A novel method for RNA extraction from FFPE samples reveals significant differences in biomarker expression between orthotopic and subcutaneous pancreatic cancer patient-derived xenografts
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Malachia Hoover1,*, Yvess Adamian1,*, Mark Brown2, Ali Maawy3, Alexander Chang1, Jacqueline Lee1, Armen Gharibi1, Matthew H. Katz4, Jason Fleming4, Robert M. Hoffman3,5, Michael Bouvet3, Robert Doebler2, Jonathan A. Kelber1
1Department of Biology, California State University Northridge, Northridge, CA, USA
2Claremont BioSolutions, Upland, CA, USA
3UCSD Moores Cancer Center and Department of Surgery, La Jolla, CA, USA
4Department of Surgery, M.D. Anderson Cancer Center, Houston, TX, USA
5AntiCancer Inc., San Diego, CA, USA
*These authors have contributed equally to this work
Jonathan A. Kelber, email: email@example.com
Keywords: FFPE RNA extraction, microHomogenizer™, pancreatic cancer, patient-derived orthotopic xenografts (PDOX) tumor microenvironment, cancer biomarkers
Received: June 16, 2016 Accepted: August 01, 2016 Published: September 01, 2016
Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies.
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