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).

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.

Abstract The PTEN and p53 tumor suppressors are the most commonly altered genes in human cancer, including prostate cancer (PCa). Loss of PTEN is associated with increased Gleason score and clinical recurrence, and the majority of human metastatic PCas have PTEN loss via multiple mechanisms. Mice with prostate-specific homozygous deletion of PTEN develop invasive PCa albeit with prolonged latency of 6-8 months. Combined PTEN/p53 inactivation in mouse prostate elicits invasive cancer by 9 weeks of age and invariable lethality by 6 months of age. Since PTEN loss results in PI3K/mTOR pathway activation, we evaluated the impact of GSK458 (PI3K/mTOR inhibitor) and GSK418 (PI3K beta/delta isoform-specific inhibitor), singly and in combination with GSK212 (MEK inhibitor), in uncastrated, prostate-specific PTEN/p53 double knockout mice (4-6 months) and PTEN mice (11-14 months), respectively, harboring advanced PCa. The drugs were administered by daily oral gavage for 3 weeks with serial 18FDG-PET/MRI imaging at baseline, 2 days, 1 week, 2 weeks and 3 weeks post-treatment respectively. GSK458 treatment of PTEN/p53 and PTEN mice results in reduction in FDG-PET uptake as early as 24 h post-treatment, with 40% tumor shrinkage by 1 week post-treatment, but rapid regrowth of 18FDG-avid tumor by 2-3 weeks post-treatment. This acquired resistance was found to be mediated in part through upregulation of multiple receptor tyrosine kinases. In contrast, PTEN/p53 and PTEN mice did not respond to GSK418, by either FDG-PET or MRI analysis. We observed increased pERK/total ERK ratio by Western blot analysis and increased p-ERK staining by immunohistochemistry in GSK458 and GSK418-treated PTEN/53 mice, respectively. Treatment of PTEN/p53 and PTEN mice with GSK212 resulted in an approx. 40% reduction in tumor volume over 3 weeks. Treatment of PTEN/p53 mice with a combination of GSK458 plus GSK212 resulted in approx. 60% reduction of tumor volume at 3 week post-treatment. Strikingly, treatment of prostate-specific PTEN only mice with GSK458 plus GSK212 combination resulted in a >90% tumor regression at 3 weeks post-treatment. These results demonstrate the potential utlitity of PI3K/MEK-directed combination therapies in the neoadjuvant setting for locally advanced disease or hormone-sensitive phase in metastatic disease, thus delaying the need for hormone ablative therapy and its associated morbidity in advanced PCa. The data underscore the value of genetically engineered mouse models to co-clinically evaluate biomarkers of response and resistance to targeted therapies, and elucidate mechanisms of acquired resistance early in clinical development. The design of “personalized” combination therapies to overcome resistance to PI3K/MEK-directed therapies in the hormone-naive and castration-resistant contexts are currently underway in multiple GEMMs and Phase Ib co-clinical trials in advanced PCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-365. doi:1538-7445.AM2012-LB-365

Abstract 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.

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 18 FDG-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 18 FDG-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 18 FDG-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 18 FDG-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