bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1527: KY-CLC-mouse, a common light chain bi-specific antibody discovery platform
Authors: Peng Hao , Hao Feng , Wu Guojin , He Feng , Ning Jinying
Published: April 2023

Bi-specific antibody (BsAb) is an attractive therapeutic antibody format for treating different diseases. The matching of light chains is the central issue of BsAb manufacturing. Here we developed a Common Light Chain mouse named KY-CLC-mouse. KY-CLC-mouse has no λ light chain expression with an identical κ light chain. This identical κ light chain was modified from a light chain that frequently appeared in mice and paired with different heavy chains. This light chain had good solubility and stability. It was also easy to humanize without post-translation modifications within CDRs. And the CDRs were modified to decrease immunogenicity. The mouse was validated by developing different monoclonal antibodies against various antigens with an identical light chain and high affinity. Mouse monoclonal antibodies against PD1, LAG-3, PD-L1, CD47, cMET, and EGFR were generated from the hybridoma of KY-CLC-mouse, with KD ranging from 0.1nM to 10nM. Humanized antibodies were derived from the above antibodies with high affinity. BsAb binding two of the above targets were generated by Knob-into-Hole (KiH) technology, such as fully humanized PD1xLAG-3 and CD47xPD-L1 BsAb. PD1xLAG-3 BsAb was simultaneously bound to PD1 and LAG-3 with a blockade of binding to their ligands, PD-L1 and MHCII. CD47xPD-L1 BsAb was simultaneously bound to CD47 and PD-L1 and inhibited their binding to ligands, SIPRα and PD1. Both BsAbs had typical mouse PK with a half-life longer than seven days. The above results indicated that KY-CLC-mouse is an efficient platform for discovering therapeutic antibodies with a common light chain.

Citation Format: Hao Peng, Feng Hao, Guojin Wu, Feng He, Jinying Ning. KY-CLC-mouse, a common light chain bi-specific antibody discovery platform [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 1527.
Abstract 1528: 6B5, an anti-human B7-H3 therapeutic antibody that enhances antibody-dependent cellular cytotoxicity and inhibits tumor growth in B7-H3-humanized mice
Authors: Chen Yunyun , Li Jiangmei , Guo Yanan , Hu Wenqi , Mao Ting , Hui Li , An W. Frank , Yang Yi , Li Feng
Published: April 2023

B7-H3 (CD276) is a type I transmembrane protein belonging to the B7 family of immune-regulatory ligands. Initially reported as a costimulatory signal of human T cells, increasing evidence suggests that it may also have coinhibitory function. TLT-2 has been identified as a potential cognate receptor for B7-H3, but others may exist. Overexpression of B7-H3 protein is found in a variety of human cancers, including lung adenocarcinomas, neuroblastomas, gliomas, and pancreatic tumors—and is associated with poor prognosis and survival. While B7-H3 can promote T cell activation and anti-tumor responses in the context of CD3 stimulation, its coinhibitory function inhibits T and NK cell activity in the tumor microenvironment. We generated 6B5, a fully human B7-H3 monoclonal antibody, from murine B7-H3 knock-out RenMab™ mice, which contain the full human immunoglobulin variable domain. 6B5 is cross-reactive with rodent and cynomolgus monkey B7-H3. It displays higher affinity for tumor cell lines than Enoblituzumab. 6B5 has no staining on normal human tissues. In vitro antibody-dependent cellular cytotoxicity (ADCC) against H520 squamous cell carcinoma cells was potentiated by 6B5 relative to Enoblituzumab and, critically, did not exhibit the hook effect at high dosage. In vivo efficacy of 6B5 in humanized B7-H3 mice bearing murine EL4 lymphomas was comparable to Enoblituzumab. ADCC enhancement in vivo was comparable to afucosylation or five-point mutations. Epitope mapping revealed non-overlapping B7-H3 binding epitopes of 6B5 and Enoblituzumab. Taken together, these data suggest that 6B5 is a promising therapeutic anti-human B7-H3 IgG monoclonal antibody that may find clinical application in a range of cancers, including current refractory types.

Citation Format: Yunyun Chen, Jiangmei Li, Yanan Guo, Wenqi Hu, Ting Mao, Li Hui, W. Frank An, Yi Yang, Feng Li. 6B5, an anti-human B7-H3 therapeutic antibody that enhances antibody-dependent cellular cytotoxicity and inhibits tumor growth in B7-H3-humanized mice [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 1528.
Abstract 1722: KRAS, BRAF, BRCA1 and EGFR mutation-specific panels using hydrogel-based 3D in vitro tumor models
Authors: Xue Bin , Schüler Julia , Harrod Christopher , Bomya Zoji , Hribar Kolin C.
Published: April 2023

Mutation-targeting therapies have become a transformative technology and in many ways a new paradigm for cancer treatment for patients. The early development of Erlotinib for non-small cell lung cancer paved the way for EGFR treatment, irrespective of tumor indication subtype. And the more recent launch of Sotorasib for KRASG12C mutation has marked a turning point for the once undruggable mutation. The advent of gene specific-treatments requires a reconfiguration of preclinical models which more effectively represent the said mutation in populations of diverse backgrounds and tumor indications. 3D models which have the ability to recreate the key hallmarks of cancer - such as growth, invasion, immune infiltration and suppression, stromal transformation, drug diffusion, as well as gene mutational signatures - are of exceptional interest to drive rapid and scalable compound efficacy screening. Here, we developed four 3D in vitro tumor panels carrying mutations in one of the following genes - KRAS, EGFR, BRCA1, and BRAF - as well as wildtype models for comparison. The tumors were grown in a hydrogel matrix in 96-well plates with stromal fibroblasts and screened with the standard of care (e.g. Sotorasib for KRASG12C, Erlotinib for EGFRmut, the PARP inhibitor Olaparib for BRCAmut, and Vemurafenib for BRAFV600E) and other known therapeutic compounds that target their respective mutations. The utility of a 3D hydrogel matrix and fibroblasts in the panels’ models were key in recreating the stromal compartment of the tumor microenvironment. Endpoints were determined within four days of treatment, including tumor size reduction and tumor killing, via high-content image analysis. The Panels’ robustness as demonstrated by the reproducible results in independent experiments suggests they can be utilized at in vitro pharmacology stages and earlier in Hit and Lead finding screens.

Citation Format: Bin Xue, Julia Schüler, Christopher Harrod, Zoji Bomya, Kolin C. Hribar. KRAS, BRAF, BRCA1 and EGFR mutation-specific panels using hydrogel-based 3D in vitro tumor models [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 1722.
Abstract 189: Developing a patient-derived organoid biobank, suitable for large scale drug screenings
Authors: Fielmich Lars-Eric , Buijs Annemarie , Mori Daniele , Viergever Bas , Draoui Nihed , Schepers Anna , Costa e Silva Mariana M. , Scholtus Mathijs , Mahmoudi Tokameh , Zuiverloon Tahlita , Derksen Merel , Gavine Paul , Boj Sylvia F. , Meijer Richard , Kranenburg Onno , Arts Janine , Vries Robert
Published: April 2023

Conventional models for preclinical drug screening offer poor predictive value for patient response, causing high attrition rates of new agents in the clinic. HUB’s proprietary Patient-Derived Organoid (PDO) Technology enables long-term expansion of primary patient material to generate ‘mini organs in a dish’ that can be used as patient avatars, thus bringing every “patient in the lab®”. We present the world’s first NMIBC PDO biobank comprising 50 PDO models from transurethral resection of bladder (TURB) biopsies collected at three hospitals. Clinical and pathological characterization of the PDOs confirmed the representation in the biobank of a range of tumor stages and differentiation grades, as well as patient treatment and follow-up. The genomic analyses of PDOs revealed mutations in known BC driver genes and genomic instability, a hallmark of cancer. Finally, transcriptomic analyses confirmed gene expression profiles familiar to BC and characterized gene fusions. Next, we successfully screened the biobank on a panel of two reference compounds for the treatment of bladder cancer and seven candidate drugs, indicating potential new leads for further development of some of the compounds. The work presented here shows the feasibility of building PDO biobanks representing a specific patient population, suitable for improving the drug development pipeline for the next generation of cancer drugs.

Citation Format: Lars-Eric Fielmich, Annemarie Buijs, Daniele Mori, Bas Viergever, Nihed Draoui, Anna Schepers, Mariana M. Costa e Silva, Mathijs Scholtus, Tokameh Mahmoudi, Tahlita Zuiverloon, Merel Derksen, Paul Gavine, Sylvia F. Boj, Richard Meijer, Onno Kranenburg, Janine Arts, Robert Vries. Developing a patient-derived organoid biobank, suitable for large scale drug screenings [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 189.
Abstract 1900: Radiotherapy-induced early adverse skin reactions: Comparative analysis of clinician- and patient-reported outcomes in a multi-racial/ethnic breast cancer population (WF-97609)
Authors: Urbanic James J. , Shaw Edward G. , Takita Cristiane , Wright Jean L. , Ip Edward H. , Lu Lingyi , Baez-Diaz Luis , Brown Doris R. , Strasser Jon , Baglan Kathy , Palmer Mark , Thakrar Anu , Curtis Amarinthia E. , Lesser Glenn , Hu Jennifer J.
Published: April 2023

Background: Adjuvant radiotherapy (RT) following surgery significantly improves breast cancer survival. However, some patients, particularly minorities, develop early adverse skin reactions (EASRs) or skin toxicities that negatively impact the quality of life (QOL). We assess RT-related EASRs and QOL using clinician-reported outcomes (CROs) and patient-reported outcomes (PROs) in a large multi-racial/ethnic breast cancer population.

Methods: This prospective study recruited 1,000 breast cancer patients undergoing RT through the Wake Forest NCI Community Oncology Research Program (NCORP) Research Base between 2011 and 2013. We used the Oncology Nursing Society (ONS) Skin Toxicity Criteria for CROs and Skindex-16 (SD-16) for PROs.

Results: This study included 405 non-Hispanic whites (NHW), 277 black/African Americans (AA), 241 HW, 62 Asian/Pacific Islanders (ASPI), and 15 others. About 42% and 15% of patients developed RT-induced ONS grade 3+ and 4+ skin toxicities, respectively. RT-induced EASRs differed significantly by race/ethnicity at mid-RT, 1-month, and 2-month post-RT, but not at the end of RT. Total SD-16 scores differ by race/ethnicity at pre-RT, 1-month, and 6-month post-RT, but not at the end of RT. Overall, PROs have a moderate but significant correlation with CROs at the end of RT.

Conclusion: RT-induced EASRs differed significantly by race/ethnicity. There was a moderate but significant correlation between PROs and CROs. However, PROs delineated a broader spectrum in capturing the impact of RT on patients’ QOL, which highlights the importance of integrating PROs in accessing RT-induced EASRs, particularly in minorities with worse RT-induced EASRs.

Trial Registration: NCT01407770 (09/02/2011)

Citation Format: James J. Urbanic, Edward G. Shaw, Cristiane Takita, Jean L. Wright, Edward H. Ip, Lingyi Lu, Luis Baez-Diaz, Doris R. Brown, Jon Strasser, Kathy Baglan, Mark Palmer, Anu Thakrar, Amarinthia E. Curtis, Glenn Lesser, Jennifer J. Hu. Radiotherapy-induced early adverse skin reactions: Comparative analysis of clinician- and patient-reported outcomes in a multi-racial/ethnic breast cancer population (WF-97609) [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 1900.
Abstract 2072: Computational modeling of metabolic variations in tumor microenvironment
Authors: Guo Tingbo , Zhu Haiqi , Wang Xiao , Wang Jia , Zhou Xinyu , Wei Yuhui , Dang Pengtao , Zhang Chi , Cao Sha
Published: April 2023

Dysregulation of metabolic pathways is a hallmark of cancer. Despite a plethora of knowledge on the core components of metabolic pathways we have gained, there are still major gaps in our understanding of the integrated behavior and metabolic heterogeneity of cells in the context of their microenvironment. Essentially, metabolic behavior can be determined by different factors and vary dramatically from cell to cell due to their high plasticity, driven by the need to cope with various dynamic metabolic requirements. Large amount of single-cell, spatial or tissue multi-omics data obtained from disease tissue has been proven to be endowed with the potential to deliver information on a cell functioning state and its underlying phenotypic switches.We have recently developed single-cell flux estimation analysis (scFEA) to predict sample-wise metabolic fluxome by using single-cell or bulk transcriptomics data. We further developed a web portal scFLUX.org, empowered by our scFEA method, expanding the analytic ability to both human and mouse metabolic networks. We also developed a probabilistic model named Michaelis-Menten-based Flux Estimation Analysis (mmFEA) for statistical inference of flux changes between conditions or cell types based on matched or unmatched gene expression data, metabolomics data, and partially observed kinetic parameters. These methods provide the following unmet capabilities to study metabolic variations in cancer: (1) reconstruction of cell or tissue specific and subcellular-resolution metabolic network, (2) estimation of cell-/sample-wise metabolic flux by considering metabolic imbalance, metabolic exchange between cells, and shifted redox, pH and energy balance in disease microenvironment, (3) a systematic evaluation of functional impact of variations in gene expression, genetic/epigenetic variations, metabolite availability and network structure on the context specific metabolic network and flux, (4) estimate the impact of each gene on flux by using a first-order derivative, (5) perturbation analysis to predict how alterations in enzymes or metabolites may affect the metabolic flux, and (6) identification of the samples and sub-network of a distinct metabolic shift.The methods were validated on matched scRNA-seq, metabolomics and fluxomics data. We demonstrated the function of this computational framework on pan-cancer transcriptomics and proteomics data, and single cell and spatial transcriptomics data.

Citation Format: Tingbo Guo, Haiqi Zhu, Xiao Wang, Jia Wang, Xinyu Zhou, Yuhui Wei, Pengtao Dang, Chi Zhang, Sha Cao. Computational modeling of metabolic variations in 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 2072.
Abstract 2078: VALIS: Virtual Alignment of pathoLogy Image Series for multi-gigapixel whole slide images
Authors: Gatenbee Chandler Dean , Baker Ann-Marie , Prabhakaran Sandhya , Robertson-Tessi Mark , Graham Trevor , Anderson Alexander R.
Published: April 2023

Interest in spatial omics is on the rise, but generation of highly multiplexed images used in many spatial analyses remains challenging, due to cost, expertise, methodical constraints, and/or access to technology. An alternative to performing highly multiplexed staining is to register collections of whole slide images (WSI), creating a collection of aligned images that can undergo spatial analyses. However, registration of WSI is two part problem, with the first being the alignment itself, and the second being the application of the transformations to huge multi-gigapixel images. To address both challenges, we have developed the Virtual Alignment of pathoLogy Image Series (VALIS) software, which enables one to rapidly and easily generate highly multiplexed images by aligning (registering) any number of multi-gigapixel whole slide images (WSI) stained using immunohistochemistry (IHC) and/or immunofluorescence (IF). Benchmarking using the publicly available 2019 ANHIR and 2022 ACROBAT Grand Challenge datasets indicates that VALIS provides state of the art accuracy, being one of the most accurate publicly available methods in the ANHIR challenge, and the most accurate opensource method in the ACROBAT challenge. VALIS is able to read, register, and save multi-gigapixel images as ome.tiff, thereby addressing the second challenge. In addition to the benchmarking datasets, the generalizability of VALIS has been tested with 273 IHC samples and 340 IF samples, each of which contained between 2-69 images per sample. In total, VALIS has therefore been tested with 5,138 images. The registered WSI tend to have low error and are completed within a matter of minutes. VALIS is written in Python, requires only few lines of code for execution, is readily available and fully documented. VALIS therefore provides a free, opensource, flexible, scalable, robust, and easy to use pipeline for rigid and non-rigid registration of multi-gigapixel WSI, facilitating spatial analyses of prospective and existing datasets,breathing new life into the countless collections of brightfield and immunofluorescence images.

Citation Format: Chandler Dean Gatenbee, Ann-Marie Baker, Sandhya Prabhakaran, Mark Robertson-Tessi, Trevor Graham, Alexander R. Anderson. VALIS: Virtual Alignment of pathoLogy Image Series for multi-gigapixel whole slide images [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 2078.
Abstract 2128: Genomic alterations in DNA mismatch repair (dMMR)
Authors: Holla Vijaykumar , Ronaghy Arash , Yang Richard K. , Patel Keyur P. , Routbort Mark J. , Overman Michael J. , Dumbrava Ecaterina E. , Shaw Kenna R. , Karp Daniel D. , Meric-Bernstam Funda
Published: April 2023

Background PD-1 inhibitors have been shown to be effective in advanced cancer patients with mismatch repair deficient tumors (dMMR) in a tumor agnostic fashion. dMMR testing by immunohistochemistry (IHC) is infrequently performed outside of colorectal and endometrial cancer. In contrast, comprehensive genomic sequencing (CGS) for somatic genomic alterations, is frequently perfomed across tumor types. We hypothesized that CGS may identify alterations in dMMR genes (MLH1, MSH2, MSH6, PMS2), and that patients with genomic MMR alterations will have higher rates of dMMR loss by IHC.

Methods From Jan 2016 to Dec 2021, 15,701 patients were identified as receiving CGS. Sequencing results were analyzed for mutations in dMMR genes, tumor type distribution and concordance with IHC results.

Results 627 (4%) of 15,701 patients had mutations in one of the dMMR genes; mutations were found across tumor types. Majority of patients with mutations in MMR genes had single nucleotide variants (SNV, 430, 68.6%) followed by frameshift/truncating (155, 24.7%), splice site (31, 4.9%) and in frame deletions/indel (11, 1.6%) mutations. A subset of the MMR gene mutant patients (n=279) had IHC testing for dMMR proteins: 99 (35.5%) demonstrated dMMR loss, including 55 (53.4%) of 103 patients with colorectal cancer, 3 (25%) of 12 patients with endometrial cancer and 41 (25%) of 164 with other tumor types. dMMR loss on IHC was found in 70 (71%) of 99 patients with frameshift/truncation/splice site/indel mutations compared with 29 (29%) of patients with SNVs (x2=8.49; p=0.00038).

Conclusion Mutations in MMR genes are found in multiple tumor types. Reflex IHC testing of patients carrying dMMR mutations inferred to be inactivating may identify patients with dMMR, expanding the pool of patients eligible for immunotherapy.

Citation Format: Vijaykumar Holla, Arash Ronaghy, Richard K. Yang, Keyur P. Patel, Mark J. Routbort, Michael J. Overman, Ecaterina E. Dumbrava, Kenna R. Shaw, Daniel D. Karp, Funda Meric-Bernstam. Genomic alterations in DNA mismatch repair (dMMR) [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 2128.
Abstract 2146: LDH as a surrogate marker for both pCR and survival in breast cancer patient
Authors: Lee In Hee , Lee Soo Jung , Lee Jeeyeon , Park Ho Yong , Jung Jin Hyang , Park Nora Jee-Young , Park Ji-Young , Chae Yee Soo , Kang Byeongju
Published: April 2023

Background: Glucose metabolism in cancer cells produces various glycolytic metabolites, which can affect cancer progression and treatment outcome. Herein we evaluated the association of glucose transporters and enzymes involved in glucose metabolism with clinic-pathological factors and outcomes in breast cancer patients who underwent neoadjuvant chemotherapy(NAC).

Methods: We selected 7 target transporters and enzymes(GLUT1, hexokinase 2, lactate dehydrogenase, MCT1, MCT4, IGF1RA, and IGF1RB) involved in glucose metabolism. Their tumoral expressions were scored both quantitively and qualitatively based on tissue microarrays and immunohistochemistry as previously described (Pinheiro et al, 2010) and then evaluated in relationships with pretreatment clinical/pathological characteristics and treatment outcomes including findings of 18F-FDG PET/CT,pathologic complete response(pCR), and relapse in breast cancer(stage IIA-IIIC) patients who underwent anthracycline or taxane-based NAC.

Results: Of the 236 enrolled patients, 145(61.4%), 44(18.6%), and 47(19.9%) were luminal, HER2-enriched, and TNBC subtypes. Pathologic CR was determined 50(21.2%) and 57(24.2%) relapses, 42(17.8%) distant relapses, and 28 deaths were observed during median follow-up duration of 64.0 (32.3-114.9) months. The expression rates of the target molecules were 76.0, 31.1, 42.1, 54.9, 67.9, 78.4, and 49.8% for GLUT1, hexokinase 2 (HK2), lactate dehydrogenase(LD), MCT1, MCT4, IGF1RA, and IGF1RB, respectively. Among target molecules in glucose metabolism, LD was significantly associated with pCR(OR=0.251; p-=0.016) and distant disease-free survival (HR= 2.29; P=0.019). Plus, LD expression shows a trend for a high SUVmax of primary tumor 18F-FDG PET/CT(OR=1.797 P=0.060).

Conclusion: Tumoral expression of lactate dehydrogenase can be considered as a predictive marker for pCR to NAC and plus prognostic marker for survival in patients with breast cancer.

Citation Format: In Hee Lee, Soo Jung Lee, Jeeyeon Lee, Ho Yong Park, Jin Hyang Jung, Nora Jee-Young Park, Ji-Young Park, Yee Soo Chae, Byeongju Kang. LDH as a surrogate marker for both pCR and survival in breast cancer patient [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 2146.
Abstract 216: Profiling single cell and spatial transcriptomes in FFPE archived tissues
Authors: Chen Jasmine Ying-Jiun , Le Quynh , Kang Chris , Tam Roy , Tai Brian , Lo Kelly , Rahmatpour Nasim , Wasdin Perry
Published: April 2023

Single cell and single nuclei RNA sequencing (scRNA-seq and snRNA-seq) have helped to identify cell type and subtypes that are present in complex tissues in both healthy samples and disease states. So far, scRNA-seq and snRNA-seq can only be performed in fresh tissues, or fresh frozen tissues. Most archived tissues are formalin-fixed, and paraffin-embedded (FFPE), making them inaccessible to the modern technology for characterization. Recently, 10x Genomics release a new reagent enabling single-cell fixed RNA characterization from fixed samples. The initial release is for fresh samples being fixed and then profiled later. However, FFPE archived tissues are not supported yet. We showed here two protocols to dissociate single cells and single nuclei from a 10-year-old FFPE human gastric tumor. We then profile them with 10x Genomics’ fixed RNA profiling kit. We further compare the data with the 10x Genomics’ Visium data obtained from the same tissues to understand the performance of these two protocols. In summary, we show that such technology will help to further characterize archived tissues that already have pathology annotation as well as clinical data and help biomarkers identification.

Citation Format: Jasmine Ying-Jiun Chen, Quynh Le, Chris Kang, Roy Tam, Brian Tai, Kelly Lo, Nasim Rahmatpour, Perry Wasdin. Profiling single cell and spatial transcriptomes in FFPE archived tissues [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 216.
Abstract 2348: Mapping the interactome of matrisome targets using extracellular proximity labeling (ePL)
Authors: Peeney David , Gurung Sadeechya , Rich Josh , Coates-Park Sasha , Liu Yueqin , Stetler-Stevenson William G.
Published: April 2023

Classical methods to investigate protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We describe a method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of matrix metalloproteinases (MMPs), the major mediators of ECM breakdown and turnover. TIMP2 is a unique family member, with a broad expression profile that is expressed in both normal and diseased tissues, even in those with minimal metalloproteinase activity. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential to the development of ECM targeted therapeutics. This knowledge may also garner understanding of therapeutic resistance and the failure of conventional and next-generation cancer therapies. Using carboxyl- and amino-terminal fusion peptides of TIMP2 with BioID2 and TurboID, we describe the TIMP2 interactome in unique tissue compartments. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell lines, and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique and comparing our findings with classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.

Citation Format: David Peeney, Sadeechya Gurung, Josh Rich, Sasha Coates-Park, Yueqin Liu, William G. Stetler-Stevenson. Mapping the interactome of matrisome targets using extracellular proximity labeling (ePL) [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 2348.
Abstract 2349: Investigating the effect of ECM stiffness on macrophage-derived exosome secretion and PDAC organoid progression
Authors: Mahgoub Bayan , Xiao Weikun , Hill Reginald
Published: April 2023

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 11%. Current treatments that mostly target the cancer cells themselves, have not been able to significantly improve survival rates, meaning there is an urgent need for novel therapies. PDAC is unique among solid tumors because it exhibits extensive desmoplasia that is characterized by an abundance of extracellular matrix (ECM) components. In recent years, an increasing body of work has shown that cancer tissue is stiffer than normal tissue by a factor of 3 or more. This change in ECM stiffness has been shown to play an important role in affecting cell to cell communication as well as cancer progression and chemoresistance. However, how ECM stiffness affects macrophages, an important immune component of the pancreatic tumor microenvironment (TME), and macrophage-derived exosome secretion is still understudied.

Preliminary studies have shown that macrophages are highly sensitive to the mechanical environment. How cell-cell information transfer through exosomes may regulate this process in the PDAC tumor microenvironment (TME) needs to be explored. Elucidating these potential changes can provide us with insights on how stiffness affects therapeutic outcome in PDAC.

Our previous studies utilizing a biomimetic model that allows us to tune stiffness, showed that increased stiffness resulted in significantly higher exosome secretion from fibroblasts. Based on this finding, we hypothesize that increasing stiffness will result in hypersecretion of macrophage-derived exosomes that increases the chemoresistance of PDAC. 2D culture of macrophages on varying stiffness plates was used to study these changes in vitro. We then examined how changes in ECM stiffness affected the function and polarization of tumor associated macrophages, as well as elucidating how exosome hypersecretion and uptake, regulate these “stiffness enhanced” macrophages’ ability to affect PDAC progression. Understanding how macrophage-derived exosome signaling changes due to ECM stiffness may provide us with insights needed to develop novel therapies.

Citation Format: Bayan Mahgoub, Weikun Xiao, Reginald Hill. Investigating the effect of ECM stiffness on macrophage-derived exosome secretion and PDAC organoid progression [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 2349.
Abstract 2357: Fibroblast activation protein (FAP) expression as a target for colorectal cancer treatment
Authors: Lippert Anna L. , Johnson Katherine A. , Pasch Cheri A. , Zhang Wei , Matkowskyj Kristina A. , LeBeau Aaron M. , Deming Dustin A.
Published: April 2023

Background: Fibroblast Activation Protein (FAP) is a protein expressed by cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) of epithelial cancers. There is potential interest in the use of FAP as a way to selectively target cancers, including colorectal cancer (CRC). Here we examine the expression of FAP in human colorectal cancers across stages and compare that expression to expression in normal colon and liver tissue. Additionally, we examine the correlation of FAP expression with key CRC clinical and biologic features to aid in potentially determining which CRC patients would be the most likely to benefit from FAP-directed therapies.

Methods: CRC and adjacent normal tissue samples spanning all stages of disease were collected across 243 patients. Immunohistochemistry (IHC) was used to evaluate FAP, MMP2, αSMA, and PDPN expression and quantified on an intensity scale from 0-3+. These scores were split into low (average score <2) and high (average score >/=2) groups. IHC was used to determine mismatch repair status by staining for MLH1, MSH2, MSH6, and PMS2. Separate samples from these patients were sequenced with the Qiagen Comprehensive Cancer Panel and analyzed for APC, KRAS, BRAF, PIK3CA, and TP53 mutations.

Results: Of 149 normal colon tissue sections, 99% had low FAP expression and 94% of those had no FAP expression. Of the 87 normal liver tissue sections, 99% had low FAP expression. In CRC sections, 58% had high FAP expression. Those patients >50 years of age had cancers with a higher rate of FAP expression (p=0.02). There was no correlation with FAP expression and sex, stage of disease, or sidedness. In cancers with BRAF V600 mutations, 91% have high FAP expression (x2 = 0.02). There is a trend for higher FAP expression in cancers possessing APC, KRAS, PIK3CA, or TP53 mutations (x2=NS). In correlating FAP with other CAF markers, MMP2 had the greatest correlation (Spearman’s Rho = 0.56). FAP is moderately associated with αSMA and PDPN (Spearman’s Rho = 0.33, 0.38).

Conclusion: FAP is an exciting therapeutic target for CRC due to its cancer selectivity. Here, we demonstrate that FAP marks a rather independent subset of CRCs, though there is particular interest in the BRAF V600 mutant cancers given the high expression and poor prognosis of this subtype. Further investigation is needed to further develop FAP-directed therapies.

Citation Format: Anna L. Lippert, Katherine A. Johnson, Cheri A. Pasch, Wei Zhang, Kristina A. Matkowskyj, Aaron M. LeBeau, Dustin A. Deming. Fibroblast activation protein (FAP) expression as a target for colorectal cancer treatment [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 2357.
Abstract 2365: Visualizing exosome structure in Dasatinib resistant colon cancer cells
Authors: Koo Hui Min , Lu Yi-Wen , Tseng Yu-Chi , Tseng Kai-Yu , Lu Yen-Tse , Chao Wei-Ting
Published: April 2023

Cells release molecules through vesicles or into specific locations through exocytosis. Extracellular vesicle is divided into ectosomes and exosomes. Exosomes has been the main mechanism of discussion in recent studies and cancer research. As Exosomes with a diameter of ~30-160 nm (with an average diameter of 100 nm) carries intracellular molecules such as DNA, RNA, miRNA, proteins, and lipids. The physiological function of exosomes is not fully understood at present, it is important to understand the role of exosome in cancer. Src is highly activated in colon cancer and Src inhibition is the potential treatment strategy in colon cancer. In this study, colon cancer cells HT-29 were used to investigate the difference sizes of exosomes that response to Src inhibitor treatment. In the preliminary results, the exosome from HT-29 cultured medium were isolated and examined by transmission electron microscopy (TEM). The micrograph showed the average size of exosome is around 100 nm. When HT-29 were treated with Dasatinib (DST) the Src inhibitor, the drug resistant (DSTR) cells were grown, the secreted exosome from DSTR were collected and demonstrated the bigger size of exosome with average diameter of 130 nm. The role of the size changes of exosome in response to Dasatinib resistant will be further investigated, however, this study provides the clues that exosome detection can be applied in monitoring drug resistance.

Citation Format: Hui Min Koo, Yi-Wen Lu, Yu-Chi Tseng, Kai-Yu Tseng, Yen-Tse Lu, Wei-Ting Chao. Visualizing exosome structure in Dasatinib resistant colon cancer cells [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 2365.
Abstract 2373: Combination treatment with pH modulating drugs and immune checkpoint inhibitors increases TALL-104 cytotoxicity in preclinical co-culture experiments
Authors: Malpass Ryan , Huntington Kelsey E. , Brown Thomas , Zhou Lanlan , Zhang Leiqing , Navaraj Arunasalam , El-Deiry Wafik S. , Maxwell Aaron
Published: April 2023

Hepatocellular carcinoma (HCC) and colorectal cancer liver metastases (CRCLM) are leading causes of death worldwide, with therapeutic options limited to surgical resection and trans-arterial chemoembolization (TACE). TACE is a minimally-invasive approach to induce cancer cell death through a local hypoxia-dependent mechanism, and intrahepatic catheterization further enables the delivery of therapeutic agents to the tumor microenvironment (TME). Phase 1 clinical trials are ongoing for treatment of primary liver cancer and uveal melanoma with liver metastases using pressure-enabled drug delivery of SD-101 in combination with systemic immune checkpoint inhibitors (ICIs). Since acidosis is associated with immunosuppression in the TME, pH modulating drugs (PMDs) may restore physiologic conditions and augment treatment response for patients undergoing TACE with immunotherapy. Pre-clinical studies using PMDs with ICIs or adoptive immune cell transfer have demonstrated increased tumor lymphocyte infiltration, increased survival, and reduced tumor volume compared to ICI treatment or T cell infusion alone. High-throughput fluorescent imaging of HCC or CRC lines co-cultured with TALL-104 cells indicates increased cancer cell death with PMD treatment alone, while imaging studies of combination PMD and ICI treatment are underway. In addition, Luminex 200 cytokine profiling of Hep3B indicates that the NHE1 inhibitor cariporide decreases HCC production of IL-8 and GDF-15 under normoxia and the proton pump inhibitor lansoprazole increases GDF-15 and MIF and decreases B2M, IL-8, CXCL10, and CXCL5. Combination PMD and ICI studies are needed to inform drug combinations and dosages in syngeneic murine models of primary liver cancer and CRCLM. Subcutaneous and orthotopic tumor models are expected to demonstrate the efficacy of local PMD treatment with systemic immunotherapy.

Citation Format: Ryan Malpass, Kelsey E. Huntington, Thomas Brown, Lanlan Zhou, Leiqing Zhang, Arunasalam Navaraj, Wafik S. El-Deiry, Aaron Maxwell. Combination treatment with pH modulating drugs and immune checkpoint inhibitors increases TALL-104 cytotoxicity in preclinical co-culture experiments [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 2373.
Abstract 2424: Development of radiation sensitivity evaluation platform in esophageal cancer patient derived organoids
Authors: Kim Jung Eun , Jeong Mi-Hyeon , Ravangnam Thunyaporn , Ju Sunghun , Lee Dong Woo , Ku Bosung , Oh Dongryul
Published: April 2023

Radiation therapy (Radiotherapy) is a highly effective treatment with wide range of cancer curing. Radiotherapy allows the cancer cells to be killed or reduced in number while protecting the majority of other organs and tissues in the body. Radiotherapy is commonly used with chemotherapy or immunotherapy or radiosensitizer drugs for enhancing the treatment efficacy. Predicting individual radiation sensitivity is very important in determining the direction of future treatment after cancer diagnosis. However, it is lack of accurate and relevant preclinical model for evaluation of radiation sensitivity.In this study, we developed the platform for radiosensitivity evaluation using the Esophageal cancer patient derived organoids. Biopsy tissues from Esophageal cancer were digested to single cells and then dispensed with ECM on pillar plate (MBD co.) to forming organoids. After organoid formation, the organoids were exposed to radiation with increasing dosage. Cell viability and radiation sensitivity were calculated by 3D Celltiter glo method and Calcein AM staining. Radiation sensitivity index was determined based on radiation response and organoid growth.We included 22 patients with ESCC treat with radiation therapy from Samsung Medical Center. After surgery, we compared radiation sensitivity index with pathological grade for sensitivity and specificity of radio sensitivity evaluation platform. The sensitivity was 75.00% (95% CI 34.91% - 86.81%) and the specificity was 71.43% (95% CI 41.90% - 91.61%). This resulted in an accuracy in this population of 72.73% (95% CI 49.78% - 89.27%).This study has a very high correlation to patient matched clinical outcomes of radiation sensitive group or non-sensitive group. This radiation sensitivity evaluation platform could contribute to precision medicine in Esophageal cancer patients and it can be helpful to make decision how to cure of cancer.

Citation Format: Jung Eun Kim, Mi-Hyeon Jeong, Thunyaporn Ravangnam, Sunghun Ju, Dong Woo Lee, Bosung Ku, Dongryul Oh. Development of radiation sensitivity evaluation platform in esophageal cancer patient derived organoids [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 2424.
Abstract 2429: AGPAT4 as a novel metabolic driver of oncogenic stemness and dedifferentiation in hepatocellular carcinoma
Authors: Ng Kai Yu , Fong Tsz-Lok , Gao Ya , Wong Tin-Lok , Gao Yuan , Yun Jing-Ping , Guan Xin-Yuan , Liu Ming , Chung Clive YS , Ma Stephanie
Published: April 2023

The liver is an unique organ in that it is responsible for many metabolic functions. During HCC development, these metabolic machineries are extensively reprogrammed to support the insatiable nutrient requirement of HCC. Cancer stem cells (CSCs) are a small and elusive subpopulation of self-renewing cancer cells with remarkable ability to initiate, propagate, and spread the malignant disease. Liver CSCs reprogram their metabolic pathways to match with the increased metabolic needs for cancer cell survival under adverse conditions. Identifying novel metabolic targets that are related to stemness can offer promising strategies for targeting CSCs and hence to kill and to control their growth. In this study, pathway enrichment analysis of genes that linked metabolism and stemness identified aberrant glycerophospholipid metabolism of which AGPAT4 ranked a top-hit. AGPAT4 upregulation in HCC is tightly correlated with aggressive clinical features, including survival, metastasis, and stemness signatures. Expression of AGPAT4 peaked during early liver progenitor development, decreased during hepatocyte maturation and increased progressively from well-differentiated to poorly differentiated HCCs. Enrichment of AGPAT4 in HCC was mediated by promoter binding of SOX9 to drive AGPAT4 transcriptional activity. AGPAT4 inhibition could mitigate tumor initiation, self-renewal, metastasis and sorafenib resistance. Mechanistic studies revealed an AGPAT4-mediated phosphatidic acid production axis to promote HCC through regulating mTOR signaling. Inhibition of Agpat4 by AAV8 shRNA reduced tumorigenicity and stemness, and sensitized HCC tumors to sorafenib. AGPAT4 overexpression was able to predict sorafenib response in the clinic. Through high-throughput screening coupled with activity-based protein profiling, a cysteine-reacting compound with high binding affinity and selectivity towards AGPAT4 was identified and found to work synergistically with sorafenib to supress HCC. To conclude, AGPAT4 is a novel metabolic driver of oncogenic stemness, dedifferentiation and metastasis in HCC. AGPAT4-induced tumor lineage plasticity may represent an Achilles heel for HCC. Inhibition of AGPAT4 may widen the therapeutic window for sorafenib treatment in the clinic.

Citation Format: Kai Yu Ng, Tsz-Lok Fong, Ya Gao, Tin-Lok Wong, Yuan Gao, Jing-Ping Yun, Xin-Yuan Guan, Ming Liu, Clive YS Chung, Stephanie Ma. AGPAT4 as a novel metabolic driver of oncogenic stemness and dedifferentiation in hepatocellular carcinoma [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 2429.
Abstract 2449: p38β promotes cancer stemness and chemoresistance in hepatocellular carcinoma
Authors: Huang Ianto Bosheng , Xie Yunong , Wong Tin Lok , Lee Terence Kin Wah , Yun Jing-Ping , Ma Stephanie Kwai Yee , Tong Man
Published: April 2023

Protein kinases control a variety of oncogenic functions through a phosphotransfer dependent signal transduction cascade in cancers where potent and selective inhibitors could be developed with favorable pharmaceutical properties. In hepatocellular carcinoma (HCC), increasing evidence suggested that cancer stemness is a driving force of therapy resistance which is tightly controlled by kinase-dependent signaling cascades. However, there are still limited druggable kinase targets that are developed to combat cancer stemness and therapy resistance for HCC treatment. To identify potential kinase targets which promote stemness, survival and therapy resistance in HCC, two datasets profiling kinase genes which are important for HCC survival and chemoresistance in HCC spheres were compared. p38 MAPK beta (p38β) was found to be a common top-ranked kinase important for these functions. Clinically, biopsies of human HCC showed high expression of total and activated forms of p38β, suggesting that p38β could be a potential therapeutic target in HCC. Functionally, we demonstrated that p38β promoted HCC tumor initiation, self-renewal and resistance towards chemotherapy by cisplatin. Using tandem affinity purification coupled with mass spectrometry, we identified BiP, which is a chaperone protein, as a novel binding partner of p38β. Subsequent mechanistic validation and mass spectrometry analysis showed that p38β directly phosphorylates BiP at a previously unrecognized phosphorylation site. Rescue experiments with BiP mutation and inhibition abrogated the stemness and chemoresistant properties observed in p38β overexpressing HCC cells. Taken together, we identified and characterized p38β and its direct phosphorylation target BiP as critical regulators for promoting cancer stemness and chemoresistance in HCC.

Citation Format: Ianto Bosheng Huang, Yunong Xie, Tin Lok Wong, Terence Kin Wah Lee, Jing-Ping Yun, Stephanie Kwai Yee Ma, Man Tong. p38β promotes cancer stemness and chemoresistance in hepatocellular carcinoma [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 2449.