Patient-derived Primary Cultures Research Articles

Opposing effects of cannabidiol (CBD) on pheochromocytoma/paraganglioma and neuroendocrine tumor cell lines and individual patient-derived primary cultures

Opposing effects of cannabidiol (CBD) on pheochromocytoma/paraganglioma and neuroendocrine tumor cell lines and individual patient-derived primary cultures

Comparison of SARS-CoV-2 variants of concern in primary human nasal cultures demonstrates Delta as most cytopathic and Omicron as fastest replicating.

Comparative analysis of infections by SARS-CoV-2 ancestral virus and variants of concern, including Alpha, Beta, Delta, and Omicron, indicated that variants were selected for efficiency in replication. In infections of patient-derived primary nasal cultures grown at air-liquid interface to model upper respiratory infection, Omicron reached the highest titers at early time points, a finding that was confirmed by parallel population sampling studies. While all infections overcame dsRNA-mediated host responses, infections with Omicron induced the strongest interferon and interferon-stimulated gene response. In both primary nasal cultures and lower respiratory cell line, infections by Delta were most damaging to the cells as indicated by syncytia formation, loss of cell barrier integrity, and nasal ciliary function.

Evaluation of the role of AXL in fusion-positive pediatric rhabdomyosarcoma identifies the small-molecule inhibitor bemcentinib (BGB324) as potent chemosensitizer.

Rhabdomyosarcoma (RMS) is a highly aggressive pediatric cancer with features of skeletal muscle differentiation. Over 80% of the high-risk patients ultimately fail to respond to chemotherapy treatment, leading to limited therapeutic options and dismal prognostic rates. The lack of response - and subsequent tumor recurrence - is driven in part by stem cell-like cells, the tumor subpopulation that is enriched after treatment, and characterized by expression of the AXL receptor tyrosine kinase (AXL). AXL mediates survival, migration and therapy resistance in several cancer types; however, its function in RMS remains unclear. In this study, we investigated the role of AXL in RMS tumorigenesis, migration and chemotherapy response, and whether targeting of AXL with small molecule inhibitors could potentiate the efficacy of chemotherapy. We show that AXL is expressed in a heterogeneous manner in patient-derived xenografts (PDXs), primary cultures and cell line models of RMS, consistent with its stem-cell-state selectivity. By generating a CRISPR/Cas9 AXL knock-out and overexpressing models, we show that AXL contributes to the migratory phenotype of RMS, but not to chemotherapy resistance. Instead, pharmacological blockade with the AXL inhibitors bemcentinib (BGB324), cabozantinib and NPS-1034 rapidly killed RMS cells in an AXL-independent manner, and augmented the efficacy of the chemotherapeutics vincristine and cyclophosphamide. In vivo administration of the combination of bemcentinib and vincristine exerted strong anti-tumoral activity in a rapidly progressing PDX mouse model, significantly reducing tumor bruden compared to single-agent treatment. Collectively, our data identify bemcentinib as a promising drug to improve chemotherapy efficacy in RMS patients.

DNA-PKcs-mediated transcriptional regulation of TOP2B drives chemoresistance in acute myeloid leukemia.

Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNA-damage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline.

Cytidine Deaminase Resolves Replicative Stress and Protects Pancreatic Cancer from DNA-Targeting Drugs.

Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.

RBIO-11. BASAL BCL-2-INTERACTING MEDIATOR OF CELL DEATH (BIM) RESPONSIVENESS CORRELATES WITH RADIOSENSITIVITY IN MENINGIOMA

Abstract INTRODUCTION Radiotherapy remains the primary adjunctive treatment for recurrent or progressive meningioma. Nevertheless, varied clinical response to radiotherapy in meningioma remains a challenge, particularly in higher grade tumors. Currently there is a paucity of predictors of radio-sensitivity. Bim peptide is an intrinsic mediator of apoptosis, and Bim-responsiveness has been postulated as a metric of general sensitivity to apoptotic stress. Herein we investigated its utility as a metric of radio-sensitivity in meningioma. METHODS In an in vitro model of meningioma, a number of immortalized human and murine meningioma cell lines and patient-derived primary cultures (WHO grades 1 + 2), were exposed to ionizing radiation at range of doses (0, 2, 4, 8, 16Gy); cells were counted (Celigo, Nexelcom) to assess for growth. Separately, cultures were irradiated at 4Gy and apoptosis was analyzed 24H later using Annexin/PI staining by flow cytometry (Attune NxT). Lastly, untreated cultures were exposed to Bim peptide as a range of doses (0.01uM-10uM); induction of apoptosis was assessed via cytochrome C staining by flow cytometry. RESULTS We were able to recapitulate a wide range of radio-sensitivities both by growth metrics and radiation-induced apoptosis. Immortalized lines showed more radiation-induced growth inhibition relative to primary cultures (p = 0.03). Activation of apoptosis in response to Bim exposure demonstrated a similarly wide range of basal apoptotic sensitivities, akin to that seen for radio-sensitivity. In fact, there was a significant correlation (Pearson, p < 0.01) between Bim-responsiveness and levels of radiation-induced apoptosis. CONCLUSIONS We identify basal state Bim-responsiveness as a potential predictor of radio-sensitivity in meningioma. Further work in this vein may shed light on mechanisms of radio-resistance in meningioma, particularly with respect to the interaction of Bim with the anti-apoptotic members of the Bcl-2 family.

Chemosensitivity of three patient-derived primary cultures of canine pericardial mesothelioma by single-agent and combination treatment.

Canine mesothelioma is a rare malignant tumor that mostly affects body cavities, such as the pericardial and pleural cavities. Chemotherapy plays a crucial role in the treatment of canine mesotheliomas. We aimed to compare the antitumor effects of single-agent and combination chemotherapeutic agents on patient-derived primary cultures of canine pericardial mesothelioma established in this study. We planned to generate xenograft models for future studies. Effusion samples were collected from three dogs with histologically diagnosed pericardial mesothelioma and used for primary culture. Cultured cells were characterized by immunostaining for pan-cytokeratin AE1/AE3, vimentin, Wilms' tumor suppressor gene 1 (WT1), and cytokeratin 5 (CK5). To assess the tumorigenic properties of cells in the effusion and generate a xenograft model, the cell suspension was injected into a severe combined immunodeficient (SCID) mouse either subcutaneously (SC) or intraperitoneally (IP). Lastly, chemosensitivity of established primary cultures against four drugs, doxorubicin, vinorelbine, carboplatin, and gemcitabine, by single-agent treatment as well as combination treatment of carboplatin at a fixed concentration, either 10 or 100 μM, and gemcitabine at different concentrations ranging from 0-1000 μM was assessed by cell viability assay. Primary cultures were successfully generated and characterized by dual positivity for AE1/AE3 and vimentin and positive staining for WT-1 and CK5, confirming the mesothelial origin of the cells. In the xenograft models, SC mouse developed a subcutaneous mass, whereas IP mouse developed multiple intraperitoneal nodules. The masses were histopathologically consistent with mesotheliomas. The chemosensitivity assay revealed that carboplatin had the highest anti-tumor effects among the four tested single-agent treatments. Furthermore, carboplatin at 100 μM combined with gemcitabine at clinically relevant doses demonstrated the augmented anti-tumor effects compared to single-agent treatment. Primary cultures and xenograft models generated in this study could be useful tools for in vitro and in vivo studies of canine mesothelioma. Carboplatin is a highly effective chemotherapeutic agent against canine mesothelioma when used as a sole agent and in combination with gemcitabine.

Evidence for the expression of vasorin in the human female reproductive tissues

Objective: To investigate the expression and localization of Vasorin (Vasn) in human female reproductive system. Methods: The presence of Vasorin was evaluated by RT-PCR and immunoblotting analyses in patient-derived endometrial, myometrial and granulosa cells (GCs) primary cultures. Immunostaining analyses were performed to detect Vasn localization in primary cultures and in ovarian and uterine tissues. Results: Vasn mRNA was detected in patient-derived endometrial, myometrial and GCs primary cultures without significant differences at the transcript level. Otherwise, immunoblotting analysis showed that Vasn protein levels were significantly higher in GCs than proliferative endometrial stromal cells (ESCs) and myometrial cells. Immunohistochemistry performed in ovarian tissues revealed that Vasn was expressed in the GCs of ovarian follicles at different stages of development with a higher immunostaining signal in mature ovarian follicles such as the antral follicle or on the surface of cumulus oophorus cells than in early-stage follicles. The immunostaining of uterine tissues showed that Vasn was expressed in the proliferative stroma endometrium while it was significantly less expressed in the secretory endometrium. Conversely, no protein immunoreactivity was revealed in health myometrial tissue. Conclusions: Our results revealed the presence of Vasn in the ovary and the endometrium. The pattern of Vasn expression and distribution suggests that this protein may have a role in the regulation of processes such as folliculogenesis, oocyte maturation, and endometrial proliferation.

AhR and HIF-1α Signaling Pathways in Benign Meningioma under Hypoxia.

The role of hypoxia in benign meningiomas is less clear than that in the malignant meningiomas. Hypoxia-induced transcription factor 1 subunit alpha (HIF-1α) and its downstream signaling pathways play a central role in the mechanism of hypoxia. HIF-1α forms a complex with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein and can compete for ARNT with aryl hydrocarbon receptor (AhR). In this work, the status of HIF-1α- and AhR-dependent signaling pathways was investigated in World Health Organization (WHO) grade 1 meningioma and patient-derived tumor primary cell culture under hypoxic conditions. mRNA levels of HIF-1α, AhR, and of their target genes as well as of ARNT and nuclear receptor coactivator NCOA2 were determined in tumor tissues from patients in whom the tumor was promptly removed either with or without prior endovascular embolization. Using the patient-derived nonembolized tumor primary cell culture, the effects of a hypoxia mimetic cobalt chloride (CoCl2) and an activator of the AhR signaling pathway benzo(α)pyrene (B[a]P) on mRNA levels of HIF-1α, AhR, and their target genes were investigated. Our findings show active functioning of AhR signaling in meningioma tissue of patients with tumor embolization and crosstalk between HIF-1α and AhR signaling in meningeal cells under hypoxia.

Effect of extended treatment with IAP inhibitor xevinapant post radiotherapy (RT) on efficacy and the tumor microenvironment (TME) in preclinical models.

6027 Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis, induced by RT and chemotherapy, and enhance antitumor immunity. In a randomized phase 2 study of patients with unresected locally advanced squamous cell carcinoma of the head and neck, xevinapant + chemoradiotherapy (CRT) significantly improved 18-month locoregional control and improved 3-year progression-free survival and 5-year overall survival vs placebo + CRT. A phase 3 confirmatory study (TrilynX NCT04459715) and a phase 3 study to evaluate xevinapant + RT in post-operative, high-risk, cisplatin-ineligible patients (XRay Vision NCT05386550) are ongoing. In these studies, we investigate 3 additional cycles of xevinapant or placebo monotherapy after completing combination treatment. Based on the role of IAPs in apoptosis, immunity, and stromal activation, continual dosing of xevinapant post RT may have additional therapeutic benefits. Methods: MC38 syngeneic tumor-bearing mice were treated with vehicle control, RT alone (3.6Gy once daily [QD], 5 days), or RT + xevinapant (100 mg/kg QD, 1, 2, or 4 weeks). To assess treatment-mediated TME modulation, immune cells in tumors were evaluated by fluorescence-activated cell sorting (FACS)–based immunophenotyping, and tumor-specific T cell responses were investigated by enzyme-linked immunosorbent spot (ELISpot) assays. To understand the effect of xevinapant on RT-induced cancer associated fibroblast (CAF) activation, CAF gene expression was profiled on cell lines and patient-derived primary cultures. Results: Xevinapant + RT increased antitumor efficacy compared with vehicle control or RT alone. Moreover, RT in combination with extended dosing of xevinapant for 4 weeks significantly improved therapeutic efficacy and prolonged survival compared with RT + shorter xevinapant dosing durations. FACS analysis demonstrated trends of increased numbers of CD8+ T cells, natural killer cells, and dendritic cells and decreased numbers of regulatory T cells in mice treated with xevinapant + RT. FACS and ELISpot further suggested an increase in antigen-specific T cell counts from baseline with combination therapy arm vs vehicle control or RT alone. Xevinapant may also suppress RT-mediated CAF activation, as indicated by downregulation of activation markers and CAF-derived secretory proteins. Conclusions: In preclinical models, extended dosing of xevinapant for 4 weeks post RT improved antitumor efficacy. Our data suggest that the effect may be, in part, modulated by TME responses, including enhanced antitumor immunity and suppressed pro-tumorigenic phenotype of CAFs. Based on these results, further evaluation is warranted to determine the mechanisms that underscore the therapeutic benefit offered by extended dosing of xevinapant.

Insights into Gene Regulation under Temozolomide-Promoted Cellular Dormancy and Its Connection to Stemness in Human Glioblastoma.

The aggressive features of glioblastoma (GBM) are associated with dormancy. Our previous transcriptome analysis revealed that several genes were regulated during temozolomide (TMZ)-promoted dormancy in GBM. Focusing on genes involved in cancer progression, Chemokine (C-C motif) Receptor-Like (CCRL)1, Schlafen (SLFN)13, Sloan-Kettering Institute (SKI), Cdk5 and Abl Enzyme Substrate (Cables)1, and Dachsous Cadherin-Related (DCHS)1 were selected for further validation. All showed clear expression and individual regulatory patterns under TMZ-promoted dormancy in human GBM cell lines, patient-derived primary cultures, glioma stem-like cells (GSCs), and human GBM ex vivo samples. All genes exhibited complex co-staining patterns with different stemness markers and with each other, as examined by immunofluorescence staining and underscored by correlation analyses. Neurosphere formation assays revealed higher numbers of spheres during TMZ treatment, and gene set enrichment analysis of transcriptome data revealed significant regulation of several GO terms, including stemness-associated ones, indicating an association between stemness and dormancy with the involvement of SKI. Consistently, inhibition of SKI during TMZ treatment resulted in higher cytotoxicity, proliferation inhibition, and lower neurosphere formation capacity compared to TMZ alone. Overall, our study suggests the involvement of CCRL1, SLFN13, SKI, Cables1, and DCHS1 in TMZ-promoted dormancy and demonstrates their link to stemness, with SKI being particularly important.

P2X7 receptor antagonism by AZ10606120 significantly reduced in vitro tumour growth in human glioblastoma

Glioblastomas are highly aggressive and deadly brain tumours, with a median survival time of 14–18 months post-diagnosis. Current treatment modalities are limited and only modestly increase survival time. Effective therapeutic alternatives are urgently needed. The purinergic P2X7 receptor (P2X7R) is activated within the glioblastoma microenvironment and evidence suggests it contributes to tumour growth. Studies have implicated P2X7R involvement in a range of neoplasms, including glioblastomas, although the roles of P2X7R in the tumour milieu remain unclear. Here, we report a trophic, tumour-promoting role of P2X7R activation in both patient-derived primary glioblastoma cultures and the U251 human glioblastoma cell line, and demonstrate its inhibition reduces tumour growth in vitro. Primary glioblastoma and U251 cell cultures were treated with the specific P2X7R antagonist, AZ10606120 (AZ), for 72 h. The effects of AZ treatment were also compared to cells treated with the current first-line chemotherapeutic drug, temozolomide (TMZ), and a combination of both AZ and TMZ. P2X7R antagonism by AZ significantly depleted glioblastoma cell numbers compared to untreated cells, in both primary glioblastoma and U251 cultures. Notably, AZ treatment was more effective at tumour cell killing than TMZ. No synergistic effect between AZ and TMZ was observed. AZ treatment also significantly increased lactate dehydrogenase release in primary glioblastoma cultures, suggesting AZ-induced cellular cytotoxicity. Our results reveal a trophic role of P2X7R in glioblastoma. Importantly, these data highlight the potential for P2X7R inhibition as a novel and effective alternative therapeutic approach for patients with lethal glioblastomas.

Stem-like signatures in human meningioma cells are under the control of CXCL11/CXCL12 chemokine activity.

Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes. Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry, and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions. Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, vasculogenic mimicry, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis. We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.

Unveiling the Genomic Basis of Chemosensitivity in Sarcomas of the Extremities: An Integrated Approach for an Unmet Clinical Need.

Myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS) can be considered as a spectrum of the same disease entity, representing one of the most common adult soft tissue sarcoma (STS) of the extremities. While MFS is rarely metastasizing, it shows an extremely high rate of multiple frequent local recurrences (50-60% of cases). On the other hand, UPS is an aggressive sarcoma prone to distant recurrence, which is correlated to a poor prognosis. Differential diagnosis is challenging due to their heterogeneous morphology, with UPS remaining a diagnosis of exclusion for sarcomas with unknown differentiation lineage. Moreover, both lesions suffer from the unavailability of diagnostic and prognostic biomarkers. In this context, a genomic approach combined with pharmacological profiling could allow the identification of new predictive biomarkers that may be exploited for differential diagnosis, prognosis and targeted therapy, with the aim to improve the management of STS patients. RNA-Seq analysis identified the up-regulation of MMP13 and WNT7B in UPS and the up-regulation of AKR1C2, AKR1C3, BMP7, and SGCG in MFS, which were confirmed by in silico analyses. Moreover, we identified the down-regulation of immunoglobulin genes in patient-derived primary cultures that responded to anthracycline treatment compared to non-responder cultures. Globally, the obtained data corroborated the clinical observation of UPS as an histotype refractory to chemotherapy and the key role of the immune system in determining chemosensitivity of these lesions. Moreover, our results confirmed the validity of genomic approaches for the identification of predictive biomarkers in poorly characterized neoplasms as well as the robustness of our patient-derived primary culture models in recapitulating the chemosensitivity features of STS. Taken as a whole, this body of evidence may pave the way toward an improvement of the prognosis of these rare diseases through a treatment modulation driven by a biomarker-based patient stratification.

Abstract 2419: A multimodel preclinical platform to evaluate photodynamic therapy as a strategy to reduce local recurrence in myxofibrosarcoma and osteosarcoma

Abstract Sarcomas are heterogeneous and rare malignant tumors that develop from connective tissues. Myxofibrosarcoma (MFS) and Osteosarcoma (OS) are the main representative subtypes of Soft tissues and Bone sarcomas. Conventional treatment consists of surgical resection and neo/adjuvant chemotherapy. However, a challenging clinical feature is a high local recurrence rate, calling for the development of novel strategies to eradicate MFS and OS. Fluorescence-guided surgery (FGS), coupled with photodynamic therapy (PDT), enables to detect and eradicate malignant tissues using a light-sensitive photosensitizer (PS). These compounds could be retained by neoplastic cells to a greater extent than by healthy tissue. Thus, when photoactivated, they lead to cancer cell death, minimizing normal tissues toxicity. The project aims to provide a preclinical proof of concept of FGS and PDT for MFS and OS, allowing us to pave the way for a more effective treatment strategy. A multimodel preclinical platform of MFS and OS comprising 2D, 3D-collagen based scaffold was developed. A MFS stabilized cell line (IM-MFS-1) and four OS cell lines (Saos-2, U-2 OS, MG-63, 143B) were used. NIH-3T3 fibroblast cell line was used as healthy counterpart. We tested a panel of synthetic PSs: Toluidine Blue, 5-Aminolevulinic Acid hydrochloride (ALA), Methylene Blue, Chlorin e6 (Ce6), Pheophorbide a (Pba) and Al(III) Phthalocyanine Chloride Tetrasulfonic Acid (AlPcS4) that can be photoactivated with light in the 630-660 nm wavelengths’ range. The PSs were ranked on (a) selective uptake by tumor cells, respect to healthy cells and (b) the cytotoxic effect, after photoactivation. PSs cell uptake was evaluated by Countess II FL automated cell counter. Cytotoxicity was evaluated by WST-1 and Presto Blue assays. For each PS we determined the highest concentration, causing less than 50% dark toxicity in IM-MFS-1, OS cell lines and fibroblast NIH-3T3, seeded in 2D and 3D collagen-based models. Then, we evaluated the cell viability upon light irradiation (λ=630 or 660 nm; 10 mW/cm^2, 5 min). For all PSs, dark toxicity was very low. However, only Ce6 was effectively uptaken by tumor cells (% uptake > 60%) and not by NIH-3T3 cells (% uptake = 27%). Moreover, upon photoactivation, Ce6 induced significantly higher cell death in cancer cells as compared to NIH-3T3 cells, e.g. 1-3% cell viability in tumor cells and 76% cell viability in NIH-3T3. Results from our multimodel platform highlight PDT as a powerful strategy for the treatment of MFS and OS. Among the screened PSs, Ce6 is the most promising, showing a remarkable selectivity in tumor cells uptake and cytotoxicity. To increase the clinical significance of our approach, we will confirm its efficacy in patient-derived primary cultures; xenograft zebrafish in vivo embryos will be used to evaluate how PDT affects the disease aggressiveness and metastatic potential. Citation Format: Chiara Spadazzi, Micaela Pannella, Chiara Bellotti, Elisa Martella, Silvia Vanni, Alessandro De Vita, Chiara Liverani, Giacomo Miserocchi, Claudia Cocchi, Chiara Calabrese, Valentina Fausti, Roberto Casadei, Federica Pieri, Ania Naila Guerrieri, Giovanni Martinelli, Greta Varchi, Enrico Lucarelli, Laura Mercatali, Toni Ibrahim. A multimodel preclinical platform to evaluate photodynamic therapy as a strategy to reduce local recurrence in myxofibrosarcoma and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2419.

Abstract 1425: TTFields reduce sensitivity in glioblastoma is associated with the functional expression of the chloride intracellular channel 1 and with voltage dependent sodium channel

Abstract Glioblastoma (GBM) is the most aggressive type of brain tumor, and existing treatments are inadequate at preventing recurrence. Tumor Treating Fields (TTFields) is a novel treatment that has been proven to extend patients' lives during the previous two decades. The antitumoral impact of TTFields involve different mechanism and is an optional add-on to standard temozolomide maintenance. Although TTFields therapy slows tumor progression initially, it does not prevent tumor relapse in most patients. We subjected patient-derived GBM primary cultures to continuous TTFields stimulation to mimic the GBM standard of care and examine the molecular pathways behind GBM sensitivity to TTFields treatment. TTFields initially limit cellular growth well. However, even under constant TTFields administration, the remaining cells regain their ability to proliferate at their own rate in long-term trials. Prolonged treatment with TTFields increases the expression of many stemness markers as well as the overexpression of different genes involved in cell cycle progression. There is also a rise in membrane ionic conductance activity. After sustained exposure to TTFields, sodium, potassium, and chloride channels are functionally increased. This influences the properties of tumor cells. Increased sodium channel functional activity, for example, promotes membrane potential depolarization. Furthermore, earlier research has shown that the membrane form of the chloride intracellular channel 1 (tmCLIC1) is important in the development of GBM. In patient-derived GBM cells exposed to TTFields, tmCLIC1 is significantly concentrated, and membrane depolarization improves its function. The evaluation of GBM tissues obtained from many patients who were treated with TTFields validates the in vitro tumor relapse model.Our findings imply that the process by which GBM cells develop a decrease in sensitivity to TTFields correlates with an increase in stemness markers and an increase in ionic channel functional expression. Citation Format: Stefania Castiglione, Gaetano Cannavale, Francesca Cianci, Ivan Verduci, Chiara Biella, Chiara Mercurio, Emanuela Pastorelli, Monica Lupi, Laura Paracchini, Laura Mannarino, Dietmar Krex, Federico Brandalise, Michele Mazzanti. TTFields reduce sensitivity in glioblastoma is associated with the functional expression of the chloride intracellular channel 1 and with voltage dependent sodium channel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1425.

Characterization and Clinical Relevance of Endometrial CAFs: Correlation between Post-Surgery Event and Resistance to Drugs.

Cancer-associated fibroblasts (CAFs) within a solid tumor can support the progression of cancer. We studied the identification and characterization of patient-derived endometrial CAFs in the context of their clinical relevance in endometrial cancers. We established patient-derived primary cultures of CAFs from surgically resected tumors (TCAF) and tumor-adjacent normal (NCAF) tissues in 53 consented patients with success rates of 97.7% and 75%, respectively. A passage of CAF was qualified by the (1) absence of CK 8,18,19, EpCAM, CD45, and CD31, and (2) presence of SMAalpha, S100A4, CD90, FAP, TE-7, CD155, PD-L1, TGFB, PDGFRA (qRT-PCR, flow cytometry, Western blot, ICC). Out of the 44 established CAFs, 31 were aggressive (having an early, i.e., 4-7 week, establishment time and/or >3 passages) compared to 13 which were non-aggressive. A post-surgery-event (PSE) was observed in 7 out of 31 patients bearing aggressive CAFs, 2 of whom were also positive for CTCs, while none of the 13 patients bearing non-aggressive CAFs had events. A positive correlation was found between patients with grade 3 (p = 0.025) as well as stage 3/4 diseases (p = 0.0106) bearing aggressive CAFs and the PSE. Finally, aggressive TCAFs from patients with PSE resisted the effects of paclitaxel and lenvatinib on the growth of HUVEC and endometrial tumor cells. Our study is the first to report a correlation between the PSE and the aggressive nature of CAFs in endometrial cancers and provides an undeniable reason to study the in-depth mechanism of CAF function towards the development of treatment resistance in endometrial cancers.

106P Synergistic effect of CDK4/6 inhibitors and standard chemotherapy sequential treatment on liposarcoma patient-derived primary culture

106P Synergistic effect of CDK4/6 inhibitors and standard chemotherapy sequential treatment on liposarcoma patient-derived primary culture

Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

EPCO-38. TYPE B ULTRA LONG-RANGE INTERACTIONS IN PFAS (TULIPS) ARE RECURRENT EPIGENOMIC FEATURES OF PFA EPENDYMOMA

Abstract Posterior Fossa Group A (PFA) ependymomas are pediatric brain tumors with extremely poor survival outcomes. As protein-coding mutations in PFA are exceedingly rare, the underlying etiology of these tumors remains elusive. Elevated CpG island methylation and depletion of H3K27me3 have been described in PFA, leading to the hypothesis that PFA may be driven by a dysregulated epigenetic state. In this study, we sought to determine how three-dimensional (3D) genome features (such as DNA loops, domains, and compartments) differ between pediatric brain tumors. We performed Hi-C sequencing on a collection of 64 patient specimens and patient-derived primary cultures that collectively span multiple subgroups of ependymoma, medulloblastoma, high-grade glioma, and non-neoplastic brain. For certain samples, we further performed RNA-seq, histone modification ChIP-seq, or whole-genome bisulfite sequencing to allow multiomic data integration. Overall, the 3D genome organization of PFA samples appeared distinct from other tumor types. We identified and defined TULIPs: a subset of type B compartments, separated by genomic distances greater than 10 Mbp, that exhibit a striking fivefold increase in reciprocal interaction strength. These TULIPs recurred at the same genomic positions across the vast majority of PFA samples with minimal representation among other tumor or non-tumor samples. TULIPs displayed enrichment for heterochromatic features such as H3K9me3 and late replication timing and were depleted of euchromatic features such as H3K27ac and protein-coding genes. By using immuno-fluorescence for H3K9me3 and oligo-FISH to label TULIP regions, we demonstrated that TULIP regions are more compact in PFA than other tumors. Finally, by applying inhibitors of H3K9 lysine methylation to PFA cultures we showed that TULIPs become more diffuse and cell viability is reduced. Altogether, this work defines TULIPs as highly recurrent epigenetic features of PFA tumors.