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

Outline

BackgroundObjectiveMethodsResultsConclusionCritiqueDiscussion

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

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 – SAGE Analysis

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’)

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’)

Result (cont’)

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

87 SAGE tags emerged

Result (cont’)

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’)

Result (cont’)

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

Result (cont’)

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

Discussion

Can results imply to other experimental samples or conditions??? (e.g. other tumor cell lines, other stromal cells)

In-vivo model???