The scope of our research is to study the mechanisms of response or resistance to standard of care immunotherapies. Our lab is trying to understand what drives a successful immune response against tumors, and if we can identify these predictors of therapy to find targets that can overcome resistance. The biggest advancement in recent years has been the development and integration of single-cell RNA sequencing techniques.
They enable us in real time to systematically study molecular features in individual cells in an unbiased manner, which is helpful in the dissection of these complex and evolving tumor ecosystems. While many preclinical models are being used to study the response mechanisms to immunotherapy, we still don't know the immunological determinants that are important in human immunity. Processing and analyzing patient samples in real time is crucial to our ability to investigate all cellular compartments within a given tumor.
Using unbiased genomic and transcriptomic techniques, our lab discovered that defects in the antigen presentation machinery are associated with immunotherapy resistance in melanoma. More recently, using single-cell approaches, we discovered unique T cell states and myeloid cell polarities associated with patient outcomes in melanoma and head and neck cancer respectively. The biggest advantage of our protocol lies in its simplicity.
It is a universal protocol adjusted for the dissociation of small and large tumor specimens from multiple types of cancers in a short period of time, ending in the generation of highly viable single-cell suspensions. To begin tumor digestion, pour the sample into a 60 millimeter tissue culture dish placed on ice. Pipette 420 microliters of media from the dish to a 1.5 milliliter microcentrifuge tube.
Using tweezers, transfer the sample tissue to the tube containing media. Cut up the sample with clean scissors to pieces that are two to three millimeters in diameter. Add appropriate volumes of digestion enzymes and media to the sample.
Vortex the tube for five seconds and incubated in a thermal mixer positioned vertically on its side. Place a 50 micrometer cell filter on top of a new 15 milliliter conical tube and prime the filter with one milliliter of fresh RPMI media. Using a 1, 000 microliter low retention white pipette tip, load the sample to the filter and add the media.
Then with the back of a syringe plunger, grind and push the sample on the filter in a twisting motion. Wash the filter with five milliliters of media and any remaining media from the dish that contain the sample. To begin, obtain the filtered and digested tumor sample.
Centrifuge the sample at 450 g for five minutes at four degrees Celsius. Aspirate the media without disturbing the pellet and resuspend the pellet in ACK lysis buffer to remove red blood cells and record the volume used. Repeat the centrifugation and the lysis steps until the sample pellet has no visible red color after centrifugation.
Then resuspend the sample pellet in fresh media. Mix 10 microliters each of trypan blue and the dissociated cells in a 1.5 milliliter tube. Load 10 microliters on each side of a hemocytometer to count the cells.
Record the cell concentration, total live cell count, viability percentage, and final media volume after count. To begin, obtain the digested tumor tissue sample and lyse the red blood cells. Centrifuge the sample at 450 g for five minutes at four degrees Celsius.
After removing the media, resuspend the pellet in one milliliter of the isolation media. After repeating the centrifugation, resuspend the pellet in 100 microliters of isolation media. Transfer 100 microliters of sample into a well of a 0.2 milliliter PCR strip tube.
In a biosafety hood, add 10 microliters each of Annexin V cocktail and biotin selection cocktail to the sample. Pipette mix the solution and incubate at room temperature for four minutes. Next, vortex the dextrin-coated magnetic particles for 30 seconds.
Mix 20 microliters of the beads and 60 microliters of isolation media with the sample. After three minutes, place the strip tube onto a 10x Genomics Magnet Separator on the high setting for five minutes at room temperature. Then transfer the liquid from the strip tube without disturbing the beads into a new 1.5 milliliter low-bind microcentrifuge tube.
Centrifuge the tube as demonstrated earlier, and resuspend the pellet in an appropriate volume of RPMI media based on the pellet size. Finally, count the cells in a hemocytometer. Following dead cell removal, the live cell count was 4.9 times 10 to the power of five per milliliter with viability above 90%
