BACKGROUND
the extracellular matrix  is an extremely complex supramolecular structure that is locally secreted by cells and influences the structure, viability and functions of cells and tissues.
epithelial-mesenchymal and epithelial-stromal interactions play important roles in both physiological and pathological settings, such as during embryonic morphogenesis  <cit> , wound healing  <cit>  and tumorigenesis  <cit> , and are accompanied by dynamic changes that generate new cell-matrix interactions  <cit> .
cell contact with a single ecm component initiates multiple signals that affect both cell behavior and gene expression  <cit> .
the tumor microenvironment plays an important role in the behavior of malignant cells.
the functional association of cancer cells with surrounding tissues changes as the malignancy progresses and is accompanied by the remodeling of the ecm  <cit> .
a severe fibrotic reaction around tumor tissues represents a host reaction to tumor growth.
fibroblasts are the main source of fibrotic tissue, which consists mainly of collagen types i and iv.
in addition to collagen, several other ecm proteins are present, including fibronectin, laminin, tenascin and proteoglycans  <cit> .
increasing evidence has demonstrated the involvement of cell surface proteoglycans in the multi-functional network of cell-cell and cell-matrix interactions that are essential for local tumor invasion and subsequent metastasis  <cit> .
syndecan- <dig> is one of the four members of the syndecan family of cell surface transmembrane heparan sulfate  proteoglycans.
it has been implicated in the formation of specialized membrane domains and functions as a direct link between the extracellular environment and the organization of the cortical cytoplasm.
in several colorectal cancer cell lines, syndecan- <dig> is highly expressed compared to normal cell lines  <cit> .
this increase appears to be critical for cancer cell behavior because it regulates adhesion and proliferation and, therefore, tumorigenic activity  <cit> .
in order to examine the relationship between tumor cells and stromal fibroblasts, we used an in vitro model to investigate the effect of ecm that is produced by stromal fibroblasts on the synthesis of glycosaminoglycans  and proteoglycans by two colorectal cancer cell lines, caco- <dig> and hct- <dig>  which have different metastatic potentials.
RESULTS
stromal fibroblast ecm influences gag synthesis by hct- <dig> colorectal cancer cells
to analyze the interactions between tumor cells and stromal fibroblast ecm, two colorectal cancer cell lines with different metastatic potentials, caco- <dig> and hct- <dig> cells, were studied.
the influence of stromal fibroblast-produced ecm on gag and proteoglycan synthesis by the cancer cells was investigated.
tumor cells were cultured for three days on top of stromal ecm and then labeled with na2so <dig>  the gags synthesized were analyzed by agarose gel electrophoresis in  <dig> -m  <dig> -diaminepropane acetate buffer and quantified as described in the methods.
the caco- <dig> colorectal cancer cell line, which has low metastatic potential, produces both chondroitin sulfate  and hs, the former being more abundant in the medium and the latter being more abundant in cell extracts .
there was no difference in gag synthesis by caco- <dig> cells in the presence or absence of stromal fibroblast ecm.
in the hct- <dig> colorectal cell line, which has high metastatic potential, the same gag distribution profile was observed, but there was a significant increase in medium, cell extract and matrix hs production when cells were cultured on top of stromal ecm .
gene expression of surface and ecm proteoglycans is modulated by stromal fibroblast ecm
proteoglycan expression was analyzed to confirm the gag synthesis results.
we investigated the expression of the family of syndecans , which have been shown to be involved in cancer-stroma interactions  <cit> .
stromal fibroblast ecm did not significantly affect the expression of syndecans in caco- <dig> cells, with the exception of syndecan- <dig>  which decreased in the presence of stromal fibroblast ecm .
many colon cancer cell lines show increased syndecan- <dig> expression, and this up-regulation seems to be crucial for their tumorigenic activity.
in contrast, colon cancer cell lines ht <dig>  caco- <dig> and dld <dig> show low syndecan- <dig> expression, and inhibition of syndecan- <dig> function in these cell lines did not affect any of their adhesion, proliferation, invasion and migration  <cit> .
in opposition, stromal fibroblast ecm decreased syndecan- <dig>  - <dig> and - <dig> expression, but significantly increased syndecan- <dig> expression in hct- <dig> cells .
of the four syndecan family members, syndecan- <dig> has been previously reported as up-regulated in several colon carcinoma cells and, further, that this increased expression is crucial for tumorigenicity  <cit> .
stromal fibroblast ecm influences the synthesis of syndecan- <dig> by hct- <dig> colorectal cancer cells
to confirm the increase in hct- <dig> syndecan- <dig> expression that was induced by stromal fibroblast ecm at the protein level, and to relate it with gags synthesis, the cells were labeled with sulfate and immunoprecipitated with an antibody against the extracellular domain of human syndecan- <dig> proteoglycan .
the results clearly indicate that the stromal fibroblast increases the amount of syndecan- <dig> in hct- <dig> cells .
furthermore, the results show the association of hs augments with this syndecan isoform.
through western blotting, a 150% increase of syndecan- <dig> in hct- <dig> cells cultured in the presence of stromal fibroblasts ecm was observed therefore corroborating the immunoprecipitation .
we also detected cell surface syndecan- <dig> levels by flow cytometry.
hct- <dig> cells were cultured for three days in the presence or absence of stromal fibroblast ecm.
as a control, cells were cultured in the presence of their own ecm.
significant levels of cell surface syndecan- <dig> were detected by flow cytometry when cells were grown on fibroblast ecm but not on their own ecm .
an increase in the expression of syndecan- <dig> as observed .
we next investigated which specific component of the ecm was responsible for stimulating the increase in syndecan- <dig> expression.
initially, fibroblasts were immunostained with anti-fibronectin, anti-laminin or anti-collagen to confirm their presence in the ecm .
the expression of fibronectin was also confirmed through western blotting, however collagen and laminin were not detected on ecm produced by fibroblasts through this method .
flow cytometry was then performed on hct- <dig> cells grown in culture dishes coated with fibronectin, laminin or collagen type i for three days .
laminin and collagen type i did not increase syndecan- <dig> expression on hct- <dig> cells over control conditions.
however, fibronectin stimulated an increase in syndecan- <dig> expression .
to further investigate this fibronectin-induced increase in syndecan- <dig> expression, hct- <dig> cells were cultured in the presence of fibronectin for different lengths of time .
surprisingly, short incubation  with fibronectin led to a remarkable increase in syndecan- <dig> expression .
overall, our results show that stromal ecm induces the expression and synthesis of syndecan- <dig> in hct- <dig> colorectal cancer cells and that the fibronectin component of ecm is mainly responsible for this effect.
taking into account the influence of fibronectin in the expression of syndecan- <dig>  we analyzed the co-localization of these molecules through immunocytochemistry.
caco- <dig> cells presented no staining for syndecan- <dig>  even in the presence of stromal fibroblasts ecm , and there was a slight increase in the staining of fibronectin, probably due to protein produced by the fibroblasts .
on the other hand, a strong staining for syndecan- <dig> was observed at the hct- <dig> cells, especially in the presence of stromal fibroblasts ecm , and co-localized with fibronectin, which confirms our previous results .
syndecan- <dig> participates in the cell adhesion, migration and organization of actin filaments in hct- <dig> cells
interactions between ecm molecules and cell surface receptors, such as heparan sulfate proteoglycans and integrins, can modulate the organization of the cytoskeleton.
therefore, we examined whether stromal fibroblast ecm regulated the organization of actin filaments in hct- <dig> cells.
cells that were cultured on fibroblast ecm showed a higher organization of cortical actin filaments, indicating reorganization of the cytoskeleton .
we next investigated whether syndecan- <dig> played a role in cell adhesion, migration and actin filament formation.
hct- <dig> cells, previously grown on top of fibroblasts ecm, were pre-incubated with an anti-syndecan- <dig> blocking antibody and then plated on stromal fibroblast ecm for different lengths of time.
cells that were pre-treated with anti-syndecan- <dig> remained isolated and did not aggregate like normal cells in this cell line.
in control cells that were pre-incubated with control igg or no antibodies, a higher organization of actin filaments was observed over time, as well as the formation of plasma membrane extensions that are indicative of stress fibers.
cells that were pre-treated with anti-syndecan- <dig> blocking antibody did not form stress fibers and remained round with cortical actin .
moreover, they were less adherent to the ecm .
previous studies have implicated syndecan- <dig> in hct- <dig> cell motility  <cit> .
to investigate this hypothesis, transmigration assays were performed.
hct- <dig> cells were pre-incubated with anti-syndecan- <dig>  human igg or no antibodies, seeded on transwell membranes coated with stromal fibroblast ecm and allowed to migrate for  <dig> h. migratory cells on the bottom chamber of the transwell were counted with an inverted microscope.
cells in which syndecan- <dig> was blocked did not migrate through the transwell membrane .
to further investigate the mechanisms involved in the increment of cell adhesion, we analyzed the expression of integrins through flow cytometry.
or results showed a three fold increase in the expression of integrins alpha- <dig> and beta- <dig> , in addition to an increase in the expression of phospho-fak.
the increase in phosphorylation of fak and src has been correlated to metastatic potential of different cancers.
src family kinase and fak activity are required for cell adhesion, spread and migration.
therefore, the signaling pathways were analyzed primarily through western blotting of cell extracts.
quantitative densitometry enabled the quantification of the levels of srcy <dig>  faky <dig> as well as of their phosphorylated form, thereby, determining the level of activation of the signaling pathways.
hct- <dig> cells that were cultured on fibroblast ecm showed an increase in phosphorylation of fak and src .
discussion
the cross-talk between cells and the ecm involves interactions between the individual components of the ecm and their respective receptors on the cell surface.
cell contact with ecm components initiates multiple signals that affect both cell behavior and gene expression  <cit> .
in particular, the behavior of tumor cells is highly influenced by cell adhesion to the ecm.
in our previous work, we demonstrated that tumor cells and stromal cells communicate through an amalgam of secreted soluble factors  <cit> .
fibroblast and prostate tumor cell cross-talk lead to fibroblast differentiation, tgf-β, and extracellular matrix down-regulation.
in this study, we found that stromal fibroblast ecm significantly increased the cell adhesion, migration and organization of actin filaments in colorectal cancer cells.
our results suggest that stromal ecm influences the amount of gags on tumor cells, leading to an increase in hs produced by hct- <dig> cancer cells.
hs proteoglycans, which are composed of a core protein and hs chains, are prevalent on the cell surface and basement membrane and have been shown to regulate various cell behaviors  <cit> .
syndecans are a family of transmembrane hs proteoglycans that bind ecm molecules and/or soluble ligands and transduce signals that influence cell adhesion, motility, proliferation, differentiation and morphogenesis  <cit> .
the hs proteoglycan-ecm interaction is differentially regulated in cancerous versus normal cells because cancer cells are less adhesive and more migratory compared to normal cells  <cit> .
this study showed that syndecan- <dig> mrna expression in a highly metastatic colorectal cancer cell line, hct- <dig>  is enhanced when the cancer cells were cultured in the presence of ecm that was produced by fibroblasts.
the augment of syndecan- <dig> was also visualized by immunoprecipitation with anti-syndecan- <dig> antibody.
in contrast, the expression of syndecans- <dig> - <dig> and - <dig> decreased.
increased syndecan- <dig> expression has been reported in several colorectal cancer cell lines with a concomitant decrease in syndecans- <dig> and - <dig>  <cit> .
interestingly, caco- <dig> cells show low syndecan- <dig> expression, and inhibition of syndecan- <dig> function in this cell line did not affect any of their adhesion, proliferation, invasion and migration  <cit> .
although derived from a colon  carcinoma, when cultured under specific conditions the cells become differentiated and polarized such that their phenotype, morphologically and functionally, resembles the enterocytes lining the small intestine  <cit> .
syndecan- <dig> has been associated with tumor suppressor function, and its expression is downregulated in a variety of cancer tissues.
similarly, syndecan- <dig>  which is mainly involved in cytoskeletal and membrane reorganization during stress fiber formation and focal adhesion in the later stages of fibroblast spreading, inhibits cell migration and tumor activity  <cit> .
consistent with this, the mrna expression levels of syndecan- <dig> and - <dig> are significantly reduced in several cancer cells, including colon carcinoma cells.
on the other hand, syndecan- <dig> is involved in the regulation of cell adhesion in several cell lines, including epithelial cells, and several reports indicate that syndecan- <dig> is normally highly expressed in cells under migratory conditions  <cit> .
these reports also indicate that syndecan- <dig> may function as a cell surface receptor in highly migratory tumor cells.
in several colorectal cancer cell lines, syndecan- <dig> is highly expressed compared to normal cell lines.
this increase appears to be critical for cancer cell behavior because it regulates adhesion and proliferation and, therefore, tumorigenic activity.
this implies that during malignant transformation, there is a shift from the expression of anti-tumorigenic syndecans  to the tumorigenic syndecan- <dig>  <cit> .
hs proteoglycans are well known to interact with specific ecm proteins  <cit> .
fibronectin contains two binding sites for heparin, named hep-i and hep-ii in the n- and c-terminal domains, respectively  <cit> , and provides structural support for cell adhesion.
fibronectin also participates in the transduction of signals that promote actin dynamics, migration and cell proliferation.
our results showed that fibronectin is mainly responsible for the increased expression and synthesis of syndecan- <dig> in hct- <dig> colorectal cancer cells that was induced by stromal fibroblast ecm.
we also observed co-localization of syndecan- <dig> and fibronectin by confocal microscopy.
therefore, in addition to binding to syndecan- <dig>  fibronectin is also able to regulate its expression.
furthermore, blocking syndecan- <dig> with a specific antibody resulted in a decrease in cell adhesion and migration, as well as the decreased organization of actin filaments during cell adhesion, highlighting an important role for syndecan- <dig> in the regulation of actin filaments.
the cytoplasmic domain of syndecan- <dig> interacts with ezrin, radixin and moesin  family proteins, which are involved in the organization of the actin cytoskeleton and the activation of focal adhesion kinase  <cit> .
kusano and colleagues demonstrated that the induction of stress fiber formation in lewis lung carcinoma-derived p <dig> cells during adhesion to fibronectin requires cooperation between integrin α5β <dig> with cell surface heparan sulfate from syndecan- <dig>  side chains with dodecasaccharide enriched in the disaccharide, idoa–glcns, are necessary for the specific interaction between syndecan- <dig> and the c-terminal heparin-binding domain of fibronectin  <cit> .
in migratory cells, forced expression of syndecan- <dig> induces filopodial extensions and results in rearrange of the actin cytoskeleton  <cit> .
our results also show that syndecan- <dig> is necessary for hct- <dig> cells to migrate through stromal ecm.
this is consistent with previous studies showing that syndecan- <dig> expression in colorectal cancer-derived ht- <dig> m <dig> epithelial cells induces a migratory phenotype  <cit>  that is associated with noticeable increases in cell proliferation, invasion and anchorage-independent growth  <cit> .
in addition to dramatic adhesion and migration changes, the contact with stromal fibroblasts ecm increased integrins alpha- <dig> and beta- <dig> expressed in hct- <dig> cancer cells.
choi and colleagues showed that overexpression of syndecan- <dig> enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells , and increased integrin alpha- <dig> expression levels.
interestingly, rie <dig> cells transfected with either syndecan- <dig> or integrin alpha- <dig> showed similar adhesion and migration patterns, and a function-blocking anti-integrin alpha- <dig> antibody abolished syndecan-2-mediated adhesion and migration  <cit> .
moreover, syndecan- <dig> is crucial for the tumorigenic activity of sarcoma cells and this function relies on fak-mediated signaling  <cit> .
fak and src are non-receptor tyrosine kinases that control a number of cellular signaling pathways, including cell motility and survival, and playing a pivotal role in integrin-linked signal transduction, such as that associated with cell migration  <cit> .
in several cell types, fak directly recruits src to the focal adhesion sites where src potentiates activation of fak through phosphorylation of additional tyrosine residues.
tyrosine phosphorylation of fak and integrin molecules creates docking sites for other proteins involved in actin cytoskeleton remodeling.
high levels of fak have been found in a variety of carcinomas, including head and neck carcinomas, ovarian carcinomas, thyroid carcinomas, and colon carcinomas  <cit> .our findings showed that the contact of colorectal cancer cells with fibroblasts ecm resulted in increased phosphorylation of fak and src.
therefore, our results further reinforce the collaboration of syndecan- <dig> and fak in colorectal tumorigenesis.
in summary, the cell surface heparan sulfate proteoglycan adhesion receptor, syndecan- <dig>  plays a critical role in regulating the tumorigenic activity of hct- <dig> colorectal cancer cells.
it can be over-expressed by the contact of cancer cells with stromal ecm, specifically fibronectin, leading to an increase in cell adhesion and migration, probably through fak-mediated signaling.
CONCLUSIONS
our results demonstrate a role for stromal ecm in regulating the expression of surface proteins in cancer cells, which are able to transduce signals that ultimately affect cell adhesion and morphology.
the use of stromal fibroblast ecm alone allowed us to study its influence on the synthesis of proteoglycans and gags by colorectal cancer cells.
although this model does not take into consideration the cross-talk between tumor and stromal cells via soluble proteins such as growth factors, contact with ecm proteins has been shown to significantly induce alterations in cancer cell behavior.
the increase of syndecan- <dig> expression in human colon cancer tissues has already been demonstrated  <cit> , and our results showed that ecm proteins can be responsible for this augmentation.
therefore, although this is an in vitro study, we attempted to model what occurs during tumorigenesis in vivo, during which tumor cells are not isolated but engage in cross-talk with cancer-associated stroma.

 the extracellular matrix is an extremely complex supramolecular structure that is locally secreted by cells and influences the structure, viability and functions of cells and tissues. epithelial-mesenchymal and epithelial-stromal interactions play important roles in both physiological and pathological settings, such as during embryonic morphogenesis, wound healing and tumorigenesis, and are accompanied by dynamic changes that generate new cell-matrix interactions. cell contact with a single ecm component initiates multiple signals that affect both cell behavior and gene expression. the tumor microenvironment plays an important role in the behavior of malignant cells. the functional association of cancer cells with surrounding tissues changes as the malignancy progresses and is accompanied by the remodeling of the ecm. a severe fibrotic reaction around tumor tissues represents a host reaction to tumor growth. fibroblasts are the main source of fibrotic tissue, which consists mainly of collagen types i and iv. in addition to collagen, several other ecm proteins are present, including fibronectin, laminin, tenascin and proteoglycans. increasing evidence has demonstrated the involvement of cell surface proteoglycans in the multi-functional network of cell-cell and cell-matrix interactions that are essential for local tumor invasion and subsequent metastasis. syndecan- is one of the four members of the syndecan family of cell surface transmembrane heparan sulfate proteoglycans. it has been implicated in the formation of specialized membrane domains and functions as a direct link between the extracellular environment and the organization of the cortical cytoplasm. in several colorectal cancer cell lines, syndecan- is highly expressed compared to normal cell lines. this increase appears to be critical for cancer cell behavior because it regulates adhesion and proliferation and, therefore, tumorigenic activity. in order to examine the relationship between tumor cells and stromal fibroblasts, we used an in vitro model to investigate the effect of ecm that is produced by stromal fibroblasts on the synthesis of glycosaminoglycans and proteoglycans by two colorectal cancer cell lines, caco- and hct- which have different metastatic potentials.  stromal fibroblast ecm influences gag synthesis by hct- colorectal cancer cells to analyze the interactions between tumor cells and stromal fibroblast ecm, two colorectal cancer cell lines with different metastatic potentials, caco- and hct- cells, were studied. the influence of stromal fibroblast-produced ecm on gag and proteoglycan synthesis by the cancer cells was investigated. tumor cells were cultured for three days on top of stromal ecm and then labeled with na2so the gags synthesized were analyzed by agarose gel electrophoresis in -m -diaminepropane acetate buffer and quantified as described in the methods. the caco- colorectal cancer cell line, which has low metastatic potential, produces both chondroitin sulfate and hs, the former being more abundant in the medium and the latter being more abundant in cell extracts. there was no difference in gag synthesis by caco- cells in the presence or absence of stromal fibroblast ecm. in the hct- colorectal cell line, which has high metastatic potential, the same gag distribution profile was observed, but there was a significant increase in medium, cell extract and matrix hs production when cells were cultured on top of stromal ecm. gene expression of surface and ecm proteoglycans is modulated by stromal fibroblast ecm proteoglycan expression was analyzed to confirm the gag synthesis results. we investigated the expression of the family of syndecans, which have been shown to be involved in cancer-stroma interactions. stromal fibroblast ecm did not significantly affect the expression of syndecans in caco- cells, with the exception of syndecan- which decreased in the presence of stromal fibroblast ecm. many colon cancer cell lines show increased syndecan- expression, and this up-regulation seems to be crucial for their tumorigenic activity. in contrast, colon cancer cell lines ht caco- and dld show low syndecan- expression, and inhibition of syndecan- function in these cell lines did not affect any of their adhesion, proliferation, invasion and migration. in opposition, stromal fibroblast ecm decreased syndecan- - and - expression, but significantly increased syndecan- expression in hct- cells. of the four syndecan family members, syndecan- has been previously reported as up-regulated in several colon carcinoma cells and, further, that this increased expression is crucial for tumorigenicity. stromal fibroblast ecm influences the synthesis of syndecan- by hct- colorectal cancer cells to confirm the increase in hct- syndecan- expression that was induced by stromal fibroblast ecm at the protein level, and to relate it with gags synthesis, the cells were labeled with sulfate and immunoprecipitated with an antibody against the extracellular domain of human syndecan- proteoglycan. the results clearly indicate that the stromal fibroblast increases the amount of syndecan- in hct- cells. furthermore, the results show the association of hs augments with this syndecan isoform. through western blotting, a 150% increase of syndecan- in hct- cells cultured in the presence of stromal fibroblasts ecm was observed therefore corroborating the immunoprecipitation. we also detected cell surface syndecan- levels by flow cytometry. hct- cells were cultured for three days in the presence or absence of stromal fibroblast ecm. as a control, cells were cultured in the presence of their own ecm. significant levels of cell surface syndecan- were detected by flow cytometry when cells were grown on fibroblast ecm but not on their own ecm. an increase in the expression of syndecan- as observed. we next investigated which specific component of the ecm was responsible for stimulating the increase in syndecan- expression. initially, fibroblasts were immunostained with anti-fibronectin, anti-laminin or anti-collagen to confirm their presence in the ecm. the expression of fibronectin was also confirmed through western blotting, however collagen and laminin were not detected on ecm produced by fibroblasts through this method. flow cytometry was then performed on hct- cells grown in culture dishes coated with fibronectin, laminin or collagen type i for three days. laminin and collagen type i did not increase syndecan- expression on hct- cells over control conditions. however, fibronectin stimulated an increase in syndecan- expression. to further investigate this fibronectin-induced increase in syndecan- expression, hct- cells were cultured in the presence of fibronectin for different lengths of time. surprisingly, short incubation with fibronectin led to a remarkable increase in syndecan- expression. overall, our results show that stromal ecm induces the expression and synthesis of syndecan- in hct- colorectal cancer cells and that the fibronectin component of ecm is mainly responsible for this effect. taking into account the influence of fibronectin in the expression of syndecan- we analyzed the co-localization of these molecules through immunocytochemistry. caco- cells presented no staining for syndecan- even in the presence of stromal fibroblasts ecm, and there was a slight increase in the staining of fibronectin, probably due to protein produced by the fibroblasts. on the other hand, a strong staining for syndecan- was observed at the hct- cells, especially in the presence of stromal fibroblasts ecm, and co-localized with fibronectin, which confirms our previous results. syndecan- participates in the cell adhesion, migration and organization of actin filaments in hct- cells interactions between ecm molecules and cell surface receptors, such as heparan sulfate proteoglycans and integrins, can modulate the organization of the cytoskeleton. therefore, we examined whether stromal fibroblast ecm regulated the organization of actin filaments in hct- cells. cells that were cultured on fibroblast ecm showed a higher organization of cortical actin filaments, indicating reorganization of the cytoskeleton. we next investigated whether syndecan- played a role in cell adhesion, migration and actin filament formation. hct- cells, previously grown on top of fibroblasts ecm, were pre-incubated with an anti-syndecan- blocking antibody and then plated on stromal fibroblast ecm for different lengths of time. cells that were pre-treated with anti-syndecan- remained isolated and did not aggregate like normal cells in this cell line. in control cells that were pre-incubated with control igg or no antibodies, a higher organization of actin filaments was observed over time, as well as the formation of plasma membrane extensions that are indicative of stress fibers. cells that were pre-treated with anti-syndecan- blocking antibody did not form stress fibers and remained round with cortical actin. moreover, they were less adherent to the ecm. previous studies have implicated syndecan- in hct- cell motility. to investigate this hypothesis, transmigration assays were performed. hct- cells were pre-incubated with anti-syndecan- human igg or no antibodies, seeded on transwell membranes coated with stromal fibroblast ecm and allowed to migrate for h. migratory cells on the bottom chamber of the transwell were counted with an inverted microscope. cells in which syndecan- was blocked did not migrate through the transwell membrane. to further investigate the mechanisms involved in the increment of cell adhesion, we analyzed the expression of integrins through flow cytometry. or results showed a three fold increase in the expression of integrins alpha- and beta-, in addition to an increase in the expression of phospho-fak. the increase in phosphorylation of fak and src has been correlated to metastatic potential of different cancers. src family kinase and fak activity are required for cell adhesion, spread and migration. therefore, the signaling pathways were analyzed primarily through western blotting of cell extracts. quantitative densitometry enabled the quantification of the levels of srcy faky as well as of their phosphorylated form, thereby, determining the level of activation of the signaling pathways. hct- cells that were cultured on fibroblast ecm showed an increase in phosphorylation of fak and src. discussion the cross-talk between cells and the ecm involves interactions between the individual components of the ecm and their respective receptors on the cell surface. cell contact with ecm components initiates multiple signals that affect both cell behavior and gene expression. in particular, the behavior of tumor cells is highly influenced by cell adhesion to the ecm. in our previous work, we demonstrated that tumor cells and stromal cells communicate through an amalgam of secreted soluble factors. fibroblast and prostate tumor cell cross-talk lead to fibroblast differentiation, tgf-β, and extracellular matrix down-regulation. in this study, we found that stromal fibroblast ecm significantly increased the cell adhesion, migration and organization of actin filaments in colorectal cancer cells. our results suggest that stromal ecm influences the amount of gags on tumor cells, leading to an increase in hs produced by hct- cancer cells. hs proteoglycans, which are composed of a core protein and hs chains, are prevalent on the cell surface and basement membrane and have been shown to regulate various cell behaviors. syndecans are a family of transmembrane hs proteoglycans that bind ecm molecules and/or soluble ligands and transduce signals that influence cell adhesion, motility, proliferation, differentiation and morphogenesis. the hs proteoglycan-ecm interaction is differentially regulated in cancerous versus normal cells because cancer cells are less adhesive and more migratory compared to normal cells. this study showed that syndecan- mrna expression in a highly metastatic colorectal cancer cell line, hct- is enhanced when the cancer cells were cultured in the presence of ecm that was produced by fibroblasts. the augment of syndecan- was also visualized by immunoprecipitation with anti-syndecan- antibody. in contrast, the expression of syndecans- - and - decreased. increased syndecan- expression has been reported in several colorectal cancer cell lines with a concomitant decrease in syndecans- and -. interestingly, caco- cells show low syndecan- expression, and inhibition of syndecan- function in this cell line did not affect any of their adhesion, proliferation, invasion and migration. although derived from a colon carcinoma, when cultured under specific conditions the cells become differentiated and polarized such that their phenotype, morphologically and functionally, resembles the enterocytes lining the small intestine. syndecan- has been associated with tumor suppressor function, and its expression is downregulated in a variety of cancer tissues. similarly, syndecan- which is mainly involved in cytoskeletal and membrane reorganization during stress fiber formation and focal adhesion in the later stages of fibroblast spreading, inhibits cell migration and tumor activity. consistent with this, the mrna expression levels of syndecan- and - are significantly reduced in several cancer cells, including colon carcinoma cells. on the other hand, syndecan- is involved in the regulation of cell adhesion in several cell lines, including epithelial cells, and several reports indicate that syndecan- is normally highly expressed in cells under migratory conditions. these reports also indicate that syndecan- may function as a cell surface receptor in highly migratory tumor cells. in several colorectal cancer cell lines, syndecan- is highly expressed compared to normal cell lines. this increase appears to be critical for cancer cell behavior because it regulates adhesion and proliferation and, therefore, tumorigenic activity. this implies that during malignant transformation, there is a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-. hs proteoglycans are well known to interact with specific ecm proteins. fibronectin contains two binding sites for heparin, named hep-i and hep-ii in the n- and c-terminal domains, respectively, and provides structural support for cell adhesion. fibronectin also participates in the transduction of signals that promote actin dynamics, migration and cell proliferation. our results showed that fibronectin is mainly responsible for the increased expression and synthesis of syndecan- in hct- colorectal cancer cells that was induced by stromal fibroblast ecm. we also observed co-localization of syndecan- and fibronectin by confocal microscopy. therefore, in addition to binding to syndecan- fibronectin is also able to regulate its expression. furthermore, blocking syndecan- with a specific antibody resulted in a decrease in cell adhesion and migration, as well as the decreased organization of actin filaments during cell adhesion, highlighting an important role for syndecan- in the regulation of actin filaments. the cytoplasmic domain of syndecan- interacts with ezrin, radixin and moesin family proteins, which are involved in the organization of the actin cytoskeleton and the activation of focal adhesion kinase. kusano and colleagues demonstrated that the induction of stress fiber formation in lewis lung carcinoma-derived p cells during adhesion to fibronectin requires cooperation between integrin α5β with cell surface heparan sulfate from syndecan- side chains with dodecasaccharide enriched in the disaccharide, idoa–glcns, are necessary for the specific interaction between syndecan- and the c-terminal heparin-binding domain of fibronectin. in migratory cells, forced expression of syndecan- induces filopodial extensions and results in rearrange of the actin cytoskeleton. our results also show that syndecan- is necessary for hct- cells to migrate through stromal ecm. this is consistent with previous studies showing that syndecan- expression in colorectal cancer-derived ht- m epithelial cells induces a migratory phenotype that is associated with noticeable increases in cell proliferation, invasion and anchorage-independent growth. in addition to dramatic adhesion and migration changes, the contact with stromal fibroblasts ecm increased integrins alpha- and beta- expressed in hct- cancer cells. choi and colleagues showed that overexpression of syndecan- enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells, and increased integrin alpha- expression levels. interestingly, rie cells transfected with either syndecan- or integrin alpha- showed similar adhesion and migration patterns, and a function-blocking anti-integrin alpha- antibody abolished syndecan-2-mediated adhesion and migration. moreover, syndecan- is crucial for the tumorigenic activity of sarcoma cells and this function relies on fak-mediated signaling. fak and src are non-receptor tyrosine kinases that control a number of cellular signaling pathways, including cell motility and survival, and playing a pivotal role in integrin-linked signal transduction, such as that associated with cell migration. in several cell types, fak directly recruits src to the focal adhesion sites where src potentiates activation of fak through phosphorylation of additional tyrosine residues. tyrosine phosphorylation of fak and integrin molecules creates docking sites for other proteins involved in actin cytoskeleton remodeling. high levels of fak have been found in a variety of carcinomas, including head and neck carcinomas, ovarian carcinomas, thyroid carcinomas, and colon carcinomas.our findings showed that the contact of colorectal cancer cells with fibroblasts ecm resulted in increased phosphorylation of fak and src. therefore, our results further reinforce the collaboration of syndecan- and fak in colorectal tumorigenesis. in summary, the cell surface heparan sulfate proteoglycan adhesion receptor, syndecan- plays a critical role in regulating the tumorigenic activity of hct- colorectal cancer cells. it can be over-expressed by the contact of cancer cells with stromal ecm, specifically fibronectin, leading to an increase in cell adhesion and migration, probably through fak-mediated signaling.  our results demonstrate a role for stromal ecm in regulating the expression of surface proteins in cancer cells, which are able to transduce signals that ultimately affect cell adhesion and morphology. the use of stromal fibroblast ecm alone allowed us to study its influence on the synthesis of proteoglycans and gags by colorectal cancer cells. although this model does not take into consideration the cross-talk between tumor and stromal cells via soluble proteins such as growth factors, contact with ecm proteins has been shown to significantly induce alterations in cancer cell behavior. the increase of syndecan- expression in human colon cancer tissues has already been demonstrated, and our results showed that ecm proteins can be responsible for this augmentation. therefore, although this is an in vitro study, we attempted to model what occurs during tumorigenesis in vivo, during which tumor cells are not isolated but engage in cross-talk with cancer-associated stroma. 
recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ecm components and the secretion of growth factors. syndecan- has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. we have examined the effect of stromal fibroblast-produced ecm on the production of proteoglycans by colorectal cancer cell lines. our results showed that in a highly metastatic colorectal cancer cell line, hct- syndecan- expression is enhanced by fibroblast ecm, while the expression of other syndecans decreased. overall, these results show that interactions between cancer cells and stromal ecm proteins induce significant changes in the behavior of cancer cells. furthermore, blocking syndecan- with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. in particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan- may have implications in the migratory behavior of highly metastatic tumor cells. of the various components of the stromal ecm, fibronectin was the most important in stimulating the increase in syndecan- expression. syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. additionally, we demonstrated an increase in the expression of integrins alpha- and beta- in addition to an increase in the expression of phospho-fak in the presence of fibroblast ecm.
 the extracellular matrix influences the structure, viability and functions of cells and tissues. recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ecm components and the secretion of growth factors. syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. syndecan- has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. we have examined the effect of stromal fibroblast-produced ecm on the production of proteoglycans by colorectal cancer cell lines.  our results showed that in a highly metastatic colorectal cancer cell line, hct- syndecan- expression is enhanced by fibroblast ecm, while the expression of other syndecans decreased. of the various components of the stromal ecm, fibronectin was the most important in stimulating the increase in syndecan- expression. the co-localization of syndecan- and fibronectin suggests that these two molecules are involved in the adhesion of hct- cells to the ecm. additionally, we demonstrated an increase in the expression of integrins alpha- and beta- in addition to an increase in the expression of phospho-fak in the presence of fibroblast ecm. furthermore, blocking syndecan- with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments.  overall, these results show that interactions between cancer cells and stromal ecm proteins induce significant changes in the behavior of cancer cells. in particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan- may have implications in the migratory behavior of highly metastatic tumor cells. colorectal cancercancer-stroma interactionproteoglycanssyndecan-2fibronectin 