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Among the many sarcoma subtypes, high-grade tumors that have failed to differentiate or undergone dedifferentiation strongly correlate with metastatic potential and poor clinical outcomes. To examine the factors that regulate cell fate, lineage plasticity, and tumor grade, we focused on two common liposarcoma variants, dedifferentiated liposarcoma (DDLS) and well-differentiated liposarcomas (WDLS), that exist at both ends of the differentiation spectrum. Since tumor grading using histology is highly subjective due to widespread tumor heterogeneity, we hypothesized that an sc/snRNA-seq-based approach would be more accurate. To determine if candidate therapies modulate differentiation, we developed a quantitative metric of differentiation using scRNA-seq. The resulting mesenchymal tissue landscape (MTL) is akin to a Waddington landscape but optimized to distinguish DDLS from WDLS. After prospectively obtaining tumors from seven chemo-naïve patients, we performed scRNA-seq on three DDLS and five WDLS specimens, totaling 59,917 cells. Classical markers were used to identify cell types. Since no pre-existing liposarcoma training sets existed, malignant cells were identified by copy number variation (CNV) inferred from the scRNA-seq data. Overexpression of MDM2, CDK4, and HMGA2, known markers of LS, were positive in clusters classified by SingleR as fibroblasts, adipocytes, and myocyte - the mesenchymal subpopulation. We also inferred CNVs and found expected amplifications in 12q13-15 in the mesenchymal subpopulation. Next, we measured cell differentiation by CytoTRACE. A subcluster of malignant fibroblast-like cells positive for MDM2 and CDK4 was the most de-differentiated. Conversely, cells labeled as adipocytes and endothelial cells were the most differentiated cells. Mapping WDLS and DDLS onto the MTL demonstrated that WDLS had more mature cells than DDLS. Surprisingly, while the pathology-assigned diagnosis falls into just two categories, our approach identified a wide range of liposarcoma cells spanning the full spectrum of possible differentiation states. Though follow-on studies are needed, our data suggest that quantitative differentiation metrics can identify malignant cells in transit from a DDLS-to-WDLS state, or possibly the reverse, whereby cells undergo dedifferentiation. Both phenomena are suspected clinically, given the frequent co-existence of WDLS and DDLS in the same tumors, and the noted emergence of DDLS from WDLS diagnosed years earlier. Summarizing our key findings, we measured the degree of differentiation in liposarcoma cells as a precursor to ongoing studies aimed at understanding the molecular mechanisms that underpin LS plasticity and differentiation. Ultimately, we aim to identify the genes and pathways linked to cell fate and discover novel biologically targeted therapies capable of pushing DDLS toward a less aggressive, slower-growing state. Citation Format: Danh Truong, Hannah C. Beird, Chia-Chin Wu, Sandhya Krishnan, Davis Ingram, Alexander Lazar, Emily Keung, Christina Roland, Wantong Yao, Robert Benjamin, Neeta Somaiah, Barry W. Feig, Joseph A. Ludwig. Defining differentiation States in well-differentiated and de-differentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6558.

Background: The majority of cancer patients remain refractory to existing cancer immunotherapies. Despite the growing evidence that dysregulated metabolism contributes to the exhaustion of tumor-infiltrating T lymphocytes (TILs) and the loss of their effector functions within the metabolically restricted tumor microenvironment (TME), actionable targets to rescue metabolic fitness and anti-tumor activity of TILs remain elusive. Sirt2 is an NAD+ dependent histone deacetylase and conflicting evidences suggest its tumor-suppressor and oncogenic roles. In inflammatory response, Sirt2 suppresses inflammation via negative regulation of NF-κB subunit; however, the role of Sirt2 in tumor immunity has not been described. Method: Human TILs from non-small cell lung cancer (NSCLC) tumor samples and matched peripheral blood T cells were analyzed for Sirt2 expression by flow cytometry. The role of Sirt2 in anti-tumor immunity was studied by in vivo B16F10 tumor challenge in Wild-type (Wt) and Sirt2 knockout (Sirt2KO) mice. The role of Sirt2 in T cell effector functions was investigated ex vivo by CFSE proliferation assay, IFN-γ ELISpot assay, intracellular staining of effector markers and LDH cytotoxicity assay on Wt versus Sirt2KO T cells. Sirt2 targets were identified using mass spectrometry (MS) and Co-immunoprecipitation analyses. T cells metabolic changes were investigated using seahorse bioanalyzer and LC-MS/MS Metabolomic profiling. Sirt2 blockade in human T cells was performed using AGK2, a Sirt2 selective inhibitor. Result: We show that Sirt2 expression is upregulated within the TME, and its upregulation in human TILs is associated with a poor clinical response to immunotherapy in a phase I clinical trial in advanced NSCLC. We also show that, Sirt2 deficiency in mice boosts T cell effector functions and tumor rejection in vivo. Our molecular and metabolomic studies revealed multiple metabolic pathways as Sirt2 targets including glycolysis, TCA-cycle, FAO and glutaminolysis. We found that Sirt2 deacetylase deficiency increased acetylation and enzymatic activity of key metabolic enzymes leading to a hyper metabolic status of T cells. Finally pharmacologic inhibition of Sirt2 in human TILs isolated from NSCLC patients enhances their metabolic fitness and effector functions. Conclusion Our findings indicate Sirt2 as a master suppressor of T cell metabolism amenable to therapeutic targeting and Sirt2 inhibition reprograms T cell metabolic fitness to optimally sustain their effector function within the metabolically challenging TME, thus, leading to an effective anti-tumor immune response. Citation Format: Imene Hamaidi, Lin Zhang, Nayoung Kim, Min Hsuan Wang, Cristina Iclozan, Bin Fang, Min Liu, John M. Koomen, Anders E. Berglund, Sean J. Yoder, Jiqiang Yao, Robert W. Engelman, Ben C. Creelan, Jose R. Conejo-Garcia, James J. Mulé, Scott J. Antonia, Sungjune Kim. Sirt2 blockade promotes T cell metabolism and restores the anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1635.

Polyploid giant cancer cells (PGCCs) are abundant in cancers after chemotherapy. PGCCs have generally been considered nonviable because of their inability to execute mitosis. However, our recently reported studies challenged this paradigm by showing that PGCCs were capable of tumor initiation. To understand the role of PGCCs in chemoresistance, we monitored paclitaxel-induced PGCCs by conventional and live-cell fluorescence time-lapse recording. We found that paclitaxel induced massive cell death but also initiated endoreplication to form PGCCs. These PGCCs continued to grow via endoreplication, endomitosis, or cytofission before generating new mononuclear or multinucleated daughter cells via nuclear fragmentation or budding. These daughter cells resumed mitosis with a new stable karyotype and exhibited drug resistance. Budding from PGCCs was also observed in chemotherapy-treated ovarian cancer specimens from patients. On the basis of our findings, we propose the existence of an amitotic giant cell cycle composed of four phases: initiation, maintenance, termination, and speciation. We believe this is the first report of fluorescent live-cell imaging of the birth of resistant cancer cells at the single-cell level in response to paclitaxel treatment. Our findings provide strong evidence that a stress-induced amitotic giant cell cycle may be a programmed event underlying chemotherapy resistance and cancer relapse. Citation Format: Na Niu, Jie Zhang, Ning Zhang, Imelda Mercado-Uribe, Fangfang Tao, Zhenbo Han, Sen Pathak, Asha Multani, Jian Kuang, Jun Yao, Robert Bast, Anil Sood, Mien-Chie Hung, Jinsong Liu. Single-cell live imaging of paclitaxel-resistant cancer cell generation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-119.

MEDI-573 is a human antibody that neutralizes the insulin-like growth factors, IGF-1 and IGF-2. IGFs are over-expressed in multiple types of cancer; their over-expression is a potential mechanism for resistance to IGF-1 receptor (IGF-1R)-targeting therapy. Effects of IGFs on cell proliferation, differentiation, and survival are mediated through their binding to and activation of IGF-1R or insulin receptor A (IR-A). In this study, we studied the anti-tumor activity and mechanism of action of MEDI-573 in models of sarcoma. MEDI-573 potently inhibited in vitro proliferation of several sarcoma cell lines, with Ewing's sarcoma cell lines being the most sensitive. This inhibition also occurred after growth stimulation with added IGF-1- and IGF-2. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGF-1R, pIR, and pAKT, and significantly blocked IGF-1- and IGF-2-induced activation of the IGF-1R and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo, and inhibition correlated with neutralization of IGF-1 and IGF-2. Combination of MEDI-573 with either rapamycin or another mTOR inhibitor, AZD2014, significantly enhanced the anti-tumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by secreted IGF-1 and IGF-2, and inhibition of IGF-1 and IGF-2 by MEDI-573 results in potent anti-tumor activity in several sarcoma models. Our data provide evidence for evaluation of MEDI-573 and mTORi combinations in clinical studies of sarcoma patients. Note: This abstract was not presented at the meeting. Citation Format: Haihong Zhong, Christine Fazenbaker, Shannon Breen, Cui Chen, Jiaqi Huang, Morehouse Chris, Yihong Yao, Robert Hollingsworth. Medi-573 alone or in combination with mammalian target of rapamycin inhibitors, targets the insulin-like growth factor pathway in sarcomas. [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 4493. doi:10.1158/1538-7445.AM2014-4493

Corrigendum to ‘‘Imaged based estimation of food volume using circular referents in dietary assessment’’ [Journal of Food Engineering 109 (2012) 76–86] Wenyan Jia , Yaofeng Yue , John D. Fernstrom , Ning Yao , Robert J. Sclabassi , Madelyn H. Fernstrom , Mingui Sun a,b,⇑ Department of Neurosurgery, University of Pittsburgh, PA, United States Department of Electrical and Computer Engineering, University of Pittsburgh, PA, United States Department of Psychiatry, University of Pittsburgh, PA, United States Computational Diagnostics Inc., Pittsburgh, PA, United States

Preparation and characterization of ZnSiP2, ZnGeP2 and ZnGeP1.8As,2 single crystalsHe -Sheng Shen, Guang -Qing Yao, Robert Kershaw, Kirby Dwight, and Aaron Wold*Department of Chemistry, Brown UniversityProvidence, Rhode Island 02912*Address all correspondenceAbstractSingle crystals of ZnSiP2, ZnGeP2 and ZnGeP1 8As 2 have been grown by several techniquesand their electronic and optical properties compared. For ZnSiP2 there are marked absorp-tion bands at 10 and 11.5 pm, and at 13 um for ZnGeP2. Upon substitution of 10 mole percentof arsenic for Phosphorus , the latter band is red -shifted by 0.3 pm. This compositionrepresents the limit of substitution of arsenic for phosphorus in this structure.IntroductionThe ternary compounds ZnSiP2 and ZnGeP2 are semiconductors which crystallize with thechalcopyrite structure (space group I42d). ZnSiP2 has been reported to melt at 1250 °C (1)and single crystals were obtained from a molten zinc or tin flux (1, 2) as well as by chemi-cal vapor transport (2 -5) using C12, CC14, ZnC12 or I2 as the transport agent. There hasbeen no report of crystal growth from the melt.Single crystals of ZnGeP2 have been grown from the melt (3, 5 -7) or from molten metalfluxes (2, 3