Immune human lymphocytes produce an acid-labile alpha-interferon.

We have described in this paper a novel human interferon (IFN) with antigenic and cross-species reactivity of alpha-IFN and physicochemical properties of gamma-IFN. This IFN is produced by normal peripheral blood mononuclear cells during an immune response but has also been associated with autoimmune disease (10). The system described here will be useful in elucidating the biological significance and cell of origin of this IFN.

A clinical study assessing the tolerability and biological effects of infliximab, a TNF-alpha inhibitor, in patients with advanced cancer.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is an important regulator of the chronic inflammation contributing to tumour progression. Infliximab, an anti-TNF-alpha monoclonal antibody was investigated in this trial of patients with advanced cancer. The primary objectives were to determine the safety profile and biological response of infliximab in a cancer population. Clinical response was a secondary objective. PATIENTS AND METHODS: Forty-one patients received infliximab at 5 mg/kg (n = 21) or 10 mg/kg (n = 20) i.v. at 0 and 2 weeks and then every 4 weeks. Post-treatment samples were measured for changes in plasma and serum TNF-alpha, CCL2, IL-6 and C-reactive protein (CRP). RESULTS: Infliximab was well tolerated with no dose-limiting toxic effects. At both doses of infliximab, neutralisation of serum TNF-alpha was observed after 1 h while plasma CCL2, IL-6 and serum CRP were decreased 24 and 48 h following infliximab administration. Seven patients experienced disease stablisation (range 10-50+ weeks). There was no evidence of disease acceleration in any patient. CONCLUSIONS: Infliximab treatment was safe and well tolerated in patients with advanced cancer. There was evidence of biological activity with baseline TNF-alpha and CCL2 being correlated with infliximab response.

Tumor necrosis factor and its receptors in human ovarian cancer. Potential role in disease progression.

The gene for tumor necrosis factor, TNF, was expressed in 45 out of 63 biopsies of human epithelial ovarian cancer. In serous tumors, there was a positive correlation between level of TNF expression and tumor grade. TNF mRNA was found in epithelial tumor cells and infiltrating macrophages, whereas TNF protein localized primarily to a subpopulation of macrophages within and in close proximity to tumor areas. mRNA and protein for the p55 TNF receptor gene localized to the tumor epithelium and tumor, but not to stromal macrophages. The p75 TNF receptor was confined to infiltrating cells. Cells expressing TNF mRNA were also found in ovarian cancer ascites and TNF protein was detected in some ascitic fluids. In 2 out of 12 biopsies of normal ovary, TNF mRNA was detected in a minority of cells in the thecal layer of the corpus luteum. Serum levels of TNF and its soluble receptor did not correlate with extent of TNF expression in matched biopsies. Northern and Southern analysis revealed no gross abnormality of the TNF gene. The coexpression of TNF and its receptor in ovarian cancer biopsies suggests the capacity for autocrine/paracrine action. TNF antagonists may have therapeutic potential in this malignancy.

The detection and localization of monocyte chemoattractant protein-1 (MCP-1) in human ovarian cancer.

Chemokines may control the macrophage infiltrate found in many solid tumors. In human ovarian cancer, in situ hybridization detected mRNA for the macrophage chemokine monocyte chemoattractant protein-1 (MCP-1) in 16/17 serous carcinomas, 4/4 mucinous carcinomas, 2/2 endometrioid carcinomas, and 1/3 borderline tumors. In serous tumors, mRNA expression mainly localized to the epithelial areas, as did immunoreactive MCP-1 protein. In the other tumors, both stromal and epithelial expression were seen. All tumors contained variable numbers of cells positive for the macrophage marker CD68. MCP-1 mRNA was also detected in the stroma of 5/5 normal ovaries. RT-PCR demonstrated mRNA for MCP-1 in 7/7 serous carcinomas and 6/6 ovarian cancer cell lines. MCP-1 protein was detected by ELISA in ascites from patients with ovarian cancer (mean 4.28 ng/ml) and was produced primarily by the cancer cells. Human MCP-1 protein was also detected in culture supernatants from cell lines and in ascites from human ovarian tumor xenografts which induce a peritoneal monocytosis in nude mice. We conclude that the macrophage chemoattractant MCP-1 is produced by epithelial ovarian cancer and that the tumor cells themselves are probably a major source. MCP-1 may contribute to the accumulation of tumor-associated macrophages, which may subsequently influence tumor behavior.

Positive interactions between human interferon and cyclophosphamide or adriamycin in a human tumor model system.

Human lymphoblastoid interferon strongly increased the anti-tumor activity of suboptimal doses of two commonly used anti-cancer drugs, cyclophosphamide and Adriamycin, on a human breast tumor xenograft growing in nude mice. A combination of human lymphoblastoid interferon with either of these agents caused regression and in some cases total disappearance of tumors at doses of drug and interferon that, used singly, were capable only of inhibiting tumor growth. The combined therapy also resulted in a greatly increased survival. Studies with interferon and cyclophosphamide indicated that the antitumor activity was greatest when the two agents were administered simultaneously rather than sequentially.

Investigation of cytokine gene expression in human colorectal cancer.

RNA was extracted from 28 samples of colorectal cancer and 26 samples of adjacent normal bowel. Northern blotting analysis showed the presence of mRNA for tumor necrosis factor (TNF) in 15 of 28 cancer samples and 6 of 26 matched normal areas. In 10 patients, TNF mRNA was found only in the tumor; in 5, TNF mRNA was seen in tumor and normal areas; and in only 1 was TNF mRNA seen in the normal, but not the malignant, area. The expression of TNF mRNA was not related to the stage of disease, degree of lymphocyte infiltration, or necrosis in the tumor. Blots were reprobed for gamma-interferon, interleukin (IL) 1 alpha and beta, IL-6, and transforming growth factor beta 1 mRNA. One tumor sample was positive for IL-1 beta, and one normal sample expressed interferon gamma mRNA. All samples had transforming growth factor beta 1 mRNA, and there was no obvious difference between levels in tumor tissues or adjacent normal areas. In situ hybridization studies with a TNF riboprobe showed that TNF mRNA was only detectable in a small minority of mononuclear and predominantly stromal cells. Immunohistochemistry on sequential sections showed that CD4- and CD8-positive lymphocytes, and macrophages, were present in the stroma. An antibody to the macrophage C3b receptor identified a minority population whose distribution corresponded closely to the cells labeled with the TNF riboprobe.

Intraperitoneal xenografts of human epithelial ovarian cancer in nude mice.

Using continuous human ovarian cancer cell lines, i.p. xenografts were successfully established in nude mice from four of four attempts. When primary tumor material was used, xenografts grew in 8 of 10 attempts. From these eight, three passageable xenograft cell lines have been established. To our knowledge, this is the first report published of such xenografts. I.p. xenografts closely mimic the clinical behavior of human ovarian cancer, and those developed from primary tumor material maintain close morphological similarity to the parent primary tumor. When expression of placental alkaline phosphatase and the tumor associated antigens defined by the monoclonal antibodies HMFG1, HMFG2, AUA1, and F36/22 by these models was determined, those i.p. xenografts derived from primary tumor material exactly matched the original tumor, while none of the xenografts derived from the cell lines expressed these antigens. These models will be useful for investigating the biology and treatment of ovarian cancer.

Therapeutic potential of tumor necrosis factor-alpha and gamma-interferon in experimental human ovarian cancer.

We have studied the activity of recombinant human gamma-interferon and recombinant human tumor necrosis factor alpha against four human ovarian cancer i.p. xenografts OS, LA, HN, and DO derived from primary tumor material. In the OS xenograft all control mice died by 42 days and therapy starting 7 days after tumor cell injection with 5 X 10(4) units recombinant human gamma-interferon or 1 microgram recombinant human tumor necrosis factor alpha alone had no significant effect on cumulative survival in three separate experiments. However, a combination of the two agents resulted in 85% cumulative survival at 150 days. This combination therapy also significantly increased survival of mice treated as late as 21 days after tumor cell injection. In the LA xenograft (where control mice were all dead by 23 days) therapy with either agent alone, or a combination, more than doubled survival time of mice. In the HN xenograft all control mice were dead at 22 days whereas either therapy alone or in combination gave +85% cumulative survival at 100 days. In a fourth xenograft, DO, survival of mice in the combination therapy group was significantly increased. Thus these two biological therapies, alone or in combination, show significant activity against human ovarian cancer cells.

Enhancement of metastatic potential by gamma-interferon.

Preincubation of murine colon 26 colon adenocarcinoma cells with gamma-interferon (IFN-gamma), but not alpha-interferon, produced a significant increase in experimental pulmonary metastases in syngeneic BALB/c and T-cell-deficient BALB/c nude mice. The enhancement was seen after as little as 1 h of exposure to 1 unit/ml of IFN-gamma and persisted for at least 72 h following removal of the cytokine. IFN-gamma exerted its effects by increasing the pulmonary retention of cells during the first 6 h following tumor cell injection. During this period all cells visualized in the lung were trapped in pulmonary capillaries. The enhancement was not due to modulations in class I major histocompatibility complex surface antigen expression; nor was it due to alterations in cell size, adhesion to components of the extracellular matrix in vitro, heterotypic or homotypic adhesion, sensitivity to lysis by activated peritoneal macrophages, osmotic fragility, enhancement of surface class II major histocompatibility complex antigen expression, or enhancement of intercellular adhesion molecule-1 (ICAM-1). Colon 26 was completely resistant to natural killer cell-mediated lysis in vitro, and IFN-gamma did not modulate the ability of colon 26 to form conjugates with isolated splenocytes. In vivo elimination of anti-asialo GM1 + cells increased pulmonary metastasis, and in such mice, there was no longer a difference in metastatic potential between control and IFN-gamma-treated cells. We conclude that low doses of IFN-gamma generated at the site of the tumor by host-infiltrating cells or during cytokine therapy could enhance the survival of tumor cells in the circulation and enhance their metastatic potential.

Augmentation of cytotoxicity of chemotherapy by human alpha-interferons in human non-small cell lung cancer xenografts.

Three human non-small lung cancer xenograft lines were used to study the activity of combinations of cytotoxic drugs with human alpha-interferons (IFNs). Statistically significant potentiation of cis-platinum (CDDP) and cyclophosphamide (CY) given weekly in a low dose was seen when human lymphoblastoid interferon (IFN-alpha nl) (2 X 10(5) mu/mouse/day) was administered simultaneously. The median tumor doubling times for CDDP in the three tumors (35, 22, and 29 days) increased to 52, 51, and 41 days when IFN-alpha nl was added. A similar though less marked effect was seen with CY (median doubling time increased from 21.5, 19.5, and 27 days to 32, 27, and 35 days with the addition of IFN-alpha nl). IFN-alpha nl alone at this dosage was shown to have some cytotoxic activity. Similar potentiation of CDDP and ifosfamide was seen in two tumors when human recombinant alpha-2 interferon was added at a lower dose (2 X 10(4) mu/mouse/day). Median doubling times for CDDP increased from 17 and 14 days to 27 and 18.5 days with the addition of human recombinant alpha-2 interferon, whereas for ifosfamide they increased from 11.5 and 14 days to 15 and 16 days. Human recombinant alpha-2 interferon in this dose had no effect as a single agent.

Antitumor activity of gamma-interferon in ascitic and solid tumor models of human ovarian cancer.

We have investigated the action of recombinant human gamma-interferon (rHuIFN-gamma) against human ovarian cancer xenografts growing as ascites or as bulky solid i.p. tumors in nude mice. Both forms of the disease responded to i.p. rHuIFN-gamma with significant increases in mouse survival time, and in 2 of 3 ascitic models the mice were cured of peritoneal disease. The activity of rHuIFN-gamma was dose and schedule dependent, and xenografts derived from 3 different patients showed a heterogeneity of response. Peak i.p. levels of rHuIFN-gamma in nude mice bearing multiple i.p. solid tumors were similar to those found in ovarian cancer patients receiving i.p. rHuIFN-gamma, but clearance was more rapid in the mice. Rat gamma-interferon had no antitumor activity at the same doses and schedules although it had some biological activity in the nude mice. Histological examination of treated tumors revealed increased necrosis and loss of cellular organization with large areas of hypocellular epithelial mucin. These changes were preceded by a fall in tumor tryptophan and a rise in tumor kynurenine. We conclude that rHuIFN-gamma has a direct dose related antitumor effect on ovarian cancer xenografts that is preceded by increased metabolism of tryptophan.

Human tumor xenografts treated with recombinant human tumor necrosis factor alone or in combination with interferons.

We have studied the activity of recombinant human tumor necrosis factor (rHuTNF) on six different human tumor xenografts derived from primary breast and bowel tumors and maintained by passage in nude mice. When 5 micrograms rHuTNF was given daily intratumorally to mice with established (approximately, 0.5 cm) tumors, total tumor regression was observed by 3-4 weeks in three of six xenograft lines. In a further two lines tumor stasis or significant slowing of growth was seen. This antitumor action was not accompanied by any consistent macroscopic change in the tumor such as necrosis, but histological examination revealed tumor cell degeneration and a large peritumoral infiltration of host inflammatory cells after 4-7 days therapy. In contrast to these data, little effect was seen when the same dose of rHuTNF was administered i.p. to nude mice bearing these tumors. In only two of six lines was any significant slowing of tumor growth seen. A 5-fold increase in the i.p. dose resulted in improved activity on only one of two xenograft lines tested. Efficacy of the i.p. rHuTNF dose could, however, be enhanced by simultaneous administration of human interferon, alpha or gamma. No obvious signs of toxicity were observed at all rHuTNF doses administered and weights of control and treated mice at the end of the experiments were comparable.

Epithelial cancer cell migration: a role for chemokine receptors?

We investigated the possibility that chemokine gradients influence migration of human ovarian epithelial tumor cells. Of 14 chemokine receptors investigated, only CXCR4 was expressed on ovarian cancer cells. CXCR4 mRNA localized to a subpopulation of tumor cells in ovarian cancer biopsies. Ovarian cancer cell lines and cells freshly isolated from ascites expressed CXCR4 protein. The CXCR4 ligand, CXCL12, was found in ascites from 63 patients. CXCL12 elicited intracellular calcium flux and directed migration and changes in integrin expression in ovarian cancer cells. CXCR4 may influence cell migration in the peritoneum, a major route for ovarian cancer spread, and could be a therapeutic target.

A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts.

We have examined the effect of a synthetic low-molecular-weight matrix metalloproteinase inhibitor, [4-(N-hydroxyamino)-2R-isobutyl-3S- (thiopen-2-ylthiomethyl)-succinyl]-L-phenylalanine-N-meth yla mide (BB-94), on human ovarian carcinoma xenografts growing in nude mice. The xenografts grew as thick intraperitoneal mucinous ascites containing free-floating tumor cell clumps. The ascites increased in volume, causing death approximately 3 weeks after introduction. Treatment with BB-94 caused resolution of ascitic disease. Tumor burden was dramatically reduced, and survival increased 5-6-fold. The increase in survival was dose dependent. The effects observed with BB-94 appeared to be due to its matrix metalloproteinase inhibiting effects, inasmuch as its inactive diastereoisomer had no effect on tumor biology. Following treatment with BB-94, free-floating clumps of tumor cells became surrounded by a capsule of host cells. These clumps of tumor cells typically formed one small (approximately 8 mm) avascular tumor of bright white appearance loosely attached to fat in the peritoneum. Tumor cells within these capsules often appeared to be necrotic. Gel substrate analysis demonstrated that activated Mr 92,000 type IV collagenase was present in the xenografts. We propose that inhibition of this enzyme causes the transition of ascites to solid tumors, concomitantly slowing tumor cell growth and allowing the development of tumor stroma.

Positive interactions between interferon and chemotherapy due to direct tumor action rather than effects on host drug-metabolizing enzymes.

The mechanism of increased antitumor activity when human lymphoblastoid interferon [HuIFN-alpha(Ly)] and the drugs cyclophosphamide and Adriamycin are used in combination on a human tumor xenograft in nude mice has been investigated. HuIFN-alpha(Ly) did not affect hepatic levels of the drug-metabolizing enzymes cytochrome P-450 or the glutathione S-transferases. In contrast, mouse interferon caused significant and differential changes in the isozymic forms of these enzymes. However, addition of mouse interferon to the HuIFN-alpha(Ly)/cyclophosphamide or Adriamycin combinations had no effect on the final result, and did not increase the toxicity of the combination therapy. These data provide evidence that the increased activity of the combination therapy is due to effects on the tumor rather than on the host. Further studies showed significant perturbations in the tumor cell cycle after in vivo combination therapy. Cyclophosphamide caused an accumulation in G2 and the addition of HuIFN-alpha(Ly), which alone caused little change in cycle distribution, delayed this G2 block and strongly increased the number of cells in S phase. A similar, although less pronounced, effect was seen with HuIFN-alpha(Ly)/Adriamycin therapy. The increase in S phase seen in combined therapy may account for the synergy seen.

Experimental validation of a flat punch indentation methodology calibrated against unconfined compression tests for determination of soft tissue biomechanics.

Mechanical characterisation of soft biological tissues using standard compression or tensile testing presents a significant challenge due to specimen geometrical irregularities, difficulties in cutting intact and appropriately sized test samples, and issues with slippage or damage at the grips. Indentation can overcome these problems but requires fitting a model to the resulting load-displacement data in order to calculate moduli. Despite the widespread use of this technique, few studies experimentally validate their chosen model or compensate for boundary effects. In this study, viscoelastic hydrogels of different concentrations and dimensions were used to calibrate an indentation technique performed at large specimen-strain deformation (20%) and analysed with a range of routinely used mathematical models. A rigid, flat-ended cylindrical indenter was applied to each specimen from which 'indentation moduli' and relaxation properties were calculated and compared against values obtained from unconfined compression. Only one indentation model showed good agreement (<10% difference) with all moduli values obtained from compression. A sample thickness to indenter diameter ratio ≥1:1 and sample diameter to indenter diameter ratio ≥4:1 was necessary to achieve the greatest accuracy. However, it is not always possible to use biological samples within these limits, therefore we developed a series of correction factors. The approach was validated using human diseased omentum and bovine articular cartilage resulting in mechanical properties closely matching compression values. We therefore present a widely useable indentation analysis method to allow more accurate calculation of material mechanics which is important in the study of soft tissue development, ageing, health and disease.

Effects of interferons and other cytokines on tumors in animals: a review.

The term cytokine describes a group of protein cell regulators involved in the control of cell growth and differentiation in embryogenesis, immunity and inflammation. They are of low molecular weight, are produced locally, and act in an autocrine or paracrine manner. In the past decade their use as cancer therapy has become a reality. Thirty years ago mice were treated with the antiviral protein interferon (IFN) which not only produced a reduction in the incidence of virus-induced tumors but also slowed the development of transplantable tumors. This was one of the first indications that cytokines can be negative regulators of cell growth. Here we outline current knowledge of the actions of IFNs and other cytokines in animal models, and draw parallels with clinical trials to illustrate the invaluable nature of this preclinical and mechanistic work.

Hypoxia down-regulates MCP-1 expression: implications for macrophage distribution in tumors.

Monocyte chemoattractant protein 1 (MCP-1) is likely to contribute to the macrophage infiltrate in human ovarian carcinomas. Although MCP-1 is predominantly expressed by the tumor parenchyma, macrophages accumulate at highest density in necrotic regions, which are associated with low oxygen tensions. Tumor necrosis factor alpha (TNF-alpha) can stimulate MCP-1 production and is also present within ovarian carcinomas. We have investigated the effect of hypoxia both on MCP-1 expression in ovarian cancer cell lines and monocyte migration. Hypoxia down-regulated TNF-alpha-induced MCP-1 mRNA and protein production by ovarian cancer cells. The effect was mimicked by cobalt chloride and desferrioxamine, consistent with a specific oxygen-sensing mechanism. Unlike antioxidants, hypoxia did not inhibit nuclear factor KB mobilization. Monocyte migration in response to MCP-1 was also diminished under hypoxic conditions. Down-regulation of MCP-1 expression and the inhibition of monocyte migration are independent effects of hypoxia that may contribute to the distribution of macrophages within ovarian tumors.

Interferon gamma induces cell cycle arrest and apoptosis in a model of ovarian cancer: enhancement of effect by batimastat.

Locoregional human IFN-gamma may have activity against refractory ovarian cancer. We investigated this further in an ovarian cancer xenograft model. Administered at clinically relevant doses, intraperitoneal IFN-gamma prolonged the survival of mice bearing multiple established peritoneal tumours, with optimal treatment giving a 3-6-fold increase in median survival time. Daily dosing, which was superior to intermittent treatment, decreased DNA synthesis and induced apoptosis in tumour cells with maximal effects after 7-21 days treatment. This was preceded by an increase in p53 protein at 48 h. The effect of IFN-gamma was not enhanced by sequential treatment with carboplatin. However, the matrix metalloprotease inhibitor, batimastat, further increased mouse survival when given after IFN-gamma. Thus IFN-gamma is cytotoxic to ovarian epithelial cells in vivo and intensive locoregional dosing over short periods is effective. Sequential administration of novel agents that perturb the host/tumour relationship may be of benefit.

In situ detection of tumour necrosis factor in human ovarian cancer specimens.

In situ hybridisation was used to study the local expression of tumour necrosis factor (TNF) mRNA in human ovarian tumours. In 8 of 14 ovarian cancers studied, a minority of cells in the epithelial areas of the tumour contained TNF mRNA. In individual high-power fields as many as 8% of cells were positive for TNF mRNA. Immunohistochemical studies on sequential sections and the morphology of the positive cells led to the conclusion that the ovarian tumour cells were transcribing the TNF gene. There was immunohistochemical evidence of the production of TNF protein by the tumour cells and TNF protein in a tumour lysate. The production of TNF by human ovarian cancer cells may influence the biology of the tumour, contribute to neoplastic progression and alter the response to therapy.