Breast cancer carcinoma-associated fibroblasts differ from breast fibroblasts in immunological and extracellular matrix regulating pathways.

Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix -related pathways were over-represented in CAFs when compared to NFs (p<0.001). Under hypoxic conditions, the expression levels of pyruvate dehydrogenase kinase-1 (PDK1) and pyruvate dehydrogenase kinase-4 (PDK4) were lower in CAFs when compared to NFs (fold changes 0.6 and 0.4, respectively). In normoxia, when compared to NFs, CAFs displayed increased expression of glucose transporter 1 (GLUT-1) and PDK1 (fold changes 1.5 and 1.3, respectively). With respect to the assessed surface markers, only CD105 was expressed differently in MSCs when compared to fibroblasts, being more often expressed on MSCs. Cells with myofibroblast features were present in both NF and CAF samples. We conclude, that CAFs differ distinctly from NFs at the gene expression level, this hypothesis was also tested in silico for other available gene expression data.

Tumor tissue inhibitor of metalloproteinases-1 (TIMP-1) in hormone-independent breast cancer might originate in stromal cells, and improves stratification of prognosis together with nodal status.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is shown to be a potential marker for poor prognosis in breast cancer, but the biology of TIMP-1 is only partially understood. In this study, TIMP-1 production was studied in a co-culture model of hormone-independent breast cancer cell lines and mesenchymal stem cells mimicking the stromal components of the tumor. In addition, the prognostic value of TIMP-1 was histologically evaluated in a clinical material of 168 patients with hormone-independent breast tumors. The hormone-independent breast cancer (BC) cell lines MDA-MB-231, M4A4 and NM2C5 did not produce TIMP-1 protein in measureable quantities. Six tested primary mesenchymal stem cell lines all produced TIMP-1. Co-culturing of mesenchymal stem cells and breast cancer cells resulted in positive immunocytochemical diffuse staining for TIMP-1 for both cell types. Culturing breast cancer cells with MSC-conditioned media resulted in a positive cytoplasmic immunoreactivity for TIMP-1, and TIMP-1 protein concentration in cell lysates increased 2.7-fold (range 1.1-4.7). The TIMP-1 mRNA levels remained unaffected in BC cells. This might suggest that breast cancer cells can take up TIMP-1 produced by stromal cells and are thus displaying cellular immunoreactivity. In addition, TIMP-1 was shown to improve stratification of prognosis in clinical material.

Long-term administration of clodronate does not prevent fracture healing in rats.

Clinicians have been concerned that fractures do not heal properly in individuals exposed to bisphosphonate treatment, a treatment that strongly affects bone metabolism. The current study attempted to clarify the long-term effects of clodronate (dichloromethylene bisphosphonate) treatment on fracture healing in growing rats. Clodronate was administered subcutaneously twice a week in a dose of 2 mg/kg or 10 mg/kg. Physiologic saline served as a control. After 24 weeks of treatment, the tibiae were fractured, and the treatment was continued for another 4 weeks and 8 weeks. At both end points the cross-sectional areas of the callus, measured by peripheral quantitative computed tomography, were greater in the clodronate-treated rats than in controls, but there were no significant differences in bone mineral density. There were no significant differences between treatments in radiologic healing, histomorphometry, or in mechanical failure load of the callus with the exception of increased tensile stiffness at a dose of 2 mg/kg at 4 weeks. Clodronate treatment does not seem to prolong the fracture healing process, even when administered on a long-term basis before the fracture. Clodronate increases the size of the callus, but has only a minor effect on its biomechanical properties. The current results suggest that long-term clodronate treatment does not inhibit fracture healing.

Clodronate prevents bone loss in aged ovariectomized rats.

The purpose of this study was to investigate the ability of clodronate to prevent ovariectomy (OVX)-induced osteopenia in aged rats. Fourteen-month-old female Sprague-Dawley rats (n = 166) were randomized into six groups. One group was sacrificed at the start of the study, four groups were ovariectomized, and one group was sham-operated (Sham). The OVX rats were given subcutaneously either vehicle (veh) or clodronate at doses of 3, 7, or 25 mg/kg once a week for 3 months, and the Sham rats were given the vehicle. At all dose levels clodronate inhibited trabecular bone loss in the distal femur and in the fourth lumbar vertebral body (L4), and decreased bone resorption as evidenced by urinary deoxypyridinoline excretion. The lowest dose of clodronate preserved serum osteocalcin and endosteal bone formation of secondary spongiosa in L4 at the level of the Sham/veh group. The OVX-induced increase in periosteal bone formation of femoral diaphysis was unaffected by two smaller doses of clodronate, but was decreased to the level of Sham rats after the highest dose. After 3 mg/kg clodronate, the percentage of femoral cortical bone area and the mean relative cortical thickness were higher compared with the OVX/veh group. There was a good positive correlation between the maximum load in three-point bending of the tibia and tibial ash weight. Normal lamellar pattern of newly formed cancellous and cortical bone was found after clodronate treatment. No signs of adverse accumulation of osteoid or any deleterious effect on mechanical strength of long bones and lumbar vertebrae were found.

A comparison of clodronate and indomethacin in the treatment of adjuvant arthritis.

OBJECTIVE AND DESIGN: The therapeutic effects of an anti-resorptive agent, clodronate, were compared with the effects of an anti-inflammatory agent, indomethacin, in rat adjuvant arthritis after therapy and after a follow-up time of two weeks. SUBJECTS: Eighty-one male Lewis rats, 6-7 weeks old, were immunized with heat-killed mycobacteria. TREATMENT: Fourteen days after immunization the animals were treated either with clodronate (50 mg/kg/day, subcutaneously), indomethacin (3 mg/kg/day, orally) or saline (controls) for two weeks. METHODS: Clinical signs of arthritis including the severity of paw swelling were assessed, biochemical variables were measured, and histological features of the non-decalcified tarsus with ankle, intertarsal and tarsometatarsal joints were evaluated for inflammatory soft-tissue, articular, and bone changes. RESULTS: The results indicated that clodronate and indomethacin suppressed significantly the intensity of inflammation, activity of beta-N-acetylglucosaminidase in inflamed hindpaw tissue, serum ICTP (cross-linked carboxyterminal telopeptide of type 1 collagen) level and bone lesions in the tibiotarsal region. The level of serum osteocalcin was also significantly decreased by clodronate. The inhibitory effect of clodronate against arthritic bone changes occurred, however, earlier and was slightly more potent than the effect of indomethacin, while indomethacin was slightly more effective in reducing paw swelling. Both drugs preserved their therapeutic effects during the follow-up time of two weeks. CONCLUSIONS: Clodronate and indomethacin have fairly similar efficacy in suppressing the intensity of joint swelling and preventing bone lesions in adjuvant arthritic rats.

Slow-release clodronate in prevention of inflammation and bone loss associated with adjuvant arthritis.

The effects of slow-release calcium clodronate on rat adjuvant arthritis were investigated using two different dosing schedules. In prophylactic treatment, calcium clodronate was given on the same day as the adjuvant injection, and in therapeutic treatment, calcium clodronate administration was delayed until the animals had active disease, to day 14 postadjuvant. Calcium clodronate was given as single i.m. injections into the thigh muscles. Arthritis index, histopathology of hindpaw, quantitative histomorphometry, bone mineral density and serum osteocalcin, alkaline phosphatase and calcium were studied. Calcium clodronate given therapeutically decreased the severity of paw swelling slightly more than prophylactic treatment, a result seen as lower scores of arthritis index. Histopathological evaluation of hindpaws showed that calcium clodronate protected against inflammation-induced bone loss and reactive bone formation in the hindpaw, but not against inflammatory changes involving articular cartilage. Quantitative histomorphometric analysis of the distal femur indicated that trabecular bone area was decreased by 86% in arthritic rats compared with normal untreated controls. Both the prophylactic and the therapeutic treatment with calcium clodronate prevented this osteopenia (P < .001). Bone mineral density measured by computed tomography was also significantly reduced in distal femoral metaphysis in adjuvant arthritic rats, but restoration to virtually normal values occurred with calcium clodronate (P < .001). In both dosing schedules, we observed a suppression of arthritis, which was associated with a decrease in paw swelling and an inhibition of the severe osteopenia in the distal femoral metaphysis. The long duration of action after a single injection of calcium clodronate indicates that the insoluble salt remains at the injection site and is released slowly into the bloodstream.

Distribution of clodronate in the bone of adult rats and its effects on trabecular and cortical bone.

Distribution of clodronate in cancellous and cortical bone of the femur and in cancellous bone of lumbar vertebrae in adult rats was examined by means of quantitative autoradiography. In addition, the effects of clodronate on cancellous and cortical bone were evaluated by bone histomorphometry. Six-month-old male rats were given a mixture of unlabeled and 14C-labeled disodium clodronate subcutaneously on 5 consecutive days at cumulative doses of 125 mg/50 microCi/kg or 250 mg/100 microCi/kg and followed up for 2, 23 or 79 days after the last dose. The highest activity of 14C-clodronate was found in the primary spongiosa of the distal femoral metaphysis and in the cortical bone of the femoral diaphysis. Radioactivity in the lumbar vertebra was found to be about half of that in the femur. No marked decrease in radioactivity was found in bone specimens taken after the follow-ups. In these specimens, however, labeled clodronate originally incorporated into the primary spongiosa was situated further away from the growth plate because of longitudinal bone growth. A cross-section of the femoral shaft showed that incorporation of clodronate was more prominent into the periosteal surface than into the endocortical surface. No marked histological effects were seen, except for an increase in the mineralized hard tissue area in the primary spongiosa of the distal femur.