1. Biplab Bose Emergence of Cellular Heterogeneity in Expression of an Oncofetal Protein Department of Biosciences & Bioengineering IIT Guwahati

2. Heterogeneity in Cancer Genetic heterogeneity within a tumor Mol Oncol. 2010, 4(3):267-83

3. Population Heterogeneity in Gene Expression cfp CFP P yfp YFP P Science, 2002, 297(5584):1183-6 Development, 2009,136:3525-30

4. Stochasticity in Gene Expression

5. Biological Implication of Heterogeneity Nat Rev Genet. 2009, 10(5):336-42 Emergence of drug resistance

6. Intracellular Signaling Pathways: Nodal/ALK4/Smad-2 Glypican-1/c-Src/MAPK Glypican-1/c-Src/AKT During Development: Patterning of the Anterior/Posterior axis Specification of mesoderm and endoderm during gastrulation In Cancer: Triggers proliferation Induction of cellular migration and invasion promotion of angiogenesis Stimulates EMT Human Cripto-1 (CR-1): an Oncofetal Protein Member of EGF-CFC family. Expressed as membrane bound molecule Over-expressed in various cancers Functional in membrane-bound and soluble form

7. Heterogeneous Expression of CR-1 Human melanoma cell linesHuman embryonal carcinoma cell lines CR-1 (+)ve : 2% - 5% Stem Cells. 2010, 28(8):1303-14 Cell Cycle. 2013 May 1;12(9):1450-6. CR-1 (+)ve : ~50% CR-1 expression is heterogeneous: two sub-populations exists CR-1 

8. Linear Circuit Positive feedback Transcriptional Circuit Affects Expression Heterogeneity

9. Current InformationHypothesis A Possible Feedback Circuit for CR-1

10. U-87 MG cells, treated with recombinant CR-1, 24 hr CR-1 Induces Its Own Expression n = 2.3713; a = 10.85 K=156.99; R 2 = 0.9747 Real-time PCR Western Blot

11. 11 Treatment with CR-1 does not effects on mRNA stability Real Time PCR: No significance difference between treated and untreated (Rank sum test, P =0.127) Treatment condition: 200 ng/ml CR1-GST, 5 µg/ml Act-D

12. The Pathway of Induction Treatment condition: Cells: U-87 MG; SB431542: 10 µM; CR1-GST or GST: 200 ng/ml; 24 hr Western Blot PCR

13. MCF-7: Expresses SMAD4 HT-29: Does not express SMAD4 The Pathway of Induction

14. Linear Circuit Positive feedback Transcriptional Circuit Affects Expression Heterogeneity

15. Treatment: U-87 MG cells treated with different conc. of recombinant CR-1, 24 hrs Induction of CR-1 expression happens only in a minority subpopulation Induction of CR-1 Expression is Heterogeneous

16. Dose-dependent Behavior of CR-1 Positive Cells Treatment: U-87 MG cells treated with different conc. of recombinant CR1, 24 hrs Overton histogram subtraction used to find CR-1 (+)ve population

17. RT-PCR Two subpopulations are sorted by FACS and gene expression was checked by RT-PCR Differential Expression of Pathway Molecules CR-1(-)veCR-1(+)ve

18. Bimodality in Expression May Be Obscured

19. Signature of Bimodality in Expression Noise When protein expression, is unimodal and increases by increase in rate of transcription Noise in CR-1 expression CR-1 expression was induced with different doses of recombinant CR-1 Mol Biosyst, 2012, 8(11):3068-76

20. Signature of Bimodality in Expression Noise “Low cells” has lower mean and variance “High cells” have high mean and variance % of “High cells” varied: 0% to 100% Simulated for three conditions: Simulated using MATLAB

21. Signature of Bimodality in Expression Noise Simulation Flow Cytometry Data

22. Biological Implication of CR-1 Positive Subpopulation Stochasticity in gene expression  Spontaneous emergence of heterogeneity  Cells in different states  Some cells having drug resistance and/ or Some cells having Stemness Ref: Cell, 2011, 146: 633–644

23. CR-1 Positive Cells May Not be Cancer Stem Cells RT-PCR: CR-1(-)veCR-1(+)ve Two subpopulations are sorted by FACS and gene expression was checked by RT-PCR

24. CR-1 Positive Cells are MDR-1 Positive Isotype ControlUntreatedCR1-GST (400 ng/ml) CR-1 and MDR-1 are co-induced in a small subpopulation: indicates co-regulation MDR-1 is an ABC-transporter involved in multidrug resistance CSC like cells isolated from U-87 MG have higher MDR-1 expression

25. Signal Cells Do Play Dice PLoS ONE 10(2): e0116748

26. Research student: Dr. Pojul Loying Collaborator: Dr. Janvie Manhas Dr. Sudip Sen, AIIMS Financial Support: DBT biplabbose@iitg.ernet.in @bose_biplab http://flowpy.wikidot.com/ FlowPy