Use of a Pfannenstiel incision in minimally invasive colorectal cancer surgery is associated with a lower risk of wound complications.

BACKGROUND: The Pfannenstiel incision, widely used in gynecological surgery, has been reported to be associated with lower rates of wound complications than midline incisions in open surgery. However, its effect on wound complications in minimally invasive surgery (MIS) is not well understood. We hypothesize that use of a Pfannenstiel incision in MIS colorectal cancer resections would be associated with fewer short-term wound complication rates. METHODS: A retrospective cohort study was performed on 171 patients who had undergone MIS colorectal cancer surgery requiring a specimen extraction/hand-access site, divided into a Pfannenstiel and a midline group depending on the type of incision used. Wound complications compared included disruption, infection, dehiscence, evisceration, and fistula formation. The Mann-Whitney U and Fisher's exact tests were used to analyze differences in risk factors between the groups. Logistic regression was performed to determine factors associated with prevention of wound complications. RESULTS: Patients in the Pfannenstiel group had significantly lower rates of wound disruption (0 vs. 13%, p = 0.02), superficial surgical site infection (7 vs. 22%, p = 0.03), and overall wound complications (13 vs. 30%, p = 0.04). Using multivariate logistic regression, Pfannenstiel incisions and colon rather than rectal resections were significant predictors of prevention of wound complications. CONCLUSIONS: The use of a Pfannenstiel incision in MIS colorectal cancer resections is associated with a decreased risk of short-term wound complications.

Early predictors of anastomotic leaks after colectomy.

BACKGROUND: An anastomotic leak after colorectal surgery is associated with significant morbidity and decreased survival. Our aim was to identify the early predictors of anastomotic leaks. METHODS: The records of patients undergoing restorative resection for colorectal disease from January 2000 to November 2005 were reviewed. Demographics, clinical events, and laboratory parameters were recorded. RESULTS: A total of 311 patients were included. An anastomotic leak was identified in 25 patients (8%). A leak was suspected and diagnosis confirmed at a mean of 10+/-1 days postoperatively. More respiratory and neurological events occurred in patients with an anastomotic leak (p<0.001). These events occurred early in the postoperative course and were usually the first signs and symptoms of a leak. More patients with a leak had absence of bowel activity by postoperative day 6 compared to patients without a leak (p<0.0001). Elevations of the white blood cell count or temperature were a late finding. CONCLUSION: The earliest clinical predictors of an anastomotic leak are pulmonary and/or neurological. Awareness of these findings might help in early diagnosis and treatment of an anastomotic leak.

Thrombospondin-1 up-regulates tumor cell invasion through the urokinase plasminogen activator receptor in head and neck cancer cells.

BACKGROUND: We have previously demonstrated that thrombospondin-1 (TSP-1) is expressed in squamous cell carcinomas of the head and neck. We have also shown that TSP-1 promotes tumor cell invasion through up-regulation of the urokinase plasminogen activator receptor (uPAR), in adenocarcinoma models. We now determined the role of TSP-1 in the regulation of uPAR expression and tumor cell invasion in squamous cell carcinoma of the head and neck cells. MATERIALS AND METHODS: KB squamous cell carcinoma of the head and neck cells were used. The effect of TSP-1 on uPAR and its ligand, urokinase plasminogen activator (uPA), expression were determined by ELISA. The effect of TSP-1 on KB tumor cell invasion was determined in a modified Boyden chamber collagen invasion assay. To determine the role of uPAR on TSP-1-mediated KB tumor cell invasion, we used the three following different strategies: (a). blocking uPAR or its ligand, uPA, with neutralizing antibodies; (b). enzymatic cleavage of uPAR with glycosylphosphatidylinositol (GPI)-specific phospholipase C; and (c). inhibition of plasminogen binding by using epsilon-aminocaproic acid. RESULTS: TSP-I up-regulated uPAR and uPA expression 3- and 4-fold, respectively. TSP-1 up-regulated KB tumor cell invasion 5-fold. Inhibition of uPAR blocked the TSP-1-mediated up-regulation of KB tumor cell invasion. CONCLUSIONS: Our data support a central role for TSP-1 in the regulation of uPAR and tumor cell invasion in squamous cell carcinomas of the head and neck cells. Furthermore, uPAR seems to play a crucial role in TSP-1-mediated squamous cell carcinoma of the head and neck tumor cell invasion.

Co-localization of macrophage inflammatory protein-3alpha (Mip-3alpha) and its receptor, CCR6, promotes pancreatic cancer cell invasion.

PURPOSE: Macrophage inflammatory protein-3alpha (Mip-3alpha) is par t of a family of chemotactic cytokines involved in recruiting inflammatory cells throughout the body. CCR6 is a G-protein-linked, seven-transmembrane receptor that is highly specific for Mip-3alpha. The role of Mip-3alpha has been well defined in several inflammatory conditions, but its role has not been well defined in neoplastic processes. Mip-3alpha has been shown to promote pancreatic cancer cell migration, but no studies have demonstrated the effect of Mip-3alpha on pancreatic cancer cell invasion. We hypothesize that Mip-3alpha and its CCR6 receptor promote pancreatic cancer cell invasion. MATERIALS AND METHODS: Immunohistochemical staining was per formed for Mip-3alpha and CCR6 in pancreatic cancer tissue and the human pancreatic cancer cell line PANC-1. RNA was isolated from PANC-1 cancer cells, and the presence of Mip-3alpha messenger RNA in PANC-1 cancer cells was determined by reverse transcriptase polymerase chain reaction. PANC-1 cancer cell invasion of type IV collagen was evaluated in the presence of Mip-3alpha and anti-CCR6 antibody with the use of a modified Boyden chamber invasion assay. RESULTS: Co-localization of Mip-3alpha and its CCR6 receptor in pancreatic cancer was confirmed using immunohistochemical staining for Mip-3alpha and its CCR6 receptor and reverse transcriptase polymerase chain reaction for Mip-3alpha. Immunohistochemical staining of pancreatic cancer tissue and the PANC-1 cancer cell line showed positive staining for Mip-3alpha and its CCR6 receptor within the cancer cells. Staining was also positive for Mip-3alpha within stromal cells adjacent to the cancer cells in pancreatic cancer tissue. Reverse transcriptase polymerase chain reaction demonstrated the presence of Mip-3alpha messenger RNA within PANC-1 cancer cells. Invasion studies showed that increasing concentrations of Mip-3alpha promoted a dose-dependent increase in pancreatic cancer cell invasion of type IV collagen. The addition of 100 ng/mL of Mip-3alpha promoted a threefold increase in pancreatic cancer cell invasion over that of the control group. Anti-CCR6 antibody inhibited Mip-3alpha-stimulated PANC-1 cancer cell invasion of type IV collagen by 63%. DISCUSSION: Co-localization of Mip-3alpha and its CCR6 receptor promotes pancreatic cancer cell invasion of type IV collagen. This finding continues to highlight the importance that inflammation plays in the progression of pancreatic cancer. As the relationship between the inflammatory and neoplastic processes involved with pancreatic cancer becomes better defined, therapies targeting the inflammatory process may help prevent pancreatic cancer invasion and metastasis.

Anaphylactic reactions to isosulfan blue dye during sentinel lymph node biopsy for breast cancer.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is an alternative to axillary dissection for many breast cancer patients. Cases of anaphylactic reaction to the isosulfan blue dye used during SLNB have recently been reported. No study on the incidence of serious anaphylactic reactions during SLNB for breast cancer has been reported. METHODS: We reviewed 639 consecutive SLNBs for breast cancer performed at our institution. Sentinel lymph node biopsy was performed using both isosulfan blue dye and technetium-99m sulfur colloid. Cases of anaphylaxis were reviewed in detail. RESULTS: Overall, 1.1% of patients had severe anaphylactic reactions to isosulfan blue requiring vigorous resuscitation. No deaths or permanent disability occurred. In patients with anaphylaxis, hospital stay was prolonged by a mean of 1.6 days. In 1 patient, the anaphylactic reaction required termination of the operation. CONCLUSIONS: Prompt recognition and aggressive treatment of anaphylactic reactions to isosulfan blue are critical to prevent an adverse outcome. Lymphatic mapping with blue dye should be performed in a setting where personnel are trained to recognize and treat anaphylaxis.

Tumour cell thrombospondin-1 regulates tumour cell adhesion and invasion through the urokinase plasminogen activator receptor.

We have previously shown that platelet-produced thrombospondin-1 up-regulates the urokinase plasminogen activator and its receptor and promotes tumour cell invasion. Although tumour cells produce thrombospondin-1 in vivo, they produce only minimal amounts of thrombospondin-1 in vitro. To determine the effect of tumour cell-produced thrombospondin-1 in the regulation of the plasminogen/plasmin system and tumour cell invasion, we studied THBS-1-transfected MDA-MB-435 breast cancer cells that overexpress thrombospondin-1. The role of urokinase plasminogen receptor in thrombospondin-1-mediated adhesion and invasion was studied by antisense inhibition, enzymatic cleavage and antibody neutralization. Tumour cell adhesion to collagen and laminin was evaluated. Tumour cell invasion was studied in a modified Boyden chamber collagen invasion assay. Tumour cell thrombospondin-1 induced a 2-7 fold increase in urokinase plasminogen activator receptor and cell-associated urokinase plasminogen activator expression and a 50-65% increase in cell-associated urokinase plasminogen activator and plasmin activities. Furthermore, tumour cell thrombospondin-1 promoted tumour cell invasion and decreased tumour cell adhesion through up-regulation of urokinase plasminogen activator receptor-controlled urokinase plasminogen activator and plasmin activities. We conclude that tumour cell-produced thrombospondin-1 may play a critical role in the regulation of tumour cell adhesion and tumour cell invasion.

Thrombospondin-1 and transforming growth factor beta-1 upregulate plasminogen activator inhibitor type 1 in pancreatic cancer.

Controlled degradation of the extracellular matrix by proteases is crucial in tumor cell invasion. We have shown that thrombospondin-1 (TSP-1), through activation of transforming growth factor beta-1 (TGF-beta1), regulates the plasminogen/plasmin protease system in breast cancer. To determine whether this occurred in other epithelial neoplasms, we studied the role of TSP-1 and TGF-beta1 in the regulation of the plasminogen/plasmin system in pancreatic cancer. ASPC-1 and COLO-357 pancreatic cancer cells were treated with TSP-1 or TGF-beta1 at varying concentrations. The TSP-1 and TGF-beta1-treated cells were also treated with either anti-TSP-1, anti-TSP-1 receptor, or anti-TGF-beta1 antibodies. Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) expression was determined by enzyme-linked immunosorbent assay. TSP-1 and TGF-beta1 promoted a dose-dependent upregulation of ASPC-1 and COLO-357 PAI-1 expression. The TSP-1 effect could be blocked with anti-TSP-1 or anti-TGF-beta1 antibodies. The TGF-beta1 effect could be blocked only with anti-TGF-beta1 antibody. Anti-TSP-1 receptor antibody blocked the TSP-1 effect on PAI-1 expression but had no effect on TGF-beta1-mediated PAI-1 expression. Neither TSP-1 nor TGF-beta1 had an effect on uPA production. We conclude that TSP-1, in a receptor-mediated process that involves the activation of TGF-beta1, upregulates PAI-1 expression in pancreatic cancer without an effect on uPA production.

Cyclooxygenase-2 alters transforming growth factor-beta 1 response during intestinal tumorigenesis.

BACKGROUND: Recent investigation suggests that cyclooxygenase-2 plays an important role in colorectal carcinogenesis. Transforming growth factor-beta1 (TGF-beta 1) is one of the most potent stimulators of cyclooxygenase-2 expression. A key step in intestinal tumorigenesis involves alteration of the normal cellular response to TGF-beta 1. We have hypothesized that overexpression of cyclooxygenase-2 alters intestinal epithelial response to TGF-beta 1. METHODS: RIE-1 cells were stably transfected with rat cyclooxygenase-2 complementary DNA in either the sense (RIE-S) or antisense (RIE-AS) orientation. Tumor cell invasion was assessed with a modified Boyden collagen type I invasion assay in the presence of TGF-beta 1, antibody to urokinase plasminogen activator (uPA), or the selective cyclooxygenase-2 inhibitor SC-58125. Expression of uPA, uPA receptor, and plasminogen activator inhibitor-1 were determined by Western blot and enzyme-linked immunosorbent assay. RESULTS: RIE-1 and RIE-AS did not invade although RIE-S cells were minimally invasive at baseline. TGF-beta 1 had no effect on RIE-1 or RIE-AS invasion; however, TGF-beta 1 significantly upregulated RIE-S cell invasion. All 3 RIE cell lines produce minimal uPA under basal conditions. TGF-beta 1 upregulated uPA production only in the RIE-S cells. Both antibody to uPA and SC-58125 reversed TGF-beta-mediated RIE-S cell invasion. SC-58125 inhibited TGF-beta-mediated RIE-S uPA production. CONCLUSIONS: These results demonstrate that overexpression of cyclooxygenase-2 alters intestinal epithelial response to TGF-beta 1, which may be a mechanism by which cyclooxygenase-2 promotes colon carcinogenesis.

Role of urokinase plasminogen activator receptor in thrombospondin 1-mediated tumor cell invasion.

We previously showed that thrombospondin 1 (TSP-1) upregulates the plasminogen/plasmin system and promotes breast tumor cell invasion. Preliminary data from our laboratory using neutralizing antibodies suggested that the upregulation in breast tumor cell invasion seen in response to TSP-1 involved the urokinase plasminogen activator receptor (uPAR). To confirm these findings in MDA-MB-231 breast cancer cells, we developed three other strategies to study the role of uPAR in tumor cell adhesion and TSP-1-mediated tumor cell invasion: (a) enzymatic cleavage of uPAR with glycosylphosphatidylinositol-specific phospholipase C; (b) inhibition at the mRNA level with a uPAR antisense construct (cells named LKAS-MDA); (c) inhibition of plasminogen binding with the lysine analogue epsilon-aminocaproic acid. Adhesion to laminin and type I and type IV collagen with and without the addition of epsilon-aminocaproic acid was studied. Tumor cell invasion was studied in a modified Boyden chamber collagen invasion assay. Antisense uPAR inhibition decreased uPAR expression by 48-66% and cell-associated urokinase plasminogen activator (uPA) by 30-68%. Additionally, antisense uPAR inhibition induced a 68-70% reduction in uPA and plasmin activities. Antisense uPAR transfection increased tumor cell adhesion by 46-53%. A similar effect was observed in epsilon-aminocaproic acid-treated MDA-MB-231 cells. TSP-1-mediated tumor cell invasion was almost completely inhibited by either antisense uPAR inhibition or treatment with phospholipase C or epsilon-aminocaproic acid. We conclude that uPAR plays a crucial role in the regulation of tumor cell adhesion and TSP-1-mediated tumor cell invasion.

Transformation of intestinal epithelial cells by chronic TGF-beta1 treatment results in downregulation of the type II TGF-beta receptor and induction of cyclooxygenase-2.

The precise role of TGF-beta in colorectal carcinogenesis is not clear. The purpose of this study was to determine the phenotypic alterations caused by chronic exposure to TGF-beta in non-transformed intestinal epithelial (RIE-1) cells. Growth of RIE-1 cells was inhibited by >75% following TGF-beta1 treatment for 7 days, after which the cells resumed a normal growth despite the presence of TGF-beta1. These 'TGF-beta-resistant' cells (RIE-Tr) were continuously exposed to TGF-beta for >50 days. Unlike the parental RIE cells, RIE-Tr cells lost contact inhibition, formed foci in culture, grew in soft agarose. RIE-Tr cells demonstrated TGF-beta-dependent invasive potential in an in vitro assay and were resistant to Matrigel and Na-butyrate-induced apoptosis. The RIE-Tr cells were also tumorigenic in nude mice. The transformed phenotype of RIE-Tr cells was associated with a 95% decrease in the level of the type II TGF-beta receptor (TbetaRII) protein, a 40-fold increase in cyclooxygenase-2 (COX-2) protein, and 5.9-fold increase in the production of prostacyclin. Most RIE-Tr subclones that expressed low levels of TbetaRII and high levels of COX-2 were tumorigenic. Those subclones that express abundant TbetaRII and low levels of COX-2 were not tumorigenic in nude mice. A selective COX-2 inhibitor inhibited RIE-Tr cell growth in culture and tumor growth in nude mice. The reduced expression of TbetaRII, increased expression of COX-2, and the ability to form colonies in Matrigel were all reversible upon withdrawal of exogenous TGF-beta1 for the RIE-Tr cells.

The effect of thrombospondin-1 and TGF-beta 1 on pancreatic cancer cell invasion.

We have previously shown that thrombospondin-1 (TSP-1) and TGF-beta 1 upregulate the urokinase plasminogen activator (uPA) and its receptor (uPAR) and promote tumor cell invasion in breast cancer. To date, the effect of TSP-1 and TGF-beta 1 on the plasminogen/plasmin system in gastrointestinal epithelial malignancies has not been investigated. In this study, we determined the effect of TSP-1 and TGF-beta 1 on uPA and uPAR expression and on tumor cell invasion in pancreatic cancer. ASPC1 human pancreatic adenocarcinoma cells were incubated for 48 h on cell-conditioned media (CCM) either alone (Control) or with the addition of either TSP-1 (40 micrograms/ml) or TGF-beta 1 (5 ng/ml). uPA and uPAR expression were determined by ELISA. ASPC1 cell invasion was determined in a modified Boyden chamber type I collagen invasion assay. The upper chamber was treated with CCM either alone (Control) or with the addition of anti-uPA (10 micrograms/ml) or anti-uPAR (10 micrograms/ml). The lower chamber was treated with CCM either alone (Control) or with the addition of either TSP-1 (40 micrograms/ml) or TGF-beta 1 (5 ng/ml). TSP-1 and TGF-beta 1 induced a twofold increase on uPAR expression but only a slight increase on total uPA. Tumor cell invasion was upregulated 3.5 to 4.5-fold by TSP-1 and TGF-beta 1, respectively. Anti-uPA and anti-uPAR antibodies completely blocked the TSP-1 and TGF-beta 1-mediated pancreatic tumor cell invasion. We conclude that TSP-1 and TGF-beta 1 mediate pancreatic tumor cell invasion through upregulation of the plasminogen/plasmin system.

Angiostatin generation by human pancreatic cancer.

BACKGROUND: Angiostatin, a proteolytic fragment of plasminogen, is a potent inhibitor of angiogenesis. In vitro, angiostatin can be generated by pancreatic elastase proteolysis of plasminogen; however, in vivo, the enzymes responsible for angiostatin production are not known. A recent study demonstrates the involvement of a serine protease in angiostatin generation. In this study we sought to determine if the human pancreatic carcinoma cell line ASPC1 produced enzymatic activity capable of converting plasminogen to angiostatin and to determine if urokinase plasminogen activator (uPA) is involved in this system. Methods. ASPC1 cells were grown to near confluence in 20% FBS-RPMI. Media were changed to serum free and cells cultured for an additional 24 h. The serum free conditioned media (SFCM) was obtained. Angiostatin generation was determined by incubating 20 microg of human plasminogen with 100 microl of SFCM for 0, 3, 8, 12, 24, and 48 h. Plasminogen cleavage was assessed in the presence of the following protease inhibitors: pefabloc, aprotinin, phosphoramidon, leupeptin, and EDTA. The effect of uPA on angiostatin generation was determined by incubating plasminogen with antibody to uPA. Angiostatin generation was determined by Western blot. RESULTS: Incubation of plasminogen with SFCM resulted in the generation of immunoreactive bands at 48 kDa corresponding to human angiostatin. Angiostatin generation by ASPC1 SFCM was time dependent; there was a significant decrease in the plasminogen substrate beginning at 3 h with complete conversion to angiostatin by 48 h. Enzymatic activity leading to angiostatin production was found to be due to a serine protease. Antibody to uPA effectively blocked angiostatin production by ASPC1 SFCM in a dose-dependent manner. CONCLUSION: Human pancreatic cancer cells express enzymatic activity which leads to the generation of angiostatin. Conversion of plasminogen to angiostatin is due to a serine protease. This serine protease is most likely uPA.

Transforming growth factor-beta 1 inhibits generation of angiostatin by human pancreatic cancer cells.

BACKGROUND: Angiostatin, a proteolytic fragment of plasminogen, is a potent inhibitor of angiogenesis. We have previously shown that the human pancreatic cancer cell line ASPC-1 produces enzymatic activity capable of generating angiostatin. In this study we sought to determine whether angiostatin production by ASPC-1 cells was regulated by the growth factor transforming growth factor-beta 1 (TGF-beta 1), a key mediator of tumor angiogenesis. METHODS: ASPC-1 cells were grown to 70% to 80% confluence in 20% fetal calf serum-RPMI. Medium was changed to serum free. TGF-beta 1 was added at concentrations of 0, 1, 5, and 10 ng/mL with or without plasminogen activator inhibitor type-1 (PAI-1) at concentrations of 0, 5, 10, 50, and 100 micrograms/mL. Cells were then cultured for an additional 24 hours. The serum-free conditioned medium was obtained. Angiostatin generation was determined by incubating 20 micrograms of plasminogen with 100 microL of serum-free conditioned medium for 0, 1, 2, 3, 6, 12, and 24 hours. Samples were run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred. The membrane was probed with a monoclonal antibody to the kringle 1-3 fragment of plasminogen and developed using enhanced chemiluminescence. RESULTS: TGF-beta 1 and PAI-1 inhibited the conversion of plasminogen into angiostatin in a time- and dose-dependent manner. Antibody to PAI-1 completely blocks TGF-beta 1 mediated angiostatin inhibition. CONCLUSIONS: TGF-beta 1 inhibits the generation of the antiangiogenic molecule angiostatin by human pancreatic cancer cells in a time- and dose-dependent manner. This effect is mediated through modulation of the plasminogen/plasmin system.

Thrombospondin-1 and its CSVTCG-specific receptor in wound healing and cancer.

Growth factors play a crucial role in the regulation of cellular proliferation and matrix degradation in wound healing and cancer. We have shown that thrombospondin 1 (TSP-1) and its cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG)-specific receptor play a key role in cell invasion and matrix degradation in different carcinomas. The present study was done to determine whether TSP-1 and its receptor show a similar pattern of expression in wound healing and cancer. Expression and localization of TSP-1 and its receptor were determined in fetal wounds, adult burn wounds, and different human malignancies by immunohistochemical staining and computerized image analysis. In healing wounds, TSP-1 was expressed in the stroma early in the process, followed by a steep decline. The TSP-1 receptor localized to neovessels and highly proliferating cells (i.e., fibroblasts, basal cells), its levels remaining relatively constant. Cancer cells and tumor-associated microvessels expressed the TSP-1 receptor, whereas TSP-1 localized predominantly to the tumor-associated stroma. These data suggest a critical role for TSP-1 and its CSVTCG-specific receptor in wound healing and cancer.

Thrombospondin-1 and transforming growth factor-beta l promote breast tumor cell invasion through up-regulation of the plasminogen/plasmin system.

BACKGROUND: Pericellular proteolysis is crucial in tumor cell invasion. The plasminogen/plasmin system is one of the main protease systems involved in cancer progression. Thrombospondin-1 (TSP-1), through activation of transforming growth factor-beta 1 (TGF-beta 1), up-regulates the main plasminogen activator, the urokinase-type plasminogen activator (uPA). The objectives of this study were to determine the role of TSP-1 and TGF-beta 1 in the localization of the plasminogen/plasmin system to the tumor cell surface by the uPA receptor (uPAR) and to determine its effect in breast tumor cell invasion. METHODS: The effect of TSP-1 and TGF-beta 1 in uPAR expression was determined in MDA-MB-231 human breast cancer cells by enzyme-linked immunosorbent assay and Western blot analysis. Their effect and the role of the plasminogen/plasmin system in breast tumor cell invasion were studied with a Boyden Chamber assay. RESULTS: uPAR expression was up-regulated more than twofold by both TSP-1 and TGF-beta 1. The effect of TSP-1 involved its receptor and the activation of TGF-beta 1 by TSP-1. Breast tumor cell invasion was up-regulated sevenfold to eightfold by both TSP-1 and TGF-beta 1 compared with the control group. Antibodies against uPA or uPAR neutralized the TSP-1- and TGF-beta 1-promoted breast tumor cell invasion. CONCLUSIONS: TSP-1, through the activation of endogenous TGF-beta 1, up-regulates the plasminogen/plasmin system and promotes tumor cell invasion in breast cancer cells.

Modulation of cranial bone healing with a heparin-like dextran derivative.

Substituted dextran polymers have been shown to bind growth factors and protect them from enzymatic degradation. Using this information, other researchers have been able to use these substituted dextrans to enhance the healing of bone in an environment where bone would otherwise not regenerate. We used substituted dextran polymers to evaluate their ability to accelerate the healing of cranial bone in a rabbit model. We were able to document a more rapid rate of healing and demonstrate micrographic evidence to support that conclusion. Possible mechanisms are postulated.

Thrombospondin and transforming growth factor-beta 1 increase expression of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in human MDA-MB-231 breast cancer cells.

BACKGROUND: Thrombospondin is a high molecular weight adhesive glycoprotein that has been shown to function in mechanisms of tumor progression. The authors' previous studies have shown that thrombospondin promotes human lung carcinoma invasion by up-regulation of the plasminogen activator system through a mechanism involving the activation of transforming growth factor-beta 1 (TGF-beta 1). In this study, a similar thrombospondin-mediated mechanism operative in breast carcinoma cells is described. METHODS: The effect of thrombospondin and TGF-beta 1 on the capacity of a line of breast carcinoma cells to activate plasminogen was measured as well as the physiologic consequences of these activities on cell adhesion and proliferation. Plasminogen activation was assessed by measuring the plasmin activity and plasminogen activator inhibitor-1 (PAI-1) levels in cell-conditioned media and the cell-associated urokinase-type plasminogen activator (uPA) levels. RESULTS: Treatment of MDA-MB-231 breast carcinoma cells with either thrombospondin or TGF-beta 1 caused increased secretion of PAI-1 with a concomitant decrease in plasmin activity, whereas cell-associated uPA expression was increased with respect to controls. Thrombospondin (40 micrograms/ml) or TGF-beta 1 (5 ng/ml) stimulated the cells to secrete 5.5- and 6.7-fold more PAI-1 than controls, respectively, and caused decreased plasmin activity in the cell culture medium. Conversely, either thrombospondin (40 micrograms/ml) or TGF-beta 1 (5 ng/ml) caused the cells to express 4.55- and 5.38-fold more uPA than controls, respectively. Thrombospondin and TGF-beta 1 induced a more flattened and spread appearance in the cells with no effect on proliferation. These effects could be reversed with antibodies to either thrombospondin or TGF-beta 1 and were not due to contamination of thrombospondin with active TGF-beta 1. CONCLUSIONS: Thrombospondin and TGF-beta 1 function similarly to increase cell-associated uPA and cell-secreted PAI-1. These data suggest that thrombospondin may not only function as an adhesive molecule, but through a mechanism involving the activation of TGF-beta 1, may modulate cell surface protease expression. In addition, these observations suggest that thrombospondin and TGF-beta 1 could promote metastasis by increasing uPA-mediated cell invasion, whereas through the action of PAI-1, also protect blood-born tumor emboli from destruction by host fibrinolytic enzymes.

Computer-assisted image analysis of tumor sections for a new thrombospondin receptor.

BACKGROUND: A cell surface receptor (50 kd) has been recently identified in malignant cells that recognizes the tumor cell adhesive domain (ie, cysteine-serine-valine-threonine-cysteine-glycine [CSVTCG]) of thrombospondin (TSP). This CSVTCG-specific TSP receptor can be considered as a new tumor marker, and its concentration on the cell surface may correlate directly with the capacity of tumor cells to invade and metastasize. MATERIALS AND METHODS: Six patients with primary, stages III and IV squamous cell carcinomas of the head and neck were studied. Tumor sections were specifically stained for this receptor with immunohistochemical techniques. The stained specimens were then subjected to computer-assisted image analysis. The area of positive staining and the heterogeneity of the pattern of staining were compared to peritumoral angiogenesis and clinical outcome of the patients. RESULTS: The results indicate that those patients with a high and homogenous positive stain score (mean +/- standard error [SE] 78 +/- 5%) for the CSVTCG-specific TSP receptor had high microvessel density and died from metastatic disease within 12 months of initial treatment (correlation coefficients = 0.95 and 1, respectively). Patients with a low and heterogenous positive stain score for receptor (mean +/- SE 8 +/- 2%; P < 0.001) had low microvessel counts and remained disease-free for at least 2 years. There was no relationship between receptor density and histologic classification of the primary tumors. CONCLUSION: The CSVTCG-specific TSP receptor, quantified through image analysis of immunohistochemical stained tissue sections, is highly predictive of clinical outcome in patients with squamous cell carcinomas of the head and neck.

Thrombospondin (TSP) and transforming growth factor beta 1 (TGF-beta) promote human A549 lung carcinoma cell plasminogen activator inhibitor type 1 (PAI-1) production and stimulate tumor cell attachment in vitro.

A growing body of evidence has recently implicated TSP and TGF-beta in the process of malignancy, such as tumor cell proliferation, tumor angiogenesis, and metastasis. The purpose of the present study was to evaluate potential mechanisms of TSP and TGF-beta in tumor cell attachment and invasion. Our results indicate that both TSP and TGF-beta promoted tumor cell attachment and spreading in the presence of plasminogen. The mechanism for these effects appeared to be due, in part, to the capacity of TSP and TGF-beta to induce tumor cell production of (PAI-1). PAI-1, which is a natural inhibitor of tumor-cell associated urokinase-type plasminogen activator (uPA) activity, inhibited activation of plasminogen to plasmin in the growth media, thereby preventing plasmin-induced detachment of cells. The TSP-promoted production of PAI-1 could be inhibited not only by anti-TSP antibodies but also by a neutralizing antibody against TGF-beta. These results suggest that TSP by a mechanism involving TGF-beta can promote cell adhesion through stimulation of tumor cell secretion of PAI-1. These data provide evidence that TSP not only has the capacity of functioning as a matrix protein to directly promote cell-substratum adhesion but that TSP can also stimulate cell adhesion and spreading by modulating cell surface protease expression through stimulation of tumor-associated production of PAI-1.

The relationship of angiogenesis to biological activity in human squamous cell carcinomas of the head and neck.

Tumor angiogenesis has recently been related to tumor growth and metastasis, which determine the clinical outcome of the patient. This study was designed to determine the relationship between angiogenesis in primary squamous cell carcinomas (SCC) of the head and neck and the development of recurrent or metastatic disease, or both. Different SCC of the head and neck were studied. Microvessels were selectively stained using a monoclonal antibody for factor VIII. Microvessel counts were performed in the tumor, in the tissues immediately adjacent, and in normal tissues of similar topographies. Microvessel counts were then correlated with clinical outcome (development of recurrent or metastatic disease, or both). Recurrent or metastatic disease, or both, developed in patients with high microvessel counts (mean, 121.25) in the tissues adjacent to the tumor 7 to 16 months after initial treatment. Those with low microvessel counts (mean, 33.75) were disease-free for 16 months to 6 years (p < 0.01). Microvessel counts inside the tumor were also higher in those in whom recurrences or metastasis, or both, developed, but were not statistically significant. In this study, angiogenesis was directly related to clinical outcome. Thus, angiogenesis may be an independent predictor of recurrent or metastatic disease, or both, which could help in the selection of patients with SCC of the head and neck for aggressive therapy.