Toremifene resistance in a rat mammary tumour model.

The effect of Toremifene on cell growth in vitro was tested on the R3230AC rat mammary adenocarcinoma as model. Toremifene differed from Tamoxifen in that it did not induce resistance; some cross-resistance was observed in Tamoxifen tolerant tumour cell lines. Toremifene does not influence P-glycoprotein expression in this model and at the levels studied.     

Tumour-stromal interactions: Phenotypic and genetic alterations in mammary stroma - implications for tumour progression

In addition to the well documented role of cytokines in mediating tissue-level interactions, it is now clear that matrix macromolecules fulfil a complementary regulatory function. Data highlighted in the present review extend the repertoire of matrix signalling mechanisms, (1) introducing the concept of 'matrikines', these defined as proteinase-generated fragments of matrix macromolecules that display cryptic bioactivities not manifested by the native, full-length form of the molecule, and (2) indicating that a previously identified motogenic factor (migration stimulating factor [MSF]) produced by foetal and cancer patient fibroblasts is a genetically generated truncated isoform of fibronectin, which displays bioactivities cryptic in all previously identified fibronectin isoforms. These observations are discussed in the context of the contribution of a 'foetal-like' stroma to the progression of breast cancer.     

Bone marrow and tumour stroma: an intimate relationship

In recent years the bone marrow has become recognized as a potential source of cells for non-haematopoietic wound healing, in some instances demonstrating surprising plasticity in providing new epithelial cells. On the other hand, the contribution of bone marrow derived cells to fibrosis and blood vessel formation is more widely acknowledged. Tumour stroma has a vital role to play in determining cancer growth and spread, and there is a growing realization that the bone marrow has a significant input into this desmoplastic response. This review focuses on the contribution of bone marrow cells to tumour stroma, highlighting the bone marrow as a potential new portal through which to direct anti-tumour therapies.     

Interstitial collagenase from rat mammary carcinoma cells: interaction with substrates and inhibitors.

Functional characteristics of the interstitial collagenase purified from the BCl rat mammary carcinoma cell line were examined and compared with literature reports of the corresponding characteristics of collagenase from non-neoplastic cells. BCl collagenase degraded soluble collagen types I, II and III at the same rate and degraded fibrillar tendon collagen with an activation energy of 75 kcal/mol; these characteristics were identical to collagenase from normal rat uterine smooth muscle cells. Degradation of fibrillar collagen by BCl collagenase was completely inhibited by rat alpha 2-macroglobulin which was concomitantly cleaved into half-fragments. BCl collagenase was also inhibited by native and recombinant tissue inhibitor of metalloproteinases, a synthetic peptide collagenase inhibitor (Z-pro-leugly-NHOH), and Zn2+. In all functional characteristics examined, BCl collagenase was the same as interstitial collagenases from non-neoplastic sources.     

A prolactin-dependent, metastasising rat mammary carcinoma as a model for endocrine-related tumour dormancy

In order to study the growth kinetics of breast tumours during long-term hormonal withdrawal, we developed a transplantable, invasive mammary carcinoma EMR-86 that originated in a female WAG/Olac rat bearing a subcutaneously implanted oestrogen pellet (EP). Outgrowth of transplanted tumours occurs only in the presence of an EP, and metastases are formed in lungs and regional lymph nodes. Subsequent EP removal induces rapid regression. However, tumours do not disappear completely, as small nodules persist. These dormant tumour remnants can be restimulated even after long periods. Because EP-stimulated tumours regressed after treatment with bromocriptine and dormant tumours in non-oestrogenized rats grew out after treatment with perphenazine, prolactin is the major growth-stimulating hormone in this model. Cell kinetics in the growing, regressing and dormant phase were studied by immunocytochemical detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in tissue sections. BrdUrd labelling indices decreased from 21.6 +/- 3.0% to less than 1% within 7 days after EP removal. After prolonged hormonal withdrawal (up to 90 days) BrdUrd-labelled tumour cells could always be demonstrated (range 0.4-0.8%), without a concomitant increase in tumour volume. Additional treatment either with bromocriptine or with ovariectomy could not significantly reduce this residual proliferative activity, as demonstrated by continuous BrdUrd labelling experiments. The results indicate that in vivo dormancy may represent a steady state of cell division and cell loss, rather than an accumulation of cells in a non-cycling G0 state.     

The phosphocholine and glycerophosphocholine content of an oestrogen-sensitive rat mammary tumour correlates strongly with growth rate

An oestrogen sensitive rat mammary tumour was grown in two groups of female and one group of male hooded rats. The male group and one of the female groups were supplemented with oestrogen. The tumours grew most rapidly in the female supplemented group. When the tumours reached 1.5 cm in diameter they were harvested and the cell cycle distribution and number of cells actively synthesising DNA (bromodeoxyuridine (BrdU) labelling index) determined in each case. Chemical extracts were prepared from each tumour and the concentration of phosphorus-containing metabolites determined using high resolution NMR spectroscopy. The concentration of phosphocholine was found to correlate strongly with the number of cells in S-phase and the number of cells labelled with BrdU, whilst a highly significant negative correlation was observed between these two parameters and glycerophosphocholine. The concentration of phosphoethanolamine did not correlate with either of these measures of proliferation rate. The concentration of glycerophosphorylethanolamine showed a weak negative correlation with the number of cells in S-phase.     

Accumulating evidence for a drug-drug interaction between methotrexate and proton pump inhibitors

Background.

A number of medications are known to interact with methotrexate through various mechanisms. The aim of this article is to apprise practitioners of a new labeling change based on the accumulating evidence for a possible drug–drug interaction between methotrexate (primarily at high doses) and proton pump inhibitors (PPIs).

Methods.

The U.S. Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) database of spontaneous adverse event reports and the published literature were searched for cases reporting an interaction between methotrexate and PPIs.

Results.

A search of the AERS database and existing literature found several individual case reports of drug–drug interactions and three additional supportive studies that suggest potential underlying mechanisms for the interaction.

Conclusion.

There is evidence to suggest that concomitant use of methotrexate (primarily at high doses) with PPIs such as omeprazole, esomeprazole, and pantoprazole may decrease methotrexate clearance, leading to elevated serum levels of methotrexate and/or its metabolite hydroxymethotrexate, possibly leading to methotrexate toxicities. In several case reports, no methotrexate toxicity was found when a histamine H₂ blocker was substituted for a PPI. Based on the reviewed data, the FDA updated the methotrexate label to include the possible drug–drug interaction between high-dose methotrexate and PPIs. Physicians should be alerted to this potential drug–drug interaction in patients receiving concomitant high-dose methotrexate and PPIs.     

Epidemiological evidence for a common mechanism for neuroblastoma and differentiated thyroid tumour.

Because genetic predisposition probably plays an important role in the aetiology of most of childhood cancers, studies of second primaries occurring after these cancers may be particularly informative about possible common genetic mechanisms in both of these cancers. We have studied the incidence of thyroid tumours occurring after cancer in childhood in a cohort of 592 children treated before 1970. Among these children, six later developed a thyroid carcinoma, and 18 developed a thyroid adenoma. Radiation doses received to the thyroid by each of the irradiated children have been estimated using individual radiotherapeutic technical records. Thyroid carcinomas and thyroid adenomas were five times more frequent after irradiation for neuroblastoma than after irradiation for any other first cancer. This ratio did not depend on sex, nor on time elapsed since irradiation, nor on dose of radiation received for the thyroid gland. This result suggests that there is a common mechanism for the occurrence of neuroblastoma and of differentiated thyroid tumour.     

Adjuvant intraoperative photodynamic therapy diminishes the rate of local recurrence in a rat mammary tumour model.

The use of photodynamic therapy (PDT) as an adjunct to curative tumour resection was investigated in a tumour recurrence model, using rat mammary adenocarcinoma BN472. Tumours were inoculated subcutaneously in 60 animals and resected after 21 days of growth. Immediately after removal, the operation site was exposed to 320-450 nm light of 0.1 W cm-2 and 60 J cm-2 after photosensitisation with either Photofrin (5 mg kg-1 i.v. 48 h before illumination) or 5-aminolaevulinic acid (ALA) (2 mg ml-1 in drinking water for 9 days). Porphyrin concentrations were measured in tissue samples. After 28 days, animals treated with adjunctive PDT had a significantly longer tumour-free interval than controls (P < 0.01); median 25 days (Photofrin), 18 days (ALA), 14 days (controls). Moreover, in the PDT groups significantly fewer rats had lymph node metastasis. A prophyrin concentration ratio between tumour and mammary tissue of 2:1 was found after Photofrin and 4:1 after ALA. The results indicate that adjuvant intraoperative PDT may be a safe and effective method of destroying residual tumour, thereby preventing locoregional tumour recurrence.     

The gross and cellular response of a rat mammary tumour to single doses of cyclophosphamide

The response of a transplanted mammary carcinoma of Wistar rats, to single intraperitoneal doses of cyclophosphamide (25–250 mg kg⁻¹) has been assayed by measurement of; delay in growth of the tumour between 9 and 25 mm; cell survival in vitro, 3 and 24 hr after in situ treatment; tritiated thymidine autoradiography and Feulgen microdensitometry of histological material. All three methods of assay indicate that at 9 mm diameter the tumour contains a subpopulation of malignant cells relatively resistant to cyclophosphamide doses in excess of 50 mg kg⁻¹. The in vitro cell survival assay suggests that these cells comprise 40–50% of the clonogenic population and are capable of considerable in situ repair of potentially lethal damage within 24 hr of treatment. Cellular repopulation from this resistant component, after 150 mg kg⁻¹ of drug, is delayed for 6 or more days and restoration to near pre-treatment status only achieved by 10 days, in this rapidly-growing tumour.     

Effects of photodynamic therapy on leucocyte-endothelium interaction: differences between normal and tumour tissue.

An inflammatory reaction is regularly noticed in irradiated tissues following photodynamic therapy (PDT). This observation is potentially associated with leucocyte-mediated tissue damage, which might further contribute to the tumoricidal effect of this therapy. The objective of our study was to investigate the effects of PDT on leucocyte-endothelium interaction in the microvasculature of tumours and normal tissue. Experiments were performed in the dorsal skinfold chamber preparation of Syrian golden hamsters bearing amelanotic melanoma A-Mel-3. The photosensitiser. Photofrin (5 mg kg-1 i.v.) was injected 24 h before laser irradiation (630 nm, 100 mW cm-2, 10 J cm-2 or 100 J cm-2). Post-capillary confluent venules (diameter 15-40 microns) of subcutaneous (s.c.) tissue or the amelanotic melanoma A-Mel-3 were observed by intravital microscopy before, 5, 30, 60 and 180 min after laser irradiation and recorded for off-line analysis. Before treatment, the number of adherent leucocytes in tumour vessels was only 22% of the number observed in vessels of s.c. tissue (P < 0.01). The maximum increase in adhering leucocytes was observed in post-capillary venules of s.c. tissue 1 h after PDT (P < 0.01). In contrast, enhanced leucocyte-endothelium interaction was missing in tumour vessels and in control groups. These results indicate that the tumour destruction observed after PDT is not mediated by leucocyte-endothelium interaction in the tumour. Induction of leucocyte adhesion in the PDT-treated normal tissue suggests a contribution to the peritumoral inflammatory response. Different maturational status or biochemical properties of tumour microvascular endothelium may explain the lack of leucocyte adherence upon PDT.     

VRP immunotherapy targeting neu: treatment efficacy and evidence for immunoediting in a stringent rat mammary tumor model

The ability to overcome intrinsic tolerance to a strict “self” tumor-associated antigen (TAA) and successfully treat pre-existing tumor is the most stringent test for anti-tumor immunotherapeutic strategies. Although this capacity has been demonstrated in various models using complicated strategies that may not be readily translated into the clinical arena, straightforward antigen-specific immunotherapeutic strategies in the most stringent models of common epithelial cancers have largely failed to meet this standard. We employed an immunotherapeutic strategy using an alphavirus-based, virus-like replicon particle (VRP), which has in vivo tropism for dendritic cells, to elicit immune responses to the non-mutated TAA rat neu in an aggressive rat mammary tumor model. Using this VRP-based immunotherapeutic strategy targeting a single TAA, we generated effective anti-tumor immunity in the setting of pre-existing tumor resulting in the cure of 36% of rats over multiple experiments, P = 0.002. We also observed down-regulation of rat neu expression in tumors that showed initial responses followed by tumor escape with resumption of rapid tumor growth. These responses were accompanied by significant anti-tumor proliferative responses and CD8+ cellular tumor infiltrates, all of which were restricted to animals receiving the anti-neu immunotherapy. Together these data, obtained in a stringent “self” TAA model, indicate that the VRP-based antigen-specific immunotherapy elicits sufficiently potent immune responses to exert immunologic pressure, selection, and editing of the growing tumors, thus supporting the activity of this straightforward immunotherapy and suggesting that it is a promising platform upon which to build even more potent strategies.     

Reproductive state of rat mammary gland stroma modulates human breast cancer cell migration and invasion

It has been established that the invasive behavior of cancer cells can be regulated by alterations in their extracellular environment. We investigated whether extracellular matrix isolated from nulliparous and postlactating (involuting) rat mammary glands differentially modulated the metastatic behavior of human breast cancer cells. Using modified Boyden chamber and three-dimensional culture assays, nulliparous mammary matrix was found to suppress motility and invasion in highly metastatic MDA-MB-435 cells, whereas involution mammary matrix supported motility and invasion in highly metastatic MDA-MB-435 cells, but not in cells with low metastatic potential. Biochemical characterization of the matrices revealed intact fibronectin (FN) and low matrix metalloproteinase activity in nulliparous mammary matrix and fragmented FN and high matrix metalloproteinase activity in the matrix isolated from involuting glands. Purified intact FN was found to inhibit cell invasiveness, whereas FN fragments enhanced cell invasiveness in a matrix metalloproteinase-dependent manner. These data suggest that physiological changes that occur in the mammary extracellular matrix as a result of reproductive status alter the in vitro parameters of metastatic potential.     

A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors

Invasion of tumor cells into the surrounding connective tissue and blood vessels is a key step in the metastatic spread of breast tumors. Although the presence of macrophages in primary tumors is associated with increased metastatic potential, the mechanistic basis for this observation is unknown. Using a chemotaxis-based in vivo invasion assay and multiphoton-based intravital imaging, we show that the interaction between macrophages and tumor cells facilitates the migration of carcinoma cells in the primary tumor. Gradients of either epidermal growth factor (EGF) or colony-stimulating factor 1 (CSF-1) stimulate collection into microneedles of tumor cells and macrophages even though tumor cells express only EGF receptor and macrophages express only CSF-1 receptor. Intravital imaging shows that macrophages and tumor cells migrate toward microneedles containing either EGF or CSF-1. Inhibition of either CSF-1- or EGF-stimulated signaling reduces the migration of both cell types. This work provides the first direct evidence for a synergistic interaction between macrophages and tumor cells during cell migration in vivo and indicates a mechanism for how macrophages may contribute to metastasis.