Abstract
Using a simple, environment friendly proteome extraction (TOP), we were able to optimize the analysis of clinical samples. Using our TOP method we analyzed a clinical cohort of microsatellite stable (MSS) and unstable (MSI-H) colorectal carcinoma (CRC). We identified a tumor cell specific, STAT1-centered, immune signature expressed by the MSI-H tumor cells. We then showed that long, but not short, exposure to Interferon-γ induces a similar signature in vitro We identified 10 different temporal protein expression patterns, classifying the Interferon-γ protein temporal regulation in CRC. Our data sheds light on the changes that tumor cells undergo under long-term immunological pressure in vivo, the importance of STAT proteins in specific biological scenarios. The data generated could help find novel clinical biomarkers and therapeutic approaches.
Highlights
Using a simple, environment friendly proteome extraction (TOP), we were able to optimize the analysis of clinical samples
We identified a tumor cell specific, signal transducers and activators of transcription 1 (STAT1)-centered, immune signature expressed by the microsatellite instability (MSI)-H tumor cells
One of the consequences is the deleterious impact of extended fixation time on the amount of protein extracted from clinical formalin-fixed and paraffin-embedded (FFPE) samples, which influences the proteomic coverage achievable by MS analysis [5, 27]
Summary
Environment friendly proteome extraction (TOP), we were able to optimize the analysis of clinical samples. Using our TOP method we analyzed a clinical cohort of microsatellite stable (MSS) and unstable (MSI-H) colorectal carcinoma (CRC). We identified a tumor cell specific, STAT1-centered, immune signature expressed by the MSI-H tumor cells. It is adequate to fix tissues for 6 h to 72 h [4] This creates a huge bias for traditional proteomics pipelines, as protein recovery seems to decrease with prolongation of fixation time, with yields dropping by ;20% for every 24 h in formalin [5]. A robust, efficient, fixation-time impervious protein extraction method will help to propel clinical molecular proteomics forward. Most CRC cases are characterized by chromosomal instability with microsatellite stability (MSS). The MSI-H tumors are typically characterized by a high rate of single-nucleotide substitutions [9]. It has been shown that MSI-H tumors show an immune active tumor microenvironment with high levels of several immune checkpoint proteins [11]
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