A novel method for RNA extraction from FFPE samples reveals significant differences in biomarker expression between orthotopic and subcutaneous pancreatic cancer patient-derived xenografts.
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.
Department of Biology, California State University Northridge, Northridge, CA, USA.
Malachia
M
Hoover
Department of Biology, California State University Northridge, Northridge, CA, USA.
Yvess
Y
Adamian
Claremont BioSolutions, Upland, CA, USA.
Mark
M
Brown
UCSD Moores Cancer Center and Department of Surgery, La Jolla, CA, USA.
Ali
A
Maawy
Department of Biology, California State University Northridge, Northridge, CA, USA.
Alexander
A
Chang
Department of Biology, California State University Northridge, Northridge, CA, USA.
Jacqueline
J
Lee
Department of Biology, California State University Northridge, Northridge, CA, USA.
Armen
A
Gharibi
Department of Surgery, M.D. Anderson Cancer Center, Houston, TX, USA.
Matthew H
MH
Katz
Department of Surgery, M.D. Anderson Cancer Center, Houston, TX, USA.
Jason
J
Fleming
UCSD Moores Cancer Center and Department of Surgery, La Jolla, CA, USA.|AntiCancer Inc., San Diego, CA, USA.
Robert M
RM
Hoffman
UCSD Moores Cancer Center and Department of Surgery, La Jolla, CA, USA.
Michael
M
Bouvet
Claremont BioSolutions, Upland, CA, USA.
Robert
R
Doebler
Department of Biology, California State University Northridge, Northridge, CA, USA.
Jonathan A
JA
Kelber
D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics*; D018740:Genetic Heterogeneity; D064593:Heterografts; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D009368:Neoplasm Transplantation / Q000379:methods*; D010190:Pancreatic Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D012313:RNA / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA; D016707:Tissue Fixation
D014408:Biomarkers, Tumor; D012313:RNA
FFPE RNA extraction; cancer biomarkers; microHomogenizer™; pancreatic cancer; patient-derived orthotopic xenografts (PDOX) tumor microenvironment