bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2943: Acoustic cell separation platform for efficient preparation of single cell/nuclei suspension from complex samples for genomics analysis
Authors: Yu Liping 
Published: June 2022

Abstract
Preparing a single cell suspension is a critical step in any solid tissue for genomics analysis as the presence of cellular debris, dead cells and free RNA will cause incorrect cell count, noise in sequencing read and possibly clogging of the microfluidic chip. Single cell preparation not only involves the tissue dissociation process but the “clean-up” step of the dissociation product. Current methods for removing dead cells and debris use magnetic beads and density gradient centrifugation, which are lengthy and costly, and often result in low cell recovery. Here we present a high-speed acoustic cell separation platform—MARS® Sample Preparation system. The system has three independent modules and each has an acoustic cell processing chip (CPC) which creates different moving trajectory of live cells, dead cells, and cellular debris in the acoustic field to achieve high efficiency of single cell separation. MARS® sample preparation provides fast, gentle, and label-free protocol to separate single cells and single nuclei from various types of organs and dissociation processes including manual enzymatic approach and automatic platform. Separated single cells and nuclei demonstrate compatibility with the downstream single cell genomics platform. We here report the separation of live single cells and nuclei from mouse brain tissue dissociation with over 99% removal of myelin and other debris, and 60%-80% recovery of cells and nuclei. Those results were obtained on the MARS® with 0.8mL/min flow rate and three samples in parallel process. Samples before and after MARS® acoustic process were analyzed with both flow cytometry and imaging-based cell counter. The performance of “clean-up” of the acoustic process was compared to conventional Percoll based centrifugation method and showed significant advantage. The results of separating kidney cells/nuclei, lung cells/nuclei, as well as spleen cells also demonstrated high debris removal and cell recovery over several samples processed in multiple batches. MARS® sample preparation platform and its acoustic technology provide a powerful new tool for single cell preparation for expanded application of single cell genomics.
Citation Format: Liping Yu. Acoustic cell separation platform for efficient preparation of single cell/nuclei suspension from complex samples for genomics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2943.
Abstract 2947: Surveying the tumor suppressive genetic network underlying chr4p deletion in TNBC
Authors: Corpo Joseph Del , Dandage Rohan , Harrington Lea , Kuzmin Elena 
Published: June 2022

Abstract
Triple negative breast cancer (TNBC) is a breast cancer subtype lacking targetable biomarkers, resulting in the worst prognosis compared to other breast cancer subtypes. TNBC is characterized by many large copy number variants that result in the deletion and amplifications of many genes, with TP53 being the only common oncogenic driver. Using TCGA data and in-depth functional genomic analysis of TNBC patient-derived xenografts (PDX), our group showed that chr4p is a recurrently deleted region in basal breast cancer, which TNBC is an enriched subtype. This correlated with poor prognosis and a highly proliferative state. Here, we set out to survey the tumor suppressive genetic network underlying the TNBC-specific chr4pdeletion. Using an arrayed CRISPR-enCas12 screening approach, I will generate a panel of mutant cell lines deleted for all protein-coding genes residing within chr4p. MCF10A series of cell lines will be used for mutant cell line construction, because it is an established normal human breast epithelial model system with a normal karyotype to ensure the diploid state ofchr4p and includes other derivatives (MCF10A(-E7-Bcl2)) that show basal anchorage independent growth in 3D to assess cell transformation. The resulting panel of single gene deletion mutant cell lines will be characterized for their effects on proliferation, apoptosis, cell transformation and senescence. Additionally, the tumor suppressive genetic interaction network of chr4p will be mapped using a multiplexed CRISPR-enCas12 screening methodology. A dual guide-RNA library will be generated for all protein-coding genes to test all pairwise combinations for tumor suppressive genetic interactions. The proliferation due to double gene deletions will be monitored and compared to single gene deletions to identify tumor suppressive interactions. This study will be the first to systematically identify tumor suppressor genetic network underlying chr4p. Ultimately, it will provide an in-depth understanding of the genetic network of large copy number variants in TNBC and insight into new avenues for precision oncology.
Citation Format: Joseph Del Corpo, Rohan Dandage, Lea Harrington, Elena Kuzmin. Surveying the tumor suppressive genetic network underlying chr4p deletion in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2947.
Abstract 2976: Associate Professor
Authors: Hua Fang , Hao Wenzhuo , Wang Lingyan , Li Shitao 
Published: June 2022

Abstract
Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including NF-κB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-κB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that plakophilin 2 (PKP2) and the linear ubiquitin chain assembly complex (LUBAC) were required for EGFR-mediated NF-κB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IκB phosphorylation and subsequent NF-κB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-κB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-κB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR, and perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.
Citation Format: Fang Hua, Wenzhuo Hao, Lingyan Wang, Shitao Li. Associate Professor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2976.
Abstract 298: Targeting KRAS mutant non-small cell lung cancer with EGFR aptamer displaying 3WJ pRNA nanoparticles
Authors: Yang Linlin , Li Zhefeng , Guo Peixuan , Williams Terence M. 
Published: June 2022

Abstract
Background and Purpose: KRAS mutations are one of the most common oncogenic mutations in human cancers, including non-small cell lung cancer (NSCLC), and have established roles in cancer pathogenesis and therapeutic resistance. Development of effective inhibitors of mutant KRAS represent a significant challenge. The field of three-way junction (3WJ) based RNA nanoparticles has advanced rapidly and shows promise as an effective in vivo siRNA delivery system. This aim of this study is to explore the potential of 3WJ-pRNA nanoparticles for specific and efficient delivery of KRASG12C siRNA to NSCLC cells harboring a KRASG12C mutation to effectively silence KRAS oncogenic activity.
Experimental design: Multifunctional EGFRapt-3WJ-siKRASG12C nanoparticles were prepared using bottom-up self-assembly approach, and characterized by agarose gel shift assay, dynamic light scattering (DLS), and temperature gradient gel electrophoresis system (TGGE). Three NSCLC cell lines, H2122 and H2030 carrying KRASG12C, and H1299 with wild type KRAS were used in this study. Binding specificity and delivery efficiency of EGFRapt-3WJ-siKRASG12C nanoparticles in NSCLC cells were evaluated in vitro and in vivo. Biological effects of KRAS mutant silencing were assessed by in vitro functional assays, and in vivo tumor xenograft experiments.
Results: Characterization of EGFRapt-3WJ-siKRASG12C by DLS showed 7.2 ± 0.5 nm average size, and TGGE indicated a melting temperature (Tm) of 51.77°C. Flow cytometry, IVIS lumina imaging assay, real-time quantitative PCR, western blot, and KRAS activity assay confirmed that EGFRapt-3WJ-siKRASG12C potently and selectively depleted cellular KRAS expression, resulting in suppression of downstream MAPK pathway signaling in KRASG12C NSCLC cells and in heterotopic tumor models. In addition, in vitro cell proliferation, migration/invasion, and chemotherapy and radiotherapy sensitivity assays, demonstrated that KRAS silencing by EGFRapt-3WJ-siKRASG12C suppressed NSCLC cell growth, migration/invasion ability, and sensitized NSCLC cells to chemotherapy or radiotherapy. In vivo, these nanoparticles led to regression of tumor growth in heterotopic models.
Conclusions: Together, this study suggests that 3WJ pRNA based platform has clinical potential to suppress mutant KRAS activity for the treatment of KRAS driven human cancers, and warrants further development.
Citation Format: Linlin Yang, Zhefeng Li, Peixuan Guo, Terence M. Williams. Targeting KRAS mutant non-small cell lung cancer with EGFR aptamer displaying 3WJ pRNA nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 298.
Abstract 3032: Selenoprotein Sepp1 determines cysteine dependence in pancreatic cancer
Authors: Amer Arslan , Alothaim Tahiyat , Tang Xiaohu 
Published: June 2022

Abstract
Pancreatic cancer (PaCa) represents the fourth leading cause of overall cancer death, despite its relatively low incidence compared to other malignancies (Lung, prostate, breast, etc.). Approximately 57,600 people were estimated to be diagnosed with pancreatic cancer in the USA, and 47,050 were estimated to die of the disease in 2020 reported by The American Cancer Society. Novel effective therapeutic strategies are urgently needed. Metabolic changes are typically associated with tumor development to support fast cell proliferation and malignant transition. Such metabolic deregulations differentiate tumor cells from normal cells and act as potential targets of anticancer therapies. Previous studies have shown cysteine dependence often presents in various cancers, since cysteine metabolism supplies glutathione - an important substrate for the cellular reactive oxygen species (ROS) cleanup cycle. Studies in breast and kidney cancers have shown that such cysteine-dependence is a prominent feature in the cancer subtypes with a mesenchymal cell phenotype, instead of in the epithelial subtype. We found that cysteine dependence exists in pancreatic cancer as well. Mesenchymal PaCa cells, in contrast to the epithelial tumor cells, appear to be more sensitive to cysteine depletion with extensive induction of ferroptosis, a programmed cell death pathway involving iron and lipid peroxidation. Gene expression analysis revealed the selenium transporter protein Sepp1 to be absent in the mesenchymal PaCa cells. Knockdown of Sepp1 expression in cysteine-independent cells renders cells dependent on cysteine. Selenium is a micronutrient required for the synthesis of selenoproteins including GPX4 and Sepp1. The addition of selenium alleviated cell reliance on cysteine and caused resistance to GPX4 inhibitor-induced ferroptosis. We observed that Sepp1 regulates the thioredoxin pathway to rescue the cells under the glutathione deficiency. Interestingly, the conditioned media assay also revealed that Sepp1 protects cells from ROS stress by a symbiotic mechanism. Our findings suggest that Sepp1 could serve as a biomarker of tumor cysteine-dependency and a potential molecular target for therapeutic strategies to overcome drug resistance in pancreatic cancer.
Citation Format: Arslan Amer, Tahiyat Alothaim, Xiaohu Tang. Selenoprotein Sepp1 determines cysteine dependence in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3032.
Abstract 3035: Proliferation induced by IL4R is regulated via Glut1 activity in metastatic breast cancer
Authors: Hargrove-Wiley Ebony , Valent Daniel , Williams Demond , Bindeman Wendy , Fingleton Barbara 
Published: June 2022

Abstract
Metabolic reprogramming is a characteristic of all cancers including breast cancer; however, the signaling pathways involved are not fully understood. Altered metabolism, like a shift to aerobic glycolysis, supports cancer cell growth and metastatic behavior. Similarly, immune signals from the tumor microenvironment can promote these same phenotypes and therefore, immune signals likely influence the metabolic state of cancer cells. More specifically, the cytokine interleukin-4 (IL4) promotes increased tumor cell survival, proliferation, and invasion upon stimulation of the type II IL4 receptor alpha (IL4Rα) which is aberrantly expressed on epithelial cancer cells. Previously, we have shown that metastatic mouse mammary cancer cells (4T1) rely on IL4 signaling for colonization of metastatic sites in the lung and liver. Additionally, these cells uptake more glucose when treated with IL4 in vitro. Here, we investigated the significance of glucose consumption on pro-tumorigenic phenotypes induced by IL4/IL4Rα. In 4T1 cells, IL4/IL4Rα signaling increased protein expression and global glycosylation of a primary glucose transporter, Glut1. In human metastatic breast cancer cells (MDA-MB-231), CRISPR-mediated knockout (KO) of Glut1 significantly reduced basal and IL4-induced glucose consumption when assayed by 2-NBDG uptake, a fluorescently labeled glucose analog. Glut1 KO reversed the proliferative effects of IL4/IL4Rα activation on human cancer cells in vitro. Glut1 depletion attenuated tumor cell dependence on glucose consumption as shown by decreased expression of Hexokinase-2, a rate-limiting enzyme in glycolysis. In addition, KO cells have elevated levels of the glutamine transporter, ASCT2, that are further enhanced by IL4 treatment which suggest a greater reliance on glutamine albeit, insufficient for cell expansion supported by IL4/IL4Rα stimulation. Our data indicate that IL4-induced glucose consumption supports the increased metabolic activity necessary for cancer cell proliferation. Targeting the IL4/IL4Rα signaling axis could be a therapeutic strategy to hinder tumor-associated metabolism and reduce tumor burden at primary and metastatic sites.
Citation Format: Ebony Hargrove-Wiley, Daniel Valent, Demond Williams, Wendy Bindeman, Barbara Fingleton. Proliferation induced by IL4R is regulated via Glut1 activity in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3035.
Abstract 3044: C. albicans infection mediates upper gastrointestinal tract malignancy independently of Il17a in an APECED-like mouse model
Authors: Zhu Feng , Phan Quynh T. , Ferre Elise M. , Willette-Brown Jami , Salcedo Rosalba , Shen Tsai-Wei , Fan Yu , Zhao Yongmei , Mehta Monika , Meerzaman Daoud , Tran Bao , Trinchieri Giorgio , Lionakis Michail S. , Filler Scott G. , Hu Yinling 
Published: June 2022

Abstract
Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) develop chronic mucocutaneous candidiasis during childhood and about 10% of senior patients develop upper GI tract squamous cell carcinoma (SCC), suggesting that C. albicans infection may be relevant to the cause of SCC development in APECED patients. In this study, we investigated the role of C. albicans infection in upper GI tract SCC development using an APECED-like IKKα kinase-dead (KA/KA) mouse model. We found that KA/KA mice developed upper GI tract SCC after oral infection of WT C. albicans weekly for three months. Interestingly, while upper GI tract SCC development in C. albicans - infected KA/KA mice is IL-17a-independent, EGFR pathway plays an important role in C. albicans infection-mediated tumorigenesis in KA/KA mice, as evidenced by significantly lower SCC incidence in the upper GI tract of KA/KA mice infected with Als3Δ/Δ C. albicans. Moreover, upper GI tract SCCs from WT C. albicans-infected KA/KA mice exhibited robust p-EGFR and Ki67 signals. However, both p-EGFR and Ki67 signals were only mildly increased in upper GI tract of Als3Δ/Δ C. albicans-infected KA/KA mice compared to WT control mice. Meanwhile, EGFR inhibition dramatically reduced C. albicans colonization in upper GI tract and the weight of stomach in KA/KA mice. Furthermore, we found that aberrant infiltrations of neutrophils and macrophages with significant inflammatory signatures were present in the upper GI tract SCCs in KA/KA mice. Interestingly, KA/KA neutrophils exhibited a defect in C. albicans killing capacity due to functional defects in ROS generation, degranulation, NETs formation, and apoptosis. We reasoned that KA/KA neutrophils’ inability in C. albicans killing capacity may further enhance the tumor-promoting microenvironment. Finally, whole genome sequencing data suggested that C. albicans infection may lead to genome instability. Currently we are investigating the role of neutrophil infiltration in C. albicans infection-mediated upper GI tract tumorigenesis.
Citation Format: Feng Zhu, Quynh T. Phan, Elise M. Ferre, Jami Willette-Brown, Rosalba Salcedo, Tsai-Wei Shen, Yu Fan, Yongmei Zhao, Monika Mehta, Daoud Meerzaman, Bao Tran, Giorgio Trinchieri, Michail S. Lionakis, Scott G. Filler, Yinling Hu. C. albicans infection mediates upper gastrointestinal tract malignancy independently of Il17a in an APECED-like mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3044.
Abstract 3082: Leveraging 3D organoids to identify molecular changes underlying HNSC initiation and development
Authors: Agee Robert , Flashner Samuel , Shimonosono Masataka , Klein-Szanto Andres , Nakagawa Hiroshi 
Published: June 2022

Abstract
Head and neck squamous cell carcinoma (HNSC) is a devastating disease accounting for 570,000 newly diagnosed cases and resulting in 344,000 deaths annually. HNSC arises from its histological precursor lesion, squamous dysplasia (HNSD); however, most patients are diagnosed with late-stage HNSC with no prior identification of pre-malignant disease. Earlier diagnosis and intervention could ameliorate the outsized burden of HNSC; therefore, there is an urgent unmet need to characterize the molecular changes underlying progression through HNSD to HNSC. To identify novel therapeutic targets or biomarkers that predict progression to HNSC, we have generated a three dimensional (3D) organoid library derived from dysplastic oral tissue harvested from mice treated with 4-Nitriquinoline N-oxide (4-NQO), a potent mimetic of tobacco smoke. Through RNA sequencing analysis of these organoids, we have identified two distinct gene expression states that accompany either early or late dysplasia. By performing gene set enrichment analysis of these two distinct gene signatures, we have identified c-Jun N-terminal Kinase (JNK) signaling as a potential biomarker that predicts or progression from HNSD to HNSC. Together, we have identified the salient transcriptional changes that accompany progression through the histological precursor lesion of HNSC. This study positions JNK signaling as a potential biomarker or therapeutic target for early intervention during the treatment of a deadly disease.
Citation Format: Robert Agee, Samuel Flashner, Masataka Shimonosono, Andres Klein-Szanto, Hiroshi Nakagawa. Leveraging 3D organoids to identify molecular changes underlying HNSC initiation and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3082.
Abstract 3107: Identifying therapeutic vulnerabilities in HR-positive, HER2-negative advanced breast cancer patient-derived xenograft models refractory to CDK4/6 inhibition
Authors: Kettner Nicole M. , Navarro-Yepes Juliana , Bui Tuyen N. , Rao Xiayu , Wang Jing , Meric-Bernstam Funda , Hunt Kelly K. , Tripathy Debu , Keyomarsi Khandan 
Published: June 2022

Abstract
Background: Clinically, the addition of CDK4/6 inhibitors (e.g. palbociclib) to endocrine therapy (ET) significantly delays progression of advanced hormone receptor-positive (HR+), HER2-negative (HER2-) breast cancer patients. However, most patients develop resistance and progress following long-term treatment. Thus, an ongoing clinical challenge has been the identification of biomarkers that predict response to CDK4/6 inhibitors. There is also an unmet need to identify actionable targets for patients who have progressed. Currently, there are no clinically useful biomarkers to predict response and/or resistance to anti-CDK4/6 therapy. Hence, the goal of our study was to identify the therapeutic vulnerabilities of CDK4/6 inhibitor resistant patient derived xenograft (PDX) models and identify key markers that longitudinally correlate with development of resistance and inform new treatment directions.
Methods: We obtained 4 different PDX models, two from patients who progressed after 2-4 months on treatment with palbociclib plus ET (i.e. intrinsic resistance) and the other two who developed resistance over time (i.e. acquired resistance) with disease progression between 12-18 months while on palbociclib plus ET. To elucidate mechanisms of resistance, we performed genome-wide expression analysis via RNA-sequencing.
Results: The molecular assessment of the PDX models revealed different transcriptomic signatures for intrinsic resistance compared to acquired resistance. We identified 3,386 upregulated and 2,829 downregulated significant differentially expressed genes (DEGs) in the acquired compared to intrinsic resistant PDX models. Further, gene set enrichment analysis (GSEA) revealed enrichment of distinct driver pathways in the acquired-compared to the intrinsic resistant models.
Conclusions: Collectively, these results have identified potential unique targets for intrinsic and acquired resistance to guide treatment strategies. Thus, our ongoing studies are geared towards identifying and targeting distinct therapeutic vulnerabilities of intrinsic and acquired resistance to palbociclib.
Citation Format: Nicole M. Kettner, Juliana Navarro-Yepes, Tuyen N. Bui, Xiayu Rao, Jing Wang, Funda Meric-Bernstam, Kelly K. Hunt, Debu Tripathy, Khandan Keyomarsi. Identifying therapeutic vulnerabilities in HR-positive, HER2-negative advanced breast cancer patient-derived xenograft models refractory to CDK4/6 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3107.