bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2506: Simultaneous DNA, RNA and protein analysis from single cells using a high-throughput microfluidic workflow for resolution of genotype-to-phenotype modalities
Authors: Mendez Pedro , Dhingra Dalia , Ooi Aik , Wang Shu , Gulati Saurabh , Beard Nigel , Manivannan Manimozhi , Björkman Jens , Kubista Mikael , Landberg Göran , Ruff David , Ståhlberg Anders
Published: September 2020

The TCGA, ICGC and other research consortiums has shown that an integral, multi-omic analysis of tumors is necessary in order to understand a more complete picture of the signaling pathways involved in development, evolution, prognosis and treatment resistance of tumors. However, in such studies, because different data modalities (“omics”) are analyzed separately, the information on how the co-existence of in the same cell is lacking. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using high-throughput droplet microfluidics on the Tapestri platform, paired with next generation sequencing. Our triomic methodology evaluates targeted protein levels, mRNA transcript levels and somatic gDNA sequence variations (SNV & CNV) from the same cell. We employ oligonucleotide-conjugated antibody panels to probe cellular surface markers and targeted RNA and DNA amplification to resolve gene expression levels and genomic variants. Cell suspensions are first stained with cocktails of oligonucleotide-antibodies. These cells are loaded onto the Mission Bio Tapestri® microfluidic cartridge for generation of the first droplet. This droplet biochemistry allows for concurrent cell lysis and release of gDNA from chromatin scaffolding. A second droplet formation event brings together a barcoded bead and multiplex PCR amplification reagents. After amplification, the combined libraries are sequenced and demultiplexed bioinformatically yielding a multi-omics readout from the single-cells. Six protein surface markers, 35 transcripts and 88 genomic regions in 68 genes were interrogated with a 50:50 cell mixture of MCF7 and GM12878 cells. These targets play key roles in cellular migration, stemness, differentiation and proliferation. We intend to further explore the relationship between genotype-to-phenotype in breast carcinoma cell lines cultured in patient-derived scaffolds mimicking tumor microenvironments under different drug regimen conditions. We report the dynamic multimodal resolution and demonstrate utility of triomics in further illuminating cellular states and biological responses.

Citation Format: Pedro Mendez, Dalia Dhingra, Aik Ooi, Shu Wang, Saurabh Gulati, Nigel Beard, Manimozhi Manivannan, Jens Björkman, Mikael Kubista, Göran Landberg, David Ruff, Anders Ståhlberg. Simultaneous DNA, RNA and protein analysis from single cells using a high-throughput microfluidic workflow for resolution of genotype-to-phenotype modalities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2506.
Abstract 2517: Ectopic expression of miR-142-3p suppresses growth, migration, colony formation and induces apoptosis by down-regulating HMGA1, HMGA2, HMGB1 and HMGB3 in human cervical cancer cells
Authors: Sharma Priyanshu , Yadav Poonam , Karunagaran Devarajan
Published: September 2020

High Mobility Group (HMG) proteins are non-histone chromosomal proteins having molecular weight less than 30 kDa. Canonical HMG family of proteins include HMGA (A1 and A2), HMGB (B1, B2, B3, and B4) and HMGN (N1, N2, N3, N4 and N5). Overexpression of HMG proteins contributing to their oncogenic properties has been reported and thus inhibition of their expression is potentially a promising therapeutic strategy in different cancers. Earlier, our laboratory demonstrated that miR-214 downregulates HMGA1 and miR-34a downregulates HMGB1 leading to the suppression of growth, proliferative and invasive properties of human cervical and colorectal cancer cells. Prompted by these results, we tried to find a single miRNA which can target multiple HMG genes. To this end, TargetScan, miRWalk, and miRDB were used to find out miRNAs targeting more than one HMG gene. We found that miR-142-3p has its seed region binding sites in the 3'UTRs of HMGA1, HMGA2, HMGB1, and HMGB3. Survival analysis with OncoLnc server revealed that low miR-142-3p expression found in cervical cancer patients correlates with their poor survival. Strengthened by this classical miRNA-target inverse relationship, we hypothesized that miRNA-142-3p may act as a tumor suppressor by targeting multiple HMG genes in cervical cancer.

Doxycycline-responsive miR-142-3p expression constructs were used to generate stable cell lines and functional assays were performed. Through dual-luciferase reporter assays, we demonstrate that all the four HMG genes are direct targets of miR-142-3p in four cervical cancer cell lines (HeLa, SiHa, C33A, and CaSki). We observed the downregulation of the HMG genes (through qRT-PCR) upon miR-142-3p overexpression in the stable cell lines. Also, a significant reduction in cell viability after 72 and 96 hours concurrent with the highest expression levels of miR-142-3p. In colony formation assay, colony-forming potential of the cell lines was found to be severely compromised upon miR-142-3p overexpression. The scratch assay was also performed and a slower rate of migration was observed in miR-142-3p overexpressing cells. Annexin V staining indicated that miR-142-3p induces cell death via apoptosis. These results suggest that miR-142-3p may act as a tumor suppressor in cervical cancer highlighting it as a potential candidate for miRNA replacement therapy.

Citation Format: Priyanshu Sharma, Poonam Yadav, Devarajan Karunagaran. Ectopic expression of miR-142-3p suppresses growth, migration, colony formation and induces apoptosis by down-regulating HMGA1, HMGA2, HMGB1 and HMGB3 in human cervical cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2517.
Abstract 2557: 15-lipoxygenase-1 suppresses linoleic acid promotion of colorectal tumorigenesis through oxidative metabolism of PI3P_linoleic acid to inhibit membranous LRP5 recycling and WNT/β-catenin signaling activation
Authors: Liu Fuyao , Zuo Xiangsheng , Liu Yi , Deguchi Yasunori , Moussalli Micheline J. , Chen Weidong , Yang Peiying , Wei Bo , Tan Lin , Lorenzi Philip L. , Gao Shen , Jaoude Jonathan C. , Mehdizadeh Amir , Valentin Lovie A. , Wei Daoyan , Shureiqi Imad
Published: September 2020

Western-type diets have been increasingly enriched with linoleic acid (LA). The impact of excess dietary LA on cancer risk remains poorly understood although some studies suggest that high LA diets promote chemically-induced colorectal carcinogenesis (CRC) in rodent models. 15-lipoxygenase-1 (15-LOX-1), the main metabolizing enzyme of LA, is transcriptionally silenced during the early stages of CRC. Whether 15-LOX-1 impacts the effects of excess LA on CRC is unknown. Herein, we report that high dietary LA promoted CRC in intestinally targeted APC mutation (ApcΔ580) and azoxymethane-induced CRC mouse models by upregulating expression of LRP5, a Wnt receptor, and aberrant β-catenin activation. These effects were negatively regulated by 15-LOX-1 when this enzyme was transgenically expressed in intestinal epithelial cells. Complementary genetic deletion studies of 15-LOX-1 in mice further supported 15-LOX-1's suppressive effects on these events. Mechanistic studies showed that 15-LOX-1 peroxidation of LA in phosphatidylinositol-3-phosphates (PI3P_LA) into PI3P_13-HODE decreased PI3P binding to SNX17 and LRP5, which inhibited LRP5 recycling from endosomes to the plasma membrane, thereby leading to an increase of LRP5 lysosomal degradation. Our findings demonstrate for the first time the importance of linoleic acid metabolism by 15-LOX-1 within complex lipids such as PI3P in regulating LRP5 membranous abundance and subsequently Wnt/β-catenin aberrant signaling activation and CRC risk. These results strongly support the need to carefully consider the potential of increasing CRC risk by enriching the diets with LA especially for individuals with low 15-LOX-1 expression.

Citation Format: Fuyao Liu, Xiangsheng Zuo, Yi Liu, Yasunori Deguchi, Micheline J. Moussalli, Weidong Chen, Peiying Yang, Bo Wei, Lin Tan, Philip L. Lorenzi, Shen Gao, Jonathan C. Jaoude, Amir Mehdizadeh, Lovie A. Valentin, Daoyan Wei, Imad Shureiqi. 15-lipoxygenase-1 suppresses linoleic acid promotion of colorectal tumorigenesis through oxidative metabolism of PI3P_linoleic acid to inhibit membranous LRP5 recycling and WNT/β-catenin signaling activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2557.
Abstract 2565: NANOG sustains ovarian cancer cell survival and disrupts ER stress signaling through BiP/PERK under glutamine depletion
Authors: Suen Astrid Man-Yee , Wong Ivy Tsz-Lo , Tse Cindy Yin-Si , Chan Ka-Kui , Wong Oscar Gee-Wan , Cheung Annie Nga-Yin
Published: September 2020

Metabolic reprogramming is a common hallmark of multiple cancers which functions to meet their high proliferation needs. High demand for the amino acid glutamine is frequently observed in many cancers. Catalyzed by glutaminase, glutamine is readily converted to glutamate which then further fuels the tricarboxylic acid cycle. In ovarian cancer, depletion of glutamine significantly inhibited cell growth suggesting that targeting glutamine metabolism might be an effective therapeutic strategy for ovarian cancer. However, the molecular mechanisms involved were not completely understood. In this study, we aim to identify molecular targets and the signaling involved in ovarian cancer cells under glutamine depletion. We observed that glutamine depletion stimulated the expression of the stem cell transcriptional factor NANOG in the serous ovarian cancer cell line OVCAR3. Stable knockdown of NANOG in OVCAR3 (shNANOG-OVCAR3) enhanced the level of reactive oxygen species under glutamine depletion. It was also found that under glutamine depletion, growth rate was not altered in shNANOG-OVCAR3 when compared to control shCtrl-OVCAR3 cells. However, shNANOG-OVCAR3 were more sensitive to glutamine depletion compared to shCtrl-OVCAR3 suggesting that NANOG is in part required to sustain growth of ovarian cancer cells under glutamine depletion. To further decipher the signaling involved in NANOG under glutamine depletion, expressions of markers of ER stress were detected by western blot. We found that shNANOG-OVCAR3 cells expressed lower levels of BiP, a master regulator of ER stress. Under glutamine depletion, knockdown of NANOG further enhanced the expression of phosphorylated PERK but did not alter the levels of cleaved ATF6 or phosphorylated IRE-1a, suggesting NANOG regulates ER stress pathway through BiP/PERK signaling in glutamine-depleted environment. Immunohistochemical studies were performed to determine the expressions of NANOG and BiP in our ovarian cancer patient cohort. NANOG expression was positively correlated with the expression of nuclear BiP. Total BiP expression was significantly higher in cancerous tissue than in benign and borderline ovarian tumor tissue. Altogether, these findings suggested a hitherto unreported role of the stem cell transcription factor NANOG in regulating ER stress through BiP/PERK signaling under glutamine depleted environment.

Citation Format: Astrid Man-Yee Suen, Ivy Tsz-Lo Wong, Cindy Yin-Si Tse, Ka-Kui Chan, Oscar Gee-Wan Wong, Annie Nga-Yin Cheung. NANOG sustains ovarian cancer cell survival and disrupts ER stress signaling through BiP/PERK under glutamine depletion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2565.
Abstract 2572: Compound C inhibits metabolic programming of ovarian cancer
Authors: Ghoneum Alia Dawlat , Gonzalez Daniela , Said Neveen
Published: September 2020

High grade serous ovarian carcinoma (HGSC) exhibits elevated energy demands to sustain rapid proliferation, survival and invasiveness. We have shown that Compound C, also known as Dorsomorphin, inhibits ovarian cancer growth, invasiveness and survival as well as interactions with mesothelial cells and macrophages. However, the effect of Compound C on metabolic programming has not been reported. The purpose of this study is to determine whether Compound C inhibits the malignant phenotypes of ovarian cancer through suppression of energy production. Therefore, we performed comprehensive metabolic profiling of ovarian cancer cells treated with Compound C. Our findings indicate that Compound C significantly inhibits ATP production in the ovarian cancer cell lines: OVCAR3, SKOV3 and IGROV1. Compound C inhibited basal and maximal mitochondrial respiration as well as the non-mitochondrial oxygen consumption in these three cell lines. Compound C had no effect on basal, induced or compensatory glycolysis. However, it significantly decreased the ratio of mitochondrial oxygen consumption rate to glycolytic proton efflux rate (mitoOCR/glycoPER), indicating a direct inhibitory effect of Compound C on mitochondrial electron transport chain and a bioenergetic shift from oxidative phosphorylation towards glycolysis. These results demonstrate for the first time, the direct inhibitory effect of Compound C on mitochondrial function as a novel mechanism of inhibition of the malignant phenotype of ovarian cancer cells.

Citation Format: Alia Dawlat Ghoneum, Daniela Gonzalez, Neveen Said. Compound C inhibits metabolic programming of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2572.
Abstract 2697: Single cell RNA sequencing and tumor cell heterogeneity in metastatic triple-negative breast cancer
Authors: Yang Dongbo , Dann Christopher , Pott Sebastian , Rosner Marsha
Published: September 2020

Tumor cell heterogeneity has been strongly implicated in metastatic progression of solid tumors such as breast cancer, leading to resistance and recurrence. We hypothesize that inhibiting metastasis will reduce tumor cell heterogeneity and thereby increase therapeutic sensitivity. To test this hypothesis, we expressed the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP or PEBP1) in triple-negative breast cancer (TNBC) cells as a means of comparing the variation in gene expression between individual cells in metastatic versus non-metastatic tumors. Xenograft tumors with or without exogenous RKIP expression were dissociated into single cell suspensions. We removed mouse as well as dead cells, and the remaining cells were loaded onto a microfluidic device (Fluidigm C1) for separation and library preparation. For each condition we performed three independent experiments yielding a total of 1569 cells. Initially, we performed controls to ensure that differences were not due to batch effects, cell cycle stage or sample quality. Clustering of single cells across all samples indicated that gene expression profiles are similar, confirming reproducibility. Gene ontology analysis of differentially expressed genes between cells with and without RKIP overexpression reveals gene sets that are important in tumor cell transitions from metastatic to non-metastatic states. These data will allow us to specifically test whether metastatic tumors are more heterogeneous than non-metastatic tumors in TNBC.

Citation Format: Dongbo Yang, Christopher Dann, Sebastian Pott, Marsha Rosner. Single cell RNA sequencing and tumor cell heterogeneity in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2697.
Abstract 2720: The GPC3 metastasis suppressor induces the activation of p38 MAPK signaling pathway promoting dormancy at the secondary organ
Authors: Pastore Magali Delgado , Guereño Macarena , Lugones Ana C. , Peters Maria G.
Published: September 2020

GPC3 is a proteoglycan involved in the control of proliferation and survival, which has been linked to cancer. In this respect, we previously demonstrated that normal breast tissues exhibit high levels of GPC3, while its expression is diminished in tumors. However, the role of the GPC3 downregulation in breast tumor progression and its molecular and cellular operational mechanisms are not fully understood. In this study we showed that GPC3 reverts the epithelial-to-mesenchymal transition (EMT) underwent by mammary tumor cells, blocks metastatic dissemination and induces dormancy at secondary site. Using genetically modified murine breast cancer cell sublines, we demonstrated that the phospho-Erk/phospho-p38 ratio is lower in GPC3 reexpressing cells, while p21, p27 and SOX2 levels are higher, suggesting a dormant phenotype. In vivo metastasis assays confirmed that GPC3 reexpressing cells reduce their metastatic ability. Interestingly, the presence of dormant cells was evidenced in the lungs of inoculated mice. Dormant cells could reactivate their proliferative capacity, remain viable as well as tumorigenic, but they reentered in dormancy upon reaching secondary site. We also proved that GPC3 inhibits metastasis through the p38 MAPK signaling pathway activation. The in vivo treatment with a pharmacological inhibitor of p38 induced an increase in cell invasion of orthotopic tumors reexpressing GPC3, as well as in spontaneous and experimental metastatic dissemination. In conclusion, our study shows that GPC3 returns mesenchymal-like breast cancer cells to an epithelial phenotype, impairing in vivo metastasis and inducing tumor dormancy through p38 MAPK signaling activation. These results help to identify the genetic determinants of dormancy and suggest the translational potential of research focusing in GPC3.

Citation Format: Magali Delgado Pastore, Macarena Guereño, Ana C. Lugones, Maria G. Peters. The GPC3 metastasis suppressor induces the activation of p38 MAPK signaling pathway promoting dormancy at the secondary organ [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2720.
Abstract 2723: XIAOPI formula inhibits breast cancer pre-metastatic niche formation via blocking TAMs/CXCL1 pathway
Authors: Wang Zhiyu , Zheng Yifeng , Wang Neng , Wang Shengqi , Yang Bowen , Lin Yi
Published: September 2020

Recent findings suggested that premetastatic niche (PMN) is a prerequisite in mediating cancer metastasis. Previously we demonstrated that XIAOPI formula could inhibit breast cancer lung metastasis via inhibiting tumor associated macrophages (TAMs)-secreted CXCL1. Herein, we aimed to explore the effects of XIAOPI formula on preventing breast cancer PMN formation and its underlying molecular mechanisms. It was found that XIAOPI formula could inhibit the proliferation of M2 phenotype macrophages and reduce CXCL1 expression in a dose-dependent manner. TAMs/CXCL1 signaling was subsequently found to stimulate the recruitment of c-Kit+/Sca-1+ HSPCs and their differentiation into CD11b+/Gr-1+ MDSCs, which were symbolic events accounting for PMN formation. Moreover, XIAOPI formula was effective in inhibiting HSPCs activation and blocking the proliferation and metastasis of breast cancer cells 4T1 induced by HSPCs and TAMs co-culture system, implying that XIAOPI was effective in preventing PMN formation in vitro. Breast cancer xenograft experiments further demonstrated that XIAOPI formula could inhibit breast cancer growth and metastasis in vivo. More importantly, XIAOPI formula significantly delayed the time of cancer cell seeding in the lung tissues. Meanwhile, the populations of HSPCs in the bone marrow and MDSCs size in both tumor and lung tissues were all remarkably declined by XIAOPI formula treatment. However, the inhibitory effects of XIAOPI formula could be relieved by CXCL1 overexpression in the TAMs. Taken together, our study provided preclinical evidence supporting the application of XIAOPI formula in preventing breast cancer PMN formation, and highlighted TAMs/CXCL1 as a potential therapeutic strategy for PMN targeting therapy.

Citation Format: Zhiyu Wang, Yifeng Zheng, Neng Wang, Shengqi Wang, Bowen Yang, Yi Lin. XIAOPI formula inhibits breast cancer pre-metastatic niche formation via blocking TAMs/CXCL1 pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2723.
Abstract 2731: Receptor for Hyaluronan Mediated Motility (RHAMM) promotes breast cancer progression
Authors: Daughters Claire Elizabeth , Knutson Todd P. , Forster Colleen , McCarthy James , Schwertfeger Kaylee , Nelson Andrew
Published: September 2020

Breast cancer remains one of the leading causes of cancer-related death in women in the U.S. with over 40,000 deaths annually. Understanding mechanisms of breast cancer progression holds great clinical utility to individualize patient therapy and to increase our portfolio of effective treatments for the different subtypes of breast cancer. We performed a focused gene expression analysis of 120 patients across all relevant hormone receptor (HR) subtypes at our institution. Increased expression of hyaluronan mediated motility receptor (HMMR, aka RHAMM) was associated with higher Nottingham grade, lymph node metastasis, and higher stage (all p<0.01). RHAMM expression was higher in more aggressive HR subtypes (triple negative and HER2+). Unsupervised hierarchical clustering of the cohort with a 29 gene signature related to RHAMM biologic activity further supported these associations with breast cancer progression/aggressiveness. Using CRISPR/Cas9, stable RHAMM -/- MCF10DCIS cell lines were created; RHAMM deletion does not affect cell proliferation, but significantly reduces in vitro Matrigel invasion and soft agar colony formation. RHAMM knockout also diminishes invasive progression of the MCF10DCIS cells in vivo, using the mouse intraductal (MIND) xenograft model; 83% of control xenografts demonstrate tissue invasion by 6 weeks, while only 50% of RHAMM-/- xenografts show any invasive component (p=0.03). Our human and model system data indicate a role for RHAMM function in breast cancer invasion. On-going work will investigate the dependency of the invasive phenotype specifically on RHAMM-hyaluronan binding; this interaction can be pharmacologically disrupted and may represent a specific intervention to limit breast cancer invasion and potentially metastasis.

Citation Format: Claire Elizabeth Daughters, Todd P. Knutson, Colleen Forster, James McCarthy, Kaylee Schwertfeger, Andrew Nelson. Receptor for Hyaluronan Mediated Motility (RHAMM) promotes breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2731.
Abstract 2746: PET-CT/FACS platform to detect the biodistribution of radioisotope 89Zr PD-1 inhibitor in syngeneic tumor models
Authors: Tang Xuzhen , Wang Fuyang , Chen Junyi , Zeng Xuanming , Liu Zhibo , Ji Qunsheng
Published: September 2020

Cancer treatment has been revolutionized by targeting the interaction between tumors and immune system. For instance, PD-1/PD-L1 mono- or combo-therapy has achieved great success in clinics, and patient responsiveness is closed associated with PD-L1 expression in cancer cells, tumor infiltration leukocytes, and drug bio-distribution in the tumor microenvironment. We established a method to label PD-1 inhibitor with radioisotope 89Zr for in-life imaging (PET-CT). Anti-PD-1 sensitive mouse colorectal cell line MC38 and resistant mouse melanoma cell line B16F10 were implanted to C57BL/6 mice, which were then treated with 89Zr PD-1 inhibitor. PET imaging was performed at multiple time points to detect the bio-distribution of anti-PD-1, while the infiltrated immune cells were identified by FCM and PD-L1 expression measured by IHC. Notable tumor uptake of 89Zr PD-1 inhibitor was observed in mouse liver and tumor grafts. The uptake in tumor increased over time and plateaued 24 hours post-injection. MC38 and B16F10 tumor grafts showed difference in uptake levels. Blood and tumor at both flanks were removed for leucocyte phenotyping, and compared with B16F10, MC-38 tumors exhibited higher percentage of total lymphocytes and cytotoxic T lymphocytes, equivalent number of dendritic cells and less myeloid cells. Further phenotyping indicates that compared to MC-38, B16F10 tumors contain higher percentage of MDSC, most of which are monocytic myeloid derived suppressor cells. With our PET-CT/FACS platform, we can evaluate immunotherapy response and microenvironment changes in different tumors in “one-mouse” system. The unique immune microenvironment (high TIL, high CD8+ T and low MDSC) may contribute to the notable and sustainable uptake of PD-1 inhibitor and the corresponding efficacy. This system can act as a good translational platform to monitor the PK-PD readout of immune therapy.

Citation Format: Xuzhen Tang, Fuyang Wang, Junyi Chen, Xuanming Zeng, Zhibo Liu, Qunsheng Ji. PET-CT/FACS platform to detect the biodistribution of radioisotope 89Zr PD-1 inhibitor in syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2746.
Abstract 2757: Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma
Authors: Martin Kelly A. , Sebastian Aimy , Hum Nicholas R. , Byers Stephen , Wheeler Elizabeth K. , Coleman Matthew A. , Loots Gabriela G.
Published: September 2020

Pancreatic ductal adenocarcinoma (PDAC) is one of the most incurable types of cancer. Cancer that develops in the acinar cells of the pancreas is typically not diagnosed until later stages, such as stage 3 or 4. As such, this form of cancer is particularly lethal with only 9% of patients reaching 5-year survival. PDAC is known to have a particularly dense extracellular matrix composed of fibroblasts, which have been previously shown to play an important role in promoting resistance to drug therapy. Characterization of the stromal networks involved in PDAC tumor development as well as protein markers of fibroblast subpopulations within the tumor stroma are critical to developing new fibroblast-targeted therapeutic approaches as well as understanding key signaling molecules that ultimately promote tumor progression and drug resistance. Single-cell RNA sequencing (scRNAseq) was utilized to analyze cancer-associated fibroblasts (CAFs) from KPC mouse-derived, MT3 subcutaneous murine allografts along with two fibroblast lines derived from human PDAC tumors: CRC-811 and IA-1340. Single cell gene expression profiling and subsequent analysis of MT3-derived CAFs resulted in the identification of three CAF subpopulations including a myofibroblast subpopulation that expressed high levels of smooth muscle actin (Acta2), which was also observed in both human samples. Fibroblast subpopulations enriched in Acta2 expression, expressed high levels of Wnt5a along with several other secreted factors including Tgfb1, Tgfb2 and Ctgf. Wnt5a is a secreted protein that activates non-canonical Wnt signaling pathways and is known to regulate normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However Wnt5a is not natively expressed in MT3 cancer cells derived from syngeneic tumors, but potentially in the stroma. We hypothesize it is exclusively derived from fibroblasts. Previously it has been shown that Wnt5a inhibition suppressed gastric cancer metastasis, therefore further validation of the role of myofibroblast-derived Wnt5a on PDAC disease progression is warranted. This study received funding by LDRD 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM Release Number: LLNL-ABS-798442.

Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas R. Hum, Stephen Byers, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2757.
Abstract 2809: Pancreatic cancer patient-derived organoids as a tool for personalized medicine
Authors: Tiriac Herve
Published: September 2020

Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient-derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we find that PDO therapeutic profiles paralleled patient outcomes and that PDOs enable longitudinal assessment of chemo-sensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemo-sensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemo-refractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection.

Citation Format: Herve Tiriac. Pancreatic cancer patient-derived organoids as a tool for personalized medicine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2809.
Abstract 2845: Role of neutrophil associated a2 isoform of V-ATPase in cancer
Authors: Ibrahim Safaa A. , Riehl Valerie , Schneiderman Sylvia , Beaman Kenneth D.
Published: September 2020

Tumors associated neutrophils (TAN) regulate the tumor microenvironment by directly promoting tumor progression and represent a potential therapeutic target for cancer treatment. Identifying neutrophil associated proteins that promotes neutrophil pro-tumorigenic properties will provide new specific targets for cancer therapy. The a2 isoform of V-ATPase (a2V) is multisubunit enzyme that is expressed on the intravesicular membranes as well as the plasma membrane of activated immune cells. Immuno-histochemical analysis shows that TAN in human cancer tissues express a2V. Isolated neutrophils from healthy donors were primed with tumor conditioned media collected from MDA MB-231 and MCF-7. Primed neutrophils showed increased gene expression of a2V while a1 and a3 isoform expression were not changed. Increased a2V in neutrophils was confirmed in the protein level by western blot and immunofluorescence staining. Flow cytometric analysis showed increased a2V surface staining on live primed neutrophils. Multiplex Luminex assay showed increased secreted protein levels of multiple pro-, anti-inflammatory cytokines and protumorigenic factors such as IL-8, VEGF and MMP-9 from primed neutrophils. Currently we are characterizing a method to knock down the a2V in human neutrophils isolated from peripheral blood and investigating its effect on neutrophil phenotypes. Together these data suggest that neutrophil associated a2V can play an important role in regulating the neutrophil phenotype. This makes it a potential candidate to target for cancer therapy.

Citation Format: Safaa A. Ibrahim, Valerie Riehl, Sylvia Schneiderman, Kenneth D. Beaman. Role of neutrophil associated a2 isoform of V-ATPase in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2845.
Abstract 2861: Targeting cancer with antibody-drug conjugates: Promises and challenges
Authors: Dean Alexis , Twomey Julianne , Zhang Baolin
Published: September 2020

Chemotherapy is inherently associated with side effects due to its poor selectivity towards tumor cells/tissues. Antibody-drug conjugate (ADC) technology promises to overcome barriers of classical chemotherapy by utilizing monoclonal antibodies to deliver potent payloads to selected target cancer cells. The antibody and cytotoxic payload are joined by a chemical linker resulting in an agent with the prospect of increased selectivity, anti-cancer activity, and improved systemic circulation. As of 2019, the US Food and Drug Administration (FDA) has approved five ADCs with cancer indications, including brentuximab vedotin for treatment of CD30-expressing lymphomas, trastuzumab emtansine for Her2-positive breast cancer, inotuzumab ozogamicin for CD22-positive acute lymphoblastic leukemia (ALL), gemtuzumab ozogamicin for CD33-positive acute myeloid leukemia (AML), and polatuzumab vedotin for diffuse large B-cell lymphoma (DLBCL). Currently, there are about 80 ADCs under clinical development in nearly 130 clinical trials. Despite initial success, there are still many challenges in the development of ADCs with respect to their safety and efficacy profiles. In this study, we aim to survey the recent efforts in the design and development of ADCs. We have focused on site-specific conjugation methods for producing homogenous ADCs with consistent drug-antibody ratio (DAR) - a challenge often observed in conventional conjugation methods. This information is further discussed with respect to the ADC interaction with target cells to elicit cytotoxicity. The survey results will be presented, giving perspective on technical considerations for ADC design towards better correlation between preclinical and clinical results and overall safer and more efficacious products.

Citation Format: Alexis Dean, Julianne Twomey, Baolin Zhang. Targeting cancer with antibody-drug conjugates: Promises and challenges [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2861.
Abstract 2878: Granzyme B functionalized nanocarriers targeting membrane-bound Hsp70 for multimodal cancer therapies
Authors: Shevtsov Maxim , Kaesler Susanne , Stangl Stefan , Yakovleva Luidmila , Tagaeva Ruslana , Marchenko Yaroslav , Nikolaev Boris , Khachatryan William , Galibin Oleg , Pitkin Emil , Multhoff Gabriele
Published: September 2020

Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as potential clinical tools for cancer theranostics. Membrane-bound 70 kDa heat shock protein (mHsp70) is ubiquitously expressed on the cell membrane of various tumor types but not on normal cells and therefore provides a tumor-specific target. The serine protease granzyme B (GrB) that is produced as an effector molecule by activated T and NK cells has been shown to specifically target mHsp70 on tumor cells. Following binding to Hsp70, GrB is rapidly internalized into tumor cells inducing the process of cellular apoptosis. Decoration of the nanoparticles surface with GrB increased the targeting properties of the nanocarriers as was shown by confocal and electron microscopies. In series of in vivo studies it was demonstrated that GrB functionalized SPIONs act simultaneously as a contrast enhancement agent when being intravenously administered for magnetic resonance imaging and induce specific tumor cell apoptosis (human U87 glioblastoma in NMRI nu/nu mice, rat C6 glioma in Wistar rats, B16 melanoma in C57/Bl6 mice). Continuous prolonged administration of GrB-SPIONs via programmable subcutaneously implanted mini-pump system iPrecio further enhanced the therapeutic benefit of magnetic conjugates. Moreover, significant therapeutic response was achieved in immunocompromised mice models (advanced U87 brain tumor, small lung cancer H1339 with brain metastasis) with a high tumor burden. Combinatorial regimens employing stereotactic radiotherapy and/or magnetic targeting with immune checkpoint inhibitors (anti-CTLA-4 monoclonal antibodies) are found to further enhance the therapeutic efficacy of GrB-SPIONs in different tumor mouse models. The study was funded by RFBR, project № 19-08-00024; DFG grant SFB824/3, Germany, TaGoNaX DFG project № 336532926, STA1520/1-1.

Citation Format: Maxim Shevtsov, Susanne Kaesler, Stefan Stangl, Luidmila Yakovleva, Ruslana Tagaeva, Yaroslav Marchenko, Boris Nikolaev, William Khachatryan, Oleg Galibin, Emil Pitkin, Gabriele Multhoff. Granzyme B functionalized nanocarriers targeting membrane-bound Hsp70 for multimodal cancer therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2878.
Abstract 2933: KLF5 and its association with HIF-1α promotes PTC progression and metastatic potential through AKT activation
Authors: Poyil Pratheeshkumar , Siraj Abdul K , Padmaja Divya , Parvathareddy Sandeep Kumar , Thangavel Saravanan , Melosantos Roxanne , Victoria Ingrid G. , Begum Rafia , Al-Dayel Fouad , Al-Kuraya Khawla S.
Published: September 2020

Thyroid cancer is the second most common malignancy among females in Saudi Arabia, with Papillary thyroid carcinoma (PTC) accounting for 80-90%. Kruppel-like factor 5 (Klf5) is a transcription factor that plays a critical role in cell transformation, proliferation, and oncogenesis. Immunohistochemical analysis of KLF5 was performed in 1219 PTC cases. KLF5 over-expression was noted in 65.1% (793/1219) of PTCs, and was significantly associated with tall-cell variant (p <0.0001), extrathyroidal extension (p = 0.0003), lymph node metastasis (p < 0.0001) and stage IV tumors (p < 0.0001). A significant association was also noted with HIF-1α over-expression (p = 0.0492). Interestingly, KLF5 over-expressing tumors showed poor disease-free survival (p = 0.0066). Functional studies in PTC cell lines showed that KLF5 co-immunoprecipitated with HIF-1α. Knockdown of KLF5 decreased the expression of HIF-1α while KLF5 was not affected by HIF-1α inhibition, suggesting that KLF5 is a functional upstream of HIF-1α. Down-regulation of KLF5 using a specific inhibitor, ML264 or siRNA inhibited cell migration, invasion, and angiogenesis. Also, the treatment of PTC cell lines with ML264 resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner. Furthermore, the silencing of KLF5 significantly decreased the self-renewal ability of spheroids generated from PTC cells. Our findings confer that KLF5 may be a potential therapeutic target for the treatment of papillary thyroid cancer.

Citation Format: Pratheeshkumar Poyil, Abdul K Siraj, Divya Padmaja, Sandeep Kumar Parvathareddy, Saravanan Thangavel, Roxanne Melosantos, Ingrid G. Victoria, Rafia Begum, Fouad Al-Dayel, Khawla S. Al-Kuraya. KLF5 and its association with HIF-1α promotes PTC progression and metastatic potential through AKT activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2933.
Abstract 2946: The prevalence of Globo H in different tumor types: Breast, pancreatic, lung, gastric, colorectal, liver, and esophageal cancers
Authors: Chen I-Ju , Yang Ming-Chen , Chen Yu-Jung
Published: September 2020

Background: Globo H (GH) is a hexassacharide glycosphingolipid overexpressed on tumor epithelial cells in several cancer types. Ongoing clinical trials of GH-targeting drugs include therapeutic vaccines, a monoclonal antibody, and an antibody drug conjugate. Consequently, the level of GH expression may provide a critical biomarker for appropriate patient selection. We present the prevalence of GH expression in various human cancers using a validated immunohistochemistry (IHC) assay.

Methods: A validated IHC assay was used to analyze a total of 562 specimens across 7 cancer types, including breast (131), lung (77), colorectal (75), gastric (73), pancreatic (72), liver (70), and esophageal cancer (64) specimens. Anti-GH monoclonal antibody (VK9) was used for IHC staining. GH expression level was assessed by certified pathologists, and results are presented using an H-score system (0 to 300). The H-score was calculated as: H-score = (% of weak intensity x 1) + (% of moderate intensity x 2) + (% of strong intensity x 3).

Results: Among the specimens tested, pancreatic cancer had the highest median H-score of 90.0 (range 0-300) with a 76.4% prevalence using cutoff of H-score ≥ 1, while liver cancer had the lowest median H-score of 0.0 (range 0 to 80) with a 17.1% prevalence. Using an H-score of 100 as a cutoff [approved in the Investigational Device Exemption (IDE) application as a pre-screening criterion of GH monoclonal antibody in the Phase 2 clinical trial OBI-888-001 (NCT03573544)], the GH prevalence of the pancreatic, esophageal, gastric, breast, lung, colorectal, and liver cancers are 50%, 17.2%, 24.7%, 13%, 10.4%, 16%, and 0%, respectively. In addition, subtypes of each cancer differ in expression profile. While immune cells are typically GH negative, some GH-positive immune cells were observed in the intra- or peri-tumor region, suggesting the shedding of GH to tumor-surrounding or infiltrated immune cells.

Conclusions: Overexpression of GH in human pancreatic, gastric, lung, colorectal, esophageal, and breast tumors renders GH a potential therapeutic target for these cancers. Furthermore, the presence of GH-positive immune cells in the intra- or peri-tumor region lends support to the proposed mechanism of cancerous cells shedding GH-ceramide to suppress normal immune functions. The heterogeneous expression of GH among different molecular subtypes may provide a biomarker in the selection of patients for GH-directed therapies. The GH IHC assay has now been validated for use and the IDE approved by the FDA for patient screening.

Citation Format: I-Ju Chen, Ming-Chen Yang, Yu-Jung Chen. The prevalence of Globo H in different tumor types: Breast, pancreatic, lung, gastric, colorectal, liver, and esophageal cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2946.
Abstract 295: Protein phosphatase, Mg2+/Mn2+ dependent 1G promotes proliferation via modulating phosphorylation of retinoblastoma protein and associates with poor outcome in hepatocellular carcinoma
Authors: Hu Wen , Guan Xin Yuan , Li Lei , Zhu Yinghui , Li Yan , Zeng Ting Ting , Wen Yun Fei
Published: September 2020

The serine/threonine protein phosphatase family has been demonstrated involved in a variety of cellular processes. Here, we identified that protein phosphatase, Mg2+/Mn2+ -dependent 1G (PPM1G) is an oncogene-associated protein in hepatocellular carcinoma (HCC), and is associated with the progression of HCC. In this study, we carried out a series of in vitro (MTT, colony formation, immunoblot analysis, IHC, and Q-PCR) as well as in vivo (Hepatocellular carcinoma cell xenograft mouse model) experiments to determine the biological roles of PPM1G in HCC. Our results showed that PPM1G expression was significantly increased in HCC tissues, compared with the adjacent non-tumorous tissues. Further analysis revealed that high expression of PPM1G is associated with advanced clinical characters including advanced clinical stage and tumor recurrence. Kaplan-Meier analysis suggested that a high level of PPM1G is correlated to unfavorable overall survival (P < 0.0001) as well as disease-free survival (P = 0.004) in patients with HCC. Furthermore, overexpression of PPM1G promoted cell proliferation while this effect can be attenuated by the knockdown of PPM1G in in vitro and in vivo studies. Notably, our findings showed that the tumor suppressor gene expression (e.g. pRb phosphorylation and p53 protein) was inhibited upon the silencing of PPM1G. Taken together, our study demonstrated that PPM1G plays an crucial role in HCC cell proliferation by modulating pRb phosphorylation as well as p53 expression. Furthermore, this study provides strong clinical implications of PPM1G in prognostic prediction in patients with HCC.

Citation Format: Wen Hu, Xin Yuan Guan, Lei Li, Yinghui Zhu, Yan Li, Ting Ting Zeng, Yun Fei Wen. Protein phosphatase, Mg2+/Mn2+ dependent 1G promotes proliferation via modulating phosphorylation of retinoblastoma protein and associates with poor outcome in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 295.
Abstract 2951: Knockdown of SCAMP3 suppresses the proliferation and emboli formation capacity of inflammatory breast cancer cells through EGFR signaling
Authors: Suarez-Arroyo Ivette J. , Reyes-Chea Jael , Martinez-Montemayor Michelle M.
Published: September 2020

Inflammatory Breast Cancer (IBC) is one of the most lethal forms of breast cancer (BC), where patients have a 43% increased risk of death compared to women with non-IBC advanced BC. Approximately 30% of triple-negative (TN) IBC cases overexpress the Epidermal Growth Factor Receptor (EGFR). EGFR amplification is associated with a poor overall survival rate compared with EGFR-negative IBC cases. We previously identified increased expression of the secretory carrier membrane protein 3 (SCAMP3) in EGFR overexpressing TN SUM149 IBC cells, tissues and in the IBC metastasis promoting structure, the tumor emboli. SCAMP3 is an endosome-associated protein known to regulate the degradation of EGFR. Thus, we aim to elucidate the role of SCAMP3 in the regulation of EGFR signaling and IBC progression. To investigate how SCAMP3 contributes to the IBC cellular response, we knocked out (KO) the expression of SCAMP3 in IBC SUM149 cells using the CRISPR/Cas9 technique. Wild type (WT) and KO IBC cells treated with EGF for 30 min in serum-starved conditions were used to assess cell viability after 24, 48, 72, and 96h, colony, and emboli formation capacity. Our results demonstrate a reduction in the proliferation, colony, and emboli formation capacity of SCAMP3 KO cells in comparison with the WT. Taken together our results show that SCAMP3 plays a promoting role in the progression of IBC through EGFR signaling modulation.

Citation Format: Ivette J. Suarez-Arroyo, Jael Reyes-Chea, Michelle M. Martinez-Montemayor. Knockdown of SCAMP3 suppresses the proliferation and emboli formation capacity of inflammatory breast cancer cells through EGFR signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2951.