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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 YungDate: March 25, 2004
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
Stromal Cells
Supporting cells for an organmade 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
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
Objective
investigate the genes expressed by tumor cells that promoted interactions between tumor cells and stromal cells
Materials
eight tumor cell lines:1. MDA-MB-231, human breast carcinoma cell line2. A-375, human melanoma cell line3. LNCaP, human prostate carcinoma4. A-549, human lung carcinoma5. PC-3, human prostate carcinoma cell line6. DU-145, human prostate carcinoma cell line7. SKOV-3 cells, human ovarian carcinoma cell line8. Mel624, melanoma cell line
-human adult dermal microvascular endothelial cells (HMVECs)
- human adult dermal fibroblasts (HDFs) comprised of myofibroblasts
Method – In vitro model
to determine whether tumor cell lines could be grouped by their ability to enable stromal invasion
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
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
Result (cont’)
MDA-MB-231 (human breast carcinoma cell line) and PC3 (human prostate carcinoma cell line):
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
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
Result (cont’)
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
Result (cont’)
T test: significant value P≤ 0.05Chi square test: significant value ≤ 0.1
99 SAGE tags emerged
Result (cont’)
a statistical comparison was carried out on the eight SAGE libraries that exceeded 20,000 tags
87 SAGE tags emerged
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
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)
Result (cont’)
in situ hybridization for BST2 and STMN3 was performed on samples of metastatic and nonmetastatic ovarian cancer
Result (cont’)
high levels of both BST2 and STMN3 were observed in the metastatic ovarian cancer tissue than the nonmetastatic ovarian cancer tissue
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
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