Extracellular Space Research Articles

Abstract 447: Anti-tumor efficacy of ompenaclid, a creatine transporter inhibitor, in KRAS mutant tumors, including NSCLC

Abstract Background: Pre-clinical studies identified creatine metabolism to be activated in KRAS mutant metastatic colorectal cancer (CRC) as a mechanism to fuel the increased energy demands of RAS mutant tumors. Metastatic colon cancer cells overexpress and subsequently release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high energy metabolite phosphocreatine. Ompenaclid is a small molecule inhibitor of the creatine transporter SLC6A8. Inhibition of SLC6A8 by ompenaclid prevents import of phosphocreatine/creatine, resulting in depletion of intracellular creatine, phosphocreatine and ATP levels. This results in tumor apoptosis and robust anti-tumor efficacy in CRC models with a wide range of KRAS mutant alleles. Ompenaclid is currently in a Phase 2 trial, in combination with FOLFIRI and bevacizumab, in patients with RAS-mutated second-line advanced/metastatic colorectal cancer. Here, we assessed efficacy of ompenaclid in tumors beyond CRC, including KRAS WT and KRAS mutant NSCLC. Methods: Athymic mice were inoculated with CRC, pancreatic, gastric or NSCLC cancer cell lines and treated with ompenaclid once tumors reached ~150 mm3, until termination. Plasma metabolites were quantified by LC-MS/MS and tumoral expression of CKB and SLC6A8 in NSCLC and CRC tumors was measured by qPCR after treatment with a control or ompenaclid-supplemented diet for ~10 days. Combination treatment with MRTX849, a KRAS G12C inhibitor, was conducted in a pancreatic MiaPaca xenograft model. Results: Our studies demonstrate ompenaclid anti-tumor efficacy in NSCLC and pancreatic xenografts. Notably, and consistent with findings in CRC, efficacy in NSCLC was more robust in RAS mutant cancer models, relative to those bearing KRAS WT tumors. Metabolite analysis of the plasma of tumor-bearing mice at baseline revealed that mice harboring RAS mutant tumors had higher levels of p-creatine and lower levels of ATP, indicating that RAS mutant tumors utilize higher amounts of ATP, which may be converted to phosphocreatine by tumoral CKB, as confirmed by qPCR analysis. This finding is consistent with metastatic RAS mutant CRC tumors utilizing extracellular ATP to generate phosphocreatine and thus exhibiting greater dependency on creatine metabolism relative to RAS WT tumors. Treatment with ompenaclid also demonstrated additive efficacy in combination with KRAS G12C inhibitors. In summary, these data support development of ompenaclid in indications beyond CRC, including NSCLC, as well as in combination with KRAS G12C inhibitors. Citation Format: PuiChi Lo, Victor Sanz Chavez, Shugaku Takeda, Masoud Tavazoie, Isabel Kurth. Anti-tumor efficacy of ompenaclid, a creatine transporter inhibitor, in KRAS mutant tumors, including NSCLC [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 447.

Abstract 6957: Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary

Abstract The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with recipient cells inducing regulation of gene expression, activation of specific signaling pathways and tumor microenvironment remodeling. We investigated the EVs released by two cell lines of cancer of unknown primary site (CUP) that we established from patient’s tumor cells, namely CUP#55 and CUP#96, as described in https://www.medrxiv.org/content/10.1101/2023.03.12.23287041v1). CUP comprises 3-5% of new cancer cases and presents with metastases of unknown or uncertain origin and no apparent primary tumor. Our CUP cell lines are both characterized by FGFR2 gene amplification. It has been recognized that tumors use circular extrachromosomal DNA (ecDNA) as a way to increase oncogenic amplification. We hypothesized that FGFR2 amplification in CUP cell lines could be associated with ecDNA generation and that this ecDNA could be loaded as cargo inside EVs. FGFR2 copy number was quantified using a probe-based droplet digital PCR assay then we identified the different nature of FGFR2 amplification in the two models using FGFR2 FISH assay: CUP#55 presented a chromosomal homogeneously staining region while CUP#96 displayed double minute chromosomes. FGFR2 DNA was detectable in the cytoplasm fraction, therefore we isolated extracellular vesicles from culture medium using an ultracentrifugation-based protocol and confirmed the presence of the entire length of FGFR2 gene inside small and large vesicles. Since ecDNA contributes to cancer genome remodeling also forming chimeric circles, we confirmed the circular nature of a fraction of FGFR2 ecDNA using the Plasmid-Safe ATP-dependent DNAse test. Finally, we tested the oncogene delivery capability of CUP EVs by administering CUP#96 and CUP#55 small and large vesicles to recipient cell lines. We observed an increase of FGFR2 DNA at 24 hours upon EV treatments, and its functional transcription in FGFR2 mRNA after 48 hours. In conclusion, we identified a model of oncogene amplification and delivery in two cell lines of cancer of unknown primary, which could explain the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Citation Format: Irene Salamon, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Giulia Gallerani, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary [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 6957.

Abstract 540: Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development

Abstract Once cancer has metastasized, it is incurable because tumors evolve resistance to all systemically administered anti-cancer therapies. Resistance to therapy has classically been attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. Extracellular vesicles (EVs) are nano-sized (40-1000 nm) vesicles that are released by all cells into the extracellular space. EVs carry diverse cargo dependent on the cell of origin and have been studied as a source of novel cancer-specific liquid biopsy biomarkers and contributors to tumor progression and metastasis. However, the paracrine function of EVs in promoting therapy resistance is under-studied, and the impact of EVs released from therapy-resistant cancer cells on surrounding therapy-sensitive cancer cells is unknown. In this study, we develop an innovative microfluidic device, TREVCo, to enable simultaneous single-cell trap, EV detection, and co-culture modules to evaluate EVs-mediated transfer of cellular information between therapy-resistant cancer cells and adjacent cells, and to characterize EVs released from resistant cells and determine their impact on the resistance phenotype of adjacent therapy-sensitive cancer cells. We utilize resistant cells from multiple prostate (PCa) cell lines that have been selected for therapy resistance by culture with increasing dosages of cancer drugs such as cisplatin, docetaxel. We use the matched untreated therapy-sensitive parental cell lines as the sensitive cells. We hypothesize a novel model of therapy resistance in lethal PCa: EVs released from resistant cells contain pro-resistance factors that are transferred to adjacent sensitive cells upon EV uptake, thus directly enabling therapeutic resistance. We will assess the impact of resistant cell-EVs at a single cell level via a novel device, TREVCo, that enables comprehensive downstream analysis. This single-cell approach enables dissection of critical EV-associated therapy-resistant factors within the large heterogeneity of resistant PCa. Co-culture of a single resistant cell and a hundred adjacent sensitive cells mimicking intra-tumoral interactions is also critical to demonstrate evidence of resistant cell secreting factors inducing a pro-resistance phenotype. These studies will be the first-in-field to assess the impact of resistant PCa cell-derived EVs at single-cell resolution. An understanding of resistant cell-derived EVs and the impact of that EV cargo on adjacent sensitive cells will provide critical insight into the acquisition of therapy resistance in lethal PCa. The novel approach to apply resistant cell-derived EVs in co-culture assay with sensitive cells will enable the screening of transmittable resistance-associated factors and provides critical opportunities for accelerating precision medicine for advanced PCa patients. Citation Format: Chi-Ju Kim, Sarah Amend, Kenneth Pienta. Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development [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 540.

Abstract 5416: Purinergic P2Y6 receptor antagonists as potential anti tumor agents

Abstract Introduction: Cancer places a tremendous strain on society in both developed and economically less developed nations. It remains the most recurring and deadliest disease despite rapid advances in treatment modalities. The major contributors are the prevalence of known risk factors, urbanization, population growth and ageing. Cancer resurgence depends on the organ in which the tumor first appears, immune response, patient’s characteristics, disease stage and tumor microenvironment (TME). Among the several inflammatory molecules found within the TMEs, the levels of adenosine triphosphate (ATP) has been found to be high. In healthy tissue, the concentration of ATP in the extracellular space (eATP) is negligible and does not accumulate significantly due to the activity of ectonucleotidases. However, tissue injury, cellular stress and chemotherapy mediated cell death can increase the levels of ATP in the TME. The eATP exerts its effect mainly through purinergic P2 receptors expressed by most malignant cells. We have previously shown that eATP promoted migration and invasion of cancer cells in vitro through increased expression of the enzyme cyclooxygenase 2 (COX-2) (Sharma et al., 2021). We showed that eATP mediated COX-2 expression is through P2 receptors (Akter et al., 2021). Accordingly, we hypothesized that blocking the P2 receptors would abolish the COX-2-mediated migration of tumor cells and tested the same in an animal model. Methodology: Tumors were generated in 8-15-week-old male C57BL/6 mice (20-25 g weight) through subcutaneous injection of syngeneic EL4 lymphoma cells in the left flank of the mice. When tumors were palpable around day 10, saline or P2 receptor, antagonists were directly injected into the center of the tumor and mice were divided into three groups: saline (vehicle control), suramin (broad-spectrum P2 receptor antagonist), and MRS 2578 (P2Y6 receptor-specific antagonist). Drugs were chosen based on prior in vitro research and a vernier caliper was used to assess the growth of the tumor every day. Behavioral tests were performed on day 8 (prior to P2 receptor injections) and days 11, 14 and 17 post-tumor injection. Mice were continuously monitored for their health and were sacrificed on day 18 with deep CO2 anesthesia and cervical dislocation. Results: We found that suramin-injected tumors had a substantial reduction in tumor volume when compared to saline-injected ones. Intra-tumoral injections of P2 receptor antagonists such as MRS-2578 also resulted in similar outcomes as suramin. In tumors treated with P2 receptor antagonists, the expression of COX-2, cell cycle proteins and epithelial-to-mesenchymal (EMT) markers, which aid in metastasis, were significantly downregulated, indicating a suppressive effect. Therefore, we propose that P2 receptor-dependent anti-inflammatory drugs (PBAIDs) could be considered a potential alternative therapeutic option for preventing cancer recurrence. Citation Format: Khagendra Ghimeray, Shilpa Sharma, Ravi Shankar Akundi. Purinergic P2Y6 receptor antagonists as potential anti tumor agents [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 5416.

Abstract 449: Catabolism of GSH by GGT supports cancer cell survival

Abstract Cancer metabolism depends on amino acid sources to fuel tumor growth and drug resistance. Using amino acid restriction to treat tumors can enhance existing therapeutic techniques. The tripeptide Glutathione (GSH), which contains cysteine, glutamate, and glycine, is often considered an antioxidant, and its potential as an amino acid pool has been overlooked. GSH is ubiquitous in the tumor microenvironment and could provide a ready source of amino acids to tumor cells through its catabolism by the enzyme γ-Glutamyl transpeptidase (GGT), which has been shown to cleave GSH and its’ conjugates in the extracellular space. It is unclear if the cleavage of GSH by GGT supports cancers in vivo. We find that inhibiting GGT with the pharmacologic agent GGsTop blocks GSH catabolism and decreases tumor growth. We show that cultured cells can be rescued from a lethal depletion of cystine upon supplementation with GSH or its catabolic product cysteinylglycine, but not under the conditions of GGT inhibition. Through pharmacokinetic methods, we identify a usable systemic dose of GGsTop and show GGT inhibition in tumor tissue. Further, we demonstrate that systemic GGT inhibition in vivo decreases tumor mass and volume. These findings support the hypothesis that GSH catabolism by GGT1 is a non-canonical source of amino acids for tumor metabolism. Understanding GSH catabolism by GGT could elucidate new metabolic pathways in tumors. Inhibiting GSH catabolism could provide new therapeutic approaches to inhibit tumor growth. Citation Format: Marco Zocchi, Fabio Hecht Castro Medeiros, Fatemeh Alimohammadi, Emily Tuttle, Gloria Asantewaa, TashJae Scales, Isaac S. Harris. Catabolism of GSH by GGT supports cancer cell survival [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 449.

Abstract 3740: Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor

Abstract Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC) targeted to carcino-embryonic antigen-related cell adhesion molecule 5 (CEACAM5) and delivering DM4, a maytansinoid cytotoxic. CEACAM5 is highly expressed in several tumor types, including non-squamous non-small cell lung cancer (NSQ NSCLC). It is currently being evaluated in patients with advanced CEACAM5-positive NSQ NSCLC, in a phase II study in combination with pembrolizumab, and a phase III trial as single agent in comparison with docetaxel. Preclinical investigations were performed to determine whether SAR408701 could be beneficially combined with immune checkpoint inhibitors. We, first, demonstrated that the tusamitamab ravtansine payload, DM4, is able to induce robust immunogenic cell death (ICD) using an in vivo vaccination assay. In this assay, immunocompetent mice were “vaccinated” by DM4-treated CT26 cells and then later rechallenged with CT26 tumor cells. Tumor rejection was observed in more than 50% of “vaccinated” mice. The ICD was characterized by the expression or release of danger-associated molecular patterns (DAMPs), and we observed DAMP modulation such as calreticulin (CRT) exposure at the cell surface of dying tumor cells, and the release of the High-Mobility Group Box 1 (HMGB1) in the extracellular space by CT26 cells in response to DM4 treatment. We also showed that DM4-treated CT26 tumor cells implanted into mice or SAR408701 treatment of MC38 tumor-bearing mice induced an elevation of plasmatic level of murine IL-6 and INFγ, revealing potential antitumor immunity modulation by the ADC and its payload. Immunohistochemistry analysis of MC38 tumors also revealed that tusamitamab ravtansine treatment induced a dose-dependent increase of CD8 T-cell infiltration in tumors correlating to dose-dependent efficacy. Finally, tusamitamab ravtansine was combined with immune checkpoint inhibitors (ICIs) in immunocompetent mice bearing MC38 tumor model. In this model, the combination of tusamitamab ravtansine with anti-PD-1 or anti-PD-L1 antibody led to complete response and, even, tumor-free survivors at 120 days post tumor implantation while single agents were inactive or transiently active. In summary, these preclinical data show that tusamitamab ravtansine could be beneficially combined with immune checkpoint and strongly support their evaluation in clinical development. Citation Format: Celine Nicolazzi, Anne-Marie Lefebvre, Nicolas Moindrot, Christelle Larois, Marion Classe, Sukhvinder Sidhu. Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor [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 3740.

Abstract 560: IGF1 induces the expression of CYR61 in metastatic prostate cancer

Abstract The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. Interestingly, CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF1). High serum levels of IGF1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. The correlation between PCa aggressiveness and circulating levels of CYR61 and IGF1 is poorly studied. Our goal was to assess the contribution of CYR61-IGF1 interaction to PCa cell proliferation. Their combined role may explain a proportion of aggressive PCa. This study was designed to characterize the role of IGF1-induced expression of CYR61 in metastatic PCa cellular models and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, migration, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. In addition, we found that IGF1 induces protein expression of CYR61 in a time-dependent manner, validated by both immunoblotting and confocal microscopy. In conclusion, these data provided evidence that CYR61 expression is induced by IGF1, and its inhibition decreased the capacity of metastatic PCa cells to proliferate, indicating a potential utilization of CYR61 inhibition in the therapy of metastatic PCa. Citation Format: Greisha L. Ortiz Hernandez, Mya Walker, Leanne Woods-Burnham, Rick A. Kittles, Carlos A. Casiano, Susan Neuhausen. IGF1 induces the expression of CYR61 in metastatic prostate 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 560.

Abstract 6168: Sex-distinct patterns of molecular pathways associated with brain tumor edema

Abstract Magnetic resonance imaging (MRI) is a centerpiece of clinical management of brain tumor patients, yet the biology underlying even commonly interpreted imaging changes is unclear. The purpose of this project was to understand what biology is associated with brain tumor edema abundance and if that association was sex distinct. We have developed a two-compartment model of MRI signal intensity to quantitatively estimate relative edema abundance from T2-weighted MRIs. Multiparametric MRIs were preprocessed with bias field correction and intensity normalization. The images were co-registered and segmented for abnormal regions and normal tissue. The segmentations and preprocessed images were utilized in the edema mathematical model to estimate the fraction of extracellular space (Fecs) and fraction of intracellular space (Fics). Through an ongoing image-localized biopsy collection protocol, we analyzed 179 samples (Female: 75; Male: 104) from 55 patients (Female: 21; Male: 34) for bulk RNA-Seq. Samples were characterized by their edema Fecs scores and underwent differential expression, gene set enrichment analysis using MSigDB hallmarks and cellular deconvolution using CIBERSORTx. Through differential expression and gene set enrichment analyses of these spatial biopsies, we have identified sex-distinct gene expression and pathways corresponding to regions of high vs low edema. Within highly edematous regions, male samples were amplified for respiratory pathways, while female samples were suppressed. Connecting changes on MRIs with molecular markers from image-localized biopsies provides an opportunity to identify the biological drivers of brain tumor associated imageable edema. Specifically, elucidating the sex-distinct patterns connecting changes on MRI with cellular populations and molecular pathways could be used to better interpret imaging of patients and interpreting treatment response. Citation Format: Pamela R. Jackson, Lee Curtin, Sara Ranjbar, Kyle W. Singleton, Maciej M. Mrugala, Richard S. Zimmerman, Bernard R. Bendok, Peter Canoll, Kristin R. Swanson. Sex-distinct patterns of molecular pathways associated with brain tumor edema [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 6168.

Abstract 6968: Extracellular vesicles as carriers of signaling factors in cancer

Abstract Extracellular vesicles (EVs) are small, membrane-bound structures released by cells. They contain various biomolecules and have emerged as mediators of cellular signaling. EVs of various origins can be isolated from body fluids and hold potential as novel biomarkers. However, their clinical implementation as biomarkers is hampered by complex isolation procedures, limited expertise in routine labs, and a lack of mechanistic understanding of signal transfer, particularly in vivo. To better understand how EVs carry oncogenic signaling factors, we performed mechanistic investigations into the role of EVs in WNT signaling as a representative cancer-associated signaling pathway, as well as ex vivo validation experiments analyzing patient-derived EVs. Hydrophobic WNT ligands can use EVs to travel the extracellular space. However, little is known about the quantities of WNT secretion onto different EV fractions and potentially other vehicles. To explore WNT secretory routes and potential functional differences, we generated fluorescently tagged marker constructs of the non-canonical ligand WNT5a, which has context-dependent tumor-suppressing or -promoting functions in cancer. In additional in vitro experiments, we tested how the WNT signaling activation state and cancer-specific WNT mutations, such as CRISPR/Cas9-induced APC truncations, quantitatively and qualitatively affect EV shedding in colorectal cancer cell lines. Further functional testing of EV-bound vs. non-EV WNT ligands is ongoing. For proof-of-concept analyses and to test EVs as biomarkers, we set up the liquid biopsy study EValuate. The project aims for cross-entity analysis of EV profiles and correlation of results to cancer presence, stage, and survival. For quality control and before pipeline implementation, we tested various EV isolation methods and compared different sample pre-processing steps. By combining multidisciplinary expertise in biophysics, molecular biology, and basic and clinical cancer research, we aim to gain a better mechanistic understanding of EV-mediated signal transfer and facilitate the integration of EVs as diagnostic and prognostic biomarkers into clinical studies. Citation Format: Antonia Schubert, Nadine Winkler, Ajaree Mongkolsittisilp, Matthias Schulz, Oksana Voloshanenko, Meike Schaffrinski, Dominique Kranz, Karin Nienhaus, Dirk Jäger, Lorenz Trümper, Judith Büntzel, Claudia Binder, Gerd Ulrich Nienhaus, Michael Boutros. Extracellular vesicles as carriers of signaling factors 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 6968.

Abstract 1781: Elucidation of glutathione related dipeptide metabolism in cancer

Abstract There is a long-standing belief that supplementation with antioxidants could potentially treat diseases like cancer. Recent year studies showed, however, that they can behave like a double-edged sword and can also increase tumor proliferation and metastatic progression. The exact mechanism behind antioxidants' impact on tumors is poorly understood. Glutathione (GSH), the most abundant antioxidant in mammalian cells, is a tripeptide containing glutamate, cysteine, and glycine. GSH levels are reported to be significantly elevated within the extracellular space of the tumor. Our preliminary findings show that the breakdown of extracellular GSH can support tumor cell survival under cystine-free conditions. Extracellular GSH catabolism releases cysteinylglycine, which can enter the cell through a dipeptide transporter and be further broken down to supply cysteine and glycine to cells. Alternatively, cysteinylglycine can also be broken down extracellularly into cysteine and glycine, allowing for the uptake of these individual peptides. The molecular mechanisms surrounding the utilization of GSH-breakdown products by the tumor cell are largely unknown. We hypothesize that the proteins responsible for the supply of cysteinylglycine to the tumor cell are required for tumor growth and survival. To test this, we will use unbiased genetic and pharmacologic screening approaches to elucidate the proteins involved in cysteinylglycine processing. Preliminarily, we have found that inhibitors of dipeptidases prevent the ability of cysteinylglycine to support tumor cell survival under cystine starvation, suggesting that tumors require downstream utilization of GSH products. By identifying the mechanisms involved in cysteinylglycine breakdown and supply to tumor cells, we will potentially reveal novel therapeutic targets for cancer treatment, including those not relied upon by normal tissues. Further, our studies will potentially provide a better understanding of how antioxidants impact tumor growth and progression. Citation Format: Fatemeh Alimohammadi, Fabio Hecht, Marco Zocchi, Emily Tuttle, Gloria Asantewaa, TashJae Scales, Nathan Ward, Gina DeNicola, Isaac Harris. Elucidation of glutathione related dipeptide metabolism 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 1781.

Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection.

Molecular chaperones are protective in neurodegenerative diseases by preventing protein misfolding and aggregation, such as extracellular amyloid plaques and intracellular tau neurofibrillary tangles in Alzheimer's disease (AD). In addition, AD is characterized by an increase in astrocyte reactivity. The chaperone HSPB1 has been proposed as a marker for reactive astrocytes; however, its astrocytic functions in neurodegeneration remain to be elucidated. Here, we identify that HSPB1 is secreted from astrocytes to exert non-cell-autonomous protective functions. We show that in human AD brain, HSPB1 levels increase in astrocytes that cluster around amyloid plaques, as well as in the adjacent extracellular space. Moreover, in conditions that mimic an inflammatory reactive response, astrocytes increase HSPB1 secretion. Concomitantly, astrocytes and neurons can uptake astrocyte-secreted HSPB1, which is accompanied by an attenuation of the inflammatory response in reactive astrocytes and reduced pathological tau inclusions. Our findings highlight a protective mechanism in disease conditions that encompasses the secretion of a chaperone typically regarded as intracellular.

Abstract 1222: Mechanisms of succinate-mediated drug resistance in ER+ breast cancer

Abstract Breast cancer is the most prevalent cancer among women worldwide with estrogen receptor-positive (ER+) cancer constituting 70% of all breast cancers. Although ER+ breast cancers initially respond well to ER-targeted therapies and CDK4/6 inhibitors, approximately 30% patients acquire resistance to targeted therapies. In addition to intrinsic resistance that tumors develop, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment, also contribute to treatment-resistance and poor survival. The role of secreted metabolites by CAFs has been poorly studied in the context of targeted drug resistance. Therefore, to investigate the mechanism of stroma-mediated resistance to CDK4/6-inhibitors, we propagated patient-derived organoids (PDOs) and CAFs from treatment-naïve and treatment-resistant ER+ breast tumors. Our data show that the CAF conditioned media containing the polar metabolite fraction stimulates resistance to CDK4/6 inhibitors. Therefore, to identify metabolites that drive resistance, we performed an untargeted metabolomics on CAF conditioned media. We observed that the CAF conditioned media was enriched for TCA cycle intermediates, including succinate. Succinate is an oncometabolite that has an essential function in energy metabolism. Aside from causing metabolic reprogramming, succinate can be transported out of the mitochondria to perform other biological processes, such as epigenetic modifications and pseudohypoxia. Furthermore, succinate in the extracellular space binds to the succinate receptor SUCNR1 and induces G-protein coupled receptor-mediated signaling in cells expressing SUCNR1. To assess mechanism of resistance that these metabolites might induce, we treated breast cancer cells with the most enriched metabolites we had identified in our metabolomics assay. These experiments identified succinate as driver of CDK4/6 inhibitor resistance in ER+ breast cancer cells. Furthermore, treating tumor cells with SUCNR1 antagonist increased their sensitivity to CDK4/6-inhibitors, showing that succinate mediated resistance is conveyed via SUCNR1. This suggests that CAF secreted succinate plays a role in supporting a pro-tumor environment by promoting drug resistance towards CDK4/6 inhibitors. Defining this mechanism of resistance will elucidate potential therapeutic targets to augment patient response to therapy. Citation Format: Jenny Hogstrom, Boryana Petrova, Camila Perea, Madelyn Ward, Gerburg Wulf, Laura Collins, Naama Kanarek, Taru Muranen. Mechanisms of succinate-mediated drug resistance in ER+ 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 1222.

Real-Time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels.

The lymphatic vasculature, now often referred to as "the third circulation," is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i). This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]iand diameter measurements continuously in real time. The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

Abstract 47: Stabilization of long CAR T cells is crucial for in vivo antitumor efficacy

Abstract Introduction: Genetically engineered T cells expressing chimeric antigen receptors (CARs) have shown substantial anti-tumor efficacy in hematological malignancies during recent years. As the field evolves to novel target epitopes, longer CAR structures are needed to reach membrane proximal epitopes for improved efficacy. Our approach to reach such epitopes is to have (non-functional) SIRPalpha based extracellular extension, spacer, to our CAR structure. We have shown, in this proof-of-concept study, that stability of the extracellular spacer of a CAR plays a pivotal role to improve in vivo anti-tumor efficacy. Various SIRPalpha-based CARs, FiCARs, targeting CD19 was studied for their function. Protein stability of the CD19 CAR structures were characterized and subsequently the killing efficacy of target cells and cytokine release in in vitro as well as in vivo anti-tumor efficacy was evaluated. Methods & Results: In vitro co-culture assays with primary CD19 CAR T and CD19 positive cancer cells had shown no significant differences between the different spacer containing FiCAR structures. A dose dependent killing efficacy was observed in all the tested structures with up to >90% killing of NALM6 and Raji cells. Comparable results for IL-2, IFN-gamma, TNF-alpha, IL-4, IL-13 and granzyme B response were seen in co-cultured assay. In the protein stability studies, modifications in the extracellular part of the CAR structure led to an increase in thermal stability (over 2 °C) and reduced/halted multimer formation. To study the FiCAR T cell function in an in vivo tumor model, NSG mice were injected i.v with Raji cells and few days later mice were injected i.v with FiCAR T cells. Anti-tumor efficacy and overall survival were studied at the end of the study (60 days), the enhanced stability of FiCAR T structure translated into improved anti-tumor efficacy and survival. Interestingly, none of the mice survived in the least stable structure, 40% survival with intermediate stability structures and over 70% survival was observed in most stable structures. Conclusion: A stabilized SIRPalpha-based CD19 FiCARs dramatically enhance anti-tumor efficacy and survival in Raji tumor model. The stabilization is critical for improved efficacy, especially in the in vivo setting. This data suggests that, in addition to the length of the CARs stability of the CAR structure appear to play a crucial role in the efficacy of the CAR T cells. The modifications presented here enhanced CD19-targeting CAR T cell efficacy. This approach requires further investigation to improve the efficacy of currently approved CAR T cells, especially CD19-CAR T cells. Citation Format: Henrik Paavilainen, Jan Koski, Veera Nikoskelainen, Manar Elmadani, Endrit Elbasani, Can Hekim, Emmi-Leena Ihantola, Virginie Kergourlay, Pirjo Vehmaan-kreula, Marie Nyman, Kiira Kalke, Markus Nurmi, Hector Monzo, Paivi Ojala, Anu Autio, Matti Korhonen, Anil Thotakura. Stabilization of long CAR T cells is crucial for in vivo antitumor efficacy [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 47.

Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry

BackgroundCalcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN.MethodsIntracellular Ca2+ levels were measured over a time frame of 0–1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0–108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109–594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595–1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann–Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test.ResultsFollowing EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells.ConclusionsThis report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.

Serine synthesis and catabolism in starved lung cancer and primary bronchial epithelial cells

Serine and glycine give rise to important building blocks in proliferating cells. Both amino acids are either synthesized de novo or taken up from the extracellular space. In lung cancer, serine synthesis gene expression is variable, yet, expression of the initial enzyme, phosphoglycerate dehydrogenase (PHGDH), was found to be associated with poor prognosis. While the contribution of de novo synthesis to serine pools has been shown to be enhanced by serine starvation, the impact of glucose deprivation, a commonly found condition in solid cancers is poorly understood. Here, we utilized a stable isotopic tracing approach to assess serine and glycine de novo synthesis and uptake in different lung cancer cell lines and normal bronchial epithelial cells in variable serine, glycine, and glucose conditions. Under low glucose supplementation (0.2 mM, 3–5% of normal plasma levels), serine de novo synthesis was maintained or even activated. As previously reported, also gluconeogenesis supplied carbons from glutamine to serine and glycine under these conditions. Unexpectedly, low glucose treatment consistently enhanced serine to glycine conversion, along with an up-regulation of the mitochondrial one-carbon metabolism enzymes, serine hydroxymethyltransferase (SHMT2) and methylenetetrahydrofolate dehydrogenase (MTHFD2). The relative contribution of de novo synthesis greatly increased in low serine/glycine conditions. In bronchial epithelial cells, adaptations occurred in a similar fashion as in cancer cells, but serine synthesis and serine to glycine conversion, as assessed by label enrichments and gene expression levels, were generally lower than in (PHGDH positive) cancer cells. In summary, we found a variable contribution of glucose or non-glucose carbon sources to serine and glycine and a high adaptability of the downstream one-carbon metabolism pathway to variable glucose supply.

When Our Best Friend Becomes Our Worst Enemy: The Mitochondrion in Trauma, Surgery, and Critical Illness.

Common for major surgery, multitrauma, sepsis, and critical illness, is a whole-body inflammation. Tissue injury is able to trigger a generalized inflammatory reaction. Cell death causes release of endogenous structures termed damage associated molecular patterns (DAMPs) that initiate a sterile inflammation. Mitochondria are evolutionary endosymbionts originating from bacteria, containing molecular patterns similar to bacteria. These molecular patterns are termed mitochondrial DAMPs (mDAMPs). Mitochondrial debris released into the extracellular space or into the circulation is immunogenic and damaging secondary to activation of the innate immune system. In the circulation, released mDAMPS are either free or exist in extracellular vesicles, being able to act on every organ and cell in the body. However, the role of mDAMPs in trauma and critical care is not fully clarified. There is a complete lack of knowledge how they may be counteracted in patients. Among mDAMPs are mitochondrial DNA, cardiolipin, N-formyl peptides, cytochrome C, adenosine triphosphate, reactive oxygen species, succinate, and mitochondrial transcription factor A. In this overview, we present the different mDAMPs, their function, release, targets, and inflammatory potential. In light of present knowledge, the role of mDAMPs in the pathophysiology of major surgery and trauma as well as sepsis, and critical care is discussed.

Noggin promotes osteogenesis in human adipose-derived mesenchymal stem cells via FGFR2/Src/Akt and ERK signaling pathway

The balance between Noggin and bone morphogenetic proteins (BMPs) is important during early development and skeletal regenerative therapies. Noggin binds BMPs in the extracellular space, thereby preventing BMP signaling. However, Noggin may affect cell response not necessarily through the modulation of BMP signaling, raising the possibility of direct Noggin signaling through yet unspecified receptors. Here we show that in osteogenic cultures of adipose-derived stem cells (ASCs), Noggin activates fibroblast growth factor receptors (FGFRs), Src/Akt and ERK kinases, and it stabilizes TAZ proteins in the presence of dexamethasone. Overall, this leads ASCs to increased expression of osteogenic markers and robust mineral deposition. Our results also indicate that Noggin can induce osteogenic genes expression in normal human bone marrow stem cells and alkaline phosphatase activity in normal human dental pulp stem cells. Besides, Noggin can specifically activate FGFR2 in osteosarcoma cells. We believe our findings open new research avenues to further explore the involvement of Noggin in cell fate modulation by FGFR2/Src/Akt/ERK signaling and potential applications of Noggin in bone regenerative therapies.

Exosomes secreted by CSFV-infected cells evade neutralizing antibody to activate innate immune responses and establish productive infection in recipient cells

Exosomes, which are small membrane-enclosed vesicles, are actively released into the extracellular space by a variety of cells. Growing evidence indicates that exosomes derived from virus-infected cells can selectively encapsulate viral proteins, genetic materials, or even entire virions. This enables them to mediate cell-to-cell communication and facilitate virus transmission. Classical swine fever (CSF) is a disease listed by the World Organisation for Animal Health (WOAH) Terrestrial Animal Health Code and must be reported to the organisation. It is caused by classical swine fever virus (CSFV) belonging to the Flaviviridae family. Recent studies have demonstrated that extracellular vesicles originating from autophagy can facilitate the antibody-resistant spread of classical swine fever virus. However, due to the extreme difficulty in achieving a complete separation from virions, the role of exosomes during CSFV infection and proliferation remains elusive. In this study, we ingeniously chose to perform immunoprecipitation (IP) targeting the CSFV E2 protein, thereby achieving the complete removal of infectious virions. Subsequently, we discovered that the purified exosomes are shown to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes secreted by CSFV-infected cells can evade CSFV-specific neutralizing antibodies, establish subsequent infection, and stimulate innate immune system after uptake by recipient cells. In summary, exosomes play a critical role in CSFV transmission. This is of great significance for in-depth exploration of the characteristics of CSFV and its complex interactions with the host.

Modification of brain conductivity in human focal epilepsy: A model-based estimation from stereoelectroencephalography.

We have developed a novel method for estimating brain tissue electrical conductivity using low-intensity pulse stereoelectroencephalography (SEEG) stimulation coupled with biophysical modeling. We evaluated the hypothesis that brain conductivity is correlated with the degree of epileptogenicity in patients with drug-resistant focal epilepsy. We used bipolar low-intensity biphasic pulse stimulation (.2 mA) followed by a postprocessing pipeline for estimating brain conductivity. This processing is based on biophysical modeling of the electrical potential induced in brain tissue between the stimulated contacts in response to pulse stimulation. We estimated the degree of epileptogenicity using a semi-automatic method quantifying the dynamic of fast discharge at seizure onset: the epileptogenicity index (EI). We also investigated how the location of stimulation within specific anatomical brain regions or within lesional tissue impacts brain conductivity. We performed 1034 stimulations of 511 bipolar channels in 16 patients. We found that brain conductivity was lower in the epileptogenic zone (EZ; unpaired median difference = .064, p < .001) and inversely correlated with the epileptogenic index value (p < .001, Spearman rho = -.32). Conductivity values were also influenced by anatomical site, location within lesion, and delay between SEEG electrode implantation and stimulation, and had significant interpatient variability. Mixed model multivariate analysis showed that conductivity is significantly associated with EI (F = 13.45, p < .001), anatomical regions (F = 5.586, p < .001), delay since implantation (F = 14.71, p = .003), and age at SEEG (F = 6.591, p = .027), but not with the type of lesion (F = .372, p = .773) or the delay since last seizure (F = 1.592, p = .235). We provide a novel model-based method for estimating brain conductivity from SEEG low-intensity pulse stimulations. The brain tissue conductivity is lower in EZ as compared to non-EZ. Conductivity also varies significantly across anatomical brain regions. Involved pathophysiological processes may include changes in the extracellular space (especially volume or tortuosity) in epileptic tissue.