A practical guide for investigating cardiac physiology using living myocardial slices.

Ex vivo multicellular preparations are essential tools to study tissue physiology. Among them, the recent methodological and technological developments in living myocardial slices (LMS) are attracting increasing interest by the cardiac research field. Despite this, this research model remains poorly perceived and utilized by most research laboratories. Here, we provide a practical guide on how to use LMS to interrogate multiple aspects of cardiac function, structure and biochemistry. We discuss issues that should be considered to conduct successful experiments, including experimental design, sample preparation, data collection and analysis. We describe how laboratory setups can be adapted to accommodate and interrogate this multicellular research model. These adaptations can often be achieved at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software, which is essential to broaden the utilization of this method. We will also highlight how current measurements can be improved to further enhance data quality and reliability to ensure inter-laboratory reproducibility. Finally, we summarize the most promising biomedical applications and envision how living myocardial slices can lead to further breakthroughs.

Label-free quantitative SWATH-MS proteomic analysis of adult myocardial slices in vitro after biomimetic electromechanical stimulation.

A special in vitro model maintained with ultrathin cardiac slices with a preserved architecture, multi-cellularity, and physiology of the heart tissue was used. In our experiments, we performed label-free quantitative SWATH-MS proteomic analysis of the adult myocardial slices in vitro after biomimetic electromechanical stimulation. Rat myocardial slices were stretched to sarcomere lengths (SL) within the physiological range of 1.8-2.2 µm. Electromechanically stimulated slices were compared with slices cultured without electromechanical stimulation (unloaded and nonstimulated-TW) on a liquid-air interface and with fresh myocardial slices (0 h-C). Quantitative (relative) proteomic analyses were performed using a label-free SWATH-MS technique on a high-resolution microLC-MS/MS TripleTOF 5600+ system (SCIEX). The acquired MS/MS spectra from the DDA LC-MS/MS analyses of the rat heart samples were searched against the UniProt Rattus norvegicus database (version of 15.05.2018) using the Paragon algorithm incorporated into ProteinPilot 4.5 (SCIEX) software. The highest number of differential proteins was observed in the TW group-121 when compared to the C group. In the 1.8 and 2.2 groups, 79 and 52 proteins present at a significantly different concentration from the control samples were found, respectively. A substantial fraction of these proteins were common for two or more comparisons, resulting in a list of 169 significant proteins for at least one of the comparisons. This study found the most prominent changes in the proteomic pattern related to mitochondrial respiration, energy metabolism, and muscle contraction in the slices that were stretched and fresh myocardial slices cultured without electromechanical stimulation.

A gastrin transcript expressed in gastrointestinal cancer cells contains an internal ribosome entry site.

As the hormone gastrin promotes gastrointestinal (GI) cancer progression by triggering survival pathways, regulation of gastrin expression at the translational level was explored. Sequence within the 5' untranslated region of a gastrin transcript expressed in GI cancer cells was investigated, then cloned into a bicistronic vector upstream of firefly luciferase and transfected into a series of GI cancer cell lines. Firefly luciferase activity was measured relative to that of a cap-dependent Renilla luciferase. A gastrin transcript that was different from that described in Ensembl was expressed in GI cancer cells. Its transcription appears to be initiated within the region designated as the gene's first intron. In GI cancer cells transfected with the bicistronic construct, firefly luciferase activity increased 8-15-fold compared with the control vector, and there was a further induction of the signal (up to 25-fold) following exposure of the cells to genotoxic stress or hypoxia, suggesting that the sequence acts as an internal ribosome entry site. These data suggest that the gastrin transcript within GI cancer cells contains an internal ribosome entry site that may allow continued expression of gastrin peptides when normal translational mechanisms are inactive, such as in hypoxia, thereby promoting cancer cell survival.

Pre-clinical evaluation of a new orally-active CCK-2R antagonist, Z-360, in gastrointestinal cancer models.

BACKGROUND: Gastrin has a role in gastrointestinal (GI) malignancy. This study provides pre-clinical evaluation of a novel, orally-active gastrin/cholecystokinin-2 receptor (CCK-2R) antagonist, Z-360. METHODS: (125)I gastrin-17 (G17) displacement and G17-stimulated calcium assays were used in classical CCK-2R-transfected cell lines. Akt phosphorylation was assessed by Western blotting. Z-360 efficacy in vivo was evaluated in three human xenograft models, and microvessel density and apoptosis in these models were investigated by immunohistochemistry. RESULTS: Z-360 inhibited (125)I G17 binding to cells expressing CCK-2R, and G17-stimulated signalling. Reduced Akt phosphorylation in an oesophageal cell-line treated with Z-360 was reversed by co-treatment with G17. Z-360 increased survival in a gastric ascites model (p=0.011) and decreased tumour growth in a hepatic metastasis model (81%, p=0.02). In an orthotopic pancreatic model, Z-360 combined with gemcitabine decreased final tumour weight compared to single agents (84%, p=0.002) and there was increased apoptosis and decreased microvessel density in ex vivo tumour tissue. CONCLUSIONS: These results show that the orally-active CCK-2R antagonist, Z-360 has high sub-nM affinity for classical CCK-2R, is well tolerated in vivo and exerts an anti-tumour effect.

Androgen receptors may act in a paracrine manner to regulate oesophageal adenocarcinoma growth.

BACKGROUND: The role of androgen receptors (ARs) in tumorigenesis, including transcription of fibroblast growth factors (FGFs), is established in prostate cancer. This study examined the role of ARs and FGFs in oesophageal adenocarcinoma (EAC), where tumour incidence in males is higher. METHODS: AR gene expression was analysed using quantitative RT-PCR; AR, fibroblast growth factor receptor-1 (FGFR-1) and fibroblast growth factor-8 isoform b (FGF-8b) protein by immunohistochemistry; and serum steroid levels (testosterone, progesterone, luteinising hormone and follicle stimulating hormone (FSH)) by immunoassay. A human oesophageal adenocarcinoma cell line was grown subcutaneously in nude mice. RESULTS: AR gene expression was of significantly higher levels than oesophageal adenocarcinomas (n=21, p=0.002) and in the squamous carcinoma line (OE21) compared with the adenocarcinoma lines (OE33 and OE19). Median serum testosterone levels in oesophageal carcinoma patients were higher than in age-matched controls (p=0.01) and reduced postoperatively, in patients undergoing curative resection (p=0.006). No significant differences were observed in hormones except FSH, where preoperative levels were significantly higher in the EAC group. AR protein was expressed in normal oesophageal squamous epithelial cells and also in the stroma of 18/23 EAC samples. FGFR-1 protein was expressed in malignant epithelium of 23/23 tumour samples. OE19 xenografts grew faster in male versus female mice (tumour weight at day 21, 1.14 g and 0.28 g, respectively, p=0.005) and had elevated FGF receptor expression. CONCLUSIONS: AR expressed in the stroma of oesophageal adenocarcinomas may induce paracrine effects following stimulation by androgens (including tumour-derived), possibly via FGFs, including FGF-8b.

The effect of jaundice on the generation of anti-gastrin antibodies in G17DT immunized patients with advanced pancreatic cancer.

AIM: The aim of this study was to determine the ability of G17DT to generate anti-gastrin antibodies in jaundiced patients with biliary obstruction due to advanced pancreatic cancer. METHODS: G17DT was administered to 41 patients with advanced pancreatic adenocarcinoma by intramuscular (i.m.) injection at a dose of 250mcg at weeks 0, 1 and 3 of the study. RESULTS: Thirty-five of 41 patients participating in the study were categorized as responders in terms of their gastrin-17 antibody response. There was no correlation between the maximum G17 antibody response and the bilirubin level at either week 0 or week 12. The median survival of patients from the time of the first injection of G17DT was 204 days with 25% of patients surviving for or=305 days. CONCLUSION: This study shows that G17DT administered to jaundiced patients with advanced pancreatic cancer is immunogenic and well tolerated.

Intracellular gastrin in human gastrointestinal tumor cells.

Flow cytometry and immunohistochemical analyses of the human gastric adenocarcinoma cell line MKN45G identified an intracellular peptide recognized by an anti-gastrin-17 (G17) antiserum but not by an anti-cholecystokinin-specific antiserum. Staining was not associated with the parental line MKN45, of which MKN45G is a clonal variant. The MKN45G cell line had elevated in vitro growth in serum-free medium in which the proliferation of MKN45G cells but not MKN45 cells was reduced to 58% of the control value by treatment with a rabbit anti-G17 antiserum. This inhibition of proliferation was reversed by preabsorbing the antiserum with excess G17. Disaggregated primary human gastric and colorectal tumors were screened for gastrin immunoreactivity by flow cytometry, and 6 of 28 colorectal and 8 of 22 gastric tumors had greater than 20% positively staining cells.

Hypergastrinemia promotes adenoma progression in the APC(Min-/+) mouse model of familial adenomatous polyposis.

Serum hypergastrinemia promotes the growth of colorectal adenocarcinoma. Some colorectal adenomas express cholecystokinin B/gastrin receptor mRNA, and thus hypergastrinemia may increase progression through the adenoma-carcinoma sequence. This was investigated in the multiple intestinal neoplasia APC(Min-/+) mouse. Serum gastrin levels in APC(Min-/+) mice were elevated 5-6-fold by oral administration of omeprazole (75 mg/kg). Terminal tumor burden was monitored by onset of anemia. A labeling index was generated by immunohistochemical detection of bromodeoxyuridine incorporation. Serum gastrin was neutralized by antigastrin antibodies raised in situ by use of a gastrin immunogen, Gastrimmune. Hypergastrinemia resulted in reduced survival of the APC(Min-/+) mice from a median survival of 13 weeks in the controls to 10 weeks following omeprazole treatment (P < 0.00001, log-rank test). The labeling indices of adenomas from the small and large intestines of omeprazole-treated mice were increased 35 and 29%, respectively (P < 0.05 and P < 0.025, respectively). Gastrimmune immunization reversed both the survival effect and the increased proliferation resulting from serum hypergastrinemia. Hypergastrinemia may promote the progression of existing premalignant colonic lesions by increasing proliferation. Clinical investigations should determine whether this occurs in the human scenario, considering the widespread use of proton pump inhibitors.

Antiserum raised against an epitope of the cholecystokinin B/gastrin receptor inhibits hepatic invasion of a human colon tumor.

Serum gastrin is known to be elevated in patients with liver-metastasizing colon cancer; thus, cholecystokinin (CCK) B/gastrin receptors may also be up-regulated. A liver-invasive model of colon cancer was established with the human colonic cell line C170HM2, which expresses the CCKB/gastrin receptor at both the gene and protein level. An antiserum has been derived that is directed against the NH2-terminal 17 amino acids of the human CCKB/gastrin receptor coupled to diphtheria toxoid. The peptide was denoted gastrin receptor protein (GRP) 1. The therapeutic effect of GRP1 antiserum was evaluated on the liver invasion of C170HM2 tumors. Biodistribution studies revealed that GRP1 antiserum localized preferentially within the liver tumors when compared with normal liver tissue (1.5-fold increase after 24 h; P < 0.05). Antiserum against GRP1 inhibited both tumor take rate and final liver tumor weight when compared with treatment with control serum in mice with an increasing tumor burden. Liver tumor weights were reduced from 0.37 to 0.10 gram (P = 0.0155), 1.25 grams to 0.76 gram (P = 0.003) and 1.89 grams to 0.76 gram (P = 0.0068, all Mann-Whitney nonparametric U test). Necrosis and apoptosis were increased in the GRP1 antiserum-treated tumors when compared with control serum-treated tumors. As shown by Western blotting, CCKB/gastrin receptor expression of C170HM2 xenografts after treatment with GRP1 antiserum shifted to a predominantly lower molecular weight form (Mr 45,000) that is known to be an internalized form of the receptor. In conclusion, targeting of the CCKB/gastrin receptor may yield a valuable therapeutic modality for the treatment of advanced colon cancer.

Inhibition of organ invasion by the matrix metalloproteinase inhibitor batimastat (BB-94) in two human colon carcinoma metastasis models.

The effect of the matrix metalloproteinase inhibitor batimastat was evaluated in two human colorectal cancer metastasis models involving: (a) the liver-invasive tumor C170HM2 and (b) the lung-invasive tumor AP5LV, both of which have been shown to express the M(r) 72,000 type IV collagenase. Batimastat at concentrations between 0.01 and 3.0 micrograms/ml had no direct cytotoxic effects on the in vitro growth of the cell lines. In the liver-invasive tumor model, batimastat administered i.p. from day 10 to termination of the therapy (day 39) at 40 mg/kg reduced both the mean number of liver tumors (35% of vehicle-treated control; P < 0.05) and the cross-sectional area of the tumors (43% of vehicle-treated control; P < 0.05). In the lung-invasive tumor model, batimastat administered daily (40 mg/kg i.p.) significantly reduced tumor weight within the lung (72% of vehicle-treated control; P < 0.05) but did not significantly affect nodule number. In the latter model, in which the take rate was unaffected, tumor cells were introduced into the lateral tail vein, and lung localization may have been a physical phenomenon not involving invasion. In the former model, tumor cells were introduced directly into the peritoneal cavity, and from there the cells adhered to and invaded the liver capsule. Because the take rate is significantly reduced, it may be that the matrix metalloproteinases are involved in this process. Batimastat may be a therapeutic modality for the treatment of colorectal cancer metastasis.

A novel, orally administered nucleoside analogue, OGT 719, inhibits the liver invasive growth of a human colorectal tumor, C170HM2.

OGT 719 is a novel p.o. bioavailable nucleoside analogue in which galactose is incorporated onto the fluoropyrimidine moiety of the cytotoxic agent 5-fluorouracil (5-FU). OGT 719 has been designed to reduce the systemic toxicity normally associated with 5-FU while retaining activity against disease localized in the liver, in which it may be preferentially localized through the asialoglycoprotein receptor (ASGP-R). We report studies confirming the activity of OGT 719 in inhibiting growth of metastatic human colorectal tumors in the liver of nude mice. The human colorectal cancer cell line C170HM2 readily forms liver metastases in vivo. Oral administration of 1500 mg/kg/day OGT 719 inhibited liver tumor burden by 95% compared with vehicle control, without any observable signs of toxicity. When the tumor burden was increased and the same OGT 719 treatment was compared with a standard clinical dose regimen of 25 mg/kg/day 5-FU/leucovorin given i.v., both treatments were equally efficacious, although 5-FU/leucovorin treatment started 7 days earlier. In contrast to 5-FU, OGT 719 is p.o. bioavailable and has a plasma half-life between 1.5 and 3 h. Several colorectal cancer cell lines express the asialoglycoprotein receptor, although no significant levels can be detected in C170HM2 cells, consistent with the observation that OGT 719 is approximately 3 log orders of magnitude less potent in vitro than 5-FU. Flux through thymidylate synthase, as measured by 3H release from [3H]dUrd, was inhibited by OGT 719 at 4 h. The notable difference in the potency of OGT 719 efficacy on C170HM2 cells in vitro and in vivo supports our model of liver-specific activation of OGT 719. As our data suggest, OGT 719 may significantly inhibit growth of metastatic colorectal tumors in the liver in vivo. This hypothesis is presently being explored in clinical trials for primary hepatocellular carcinoma and colorectal liver metastases.

Gastrimmune raises antibodies that neutralize amidated and glycine-extended gastrin-17 and inhibit the growth of colon cancer.

The effect of gastrin neutralization was evaluated on the in vivo growth of the rat colon line, DHDK12, which expressed cholecystokinin B/gastrin receptors and secreted glycine-extended gastrin-17 (G17). Gastrin neutralization was achieved by administration of the immunogen, Gastrimmune, which is composed of the amino terminal portion of G17 linked to a diphtheria toxoid. A rat-specific version of Gastrimmune was used to preimmunize rats, with control animals receiving diphtheria toxoid only. The antibodies raised neutralized both carboxy-amidated and glycine-extended G17. The tumor was implanted into the muscle layer of the abdominal wall, and rats immunized with Gastrimmune had significantly reduced median cross-sectional tumor areas (70.2% reduction; P = 0.005) and weights (56.5% reduction; P = 0.0078)) when compared to control rats. Histological analysis revealed that the tumors had an enhanced degree of necrosis, with the area of viable tumor in the Gastrimmune-immunized rat reduced to 40.3% compared to 58.6% in the control rats (P = 0.003). Immunization with Gastrimmune raised antibodies that inhibited the growth of a rat colon tumor. This could have been mediated by neutralization of both serum G17 and cell-associated precursor gastrin molecules.

Inhibition of matrix metalloproteinase activity and growth of gastric adenocarcinoma cells by an angiotensin converting enzyme inhibitor in in vitro and murine models.

INTRODUCTION: Angiotensin converting enzyme (ACE) shares structural homology with the matrix metalloproteinase family of proteolytic enzymes (MMPs) responsible for degradation of the extracellular matrix (ECM). ACE inhibitors have been reported to protect against cancer in patients. The aim of this study was to determine whether the ACE inhibitor, captopril, could impair the activity of MMPs and impact on tumour invasion and growth in a cell line and murine model. METHODS: For proof of principle, the protein activity of human MMP-2 and MMP-9 produced by the HT1080 fibrosarcoma cell line was detected using gelatin zymography. Gene expression was determined by real time reverse transcriptase PCR and tumour cell invasion using Matrigel invasion chambers. The effect of captopril on the in vivo growth of MGLVA-1 human gastric adenocarcinoma xenografts was evaluated in a nude mouse model. RESULTS: Captopril inhibited activity of secreted MMP-9 and MMP-2, however, gene expression in HT1080 remained unaltered. Invasion of HT1080 cells was inhibited by 48% (p<0.001). Tumour size was reduced by 40-50% with 0.4 mg/ml captopril (p<0.01) and when combined with cisplatin the inhibition increased to 71% (p<0.05). DISCUSSION: ACE inhibitors inhibit the activity of secreted MMP-2 and -9 by a mechanism similar to synthetic MMP inhibitors. ACE inhibitors have previously been shown to inhibit tumour growth, however; this is the first study to demonstrate inhibition of a human gastric xenograft, both alone and in combination with cisplatin. These results support further investigation into the anticancer effects of ACE inhibitors.

Radiation induced MMP expression from rectal cancer is short lived but contributes to in vitro invasion.

AIMS: Matrix metalloproteinase (MMP) activity is increased after radiation. The aims of this study were to assess the time course of this increase and its effects on malignant cell invasion. METHODS: Colorectal cancer (HCT 116, LoVo, C 170 HM 2, CaCO-2), fibroblast (46-BR.IGI, CCD-18 Co) and fibrosarcoma (HT1080) cell lines were irradiated at 4 gray (4 Gy) and matrix metalloproteinase gene and protein expression examined over a 96 h period by real time polymerase chain reaction and gelatin zymography. Invasion was assessed on Matrigel. Human rectal tumour MMP expression was compared before and after long course radiotherapy. RESULTS: Radiation increased MMP gene expression of tumour cell lines, and resulted in increased MMP protein activity in the HT1080 line. HT1080 and HCT 116 in monoculture and LoVo in co-culture were more invasive after radiation at 48 h in vitro, but long course radiotherapy did not result in a consistent increase in MMP expression from human rectal tumour biopsies. CONCLUSIONS: Radiation results in increased MMP expression for a limited time period. This results in an early increase in cell line invasion. Further clinical research is required to clarify if MMP inhibition given perioperatively following radiotherapy decreases local recurrence rates.

Phase I/II study of G17-DT, an anti-gastrin immunogen, in advanced colorectal cancer.

Gastrin is a growth factor for colorectal cancer, and therefore, anti-gastrin hormone therapy has a potential role in treatment of this disease. The gastrin immunogen gastrin-17-diphtheria toxoid (G17-DT; Gastrimmune) produces anti-G17 antibodies that have been shown to be effective in the treatment of colorectal carcinoma in preclinical models. Fifty patients with advanced colorectal cancer were treated with G17-DT in a multicenter, sequential group, open label Phase I/II study. Primary injections with two booster doses were given by i.m. injection. The main aim of the study was to assess the safety and efficacy of the production of antigastrin antibodies. Locally developed and standard WHO toxicity measurements with RIA and Scatchard analysis for antibody assessment were used. One center measured tumor response radiologically. Eighty % of patients produced a measurable antibody response. Antibodies of high affinity (median Kd, 0.295 nM; interquartile range, 0.16-0.41 nM) were detected between 4 and 12 weeks after primary injection. The antigen binding capacity was high at 2.8 x 10(-9) M (interquartile range, 5.1 x 10(-10) to 7.25 x 10(-9) M). The treatment was well tolerated with no systemic side effects seen. Myalgia at the injection site was seen in 46% of patients with severe pain caused by the formation of a sterile abscess seen in 14% of patients. The abscesses were all drained under ultrasound guidance, and the patients recovered fully within 6 weeks. No radiological responses were seen, but two patients had stable disease. G17-DT immunization produces anti-G17 antibodies in patients with advanced colorectal cancer. The antibodies were of an affinity high enough to compete with the cholecystokinin B/gastrin receptor for G17 binding with adequate capacity to neutralize postprandial gastrin surges. Additional dose-ranging studies have been performed in patients with gastric cancer using 100- and 200-microg doses of G17-DT formulated without adjuvant and the emulsifier aluminum monostearate. In addition, the effect of immunizing at different time intervals has been determined.

Oestrogen and progesterone receptors in gastrointestinal cancer cell lines.

Expression of sex steroid receptors by gastrointestinal (GI) tumours may indicate a role for hormonal manipulation in their management. A panel of seven established cell lines derived from primary human GI tumours were assayed for oestrogen and progesterone receptor (ER and PgR) expression by ligand binding, immunocytochemistry and enzyme-immunoassay methods. It was possible to demonstrate very low levels of both ER and PgR in the GI cell lines using enzyme immunoassay (EIA), with levels of PgR generally higher than those for ER. Neither ER nor PgR were detected in cytosols made from the GI cell lines in a classical dextran coated charcoal ligand binding assay. Similarly, immunohistological analysis (Abbott ERICA, PgRICA) of cultured cell preparations or of frozen and paraffin sections of xenograft tumour failed to demonstrate receptors. This confirms that low PgR levels are measurable by EIA in GI tumour cell lines. Upregulation of PgR expression in tumours by exposure to oestradiol in vitro was not observed.

The effect of onapristone, a progesterone antagonist, on the growth of human gastrointestinal cancer xenografts.

Onapristone is a progesterone antagonist which inhibits the growth of mammary tumours in mice. The effect of Onapristone and concomitant oestrogen (E2) supplements on the growth of five human gastrointestinal cancer xenografts was examined. E2 stimulated RD19 (gastric tumour) tumour growth in female mice and tumours grew less well when also treated with Onapristone (P < 0.05). Onapristone had no effect on male mice bearing RD19 tumours or mice of either sex bearing MKN45G (gastric) tumours. PAN-1 (pancreatic) tumours were significantly stimulated by E2 (by 64% of control, P = 0.02) and Onapristone treatment inhibited E2 stimulated growth (52% reduction of E2 control, P > 0.05). C146 (colorectal) tumour growth was not stimulated by E2 nor inhibited by Onapristone. E2 stimulated formation of AP5LV (colorectal) tumour nodules (in lungs) (mean 38-52, P = 0.001). Onapristone significantly reduced the number of nodules (mean 32, P < 0.05) only in female mice not given E2. Xenografts of some GI tumour cell lines grow at different rates in male and female mice. E2 may cause additional growth stimulation and E2 stimulated growth can be reversed by Onapristone to basal levels.

Effect of preoperative radiotherapy on matrilysin gene expression in rectal cancer.

Matrilysin, a member of matrix metalloproteinase family, is believed to play a significant role in the growth and proliferation of colon cancer cells. Overexpression of the matrilysin gene has been shown to correlate with Dukes' stage and increased metastatic potential in colorectal cancer. The aim of this study was to evaluate the effect of preoperative high-dose radiotherapy (25 Gy in five fractions over 5 days) on matrilysin (MMP-7) gene expression, in patients with resectable rectal cancer, by a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Biopsy samples of tumour (n=30) and distant normal mucosa (n=12) from 15 patients were obtained pre- and post-radiotherapy. Messenger (m)RNA was extracted from all of the tissue samples and reverse transcribed to double-stranded cDNA. Quantitative RT-PCR was performed to study the effect of preoperative radiotherapy on matrilysin gene expression in both the tumour and normal mucosal specimens. Matrilysin mRNA values were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample. In 14 out of 15 cases, matrilysin mRNA was detected in the cancerous tissue. Although all six normal mucosal specimens expressed matrilysin mRNA, the levels were approximately 10-fold lower compared with those seen in the paired tumour samples. Preoperative radiotherapy led to a significant 6- to 7-fold increase (P=0.001) in the expression of matrilysin mRNA in rectal cancer tissue. In contrast, there was no significant change in the matrilysin mRNA expression of normal mucosal specimens post-radiotherapy. Preoperative high-dose radiotherapy upregulates matrilysin gene expression in rectal cancer. Matrilysin inhibition may be a useful preventive or therapeutic adjunct to radiotherapy in rectal cancer.

A hepatic invasive human colorectal xenograft model.

A hepatic invasive human colorectal xenograft model was derived in nude mice by selection through the liver of the parental cell line, C170. Following intraperitoneal injection, tumours selectively grew on the liver in > 80% of the animals within 15-20 days. The liver-invading xenograft line, renamed C170HM2, had a significantly greater expression of the Lewisx antigen compared to C170 (mean linear fluorescence per cell > 1000 compared with 500 for C170, P < 0.02). C170HM2 had significantly elevated proliferation (when compared with C170) in the presence of epidermal (P < 0.001) and basic fibroblast growth factor (P < 0.001). C170HM2 also mitogenically responded to type I collagen (derived from rat tails), unlike C170. C170HM2 tumours when invading the liver expressed both interstitial collagenase and gelatinase activity at the invading edge.