing the receptor but not to control cells lacking the receptor and can immunoprecipitate EGFR from cell lysates. Some VHH domains have cross-specificity with existing anti-EGFR monoclonal antibodies and have reasonably high (nM) affinities. The llama-VHH domain library is also potentially a rich source of targeting agents directed toward other tumor cell receptors. ( Journal of Biomolecular Screening 2009:77-85)

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Lu, Rong, Qiang Chen, Xiaoying Liu, Songfei Shen, Zhangchi Pan, and Chunmei Shi. "Detection of circulating stage III–IV gastric cancer tumor cells based on isolation by size of epithelial tumor: using the circulating tumor cell biopsy technology." Translational Cancer Research 8, no. 4 (August 2019): 1342–50. http://dx.doi.org/10.21037/tcr.2019.07.32.

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Lee, Ada, Juhee Park, Minji Lim, Vijaya Sunkara, Shine Young Kim, Gwang Ha Kim, Mi-Hyun Kim, and Yoon-Kyoung Cho. "All-in-One Centrifugal Microfluidic Device for Size-Selective Circulating Tumor Cell Isolation with High Purity." Analytical Chemistry 86, no. 22 (October 30, 2014): 11349–56. http://dx.doi.org/10.1021/ac5035049.

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Hosokawa, Masahito, Hirotsugu Kenmotsu, Yasuhiro Koh, Tomoko Yoshino, Takayuki Yoshikawa, Tateaki Naito, Toshiaki Takahashi, et al. "Size-Based Isolation of Circulating Tumor Cells in Lung Cancer Patients Using a Microcavity Array System." PLoS ONE 8, no. 6 (June 28, 2013): e67466. http://dx.doi.org/10.1371/journal.pone.0067466.

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Ahmed, Metages Gashaw, Mahlet Fasil Abate, Yanling Song, Zhi Zhu, Feng Yan, Yao Xu, Xiaomin Wang, Qingbiao Li, and Chaoyong Yang. "Isolation, Detection, and Antigen-Based Profiling of Circulating Tumor Cells Using a Size-Dictated Immunocapture Chip." Angewandte Chemie International Edition 56, no. 36 (July 26, 2017): 10681–85. http://dx.doi.org/10.1002/anie.201702675.

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Ahmed, Metages Gashaw, Mahlet Fasil Abate, Yanling Song, Zhi Zhu, Feng Yan, Yao Xu, Xiaomin Wang, Qingbiao Li, and Chaoyong Yang. "Isolation, Detection, and Antigen-Based Profiling of Circulating Tumor Cells Using a Size-Dictated Immunocapture Chip." Angewandte Chemie 129, no. 36 (July 26, 2017): 10821–25. http://dx.doi.org/10.1002/ange.201702675.

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Kurian, Talitha Keren, Soumyabrata Banik, Dharshini Gopal, Shweta Chakrabarti, and Nirmal Mazumder. "Elucidating Methods for Isolation and Quantification of Exosomes: A Review." Molecular Biotechnology 63, no. 4 (January 25, 2021): 249–66. http://dx.doi.org/10.1007/s12033-021-00300-3.

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AbstractExosomes are the smallest extracellular vesicles present in most of the biological fluids. They are found to play an important role in cell signaling, immune response, tumor metastasis, etc. Studies have shown that these vesicles also have diagnostic and therapeutic roles for which their accurate detection and quantification is essential. Due to the complexity in size and structure of exosomes, even the gold standard methods face challenges. This comprehensive review discusses the various standard methods such as ultracentrifugation, ultrafiltration, size-exclusion chromatography, precipitation, immunoaffinity, and microfluidic technologies for the isolation of exosomes. The principle of isolation of each method is described, as well as their specific advantages and disadvantages. Quantification of exosomes by nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing, electron microscopy, dynamic light scattering, and microfluidic devices are also described, along with the applications of exosomes in various biomedical domains.

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Guibert, Nicolas Marie, Myriam Delaunay, Amellie Lusque, Sandrine Gouin, Nadia Boubekeur, Isabelle Rouquette, Estelle Clermont, et al. "Monitoring PD-L1 expression on circulating tumor cells (CTCs) in patients with NSCLC treated with PD1 inhibitors." Journal of Clinical Oncology 36, no. 5_suppl (February 10, 2018): 147. http://dx.doi.org/10.1200/jco.2018.36.5_suppl.147.

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147 Background: Inhibitors of the immune checkpoint PD-1/PD-L1 have become a standard of care in NSCLC. Patient selection, currently based on PD-L1 expression in tumor tissue, is limited by its temporal and spatial heterogeneity. We hypothesized that monitoring PD-L1 staining of circulating tumor cells (CTCs) could represent a valuable non-invasive biomarker. Methods: Up to 3 blood samples were prospectively collected from patients with advanced NSCLC: i) pretreatment (nivolumab), ii) first follow-up, iii) progression. CTCs were isolated from 10 mL of blood using cell size-based technology (ISET, Rarecells). PD-L1 expression was assessed by immunofluorescence on CTCs and immunohistochemistry on tissue. Results: 162 samples from 96 patients were collected. PD-L1 expression could be assessed pretreatment on tissue and CTCs in 72% and 93%, respectively; and was ≥1% in 37% and 83%; ≥5% in 35% and 79%, respectively. No correlation between tissue and CTCs PD-L1 expressions was observed (Spearman coefficient correlation = 0.04, p = 0.77). At baseline, each 10/7.5 ml increase in CTCs count was associated with increased risk of death and progression (HR[95%CI]: 1.06 [1.005;1.117] p = 0.03 for OS and HR[95%CI]: 1.05 [1.01;1.10] p = 0.02 for PFS). The presence of PD-L1(+)CTC (≥1%) had no prognostic impact (OS: p = 0.89 and PFS: p = 0.55), but pretreatment PD-L1(+)CTCs were more frequent in the “non-responders” group (PFS < 6 months) (p = 0.04). Median CTC count was 30 (n = 96), 68.3 (n = 44) and 50.3 (n = 22) pretreatment, at the first follow-up and at progression, respectively. The changes in CTC count at the first follow-up had no impact on PFS (p = 0.45) or OS (p = 0.68). 96% of patients had PD-L1(+)CTCs at progression. Further analyses are ongoing to assess the prognostic value of the persistence of CTCs and PD-L1(+)CTCs at the first follow up and at progression. Conclusions: Analysis of PD-L1 expression on CTCs is highly feasible. PD-L1 expressions on tissue and CTCs are discordant, CTCs being more likely positives, suggesting false negatives occurring in small biopsies. Further analyses of the kinetics of this biomarker throughout ICI treatment, along with other circulating biomarkers, are ongoing. Clinical trial information: NCT02827344.

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Lee, Ada, Juhee Park, Minji Lim, Vijaya Sunkara, Shine Young Kim, Gwang Ha Kim, Mi-Hyun Kim, and Yoon-Kyoung Cho. "Correction to All-in-One Centrifugal Microfluidic Device for Size-Selective Circulating Tumor Cell Isolation with High Purity." Analytical Chemistry 90, no. 5 (February 15, 2018): 3637. http://dx.doi.org/10.1021/acs.analchem.8b00591.

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Yagi, Satomi, Yasuhiro Koh, Hiroaki Akamatsu, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Keiichiro Akamatsu, et al. "Development of an automated size-based filtration system for isolation of circulating tumor cells in lung cancer patients." PLOS ONE 12, no. 6 (June 22, 2017): e0179744. http://dx.doi.org/10.1371/journal.pone.0179744.

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Chen, Hongmei, Zhifeng Zhang, and Bin Wang. "Size- and deformability-based isolation of circulating tumor cells with microfluidic chips and their applications in clinical studies." AIP Advances 8, no. 12 (December 2018): 120701. http://dx.doi.org/10.1063/1.5072769.

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Waheed, Waqas, Anas Alazzam, Bobby Mathew, Nicolas Christoforou, and Eiyad Abu-Nada. "Lateral fluid flow fractionation using dielectrophoresis (LFFF-DEP) for size-independent, label-free isolation of circulating tumor cells." Journal of Chromatography B 1087-1088 (June 2018): 133–37. http://dx.doi.org/10.1016/j.jchromb.2018.04.046.

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Hofman, Véronique, Christelle Bonnetaud, Marius I. Ilie, Philippe Vielh, Jean Michel Vignaud, Jean François Fléjou, Sylvie Lantuejoul, et al. "Preoperative Circulating Tumor Cell Detection Using the Isolation by Size of Epithelial Tumor Cell Method for Patients with Lung Cancer Is a New Prognostic Biomarker." Clinical Cancer Research 17, no. 4 (November 23, 2010): 827–35. http://dx.doi.org/10.1158/1078-0432.ccr-10-0445.

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De Giorgi, Vincenzo, Pamela Pinzani, Francesca Salvianti, John Panelos, Milena Paglierani, Agata Janowska, Marta Grazzini, et al. "Application of a Filtration- and Isolation-by-Size Technique for the Detection of Circulating Tumor Cells in Cutaneous Melanoma." Journal of Investigative Dermatology 130, no. 10 (October 2010): 2440–47. http://dx.doi.org/10.1038/jid.2010.141.

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Feng, Ruonan, Ruixue Wang, Jessica Hong, Christopher M. Dower, Brad St Croix, and Mitchell Ho. "Isolation of rabbit single domain antibodies to B7-H3 via protein immunization and phage display." Antibody Therapeutics 3, no. 1 (January 2020): 10–17. http://dx.doi.org/10.1093/abt/tbaa002.

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Abstract Single domain antibodies have certain advantages including their small size, high stability and excellent tissue penetration, making them attractive drug candidates. Rabbit antibodies can recognize diverse epitopes, including those that are poorly immunogenic in mice and humans. In the present study, we established a method to isolate rabbit VH single domain antibodies for potential cancer therapy. We immunized rabbits with recombinant human B7-H3 (CD276) protein, made a phage-displayed rabbit VH single domain library with a diversity of 7 × 109, and isolated two binders (A1 and B1; also called RFA1 and RFB1) from phage panning. Both rabbit VH single domains exhibited antigen-dependent binding to B7-H3-positive tumor cell lines but not B7-H3 knockout tumor cell lines. Our study shows that protein immunization followed by phage display screening can be used to isolate rabbit single domain antibodies. The two single domain antibodies reported here may have potential applications in cancer immunotherapy.

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Philippron, Annouck, Lieven Depypere, Steffi Oeyen, Bram De Laere, Charlotte Vandeputte, Philippe Nafteux, Katleen De Preter, and Piet Pattyn. "Evaluation of a marker independent isolation method for circulating tumor cells in esophageal adenocarcinoma." PLOS ONE 16, no. 5 (May 7, 2021): e0251052. http://dx.doi.org/10.1371/journal.pone.0251052.

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Objective The enrichment of circulating tumor cells (CTCs) from blood provides a minimally invasive method for biomarker discovery in cancer. Longitudinal interrogation allows monitoring or prediction of therapy response, detection of minimal residual disease or progression, and determination of prognosis. Despite inherent phenotypic heterogeneity and differences in cell surface marker expression, most CTC isolation technologies typically use positive selection. This necessitates the optimization of marker-independent CTC methods, enabling the capture of heterogenous CTCs. The aim of this report is to compare a size-dependent and a marker-dependent CTC-isolation method, using spiked esophageal cells in healthy donor blood and blood from patients diagnosed with esophageal adenocarcinoma. Methods Using esophageal cancer cell lines (OE19 and OE33) spiked into blood of a healthy donor, we investigated tumor cell isolation by Parsortix post cell fixation, immunostaining and transfer to a glass slide, and benchmarked its performance against the CellSearch system. Additionally, we performed DEPArray cell sorting to infer the feasibility to select and isolate cells of interest, aiming towards downstream single-cell molecular characterization in future studies. Finally, we measured CTC prevalence by Parsortix in venous blood samples from patients with various esophageal adenocarcinoma tumor stages. Results OE19 and OE33 cells were spiked in healthy donor blood and subsequently processed using CellSearch (n = 16) or Parsortix (n = 16). Upon tumor cell enrichment and enumeration, the recovery rate ranged from 76.3 ± 23.2% to 21.3 ± 9.2% for CellSearch and Parsortix, respectively. Parsortix-enriched and stained cell fractions were successfully transferred to the DEPArray instrument with preservation of cell morphology, allowing isolation of cells of interest. Finally, despite low CTC prevalence and abundance, Parsortix detected traditional CTCs (i.e. cytokeratin+/CD45-) in 8/29 (27.6%) of patients with esophageal adenocarcinoma, of whom 50% had early stage (I-II) disease. Conclusions We refined an epitope-independent isolation workflow to study CTCs in patients with esophageal adenocarcinoma. CTC recovery using Parsortix was substantially lower compared to CellSearch when focusing on the traditional CTC phenotype with CD45-negative and cytokeratin-positive staining characteristics. Future research could determine if this method allows downstream molecular interrogation of CTCs to infer new prognostic and predictive biomarkers on a single-cell level.

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Kim, Jinhyun, Hoyoon Lee, KyongHwa Park, and Sehyun Shin. "Rapid and Efficient Isolation of Exosomes by Clustering and Scattering." Journal of Clinical Medicine 9, no. 3 (February 28, 2020): 650. http://dx.doi.org/10.3390/jcm9030650.

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Tumor-derived extracellular vesicles (EVs) have become important biomarkers of liquid biopsies for precision medicine. However, the clinical application of EVs has been limited due to the lack of EV isolation practical technology applicable to clinical environments. Here, we report an innovative EV isolation method, which is quick and simple, and facilitates high-yield and high-purity EV isolation from blood. Introducing a cationic polymer in plasma resulted in rapid clustering of anionic EVs and a chaotropic agent can separate EVs from these clusters. Isolated EVs were characterized in terms of size distribution, morphology, surface protein markers, and exosomal RNA. Through performance comparison with various methods, including ultracentrifugation (UC), the present method delivered the highest recovery rate (~20 folds that of UC) and purity ratio (3.5 folds that of UC) of EVs in a short period of time (<20 min). The proposed method is expected to be used in basic and applied research on EV isolation and in clinical applications.

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Heider, Katrin, Jonathan C. M. Wan, James Hall, Jelena Belic, Samantha Boyle, Irena Hudecova, Davina Gale, et al. "Detection of ctDNA from Dried Blood Spots after DNA Size Selection." Clinical Chemistry 66, no. 5 (April 8, 2020): 697–705. http://dx.doi.org/10.1093/clinchem/hvaa050.

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Abstract Background Recent advances in the study and clinical applications of circulating tumor DNA (ctDNA) are limited by practical considerations of sample collection. Whole-genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations and fragmentation patterns. We hypothesized that low-depth/shallow WGS (sWGS) data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may remove contaminating genomic DNA from small blood volumes. Dried blood spots have practical advantages for sample collection, may facilitate serial sampling, and could support novel study designs in humans and animal models. Methods We developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots using filter paper cards and bead-based size selection. DNA extracted and size-selected from dried spots was analyzed using sWGS and polymerase chain reaction (PCR). Results Analyzing a 50 μL dried blood spot from frozen whole blood of a patient with melanoma, we identified ctDNA based on the presence of tumor-specific somatic copy-number alterations, and found a fragment-size profile similar to that observed in plasma DNA. We found alterations in different chromosomes in blood spots from 2 patients with high-grade serous ovarian carcinoma. Extending this approach to serial dried blood spots from mouse xenograft models, we detect tumor-derived cell-free DNA and identified ctDNA from the originally grafted ascites. Conclusion Our data suggest that ctDNA can be detected and monitored in dried blood spots from archived and fresh blood samples, enabling new approaches for sample collection and novel study/trial designs for both patients and in vivo models.

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Bordas, Marie, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K. Maaß, and Martina Seiffert. "Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues." International Journal of Molecular Sciences 21, no. 15 (August 4, 2020): 5586. http://dx.doi.org/10.3390/ijms21155586.

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Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches—(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols.

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To, Van Vinh, Thi Phuong Tuyen Dao, Van Binh Pham, Duy Hien Tong, and Van Hieu Tran. "Application of silicon nitride membrane filters in circulating tumor cell capture." Science and Technology Development Journal 17, no. 2 (June 30, 2014): 35–46. http://dx.doi.org/10.32508/stdj.v17i2.1313.

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Circulating tumor cells (CTCs) have been recognized as holding extraordinary potential for disease management in cancer patients including prognostic, therapy, and monitoring disease progression. Sensitive and quick detection of CTC could enable the approach to patients with early-stage and metastatic cancer. The technical challenge in this field consists of finding rare tumor cells (just a few CTCs in 1 ml of blood) and being able to distinguish them from epithelial non-tumor cells and leukocytes. The current methodologies have significant limitations such as low capture efficiency, cannot capture live cells and time consuming. This paper presents the development of a new generation of microfilter for size-based isolation of CTCs in epithelial cancer using silicon nitride membrane filters 0.5 cm by 0.5 cm square sheets with slit shaped pores of 5μm by 15μm. We evaluated the sensitivity and efficiency of CTCs capture in a model system using the MCF-7 cells (breast cancer cells) spiked in the blood from the healthy donors. Preliminary results this research shown that silicon nitride membrane filter is a very good candidate to be CTCs detection platfo.

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Lianidou, Evi S., and Athina Markou. "Circulating Tumor Cells in Breast Cancer: Detection Systems, Molecular Characterization, and Future Challenges." Clinical Chemistry 57, no. 9 (September 1, 2011): 1242–55. http://dx.doi.org/10.1373/clinchem.2011.165068.

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BACKGROUND Circulating tumor cell (CTC) analysis is a promising new diagnostic field for estimating the risk for metastatic relapse and metastatic progression in patients with cancer. CONTENT Different analytical systems for CTC isolation and detection have been developed as immunocytochemical and molecular assays, most including separation steps by size or biological characteristics, such as expression of epithelial- or cancer-specific markers. Recent technical advancements in CTC detection and characterization include methods based on multiplex reverse-transcription quantitative PCR and approaches based on imaging and microfilter and microchip devices. New areas of research are directed toward developing novel assays for CTC molecular characterization. QC is an important issue for CTC analysis, and standardization of micrometastatic cell detection and characterization methodologies is important for the incorporation of CTCs into prospective clinical trials to test their clinical utility. The molecular characterization of CTCs can provide important information on the molecular and biological nature of these cells, such as the status of hormone receptors and epidermal and other growth factor receptor family members, and indications of stem-cell characteristics. This information is important for the identification of therapeutic targets and resistance mechanisms in CTCs as well as for the stratification of patients and real-time monitoring of systemic therapies. SUMMARY CTC analysis can be used as a liquid biopsy approach for prognostic and predictive purposes in breast and other cancers. In this review we focus on state-of-the-art technology platforms for CTC isolation, imaging, and detection; QC of CTC analysis; and ongoing challenges for the molecular characterization of CTCs.

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Huang, Qinqin, Bo Cai, Bolei Chen, Lang Rao, Zhaobo He, Rongxiang He, Feng Guo, et al. "Circulating Tumor Cell Isolation: Efficient Purification and Release of Circulating Tumor Cells by Synergistic Effect of Biomarker and SiO2@Gel-Microbead-Based Size Difference Amplification (Adv. Healthcare Mater. 13/2016)." Advanced Healthcare Materials 5, no. 13 (July 2016): 1525. http://dx.doi.org/10.1002/adhm.201670063.

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Derler, Rupert, Nikola Kitic, Tanja Gerlza, and Andreas J. Kungl. "Isolation and Characterization of Heparan Sulfate from Human Lung Tissues." Molecules 26, no. 18 (September 10, 2021): 5512. http://dx.doi.org/10.3390/molecules26185512.

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Glycosaminoglycans are a class of linear, highly negatively charged, O-linked polysaccharides that are involved in many (patho)physiological processes. In vitro experimental investigations of such processes typically involve porcine-derived heparan sulfate (HS). Structural information about human, particularly organ-specific heparan sulfate, and how it compares with HS from other organisms, is very limited. In this study, heparan sulfate was isolated from human lung tissues derived from five donors and was characterized for their overall size distribution and disaccharide composition. The expression profiles of proteoglycans and HS-modifying enzymes was quantified in order to identify the major core proteins for HS. In addition, the binding affinities of human HS to two chemokines—CXCL8 and CCL2—were investigated, which represent important inflammatory mediators in lung pathologies. Our data revealed that syndecans are the predominant proteoglycan class in human lungs and that the disaccharide composition varies among individuals according to sex, age, and health stage (one of the donor lungs was accidentally discovered to contain a solid tumor). The compositional difference of the five human lung HS preparations affected chemokine binding affinities to various degrees, indicating selective immune cell responses depending on the relative chemokine–glycan affinities. This represents important new insights that could be translated into novel therapeutic concepts for individually treating lung immunological disorders via HS targets.

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Journal articles on the topic 'ISET (Isolation by SizE of Tumor'

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