Journal Article

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

Whiteley, Gordon, Borgel, Suzanne, Navas, Tony, Tomaszewski, Joseph E., Hiltke, Tara, Kinders, Robert J., Lee, Young H., Doroshow, James H., Takebe, Naoko, Pfister, Thomas D., Parchment, Ralph E., Hollingshead, Melinda G., Bottaro, Donald P., Aziz, Amina, Colantonio, Simona, Saul, Richard G., Kaczmarczyk, Jan, Dieckman, Lynda, Alcoser, Sergio Y.

2018

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: AC133 Antigen - metabolism / Animal models / Animals / Antibodies / Antibodies, Monoclonal - biosynthesis / Antibodies, Monoclonal - pharmacology / Antineoplastic Agents - pharmacology / Antineoplastic Agents - therapeutic use / Benzamides - pharmacology / Benzamides - therapeutic use / Biology and Life Sciences / Biomarkers / Breast cancer / Cancer / Cancer research / Cancer therapies / Carcinoma, Non-Small-Cell Lung - metabolism / Carcinoma, Non-Small-Cell Lung - pathology / Cell Line, Tumor / Drug resistance / Enzyme inhibitors / Epithelial-Mesenchymal Transition - drug effects / Epitopes / Female / Fluorescent antibody technique / Focal adhesion kinase / Gene Knock-In Techniques / Glycosylation / Hepatocyte growth factor / Hepatocyte Growth Factor - genetics / Humans / Immunofluorescence / Immunohistochemistry / Immunoprecipitation / Indicators and Reagents / Laboratories / Localization / Lung cancer / Lung diseases / Lung Neoplasms - metabolism / Lung Neoplasms - pathology / Mass spectrometry / Mass spectroscopy / Medical prognosis / Medical research / Medicine and Health Sciences / Melanoma / Mesenchyme / Metastases / Metastasis / Mice / Mice, Transgenic / Microscopy / Molecular chains / Neoplastic Stem Cells - drug effects / Neoplastic Stem Cells - metabolism / Neoplastic Stem Cells - pathology / Non-small cell lung carcinoma / Pancreatic cancer / Phenotype / Proteins / Pyrazines - pharmacology / Pyrazines - therapeutic use / Quantitation / Reagents / Research and Analysis Methods / Science & Technology - Other Topics / Selectivity / Sox9 protein / Stem cell transplantation / Stem cells / Sulfonamides - pharmacology / Sulfonamides - therapeutic use / Transcription factors / Transgenic mice / Triple Negative Breast Neoplasms - drug therapy / Triple Negative Breast Neoplasms - pathology / Tumor cells / Tumor Microenvironment - drug effects / Tumors / Xenograft Model Antitumor Assays / Xenografts / Xenotransplantation