bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract LB-038: Development and characterization of cancer cell line exosomes as reference standards in cancer research
Authors: Paul Siddhartha , Mahashetty Nanda , Wells Joy , Yin Dezhong , Tian Fang 
Published: August 2019

Abstract
Exosomes are subcellular particles 50-200 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases, including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor-derived exosomes have been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for the isolation of large quantities of highly purified, ready to use exosomes from cell culture supernatant. Exosomes from a variety of cancer cell lines representing the most prevalent cancer types, including prostate (LNCaP), colorectal (HCT116), breast (MDA-MB-231, MCF-7), lung (A549), cervical (HeLa), and glioblastoma (U87-MG) were isolated and characterized in this study. We employed a stringent quality control approach in order to define and characterize these exosomes. Our method generated a high yield (&gt;1×1010 exosomes/mL) and average protein equivalent of 2 mg/mL. Our data demonstrated expression of a number of different characteristic exosomal proteins including tetraspanin, which was confirmed through western blot and PCR. Isolated exosomes had a median size of 102 nm through nanoparticle tracking analysis. Western blot data revealed cell line specific protein marker expression of CD63, CD81, flotillin 1, Alix and TSG101 protein. We assessed the functionality of these purified exosomes through cellular uptake, migration assay, and anchorage independent growth assay in this study. We also evaluated exosomal RNA from these purified exosomes for the presence of common mutations associated with cancer through PCR. Here we report that exosomes isolated by our method not only had high exosome yield and quality, but also retained relevant functionality and thereby make ideal reference standards/controls in the field of cancer research and diagnostics development.
Citation Format: Siddhartha Paul, Nanda Mahashetty, Joy Wells, Dezhong Yin, Fang Tian. Development and characterization of cancer cell line exosomes as reference standards in cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-038.
Abstract LB-050: Memory sequencing reveals heritable single cell gene expression programs associated with therapy resistance
Authors: Shaffer Sydney , Emert Benjamin , Torre Eduardo , Raj Arjun 
Published: August 2019

Abstract
Single cancer cells can have substantial fluctuations in gene expression from non-genetic factors. However, it is unclear how long these fluctuations exist and whether they extend beyond the length of a single-cell division. Current technologies for gene expression measurements in single cells provide just one measurement of each cells’ transcriptome and fail to capture how these gene expression fluctuations change with time. We developed a new method combining Luria and Delbruck's fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several cell divisions. MemorySeq revealed multiple gene modules that express together in rare cells within otherwise homogeneous clonal populations. Further, we found that these rare cell subpopulations are associated with biologically distinct phenotypes in multiple different cancer cell lines. In melanoma, we identified a rare subpopulation of cells expressing EGFR, NGFR, and AXL, and demonstrated that these cells are resistant to BRAF and MEK inhibitors. In triple-negative breast cancer cells, we identified a different gene expression signature consisting of CA9, and found that this subpopulation is resistant to paclitaxol. We validated these findings in melanoma cell lines by tagging the endogenous locus of MemorySeq gene, NGFR, with a fluorescent protein to directly visualize gene expression fluctuations in individual cells. Broadly, we believe that memory is an essential property of variability that will have phenotypic consequences for cancer cells. Thus, we anticipate that MemorySeq can be extended to uncover the transcriptional states of rare cells driving many phenotypes in cancer including drug resistance, differentiation, invasion, and metastasis.
Citation Format: Sydney Shaffer, Benjamin Emert, Eduardo Torre, Arjun Raj. Memory sequencing reveals heritable single cell gene expression programs associated with therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-050.
Abstract LB-052: Detection of rare, deleterious clonal mutations during unrelated allogeneic hematopoietic stem cell transplants using an error correction pipeline for NGS
Authors: Wong Wing Hing , Young Andrew , Druley Todd 
Published: August 2019

Abstract
While next-generation sequencing (NGS) has transformed our ability to characterize the genome, it is limited in detection of rare mutations due to a high error rate of ~0.5-2.0%. We have developed an error correction pipeline and previously demonstrated that our process is capable of detecting variants as rare as 1:10,000 or 0.0001 VAF. Using our error correction pipeline, we have recently identified leukemia-related hematopoietic clones in a cohort of young healthy bone marrow donors (median age 26). In the context of allogeneic hematopoietic stem cell transplantation (HSCT), we sought to determine whether these detected leukemia-related clones of donor origin selectively engraft and correlate with transplant-related morbidities in recipients post-HSCT. Genomic profiles of pre-transplant donor samples were compared to the genomic profiles of corresponding recipient samples post-HSCT. Results show that two-thirds of the healthy donor samples harbor deleterious hematopoietic clones and that these specific clones are significantly more likely to engraft compared to benign clones. We also found that 75% of recipients who had at least one persistently engrafted, deleterious mutation developed chronic GvHD versus 50% of those without persistently engrafted clones with deleterious mutations. In comparing the results from our error correction pipeline and conventional short tandem repeat (STR) testing for donor-patient chimerism, we showed that our method provides a highly sensitive approach to both screening for donors without deleterious clones and monitoring residual disease in recipients following hematopoietic stem cell transplants (HSCT). Furthermore, the clinical implications of these results warrant a large-scale study using our error correction pipeline to detect rare variants in HSCT.
Citation Format: Wing Hing Wong, Andrew Young, Todd Druley. Detection of rare, deleterious clonal mutations during unrelated allogeneic hematopoietic stem cell transplants using an error correction pipeline for NGS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-052.
Abstract LB-063: Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics
Authors: Avrutskaya Anya , Sonego Fabiane , Hauser Jacob , O’Koren Emily , Martin Gaëlle , Chaix Julie , Ball Robin , Bui Thi , Belle Ian , Hall Chassidy , Thiam Kader , Marval Paula L. Miliani De 
Published: August 2019

Abstract
Over the past decade there has been an increasing demand in the use of syngeneic models for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. During tumor development, immune cells can became unresponsive to the presence of tumor cells due to chronic activation and expression of the programmed cell death protein-1 (PD-1) or the T-lymphocyte-associated antigen 4 (CTLA4) in T-cells or the presence of FoxP3+ Treg cells resulting in tumor immune-tolerance. Our previous studies have demonstrated that murine anti-PD-1 and CTLA-4 therapy can effectively re-activate the antitumor response against multiple syngeneic tumor models. While these models proved instrumental for evaluating murine immune-checkpoint inhibitors (ICI), there is a clear need for additional mouse models to evaluate the efficacy of ICI specific for human targets. To address this need, we describe the development of a humanized PD-1 knock-in (KI) mouse model. This mouse model has the advantage of expressing the human PD-1 protein in the context of a fully functional immune system. We also show the response to clinically relevant immune checkpoint inhibitors in two preclinical tumor models. We evaluated the anti-tumor activity of pembrolizumab in the MC38 colorectal carcinoma and the GL261 glioblastoma models. We observed significant tumor growth inhibition and growth delay in the MC38 tumor model when treated with pembrolizumab monotherapy, but not when treated with the murine counterpart (anti-PD-1 clone RPM1-14). To extend our validation studies to other tumor models, we implanted GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembrolizumab compared to both the control group and the group treated with murine anti-PD-1 antibody. The immune profile from control and treated these animals were also characterized in these studies by flow cytometry. In summary, the results shown here underscore the value of the humanized PD-1 knock-in (KI) mouse model as a tool to evaluate human specific immune-checkpoint based therapeutics alone and in combination with other agents.
Citation Format: Anya Avrutskaya, Fabiane Sonego, Jacob Hauser, Emily O’Koren, Gaëlle Martin, Julie Chaix, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Kader Thiam, Paula L. Miliani De Marval. Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-063.
Abstract LB-071: Functional NK cells generated from human iPS cells with 3D-bioreactor for immuno-oncology
Authors: Feng Qiang , Zhang Miao-yun , Lu Shi-Jiang 
Published: August 2019

Abstract
NK cells are cytotoxic cells critical for innate immune system. Early investigation using NK cells carrying CAR specific targeting tumor antigens have shown promising antitumor activities. CAR-T therapy requires either autologous or MHC matching cells to avoid graft-verse-host disease (GVHD), whereas allogeneic NK cells showed substantial anti-tumor activity without GVHD. Therefore, the “ready-to-kill” NK population is regarded as the ideal Off-The-Shelf immune cell therapy products. The development of NK-based immunotherapy, however, has been limited by unsatisfactory sources of human NK cells. Human induced pluripotent stem cells (iPSCs) offer unlimited source for manufacturing NK cells suitable for immunotherapy. Previously NK cell derivation methods from human iPS cells used feeder cells with multiple scale-up limited processes which are not suitable for industrial scale production. In this study we described a novel 3D-bioreactor platform that can continuously generate highly pure NK cells with strong cytotoxic activity for 2 weeks. First, undifferentiated human iPS cells were cultured as 3D-spheres with mesoderm inducing conditions in bioreactor, which converted 60-70% iPS cells into hemogenic endothelial progenitor (HEP, CD31+CD144+CD34+) in a week. HEP cells were then cultured under NK cell differentiation condition for up to 35 days. Starting from day 20, NK cells were started to release from these 3D-spheres which can be harvested daily by centrifugation. These iPS-NK cells can be cryopreserved for long time. Human iPS-NK cells display a distinctively homogenous morphology. Over 95% of collected cells express CD56, NKG2D, NKp44 and NKp46 markers; Approximately 30-50% of CD56+ iPS-NK cells express KIR2DL/DS1 and KIR2DS4 receptors; CD56+ PSC-NK cells do not express TCR and CD3, and CD19, which are specific markers for T and B cells, respectively. Surprisingly, we discovered that more than 80% CD56+ iPS-NK cells also expressed CD8 as compared to ≈30% of peripheral blood CD56+ NK cells, the increase of which is associated with disease regression in AIDS patients. More importantly, when iPS-NK cells and K562 leukemia cells were mixed together, even at a ratio of 1 to 1, more than 80% of leukemia cells were killed within hours as demonstrated both by flow cytometry analysis and time-lapse record. With the establishment of permanent master iPS-CAR cell lines, our novel technology platform will provide inexhaustible cell sources for the generation of truly off-the-shelf CAR-NK cells suitable for treatment of a large numbers of patients, which can revolutionize the field of immuno-oncology.
Citation Format: Qiang Feng, Miao-yun Zhang, Shi-Jiang Lu. Functional NK cells generated from human iPS cells with 3D-bioreactor for immuno-oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-071.
Abstract LB-074: Priming the tumor microenvironment with epigenetic modifiers to overcome resistance to immune checkpoint inhibitors
Authors: Knox Tessa J. , Sahakian Eva , Banik Debarati , Hadley Melissa , Palmer Erica , Kim Jennifer , Noonepalle Satish , Powers John , Gracia-Hernandez Maria , Oliveira Vasco , Cheng Fengdong , Chen Jie , Barinka Cyril , Pinilla-Ibarz Javier , Lee Norman , Kozikowski Alan , Villagra Alejandro 
Published: August 2019

Abstract
Histone deacetylases (HDACs) are involved in diverse cellular regulatory mechanisms including functions outside the chromatin environment. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor immunogenicity and the functional activity of specific immune cells. In particular, the selective inhibition of HDAC6 has been reported to decrease tumor growth in several malignancies. However, there is still no clarity about the cellular components mediating this effect. In this study, we evaluated the immunological modulation of the HDAC6i Nexturastat A in combination with anti-PD-1 checkpoint blockade therapy and the use of this HDAC6i as a priming agent to facilitate the transition of the tumor microenvironment from “cold” to “hot” in order to more specifically augment immune check-point blockade therapies. This combination of Nexturastat A and anti-PD-1 therapy demonstrated a significant reduction of tumor growth in syngeneic melanoma tumor animal models. Additionally, we observed a complete neutralization of the up-regulation of PD-L1 and other immunosuppressive pathways induced by the treatment with anti-PD-1 blockade. This combination also showed that the pre-treatment with selective HDAC6i induced major changes in the tumor microenvironment such as enhanced infiltration of immune cells, increased central and effector T cell memory, and a significant reduction of pro-tumorigenic M2 macrophages. The evaluation of the effect of HDAC6i on individual immune components suggest that the in vivo anti-tumor activity of HDAC6i is mediated by its effect on tumor cells and tumor associated macrophages, and not directly over T cells. Overall, our results indicate that selective HDAC6i could be used as immunological priming agents to sensitize immunologically “cold” tumors and subsequently improve ongoing immune check-point blockade therapies.
Note: This abstract was not presented at the meeting.
Citation Format: Tessa J. Knox, Eva Sahakian, Debarati Banik, Melissa Hadley, Erica Palmer, Jennifer Kim, Satish Noonepalle, John Powers, Maria Gracia-Hernandez, Vasco Oliveira, Fengdong Cheng, Jie Chen, Cyril Barinka, Javier Pinilla-Ibarz, Norman Lee, Alan Kozikowski, Alejandro Villagra. Priming the tumor microenvironment with epigenetic modifiers to overcome resistance to immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-074.
Abstract LB-078: New generation of oncolytic herpesviruses embodying immunotherapeutic genes encoding IL-12 and anti-PD-1 antibody
Authors: Zhou Grace , Ni Dongyao , Yan Runbin , Chen Xiaoqing , Liu Xianjie , Ma Jie , Tang Yuxin 
Published: August 2019

Abstract
Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpes simplex viruses (oHSV) have a major impact on cancer therapy marred by rare failures of healthy organs. Here we report the properties of 3 families of oHSV expressing no immunomodulatory genes (T1 series), murine or human IL-12 (T2 series) and murine or human IL-12 and anti-PD-1 antibody (T3 series). Thus insertion of the genes encoding PD-1 Ab and IL-12 significantly augmented the oncolytic activity of oHSV bereft of immune-stimulatory genes (T1 series). In syngeneic mice the murine T3 oHSV induced significant intratumoral accumulation of IL-12, PD-1 Ab and IFN-γ and was effective against a variety of murine tumors. Thus T3 oHSV concentrates the immunomodulatory products in the environment of the tumors thereby reducing the risk of toxicity associated with systemic administration. T3 oHSV injected into tumors was more effective than systemic administration of anti PD-1 Ab and IL12 alone or in combination with intratumoral injection of T1 oHSV. T3 induces local and systemic immune response characterized by cytokine signature.
Citation Format: Grace Zhou, Dongyao Ni, Runbin Yan, Xiaoqing Chen, Xianjie Liu, Jie Ma, Yuxin Tang. New generation of oncolytic herpesviruses embodying immunotherapeutic genes encoding IL-12 and anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-078.
Abstract LB-085: PTPN11 plays oncogenic roles and is a therapeutic target for BRAF wild-type melanomas
Authors: Hill Kristen , Messina Jane , Wu Jie , Chalfant Charles E. , Kim Minjung 
Published: August 2019

Abstract
Melanoma is one of the most highly mutated cancer types. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase (PTP) that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten and Cdkn2a null mice, tet-inducible and melanocyte-specific PTPN11 E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11 E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence, and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRAS Q61K-mutant melanoma. Using a quantitative tyrosine phosphoproteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study identifies PTPN11 as an oncogenic driver and a novel and actionable therapeutic target for BRAF wild-type melanoma.
Citation Format: Kristen Hill, Jane Messina, Jie Wu, Charles E. Chalfant, Minjung Kim. PTPN11 plays oncogenic roles and is a therapeutic target for BRAF wild-type melanomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-085.
Abstract LB-095: Aurora-A is elevated in cancer cells due to inactivation of EF1A2-PTEN-GSK3b regulated late S-G2 phase specific degradation pathway
Authors: Treekitkarnmongkol Warapen , Solis Luisa Maren , Kai Kazuharu , Sasai Kaori , Wistuba Ignacio I. , Sahin Aysegul A. , Wong Kwong K. , Thompson Alastair M. , Jltsumori Yoshimi , Lee Jonathan M. , Qin Lidong , Bawa-Khalfe Tasneem , Rad Roland , Abbott Catherine M. , Katayama Hiroshi , Sen Subrata 
Published: August 2019

Abstract
Elevated expression of Aurora kinase-A oncoprotein is detected in a subset of human cancers across different organ sites. The mechanism(s) underlying over expression of the protein in cancer cells have not been well elucidated. This study demonstrates that inactivation of a novel mechanism of EF1A2 regulated PTEN-GSK3β-SCF complex mediated Aurora-A protein degradation pathway in S-G2 phases of mitosis leads to enhanced stability/steady state level of Aurora-A in cancer cells. EEF1A2 regulates interaction of Aurora-A with activated PTEN, GSK3β, Neddylated CUL1 and FBXW7, promoting ubiquitination mediated degradation of Aurora-A that is rescued in EEF1A2 knockdown cells. Ectopic expression of EEF1A2, on the other hand, enhances formation of the complex with GSK3β mediated inactivating phosphorylation and reduction in Aurora-A protein, recapitulating the S-G2 phase events. Genetic ablation of EEF1A2 and PTEN in both in vitro cell lines and in vivo mouse models validated the results. Aurora-A over expressing mouse models of mammary carcinoma and human breast cancer tissue samples also corroborated the findings. Therapeutic targeting of PTEN-GSK3β-AKT pathway may, therefore, be effective in treating a subset Aurora-A over expressing cancers.
Citation Format: Warapen Treekitkarnmongkol, Luisa Maren Solis, Kazuharu Kai, Kaori Sasai, Ignacio I. Wistuba, Aysegul A. Sahin, Kwong K. Wong, Alastair M. Thompson, Yoshimi Jltsumori, Jonathan M. Lee, Lidong Qin, Tasneem Bawa-Khalfe, Roland Rad, Catherine M. Abbott, Hiroshi Katayama, Subrata Sen. Aurora-A is elevated in cancer cells due to inactivation of EF1A2-PTEN-GSK3b regulated late S-G2 phase specific degradation pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-095.