? The matrix metalloproteinases (MMPs) have a long link with malignancy. Recently stromal cells of the host have been implicated as sources of MMPs, which appear to directly contribute to tumor progression. Tumor-stroma interrelationships generally need to be examined in vivo. Various mouse systems have comprised the major in vivo models for studying tumor-and host-derived MMPs and although they provide unique genetic approaches for study of tumor progression, they have a number of experimental limitations. Our laboratory has utilized an alternative in vivo tumor progression model, namely a human tumor/chick embryo system that is both rapid and quantitative. Recently, in this model we have utilized human-specific, real time PCR to detect small numbers of disseminating human tumor cells, allowing for isolation of HT1080 human tumor cell variants, that intravasate and metastasize al greatly enhanced rates. We also had been examining separately, the MMPs expressed by chick embryo cells and human tumor cells. We propose to combine our MMP studies with our rapid, quantitative tumor progression model to initiate an in-depth study of tumor/stroma interrelationships.
The specific aims of this renewal application are:
Aim 1 : To Examine the Differential Expression of Tumor (Human) and Host (Chicken) MMPs During HT1080 Tumor expansion In Vivo. In situ hybridization, quantitative PCR, immunocytochemistry and Western blotting, all using human-or avian-specific probes, will be employed to determine the in vivo levels (mRNA and protein) of eight human MMPs and four chicken MMPs in expanding tumor tissue and responding host tissue.
Aim 2 : To Asses; the Spatial, Temporal and Enzyme Nature of the In Vivo Activation Pathways of Select MMPs of Tumor/Stroma Origin. The focus initially will be on the activation of tumor-derived MMP-9 as the prototype, pathway, but the activation of other proMMPs also will be examined.
Aim 3 : To Determine the Mechanistic Involvement of Individual MMPs to Tumor Dissemination and proMMP activation In Vivo. Approaches to specifically downregulate the expression and catalytic activity of selected tumor and stromal MMPs will be developed using siRNA and subtractive immunization methods respectively. The precise contributory role of individual tumor and stromal MMPs will be elucidated.
Aim 4 : To Identify the In Vivo Substrates of the MMPs that Contribute to Tumor Progression. A comparison of highly disseminating tumors with non-disseminating tumors will be carried out to identify unique substrates that are cleaved in tandem with tumor dissemination. Biochemical, immunological and proteomic approaches will be used to identify the putative substrates. ? ? ?
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