1. Identification of Genes Expressed in Malignant Cells That Promote Invasion Authors: Jennifer Walter-Yohrling, Xiaohong Cao, Michele Callahan, William Weber, Sharon Morgenbesser, Stephen L. Madden, Clarence Wang, and Beverley A. Teicher Published in Cancer Research, December 15, 2003 Presented by Rossitta Yung Date: March 25, 2004

2. Outline Background Objective Methods Results Conclusion Critique Discussion

3. Cancer Malignant tumor or forms of new tissue cells that lack a controlled growth pattern Invade and destroy normal tissue cells Tend to spread to other parts of the body

4. Stromal Cells Supporting cells for an organ made up of connective tissues (except brain and spinal cord) E.g. endothelial cells – form cellular sheets that cover line its cavities of heart, blood vessels and serous of the body, originating from mesoderm E.g. myofibroblasts – responsible for wound closure after tissue injury

5. Stromal cells (cont’) Actively contribute to tumor growth, invasion and metastasis Often comprise a major portion of solid tumors E.g. myofibroblasts: - ECM proteins  host desmoplastic response - GFs  involved in tumor angiogenesis

6. Objective investigate the genes expressed by tumor cells that promoted interactions between tumor cells and stromal cells

7. Materials eight tumor cell lines: 1. MDA-MB-231, human breast carcinoma cell line 2. A-375, human melanoma cell line 3. LNCaP, human prostate carcinoma 4. A-549, human lung carcinoma 5. PC-3, human prostate carcinoma cell line 6. DU-145, human prostate carcinoma cell line 7. SKOV-3 cells, human ovarian carcinoma cell line 8. Mel624, melanoma cell line -human adult dermal microvascular endothelial cells (HMVECs) - human adult dermal fibroblasts (HDFs)  comprised of myofibroblasts

8. Method – In vitro model to determine whether tumor cell lines could be grouped by their ability to enable stromal invasion

9. Method – In vitro model Added Matrigel to each well of a 24-well plate Polymerized Removed a ~1mm plug Filled with tumor cells Added labeled endothelial cells (green) and myofibroblasts (red) Captured both fluorescent and bright field images

10. Result

11. Result (cont’) High [fluorescence] Stromal Invasion Positive Low [fluorescence] Stromal Invasion Negative MDA-MB-231, human breast carcinoma cell line A-549, human lung carcinoma SKOV-3 cells, human ovarian carcinoma cell line LNCaP, human prostate carcinoma A-375, human melanoma cell line PC-3, human prostate carcinoma cell line Mel624, melanoma cell line DU-145, human prostate carcinoma cell line

12. Result (cont’) MDA-MB-231 (human breast carcinoma cell line) and PC3 (human prostate carcinoma cell line):

13. Method – SAGE Analysis to obtain a comprehensive, unbiased comparison of gene expression between the human tumor cell lines that underwent efficient invasion by the myofibroblasts and endothelial cells with those cell lines that did not

14. Method – SAGE Analysis

15. Method – Construction and Analysis of SAGE library Retrieved from either the Genzyme proprietary database or the public Gene Expression Omnibus database Normalized tag counts to 50,000 total library counts for each library GeneSpring software: an initial hierarchical clustering

16. Result (cont’)

17. 2 statistical tests were used to extract tags with significant differential expression 1. T test - For group comparison - Unable to eliminate tags with very low counts in one group and zeros in the other group 2. Chi square test - For the comparison of the averages of each group

18. Result (cont’) T test: significant value P≤ 0.05 Chi square test: significant value ≤ 0.1 99 SAGE tags emerged

19. Result (cont’)

20. a statistical comparison was carried out on the eight SAGE libraries that exceeded 20,000 tags 87 SAGE tags emerged

21. Result (cont’)

22. Method and Result – Quantitative PCR and In Situ Hybridization to validate the differential gene expression profiles identified by analysis of the SAGE libraries 30 genes were selected for real-time PCR examination in the eight tumor cell lines 9 of the genes analyzed by real-time PCR showed good correlation with normalized SAGE tag counts

23. Result - Strongest Correlation: bone marrow stromal antigen 2 (BST2 or HM1.24) stathmin-like 3 (STMN3) tumor necrosis factor receptor superfamily member 5 (TNFRSF5) hepatocyte growth factor tyrosine kinase substrate (HGS or HRS)

24. Result (cont’)

25. in situ hybridization for BST2 and STMN3 was performed on samples of metastatic and nonmetastatic ovarian cancer

26. Result (cont’)

27. high levels of both BST2 and STMN3 were observed in the metastatic ovarian cancer tissue than the nonmetastatic ovarian cancer tissue

28. Conclusion the combination of the stromal invasion assay amd SAGE analysis appears a useful tool for identifying genes involved in tumor-stromal interactions the genes identified through this analysis may be useful as therapeutic and/or diagnostic targets

29. Critique No mention about the selection of tumor cell lines for the experiment Figure 1: no mention about the significance of bright field images How do they perform the two statistical tests??? How do they select the 30 genes for RT-PCR??? Why do they choose BST2 and STMN3 for in situ hybridization??? Personally, I am not familiar with the histochemical pictures in Figure 6

30. Discussion Can results imply to other experimental samples or conditions??? (e.g. other tumor cell lines, other stromal cells) In-vivo model???