TY - JOUR
T1 - COL2A1 is a novel biomarker of melanoma tumor repopulating cells
AU - Talluri, Bhavana
AU - Amar, Kshitij
AU - Saul, Michael
AU - Shireen, Tasnim
AU - Konjufca, Vjollca
AU - Ma, Jian
AU - Ha, Taekjip
AU - Chowdhury, Farhan
N1 - Publisher Copyright:
© 2020 by the authors.
PY - 2020/9
Y1 - 2020/9
N2 - Soft 3D-fibrin-gel selected tumor repopulating cells (TRCs) from the B16F1 melanoma cell line exhibit extraordinary self-renewal and tumor-regeneration capabilities. However, their biomarkers and gene regulatory features remain largely unknown. Here, we utilized the next-generation sequencing-based RNA sequencing (RNA-seq) technique to discover novel biomarkers and active gene regulatory features of TRCs. Systems biology analysis of RNA-seq data identified differentially expressed gene clusters, including the cell adhesion cluster, which subsequently identified highly specific and novel biomarkers, such as Col2a1, Ncam1, F11r, and Negr1. We validated the expression of these genes by real-time qPCR. The expression level of Col2a1 was found to be relatively low in TRCs but twenty-fold higher compared to the parental control cell line, thus making the biomarker very specific for TRCs. We validated the COL2A1 protein by immunofluorescence microscopy, showing a higher expression of COL2A1 in TRCs compared to parental control cells. KEGG pathway analysis showed the JAK/STAT, hypoxia, and Akt signaling pathways to be active in TRCs. Besides, the aerobic glycolysis pathway was found to be very active, indicating a typical Warburg Effect on highly tumorigenic cells. Together, our study revealed highly specific biomarkers and active cell signaling pathways of melanoma TRCs that can potentially target and neutralize TRCs.
AB - Soft 3D-fibrin-gel selected tumor repopulating cells (TRCs) from the B16F1 melanoma cell line exhibit extraordinary self-renewal and tumor-regeneration capabilities. However, their biomarkers and gene regulatory features remain largely unknown. Here, we utilized the next-generation sequencing-based RNA sequencing (RNA-seq) technique to discover novel biomarkers and active gene regulatory features of TRCs. Systems biology analysis of RNA-seq data identified differentially expressed gene clusters, including the cell adhesion cluster, which subsequently identified highly specific and novel biomarkers, such as Col2a1, Ncam1, F11r, and Negr1. We validated the expression of these genes by real-time qPCR. The expression level of Col2a1 was found to be relatively low in TRCs but twenty-fold higher compared to the parental control cell line, thus making the biomarker very specific for TRCs. We validated the COL2A1 protein by immunofluorescence microscopy, showing a higher expression of COL2A1 in TRCs compared to parental control cells. KEGG pathway analysis showed the JAK/STAT, hypoxia, and Akt signaling pathways to be active in TRCs. Besides, the aerobic glycolysis pathway was found to be very active, indicating a typical Warburg Effect on highly tumorigenic cells. Together, our study revealed highly specific biomarkers and active cell signaling pathways of melanoma TRCs that can potentially target and neutralize TRCs.
KW - 3D-fibrin gel
KW - Biomarkers
KW - Melanoma
KW - RNA-sequencing
KW - Tumor repopulating cells
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U2 - 10.3390/BIOMEDICINES8090360
DO - 10.3390/BIOMEDICINES8090360
M3 - Article
AN - SCOPUS:85092132555
SN - 2227-9059
VL - 8
JO - Biomedicines
JF - Biomedicines
IS - 9
M1 - 360
ER -