141 A novel liquid nitrogen-free snap freezer DISCUSSION Snap freezing of core needle biopsies by quenching in liquid nitrogen (LN2) is the golden standard to preserve tumor biology and allow profiling for precision medicine purposes at the DNA, RNA and (phospho)protein level, but the use of LN2 has several disadvantages. We have previously developed a LN2-independent, electrically powered and mobile snap freezer with adjustable cold sink temperature13. Comparing the novel snap freezer with the golden standard of LN2 quenching, we here show that MS-based phosphoproteomic and transcriptomic profiles of cancer cell line K562 and human liver biopsies are preserved (Figure 2 and 3). Phosphoproteome differences between individual patients were larger than potential differences induced by either freezing method (Figure 3A). Gene expression profiling by RNA sequencing corroborated these findings (Figure 3B). These findings are important, because MS-based phosphoproteomics and RNA sequencing profiles are sensitive to variation induced by differences in pre-analytical handling that impact tissue integrity. Ultimately, such variations would hamper extrapolation and implementation of research findings to the general patient population27,28. In particular, cold ischemia time can alter the (phospho)proteome and transcriptome29-31. While DNA in tumor tissue remains stable after one hour of cold ischemia time32,33, earlier studies describe multiple examples of altered protein and mRNA expression within 15-30 minutes and phosphorylation as early as after 5 minutes of cold ischemia time34-37. Remarkably, MS-based phosphotyrosine (pY)-phosphoproteomic profiles from acute myeloid leukemia samples were recently shown to remain relatively stable after a 4-hour delay of sample processing38. These results may indicate that the impact of pre-analytical variation may differ for hematological specimens vs solid tumor biopsies, but need further confirmation. In general, standard methods resulting in reliable results with minimal variation are prerequisites for application in precision oncology. Here, we found that the novel snap freezer is fulfilling this requirement by showing that molecular profiles of cell lines and individual patients’ biopsies were maintained. In addition, the effect of freezing rate differences on the phosphoproteomic profile of a cancer cell line was evaluated. Freezing rates that are too low will damage the cell membrane, likely due to increased solute concentration caused by volume reduction of liquid surrounding the cells39, while ultra-rapid cooling may lead to damage through devitrification and ice crystal formation upon storage including the Leidenfrost phenomenon20. We here found that differences in freezing rate up to 23 seconds to a goal temperature of -73 °C did not induce significant changes in phosphoproteomic profiles (Figure 4) indicating that a freezing rate faster than achieved with the snap freezer and with LN2 is unnecessary. Increasing the freezing rate by overcoming the Leidenfrost effect will not further improve preservation of the molecular profile of a biological sample. Together, these results imply that this snap freezer is of valid use in clinical setting, eliminating the need for harmful coolants and preventing technical and practical challenges of LN2 for cryopreservation. Alternative snap freezing solutions have been developed to circumvent the limitations of liquid nitrogen, but each of them has limitations in terms of mobility or cooling performance40,41. 5
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