Novel antibody reagents for characterization of drug- and tumor microenvironment-induced changes in epithelial-mesenchymal transition and cancer stem cells

Abstract

The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation−mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.

Clinical Evolution of Epithelial–Mesenchymal Transition in Human Carcinomas

“…#AF1997; R&D Systems). CSC multiplex IFA consisting of CD133, CD44v6, and NANOG (panel B) was performed using unconjugated rabbit monoclonal CD133 (47-10) antibody developed by our group (25), mouse monoclonal anti-CD44v6 antibody, and goat polyclonal antibody to human NANOG (AF1997). For both panels, 10 mg/mL of donkey antirabbit-AF647, anti-mouse-AF488, and anti-goat-AF555 (all from Life Technologies) were used as secondary antibodies.…”

LETM1 is a potential biomarker of prognosis in lung non-small cell carcinoma

“…NSCLC cells with CSC characteristics are enriched within populations with specific cell markers such as CD44, CD166, ALDH1A1, Sox2, Oct4, Nanog, and CD133, which also contribute directly to the CSC properties. These markers may be associated with carcinogenesis and tumor progression, and may also play an important role in maintaining the stemness phenotype of CSCs [8–11]. Therefore, studies on CSCs and a better understanding of CSC biology in lung cancer will provide a basis for developing novel diagnostic and therapeutic strategies.…”

FAST1 Predicts Poor Survival of Renal Carcinoma and Promotes Its Progression Through the TGF-β/Smad Pathway

“…23 In terms of its functions, EMT plays a vital role in modulating embryonic development, wound healing, cancer cell metastasis, and drug resistance. [24][25][26][27] It is worth noting that in non-small cell lung cancer (NSCLC), UBE2T enhances the degradation of FOXO1 in a ubiquitination-dependent manner, thus repressing EMT of NSCLC cells. 28 Similarly, this study found that FAST1 up-regulation led to up-regulated N-cadherin and Snail but inhibited E-cadherin and…”