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PDF file - 319K, Target inhibition after treatments with NVP-BKM120 in vivo. Tumor-bearing MMTV-CreBRCA1f/fp53+/- mice were biopsied before treatment, and tumor tissues harvested within 3 hours after the last treatment. Tissues were fixed and processed for immunohistochemistry with anti-phospho AKT (S473) antibodies. 400 x magnification

PDF file - 526K, FDG-uptake and CT-PET scanning of mice before and after 2 weeks of treatment with NVP-BKM120. Tumor-bearing female MMTV-CreBRCA1f/fp53+/- mice were imaged with PET-CT scans before and after treatments with NVP-BKM120 at 50 mg/kg/day. Yellow arrows in the upper panel point to the dominant mass in each mouse before treatment. Synchronous additional tumors are indicated with green arrows. The lower panel displays the same mice imaged within 3 hours of treatment with a two-week course of NVP-BKM120 at 50 mg/kg/day. Percent decrease of FDG-uptake in the dominant mass was determined as described in Materials and Methods

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PDF file - 71K, In vitro responses of BRCA1-mutant breast cancer cell lines to treatments with NVP-BKM120 (1 muM), Olaparib (10 muM) or their combination. Cells were seeded in quadruplicate in 96-well plates and treated for 7 days as indicated. A. HCC1937 parental cells, B. SUM149 cells, C. HCC1937 cells stably transfected with vector control or a PTEN expression construct (D)

PDF file - 137K, Effects of the combination of NVP-BKM120 and Olaparib (left pair, treated with NVP-BKM120 at 30 mg/kg/day and Olaparib at 50 mg/kg/day) and of Olaparib alone (right pair, 50 mg/kg/day). Mice were imaged before and after 3 daily treatments, the repeat scan was obtained within 3 hours after the third treatment. The mouse on the right had multiple synchronous primary tumors, labeled in the before and after images with blue, orange and yellow arrows. Percent FDG-uptake was 65% (tumor with orange arrow), 55% (tumor with blue arrow) and 64% (tumor with yellow arrow). The mouse in the image pair on the right that was treated with Olaparib carried only one macroscopically detectable tumor (red arrow). Olaparib treatments increased FDG-uptake by 75% in this tumor. Note that in the after-Olaparib-treatment image (far right) several hotspots in the upper thorax are visible which upon necroscopy were found to be tumors of less than 2 mm in diameter

PDF file - 159K, Erk phosphorylation after treatments with NVP-BKM120 in vivo. Tumor tissues were harvested from MMTV-CreBRCA1f/fp53+/- mice within 3 hours after the last treatment with NVP-BKM120. Tissues were fixed and processed for immunohistochemistry with anti-phospho ERK (Thr202/Tyr204) antibodies. 400 x magnification

PDF file, 59K, Supplementary Figure S1. A typical example of Bilateral Inguinal Lymph node size 8 days after vaccination with the native peptide QQNPSYDSV (#14) or its modified derivative YLNPSVDSV (#24).

Purpose: The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types.Design: Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro.Results: EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askin's Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing.Conclusions: These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development. Clin Cancer Res; 18(19); 5341–51. ©2012 AACR.

Methylene blue (MB) is a near-infrared fluorophore that provides a stable visual map of skin perfusion after intravenous injection. We explored the capability of MB to predict submental flap postoperative outcome using a single intraoperative measurement. Submental flaps were created in N = 15 pigs and imaged using the FLARE imaging system immediately after surgery and at 72 hours. Using the first 3 pigs, optimal MB dosing was found to be 2.0 mg/kg. Training and validation sets of 6 pigs each were then used for receiver operating characteristic analysis. In the training set, a contrast-to-background ratio (CBR) threshold of 1.24 provided the highest sensitivity and specificity to predict tissue necrosis at 72 hours. In the validation set, this threshold provided a prediction sensitivity of 95.3% and a specificity of 98.0%. We demonstrate that a single intraoperative near-infrared measurement can predict submental flap outcome at 72 hours.

Preserving the nipple-areolar complex with a nipple-sparing mastectomy improves cosmesis compared with skin-sparing mastectomy. However, complications such as necrosis of the nipple-areolar complex significantly affect cosmetic outcome. Many factors influence nipple-areolar perfusion, and no consensus currently exists on optimal incisional choice. This study evaluates 2 nipple-sparing mastectomy incision models using near-infrared fluorescence to assess perfusion quantitatively. The periareolar and radial incisions were compared with 2 control models in Yorkshire pigs (N = 6). Methylene blue and indocyanine green were injected intravenously, and near-infrared fluorescence images were recorded at 3 time points: before surgery, immediately after (0 hour), and 3 days postoperatively. Contrast-to-background ratio was used to assess perfusion. At 72 hours, radial incisions showed a statistically significantly higher perfusion compared with periareolar incisions (P < 0.05). Based on our findings, radial incisions for nipple-sparing mastectomy may be preferable due to higher perfusion; however, clinical trials are necessary for further assessment.

Purpose: The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types. Design: Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro. Results: EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askin's Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing. Conclusions: These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development. Clin Cancer Res; 18(19); 5341–51. ©2012 AACR.

Brown adipose cells are specialized to dissipate chemical energy in the form of heat, as a physiological defence against cold and obesity. PRDM16 (PR domain containing 16) is a 140 kDa zinc finger protein that robustly induces brown fat determination and differentiation. Recent data suggests that brown fat cells arise in vivo from a Myf5-positive, myoblastic lineage by the action of PRDM16 (ref. 3); however, the molecular mechanisms responsible for this developmental switch is unclear. Here we show that PRDM16 forms a transcriptional complex with the active form of C/EBP-beta (also known as LAP), acting as a critical molecular unit that controls the cell fate switch from myoblastic precursors to brown fat cells. Forced expression of PRDM16 and C/EBP-beta is sufficient to induce a fully functional brown fat program in naive fibroblastic cells, including skin fibroblasts from mouse and man. Transplantation of fibroblasts expressing these two factors into mice gives rise to an ectopic fat pad with the morphological and biochemical characteristics of brown fat. Like endogenous brown fat, this synthetic brown fat tissue acts as a sink for glucose uptake, as determined by positron emission tomography with fluorodeoxyglucose. These data indicate that the PRDM16-C/EBP-beta complex initiates brown fat formation from myoblastic precursors, and may provide opportunities for the development of new therapeutics for obesity and type-2 diabetes.

Biodegradable scaffolds could revolutionize tissue engineering and regenerative medicine; however, in vivo matrix degradation and tissue ingrowth processes are not fully understood. Currently a large number of samples and animals are required to track biodegradation of implanted scaffolds, and such nonconsecutive single-time-point information from various batches result in inaccurate conclusions. To overcome this limitation, we developed functional biodegradable scaffolds by employing invisible near-infrared fluorescence and followed their degradation behaviors in vitro and in vivo. Using optical fluorescence imaging, the degradation could be quantified in real-time, while tissue ingrowth was tracked by measuring vascularization using magnetic resonance imaging in the same animal over a month. Moreover, we optimized the in vitro process of enzyme-based biodegradation to predict implanted scaffold behaviors in vivo, which was closely related to the site of inoculation. This combined multimodal imaging will benefit tissue engineers by saving time, reducing animal numbers, and offering more accurate conclusions.

There is a need to improve treatments for metastatic breast cancer. Here, we show the activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in a MMTV-CreBrca1f/fTrp53+/− mouse model of breast cancer. When treated with the pan-class IA PI3K inhibitor NVP-BKM120, tumor doubling was delayed from 5 to 26 days. NVP-BKM120 reduced AKT phosphorylation, tumor cell proliferation, and angiogenesis. Resistant tumors maintained suppression of AKT phosphorylation but exhibited activation of the MAPK pathway at the “pushing margin.” Surprisingly, PI3K inhibition increased indicators of DNA damage, poly-ADP-ribosylation (PAR), and γ-H2AX, but decreased Rad51 focus formation, suggesting a critical role of PI3K activity for Rad51 recruitment. The PARP inhibitor olaparib alone attenuated tumor growth modestly; however, the combination of NVP-BKM120 and olaparib delayed tumor doubling to more than 70 days in the mouse model and more than 50 days in xenotransplants from human BRCA1-related tumors, suggesting that combined PI3K and PARP inhibition might be an effective treatment of BRCA1-related tumors. Significance: Current treatment options for triple-negative breast cancer are limited to chemotherapeutic regimens that have considerable toxicity and are not curative. We report here that the combination of a PI3K inhibitor with a PARP inhibitor provides in vivo synergy for treatment of an endogenous mouse model for BRCA1-related breast cancers, making this a candidate combination to be tested in human clinical trials. Cancer Discov; 2(11); 1048–63. ©2012 AACR. Read the Commentary on this article by Rehman et al., p. 982. This article is highlighted in the In This Issue feature, p. 961

Increasing the number of pinholes in small-animal SPECT significantly improves its count sensitivity. When the detector(s) are small, however, overlapping of projections (multiplexing) from different pinholes is unavoidable and can amplify noise in reconstructed images. We have evaluated the performance of two multi-pinhole systems, one with and one without multiplexing, for a prototypical tumor-imaging task. We prepared seven beads (∼1.8-mm diameter) to mimic tumors labeled with Tc-99m. A uniform gelatin phantom was used to simulate normal background tissue. The tumor-to-normal tissue ratio was ∼6:1, and each bead contained ∼1 μCi. The first scanner, equipped with two 0.8-mm pinholes on each of three heads (HMS-0.8), acquired only non-overlapping projections. We also scanned the phantom using 1.6mm pinholes (HMS-1.6) The second scanner had 9 pinholes on each of four heads, and allowed multiplexing. To compensate for decay, the phantom was scanned for 50 min on HMS-0.8, 58 min on HMS-1.6, 82 min on NanoSPECT/CT with 1.4 mm pinholes (Nano-1.4), and 102 min with 1.0 mm pinholes (Nano-1.0). A total of 30 (24) angular projections were acquired with HMS (Nano); these were reconstructed using 10 OSEM subsets for HMS, and 4 subsets for Nano. The mean voxel value in each sphere, and the mean and standard deviation in a large VOI in the background, were used to compute the signal-to-noise ratio (SNR) and contrast of each bead. The relative noise in the background was also calculated. The systems with and without multiplexing yielded similar image quality and average bead SNR, especially for HMS-0.8 and Nano-1.0. Both systems yielded very similar SNR values, despite the fact that the multiplexed system acquired data using 36 pinholes, while the non-multiplexed system had only 6 pinholes. The multiplexed acquisition did not seem to adversely affect the image contrast of the spherical tumors.

The development of novel diagnostic agents for the detection of breast cancer microcalcifications requires a reliable animal model. Based on previous work from our group, we hypothesized that a single systemic injection of recombinant bone morphogenetic protein-2 (rBMP-2) could be used to create such a model. The cDNA encoding mature human BMP-2 was expressed in BL21(DE3) bacteria, purified to homogeneity, and refolded as a dimer. Bioactivity was confirmed using a C2C12 alkaline phosphatase assay. rBMP-2 was radiolabeled with (99m)Tc, and its biodistribution and clearance were quantified after both intravenous (IV) and intraperitoneal (IP) injection. Fischer 344 rats bearing syngeneic R3230 breast tumors received a single intraperitoneal injection of rBMP-2 at a specified dose. Tumor microcalcification was quantified over time using micro-single photon emission computed tomography (SPECT) and microcomputed tomography (CT). rBMP-2 could be expressed in E. coli at high levels, isolated at >95% purity, and refolded to a bioactive dimer. Beta-phase half-life was 30.5 min after IV administration and 47.6 min after IP administration. Renal excretion was the primary mode of clearance. A single IP injection of >or=50 microg rBMP-2 when tumors were not yet palpable resulted in dose-dependent microcalcification in 8 of 8 R3230 tumors. No calcification was found in control tumors or in normal tissues and organs of animals injected with rBMP-2. Tumor calcification increased progressively between weeks 2 and 4 post-rBMP-2 injection. A single IP injection of rBMP-2 in rats bearing a syngeneic breast cancer will produce dose-dependent and time-dependent microcalcifications. This animal model lays the foundation for the development of novel diagnostic radiotracers for breast cancer.

Microcalcification is a hallmark of breast cancer and a key diagnostic feature for mammography. We recently described the first robust animal model of breast cancer microcalcification. In this study, we hypothesized that high-resolution computed tomography (CT) could potentially detect the genesis of a single microcalcification in vivo and quantify its growth over time. Using a commercial CT scanner, we systematically optimized acquisition and reconstruction parameters. Two ray-tracing image reconstruction algorithms were tested: a voxel-driven “fast” cone beam algorithm (FCBA) and a detector-driven “exact” cone beam algorithm (ECBA). By optimizing acquisition and reconstruction parameters, we were able to achieve a resolution of 104 μm full width at half-maximum (FWHM). At an optimal detector sampling frequency, the ECBA provided a 28 μm (21%) FWHM improvement in resolution over the FCBA. In vitro, we were able to image a single 300 μm X 100 μm hydroxyapatite crystal. In a syngeneic rat model of breast cancer, we were able to detect the genesis of a single microcalcification in vivo and follow its growth longitudinally over weeks. Taken together, this study provides an in vivo “gold standard” for the development of calcification-specific contrast agents and a model system for studying the mechanism of breast cancer microcalcification.

The PTEN and p53 tumor suppressors are among the most commonly inactivated or mutated genes in human cancer, including prostate cancer. Loss of PTEN is associated with increased pathologic Gleason score and risk of clinical recurrence, and 20-60% of human metastatic prostate cancers have loss of heterozygosity at the PTEN locus, resulting in hyperactivation of the PI3K/mTOR pathway. Mice with germline heterozygosity for PTEN have been shown to develop prostate intraepithelial neoplasia (PIN) at a high rate (>60%) and mice with prostate specific homozygous deletion of PTEN develop invasive prostate cancer, albeit with prolonged latency of approx. 6 months. Combined inactivation of PTEN and p53 in mouse prostate elicits invasive cancer by 9 weeks of age and invariable lethality by 7 months of age. There are several PI3K pathway-directed therapies currently in Phase I clinical trials, but the underlying tumor genetic signature of patients most likely to respond to these therapies is largely unknown. To understand the significance of targeting the PI3K/mTOR pathway in advanced prostate cancer driven by PTEN +/− p53 loss, we evaluated the impact of GSK458 (a dual PI3K/mTOR inhibitor) in prostate-specific PTEN/p53 double knockout mice and prostate-specific PTEN-knockout mice. The mice were imaged by synchronized 18FDG-PET and T2-weighted MRI, respectively, for baseline tumor metabolic and volumetric assessment prior to drug administration. GSK458 was administered at 3 mg/kg by daily oral gavage for 3 weeks with serial 18FDG-PET and T2-weighted MRI imaging at 2 days, 1 week, 2 weeks and 3 weeks after initiation of treatment, followed by sacrifice, prostate harvest and standard hisopathologic and immunohistochemical staining. GSK458 treatment of PTEN/p53-deficient and PTEN-deficient mice results in target inhibition, based on pharmacodynamic assessment by 18FDG-PET uptake. MRI and histopathologic analysis demonstrate that there is a significant reduction, but not complete regression of tumor burden in both intraepithelial and poorly differentiated atypical components within stroma and partial stromal collapse following 3 weeks of GSK458 treatment. These data highlight the feasibility of monitoring dual pharmacodynamics/antitumor effects of PI3K-directed therapies using 18FDG-PET/MRI imaging and underscore the utility of genetically engineered mouse models to predict response to targeted therapies in genetically stratified human clinical trials. The evaluation of PI3K-isoform specific inhibitors and the design of rational combinations to overcome de novo and acquired resistance mechanisms to PI3K-directed therapies are currently being explored in multiple genetically engineered mouse model systems. 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 LB-418. doi:10.1158/1538-7445.AM2011-LB-418