bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4319: The need for new publication standards in cancer genomics, lessons from curating COSMIC database
Authors: Holmes Alexander , Ward Sari A. , Sondka Zbyslaw
Published: April 2023

A consequence of the progress in cancer genomics is the exponential growth in data produced by research and clinical projects. Given the limited capacity within a scientific manuscript to share experimental data, various approaches have been developed: some data are simply excluded from the publication, moved to the supplementary materials, external repositories are used, and big research programmes present their integrated results on dedicated websites. This last approach has been extremely successful in generating comprehensive and unbiased datasets used by legions of scientists to discover new mechanisms driving cancer, biomarkers and drug targets. However, journal publications are still delivering more data globally, especially from rare cancers and underrepresented populations. To bring the full potential of these data and to make all this knowledge fully available, several challenges need to be addressed. COSMIC (Catalogue of Somatic Mutations in Cancer) has an in-house expert curation team that extracts information about genetic variants, their significance, patients and their response to therapy from publications and integrates it into a standardised cross-referenced database available to the scientific community. Although curators are able to extract data presented in non-standard forms (e.g. manuscript figures), interpret and translate between various data formats, they cannot extract data that are not included in the publication. Approximately one third of manuscripts that are curateable are discarded due to the absence of data generated in the research process but not included in the publication. This clearly shows an urgent need for bringing researchers, publishers, and curators together to develop new publication standards that would allow communities to fully exploit the potential of published data and make them re-usable for further discoveries. We would like to propose several suggestions to start this discussion: - Use accepted naming conventions and standards to make your data interoperable, think about your work as part of the world heritage - Include all data you generate; even if not directly relevant to the manuscript topic, they may be invaluable for somebody else’s research - Share full data and use service providers that share all the data with you - When possible, share the data per patient or sample, not per cohort - Publishing a manuscript is not an endpoint, there’s a huge potential in your data to be further utilised to fight cancer

Citation Format: Alexander Holmes, Sari A. Ward, Zbyslaw Sondka. The need for new publication standards in cancer genomics, lessons from curating COSMIC database. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4319.
Abstract 4320: A novel digitally directed tissue microdissection platform integrates digital pathology with molecular analysis: Case study in metastatic colorectal cancer
Authors: Butler John H. , Chaudhuri Bidhan , Konigsfeld Katie , Pai Rish
Published: April 2023

Artificial intelligence (AI) based digital pathology has been getting rapid adoption over the last 2 to 3 years

These AI based digital pathology models can analyze samples at high resolution and scale

Retaining that resolution at scale through molecular analysis is key to the discovery of novel biomarkers

We have developed a technology that can integrate AI-supported digital pathology (DP) algorithms to identify informative tissue regions for selective microdissection to improve molecular analysis of FFPE material

The system is capable of extracting DNA, RNA or proteins with cellular (10 micron) resolution from FFPE tissue sections. The samples are ready for use in off-the-shelf molecular analysis workflows (qPCR, Next Gen Sequencing, etc)

The system decreases the tumor cellularity requirements for clinical analysis by extracting only the area enriched for informative biomarkers

To demonstrate the capability of the system we used an AI based digital pathology model that was developed to identify multiple cellular phenotypes from a H&E stained colorectal cancer FFPE tissue sections

We used the digital pathology output to guide the microdissection of RNA from tumor buds, carcinoma, immature stroma and inflammatory stroma in a paired designed RNA seq experiment

The data showed that the system is capable of enriching the tumor-bud tissue content from<1% in the tissue to ~50% purity in the crude lysate

Bioinformatic analysis of the RNAseq signatures showed that the tumor buds had a distinct RNAseq signature relative to the other three cell types and that the gene expression pattern was consistent with EMT pathways. The methodology is scalable, robust, and simpler than current microdissection techniques. This technology can be used to develop novel tests by integrating with AI algorithms, targeting tissue enriched for predictive biomarkers, and enabling low tumor cellularity samples to be analyzed with high precision.

Citation Format: John H. Butler, Bidhan Chaudhuri, Katie Konigsfeld, Rish Pai. A novel digitally directed tissue microdissection platform integrates digital pathology with molecular analysis: Case study in metastatic colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4320.
Abstract 4416: IL-1 blockade prevents cardiac toxicity and improves immunotherapy efficacy
Authors: Talele Nilesh P.
Published: April 2023

Background: Immune checkpoint blockers (ICBs) have revolutionized cancer treatment, but they are often associated with severe immune related adverse events (irAEs). These severe irAEs are more often seen in patients with obesity or concomitantly treated with cytotoxic therapies.

Methods: We aimed to understand the mechanisms of ICB-induced irAEs, in the context of obesity and ICB/chemotherapy combinations. We developed a mouse model of cardiac irAEs, which is the most fatal type of irAE in ICB-treated cancer patients, with clinically relevant features: (i) an ICB-resistant cancer (pancreatic ductal adenocarcinoma or PDAC), (ii) obesity induced with high-fat diets, and (iii) a combination treatment of ICB (α-PD1 + α-CTLA4) and chemotherapy (FOLFIRINOX).

Results: Our FDA and single institution retrospective analyses indicate that patients treated with ICB had greater relative risk of developing myocarditis as compared to other anti-cancer therapies. Mice with orthotopic PDAC and obesity developed irAEs after treatment with ICB and chemotherapy as compared to chow diet. These irAEs recapitulated those observed in patients with cancer and obesity, including cardiac dysfunction consistent with myocarditis, cardiac fibrosis, and increased circulating levels of interleukin-1 beta (IL-1b). IL-1β blockade prevented myocarditis and reduced cardiac fibrosis after immunotherapy. Importantly, IL-1β blockade also enhanced the anti-tumor effects of ICB + FOLFIRINOX combination therapy, and increased mouse survival.

Conclusions: We developed a translationally relevant 'triple hit' mouse model and discovered that IL-1β mediates ICB-induced cardiotoxicity. In addition, we found that IL-1β blockers, which are already used in the clinic for cardiology indications, both reduce adverse events and simultaneously enhance the antitumor effects triggered by immunotherapy.

Citation Format: Nilesh P. Talele. IL-1 blockade prevents cardiac toxicity and improves immunotherapy efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4416.
Abstract 4472: A mouse model for GvHD: an excellent tool for evaluating the efficacy of anti-GvHD drugs and testing mechanisms of immunoregulation
Authors: Wu Meirong , Zhang Shiru , Zhu Fang , Sun Hongyan , Xu Jianming , Ju Cunxiang , Wang Hongyu , Chen Santi Suryani , Li Zhiying , Moore Mark Wade , Zhao Jing , Gao Xiang
Published: April 2023

Hematological malignancies, including leukemia, lymphoma and multiple myeloma, account for about 7% of all newly diagnosed cancers. Allogeneic hematopoietic stem cell transplantation (HSCT) is a traditional treatment for hematologic malignancies. Currently, CAR-T cell therapy has made a major breakthrough in the treatment of hematologic malignancies. However, the occurrence of acute Graft-versus-Host Disease (GvHD) represents a major obstacle for HSCT and limits the application of CAR-T cell therapy.

GemPharmatech established and characterized two murine models for anti-GvHD drug evaluation. In the first model, bone marrow from C57BL/6 donor mice was transplanted into a sublethally-irradiated BALB/c recipient mouse. In this model, T cell infiltration, tissue damage, and systemic inflammation were observed, similar to the clinical observations of patients receiving allogeneic HSCT. Efficacy evaluation of anti-GVHD drugs have been carried out based on this model. Our results showed that treatment with the anti-GVHD drug Ibrutinib, the survival rate and the lifespan of the mice was significantly prolonged, and the GvHD score was reduced by about 70% compared with the placebo group.

In the second model, human Peripheral Blood Mononuclear cells (PBMCs) were transplanted into irradiated severe immunodeficient NCG mice. The cohort was irradiated to accelerate GvHD occurrence. The reconstructed human T cells will recognize and attack mouse tissues to cause GvHD. Our results showed that hCD45+ cells infiltrated into heart, liver, and spleen of huPBMC-NCG mice. This model has also been successfully used for anti-GvHD efficacy evaluation. Upon treatment with an anti-GVHD drug, the lifespan of huPBMC-NCG mice was significantly extended and weight loss was alleviated.

Overall, the above-mentioned GvHD models show great potential for preclinical evaluation of anti-GvHD drug development in allogeneic HSCT.

Citation Format: Meirong Wu, Shiru Zhang, Fang Zhu, Hongyan Sun, Jianming Xu, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. A mouse model for GvHD: an excellent tool for evaluating the efficacy of anti-GvHD drugs and testing mechanisms of immunoregulation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4472.
Abstract 448: Computational discovery and experimental validation of splicing-derived neoantigens
Authors: Zumrut Hasan Ekrem , Anderson Kendall , Manzanares Miguel , Smith Shaleigh , Gera Sakshi , Habib Nayab , Shah Sanjana , Trahan Devon , Akerman Martin , Arun Gayatri
Published: April 2023

RNA splicing is a central step in the regulation of gene expression, leading to the accurate assembly of viable mRNA isoforms. Errors in the splicing process are common in cancer and lead to the production of tumor-specific mRNAs that often encode for neoantigen peptides. Such neoantigens cannot be discovered using conventional DNA-based tools; they require specialized software for alternative splicing analysis using RNA-seq data. Envisagenics has developed SpliceIOTM, a proprietary software platform for splicing-derived neoantigen discovery. SpliceIO uses RNA-seq data to uncover extracellular neoantigens encoded by transmembrane proteins or presented to immune cells through the MHC complex. Using SpliceIO, we have analyzed roughly 8,000 RNA-seq samples from 4 different cancers and 14 normal tissue types. Each dataset first undergoes an unsupervised clustering analysis using SpliceSliceTM to stratify patients into subpopulations based on similar aberrations in splicing machinery. We then leverage SpliceIO to identify splicing-derived, tumor-specific neoantigens enriched in these cohorts. SpliceIO incorporates features, such as protein localization, MHC binding affinity, epitope accessibility and cancer relevance of the gene to help select tumor specific targets with the highest therapeutic potential. Here, we summarize the discovery and validation of novel splicing-derived neoantigens identified using SpliceIO. To validate neoantigen candidates, we performed isoform expression quantification at the RNA and protein levels using PCR, RNA-FISH, western blot and mass spectrometry. Our experiments confirmed several neoantigen candidates encoded by tumor-specific RNA isoforms, that are translated and localized to the cellular membrane. Interestingly, one of the candidates is an endoplasmic reticulum protein that exhibits cassette exon skipping in approximately 24% of TNBC tumors. Fluorescence microscopy experiments demonstrate that the TNBC-specific isoform of the protein is localized to plasma membrane but not the ER, suggesting that alternative splicing can alter protein localization. Together, our results demonstrate the potential of RNA splicing analysis for the discovery of novel immunotherapeutic targets.

Citation Format: Hasan Ekrem Zumrut, Kendall Anderson, Miguel Manzanares, Shaleigh Smith, Sakshi Gera, Nayab Habib, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Computational discovery and experimental validation of splicing-derived neoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 448.
Abstract 4531: Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study
Authors: Luo Zhen , Wang Xiao , Zeng Xuehuo , Yi Jiaqing , Burns Tegan , Liu Herman , Zhang Xinyu , Liu Wenyu , Hsu Karl , Gong Hua
Published: April 2023

ZL-1211, a humanized monoclonal IgG1 antibody targeting CLDN18.2, induces cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We developed ex vivo ADCC and CDC assays to study the mechanism of actions of ZL-1211. Our preclinical data has demonstrated greater antitumor activities than the clinical leading benchmark via enhanced ADCC by mutation in Fcγ domain and potent CDC activities. We used those ex vivo assays to further profile the clinical specimens from patients collected at baseline (pre-infusion) and the following cycles (post-infusion) in our phase 1 study (NCT05065710). Preliminary results in clinical samples indicated that baseline ADCC and CDC capabilities were correlated with anti-tumor activity of ZL-1211 in patients with CLDN18.2-positive locally advanced or metastatic solid tumors, suggesting probable predictive biomarkers. Results also showed that changes in circulating ZL-1211 mediated ADCC and CDC activities were dose-dependent and in accordance with the pharmacokinetic (PK) profile. In addition, we performed differential gene expression analysis and real-time PCR to compare gene expression in ZL-1211 responder and non-responder PDX models and CCLE cell lines. Our findings reveal that several immune-related genes may serve to predict the responses to ZL-1211 treatment. Other potential predictive biomarkers such as Fcγ receptor CD16A polymorphism and baseline stromal CD8 expression were also evaluated in clinical samples. Taken together, analytical approaches developed and validated from preclinical studies were successfully translated to characterize clinical samples in the current Phase 1 trial. The exploratory analysis indicated the clinical relevance of multiple biomarkers and their potential as predictive biomarkers of ZL-1211.

Citation Format: Zhen Luo, Xiao Wang, Xuehuo Zeng, Jiaqing Yi, Tegan Burns, Herman Liu, Xinyu Zhang, Wenyu Liu, Karl Hsu, Hua Gong. Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4531.
Abstract 4632: Spatial whole transcriptome analysis of differential expression for biomarker discovery in colorectal cancer
Authors: Cheung Elim , Sundaram Vidyodhaya , Zhang Baowen , Zhang Lutong , Lu Tong
Published: April 2023

Background: Colorectal cancer (CRC) is one of the leading causes of cancer death, which is frequently caused by genetic or epigenetic changes. The study of tumor microenvironment (TME) profiling may provide a better knowledge of spatial gene expression patterns of known CRC markers, visualize the cell composition and localization, and serve to aid the understanding of the mechanisms of tumor formation. BioChain Institute, Inc. is currently investigating the TME profiling of CRC through tissue-wide, whole transcriptome analysis for the ability to discover potential therapeutic targets.

Methods: BioChain’s formalin-fixed paraffin-embedded (FFPE) tissue samples from 3 primary colon adenocarcinoma tumors (PT), and their matched adjacent normal tissues (PN), were used to investigate the TME. The images of Hematoxylin and Eosin (H&E)-stained tissue sections were annotated by our pathologist. The spatial whole transcriptome was analyzed using 10x Genomics Visium Spatial Gene Expression for FFPE. This assay provided about 5,000 spatially barcoded spots with mRNA-binding oligonucleotides to capture the gene expression. The integration of the imaging and sequencing data enabled a spatially resolved transcriptome of CRC heterogeneity.

Results: The annotations of both PT and PN tissue samples were found to be correlated with the spatial gene expression clustering. With the overlaying of total gene counts for each spot on the H&E-stained tissue image, about 16,000-17,000 genes were detected in each sample. The median of ~5,500 and ~1,600 genes per spot were expressed in PT and PN samples respectively. The study of gene expression levels revealed the overexpression of several well-known CRC markers (APOE, SCD, IGFBP3, TIMP1, SPARC) and the down-regulation of numerous markers (DES, CA1, KLF4) in PT samples significantly.

Conclusion: Our spatial analysis results provide insight into tumor heterogeneity, spatial organization of cells within the TME and biomarker identification. BioChain is in a unique position to facilitate researchers with a plethora of oncologic and other diseased tissue types, as well as the spatial whole transcriptome analysis. Our study enables the understanding of the TME, discovery of potential diagnostic and prognostic targets, especially for the development of personalized therapy.

Citation Format: Elim Cheung, Vidyodhaya Sundaram, Baowen Zhang, Lutong Zhang, Tong Lu. Spatial whole transcriptome analysis of differential expression for biomarker discovery in colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4632.
Abstract 4640: CAF contributes to cancer progression through its immunomodulatory role within the tumor microenvironment
Authors: Núñez Muriel Antonieta , Corrales Oreste , Velasquez Victoria , Ernst Daniel , Viviana Montecinos , Nualart Francisco , Yuseff María Isabel , Cerda-Infante Javier
Published: April 2023

Introduction: Cancer progression and metastatic spread is modulated by the tumor microenvironment. Cancer-associated fibroblasts (CAF) are the most abundant cell type within the tumor microenvironment. Little is known about the specific mechanisms by which CAF would exert its cancer progression, through the high secretory capacity of soluble pro-tumorigenic molecules and remodeling of the extracellular matrix. Recent studies in our laboratory have shown for the first time that CAF derived from patients without metastasis express a different genetic profile than those with metastases (mCAF). This study seeks to evaluate the contribution of mCAF to tumor progression through its immunomodulatory role.

Methodology: mCAFs were obtained from patients with metastatic disease and BAF from benign patients. The fibroblasts were functionally characterized by the generation of fibroblast-derived matrices (FDM) and secretory profiles by cytokine array. CD8 T lymphocytes were obtained from healthy patients and treated with fibroblast-secretome. Markers were studied by flow cytometry: activation (CD25 and CD69) and repression (LAG3 and PD1). The migration of CD8 T lymphocytes was studied by transwell and agarose drop migration assay.

Results and conclusion: FDM of mCAF are different in their composition and organization. Furthermore, the secretome of each type of fibroblast presents a differential effect on the activation of CD8 T cells according to the markers highlighted. Regarding migration, it has been observed that the mCAF-secretome can attract lymphocytes. However, it would lead them to apoptosis. Our results suggest that mCAF, within the tumor microenvironment, has an immunomodulatory role on CD8 T lymphocytes.

Acknowledgment: Beca Doctorado Nacional ANID 21181427

Citation Format: Muriel Antonieta Núñez, Oreste Corrales, Victoria Velasquez, Daniel Ernst, Montecinos Viviana, Francisco Nualart, María Isabel Yuseff, Javier Cerda-Infante. CAF contributes to cancer progression through its immunomodulatory role within the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4640.
Abstract 4688: Comprehensive analysis of natural killer cell-associated markers using MultiOmyxTM immunofluorescence assay
Authors: Parnell Erinn A. , Fei Jiong , Nunns Harry , Leones Eric , Yeung Elaine , Russell Blair , Sahafi Flora , Au Qingyan
Published: April 2023

As a promising alternative platform for cellular immunotherapy, natural killer cells (NK) have recently gained attention as an important type of innate immune regulatory cell. NK cell immunotherapy approaches have been translated into clinical applications, and clinical trials of NK cell infusion in patients with hematological malignancies (HM) and solid tumors have thus far yielded many encouraging clinical results. Understanding the pattern of NK expression and the relationship to different states of NK cells may have direct relevance for immune responses in cancer. Approaches capable of simultaneously detecting NK cells with detailed information on NK differentiation state, however, remain limited. In this study, we used MultiOmyx hyperplexed immunofluorescence (IF) assay to classify and characterize the spatial arrangement of NK cell markers in a pan-cancer cohort including 9 tissue microarrays (TMAs) from breast, head and neck, prostrate, colon, bladder, lung, kidney, ovarian and melanoma indications. The panel includes CD3, CD4, CD8, CD16, CD45, CD56, CD57, CD137, FoxP3, Granzyme B, HLA-E, NKG2A, NKP46 and tumor segmentation marker. The panel enables the detection of NK cells expressing CD56 and/or NKP46 in the 9 different tumor indications. We also studied the expression of activating and inhibiting receptors, such as CD16 and NKG2A, in NK cell population. The active cytotoxic subsets of mature NK cells were evaluated using co-expression of NK cell surface markers with CD57 and the cytotoxic molecule expression in NK cells was assessed using co-expression with Granzyme B. Using proprietary deep-learning-based image analysis, we were able to quantify the densities of these different NK cell population and study the prevalence of these NK cells in different cancer indications included in this study. Many strategies have been developed for exploiting NK-mediated anti-tumor activities. CAR-NK cell therapy and antibodies that directly target NK cell inhibitory receptors such as NKG2A and TIGIT, are currently being evaluated in the clinical trials. The MultiOmyx NK cell panel reported in this study enables the comprehensive profiling of the NK population and can provide greater understanding of NK cell biology during cancer progression. The panel can be further used to explore the efficacy of the NK cell-based immune therapy.

Citation Format: Erinn A. Parnell, Jiong Fei, Harry Nunns, Eric Leones, Elaine Yeung, Blair Russell, Flora Sahafi, Qingyan Au. Comprehensive analysis of natural killer cell-associated markers using MultiOmyxTM immunofluorescence assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4688.
Abstract 4691: Spatially resolved transcriptomics deconvolutes histological prognostic subgroups in patients with colorectal cancer and synchronous liver metastases
Authors: Wood Colin S. , Pennel Kathryn A. , Leslie Holly , Legrini Assya , Cameron Andrew J. , Horgan Paul , Edwards Joanne , Steele Colin W. , Jamieson Nigel B.
Published: April 2023

Patients demonstrating strong immune responses to primary colorectal cancer (CRC) have a survival benefit following surgery, while those with predominantly stromal microenvironments do poorly. Biomarkers to identify patients with colorectal cancer liver metastases (CRLM) who have good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary CRC and CRLM, and to interrogate the underlying functional biology that drives disease progression. Patients undergoing synchronous resection of primary CRC and CRLM underwent detailed histological assessment, panel genomic and bulk transcriptomic assessment, immunohistochemistry (IHC) and GeoMx Spatial Transcriptomics (ST) analysis. Integration with genomic features, pathway enrichment analysis and immune deconvolution were performed. High-immune metastases were associated with improved cancer specific survival (HR, 0.36, P=0.01). Bulk transcriptomic analysis was confounded by stromal content but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for Type II Interferon signaling (NES=-2.05 P.Adj<0.005) and MHC-Class II Antigen Presentation (NES=-2.09 P.Adj<0.005). In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T-cells and neutrophils with enrichment of Notch (NES=2.2 P.Adj=0.022) and TGF-β (NES=2.2 P.Adj=0.02) signaling pathways at the metastatic tumor center. In summary, histological assessment can stratify outcome in patients undergoing synchronous resection of CRLM and ST analysis has revealed significant intra-tumoral and inter-lesional heterogeneity with underlying transcriptomic programs identified in driving each phenotype.

Citation Format: Colin S. Wood, Kathryn A. Pennel, Holly Leslie, Assya Legrini, Andrew J. Cameron, Paul Horgan, Joanne Edwards, Colin W. Steele, Nigel B. Jamieson. Spatially resolved transcriptomics deconvolutes histological prognostic subgroups in patients with colorectal cancer and synchronous liver metastases. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4691.
Abstract 4698: Reproducibility in spatial biology: reducing variables to improve the reliability of insight generation
Authors: Arvidson Lisa , Prioli Reginaldo , Jensen Samuel , Hoying James B. , Golway Michael W. , Smith Michael J. , White Katie O. , Heil-Chapdelaine Richard A. , Huang Chi-Chou , Phan Tuan H. , Sasaki Hideki , Angus-Hill Melinda
Published: April 2023

There is an ongoing crisis in reproducibility in scientific studies, and studies in spatial biology are no exception. Cell DIVETM Multiplex imaging solution (Leica Microsystems) provides reliable workflow solutions to minimize variability from study to study. Cell DIVE allows probing and imaging of dozens of biomarkers on a whole tissue section with an iterative staining and dye inactivation workflow. At its core, Cell DIVE is designed to provide methods reproducibility, from tissue preparation, antigen retrieval, and sample imaging and slide storage. Cell DIVE is designed to work with directly conjugated primary antibodies, another source of variability. However, conjugated antibodies from Cell Signaling Technology (CST) are rigorously validated using stringent acceptance criteria to reduce variability. In addition, the use of recombinant antibodies, the consistent conjugate brightness and antibody degree-of-labeling reduce lot to lot variability, ensuring reliable conjugated antibodies for spatial biology studies. High resolution imaging results are obtained by consistent round to round imaging, consistent calibration and corrections, and reduction of human error using the BAB 200 liquid handling solution (Advanced Solutions Life Sciences). We present here, an iterative biomarker study using adjacent tissue sections probed with distinct lots of CST antibody panels, imaged in temporally separated batches using a Cell DIVE imager fitted with a BAB200 liquid handler. Following robust analysis (segmentation, phenotyping and statistical analysis), we report the reproducibility findings across parameters. Methods and results reproducibility in spatial biology is essential for reliable insight generation, giving confidence in the quality of future studies aimed at improving patient outcomes.

Citation Format: Lisa Arvidson, Reginaldo Prioli, Samuel Jensen, James B. Hoying, Michael W. Golway, Michael J. Smith, Katie O. White, Richard A. Heil-Chapdelaine, Chi-Chou Huang, Tuan H. Phan, Hideki Sasaki, Melinda Angus-Hill. Reproducibility in spatial biology: reducing variables to improve the reliability of insight generation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4698.
Abstract 4699: Pan-cancer single-cell analysis reveals the interactions of SPP1+MMP12+ macrophages with metastasis-initiating cells that drives metastasis
Authors: Zhang Baifeng , Lai-wan Kwong Dora , Guan Xin-Yuan
Published: April 2023

Advanced understanding of cancer immunology has contributed to a significant decline in cancer deaths in the past few decades. However, there are still vast majority of patients with no effective response for current immunotherapy drugs because of some innate immune cells, particularly macrophages and neutrophils, promoting all steps of malignant progression to metastasis. Therefore, the development of new strategies against these pro-metastatic non-tumor components in the tumor microenvironment is in need. Tumor-associated macrophage (TAMs), as the critical component of tumor microenvironment (TME), display diverse and even opposing function phenotypes, but without systematic investigation on their ubiquitous characteristics across different cancer types. Here, we perform pan-cancer analysis on 92 samples across 6 solid cancer types to profile the TME of normal, primary tumor, metastatic lymph node and distant metastasis at single-cell resolution, illustrating the commonalities/plasticity of heterogenous TAMs. We uncover 21 distinct subtypes of TAMs with varying abundance and biological function in different tissue microenvironment, providing evidence for the plasticity of TAMs. In particular, the enrichment of SPP1+MMP12+ macrophages in the TME of lymph node metastasis, likely interacting with metastasis-initiating cells (MICs), is associated with promoting malignant progression to metastasis and survival stratifications. Multi-color immunofluorescence (mIF) staining for relevant markers is being performed in mouse models and clinical samples for comprehensive validation. Our study comprehensively describes the characteristics and dynamics of TAMs and highlights their heterogeneity and plasticity depending on their surrounding microenvironment.

Citation Format: Baifeng Zhang, Dora Lai-wan Kwong, Xin-Yuan Guan. Pan-cancer single-cell analysis reveals the interactions of SPP1+MMP12+ macrophages with metastasis-initiating cells that drives metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4699.
Abstract 4710: Novel 3D cytotoxicity assay to assess the impact of chimeric antigen receptor (CAR) domain design on the tumor infiltration and cytotoxicity efficacy of CAR T-cell therapies for solid tumors
Authors: Johnston Adrian , Wan Zeqi , Chen Tina , Lim Yeongseo , Lee Cameron , Du Wenxuan , Philip Jude , Wirtz Denis
Published: April 2023

CAR T-cell therapy is a popular topic of discussion in and out of the scientific community. Already with multiple therapies FDA-approved for certain blood cancers, focus has rightly shifted toward FDA approval for solid tumor indications. Yet, advancing past clinical trials to FDA approval has yet to occur and has proved challenging. Of course, multiple factors are at play as to why, but we aim to use-in house expertise to elucidate one, namely CAR T-cell tumor infiltration and tissue migration and surveillance efficacy. Migration is a central parameter ignored in traditional 2D cytotoxicity assays, but there is no tumor cell killing by T cells if there is no T cell migration. We developed a novel first-of-its-kind organoid-based 3D cytotoxicity assay using multicompartment organoids and the most abundant extracellular matrix protein in human organs, collagen. We suspend solid tumor organoids in the center of CAR T-cell/collagen gel mixtures that are solidified then imaged in intervals in the span of two weeks by live-cell time-lapse reflection/fluorescence confocal microscopy, after which standard luciferase-based readouts of quantitative specific lysis is determined. Our assay allows high-resolution tracking of CAR T-cell 3D movements through collagen fibers surrounding solid tumor organoids to determine their random and directional migration as well as their surveillance and infiltration of solid tumor organoids. We will describe the differences between currently in clinical trial mesothelin-specific CAR T-cell therapies that differ in their scFv, hinge, transmembrane, and costimulatory domains; and comparing alongside the traditional 2D luciferase-based cytotoxicity assay, we will underline how 3D migration and infiltration around and into mesothelin-expressing ovarian cancer cell organoids allow the determination of CAR T-cell therapy efficacy of different CAR T clones in a lab’s arsenal for a given solid tumor through migration, infiltration, and subsequent cytotoxic capacity that is absent in traditional 2D cytotoxicity assays. CAR T-cell therapy vector design and cloning can thus be tuned for optimal migration and infiltration using our novel 3D cytotoxicity assay.

Citation Format: Adrian Johnston, Zeqi Wan, Tina Chen, Yeongseo Lim, Cameron Lee, Wenxuan Du, Jude Philip, Denis Wirtz. Novel 3D cytotoxicity assay to assess the impact of chimeric antigen receptor (CAR) domain design on the tumor infiltration and cytotoxicity efficacy of CAR T-cell therapies for solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4710.
Abstract 4734: BRD8-driven EP400 complex hijacks H2AZ to maintain proliferation in glioblastoma
Authors: Sun Sherine Xueqin , Klingbeil Olaf , Vakoc Christopher , Mills Alea
Published: April 2023

Glioblastoma (GBM) is the most notorious primary brain tumor. The median survival of GBM patients is only about one year and about 95% patients succumb after five years. This gloomy picture has not been improved for decades, even with extensive treatments including surgery, radiotherapy, and chemotherapy. Hence, a better understanding of the mechanisms underlying GBM development may provide new therapeutic opportunities. Primary GBM generally harbors a low mutational load when compared to other human cancers. Even P53—the guardian of the genome that is essentially disabled in nearly all human cancers, remains unmutated in about 71% of GBM cases. Moreover, these P53 wildtype GBM cases are as aggressive as those with P53 mutations. To explore the mechanism by which P53 wildtype GBM thrive in the presence of intact P53, we performed CRISPR screens in a panel of human cancer cell lines using an sgRNA library specifically targeting the functional domains of ~200 chromatin regulators. We discovered that BRD8’s bromodomain—a druggable domain as shown by the tremendous success in preclinical and clinical trial in diverse cancers—is a vulnerability specifically in P53 wildtype GBM cases. Our mechanistic studies demonstrate that BRD8 functions through the EP400 chromatin remodeling complex and hijacks the histone variant H2AZ at P53 target loci, enforcing a compact chromatin state that blocks P53’s accessibility to its targets. We show that targeting the BRD8 bromodomain releases H2AZ, opens up chromatin, engages P53-mediated transactivation, and triggers growth arrest. Consistent with these findings, BRD8 is highly expressed with H2AZ in patient-derived proliferating GBM cells, and is inversely correlated with the expression of P53 targets. Our work solves a long-standing mystery in P53 wildtype GBM, and presents a promising therapeutic target for the majority of GBM patients.

Citation Format: Sherine Xueqin Sun, Olaf Klingbeil, Christopher Vakoc, Alea Mills. BRD8-driven EP400 complex hijacks H2AZ to maintain proliferation in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4734.
Abstract 477: Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers
Authors: Xiao Fei , Wang Kailiang , Wang Xinjuan , Li Huijuan , Hu Zhilong , Huang Wei , Ren Xiaoming , Feng Teng , Yao Lili , Lin Jing , Li Chunlai , Mei Liufeng , Zhang Zhuanzhuan , Liu Yangyang , Chen Xi , Zhu Xiaotian , Zhong Wenge , Xie Zhi
Published: April 2023

Background: KRAS is the most frequently mutated oncogene with high prevalence in non-small cell lung cancers (NSCLC), colorectal cancers (CRC), and pancreatic cancers (PAC). FDA currently approved sotorasib provides breakthrough therapy for cancer patients with KRASG12C mutation, however there is still high unmet medical need for new agents that target a broader KRAS mutated tumors. A new opportunity emerges to develop a pan KRAS inhibitor by suppressing the upstream guanine nucleotide exchange factor (GEF) protein son of sevenless 1 (SOS1). SOS1 is a key activator of KRAS and facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state. Binding to its catalytic domain, small molecule SOS1 inhibitor prevents KRAS activation and suppresses cancer cell proliferation.

Material and Methods: Regor’s unique Computer Accelerated Rational Design (CARD) technology platform was applied to identify potent and selective SOS1 inhibitors. Biochemical assays and cellular assays were utilized to drive the structure-activity relationship (SAR). In vitro and in vivo target engagement were confirmed. In vivo efficacy study data were generated using lung and pancreatic cancer xenograft mouse models with KRAS mutation.

Results: In vitro, RGT-018 blocked the interaction of KRAS::SOS1 with high selectivity, and inhibited proliferation of a broad spectrum of mutant KRAS-driven cancer cells as a single agent. Robust anti-proliferation activity was observed when RGT-018 was combined with MEK, KRASG12C, EGFR or CDK2/4/6 inhibitors in vitro. Oral administration of RGT-018 inhibited tumor growth and suppressed KRAS signaling pathway activation in tumor xenografts. Furthermore, combination with MEK or KRASG12C inhibitors led to profound tumor regression.

Conclusions: The pharmacological properties of RGT-018 represent an attractive drug candidate with oral bioavailability for combination with targeted agents to treat a broader patient population driven by mutant KRAS.

Citation Format: Fei Xiao, Kailiang Wang, Xinjuan Wang, Huijuan Li, Zhilong Hu, Wei Huang, Xiaoming Ren, Teng Feng, Lili Yao, Jing Lin, Chunlai Li, Liufeng Mei, Zhuanzhuan Zhang, Yangyang Liu, Xi Chen, Xiaotian Zhu, Wenge Zhong, Zhi Xie. Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 477.
Abstract 4781: New generation of fluorescent probes for cell-based measurements of caspase activation and mitochondrial superoxide
Authors: Mandavilli Bhaskar S. , Beacham Daniel , Hu Yi Zhen , Yang Jongtae , Chen Aimei
Published: April 2023

Cell health and stress readouts are critical indicators of altered or impaired function in normal and diseased states of cells, and work has been underway to develop improved small molecule sensor dyes compatible with traditional imaging and High Content Analysis (HCA) interrogation of apoptotic and mitochondrial stress pathways.

The CellEvent™ Caspase Green dye effectively reports caspase activation, but suffers complications in assay configuration when attempting to multiplex with the Green Fluorescent Protein (GFP), calcein, or other 488 laser line tools in fluorescence microscopy. Here, we describe the testing and functional characterization of a new candidate molecule for measuring apoptosis in living cells. Our sensor is comprised of a fluorogenic reporter dye that is liberated from a DEVD peptide substrate by caspase activation, but operates in the Texas Red, 590nm excitation band, with an emission peak near 610 nm, permitting easy multiplex with GFP or calcein stained neurons in both traditional and HCA microscopy configurations. Similarly, mitochondrial superoxide accompanying cell stress is probed in microscopy with the MitoSOX™ Red Mitochondrial Superoxide Indicator dye, which localizes to mitochondria and reports superoxide generation, ignoring other Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). This dye has an unusually long Stokes’ shift, requiring specialized microscopy and HCA filters that excite at 405nm, and capture emission at 610nm for specific superoxide detection. This unconventional spectroscopic profile prevents the dye’s use on many imaging platforms and promotes phototoxicity. To this end, our team has produced a dye with the same level of specificity for superoxide that will operate in one of the traditional fluorescence microscopy channels. Our candidate dye, here named MitoSOX™ Green Mitochondrial Superoxide Indicator also localizes to mitochondria of live cells and selectively reports superoxide generation, while ignoring other ROS and RNS species in ex vivo testing. With an Excitation/Emission profile in the GFP/FITC microscopy channel, a series of comparative studies in immortalized and neural cells are shown, highlighting photostability, specificity and signal amplitude from the dye. These reagents are research use only, not for diagnostic purposes

Citation Format: Bhaskar S. Mandavilli, Daniel Beacham, Yi Zhen Hu, Jongtae Yang, Aimei Chen. New generation of fluorescent probes for cell-based measurements of caspase activation and mitochondrial superoxide. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4781.
Abstract 4786: Imipridones induce lipid peroxidation and oxidative stress in gastrointestinal malignancies
Authors: Cruz Arielle De La , Srinivasan Praveen R. , George Andrew , Prabhu Varun V. , Pinho-Schwermann Maximilian P. , El-Deiry Wafik S.
Published: April 2023

Colorectal and pancreatic cancer collectively comprise 20% of all cancer-related deaths in the United States. Despite therapeutic advancements, the current standard of care often results in significant toxicity. Therefore, there is an urgent need to develop more efficacious novel therapies. The imipridones, ONC201/TIC10, ONC206, and ONC212, are novel small-molecules that bind mitochondrial ClpP and induce activation of the integrated stress response (ISR), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling, and oxidative stress. Each has previously demonstrated antitumor effects in a variety of preclinical in vitro and in vivo cancer subtypes, including colorectal cancer, with limited toxicity. Reactive oxidation species (ROS) are natural biproducts of cellular oxidative metabolism and play important roles in the modulation of cell death pathways, differentiation, and immune response. Using varying doses of ONC201, ONC206, and ONC212, we observed increased lipid peroxidation in several colorectal and pancreatic cancer cell lines using the C-11 BODIPY assay which measures lipid-specific peroxides. Lipid peroxidation was reversed by the ferroptosis inhibitor liproxstatin-1, suggesting that in addition to inducing apoptosis, imipridones may be involved in ferroptosis induction. Future directions include mechanistic work, investigation of the effect of imipridone-induced lipid peroxidation on immune cell cytotoxicity, and understanding the relative importance of lipid peroxidation in the clinical efficacy of imipridones.

Citation Format: Arielle De La Cruz, Praveen R. Srinivasan, Andrew George, Varun V. Prabhu, Maximilian P. Pinho-Schwermann, Wafik S. El-Deiry. Imipridones induce lipid peroxidation and oxidative stress in gastrointestinal malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4786.