This work was supported by research grants from AstraZeneca (London, United-Kingdom), Biolidics (Singapore) and the Ligue Contre le Cancer (Saone et Loire, France). The authors thank AstraZeneca and Biolidics companies for their financial support.

Samples were prospectively collected within the framework of the CIRCAN (&#34;CIRculating CANcer&#34;) cohort based at the Lyon University Hospital following patient written consent. This study was integrated into the CIRCAN_ALL cohort. The study CIRCAN_ALL was recognized as non-interventional by the CPP South-East IV dated 04/11/2015 under the reference L15-188. An amended version was recognized as non-interventional on 20/09/2016 under reference L16-160. The CIRCAN_ALL study was declared to the IT and freedom correspon...

<ol>
	<li>Gandhi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pembrolizumab+plus+Chemotherapy+in+Metastatic+Non-Small-Cell+Lung+Cancer.">Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer.</a> The New England Journal of Medicine. 378 (22), 2078-2092 (2018).</li><li>Paz-Ares, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pembrolizumab+plus+Chemotherapy+for+Squamous+Non-Small-Cell+Lung+Cancer.">Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer.</a> The New England Journal of Medicine. 379 (21), 2040-2051 (2018).</li><li>Langer, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboplatin+and+pemetrexed+with+or+without+pembrolizumab+for+advanced,+non-squamous+non-small-cell+lung+cancer:+a+randomised,+phase+2+cohort+of+the+open-label+KEYNOTE-021+study.">Carboplatin and pemetrexed with or without pembrolizumab for advanced, non-squamous non-small-cell lung cancer: a randomised, phase 2 cohort of the open-label KEYNOTE-021 study.</a> The Lancet Oncology. 17 (11), 1497-1508 (2016).</li><li>Hellmann, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Mutational+Burden+and+Efficacy+of+Nivolumab+Monotherapy+and+in+Combination+with+Ipilimumab+in+Small-Cell+Lung+Cancer.">Tumor Mutational Burden and Efficacy of Nivolumab Monotherapy and in Combination with Ipilimumab in Small-Cell Lung Cancer.</a> Cancer Cell. 33 (5), 853-861 (2018).</li><li>Chouaid, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+and+clinical+impact+of+re-biopsy+in+advanced+non+small-cell+lung+cancer:+a+prospective+multicenter+study+in+a+real-world+setting+(GFPC+study+12-01).">Feasibility and clinical impact of re-biopsy in advanced non small-cell lung cancer: a prospective multicenter study in a real-world setting (GFPC study 12-01).</a> Lung cancer. 86 (2), 170-173 (2014).</li><li>Nosaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-biopsy+status+among+non-small+cell+lung+cancer+patients+in+Japan:+A+retrospective+study.">Re-biopsy status among non-small cell lung cancer patients in Japan: A retrospective study.</a> Lung cancer. 101, 1-8 (2016).</li><li>Uozu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+tissue+re-biopsy+in+non-small+cell+lung+cancers+resistant+to+previous+epidermal+growth+factor+receptor+tyrosine+kinase+inhibitor+therapies.">Feasibility of tissue re-biopsy in non-small cell lung cancers resistant to previous epidermal growth factor receptor tyrosine kinase inhibitor therapies.</a> BMC Pulmonary Medicine. 17 (1), 175 (2017).</li><li>Kim, T. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+re-biopsy+and+EGFR+mutation+analysis+in+patients+with+non-small+cell+lung+cancer.">Feasibility of re-biopsy and EGFR mutation analysis in patients with non-small cell lung cancer.</a> Thoracic Cancer. 9 (7), 856-864 (2018).</li><li>Nicolazzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+PD-L1+positive+circulating+tumor+cells+in+non-small+cell+lung+cancer+patients+treated+with+the+PD-1+inhibitor+Nivolumab.">Monitoring PD-L1 positive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor Nivolumab.</a> Scientific Reports. 6, 31726 (2016).</li><li>Guibert, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-L1+expression+in+circulating+tumor+cells+of+advanced+non-small+cell+lung+cancer+patients+treated+with+nivolumab.">PD-L1 expression in circulating tumor cells of advanced non-small cell lung cancer patients treated with nivolumab.</a> Lung cancer. 120, 108-112 (2018).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-L1+Expression+in+Circulating+Tumor+Cells+Increases+during+Radio(chemo)therapy+and+Indicates+Poor+Prognosis+in+Non-small+Cell+Lung+Cancer.">PD-L1 Expression in Circulating Tumor Cells Increases during Radio(chemo)therapy and Indicates Poor Prognosis in Non-small Cell Lung Cancer.</a> Scientific Reports. 9 (1), 566 (2019).</li><li>Hao, S. -. J., Wan, Y., Xia, Y. -. Q., Zou, X., Zheng, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-based+separation+methods+of+circulating+tumor+cells.">Size-based separation methods of circulating tumor cells.</a> Advanced Drug Delivery Reviews. 125, 3-20 (2018).</li><li>Williams, A., Balic, M., Datar, R., Cote, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-based+enrichment+technologies+for+CTC+detection+and+characterization.+Recent+results+in+cancer+research.">Size-based enrichment technologies for CTC detection and characterization. Recent results in cancer research.</a> Fortschritte der Krebsforschung. Progres dans les recherches sur le cancer. 195, 87-95 (2012).</li><li>Garcia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+Circulating+Tumor+DNA+(ctDNA)+in+Plasma+of+non-small+cell+lung+cancer+(NSCLC)+patients,+Monitoring+of+EGFR+p.T790M+mutated+allelic+fraction+using+BEAMing+Companion+Assay+and+Evaluation+in+future+application+in+mimicking+Circulating+Tumors+Cells+(mCTC).">Profiling of Circulating Tumor DNA (ctDNA) in Plasma of non-small cell lung cancer (NSCLC) patients, Monitoring of EGFR p.T790M mutated allelic fraction using BEAMing Companion Assay and Evaluation in future application in mimicking Circulating Tumors Cells (mCTC).</a> Cancer Medicine. , (2019).</li><li>Garcia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+pre-analytical+conditions+and+comparison+of+the+performance+of+several+digital+PCR+assays+for+the+detection+of+major+EGFR+mutations+in+circulating+DNA+from+non-small+cell+lung+cancers:+the+CIRCAN_0+study.">Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study.</a> Oncotarget. 8 (50), 87980-87996 (2017).</li><li>Lustberg, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+atypical+cell+populations+are+present+in+blood+of+metastatic+breast+cancer+patients.">Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients.</a> Breast Cancer Research. 16 (2), 23 (2014).</li><li>Ilie, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Sentinel"+circulating+tumor+cells+allow+early+diagnosis+of+lung+cancer+in+patients+with+chronic+obstructive+pulmonary+disease.">"Sentinel" circulating tumor cells allow early diagnosis of lung cancer in patients with chronic obstructive pulmonary disease.</a> PLoS ONE. 9 (10), 111597 (2014).</li><li>Khoo, B. 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Two major points were raised in the present study, the first with regards to performance of the workflow for its transfer to clinical applications, and the second concerning the decrease in subjectivity for the analysis of fluorescence images obtained.
A performant and optimized workflow for CTC enumeration was initially determined using customizable IF assay after cell enrichment via a CTC label-free microfluidic system (spiral microfluidic device). Using this workflow, a pilot study confirme...

Tumor antigen-specific cytotoxic T-lymphocytes (CTLs) play a crucial role in the response to cancers through a process known as cancer &#34;immune surveillance&#34;. Their anti-tumor functions are enhanced by immune checkpoint blockade antibodies such as CTLA-4 inhibitors and PD-1/PD-L1 inhibitors. In non-small cell lung cancer (NSCLC), anti-PD-1/PD-L1 therapies result in response rates ranging from 0%-17% in patients with PD-L1-negative tumors and 36%-100% in those expressing PD-L1. The robust responses to PD-1/PD-L1 blockade observed in melanoma and NSCLC are shown by evidence of improved overall response rate (RR), durable clinical benefits, and progression-free survival (PFS). Currently, anti-PD1 treatments are the standard of care in second-line NSCLC treatment with nivolumab regardless of PD-L1 expression and with pembrolizumab in patients expressing PD-L1 &#8805;1%. In first-line treatment, standard of care is pembrolizumab alone in patients with NSCLC expressing PD-L1 &#8805;50% and can be potentially enhanced with chemotherapy (platin and doublet drug depending on histologic subtype)1,2.
However, such an approach to patient management is debatable3, since PD-L1 expression in tumor cells by immunohistochemistry (IHC) is probably not the most ideal companion biomarker. Others such as tumor mutation burden4 (TMB), microsatellite instability (MSI), and/or microbiota are possibly interesting in this setting either alone or in combination. NSCLC are known to be heterogeneous tumors, either spatially (from a tumor site to another one) or temporally (from diagnosis to recurrence). Patients with NSCLC are usually fragile, and iterative invasive tissue biopsies may be an issue. Indeed, re-biopsy rate at first progression ranges from 46%-84% depending on series, and successful re-biopsy (meaning with histological and full molecular analysis) ranges from 33%-75%. This means that 25%-67% of patients cannot receive a comprehensive re-biopsy analysis during first progression5,6,7,8.
The advent of &#34;liquid biopsies&#34; has thus generated considerable enthusiasm in this particular setting, as it enables crucial reassessment of molecular alterations during disease progression by examining circulating free DNA (cfDNA) derived from circulating tumor cells (CTCs). These live cells are released from the tumor into the bloodstream, where they circulate freely. Although not routinely used, the analysis of CTCs appears to be highly promising in the case of molecular and phenotypic characterization, prognosis, and predictive significance in lung cancer (via DNAseq, RNAseq, miRNA and protein analysis). Indeed, CTCs likely harbor phenotypic characteristics of the active disease rather than the initial markers (detected on tissue biopsies at diagnosis). Furthermore, CTCs bypass the problem of spatial heterogeneity of the tumor tissue, which may be a crucial issue in small biopsies. Consequently, PD-L1 expression on CTCs may potentially shed light on the discrepancies derived from its use as a predictive biomarker using tumor tissue.
Recently, PD-L1 expression has been tested in CTCs of NSCLC. Almost all of the patients tested9 were PD-L1 positive, complicating the interpretation of the result and its clinical use. Overall, PD-L1-positive CTCs were detected in 69.4% of samples from an average of 4.5 cells/mL10. After initiation of radiation therapy, the proportion of PD-L1-positive CTCs increased significantly, indicating upregulation of PD-L1 expression in response to radiation11. Hence, PD-L1 CTCs analysis may be used to monitor dynamic changes of the tumor and immune response, which may reflect the response to chemotherapy, radiation, and likely immunotherapy (IT) treatments.
To date, CTCs isolation and PD-L1 characterization rely on various methods such as anti-EpCAM-coated magnetic bead-based CTC capturing, enrichment-free based assay, and size-based12,13 CTC capture assays. However, CTCs were only detected in 45%-65% of patients with metastatic NSCLC, thus limiting their ability to provide any information for more than half of metastatic NSCLC patients. In addition, CTC count was low in most of these studies using size-based approach10. Furthermore, this method has led to discrepancies such as the detection of CD45(-)/DAPI(+) cells with &#34;cytomorphological patterns of malignancy&#34;&#160;in the bloodstream of healthy donors. These concerns highlight the need for a highly sensitive method of CTC collection associated with immune-phenotyping of atypical CD45(-) cells from healthy whole blood using additional cancer biomarkers (i.e., TTF1, Vimentin, EpCAM, and CD44) in NSCLC.
Consequently, we evaluated a spiral microfluidic device that uses inertial and Dean drag forces to separate cells based on size and plasticity through a microfluidic chip. The formation of Dean vortex flows present in the microfluidic chip results in larger CTCs located along the inner wall and smaller immune cells along the outer wall of the chip. The enrichment process is completed by siphoning the larger cells into the collection outlet as the enriched CTC fraction. This method is particularly sensitive and specific (detection of around 1 CTC/mL of whole blood)14 and can be associated with customized immunofluorescence (IF) analyses. These tools will enable setting up of a positive threshold for clinical interpretation. A workflow is thus described that enables biologists to isolate and immunophenotype CTCs with a high rate of recovery and specificity. The protocol describes optimal use of the spiral microfluidic device to collect CTCs, the optimized IF assays that can be customized according to cancer type, and use of free open-source software for measuring and analyzing cell images to perform a semi-automatic numeration of the cells according to fluorescent staining. In addition, microscope multiplexing can be carried out depending on the number of fluorescent filters/markers available.

<table border="0" cellpadding="0" cellspacing="0" style="width:1004px;" width="1004">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">4&#39;,6-diamidino-2-ph&#233;nylindole (DAPI)</td>
			<td style="width:148px;">Ozyme</td>
			<td style="width:105px;">BLE 422801</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:376px;">BD Facs Clean &#8211; 5L</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:105px;">340345</td>
			<td style="width:375px;">Bleach-based cleaning agent. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Bleach 1% Cleaning Solution 100 mL</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F016012</td>
			<td style="width:375px;">Bleach. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Bovine Serum Albumin (BSA) 7.5%</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:105px;">A8412</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">CD45 monoclonal antibody (clone HI30) Alexa Fluor 647</td>
			<td style="width:148px;">BioLegend</td>
			<td style="width:105px;">BLE304020</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">CellProfiler Software</td>
			<td style="width:148px;">Broad Institute</td>
			<td style="width:105px;"></td>
			<td style="width:375px;">Image Analysis Software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Centrifuge device</td>
			<td style="width:148px;">Hettich</td>
			<td style="width:105px;">4706</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Centrifuge tube 50 mL</td>
			<td style="width:148px;">Corning</td>
			<td style="width:105px;">430-829</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Centrifuge Tube 15 mL</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F001004-25</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">ClearCell FX-1 System</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F011002</td>
			<td style="width:375px;">Spiral microfluidic device. Storage conditions: Room temperature</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:376px;">Coulter Clenz Cleaning Agent &#8211; 5L</td>
			<td style="width:148px;">Beckman Coulter</td>
			<td style="width:105px;">8448222</td>
			<td style="width:375px;">All-purpose cleaning reagent. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">CTChip FR1S</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-FR001002</td>
			<td style="width:375px;">Microfluidic chip. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Cytospin 4</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">A78300003</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Diluent Additive Reagent &#8211; 20 mL</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F016009</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:376px;">EZ Cytofunnels</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">A78710003</td>
			<td style="width:375px;">Sample chamber with cotton. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">FcR blocking Agent</td>
			<td style="width:148px;">Miltenyi Biotec</td>
			<td style="width:105px;">130-059-901</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Fetal Calf Serum (FCS)</td>
			<td style="width:148px;">Gibco</td>
			<td style="width:105px;">10270-106</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Fluoromount</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:105px;">F4680</td>
			<td style="width:375px;">Mounting solution. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Fungizone - 50 mg</td>
			<td style="width:148px;">Bristol-Myers-Squibb</td>
			<td style="width:105px;">90129TB29</td>
			<td style="width:375px;">Anti-fungal reagent. Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">FX1 Input Straw with lock cap</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F013005</td>
			<td style="width:375px;">Straw. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">KovaSlide</td>
			<td style="width:148px;">Dutscher</td>
			<td style="width:105px;">50126</td>
			<td style="width:375px;">Chambered slide. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">PanCK monoclonal antibody (clone AE1/AE3) Alexa Fluor 488</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">53-9003-80</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Paraformaldehyde 16%</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">11490570</td>
			<td style="width:375px;">Fixation solution. Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">PD-L1 monoclonal antibody (clone 29E2A3) - Phycoerythrin</td>
			<td style="width:148px;">BioLegend</td>
			<td style="width:105px;">BLE329706</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Petri Dish</td>
			<td style="width:148px;">Dutscher</td>
			<td style="width:105px;">632180</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:148px;">Ozyme</td>
			<td style="width:105px;">BE17-512F</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Phosphate Buffered Saline Ultra Pure Grade 1X &#8211; 1L</td>
			<td style="width:148px;">1st Base Laboratory</td>
			<td style="width:105px;">BUF-2040-1X1L</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Pluronic F-68 10%</td>
			<td style="width:148px;">Gibco</td>
			<td style="width:105px;">24040-032</td>
			<td style="width:375px;">Anti-binding solution. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Polylysine slides</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">J2800AMNZ</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Polypropylene Conical Tube 50 mL</td>
			<td style="width:148px;">Falcon</td>
			<td style="width:105px;">352098</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">RBC Lysis Buffer &#8211; 100 mL</td>
			<td style="width:148px;">G Biosciences</td>
			<td style="width:105px;">786-649</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">RBC Lysis Buffer &#8211; 250 mL</td>
			<td style="width:148px;">G Biosciences</td>
			<td style="width:105px;">786-650</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Resuspension Buffer (RSB)</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F016003</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Shandon Cytopsin4 centrifuge</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">A78300003</td>
			<td style="width:375px;">Dedicated centrifuge. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Silicon Isolator</td>
			<td style="width:148px;">Grace bio-Labs</td>
			<td style="width:105px;">664270</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Sterile Deionized Water &#8211; 100 mL</td>
			<td style="width:148px;">1st Base Laboratory</td>
			<td style="width:105px;">CUS-4100-100ml</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Straight Fluorescent microscope Axio Imager D1</td>
			<td style="width:148px;">Zeiss</td>
			<td></td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Surgical Sterile Bag</td>
			<td style="width:148px;">SPS Laboratoires</td>
			<td style="width:105px;">98ULT01240</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Syringe BD Discardit II 20 mL sterile</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:105px;">300296</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Syringe Filter 0.22 &#181;m 33 mm sterile</td>
			<td style="width:148px;">ClearLine</td>
			<td style="width:105px;">51732</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Zen lite 2.3 Lite Software</td>
			<td style="width:148px;">Zeiss</td>
			<td style="width:105px;"></td>
			<td style="width:375px;">Microscope associated software</td>
		</tr>
	</tbody>
</table>

semi-automatic pd-l1 characterization and enumeration of circulating tumor cells from non-small cell lung cancer patients by immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The first pre-requisite was to obtain uncontaminated (infectious agent-free) collections of CTCs for tissue culture and avoid IF background generated. The decontamination protocol enabled cleaning of all the pipes and pumps, and it resulted in the collection of CTCs with a good recovery rate without bacterial contamination. The enriched samples were compared without and with the decontamination protocol workflow of the spiral microfluidic device. To validate the decontamination protocol, the A549 cell line was used in ab...

Watch this Scientific Journal Video about Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence at JoVE.com

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Based on microfluidic technology, the isolation and characterization of CTCs from normal blood cell do not require the use of specific CTC biomarkers, and is compatible with cell culture. This protocol facilitates a high rate of CTC extraction, and provides an extensive cytological analysis of the CTCs, by evaluating malignant cytomorphological characteristics. PD-L1 expression by CTCs has a predictive value in lung cancer management.Improvement of its detection using this test could promote a better patient management in the long term. Other application of this protocol include detecting genetic aberrations using FISH, or conducting transcriptomic analysis on CTCs, as well as isolating cells of fetal origin in mothers. Demonstrating the procedure will be Julie Balandier, a technician from our laboratory.For pre-analytical circulating tumor cell enrichment, first collect 7.5 milliliters of blood into a potassium EDTA tube, and keep the sample under gentle agitation to avoid cell sedimentation and clotting. Within six hours of collection, use a serological pipette under a microbiological safety cabinet, to transfer up to 7.5 milliliters of whole blood into a new 50 milliliter centrifuge tube, and centrifuge the sample for 10 minutes at 1600 times G, at room temperature. At the end of the centrifugation, use a transfer pipette to collect the plasma fraction without disturbing the buffy coat, and dilute the plasma with an equivalent volume of PBS, up to 7.5 milliliters.Next, carefully add red blood cell lysis buffer to the blood sample, to a final volume of 30 milliliters, and gently invert the blood collection tube three times, before placing the tube at room temperature for 10 minutes. At the end of the incubation, collect the cells by centrifugation, and carefully remove all but the last four to five milliliters of supernatant. Use a filtered micropipette to remove the remaining supernatant, and use a P1000 micropipette with a filtered tip to add one milliliter of resuspension buffer down the wall of the tube.To avoid introducing bubbles, resuspend the cell pellet with gentle pipetting, until the sample is homogenous. When the pellet has been resuspended, add an additional three milliliters of resuspension buffer to the wall of the tube, without introducing bubbles, and gently mix the cells again. For spiral microfluidic device circulating tumor cell enrichment, first load a new spiral microfluidic chip onto the device, and load one empty 50 milliliter centrifuge tube into each of the input and output ports.Click prime to prime the spiral microfluidic device for three minutes. Removing the input and output tubes at the end of the cycle. Load the resuspended blood sample into the input port, and load a clear 15 milliliter conical tube into the output port.Then click run, and select program three to initiate the 31 minute circulating tumor cell enrichment program. At the end of the cycle transfer the output tube to the centrifuge, and use a five milliliter serological pipette to remove all but the last two milliliters of the supernatant. Then use a micropipette to remove all but the last 100 microliters of supernatant.For immunofluorescence staining, count the cells in a hemocytometer, and dilute the enriched sample to a one times ten to fifth cells per 100 microliters of 0.2%anti-binding solution concentration. Next, use a cotton swab soaked with 50 microliters of anti-binding solution to moisten the contour of the sample chamber, and place a polylysine glass slide in the sample chamber. Close the chamber, and pipette up and down three times to coat a micropipette tip with the binding solution before resuspending the sample in the last 100 microliters of the supernatant.Transfer the cell solution to the sample chamber and cytospin the sample in a dedicated centrifuge at 400 rotations per minute for four minutes, at a low acceleration. At the end of the centrifugation, place a silicone isolator around the area of deposition and let the slide dry under a microbiological safety cabinet for two minutes, then fix the cytospun sample with 100 microliters of 4%paraformaldehyde for 10 minutes at room temperature, followed by three two minute washes with 200 microliters of PBS per wash, at room temperature. After the last wash, block any non-specific binding with 30 minute incubation in blocking reagent at room temperature, followed by labeling with 100 microliters of the antibody solution of interest.Place the labeled slide in a 100 by 15 milliliter petri dish on a piece of absorbent paper moistened with two milliliters of sterile water, and place the dish closed, at four degrees Celsius overnight, protected from light. The next morning, wash the sample three times with PBS as demonstrated, and mount the sample with 10 microliters of an appropriate mounting solution and a glass cover slip. Then seal the cover slip with clear nail polish.To image the cytospun samples, load the slide onto a straight fluorescent microscope with an X Y motorized platform, and select a 20x objective and the appropriate channels according to the fluorophores used for the antibody labeling. Turn on the mercury lamp and adapt the microscope and associated software to a semi-automized shoot. When the lamp is ready, in the acquisition menu, define the four channels, set the exposure time, and define the tiles to scan.Click tiles, and advanced experiment, and define the area to scan. Then adjust the focus on the screen, and click start experiment. At the end of the experiment, export the TIF files for each channel, and specifically name the file to include the sample, number of times, dye, and number of sub-tiles.For image analysis, open the image analysis software from the Broad Institute website, and click file, pipeline from file, and analysis four channels CTC. Drop files into the file list, and update the metadata to group the files by tiles. Then click analyze images to open the spreadsheet file corresponding to the measure intensity parameters.Without the optimized decontamination protocol, high bacterial contamination is observed in tissue cultures of enriched A549 cell lines after only 24 hours, causing death and cytomorphological changes in the eukaryotic cells. In contrast, after the cleaning protocol, living A549 cells are obtained in 2D cultures after 10 hours of tissue culture and medium removal, under 3D culture conditions, and within patient samples. Using a liquid immunofluorescent staining assay or an immunofluorescent staining assay on polylysine coated slides with cytospin, a clear distinction in the number of nuclei enumerated between the two assays can be observed.Without the cytospin step, it is difficult to differentiate the white blood cells from the tumor cells, due to the blurry outlines in the non-cytospin cell preps. Here different representative findings from different patient samples can be observed, with the residual count of the white blood cells in particular determined to be strongly variable, and dependent on the whole blood sample. A high variability was also observed in the circulating tumor cells sub-populations obtained.Each cell on the cytospin is identified by the image analysis software, and enables the tracking of cell and manually to confirm the results as necessary. Indeed, a pilot analysis demonstrated a concordance between the manual enumeration and the image analysis software enumeration. The setup of the in-house designed damp petri dish for the protein-antibody hybridization is critical, as the device remains sufficiently damp throughout the entire incubation time.

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Research

JoVE Journal

Cancer Research

10.4K vistas.  Hospices Civils de Lyon. Basado en tecnología microfluídica, el aislamiento y caracterización de los CTC de las células sanguíneas normales no requieren el uso de biomarcadores específicos de CTC, y es compatible con el cultivo celular. Este protocolo facilita una alta tasa de extracción de CTC, y proporciona un análisis citológico extenso de los CTC, mediante la evaluación de las características citomorfológicas malignas. La expresión PD-L1 por CTCs tiene un valor predictivo en el manejo del cáncer de ...