Animal Models Of Bone Metastasis Research Articles

Development of a New Focal Mouse Model of Bone Metastasis in Renal Cell Carcinoma.

Developing animal models of bone metastasis in renal cell carcinoma (RCC) is challenging as immunodeficient mice are required. The aim of this study was to develop a simple immune model of RCC bone metastasis. RENCA tumor cells were injected into the right femurs of BALB/c mice. Sixty mice were grouped into each twenty-mouse group according to the tumor cell concentration, and the presence or absence and extent of bone metastasis in the total length of the femur were compared using hematoxylin and eosin staining of the excised tissues. Bone metastasis was significantly higher in the high concentration group than in the other groups (p<0.05), with 10 mice developing bone metastasis at two weeks and nine mice developing bone metastasis at three weeks. The extent of bone metastasis was significantly greater in the high concentration group than in the other groups (p<0.05). Multiple logistic regression analysis was performed to examine the factors influencing bone metastasis, and only the high concentration was a significant factor (p<0.05). We developed a normal immunity mouse model of local bone metastasis from RCC. This model could prove valuable for research into the treatment of bone metastases in RCC.

Mouse Models of Tumor Bone Metastasis and Invasion for Studying CCN Proteins.

Bone metastasis and bone destruction are common occurrences in human malignancies, including breast, prostate, and lung cancer, and are associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis and bone destruction are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the left cardiac ventricle. In addition, we introduce mouse model of the bone destruction by injection of SAS oral squamous cell carcinoma cells into the bone marrow space of the right tibial metaphysis. These assays can be applied to studies on roles of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) protein in tumor metastasis and development of treatment strategies targeting CCN proteins.

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model.

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Optimization of a syngeneic murine model of bone metastasis

Many cancers metastasize to the bones, particularly in cases of breast and prostate cancers. Due to the “vicious cycle” of cancer cells inducing bone resorption, which promotes further tumor growth, they are difficult to treat and may lead to extreme pain. These factors increase the urgency for emerging therapeutics that target bone metastases more specifically and effectively. Animal studies are essential to the development of any therapeutics, but also require robust animal models of human diseases. Robust animal models are often challenging to develop in the case of bone metastasis studies. Previous methods to induce bone metastasis include intracardiac, intravenous, subcutaneous via mammary fat pad, and intraosseous cancer cell injections, but these methods all have limitations. By contrast, the caudal artery route of injection offers more robust bone metastasis, while also resulting in a lower rate of vital organ metastases than that of other routes of tumor implantation. A syngeneic animal model of bone metastasis is necessary in many cancer studies, because it allows the use of immunocompetent animals, which more accurately mimic cancer development observed in immunocompetent humans. Here we present a detailed method to generate robust and easily monitored 4T1-CLL1 syngeneic bone metastases with over 95% occurrence in BALB/c mice, within two weeks. This method can potentially increase consistency between animals in bone cancer metastasis studies and reduce the number of animals needed for studying bone metastases in mice.

Castration Determines the Efficacy of ETAR Blockade in a Mouse Model of Prostate Cancer Bone Metastasis.

Bone metastasis is a painful complication of advanced prostate cancer. Endothelin-1 is a tumor-secreted factor that plays a central role in osteoblast activation and the osteosclerotic response of prostate cancer metastatic to bone. Antagonists that block the activation of the endothelin A receptor (ETAR), located on osteoblasts, reduce osteoblastic bone lesions in animal models of bone metastasis. However, ETAR antagonists demonstrated limited efficacy in clinical trials of men with advanced prostate cancer who also received standard androgen deprivation therapy (ADT). Previous data from our group suggested that, in a mouse model, ETAR antagonists might only be efficacious when androgen signaling in the osteoblast is lowered beyond the ability of standard ADT. This notion was tested in a mouse model of prostate cancer bone metastasis. Castrated and sham-operated male athymic nude mice underwent intracardiac inoculation of the ARCaPM castration-resistant prostate cancer cell line. The mice were then treated with either the ETAR antagonist zibotentan or a vehicle control to generate four experimental groups: vehicle+sham (Veh+Sham), vehicle+castrate (Veh+Castr), zibotentan+sham (Zibo+Sham), and zibotentan+castrate (Zibo+Castr). The mice were monitored radiographically for the development of skeletal lesions. The Zibo+Castr group had significantly longer survival and a single incidental lesion. Mice in the Zibo+Sham group had the shortest survival and the largest number of skeletal lesions. Survival and skeletal lesions of the Veh+Sham and Veh+Castr groups were intermediate compared with the zibotentan-treated groups. We report a complex interaction between ETAR and androgen signaling, whereby ETAR blockade was most efficacious when combined with complete androgen deprivation.

Models of Prostate Cancer Bone Metastasis.

More than 80% of patients with advanced prostate cancer (PCa) experience bone metastasis, which negatively impacts overall survival and patient quality of life. Various mouse models have been used to study the mechanisms of bone metastasis over the years; however, there is currently no model that fully recapitulates what happens in humans because bone metastasis rarely occurs in spontaneous PCa mouse models. Nevertheless, animal models of bone metastasis using several different tumor inoculation routes have been developed to help study bone metastatic progression, which occurs particularly in late-stage PCa patients. This chapter describes the protocols commonly used to develop models of bone metastatic cancer in mice using different percutaneous injection methods (Intracardiac and Intraosseous). These models are useful for understanding the molecular mechanisms of bone metastatic progression, including tumor tissue tropism and tumor growth within the bone marrow microenvironment. Betterunderstanding of the mechanisms involved in these processes will clearly lead to the development of new therapeutic strategies for PCa patients with bone metastases.

Abstract 1086: Phosphoproteomic analysis of signaling pathways driving bone metastasis in prostate cancer

Abstract Osteoblasts play a fundamental role in prostate cancer bone metastasis, whereby dysregulated bone remodelling results in bone pain, fractures and increased mortality. Although it remains unclear how early on in the disease pathway prostate cancer cells populate the endosteal niche, osteoblasts almost certainly play a role in the homing of tumor cells to this microenvironment. In support of this, prostate cancer cells have been shown to home to osteoblast-rich areas in animal models of bone metastasis and human prostate cancer bone metastases are typically osteoblastic on radiography. We have shown that conditioned medium from mature, mineralising human hFOB1.19 osteoblasts (hFOB-CM) is rich in pro-inflammatory cytokines including IL-6, IL-8 and various chemokine ligands. hFOB-CM promotes a strong migratory and invasive phenotype in a panel of prostate cancer cell lines (including androgen-sensitive and -insensitive lines), with invading cells displaying increased actomyosin contractility and amoeboid-like traits, namely a rounded cell morphology, cytoskeletal reorganization and phosphorylation of myosin light chain. We now aim to comprehensively interrogate the intracellular signaling pathways driving endosteal homing in prostate cancer cells by systems-wide ITRAQ mass spectrometry-based phosphoproteomic analysis of prostate cancer cells treated with hFOB-CM. This approach will identify individual signaling pathways or combinations of pathways to target with existing kinase inhibitors to inhibit osteoblast-driven invasion and endosteal homing during prostate cancer metastasis. Ultimately, targeting tumor cell responses to stromal signals may prove critical in the search for better therapies to treat this frequently lethal disease. Citation Format: Benjamin Abbott, Andrew Pierce, Anthony D. Whetton, Paul A. Townsend. Phosphoproteomic analysis of signaling pathways driving bone metastasis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1086.

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaPM cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis.

Establishment of mouse model of bone metastasis of prostate cancer and breast cancer via femoral artery injection of tumor cells

To explore an efficient and reliable method for establishing an animal model of bone metastasis of tumors. Male C57BL/6J mice were randomized into 4 equal groups to receive injections of normal saline or prostate cancer RM-1 cell suspension via the femoral artery or the external iliac artery, and breast cancer 4T1-luc cells were injected in 15 female BALB/c mice via the femoral artery. The operation time, postoperative survival rate, and the tumor formation rates in the mice with two different injection methods were compared. The tumor metastasis in the mice was evaluated with in vivo imaging. The mean time for hemostasis time was 2.53∓1.75 min in mice receiving tumor cell injection via the femoral artery, significantly shorter than that in mice with injections via the external iliac artery (4.70∓1.63 min; P<0.05); the mean operation time was 14.67∓2.16 min and 22.47∓3.50 min in the two groups, respectively (P<0.05). At 21 days after the operation, the survival rate was 93.3% in femoral artery injection group, significantly higher than that in external iliac artery injection group (66.7%;P<0.05). The tumor metastasis rate was 100% in both groups. Injection of the tumor cells via the femoral artery is more suitable for establishing mouse models of bone metastasis of cancers.

Evaluation of antitumor activity and cardiac toxicity of a bone-targeted ph-sensitive liposomal formulation in a bone metastasis tumor model in mice

Chemotherapy for bone tumors is a major challenge because of the inability of therapeutics to penetrate dense bone mineral. We hypothesize that a nanostructured formulation with high affinity for bone could deliver drug to the tumor while minimizing off-target toxicity. Here, we evaluated the efficacy and toxicity of a novel bone-targeted, pH-sensitive liposomal formulation containing doxorubicin in an animal model of bone metastasis. Biodistribution studies with the liposome showed good uptake in tumor, but low accumulation of doxorubicin in the heart. Mice treated with the bone-targeted liposome formulation showed a 70% reduction in tumor volume, compared to 35% reduction for free doxorubicin at the same dose. Both cardiac toxicity and overall mortality were significantly lower for animals treated with the bone-targeted liposomes compared to free drug. Bone-targeted, pH-sensitive, doxorubicin containing liposomes represent a promising approach to selectively delivering doxorubicin to bone tumors while minimizing cardiac toxicity.

Analysis of Pathological Activities of CCN Proteins in Bone Metastasis.

Bone metastasis is a common occurrence in human malignancies, including breast, prostate, and lung cancer, and is associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the heart. This assay can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

Humanised xenograft models of bone metastasis revisited: novel insights into species-specific mechanisms of cancer cell osteotropism

The determinants and key mechanisms of cancer cell osteotropism have not been identified, mainly due to the lack of reproducible animal models representing the biological, genetic and clinical features seen in humans. An ideal model should be capable of recapitulating as many steps of the metastatic cascade as possible, thus facilitating the development of prognostic markers and novel therapeutic strategies. Most animal models of bone metastasis still have to be derived experimentally as most syngeneic and transgeneic approaches do not provide a robust skeletal phenotype and do not recapitulate the biological processes seen in humans. The xenotransplantation of human cancer cells or tumour tissue into immunocompromised murine hosts provides the possibility to simulate early and late stages of the human disease. Human bone or tissue-engineered human bone constructs can be implanted into the animal to recapitulate more subtle, species-specific aspects of the mutual interaction between human cancer cells and the human bone microenvironment. Moreover, the replication of the entire "organ" bone makes it possible to analyse the interaction between cancer cells and the haematopoietic niche and to confer at least a partial human immunity to the murine host. This process of humanisation is facilitated by novel immunocompromised mouse strains that allow a high engraftment rate of human cells or tissue. These humanised xenograft models provide an important research tool to study human biological processes of bone metastasis.

Abstract 5595: Imaging biomarker development for treatment efficacy for prostate cancer to the bone.

Abstract Approximately 70% of patients with late stage cancers metastasize to the bone. RECIST identifies these tumors as “unmeasureable” for therapeutic response assessment. Cabozantinib (CABO), a tyrosine kinase inhibitor of MET and VEGFR2, has shown promise as an effective therapy for metastatic prostate cancer to the bone. Patients treated with CABO showed a reduction in [Tc99m]-bone scan signal suggesting tumor response to treatment. Nevertheless, a return in signal to pre-treatment levels was observed in these patients following therapy completion. It has yet to be determined if the response observed in bone scans results from tumor death, changes in bone metabolism or vascular alterations in the tumor-stromal microenvironment resulting in reduced transport of the imaging tracer. The study objective was to assess tumor and bone response to CABO in an animal model of bone metastasis using a quantitative multi-modal imaging approach. Twenty-seven male SCID mice were implanted with PC3 cell line in the right tibia. PC3 cells were transfected with a plasmid expressing a bioluminescence imaging (BLI) reporter for measuring apoptosis. When tumor volumes reached 10mm3 by MRI, mice were distributed into 2 groups: CABO at 30 mg/kg (N=13) and vehicle (N=14). Treatments were delivered by oral gavage once a day for three weeks. Starting pre-treatment, MRI and BLI were acquired every third day and CT every week. Tumor and bone volumes were monitored by manually contouring volumes of interest (VOI) on anatomical MRI and CT images, respectively. The apparent diffusion coefficient (ADC), an indirect measure of tumor cellularity, was calculated from diffusion MRI. Mice treated with CABO were found to have significantly higher ADC values and slower growth profile than controls (p&amp;lt;0.05) suggesting a drop in tumor cellularity. Following one week of treatment, BLI signal increased 7 fold over baseline values indicating a delay in tumor apoptosis. In contrast, ADC values and apoptosis activation were unchanged in the controls resulting in significant differences between groups (p&amp;lt;0.05). As determined by CT, control mice had significantly more loss in bone volume than what was observed in the CABO treated mice, with differences first observed at day 14 post-treatment initiation (p&amp;lt;0.05). CABO was found to activate cell death through apoptosis (BLI) resulting in reduced tumor cellularity (ADC) and inhibited tumor growth (anatomical MRI). Increased tumor burden may have reduced osteolytic processes causing less bone loss over controls. Quantitative diffusion MRI and CT, backed by preclinical optical techniques, provide highly sensitive temporal information on the tumor-stromal microenvironment and their interaction to a targeted therapeutic intervention. Translatable quantitative imaging techniques, i.e. MRI and CT, may provide individualized medicine to patients suffering from metastatic prostate cancer to the bone. Citation Format: Joshua S. George, Craig J. Galban, Jean-Christophe Brisset, Benjamin Hoff, Stephanie Galban, Alnawaz Rehemtulla, Brian D. Ross. Imaging biomarker development for treatment efficacy for prostate cancer to the bone. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5595. doi:10.1158/1538-7445.AM2013-5595

Development of Radiopharmaceuticals for Diagnosis and Therapy of Metastatic Bone Cancer

Rhemium-186-1-hydroxyethylidene-1,1-diphosphonate ((186)Re-HEDP) has been used for the palliation of metastatic bone pain. However, delayed blood clearance and high gastric uptake of radioactivity have been observed upon injection, due to the instability of (186)Re-HEDP. We designed, synthesized and evaluated a stable (186)Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate, [[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl] carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate] oxorhenium(V) ((186)Re-MAG3-HBP). The stability of (186)Re-MAG3-HBP and (186)Re-HEDP in phosphate buffer were compared. No measurable decomposition of (186)Re-MAG3-HBP occurred, while only approximately 30% of (186)Re-HEDP remained intact 24 hours post-incubation. In biodistribution experiments, the radioactivity level of (186)Re-MAG3-HBP in bone was significantly higher than that of (186)Re-HEDP. Blood clearance of (186)Re-MAG3-HBP was faster than that of (186)Re-HEDP. In addition, the gastric accumulation of (186)Re-MAG3-HBP radioactivity was lower. To evaluate the therapeutic effects of (186)Re-MAG3-HBP, an animal model of bone metastasis was prepared. In the rats treated with (186)Re-HEDP, tumor growth was comparable to that in untreated rats. In contrast, when (186)Re-MAG3-HBP was administered, tumor growth was significantly inhibited. Bone pain was attenuated by treatment with (186)Re-MAG3-HBP or (186)Re-HEDP, but (186)Re-MAG3-HBP tended to be more effective. These results indicate that (186)Re-MAG3-HBP could be useful as a therapeutic agent of metastatic bone pain. Moreover, based on the similar concept, we designed, synthesized, and evaluated a (99m)Tc-6-hydrazinopyridine-3-carboxylic acid-conjugated bisphosphonate ((99m)Tc-HYNIC-HBP) as a bone scintigraphic agent. (99m)Tc-HYNIC-HBP gave higher levels of radioactivity in bone than (99m)Tc-HMDP. There was no significant difference in clearance from blood between (99m)Tc-HYNIC-HBP and (99m)Tc-HMDP. Consequently, (99m)Tc-HYNIC-HBP showed a higher bone-to-blood ratio than (99m)Tc-HMDP. The findings indicate that (99m)Tc-HYNIC-HBP holds great potential for bone scintigraphy.

Non-invasive MicroCT Imaging Characterization and In Vivo Targeting of BB2 Receptor Expression of a PC-3 Bone Metastasis Model

A devastating progression of human prostate cancer is the development of bone metastasis. Animal models of bone metastasis induced by inoculating human prostate cell lines into mice are well established. Here, we report the characterization of a mouse model of prostatic bone metastasis using non-invasive microCT and targeted microSPECT imaging of bone tumors using the bombesin receptor (BB2r)-avid radiolabeled peptide, (111)In-DOTA-8-Aoc-BBN[7–14]NH(2). Immunocompromised mice were inoculated with human prostate cancer cells by intracardiac injection. Metastatic lesion development was monitored by serially imaging mice weekly with microCT. Mice with CT imaging-confirmed bone lesions were administered (111)In-DOTA-8-Aoc-BBN[7–14]NH(2) for microSPECT imaging of BB2r expressing lesions. Metastatic bone lesions as small as 0.3 mm in diameter were detected by microCT image analysis as early as 21 days after tumor cell inoculation and had wide anatomical distribution. MicroSPECT imaging using (111)In-DOTA-8-Aoc-BBN[7–14]NH(2) successfully targeted BB2r expressing metastatic bone lesions of the tibia at day 29. MicroCT imaging can accurately and non-invasively follow the onset and progression of metastatic bone lesions in mouse models of prostate cancer. Micro-CT coupled with BB2r Micro-SPECT imaging affords the opportunity to obtain a combined receptor/anatomic map of metastatic bone lesion status in this mouse model.

An allogenic site-specific rat model of bone metastases for nuclear medicine and experimental oncology

Bone metastases are a major problem in several tumor entities affecting the therapeutic decision and the patient's prognosis. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are promising techniques for identifying bone tumors using gamma- or positron-emitting labeled radiotracers, but the same tracers if labeled with beta-emitters may also be used to apply therapeutic radionuclides for localized irradiation. For the tracer development specifically accumulating in osseous lesions, animal models of bone metastasis are needed. A technique was developed for tumor cell injection into the circulation of the hind limb of rats. For tumor implantation, the arteria epigastrica caudalis superficialis (a branch of the femoral artery) was cannulated, and 2×10(5) cells were injected. By using the allogenic Walker 256 mammary carcinoma cell line, isolated bone metastases were induced. For visualizing of the tumor growth, PET with 18F-fluoride was performed weekly on a μ-PET system. After 2-3 weeks, tumor invasion was confirmed by histology. Three weeks after tumor cell inoculation, PET images showed signs of bone metastases in 9 out of 11 animals. The tumors were located either in the proximal tibia/fibula or in the distal femur. At this time, the animals showed no restrictions in mobility. The tumors grew constantly over time. The final histological analysis showed tumors growing invasively into the bone matrix. With this model, new SPECT or PET tracers can be evaluated for their potency of accumulating in bone metastases in vivo and to determine which are therefore suitable for diagnosis and/or therapy.

Abstract 2255: In vivo study of serum cancer biomarkers and pain-induced bone metastasis of human prostate tumor in Nude rats

Abstract Bone is one of the most frequent sites of spread for many cancers such as breast, prostate, lung or kidney. Management of metastatic bone disease and associated pain can control the symptoms and prevent further complications such as the decrease of quality of life, pathological fracture or compression of the spinal cord. Until recently, investigation of novel drugs in the treatment of cancer-induced bone pain was hampered by a lack of suitable models. The aim of this study was to characterize biochemical markers and behavioral pain responses in an experimental animal model of bone metastases induced by intracardiac injection of PC-3 human prostate cancer cells in Nude rats. Firstly, potential differences of nociceptive responses between healthy Nude rats (classically used in cancer studies) and healthy SD rats (routinely used in pain studies) were assessed in 2 mechanical tests (electronic Von Frey and paw pressure tests) and 2 thermal tests (plantar and acetone tests). Pharmacological response to morphine was also assessed in both strains using the paw pressure test. Secondly, Nude rats were intracardiacally inoculated with PC3 cancer cells or RPMI medium and nociceptive responses were measured between tumor-bearing and non tumor-bearing Nude rats. Behavioral tests were carried out once a week during 4 consecutive weeks. In a satellite group, 15 days after tumor cell injection, 5 Nude rats received 4 intravenous injections of paclitaxel (5 mg/kg every 4 days). Development of bone metastases was monitored by ELISA quantification of serum biomarkers such as CTX-1 and TRACP 5b (bone resorption) and PINP (bone formation). Nociceptive tests in healthy SD and Nude rats did not demonstrate any major difference of hypersensitivity between both strains. Morphine (3 mg/kg, s.c.) induced a similar and classically observed analgesic effect in both strains. Any significant difference between Nude rats inoculated or not with cancer cells was observed neither in mechanical nor in thermal hyperalgesia assays. CTX-1 and TRACP-5b levels were significantly higher in tumor-bearing Nude rats than in non tumor-bearing ones. PINP level was lower in Nude rats injected with PC-3 cells than in non injected-rats. Paclitaxel significantly increased the life span of tumor-bearing Nude rats (ILS &amp;gt; 120%) and affected the kinetic of the 3 serum biomarkers. At sacrifice, CTX-1 and TRACP-5b serum levels were decreased by 51% and 95% respectively, and PINP level was increased by 325% compared to the non-treated rats. These results indicated that intracardiac injection of PC-3 human prostate cancer cells in Nude rats associated with bone formation/resorption serum biomarkers monitoring is a useful experimental in vivo model for bone metastases study and anti-cancer drug evaluation. Surprisingly, pain induced by experimental bone metastases was not demonstrated in this model with classical painful assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2255.

Cancer cell lines release glutamate into the extracellular environment

Bone is one of the most frequent sites for metastasis of breast and prostate cancers. Bone metastases are associated with pathologic changes in bone turnover and severe pain. The mechanisms that trigger these effects are not well understood, but it is postulated that tumour cells release factors which interfere with signalling processes critical to bone homeostasis. We have identified that several cancer cell lines known to cause bone disruption in animal models of bone metastasis appear to secrete glutamate into their extracellular environment in vitro. Although these cells also express specific glutamate receptors, the implications of this potentially disruptive chemical signal are discussed in relation to normal glutamate-dependent communication processes in bone and a possible mechanistic connection is made between tumour cell glutamate release and the development of pathological changes in bone turnover.

Unexpected Preferential Brain Metastases with a Human Breast Tumor Cell Line MDA-MB-231 in BALB/c Nude Mice

Animal models are useful tools to study etiology, progress, and new treatments of disease and are an approximation of human disease for experimental study. Intracardiac injection of the human estrogen-independent breast cancer cell line MDA-MB-231 in nude mice is a well-characterized animal model of bone metastasis mainly used to study new treatments for late-stage breast cancer. According to the published literature, this model should produce radiologically distinguishable bone tumors within 17 days after injection. Mice should develop complications such as cachexia, paraplegia, and morbidity within 28 days and require euthanasia within 35 days after injection. We report a study in which injection of MDA-MB-231 cell line led to brain rather than bone metastasis. Unexpected alterations in biological behavior are an important confounding variable in the use of tumor cell lines, and the occurrence and cause of such variants is poorly documented.

Interactions between microenvironment and cancer cells in two animal models of bone metastasis

The preferential proliferation of cancer cells in the bone microenvironment is poorly characterised. Expression pattern of bone marrow and other organ microenvironment in contact with osteolytic (Walker W256) and osteoblastic (MatLyLu MLL) metastases were investigated. Fisher and Copenhagen rats received, respectively, W256 and MLL cells injection. Bone and soft tissues were analysed by immunochemistry for DKK1, cathepsin K, RANKL, MCSF or IL6 expression. Tartrate-resistant acid phosphatase (TRAcP)-positive cells were detected by a histoenzymatic technique. In bone, expressions of MCSF and DKK1 were shown in stromal cells of the bone marrow, in contact with metastatic foci of both tumours. Many stromal cells were found RANKL positive in the vicinity of the tumours. Cells expressing cathepsin K and multinucleated TRAcP+ cells were found in direct contact with trabeculae but also in bone marrow spaces near metastatic cells. In extraosseous tumours, cells in contact with malignant cells did not expressed DKK1, MCSF, cathepsin K and IL6. Some RANKL+ cells were found in the periphery of subcutaneous tumours but may represent Langerhans cells. Abnormal presence of TRAcP+ cells was never observed in the vicinity of malignant cells. Interaction between stromal and cancer cells induces the expression on the formers of characteristics leading to osteoclastogenesis only in the bone microenvironment.