Tony Navas1, Thomas D. Pfister1, Simona Colantonio2, Amina Aziz3, Lynda Dieckman3, Richard G. Saul2, Jan Kaczmarczyk2, Suzanne Borgel4, Sergio Y. Alcoser4, Melinda G. Hollingshead4, Young H. Lee5, Donald P. Bottaro5, Tara Hiltke6, Gordon Whiteley2, Naoko Takebe7, Robert J. Kinders1, Ralph E. Parchment1, Joseph E. Tomaszewski7, James H. Doroshow7,8
- Clinical Pharmacodynamics Biomarker Program, Applied/Developmental Research Directorate, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland,United States of America
- Antibody Characterization Laboratory, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland, United States of America
- Biosciences Division, Argonne National Laboratory, Argonne, Illinois, United States of America
- Biological Testing Branch, Developmental Therapeutics Program, National Cancer Institute at Frederick, Frederick, Maryland, United States of America
- Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
- Office of Cancer Clinical Proteomics Research, Center for Strategic Scientific Initiatives, National Cancer Institute, Bethesda, Maryland, United States of America
- Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland, United States of America
- Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
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.
CovalX Technology Used (Click each option to learn more)
Immunoprecipitation-mass spectrometry was used to verify binding selectivity of the target proteins associated with cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) in tumors. Samples were obtained from three month old New Zealand white rabbits that were immunized through 4/5 subcutaneous injections and two test bleeds each. Titers for each target protein group were determined by ELISA and confirmed by the NCI.
Monoclonal antibodies were diluted in 1X PBS with 500 μm octyl-β-glucoside (“PBS/BOG”) to 50 ng/μL while test proteins/peptides were diluted to 100 ng/μL (protein) pr 25 ng/μL (peptide) in PBS/BOG. Solutions were created using a magnetic bead suspension diluted 1/4 in PBS/BOG and 200 μL of diluted suspension in each well of a 96-well plate. 200 μL of antibodies per well were incubated with Protein A beads at room temperature for 1 hour before being washed three times and the addition of 200 μL of antigen solution per well. The plate was incubated overnight at 4 °C and then the beads were washed three times with 200 μL of PBS/BOG and then three washes of HPLC-grade water. The bound peptides or proteins were extracted from the beads by adding 20 μL of 0.2% trifluoroacetic acid solution in HPLC-grade water. These solutions were then spotted onto the MALDI target and mixed on the plate with matrix solution, CHCA for peptides or SA for proteins. The plates were dried at room temperature and then analyzed. The protein analysis was performed using a MALDI TOF mass spectrometer modified with a CovalX HM1 detection system.
Epitope sequences were found for each of the antibodies which proved that the chosen reagents are useful when trying to identify differences in protein expression. These epitope identifications allow for a more precise analysis of protein expression for each of the antibodies studied in contrast to the existing, commercially available antibodies where the epitopes are not known.