Analysis of the interaction of extracellular matrix and phenotype of bladder cancer cells

Abstract

BackgroundThe extracellular matrix has a major effect upon the malignant properties of bladder cancer cells both in vitro in 3-dimensional culture and in vivo. Comparing gene expression of several bladder cancer cells lines grown under permissive and suppressive conditions in 3-dimensional growth on cancer-derived and normal-derived basement membrane gels respectively and on plastic in conventional tissue culture provides a model system for investigating the interaction of malignancy and extracellular matrix. Understanding how the extracellular matrix affects the phenotype of bladder cancer cells may provide important clues to identify new markers or targets for therapy.MethodsFive bladder cancer cell lines and one immortalized, but non-tumorigenic, urothelial line were grown on Matrigel, a cancer-derived ECM, on SISgel, a normal-derived ECM, and on plastic, where the only ECM is derived from the cells themselves. The transcriptomes were analyzed on an array of 1186 well-annotated cancer derived cDNAs containing most of the major pathways for malignancy. Hypervariable genes expressing more variability across cell lines than a set expressing technical variability were analyzed further. Expression values were clustered, and to identify genes most likely to represent biological factors, statistically over-represented ontologies and transcriptional regulatory elements were identified.ResultsApproximately 400 of the 1186 total genes were expressed 2 SD above background. Approximately 100 genes were hypervariable in cells grown on each ECM, but the pattern was different in each case. A core of 20 were identified as hypervariable under all 3 growth conditions, and 33 were hypervariable on both SISgel and Matrigel, but not on plastic. Clustering of the hypervariable genes showed very different patterns for the same 6 cell types on the different ECM. Even when loss of cell cycle regulation was identified, different genes were involved, depending on the ECM. Under the most permissive conditions of growth where the malignant phenotype was fully expressed, activation of AKT was noted. TGFβ1 signaling played a major role in the response of bladder cancer cells to ECM. Identification of TREs on genes that clustered together suggested some clustering was driven by specific transcription factors.ConclusionThe extracellular matrix on which cancer cells are grown has a major effect on gene expression. A core of 20 malignancy-related genes were not affected by matrix, and 33 were differentially expressed on 3-dimensional culture as opposed to plastic. Other than these genes, the patterns of expression were very different in cells grown on SISgel than on Matrigel or even plastic, supporting the hypothesis that growth of bladder cancer cells on normal matrix suppresses some malignant functions. Unique underlying regulatory networks were driving gene expression and could be identified by the approach outlined here.

Suppression and Activation of the Malignant Phenotype by Extracellular Matrix in Xenograft Models of Bladder Cancer: A Model for Tumor Cell “Dormancy”

“…The findings confirm our previous hypothesis that the ECM exerts a suppressive effect in vivo based on loss of malignant properties on SISgel in vitro [23]. The suppressive effect of SISgel is not due to the dominant protein, collagen I, or to absence of some factor in SISgel necessary for malignant grown because tumors formed with co-injected collagen I grew nearly as well as they grew when co-injected with Matrigel.…”

“…Earlier research in our labs suggested that, unlike mammary epithelial cells, α6β4 integrins were not involved [24]. Microarray and proteomic studies implicated complex networks involving TGFβ, cMYC and a series of transcription factors [23], [25], [26]. Because a consistent gene signature could not be identified, it is likely that a protein switch regulates the conversion between the malignant and suppressed phenotypes.…”

Modeling of oxygen transport across tumor multicellular layers

“…Like spheroids, the MCL can be used to study the effects of growth conditions, ambient oxygen levels, media glucose concentration, or other media factors on tumor oxygen metabolism. In addition, the impact of different growth substrates on tumor oxygen consumption can be studied in the MCL, since, unlike spheroids, MCL form a 3-D layer of parenchyma on an underlying extracellular matrix, which has been shown to impact tumor phenotype (Birgersdotter et al, 2005,Dozmorov et al, 2006. Another powerful use of the MCL model involves combining quantitative oxygenation and drug transport studies.…”

“…First, it would likely expand the number of tumor cell lines that could be studied in vitro. Second, the impact of different types of extracellular matrix on tumor metabolism could be investigated, since, unlike spheroids, MCL grow on an underlying substrate, and extracellular matrix has been shown to influence tumor phenotype (Birgersdotter et al, 2005,Dozmorov et al, 2006. Third, it would permit drug transport and oxygenation to be quantitatively studied in the same in vitro model, which would be valuable for drugs whose uptake is known to be oxygen dependent (Hicks et al, 1998).…”

The Role of Structural Extracellular Matrix Proteins in Urothelial Bladder Cancer (Review)

“…In urothelial carcinoma of the bladder (UC), the role of ECM proteins has been widely studied to assess invasive behavior. Gene expression in bladder cancer cell lines has been shown to depend on the matrix used for cultivation (Deen et al 1994; Ioachim et al 2005; Dozmorov et al 2006). The following review will focus on the role of structural proteins in UC, which have been suggested to be of prognostic value (Table 1).…”