Female Athymic Nude Mice Research Articles

FABP5 promotes cell growth, invasion and metastasis in cervical cancer

Objective: To investigate the functions of FABP5 in the carcinogenesis and development of cervical cancer. Methods: The expression of FABP5 was detected in several cervical cancer cell lines (C33A, Siha, Caski, HeLa and HCC94), 206 cases of cervical cancer tissues with stage Ⅰa2-Ⅱa2 and 40 cases of normal cervical tissues by real-time PCR and Western blotting. Then, the cells were infected with lentivirus-mediated siRNA-targeting FABP5. CCK-8 cell proliferation, colony formation, wound healing and transwell assays were used to investigate the effects of FABP5 on in vitro cell proliferation, migration and invasion. And in vivo xenograft model and lung metastasis model were used to observe the transplanted tumor growth and metastasis in female athymic nude mice. Furthermore, the total protein and RNA were extracted from the primary xenografts to determine the expression levels of FABP5, metalloproteinase-2 and metalloproteinase-9 using Enzyme linked immunosorbent assay (ELISA), real-time PCR and Western blotting. Results: FABP5 expression was found to be significantly unregulated in cervical cancer tissues than that in normal cervical tissues (P<0.05). Compared with the Siha-NC group and uninfected group, the expression of FABP5 mRNA and protein in Siha-FABP5-RNAi group was significantly inhibited along with the decrease of cell proliferation, colony formation, wound healing and invasion ability. The clone formation rates of Siha cells in uninfected group, Siha-NC group and Siha-FABP5-RNAi group were (84.6±4.5)%, (84.6±5.1)% and (21.2±2.6)%, respectively. Moreover, the transwell assay showed that invasive cells in three groups were (72.8±4.7)/HPF, (72.6±3.3)/HPF and (21.4±2.3)/HPF, respectively. All of the difference was statistically significant (P<0.05). Furthermore, FABP5 silencing significantly reduced tumor growth and lung metastases in nude mice in vivo (P<0.001). The subcutaneously xenografted volume in uninfected group, Siha-NC group and Siha-FABP5-RNAi group was (921.4±63.0) mm(3,) (1 021.4±56.0) mm(3) and (139.6±36.0) mm(3,) respectively. The real-time quantitative PCR results showed that the relative expression levels of MMP-2 and MMP-9 mRNA were 1.00±0.10 and 1.00±0.10, 1.00±0.10 and 1.00±0.10 as well as 0.34±0.13 and 0.38±0.17 in xenografted tumor tissues of uninfected group, Siha-NC group and Siha-FABP5-RNAi group, respectively. MMP-2 and MMP-9 was significantly downregulated after FABP5 inhibition(P<0.05). Additionally, the protein expression trend of MMP-2 and MMP-9 in three groups was consistent with the mRNA levels. Conclusion: FABP5 might promote the carcinogenesis and metastasis of cervical cancer via up-regulating MMP-2 and MMP-9.

18F-Fluoroestradiol PET Imaging of Activating Estrogen Receptor-α Mutations in Breast Cancer.

The purpose of this study was to determine the effect of estrogen receptor-α gene (ESR1) mutations at the tyrosine (Y) 537 amino acid residue within the ligand binding domain on 18F-fluoroestradiol (18F-FES) binding and in vivo tumor uptake compared with wild-type (WT)-estrogen receptor α (ER). Methods: ER-negative MDA-MB-231 breast cancer cells were used to generate stable cell lines that express WT-ER, Y537S, or Y537C mutant ER. Receptor expression and localization were confirmed by Western blot and immunofluorescence, respectively. ER transcriptional function was measured using an estrogen response element-luciferase reporter gene assay and quantitative polymerase chain reaction analysis of ER-regulated endogenous target genes. Saturation binding and competition assays were performed to determine equilibrium dissociation constant (Kd) and half maximal inhibitory concentration (IC50) values. 18F-FES uptake was measured in tumor xenografts grown in female athymic nude mice by small-animal PET/CT imaging and tissue biodistribution using 5.55 MBq (150 μCi) of 18F-FES. A 10-fold-lower injected dose of 0.555 MBq (15 μCi) of 18F-FES was also used for tissue biodistribution. Statistical significance was determined using ANOVA. Results: Y537S and Y537C mutations resulted in increased ER transcriptional activity in the absence of estrogen compared with WT-ER (11.48 ± 2.42 fold; P = 0.0002, and 5.89 ± 0.94 fold; P = 0.04, respectively). Constitutive ER activation of two target genes (PGR and TFF1) in the absence of estrogen was also observed in Y537S- and Y537C-ER cells compared with WT-ER. Kd values for 18F-FES were 0.98 ± 0.54 nM for Y537S-ER (P = 0.27) and 0.24 ± 0.03 nM for Y537C-ER (P = 0.95) compared with 0.07 ± 0.03 nM for WT-ER. IC50 values were 0.22 ± 0.09 nM for Y537S-ER (P = 0.97), 0.18 ± 0.09 nM for Y537C-ER (P = 0.99), and 0.19 ± 0.11 nM for WT-ER. Tumor xenografts expressing Y537S-ER (mean percentage injected dose per gram, 1.45 ± 0.06; P = 0.77) and Y537C-ER (2.09 ± 0.20; P = 0.21) had similar 18F-FES uptake compared with WT-ER (1.68 ± 0.12). Comparable 18F-FES uptake between Y537S-, Y537C-, and WT-ER xenografts was also observed using a 10-fold-lower injected dose with the tissue biodistribution assay. Conclusion: Since tumoral uptake of 18F-FES is not significantly impacted by Y537S-ER or Y537C-ER mutations, the potential diagnostic utility of 18F-FES PET imaging is expected to be equally valid for patients with or without these activating ESR1 mutations.

Abstract PD7-11: Therapeutic strategy for ESR1 mutation driven-endocrine resistance in ER-positive breast cancers

Abstract Background: Endocrine therapy is used in estrogen receptor (ER)-positive breast cancers, however, 25% of these patients are at risk of distant relapse and the development of acquired endocrine resistance. Recently mutations in the ER gene (ESR1) have been validated to be acquired during the development of endocrine resistance. The most frequent ESR1 mutation, Y537S, promotes ligand-independent ER activity and emerges subclonally during aromatase inhibitor treatment. In this study, we examined the effects of the Y537S ESR1 mutation on cell cycle signaling and therapeutic response to a novel checkpoint inhibitor. Material and Methods: MCF-7 cells expressing the Y537S ESR1 mutation were generated by CRISPR-Cas9 knock-in techniques. Cells were incubated in steroid deprived conditions. Cell cycle analysis and apoptosis were examined by flow cytometry annnexin-V assays. Proliferation was analyzed by BrdU incorporation. Cell cycle checkpoint kinases were examined by western blot analysis. Cell growth was analyzed using soft agar and MTT assays. Replication stress was identified by RPA32 and gamma-H2AX foci formation assay. For in vivo studies, MCF-7 ESR1 Y537S mutant cells were injected into female athymic nude mice with 17β-estradiol (E2) supplemented water. When tumors reached 350 mm3, tamoxifen (20 mg/kg; s.c.; three times a week), fulvestrant (200 mg/kg; s.c; once a week) and/or PF477736 Chk1 inhibitor (7.5 mg/kg; i.p.; twice a day and twice a week) was treated without E2. Results: ESR1 Y537S mutant cells accumulated approximately 5 fold in S phase and 1.7 fold in G2/M phase compared to control cells in estrogen-deprived (ED) conditions. BrdU incorporation also increased about 2.5-fold, however, apoptosis was decreased about 60 % compared with wild-type ER parental cells. ESR1 Y537S mutant cells induced significant replication stress, showing increased RPA32 foci together with increased gamma H2AX foci, a marker of DNA double-stranded breaks. ChIP-seq analysis revealed binding sites on ATR and CHEK1 genomic locations. ATR/Chk1-mediated checkpoint signaling was activated in ESR1 Y537S mutant cells, and was repressed with fulvestrant, tamoxifen, or ESR1 siRNA treatment. The Chk1 inhibitor, PF477736, sensitized MCF-7 expressing the ESR1 Y537S mutation to endocrine treatments such as fulvestrant, tamoxifen, and the ER degrader AZD9496 in cell proliferation assays. In MCF-7 ESR1 Y537S mutant xenograft and patient derived mouse models, tamoxifen treatment combined with the Chk1 inhibitor PF477736 repressed primary xenograft tumor doubling times (P=0.038, Wilcoxon test). Treatment of mutant tumors with PF477736 together with fulvestrant significantly inhibited the frequency of distant lung metastases by 80% (P=0.0031, t-test), suggesting that these combinations may be useful in second line treatment of metastatic breast cancer patients resistant to endocrine therapies. Conclusion: These preclinical results suggest that ESR1 mutant tumors have a therapeutic vulnerability to combination endocrine therapy with cell cycle checkpoint kinase inhibitors. These data demonstrate that this new therapeutic approach may be useful to restore endocrine sensitivity in metastatic breast cancer patients with ESR1 mutation driven-endocrine resistance. Citation Format: Kim J-A, Dustin D, Gu G, Corona-Rodriguez A, Edwards D, Coarfa C, Keyomarsi K, Fuqua SA. Therapeutic strategy for ESR1 mutation driven-endocrine resistance in ER-positive breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-11.

Responsiveness to basement membrane extract as a possible trait for tumorigenicity characterization

Immortalized cell lines used to produce vaccines are expected to be described in terms of their tumorigenicity. However, current in vivo tumorigenicity assays can be time-consuming and results can be equivocal, especially for weakly tumorigenic cells. Basement membrane extract (BME) derived from the Engelbreth-Holm-Swarm mouse tumor, such as Matrigel and Cultrex, consists of laminin, collagen IV, entactin, heparan sulfate, and proteoglycans, as well as biologically active peptides and growth factors. For nearly three decades, BME has been used in cancer research to enhance tumorigenicity assays (both tumor “take” as well as tumor growth are substantially improved). We assessed the feasibility of using BME to facilitate the evaluation of vaccine cell substrate tumorigenicity. Vero cells (WHO 10-87) were serially passaged and banked at every ten passages beginning with p140; for the present study, low-passage Vero cells (Vero LP, originating from cells banked at p140) and high-passage Vero cells (Vero HP, originating from cells banked at p250) were used. In addition, Vero TPX2 and Vero NM1, cell lines established from tumors formed in nude mice by Vero HP cells, as well as other cell lines relevant to vaccine production (HeLa, MDCK, 293, and ARPE-19), were assessed. Female adult athymic nude mice were injected subcutaneously with cells in the absence or presence of BME. We observed that the tumorigenicity of ARPE-19 cells as well as Vero cells below passage 258 (Vero LP and Vero HP; previously characterized as non-tumorigenic or weakly tumorigenic, respectively) was not enhanced by BME. In contrast, BME shortened the latency and decreased the tumor-producing cell dose of HeLa, 293, and MDCK cells as well as the tumorigenic Vero derivatives TPX2 and NM1. Thus, responsiveness to BME may reflect the status of the neoplastic process and possibly serve as a useful trait for better defining the tumorigenic phenotype of cells.

Chemotherapy‐induced loss of bone and muscle mass in a mouse model of breast cancer bone metastases and cachexia

Chemotherapy used to treat malignancy can lead to loss of skeletal muscle mass and reduced force production, and can reduce bone volume in mice. We have shown that bone-muscle crosstalk is a key nexus in skeletal muscle function and bone homeostasis in osteolytic breast cancer bone metastases. Because chemotherapy has significant negative side effects on bone mass, and because bone loss can drive skeletal muscle weakness, we have examined the effects of chemotherapy on the musculoskeletal system in mice with breast cancer bone metastases. Six-week-old Female athymic nude mice were inoculated with 105 MDA-MB231 human breast cancer cells into the left ventricle and bone metastases were confirmed by X-ray. Mice were injected with carboplatin at a dose of 60mg/kg once per week starting 4 days after tumor inoculation. Skeletal muscle was collected for biochemical analysis and extensor digitorum longus (EDL) whole muscle contractility was measured. The femur and tibia bone parameters were assessed by microCT and tumor burden in bone was determined by histology. Healthy mice treated with carboplatin lose whole body weight and have reduced individual muscle weights (gastrocnemius, tibialis anterior (TA), and EDL), reduced trabecular bone volume (BV/TV), and reduced EDL function. Mice with MDA-MB-231 bone metastases treated with carboplatin lose body weight, and have reduced EDL function as healthy mice treated with carboplatin. Mice with MDA-MB-231 bone metastases plus carboplatin do have reduced proximal tibia BV/TV compared to carboplatin alone, but carboplatin does reduce tumor burden in bone. Our data shows that carboplatin treatment, aimed at reducing tumor burden, contributes to cachexia and trabecular bone loss. The muscle atrophy and weakness may occur through bone-muscle crosstalk and would lead to a feed-forward cycle of musculoskeletal degradation. Despite anti-tumor effects of chemotherapy, musculoskeletal impairment is still significant in mice with bone metastases.

Development of an Image-Guided Orthotopic Xenograft Mouse Model of Endometrial Cancer with Controllable Estrogen Exposure.

Endometrial cancer (EC) is the most common gynaecological malignancy in Western society and the majority of cases are estrogen dependent. While endocrine drugs proved to be of insufficient therapeutic value in the past, recent clinical research shows promising results by using combinational regimens and pre-clinical studies and identified potential novel endocrine targets. Relevant pre-clinical models can accelerate research in this area. In the present study we describe an orthotopic and estrogen dependent xenograft mouse model of EC. Tumours were induced in one uterine horn of female athymic nude mice using the well-differentiated human endometrial adenocarcinoma Ishikawa cell line—modified to express the luciferase gene for bioluminescence imaging (BLI). BLI and contrast-enhanced computed-tomograph (CE-CT) were used to measure non-invasive tumour growth. Controlled estrogen exposure was achieved by the use of MedRod implants releasing 1.5 μg/d of 17β-estradiol (E2) in ovariectomized mice. Stable E2 serum concentration was demonstrated by LC-MS/MS. Induced tumours were E2 responsive as increased tumour growth was observed in the presence of E2 but not placebo, assessed by BLI, CE-CT, and tumour weight at sacrifice. Metastatic spread was assessed macroscopically by BLI and histology and was seen in the peritoneal cavity, in the lymphovascular space, and in the thoracic cavity. In conclusion, we developed an orthotopic xenograft mouse model of EC that exhibits the most relevant features of human disease, regarding metastatic spread and estrogen dependency. This model offers an easy to manipulate estrogen dosage (by simply adjusting the MedRod implant length), image-guided monitoring of tumour growth, and objectively measurable endpoints (including tumour weight). This is an excellent in vivo tool to further explore endocrine drug regimens and novel endocrine drug targets for EC.

Abstract 2172: Dying tumor cells (tumor debris) stimulate the growth of 4T1 breast cancer tumor in female athymic nude mice

Abstract Current cancer therapies are based on chemotherapy and radiation. These therapeutic approaches can initially reduce the tumor mass or tumor burden by killing tumor cells. It has been postulated that the resulting dying tumor cells, or tumor debris, can act as sources of tumor stimulation on surviving cells leading to reappearance of the cancer, and thereby drastically reducing the survival rate. In our study we investigated whether 4T1 debris induced by chemotherapeutic agent docetaxel would affect breast tumor growth in female nude mice. Female athymic nude mice were divided into three groups: Group 1 was inoculated with 4T1 cells; Group 2 with 4T1 cells together with their debris and Group 3 with debris alone. All mice were fed a regular diet. After four weeks, the mice were sacrificed; tumors were excised, weighed and processed for histology and immunohistochemistry for inflammation parameters. Tumor weight significantly increased in Group 2 mice compared to the control Group 1. Group 3 mice developed no tumors. Although the tumor histology for both Groups 1 and 2 were similar, there were significant differences in inflammatory parameters in the two groups. Tumors developed in Group 2 mice had intense staining patterns for TNF-alpha, IL-6, Ki-67, i-Nos and VEGF compared to tumors in the control Group 1. Our results show that dying cells from docetaxel treatment, or tumor debris, had stimulatory effect on growth of breast cancer 4T1 cells in nude mice which was associated with increased inflammation. This study represents a novel method to evaluate the effects of chemotherapy or radiation on surviving tumor cells and to investigate the effect of various inhibitors on the growth recurrence. Citation Format: M Waheed Roomi, Aleksandra Niedzwiecki, Matthias Rath. Dying tumor cells (tumor debris) stimulate the growth of 4T1 breast cancer tumor in female athymic nude mice [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 2172.

Abstract 4172: Radiation-induced blood-tumor barrier permeability in a preclinical model of lung cancer brain metastasis

Abstract BACKGROUND: Lung cancer has the highest mortality rate among all cancers and accounts for over half of newly diagnosed brain metastases each year. Standard therapeutic advancements are prolonging lives of lung cancer patients with each new approved formulation, however patients with brain metastases have limited options and no curative measures currently exist. Current treatment modalities for brain metastases from primary lung cancer include differing methodologies of radiotherapy (whole brain radiotherapy and/or stereotactic radiosurgery), systemic chemotherapies, and neurosurgery. Radiation in many cases is not ablative, but palliative. Herein we demonstrate the ability of radiotherapy to influence blood-tumor barrier permeability in a novel preclinical model of lung cancer brain metastasis. METHODS: Athymic female nude mice were intracardially injected with our novel PC-9-BR brain specific lung cancer cell line. Tumors were allowed to develop and monitored using bioluminescent imaging. Using the Xstrahl XenX small animal irradiator, we were able to deliver 26 Gy to the right hemisphere of the immobilized mouse. 4 hours post radiotherapy, mice were perfused with the passive permeability marker Oregon Green, followed by an indocyanine green (bound to albumin) washout to infer vascular space. Brains were then extracted, snap frozen in isopentane (-50°C), and sliced to 20μm. Florescence was quantified using a MVX microscope and cellsense software. RESULTS: Analysis of irradiated brain regions demonstrated statistically significant more accumulation of the passive permeability marker, Oregon Green, compared to the contralateral hemisphere. CONCLUSIONS: Radiation induced permeability was witness in mice after a single dose of 26 Gy when compared to non-irradiated tumor tissue 4 hours post treatment. FURTURE DIRECTIVES: Experiments utilizing ionizing radiation to simulate treatment of CNS metastases could reveal better adjunctive chemotherapeutic regimens and or provide insight to the mechanism underlying this enhanced permeability. Citation Format: Samuel A. Sprowls, Neal Shah, Afroz S. Mohammad, Marc Pinti, Rachel M. Tallman, Devin U. John, Paul R. Lockman. Radiation-induced blood-tumor barrier permeability in a preclinical model of lung cancer brain 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 4172.

Abstract 1263: TGF-ß inhibition as a novel strategy to prevent breast cancer brain metastases

Abstract Background: Incidence of breast cancer brain metastases (BCBM) is on the rise due to improved management of systemic disease and ranges from 15-20% in women with disseminated breast cancer. Common chemotherapeutic strategies have limited efficacy for brain metastases due to the presence of the blood brain barrier (BBB). Prevention of metastasis after diagnosis of primary breast tumors may lead to new treatment strategies. TGF-ß is a biomolecule in the metastatic pathway and inhibition of its expression can be achieved by treatment with the antihypertensive agent Losartan. Herein we hypothesized that treatment with Losartan will decrease metastatic burden and improve survival in a preclinical model BCBM. Methods: Athymic female nude mice were separated into four groups as follows: Vehicle, (saline, 50µL/day), SB431542 (Inhibitor, 5mg/kg/day), Low Dose Losartan (10mg/kg/day) and High Dose Losartan (25mg/kg/day). On day 7 mice were intracardially injected with 175,000 cells of either JIMT-1-Br-LUC-GFP (HER2+) or MDA-MB-231Br-LUC (TNBC) breast cancer cell lines. Tumor burden was monitored once weekly utilizing bioluminescent imaging. Mice were allowed to progress until neurological symptoms arose. Following euthanasia, brains were extracted, sectioned and analyzed to determine number of metastatic lesions and size. Results: With regard to survival, no significance was shown in the JIMT-1 (HER2+) metastatic model. The MDA-MB-231Br (TNBC) model demonstrated an increase in survival in all treatment groups compared to vehicle. End time-point analyses showed no difference in metastases number in either model, however, the low dose treatment group in the MDA-MB-231Br model had a smaller brain burden of metastases than high dose treatment as well as inhibitor treatment. Conclusions: Losartan reduced metastatic seeding in the MDA-MB-231Br (TNBC) brain metastasis model, but had little to no effect in the JIMT-1 (HER2+) model. Citation Format: Mark V. Pinti. TGF-ß inhibition as a novel strategy to prevent breast cancer brain metastases [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 1263.

Abstract 736: Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity

Abstract Although four ADCs have been approved and over sixty others are in development, the majority contain payloads belonging to two classes; tubulin inhibitors and DNA cross-linkers. Challenges in the development of ADCs include managing off-target toxicity and hydrophobicity. Some payload classes (e.g., PBD dimers) are notably hydrophobic leading to problems (e.g., aggregation) during conjugation. Thus, there is interest in developing novel payloads which retain the potency of DNA cross-linkers but have lower hydrophobicity and a wider therapeutic window when part of an ADC. The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA mono-alkylating ADC payload which contain a polyheterocyclic chain with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). The lead PDD payload, FGX-2-62, has a different sequence-selectivity profile to other DNA-interactive agents, spanning 8-9 base-pairs compared to 6-7 for a PBD dimer, and DNA footprinting experiments indicate a preference for 5'-XGXWWWWXX-3' sequences (X is any base; W is A/T). Transcription factor array studies have shown that the molecule inhibits DNA-binding of oncogenic transcription factors (e.g., NF-κB and GATA). In in vitro cell line studies, FGX-2-62 has low pM cytotoxicity in a diverse cell line panel, including stem cells, cells from both solid and blood cancers (e.g., 9 pM in HL-60) and MDR-resistant tumours, and arrests the cell cycle at the G0/G1 phase compared to G2-M arrest for PBD dimers. It is compatible with attachment to most linker technologies, and is significantly less hydrophobic than other payload classes. Initial MTD studies were carried out by separately conjugating (with negligible aggregation) FGX-2-62 and the PBD dimer Talirine to a THIOMAB® version of trastuzumab (DAR = 2). In female athymic nude mice, a greater tolerance was observed for the THIOMAB®-(FGX-2-62) ADC compared to the THIOMAB®-PBD dimer (i.e, MTD &amp;gt;8 mg.kg-1 versus 4 mg.kg-1). In an efficacy study, FGX-2-62 was conjugated to a cancer stem cell-targeting IgG1 antibody (Bstrongximab) with DAR 1.9. Initial evaluation afforded IC50 values of 0.67 nM and 0.47 nM in two antigen positive cell-lines, and an MTD of 6 mg.kg-1 in mice. In an antigen-positive embryonal carcinoma stem cell CDX mouse model, complete regression was observed at a dose of 2 mg.kg-1 (Q7Dx3). In a cholangiocarcinoma PDX model, complete tumour regression was observed out to 80 days (when experiment was terminated) at a dose of 5 mg.kg-1 (Q7Dx3), with no observed toxicity. The favourable hydrophobicity profile of the PDDs and ease of conjugation, along with their novel mechanism of action, significant in vitro cytotoxicity, in vivo efficacy and tolerability in MTD studies suggest that they represent a promising new class of ADC payloads. Citation Format: Nicolas Veillard, Paolo Andriollo, Julia Mantaj, Keith R. Fox, K Miraz Rahman, George Procopiou, Francesco Cascio, David B. Corcoran, Ilona Pysz, Patricia A. Cooper, Steven D. Shnyder, Yawen Ju, Edwin Tan, William M. Schopperle, Paul J. Jackson, David E. Thurston. Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity [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 736.

AGS cell line xenograft tumor as a suitable gastric adenocarcinoma model: growth kinetic characterization and immunohistochemistry analysis.

Objective(s):Gastric cancer is the third leading cause of cancer-related death worldwide. The overall survival rate of patients is poor because gastric cancers are usually diagnosed at the late stages. Therefore, further research is needed and appropriate research tools are required to develop novel therapeutic approaches.Materials and Methods:Eight female athymic nude mice with a C57BL/6 background were used in this study. AGS cells were inoculated into the flank. The tumor volumes were calculated and growth curves were drawn. When the volume of the tumors reached 1000 mm3, the animals were humanely euthanized with CO2 gas. After harvesting, tumors were analyzed with Hematoxylin and Eosin (H&E) and immunohistochemistry (IHC). A pathologist confirmed tumor entity through H&E staining. Tumors were evaluated for expression of HER-2, P53, Ki-67, CD34, cytokeratin 8 (CK8), vimentin, estrogen receptor (ER), and progesterone receptor (PR) utilizing immunohistochemistry.Results:The tumor take rate was 62.5%, mean doubling time was 40.984 d, and the latency period was 30.62 days. The H&E staining results showed highly malignant hyperchromatin epithelial cells. IHC assessment showed the mutation status of P53 gene. The expression score of the CK8 protein in the tumor cells was +3. Vimentin protein was not expressed and changes in mesenchymal phenotype were not observed. Ki-67 IHC indicated that the proliferation rate was >43% and angiogenesis was defined as high MVD.Conclusion:The respective AGS xenograft model provides an opportunity to understand the pattern of tumor growth as well as to evaluate new gastric cancer therapies in in vivo studies.

Abstract 2913: Cancer growth under check: Anti-vacuolar ATPase "a2" antibody as novel therapy against ovarian cancer

Abstract Tumor growth and metastasis involves complex crosstalk among cancer cells and immune infiltrates. The balance of pro- and antitumor infiltrating immune cells can impact the outcomes of tumor growth. Vacuolar-ATPase (V-ATPase) proton pump promotes cancer growth and metastasis by regulating the pH-associated cancer cell-signaling and the tumor microenvironment. V-ATPase is overexpressed on cancer cell surface in a wide array of tumor types including ovarian cancer (OVCA) as well as on inflammatory cells recruited to the tumor microenvironment. This makes V-ATPase an important target for anticancer therapy. We have created a monoclonal antibody that specifically recognizes the plasma membrane isoform of V-ATPase (a2v) present distinctly on malignant cells and absent on normal cells. The purpose of this study is to investigate the in vivo effect of anti-VATPase-a2v antibody (Mab; 2C1) on the growth of human ovarian cancer (OVCA). The antitumor effects were evaluated in female athymic nude mice xenograft model. Mice were injected with human OVCA cell line (A2780;0.4X106 cells; s.c in upper flank) and the tumor growth was compared to mice that were simultaneously given a single dose of MAb (2C1; 300µg in PBS) or an isotype control antibody(mouse IgG1A). The 2C1 treatment resulted in delayed tumor growth (2.4 fold, p&amp;lt; 0.05), with no apparent in vivo toxicity. Histologic staining of 2C1-treated tumors revealed a loose tumor matrix with higher immune-infiltration compared to control. There was a marked decrease in cancer cell numbers in 2C1-treated tumor tissues compared to control using CA125 cancer-antigen staining. In in vitro assays, there was no measurable cytotoxic effect of 2C1 on OVCA cell proliferation, indicating the role of tumor microenvironment in delayed tumor growth. We further investigated the infiltrated immune population in 2C1-treated tumors. Interestingly, we observed an overall increased total leukocyte population (CD45; p=0.028) and a higher expression of iNOS (IHC-score 2C1 treated tumor= 11.3, control =7.6; p=0.04), suggesting an antitumor response. The total F4/80 macrophage population was higher in anti-VATPase-a2v antibody treated cells (absolute numbers in 2C1 treated tumor=245.2, control tumor=193.1; p=0.04). In 2C1 treated mice, tumor-associated macrophages displayed antitumor properties with correlated iNOS expression. Further, a marked increase in the number of Ly6G positive cells (neutrophil marker) was observed in 2C1 treated tumors, further suggesting an elevated antitumor response. Studies are under way to characterize other immune infiltrated populations that may contribute to antitumor response in 2C1 treated tumor tissues. In conclusion, the study demonstrates that the anti-VATPase "a2" antibody is an effective form of treatment in ovarian cancer. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Shayna Levine, Manoranjan Sahoo, Safaa A. Ibrahim, Alice Gilman-Sachs, Kenneth D. Beaman. Cancer growth under check: Anti-vacuolar ATPase "a2" antibody as novel therapy against ovarian 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 2913.

Abstract 2171: Efficacy of chemotherapeutic agents in brain metastases of lung cancer model

Abstract Background: Lung cancer is not only the second most common cancer in the United States, but it is also the highest cause of cancer-related death worldwide. In 2017, it is estimated that there will be over 200,000 new cases and 150,000 deaths as a result of lung cancer. The associated high mortality can in part be attributed to the high rate of primary lung cancer metastasis to the brain. Median survival for untreated patients with metastatic brain lesions is approximately 4 to 9 months, while treated patients still typically die within two years. Treatment options are limited for patients with brain metastases due in part to chemotherapeutic ineffectiveness, as many agents used to treat the primary tumor are not able to penetrate the blood-brain barrier (BBB). This study aims to investigate the efficacy of various chemotherapies on brain metastases of lung cancer via bioluminescence imaging data as a surrogate for tumor burden and overall survival data. Methods: Female athymic nude mice underwent intracardiac injection with brain-seeking PC-9 lung cancer cells transfected with Luc-2 and TdTomato. Tumor burden was monitored using bioluminescent imaging biweekly. Upon tumor burden establishment, mice were administered either Doxil (peglylated liposomal doxorubicin) (6 mg/kg), irinotecan (50 mg/kg), gemcitabine (60 mg/kg), docetaxel (10 mg/kg), eribulin (1.5 mg/kg), vinorelbine (10 mg/kg), cisplatin (5 mg/kg) + etoposide (10 mg/kg), cisplatin (5 mg/kg) + pemetrexed (100 mg/kg), osimertinib (25 mg/kg), or gefitinib (6.25 mg/kg). Upon sacrifice, brains were perfused to visualize tumors and permeability using fluorescent microscopy. Survival data were collected and plotted on a Kaplan-Meier curve for statistical analyses. Results: Bioluminescence for nontargeted chemotherapy was nearly equal to that of vehicle. Osimertinib (25 mg/kg) demonstrated immediate decreased tumor burden and increased survival. Vehicle and all nontargeted agents had a survival of 42 +/- 3 days. Images taken of metastatic lesions indicate permeability has increased compared to normal brain, but is not as drastic as expected (1.5 to 1.8 fold increase) and displays heterogeneity between tumors. Conclusion: The efficacy of chemotherapies in brain metastases of lung cancer is not necessarily associated with increased permeability. No smaller nontargeted agent showed improved survival or superior tumor control compared to vehicle, while the third-generation EGFR inhibitor osimertinib showed superior tumor burden control and survival in vivo. Future directions aim to investigate methods to increase drug permeation into tumors so that they may overcome the BBB and accumulate to cytotoxic levels in tumors. Citation Format: Rachel M. Tallman, Neal Shah, Sam Sprowls, Mark Pinti, Afroz Mohammad, Paul Lockman, Pushkar Saralkar. Efficacy of chemotherapeutic agents in brain metastases of lung cancer model [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 2171.

Abstract 2683: The olive-based oleocanthal as a dual HER2-MET inhibitor for the control of breast cancer recurrence

Abstract Dysregulation of the receptor tyrosine kinases (RTKs) HER2 and c-Met correlate well with poor breast cancer (BC) prognosis and invasive aggressive profile. c-Met amplification proved to be the HER2-dependent tumors' inevitable escape mechanism from the anticancer effects of targeted therapies including trastuzumab, cetuximab, and small-molecule RTK inhibitors like lapatinib, gefitinib, and erlotinib. Dual HER2-Met inhibition is highly expected to be effective and less likely to develop resistance. This study reports the novel dual HER2-Met inhibitory molecular mechanism for the extra virgin olive oil-derived phenolic secoiridoid (-)-oleocanthal (OC). OC showed typical type-I binding mode at c-Met ATP kinase domain. OC aldehydes, ester, and phenol groups showed critical interactions with the critical activation loop ASP1222/TYR1230 and the hinge region PRO1158/MET1160. In addition, OC overlapped two out of the three lapatinib critical interactions at the HER2 kinase domain, the hinge region MET801 and PHE864; besides, OC uniquely targeted the hinge region's THR862 and SER783, justifying unique binding mode. OC showed low-µM level inhibitory activities against both c-Met and HER2 kinases in cell-free Z-LYTE assays. In vitro, OC induced dose- and time-dependent inhibition of the proliferation and migrations of several BC cells including BT-474, SK-BR-3, MDA-MB-231, MCF-7, MDA-MB-468 at low μM IC50 dose range. This effect was associated with selective blockade of HER2 and Met receptors activation in response to their natural ligands HGF and EGF, respectively, confirmed by Western blotting, flow cytometry and immunocytochemistry studies. OC potently induced autophagy via the upregulation of LCA/B, Atg-3, Atg-7, Atg-16L within 6-12 h of SKBR-3 cells treatment. OC had no effect on the viability of the nontumorigenic human MCF-12A mammary epithelial and neuronal Schwann RSC 96 cells. In vivo, 5-10 mg/kg oral or ip dose range of OC potently inhibited 65-90% of the growth of the HER2-dependent BT-474 and the c-Met-dependent MDA-MB-231 BC cells in female athymic nude mice xenograft models. Orally, 10 mg/kg dose of OC prevented over 90% of local and regional recurrences of both tumor phenotypes after primary tumor surgical excision in orthotopic xenograft mice models. This was further confirmed by significant reductions of Ki-67, CD31, pHER2 and p-c-Met levels in treated animal tumors by IHC studies. OC is a novel dual c-Met-HER2 inhibitory dietary supplement lead with excellent potential for translational use to prevent and control breast malignancies with aberrant c-Met or HER2 activities. Supported by NCI Award R15CA167475 and Louisiana Board of Regents ITRS Award LEQSF (2017-20)-RD-B-07. Citation Format: Abu Bakar Siddique, Hassan Ebrahim, Mohamed Mohyeldin, Seetharama D. Jois, Khalid A. El Sayed. The olive-based oleocanthal as a dual HER2-MET inhibitor for the control of breast cancer recurrence [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 2683.

Oncolytic Tanapoxvirus Expressing Interleukin-2 is Capable of Inducing the Regression of Human Melanoma Tumors in the Absence of T Cells.

Oncolytic viruses (OVs), which preferentially infect cancer cells and induce host anti-tumor immune responses, have emerged as an effective melanoma therapy. Tanapoxvirus (TANV), which possesses a large genome and causes mild self-limiting disease in humans, is potentially an ideal OV candidate. Interleukin-2 (IL-2), a T-cell growth factor, plays a critical role in activating T cells, natural killer (NK) cells and macrophages in both the innate and adaptive immune system. We aimed to develop a recombinant TANV expressing mouse IL-2 (TANVΔ66R/mIL- 2), replacing the viral thymidine kinase (TK) gene (66R) with the mouse (m) mIL-2 transgene resulting in TANVΔ66R/mIL-2. Human melanoma tumors were induced in female athymic nude mice by injecting SKMEL- 3 cells subcutaneously. Mice were treated with an intratumoral injection of viruses when the tumor volumes reached 45 ± 4.5 mm3. In cell culture, expression of IL-2 attenuated virus replication of not only TANVΔ66R/ mIL-2, but also TANVGFP. It was demonstrated that IL-2 inhibited virus replication through intracellular components and without activating the interferon-signaling pathway. Introduction of mIL-2 into TANV remarkably increased its anti-tumor activity, resulting in a more significant regression than with wild-type (wt) TANV and TANVΔ66R. Histopathological studies showed that extensive cell degeneration with a significantly increased peri-tumor accumulation of mononuclear cells in the tumors treated with TANVΔ66R/mIL-2, compared to wtTANV or TANVΔ66R. We conclude that TANVΔ66R/mIL-2 is potentially therapeutic for human melanomas in the absence of T cells, and IL-2 expression resulted in an overall increase of therapeutic efficacy.

Genetic profiling of young and aged endothelial progenitor cells in hypoxia

Age is a major risk factor for diseases caused by ischemic hypoxia, such as stroke and coronary artery disease. Endothelial progenitor cells (EPCs) are the major cells respond to ischemic hypoxia through angiogenesis and vascular remodeling. However, the effect of aging on EPCs and their responses to hypoxia are not well understood. CD34+ EPCs were isolated from healthy volunteers and aged by replicative senescence, which was to passage cells until their doubling time was twice as long as the original cells. Young and aged CD34+ EPCs were exposed to a hypoxic environment (1% oxygen for 48hrs) and their gene expression profiles were evaluated using gene expression array. Gene array results were confirmed using quantitative polymerase chain reaction, Western blotting, and BALB/c female athymic nude mice hindlimb ischemia model. We identified 115 differentially expressed genes in young CD34+ EPCs, 54 differentially expressed genes in aged CD34+ EPCs, and 25 common genes between normoxia and hypoxia groups. Among them, the expression of solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) increased the most by hypoxia in young cells. Gene set enrichment analysis indicated the pathways affected by aging and hypoxia most, including genes “response to oxygen levels” in young EPCs and genes involved “chondroitin sulfate metabolic process” in aged cells. Our study results indicate the key factors that contribute to the effects of aging on response to hypoxia in CD34+ EPCs. With the potential applications of EPCs in cardiovascular and other diseases, our study also provides insight on the impact of ex vivo expansion might have on EPCs.

INTRACRANIAL TARGETING OF GLIOBLASTOMA MULTIFORME WITH COLD ATMOSPHERIC PLASMA

Glioblastoma multiforme (GBM) is a highly malignant aggressive neoplasm of the primary central nervous system characterized by rapid growth, extensive angiogenesis, and resistance to current therapies. As the most common malignant primary brain tumor, GBM accounts for 45% of all brain tumors with over 20,000 new diagnoses per year in the U.S. Due to the highly aggressive nature of GBM, the median survival is limited to 16–19 months, with less than 30% of patients alive at two years after diagnosis. In this context, GBM treatment strategies remain largely palliative despite the advancement of multi‐modal therapies. Thus, it is necessary to develop novel tools that can target proliferating tumor cells and enhance existing therapies. Conventional lasers in medical devices are based on the thermal interaction with tissues, which lead to necrosis and permanent tissue damage. In contrast, cold atmospheric plasma (CAP) has recently emerged as a novel therapeutic approach for targeting of cancerous tissue. Indeed, recent findings suggest that CAP jet interactions with tissue may allow for cell death without necrosis. However, studies to date have been limited primarily to subcutaneous implantation of tumors. While beneficial, this approach does not replicate the complex environment of the brain (i.e. GBM). Here, we developed a novel approach to target CAP to intracranial GBM tumors. This new device, termed μCAP, consists of a Pyrex syringe through which CAP, employing helium gas, is supplied via the implanted endoscopic cannula. We first performed a set of experiments to test the influence of μCAP on normal brain parenchyma. Female athymic Foxn1nu nude mice underwent intracranial μCAP injection to the frontal lobe (15s total). Helium alone was administered as a control. Histological examination (Nissl staining) of the frontal lobe 7 days later revealed a similar number of apoptotic cells surrounding the injection site between μCAP and control treated animals (9±3 vs. 6±1 apoptotic cells/μm2, control vs. μCAP, n=2–3, p&gt;0.05). Similarly, no evidence of glia infiltration at the injection site was apparent. Next, nude mice underwent implantation of U87 glioblastoma cells (105 cells) into the frontal lobe and were simultaneously instrumented with a custom endoscopic cannula. Tumors were allowed to develop for 7 days and mice were then treated intracranially with μCAP (15s total) or helium control. Using in vivo bioluminescence imaging (Figure), the tumor volume in control animals increased nearly 1000% over the course of a week, whereas μCAP treated tumor volumes remained at baseline levels (day 7: 1035±773 vs. 172±107 radiance %baseline, control vs. μCAP, n=3, p&gt;0.05). These findings indicate that CAP has a minimal effect on healthy brain tissue, and further provide the first evidence for the potential of CAP to inhibit intracranial GBM tumor growth.Support or Funding InformationGW Institute for Biomedical Engineering Interdisciplinary Research Fund; US Patent Innovations, LLCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract LB-B19: Re-sensitizing Pancreatic Cancer, targeted siRNA inhibition of HuR

Abstract We have demonstrated that 3DNA nanocarriers can specifically, effectively, and safely deliver siRNA oligos targeted against a pro-survival hub, HuR (the ELAVL1 gene) in an in vivo, pre-clinical model of pancreatic cancer. Pancreatic cancer remains one of the deadliest cancers with an overall 5 year survival rate of 9%; and is on pace to become the second leading cause of cancer related deaths in the U.S. by 2020. Pancreatic cancer is so deadly, in part, because it is highly refractory to standard chemotherapeutics. In fact, new standard of care regiments such as FOLFIRINOX have only improved survival by a few months. Our previous work has established HuR as a target in pancreatic cancer. HuR is an RNA binding protein that is primarily retained in the nucleus, but upon exposure to cancer associated stressors translocates to the cytoplasm. In the cytoplasm, HuR functions to bind, stabilize, and up-regulate expression of pro-survival target mRNAs. CRISPR-mediated deletion of HuR causes a xenograft lethal phenotype in vivo and increases sensitivity to a variety of drugs, particularly PARP inhibitor therapy, enhancing Olaparib therapy over 20-fold in vitro. In an independent doxycycline inducible knockdown model, HuR depletion combined with Olaparib treatment resulted in a 9.3 fold (p&amp;lt;.001) decrease in tumor volume in vivo. Based on these findings and our previous published work, we have launched into a line of investigation attempting to target HuR in pancreatic cancer cells. We have delivered siHuR oligos tethered to 3DNA nanostructures (Genisphere, LLC) in a targeted fashion using either a folic acid targeting moiety, a transferrin receptor targeted antibody, or an EGFR targeted antibody (i.e., siHuR nanotherapy). Additionally, we are working to combine siHuR nanotherapy with conventional therapeutics. 3DNA nanocarriers are constructed using stable DNA strands assembled and crosslinked in a stepwise fashion, to create a layered nanoscaffold capable of attaching multiple different targeting moieties and deliverables. The siRNA oligos hybridized to the 3DNA were modified in order to withstand systemic RNases during treatment. We first validated the modified siHuR nanotherapy in vitro by evaluating protein and RNA levels of HuR. Second, we validated the expression of our intended targeting moieties in both human cell lines and a tissue microarray consisting of patient derived pancreatic cancer tumors. For the mouse studies, athymic female nude mice were injected with 4 million MIA PaCa-2 cells per flank, and randomized to treatment arms when tumors reached 100mm3. Mice were then treated intraperitoneally twice weekly with either siHuR or siControl nanotherapies (3ug siRNA per treatment) with one of the three targeting moieties. We demonstrated efficacy of systemic targeted delivery of siHuR nanotherapy which improved average overall survival by 35% (p=.012) compared to the control arm, with an increase in survival using a Kaplan-Meier survival analysis (p=.01). We are currently setting up treatments with siHuR nanotherapy in conjunction with a targeted treatment strategy, Olaparib, which has previously been shown to work in conjunction with dox-inducible HuR silencing (i.e.,shRNA targeting) in vivo. Herein, we describe the development of siHuR nanotherapy that, as a monotherapy or most likely with clinically available drugs, may improve outcomes for patients with pancreatic cancer. Citation Format: Chris W. Schultz, Kevin O'Hayer, Kathryn M. Bormes, Teena Dhir, Samantha Brown, Avinoam Nevlar, Saswati Chand, Henry Thomsett, Wei Jiang, Jessica Bowers, Kelly Rhodes, Michael Pishvaian, Robert Getts, Jonathan Brody. Re-sensitizing Pancreatic Cancer, targeted siRNA inhibition of HuR [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B19.

Sex as a Biologic Variable in Preclinical Imaging Research: Initial Observations with 18F-FLT.

The study objective was to investigate whether sex influences 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) uptake and tissue distribution in mouse models of cancer. Methods:18F-FLT biodistribution was measured in 3 strains of male and female mice (129S6/SvEv, athymic nude, and BALB/c). 18F-FDG biodistribution was measured for comparison. 18F-FLT uptake was also measured in female 129S6/SvEv mice bearing estrogen-dependent SSM3 mouse mammary tumors, male athymic nude mice bearing androgen-dependent CWR22 prostate cancer xenografts, and male and female athymic nude mice bearing estrogen-independent MDA-MB-231 human breast cancer xenografts. Ki-67 expression was assayed by immunohistochemistry. PET/CT imaging was performed to visualize 18F-FLT biodistribution and to determine pharmacokinetics. Results: Greater 18F-FLT activity was observed in blood, liver, muscle, heart, kidney, and bone in female than male mice. Pharmacokinetic analysis demonstrated higher early renal 18F-FLT activity and greater accumulation of 18F-FLT in the urinary bladder in male than female mice. The differential pattern of 18F-FLT biodistribution between the sexes seen with 18F-FLT was not observed with 18F-FDG. Increased tumoral 18F-FLT uptake compared with muscle was observed in both the SSM3 mammary tumors (2.4 ± 0.17 vs. 1.6 ± 0.14 percentage injected dose [%ID]/g at 2 h after injection, P = 0.006) and the CWR22 prostate cancer xenografts (0.34 ± 0.08 vs. 0.098 ± 0.033 %ID/g at 2 h after injection, P = 0.03). However, because of higher nonspecific muscle uptake in female mice, tumor-to-muscle uptake ratios were greater for CWR22 tumors than for SSM3 tumors (4.2 ± 0.78 vs. 1.5 ± 0.049 at 2 h after injection, P = 0.008). Sex-dependent differences in 18F-FLT uptake were also observed for MDA-MB-231 xenografts (tumor-to-muscle ratio, 7.2 ± 0.9 for female vs. 16.9 ± 8.6 for male, P = 0.039). Conversely, greater tumoral Ki-67 staining was observed in female mice (71% ± 3% for female vs. 54% ± 2% for male, P = 0.009), and this finding more closely matched the relative differences in absolute 18F-FLT tumor uptake values (4.5 ± 0.99 %ID/g for female vs. 1.9 ± 0.30 %ID/g for male, P = 0.03). Conclusion: Depending on whether female or male mice are used, differences in biodistribution and nonspecific tissue uptake can adversely affect quantitative measures of 18F-FLT uptake. Thus, sex is a potential variable to consider in defining quantitative imaging metrics using 18F-FLT to assess tumor proliferation.

Effect of 17β-estradiol on estrogen receptor negative breast cancer cells in an osteolytic mouse model

17β-Estradiol (E2) promotes metastasis of triple negative breast cancer cells to bone. Recent studies show many triple negative breast cancer cell lines lacking the 66 kDa estrogen receptor (ER) alpha (ERα66) or its splice variant ERα46, express another splice variant, ERα36 associated with membrane-mediated rapid actions of the hormone. qPCR and western blot confirmed that MCF7 cells possessed ERα splice variants ERα66, ERα46 and ERα36, while ER-negative breast cancer cells MDA-MB-231 possessed only ERα36. MDA-MB-231 breast cancer cells were implanted into medullary canals of ovariectomized female athymic nude mice femurs (N = 8 mice/treatment). To examine the effect of E2 on osteolysis, mice were treated with 0.72 mg E2 or placebo via 60 day release osmotic pumps implanted subcutaneously. Legs were examined by Faxitron through the course of the study, and by microCT and histology after 8 weeks. Greater occurrence of osteolysis and pathologic fracture was observed in E2-treated animals compared to placebo, and microCT demonstrated less bone volume remaining in MDA-MB-231 treated legs compared to contralateral control legs, as well as E2-treated animals compared to placebo. E2-treated animals had significantly greater tumor volume compared to placebo. Large nests of anaplastic tumor cells with eroded cortical margin were observed in E2-treated animals compared to placebo. MDA MB 231 breast cancer cells positive for ERα36 but negative for ERα46/66 had enhanced osteolysis, pathologic fractures, and tumor volume in an in vivo osteolytic mouse model when treated with 17β-estradiol compared to placebo, demonstrating a role for ERα36 in bone tumor progression.