Activation of the spinal EGFR signaling pathway in a rat model of cancer-induced bone pain with morphine tolerance.

Cancer-induced bone pain (CIBP) is considered to be one of the most difficult pain conditions to treat. Morphine, an analgesic drug, is widely used in clinical practice, and long-term use of morphine can lead to drug tolerance. Recent reports have suggested that inhibitors of epidermal growth factor receptor (EGFR) may have analgesic effects in cancer patients suffering from pain. Therefore, we sought to determine whether EGFR signaling was involved in morphine tolerance (MT) in a rat model of cancer-induced bone pain. In this study, Walker 256 mammary gland carcinoma cells were inoculated into the tibias of rats to provoke cancer-induced bone pain. Then, morphine was intrathecally administered twice daily for seven consecutive days to induce drug tolerance. We observed sustained increased in the protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 during the development of morphine tolerance in rats with cancer-induced bone pain by western blotting. The EGFR level was significantly increased in the MT and CIBP + MT groups, and EGFR was colocalized with markers of microglia and neurons in the spinal cords of rats. Inhibition of EGFR by a small molecule inhibitor markedly attenuated the degree of morphine tolerance and decreased the number of microglia, and the protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 were also reduced. In summary, our results suggest that the activation of the EGFR signaling pathway in spinal microglia plays an important modulatory role in the development of morphine tolerance and that inhibition of EGFR may provide a new therapeutic option for cancer-induced bone pain.

SAP102 contributes to hyperalgesia formation in the cancer induced bone pain rat model by anchoring NMDA receptors.

The pathogenesis of cancer induced bone pain (CIBP) is extremely complex, and glutamate receptor dysfunction plays an important role in the formation of CIBP. Synapse-associated protein 102 (SAP102) anchors glutamate receptors in the postsynaptic membrane. However, its effect on hyperalgesia formation in CIBP has not been clarified. This study investigated the role of SAP102 in the formation of hyperalgesia in rats with CIBP SAP102 is present in spinal dorsal horn neurons, but not in astrocytes or microglia. NMDAR-NR2B is localized with neurons. In addition, SAP102 and NMDAR-NR2B expression levels in spinal dorsal horn tissues were detected by Western blot and co-immunoprecipitation. Intrathecal injection of lentiviral vector of RNAi to knockdown SAP102 expression in the spinal dorsal horn significantly attenuated abnormal mechanic pain when compared to non-coding lentiviral vector. These findings indicate that SAP102 can anchor NMDA receptors to affect hyperalgesia formation in bone cancer pain.

cGMP and cGMP-dependent protein kinase I pathway in dorsal root ganglia contributes to bone cancer pain in rats.

STUDY DESIGN: A prospective, randomized experimental research. OBJECTIVE: To demonstrate the role of cGMP (cyclic guanosine monophosphate)-cGKI (cGMP-dependent protein kinase I) pathway in dorsal root ganglia (DRG) in bone cancer pain. SUMMARY OF BACKGROUND DATA: Treating bone cancer pain continues to possess a major clinical challenge because the specific cellular and molecular mechanisms underlying bone cancer pain remain elusive. cGMP and cGMP-dependent protein kinases pathway in DRG plays important role in nerve injury-induced hyperexcitability of DRG neurons, as well as neuropathic pain, however, whether this pathway participates in bone cancer pain is unknown. METHODS: The rat model of bone cancer pain was produced by intramedullary injection of rat breast cancer cells (Walker 256) into right tibia. Thermal hyperalgesia and mechanical allodynia were measured before and after administration of inhibitor of cGMP-cGKs pathway (Rp-8-pCPT-cGMPS). Immunofluorescence and reverse transcription-polymerase chain reaction were used to reflect expression of cGKI in DRG neurons, whereas the concentration of cGMP in DRG was tested using enzyme-linked immunosorbent assay method. Whole-cell patch clamp was used to record the hyperexcitability of small neurons in DRG with or without cGKs inhibitor after tumor cell implantation (TCI). RESULTS: TCI treatment significantly increased the concentration of cGMP in DRG and activity of cGKs in DRG and the spinal cord. TCI treatment also induced upregulation of cGKI messenger ribonucleic acid and protein in DRG, as well as enhanced hyperexcitability in DRG neurons. Spinal administration of Rp-8-pCPT-cGMPS, cGMP-cGKs inhibitor, significantly suppressed TCI-induced activation of cGMP-cGKI signaling, and hyperexcitability of DRG neurons. Meanwhile, in vivo intrathecal delivery of the Rp-8-pCPT-cGMPS significantly prevented and suppressed TCI-induced hyperalgesia and allodynia. CONCLUSION: From these results, we confirm that TCI treatment activates cGMP-cGKI signaling pathway and continuing activation of this pathway in DRG is required for hyperalgesia and/or hyperalgesia and allodynia after TCI treatment. LEVEL OF EVIDENCE: N/A.

[The evaluation of metastatic damage of the liver of the Wistar rats under conditions of spontaneous development of the Walker 256 carcinosarcoma].

There was held micro- and ultrastructural study of the liver of the Wistar rats in the dynamics of development of transplantable Walker 256 carcinosarcoma. The regularities of the progression of the tumor process in the form of liver metastases with appearance of intralobular metastases. In the early stages of tumor development it was marked activation of cytotoxic function of the liver with necrosis of tumor lesions accompanied by a decrease of the structural density of metastases. In advanced stages of development of the Walker 256 carcinosarcoma an estimation of parameters of tumor invasion in the liver showed an intensification of these processes with increased severity of inflammatory reactions.

Intrathecal injection of anti-CX3CR1 neutralizing antibody delayed and attenuated pain facilitation in rat tibial bone cancer pain model.

This study was designed to investigate the effect of intrathecal injection of anti-CX3CR1 neutralizing antibody on pain behaviors in the rat tibial bone cancer pain model. Syngeneic Walker 256 mammary gland carcinoma cells were injected into the tibia medullary cavity to establish the rat model of bone cancer pain. Ambulatory pain, mechanical hindpaw withdrawal threshold, and latency of paw withdrawal to a thermal stimulus were observed. Haematoxylin/eosin staining was used to observe the bone damage on day 21. Intrathecal injection of anti-CX3CR1 neutralizing antibody both delayed the development of ambulatory pain and hyperalgesia and attenuated established pain facilitation, but had no effects on destruction of bone. Our results suggest that intrathecal injection of anti-CX3CR1 neutralizing antibody delayed and attenuated pain facilitation in the rat tibial bone cancer pain model.

Bilateral downregulation of Nav1.8 in dorsal root ganglia of rats with bone cancer pain induced by inoculation with Walker 256 breast tumor cells.

BACKGROUND: Rapid and effective treatment of cancer-induced bone pain remains a clinical challenge and patients with bone metastasis are more likely to experience severe pain. The voltage-gated sodium channel Nav1.8 plays a critical role in many aspects of nociceptor function. Therefore, we characterized a rat model of cancer pain and investigated the potential role of Nav1.8. METHODS: Adult female Wistar rats were used for the study. Cancer pain was induced by inoculation of Walker 256 breast carcinosarcoma cells into the tibia. After surgery, mechanical and thermal hyperalgesia and ambulation scores were evaluated to identify pain-related behavior. We used real-time RT-PCR to determine Nav1.8 mRNA expression in bilateral L4/L5 dorsal root ganglia (DRG) at 16-19 days after surgery. Western blotting and immunofluorescence were used to compare the expression and distribution of Nav1.8 in L4/L5 DRG between tumor-bearing and sham rats. Antisense oligodeoxynucleotides (ODNs) against Nav1.8 were administered intrathecally at 14-16 days after surgery to knock down Nav1.8 protein expression and changes in pain-related behavior were observed. RESULTS: Tumor-bearing rats exhibited mechanical hyperalgesia and ambulatory-evoked pain from day 7 after inoculation of Walker 256 cells. In the advanced stage of cancer pain (days 16-19 after surgery), normalized Nav1.8 mRNA levels assessed by real-time RT-PCR were significantly lower in ipsilateral L4/L5 DRG of tumor-bearing rats compared with the sham group. Western-blot showed that the total expression of Nav1.8 protein significantly decreased bilaterally in DRG of tumor-bearing rats. Furthermore, as revealed by immunofluorescence, only the expression of Nav1.8 protein in small neurons down regulated significantly in bilateral DRG of cancer pain rats. After administration of antisense ODNs against Nav1.8, Nav1.8 protein expression decreased significantly and tumor-bearing rats showed alleviated mechanical hyperalgesia and ambulatory-evoked pain. CONCLUSIONS: These findings suggest that Nav1.8 plays a role in the development and maintenance of bone cancer pain.

Effect of alpha tocopherol acetate in Walker 256/B cells-induced oxidative damage in a rat model of breast cancer skeletal metastases.

The pathophysiological changes and the oxidative-antioxidative status were evaluated in the bone microenvironment of rat inoculated with Walker 256/B mammary gland carcinoma cells, and used alpha-tocopherol acetate (ATA) as a countermeasure. Walker 256/B cells were injected into the right femora of aged male rats. Animals were randomized into three groups: 12 rats were injected with saline (control group); 14 rats were injected with Walker 256/B cells (5x10(4)) in the medullar cavity (W256 group); 14 rats were inoculated with Walker 256/B cells and treated with ATA (45mg/kg BW) (W256+ATA group). After 20 days, rats were euthanized and the femurs were radiographed. Micro architectural parameters were measured by microcomputed tomography and histology. Serum, bone and bone marrow were evaluated for oxidative damage. In parallel, cell cultures were done in the presence of ATA and ROS were measured by fluorescence; apoptotic cells were determined in parallel. W256 groups had osteolytic damages with marked resorption of cortical and trabecular bone. W256+ATA animals presented marked osteosclerotic areas associated with tumor necrosis areas inside the bone cavity. Levels of lipid peroxidation and protein oxidation were found to increase in W256 rats; a significant reduction in SOD and GSH-p activities was also observed. W256+ATA group had significantly reduced oxidative damage, but not reversed back to the control levels. The present study shows that Walker 256/B cells induce skeletal metastases associated with oxidative damage in the bone microenvironment. ATA reduced the oxidative stress damage, enhanced osteosclerosis and tumor cell apoptosis both in vitro and in vivo.

Pharmacologic inhibitors of IkappaB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and prevent osteolytic bone metastasis in vivo.

The NF-kappaB signaling pathway is known to play an important role in the regulation of osteoclastic bone resorption and cancer cell growth. Previous studies have shown that genetic inactivation of IkappaB kinase (IKK), a key component of NF-kappaB signaling, inhibits osteoclastogenesis, but the effects of pharmacologic IKK inhibitors on osteolytic bone metastasis are unknown. Here, we studied the effects of the IKK inhibitors celastrol, BMS-345541, parthenolide, and wedelolactone on the proliferation and migration of W256 cells in vitro and osteolytic bone destruction in vivo. All compounds tested inhibited the growth and induced apoptosis of W256 cells as evidenced by caspase-3 activation and nuclear morphology. Celastrol, BMS-345541, and parthenolide abolished IL1beta and tumor necrosis factor alpha-induced IkappaB phosphorylation and prevented nuclear translocation of NF-kappaB and DNA binding. Celastrol and parthenolide but not BMS-345541 prevented the activation of both IKKalpha and IKKbeta, and celastrol inhibited IKKalpha/beta activation by preventing the phosphorylation of TAK1, a key receptor-associated factor upstream of IKK. Celastrol and parthenolide markedly reduced the mRNA expression of matrix metalloproteinase 9 and urinary plasminogen activator, and inhibited W256 migration. Administration of celastrol or parthenolide at a dose of 1 mg/kg/day suppressed trabecular bone loss and reduced the number and size of osteolytic bone lesions following W256 injection in rats. Histomorphometric analysis showed that both compounds decreased osteoclast number and inhibited bone resorption. In conclusion, pharmacologic inhibitors of IKK are effective in preventing osteolytic bone metastasis in this model and might represent a promising class of agents to the prevention and treatment of metastatic bone disease associated with breast cancer.

Transient increased expression of VEGF and MMP-1 in a rat liver tumor model after hepatic arterial occlusion.

BACKGROUND/AIMS: This study investigates the influence of hepatic arterial occlusion (HAO) on blood perfusion of transplanted cancer in rat's liver, and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-1 (MMP-1) and explores the mechanisms involved in transcatheter arterial embolization (TAE)-induced metastasis of liver cancer preliminarily. METHODOLOGY: Walker 256 carcinosarcoma was transplanted into rat's liver to create the liver cancer model. Hepatic arterial ligation (HAL) was used to block the hepatic arterial blood supply and simulate TAE. Rats bearing tumor were divided into three groups: control, laparotomy control, and HAL groups. Blood perfusion of tumor was analyzed by a Hoechst 33342 labeling assay. The level of serum VEGF was assayed by ELISA; and the expression of VEGF and MMP-1 mRNA was detected by in situ hybridization. RESULTS: Two days after HAL, the number of Hoechst 33342 labeled cells which represent the blood perfusion of the tumor directly and hypoxia of tumor indirectly in the HAL group decreased significantly compared with that in the control group (329.1+/-29.3 vs. 383.6+/-19.2, P<0.01). The level of serum VEGF in the HAL group increased significantly compared with that of the control group (92.5+/-43.9 pg/mL vs. 54.9+/-19.3 pg/mL, P<0.05). The expression of VEGF and MMP-1 mRNA in the tumor tissue of the HAL group increased significantly compared with that of the control and the laparotomy control groups (P<0.05). The blood perfusion data of the tumor, represented by number of Hoechst 33342 labeled cells, showed an inverse correlation with the expression of VEGF mRNA in tumor tissue (P<0.05). While 6 days after HAL, the blood perfusion of tumor in HAL group decreased and the expression of VEGF and MMP-1 increased only slightly, not significantly, compared with that in the control group. CONCLUSIONS: In conclusion, blockage of hepatic arterial blood supply results in transient decreased blood perfusion and increased expression of metastasis-associated genes VEGF and MMP-1 of transplanted liver cancer in rats. Decreased blood perfusion and hypoxia may be the major reason for up-regulated expression of VEGF. Better understanding of the mechanisms involved with TAE-induced metastasis may lead to the enhancement of the long-term effects of TAE for liver cancer.

Influence of hepatic arterial blockage on blood perfusion and VEGF, MMP-1 expression of implanted liver cancer in rats.

AIM: To investigate the influence of hepatic arterial blockage on blood perfusion of transplanted cancer in rat liver and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-1 (MMP-1), and to explore the mechanisms involved in transarterial embolization (TAE)-induced metastasis of liver cancer preliminarily. METHODS: Walker 256 carcinosarcoma was transplanted into rat liver to establish the liver cancer model. Hepatic arterial ligation (HAL) was used to block the hepatic arterial blood supply and simulate TAE. Blood perfusion of tumor in control, laparotomy control, and HAL group was analyzed by Hoechst 33342 labeling assay, the serum VEGF level was assayed by ELISA, the expression of VEGF and MMP-1 mRNA was detected by in situ hybridization. RESULTS: Two days after HAL, the number of Hoechst 33342 labeled cells which represent the blood perfusion of tumor directly and hypoxia of tumor indirectly in HAL group decreased significantly compared with that in control group (329+/-29 vs 384+/-19, P<0.01). The serum VEGF level in the HAL group increased significantly as against that of the control group (93 ng.L(-1)+/-44 ng.L(-1) vs 55 ng.L(-1)+/-19 ng.L(-1), P<0.05). The expression of VEGF and MMP-1 mRNA in the tumor tissue of the HAL group increased significantly compared with that of the control and the laparotomy control groups (P<0.05). The blood perfusion data of the tumor, represented by the number of Hoechst 33342 labeled cells, showed a good linear inverse correlation with the serum VEGF level (r=-0.606, P<0.05) and the expression of VEGF mRNA in the tumor tissue ( r =-0.338, P<0.01). CONCLUSION: Blockage of hepatic arterial blood supply results in decreased blood perfusion and increased expression of metastasis-associated genes VEGF and MMP-1 of transplanted liver cancer in rats. Decreased blood perfusion and hypoxia may be the major cause of up-regulated expression of VEGF.

An experimental approach of the Doppler perfusion index of the liver in detecting occult hepatic metastases: histological findings related to the hemodynamic measurements in Wistar rats.

Image-directed colour Doppler sonography has been successfully introduced for the detection of hepatic haemodynamic changes in the presence of liver metastases. The aim of our study was to correlate these haemodynamic changes with the liver histology at the time of measurement. We experimentally induced liver metastases in 30 male Wistar rats by inoculating Walker 256 tumour subcutaneously. The animals were assigned into three groups of ten and were studied sonographically at 4, 7 and 15 days after tumour implantation. Another group of ten normal animals were used as controls. Portal vein and hepatic artery measurements included resistance index (PVRI, HARI) and flow volume (PVFV, HAFV). Doppler perfusion index (DPI) of the liver was calculated as the ratio of HAFV/PVFV + HAFV. Liver histology followed each Doppler measurement. Metastases were first encountered on day 4, as small groups of cells in the connective tissue of the porta hepatis and the portal triads without apparent vascular association. Distinct elevation of HAFV and DPI was recorded in comparison with the controls (p = 0.0004 and p = 0.0005, respectively). PVFV reduction was subtle. Up to day 15 there were no significant changes in the measurements. Our data suggest that HAFV and DPI can efficiently detect early liver metastases and this is in accordance with existing clinical reports. Haemodynamic changes seem to originate from the early non-vascular phase of the metastases.

Effects of fluoxetine on the development of lung metastases induced by operative stress in rats.

Experiments were performed in order to evaluate the effects of fluoxetine, a selective inhibitor of neural serotonin transporter antidepressant, on the development lung metastases in rats subjected to laparotomy and injected (i.v.) with 10(4) Walker 256 (W-256) carcinosarcoma cells. The number of metastatic nodules on the surface of the lungs, as well as the percentage-area of metastases in the frontal section through pulmonary hilus were increased in rats subjected to sham-surgery or laparotomy. Treatment with fluoxetine (5 mg/kg) partially reversed those adverse effects of surgery, but the difference was clearer when it was administered before surgery was performed. Survival periods were also assessed and fluoxetine was found to decrease the lethality of rats exposed to surgery.

Variability and discontinuity of the pathognomonic systemic effects caused by Walker 256 tumor progression in rats.

Cancer pathognomonic systemic effects (PSE) have high individual variability. For this reason present data were collected daily and synchronized considering four main points: inoculation day, onset of PSE, aggravation and death. The subclinical period free of PSE ranged between 15.7 +/- 2.2 days, the clinical period was less variable, 8.9 +/- 0.5 days, divided in a moderate and a grave phase of nearly the same length. PSE involved disturbances of fundamental homeostatic regulations: appetite, sodium, water, immune, etc. PSE triggering correlated highly with survival (r2 = 0.95, P < 0.01), but poorly with primary tumor growth, and it was anticipated by metastases from 20.5 +/- 2.6 to 10.6 +/- 1.1 days (P < 0.01). After multifocal simultaneous inoculations, PSE triggering was anticipated to 4.2 +/- 0.2 days (marked reduction of individual variability), in the presence of small total-tumor masses, absence of macroscopic metastases, and without changes in the following clinical period features. PSE triggering seems to be a major prognostic indicator probably related to multifocal tumor growth.

Increased growth rate and tumor burden of spontaneously metastatic Walker 256 cancer cells in the skeleton of bisphosphonate-treated rats.

We have studied the effect of 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) on the morphology of rat bone and the metastatic behavior of Walker 256 (W256) cancer cells in the rat skeleton. Male Fischer rats (150-175 g) received s.c. injections for 7 days with APD (0.5 mg/kg body weight/day) (+ APD; n = 20) or with vehicle (-APD; n = 20). Subsequently, 10 + PD and 10 -APD rats received i.m. injections with W256 cells (+ W256), and the remaining rats received injections of vehicle (-W256). All rats were killed 14 days later. Trabecular bone volume was increased by 46 +/- 3% by APD treatment alone and was decreased by 56 +/- 7% (SEM) by W256 tumor burden alone. After 14 days of tumor burden, + APD/+ W256 rats had 3-fold more trabecular bone than did -APD/+W256 rats. Despite this bone-sparing effect, APD treatment of +W256 rats was associated with a 2.6-fold increase in skeletal tumor burden, while metastatic tumor burden in the liver, lungs, and kidneys was unaffected. The increased skeletal tumor burden in + APD/+ W256 rats was accompanied by an increase in the growth rate of W256 cells located in bone. Independent of APD treatment, W256 cells located adjacent to trabecular bone surfaces had greater growth rates than did W256 cells in the marrow, located > 50 microns from trabecular bone. In summary, the APD-induced increase in trabecular bone volume in rats is associated with a selective increase in skeletal tumor burden and an increased growth rate of W256 cells in the skeleton.

Prophylactic treatment of skeletal metastases, tumor-induced osteolysis, and hypercalcemia in rats with the bisphosphonate Cl2MBP.

BACKGROUND: The purpose of this study was to investigate the influence of a prophylactic bone protective treatment with the bisphosphonate dichloromethane/diphosphonic acid (Cl2MBP) in an experimental model of osteolysis with intraosseous implantation of the Walker carcinosarcoma 256 B. METHODS: The biphosphonate was given as a high-dose, short-term treatment (30 mg/5 days sc) followed by a treatment-free interval (1-7 weeks) or as a low-dose, long-term prophylactic treatment (2.5 or 5.0 mg/day/3 weeks sc). Osteolysis was measured with a radiographic and histologic grading system. RESULTS: The high-dose short-term prophylactic treatment was shown clearly to inhibit tumor osteolysis. The osteoprotective effect decreases with increasing length of the treatment-free interval. A similar positive result could be achieved following the low-dose long-term prophylactic treatment. Dosage could not be shown to influence the inhibition of tumor osteolysis in the long-term bone protective treatment. A possible direct influence of the treatment on tumor growth could be ruled out. The prophylactic treatment does not inhibit body weight increase. Animals treated prophylactically showed less weight loss than the controls after tumor implantation. CONCLUSIONS: These results show that a prophylactic treatment with Cl2MBPs protects the skeleton effectively against tumor osteolysis.

Stimulation of bone resorption results in a selective increase in the growth rate of spontaneously metastatic Walker 256 cancer cells in bone.

To test the hypothesis that bone metastasis is related to the rate of bone remodeling, we have examined the effect of enhanced bone resorption on the growth of spontaneously metastatic Walker 256 (W256) cancer cells. Bone resorption was stimulated in male Fischer rats by injecting Rice H-500 Leydig tumor cells subcutaneously. The resorptive response of the skeleton was confirmed in a pilot study by evaluating parameters of bone morphometry after 4, 7 and 10 days of tumor burden. The distal femoral epiphyses had 35 +/- 10% more osteoclast surface, 83 +/- 11% less osteoblast surface, and 46 +/- 5% less trabecular bone after 10 days of tumor burden, compared to non-tumor-bearing controls. To evaluate the effect of Leydig tumor-induced bone resorption on the growth response of W256 cells, 20 rats were injected intramuscularly with 2 x 10(7) W256 cells, and 20 rats were vehicle-injected. Two days later, 10 rats from each group were injected subcutaneously with Leydig tumor cells. Twelve days after W256/vehicle injection, rats were injected with [3H]thymidine, killed 2 h later, and their femurs, liver, lungs and kidneys were processed for histology. In rats injected with Leydig tumor cells only, enhanced bone resorption was confirmed by a 40 +/- 4% increase in serum calcium concentration, a 48 +/- 8% decrease in trabecular bone content, and a 72 +/- 15% decrease in osteoblast surface, compared with non-tumor-bearing rats. Metastatic W256 cells adjacent to trabecular bone in Leydig tumor-bearing rats had a 56 +/- 18% greater relative [3H]thymidine labeling index (TdR) than did W256 cells in the bones of non-Leydig tumor-bearing rats. The TdRs of W256 cells in the liver, lungs, and kidneys were not affected by Leydig tumor burden. In this model, enhanced bone resorption was associated with the selective growth promotion of metastatic W256 cells in bone, suggesting the existence of a bone-derived factor which is mitogenic to W256 cells.

A quantitative model for spontaneous bone metastasis: evidence for a mitogenic effect of bone on Walker 256 cancer cells.

A new model for the study of spontaneous bone metastasis has been developed which allows for the quantification of metastatic tumor burden and cancer cell growth rate, and which describes the progressive changes in bone morphology. Walker 256 (W256) cells or vehicle were injected into the left upper thigh muscle of male Fischer rats, which were killed 7, 10 or 14 days later. By day 7, metastases had appeared in the distal femur, in the glomeruli of the kidney, and diffusely throughout the liver and lungs. The extent of tumor burden in these organs increased over time. In the femur, 14 days of tumor burden was associated with a 53 +/- 10% decrease in trabecular bone content, a 61 +/- 15% increase in osteoclast surface, and a 95 +/- 10% decrease in osteoblast surface, as compared with non-tumor-bearing controls. By autoradiography, metastatic tumor cells in all organs were determined to have greater growth rates than did cells in the primary tumor. However, within the femur, W256 cells located adjacent to trabecular bone surfaces had a 33 +/- 7% greater growth rate than did W256 cells located > 50 microns from bone surfaces (P < 0.05), suggesting a mitogenic effect of bone.

Inflammatory cell infiltrates vary in experimental primary and metastatic brain tumors.

We have studied the cellular immune response that accompanies primary and metastatic brain cancers induced experimentally in rats by inoculation of RG-2 glioma and Walker 256 (W256) carcinoma cells, respectively. The inflammatory cell infiltrates were characterized with lectin histochemistry to visualize microglial cells and macrophages and with immunohistochemistry, using a panel of monoclonal antibodies, to detect major histocompatibility complex (MHC), lymphocytic, and macrophage antigens. The metastatic tumor was composed of a loose stroma with multiple, often large, necrotic areas, whereas the RG-2 glioma was composed of a dense collection of tumor cells showing only rare necrotic foci. Both tumor types were heavily infiltrated with microglia and/or macrophages, and these were positive for MHC Class II (Ia) antigens. Expression of MHC Class I antigens was absent from RG-2 glioma cells, but it was present in W256 metastatic carcinoma cells. The metastatic tumor was also characterized by a much heavier infiltrate of lymphocytes, as shown by the presence of cells positive for CD4, CD8, and leukocyte common antigens. These lymphocytic markers were absent from reactive microglia in the W256 carcinoma, whereas they were present in the RG-2 glioma. Polymorphonuclear leukocytes were seen only in the metastatic tumor. Our study delineates differences between the inflammatory cell infiltrates found in metastatic brain tumors and those found in primary brain tumors. The differences in cell composition and immunophenotype may indicate a more effective antitumor response in the metastatic tumor that could account for the observed tissue destruction.

[The anti-tumor efficacy of natural killer (NK) cells and interleukin 2 against experimental pulmonary metastasis in rats].

Rat spleen cells with NK activity were enriched by discontinuous percoll density gradient centrifugation. Morphologically, about 50% were large granular lymphocytes (LGL), and the majority of these were OX8+ and Asialo GM1+. Cytotoxicity assays in vitro showed that the activity of enriched NK cells was significantly higher than that of unseparated rat spleen cells. Intravenous administration of enriched NK cells plus intraperitoneal crude IL2 led to the successful regression of pulmonary metastasis Walker 256 carcinosarcoma in rats. The effect was much stronger than that caused by unseparated spleen cells plus IL2. The results indicate that adoptive therapy using NK cells instead of spleen cells, in combination with exogenous IL2, might improve the efficiency of tumor immunotherapy.

Imaging peripheral benzodiazepine receptors in brain tumors in rats: in vitro binding characteristics.

Peripheral benzodiazepine binding constants for transplanted RG-2 gliomas and HD and LK Walker 256 tumors (metastatic breast carcinoma) were determined in Wistar rats using autoradiography. In addition, Kd and Bmax parameters for peripheral benzodiazepine receptors on RG-2 tumors were directly visualized using digital image analysis of autoradiograms. High specific binding of [3H]PK11195, a selective peripheral benzodiazepine ligand, had excellent topographical correlation to areas of histologically verified tumor. Scatchard analysis suggested a single class of peripheral binding sites with similar binding affinities in RG-2 and LK Walker 256 tumors and normal cortex. Bmax was 20-fold greater in glial tumors and 11.6- and 10.6-fold greater in LK and HK Walker 256 tumors, respectively, compared to normal cortex. The location of metastatic tumors, either intracerebrally or subcutaneously, did not effect their Kd or Bmax values. Kd and Bmax values for RG-2 tumors were similar whether determined densitometrically or by direct visualization with image analysis. Binding parameters within normal brain were difficult to visualize by image analysis due to the low level of specific binding. The ability to label specifically intracerebral tumor cells and to characterize the binding parameters shown in this study suggest that peripheral benzodiazepine receptor ligands could be utilized by PET to analyze directly a variety of tumors in humans.