Limits...
Increased fibrosis and interstitial fluid pressure in two different types of syngeneic murine carcinoma grown in integrin β3-subunit deficient mice.

Friman T, Gustafsson R, Stuhr LB, Chidiac J, Heldin NE, Reed RK, Oldberg A, Rubin K - PLoS ONE (2012)

Bottom Line: IFP was elevated and ECV lowered in syngeneic CT26 colon and LM3 mammary carcinomas grown in integrin β(3)-deficient compared to wild-type BALB/c mice.The fibrotic extracellular matrix (ECM) did not correlate with increased macrophage infiltration in integrin β(3)-deficient mice bearing CT26 tumors, indicating that the fibrotic phenotype was not mediated by increased inflammation.In conclusion, we report that integrin β(3)-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

ABSTRACT
Stroma properties affect carcinoma physiology and direct malignant cell development. Here we present data showing that α(V)β(3) 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 β(3)-deficient compared to wild-type BALB/c mice. Integrin β(3)-deficiency had no effect on carcinoma growth rate or on vascular morphology and function. Analyses by electron microscopy of carcinomas from integrin β(3)-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 β(3)-deficient mice. The fibrotic extracellular matrix (ECM) did not correlate with increased macrophage infiltration in integrin β(3)-deficient mice bearing CT26 tumors, indicating that the fibrotic phenotype was not mediated by increased inflammation. In conclusion, we report that integrin β(3)-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.

Show MeSH

Related in: MedlinePlus

Pericyte coverage in CT26 carcinomas grown in integrin β3-deficient and WT mice.A) The total fraction of pxl. positive for NG2 or α-SMA were quantitated. B) The amount of CD31 and NG2 double positive pixels (pxl.) and CD31 and α-SMA double positive pixels were quantitated and presented as the fraction of CD31 pixels that were also NG2 or α-SMA positive. C) Representative immunofluorescence images of CD31 double stained with either NG2 or α-SMA in CT26 carcinoma sections from both genotypes (× 200 magnification). D) Leakage of Evans blue dye was assessed in integrin β3-deficient (n = 4) and WT mice (n = 5) bearing CT26 carcinomas. Leakage was corrected for the tumor plasma volume. Data are presented as leaked volume plasma per tumor tissue dry weight. Bar is 50 µm. * indicates p<0.05 and ** indicates p<0.01 by Mann-Whitney test. Error bars are SEM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316610&req=5

pone-0034082-g005: Pericyte coverage in CT26 carcinomas grown in integrin β3-deficient and WT mice.A) The total fraction of pxl. positive for NG2 or α-SMA were quantitated. B) The amount of CD31 and NG2 double positive pixels (pxl.) and CD31 and α-SMA double positive pixels were quantitated and presented as the fraction of CD31 pixels that were also NG2 or α-SMA positive. C) Representative immunofluorescence images of CD31 double stained with either NG2 or α-SMA in CT26 carcinoma sections from both genotypes (× 200 magnification). D) Leakage of Evans blue dye was assessed in integrin β3-deficient (n = 4) and WT mice (n = 5) bearing CT26 carcinomas. Leakage was corrected for the tumor plasma volume. Data are presented as leaked volume plasma per tumor tissue dry weight. Bar is 50 µm. * indicates p<0.05 and ** indicates p<0.01 by Mann-Whitney test. Error bars are SEM.

Mentions: CD31 immunostaining showed an abundance of vessels in the viable zone of CT26 carcinomas grown in both integrin β3-deficient and WT mice but no significant differences in vessel density and morphology as assessed by stereology (Figure 4). Also, qRT-PCR analyses of mRNA expression from integrin β3-deficient and WT tumors showed equal levels of VEGF receptor-2 transcripts (Figure S4). Antibodies to the chondroitin sulfate proteoglycan NG2 were used to visualize activated pericytes. NG2 is known to be selectively expressed by pericytes in the vasculature of tumors [22]. Blood vessels in carcinomas grown in integrin β3-deficient mice displayed reduced NG2 positive staining and less co-localization between NG2 and CD31 (Figure 5A–C), but there were no significant difference in vascular coverage by α-SMA staining (Figure 5B and 5C). Hence, the pericyte coverage were similar between CT26 carcinomas grown in integrin β3-deficient and WT mice, however the phenotype of the pericytes differed between these two conditions. Additionally, α-SMA staining was not only confined to the vascular compartment, since also the extravascular compartment of the tumor stroma was positive for α-SMA staining (Figure 5C). There was no difference in total α-SMA staining in tumors from β3-deficient or WT mice (Figure 5A). Staining with reticular fibroblast marker showed no difference between the genotypes (data not shown), which supports equal content of carcinoma-associated fibroblasts. Vascular leakage of plasma proteins as determined by the modified Miles' assay, was similar in CT26 carcinomas grown in integrin β3-deficient and WT mice (Figure 5D). Moreover, the plasma volumes (PV) in CT26 carcinomas in the two genotypes were not significantly different, further supporting the hypothesis that vascular function was not affected by integrin β3-deficiency (Table 1). The extracellular volume (ECV) was, however, significantly lower in carcinomas grown in integrin β3-deficient mice (p<0.04), whereas total tissue water (TTW) was similar (Table 1). Collectively, the data show that the lack of integrin β3-subunit in tumor stroma has little effect on vascular function in CT26 carcinoma.


Increased fibrosis and interstitial fluid pressure in two different types of syngeneic murine carcinoma grown in integrin β3-subunit deficient mice.

Friman T, Gustafsson R, Stuhr LB, Chidiac J, Heldin NE, Reed RK, Oldberg A, Rubin K - PLoS ONE (2012)

Pericyte coverage in CT26 carcinomas grown in integrin β3-deficient and WT mice.A) The total fraction of pxl. positive for NG2 or α-SMA were quantitated. B) The amount of CD31 and NG2 double positive pixels (pxl.) and CD31 and α-SMA double positive pixels were quantitated and presented as the fraction of CD31 pixels that were also NG2 or α-SMA positive. C) Representative immunofluorescence images of CD31 double stained with either NG2 or α-SMA in CT26 carcinoma sections from both genotypes (× 200 magnification). D) Leakage of Evans blue dye was assessed in integrin β3-deficient (n = 4) and WT mice (n = 5) bearing CT26 carcinomas. Leakage was corrected for the tumor plasma volume. Data are presented as leaked volume plasma per tumor tissue dry weight. Bar is 50 µm. * indicates p<0.05 and ** indicates p<0.01 by Mann-Whitney test. Error bars are SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316610&req=5

pone-0034082-g005: Pericyte coverage in CT26 carcinomas grown in integrin β3-deficient and WT mice.A) The total fraction of pxl. positive for NG2 or α-SMA were quantitated. B) The amount of CD31 and NG2 double positive pixels (pxl.) and CD31 and α-SMA double positive pixels were quantitated and presented as the fraction of CD31 pixels that were also NG2 or α-SMA positive. C) Representative immunofluorescence images of CD31 double stained with either NG2 or α-SMA in CT26 carcinoma sections from both genotypes (× 200 magnification). D) Leakage of Evans blue dye was assessed in integrin β3-deficient (n = 4) and WT mice (n = 5) bearing CT26 carcinomas. Leakage was corrected for the tumor plasma volume. Data are presented as leaked volume plasma per tumor tissue dry weight. Bar is 50 µm. * indicates p<0.05 and ** indicates p<0.01 by Mann-Whitney test. Error bars are SEM.
Mentions: CD31 immunostaining showed an abundance of vessels in the viable zone of CT26 carcinomas grown in both integrin β3-deficient and WT mice but no significant differences in vessel density and morphology as assessed by stereology (Figure 4). Also, qRT-PCR analyses of mRNA expression from integrin β3-deficient and WT tumors showed equal levels of VEGF receptor-2 transcripts (Figure S4). Antibodies to the chondroitin sulfate proteoglycan NG2 were used to visualize activated pericytes. NG2 is known to be selectively expressed by pericytes in the vasculature of tumors [22]. Blood vessels in carcinomas grown in integrin β3-deficient mice displayed reduced NG2 positive staining and less co-localization between NG2 and CD31 (Figure 5A–C), but there were no significant difference in vascular coverage by α-SMA staining (Figure 5B and 5C). Hence, the pericyte coverage were similar between CT26 carcinomas grown in integrin β3-deficient and WT mice, however the phenotype of the pericytes differed between these two conditions. Additionally, α-SMA staining was not only confined to the vascular compartment, since also the extravascular compartment of the tumor stroma was positive for α-SMA staining (Figure 5C). There was no difference in total α-SMA staining in tumors from β3-deficient or WT mice (Figure 5A). Staining with reticular fibroblast marker showed no difference between the genotypes (data not shown), which supports equal content of carcinoma-associated fibroblasts. Vascular leakage of plasma proteins as determined by the modified Miles' assay, was similar in CT26 carcinomas grown in integrin β3-deficient and WT mice (Figure 5D). Moreover, the plasma volumes (PV) in CT26 carcinomas in the two genotypes were not significantly different, further supporting the hypothesis that vascular function was not affected by integrin β3-deficiency (Table 1). The extracellular volume (ECV) was, however, significantly lower in carcinomas grown in integrin β3-deficient mice (p<0.04), whereas total tissue water (TTW) was similar (Table 1). Collectively, the data show that the lack of integrin β3-subunit in tumor stroma has little effect on vascular function in CT26 carcinoma.

Bottom Line: IFP was elevated and ECV lowered in syngeneic CT26 colon and LM3 mammary carcinomas grown in integrin β(3)-deficient compared to wild-type BALB/c mice.The fibrotic extracellular matrix (ECM) did not correlate with increased macrophage infiltration in integrin β(3)-deficient mice bearing CT26 tumors, indicating that the fibrotic phenotype was not mediated by increased inflammation.In conclusion, we report that integrin β(3)-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

ABSTRACT
Stroma properties affect carcinoma physiology and direct malignant cell development. Here we present data showing that α(V)β(3) 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 β(3)-deficient compared to wild-type BALB/c mice. Integrin β(3)-deficiency had no effect on carcinoma growth rate or on vascular morphology and function. Analyses by electron microscopy of carcinomas from integrin β(3)-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 β(3)-deficient mice. The fibrotic extracellular matrix (ECM) did not correlate with increased macrophage infiltration in integrin β(3)-deficient mice bearing CT26 tumors, indicating that the fibrotic phenotype was not mediated by increased inflammation. In conclusion, we report that integrin β(3)-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.

Show MeSH
Related in: MedlinePlus