Susceptibility Of Tumor Cells Research Articles

Intracellular zinc protects tumours from T cell-mediated cytotoxicity.

Tumour immune evasion presents a significant challenge to the effectiveness of cancer immunotherapies. Recent advances in high-throughput screening techniques have uncovered that loss of antigen presentation and cytokine signalling pathways are central mechanisms by which tumours evade T cell immunity. To uncover additional vulnerabilities in tumour cells beyond the well-recognized antigen presentation pathway, we conducted a genome-wide CRISPR/Cas9 screen to identify genes that mediate resistance to chimeric-antigen receptor (CAR)-T cells, which function independently of classical antigen presentation. Our study revealed that loss of core-binding factor subunit beta (CBFβ) enhances tumour cell resistance to T cell killing, mediated through T cell-derived TNF. Mechanistically, RNA-sequencing and elemental analyses revealed that deletion of CBFβ disrupts numerous pathways including those involved in zinc homoeostasis. Moreover, we demonstrated that modulation of cellular zinc, achieved by supplementation or chelation, significantly altered tumour cell susceptibility to TNF by regulating the levels of inhibitor of apoptosis proteins. Consistent with this, treatment of tumour cells with a membrane-permeable zinc chelator had no impact on tumour cell viability alone, but significantly increased tumour cell lysis by CD8+ T cells in a TNF-dependent but perforin-independent manner. These results underscore the crucial role of intracellular zinc in regulating tumour cell susceptibility to T cell-mediated killing, revealing a novel vulnerability in tumour cells that might be exploited for the development of future cancer immunotherapeutics.

A Two-Pronged Nanostrategy of Iron Metabolism Disruption to Synergize Tumor Therapy by Triggering the Paraptosis-Apoptosis Hybrid Pathway.

Iron metabolism has emerged as a promising target for cancer therapy; however, the innate metabolic compensatory capacity of cancer cells significantly limits the effectiveness of iron metabolism therapy. Herein, bioactive gallium sulfide nanodots (GaSx), with dual functions of "reprogramming" and "interfering" iron metabolic pathways, were successfully developed for tumor iron metabolism therapy. The constructed GaSx nanodots ingeniously harness hydrogen sulfide (H2S) gas, which is released in response to the tumor microenvironment, to reprogram the inherent transferrin receptor 1 (TfR1)-ferroportin 1 (FPN1) iron metabolism axis in cancer cells. Concurrently, the gallium ions (Ga3+) derived from GaSx act as a biochemical "Trojan horse", mimicking the role of iron and displacing it from essential biomolecular binding sites, thereby influencing the fate of cancer cells. By leveraging the dual mechanisms of Ga3+-mediated iron disruption and H2S-facilitated reprogramming of iron metabolic pathways, GaSx prompted the initiation of a paraptosis-apoptosis hybrid pathway in cancer cells, leading to marked suppression of tumor proliferation. Importantly, the dysregulation of iron metabolism induced by GaSx notably increased tumor cell susceptibility to both chemotherapy and immune checkpoint blockade (ICB) therapy. This study underscores the therapeutic promise of gas-based interventions and metal ion interference strategies for the tumor metabolism treatment.

Ebselen: A Review on its Synthesis, Derivatives, Anticancer Efficacy and Utility in Combating SARS-COV-2.

Ebselen is a selenoorganic chiral compound with antioxidant properties comparable to glutathione peroxidase. It is also known as 2-phenyl-1,2-benzisoselenazol-3(2H)-one. In studies examining its numerous pharmacological activities, including antioxidant, anticancer, antiviral, and anti- Alzheimer's, ebselen has demonstrated promising results. This review's primary objective was to emphasize the numerous synthesis pathways of ebselen and their efficacy in fighting cancer. The data were collected from multiple sources, including Scopus, PubMed, Google Scholar, Web of Science, and Publons. The starting reagents for the synthesis of ebselen are 2-aminobenzoic acid and N-phenyl benzamide. It was discovered that ebselen has the ability to initiate apoptosis in malignant cells and prevent the formation of new cancer cells by scavenging free radicals. In addition, ebselen increases tumor cell susceptibility to apoptosis by inhibiting TNF-α mediated NF-kB activation. Ebselen can inhibit both doxorubicin and daunorubicin-induced cardiotoxicity. Allopurinol and ebselen administered orally can be used to suppress renal ototoxicity and nephrotoxicity. Due to excessive administration, diclofenac can induce malignancy of the gastrointestinal tract, which ebselen can effectively suppress. Recent research has demonstrated ebselen to inhibit viral function by binding to cysteinecontaining catalytic domains of various viral proteases. It was discovered that ebselen could inhibit the catalytic dyad function of Mpro by forming an irreversible covalent bond between Se and Cys145, thereby altering protease function and inhibiting SARS-CoV-2. Ebselen may also inhibit the activation of endosomal NADPH oxidase of vascular endothelial cells, which is believed to be required for thrombotic complications in COVID-19. In this review, we have included various studies conducted on the anticancer effect of ebselen as well as its inhibition of SARS-CoV-2.

Abstract PO4-11-03: Impact of Short Term Fasting (STF) on Fatigue developing in primary breast cancer (BC) patients receiving the Henderson or Sparano Scheme in the prospective multicentre FIT2- cohort

Abstract Background: Experimentally, it has been shown that short-term fasting (STF) induces a “protective mode” in healthy cells against chemotherapeutic toxicity while it enhances the susceptibility of tumor cells to chemotherapy. In healthy cells, fasting triggers distinct protective metabolic and gene expression changes, such as upregulation of DNA repair mechanisms, autophagy, and downshift of metabolic pathways including IGF-1-dependent signaling. This study was conducted to assess feasibility, quality of life (QoL) including chemotherapy-induced fatigue (CTF) comparing STF around each chemotherapy (4xAC or 4xEC) to a plant-based low-sugar diet (PBD). Methods: Newly diagnosed BC-patients treated by the Henderson or Sparano scheme were included in this randomised, controlled, multicenter study. CTF was assessed by the FACIT-F questionnaire before (day 0) and after chemotherapy (day 7) as well as at months 4 (V1) and 6 (V2). Results: Of the included n=106 patients, 52 were randomized to STF and 54 to PBD. n=90 (84.9%) patients obtained AC/EC+Pac and 16 (15.1%) AC/EC+Doc. FACIT-F showed no significant difference at baseline (STF 42.8; PBD 41.3; p=0.708). The differences between the corresponding FACIT-F Trial Outcome Index resp. Total FACIT-F at cycle 4 day 7 were 13.1 (SE 4.7) resp. 15.8 (SE 5.6), both in favor of STF (statistically significant (p < 0.007) and clinically relevant (minimal important difference (MID) > 5). This difference was visible for all breast cancer subtypes, i.e. luminal A & B, as well as HER2 overexpressed and triple negative tumors, with the STF group showing no clinically relevant fatigue after 4 cycles of chemotherapy (FACIT-F > 34), across all subtypes. The opposite was true for the PBD group (FACIT-F < 34) across all subtypes. FACIT-F (22.7) for PBD in HER2 overexpressed and triple negative tumors was significantly worse than those in luminal A and B patients (FACIT-F= 29.1; difference 6.4 > MID=3). STF was well tolerated and there were no serious adverse effects. Conclusion: STF during chemotherapy is well tolerated and appears to improve QoL including fatigue during chemotherapy compared to a plant-based low-sugar diet, independent of intrinsic subtypes. Citation Format: Daniela Koppold, Christian Kessler, Manfred Wischnewsky, Ixquic Alvarado, Nico Steckhan, Cornelia Kempter, Marion Paul, Barbara Brückner, Dorothea Fischer, Aysen Wunschel, Rainer Stange, Etienne Hanslian, Michael Jeitler, Andreas Michalsen. Impact of Short Term Fasting (STF) on Fatigue developing in primary breast cancer (BC) patients receiving the Henderson or Sparano Scheme in the prospective multicentre FIT2- cohort [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-11-03.

Targeting sinonasal undifferentiated carcinoma with a combinatory immunotherapy approach

PurposeSinonasal undifferentiated carcinoma (SNUC) is a rare, aggressive malignancy of the sinonasal cavity with poor prognosis and limited treatment options. To investigate the potential for SNUC sensitivity to combinatory immunotherapy, we performed in vitro studies with SNUC cell lines and used multi-spectral immunofluorescence to characterize the in vivo patient SNUC tumor immune microenvironment (TIME). Experimental designHuman-derived SNUC cell lines were used for in vitro studies of tumor cell susceptibility to natural killer (NK) cell-based immunotherapeutic strategies. Tumor samples from 14 treatment naïve SNUC patients were examined via multi-spectral immunofluorescence and clinical correlations assessed. ResultsAnti-PD-L1 blockade enhanced NK cell lysis of SNUC cell lines ∼5.4 fold (P ≤ 0.0001). This effect was blocked by a CD16 neutralizing antibody demonstrating activity through an antibody-dependent cellular cytotoxicity (ADCC) mediated pathway. ADCC-dependent lysis of SNUC cells was further enhanced by upregulation of PD-L1 on tumor cells by exogenous interferon-gamma (IFN-γ) administration or interleukin-15 (IL-15) stimulated IFN-γ release from NK cells. Combination treatment with anti-PD-L1 blockade and IL-15 superagonism enhanced NK-cell killing of SNUC cells 9.6-fold (P ≤ 0.0001). Untreated SNUC patient tumor samples were found to have an NK cell infiltrate and PD-L1+ tumor cells at a median of 5.4 cells per mm2. A striking 55.7-fold increase in CKlow tumor cell/NK cell interactions was observed in patients without disease recurrence after treatment (P = 0.022). Patients with higher CD3+CD8+ in the stroma had a significantly improved 5-year overall survival (P = 0.0029) and a significant increase in CKlow tumor cell/CD8+ cytotoxic T cell interactions was noted in long-term survivors (P = 0.0225). ConclusionThese data provide the pre-clinical rationale for ongoing investigation into combinatory immunotherapy approaches for SNUC.

GSH-responsive degradable nanodrug for glucose metabolism intervention and induction of ferroptosis to enhance magnetothermal anti-tumor therapy

The challenges associated with activating ferroptosis for cancer therapy primarily arise from obstacles related to redox and iron homeostasis, which hinder the susceptibility of tumor cells to ferroptosis. However, the specific mechanisms of ferroptosis resistance, especially those intertwined with abnormal metabolic processes within tumor cells, have been consistently underestimated. In response, we present an innovative glutathione-responsive magnetocaloric therapy nanodrug termed LFMP. LFMP consists of lonidamine (LND) loaded into PEG-modified magnetic nanoparticles with a Fe3O4 core and coated with disulfide bonds-bridged mesoporous silica shells. This nanodrug is designed to induce an accelerated ferroptosis-activating state in tumor cells by disrupting homeostasis. Under the dual effects of alternating magnetic fields and high concentrations of glutathione in the tumor microenvironment, LFMP undergoes disintegration, releasing drugs. LND intervenes in cell metabolism by inhibiting glycolysis, ultimately enhancing iron death and leading to synthetic glutathione consumption. The disulfide bonds play a pivotal role in disrupting intracellular redox homeostasis by depleting glutathione and inactivating glutathione peroxidase 4 (GPX4), synergizing with LND to enhance the sensitivity of tumor cells to ferroptosis. This process intensifies oxidative stress, further impairing redox homeostasis. Furthermore, LFMP exacerbates mitochondrial dysfunction, triggering ROS formation and lactate buildup in cancer cells, resulting in increased acidity and subsequent tumor cell death. Importantly, LFMP significantly suppresses tumor cell proliferation with minimal side effects both in vitro and in vivo, exhibiting satisfactory T2-weighted MR imaging properties. In conclusion, this magnetic hyperthermia-based nanomedicine strategy presents a promising and innovative approach for antitumor therapy.Graphical

Abstract 729: IDO1 inhibition enables IFNγ-elicited responsiveness of pulmonary breast cancer metastases to hypoxia-directed tumoricidal agents

Abstract In mouse models of lung carcinoma and pulmonary breast cancer metastasis, genetic ablation of the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) resulted in restricted tumor outgrowth associated with attenuated induction of the inflammatory cytokine IL6 (interleukin 6). Utilizing the 4T1 breast cancer metastasis model to further elucidate the mechanistic basis underlying IDO1’s pro-tumor activity in the lungs established that IDO1 acts as an integral determinant of a tumor-promoting inflammatory state that supports neovascular retention. Ido1−/− (nullizygous) mice exhibited impaired pulmonary metastases neovascularization and outgrowth that was driven by IFNγ (interferon gamma) in the absence of IDO1-dependent IL6 production. Hypoxia was likewise elevated, and these stress conditions increased the susceptibility of the metastatic tumor cells to cytotoxicity associated with administration of GSK2656157, an inhibitor of PERK (protein kinase RNA-like ER kinase), which acts as a primary sensor for activating the UPR (unfolded protein response) survival pathway. In WT (wild type) mice with established 4T1 lung metastases, administration of the IDO1 inhibitors epacadostat, navoximod or indoximod each similarly resulted in rapid collapse of the metastatic tumor neovasculature and concomitantly increased intratumoral hypoxia and sensitivity to PERK inhibition. Therefore, while IDO1 inhibition does not directly promote effective tumor cell killing in this context, the disruption of blood supply due to IFNγ-mediated neovascular regression subjects the tumor cells to hypoxic environmental stress, raising the possibility for therapeutic intervention with selectively targeted agents. Combining chemotherapy with immune checkpoint blockade (ICB) is recognized as an effective strategy for treating lung cancer, with first-line therapy for some patients including the alkylating agent carboplatin in conjunction with pembrolizumab. The challenge remains, however, that chemotherapy causes systemic side effects that can negatively impact quality of life and limit efficacy. Evofosfamide is an alkylating prodrug that was designed to be selectively activated in regions of hypoxia, thereby limiting systemic exposure. We report that enhanced metastatic tumor cell killing with evofosfamide can be achieved by co-administration of an IDO1 inhibitor. Elevated intratumoral hypoxia has also previously been linked to increased expression levels of PDL1, a favorable indicator of ICB responsiveness, and we have confirmed the upregulation of PDL1 in 4T1 lung metastases following IDO1 inhibitor administration. Our data in the lung metastasis model support future studies to evaluate whether IDO1 inhibition can be effectively combined with evofosfamide and anti-PD1 treatment to improve therapeutic outcomes. Citation Format: Shih-Chun Shen, Souvik Dey, James B. DuHadaway, Erika Sutanto-Ward, George C. Prendergast, Alexander J. Muller. IDO1 inhibition enables IFNγ-elicited responsiveness of pulmonary breast cancer metastases to hypoxia-directed tumoricidal 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 729.

An IRF2-Expressing Oncolytic Virus Changes the Susceptibility of Tumor Cells to Antitumor T Cells and Promotes Tumor Clearance.

IFN regulatory factor 1 (IRF1) can promote antitumor immunity. However, we have shown previously that in the tumor cell, IRF1 can promote tumor growth, and IRF1-deficient tumor cells exhibit severely restricted tumor growth in several syngeneic mouse tumor models. Here, we investigate the potential of functionally modulating IRF1 to reduce tumor progression and prolong survival. Using inducible IRF1 expression, we established that it is possible to regulate IRF1 expression to modulate tumor progression in established B16-F10 tumors. Expression of IRF2, which is a functional antagonist of IRF1, downregulated IFNγ-induced expression of inhibitory ligands, upregulated MHC-related molecules, and slowed tumor growth and extended survival. We characterized the functional domain(s) of IRF2 needed for this antitumor activity, showing that a full-length IRF2 was required for its antitumor functions. Finally, using an oncolytic vaccinia virus as a delivery platform, we showed that IRF2-expressing vaccinia virus suppressed tumor progression and prolonged survival in multiple tumor models. These results suggest the potency of targeting IRF1 and using IRF2 to modulate immunotherapy.

Abstract 2589: Novel workflow to generate natural killer cell sensitivity prediction model based on the epithelial mesenchymal transition related reprogramming at a single cell resolution

Abstract Single cell technology allows us to examine cell heterogeneity and identify rare cell populations; however, the high cost of single cell RNAseq limits its adoption into clinical and research environments. As an inexpensive alternative, immunofluorescent assays analyzing cell morphologies have been used in high content screening to examine cell phenotype and response to external stresses. Natural Killer cells (NK) are the bodies first line of defense and gained traction as a potential target in treating cancer. A study showed that cancer cells undergoing the process epithelial mesenchymal transition (EMT) increase their vulnerability to NK mediated cytotoxicity. We hypothesize that the EM morphology change can be correlated with NK sensitivity. Our goal is to develop an image-based, cost-effective, and efficient workflow capable of extracting cancer cell morphological features to predict its NK sensitivity at the single-cell level. To optimize this workflow, we use A549 cell line cells treated with transforming growth factor- β (TGF-β) as a representative models of cells with various EM phenotypes as NK sensitivities. We utilized a Cell Painting inspired novel image based live cell cytotoxicity assay. To capture cell morphological features, we optimized an organelle specific and live cell compatible staining panel that targets the nucleus, nucleic acid, cell membrane, F-actin, and mitochondria. The cell viability is assessed by Propidium Iodide, which stains dead cell nuclei. Our image analysis pipeline can extract the morphological features and track the cells of interest during the experiment and assess their viability. Subsequently, the morphology and viability data will be processed by a classification algorithm that could discern morphological features of cancer cells that indicate high NK vulnerability. We started with treating A549 cells with 5ng/mL TGF-β for 0, 24, 48, and 72 hours, and successfully applied the organelle staining panel. We observed distinct cell morphologies at each timepoint of TGF-β treatment, with cells shifting from a cobblestone (no TGF-β) to a spindle-like (72 hours TGF-β) morphology. Notably, the F-actin signal was significantly overexpressed in the 72 hours TGF-β treated A549 cells. A CellProfiler based preliminary image analysis pipeline was established for cell segmentation, cell features extraction, and cell viability tracking with quality control filters. Further optimization for cell segmentation, viability tracking, and classification algorithms will be needed to enhance the robustness of the workflow. Our established workflow demonstrates the feasibility of live cell functional assay with organelle staining. This workflow could be adopted to correlate and predict single tumor cell susceptibility to cytotoxic immune cells, with the goal of developing a therapeutic index. Citation Format: Yin Zou, Harrison Ball, Cade Harris, Nithya Ramnath, Venkateshwar Keshamouni, Sunitha Nagrath. Novel workflow to generate natural killer cell sensitivity prediction model based on the epithelial mesenchymal transition related reprogramming at a single cell resolution [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 2589.

New Insights into Chemoresistance Mediated by Mdm2 Inhibitors: The Benefits of Targeted Therapy over Common Cytostatics.

The inhibition of the Mdm2-p53 protein-protein interaction is a promising strategy for anticancer therapy. However, the problem of developing secondary chemoresistance in tumors treated with such drugs has not yet been sufficiently studied. In this work, we compared the properties of a drug-resistant cell line obtained during long-term cultivation in the presence of an Mdm2 inhibitor, Nutlin-3a, with a similarly obtained line insensitive to the cytostatic drug paclitaxel. We first confirmed the higher safety levels of Mdm2 inhibitors when compared with cytostatics in terms of the development of secondary chemoresistance. We showed that Nutlin-3a affects both the targeted p53-mediated cellular machinery and the universal ABC-mediated efflux mechanism. While both targeted and general defense mechanisms are activated by the Mdm2 inhibitor, it still increases the susceptibility of tumor cells to other drugs. The results obtained indicate that the risks of developing chemoresistance under the therapy with a targeted agent are fundamentally lower than during cytotoxic therapy.

Synergic actions of botulinum neurotoxin A and oxaliplatin on colorectal tumour cell death through the upregulation of TRPM2 channel-mediated oxidative stress.

Botulinum neurotoxin A (BoNT) is being shown to have anticancer action as a potential adjuvant treatment. The transient receptor potential (TRP) melastatin 2 (TRPM2) stimulator action of BoNT was reported in glioblastoma cells, but not in colorectal cancer (HT29) cells. By activating TRPM2, we evaluated the impacts of BoNT and oxaliplatin (OXA) incubations on oxidant and apoptotic values within the HT29 cells. Control, BoNT (5 IU for 24 h), OXA (50 μM for 24 h) and their combinations were induced. We found that TRPM2 protein is upregulated and mediates enhanced BoNT and OXA-induced Ca2+ entry in cells as compared to control cells. The increase of free reactive oxygen species (ROS), but the decrease of glutathione is the main ROS responsible for TRPM2 activation on H29 exposure to oxidative stress. BoNT and OXA-mediated Ca2+ entry through TRPM2 stimulation in response to H2 O2 results in mitochondrial Ca2+ overload, followed by mitochondrial membrane depolarization, apoptosis and caspase-3/-8/-9, although they were diminished in the TRPM2 antagonist groups (N-(p-amylcinnamoyl)anthranilic acid and carvacrol). In conclusion, by increasing the susceptibility of HT29 tumour cells to oxidative stress and apoptosis, the combined administration of BoNT and OXA via the targeting of TRPM2 may offer a different approach to kill the tumour cells.

Lung endothelium exploits susceptible tumor cell states to instruct metastatic latency

In metastasis, cancer cells travel around the circulation to colonize distant sites. Due to the rarity of these events, the immediate fates of metastasizing tumor cells (mTCs) are poorly understood while the role of the endothelium as a dissemination interface remains elusive. Using a newly developed combinatorial mTC enrichment approach, we provide a transcriptional blueprint of the early colonization process. Following their arrest at the metastatic site, mTCs were found to either proliferate intravascularly or extravasate, thereby establishing metastatic latency. Endothelial-derived angiocrine Wnt factors drive this bifurcation, instructing mTCs to follow the extravasation–latency route. Surprisingly, mTC responsiveness towards niche-derived Wnt was established at the epigenetic level, which predetermined tumor cell behavior. Whereas hypomethylation enabled high Wnt activity leading to metastatic latency, methylated mTCs exhibited low activity and proliferated intravascularly. Collectively the data identify the predetermined methylation status of disseminated tumor cells as a key regulator of mTC behavior in the metastatic niche.

Study of Cytotoxic Responses of Anticancer Drug Cyclophosphamide

It is widely accepted that cancer, the second leading cause of death, is a morbidity with big impacts on the global health. In the last few years, chemo- therapeutic treatment continually induces alone most lengthy consequents, which is extremely harmful for the physiological and psychological health of the patients. In the present research, we discuss the recent techniques for employed for extraction, and quantitative determination of such compounds in pharmaceutical, and biological specimens. In the frame of this information, this review aims to provide basic principles of chromatography, spectroscopy. Anticancer drugs research and development have been largest market area in pharmaceuticals industry in terms of the numbers of project and clinical trials and spending. Our goals to improve cancer treatment by increasing our understanding of the mechanism by which anti-cancer treatments kill susceptible tumor cells. This articles provides an overview of current knowledge of anticancer drug their pharmacology, mechanism of action, uses, side effects,precautions, and contraindication. This mini review outlined the current status of anticancer drugs development and hinted the opinions of how to further increase the accuracy and efficacy of discovery for cancer treatment.

Engineering redirected NF-κB/OIP5 expression programs to enhance tumor responses to chemotherapy in bladder cancer

Nuclear factor kappa-B (NF-κB), a pivotal transcriptional regulator, plays a crucial role in modulating downstream genes implicated in tumor drug resistance. We establish a programmable system within bladder cancer cells to tailor drug responses by employing a synthetic clustered regularly interspaced short palindromic repeats (CRISPR)-based expression strategy that emulates natural transcriptional regulators. Our investigation uncovers the functional significance of Opa-interacting protein 5 (OIP5), upregulated upon NF-κB activation, as a key regulator governing drug-resistance to vincristine (VCR) treatment in bladder cancer. Through engineered guide RNAs (sgRNAs) targeting OIP5 to integrate NF-κB aptamers, we construct a modular scaffold RNA that encodes both the target locus and regulatory functionality. This engineered CRISPR scaffold RNA effectively responds to VCR stimulus by binding with activated NF-κB. Intriguingly, it redirects NF-κB to attenuate OIP5 expression—a reversal of its original role—while concurrently obstructing multiple NF-κB-mediated drug resistance pathways. This dual action thwarts drug resistance development. Further enhancing therapeutic potential, we develop a versatile nanoparticle system capable of co-delivering CRISPR scaffold RNAs and VCR. This synergistic approach demonstrates potent anti-tumor effects in both in vitro and in vivo settings. Our nanoparticle-mediated combination presents a compelling proof-of-concept, showcasing the utility of engineered CRISPR-based synthetic expression programs to reconfigure cellular drug responses and heighten tumor cell susceptibility to chemotherapy.

EPCO-30. UNCOVERING MECHANISMS OF RESISTANCE IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG)

Abstract Pediatric high-grade gliomas (pHGGs) are currently the leading cause of cancer-related deaths in children, remaining incurable in most cases. A molecular subgroup of pHGGs harbor G34R mutations in the histone H3.3, although the mechanisms of the H3.3-G34R/V are not well established. In most of the patients, the tumors resist radiation and relapse after standard of care (SOC). We aim to study the molecular mechanisms mediating resistance in these tumors. Knowing why certain tumors resist treatment allows us to implement targeted therapies. We performed single cell RNA sequencing on 3 non-treated (NT) glioma bearing mice, 3 mice resistant to treatment (R), and 3 mice susceptible to treatment (S). We isolated the tumor tissue (including tumor microenvironment) and processed the cells for single cell RNA sequencing. Using R, the single-cell RNA sequencing data was converted to Seurat datasets for analysis and subsequently combined and clustered using UMAP. We used the ScType and SingleR software tools to annotate cell types. After cell annotation, Gene Set Enrichment Analysis (GSEA) was performed between R and S tumors for each cell cluster as well as NT vs Treated. GSEA showed that susceptible tumor cells showed increased immune responses when compared to resistant cells. Additionally, resistant cells showed increased neuronal phenotypes as well as suppression of the ERK 1/2 cascade pathway in comparison to susceptible cells. There were also numerous differences in immune cell subpopulations, which may help uncover mechanisms of resistance. For example, certain immune cells show signs of increased epigenetic modifications in resistant vs. susceptible glioma cells. Our results indicate that the resistance of pHGG to treatment is associated with the establishment of immunosuppressive signals from tumor cells, and the suppression of tumor growth mediated by downregulation of the MAPK/ERK cascade.

3D vascularized microphysiological system for investigation of tumor-endothelial crosstalk in anti-cancer drug resistance

Despite the advantages of microfluidic system in drug screening, vascular systems responsible for the transport of drugs and nutrients have been hardly considered in the microfluidic-based chemotherapeutic screening. Considering the physiological characteristics of highly vascularized urinary tumors, we here investigated the chemotherapeutic response of bladder tumor cells using a vascularized tumor on a chip. The microfluidic chip was designed to have open-top region for tumor sample introduction and hydrophilic rail for spontaneous hydrogel patterning, which contributed to the construction of tumor-hydrogel-endothelium interfaces in a spatiotemporal on-demand manner. Utilizing the chip where intravascularly injected cisplatin diffuse across the endothelium and transport into tumor samples, chemotherapeutic responses of cisplatin-resistant or -susceptible bladder tumor cells were evaluated, showing the preservation of cellular drug resistance even within the chip. The open-top structure also enabled the direct harvest of tumor samples and post analysis in terms of secretome and gene expressions. Comparing the cisplatin efficacy of the cisplatin-resistant tumor cells in the presence or absence of endothelium, we found that the proliferation rates of tumor cells were increased in the vasculature-incorporated chip. These have suggested that our vascularized tumor chip allows the establishment of vascular-gel-tumor interfaces in spatiotemporal manners and further enables investigations of chemotherapeutic screening.

Single-cell molecular profiling using ex vivo functional readouts fuels precision oncology in glioblastoma.

Functional profiling of freshly isolated glioblastoma (GBM) cells is being evaluated as a next-generation method for precision oncology. While promising, its success largely depends on the method to evaluate treatment activity which requires sufficient resolution and specificity. Here, we describe the 'precision oncology by single-cell profiling using ex vivo readouts of functionality' (PROSPERO) assay to evaluate the intrinsic susceptibility of high-grade brain tumor cells to respond to therapy. Different from other assays, PROSPERO extends beyond life/death screening by rapidly evaluating acute molecular drug responses at single-cell resolution. The PROSPERO assay was developed by correlating short-term single-cell molecular signatures using mass cytometry by time-of-flight (CyTOF) to long-term cytotoxicity readouts in representative patient-derived glioblastoma cell cultures (n = 14) that were exposed to radiotherapy and the small-molecule p53/MDM2 inhibitor AMG232. The predictive model was subsequently projected to evaluate drug activity in freshly resected GBM samples from patients (n = 34). Here, PROSPERO revealed an overall limited capacity of tumor cells to respond to therapy, as reflected by the inability to induce key molecular markers upon ex vivo treatment exposure, while retaining proliferative capacity, insights that were validated in patient-derived xenograft (PDX) models. This approach also allowed the investigation of cellular plasticity, which in PDCLs highlighted therapy-induced proneural-to-mesenchymal (PMT) transitions, while in patients' samples this was more heterogeneous. PROSPERO provides a precise way to evaluate therapy efficacy by measuring molecular drug responses using specific biomarker changes in freshly resected brain tumor samples, in addition to providing key functional insights in cellular behavior, which may ultimately complement standard, clinical biomarker evaluations.

Abstract 667: Single-cell functional genomics of natural killer cell evasion by tumor cells

Abstract Background: Natural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells using single-cell and genome-scale functional genomics screens. Methods: We performed multiplexed single-cell RNA-seq (scRNA-seq) on co-cultures of NK cells and 26 cell lines representing diverse blood cancers. Using screens of pooled DNA-barcoded cell lines (PRISM), we quantified the sensitivity of over 60 blood cancer cell lines and integrated the results with CCLE multi-omics to uncover molecular correlates of tumor cell susceptibility to NK cells. We performed 12 genome-scale CRISPR loss-of-function and gain-of-function screens of cancer-cell intrinsic NK cell resistance mechanisms across 7 blood cancer cell lines. Finally, we investigated the mechanisms-of-action of 65 genome-scale screen hits using CRISPR screens with scRNA-seq readout in both tumor and NK cells. Results: At single-cell resolution, interaction of NK and cancer cells induced distinct activation states in both cell types depending on the cancer cell lineage and molecular phenotype. NK cells transitioned either into an activated state characterized by 4-1BB, GITR, TIM-3, and TIGIT or a state marked by type I interferon signature. Tumor cells responded to NK cell attack by activating interferon gamma (IFNy) signaling, inducing MHC class I. The activation states correlated with sensitivity to NK cells, ranging from more sensitive myeloid to more resistant B-lymphoid cancers. Molecular correlates of increased sensitivity included expression of activating receptor ligands NCR3LG1, PVR, and ULBP1, whereas TRAF3 mutations correlated with resistance. CRISPR screens uncovered cancer cell-intrinsic genes driving sensitivity and resistance, including antigen presentation and death receptor signaling mediators and adhesion molecules. The screens identified new ​​blood cancer-specific NK cell inhibitory regulators (SELPLG, SPN, and MYB ) and genes previously underappreciated in NK cell evasion, including protein fucosylation and transcriptional regulators (e.g. GFI1B). CRISPR screens with scRNA-seq readout identified MHC-I, IFNy, and NF-κB regulation as underlying mechanisms. Cancer cell knockout of positive regulators of NK cell response (CD58, NCR3LG1) induced an inactive NK cell state, providing experimental evidence how cancer cell-intrinsic genetic alterations can shape the molecular profile of attacking immune cells to promote immune evasion. Conclusions: By integrating diverse functional genomics screens and patient genomic profiles, we provide a comprehensive landscape of potential biomarkers and functionally validated genetic mechanisms which influence how NK cells recognize and kill malignant cells. The results offer a roadmap to facilitate development of NK-cell based immunotherapy for blood cancers and beyond. Citation Format: Olli Dufva, Sara Gandolfi, Jani Huuhtanen, Olga Dashevsky, Khalid Saeed, Jay Klievink, Petra Nygren, Jonas Bouhlal, Jenni Lahtela, Anna Näätänen, Bishwa Ghimire, Tiina Hannunen, Pekka Ellonen, Hanna Duàn, Jason Theodoropoulos, Essi Laajala, Jouni Härkönen, Petri Pölönen, Merja Heinäniemi, Shizuka Yamano, Ryosuke Shirasaki, David Barbie, Jennifer Roth, Rizwan Romee, Michal Sheffer, Harri Lähdesmäki, Dean A. Lee, Ricardo De Matos Simoes, Matti Kankainen, Constantine S. Mitsiades, Satu Mustjoki. Single-cell functional genomics of natural killer cell evasion by tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 667.

Accumulated cholesterol protects tumours from elevated lipid peroxidation in the microenvironment

Elevated lipid peroxidation (LPO), usually present in the tumour microenvironment (TME), is profoundly implicated in antitumour immunity and may be targeted for the development of new antitumour therapies. However, tumour cells may also rewire their metabolism to survive elevated LPO. Here, we report a novel and nonantioxidant mechanism by which tumour cells benefit from accumulated cholesterol to restrain LPO and ferroptosis, a nonapoptotic form of cell death characterized by accumulated LPO. Modulating cholesterol metabolism, especially LDLR-mediated cholesterol uptake, shifted the susceptibility of tumour cells to ferroptosis. Elevation of cellular cholesterol content specifically restrained LPO triggered by GSH-GPX4 inhibition or oxidizing factors in the TME. Furthermore, depletion of TME cholesterol by MβCD efficiently enhanced the antitumour efficacy of ferroptosis in a mouse xenograft model. Distinct from the antioxidant effect of its metabolic intermediates, the protective role of cholesterol was ascribed to its ability to decrease membrane fluidity and promote lipid raft formation, which affects the diffusion of LPO substrates. A correlation between LPO and lipid rafts was also found in tumour tissues from renal cancer patients. Together, our findings have identified a general and nonsacrificial mechanism by which cholesterol suppresses LPO, which can be exploited to enhance the efficacy of ferroptosis-based antitumour strategies.

Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer

BackgroundThe rich biodiversity of medicinal plants and their importance as sources of novel therapeutics and lead compounds warrant further research. Despite advances in debulking surgery and chemotherapy, the risks of recurrence of ovarian cancer and resistance to therapy are significant and the clinical outcomes of ovarian cancer remain poor or even incurable. ObjectiveThis study aims to investigate the effects of leaf extracts from a medicinal plant Leea indica and its selected phytoconstituents on human ovarian cancer cells and in combination with oxaliplatin and natural killer (NK) cells.MethodsFresh, healthy leaves of L. indica were harvested and extracted in 70% methanol by maceration. The crude extract was partitioned with n-hexane, dichloromethane and ethyl acetate. Selected extracts and compounds were analyzed for their effects on cell viability of human ovarian cancer cells, NK cell cytotoxicity, and stress ligands expression for NK cell receptors. They were also evaluated for their effects on TNF-α and IL-1β production by enzyme-linked immunosorbent assay in lipopolysaccharide-stimulated human U937 macrophages.ResultsLeaf extracts of L. indica increased the susceptibility of human ovarian tumor cells to NK cell-mediated cytotoxicity. Treatment of cancer cells with methyl gallate but not gallic acid upregulated the expression of stress ligands. Tumor cells pretreated with combination of methyl gallate and low concentration of oxaliplatin displayed increased levels of stress ligands expression and concomitantly enhanced susceptibility to NK cell-mediated cytolysis. Further, NK cells completely abrogated the growth of methyl gallate-pretreated ovarian cancer cells. The leaf extracts suppressed TNF-α and IL-1β production in human U937 macrophages. Methyl gallate was more potent than gallic acid in down-regulating these cytokine levels.ConclusionsWe demonstrated for the first time that leaf extracts of L. indica and its phytoconstituent methyl gallate enhanced the susceptibility of ovarian tumor cells to NK cell cytolysis. These results suggest that the combined effect of methyl gallate, oxaliplatin and NK cells in ovarian cancer cells warrants further investigation, for example for refractory ovarian cancer. Our work is a step towards better scientific understanding of the traditional anticancer use of L. indica.