Volume 17, Issue 6, Pages (June 2015)

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Volume 17, Issue 6, Pages 473-480 (June 2015) In Vivo Loss of Function Screening Reveals Carbonic Anhydrase IX as a Key Modulator of Tumor Initiating Potential in Primary Pancreatic Tumors Nabendu Pore, Sanjoo Jalla, Zheng Liu, Brandon Higgs, Claudio Sorio, Aldo Scarpa, Robert Hollingsworth, David A. Tice, Emil Michelotti Neoplasia Volume 17, Issue 6, Pages 473-480 (June 2015) DOI: 10.1016/j.neo.2015.05.001 Copyright © 2014 The Authors Terms and Conditions

Figure 1 Schematic design of in vivo pooled shRNA screening strategy to identify novel targets for cancer metabolism. (A) Outline of the experimental design. (B) Percentage frequency of shRNA hairpins to selected targets present in tumors normalized with respect to the starting pool (injected cell population). Neoplasia 2015 17, 473-480DOI: (10.1016/j.neo.2015.05.001) Copyright © 2014 The Authors Terms and Conditions

Figure 2 Increased expression of CAIX in primary pancreatic (PDX15) TICs versus non-TICs. (A) Assessment of CAIX cell surface levels in tumor initiating (EpCAM+/CD44+/CD24+) and non-tumor initiating (EpCAM+/CD44−/CD24−) populations. (B) Graphical representation of A. The graph describes median fluorescence of TIC and non-TIC populations. (C) Relative mRNA expression of TIC and non-TIC populations by TaqMan analysis. Neoplasia 2015 17, 473-480DOI: (10.1016/j.neo.2015.05.001) Copyright © 2014 The Authors Terms and Conditions

Figure 3 Increased tumor initiating potential of CAIX-enriched primary pancreatic tumor cells (PDX15). EpCAM+CAIX+ and EpCAM+CAIX− sorted cells from PDX15 tumors were implanted at indicated numbers in RAG2 KO mice. (A) Growth curve of EpCAM+CAIX+ and EpCAM+CAIX− phenotypes in RAG2 KO mice. Mice were implanted with 5000 cells of each population and were euthanized when tumor volume reached ~2000 mm3. (B) The gating strategy used to sort cells representing CAIX(+) or CAIX(−) phenotype. Neoplasia 2015 17, 473-480DOI: (10.1016/j.neo.2015.05.001) Copyright © 2014 The Authors Terms and Conditions

Figure 4 Silencing of CAIX expression by shRNAs significantly reduces tumor forming potential of PDX15. (A) Growth characteristics of CAIX-enriched EpCAM+ cells (5000 cells per mouse) that were transduced with either CAIX shRNAs (shRNA1 or shRNA2) or NT-shRNA. Neoplasia 2015 17, 473-480DOI: (10.1016/j.neo.2015.05.001) Copyright © 2014 The Authors Terms and Conditions