bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5421: Characterizing functional cell migration states with highly multiplexed immunofluorescence imaging
Authors: Leineweber William Dee , Finkelstein Alice , Lundberg Emma
Published: March 2024

Cancer cell migration is a necessary step in the metastatic cascade, but there are heterogeneous cellular mechanisms to achieve this behavior. This study leveraged live-cell tracking and end-point spatial proteomics to better characterize what underlying molecular states drive migration heterogeneity. U2OS osteosarcoma stably expressing the FUCCI cell cycle reporter system were tracked for 20 hours on a Leica DMI8 widefield microscope. Immediately after, cells were fixed and prepared for highly-multiplexed immunofluorescence imaging on a Canopy CellScape instrument. Initial results from these experiments demonstrate that transferring the sample between these two systems is feasible and results in minimal loss of cells despite the multiple staining cycles. Unlike standard marker panels intended to differentiate between cell types, the initial panel used in this study includes markers that differentiate between migration modes. Even within one cell line, unsupervised clustering identified cell clusters for vimentin high/E-cadherin low and E-cadherin high/vimentin low, indicated an epithelial to mesenchymal transition (EMT) axis in the dataset. Furthermore, the spatial relationships between the cell neighborhoods show largely heterogeneous distributions of cell states, with clusters of more homogeneous cell states arising in more densely-packed regions of the coverslip. Further analysis of how single cell trajectories correlate to these cell states, as well as how cell cycle dynamics impact both migration and molecular cell states are in progress. Results from these analyses will shed new light on the “go vs. grow” hypothesis by directly testing how cell speed, persistence, and overall invasion correspond to cell cycle stage and progression. Furthermore, the high-resolution detail from the multiplexed imaging will allow for subcellular profiling of dynamic proteins, such as beta catenin, and directly link it to a larger protein and migration state. Overall, this experimental approach represents an important bridging between functional measurements and end-point omics technologies that will be crucial to gain actional insights amid the boom in multiomics.

Citation Format: William Dee Leineweber, Alice Finkelstein, Emma Lundberg. Characterizing functional cell migration states with highly multiplexed immunofluorescence imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5421.
Abstract 5426: SMC2 orchestrates non-small cell lung cancer proliferation, invasion, and metastasis by modulating the ERK/AKT/NF-κB pathways
Authors: He Yan , Luo Zhenyu , Wang Yiyao , Xiong Shan , Wan Rui , Zhang Xue , Bai Hua , Wang Jie
Published: March 2024

Chromatin instability has been associated with the expression of structural maintenance of chromosomes (SMC) family members and is a common feature in many tumors. SMC family members have been associated with poor outcomes in several cancer types. However, little is known about their functions and mechanisms in promoting cancer progression, particularly in non-small cell lung cancer (NSCLC). Here, we evaluated the expression of SMC family members in the tumor microenvironment of NSCLC specimens using single-cell RNA sequencing analyses. After a survival analysis, it was found that the sole factor associated with a poor prognosis for NSCLC was member SMC2 expression. Immunohistochemistry was used with data from the Human Protein Atlas, the Cancer Genome Atlas, and the Gene Expression Omnibus databases to confirm the overexpression of SMC2 in NSCLC. We demonstrated that SMC2 regulates not only the fluctuations in the critical states of tumor cells, which are defined by different transcriptome and metabolic properties but also coordinates the immunological components and cell-to-cell communications of the immune milieu, potentially through the pleiotrophin and the Galectin pathways. In vitro and in vivo evidence support the pro-metastatic role of SMC2 in NSCLC. Subsequent investigations showed that SMC2 increases NSCLC progression and metastasis via modulating the ERK/AKT/NF-κB pathway and the epithelial-mesenchymal transition process. Our research clarifies the role and potential mechanisms of SMC2 in NSCLC, providing new treatment options and valuable insights for the management of NSCLC.

Citation Format: Yan He, Zhenyu Luo, Yiyao Wang, Shan Xiong, Rui Wan, Xue Zhang, Hua Bai, Jie Wang. SMC2 orchestrates non-small cell lung cancer proliferation, invasion, and metastasis by modulating the ERK/AKT/NF-κB pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5426.
Abstract 5439: Metastasis-enhancing protein KITENIN confers temozolomide resistance in glioblastoma with unmethylated MGMT via upregulation of cancer stem cell makers
Authors: Ahn Eun-Jeong , Kim Yeong Jin , Jung Tae-Young , Lee Jae-Hyuk , Rhee Joon Haeng , Kim Kyung-Keun , Kim Sung Sun , Kim Nah Ihm , Kim Hangun , Moon Kyung-Sub , Lee Kyung-Hwa
Published: March 2024

Background: Elevated expression of cancer stem cell (CSC) markers is linked with progression, poor survival, and treatment resistance in glioblastoma (GBM). Our prior work identified KAI1 COOH-terminal interacting tetraspanin (KITENIN) as key to GBM progression. We investigated its influence on temozolomide (TMZ) resistance in GBM through modulation of CSC markers.

Methods: We examined KITENIN and CSC markers in TMZ-resistant GBM cells and patient-derived GBM cells, and assessed therapeutic responses to TMZ in mice implanted with KITENIN-modulated GBM cells. The correlation of these markers with clinical outcomes was analyzed using our cohort and The Cancer Genome Atlas (TCGA) data.

Results: High KITENIN expression correlated positively with elevated CSC marker levels in human GBM samples and KITENIN-modulated GBM cells. Inhibition of KITENIN using usnic acid reduced tumor progression by decreasing CSC marker expression. Our data and TCGA analyses linked co-directional increase of these factors with tumor recurrence and shorter survival in GBM patients with unmethylated MGMT. This increase was also observed in TMZ-resistant cell lines with unmethylated MGMT. Implantation of KITENIN-overexpressing cells led to TMZ resistance in our mouse model, associated with co-directionally increased CD44 and ALDH1A1 expression. The link between KITENIN and CD44/ALDH1A1 could be inhibited by DKC2511, a novel KITENIN inhibitor, and T5244, a c-Fos/AP-1 inhibitor.

Conclusion: KITENIN might contribute to TMZ resistance in GBM patients with unmethylated MGMT, potentially through AP-1 activation and subsequent upregulation of CSC markers. Hence, targeting KITENIN could provide a novel strategy to overcome TMZ resistance in GBM patients with unmethylated MGMT.

Citation Format: Eun-Jeong Ahn, Yeong Jin Kim, Tae-Young Jung, Jae-Hyuk Lee, Joon Haeng Rhee, Kyung-Keun Kim, Sung Sun Kim, Nah Ihm Kim, Hangun Kim, Kyung-Sub Moon, Kyung-Hwa Lee. Metastasis-enhancing protein KITENIN confers temozolomide resistance in glioblastoma with unmethylated MGMT via upregulation of cancer stem cell makers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5439.
Abstract 5443: Identifying key regulators of glioblastoma cancer stem cells
Authors: Wang Tao , Xie Xuanhua , Parada Luis F.
Published: March 2024

Glioblastoma multiforme (GBM) has a dire prognosis that has had little progress over the past three decades. The existence of a unique population of stem-like gene signature baring quiescent cells (GSCs) within GBM are important for GBM tumorigenesis, therapy-resistance and recurrence. Our lab has developed one of the most faithful genetically engineered mouse models, encompassing a Nestin promoter-enhancer driven CreERT2-H2BeGFP-hDTR cassette (hereafter as CGD), that allows us to accurately capture and isolate GSCs in mouse GBM. The next logical step is to leverage these tools to uncover therapeutic vulnerabilities of GSCs and make a meaningful impact on GBM patient outcomes. To our knowledge, very few studies have explored the functional regulators of GSCs using a CRISPR/Cas9 screening approach. Our GSC signature provides a strong foundation for performing targeted in vitro or in vivo screens. In this study, to identify functionally important genes that may regulate GSC self-renewal and tumorigenic properties, an in vitro CRISPR knockout screens using a gene library derived from the GSC signature. The viral library was applied to low passage primary cultures of FACS sorted CGD_GFP+ and CGD_GFP- GBM cells developed from CGD; Nf1f/+;Trp53f/f;Ptenf/+ mice, respectively. The result of our in vitro CRISPR knock-out screen demonstrated guide depletion of Atp1b2-sgRNA in CGD_GFP+ but not in CGD_GFP- cells. The main objective of this study is to utilize in vitro and in vivo functional assays to test the difference between Atp1b2 WT and Atp1b2 KO GSCs and Atp1b2 WT vs KO GSC-derived tumors. Having a targeted candidate gene list is also particularly helpful for designing an in vivo CRISPR knockout screen, which is more physiological relevant. Therefore, I propose to use the candidate gene list derived from the in vitro screen as a starting point. The in vivo screen can provide valuable insights for the selection of the most optimal candidates for anti-GSC therapies.

Citation Format: Tao Wang, Xuanhua Xie, Luis F. Parada. Identifying key regulators of glioblastoma cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5443.
Abstract 5444: EGFR missense mutation(s) induce OLIG2 expression to regulate glioma stem cells maintenance and therapeutic resistance
Authors: Weaver Sidney , Miller Aunay , Blomquist Mylan , McNamara James , Orndorff Keely , Sereduk Chris , Kloss Jean , Loftus Joseph , Mehta Shwetal , Tran Nhan L.
Published: March 2024

Glioblastoma (GBM) tumors contain a subpopulation of glioma stem cells (GSC) which can escape therapeutic effects through self-renewal and invasion. Epidermal growth factor receptor (EGFR) mutations are the most common alteration in GBM (50%). One example includes extracellular domain missense mutations like G598V, A289V, and R108K (10-15%). These extracellular domain missense mutations increase ligand binding affinity, inducing a highly active receptor. Earlier reports have shown these EGFR missense mutations exhibit therapeutic resistance; however, the molecular mechanism of these missense mutations in GBM remains unknown. In this study, we show high oligodendrocyte transcription factor 2 (OLIG2) expression in GSCs with EGFR-G598V and EGFR-A289V missense mutations. OLIG2 is universally expressed in glioma and essential for reprogramming GBM cells into stem-like tumor cells. Here, we report GSCs with these point mutations display increased self-renewal, growth, invasion, and therapeutic resistance. In addition, EGFR-G598V and EGFR-A289V trigger distinct downstream signaling pathways in GSCs to regulate OLIG2 function. These missense mutation(s) also represent different pathway-based GBM subclassifications, where A289V induces a Neuronal subtype and G598V induces a Glycolytic/Plurimetabolic subtype. We hypothesize that GBM tumors harboring EGFR missense mutations (G598V, A289V) influence stem cell properties via multiple pathways to evade therapeutic effects and inhibiting these different signaling pathways will decrease therapeutic resistance in GBM. Understanding the mechanism of these EGFR mutations may lead to improved therapeutic strategies for GBM patients.

Citation Format: Sidney Weaver, Aunay Miller, Mylan Blomquist, James McNamara, Keely Orndorff, Chris Sereduk, Jean Kloss, Joseph Loftus, Shwetal Mehta, Nhan L. Tran. EGFR missense mutation(s) induce OLIG2 expression to regulate glioma stem cells maintenance and therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5444.
Abstract 5446: The role of snoRNAs in GBM cells, GSCs, neuronal precursor cells (NPCs), astrocytes, and normal brain tissues
Authors: Chatterjee Sujash , Kuersten Scott , Middle Christina , Roos Morgan , Galante Pedro A. , Guardia Gabriela , Penalva Luiz
Published: March 2024

rRNA modification are a crucial step in ribosome assembly and function. Small nucleolar RNAs select which nucleotides in rRNAs get modified. Typically, C/D box snoRNAs are implicated in 2’-O-ribose methylation (2’Omet) while H/ACA box snoRNAs are responsible for pseudo-uridylations. Alterations in snoRNA expression have been described in multiple tumor types while distinct snoRNAs have been linked to cancer-relevant phenotypes. Our initial study on snoRNA expression using a dedicated Ampliseq platform revealed a specific glioma stem cell (GSC) signature. We propose this snoRNA signature creates a unique rRNA modification pattern that shapes the ribosome, favoring the translation of genes in pathways critical for GSC growth and survival. We will expand our study and characterize the expression profile of snoRNAs and 2’Omethylation in a large panel of GBM cells, GSCs, neuronal precursor cells (NPCs), astrocytes, and normal brain tissues. Next, we will select snoRNAs displaying differential expression in GSCs and determine if changes in their levels affect proliferation, viability, response to temozolomide and radiation, and translation. We and others have observed that the integrity of protein complexes that regulate ribosomal biogenesis depends on PolyADP-ribosylation (PARylation)and PAR-binding. In agreement, genomic screenings determined that multiple genes regulating ribosome biogenesis confer sensitivity to PARP inhibitors. These findings offer a therapeutic opportunity. We will investigate the functional impact of PARylation on rRNA modification by determining the effect of PARP inhibitors on snoRNP assembly and 2’Omethylation. Finally, we will test the hypothesis that PARP and ribosome biogenesis inhibitor combinations would be more effective than PARP inhibition alone in treating GBM.

Citation Format: Sujash Chatterjee, Scott Kuersten, Christina Middle, Morgan Roos, Pedro A. Galante, Gabriela Guardia, Luiz Penalva. The role of snoRNAs in GBM cells, GSCs, neuronal precursor cells (NPCs), astrocytes, and normal brain tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5446.
Abstract 5483: Macrospatial ecotype heterogeneity in early-stage breast cancer identified by spatial nucleus barcoding
Authors: Wang Kaile , Xiao Zhenna , Lin Yiyun , Wei Runmin , Bai Shanshan , Ye Jie , Ye Rui , Hu Min , Thennavan Aatish , Sei Emi , Thompson Alastair , Krishnamurthy Savitri , Navin Nicholas
Published: March 2024

Ductal carcinoma in situ (DCIS), the predominant form of early-stage breast cancer and a precursor to invasive breast cancer, remains inadequately understood in terms of spatial microenvironment reprogramming. In this study, we developed a Spatial Nucleus Barcoding (SNuBar) approach for single-nucleus RNA sequencing (SNuBar-RNA) that utilizes a single oligonucleotide adaptor to preserve spatial information. The accuracy and scalability of SNuBar was validated using cell line mixture experiments. We then applied SNuBar-RNA to investigate the macrospatial ecotype heterogeneity in early-stage breast cancer. By analyzing single cells from different macrodissected spatial regions in normal breast tissues, matched normal and DCIS tissues, we identified 13 major cell types and 43 distinct cell states, co-localizing in diverse combinations across four primary topographic areas (adipose-enriched, normal epithelial, activated stroma and tumor). Spatial analysis revealed the reprogramming of vascular and myoepithelial cells in localized zones near premalignant cells, while fibroblasts, T-cells, and myeloid cells exhibited reprogramming across broader fields. Validation using the MERSCOPE assay confirmed stromal heterogeneity between tumor and normal areas. Collectively, our data shows that many diverse cell types are reprogrammed in spatially-defined areas in the early-stage breast cancer microenvironment, relative to the normal breast tissue regions.

Citation Format: Kaile Wang, Zhenna Xiao, Yiyun Lin, Runmin Wei, Shanshan Bai, Jie Ye, Rui Ye, Min Hu, Aatish Thennavan, Emi Sei, Alastair Thompson, Savitri Krishnamurthy, Nicholas Navin. Macrospatial ecotype heterogeneity in early-stage breast cancer identified by spatial nucleus barcoding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5483.
Abstract 5487: Improved spatial biology analysis of the tumor microenvironment with the next generation of the MultiOmyxTMplatform
Authors: Lazare Michael , Nunns Harry , Moghaddam Fatemeh Jelvhei , Jin Xiaowei , Todorov Courtney , Leones Eric , Thio Marianne , Sahafi Flora , Parnell Erinn , Au Qingyan
Published: March 2024

Multiplexed Immunofluorescence is a powerful tool for spatially characterizing and phenotyping cells within the tumor microenvironment. MultiOmyx has been one of the leading platforms for generating multiplexed immunofluorescence data to support translational and clinical research for more than 10 years. However, MultiOmyx and other similar platforms are often hampered by a limited imaging area due to either restricted staining areas or being cost prohibitive due to excessive imaging times. Here, we demonstrate the capabilities of an improved MultiOmyx platform which can be used to generate whole-tissue data from the iterative MultiOmyx multiplexing process using the CyteFinder® II microscope and a customized software package developed by RareCyte, Inc. The new NeoLYTX image analysis pipeline in conjunction with the whole-tissue image output allows for improved interaction with pathologists, better histological context, and unbiased spatial analysis. In this study we utilized a 16-marker panel consisting of CD3, CD4, CD8, FoxP3, CD20, CD68, CD163, HLA-DR, PD-1, PD-L1, CTLA-4, CD34, Vimentin, SOX10, Ki67, and PanCK to generate immune profiles for colorectal carcinoma, prostate carcinoma, non-Small cell lung cancer, and triple negative breast cancer samples. We showed concordance with the current MultiOmyx imager - the IN Cell 2200 - for each of the markers in the panel. Additionally, we demonstrated that the updates made to the proprietary NeoLYTX image analysis pipeline are able to facilitate the significantly larger data sets that will be generated with the new MultiOmyx platform. This study successfully demonstrates the capability of the improved MultiOmyx system to characterize the distribution of T-cell and myeloid phenotypes in the tumor microenvironment using the proven MultiOmyx workflow, now enhanced by whole slide images and analysis.

Citation Format: Michael Lazare, Harry Nunns, Fatemeh Jelvhei Moghaddam, Xiaowei Jin, Courtney Todorov, Eric Leones, Marianne Thio, Flora Sahafi, Erinn Parnell, Qingyan Au. Improved spatial biology analysis of the tumor microenvironment with the next generation of the MultiOmyxTMplatform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5487.
Abstract 5491: Mapping ovarian cancer spatial organization uncovers immune evasion drivers at the genetic, cellular, and tissue level
Authors: Aguirre Karmen , Yiwen-Yeh Christine , Laveroni Olivia
Published: March 2024

Immune exclusion and evasion are central barriers to the success of immunotherapies and cell therapies in solid tumors. Here we applied single cell spatial and perturbational transcriptomics alongside clinical,histological, and genomic profiling to elucidate immune exclusion and evasion in high-grade serous tubo-ovarian cancer (HGSC). Using high-plex spatial transcriptomics we profiled more than 1.3 million cells from 95 tumors and 60 patients, revealing generalizable principles in HGSC tumor tissue organization.Our data demonstrates that effector T cells resist stroma-mediated trapping and sequestration. However, upon infiltration into the tumor, T cells, as well as Natural Killer (NK) cells, preferentially co-localize only with a subset of malignant cells that manifest a distinct transcriptional cell state. The latter consists of dozens of co-regulated genes and is repressed under various copy number alterations. Performing CRISPR Perturb-seq screens in ovarian cancer cells, we identified functionally diverse genetic perturbations - including knockout of the insulin sensing repressor PTPN1 and the epigenetic regulator ACTR8 - that de-repress the proposed immunogenic malignant cell state identified in patients and indeed sensitize ovarian cancer cells to T cell and NK cell cytotoxicity. Taken together, our study uncovered a profound connection between somatic genetic aberrations, malignant cell transcriptional dysregulation, and immune evasion at the cellular and tissue level, allowing us to identify targets that reprogram malignant cell states as an avenue to unleash anti-tumor immune responses.

Citation Format: Karmen Aguirre, Christine Yiwen-Yeh, Olivia Laveroni. Mapping ovarian cancer spatial organization uncovers immune evasion drivers at the genetic, cellular, and tissue level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5491.
Abstract 5513: Sexual differences in the aged melanoma tumor microenvironment dictates metastasis and therapeutic responses
Authors: Chhabra Yash , Fane Mitchell , Pramod Sneha , Hueser Laura , Zabransky Daniel , Wang Vania , Kumah Edwin , Carey Alexis , Harper Elizabeth , Rocha Murilo Ramos , Weeraratna Ashani
Published: March 2024

There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear; while biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the microenvironment surrounding the tumor, contextually in aging, has been overlooked. We find that skin fibroblasts undergo age-mediated changes in their proliferation, senescence, ROS levels and stress response that vary with sex. We find that aged male fibroblasts selectively drive an invasive and slow-cycling phenotype in melanoma cells in vitro by increasing AXL expression. This is also evident in syngeneic mouse models where metastasis is increased in aged male mice. Mechanistically, intrinsic aging in male fibroblasts coupled with elevated ROS promotes EZH2 decline thereby increasing BMP2 secretion, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of the invasive phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. Our data provides an integrated view of how age and sex of the host contributes to melanoma progression and therapy responses. Bridging this knowledge gap will improve patient stratification and assist in tailoring the therapy to the individual.

Citation Format: Yash Chhabra, Mitchell Fane, Sneha Pramod, Laura Hueser, Daniel Zabransky, Vania Wang, Edwin Kumah, Alexis Carey, Elizabeth Harper, Murilo Ramos Rocha, Ashani Weeraratna. Sexual differences in the aged melanoma tumor microenvironment dictates metastasis and therapeutic responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5513.
Abstract 5517: Chronic stress causes metastasis via neutrophil-mediated changes to the microenvironment
Authors: He Xue-Yan , Gao Yuan , Ng David , Michalopoulou Evdokia , George Shanu , Adrover Jose M. , Sun Lijuan , Albrengues Jean , Daßler-Plenker Juliane , Han Xiao , Wan Ledong , Wu Xiaoli S. , Shui Longling S. , Huang Yu-Han , Liu Bodu , Su Chang , Spector David L. , Vakoc Christopher R. , VanAelst Linda , Egeblad Mikala
Published: March 2024

Chronic stress is associated with increased risk of metastasis and poor survival in cancer patients, yet the reasons are unclear. We show that chronic stress increased lung metastasis from disseminated cancer cells 2- to 4-fold in mice. Chronic stress significantly altered the lung microenvironment, with fibronectin accumulation, reduced T cell infiltration, and increased neutrophil infiltration. Depleting neutrophils abolished stress-induced metastasis, indicating their critical role. Chronic stress shifted neutrophils’ normal circadian rhythm, and, via glucocorticoid release, caused increased neutrophil extracellular trap (NET) formation. Importantly, in mice with neutrophil-specific glucocorticoid receptor deletion, chronic stress failed to increase NETs and metastasis. Furthermore, digesting NETs with deoxyribonuclease (DNase) I prevented chronic stress-induced metastasis. Together, our data show that glucocorticoids released during chronic stress cause NET formation and establish a metastasis-promoting microenvironment. Therefore, NETs could be targets for preventing metastatic recurrence in cancer patients, many of whom will experience chronic stress due to their disease.

Citation Format: Xue-Yan He, Yuan Gao, David Ng, Evdokia Michalopoulou, Shanu George, Jose M. Adrover, Lijuan Sun, Jean Albrengues, Juliane Daßler-Plenker, Xiao Han, Ledong Wan, Xiaoli S. Wu, Longling S. Shui, Yu-Han Huang, Bodu Liu, Chang Su, David L. Spector, Christopher R. Vakoc, Linda VanAelst, Mikala Egeblad. Chronic stress causes metastasis via neutrophil-mediated changes to the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5517.
Abstract 5568: Understanding the role of immune exclusion in mammary tumor growth
Authors: Mitra Payal , Azpurua Jorge , Chiang Huai-Chin , Li Rong
Published: March 2024

Multiple solid tumor types, including triple-negative breast cancer (TNBC), respond poorly to immunotherapy due to low infiltration of CD8+ T cells into the tumor core. The extracellular matrix (ECM) and its components such as collagen have been found to contribute to immune cell exclusion. Discoidin domain receptor 1 (DDR1) is a collagen receptor known to be aberrantly expressed in TNBC among multiple solid tumor types. We have uncovered a previously unappreciated function of DDR1 in immune exclusion through shaping a collagen fiber-based tumor defense against anti-tumor immunity (Sun et al, Nature 599, 673-678 (2021)). Our study showed that deletion of DDR1 in mouse mammary carcinoma cell lines such as E0771 and M-Wnt (syngeneic with C57BL/6 strain background) promotes infiltration of CD8+ T cells and reduces tumor growth in an immunocompetent mouse model. Extending the published study to other immunocompetent mouse strain backgrounds, we observed that deletion of DDR1 in EMT6 cells, another immune excluded TNBC mouse model in the BALB/c strain, increased the infiltration of CD8+ T cells to the tumor core and reduced tumor growth. Data concerning the DDR1-dependent spatial architecture of the mammary tumor microenvironment will be presented. Identifying specific immune and stromal cell populations and proteins spatially modified in the presence or absence of DDR1 may aid in development of new stand-alone or combination anti-cancer treatments with anti DDR1 antibody.

Citation Format: Payal Mitra, Jorge Azpurua, Huai-Chin Chiang, Rong Li. Understanding the role of immune exclusion in mammary tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5568.
Abstract 5612: MRE11 function in DNA damage response is regulated by SIRT2 deacetylation
Authors: Essien Mark , Sesay Fatmata , Zhang Hui , Jung Andrew , Yu David
Published: March 2024

MRE11 is known to play a role in DNA damage repair through the MRE11-RAD50-NBS1 (MRN) complex and homologous recombination. However, the upstream signaling mechanisms regulating MRE11 in DNA repair are unclear. Here, we aimed to determine the extent to which MRE11 is regulated by deacetylation by the SIRT2 sirtuin deacetylase and tumor suppressor protein. We overexpressed MRE11 in HEK293T cells, induced DNA damage through radiation treatment, and performed a series of co-immunoprecipitation (co-IP) studies with and without ionizing radiation (IR). Additionally, we evaluated the acetylation of MRE11 with and without the presence of SIRT2. In a series of functional experiments, immunofluorescence staining assays were performed in U2OS-265 Fok1 cells with SIRT2 knockdown to determine the effect of on MRE11 recruitment to DNA damage sites. Co-IP studies and in vitro DNA pulldown assays were performed to determine the role of SIRT2 in impacting MRE11 interaction with RAD50 and NBS1 and binding to DNA damage. We also examined for IR-induced RPA70 foci formation and downstream ATM-dependent signaling. We found that SIRT2 interacts with and deacetylates MRE11. Furthermore, SIRT2 deacetylation facilitated MRE11 binding to DNA but not interaction with RAD50 and NBS1. Finally, SIRT2 deacetylation facilitated DNA end resection and ATM-dependent phosphorylation of KAP1. In conclusion, our results define a role for SIRT2 in directing MRE11 localization and binding to DNA to promote DNA end resection and ATM-dependent signaling through deacetylation.

Citation Format: Mark Essien, Fatmata Sesay, Hui Zhang, Andrew Jung, David Yu. MRE11 function in DNA damage response is regulated by SIRT2 deacetylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5612.
Abstract 5613: Study of long non-coding RNA TUG1 kinetics under replication stress conditions in cancer cell
Authors: Fujita Tomoya , Suzuki Miho , Masui Osamu , Shinjo Keiko , Kondo Yutaka
Published: March 2024

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that are not translated into proteins. In contrast to protein-coding mRNAs, certain lncRNAs carry out their functions within the nucleus. A detailed elucidation of the kinetics of lncRNAs contributes to a comprehensive understanding of their bona fide biological characteristics. Consequently, advancements in the in vivo visualization of lncRNAs have continued. Currently, various techniques, including RNA imaging technologies, have been employed to facilitate real-time tracking and visualization of RNA molecules within living cells. Taurine upregulated gene 1 (TUG1) is an oncogenic lncRNA, playing a pivotal role in cancer cells by resolving R-loops formed at stalled replication forks, particularly under replication stress conditions such as Hydroxyurea (HU) treatment. Despite its significance, the dynamic regulation of TUG1 under replication stress conditions remains largely unknown. In this study, we developed a stem loop-based live imaging system enabling real-time visualization of TUG1, providing insights into its subcellular localization and dynamic roles in physiological contexts. Upon treatment with HU, a chemical that interferes with replication, TUG1 expression was rapidly upregulated in the nucleus within 20 minutes. Additionally, we observed TUG1 molecules relocating close to the nuclear membrane. Considering the reported relocation of collapsed forks to the nuclear pore complex, this suggests that TUG1 may be involved in this relocation structure. These data indicate that TUG1 is associated with the fork restore mechanism occurring at the relocated collapsed forks, and the current live-cell imaging system holds potential for further uncovering the critical roles of TUG1 in cancer cells. Our stem loop-based imaging may contribute to a more precise understanding of RNA dynamics in live cells.

Citation Format: Tomoya Fujita, Miho Suzuki, Osamu Masui, Keiko Shinjo, Yutaka Kondo. Study of long non-coding RNA TUG1 kinetics under replication stress conditions in cancer cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5613.
Abstract 5645: HuR regulates aerobic glycolysis in triple negative breast cancer (TNBC)
Authors: Dev J R Arundhathi , Gogia Ajay , Mathur Sandeep R. , Deo S.V.S. , Prasad Chandra Prakash
Published: March 2024

Introduction: The RNA-binding protein Human Antigen R (HuR/ELAVL1) is ubiquitously expressed and exerts post-transcriptional regulation on mRNAs characterized by U/AU-rich sequences in their 3' UTR. Elevated cytoplasmic expression of HuR has been associated with various malignancies, influencing key cellular processes such as proliferation, apoptosis, angiogenesis, and metastasis. The involvement of HuR influences prognostic outcomes and hence is a potential biomarker and therapeutic target in cancer research. The present study investigates the oncogenic role of HuR in TNBCs, an aggressive subtype of breast cancer.

Methods: Immunohistochemistry was performed on TNBC patient tumor tissue (n=40) to asses the expressions of HuR and MMP9. Human TNBC cell lines MDA-MB-231 and MDA-MB-468 were used for in vitro studies after confirming cytoplasmic HuR accumulation in these cells via Immunofluorescence. The RNA binding activity of HuR was disrupted using CMLD2, a specific inhibitor of HuR, and subsequent physiological changes were assessed through functional assays. An exploratory investigation of HuR-moderated metabolic changes was performed employing NANOSTRING nCounter XT gene expression assay using metabolic pathways panel. Investigation and/or validation of molecular changes were done via immunoblotting.

Results: Meta-analysis using Liu-2014 TNBC dataset correlated high HuR protein expression with reduced patient survival (p=0.028). HuR (77%) and MMP9(55%) were found to be overexpressed in TNBC tissues. Inhibiting HuR with CMLD2 resulted in the downregulation of the protein and its downstream targets CDK2, MMP9, and β catenin. HuR inhibition compromised proliferation, migration, Matrigel invasion and EMT (downregulated E-Cadherin, N-Cadherin, and Vimentin) in TNBC cell lines. Further, HuR inhibition resulted in decreased clonogenicity in 2D and 3D TNBC cultures. Transcriptomics studies showed that HuR inhibition in MDA-MB-231 altered the profile of glycolytic genes in addition to other pathways such as cell cycle and DNA damage repair. In accordance, the expression of glycolytic proteins was reduced upon CMLD2 treatment. Similar results were obtained on HuR knockdown by siRNA suggesting that HuR-mediated effects on TNBC progression might be in part through its modulation of aerobic glycolysis.

Conclusion: Overall, our results demonstrate HuR as a positive regulator of aerobic glycolysis in TNBCs and the ability of CMLD2 to inhibit HuR provides a rationale to consider it as a potential anticancer agent for TNBCs.

Citation Format: Arundhathi Dev J R, Ajay Gogia, Sandeep R. Mathur, S.V.S. Deo, Chandra Prakash Prasad. HuR regulates aerobic glycolysis in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5645.
Abstract 5672: Transcriptomic profiling of lung adenocarcinoma from never-smokers reveals molecular subtypes with clinical implications
Authors: Zhao Wei , Zhang Tongwu , Hoang Phuc H. , Sang Jian , Landi Maria Teresa
Published: March 2024

Lung cancer is the leading cause of cancer mortality worldwide and about 10-25% of lung cancers are from never-smokers (LCINS). Previous genomic studies of LCINS identified multiple genomic subtypes with different genetic drivers and evolutionary processes. To dissect the profile of LCINS cell states and tumor microenvironment, and their relationship with genomic lesions, we sequenced and assembled a large RNA-seq data set of 685 samples of lung adenocarcinoma from never-smokers and integrated them with whole genome sequencing data, pathological features and clinical outcomes from the same subjects. We identified three transcriptomic subtypes defined by distinct gene expression patterns. The three subtypes were associated with different pathway activities and showed remarkable heterogeneity in cell composition, lineage fidelity and pathological features. The genomic driver events and mutational signatures were significantly enriched in specific subtypes. Clinical outcomes also differed across the subtypes. For example, one subtype had prolonged overall survival and was associated with predicted response to immune checkpoint blockade. This study emphasizes the importance of transcriptome-based classification of LCINS, which has profound clinical implications beyond those provided by genomic and pathological assessment.

Citation Format: Wei Zhao, Tongwu Zhang, Phuc H. Hoang, Jian Sang, Maria Teresa Landi. Transcriptomic profiling of lung adenocarcinoma from never-smokers reveals molecular subtypes with clinical implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5672.
Abstract 5694: Elucidating areca nut-induced miRNA-mRNA regulatory networks in head and neck cancer pathogenesis
Authors: Huang Hung-Han , You Guo-Rung , Lin Kai-Hsin , Chen Yin-Ju , Cheng Ann-Joy
Published: March 2024

Areca nut has been considered a high-risk carcinogen for head and neck cancer (HNC) in Southeast Asia, while its molecular effects, especially on the miRNA regulatory roles, remain elusive. This study characterized miRNA-mRNA networks associated with areca nut-related HNC. We integratively analyzed the areca nut-induced mRNA profile and TCGA-HNSC gene set. A total of 2575 genes were identified, with 1971 upregulated and 604 downregulated in cancers. The KEGG pathway analysis revealed that these genes were enriched in the cellular functions related to motility and stress response. Experimental studies confirmed that arecoline promoted cell migration, invasion, and chemo-radioresistance. We further cross-analyzed the miRNAs induced by areca nut and dysregulated in TCGA-HNSC data and defined 71 molecules affected, with 35 upregulation and 36 downregulation. Using the multimiR tool to map potential mRNA-miRNA interactions, we uncovered that these miRNAs were connected to 2173 genes. These molecules participated in several oncogenic mechanisms, such as ErbB and PI3K-Akt signaling, cell adhesion, and DNA damage response. We endeavored to establish miRNA-mRNA interaction networks with the functions of cellular motility and stress response via the Cytoscape analytical method. Our data revealed nexus comprising 41 miRNAs with 83 mRNAs, and 38 miRNAs with 66 mRNAs, respectively, co-regulate these two malignant functions. A key miRNA, miR-410-3p, downregulated in HNC and decreased following areca nut treatment, was studied. Ectopic expression of miR-410-3p attenuated areca nut-induced cell invasion and exerted modulatory effects on its target oncogenes, CRK. Thus, our study delineates a comprehensive view of the miRNA-mRNA interplay altered by areca nut, illustrating a sophisticated mechanism of carcinogenesis and suggesting new avenues for diagnostic and therapeutic strategies in HNC.

Citation Format: Hung-Han Huang, Guo-Rung You, Kai-Hsin Lin, Yin-Ju Chen, Ann-Joy Cheng. Elucidating areca nut-induced miRNA-mRNA regulatory networks in head and neck cancer pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5694.
Abstract 5733: Development of a topoisomerase 1 inhibitor platform technology for efficacious and well tolerated ADCs
Authors: Lyski Ryan , Bou Lauren , Sigurjonsson Johann , Meyer David , Neace Calvin , Ortiz David , Snead Katie , Smith Kaleb , Jin Steven , Stevens Nicole , Cochran Julia , Alizadeh Kaveh , Yeddula Narayana , McKinney Erica , Peterson Adam , Dotloe Lindsay , Simmons Jessica , Carosino Christopher , Emmerton Kim , Everds Nancy , Jeffrey Scott , Senter Peter
Published: March 2024

Topoisomerase 1 (TOP1) inhibitors are a class of cytotoxic small molecules that have demonstrated effectiveness as therapeutic agents in cancer. However, the broad clinical application has been hindered by poor pharmacokinetic profiles and off-target toxicities. Antibody-drug conjugates (ADCs) enable the targeted delivery of cytotoxic payloads by specifically targeting cell-surface antigens expressed on cancers reducing systemic exposure and toxicity. TOP1-ADCs have received FDA approval for HER2- (trastuzumab-deruxtecan) and Trop2- (sacituzumab-govitecan) expressing solid tumors, demonstrating the promise for this drug class to improve the lives of patients. We have developed a novel drug-linker using a potent TOP1 inhibitor with desirable ADME properties, and a hydrophilic glycoside linker as a platform technology enabling ADCs with favorable physicochemical and biological properties. The TOP1 inhibitor payload was optimized for potency, with reduced P-glycoprotein efflux and enhanced permeability. The hydrophilicity of the linker facilitates the development of highly loaded ADCs (8 drugs/mAb) with high plasma stability, low aggregation, and pharmacokinetics similar to parental antibody. The resultant ADCs are potent and immunologically specific with activity in multidrug-resistant tumor models and bystander activity in heterogenous antigen models. Human cancer cell derived xenograft models demonstrated potent and specific ADC anti-tumor activity with several targets and indications. Improved activity was observed compared to corresponding govitecan- and deruxtecan-based ADCs. ADCs prepared from this drug-linker technology were well tolerated in rats after repeat administration of 60 mg/kg every 4 days for 4 doses. The excellent anti-tumor activity in several models and favorable safety profile demonstrates this platform TOP1 drug-linker technology has the potential for development as ADCs against many cancer targets.

Citation Format: Ryan Lyski, Lauren Bou, Johann Sigurjonsson, David Meyer, Calvin Neace, David Ortiz, Katie Snead, Kaleb Smith, Steven Jin, Nicole Stevens, Julia Cochran, Kaveh Alizadeh, Narayana Yeddula, Erica McKinney, Adam Peterson, Lindsay Dotloe, Jessica Simmons, Christopher Carosino, Kim Emmerton, Nancy Everds, Scott Jeffrey, Peter Senter. Development of a topoisomerase 1 inhibitor platform technology for efficacious and well tolerated ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5733.
Abstract 5741: siteDAR, complete antibody drug conjugate, ADC characterization
Authors: Martin Warham L. , Senseney Michael , Kusuma Bhaskar , Dugal-Tessier Julien , Jain Nareshkumar
Published: March 2024

Antibody drug conjugate characterization currently extends to the global determination of the drug -to- antibody ratio (DAR). However, there can be confounding batch variability in pharmacological outcome from ADCs of the same drug, antibody, and DAR. Previously, DAR was obtained by quantifying the extent of modification of the intact antibody or its protein subunits. The coupling chemistry is used to infer which type of amino acids were modified but generally no specific sites of modification are identified and if identification is obtained, it is not quantitative. Borrowing the rigorously reproducible proteolytic digestion methods from multi-atribute mapping (MAM) used in monoclonal antibody manufacture, and compiling a comprehensive quantitative assignment of every abundant peptide in the ADC digest we can both calculate a DAR from the ion volume ratios and a quantitative map of the specific modified sites. This method, termed “siteDAR”, is applied here to both commercial Kadcyla® (ado-trastuzumab emtansine) and in-house preparations of ado-trastuzumab emtansine. We show that preparations of Kadcyla® with the same DAR can have significantly different quantitative maps of modification sites. We use siteDAR analysis in our ADC design cycle to characterize an ADC, and have developed an ADC synthesis method that was quantitatively verified, and had increased biosimilarity with commercially available Kadcyla®. siteDAR for commercial Kadcyla siteDAR for initial in-house Kadcyla Initial biosimilarity

Citation Format: Warham L. Martin, Michael Senseney, Bhaskar Kusuma, Julien Dugal-Tessier, Nareshkumar Jain. siteDAR, complete antibody drug conjugate, ADC characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5741.
Abstract 5743: Liposome platform enables X-ray induced photodynamic therapy treatment against human triple negative breast cancer cells
Authors: Yang Biyao , Sang Rui , Li Yi , Goldys Ewa , Deng Wei
Published: March 2024

In this study, we employed X-ray induced photodynamic therapy (X-PDT) for the treatment on triple negative breast cancer (TNBC) cells. To do this, we rationally developed a liposome delivery system co-loaded with protoporphyrin IX (PPIX) and perfluorooctyl bromide (PFOB). Low-dose X-ray at 2Gy was employed to activate PPIX for reactive oxygen species (ROS) generation, and the co-loading of PFOB provided additional oxygen to augment ROS production. The highly toxic ROS triggered TNBC cell death. In vitro X-PDT effects including intracellular ROS generation, cytotoxicity, cell viability and apoptosis/necrosis assay in TNBC cells were studied. Our results indicate that the nanocarriers effectively induced X-PDT effect with very low dose radiation, which makes it possible to damage cancer cells. Our strategy may offer a paradigm-shifting treatment alternative for TNBC patients who need neoadjuvant radiotherapy but wish to avoid long term detrimental effect on functional outcome by undergoing X-PDT using only a fraction of the conventional radiotherapy.

Citation Format: Biyao Yang, Rui Sang, Yi Li, Ewa Goldys, Wei Deng. Liposome platform enables X-ray induced photodynamic therapy treatment against human triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5743.