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PDF file - 1004K, S1. The Twist box domain is required for full Twist1 transcriptional activity. S2. Quantification of immunofluorescence from isogenic prostate cancer cell lines stably expressing Twist1 and Twist1-F191G. S3. The Twist box domain is required for full Twist1-induced EMT marker phenotypes of prostate cancer cells. S4. Twist1 overexpression induces temporal changes in the material properties of prostate cancer cells during their migration in a wound healing assay. S5. The overexpression of Twist1 or Twist1-F191G does not increase cellular proliferation of prostate cancer cells in vitro. S6. The Twist box domain is required for full Twist1-induced cellular migration in PC3 cells. S7. Twist1 overexpression increases cell traction forces of individual androgenindependent PC3 prostate cancer cells. S8. Twist1 overexpression confers radioresistance to prostate cancer cells which is attenuated by mutation of the Twist box domain. S9. The Twist box domain is required for Twist1-induced soft agar anchorageindependent growth of 22Rv1 prostate cancer cells. S10. Twist1 overexpression does not confer prostate cancer cells increased primary tumorigenicity and slows primary tumor cell growth in vivo. S11. The Twist box domain is required for full Twist1-induced expression of Hoxa9/HOXA9.

PDF file - 116K, Supplemental Table S2 - Myc-CaP + Twist1-F191G versus Myc-CaP + Vector Differentially.

PDF file - 384K, Supplemental Table S1 - Myc-CaP + Twist1 versus Myc-CaP + Vector Differentially.

Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.

Supplementary Figure Legends from Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

Supplementary Figure 1 from Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

Supplementary Figure 2 from Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

Objectives: This study aims to investigate whether endocan levels in the peripheral blood change after the removal of primary breast tumors and allow predicting postoperative prognosis for breast cancer. Significance: An early prediction of postoperative recurrence and metastases with liquid biopsy will lead to better management and quality of life of breast cancer patients. Background: Cancer recurrence comes down to a poor prognosis in breast cancer patients. Although removal of primary breast cancer can achieve a successful outcome, metastasis and local recurrence remain a critical clinical problem in later years. Although the overall survival of breast cancer patients varies among molecular subtypes, the individual patients have different prognostic outcomes; some patients relapse within a few months, and others have not seen any recurrence for several years. Our previous study indicated that endocan had the potential to be a prognostic biomarker for triple-negative breast cancer. It is critical to show changes in endocan levels in the peripheral blood after the surgical excision of the primary tumor, which, in turn, will corroborate our hypothesis that plasma endocan is a beneficial biomarker for a postoperative prognosis for breast cancer patients. We included all the breast cancer subtypes in this study as the information on endocan levels in the peripheral blood from breast cancer patients has been limited thus far. Methods: To detect metastatic recurrence of breast cancer cells noninvasively, MDA-MB-231BR was transfected with the mVenus-Akaluc gene. MDA-MB-231BR spontaneously overexpresses the ESM1 gene. MDA-MB-231BR/mVenus-Akaluc was inoculated into the mammary fat pad of female athymic nu/nu mice and NSG mice. Once tumors top 200 mm3, they were surgically removed. Metastatic recurrence was visualized by the IVIS imager. Blood was withdrawn from the tail of each mouse before and after the resection of the primary tumors in a timely manner, and plasma samples were stored at -80°C until usage. Endocan in plasma samples was quantitated with a commercial ELISA kit. Tumor burden was estimated from luminescence signals of images obtained by IVIS. We obtained IRB approval from both institutions for the following research. Blood samples were collected from breast cancer patients who had given informed consent at the Breast Cancer, Showa University Hospital, and the plasma samples have been stored at -80°C until usage. Endocan in plasma sample was determined with a commercial ELISA kit. Results and discussion: Before removing primary breast tumors, the median endocan level in plasma was 0.73 ng/mL (range, 0.38-1.54 ng/mL) in nude mice. After the removal, endocan remained below the detection limits even though small metastases were detected by IVIS imager at later time points. These data indicate that loss of primary tumors results in an out-of-detection range in endocan levels in the blood. In contrast, NSG mice displayed inconsistent results: out of eight mice, three mice showed a decrease in endocan, three had an increase in endocan, and two had no change three days postoperatively. IVIS images of NSG mice indicated that distant metastases were already detected even when some mice underwent tumor resection. In NSG mice, the occurrence of tumor metastases was much earlier, and metastatic tumor burdens were much more significant than in nude mice, which might lead to a different outcome. Endocan levels in plasma from breast cancer patients at the postoperative period were not always dropped, which was similar to the results obtained from NSG mice. The rationale behind the inconsistent kinetics of endocan remains to be clarified. Conclusion: Our results from NSG mice and clinical data in patients’ plasma may have more relevance to prognostic outcomes in breast cancer patients. Citation Format: Kentaro Daiki, Yoko Kanada, Aya Nagata, Seigo Nakamura, Yoshinori Kato. Kinetics of endocan in the peripheral blood before and after the resection of primary breast tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-05-12.

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial–mesenchymal transition (EMT) that promotes cancer metastasis. Structure–function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Implications: Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential. Mol Cancer Res; 11(11); 1387–400. ©2013 AACR.

Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.

The HER2 receptors are overexpressed in ∼25% of breast cancer patients and characterized by poor internalization capability. The humanized monoclonal antibody, trastuzumab (Tz), is used as a first-line treatment for HER2(+) breast cancers. However, approximately 50% of patients with HER2(+) disease do not benefit from trastuzumab or the disease becomes refractory to the drug. We have demonstrated a target-specific intracellular delivery of therapeutics using new two-step/two-component drug delivery system driven by click chemistry that results in the in situ complexation and rapid internalization. Two delivery strategies based on azide (Az)/dibenzylcyclooctyne (DBCO), (Click-1) and trans-cyclooctene (TCO)/tertrazine (Tt), (Click-2) bioorthogonal click reactions were tested in human HER2(+) breast cancer mouse models. Trastuzumab was functionalized with Az or TCO groups and labeled with NIR CF-680 dye, to prepare Tz(Az/TCO)a(CF-680)b as the pretargeting component. Albumin substituted with paclitaxel (Px), functionalized with DBCO or Tt, and labeled with NIR CF-750 dye to prepare Alb(DBCO/Tt)x(Px)y(CF-750)z was used as the second delivery component. BT-474 HER2(+) breast cancer models were grown in athymic nude mice. Five groups of mice (a) untreated/PBS (b) first-component without Az, control for Click-1 (c) treated by Click-1 (d) first-component without TCO, control for Click-2, and (e) treated by Click-2 were used for the study. On day 0 mice were imaged with in vivo Xenogen optical imaging system and injected with the first pretargeting component followed by the second drug delivery component after 6h. Similarly, the second dose was administered on day 7. Mice were imaged at days 0 and 7 after administration of therapy to confirm the accumulation of components in tumors. Tumor volumes were measured every forth day up to day 20. After 21 days mice were sacrificed and tumors were extracted and analyzed by H&E staining for possible necrosis. Accumulation of the pretargeting and delivery components were observed in mice treated under Click-1 and Click-2 conditions compared to the corresponding controls and untreated mice. However, the accumulation was significantly higher in Click-2 group in comparison to all other groups. In addition, both pretargeting and delivery components were retained in the tumor for much longer time in Click-2 group than in other groups. We observed a tumor shrinking and significant decrease of tumor growth rate in Click-2 group. The results were confirmed by H&E immunohistochemistry analysis showing significant necrotic area in the Click-2 treated groups. Citation Format: Sudath Hapuarachchige, Wenlian Zhu, Yoshinori Kato, Dmitri Artemov. Bioorthogonal, two-component drug delivery in HER2(+) breast cancer mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2014-4572

Antiangiogenic therapy targeting VEGF/VEGFR signaling is an important strategy for cancer treatment, which is currently used in mono-therapy or combination therapy settings. However, the emerging resistance to antiangiogenic therapy as well as varied clinical response calls for the development of early, preferably noninvasive, markers of treatment response. In this study we used a recently developed molecular imaging tracer, site-specifically 99mTc-labeled engineered single-chain VEGF derivative, to monitor the VEGF receptor expression during the course of sunitinib antiangiogenic therapy in orthotopic human breast cancer model MDA-MB-231. The treatment protocol included two cycles of therapy delivered daily for 5 days (80mg/kg, orally) with a 5-day vacation between the cycles. Micro SPECT/CT imaging was performed longitudinally up to day 16. Animals in the control group received vehicle solution only. Tumors were excised and stained for the expression of CD31 endothelial cell marker. Experimental results suggest a highly heterogeneous vascular regression response to the first round of treatment, with significantly decreased tracer uptake and CD31 prevalence in some but not all tumors. As judged by the increase in tracer uptake and CD31 prevalence, a significant vascular rebound took place in all tumors after the vacation period. Judging by the same criteria, the second round of treatment was significantly less effective than the first round. Taken together, these results suggests that noninvasive molecular imaging of VEGF receptors reflects vascular regression and rebound in individual tumors and can provide clinically relevant information for monitoring and scheduling antiangiogenic therapy. Citation Format: Wenlian Zhu, Yoshinori Kato, Ronnie C. Mease, Susanta Sarkar, Marina V. Backer, Joseph M. Backer, Dmitri Artemov. Molecular imaging of VEGFR expression modulation by antiangiogenic therapy in mouse model of human breast cancer. [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 3926. doi:10.1158/1538-7445.AM2013-3926

The Twist1 gene has diverse roles during development and pathologic states such as cancer. Twist1 is best known for its roles in cancer by inducing an epithelial-mesenchymal transition (EMT) transcriptional program implicated in facilitating tumorigenesis, tumor progression and treatment resistance. Twist1 is a bHLH transcription factor that has both repressor and transactivation functions, but the importance of these different activities for Twist1 cancer phenotypes are unknown. We hypothesized Twist1 may mediate these various functions using distinct structural domains and/or motifs. We disrupted the putative transactivation domain in the Twist box of Twist1 by mutating a critical phenylalanine residue (F191) to glycine. We then created stable isogenic prostate cancer cell lines overexpressing wildtype and F191G versions of Twist1. We assessed the role of the Twist box using in vitro and in vivo assays, which mimic the various stages of cancer progression to metastasis. These include loss of homotypic cell-cell contacts, cell migration and invasion, anoikis resistance and soft agar colony formation. We also observed biophysical cell traction forces on a fabricated substratum and finally performed experimental lung metastasis assays. The overexpression of Twist1 in prostate cancer cells lead to an EMT biomarker phenotype and the F191G mutant lacked expression of some of these markers. The F191G mutant was deficient for transcriptional activity using promoter reporter based assays. Using single cell measurements we found that Twist1 expressing Myc-CaP cells exert more force on the substratum than vector control cells. Additional in vitro assays suggest Twist1 can confer cellular properties associated with increased tumor aggressiveness including increased migration/invasion, cell death/anoikis resistance and in vitro tumorigenic potential by soft agar colony formation. The Twist box mutant, F191G, displayed compromised activity compared to wildtype Twist1 in many of the in vitro assays described above revealing that the Twist box is necessary for many of the pro-metastatic functions of Twist1. We compared the gene expression profile of Twist1 and F191G overexpressing prostate cancer cells by microarray and observed that the F191G mutant had an expression profile that was similar to wildtype Twist1 but attenuated. Lastly, Twist1 overexpression compared to vector control prostate cancer cells showed an increased frequency of metastatic lung tumors using the experimental lung metastasis assay. Interestingly, Twist1 overexpression also resulted in the appearance of extra-thoracic metastases. The F191G mutant was less able to confer prostate cancer cells the ability to colonize metastatic lesions in the lung and resulted in no extra-thoracic metastases. Our results show that F191G mutation behaves as loss of function and is necessary for Twist1-induced metastasis of prostate cancer cells. Citation Format: Rajendra P. Gajula, Sivarajan T. Chettiar, Russell D. Williams, Saravanan Thiyagarajan, Yoshinori Kato, Khaled Aziz, Ruoqi Wang, Nishant Gandhi, Aaron T. Wild, Farhad Vesuna, Jinfang Ma, Tarek Salih, Jessica Cades, Elana Fertig, Shyam Biswal, Timothy F. Burns, Christine Chung, Charles M. Rudin, Venu Raman, Joseph M. Herman, Russell K. Hales, Steven An, Phuoc T. Tran. The Twist box is required for Twist1-induced prostate cancer 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 1493. doi:10.1158/1538-7445.AM2013-1493

Minimally invasive cancer therapy with nanostructures such as hyperthermia treatment with superparamagnetic iron oxide (SPIO) nanoparticles that has been recently demonstrated, represents novel highly important strategy for cancer management. In this study we have demonstrated that oscillating gradients of magnetic field induce significant non-thermal damage to cells labeled with magnetic SPIO nanoparticles. Iron oxide labeling was achieved either by specific cell surface labeling with biotinylated trastuzumab antibody followed by streptavidin-conjugated SPIO nanoparticles (MiltenyiBiotec) at 4 °C to prevent internalization, or by internalization of BNF-starch nanoparticles (Micromod Partikeltechnologie) using standard cell transfection reagents. Both protocols resulted in an efficient cell labeling with SPIO confirmed by Prussian blue staining. The novel treatment procedure, called GIFT (gradient-induced Fe therapy), was implemented on a small animal Bruker Biospec 9.4T MRI scanner equipped with G060 gradient system. SPIO nanoparticles can also serve as MRI contrast agent thus enabling image-guided delivery of therapy. Cell samples or mice were positioned in the center of the gradient system of the magnet, and cycling gradients with the amplitude of Gmax = 93.5 G/cm were applied for variable periods of time. The sample temperature was stabilized by a home-built temperature controlled chamber. The treatment resulted in an efficient destruction of both surface labeled and SPIO loaded human breast cancer HER2/neu positive BT-474 cells. We also obtained preliminary results for in vivo GIFT therapy with human breast cancer MDA-MB-231/luc models expressing firefly luciferase. Cancer cells prelabeled with BNF nanoparticles were inoculated orthotopically and GIFT and T2 MR imaging were initiated starting from 48h after the tumor inoculation. The tumor viability following the treatment was longitudinally evaluated by bioluminescence imaging (BLI) and by histological endpoints. GIFT therapy resulted in significant reduction of BLI intensities from SPIO prelabeled tumors, whereas no effect was observed for unlabeled tumors or when no gradients were applied. We hypothesize that variable gradients induce structural damage to iron-oxide labeled cells in the presence of strong polarizing magnetic field and that the potential mechanism may include mechanical destruction of cell membranes and/or organelles by magnetically polarized nanoparticles or their aggregates and is not related to hyperthermia induced by the radiofrequency. GIFT can be implemented on a standard MRI system that makes the technology applicable for clinical use. Citation Format: Sudath Hapuarachchige, Yoshinori Kato, Dmitri Artemov. Specific destruction of iron oxide labeled cancer cells by variable gradient magnetic field. [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 727. doi:10.1158/1538-7445.AM2013-727

Purpose: The Twist1 gene has diverse roles during development and pathologic states such as cancer. Twist1 is a bHLH transcription factor that has both repressor and transactivation functions. Twist1 is known to repress transcription by several mechanisms and is therefore considered to mediate its function mainly through transcriptional repression. The Twist1 transactivation domain has been reported but the functional significance of this domain is still unclear. In cancer, Twist1 is best known for its roles in facilitating tumor progression by inducing an epithelial-mesenchymal transition (EMT) transcriptional program implicated in facilitating tumorigenesis, tumor progression and treatment resistance. We hypothesized Twist1 may mediate these various functions using distinct structural domains and/or motifs. Here we have investigated the role of the Twist box domain of Twist1 in prostate cancer. Methods: We disrupted the putative transactivation domain (TD) of Twist1 by mutating a critical phenylalanine residue (F191) to glycine. We then created stable isogenic prostate cancer cell lines overexpressing wildtype and F191G versions of Twist1. We assessed the role of the Twist box using in vitro and in vivo assays, which mimic the various stages of cancer progression to metastasis. These include loss of homotypic cell-cell contacts, cell migration and invasion, anoikis resistance and soft agar colony formation. We also observed single-cell biophysical traction forces on a fabricated substratum and finally performed experimental lung metastasis assays. Results: The overexpression of Twist1 in prostate cancer cells lead to an EMT biomarker phenotype and the F191G mutant lacked expression of some of these markers. The F191G mutant was deficient for transcriptional activity using promoter reporter based assays. Using single cell measurements we found that Twist1 overexpressing prostate cancer cells exert more force on the substratum than vector control cells. Additional in vitro assays suggest Twist1 can confer cellular properties associated with increased tumor cell aggressiveness including increased migration/invasion, cell death/anoikis resistance and in vitro tumorigenic potential by soft agar colony formation. The Twist box mutant, F191G, displayed compromised activity compared to wildtype Twist1 in many of the in vitro assays described above revealing that the Twist box is necessary for many of the pro-metastatic functions of Twist1. We compared the gene expression profile of Twist1 and F191G overexpressing prostate cancer cells by microarray and observed that the F191G mutant had an expression profile that was similar to wildtype Twist1 but attenuated in key gene sets. Lastly, Twist1 overexpression compared to vector control prostate cancer cells showed an increased frequency of metastatic lung tumors using the experimental lung metastasis assay. Interestingly, Twist1 overexpression also resulted in the appearance of extra-thoracic metastases. The F191G mutant was less able to confer prostate cancer cells the ability to colonize metastatic lesions in the lung and resulted in no extra-thoracic metastases. Conclusions: Our results show that the Twist1-F191G mutant behaves as a loss of function and consequently that the Twist1 box is necessary for Twist1-induced metastasis of prostate cancer cells. Citation Format: Rajendra Gajula, Sivarajan Chettiar, Russell Williams, Saravanan Thiyagarajan, Khaled Aziz, Nishant Gandhi, Aaron Wild, Tarek Salih, Yoshinori Kato, Jessica Cades, Elana Fertig, Christine Chung, Joseph Herman, Russell Hales, Charles Rudin, Steven An, Phuoc T. Tran. The Twist box domain is required for Twist1-induced metastasis of prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B45.

Purpose: Olive oil has been reported to play a beneficial role in cancer prevention and treatment. Currently, studies published on the anticancer effects of olive oil and oleic acid, one of the main components of olive oil, have been limited to in vitro cell studies or to simple trophology following oral administration in vivo. The effects of these natural compounds on cancer cells/tissues after parenteral delivery are largely unknown, and deserve investigation. Meanwhile, olive oil can also serve as a nanoemulsion, which can encapsulate drug molecules and be used as a drug carrier. Therefore, the development of bioactive nanocarriers that, ideally, would also have synergistic anticancer activity with cargo drugs, will introduce a new concept of drug carriers, which would have clinical significance for cancer therapy. Concept: Oleic acid, one of the main components of olive oil, was found to enhance the effectiveness of vinorelbine, a semi-synthetic vinca alkaloid, against various breast and prostate cancer cell lines. Oleic acid was found to dramatically reduce cellular levels of the receptor tyrosine kinase ErbB2, produced by the breast cancer oncogene Her-2/neu. Olive oil also contains at least 30 phenolic compounds, which are strong antioxidants and radical scavengers. If a cargo drug would have a synergistic effect with the components of olive oil and is then encapsulated into the olive oil nanoemulsion, the efficacy is expected to be stronger than that of conventional nanocarrier-based agents. If successful, this could go beyond the conventional concept of nanocarriers, i.e., an inert substance would no long be a prerequisite for drug carriers. Methods: In this study, olive oil was used as a drug carrier, and vinblastine (VIN) was used as an anticancer agent. Olive oil nanoemulsions were prepared using a sonication method, and the size distribution and VIN content of the nanoemulsions were characterized with dynamic light scattering and a spectrophotometer, respectively. Orthotopic xenograft tumor models were grown in female SCID mice by injecting human breast carcinoma MCF-7 cells or SK-BR-3 cells into the mammary fat pad of the animal. Animals received intravenous injection of (i) saline, (ii) vinblastine sulfate (5 mg/kg), (iii) olive oil nanoemulsions, or (iv) VIN-loaded olive oil nanoemulsions at days 1, 4, and 7. Tumor volumes and body weights were monitored twice a week, up to 30 days. Results/Discussion: The hydrodynamic size of olive oil nanoemulsions was 6 nm. Although vinblastine sulfate dramatically reduced the tumor volume, the drug led to lethal toxicity (6 of 6 mice). The olive oil nanoemulsion alone did not affect the tumor growth of MCF-7 xenografts. Studies of the anticancer activity of VIN-loaded olive oil nanoemulsion, an anticancer activity using SK-BR-3 xenografts, and the biodistribution of olive oil nanoemulsions are currently under investigation. Acknowledgment: Supported by The Nagai Foundation, Tokyo. 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 384. doi:10.1158/1538-7445.AM2011-384

Purpose: Anticancer agents released from nanocarriers have to reach cancer cells in order to demonstrate their cytotoxic activity. The distribution pattern of anticancer agents can be affected by the tumor microenvironment which varies among different types of tumors. The purpose of this study is to delineate the relationship between drug transport and tumor microenvironmental factors, such as extracellular matrix (ECM) integrity. Such studies provide insight into the factors that hinder the accessibility of cargo molecules to cancer cells. Hypothesis: In tumors with a “permissive” ECM, small molecules are likely to diffuse farther after release from nanocarriers, while they are likely to be hindered in the tumors with denser ECM. Non-invasive monitoring of drug release and subsequent intratumoral distribution of the released drug can be visualized using a dual MR contrast technique capable of distinguishing between released and intact molecules in vivo. Methods: GdDTPA was used as a surrogate tracer for a water-soluble, small molecular-weight drug. Liposomes were used as the model nanocarrier in this study. Xenografts derived from the highly-invasive and metastatic MDA-MB-231 human breast carcinoma cell line, and weakly-metastatic MCF-7 human breast carcinoma cells were used. Liposomes encapsulating GdDTPA, iron particles, and rhodamine (Lip-Gd/Fe/Rho) were prepared using a sonication method, followed by extrusion. Orthotopic xenografts were grown in female SCID mice by injecting 3×106 of cancer cells into the mammary fat pad of the animal. All MR studies were carried out on a horizontal bore Bruker Biospec 9.4T spectrometer. MR images were acquired before, 30 min, and 23 hrs post-i.v. administration of Lip-Gd/Fe/Rho. To identify iron particles, a 3D fast low-angle shot (FLASH) sequence was used, while a 3D fast spin echo (RARE: Rapid Acquisition with Relaxation Enhancement) sequence with an effective echo time of 50 ms and five different repetition times was acquired to track GdDTPA released from liposomes. After completion of the MRI study, tumors were excised, fixed and were cut into 10μm sections for imaging with second harmonic generation multiphoton microscopy. Region of interest analysis were carried out on the MRI data. Results/Discussion: In vivo MRI results demonstrated that a decrease in T1 of the tumors was observed following i.v. administration of the Lip-Gd/Fe/Rho, indicating successful delivery and degradation of liposomes in the tumor. The comparison of differences in liposome delivery and its correlation with collagen-I density in the ECM of MDA-MB-231 and MCF-7 tumors is currently underway. Our results demonstrate the feasibility if using complementary imaging techniques to characterize the factors affecting intratumoral distribution of drug molecules after a release from nanocarriers. Acknowledgment: Supported by NIH grant R21 EB008162 (YK). 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 5219.

Background: Estrogen receptor alpha (ER) plays a crucial role in breast cancer development and progression due to its role as an effector/mediator of the mitogenic hormone estrogen. However, the role of transcriptional regulation of ER in tumor progression is not completely characterized. While defining the role of the basic helix loop helix transcription factor Twist (TWIST1) in breast cancer development, we identified ER as a downstream target of Twist in breast cells. Methods: A knock-up and knock-down approach in Twist negative and positive cell lines was used in order to characterize the regulation of ER by Twist. We analyzed ER expression by immunoblotting, ER transcriptional activity by promoter assays, DNA binding by chromatin immunoprecipitation, and estrogen independent growth in the presence of stripped serum or the estrogen antagonist tamoxifen (TAM). Additionally, ER promoter methylation was measured by methylation specific quantitative PCR (MS-qPCR). Using orthotopic xenografts in SCID mice, tumor growth was determined in presence of TAM and assayed for vascular permeability and volume by functional magnetic resonance imaging. Finally, the relationship between Twist and ER expression in breast tumors was evaluated by quantitative reverse-transcription PCR (qRTPCR). Results: Twist transcriptionally down-regulated ER expression by direct promoter modulation. Twist over-expression caused estrogen independence and TAM resistance, and induced hypermethylation of the ER promoter. In vivo, Twist expression resulted in estrogen independent growth and resistance to TAM. Twist over-expressing tumor xenografts exhibited high vascular volume and permeability. Moreover, Twist and ER expression were inversely correlated in primary breast cancer patient samples. Conclusion: The regulation of ER by Twist might be an underlying mechanism for ER negativity seen in breast tumors and could have important clinical implications for ER negative breast cancers. 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 3906.