bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2794: A novel HNSCC organoid-on-a-chip platform for preclinical investigation and drug screening
Authors: Bonilla Silvia , Driehuis Else , Lanz Henriëtte , Clevers Hans , Joore Jos , Queiroz Karla , Vulto Paul 
Published: September 2020

Abstract
Head and neck squamous cell carcinoma (HNSCC) cancer is a broad category of tumor types arising from the inner lining of various anatomic structures including the oral cavity, pharynx, larynx and salivary glands. Treatment often involves an intensive combination of surgery, radiotherapy and chemotherapy. Despite this, tumor recurrence rates remain high and survival rates are relatively poor. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate®, a microfluidic based 3D-culture system, and HN cancer-derived organoids. MIMETAS develops Organ-on-a-Chip-based models for evaluation of new medicines. Our unique microfluidic technology enables testing of compounds on miniaturized 3D organ models in high-throughput. Hereby we show the establishment of HNSCC cancer-derived organoids in 2-lane OrganoPlate®, and its usefulness for phenotypic drug screenings. The aim of the study is to capture differential sensitivity of HN organoid lines to either a common platinum-based chemotherapy or to a targeted drug still not used in the clinic. Organoid lines (T1, T3 and T5) were embedded in the 2-lane OrganoPlate® as single cells in an extracellular matrix gel. At day 2, cultures were treated with cisplatin or PARP-inhibitor niraparib for 120 hours. Additionally, cultures exposed to high doses of cisplatin were evaluated after one week of recovery in order to determine treatment failure and recapitulate potential patient relapse.
Drug response was evaluated by assessment of morphology (phase contrast), cell viability (total nuclei count, alamar blue assay), proliferation (EdU incorporation) and DNA damage (phosphorylated histone H2AX). Organoid cultures grow well under medium perfusion in the 2-lane OrganoPlate® and selective sensitivity to cisplatin and niraparib is revealed by the used readouts. The high-throughput, microfluidic 2-lane OrganoPlate® platform offers an attractive method for growing HNSCC cancer-derived organoids, supporting development of individualized tumour models for phenotypic drug screenings.
Citation Format: Silvia Bonilla, Else Driehuis, Henriëtte Lanz, Hans Clevers, Jos Joore, Karla Queiroz, Paul Vulto. A novel HNSCC organoid-on-a-chip platform for preclinical investigation and drug screening [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 2794.
Abstract 2832: Multiplex fluorescent imaging of the metastatic melanoma microenvironment reveals spatially-dependent macrophage interactions modulating tumor-infiltrating T-cells
Authors: Wang Victor G. , Martinek Jan , Gulati Ananya , Boruchov Hannah , Ray Kelly , Palucka Karolina , Chuang Jeffrey H. 
Published: September 2020

Abstract
Tumor-infiltrating lymphocytes (TIL) hold strong prognostic value in determining patient survival as well as response to immune checkpoint blockade across many tumor types, especially metastatic melanoma. Tumors often evolve mechanisms to prevent TIL access and/or function to allow for continued growth. One such mechanism is the recruitment and reprogramming of tumor-associated macrophages (TAM) which suppress TILs through a variety of pathways. The interactions between TAMs and TILs that result in a suppressive tumor microenvironment (TME) have been studied in vitro and with spatially-agnostic sequencing, leaving the location-specific contributions underexplored. Interrogating the TAM-TIL interactions in an intact TME will uncover novel mechanisms responsible for TAM function and may be key to reversing TIL immunosuppression, improving anti-tumor immune responses, survival, and potentially therapy response. We use histocytometry, a multiplex quantitative tissue imaging method capable of capturing whole-slide tumor sections, to explore the human metastatic melanoma TME in situ and resolve the spatial contributions of TAM-TIL interactions. We demonstrate a T-cell preference for physical contact with phagocytic TAMs in the tumor but non-phagocytic TAMs in the stroma. We further model immune synapses from histocytometry to probe the function underlying the TAM-TIL preference based on the directionality of signaling components. The model suggests T-cells in contact with macrophages in the tumor are communicating more frequently with the phagocytic TAMs as compared to their non-phagocytic counterparts, indicating that this interaction extends beyond a simple proximity preference. In addition, proliferating T-cells communicate more frequently with macrophages than non-proliferating T-cells but this effect limited to the intratumor space. Together we demonstrate the functional consequences of physical TAM-TIL interactions with unprecedented spatial resolution. This work unravels important TAM interactions that shape the TME which have not been previously appreciated, providing novel insight into the forces modifying T-cell function in the TME and revealing new TAM biology to explore further.
Citation Format: Victor G. Wang, Jan Martinek, Ananya Gulati, Hannah Boruchov, Kelly Ray, Karolina Palucka, Jeffrey H. Chuang. Multiplex fluorescent imaging of the metastatic melanoma microenvironment reveals spatially-dependent macrophage interactions modulating tumor-infiltrating T-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 2832.
Abstract 2836: Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages
Authors: Shephard Miranda , Pease Nicholas , Vinnedge Lisa M. Privette 
Published: September 2020

Abstract
Breast cancer is the second leading cause of cancer deaths among women. DEK is a known oncoprotein found to be highly expressed in more than 60% breast cancers and is found to be an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To elucidate the oncogenic functions of DEK, we performed RNA-seq analysis on isogenic Dek-knockout and complemented murine breast cancer cells, which indicated dysregulation of immune signaling. Among the target genes identified and confirmed was an upregulation of thymic stromal lymphopoietin (TSLP) in Dek expressing murine breast cancer cells. TSLP was previously shown by Han et al to amplify the alternative (M2) activation of macrophages. M2-like macrophages are tumor promoting, they recycle iron for cell growth and encourage tissue remodeling and repair activities like angiogenesis. To test the immune modulating functions of Dek-expressing breast cancer cells, we treated bone marrow derived macrophages (BMDM) with tumor cell conditioned media. We found that in vitro, Dek expressing cancer cells induced an M2-like polarization of macrophages, as determined by the expression of M2-associated genes, enhanced migration, and iron recycling phenotypes, which was accompanied by inhibited ERK1/2 signaling. We found this phenotypic trend to be true in vivo, as well. In sectioned mammary tumors from MMTV-RontgDek+/+ and MMTV-RontgDek−/− mice, we see lower levels of iron staining in Dek+/+ tumors than in Dek−/− tumors using Prussian blue staining that co-localized with F4/80 macrophage marker staining. This suggested that Dek expression in the tumors induced an iron recycling, M2-like phenotype in tumor associated macrophages. Furthermore, we observed increased TSLP expression and angiogenesis, as determined by CD31 staining, in Dek-expressing tumors in vivo. These findings suggest that tumor Dek expression may promote breast cancer progression by inducing M2-like macrophage polarization in a murine breast cancer model.
Citation Format: Miranda Shephard, Nicholas Pease, Lisa M. Privette Vinnedge. Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages [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 2836.
Abstract 2842: Neutrophil migration towards tumor-conditioned media correlates with the malignant potential of breast tumor cells
Authors: Hein Lauren , SenGupta Shuvasree , Xu Yang , Parent Carole 
Published: September 2020

Abstract
During tumor development, neutrophils and other immune cells infiltrate the tumor niche. Neutrophils have been shown to have primarily pro-tumorigenic effects, and high levels of circulating neutrophils are indicative of a poor prognosis. Tumor-associated neutrophils (TANs) migrate to tumors by sensing chemical gradients produced by tumor cells; however, the mechanisms by which tumors and TANs interact and impact each other remain to be determined. It has been shown that malignant breast tumors, such as triple-negative breast tumors, harbor more TANs compared to less aggressive breast tumors. We hypothesize that neutrophil recruitment to the tumor niche depends on tumor-secreted, chemotactic factors that are primarily produced by malignant breast tumors. To test this, we used a panel of breast cancer cells grown in monolayers and more physiologically-relevant tumor spheroids, which were formed over the course of four days using the hanging-drop method. We characterized the size and morphology of the spheroids, which were generated from the poorly metastatic BT474 cells, as well as the highly malignant BT549 cells, MDA-MB-231 cells, and M4 cells of the MCF-10A cell series. We then compared the ability of tumor-conditioned media (TCM) isolated from either monolayers or spheroids to stimulate the migration of primary human neutrophils using transwell assays. We found that, compared to TCM isolated from control or poorly metastatic cell lines, the TCM derived from highly malignant cancer cells robustly stimulated the migration of neutrophils. These data indicate that breast cancer cells secrete factors with unique profiles that have varying potentials to recruit neutrophils. We are in the process of identifying specific tumor-secreted factors in the TCM using mass spectrometry. Additionally, we are assessing how the presence of neutrophils affects cancer cell migration and invasion by utilizing a coculture system in which tumor spheroids and primary neutrophils are imaged in real time. We reason that understanding the role of TANs during breast cancer progression will provide insight into novel therapeutic approaches, particularly in the context of highly metastatic forms of the disease.
Citation Format: Lauren Hein, Shuvasree SenGupta, Yang Xu, Carole Parent. Neutrophil migration towards tumor-conditioned media correlates with the malignant potential of breast tumor 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 2842.
Abstract 2858: Comparative surface proteomic analysis of human breast cancer cell lines
Authors: Benton Betsy , Snovida Sergei , Herting Katherine , Opperman Kay , Kaboord Barbara 
Published: September 2020

Abstract
Enrichment of cell surface proteins is commonly performed by biotinylation using amine-based chemistry; however, this method is often associated with intracellular labeling and difficulty distinguishing the true cell surface proteins from background contaminants. We developed a robust cell surface protein isolation method using amine-based chemistry that is compatible with mass spectrometry (MS). We applied this methodology to evaluate the surface proteomes of three breast cancer cell lines, including hormone responsive MCF-7 cells, and two triple negative breast cancer cell lines (TNBC), BT549 and Hs5789T. Thermo Scientific™ Tandem Mass Tag (TMT) Reagents, combined with high pH reverse phase fractionation of the labeled peptides, were used to multiplex protein identification and quantitative analyses by tandem MS. A heat map of enriched proteins from the three cell lines showed clearly distinct banding patterns of clustered protein groups. Relative quantitative differences in several classes of enriched proteins were observed, including tyrosine kinases, G-Protein-Coupled Receptors (GPCRs), and cell adhesion proteins. The tyrosine kinase EGFR was found to be more highly expressed in TNBC cell lines compared with MCF-7 cells. In contrast, the receptor tyrosine-protein kinase erbB-3 was found to be significantly higher in the MCF-7 cells compared with the TNBC cells. Several GPCRs were identified and found to vary across the three cell lines, including Frizzled 7 which is an emerging target for cancer therapy. Multiple cell adhesion proteins were also identified, including neural cell adhesion molecule L1 which promotes migration of MCF7 cells. Where possible, the data set was found to be consistent with literature reports. In conclusion, we have developed a highly sensitive method for cell surface proteomic analyses that can be effectively used to identify differences in cell types as well as for biomarker discovery and potential drug therapy targets.
Citation Format: Betsy Benton, Sergei Snovida, Katherine Herting, Kay Opperman, Barbara Kaboord. Comparative surface proteomic analysis of human breast cancer cell lines [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 2858.
Abstract 286: Regulation of APOBEC3B gene expression through the Rb/E2F pathway
Authors: Roelofs Pieter A. , Brown William L. , Span Paul N. , Martens John W. , Goh Chai Y. , Chua Boon H. , Kappei Dennis , Harris Reuben S. 
Published: September 2020

Abstract
The propensity of tumors to increase genomic diversity allows for a bottleneck-type adaptation to anti-cancer treatments. The genomic alterations fueling these adaptations in many different tumor types are, in part, brought about by ongoing endogenous activity of the DNA cytosine deaminase APOBEC3B (A3B). The ability of A3B to convert DNA cytosine bases into uracils has been shown to facilitate C-to-T transitions, C-to-G transversions, and DNA breakage. While low in healthy tissues, A3B expression and activity is elevated in tumors and may increase further throughout metastatic disease. However, molecular mechanisms responsible for transcriptional upregulation of A3B in tumor cells are poorly understood. Here, we investigate the possibility that the Rb/E2F pathway, which is often dysregulated in breast cancer, contributes to A3B gene expression. First, we cloned the A3B promoter upstream of a luciferase reporter and used a combination of deletion and site-directed mutagenesis to demonstrate that only one out of five in silico predicted E2F binding sites is functionally relevant, as disrupting this site caused constitutive reporter activation. Moreover, expression of the BK polyomavirus T-antigen, which inactivates Rb, induced the reporter and additional activation was not observed by mutating the functional E2F binding site. Second, SILAC-labelled nuclear protein extracts were subjected to affinity purification coupled to quantitative mass spectrometry to compare transcriptional complexes that bound wild-type promoter sequences, but not sequences with mutant E2F binding site promoter elements. These proteomics experiments showed that proteins belonging to the DREAM and PRC1.6-complexes strongly bind the wild-type but not the mutant E2F binding sites. Third, RNAi-mediated knock-down of candidate E2F family members implicated E2F6, and to a lesser extend E2F4, which are components of the PRC1.6 and DREAM complexes, respectively, in A3B gene repression. Taken together, these studies indicate that A3B expression may be suppressed by the concerted effort of two different E2F binding complexes and that Rb inactivation, by viral T-antigen here and genetic mutation as often seen in breast cancer, is able to upregulate A3B gene expression, promote genomic DNA mutagenesis, and fuel tumor evolution.
Citation Format: Pieter A. Roelofs, William L. Brown, Paul N. Span, John W. Martens, Chai Y. Goh, Boon H. Chua, Dennis Kappei, Reuben S. Harris. Regulation of APOBEC3B gene expression through the Rb/E2F 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 286.
Abstract 2931: VGF as a theranostic marker in neuroendocrine lung cancer
Authors: Yang Li-Hao , Yu Chia-Cherng , Chou Yu-Ting 
Published: September 2020

Abstract
Activating EGFR mutations are predictive markers for response in lung cancer patients under the treatment of EGFR-tyrosine kinase inhibitors (TKIs), whereas acquired resistance to EGFR-TKIs inevitably occurs in patients. Neuroendocrine transformation is one of the mechanisms behind the development of acquired resistance to EGFR-TKIs. In this study, we report that VGF, a secreted factor highly expressed in neurons and neuroendocrine tissues, is a potential theranostic marker in neuroendocrine lung tumors. Gene expression profiling analysis revealed that VGF levels were elevated during the differentiation of neuronal stem cells to neurons with surged H3K27ac modification at the VGF locus. We observed that VGF was highly expressed in lung neuroendocrine cancer cells. Moreover, VGF expression was low in lung adenocarcinoma but was enriched during EGFR-TKI selection. Correlation analysis showed that VGF was positively associated with chromogranin A, chromogranin B, and synaptophysin expression in lung tumors. ChIP-qPCR assays showed that the signal of H3K27ac modification at the VGF locus was increased in EGFR-TKI resistant cells compared to their sensitive counterparts. Clonogenic analysis and annexin-V staining assays revealed that VGF silencing attenuated cellular grown with increased apoptosis in neuroendocrine lung cancer cells as well as in EGFR-TKI resistant cells, indicating the potential of VGF as a therapeutic target. Kaplan-Meier analysis showed that high VGF expression predicted poor survival outcome in patients with lung tumors. Our findings provide an insight into the oncogenic role of VGF and suggest the potential of VGF as a theranostic for lung cancer.
Citation Format: Li-Hao Yang, Chia-Cherng Yu, Yu-Ting Chou. VGF as a theranostic marker in neuroendocrine lung 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 2931.
Abstract 2939: Canonical and noncanonical survivin isoforms translocate to the plasma membrane and are secreted in exosomes
Authors: Figel Sheila , Birkemeier Meaghan , Dharma Sanam , Steinmetz Emma , Ciesielski Michael , Fenstermaker Robert 
Published: September 2020

Abstract
Background: Previously we found that survivin is expressed on the surface of cancer cells in vitro, and is targetable using certain survivin-specific antibodies. The survivin gene is alternatively spliced into two alternative isoforms, ΔEX3 and 2B, which diverge structurally from the canonical isoform in their C-termini but retain a conserved BIR (Baculovirus IAP Repeat) domain.
Methods: In order to characterize plasma membrane survivin (PM-SVN), tagged proteins representing canonical survivin (isoform 1), Δex3 and 2B were transiently expressed in multiple cancer cell line backgrounds. Relative surface expression was measured by flow cytometry staining of intact cells. Mutant constructs were used to determine the structural requirements for translocation. Imaging flow cytometry (Imagestream) was used to visualize survivin isoform localization, and to detect co-localization on the cell surface. Secreted exosomes were examined for expression of survivin isoforms.
Results: Isoforms 1, Δex3 and 2B could be detected on the surface of HEK293T embryonic kidney, HeLa cervical adenocarcinoma, U87 glioblastoma, and A1207 glioblastoma cells. PM-SVN expression was variable, however Δex3 showed robust membrane expression across all cell lines. Deletion of a predicted transmembrane sequence in Δex3's unique C-terminus did not impair its translocation, and deletion of the first 10 amino acids, thought to be required for dimerization, disrupted surface expression only in HEK293T cells. Imaging flow cytometry analysis of cells revealed polar localization of PM-SVN, with specific punctate areas seen in 13-20% of cells. Staining with antibodies targeting both isoform 1 and Δex3 showed co-immunofluorescence in cell membrane puncta, indicating the formation of heterodimers. Stressing cells by heat shock increased the proportion of PM-SVN in 293T and A1207 cells; in 293T, this was accompanied by enrichment of survivin protein in secreted exosomes.
Conclusions: In addition to canonical survivin, isoforms Δex3 and 2B are displayed on the surface of cancer cells, and can be secreted as exosomal cargo. Membrane translocation may be stress-induced and appears to require the conserved BIR domain. PM-SVN shows a polar distribution on the cell membrane, and isoforms 1 and Δex3 heterodimerize within these puncta.
Citation Format: Sheila Figel, Meaghan Birkemeier, Sanam Dharma, Emma Steinmetz, Michael Ciesielski, Robert Fenstermaker. Canonical and noncanonical survivin isoforms translocate to the plasma membrane and are secreted in exosomes [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 2939.