EWS-FLI-1-targeted cytotoxic T-cell killing of multiple tumor types belonging to the Ewing sarcoma family of tumors.

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.

Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1-related breast cancer.

UNLABELLED: 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-CreBrca1(f/f)Trp53(+/-) 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.

Real-time monitoring of tumorigenesis, dissemination, & drug response in a preclinical model of lymphangioleiomyomatosis/tuberous sclerosis complex.

BACKGROUND: TSC2-deficient cells can proliferate in the lungs, kidneys, and other organs causing devastating progressive multisystem disorders such as lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC). Preclinical models utilizing LAM patient-derived cells have been difficult to establish. We developed a novel animal model system to study the molecular mechanisms of TSC/LAM pathogenesis and tumorigenesis and provide a platform for drug testing. METHODS AND FINDINGS: TSC2-deficient human cells, derived from the angiomyolipoma of a LAM patient, were engineered to co-express both sodium-iodide symporter (NIS) and green fluorescent protein (GFP). Cells were inoculated intraparenchymally, intravenously, or intratracheally into athymic NCr nu/nu mice and cells were tracked and quantified using single photon emission computed tomography (SPECT) and computed tomography (CT). Surprisingly, TSC2-deficient cells administered intratracheally resulted in rapid dissemination to lymph node basins throughout the body, and histopathological changes in the lung consistent with LAM. Estrogen was found to be permissive for tumor growth and dissemination. Rapamycin inhibited tumor growth, but tumors regrew after the drug treatment was withdrawn. CONCLUSIONS: We generated homogeneous NIS/GFP co-expressing TSC2-deficient, patient-derived cells that can proliferate and migrate in vivo after intratracheal instillation. Although the animal model we describe has some limitations, we demonstrate that systemic tumors formed from TSC2-deficient cells can be monitored and quantified noninvasively over time using SPECT/CT, thus providing a much needed model system for in vivo drug testing and mechanistic studies of TSC2-deficient cells and their related clinical syndromes.

Mice with a targeted deletion of the type 2 deiodinase are insulin resistant and susceptible to diet induced obesity.

BACKGROUND: The type 2 iodothyronine deiodinase (D2) converts the pro-hormone thyroxine into T3 within target tissues. D2 is essential for a full thermogenic response of brown adipose tissue (BAT), and mice with a disrupted Dio2 gene (D2KO) have an impaired response to cold. BAT is also activated by overfeeding. METHODOLOGY/PRINCIPAL FINDINGS: After 6-weeks of HFD feeding D2KO mice gained 5.6% more body weight and had 28% more adipose tissue. Oxygen consumption (V0(2)) was not different between genotypes, but D2KO mice had an increased respiratory exchange ratio (RER), suggesting preferential use of carbohydrates. Consistent with this, serum free fatty acids and ß-hydroxybutyrate were lower in D2KO mice on a HFD, while hepatic triglycerides were increased and glycogen content decreased. Neither genotype showed glucose intolerance, but D2KO mice had significantly higher insulin levels during GTT independent of diet. Accordingly, during ITT testing D2KO mice had a significantly reduced glucose uptake, consistent with insulin resistance. Gene expression levels in liver, muscle, and brown and white adipose tissue showed no differences that could account for the increased weight gain in D2KO mice. However, D2KO mice have higher PEPCK mRNA in liver suggesting increased gluconeogenesis, which could also contribute to their apparent insulin resistance. CONCLUSIONS/SIGNIFICANCE: We conclude that the loss of the Dio2 gene has significant metabolic consequences. D2KO mice gain more weight on a HFD, suggesting a role for D2 in protection from diet-induced obesity. Further, D2KO mice appear to have a greater reliance on carbohydrates as a fuel source, and limited ability to mobilize and to burn fat. This results in increased fat storage in adipose tissue, hepatic steatosis, and depletion of liver glycogen in spite of increased gluconeogenesis. D2KO mice are also less responsive to insulin, independent of diet-induced obesity.

High-resolution computed tomography of single breast cancer microcalcifications in vivo.

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 × 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.

A dose- and time-controllable syngeneic animal model of breast cancer microcalcification.

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.