Novel antibody reagents for characterization of drug- and tumor microenvironment-induced changes in epithelial-mesenchymal transition and cancer stem cells
[6];
[4];
[1];
- Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland (United States). Clinical Pharmacodynamics Biomarker Program, Applied/Developmental Research Directorate
- Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland (United States). Antibody Characterization Laboratory
- Argonne National Laboratory, Argonne, Illinois (United States). Biosciences Division
- Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland (United States). Antibody Characterization Laboratory
- National Cancer Institute at Frederick, Frederick, Maryland (United States). Biological Testing Branch, Developmental Therapeutics Program
- National Cancer Institute, Bethesda, Maryland (United States). Urologic Oncology Branch, Center for Cancer Research
- National Cancer Institute, Bethesda, Maryland (United States). Office of Cancer Clinical Proteomics Research, Center for Strategic Scientific Initiatives
- National Cancer Institute, Bethesda, Maryland (United States). Division of Cancer Treatment and Diagnosis
- Center for Cancer Research, National Cancer Institute, Bethesda, Maryland (United States). Division of Cancer Treatment and Diagnosis, National Cancer Institute
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.
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- AC02-06CH11357
- OSTI ID:
- 1627864
- Journal Information:
- PLoS ONE, Vol. 13, Issue 6; ISSN 1932-6203
- Publisher:
- Public Library of Science
- Country of Publication:
- United States
- Language:
- English
Similar Records
Bovine lactoferrin reverses programming of epithelial-to-mesenchymal transition to mesenchymal-to-epithelial transition in oral squamous cell carcinoma
Enhancement of cancer stem-like and epithelial−mesenchymal transdifferentiation property in oral epithelial cells with long-term nicotine exposure: Reversal by targeting SNAIL