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Background: This study investigated the expression and localization of cathepsins B, D, and G in relationship to the embryonic stem cell (ESC)-like population we have previously identified in micro...

Aim: We have previously identified and characterized cancer stem cell (CSC) subpopulations in liver metastasis from colon adenocarcinoma (LMCA). In this study we investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D and G was performed on 4μm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients. Immunofluorescence (IF) IHC staining was performed on three representative samples of LMCA from the original cohort of nine patients, to determine the localization of these cathepsins in relation to the CSC subpopulations. NanoString mRNA analysis and Western Blotting (WB) were used to examine the transcript and protein expression of these cathepsins, respectively. Enzyme activity assays were utilized to determine their functional activity. Data acquired from counting of cells staining positively of the cathepsins on the DAB IHC-stained slides and from Nanostring mRNA analysis were subjected to statistical analyses to determine significance. Results: DAB IHC staining demonstrated expression of cathepsins B, D and G within LMCA. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4- cells within the tumor nests and the OCT4+ CSC subpopulation within the peritumoral stroma. NanoString mRNA analysis showed significantly greater transcript expression of cathepsin B and cathepsin D, compared to cathepsin G. WB confirmed expression of cathepsin B and cathepsin D proteins, while cathepsin G was below detectable levels. Enzyme activity assays showed functional activity of cathepsin B and cathepsin D. Conclusion: Our study demonstrated the novel finding of the expression of cathepsin B, cathepsin D, and possibly cathepsin G by the putative CSC subpopulations within LMCA.

Background Fifty percent of colorectal cancer (CRC) patients develop liver metastasis. This study identified and characterized cancer stem cells (CSCs) within colon adenocarcinoma metastasis to the liver (CAML). Methods 3,3-Diaminobenzidine immunohistochemical (IHC) staining was performed on nine CAML samples for embryonic stem cell (ESC) markers OCT4, SOX2, NANOG, c-Myc, and KLF4. Immunofluorescence (IF) IHC staining was performed to investigate coexpression of two markers. NanoString mRNA expression analysis and colorimetric in situ hybridization (CISH) were performed on four snap-frozen CAML tissue samples for transcript expression of these ESC markers. Cells stained positively and negatively for each marker by IHC and CISH staining were counted and analyzed. Results 3,3-Diaminobenzidine IHC staining, and NanoString and CISH mRNA analyses demonstrated the expression of OCT4, SOX2, NANOG, c-Myc, and KLF4 within in all nine CAML samples, except for SOX2 which was below detectable levels on NanoString mRNA analysis. IF IHC staining showed the presence of a SOX2+/NANOG+/KLF4+/c-Myc+/OCT− CSC subpopulation within the tumor nests, and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4− CSC subpopulation and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4+ CSC subpopulation within the peritumoral stroma. Conclusion The novel finding of three CSC subpopulations within CAML provides insights into the biology of CRC.