Increased Fibrosis and Interstitial Fluid Pressure in Two Different Types of Syngeneic Murine Carcinoma Grown in Integrin β3-Subunit Deficient Mice

Stroma properties affect carcinoma physiology and direct malignant cell development. Here we present data showing that αVβ3 expressed by stromal cells is involved in the control of interstitial fluid pressure (IFP), extracellular volume (ECV) and collagen scaffold architecture in experimental murine carcinoma. IFP was elevated and ECV lowered in syngeneic CT26 colon and LM3 mammary carcinomas grown in integrin β3-deficient compared to wild-type BALB/c mice. Integrin β3-deficiency had no effect on carcinoma growth rate or on vascular morphology and function. Analyses by electron microscopy of carcinomas from integrin β3-deficient mice revealed a coarser and denser collagen network compared to carcinomas in wild-type littermates. Collagen fibers were built from heterogeneous and thicker collagen fibrils in carcinomas from integrin β3-deficient mice. The fibrotic extracellular matrix (ECM) did not correlate with increased macrophage infiltration in integrin β3-deficient mice bearing CT26 tumors, indicating that the fibrotic phenotype was not mediated by increased inflammation. In conclusion, we report that integrin β3-deficiency in tumor stroma led to an elevated IFP and lowered ECV that correlated with a more fibrotic ECM, underlining the role of the collagen network for carcinoma physiology

Diverse Bone Morphogenetic Protein Expression Profiles and Smad Pathway Activation in Different Phenotypes of Experimental Canine Mammary Tumors

BACKGROUND:BMPs are currently receiving attention for their role in tumorigenesis and tumor progression. Currently, most BMP expression studies are performed on carcinomas, and not much is known about the situation in sarcomas. METHODOLOGY/PRINCIPAL FINDINGS:We have investigated the BMP expression profiles and Smad activation in clones from different spontaneous canine mammary tumors. Spindle cell tumor and osteosarcoma clones expressed high levels of BMPs, in particular BMP-2, -4 and -6. Clones from a scirrhous carcinoma expressed much lower BMP levels. The various clones formed different tumor types in nude mice but only clones that expressed high levels of BMP-6 gave bone formation. Phosphorylated Smad-1/5, located in the nucleus, was detected in tumors derived from clones expressing high levels of BMPs, indicating an active BMP signaling pathway and BMP-2 stimulation of mammary tumor cell clones in vitro resulted in activation of the Smad-1/5 pathway. In contrast BMP-2 stimulation did not induce phosphorylation of the non-Smad pathway p38 MAPK. Interestingly, an increased level of the BMP-antagonist chordin-like 1 was detected after BMP stimulation of non-bone forming clones. CONCLUSIONS/SIGNIFICANCE:We conclude that the specific BMP expression repertoire differs substantially between different types of mammary tumors and that BMP-6 expression most probably has a biological role in bone formation of canine mammary tumors

Collagen-binding proteoglycan fibromodulin can determine stroma matrix structure and fluid balance in experimental carcinoma.

Research on the biology of the tumor stroma has the potential to lead to development of more effective treatment regimes enhancing the efficacy of drug-based treatment of solid malignancies. Tumor stroma is characterized by distorted blood vessels and activated connective tissue cells producing a collagen-rich matrix, which is accompanied by elevated interstitial fluid pressure (IFP), indicating a transport barrier between tumor tissue and blood. Here, we show that the collagen-binding proteoglycan fibromodulin controls stroma structure and fluid balance in experimental carcinoma. Gene ablation or inhibition of expression by anti-inflammatory agents showed that fibromodulin promoted the formation of a dense stroma and an elevated IFP. Fibromodulin-deficiency did not affect vasculature but increased the extracellular fluid volume and lowered IFP. Our data suggest that fibromodulin controls stroma matrix structure that in turn modulates fluid convection inside and out of the stroma. This finding is particularly important in relation to the demonstration that targeted modulations of the fluid balance in carcinoma can increase the response to cancer therapeutic agents

Immunohistochemical staining for phosphorylated Smad-1 and –5 (P-Smad-1/5).

<p>(A–C) Tumors generated by clones from the cell lines CMT-U309 (spindle cell); (D–F) CMT-U353 B (osteosarcoma); (G–I) CMT-U353 H4 (scirrhous carcinoma). (A–C) Spindle cell tumors generated by CMT-U309, clone C6. (A) Spindle cell tumor with an area of formed bone (*). Cells adjacent to the bone area and spindle cells with a distance to the formed bone showed clear positive staining (arrows). (B) Spindle cell tumors with strongly positive cells evenly distributed in the tumor (arrows). (C) Antibody specificity test: the signal was abolished when the antibody was incubated with blocking peptide. (D–E) Osteosarcomas formed by CMT-U353 B, clone 2 (D) and 7 (E). P-Smad-1/5 was detected in the cell dense border of the osteosarcomas. Cells closer to the centre of the tumor produced osteoid (*). (F) Spindle cell tumor generated by CMT-U353 B, clone 6. Bone formation was not seen in any tumors from this clone. P-Smad-1/5 was detected at lower levels than in tumors from clone 2 and 7, and was evenly distributed in the tumors (arrows). (G–H) Spindle cell tumors generated by clones from the scirrhous carcinoma: CMT-U353 H4, clone 6 (G), clone 9 (H) and clone 10 (I). Evenly distributed cells positive for P-Smad-1/5 were seen in the tumors (arrows). Note that positive cells show a predominantly nuclear staining.</p

Immunohistochemical analysis for BMP-6.

<p>BMP-6 staining was performed in tumors generated by clones from the CMT-U309 (spindle cell), CMT-U353 B (osteosarcoma) and CMT-U353 H4 (scirrhous carcinoma) cell lines. (A–B) Spindle cell tumors generated by CMT-U309, clone C6. (A) Spindle cell tumor with an area of formed bone (*). Cells adjacent to the bone area and spindle cells further away from the formed bone showed strong positive cytoplasmic staining (arrows). (B) Spindle cell tumors with cells strongly positive for BMP-6, evenly distributed in the tumors. (C) Osteosarcoma formed by CMT-U353 B, clone 2. BMP-6 was detected in the cell dense border of the osteosarcoma and some cells showed membranous accentuation of the staining (arrow). (D) Spindle cell tumor generated by CMT-U353 B, clone 6. Faint staining for BMP-6 was seen throughout the tumors, with only a few cells with stronger staining and clear cytoplasmic positivity (arrow). (E–F) Spindle cell tumors generated by CMT-U353 H4, clone 6 (E) and 9 (F) were positive for BMP-6. Tumor cells showed cytoplasmic positivity and some cells had membranous accentuation of the staining (arrow).</p

The effect of BMP-2 on phosphorylation of Smad-1, Smad-5 and p38 in mammary tumor clones.

<p>CMT-U353 H4 (clone 12), CMT-U309 (clone 4) and HTh 74 (positive control for Smad-1/5 phosphorylation) cells were stimulated with BMP-2 (as indicated) in vitro and phosphorylation of Smad-1/5 (P-Smad-1, -5) and p38 (P-p38) was analyzed by Western blot. Immunoblot analysis for total Smad-5, total p38 and β-Actin was performed as loading controls.</p

RPA analysis of expressed BMP mRNA in canine mammary clones.

<p>(A) Spindle cell clones (CMT-U309), (B) osteosarcoma clones (CMT-U353 B) and (C) scirrhous carcinoma clones (CMT-U353 H4).</p

Immunohistochemical analysis for BMP-2/4.

<p>Staining for BMP-2/4 was performed in tumors generated by clones from the CMT-U309 (spindle cell), CMT-U353 B (osteosarcoma) and CMT-U353 H4 (scirrhous carcinoma) cell lines. (A–B) Spindle cell tumors generated by CMT-U309, clone C6. (A) Spindle cell tumor with an area of formed bone (*). Cells adjacent to the bone area and spindle cells further away from the formed bone showed strong positive cytoplasmic staining. (B) Spindle cell tumors with cells strongly positive for BMP-2/4, evenly distributed in the tumors. (C) Osteosarcoma formed by CMT-U353 B, clone 2. BMP-2/4 was detected in the cell dense border of the osteosarcoma as well as in cells adjacent to bone (*). (D) Spindle cell tumor generated by CMT-U353 B, clone 6. Strong staining for BMP-2/4 was seen throughout the tumors. (E–F) Spindle cell tumors generated by CMT-U353 H4, clone 6 (E) and 9 (F) stained less intensely for BMP-2/4.</p

Tumors from cloned cell lines CMT-U309, CMT-U353 B and CMT-U353 H4 inoculated in mice.

<p>Tumors from cloned cell lines CMT-U309, CMT-U353 B and CMT-U353 H4 inoculated in mice.</p

Inhibition of carcinoma cell-derived VEGF reduces inflammatory characteristics in xenograft carcinoma

The stroma of carcinomas shares several characteristics with inflamed tissues including a distorted vasculature, active angiogenesis and macrophage infiltration. In addition, the tumor interstitial fluid pressure (P-IF) of the stroma is pathologically elevated. We show here that bevacizumab [rhuMab vascular endothelial growth factor (VEGF), Avastin], a monoclonal antibody to VEGF, at a dose of 5 mg/kg modulated inflammation in KAT-4 xenograft human anaplastic thyroid carcinoma tissue. At this dose, bevacizumab reduced the density of macrophages, MHC class II antigen expression by macrophages and II-1 beta mRNA expression. Furthermore, bevacizumab lowered tumor extracellular fluid volume, plasma protein leakage from tumor vessels, the number of CD31positive structures and tumor P-IF. The tumor plasma volume and the number of alpha-smooth muscle actin-positive vessels, however, remained unchanged. Our data suggest that carcinoma cellderived VEGF either directly or indirectly participates in maintaining an inflammatory microenvironment in experimental KAT4 carcinoma. Furthermore, our data indicate that the reduction of inflammation resulting in reduced vascular permeability and decrease in the tumor extracellular fluid volume by bevacizurnab contributes to reduced tumor P-IF. (c) 2006 Wiley-Liss, Inc