Comprehensive investigation of stool metaproteomes among patients with precancerous lesions and colorectal cancer

INTRODUCTION: Colorectal cancer (CRC) is the 2nd most fatal cancer in the U.S.; most benefits of CRC screening are from detection of advanced pre-cancerous lesions (APLs). With a goal to improve non-invasive stool test APL sensitivity, we sought novel APL signatures in stool metaproteomes (host- and gut microbiome-derived proteins) among cancer-free controls (CTRL), APL, and CRC patients. Since dysbiosis in gut microbiota is associated with CRC initiation, we also examined the microbial source of differentially expressed proteins (DEPs). METHODS: Mass spectrometry was performed on archival stool supernatants (n=63 patients; 15 CTRL, 32 APL [16 adenomas & 16 sessile serrated lesions], 16 CRC). A sectioning and database-enrichment strategy was applied for peptide-spectrum matching. Microbial and human-derived proteins were searched against the Integrated Gene Catalogue (IGC) and UniProt human reference proteome. DEPs were identified using thresholds of P < 0.05 and fold-change ≥ 1.5. Pathway annotation of human DEPs was performed with the REVIGO webserver, while gut microbial DEPs were functionally categorized using the KEGG Automatic Annotation Server (KAAS). To assess the taxonomic distribution of microbial DEPs, sequences were aligned against the NCBI non-redundant database using BLASTP. RESULTS: Mass spectrometry detected 879 human and 32,514 microbiome-derived proteins. When limiting detection to proteins present in ≥ 75% of patients, only 117 human and 204 microbial proteins remained. There were 17 (4,875), 288 (9,638), and 63 (3,053) unique human (microbial) proteins detected in CTRL, APL, and CRC groups, respectively. When the protein presence cutoff was set at >30% of patients, there were 52 (13 human [11 upregulated, 2 downregulated], 39 microbial [28 upregulated, 11 downregulated]) DEPs detected for the CRC + APL vs CTRL comparison. Some pathways associated with upregulated human DEPs included: inflammatory response, cellular response to iron, and endothelial cell migration. The top upregulated microbial DEPs were derived from the Phocaeicola, Lachnospiraceae, and Oscillospiraceae genera. The top downregulated microbial DEPs belonged to the Bacteroides, Eubacterium, Agathobacter, Coprococcus, and Blautia genera. CONCLUSION: This work highlights the biological diversity and molecular heterogeneity of the stool proteome. We identified several host and microbial protein markers that differentiate APLs and early CRC from controls. These results provide a foundation for future validation studies, which are necessary to determine the clinical utility of these protein biomarkers prior to their integration into a noninvasive stool test.