Tumor mass twelve times following subcutaneous implantation of Lewis Lung (LL2) tumor cells into mice dealt with with 3x 1mg tamoxifen 1 week prior to tumor cell implantation. (B) Overall Ligustilide number of angiogenic islets harvested from RIP-Tag mice at week a 1282512-48-4 hundred and one.number of angiogenic tumors at one hundred and one weeks of age. We located that deletion of FN diminished (~thirty%) the complete quantity of angiogenic islets (Fig. 3A-3C, P<0.004). To determine the effect on final tumor mass, we weighed all of the tumors from the pancreas of each individual mouse at 123 weeks. Surprisingly, we saw no significant effect on final tumor mass (Fig. 3D). To confirm the efficiency of gene deletion, we examined the mT/mG Cre reporter in RosaCreER mT/mG FN f/f RIP-Tag mice. We found that excision in the islets and adjacent pancreas was robust 1 week after the first tamoxifen treatment (Fig. 3E). Initially, we had performed analysis with continuous weekly tamoxifen treatment, to prevent outgrowth of cells with intact FN floxed sites. However, analysis of the largest tumors from mice with either a single (5x 1mg, over one week) or continuous (5x 1mg + 1mg weekly thereafter) tamoxifen treatments showed no significant difference in final tumor mass (Fig. 3D), or mT/mG Cre-reporter activation at endpoint (Fig. 3E), suggesting that outgrowth of cells with intact FN floxed sites does not explain the maintenance of tumor growth. To confirm the ablation of tumor FN, we examined sections of tumors from Rosa-CreER mT/mG FN f/f RIP-Tag mice and their Cre-negative or tamoxifen-negative controls at 123 weeks. We found the characteristic vascular pattern of FN staining was absent in the RosaCreER mT/mG FN f/f RIP-Tag mice with either single or continuous Tam treatment (Fig. 3E). Quantification of the stained areas revealed a nearly complete ablation of FN in the RosaCreER mT/mG FN f/f RIP-Tag tumors (Fig. 3F). We asked whether there was any difference in the depletion of DOC-soluble FN versus the DOC-insoluble, or "fibrillar" FN. We found that, consistent with the histological immunofluorescence staining, both total and soluble FN were depleted by 12 days after the first tamoxifen Fig 3. RIP-Tag Tumor growth in the absence of Fibronectin. (A&B) Isolated pancreatic tumors from individual mice. (C) Numbers of angiogenic islets isolated from individual mice. (D) Total mass of tumors isolated from individual mice. (E) Immunofluorescence staining for FN in the pancreas or in tumors in mT/mG reporter mice. (F) Percentage of the total tumor area covered by FN staining in individual mice. Each dot represents the average of three fields from a single mouse. Non-specific control represents the staining from pre-immune serum. (G) Western blot of equal tissue loads (by wet mass) of total pancreas or tumor, and 1% DOC-soluble and insoluble protein. (H) Remaining FN genomic DNA after Cre-mediated deletion, as determined by qPCR relative to an unaffected genomic location. (I) Fold-reduction in FN mRNA expression relative to 18s. Each point represents the results from the largest tumor in each mouse. Scale bars (A&B) = 1mm, (E) 7 week = 50m, 13 week = 100m. p value <0.01.treatment, and that the levels of total and soluble FN remained low to undetectable in most Rosa-CreER FN f/f RIP-Tag tumors up to 13 weeks (N = 3) (Fig. 3G). FN was not entirely removed from the insoluble pool. It is not clear whether the remaining insoluble FN is derived from existing matrix, prior to FN excision, or FN deposited subsequent to FN excision and tumor growth. We next asked whether FN expression by the tumor cells themselves was also reduced. First, we examined the efficiency in the deletion of FN in the tumors by quantitative PCR. We found that Cre activity resulted in a reduction of intact FN alleles by 405% with a single period of Tamoxifen treatment and 500% with continuous Tamoxifen treatments (Fig. 3H).
