Fractionated radiotherapy initiated at the early stage of bone metastasis is effective to prolong survival in mouse model

Breast cancer metastasis to bone results in significant morbidity and poor prognosis. Radium-223 dichloride is an alpha-emitting calcium mimetic that localizes to bone and provides targeted radiation therapy. A phase III clinical study on prostate cancer patients with bone metastases showed that radium-223 dichloride improved overall survival (ALSYMPCA, Parker et al. ECCO/ESMO 2011). We have previously reported that radium-223 decreases osteolysis and tumor burden in bone in a mouse model of breast cancer bone metastasis in preventive and micro-metastatic settings (Suominen et al. AACR Annual Meeting 2012), as well as, in mice with established bone metastases (Suominen et al. AACR Annual Meeting 2011). Here, we investigated the effects of radium-223 dichloride monotherapy compared to and in combination with either doxorubicin or zoledronic acid on survival in a mouse model of established breast cancer bone metastasis. Human MDA-MB-231(SA)/GFP cells were inoculated intracardially into nude mice, and 15 days later, a single dose of vehicle, radium-223 dichloride (300 kBq/kg, iv injection) and/or zoledronic acid (0.1 mg/kg, sc injection) was administered. Doxorubicin (5 mg/kg, ip injection) was administered once weekly. Radium-223 dichloride monotherapy extended time to sacrifice (P = 0.039), unlike doxorubicin or zoledronic acid monotherapy which did not improve survival as compared to the vehicle group. Radium-223 dichloride in combination with zoledronic acid (P = 0.004) or doxorubicin (P < 0.001) also extended time to sacrifice as compared to the vehicle but did not provide additional survival benefit as compared to the radium-223 dichloride monotherapy. Histological examination revealed that radium-223 dichloride treatment induced tumor cell necrosis in bone metastases. Therefore, the effect of radium-223 dichloride in inducing double-strand breaks in cancer cells was evaluated by immunohistochemical staining of γ-H2AX molecules. A 3-fold increase in the number of tumor cells with double-strand breaks in the radium-223 dichloride-treated as compared to the vehicle control mice was observed (P < 0.001). This finding supports our previous observations that radium-223 dichloride has an effect on both tumor cells and osteoclasts. In conclusion, radium-223 dichloride therapy alone or in combination with doxorubicin or zoledronic acid increases survival in breast cancer bone metastasis mouse model via dual action by targeting tumor growth and osteolysis, both important players in the destructive vicious cycle of bone metastasis. Our findings strongly support the development of radium-223 dichloride for the treatment of patients with bone metastatic breast cancer. Citation Format: Mari I. Suominen, Jukka P. Rissanen, Rami Kakonen, Katja M. Fagerlund, Esa Alhoniemi, Dominik Mumberg, Karl Ziegelbauer, Jussi M. Halleen, Sanna-Maria Kakonen, Arne Scholz. Radium-223 dichloride monotherapy and combination therapy with zoledronic acid or doxorubicin improve survival in a mouse model of breast cancer bone metastasis. [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 LB-105. doi:10.1158/1538-7445.AM2013-LB-105

Bone metastases are a significant clinical problem in many major cancers, especially in breast and prostate cancer where 70-90% of advanced patients develop bone metastases. Myeloma bone disease is associated with similar clinical problems than bone metastases, including increased risk of fractures and bone pain that decrease the quality of life. Current cancer therapies can only partially decrease tumor growth, resulting in only 5% of bone metastatic patients being alive 5 years after the diagnosis. Bone metastases are therefore a high unmet medical need with a high demand for effective therapies. Lack of appropriate preclinical bone metastasis models that would exhibit the same clinical features that are observed in bone metastatic patients has made it difficult to advance therapy development at early stages. This study describes the establishment of three preclinical bone metastasis models, a breast cancer model using 4T1 mouse triple-negative breast cancer cells in BALB/c mice, a prostate cancer model using RM-1 mouse androgen-insensitive prostate cancer cells in C57BL/6 mice, and a multiple myeloma model using human RPMI 8226 cells in immunodeficient NPG mice. The cancer cells were inoculated intratibially into the bone marrow to model tumor growth in bone. Tumor growth was monitored by bioluminescence imaging (BLI), cancer-induced bone changes by X-ray imaging, and bone pain by Von Frey filaments (mechanical allodynia). In the 4T1 breast cancer model, 100% of the mice had bone metastases at day 7, and maximum study duration was 21 days. Osteolytic bone lesions were clearly observed and bone pain was detected at day 7. In the RM-1 prostate cancer model, 83% of the mice had bone metastases at day 7, and 100% of the mice at day 14, and maximum study duration was 28 days. Bone pain was observed at day 7, and osteolytic-mixed bone metastases were visible at day 14. In the RPMI 8226 multiple myeloma model, 100% tumor take rate was detected at day 7. Osteolytic bone metastases were visible at day 21, and maximum study duration was 100 days. We have established a clinically relevant Bone Metastasis Technology Platform (BMTP©) that currently includes preclinical bone metastasis models for breast and prostate cancer and multiple myeloma. These models have clinical features that are similar to those observed in bone metastatic patients. In preclinical models established in BMTP, tumor burden is monitored by BLI, the type and extent of cancer-induced bone loss is visualized by X-ray imaging, and bone pain is analyzed to provide a clinically relevant readout about the quality of life. We conclude that BMTP is a clinically relevant translational tool for evaluating efficacy of cancer therapies on bone metastasizing cancers. Citation Format: Tiina E. Kähkönen, Jie Wen, Ru Yang, Yuyang Xu, Michael Zhang, Jussi M. Halleen. Bone Metastasis Technology Platform: Establishing clinically relevant bone metastasis models for breast and prostate cancer and multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3927.

Oncolytic Adenovirus Expressing Soluble TGFβ Receptor II-Fc-mediated Inhibition of Established Bone Metastases: A Safe and Effective Systemic Therapeutic Approach for Breast Cancer

Bone metastases still present an unmet need for effective treatment. One preclinical study has suggested a beneficial effect with sequential treatment of doxorubicin (DOX) and zoledronic acid (ZOL). Furthermore, bisphosphonates are used as adjuvant therapy, but controversy of their anti-tumor effects remains. We tested clinically relevant doses of DOX, ZOL and their sequential combination in a treatment setting in a widely used nude mouse model of breast cancer bone metastasis. GFP-transfected MDA-MB-231(SA) human breast cancer cells were inoculated into five-week-old female nude mice via left cardiac ventricle. Subsequent tumor growth and development of osteolytic bone lesions were quantitated by fluorescence imaging and radiography, respectively, on days 14 and 25. The mice were randomized to four groups (n=8/group) on day 14 according to body weight and the presence of osteolytic lesions. Starting on day 15, groups received either weekly dose of vehicle (control group), DOX 2.5 mg/kg i.p. weekly, ZOL 0.1 mg/kg s.c. on day 15 and vehicle weekly, or weekly dose of DOX and a single injection of ZOL 24 hrs after the first dose of DOX (combination group). Bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b) was determined in serum samples collected on days 1, 9, 17 and 24. Bone samples were collected on day 25, and tumor area, trabecular bone area and apoptotic index (TUNEL and apoptotic morphology) were quantitated by histomorphometry. Statistical analysis was performed using One-way ANOVA followed by Dunnett's test or Kruskal-Wallis test followed by Mann-Whitney U-test. All animals in the control and DOX groups had lesions at sacrifice, whereas 12.5% of the mice in the ZOL group and 42.9% in the combination group had lesions. Osteolytic lesion area at sacrifice was significantly lower in both groups treated with ZOL compared to the control group, with no increase from day 14. Lesion area in the DOX-treated group was not different from control. Whole body tumor burden or the tumor area in hind limbs did not differ between the groups, indicating no inhibition on tumor growth. Apoptotic index was increased in groups treated with DOX. ZOL inhibited the increase in serum TRACP 5b, although less effectively in the combination group. Taken together, clinically relevant doses of DOX, ZOL or a sequential combination treatment of these two were not able to inhibit tumor growth in the established disease in this aggressive model of breast cancer bone metastasis. However, DOX increased the apoptosis of cancer cells and ZOL potently inhibited cancer-induced osteolysis, but the sequential treatment did not have additive affects on either tumor growth or osteolytic lesion area. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5394.

Override of the Osteoclast Defect in Osteopontin-Deficient Mice by Metastatic Tumor Growth in the Bone

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.

Overcoming tumor tolerance and immune suppression remains a formidable obstacle to the development of immunotherapeutics that harness the immune system to eradicate breast cancer. We have developed a syngeneic mouse model to study mechanisms of tumor tolerance and immune suppression during breast cancer bone metastasis. Comparative analysis of low metastatic vs. highly bone metastatic tumor cells isolated from mice revealed several important genetic alterations related to immune control of tumor progression. Of these, a significant downregulation of cd1d1 was observed in the highly bone metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells that bridge the innate and adaptive immune response to cancer, known as invariant natural killer T (iNKT) cells. Activated iNKT cells mediate antitumor immunity in cell culture and in mouse models in vivo. Clinically, iNKT levels are significantly reduced in solid tumors, and this reduction is correlated with a poor prognosis in many types of cancers, including breast cancer. It is well known that the majority of advanced breast cancer patients present with bone metastases, and NKT cells have been shown to comprise a significant portion of the T cells found in healthy bone marrow. We hypothesize that downregulation of CD1d by breast cancer cells enables evasion of iNKT immune surveillance and inhibition of iNKT-mediated antitumor immunity, and may be an important prerequisite for the ability of breast cancer cells to colonize bone. In this study, we demonstrate in a mouse model of breast cancer bone metastasis that tumor cells expressing CD1d can induce direct iNKT-mediated cytolysis in a CD1d-dependent manner in vitro, and demonstrate increased CD1d-dependent iNKT-mediated antitumor immunity in vivo. Using transgenic knockout (KO) models, we show that the ability of CD1d-expressing tumor cells to metastasize to bone is dependent on the presence of CD1 d-restricted NKT cells. The results of this study further support research into therapeutic strategies that boost iNKT-mediated antitumor immunity for the treatment of advanced metastatic breast cancer. In addition, we propose the use of our novel mouse model of breast cancer bone metastasis as an ideal system for conducting further studies into mechanisms of immune regulation of breast cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B21.

Transforming growth factor β (TGF-β) is a key regulator of several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparin sulfate which is a member of the glycosaminoglycan family of carbohydrates. Our subsequent studies with highly sulfated heparin-like glycosaminoglycans (HLGAGs) with low anti-coagulant activity indicated that a high-molecular-weight E. coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. We then evaluated the therapeutic potential of K5-NSOS and a low-molecular-weight synthetic heparin (fragmin) in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were then administered with K5-NSOS, fragmin, or vehicle once daily for 4 weeks. Both HLGAGs increased body weight, decreased osteolytic lesion area, and reduced tumor burden in bone. Our in vitro studies showed that both K5-NSOS and fragmin inhibited adhesion of breast cancer cells to ICAM-1, and K5-NSOS also inhibited adhesion to E- and P-selectin. Furthermore, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. Our data support and further clarify the critical role of heparan sulfate glycosaminoglycans in breast cancer metastasis and indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent with low anticoagulant activity that could be further optimized as an anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 845. doi:10.1158/1538-7445.AM2011-845

Bone metastases are associated with increased morbidity and poor prognosis in breast cancer patients. Radium-223 dichloride is a calcium mimetic that localizes to bone, providing targeted therapy for skeletal metastasis. We investigated the mode of action of radium-223 dichloride using breast cancer cell, osteoclast, and osteoblast cultures as well as a mouse model of breast cancer bone metastasis. A single dose of radium-223 dichloride was used in three different settings mimicking the prevention or treatment of bone metastasis. Disease progression was monitored using fluorescence and radiographic imaging and histological analyses. The effect of radium-223 dichloride alone and in combination with doxorubicin or zoledronic acid on survival of mice was analyzed by Kaplan-Meier methods. All statistical tests used were two-sided. Radium-223 dichloride incorporated into bone matrix and inhibited proliferation of breast cancer cells and differentiation of osteoblasts and osteoclasts (all P values < .001) in vitro. In an established bone metastasis setting, radium-223 dichloride prevented tumor-induced cachexia (0/14 vs 7/14 control mice) and decreased osteolysis by 56% and tumor growth by 43% (all P values < .05). Radium-223 dichloride induced double-strand DNA breaks in cancer cells in vivo. Finally, radium-223 dichloride extended survival as a monotherapy (29.2 days, 95% confidence interval [CI] = 26.6 to 31.8 days, P = .039) and in combination with zoledronic acid (31.4 days, 95% CI = 28.8 to 34.0 days, P = .004) or doxorubicin (31.5 days, 95% CI = 29.5 to 33.5 days, P < .001) compared to the vehicle group (24.9 days, 95% CI = 23.4 to 26.4 days). Similar but even more pronounced effects were observed when radium-223 dichloride was administered in a preventive or micrometastatic setting. Our findings strongly support the development of radium-223 dichloride for the treatment of breast cancer patients with or at high risk of developing bone metastases.

&lt;div&gt;Abstract&lt;p&gt;TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-&lt;i&gt;O&lt;/i&gt;-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β–induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight &lt;i&gt;Escherichia coli&lt;/i&gt; K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β–induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts &lt;i&gt;in vitro&lt;/i&gt;. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate &lt;i&gt;in vitro&lt;/i&gt; siRNA screening results toward &lt;i&gt;in vivo&lt;/i&gt; therapeutic concepts. &lt;i&gt;Mol Cancer Res; 10(5); 597–604. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.

&lt;div&gt;Abstract&lt;p&gt;TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-&lt;i&gt;O&lt;/i&gt;-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β–induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight &lt;i&gt;Escherichia coli&lt;/i&gt; K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β–induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts &lt;i&gt;in vitro&lt;/i&gt;. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate &lt;i&gt;in vitro&lt;/i&gt; siRNA screening results toward &lt;i&gt;in vivo&lt;/i&gt; therapeutic concepts. &lt;i&gt;Mol Cancer Res; 10(5); 597–604. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Bone metastases cause severe skeletal morbidity including fractures and hypercalcemia. Tumor cells in bone induce activation of osteoclasts, which mediate bone resorption and release of growth factors from bone matrix, resulting in a "vicious cycle" of bone breakdown and tumor proliferation. Receptor activator of NF-kappaB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival, and is blocked by a soluble decoy receptor, osteoprotegerin (OPG). In human malignancies that metastasize to bone, dysregulation of the RANK/RANKL/OPG pathway can increase the RANKL:OPG ratio, a condition which favors excessive osteolysis. In a mouse model of bone metastasis, RANKL protein levels in MDA-MB-231 (MDA-231) tumor-bearing bones were significantly higher than tumor-free bones. The resulting tumor-induced osteoclastogenesis and osteolysis was dose-dependently inhibited by recombinant OPG-Fc treatment, supporting the essential role for RANKL in this process. Using bioluminescence imaging in a mouse model of metastasis, we monitored the anti-tumor efficacy of RANKL inhibition on MDA-231 human breast cancer cells in a temporal manner. Treatment with OPG-Fc in vivo inhibited growth of MDA-231 tumor cells in bony sites when given both as a preventative (dosed day 0) and as a therapeutic agent for established bone metastases (dosed day 7). One mechanism by which RANKL inhibition reduced tumor burden appears to be indirect through inhibition of the "vicious cycle" and involved an increase in tumor cell apoptosis, as measured by active caspase-3. Here, we demonstrate for the first time that OPG-Fc treatment of mice with established bone metastases resulted in an overall improvement in survival.

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.

Breast cancer expressing estrogen receptor (ER) and progesterone receptor (PR) is classified as hormone receptor positive, and triple-positive breast cancer expresses also human epidermal growth factor receptors (HER2). Despite the hormonal and HER2 targeted treatments, breast cancer metastasizes to bone in high frequency and may develop resistance against the used treatment. Prevention and treatment of bone metastases is challenging and moreover, hormones are strong regulators of both bone and immune system. Aim of the present study was to verify and compare ER, PR, HER2 and also HER3 status in preclinical primary and bone metastasis breast cancer models utilizing immunodeficient and human immune system engrafted mice. BT-474 human breast cancer cells were inoculated orthotopically into mammary fat pad of placebo or 17β-estradiol (E2) supplemented female immunodeficient NOG mice. In a bone tumor study, BT-474 cells were inoculated into the tibia of female NOG or humanized NOG mice (HSCFTL-NOG-F mice, Taconic Biosciences). Tumor growth was followed for 8 weeks and histopathological tumor evaluation and immunohistochemical stainings for ER, PR, HER2 and HER3 were performed. Orthotopic tumor growth of BT-474 was hormone dependent and only minor growth was observed in the absence of E2. In the presence of E2 supplement, the orthotopic tumor expressed ER, PR and HER2/HER3. However, in the absence of E2 supplement there was reduced PR expression but no major changes in the ER and HER2/HER3 expression. In contrast, when breast cancer cells were inoculated into the tibia, tumor growth was observed also without E2 supplement. In this case, tumor in the bone was positive for ER and HER2/HER3 but negative for PR. No significant changes were observed between immunodeficient and humanized mice regarding intratibial tumor growth or ER, PR and HER2/HER3 expression. As a summary, estrogen supplementation is needed to support breast cancer BT-474 tumor growth when cancer cells are inoculated orthotopically into mammary fat pad. In contrast, BT-474 tumor growth was observed in bone also in the absence of supplied E2 in immunodeficient and humanized mice. ER and HER2/HER3 expression was observed in primary and bone tumors, but PR expression was significantly reduced if no estrogen supplement was used. Taking together, when developing new therapies against breast cancer, treatment targets in preclinical models should be carefully verified. Focus should be addressed not only on primary tumor but also on bone metastasis where cancer cells are under influence of different tumor microenvironment and may express differently hormone receptors and HER2/HER3. While hormones influence breast cancer progression, they also regulate bone turnover and immune system, and therefore humanized mouse models provide an essential platform for novel therapy development. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni H. Mäki-Jouppila, Azusa Tanaka, Michael Seiler, Teppo Haapaniemi, Jenni Bernoulli. Hormone receptor and HER2/HER3 expression in preclinical breast cancer models of primary tumor and bone metastasis [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 3859.