Comprehensively characterizing the tumor microenvironment is essential in immuno-oncology research. This assay is the first to use RNA-based health expression models to measure immune cells in solid tumor tissue. This assay enables researchers to gain highly sensitive and specific measurements of the immune contexture in FFB solid tumor tissues and to combine these results into a multi-dimensional biomarker.
All cancer treatments, not just immunotherapies, elicit an immune response at the site of the solid tumor. Measuring this immune response is important for understanding disease progression and therapy responses. This protocol combines traditional RNA library preparation techniques with targeted capture.
While time-consuming, following the wash steps and monitoring the quality control checkpoints as suggested is very important for success. For highly degraded RNA from formalin-fixed paraffin embedded samples, assemble the first strand synthesis reaction on ice according to the table. Thoroughly mix the reactions by pipetting up and down several times before briefly spinning down the samples in a microcentrifuge.
At the end of the centrifugation, immediately place the samples into a preheated thermal cycler following program number three from the table. For high-quality intact RNA, assemble the first strand synthesis reaction on ice in a nuclease-free PCR tube as outlined in the tables. To begin this procedure, mix 200 nanograms of the barcoded library of interest with two micrograms of COT-1 DNA and two microliters of blocking oligos in a nuclease-free PCR tube.
Then use a vacuum concentrator set to 30 to 45 degrees Celsius to dry the contents of the tube. After hybridization, remove the samples from the thermal cycler and set the thermal cycler to incubate at 65 degrees Celsius with the heated lid set to 70 degrees Celsius. Use a multichannel pipette to transfer 17 microliters of fully homogenized beads to the samples and pipette thoroughly up and down 10 times.
To bind the DNA to the beads, place the tubes into the thermal cycler following program 10 briefly removing the stripped tubes every 10 to 12 minutes for three seconds of gentle vortexing to keep the beads resuspended. Immediately following the binding incubation, remove one strip of tubes from the thermal cycler and add 100 microliters of preheated wash buffer one to the tubes. Pipette thoroughly up and down 10 times and place the tubes on a magnetic rack to allow the beads to fully separate from the wash solution before aspirating the unbound DNA containing supernatant.
Remove the tubes from the rack and add 150 microliters of preheated stringent wash buffer with thorough mixing to completely resuspend the beads taking care to avoid bubbles. Place the tubes back into the thermal cycler at 65 degrees Celsius for five minutes before placing the tubes back onto the magnetic rack to allow the beads to fully separate from the supernatants. Discard the unbound DNA containing supernatants, wash the beads once more with preheated stringent wash buffer as just demonstrated for a total of two stringent washes.
After the last wash, remove the supernatant and add 150 microliters of room temperature wash buffer one to the tubes. Pipette the wash 10 to 20 times to completely resuspend the beads and incubate the samples with the lid sealed for two minutes alternating between vortexing for 30 seconds and resting for 30 seconds. At the end of the incubation, centrifuge briefly and place the tubes on the magnetic rack and remove the supernatant once the beads have fully adhered to the magnet.
Centrifuge the tubes briefly with the caps closed and return the samples to the magnetic rack. Use a 10 microliter pipette to remove any residual wash buffer and add 150 microliters of room temperature wash buffer two. Pipette the wash 10 to 20 times to completely resuspend the beads and incubate the samples with the lids sealed for two minutes alternating between vortexing for 30 seconds and resting for 30 seconds.
After a brief centrifugation, transfer the entire volume of beads from each tube into clean nuclease-free PCR tubes and place the tubes onto the magnetic rack. After discarding the unbound DNA containing supernatants, briefly centrifuge the samples again and use a 10 microliter pipette to remove any residual wash buffer from the beads while they're in the magnetic rack. Add 150 microliters of wash buffer three to the tubes and pipette up and down 10 to 20 times to completely resuspend the beads.
Incubate the tube for two minutes alternating between 30 seconds of gentle vortexing and 30 seconds of rest before briefly centrifuging. Place the tubes onto the magnetic rack to aspirate the supernatants and briefly centrifuge the bead samples again. Use a 10 microliter pipette to remove any residual wash buffer from the beads while they are in the magnetic rack before removing the tubes from the rack and resuspending the beads in 20 microliters of nuclease-free water per tube.
Then pipette the beads 10 times to ensure that any beads stuck to the side of the tubes have been resuspended. The presence of adapter dimers should be evaluated to determine if additional cleanup is necessary. For example, this electropherogram shows an acceptable level of adapter dimer which appears as a sharp peak around 128 base pairs while this electropherogram shows an unacceptable level of adapter dimer.
The immune profiles pre and post-treatment may be used to understand the effects of a specific therapy on the tumor microenvironment as in this representative analysis for which the changes in percentage for each immune cell of interest and the total immune content are shown pre and post-chemotherapy for a single patient. In this analysis, samples between patient groups were compared according to their time to disease progression following treatment. A subset of the patients showed disease recurrence after 18 months while another subset progressed faster in 18 or fewer months.
The median delta value could then be compared for each sample to identify putative biomarkers of disease progression. For example, here two immune escape genes were identified as statistically significant differentiators of the sample groupings. Because the individual gene biomarkers were robust with clear statistical significance, the multi-dimensional biomarker did not add significant predictive value.
Be sure to perform the heated washes one strip at a time to prevent sample cooling and remember to transfer the beads to fresh tubes to avoid off-target contamination. This protocol demonstrates the use of gene expression models to quantify immune cells and tumor tissue. New models are being developed to measure cell states such as exhaustion or activation.
