bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6797: CCR5/CCL4 axis in lung cancer clinicopathological significance and its therapeutic application
Authors: Singh Santosh K. , Singh Rajesh
Published: March 2024

Lung cancer (LC) is the most prevalent type of cancer globally and the leading cause of cancer-related deaths. Based on the histology and type of treatments, LC is divided into small-cells and non-small cell lung cancer (NSCLC) carcinomas, which are more aggressive and less responsive to chemotherapy. Since LC is known to be a highly heterogeneous cell population, treatment is challenging. It has been observed that chemokine receptor-ligand interaction is essential for cancer cells homing to distant sites. Most malignancies, including LC, have established disease status indicators concerning elevated levels of CCL4, a structurally unique chemokine, and its receptor (CCR5). Therefore, treatment strategies that inhibit the LC cell's tumor environment may improve patients' quality of life. However, the CCR5/CCL4 interface in LC invasion and metastasis is poorly understood. In this study, we examined the expression and function of the CCR5/CCL4 axis in the development and invasiveness of LC patient's clinical tissues and cell lines. We conducted immunohistochemistry (IHC) dual staining of CCR5/CCL4 antibodies in LC tissues. Our findings demonstrated significantly (p < 0.001) higher expression of CCR5 and CCL4 in advanced-stage malignant tissues than in normal tissues. The in-vitro analysis of the human LC cell lines NCI-H1437, NCI-H1573, and NCI-H1975 revealed CCL4 activation expressing higher CCR5, determined by immunofluorescence, western blots, flow cytometry, 3D-invasion assay, and RT-qPCR methods. Further, a calcium mobilization assay confirmed the direct interaction between CCR5 and CCL4. However, treating with Maraviroc, a small molecule inhibitor of CCR5, to LC cells blocks their ability to proliferate, demonstrating the crucial role that CCR5-CCL4 chemotaxis plays in LC metastasis. Our findings show both the biological and clinical significance of CCR5 and CCL4 expression, which may serve as potential indicators for metastatic LC. Therefore, inhibiting the CCR5/CCL4 interaction may offer a more effective therapeutic approach for managing the progression of LC.

Citation Format: Santosh K. Singh, Rajesh Singh. CCR5/CCL4 axis in lung cancer clinicopathological significance and its therapeutic application [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 6797.
Abstract 681: Rewiring collagen architecture using a highly potent bi-thiazole inhibitor of LOX mediates chemosensitization in triple-negative breast cancer
Authors: Cetin Metin , Saatci Ozge , Rezaeian Abdol-Hossein , Rao Chintada N. , Beneker Chad , Taylor Harrison , Pederson Breanna , Chatzistamou Ioulia , Buckley Brian , Lessner Susan , Angel Peggi , McInnes Campbell , Sahin Ozgur
Published: March 2024

Lysyl oxidase (LOX) is the founding member of the LOX family proteins and mediates the conversion of lysine residues in collagen I and elastin precursors into highly reactive aldehydes, leading to crosslinking of extracellular matrix (ECM). LOX is upregulated in aggressive tumors with high stiffness, and its higher expression correlates with metastasis, therapy resistance, and worse clinical outcome; however, relatively LOX-specific, orally bioavailable inhibitors are still lacking. Here we discovered bi-thiazole derivatives as potent LOX inhibitors by combining a robust screening platform for drug-like molecules, cell- and recombinant protein-based assays. Structure-activity relationship analysis further identified a potent and relatively LOX-specific lead compound (LXG6403) that reduces collagen content and crosslinking, and fibronectin assembly, leading to increased chemoresponse in triple-negative breast cancer (TNBC) cell lines and PDX organoids in 3D collagen. LXG6403 shows favorable pharmacokinetic properties, reduces collagen content and crosslinking, as demonstrated by the state-of-the-art MALDI-MSI and MP-SHG analyses, respectively. This leads to better drug penetration, inhibition of FAK signaling, and induction of ROS generation/DNA damage, leading to G1 arrest and apoptosis in chemoresistant TNBC PDXs. Overall, our novel, potent, tolerable bi-thiazole LOX inhibitor enhances chemoresponse in TNBC, the deadliest breast cancer subtype.

Citation Format: Metin Cetin, Ozge Saatci, Abdol-Hossein Rezaeian, Chintada N. Rao, Chad Beneker, Harrison Taylor, Breanna Pederson, Ioulia Chatzistamou, Brian Buckley, Susan Lessner, Peggi Angel, Campbell McInnes, Ozgur Sahin. Rewiring collagen architecture using a highly potent bi-thiazole inhibitor of LOX mediates chemosensitization in triple-negative breast cancer [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 681.
Abstract 6830: Xenium in situ analysis and reproducibility study of multiple carcinomas for clinical studies
Authors: Cheung Elim , Zhang Baowen , Zhang Lutong , Lu Tong , Gakhar Rikita , Sundaram Vidyodhaya
Published: March 2024

10x Genomics’ Xenium is the newest in situ spatial transcriptomics platform offering sub-cellular resolution in frozen and FFPE tissue sections for a deeper study of cellular mechanisms and interactions. Mapping RNA targets at high specificity with fully customizable assays and quick workflow, it uses rolling circle amplification and interpretation-ready data is available immediately after the run. At BioChain Institute, Inc. (BioChain), we are conducting validation studies for reproducibility of results with the Xenium platform using BioChain’s oncology biospecimen.FFPE tissues from BioChain’s repository were screened by our pathologist for tumor content above 30%. Four carcinoma tissues from Breast, Colon, Lung, and Kidney with high DV200 scores were selected. An array was constructed with these tissues to fit the Xenium slide’s imageable area. Two serial sections were used for the Xenium run per 10x Genomics’ protocol. The Off-the-shelf Human Multi-Tissue and Cancer Panel was used for targeting 377 genes. H&E staining and imaging was performed, and the image was integrated with the data on the Xenium platform. The results were found to be reproducible between two different operators. For example, in the breast section, over 96,000 cells were detected and median transcripts per cell were 70 in both the runs. There were 20 clusters representing different cell types/states, 99% transcripts were within cells, and ~85% of the transcripts were found to be high quality. Furthermore, we are analyzing the expression of unique tumor type and immune transcripts to map the heterogeneity of the tissues, as well as sensitivity, and standard deviation between the runs. The Xenium platform is a powerful tool for the sub-cellular spatial transcriptomics study of tissues with targeted gene panels. However, one of the four tissues used here failed to show enough transcripts. Further investigation is underway to examine the issue and conclude best practices thereafter. Our future studies will include accuracy and concordance validation with Xenium run on different days and Visium whole transcriptome assay post- Xenium.

Citation Format: Elim Cheung, Baowen Zhang, Lutong Zhang, Tong Lu, Rikita Gakhar, Vidyodhaya Sundaram. Xenium in situ analysis and reproducibility study of multiple carcinomas for clinical studies [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 6830.
Abstract 6839: Comprehensive transcriptomic analysis of acquired resistance after immune-checkpoint inhibitors in non-small cell lung cancer
Authors: Joe Cheol Yong , Bang Yeong Hak , Park Sehhoon , Jung Hyun Ae , Sun Jong-Mu , Choi Yoon-La , Ahn Jin Seok , Ahn Myung-Ju , Lee Se-Hoon
Published: March 2024

Background: Treatment of lung cancer has changed dramatically with the use of immune checkpoint inhibitors (ICI). Nevertheless, most patients underwent drug resistance even though they initially showed a response to ICI, and there is an unmet need to delineate the mechanism.

Method: We analyzed RNA sequencing data of paired pre- and post-ICI samples from 31 NSCLC patients. Among them, we identified 17 patients who underwent acquired resistance (AR) who initially showed partial or complete response or stable disease lasting more than six months, and the remaining 14 patients who showed primary resistance (PR) to ICI. The analysis was performed using a tumor microenvironment (TME) classifier, classifying immune-enriched [IE], immune-enriched and fibrotic [IE/F], fibrotic [F] and immune-desert [D] subtype, and CIBERSORTx, the immune cell deconvolutional analysis tool.

Results: Most patients with AR were initially classified as IE or IE/F subtypes (N=12, 70.6%), but six out of 14 with PR (42.8%) showed IE or IE/F subtypes. Among 12 patients with initial IE or IE/F subtypes, seven (58.3%) patients turned into the D subtype after AR to ICI. Most patients initially classified as D or F subtypes (N=5, 29.4%) maintained their subtypes (N=4, 80%) after AR.When assessing the pre-ICI samples, memory B cell was enriched, and M2 macrophage and regulatory T cell were depleted in AR patients compared to PR. The memory B cell and resting memory CD4+T cell decreased, and M2 macrophage increased after AR to ICI. These immune cell composition changes were not observed in PR patients.

Conclusions: We observed differences in TME phenotype and immune cell composition before ICI between AR & PR patients. The TME change from immune-enriched to desert and cell composition change of increased M2 macrophages and decreased memory B and resting CD4+ T cells after AR to ICI could be significant mechanisms of AR to ICI.

Citation Format: Cheol Yong Joe, Yeong Hak Bang, Sehhoon Park, Hyun Ae Jung, Jong-Mu Sun, Yoon-La Choi, Jin Seok Ahn, Myung-Ju Ahn, Se-Hoon Lee. Comprehensive transcriptomic analysis of acquired resistance after immune-checkpoint inhibitors in non-small cell lung cancer [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 6839.
Abstract 6840: Automated preparation of single nuclei from FFPE samples for snRNA-Seq
Authors: Jovanovich Stevan , Pereira Nate , Meyer Danielle , Snopkowski Catherine , Masilionis Ignas , Meškauskaitė Brigita , Takizawa Meril , Abou-Mrad Zaki , Yu Kenny , Tabar Viviane , Luckett Kathleen , Ganesh Karuna , Khan Nabiha , Chaligne Ronan
Published: March 2024

S2 Genomics developed the Singulator™ 100 and 200 Systems to automate the processing of fresh, frozen, or OCT tissues into single cell or single nuclei suspensions using disposable cartridges, reagents, and customizable protocols. The systems have been used to prepare cells or nuclei from a wide range of tissues and organisms, including human, mouse, chicken, insects, snails, zebrafish, and plants.

Formalin-fixed, paraffin-embedded (FFPE) tissues are the preferred format for pathologists and as many as one billion FFPE slides are in repositories. FFPE tissue samples have traditionally been difficult to study with molecular techniques due to the chemical modifications caused by the formalin fixation, which can lead to degradation and sequestration of RNA and DNA. Recent advances in single cell/nuclei RNA-Seq library preparation technology have made it possible to overcome these challenges and generate high-quality single cell or nuclei genomic data from FFPE samples. Protocols have been released for processing previously deparaffinized and rehydrated FFPE samples on the Singulator 100 or 200 into single nuclei.

We now report the development of a fully automated workflow for the deparaffinization and rehydration of FFPE tissues in about 40 min, with an optional enzymatic treatment of 45 min, and processing of FFPE tissues into singulated cells or nuclei on prototype Singulator 200 systems. Data will be presented for the optimization of process parameters for cell or nuclei recovery and RNA quality for six tissues. Finally, single nuclei sequencing data will be presented for snRNA-Seq libraries from healthy and cancerous human FFPE samples including glioblastomas and colorectal cancer. These data demonstrate the successful recovery of genomic materials from FFPE samples using an automated sample preparation system for single cell/nuclei transcriptomics.

Citation Format: Stevan Jovanovich, Nate Pereira, Danielle Meyer, Catherine Snopkowski, Ignas Masilionis, Brigita Meškauskaitė, Meril Takizawa, Zaki Abou-Mrad, Kenny Yu, Viviane Tabar, Kathleen Luckett, Karuna Ganesh, Nabiha Khan, Ronan Chaligne. Automated preparation of single nuclei from FFPE samples for snRNA-Seq [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 6840.
Abstract 6841: Mutation specific mechanisms of resistance to oncogenic KRAS inhibition
Authors: Barravecchia Ivana , Lasse-Opsahl Emily , Cavanaugh Sophia , Rober Lily , Baliira Rachael , Robotti Marzia , Hinshaw Rachael , Lee Jennifer M. , Zhang Yaqing , di Magliano Marina Pasca , Galbán Stefanie
Published: March 2024

Mechanisms of resistance to inhibitors against mutant KRAS are linked to the remodeling of the tumor microenvironment (TME). Understanding this remodeling process during intervention and tumor recurrence will likely guide the development of future therapeutic treatment paradigms with long-term responses. 56% of never-smokers with non-small cell lung cancer have KRASG12D mutant cancer and respond little to immune checkpoint therapy (ICI) compared to smokers, who often have mutations in KRASG12C. Our analysis of the TME in a unique KrasG12D inducible and reversible mouse model of NSCLC using single cell RNA sequencing data shows KrasG12D-dependent control of the TME, particularly of the expression of PDL1 in a subset of myeloid cells. PDL1 expression was found to be KrasG12D dependent as it was reduced significantly when KrasG12D was turned ON in mouse lungs. Interestingly, when KrasG12D was turned OFF, the expression of PDL1 increased, indicating mechanisms of resistance. Furthermore, the cytokine expression and secretion of IL6, a known regulator of PDL1 expression, increased in lung tissue and lung cancer cells derived from this model upon genetic and chemical (MRTX1133) Kras inhibition. Moreover, the mechanisms of resistance to oncogenic Kras inhibition appear to be mutant specific. Transcriptome and secretome analyses in human and murine KrasG12D or KrasG12C mutant lung cancer cells show distinct cytokine regulation upon KRAS inhibition. Identification of mechanisms of resistance like the induction of PDL1 expression upon KrasG12D inhibition may provide a strong rationale for co-treatment of KRAS inhibitors with ICI for KrasG12D never-smokers with NSCLC.

Citation Format: Ivana Barravecchia, Emily Lasse-Opsahl, Sophia Cavanaugh, Lily Rober, Rachael Baliira, Marzia Robotti, Rachael Hinshaw, Jennifer M. Lee, Yaqing Zhang, Marina Pasca di Magliano, Stefanie Galbán. Mutation specific mechanisms of resistance to oncogenic KRAS inhibition [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 6841.
Abstract 6892: Murine ex vivo platform: A powerful tool for biomarker assessment and high throughput screening of translationally relevant in vivo tumor models for PD and efficacy studies
Authors: Bachawal Sunitha , Leung Janet , Mandawe Remie , Garza Malia , ly Kimberly , Stefanson Ofir , Lohre Jack , Kamali Diba , Doan Ngan , King Benjamin , Kim Elliot , Hegde Ganapathi , Liao Xiaoyun , Liu Scot , Kim Jeong , Vigdal Amanda Mikels , Mukhopadhyay Anandaroop
Published: March 2024

Ex vivo model systems using tumor biopsies from patients are often utilized to develop predictive biomarkers. However, these readouts are often proximal and do not inform on downstream mechanisms (distal pharmacodynamics) and their relationship to anti-tumor efficacy. In vivo murine models represent a powerful tool to bridge this gap. However, selection of an appropriate translationally relevant murine model can often be a time-consuming process given that the complexity of the tumor microenvironment (TME). Additionally, in vivo models are generally not amenable to high throughput screening. Here, we demonstrate that ex vivo mouse tumor platforms can be used for rapid screening of treatment responsive and translationally relevant tumor models. These models can be used to interrogate the impact of a therapeutic on anti-tumor efficacy in a long-term in vivo study, thus allowing for the establishment of biomarker/response relationships in a rational and rapid manner. To operationalize this platform, ex vivo cultures of dissociated tumor cells (DTC) or tumor tissue slices (histoculture) were first optimized for maximum viability and/or preservation of tissue architecture. To allow for high throughput screening, feasibility of freezing DTCs or tumor chunks prior to sectioning was also explored. Finally, both ex vivo tumor cultures and in vivo tumors were treated with a Innate agonist as a proof of concept to demonstrate functional equivalency. In conclusion, this work provides a novel way of screening responsive, translationally relevant murine tumor models in a high throughput and cost-effective manner. These efforts have the potential benefits of saving time and animal usage, while also maximizing the probability of successfully evaluating a therapeutic’s benefit.

Citation Format: Sunitha Bachawal, Janet Leung, Remie Mandawe, Malia Garza, Kimberly ly, Ofir Stefanson, Jack Lohre, Diba Kamali, Ngan Doan, Benjamin King, Elliot Kim, Ganapathi Hegde, Xiaoyun Liao, Scot Liu, Jeong Kim, Amanda Mikels Vigdal, Anandaroop Mukhopadhyay. Murine ex vivo platform: A powerful tool for biomarker assessment and high throughput screening of translationally relevant in vivo tumor models for PD and efficacy studies [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 6892.
Abstract 6900: Patient-derived xenografts from circulating cancer stem cells and their potential utility in personalized medicine of pancreatic cancer
Authors: Pizon Monika , Schott Dorothea , Ettner-Sittner Andreas , Wagner Benedikt J. , Pachmann Katharina , Aung Thiha , Hackl Christina , Haerteis Silke
Published: March 2024

Background: Pancreatic cancer carries a poor prognosis in the majority of patients, with an overall 5-year survival rate of less than 10%. Therefore, the development of new therapies and preclinical models is crucial and urgently needed. Circulating cancer stem cells (cCSCs) are a subset of circulating cancer cells that have stem cell-like properties and play a significant role in tumor progression, metastasis, and treatment resistance. Patient-derived xenografts (PDX) reflect the genotypic and phenotypic landscape of patients' cancers and are useful for drug screening, biomarker development, and preclinical evaluation of personalized medicine strategies. In this study, we describe a fast, simple, and cost effective method to generate PDX on Chorioallantoic Membrane (CAM) from circulating cancer stem cells.

Methods: In this study, primary cultures from circulating cancer stem cells were established using sphere-forming assays from 30 patients with newly diagnosed pancreatic cancer. Subsequently, tumorspheres were transplanted onto the CAM membrane of fertilized chicken eggs to form patient- derived xenografts.

Results: For the identification of cCSCs from peripheral blood of pancreatic cancer patients, an innovative in vitro sphere-forming assay was established. The number of tumorspheres was correlated with clinicopathological parameters like tumor size, lymph node involvement and grading status of the primary tumor. Implantation of tumorspheres onto the CAM was successful in ~90 % of the applied samples. The histological analysis suggests that the PDX tumors were morphologically and pathologically identical to primary tumor tissue.

Conclusions: The number of tumorspheres was associated with clinicopathological parameters. Further follow-up is needed to evaluate the prognostic significance of tumorsphere detection in pancreatic cancer. Generation of PDX from cCSCs on CAM has a high success rate and may become an integral part of the drug development arena, including drug screening and biomarker development.

Citation Format: Monika Pizon, Dorothea Schott, Andreas Ettner-Sittner, Benedikt J. Wagner, Katharina Pachmann, Thiha Aung, Christina Hackl, Silke Haerteis. Patient-derived xenografts from circulating cancer stem cells and their potential utility in personalized medicine of pancreatic cancer [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 6900.
Abstract 693: The effect of butyrophilin 1A1 blockade in combination with radiation treatment in tumor models
Authors: Li Nan , Braun Chen , Kim Young-Seung , Park Andrew , Yoo Stephen S , Lin Steven H.
Published: March 2024

Expression of radiation-induced immune escape molecules, including PD-L1, contributes to the efficacy of cancer immunotherapy. We used an unbiased in vivo gene knockdown screen in naïve T-cells and introduced them into irradiated host tumors to identify novel immunosuppressive genes activated during radiation. From the screen, we found that butyrophilin 1A1 (BTN1A1) knockdown causes T-cell expansion in irradiated tumors. This points to the potential immune checkpoint role of BTN1A1. We validated that BTN1A1 expression is inducible in activated T-cells and that blocking BTN1A1 contributes to T-cell activation. We tested an anti-mouse BTN1A1 antibody (STC109) in mouse colon tumor models (CT26 tumors), we observed partial anti-tumor efficacy using single agent STC109, which was enhanced by the combination of STC109 with radiation. We also got similar result in in mouse lung tumor models (LLC tumors) using anti-mouse BTN1A1 antibody, and the combination treatment showed an improved suppression of tumor growth and lung metastasis compared to single agent. Moreover, our data shown that knockout of BTN1A1 in 4T1 tumor models shown dramatic defect of tumor growth, and the BTN1A1 ko tumors are more sensitive to radiotherapy. These findings indicate the role of BTN1A1 in tumor immune response from radiation stress, and that anti-BTN1A1 has strong potential to enhance efficacy of radiotherapy for cancer treatment.

Citation Format: Nan Li, Chen Braun, Young-Seung Kim, Andrew Park, Stephen S Yoo, Steven H. Lin. The effect of butyrophilin 1A1 blockade in combination with radiation treatment in tumor models [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 693.
Abstract 6960: Lysosome inhibition suppress collective cell migration through caveolin-1 mediated E-cadherin stabilization in colon cancer cells
Authors: Ching Yu-Chien , Chen Hui-Chun , Wang Yu-Chun , Cheng Chun-Pu , Ku Chia-En , Lee Shin-Ru , Chao Wei-Ting
Published: March 2024

In tumor nest growth and collective cell migration, the dynamics of E-cadherin is critical, and been shown to be regulated by cell vesicle traffic system. Caveolin-1 is the vesicle protein to regulate caveolae associated endocytosis, many of the membrane receptors are shown been regulated by caveolin-1 in cancer progression, however, the role is still controversial. This study demonstrated the effect of lysosome inhibition in caveolin-1 mediated E-cadherin turnover in the cell cohort of colon cancer cells. HT-29 colon cancer cells were used in this study, the cells were treated with chloroquine to inhibit lysosome activity. The cell spheroid from HT-29 cells were also collected according to cell adhesion ability. Caveolin-1 and E-cadherin were immuno-stained and revealed by confocal microscopy. In the result of immunostaining, caveolin-1 was showed the co-localization with E-cadherin at cell-cell contacts. When cells were treated with chloroquine, the lysosome inhibitor, the collective cell migration of HT-29 cells were suppressed, and stable caveolin-1 distribution was found accumulated on the cell-cell contacts., meanwhile, chloroquine has no effect on clathrin distribution at either cell-cell contacts or cell leading edge. Furthermore, the distribution of caveolin-1 at cell-cell contacts on cell spheroid that derived from HT-29 cells was decreased. The results of these study suggested lysosome plays important role in mediating caveolin-1 regulated E-cadherin dynamics in collective cell migration, but not in the cell spheroid formation. The results of these study demonstrated chloroquine can be the potential inhibitor for collective cell migration in colon cancer cells through caveolin-1 mediated vesicle transport.

Citation Format: Yu-Chien Ching, Hui-Chun Chen, Yu-Chun Wang, Chun-Pu Cheng, Chia-En Ku, Shin-Ru Lee, Wei-Ting Chao. Lysosome inhibition suppress collective cell migration through caveolin-1 mediated E-cadherin stabilization in colon 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 6960.
Abstract 6969: Determining the role of obesity in breast cancer progression
Authors: Perlman Alexandra , Piekny Alisa , Kuzmin Elena , Santosa Sylvia
Published: March 2024

Breast cancer, especially triple negative breast cancer, impacts a large proportion of the female population. Approximately ten to fifteen percent of all breast cancer diagnoses will be triple negative. Triple negative breast cancer lacks receptors that are used as therapeutic targets for other forms of breast cancer and has the poorest prognosis. Notably, the age of onset has also decreased, which correlates with the rise in prevalence of obesity. In addition, past studies revealed that maintaining a high BMI from childhood correlates with higher cancer mortality in adulthood. However, the mechanisms by which obesity contributes to cancer progression are not clear. Adipose tissue from different locations (visceral vs. subcutaneous, abdominal vs. femoral) and from patients with obesity from childhood rather than as adults could have different molecular characteristics. Further, depending on the genetic background, different profiles may be more susceptible to environmental cues that could drive their progression. To address this, we are using conditioned media collected from tissues that reflect these differences and determining how they affect TNBC cell lines with distinct genetic profiles. Preliminary data revealed that adipose tissue from both the abdominal and femoral regions of high BMI individuals caused an increase in the proliferation rate of TNBC cell lines. To determine if the adipocytes vs. other cell types in the adipose tissue drive this growth, we are co-culturing mature adipocytes from these tissues with the TNBC cell lines. Recent studies by other groups showed that adipocyte-derived extracellular vesicles could drive the expression of EMT genes in cancer cells. We will purify EVs from the adipocytes and characterize differences in their molecular signatures for different patients to determine which factors cause the change in TNBC progression and correlate this with different fat depots and/or age of onset. We will also measure the effect of adipose tissue and/or adipocytes on other cancer hallmarks including metastasis (migration and invasion) and changes in metabolism. These studies should reveal how adipocytes confer changes in TNBC progression, and how to consider obesity in different treatment regimes.

Citation Format: Alexandra Perlman, Alisa Piekny, Elena Kuzmin, Sylvia Santosa. Determining the role of obesity in breast cancer progression [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 6969.
Abstract 697: Enhanced tumor control and survival in solid tumor models following combined low dose radiotherapy and T cell therapy
Authors: Puebla Nahum , Srivastava Gitika , Nizzero Sara , Fowlkes Natalie Wall , Barsoumian Hampartsoum , Hu Yun , Leuschner Carola , Riad Thomas , Loparic Marko , Plodinec Marija , Welsh James
Published: March 2024

Background: Solid tumors remain a significant challenge in cancer therapy due to their resistance to conventional treatments. Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising approach for treating solid tumors, but its effectiveness is often limited by the poor infiltration of CAR-T cells into the tumor microenvironment. Thus, enhancing a robust infiltration of chimeric antigen receptor T cells into solid tumors is essential for an effective antitumor response and therapeutic success.

Methods: In this study, we investigated the combined effects of multiple doses of low-dose radiotherapy (LD-RT) and CAR T-cell therapy on tumor growth and survival using two models of NSG mice implanted with gastric carcinoma (GSU) or pancreatic adenocarcinoma (CAPAN-2) cells. The engineered CAR-T cells were designed to target guanyl cyclase-C (GCC) or mesothelin, respectively. Furthermore, our study employed atomic force microscopy (AFM) in a small cohort to investigate the impact of LD-RT and CAR-T cell therapy on tumor stiffness and plasticity.

Results: The synergistic effect of combining low-dose radiotherapy (LD-RT) with T-cell therapy resulted in enhanced tumor control and prolonged survival in mice with tumors, surpassing the outcomes observed with either monotherapy alone. This combined treatment holds promise for improving responses in patients with solid tumors by leveraging LD-RT to enhance the intratumoral infiltration of effector T cells. Our findings suggest that the nanomechanical signature may be a potential biomarker for LD-RT combined CAR T cell therapy in solid tumors.

Conclusions: Our findings substantially contribute to treating solid tumors using cell therapies, highlighting the potential for improved infiltration of adoptively transferred T cells following radiotherapy (RT), which can transform existing therapeutic strategies for solid tumors while minimizing toxicity.

Citation Format: Nahum Puebla, Gitika Srivastava, Sara Nizzero, Natalie Wall Fowlkes, Hampartsoum Barsoumian, Yun Hu, Carola Leuschner, Thomas Riad, Marko Loparic, Marija Plodinec, James Welsh. Enhanced tumor control and survival in solid tumor models following combined low dose radiotherapy and T cell therapy [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 697.
Abstract 6972: Programmed death-ligand 1 (PD-L1) upregulates c-MET phosphorylation via protein tyrosine phosphatase and contributes to the development of MET amplification in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer
Authors: Huang Derek De-Rui , Hsu Chia-Chi , Hsu Wei-Hsun , Liao Bin-Chi , Yang James Chih-Hsin
Published: March 2024

High expression of programmed death-ligand 1(PD-L1) is associated with poor survival outcomes in epidermal growth factor receptor (EGFR) mutation non-small cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors (EGFR-TKIs). In this study we aimed to determine how increased PD-L1 affects the signal transduction of EGFR-mutant NSCLC cells. We first established PD-L1 overexpression EGFR mutant NSCLC cells, and discovered that PD-L1 overexpression cells had upregulated c-MET phosphorylation compared with parental cells using receptor tyrosine kinase array study. Subsequently, we demonstrated that knocking out PD-L1 (CD 274) in EGFR mutant NSCLC cells resulted in down-regulation of c-MET phosphorylation. Since protein tyrosine phosphatases (PTP) are known key regulators for c-MET phosphorylation and dephosphorylation, the activity of PTP was assessed. In PD-L1 overexpression EGFR mutant NSCLC cells, the PTP activity was decreased, whereas in PD-L1 knock-out cells, the PTP activity was increased. Additionally, the downregulation of c-MET phosphorylation in PD-L1 knock-out cells could be restored by adding PTP inhibitor (Na3VO4). The gene expression analysis showed that PTP expression was down-regulated in PD-L1 overexpression cells, indicating that PD-L1 upregulates c-MET phosphorylation by reducing PTP expression. We further established xenograft animal models using NSCLC cells with strong baseline c-MET phosphorylation to determine the effect of c-MET phosphorylation on osimertinib (a 3rd-generation EGFR-TKI) treatment. We found that tumor cells with strong c-MET phosphorylation were prone to develop MET amplification as acquired resistance under osimertinib, which could be overcome with combination of osimertinib and tepotinib, a c-MET inhibitor. Taken together, our results expand our understanding of PD-L1’s role beyond an immune checkpoint in EGFR mutant NSCLC. PD-L1-regulated MET alterations could be a crucial mechanism leading to poor treatment outcome of EGFR-TKIs in PD-L1 overexpression EGFR mutant NSCLC.

Citation Format: Derek De-Rui Huang, Chia-Chi Hsu, Wei-Hsun Hsu, Bin-Chi Liao, James Chih-Hsin Yang. Programmed death-ligand 1 (PD-L1) upregulates c-MET phosphorylation via protein tyrosine phosphatase and contributes to the development of MET amplification in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer [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 6972.
Abstract 6986: Oncogenic CARD11 enhances marginal zone B cell survival by potentiating the non-canonical NF-κB pathway
Authors: Ghosh Anushka , Pomerantz Joel
Published: March 2024

Dysregulation of antigen receptor-mediated signaling contributes to the oncogenesis of lymphoid malignancies. The activated B cell-like (ABC) subtype of Diffuse Large B cell Lymphoma (DLBCL) is characterized by receptor-independent constitutive activation of the canonical nuclear factor-kappaB (NF-κB) pathway, which is essential for the proliferation and survival of tumor cells. Caspase recruitment domain-containing protein 11 (CARD11) is a scaffolding protein that regulates B and T cell receptor signaling to canonical NF-κB activation downstream for adaptive immune response. Oncogenic gain-of-function (GOF) CARD11 mutations bypass normal pathway regulation and result in a hyperactive CARD11 that signals constitutively. Although present in ~10% of human ABC-DLBCL biopsies, we do not have a complete mechanistic understanding of how CARD11 GOF mutations drive abnormal B cell proliferation to cause lymphoma. We generated a mouse expressing the DLBCL-associated GOF mutation C49Y from the endogenous CARD11 locus. GOF CARD11 was sufficient to specifically drive B cell expansion in the mice with no significant effect on CD4+ or CD8+ T cells or NK cells. Quantification of splenic lymphocytes revealed that CARD11C49Y/C49Y mice had ~5-fold more splenic B cells when compared to CARD11+/+ mice and ~13-fold more marginal zone (MZ) B cells. CARD11C49Y/C49Y MZ B cells proliferated and survived much better than CARD11+/+ following stimulation with αIgM and importantly exhibited significant survivability prior to stimulation. Bulk mRNA sequencing revealed genes uniquely dysregulated in CARD11C49Y/C49Y MZ B cells including those associated with non-canonical NF-κB pathway signaling. CARD11C49Y/C49Y MZ B cells exhibited elevated levels of the p100 precursor NF-kB subunit leading to enhanced production of nuclear p52 following BAFF treatment. CARD11C49Y/C49Y MZ B cells also exhibited elevated levels of TRAF2, TRAF3 and cIAP1/2, components of a complex that degrades the NIK kinase that is responsible in normal cells for inducing p100 processing. Our results reveal an unexpected ability of CARD11 GOF mutants to potentiate the non-canonical NF-κB pathway through p100 accumulation, which likely contributes to the dysregulated B cell expansion observed in DLBCL cases that harbor CARD11 GOF variants.

Citation Format: Anushka Ghosh, Joel Pomerantz. Oncogenic CARD11 enhances marginal zone B cell survival by potentiating the non-canonical NF-κB pathway [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 6986.
Abstract 6992: Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma
Authors: LIu Chung Ji , Lin Li-Han , Chang Kuo-Wei , Cheng Hui-Wen
Published: March 2024

Background:Oral squamous cell carcinoma (OSCC) stands as a prevalent malignancy worldwide. Profilin 2 (PFN2), an actin-binding protein regulating actin polymerization dynamics and associated with cell motility, has recently gained attention as a significant regulator in various cancers. However, the clinical significance and biological function of PFN2 in OSCC remain debatable. This study aims to elucidate the role of PFN2 in OSCC.

Methods:We assessed PFN2 expression in OSCC and adjacent normal oral mucosa tissues through Q-RT-PCR and immunohistochemistry analysis. Associations between PFN2 expression, clinicopathological features, and OSCC prognosis were analyzed. Cell proliferation, invasion, and migration effects were examined via MTT and Transwell assays. Western blotting assessed the protein expression related to epithelial–mesenchymal transition (EMT). Additionally, we established OSCC xenografts to determine tumorigenicity in vivo using female Nude mice.

Results:PFN2 RNA and protein expression were significantly increased in OSCC compared to normal oral mucosa. PFN2 expression positively correlated with lymphovascular invasion and cervical lymph node metastasis. MTT and Transwell assays demonstrated that PFN2 promoted OSCC cell proliferation, migration, invasion, and endothelial invasion. Downregulation of PFN2 induced an EMT phenotype, including an increase in the expression of the epithelial marker E-cadherin and a decrease in the expression of mesenchymal markers Vimentin, Snail, and Slug in OSCC cells in vitro. Similarly, OSCC cells with reduced PFN2 expression exhibited weaker tumorigenicity in vivo.

Conclusions:Our findings unveil a novel role for PFN2 in promoting OSCC progression and metastasis, suggesting its potential as an early biomarker for high-risk populations. Targeting PFN2 may offer a promising therapeutic strategy for OSCC treatment.

Citation Format: Chung Ji LIu, Li-Han Lin, Kuo-Wei Chang, Hui-Wen Cheng. Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma [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 6992.
Abstract 6996: A role for the cell adhesion molecule NCAM1 in regulating activity of the receptor tyrosine kinase RET in cancer
Authors: Hussain Montdher H. , Hyndman Brandy D. , Vizeacoumar Frederick S. , Crupi Mathieu J. , Vizeacoumar Franco J. , Mulligan Lois M.
Published: March 2024

RET (REarranged during Transfection) is a receptor tyrosine kinase with essential roles in early development; however, genetic alterations lead to aberrant RET activity that contributes to oncogenic processes in several cancers. While some of the mechanisms by which RET mediates these processes are known, the network of proteins facilitating its activity remains largely undiscovered. To address this, we performed a genome-wide synthetic dosage lethal screen which identified NCAM1 (Neural Cell Adhesion Molecule 1) as one potential candidate modulating RET activity. Using an NCAM1 knockdown (KD) in SH-SY5Y neuroblastoma cells, we show that loss of NCAM1 reduces total protein levels of RET with no effect on RET transcription. Despite reduced abundance of RET, we show that the mature form of RET has a prolonged half-life in the absence of NCAM1. Assessing the functional outcomes of reduced RET protein levels, we observe reduced activation of RET by its extracellular ligand GDNF (Glial cell line-derived neurotrophic factor) and a subsequent reduction in activation of proliferative signaling pathways downstream of RET. As a result, RET-mediated proliferation is diminished in the absence of NCAM1, with reduced viability of NCAM1 KD SH-SY5Y observed in response to GDNF. Together, our data suggest that the synthetic lethal relationship between RET and NCAM1 may arise from a role for NCAM1 in regulating total RET protein available for activation, thereby reducing RET-mediated cell viability and proliferation. Our work has uncovered a role for NCAM1 in modulating RET activity in cancer and validates the use of our synthetic dosage lethal approach in uncovering proteins facilitating oncogenic RET activity.

Citation Format: Montdher H. Hussain, Brandy D. Hyndman, Frederick S. Vizeacoumar, Mathieu J. Crupi, Franco J. Vizeacoumar, Lois M. Mulligan. A role for the cell adhesion molecule NCAM1 in regulating activity of the receptor tyrosine kinase RET in cancer [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 6996.
Abstract 7018: Application of HiTIMED and cell type adjusted epigenome-wide models identifies driver tumor DNA methylation alterations in multiple cancers
Authors: Vlasac Irma M. , Zhang Ze , Salas Lucas A. , Christensen Brock C.
Published: March 2024

DNA methylation (DNAm) is an epigenetic mark crucial in lineage specification and cellular differentiation, resulting in distinct patterns by cell type. These patterns can serve as biomarkers in deconvolution approaches inferring cell proportions from composite DNAm signals in biospecimens like blood and tumor tissue. Adjusting for cellular heterogeneity is critical to avoid confounding by cell type. The HiTIMED (hierarchical tumor immune microenvironment epigenetic deconvolution) algorithm provides high-resolution tumor microenvironment profiling, up to 17 cell types, including tumor, myeloid, and angiogenic compartments.

We analyzed bladder, prostate, and uterine cancers, applying HiTIMED on the tumor (n=1,341) and normal adjacent tissue (n=105), adjusting for age, sex, and cell types. DNAm data from The Cancer Genome Atlas (TCGA) was used, and HiTIMED was applied to deconvolute various cell proportions.

Linear model comparisons to identify significantly differentiated loci with/without cell type adjustment are summarized in Table 1. We demonstrate greater than 99% reduction in both hypermethylated and hypomethylated loci in all cancer types after adjustment for cell type. Our study demonstrates HiTIMED's applicability to deconvolve the tumor microenvironment in multiple cancer types and demonstrates the importance of adjusting for cell type in epigenome-wide association studies to identify differentially methylated loci, offering greater biological insight into the etiology of specific cancer types.

Significant differentially methylated loci in tumor versus normal adjacent tissue, FDR <0.05 Cancer type Linear model Hypermethylated in tumor (logFC >2.0) Hypomethylated in tumor (logFC < -2.0) Bladder cancer n = 21, normal adjacent tissue n = 413 tumor Model 1 (Cancer Status, Age, Sex) 4495 19,936 Model 2: (Cancer Status, Sex, age, six cell types) 27 39 Prostate adenocarcinoman = 50, normal adjacent tissuen = 499 tumor Model 1 (Cancer Status, Age) 8988 2207 Model 2: (Cancer Status, age, six cell types) 2 4 Uterine carcinoman = 34 normal adjacent tissuen = 429 tumor Model 1 (Cancer Status, Age) 11,698 16,249 Model 2: (Cancer Status, age, six cell types) 250 8245

Citation Format: Irma M. Vlasac, Ze Zhang, Lucas A. Salas, Brock C. Christensen. Application of HiTIMED and cell type adjusted epigenome-wide models identifies driver tumor DNA methylation alterations in multiple cancers [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 7018.
Abstract 705: miR-21-5p induces radioresistance through reinforced DNA damage response in oral squamous cell carcinoma
Authors: Liu Qi , Hu Weitao , Li Xinghan , Deng Yongqiang , Ma Lin
Published: March 2024

Cancers arising from oral cavity and oropharynx are commonly treated with radiotherapy, whereas tumor radioresistance results in poor outcome. The prerequisite for developing more effective interventions for these patients is that the molecular mechanisms leading to a radioresistant phenotype in these tumors are clear to researchers. Unfortunately, the radioresistant mechanisms remain elusive to date. We recently identified that a highly conserved and cancer-related microRNA, i.e. miR-21-5p, plays an important role in radioresistance of oral squamous cell carcinoma (OSCC). We analyzed the RNA-sequencing data of 290 tissue samples from TCGA database, and found the expression of miR-21-5p was significantly upregulated in many OSCC samples (N=271) when comparing to adjacent normal tissues (N=19). The difference was further confirmed on our freshly collected OSCC tissues from patients in operation room. We performed RNA-sequencing analysis in 11 OSCC samples and their adjacent normal tissues. Among the 2106 differentially expressed genes between OSCC and normal control groups, we found that several important DNA repair genes (RAD50 double strand break repair protein gene, RAD50; DNA Polymerase Theta, POLQ; Ataxia Telangiectasia and Rad3-related, ATR) were significantly overexpressed in OSCC samples (P<0.05), while positive correlations between miR-21-5p expression and DNA repair genes RAD51 and XRCC4 were identified in the OSCC cohort. Notably, ATR is an important DNA damage response gene, while XRCC4, RAD51, and POLQ are involved in DNA double strand break (DSB) repair through non-homologous end joining, homologous recombination, and microhomology-mediated end joining, respectively. OSCC cell lines were further recruited to study the relations of miR-21-5p with radiation-induced DSB repair. By analyzing kinetics of 53BP1 foci, a biomarker for detecting DSBs, we found that anti-miR-21-5p transfected cells showed more residual foci, indicating of unfinished repair of DSBs, at 5 hours and 18 hours post irradiation. The deficient DSB repair resulted from anti-miR-21-5p transfection were further confirmed by single cell gel electrophoresis with comet assay. Consistently, the same treatment resulted in fewer clonogenic survivors in colony formation assay, demonstrating increased radiosensitivity of OSCC cells when miR-21-5p was antagonized. This work provided novel insights about the mechanism of miR-21-5p-induced radioresistance in OSCC, which may give rise to novel therapeutic strategy for improved treatment of OSCC patients.

Citation Format: Qi Liu, Weitao Hu, Xinghan Li, Yongqiang Deng, Lin Ma. miR-21-5p induces radioresistance through reinforced DNA damage response in oral squamous cell carcinoma [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 705.