bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 1689: Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts
Authors: Landua John , Gong Ping , Lewis Michael T. 
Published: June 2022

Abstract
Classical markers of tumor-initiating cells (TIC), aldehyde dehydrogenase 1 (ALDH1) activity and the cell-surface markers, CD44pos/CD24low/neg, show insufficient cell type specificity to identify TIC definitively, nor do they allow robust enrichment by FACS. As a consequence, analyses of TIC function and gene expression to date are not definitive. Identification and characterization of the TIC subpopulation is critically important in breast cancer since these cells are thought to mediate treatment resistance, as well as local and distant recurrence. This is particularly critical for “triple negative” breast cancer (TNBC), since currently there are no targeted therapies available for this clinical subtype. Lentiviral signaling reporter vectors allow purification of enriched TIC subpopulations thereby facilitating single cell analyses to identify distinct sets of cells with TIC features. We used a collection of fluorescent lentiviral reporters that enable visualization and flow cytometric enrichment of cells undergoing STAT3-, GLI-, WNT-, or OCT4/SOX2-mediated transcriptional activity. Our collection of patient derived xenograft (PDX) models of TNBC were infected with the lentiviral reporters, re-transplanted, and screened for reporter activity upon regrowth. TIC function of reporter-expressing cells relative to negative cells was assayed by mammosphere formation and limiting dilution transplantation. Reporter expressing cells with enriched TIC function were then analyzed by single cell RNAseq for expression of classical markers to identify the subset of cells most likely to represent the TIC subpopulation. Our results indicate that STAT3 and GLI transcriptional activity can serve as uniquely PDX-dependent indicators for TIC function for a subpopulation of cancer cells. Also, multiple potential TIC signaling pathways, including STAT3, GLI, WNT, and OCT4/SOX2, can be active in a single PDX model, but do not necessarily associate with TIC function, indicating TIC diversity in TNBC. These data in conjunction with treatment response and single-cell RNA-seq sheds light on the molecular diversity of TIC populations in TNBC, identifies candidate targets for therapeutic intervention, and will indicate whether TIC diversity is associated with differential response to different chemotherapeutic agents.
Citation Format: John Landua, Ping Gong, Michael T. Lewis. Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1689.
Abstract 1717: Single-cell RNA sequencing reveals insights into the intra-tumoral heterogeneity and tumor microenvironment of ocular adnexal sebaceous carcinoma
Authors: Gallo Ryan Alexander , Zhang Michelle G. , Tse David T. , Pelaez Daniel , Rong Andrew J. 
Published: June 2022

Abstract
Sebaceous carcinoma (SC) is a malignant neoplasm that predominantly arises from the sebaceous glands of the ocular adnexa. It is the second most common malignant eyelid neoplasm in the United States. No standardized treatment exists, but surgical excision is the mainstay of therapy. Therapeutic advances have been stagnant largely due to a limited understanding of SC tumor biology. We performed single-cell RNA sequencing to profile primary ocular adnexal SC and gain insights into its tumor microenvironment. Ocular adnexal SC has extensive intra-tumoral heterogeneity at the transcriptional level. Dimensionality reduction via principal component analysis was performed to identify major cell types. 8 clusters were detected, including clusters for cancer epithelial cells and various immune cells. Transcriptional programs were related to pathways in cancer, immune response, and antigen processing and processing, among others. We identified unique subpopulations of tumor-infiltrating immune cells, including plasma B cells, GNLY+ natural killer cells, CD8+ T cells, memory T cells, and CD20+ B cells. This complex ecosystem of tumor and immune cells provides new insights into SC biology. Additional patient samples we be analyzed in the immediate future. Follow-up mechanistic studies are needed to clarify the roles of the identified immune cells and how they can be exploited for immunotherapeutic approaches.
Citation Format: Ryan Alexander Gallo, Michelle G. Zhang, David T. Tse, Daniel Pelaez, Andrew J. Rong. Single-cell RNA sequencing reveals insights into the intra-tumoral heterogeneity and tumor microenvironment of ocular adnexal sebaceous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1717.
Abstract 1719: Investigating intra-tumor heterogeneity using multiplexed immunohistochemistry & deep learning: A new approach to spatially map the tumor microenvironment
Authors: Noureddine Kouther , Gallagher Paul , Guillaud Martial , MacAulay Calum 
Published: June 2022

Abstract
The tumor microenvironment (TME) is a highly complex mixture containing epithelium, stroma and a diverse network of immune cells & the spatial organization of these immune cells within the TME reflects a crucial process in anti-tumor immunity. At present, the usual standard of care for assessing if a patient has cancer, its stage and its likely future biological behaviour is visual examination of one or more stained sections. Although recent advances in multiple immunostaining have enabled characterization of several parameters on a single tissue section. For a higher dimensional chromogen based methodology, we have developed a multiplexed IHC (mIHC) procedure combining multiple labels per round with several sequential rounds, enabling analysis of complex immune cell populations on a single slide through consecutive cycles of staining, destaining & hyperspectral imaging. By integrating serial imaging, sequential labeling & image registration, we are able to spatially map the TME. The process presented is using absorption microscopy, enabling these images to be done in a reasonable time frame.
Robust, accurate, segmentation of cell nuclei for overlapping nuclei is one of the most significant unsolved issues in digital pathology. Using the counterstain concentration images from our mIHC methodology, we have trained a deep learning segmentation method to accurately segment individual cell nuclei within overlapping clusters of nuclei. By combining a multiplexed IHC technique which enables the detection of multiple markers on a single slide with deep learning segmentation methods to segment every individual cell nuclei in tissue sections with an accuracy comparable to human annotation. We can analyze the cell-cell interactions between immune and tumour cells, enhancing our ability to perform molecularly based single cell analysis of multiple cell types simultaneously within the tissue. These two techniques joined can be scaled up to the entire tissue section level, improving our understanding of the biological aggressiveness of specific cancers & enabling an accurate spatial cell level representation of the tissue.
Citation Format: Kouther Noureddine, Paul Gallagher, Martial Guillaud, Calum MacAulay. Investigating intra-tumor heterogeneity using multiplexed immunohistochemistry & deep learning: A new approach to spatially map the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1719.
Abstract 1740: Determining the intracellular fate of ligand-conjugated therapeutics using nanogold labeling
Authors: Sohal Ikjot S. , Abdelaal Ahmed M. , Kasinski Andrea L. 
Published: June 2022

Abstract
Recent developments in ligand-conjugated RNAi therapeutics have led to improved stability of RNAi molecules, reducing effective therapeutic dose, and avoiding delivery vehicle-associated toxicity. However, as our group has shown in previous studies with FolamiR (Folate-conjugated miR-34a), endosomal entrapment of the therapeutic molecule is a rate-limiting factor in RNAi activity. To improve endosomal release of ligand-conjugates, several approaches are being explored such as incorporation of endosomal escape agents (nigericin), chemical modification to enhance stability of miRNA/siRNA within the acidic compartments, and modification of ligand-receptor interaction to enable adequate release from the receptor upon internalization. Current fluorescent microscopy techniques are only able to approximately estimate the cellular fate of FolamiR without providing enough resolution to determine the endosomal stage, receptor disengagement and other dynamics involved in the endosomal entrapment/release of FolamiR. To overcome this, we developed Folate-conjugated nanogold construct (FolaGold) that mimics FolamiR molecule. The nanogold in FolaGold is used as a proxy for miR-34a and electron microscopy can be performed to pinpoint the cellular fate of nanogold including receptor disengagement process, the endosomal stage as well as cytosolic enrichment at the nanometer resolution. In this study, FolaGold is validated to bind to folate receptor in a competition assay and is confirmed to be present in specific endosomal compartments in MB231 cells, unlike unconjugated nanogold. We further plan to use FolaGold to determine changes in intracellular fate of nanogold upon incorporation of endosomal escape agents (nigericin) and modification of ligand-receptor interaction by using a lower affinity binding ligand that facilitates ligand-receptor disengagement following internalization. While we are using nanogold labeling to determine the intracellular fate of ligand-conjugated RNAi therapeutics, the labeling can also help improve our understanding of endosomal release dynamics and activity of other ligand-conjugate based therapeutics.
Citation Format: Ikjot S. Sohal, Ahmed M. Abdelaal, Andrea L. Kasinski. Determining the intracellular fate of ligand-conjugated therapeutics using nanogold labeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1740.
Abstract 1752: Switchable binding of TecApta to Atezolizumab provides the possibility of enhancing the therapeutic effects of this immune checkpoint inhibitor
Authors: Atefi Mohammad Sadegh , Takahashi Sola , Sich David , Grondin Jessica , Doueiri Rami , Bourgeois Lindsay , Olsem Gary , Futamura Akiko , Yang Julie , Oliva Joan , Elshimali John , Pereira Carlos 
Published: June 2022

Abstract
One of the main goals of cancer therapeutics is to develop smart molecules that can deliver drugs or immunogens to cancer cells within the tumor, while avoiding the toxicity to normal cells. Lately, immune checkpoint inhibitors have shown significant effects in the field of cancer treatment. Atezolizumab is an immune check point inhibitor specific to PD-L1. In order to potentiate anti-tumor aspects of Atezolizumab, we have developed TecApta, a switchable single strand DNA oligonucleotide aptamer that specifically binds to this antibody with a high affinity and specificity. However, TecApta is detached from Atezolizumab upon binding of this antibody to its target PD-L1. This switchable aspect of TecApta makes it a suitable drug carrier for targeting cancer cells within the tumor microenvironment. We have developed modified versions of TecApta which include either the chemotherapy drug, gemcitabine, or the immune activator, TLR9A. Based on our in vitro data, both versions are biologically active. The gemcitabine sensitive cell line MDA-MB-231/Luc exhibits similar IC50s (20nM) for both gemcitabine-linked-TecApta (GMZ-TecApta) and the free unbound gemcitabine. Moreover, the same concentrations of TLR9A-linked-TecApta (TLR9A-TecApta) and the free TLR9A show similar effects in inducing TLR9 receptors in a cell-based assay. These results suggest that possibly GMZ-TecApta and TLR9A-TecApta can be carried by Atezolizumab and released within the tumor microenvironment, where they may induce apoptosis or aggravate an immune reaction, respectively. Animal studies will be conducted in the future to investigate the possibility of such favorable outcomes.
Citation Format: Mohammad Sadegh Atefi, Sola Takahashi, David Sich, Jessica Grondin, Rami Doueiri, Lindsay Bourgeois, Gary Olsem, Akiko Futamura, Julie Yang, Joan Oliva, John Elshimali, Carlos Pereira. Switchable binding of TecApta to Atezolizumab provides the possibility of enhancing the therapeutic effects of this immune checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1752.
Abstract 176: Development of PDX-derived spheroids using LifeGel, an innovative 3D cell culture technology
Authors: Pietrzyk Agnieszka , Indersie Emilie , Deas Olivier , Judde Jean-Gabriel , Krzykawski Marcin , Cairo Stefano 
Published: June 2022

Abstract
One of the biggest challenges in preclinical oncology is the lack of models faithfully recapitulating the physiological and pathophysiological features of the native tumor. PDXs (Patient-Derived Xenografts), developed in immune-compromised mice, have proven their relevance in the study of aberrations leading to the development and progression of cancer, to the mechanisms linked to tumor resistance and to the identification of novel therapies. Despite being a fundamental step to generate new knowledge on cancer, the use of PDXs shows some technical disadvantages such as long engraftment time and/or low growth rate, thus raising economic and ethical concerns. Huge efforts are being done to develop cell-based alternatives systems reproducing the tumor complexity combined with suitable methods. To this aim, we have developed 50+ PDX-derived cell lines (PDXDCs) from our proprietary 200+ PDX collection. These lines are currently used as two-dimensional (2D) cultures to perform drug screening in an affordable, timewise, and cost-effective way. While well designed, drug-tailored 2D screenings can predict drug behavior in vivo, they present limitations as they are unable to reproduce cell-cell and cell-extracellular matrix interactions, two important parameters that contribute to the regulation of drug accessibility and oxygen diffusion in vivo. To encompass those limitations and give access to a wider range of experimental methods we have generated three-dimensional (3D) cell culture models by taking advantage of LifeGel technology, consisting of protein-based hydrogels that modulate matrix density and stiffness to mimic the organ of origin-specific extracellular environment. Thanks to its versatility we successfully developed spheroid cultures from PDXDCs from various indications, including breast cancer cells from different histological subtypes. Tumor cells plated on LifeGel adopted 3D spheroid-like structures in a few days. These structures showed heterogeneous morphology and size depending on the tumor type and the hydrogel composition. As spheroids can be passaged in vitro by digestion, we could easily expand them and perform drug screening that will be presented and compared to 2D and in vivo results. The aim of this project is to develop a collection of PDX-derived spheroids recapitulating PDXs characteristics. This collection will give the opportunity to perform high-content drugs screening to select the best candidate drugs to be retained for further preclinical in vivo studies.
Citation Format: Agnieszka Pietrzyk, Emilie Indersie, Olivier Deas, Jean-Gabriel Judde, Marcin Krzykawski, Stefano Cairo. Development of PDX-derived spheroids using LifeGel, an innovative 3D cell culture technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 176.
Abstract 1786: SM-88, D/L-alpha-metyrosine, is a novel anti-cancer agent in estrogen receptor positive breast cancer
Authors: Demas Diane M. , Collins Julie , Shajahan-Haq Ayesha N. 
Published: June 2022

Abstract
Breast cancer is the second most common cause of death from cancer in women in the United States after lung cancer. Estrogen receptor positive (ER+) breast cancer comprises 70% of all breast cancer cases. Antiestrogens and CDK4/6 inhibitors are now part of standard of care therapies for advanced and metastatic ER+ breast tumors. Unfortunately, resistance to these therapies is prevalent and metastatic breast cancer remains an incurable disease. Since drug resistant cancer cells often have deregulated metabolic pathways, we tested the efficacy of a novel dysfunctional tyrosine, SM-88 (D,L-alpha-metyrosine; racemetyrosine), alone or in combination with sub-toxic doses of conditioning agents, methoxsalen, phenytoin, and sirolimus (MPS) in resistant cell models of ER+ breast cancer. In normal cells, tyrosine, a non-essential amino acid, is made from phenylalanine and not readily taken up by cells. Therefore, it is hypothesized that SM-88 uptake will be increased in cancer cells compared to normal cells. To evaluate whether SM-88 is a potential anti-cancer agent for drug resistant ER+ tumors, we used antiestrogens and CDK4/6 inhibitors sensitive (parental MCF7 and T47D cell lines) or resistant (MCF7-R and T47D-R) cells in this study. We show that cell proliferation was inhibited in both sensitive and resistant ER+ breast cancer cells at 72 h, however, the IC50 of SM-88 as a monotherapy was higher in resistant cells compared with sensitive cells, 5mM versus 2 mM, respectively. Combination of SM-88 and MPS had an additive effect and lowered the IC50 in both resistant and sensitive cells, 2mM versus 1mM, respectively. Both LAT1 and CD98, known transporters of tyrosine, were expressed in sensitive and resistant cells. Furthermore, SM-88 as a single agent or in combination with MPS resulted in the induction of incomplete autophagy in both sensitive and resistant cells. Apoptosis and DNA damage was noted in p53-wildtype MCF7 and MCF7-R but not in p53-mutant T47D and T47D-R cells with SM-88 monotherapy or in combination with MPS. Ongoing work is focused on further defining the biochemical pathways in drug sensitive and resistant cells that will further support the current clinical use of SM-88 in advanced ER+ breast cancer (ClinicalTrials.gov Identifier: NCT04720664). Collectively, we show that SM-88 is potentially a novel anti-cancer agent in drug resistant ER+ breast cancer.
Citation Format: Diane M. Demas, Julie Collins, Ayesha N. Shajahan-Haq. SM-88, D/L-alpha-metyrosine, is a novel anti-cancer agent in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1786.
Abstract 184: High-throughput chemotherapeutic drug screening of tumor spheroids with individual spheroid results using image cytometry
Authors: Bell Jordan , Mukundan Shilpaa , Teryek Matthew , Lin Bo , Parekkadan Biju , Chan Leo 
Published: June 2022

Abstract
Three-dimensional cancer models have gained popularity for in vitro studies of chemotherapeutic compounds by providing a more physiologically relevant analog of gas, nutrient, and drug diffusion throughout the tumor microenvironment. Some 3D assays are performed to study individual spheroids over time, where a majority of these assays rely on maintaining a single spheroid in each well of a 96-well round-bottom ultra-low attachment plate, limiting the number of spheroids in a study. Other assays may gather population-level data from large ensembles of spheroids grown together, but the information about individual differences amongst the spheroids is lost. Important kinetic information may also be lost for destructive endpoint assays such as MTS or MTT. Here, we describe the development of a 3D image cytometry assay that is capable of generating kinetic data for thousands of breast cancer spheroids at the individual level. T47D spheroids are grown and maintained in a 24-well Aggrewell࣪400 plate and imaged using the Celigo image cytometer. Each well contains more than 1000 subwells that both aid in spheroid formation and constrain each spheroid to a specific location. Using the spheroid location data, we are able to track and monitor the growth of each spheroid over 7 days. Furthermore, we investigate the dose-dependent effects on spheroid viability of 6 anti-cancer drugs (Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen) using calcein AM and propidium iodide (PI). To validate the viability measurement results, we utilize the CellTiter96® MTS assay as an orthogonal method to compare the dose-dependent trends using both the calcein AM and PI fluorescence intensities as well as the spheroid sizes. This work may lay a foundation for the investigation of other spheroids, organoids, or tissue samples, significantly increasing the number of spheroids analyzed per condition, improving the statistical analysis, and adding more parameters to further analyze the spheroids. These improvements may be especially helpful for spheroids grown from patient-derived or otherwise heterogeneous cell populations
Citation Format: Jordan Bell, Shilpaa Mukundan, Matthew Teryek, Bo Lin, Biju Parekkadan, Leo Chan. High-throughput chemotherapeutic drug screening of tumor spheroids with individual spheroid results using image cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 184.