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 correspondent of the Hospices Civils de Lyon on 01/12/2015, under the reference 15-131. Blood collection was performed when physicians observed the earliest indication of tumor progression.
NOTE: Use all the reagents and materials outlined in Table of Materials with the respective storage conditions for pre-analytical sample preparation and immunofluorescence assay. Substituting reagents and/or modifying storage conditions could result in suboptimal assay performance.
1. Decontamination of Spiral Microfluidic Device
NOTE: Decontamination of the spiral microfluidic device is a requirement to remove all immunofluorescence background generated from bacteria contamination, explore the cytomorphology of CTCs, and be able to differentiate them from normal immune cells. The protocol is optimized for blood samples collected in K2EDTA tubes within 6 h after blood sampling and enriched using the spiral microfluidic device in clean conditions. Using this assay for other types of samples (other biological fluids) may require additional optimization. This decontamination protocol should be done once per week.
<ol>
	<li>Preparation of reagents
	<ol>
		<li>Preparation of the diluent buffer
		<ol>
			<li>Sterilize 20 mL of diluent additive reagent using a 0.22 &#181;m syringe filter and add directly to 1 L of 1x phosphate buffer saline (PBS) ultra-pure grade (Table of Materials).</li>
		</ol>
		</li>
		<li>Sterilization of reagents and the input straw
		<ol>
			<li>Sterilize the RBC lysis buffer and resuspension buffer (RSB; Table of Materials) using a 0.22 &#181;m syringe filter and stock in a new 50 mL polypropylene conical tube for each solution.</li>
			<li>Sterilize the input straw by incubating at room temperature (RT) for 1 h in the all-purpose cleaning reagent (Table of Materials). Transfer the straw to the bleach-based cleaning agent (Table of Materials) and incubate at RT for 1 h.</li>
			<li>Rinse the input straw twice with sterile PBS for 1 h each and store the sterilized input straw in a surgically sterile bag (Table of Materials).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Decontaminating the spiral microfluidic device 
	<ol>
		<li>Disinfection using the all-purpose cleaning reagent
		<ol>
			<li>Disconnect the diluent bottle cap from the diluent port of the spiral microfluidic device by unscrewing the brown screw. Under a microbiological safety cabinet, transfer up to 250 mL of all-purpose cleaning reagent (Table of Materials) into a new empty bottle.</li>
			<li>Screw the diluent bottle cap and straw to the bottle of 250 mL all-purpose cleaning reagent under a microbiological safety cabinet. Attach this bottle to the diluent port of the spiral microfluidic device by screwing back the brown screw.</li>
			<li>Transfer up to 100 mL of bleach (1% final concentration; Table of Materials) to the waste container supplied in the run kit (Figure 1A).</li>
			<li>Load a new sterile input straw onto the spiral microfluidic device (Table of Materials) in the input port. Load a new 50 mL centrifuge tube in the input port. Load a new 50 mL centrifuge tube in the output port.</li>
			<li>Proceed to prime the spiral microfluidic device by clicking on Prime on the spiral microfluidic device (3 min). Remove the input tube after the prime is completed.</li>
			<li>Transfer up to 15 mL of all-purpose cleaning reagent to a new 50 mL centrifuge tube with a serological pipette under a microbiological safety cabinet and attach the tube to the input port of the spiral microfluidic device.</li>
			<li>Before starting the run, check that the solution is free of excessive bubbles. If bubbles are present, remove them by slow aspiration with a pipette.</li>
			<li>Load a decontamination microfluidic chip in the spiral microfluidic device. Run a Program 3 on the spiral microfluidic device by clicking on Run and selecting the Program 3 (31 min). 
			NOTE: The Program 3 of the spiral microfluidic device enables a rapid enrichment of CTCs in 31 min.</li>
			<li>Continue on with the spiral microfluidic device&#39;s cleaning step using the remaining volume of the all-purpose cleaning reagent in the input tube.</li>
			<li>Discard the input tube after cleaning step is completed, leaving behind the input straw.</li>
		</ol>
		</li>
		<li>Decontamination using the bleach-based cleaning agent
		<ol>
			<li>Disconnect the all-purpose cleaning reagent bottle cap from the diluent port of the spiral microfluidic device by unscrewing the brown screw. Under a microbiological safety cabinet, transfer up to 250 mL of bleach-based cleaning agent (Table of Materials) in a new empty bottle (Figure 1A).</li>
			<li>Screw the all-purpose cleaning reagent bottle cap and straw to the bottle containing the Bleach-based cleaning agent under a microbiological safety cabinet. Attach this bottle to the diluent port of the spiral microfluidic device by screwing back the brown screw.</li>
			<li>Transfer up to 15 mL of bleach-based cleaning agent to a new 50 mL centrifuge tube input tube using a serological pipette under a microbiological safety cabinet. Load the 50 mL centrifuge tube input position. Load an empty tube in output position.</li>
			<li>Before processing the run, check that the sample is free of excessive bubbles and if any are present, remove the bubbles by aspirating them slowly with a pipette.</li>
			<li>Run Program 3 by clicking on Run and selecting the Program 3 (31 min). After the run, proceed directly to the cleaning step using the remaining volume of bleach-based cleaning agent in the input tube.</li>
			<li>Discard the input and output tubes.</li>
		</ol>
		</li>
		<li>Rinse the spiral microfluidic device.
		<ol>
			<li>Disconnect the bleach-based cleaning agent bottle cap from the diluent port of the spiral microfluidic device by unscrewing the brown screw. Under a microbiological safety cabinet, transfer the straw from the bottle containing the bleach-based cleaning agent to the new bottle containing the diluent buffer. Screw the bottle to the spiral microfluidic device.</li>
			<li>Transfer up to 15 mL of sterilized water (Table of Materials) to a new 50 mL centrifuge tube input tube using a serological pipette under a microbiological safety cabinet. Load the 50 mL centrifuge tube input position. Load an empty tube in output position.</li>
			<li>Before processing the run, check that the sample is free of excessive bubbles and if any are present, remove the bubbles by aspirating them slowly with a pipette.</li>
			<li>Run Program 3 by clicking on Run and selecting the Program 3 (31 min). After the run, proceed directly to the cleaning step using the remaining volume of sterilized water in the input tube.</li>
			<li>Discard the input and output tubes.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Maintenance to Keep the Spiral Microfluidic Device Bacteria-free
NOTE: The routine maintenance should be done at the end of the day during the last cleaning step.
<ol>
	<li>Transfer up to 7 mL of bleach-based cleaning agent into the new 50 mL centrifuge tube using a serological pipette under a microbiological safety cabinet. Screw the bleach-based cleaning tube in input port of the spiral microfluidic device.</li>
	<li>Before processing the clean run, check that the input sample is free of excessive bubbles and if any are present, remove the bubbles by aspirating them slowly with a pipette.</li>
	<li>Run the clean on the spiral microfluidic device.</li>
</ol>
3. Pre-analytical Enrichment of CTC from Patient Blood Samples
<ol>
	<li>Collect 7.5 mL of blood in the K2EDTA tube and keep under gentle agitation to avoid cell sedimentation and clotting. Process within 6 h. 
	NOTE: If the blood is collected in a cell-free DNA blood collection tube containing preservative, store at 4 &#176;C until processing. Ensure that the blood sample, RBC lysis buffer, and RBS buffer are at RT before proceeding with the enrichment step.</li>
	<li>Transfer up to 7.5 mL of whole blood to a new 50 mL centrifuge tube input tube using a serological pipette under a microbiological safety cabinet.</li>
	<li>Centrifuge at 1,600 x g for 10 min at RT. Collect the plasma fraction with a pipette without disturbing the buffy coat. Replace the plasma fraction by adding directly equivalent volume of PBS up to 7.5 mL.</li>
	<li>Gently add RBC lysis buffer (Table of Materials) to blood sample to a final volume of 30 mL (for a K2EDTA tube) or 37.5 mL (for a cell-free DNA blood collection tube). Gently invert the blood collection tube 10x and incubate for 10 min at RT. 
	NOTE: The blood sample turns darker red during RBC lysis. If no change (from dark red and opaque) is observed after 10 min, gently invert the tube 3x and leave to stand for another 5 min maximum. Do not leave the sample in RBC lysis buffer for more than 15 min because it can compromise the sample quality and assay performance.</li>
	<li>Centrifuge the lysed blood sample at 500 x g for 10 min at RT, with centrifuge brakes on (or highest deceleration speed). Use a Pasteur pipette or serological pipette to gently remove the supernatant until the volume reaches the 4-5 mL mark. Then, use filtered micropipette tips to remove the remaining supernatant.</li>
	<li>Using a P1000 micropipette with a filtered tip, add 1.0 mL of RSB to the wall of the 50 mL centrifuge tube input tube. To avoid introducing bubbles into the mix, resuspend the cell pellet by gently pipetting up and down until the sample is homogeneous.</li>
	<li>Add an additional 3 mL of RSB to the wall of the 50 mL centrifuge tube input tube (total volume 4 mL). Avoid introducing bubbles into the mix. Gently mix the cell suspension by gently pipetting up and down. 
	NOTE: In the unlikely event that regular pipetting is unable to break down cell clumps (defined by being visible or blocking the pipette tip), filter the sample through a 40 &#956;m cell strainer to remove any clumps. Add 150 &#956;L of RSB to the sample to make up for volume loss from filtering. Note that this method is to be used sparingly and only when large clumps are observed.</li>
	<li>Before proceeding to the enrichment step, check that the sample is free of excessive bubbles and if any are present, remove the bubbles and take care not to discard any sample. If tiny bubbles are present, their removal is not required.</li>
	<li>Process the sample on the spiral microfluidic device.</li>
</ol>
4. Enrichment of CTCs from Patient Whole Blood with the Spiral Microfluidic Device
<ol>
	<li>Load a new spiral microfluidic chip. Load two empty 50 mL centrifuge tubes in input and output ports.</li>
	<li>Run a prime by clicking on Prime on the spiral microfluidic device (3 min). Remove the input and output tubes and load the sample to be processed in input port.</li>
	<li>Load a clear 15 mL conical tube in output port to collect enriched CTCs. Run Program 3 by clicking on Run and selecting the Program 3 (31 min).</li>
	<li>Unload the output tube and centrifuge at 500 x g for 10 min (acceleration: 9; deceleration: 5). With a 5 mL serological pipette, remove supernatant stopping at the 2 mL mark on the conical 15 mL tube. With a micropipette, remove supernatant stopping at 100 &#181;L mark on the conical 15 mL tube. Process the enriched sample directly for immunofluorescence staining.</li>
</ol>
5. Immunofluorescence Staining
<ol>
	<li>Enumerate on a chambered slide with a hemocytometer-type grid the number of cells per mL. Dilute the enriched sample with 0.2% anti-binding solution (Table of Materials) to a concentration reaching 100,000 cells/100 &#181;L per cytospin.</li>
	<li>Moisten the contour of the sample chamber using cotton (Table of Materials) with 50 &#181;L of 0.2% anti-binding solution. Place a polylysine glass-slide in the sample chamber and close.</li>
	<li>Coat a tip with 0.2% anti-binding solution by pipetting up and down 3x. Resuspend the enriched sample and transfer the cell solution into the sample chamber. Centrifuge with a dedicated centrifuge (Table of Materials) at 400 rpm for 4 min (acceleration low).</li>
	<li>Place a silicon isolator around the area of deposition. Let dry the glass-slide under a microbiological safety cabinet for 2 min.</li>
	<li>Prepare the fixation solution by diluting 1 mL of 16% paraformaldehyde (PFA) with 3 mL of sterile PBS. Add 100 &#181;L of fixation solution (4% PFA) per sample and incubate at RT for 10 min. Remove fixation solution and perform three washes with 200 &#181;L of PBS and incubate at RT each for 2 min. 
	CAUTION: Use PFA under a chemical safety cabinet to prevent inhalation.</li>
	<li>Prepare the saturation solution by diluting the fetal bovine serum (FBS) at 5%, Fc receptor (FcR) blocking reagent at 5%, and bovine serum albumin (BSA) at 1% in sterile PBS (Table of Materials). Add 100 &#181;L of saturation solution per sample and incubate for 30 min at RT. Remove saturation solution.</li>
	<li>Add 100 &#181;L of antibody solution per sample (CD45 antibody 1/20; PanCK antibody 1/500; PD-L1 antibody 1/200; Qsp saturation solution 100 &#181;L) (Table of Materials). Place the polylysine glass-slide in a 100 mm x 15 mm Petri dish. Moisten an absorbent paper with 2 mL of sterile water and close the Petri dish with the lid. Place at 4 &#176;C overnight and protect from the light.</li>
	<li>Remove the antibody mix and perform 3 washes with 200 &#181;L of PBS incubating each wash for 2 min. Let the sample dry for 5 min and protect it from the light. Place 10 &#181;L of mounting solution (Table of Materials) in the area of deposition and cover with a microscope coverslip without making a bubble. Seal the coverslip with nail polish.</li>
</ol>
6. Acquisition of Immunofluorescent Images with Straight Fluorescent Microscope and Associated Software
<ol>
	<li>Use a straight fluorescent microscope with an X/Y motorized platform. Use a 20x objective to take 8-bit RGB tiff images in four channels corresponding to DNA dye (4&#39;,6-diamidino-2-ph&#233;nylindole [DAPI]), PanCK dye (fluorescein isothiocyanate [FITC]), PD-L1 dye (CY3), and CD45 dye (CY5). Turn on the mercury lamp 15 min before use, and adapt the microscope and associated software to semi-automatized shoot.</li>
	<li>Place the glass-slide on the platform.</li>
	<li>In acquisition menu, define the four channels and set up the exposure time (DAPI: 15 ms, FITC [PanCK]: 500 ms; CY3 [PD-L1]: 800 ms; CY5 [CD45]: 1,000 ms). Define the tiles to scan. Click Tiles. In Advanced experiment, define the area to scan.</li>
	<li>Adjust the focus on the screen. Click Start experiment.</li>
	<li>Export TIF files of each channel and specifically name the image file with this information: Sample_NumberofTilesRegion_dye_NumberOfSubtiles.tif (e.g., Sample1_TR1_c1m01). Name dye as follows: the DAPI channel is c1, FITC channel is c2, CY3 channel is c3, and CY5 channel is c4.</li>
</ol>
7. Analysis of Immunofluorescent Images with Image Analysis Software
<ol>
	<li>Download and install the free image analysis software from the Broad Institute website. Accept all default during installation. Open the image analysis software and click File | Pipeline from file | Analysis_4channels_CTC.cppipe. 
	NOTE: The pipeline converts RGB color images into grayscale, removes artifacts by smoothing images with a median filter, identifies nuclei and cytoplasm, quantifies fluorescence intensities of each channel, and exports them into an excel file.</li>
	<li>Drop files in the file list. Update the metadata to group the files by tiles. 
	NOTE: All the instructions to group images are specified in the software. Name files appeared in NamesAndTypes module and files are grouped according to the number of the tile and channel per samples.</li>
	<li>Click View Output settings and specify a correct default output. Click Analyze Images. Open the spreadsheet file corresponding to measure_intensity parameters.</li>
</ol>

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Jean-Philippe Aurel and Kathryn Weiqi Li are employees of Biolidics company that produces instruments used in this article. The other authors have nothing to disclose.

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.

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		<col />
		<col />
		<col />
		<col />
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	<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 ...

semi-automatic-pd-l1-characterization-enumeration-circulating-tumor

Research

JoVE Journal

Cancer Research

10.5K Views.  Hospices Civils de Lyon. 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.

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

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...