F Savage

Detection of free malignant cells in the peritoneal cavity before and after resection of colorectal cancer

PURPOSE: This study was designed to select the best monoclonal antibody to stain malignant cells in peritoneal wash fluid, and to investigate the incidence of free malignant cells in preresection and postresection colorectal cancer peritoneal washings using a combination of conventional cytology and immunocytochemistry. METHODS: Peritoneal washings were taken from 35 consecutive patients undergoing colorectal cancer resection. RESULTS: Malignant cells were isolated on a density gradient and identified by conventional cytology and an indirect immunoperoxidase stain. Malignant cells were identified in peritoneal washings from 15 patients (preresection only n=3, postresection only n=4, both n= 8). The origin of free malignant peritoneal cells in 11 preresection-positive washings must be the serosa. The origin of these cells in the four postresection-positive patients is uncertain: serosal and luminal spillage were considered unlikely and no circulating cells were found in the mesenteric vessels near the tumor. CONCLUSION: Tumor cells may have leaked out from lymphatics cut during the dissection.

Role of matrix metalloproteinases in healing of colonic anastomosis

PURPOSE: The aim of this study was to compare the distribution of the matrix metalloproteinases (MMPs) during anastomotic healing in a normal colon with that in an ischemic colon in a rabbit. This family of enzymes degrades all components of connective tissue and has been implicated as a cause of anastomotic dehiscence. METHODS: A left-sided anastomosis was formed in the distal colon of one group of rabbits, and in the other group, 9 cm of distal colon was made ischemic before resection and anastomosis 12 hours later. Tissues from the anastomosis and sites around the colon were removed at 12 hours, 1 day, and 3 days after anastomosis and, also, at 7 days in the normal group. Distribution of the MMPs and their inhibitor, tissue inhibitor of metalloproteinases (TIMP), was localized by indirect immunofluorescence. RESULTS: In rabbits having only an anastomosis, the MMPs and TIMP-1 were, at all times, seen solely in the anastomotic segment and were strictly confined to the immediate vicinity of the suture line. While in rabbits with an ischemic colon before anastomosis, the MMPs initially extended several centimeters proximally and distally from the suture line. By the third day, however, there were only minor differences between the two models. CONCLUSION: Distribution of the MMPs and TIMP-1 in normal healing is consistent with a role in the remodeling of colonic anastomosis, but when healing of the colon is compromised, these en2ymes are more widespread and may contribute to anastomotic dehiscence.

The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs), is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumour cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

Altered endothelin receptor subtypes in colorectal cancer.

Background The vasoactive peptide endothelin-1 (ET-1) acts via two endothelin receptor subtypes, ETA (ETAR) and ETB (ETBR). ET-1 and ETAR are overexpressed in colorectal cancer tissues. In vitro, ET-1 acting via ETAR, is a mitogen for colorectal cancer cells. To identify other potential stimulatory loops, we investigated the distribution and cell-specific localization of both ETAR and ETBR in tissue sections from patients with colorectal cancer. Methods Frozen sections from specimens of colorectal cancer (n=9) and normal colon (n=9) were cut and subjected to either (i) autoradiography or (ii) a combination of cell type-specific immunohistochemistry, using antibodies against fibroblasts (AS02), endothelial cells (CD31) or nerve fibres (NF200) and in-vitro receptor microautoradiography, using ETAR-specific and ETBR-specific radioligands. Results ETARs were upregulated in all cell types, apart from nerve, in cancer compared with normal colon (1 : 1.59 normal to cancer). Specifically, ETAR binding was highest in cancer-associated blood vessels and fibroblasts and to a lesser extent in epithelial cancer cells. In contrast, ETBRs were the predominant receptors in normal colon (1 : 0.59 normal to cancer) and were markedly down-regulated in cancer-associated blood vessels, fibroblasts and to a lesser extent in epithelial cells. Nerve colocalization was demonstrated, but remained unchanged for all tissues. Conclusion The shift in ET receptor binding observed in epithelial cancer cells and cancer-associated fibroblasts and endothelial cells may favour ET-1 signals contributing to colorectal cancer growth and neovascularization via ETAR. This may provide the basis for therapeutic use of specific ETAR antagonists as adjuvant treatment of colorectal cancer.

Regulation of matrix metalloproteinases in a model of colonic wound healing in a rabbit

PURPOSE: Matrix metalloproteinases occur in the colon at an anastomosis but not in the normal colon. Matrix metalloproteinase synthesis can be regulated by cytokines, for example interleukin-1 beta and growth factors, such as transforming growth factor beta and basic fibroblast growth factor. The aim of this study was to investigate the regulation of matrix metalloproteinases at an anastomosis by identifying the cell types that synthesize matrix metalloproteinases, examining factors that might regulate their synthesis, determining whether they occur in an active form, and assessing the effect of suture type on these parameters. METHODS: An anastomosis was formed in the distal colon of rabbits using either polyglactin or polydioxanone and the animals were killed six hours or seven days later. The distribution of matrix metalloproteinases and cytokines and the cell types were assessed by immunohistochemistry. Matrix metalloproteinase-2, matrix metalloproteinase-3, and matrix metalloproteinase-9 were detected also by zymography. RESULTS: Immunohistochemistry showed that matrix metalloproteinases were restricted to the suture line. Although zymography demonstrated that matrix metalloproteinase-2 was present mainly in an active form, matrix metalloproteinase-9 and matrix metalloproteinase-3 were present in the pro-form. The active form of matrix metalloproteinase-3 occurred more often in the polydioxanone-sutured rabbits. With the exception of matrix metalloproteinase-9, the matrix metalloproteinases were synthesized by fibroblasts. Interleukin-1 beta and transforming growth factor beta were more widespread than in the normal colon and were localized adjacent to the matrix metalloproteinases. Basic fibroblast growth factor was also more widespread postoperatively but occurred deeper in the anastomosis than the matrix metalloproteinases. CONCLUSIONS: This study has shown that interleukin-1 beta and transforming growth factor beta may regulate the synthesis of the matrix metalloproteinases by fibroblasts and that minor differences that occur in the matrix metalloproteinase profile are dependent on the suture type.

Efficacy of the Specific Endothelin A Receptor Antagonist Zibotentan (ZD4054) in Colorectal Cancer: A Preclinical Study

Endothelin 1 (ET-1) is overexpressed in cancer, contributing to disease progression. We previously showed that ET-1 stimulated proliferative, migratory, and contractile tumorigenic effects via the ETA receptor. Here, for the first time, we evaluate zibotentan, a specific ETA receptor antagonist, in the setting of colorectal cancer, in cellular models. Pharmacologic characteristics were further determined in patient tissues. Colorectal cancer lines (n = 4) and fibroblast strains (n = 6), isolated from uninvolved areas of colorectal cancer specimens, were exposed to ET-1 and/or ETA/B receptor antagonists. Proliferation (methylene blue), migration (scratch wounds), and contraction (gel lattices) were assessed. Receptor distribution and binding characteristics (Kd, Bmax) were determined using autoradiography on tissue sections and homogenates and cytospun cells, supported by immunohistochemistry. Proliferation was inhibited by ETA (zibotentan > BQ123; P < 0.05), migration by ETB > ETA, and contraction by combined ETA and ETB antagonism. Intense ET-1 stromal binding correlated with fibroblasts and endothelial cells. Colorectal cancer lines and fibroblasts revealed high density and affinity ET-1 binding (Bmax = 2.435 fmol/1 × 106 cells, Kd = 367.7 pmol/L; Bmax = 3.03 fmol/1 × 106 cells, Kd = 213.6 pmol/L). In cancer tissues, ETA receptor antagonists (zibotentan; BQ123) reduced ET-1 binding more effectively (IC50: 0.1–10 μmol/L) than ETB receptor antagonist BQ788 (∼IC50, 1 mmol/L). ET-1 stimulated cancer-contributory processes. Its localization to tumor stroma, with greatest binding/affinity to fibroblasts, implicates these cells in tumor progression. ETA receptor upregulation in cancer tissues and its role in tumorigenic processes show the receptors importance in therapeutic targeting. Zibotentan, the most specific ETA receptor antagonist available, showed the greatest inhibition of ET-1 binding. With its known safety profile, we provide evidence for zibotentans potential role as adjuvant therapy in colorectal cancer. Mol Cancer Ther; 12(8); 1556–67. ©2013 AACR.

Effect of methylprednisolone on the ulceration, matrix metalloproteinase distribution and eicosanoid production in a model of colitis in the rabbit.

This study has examined the response of a rabbit model of inflammatory bowel disease to methylprednisolone. Colitis was induced in the colon of rabbits with 40 mg trinitrobenzenesulphonic acid in 25% ethanol (TNBS). The effect of methylprednisolone (0.5 mg/kg/day) on the development of colitis was determined at one week, by examining the colons macroscopic and microscopic appearance, the distribution of matrix metalloproteinases (MMPs) and by measuring eicosanoid production. Although there was no difference in the area of ulcerated colonic tissue in the treated and untreated TNBS animals, the increase in polymorphonuclear leucocytes was significantly reduced in TNBS rabbits given methylprednisolone. The only difference in the distribution of MMPs was a reduction in the number of polymorphonuclear leucocytes containing gelatinase B. The release of immunoreactive PGE2 and LTB4, but not 6‐keto PGF1α, was increased in the TNBS animals and was unchanged by methylprednisolone. These results show that methylprednisolone does not modify the injury produced by TNBS in this model despite reducing the infiltration of polymorphonuclear leucocytes. Hence it suggests that these cells do not contribute to the injury observed, are not the source of the eicosanoids and that gelatinase B is not required in the healing process in this model.

Protease expression in experimental colitis

Extracellular matrix (ECM) is degraded by matrix metalloproteinases, collagenase, stromelysin and gelatinase, whose activity is strictly controlled by tissue inhibitor of metalloproteinase (TIMP). Excessive enzyme activity could lead to tissue destruction in inflammatory bowel disease (IBD).Using a rabbit model of chronic colitis we investigated the temporal and spatial distribution of these enzymes by immunolocalisation. 72 kD intracellular gelatinase was observed 3 h after initiation of colitis. At 6 h and 12 h, collagenase and, to a lesser extent, 72 kD and 95 kD gelatinase and stromelysin were all observed on the ECM in regions of mucosal ulceration. TIMP, however, was absent at these earlier times suggesting uncontrolled degradation of ECM, but by 24 h, it was expressed in mucosa adjacent to areas of ulceration. At 72 h and after one week, expression of collagenase declined and from two weeks until the ulcers resolved, stromelysin and gelatinase were found at the junction of normal and ulcerated tissue. TIMP expression remained constant until ulceration had healed at 6 weeks. In colon from animals killed at 0 h, no enzyme or TIMP expression was observed.Collagenase appears to be associated with the acute phase of ulcer formation, whereas stromelysin and gelatinase are predominant during healing.

THE CHEMOATTRACTANT ACTIVITY OF THE VITREOUS TO HUMAN SCLERAL FIBROBLASTS FOLLOWING RETINAL-DETACHMENT AND PROLIFERATIVE VITREORETINOPATHY

The results are presented of a migration assay of scleral fibroblasts to 25% diluted vitreous samples from 27 patients with complex retinal detachments divided into three groups: group 1, no proliferative vitreoretinopathy (PVR); group 2, early PVR; and group 3, advanced PVR. There was a statistically significantly greater migration with vitreous samples from group 3 than with group 1 (p less than 0.0001) but no significant correlation for migration with the age of patients, duration of retinal detachment, or number of retinal procedures undertaken. Analysis of vitreous by gel electrophoresis showed that cellular migration was proportional to the number of peptide bands. Vitreous fibronectin levels were measured and there was a positive correlation between fibronectin in the vitreous samples and the migration of fibroblasts to the vitreous (r = 0.68, p less than 0.004).

The requirement for integrins in the adhesion of human colonic fibroblasts to extracellular matrix components

PURPOSE. Cell-matdx interactions mediated by integrins may be important in fibroblast adhesion, migration, proliferation and matrix remodeling during colonic healing and in pathological conditions that result in fibrosis and stricture formation. The aim of this study was to define the integrins required for adhesion of human colonic fibroblasts to collagen I, the most abundant collagen in normal colon, and to fibronectin an important component of wound provisional matrix. METHODS. Human colonic fibroblast cultures were obtained by enzymatic digestion of colonic mucosa. Cell suspensions (4xl(P cells/ml) were incubated in the presence or absence of antibody to the integrins ~, ,z2, ~3, =4, <zs or ~, before adding 4x10 ~ sells/ well to microtitre plates coated with collagen I or fibronectin. After incubation for 1 hour nonadherent cells were removed by washing. Adherent cells were fixed and quantified using the methylene blue assay. The results are expressed as median absorbance in arbitrary units for test wells, A,b. and controls A:. Statistical analysis was by the Wilcoxon signed rank test. RESULTS. Adhesion to collagen was inhibited by antibody to the/~ integrin subonit, A= = 0.08, Ac = 0.98 (n = 20, p<O.OOO1)and by antibody to the ~ integrin subunit, ~ = 0.02, A~ = 0.78 (n = 9,p = 0.0039). Adhesion to fibronectin was partially inhibited by antibody to the ~ subonlt, /~b=0.83, A~= 1.06 (n=13, p=O.O061) and by antibody to ~, absorbance A= =1.06, A~ =1.21 (n=9, p=0.0039). No significant inhibition of adhesion to collagen or fibronectin was observed in the presence of antibodies to the e2, ,z3, or ~ integrins. CONCLUSIONS. The major determinant of adhesion of human colonic fibroblasts to collagen 1 is the ~/~ integrin. Only partial inhibition of adhesion of colonic fibrohlasts to fibronsctin could be achieved using antibodies to ~z5 or ~ suggesting that other cell adhesion molecules are involved in this interaction.