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

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.