3D Spheroid Model Research Articles

MANCR lncRNA Modulates Cell-Cycle Progression and Metastasis by Cis-Regulation of Nuclear Rho-GEF.

A significant number of the genetic alterations observed in cancer patients lie within nonprotein-coding segments of the genome, including regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in breast cancer (BrCa), but the functional implications of this altered expression remain to be elucidated. By performing transcriptome screen in a triple negative BrCa (TNBC) isogenic 2D and 3D spheroid model, we observed aberrant expression of >1000 lncRNAs during BrCa progression. The chromatin-associated lncRNA MANCR shows elevated expression in metastatic TNBC. MANCR is upregulated in response to cellular stress and modulates DNA repair and cell proliferation. MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion, and wound healing invitro, and reduced metastatic lung colonization in xenograft experiments invivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint response. MANCR promotes the transcription of NET1A, a Rho-GEF that regulates DNA damage checkpoint and metastatic processes in cis, by differential promoter usage. Experiments suggest that MANCR regulates the expression of cancer-associated genes by modulating the association of various transcription factors and RNA-binding proteins. Our results identified the metastasis-promoting activities of MANCR in TNBC by cis-regulation of gene expression.

Anti-Cancer Properties of Two Intravenously Administrable Curcumin Formulations as Evaluated in the 3D Patient-Derived Cancer Spheroid Model.

Curcumin (Cur) is a heavily used complementary derived drug from cancer patients. Spheroid samples derived from 82 patients were prepared and treated after 48 h with two Cur formulations (CurA, CurB) in mono- and combination therapy. After 72 h, cell viability and morphology were assessed. The Cur formulations had significant inhibitory effects of -8.47% (p < 0.001), CurA of -10.01% (-50.14-23.11%, p = 0.001) and CurB of -6.30% (-33.50-19.30%, p = 0.006), compared to their solvent controls Polyethylene-glycol, β-Cyclodextrin (CurA) and Kolliphor-ELP, Citrate (CurB). Cur formulations were more effective in prostate cancer (-19.54%) and less effective in gynecological non-breast cancers (0.30%). CurA showed better responses in samples of patients <40 (-13.81%) and >70 years of age (-17.74%). CurB had stronger effects in metastasized and heavily pretreated tumors. Combinations of Cur formulations and standard therapies were superior in 20/47 samples (42.55%) and inferior in 7/47 (14.89%). CurB stimulated chemo-doublets more strongly than monotherapies (-0.53% vs. -6.51%, p = 0.022) and more effectively than CurA (-6.51% vs. 3.33%, p = 0.005). Combinations of Cur formulations with Artesunate, Resveratrol and vitamin C were superior in 35/70 (50.00%) and inferior in 16/70 (22.86%) of samples. Cur formulations were significantly enhanced by combination with Artesunate (p = 0.020). Cur formulations showed a high variance in their anti-cancer effects, suggesting a need for individual testing before administration.

Design, synthesis, and optimization of novel PD-L1 inhibitors and the identification of a highly potent and orally bioavailable PD-L1 inhibitor

In this paper we report the discovery of structurally novel and highly potent programmed cell death-ligand 1 (PD-L1) inhibitors targeting surface and intracellular PD-L1. A ring fusion design utilizing dimethoxyphenyl indazole derivatives was used, followed by structural extension, which further improved potency by inducing the formation of additional symmetrical interactions within the PD-L1 binding site, leading to the discovery of novel and highly active tetra-aryl-scaffold inhibitors. Key optimizations involved polar tail chain modifications that improve potency and minimize cell cytotoxicity. In addition, druggability issues that exist outside the rule-of-five chemical space were addressed. CB31, a representative compound, was found to exhibit outstanding activity in blocking programmed cell death-1 (PD-1)/PD-L1 interactions (IC50 = 0.2 nM) and enhancing T-cell functions, with minimal cell cytotoxicity. CB31 also displayed favorable oral pharmacokinetic properties, consistent with its high passive permeability and insusceptibility to efflux transporters, as well as its high metabolic stability. Additionally, CB31 demonstrated mechanistically differentiated features from monoclonal antibodies by inducing PD-L1 internalization, intracellular retention of PD-L1 with altered glycosylation pattern, and PD-L1 degradation. It also demonstrated greater effects on tumor size reduction and tumor cell killing, with enhanced T-cell infiltration, in a 3D tumor spheroid model. Overall, results show that CB31 is a promising small-molecule PD-L1 inhibitor that can inhibit PD-1/PD-L1 interactions and promote PD-L1 degradation.

Hyaluronic acid-coated silver nanoparticles releasing Doxorubicin for combinatorial antitumor therapy

Although various nanocarriers have been developed to deliver anticancer drugs to the target tumors, it is still remaining great challenges, including burst release, non-specific targetability and insufficient therapeutic efficacy. Herein, we prepared a multifunctional nanoparticle composed of Ag core and hyaluronic acid shell (Ag NPs@HA) for stimultaneously intracellular delivery of Doxorubicin (DOX) and Ag NPs to improve the anticancer therapeutic efficacy. For our approach, Ag NPs was simply synthesized via the redox reaction between Ag+ ions and catechol group of functional HA, and DOX was subsequently loaded into the HA-coated Ag NPs through the interaction between the remaining catechol groups and DOX (Ag NPs@HA/DOX). The particles exhibited uniform morphology, good biocompatibility to normal cells, and pH-sensitive drug release. Notably, thanks to the specific interaction of HA and CD44 receptor-overexpressing cancer cells, Ag NPs@HA/DOX could dramatically improve the anti-tumor efficacy in vitro as well as in vivo mimicking 3D spheroid model, compared to the free DOX. Moreover, Ag NPs@HA/DOX exhibited superior tumor supression in vivo on a HELA-xenografted mouse model, while minimizing the systemic toxicity of free DOX. From the obtained results, we suggest Ag NPs@HA as potential nanocarrier for targeting delivery of various therapeutic drugs in anticancer therapy.

Malignancy progression and treatment efficacy estimation of osteosarcoma patients based on in vitro cell culture model and analysis

BackgroundOsteosarcoma (OS) usually happens in patients under 20 years old and is notorious for its low survivorship and limb loss. Personalized medicine is a viable approach to increase the efficiency of chemotherapy which is the main prognostic factor for survivorship after surgical treatment. MethodsIn this five-year prospective observational study, we collected primary cells of osteosarcoma from 15 patients, and examined the correlation between clinical characters of patients and cell properties characterized using various in vitro assays. The properties including genes expression, pro-angiogenic capability and anti-cancer drug response are characterized respectively by using RT-PCR, tube formation assay, osteogenesis assay and drug testing on 3D tumor spheroid model. ResultThe results suggest that OS patients with higher MMP9 expression levels have higher probability to develop skip metastasis (p = 0.041). The 3D tumor spheroid test based on the median lethal dose from 2D culture provides some prognostic value. Patients do not response well to methotrexate (MTX) show higher percentage of high pathology grade (p = 0.009) and lung metastasis (p = 0.044). Also, patients respond well to ifosfamide (IFO) have higher probability to achieve high tumor necrosis rate (p = 0.007). ConclusionThe association between cell properties and clinical characters of patients provided by our data can act as potential prognostic factors to help physicians to develop effective personalized chemotherapy for osteosarcoma treatments.

Asparagine synthetase and G-protein coupled estrogen receptor are critical responders to nutrient supply in KRAS mutant colorectal cancer.

Survival differences exist in colorectal cancer (CRC) patients by sex and disease stage. However, the potential molecular mechanism(s) are not well understood. Here we show that asparagine synthetase (ASNS) and G protein-coupled estrogen receptor-1 (GPER1) are critical sensors of nutrient depletion and linked to poorer outcomes for females with CRC. Using a 3D spheroid model of isogenic SW48 KRAS wild-type (WT) and G12A mutant (MT) cells grown under a restricted nutrient supply, we found that glutamine depletion inhibited cell growth in both cell lines, whereas ASNS and GPER1 expression were upregulated in KRAS MT versus WT. Estradiol decreased growth in KRAS WT but had no effect on MT cells. Selective GPER1 and ASNS inhibitors suppressed cell proliferation with increased caspase-3 activity of MT cells under glutamine depletion condition particularly in the presence of estradiol. In a clinical colon cancer cohort fromThe Cancer Genome Atlas, both high GPER1 and ASNS expression were associated with poorer overall survival for females only in advanced stage tumors. These results suggest KRAS MT cells have mechanisms in place that respond to decreased nutrient supply, typically observed in advanced tumors, by increasing the expression of ASNS and GPER1 to drive cell growth. Furthermore, KRAS MT cells are resistant to the protective effects of estradiol under nutrient deplete conditions. The findings indicate that GPER1 and ASNS expression, along withthe interaction between nutrient supply and KRAS mutations shed additional light on the mechanisms underlyingsex differences in metabolism and growth in CRC, and have clinical implications in the precision management of KRAS mutant CRC.

Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma.

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

Targeted lipid-coated ZnO nanoparticles coupled with ultrasound: A sonodynamic approach for the treatment of osteosarcoma as 3D spheroid models

Bone cancer is a rare disease with limited treatment options. Surgery and chemotherapy frequently fail to cure the disease because metastases can develop in the former, and the latter often lacks specificity. Innovative therapeutic treatments are therefore needed to address this rare disease, which typically affects children and adolescents. Physical stimulation to remotely activate a therapeutic agent may represent a promising solution, especially for deep-seated and/or distributed tumors. In this regard, sonodynamic therapy gathers importance: it offers a valid solution to the limitations of photodynamic therapy, such as limited tissue penetration, but retains the advantages of remotely activatable cell death. In this work, we developed lipidic coated iron-doped zinc oxide nanoparticles with superior biocompatibility as remotely activated sonosensitizers for osteosarcoma sonodynamic therapy and an active targeting mechanism addressing the erythropoietin-producing hepatocellular receptor-2 overexpressed in bone cancer cells. The positively charged zinc oxide NPs are coated with a negatively charged phospholipidic shell, which significantly enhances the biocompatibility and hemocompatibility of the nanoconstruct. Most importantly, the lipidic shell is modified by grafting a targeting peptide onto it, increasing cellular uptake towards osteosarcoma cells and demonstrating the potential to address various tissues with the same system. Reactive Oxygen Species (ROS) are effectively generated upon ultrasound stimulation and measured through electron paramagnetic resonance spectroscopy. The nanoconstructs are evaluated in vitro on 3D osteosarcoma spheroids, to demonstrate the effectiveness of the combination of NPs and ultrasound stimulation in suppressing the growth of bone osteosarcoma. The biocompatibility, targeting capabilities, and potential flexibility of the nanoparticles here described open up avenues for an effective and remotely-activated cancer therapy.

Induction of Cell Death by Bifidobacterium infantis DS1685 in Colorectal and Breast Cancers via SMAD4/TGF-Beta Activation.

Therapeutic advancements in treatments for cancer, a leading cause of mortality worldwide, have lagged behind the increasing incidence of this disease. There is a growing interest in multifaceted approaches for cancer treatment, such as chemotherapy, targeted therapy, and immunotherapy, but due to their low efficacy and severe side effects, there is a need for the development of new cancer therapies. Recently, the human microbiome, which is comprised of various microorganisms, has emerged as an important research field due to its potential impact on cancer treatment. Among these microorganisms, Bifidobacterium infantis has been shown to significantly improve the efficacy of various anticancer drugs. However, research on the role of B. infantis in cancer treatment remains insufficient. Thus, in this study, we explored the anticancer effect of treatment with B. infantis DS1685 supernatant (BI sup) in colorectal and breast cancer cell lines. Treatment with BI sup induced SMAD4 expression to suppress cell growth in colon and breast cancer cells. Furthermore, a decrease in tumor cohesion was observed through the disruption of the regulation of EMT-related genes by BI sup in 3D spheroid models. Based on these findings, we anticipate that BI sup could play an adjunctive role in cancer therapy, and future cotreatment of BI sup with various anticancer drugs may lead to synergistic effects in cancer treatment.

A NSC-34 cell line-derived spheroid model: Potential and challenges for in vitro evaluation of neurodegeneration.

Three-dimensional (3D) spheroid models aim to bridge the gap between traditional two-dimensional (2D) cultures and the complex in vivo tissue environment. These models, created by self-clustering cells to mimic a 3D environment with surrounding extracellular framework, provide a valuable research tool. The NSC-34 cell line, generated by fusing mouse spinal cord motor neurons and neuroblastoma cells, is essential for studying neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), where abnormal protein accumulation, such as TAR-DNA-binding protein 43 (TDP-43), occurs in affected nerve cells. However, NSC-34 behavior in a 3D context remains underexplored, and this study represents the first attempt to create a 3D model to determine its suitability for studying pathology. We generated NSC-34 spheroids using a nonadhesive hydrogel-based template and characterized them for 6 days. Light microscopy revealed that NSC-34 cells in 3D maintained high viability, a distinct round shape, and forming stable membrane connections. Scanning electron microscopy identified multiple tunnel-like structures, while ultrastructural analysis highlighted nuclear bending and mitochondria alterations. Using inducible GFP-TDP-43-expressing NSC-34 spheroids, we explored whether 3D structure affected TDP-43 expression, localization, and aggregation. Spheroids displayed nuclear GFP-TDP-43 expression, albeit at a reduced level compared with 2D cultures and generated both TDP-35 fragments and TDP-43 aggregates. This study sheds light on the distinctive behavior of NSC-34 in 3D culture, suggesting caution in the use of the 3D model for ALS or TDP-43 pathologies. Yet, it underscores the spheroids' potential for investigating fundamental cellular mechanisms, cell adaptation in a 3D context, future bioreactor applications, and drug penetration studies. RESEARCH HIGHLIGHTS: 3D spheroid generation: NSC-34 spheroids, developed using a hydrogel-based template, showed high viability and distinct shapes for 6 days. Structural features: advanced microscopy identified tunnel-like structures and nuclear and mitochondrial changes in the spheroids. Protein dynamics: the study observed how 3D structures impact TDP-43 behavior, with altered expression but similar aggregation patterns to 2D cultures. Research implications: this study reveals the unique behavior of NSC-34 in 3D culture, suggests a careful approach to use this model for ALS or TDP-43 pathologies, and highlights its potential in cellular mechanism research and drug testing applications.

Photophysical Characterization and In Vitro Evaluation of α-Mangostin-Loaded HDL Mimetic Nano-Complex in LN-229 Glioblastoma Spheroid Model.

Cytotoxic activity has been reported for the xanthone α-mangostin (AMN) against Glioblastoma multiforme (GBM), an aggressive malignant brain cancer with a poor prognosis. Recognizing that AMN's high degree of hydrophobicity is likely to limit its systemic administration, we formulated AMN using reconstituted high-density lipoprotein (rHDL) nanoparticles. The photophysical characteristics of the formulation, including fluorescence lifetime and steady-state anisotropy, indicated that AMN was successfully incorporated into the rHDL nanoparticles. To our knowledge, this is the first report on the fluorescent characteristics of AMN with an HDL-based drug carrier. Cytotoxicity studies in a 2D culture and 3D spheroid model of LN-229 GBM cells and normal human astrocytes showed an enhanced therapeutic index with the rHDL-AMN formulation compared to the unincorporated AMN and Temozolomide, a standard GBM chemotherapy agent. Furthermore, treatment with the rHDL-AMN facilitated a dose-dependent upregulation of autophagy and reactive oxygen species generation to a greater extent in LN-229 cells compared to astrocytes, indicating the reduced off-target toxicity of this novel formulation. These studies indicate the potential therapeutic benefits to GBM patients via selective targeting using the rHDL-AMN formulation.

Mechanistic investigation of the cytotoxicity of new Ce(IV) and Zn(II) complexes containing pyridinedicarboxylic acid derivatives against the SW480 cell line

Two cerium(IV) (C1) and zinc(II) (C2) complexes formulated as (aeea)2[Ce(pydc)3].5H2O (where pydc is pyridine-2,6-dicarboxylate and aeea is aminoethylethanolamine) and [Zn(hpydc)(dmp)].2H2O (where hpydc is 4-hydroxy-pyridine-2,6-dicarboxylate and dmp is 2,9-dimethyl-1,10-phenanthroline) were obtained by a one-pot reaction of organic ligands and the metal salts Ce(NO3)2.6H2O and Zn(NO3)2.6H2O. The complexes were characterized by spectroscopic methods and single-crystal X-ray diffraction. The MTT assay was performed for the compounds against four cell lines, including human hepatocellular carcinoma (BEL-7404), primary human colon cancer (SW480), metastatic human colon cancer (SW620) and normal human colon tissue (CCD841CoN), which showed the highest sensitivity to C1 (IC50= 1.89 μM, MAE= 72.46%) (where MAE represents the maximal antiproliferative effect). C1 demonstrated its ability as a redox-active compound to generate high levels of reactive oxygen species (ROS) in this cell line. Mechanistic investigation of the efficacy of the compounds on SW480 cells showed that C1 can induce bimodal cell death by both (intrinsic and extrinsic) apoptosis and autophagy, whereas only the intrinsic apoptosis pathway was detected when the aforementioned cells were treated with C2. SW480 cells treated with C1 showed remarkable morphological changes as well as a reduction in tumor size in the SW480 3D spheroid model.

Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

BackgroundIn the treatment of oral cavity cancer, margin status is one of the most critical prognostic factors. Positive margins are associated with higher local recurrence and lower survival rates. Therefore, the universal goal of oral surgical oncology is to achieve microscopically clear margins. Near-infrared fluorescence guided surgery (FGS) could improve surgical resection using fluorescent probes. αVβ6 integrin has shown great potential for cancer targeting due to its overexpression in oral cancers. Red fluorescent contrast agent IRDye 680 coupled with anti-αVβ6 peptide (IRDye-A20) represents an asset to improve FGS of oral cancer. This study investigates the potential of IRDye-A20 as a selective imaging agent in 3D three-dimensional tongue cancer cells.MethodsαVβ6 integrin expression was evaluated by RT-qPCR and Western Blotting in 2D HSC-3 human tongue cancer cells and MRC-5 human fibroblasts. Targeting ability of IRDye-A20 was studied in both cell lines by flow cytometry technique. 3D tumor spheroid models, homotypic (HSC-3) and stroma-enriched heterotypic (HSC-3/MRC-5) spheroids were produced by liquid overlay procedure and further characterized using (immuno)histological and fluorescence-based techniques. IRDye-A20 selectivity was evaluated in each type of spheroids and each cell population.ResultsαVβ6 integrin was overexpressed in 2D HSC-3 cancer cells but not in MRC-5 fibroblasts and consistently, only HSC-3 were labelled with IRDye-A20. Round shaped spheroids with an average diameter of 400 μm were produced with a final ratio of 55%/45% between HSC-3 and MRC-5 cells, respectively. Immunofluorescence experiments demonstrated an uniform expression of αVβ6 integrin in homotypic spheroid, while its expression was restricted to cancer cells only in heterotypic spheroid. In stroma-enriched 3D model, Cytokeratin 19 and E-cadherin were expressed only by cancer cells while vimentin and fibronectin were expressed by fibroblasts. Using flow cytometry, we demonstrated that IRDye-A20 labeled the whole homotypic spheroid, while in the heterotypic model all cancer cells were highly fluorescent, with a negligible fluorescence in fibroblasts.ConclusionsThe present study demonstrated an efficient selective targeting of A20FMDV2-conjugated IRDye 680 in 3D tongue cancer cells stroma-enriched spheroids. Thus, IRDye-A20 could be a promising candidate for the future development of the fluorescence-guided surgery of oral cancers.Graphical

Downregulation of chemoresistance by claudin-14 silencing in human colorectal cancer cells

An exceptional expression of claudins (CLDNs), tight junction (TJ) proteins, is observed in various solid cancer tissues. However, the pathophysiological roles of CLDNs have not been clarified in detail. CLDN14 is highly expressed in human colorectal cancer (CRC) tissues and cultured cancer epithelial cells. We found CLDN14 silencing decreased cell viability without affecting spheroid size in the three-dimensional (3D) spheroid model of DLD-1 cells derived from human CRC. Mitochondria activity and oxidative stress level were reduced by CLDN14 silencing. Furthermore, CLDN14 silencing decreased the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target antioxidative genes. CLDN14 was colocalized with ZO-1, a scaffolding protein in the TJ. CLDN14 silencing induced the disruption of TJ barrier such as the reduction of transepithelial electrical resistance and elevation of fluxes of small molecules including glucose in two-dimensional (2D) cultured model,. The depletion of glucose induced the elevation of ROS generation, mitochondria activity, and Nrf2 expression. These results suggest that CLDN14 increases Nrf2 expression in spheroids mediated via the formation of paracellular barrier to glucose. The cytotoxicities of doxorubicin, an anthracycline anticancer drug, and oxaliplatin, a platinum-based agent, were augmented by an Nrf2 activator in 2D cultured cells. The anticancer drug-induced toxicity was enhanced by CLDN14 silencing in 3D spheroids. We suggest that CLDN14 may potentiate chemoresistance mediated by the suppression of paracellular glucose permeability and activation of the Nrf2 signaling pathway in CRC cells.

Lung decellularized matrix-derived 3D spheroids: Exploring silicosis through the impact of the Nrf2/Bax pathway on myofibroblast dynamics

Silicosis is an occupational respiratory disease caused by long-term inhalation of high concentrations of free silica particles. Studies suggest that oxidative stress is a crucial initiator of silicosis fibrosis, and previous studies have linked the antioxidative stress transcription factor known as Nrf2 to fibrosis antagonism. Myofibroblasts play a pivotal role in tissue damage repair due to oxidative stress. Unlike physiological repair, myofibroblasts in fibrosis exhibit an apoptosis-resistant phenotype, continuously synthesising and secreting significant amounts of collagen and other extracellular matrices, which could be a direct cause of silicosis fibrosis. However, the relationship and mechanism of action between oxidative stress and myofibroblast apoptosis resistance remain unclear. In this study, a new 3D cell culture model using mice lung decellularised matrix particles and fibroblasts was developed, simulating the changes in myofibroblasts during the development of silicotic nodules. Western Blot results indicate that silica stimulation leads to increased collagen deposition and decreased apoptosis-related protein Bax and oxidative stress-related protein Nrf2 in the 3D spheroid model. Immunofluorescence experiments reveal co-localisation in their expression. In Nrf2 overexpressing spheroids, Bax exhibits significant upregulation. In the Nrf2 knockout spheroids, Bax is also significantly downregulated; after intervention with Bax inhibitors, a significant downregulation of Bax-induced apoptosis was also detected in the Nrf2-overexpressed spheroids. In contrast, Bax-induced apoptosis showed a significant upregulation trend in Nrf2-overexpressed spheroids after intervention with Bax agonists. The results demonstrate that the spheroid model can mimic the development process of silicotic nodules, and silica stimulation leads to an apoptosis-resistant phenotype in myofibroblasts in the model, acting through the Nrf2/Bax pathway. This research establishes a new methodology for silicosis study, identifies therapeutic targets for silicosis, and opens new avenues for studying the mechanisms of silicosis fibrosis.

Multimodal Phasor Approach to study breast cancer cells invasion in 3D spheroid model.

We implemented a multimodal set of functional imaging techniques optimized for deep-tissue imaging to investigate how cancer cells invade surrounding tissues and how their physiological properties change in the process. As a model for cancer invasion of the extracellular matrix, we created 3D spheroids from triple-negative breast cancer cells (MDA-MB-231) and non-tumorigenic breast epithelial cells (MCF-10A). We analyzed multiple hallmarks of cancer within the same spheroid by combining a number of imaging techniques, such as metabolic imaging of NADH by Fluorescence Lifetime Imaging Microscopy (NADH-FLIM), hyperspectral imaging of a solvatochromic lipophilic dye (Nile Red) and extracellular matrix imaging by Second Harmonic Generation (SHG). We included phasor-based bioimage analysis of spheroids at three different time points, tracking both morphological and biological properties, including cellular metabolism, fatty acids storage, and collagen organization. Employing this multimodal deep-imaging framework, we observed and quantified cancer cell plasticity in response to changes in the environment composition.

Myoferlin: A Potential Marker of Response to Radiation Therapy and Survival in Locally Advanced Rectal Cancer

PurposePatients with locally advanced rectal cancer often require neo-adjuvant chemoradiotherapy to downstage the disease, but the response is variable with no predictive biomarkers. We have previously revealed through proteomic profiling that myoferlin is associated with response to radiotherapy. The aims of this study were to further validate this finding and explore the potential for myoferlin to act as a prognostic and/or therapeutic target. Materials and MethodsImmunohistochemical analysis of a tissue microarray for 111 patients was used to validate the initial proteomic findings. Manipulation of myoferlin was achieved using siRNA, a small molecular inhibitor (wj460) and a CRISPR-Cas9 knockout cell line. Radiosensitisation following treatment was assessed using 2D clonogenic assays, 3D spheroid models and patient derived organoids. Underlying mechanisms were investigated using electrophoresis, immunofluorescence and immunoblotting. ResultsAnalysis of both the diagnostic biopsy and tumour resection samples confirmed that low myoferlin expression correlated with a good response to neoadjuvant LCRT. High myoferlin expression was associated with spread to local lymph nodes and worse 5-year survival (p = 0.01, HR 3.5, 95%CI [1.27, 10.04]). This was externally validated using the S:CORT database. Quantification of myoferlin using immunoblotting in immortalised colorectal cancer cell lines and organoids demonstrated that high myoferlin expression was associated with increased radioresistance. Biological and pharmacological manipulation of myoferlin resulted in significantly increased radiosensitivity across all cell lines in 2D and 3D models. Following irradiation, myoferlin knockdown cells had a significantly impaired ability to repair DNA double strand breaks. This appeared to be mediated via non-homologous end-joining. ConclusionsWe have confirmed that high expression of myoferlin in rectal cancer is associated with poor response to neoadjuvant therapy and worse long-term survival. Furthermore, the manipulation of myoferlin led to increased radiosensitivity in vitro. This suggests that myoferlin could be targeted to enhance the sensitivity of rectal cancer patients to radiotherapy and further work is required.

Deciphering Natural Killer Cell Cytotoxicity Against Medulloblastoma in vitro and in vivo: Implications for Immunotherapy.

Medulloblastoma (MB) is the most prevalent paediatric brain tumour. Despite improvements in patient survival with current treatment strategies, the quality of life of these patients remains poor owing to the sequelae and relapse risk. An alternative, or, in addition to the current standard treatment, could be considered immunotherapy, such as Natural Killer cells (NK). NK cells are cytotoxic innate lymphoid cells that play a major role in cancer immunosurveillance. To date, the mechanism of cytotoxicity of NK cells, especially regarding the steps of adhesion, conjugation, cytotoxic granule polarisation in the cell contact area, perforin and granzyme release in two and three dimensions, and therapeutic efficacy in vivo have not been precisely described. Each step of NK cytotoxicity against the three MB cell lines was explored using confocal microscopy for conjugation, Elispot for degranulation, flow cytometry, and luminescence assays for target cell necrosis and lysis and mediators released by cytokine array, and then confirmed in a 3D spheroid model. Medulloblastoma-xenografted mice were treated with NK cells. Their persistence was evaluated by flow cytometry, and their efficacy in tumour growth and survival was determined. In addition, their effects on the tumour transcriptome were evaluated. NK cells showed variable affinities for conjugation with MB target cells depending on their subgroup and cytokine activation. Chemokines secreted during NK and MB cell co-culture are mainly associated with angiogenesis and immune cell recruitment. NK cell cytotoxicity induces MB cell death in both 2D and 3D co-culture models. NK cells initiated an inflammatory response in a human MB murine model by modulating the MB cell transcriptome. Our study confirmed that NK cells possess both in vitro and in vivo cytotoxic activity against MB cells and are of interest for the development of immunotherapy.

Next generation CD44v6-specific CAR-NK cells for the treatment of solid tumors.

e14518 Background: There is a medical need to develop new and effective therapies against solid tumors. CD44v6, an adhesion molecule that is implicated in tumorigenesis and metastases, was identified as a suitable target in solid tumors such as triple negative breast cancer (TNBC) or glioblastoma. Methods: We have developed a next-generation CAR targeting CD44v6 that incorporates IL-15 superagonist and checkpoint inhibitor molecules to overcome the immunosuppressive microenvironment (TME). NK cells were used due to their potential for allogeneic usage. Results: CD44v6 CAR-NK cells demonstrated effective cytotoxicity against TNBC and glioblastoma in 3D spheroid models. The IL-15 superagonist was specifically released upon recognition of CD44v6 on TNBC and contributed to the cytotoxic attack. Competitive inhibition of PD1 neutralized inhibition by PD1 ligands. We have validated iPSC-derived CD44v6 CAR-NK cells. Conclusions: Next generation CD44v6 CAR-NK cells are resistant to TME immunosuppression and offer a new therapeutic option for the treatment of solid tumors.