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Jenny Chang Lab by Bhuvanesh Dave PhD

Lentiviral shRNA screen to test the validity of a gene signature of breast cancer stem cells using high throughput mammosphere assays. Jenny Chang Lab by Bhuvanesh Dave PhD. Tumorigenic Cells: Are we missing the target? Jones et al. JNCI 96:583, 2004.

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Jenny Chang Lab by Bhuvanesh Dave PhD

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  1. Lentiviral shRNA screen to test the validity of a gene signature of breast cancer stem cells using high throughput mammosphere assays Jenny Chang Lab by Bhuvanesh Dave PhD

  2. Tumorigenic Cells: Are we missing the target? Jones et al. JNCI 96:583, 2004

  3. Tumorigenic Cells: Are we missing the target? Jones et al. JNCI 96:583, 2004

  4. Tumorigenic Cells: Are we missing the target? Jones et al. JNCI 96:583, 2004

  5. A Model For “Cancer Stem Cells” In Treatment Resistance and Disease Recurrence

  6. Chemotherapy Increases the Proportion of CD44+/CD24low/- Cells and Mammosphere-Initiating Cells A B CD44+/CD24- Mammosphere Formation Efficiency n=31 n=31 Li et al (2008) JNCI 100:672-9

  7. The Proportion of CD44+/CD24- Cells Correlates With Mammosphere Initiation Potential Mammospheres Are Enriched For Mammosphere-initiating Cells Compared To The Tumor of Origin

  8. Gene Expression Signature of Chemoresistant Mammosphere-initiating Cells

  9. The “Signature” Is Preferentially Manifest in Claudin-Low Tumors

  10. Design of GIPZ shRNAmir Library

  11. Choosing a Cell Line • In order to confirm our patient derived gene signature we looked for Claudin Low like cell lines • Based on Joe gray’s dataset we narrowed it down to MDA-MB231, SUM-159PT & HS578T • We chose SUM159PT because it possessed the CD44/24 population, which had been shown to have increased tumorigenic potential.

  12. Lentiviral Screen using open bioystems library and our gene signature We started with 493 genes and 1120 shRNA’s for this Lentiviral screen.

  13. Library Cherry Picking of clones Purification of DNA Transfection Infection Mammospheres Day 1 transfect Day 3 collect Day 4 collect Visualize and count Spin and Resuspend in MEGM+ PKH26 dye on Day4 Trypsinize mammospheres HBSS + serum Visualize and count Screening for CSC drug targets using shRNA for gene signature Phase I Phase II

  14. SUM159PT cells-Mammospheres plated 081408 and photographed 082008 96 Well 24 Well 6 Well 20x 20x 10x

  15. Testing Controls • Testing of Controls to Determine the Set of Controls to be used in the Screen - Infectivity - Impact on Mammosphere formation.

  16. Experiments • Conduct the screen using SUM159PT cells with the aforementioned controls and genes derived from our signature • Add some of the Hedgehog and Notch sigaling pathway regulators to that gene list in order to test for things that have been the usual suspects in the cancer stem field. • We added 40 genes from the ALDH1 derived signature from Dr. Max Wicha’s Group. • Confirm reduction of gene expression of the control genes by real time PCR which will be eventually followed by western blots.

  17. Results • We conducted this screen in 14x96 well plates each time (8 times) • We collected information on MS formation information for primary and secondary MS using Gelcount from Oxford Optronix • We have currently analyzed the primary MS data and we got 151 unique shRNAs where MS number changes significantly p=0.05

  18. Results of Primary MS formation screen depicted as a Z-score of all the genes tested in the screen Z Score

  19. Candidate Regulators p<0.05 128 shRNA’s representing 108 unique genes showed significant alteration in the MS formation Efficiency in SUM159

  20. Increasing MSFE Decreasing MSFE

  21. BT549 p<0.05

  22. Candidate Regulators p<0.05 BT549

  23. Increasing MSFE Decreasing MSFE BT549

  24. Future Directions • Do real time PCR of all the targets. • Liposomal delivery of shRNA in cells/animals using nanoparticles • Target two tumor lines based on five target gene identified from the combination of these two cell lines.

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