tbuckley/cabot-search huggingface dataset:
Abstract 5308: RePROBE: A simple and customizable multiplex immunohistochemistry technique
Authors: Takashi Semba, Atsuko Yonemura, Huaitao Wang, Yilin Tong, Masaya Yamazaki, Takatsugu Ishimoto
Journal: Cancer Research (2025)

Abstract Background: Multiplex immunostaining techniques have been actively developed and have recently become more prevalent in cancer research. However, most of these techniques require specialized instruments and customized primary antibodies. Here, we established a simple and versatile workflow for multiplex immunohistochemistry (IHC) named RePROBE (Repetitive Primary antibody Replacement and Overlaying images from a Broad range of fluorescence imaging Equipment) that can be readily adopted in many laboratories, providing deeper insight into tumor organization and spatial molecular biology in the tumor microenvironment (TME). Methods: Formalin-fixed paraffin-embedded (FFPE) sections of various tissue types, such as resected tumors of cancer patients, patient-derived xenografts, and syngeneic mouse tumors, were subjected to the RePROBE workflow. Commercially available primary and secondary antibodies were used for fluorescence immunostaining with routine indirect IHC methods. Images were acquired by multiple imaging platforms, including fluorescence microscopes, confocal microscopes, and whole slide image scanners. Antibody complexes were removed by heat-based stripping after imaging, and the slides were subjected to the next cycle. All image processing and analysis were performed by open-source software. Results: We confirmed that the RePROBE workflow worked across different types of tissue sections and imaging modalities and achieved up to 36-plex on human gastric cancer samples and more than 20-plex on syngeneic mouse tumors. The multiplex IHC images derived from the RePROBE workflow were able to be analyzed in downstream pipelines, such as QuPath, Cellpose, and MCMICRO. Heat-based stripping successfully removed most antibodies with some exceptions. Although some loss of cells was observed (less than 7%) after cycle 1, significant cell loss was not observed after cycle 2 up to cycle 10 in most of the samples. We also found that the cell loss was dependent on tissue type and samples. Conclusions: The RePROBE workflow enabled up to 36-plex whole-slide single-cell resolution imaging and spatial phenotyping analysis, revealing the cell population and spatial organization of tumor cells, CAFs, and immune cells in TME. Citation Format: Takashi Semba, Atsuko Yonemura, Huaitao Wang, Yilin Tong, Masaya Yamazaki, Takatsugu Ishimoto. RePROBE: A simple and customizable multiplex immunohistochemistry technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5308.
Abstract 4135: AKT1 mediates PTEN-loss induced intrahepatic cholangiocarcinoma development in mice
Authors: Ielyzaveta Slarve, Yining Ding, Yushan Wang, Qi Tang, Zifei Xu, Lina He, Bangyan Stiles
Journal: Cancer Research (2025)

Introduction: Liver cancer is the third most common cause of cancer-related mortality worldwide. Mice with liver-specific Pten loss (PtenloxP/loxP; albumin [Alb]-Cre-/+; LiPten) develop predominantly intrahepatic cholangiocarcinoma (ICC) and some hepatocellular carcinoma (HCC) by 12 months of age. Pten loss results in activation of the serine/threonine kinase AKT. Of the three AKT isoforms identified to date, AKT1 isoform is implicated in regulation of cell growth, proliferation, and survival. The present study investigated the role of AKT1 in mediating PTEN-loss induced liver cancer development. Methods: To generate mice with liver-specific Pten and Akt1 deletion (PtenloxP/loxP; Akt1loxP/loxP; Alb-Cre-/+; LiPtenA1), LiPten mice were crossed with mice that have liver-specific Akt1 loss (Akt1loxP/loxP; Alb-Cre-/+; LiA1). Whole livers from 12-month-old WT, LiA1, LiPten, and LiPtenA1 mice were analyzed for tumor prevalence and morphology via hematoxylin and eosin staining, and for markers of HCC and ICC via qPCR and immunofluorescence. Results: All male LiA1 mice displayed WT-like phenotype. Like LiPten mice, all male LiPtenA1 mice develop steatosis followed by tumors by 12 months of age. Compared to LiPten mice, LiPtenA1 animals displayed predominantly HCC phenotype based on morphology, increased HCC markers (Hnf4a, Alb), and decreased ICC markers (Sox9, Spp1, Hes1, Ck19). Conclusions: AKT1 loss in LiPten mice did not prevent development of steatosis and liver tumorigenesis but resulted in predominantly HCC rather than ICC phenotype, suggesting the role of AKT1 in mediating ICC development. These results contribute to the understanding of the role of AKT isoforms in regulating liver tumorigenesis, and further investigation into signaling mechanisms underlying these observations is required. Citation Format: Ielyzaveta Slarve, Yining Ding, Yushan Wang, Qi Tang, Zifei Xu, Lina He, Bangyan Stiles. AKT1 mediates PTEN-loss induced intrahepatic cholangiocarcinoma development in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4135.
Abstract 6359: MUTE-Seq: An ultrasensitive method for detecting low-frequency mutations in cfDNA with engineered advanced-fidelity FnCas9
Authors: Sunghyeok Ye, Jin-Soo Kim, Ki-Yeon Kim, Yoon-Ho Won, Taegun Park, Sungjae An, Haerin Jeong, Hee-Joon Chung, In Seon Lee, Jae Won Rhee, Raehee Kang, Chanyoung Kang, Myoung-Hee Kang, Mi Young Kim, Jeong-An Gim, Yonggoo Kim, Song Cheol Kim, Sungho Lee, Junho K. Hur, Junseok W. Hur
Journal: Cancer Research (2025)

Abstract In this study, we present the development of the Mutation tagging by CRISPR-based Ultra-precise Targeted Elimination in Sequencing (MUTE-Seq) method. We engineered a highly precise advanced-fidelity FnCas9 variant, named FnCas9-AF2, to effectively discriminate single-base mismatches at all positions of the single guide RNA (sgRNA) target sequences. FnCas9-AF2 exhibited significantly lower off-target effects compared to existing high-fidelity CRISPR-Cas9 variants. MUTE-Seq leverages FnCas9-AF2 for the enrichment of mutant DNA through the exclusive cleavage of perfectly matched wild-type DNA, allowing for sensitive detection of low-frequency cancer-associated mutant alleles. MUTE-Seq enabled sensitive monitoring of minimal residual disease (MRD) from the bone marrow of patients with Acute Myeloid Leukemia (AML). Furthermore, MUTE-Seq was applied in a multiplexed manner on cell-free DNA (cfDNA) from patients diagnosed with non-small cell lung cancer (NSCLC) and pancreatic cancer. This approach demonstrated a significant improvement in the sensitivity of simultaneous mutant detection and highlighted its clinical utility for early-stage cancer patients with extremely low levels of circulating tumor DNA (ctDNA). We anticipate that the FnCas9-AF2-based MUTE-Seq could offer a valuable clinical tool to facilitate improved molecular diagnosis, prognosis evaluation, and treatment planning for cancers in various stages. Citation Format: Sunghyeok Ye, Jin-Soo Kim, Ki-Yeon Kim, Yoon-Ho Won, Taegun Park, Sungjae An, Haerin Jeong, Hee-Joon Chung, In Seon Lee, Jae Won Rhee, Raehee Kang, Chanyoung Kang, Myoung-Hee Kang, Mi Young Kim, Jeong-An Gim, Yonggoo Kim, Song Cheol Kim, Sungho Lee, Junho K. Hur, Junseok W. Hur. MUTE-Seq: An ultrasensitive method for detecting low-frequency mutations in cfDNA with engineered advanced-fidelity FnCas9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6359.
Abstract 2017: Eukaryotic translation initiator factor 4E in breast cancer progression and prognosis
Authors: Dat Nguyen, Angelo Laranjeira, John Yu, Stephen M. Hewitt, Sherry X. Yang
Journal: Cancer Research (2025)

Abstract The network of the eukaryotic translation initiator factor 4E (eIF4E), a mRNA cap-binding protein that is critical in the translation initiation of protein synthesis and the downstream effector of PI3K/AKT/mTOR and RTK/RAS/ERK signaling pathways, is implicated in tumorigenesis and tumor progression through regulation of cell proliferation, angiogenesis and metastasis. Targeting eIF4E with e.g. eIF4E/cap interaction antagonists, siRNA and antisense oligonucleotides is in preclinical and early clinical development and may evolve as one of the effective cancer treatments. However, there has been a dearth of data of eIF4E translational research for tumor progression clinically and prognosis. In this study, we investigated the expression of eIF4E by immunohistochemistry, and its association with clinicopathological factors and prognosis in a cohort of 710 cases of nonmetastatic breast cancer. eIF4E was expressed in 50% formalin-fixed and paraffin-embedded primary invasive breast cancer. The cap-binding protein in high-grade tumors was more prominent in the eIF4E-positive tumors than eIF4E-negative tumors (p=0.0005). Its expression was associated with larger tumor size (p=0.0001) and node-positive disease (p=0.006). In addition, there was more estrogen receptor/progesterone receptor negativity in eIF4E-positive than eIF4E-negative tumors. Notably, eIF4E demonstrated a significance towards association with both overall survival and recurrence-free survival (p=0.04 and p=0.03 by Gehan-Breslow-Wilcoxon test). The significance in the survival and recurrence outcomes was evident within 10 years of follow-up and the difference gradually diminished with long-term follow-up after 10 years. Taken together, eIF4E is linked to clinical tumor progression through its association with larger tumor size and nodal involvement as well as with high tumor grade. It has a role for prognosis in patients who were diagnosed with nonmetastatic breast cancer particularly within 10 years of clinical follow-up. Citation Format: Dat Nguyen, Angelo Laranjeira, John Yu, Stephen M. Hewitt, Sherry X. Yang. Eukaryotic translation initiator factor 4E in breast cancer progression and prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2017.
Abstract 5252: 2D and 3D spatial characterisation of tertiary lymphoid structures in head and neck cancer patients receiving immune checkpoint blockade therapy
Authors: Meg L. Donovan, Habib Sadeghirad, Naomi Berrell, James Monkman, Vahid Yaghoubi Naei, Chin Wee Tan, Joseph Yunis, Zoe West, Majid E. Warkiani, Rahul Ladwa, Brett G. Hughes, Jessica Da Gama Duarte, Arutha Kulasinghe
Journal: Cancer Research (2025)

Tertiary lymphoid structures (TLS) play an important role in the tumor microenvironment (TME) of Head and Neck Cancer (HNC) and have been linked to improved response to immunotherapy. Moreover, HNC patients with human papillomavirus (HPV)+ tumors have better survival compared to those with HPV- tumors, although both groups have better clinical outcomes when the tumors are TLS-rich. How TLS composition, characteristics, and signatures differ between response to treatment in relation to HPV status is still unclear. To investigate this, whole-tissue samples from 55 HNC patients were collected across two separate sites in Brisbane, Australia (The Princess Alexandra Hospital and The Royal Brisbane and Women’s Hospital) prior to immune checkpoint therapy (ICI). Using a multi-pathway panel, covering the ‘hallmarks of cancer’ and ‘TLS-associated proteins,’ we profiled 53 spatially resolved biomarkers using both a PhenoCycler-Fusion panel and 7-plex PSP panel (Akoya Biosciences). Using spatial proteomics, the TLS were assessed for their composition, activity, maturity, and distance metrics from the tumor. Using spatial transcriptomics, we profiled 96 TLS using whole transcriptome profiling (Bruker Spatial Biology). In addition, in patient samples abundant with TLS, we profiled 50 serial sections to obtain and profile the volumetric 3D data. Our study identified various degrees of TLS maturity, proteomic, and transcriptomic profiles associated with TLS localisation within the TME and proximity to the tumor. In addition, we identified TLS signatures associated with clinical endpoints such as response and resistance to ICI therapy. Taken together, this research highlights the multifaceted role of TLS in modulating the immunogenic landscape of HNC, influencing immunotherapy efficacy, and potentially serving as a biomarker for patient stratification and therapeutic strategies. Citation Format: Meg L. Donovan, Habib Sadeghirad, Naomi Berrell, James Monkman, Vahid Yaghoubi Naei, Chin Wee Tan, Joseph Yunis, Zoe West, Majid E. Warkiani, Rahul Ladwa, Brett G. Hughes, Jessica Da Gama Duarte, Arutha Kulasinghe. 2D and 3D spatial characterisation of tertiary lymphoid structures in head and neck cancer patients receiving immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5252.
Abstract 5253: Immune landscape analysis of solid tumors <i>via</i> spatially guided cell isolation for enhanced immunotherapeutic development
Authors: Elie Abi Khalil, Yi-Chien Wu, Jingtian Zheng, Joshua Plank, Alexander Lippert, Steve Lee
Journal: Cancer Research (2025)

Abstract The tumor-immune microenvironment (TIME) in solid tumors is a complex ecosystem consisting of diverse interactions between cellular populations. Deeper investigation into the TIME reveals a prominent role of immune cells in regulating tumor progression- a novel phenomenon that has encouraged the evaluation of the crosstalk between cell types. The development of spatial omic technologies has thus enabled the exploration of these nuances at previously inaccessible resolutions, but the currently available platforms come at the cost of introducing bias, excluding molecular information, and incompatibility with single cell technologies. Here, we leverage a photoactivatable (PA) dye Janelia-Fluor-646 in combination with a digital light processing (DLP) microscope to address these issues by developing a platform that enables the spatially guided isolation of cell populations from a region of interest (ROI) for downstream multiomic and single cell analyses. As proof of concept, we show that ROIs identified via immunofluorescence could successfully be barcoded in mouse spleen and tumor sections using the dye and distinguished from the background by fluorescence activated cell sorting (FACS). We also demonstrate the ability to extract high quality DNA, RNA, and protein- as well as maintain high cell viability post-sorting. We have also developed a pipeline for the analysis of the sequenced RNA to identify differential patterns of gene expression present in immune cells from spatially distinct regions. Moving forward, we hope to establish this as a platform for immune landscape analysis within the TIME to uncover unique targets that could be explored within the context of tumor-immune interactions as well as immunotherapeutic development. Our findings will also help us generate the first single-cell atlas of niche-specific immune cells. Citation Format: Elie Abi Khalil, Yi-Chien Wu, Jingtian Zheng, Joshua Plank, Alexander Lippert, Steve Lee. Immune landscape analysis of solid tumors via spatially guided cell isolation for enhanced immunotherapeutic development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5253.
Abstract 4725: Engineering a novel serine-degrading enzyme for targeted cancer therapy
Authors: Ladan Mashouri, Ebru Cayir, Sarita Bhetawal, Daniel J Diaz, Vipin Rawat, Jonathan Coloff, George Georgiou, Everett Stone
Journal: Cancer Research (2025)

Abstract The non-essential amino acid L-serine (serine) is the second most consumed amino acid by cancer cells, supporting their survival and proliferation by contributing to the biosynthesis of nucleotides, lipids, and proteins. Serine-auxotrophic tumors such as luminal breast cancers have compromised serine synthesis pathways, thus rely on extracellular serine, making them vulnerable to serine depletion therapies. While dietary serine restriction, the only current method to reduce serine availability for tumors in vivo, has shown limited success in reducing tumor growth, its application is hindered by poor patient compliance and an inadequate reduction of circulating serine. To overcome these limitations, we developed an engineered human serine/threonine dehydratase (eSDH) enzyme that irreversibly depletes over 95% of circulating serine and threonine without requiring dietary changes. Preclinical studies show that eSDH treatment significantly inhibits the growth of serine-auxotrophic tumors. To enhance the therapeutic potential of eSDH and reduce off-target effects, particularly the depletion of the essential amino acid threonine, we focused on improving the specificity of eSDH for serine. Through iterative structure-based engineering, we developed a new SDH variant with over 20 times higher selectivity for serine toward threonine. Both in vitro and in vivo studies have confirmed that this novel SDH variant depletes serine while preserving threonine in cell culture media and serum. Current efforts are focused on improving the stability of the new SDH variant to extend its half-life in serum and optimize dosing for greater therapeutic efficiency. This advancement allows us to target serine-auxotrophic cancers more precisely while minimizing the impact on normal cells. The broader impact of this work lies in establishing enzymatic serine depletion as a more effective and feasible alternative to dietary restriction for cancer therapy. This approach could provide a new therapeutic strategy for treating various serine-auxotrophic tumors, offering better patient compliance and fewer side effects. Citation Format: Ladan Mashouri, Ebru Cayir, Sarita Bhetawal, Daniel J Diaz, Vipin Rawat, Jonathan Coloff, George Georgiou, Everett Stone. Engineering a novel serine-degrading enzyme for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4725.
Abstract 2093: Evaluation of IDH1, EGFR, IGF1R and Ki67 biomarkers in glioblastoma using Lunaphore COMET platform
Authors: Jennifer C. Jones, Ashley Oliver, Jodi Hagen, Kristine Trueman, Grace Leland, Alexander Kalyuzhny
Journal: Cancer Research (2025)

Abstract Glioblastoma (GBM) is the most aggressive primary neuroepithelial tumor diagnosed in the United States with approximately 14, 000 diagnoses each year. Patients with GBM have a poor prognosis and only 5% of these patients survive for more than 5 years. Diagnosis of GBM requires tumor biopsies with consideration of histopathological and molecular characteristics. Typically, IHC is done using a single-biomarker HRP-DAB detection technique which is not suitable for the simultaneous spatial analysis of multiple biomarkers. Specific markers involved with the histopathology of glioblastomas include Isocitrate Dehydrogenase 1 (IDH1), Epidermal Growth Factor Receptor (EGFR), Insulin Like Growth Factor 1 Receptor (IGF-1R), and a cell proliferating marker, Ki67. A deeper understanding of the pathology of GBM has the potential to improve early diagnosis and treatment options for patients. The complex nature of the tumor microenvironment (TME) makes spatial multiplex immunohistochemistry (IHC) and immunofluorescence (IF) ideal for visualizing biomarker expression and cellular localization. Emerging automated staining and imaging platforms rely on validated antibodies for accurate marker detection. Lunaphore COMET™ is a fully-automated, high-throughput, hyperplex platform for spatial analysis of Formalin Fixed Paraffin Embedded (FFPE) and frozen tissues. It performs sequential immunofluorescence (seqIF™) assays through cycles of staining, imaging, and elution. Antibody specificity, sensitivity, and availability for multiplex IF is a critical success factor for TME characterization. We use a systematic process to qualify antibodies for use on COMET™ which includes specificity on a positive tissue sample, sensitivity using different antibody dilutions and elution efficiency using secondary antibody only incubation. In this study, we evaluated IDH1, EGFR, IGF-1R and Ki67 expression in GBM on COMET™. This will streamline multiplex panel development for spatial analysis of GBM to understand biomarker expression at the single-cell level. Citation Format: Jennifer C. Jones, Ashley Oliver, Jodi Hagen, Kristine Trueman, Grace Leland, Alexander Kalyuzhny. Evaluation of IDH1, EGFR, IGF1R and Ki67 biomarkers in glioblastoma using Lunaphore COMET platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2093.