3D Spheroid Model Research Articles

P-250 A novel small molecule inhibits enzyme associated with colorectal cancer

Colorectal cancer (CRC) represents one of the most common malignant tumor with high morbidity worldwide. N6-methyladenosine (m6A) methylation, is the most frequently observed RNA modifications that tremendously contribute to cancer initiation, progression, and resistance. METTL3 (Methyltransferase like 3), is a predominant catalytic enzyme in the m6A methyltransferase system. METTL3 has been implicated in carcinogenesis in various cancers including colon cancer by stabilizing oncogenic mRNAs in an m6A-dependent manner which is why it is considered as an attractive target for cancer therapy. Here, we identified STM2457, as a selective small-molecule METTL3 inhibitor that exerts promising anticancer activity in CRC. We detected the mRNA and protein expression of METTL3 and its target oncogenes in human colorectal cancer cell line panel. The anti-tumor activities of STM2457 in human colorectal cancer cell line panel were investigated by MTT, wound healing, colony formation, western blot assays, and 3D spheroid model. m6A level of mRNA was measured by an m6A-RNA methylation quantification kit. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to screen the target genes of METTL3. We discovered that STM2457 treatment exerted potential anticancer effects in CRC cells. Pharmacological inhibition of METTL3 by STM2457 reduced cellular proliferation, invasion, colony formation, and tumorsphere formation of CRC cells. STM2457 treatment reduced m6A level in CRC cells. Mechanistically, MeRIP-seq illustrated that SLC2A1 (Solute Carrier Family 2 Member 1) as a target of METTL3. The TCGA data analysis showed that SLC2A1 mRNA and protein were highly expressed in colorectal cancer tissues. In addition, the highly expressed SLC2A1 mRNA associated with metastasis and poor survival in CRC patients. Thus, the METTL3/m6A/SLC2A1 axis accelerated colorectal carcinogenesis. The data show that the blockade of METTL3/m6A/SLC2A1 axis by a potent and novel small-molecule inhibitor, STM2457 is a promising approach for treatment of CRC.

Synthesis and characterization of polyimidazolium species on silica nanoparticles: Study on cytotoxicity and removal of nitrate from water

AbstractA nanocomposite containing imidazolium based on modified silica nanoparticles (polyimidazolium based on nano silica particles [PIZNS]) was prepared to remove nitrate from water. The prepared compounds were fully characterized by Fourier transform infrared, 1H‐NMR, field emission scanning electron microscopy, energy dispersive X‐ray (EDX) and thermogravimetric analyzes. The ability of PIZNS in nitrate ions adsorption was tested under different operating conditions. The maximum removal efficiency and adsorption capacity (Qmax) of the PIZNS were obtained 90.56% and 37.73 mg g−1, respectively (initial nitrate ion concentration of 50 mg/L, at pH = 7, 30 mg adsorbent, contact time of 30 min). To evaluate the selectivity coefficient, the removal of nitrate ion was determined in the presence of competitive sulfate, bicarbonate, chloride, and phosphate ions by adsorbent. Further, kinetic and isotherm investigation showed best fit by second order and Langmuir models, respectively. In addition, the anti‐cancer properties and subsequently the cytotoxicity effect of the PIZNS was assayed in vitro using cisplatin as a standard against three cancer cell lines including PC12, HL60, and βTC. The most potent inhibition effect was observed toward HL60 cells by PIZNS. Thus, the collapse of mitochondrial membrane potential was evaluated in these cells. In addition, the cytostatic effect of this polymer was depicted in the HL60 3D spheroid model.

Controlling the Biological Behaviors of Polymer-Coated Upconverting Nanoparticles by Adjusting the Linker Length of Estrone Ligands.

The estrone ligand is used for modifying nanoparticle surfaces to improve their targeting effect on cancer cell lines. However, to date, there is no common agreement on the ideal linker length to be used for the optimum targeting performance. In this study, we aimed to investigate the impact of poly(poly ethylene glycol methyl ether methacrylate) (PPEGMEMA) linker length on the cellular uptake behavior of polymer-coated upconverting nanoparticles (UCNPs). Different triblock terpolymers, poly(poly (ethylene glycol) methyl ether methacrylate)-block-polymethacrylic acid-block-polyethylene glycol methacrylate phosphate (PPEGMEMAx-b-PMAAy-b-PEGMP3: x = 7, 15, 33, and 80; y = 16, 20, 18, and 18), were synthesized with different polymer linker chain lengths between the surface and the targeting ligand by reversible addition-fragmentation chain transfer polymerization. The estrone ligand was attached to the polymer via specific terminal conjugation. The cellular association of polymer-coated UCNPs with linker chain lengths was evaluated in MCF-7 cells by flow cytometry. Our results showed that the bioactivity of ligand modification is dependent on the length of the polymer linker. The shortest polymer PPEGMEMA7-b-PMAA16-b-PEGMP3 with estrone at the end of the polymer chain was found to have the best cellular association behavior in the estrogen receptor (ER)α-positive expression cell line MCF-7. Additionally, the anticancer drug doxorubicin•HCl was encapsulated in the nanocarrier to evaluate the 2D and 3D cytotoxicity. The results showed that estrone modification could efficiently improve the cellular uptake in ERα-positive expression cell lines and in 3D spheroid models.

Metabolic flux analysis of 3D spheroids reveals significant differences in glucose metabolism from matched 2D cultures of colorectal cancer and pancreatic ductal adenocarcinoma cell lines

BackgroundMost in vitro cancer cell experiments have been performed using 2D models. However, 3D spheroid cultures are increasingly favored for being more representative of in vivo tumor conditions. To overcome the translational challenges with 2D cell cultures, 3D systems better model more complex cell-to-cell contact and nutrient levels present in a tumor, improving our understanding of cancer complexity. Despite this need, there are few reports on how 3D cultures differ metabolically from 2D cultures.MethodsWell-described cell lines from colorectal cancer (HCT116 and SW948) and pancreatic ductal adenocarcinoma (Panc-1 and MIA-Pa-Ca-2) were used to investigate metabolism in 3D spheroid models. The metabolic variation under normal glucose conditions were investigated comparing 2D and 3D cultures by metabolic flux analysis and expression of key metabolic proteins.ResultsWe find significant differences in glucose metabolism of 3D cultures compared to 2D cultures, both related to glycolysis and oxidative phosphorylation. Spheroids have higher ATP-linked respiration in standard nutrient conditions and higher non-aerobic ATP production in the absence of supplemented glucose. In addition, ATP-linked respiration is significantly inversely correlated with OCR/ECAR (p = 0.0096). Mitochondrial transport protein, TOMM20, expression decreases in all spheroid models compared to 2D, and monocarboxylate transporter (MCT) expression increases in 3 of the 4 spheroid models.ConclusionsIn this study of CRC and PDAC cell lines, we demonstrate that glucose metabolism in 3D spheroids differs significantly from 2D cultures, both in terms of glycolytic and oxidative phosphorylation metrics. The metabolic phenotype shift from 2D to 3D culture in one cell line is greater than the phenotypic differences between each cell line and tumor source. The results herein emphasize the need to use 3D cell models for investigating nutrient utilization and metabolic flux for a better understanding of tumor metabolism and potential metabolic therapeutic targets.

Bioactivity of Novel Pyrazole-Thiazolines Scaffolds against Trypanosoma cruzi: Computational Approaches and 3D Spheroid Model on Drug Discovery for Chagas Disease.

Chagas disease, a century-old disease that mainly affects the impoverished population in Latin America, causes high morbidity and mortality in endemic countries. The available drugs, benznidazole (Bz) and nifurtimox, have limited effectiveness and intense side effects. Drug repurposing, and the development of new chemical entities with potent activity against Trypanosoma cruzi, are a potential source of therapeutic options. The present study describes the biological activity of two new series of pyrazole-thiazoline derivatives, based on optimization of a hit system 5-aminopyrazole-imidazoline previously identified, using structure–activity relationship exploration, and computational and phenotype-based strategies. Promising candidates, 2c, 2e, and 2i derivatives, showed good oral bioavailability and ADMET properties, and low cytotoxicity (CC50 > 100 µM) besides potent activity against trypomastigotes (0.4–2.1 µM) compared to Bz (19.6 ± 2.3 µM). Among them, 2c also stands out, with greater potency against intracellular amastigotes (pIC50 = 5.85). The selected pyrazole-thiazoline derivatives showed good permeability and effectiveness in the 3D spheroids system, but did not sustain parasite clearance in a washout assay. The compounds’ mechanism of action is still unknown, since the treatment neither increased reactive oxygen species, nor reduced cysteine protease activity. This new scaffold will be targeted to optimize in order to enhance its biological activity to identify new drug candidates for Chagas disease therapy.

Novel HDL mimicking targeted drug delivery system for the treatment of Ewing Sarcoma

PurposeSince their discovery as antitumor agents, anthracycline drugs such as doxorubicin have remained mainstays as an important class of chemotherapy for the treatment of a wide range of cancers including pediatric malignancies like Ewing Sarcoma (EWS). Unfortunately, their usage is limited due to cumulative dose‐dependent cardiotoxicity. Targeted drug delivery could be an important option to circumvent this problem.Experimental designOur laboratory has developed a novel reconstituted HDL drug delivery system comprised of Myristic acid conjugated Apo AI mimicking 5A peptide (Myr5A). We used a hydrophobic derivative of doxorubicin, N‐Benzyladriamycin‐14‐valerate (AD198) to engineer formulations of Myr5A‐AD198. Physico‐chemical characterization including size, zeta potential and entrapment efficiency was determined. Cytotoxic effectiveness of this formulation was tested in Ewing Sarcoma cells A673 and Cardiomyocytes, H9c2 in 2D and 3D spheroid model using CCK‐8 assays. Using Myr5A based formulation‐containing BODIPY; mechanism of uptake of payload was demonstrated by imaging. In addition, we investigated Biodistribution and payload retention in EWS tumors in xenograft NOG mice with EWS tumor model.ResultsFormulation of the Myr5A‐AD198 was stable and non‐leaky with payload entrapment efficiency of 47.6 ± 3.2%. The nanoparticles were spherical with average size of 47 ± 12 nm as observed by transmission electron microscopy (TEM). Cytotoxicity assays demonstrated that IC50 values of Myr5A‐AD198 formulation was 2.8 ± 0.4 times more effective than free AD198 in EWS cells. On the other hand, 3 times more dose was required for Cardiomyocytes with Myr5A‐AD198 formulation as compared to free AD198. Blocking the Scavenger Receptor type B1 (SR‐B1) receptors, resulted in inhibition of the uptake of payload in Myr5A formulation. Subcutaneous injection of Myr5A‐IR 780 dye in xenograft NOG mice indicated the retention of payload for up to 72 hours in tumor.ConclusionThese preclinical studies indicated a great translational potential of Myr5A‐AD198 formulation for the treatment of EWS patients due to its selective uptake mechanism and cardio‐protective effect. Longer retention of the drug in tumors as well as lower dose requirement compared to free drug gives an additional advantage. In future, the Myr5A based technology shows promise for personalized treatment options in EWS and other type of cancers as well.

Target Discovery Platform Identifies Cardioprotective Genes Using Hibernation Omics Data

Over the course of their evolution, many mammals have naturally solved a number of human biomedical problems. The genes underlying their protective strategies are likely neither novel nor species‐specific, but rather shared among all mammals, including humans. Identifying and validating these genes could therefore lead to new therapeutic treatments for human clinical indications. To this end, we developed a target discovery platform which mines multi‐omics data from mammals resistant to human pathologies in order to identify novel therapeutic genetic targets. More specifically, our platform is built upon transcriptomic and epigenomic data from bio‐banked 13‐lined ground squirrel (13‐LGS) tissues collected at precise seasonal and physiological time‐points across their annual cycle of hibernation. Our platform also incorporates myriad genomic, transcriptomic, and proteomic datasets publicly available for this and other disease‐resistant species, as well as human genomics and transcriptomics data. Here we sought to identify novel genetic targets protective against cardiac fibrosis. While the hearts of 13‐LGS experience repeated bouts of ischemia‐reperfusion during hibernation, long‐term fibrotic scarring resulting from this type of injury is not detected. To identify protective targets, we first generated RNASeq data from hearts of 13‐LGS across 11 different seasonal and physiological groups (n≥5 per group). After obtaining transcript abundance counts for each sample library, we performed weighted correlation gene network analysis (WGCNA) to identify modules of transcripts co‐expressed among the different physiological groups. One resulting WGCNA module contained TGFB1 (transforming growth factor beta 1), a known central mediator of fibrogenesis. Knockdown (KD) of this module’s hub gene, designated here as FAUN211G, as well as the second top‐ranking gene, designated FAUNA212G, significantly decreased collagen deposition in an in vitro 2D model of cardiac fibrosis using primary human cardiac fibroblasts. We next perturbed these genes in an in vitro 3D spheroid model of human cardiac fibrosis and detected improved parameters of cardiac function. Finally, we tested small molecule inhibitors on FAUNA211G in both 2D and 3D in vitro models; we identified significant reductions in myofibroblast activation, proliferation and extracellular matrix deposition. Both FAUNA211G and FAUNA212G were previously unknown to be involved in cardiac fibrosis. In sum, we demonstrate that network analysis of the hibernator’s transcriptome is a strategy to identify novel target genes for treatment of human clinical indications.

Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models.

The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC.

Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple-Negative Breast Cancer Cells.

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxia-responsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxia-responsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes' ability to target the αvβ3 integrin, overexpressed on triple-negative breast cancer cells' surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 μM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells' survival.

A three-dimensional human brain spheroid model for studying sonoporation-induced drug penetration beyond the blood-brain barrier

Ultrasound (US)-targeted microbubble (MB) cavitation (UTMC) facilitates delivery of cell-impermeant drugs across the blood brain barrier (BBB). The effects of UTMC on extravascular cells, and the extent of payload penetration, are unknown. Spheroids were generated using neurons and astrocytes derived from human iPSCs, primary human brain- microglia, endothelial cells (ECs) and pericytes, and placed in a water tank with lipid MBs. US (1 MHz frequency, 250 kPa peak negative pressure, 10 μs pulse duration, 10 ms pulse interval) was delivered for 10 s. Immunostaining showed ECs and pericytes at the spheroid periphery, with high expression of membranous ZO-1. BBB functionality was confirmed with histamine treatment, which increased permeability to Texas red dextran (TRD) (28.8% increase, p = 0.0015). TRD penetrated up to 100 μm beyond the BBB upon UTMC (12% increase compared to 0 kPa control). Inhibition of endothelial nitric oxide synthase with L-NAME reduced UTMC-induced TRD penetration (19% decrease, p < 0.05). Sonoporation, as shown by uptake of propidium iodide, occurred in cells beyond the BBB. In our novel 3D spheroid model with intact BBB, UTMC caused nitric-oxide dependent BBB breach. This is the first report of UTMC causing “remote” sonoporation of cells not in direct contact with MBs and bears further study.

Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5–7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

Abstract P4-01-12: Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models

Abstract Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.

Before in vivo studies: In vitro screening of sphingomyelin nanosystems using a relevant 3D multicellular pancreatic tumor spheroid model

Sphingomyelin nanosystems have already shown to be promising carriers for efficient delivery of anticancer drugs. For further application in the treatment of pancreatic tumor, the investigation on relevant in vitro models able to reproduce its physio-pathological complexity is mandatory. Accordingly, a 3D heterotype spheroid model of pancreatic tumor has been herein constructed to investigate the potential of bare and polyethylene glycol-modified lipid nanosystems in terms of their ability to penetrate the tumor mass and deliver drugs. Regardless of their surface properties, the lipid nanosystems successfully diffused through the spheroid without inducing toxicity, showing a clear safety profile. Loading of the bare nanosystems with a lipid prodrug of gemcitabine was used to evaluate their therapeutic potential. While the nanosystems were more effective than the free drug on 2D cell monocultures, this advantage, despite their efficient penetration capacity, was lost in the 3D tumor model. The latter, being able to mimic the tumor and its microenvironment, was capable to provide a more realistic information on the cell sensitivity to treatments. These results highlight the importance of using appropriate 3D tumor models as tools for proper in vitro evaluation of nanomedicine efficacy and their timely optimisation, so as to identify the best candidates for later in vivo evaluation.

Doxorubicin sensitizes breast cancer cells to natural killercells in connection with increased Fas receptors.

Breast cancer (BC) is the most common cancer in women. Although standard treatments are successful in patients with BC diagnosed at an early stage, an alternative treatment is required for patients with advanced-stage disease who do not respond to these treatments. The concept of using chemotherapy to sensitize cancer cells to become susceptible to immunotherapy was recently introduced and may be used as an alternative treatment for BC. The chemotherapeutic drug doxorubicin has been reported to sensitize cancer cells; however, the efficacy to sensitize the solid spheroids, in addition to its underlying mechanism regarding how doxorubicin sensitizes BC, has not previously been explored. In the present study, the effectiveness of a combined treatment of doxorubicin and natural killer-92 (NK-92) cells against BC in either 2D or 3D spheroid models, and its association with Fas receptor (FasR) expression, was demonstrated. The BC (MCF7) cell line expressing a higher level of FasR was more sensitive to NK-92 cell killing than the MDA-MB-231 cell line, which expressed a lower level of FasR. A sublethal dose of doxorubicin caused a significant improvement in NK cytotoxicity. Concordantly, a significant reduction in cell viability was observed in the doxorubicin-treated MCF7 spheroids. Notably, flow cytometric analysis revealed significantly increased FasR expression in the MCF7 cells, suggesting the underlying sensitization mechanism of doxorubicin in BC was related to the FasR upregulation. The present findings supported the use of combined doxorubicin and NK immunotherapy in BC treatment.

Oxidative Stress Modulation by Carnosine in Scaffold Free Human Dermis Spheroids Model: A Proteomic Study.

Carnosine is an endogenous β-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.

Short-term tetrabromobisphenol A exposure promotes fibrosis of human uterine fibroid cells in a 3D culture system through TGF-beta signaling.

Tetrabromobisphenol A (TBBPA), a derivative of BPA, is a ubiquitous environmental contaminant with weak estrogenic properties. In women, uterine fibroids are highly prevalent estrogen‐responsive tumors often with excessive accumulation of extracellular matrix (ECM) and may be the target of environmental estrogens. We have found that BPA has profibrotic effects in vitro, in addition to previous reports of the in vivo fibrotic effects of BPA in mouse uterus. However, the role of TBBPA in fibrosis is unclear. To investigate the effects of TBBPA on uterine fibrosis, we developed a 3D human uterine leiomyoma (ht‐UtLM) spheroid culture model. Cell proliferation was evaluated in 3D ht‐UtLM spheroids following TBBPA (10−6–200 µM) administration at 48 h. Fibrosis was assessed using a Masson's Trichrome stain and light microscopy at 7 days of TBBPA (10−3 µM) treatment. Differential expression of ECM and fibrosis genes were determined using RT² Profiler™ PCR arrays. Network and pathway analyses were conducted using Ingenuity Pathway Analysis. The activation of pathway proteins was analyzed by a transforming growth factor‐beta (TGFB) protein array. We found that TBBPA increased cell proliferation and promoted fibrosis in 3D ht‐UtLM spheroids with increased deposition of collagens. TBBPA upregulated the expression of profibrotic genes and corresponding proteins associated with the TGFB pathway. TBBPA activated TGFB signaling through phosphorylation of TGFBR1 and downstream effectors—small mothers against decapentaplegic ‐2 and ‐3 proteins (SMAD2 and SMAD3). The 3D ht‐UtLM spheroid model is an effective system for studying environmental agents on human uterine fibrosis. TBBPA can promote fibrosis in uterine fibroid through TGFB/SMAD signaling.

Magnetoelectric core-shell CoFe2O4@BaTiO3 nanorods: their role in drug delivery and effect on multidrug resistance pump activity in vitro.

Nanoparticle mediated targeted drug delivery has become a widespread area of cancer research to address premature drug delivery problems. We report the synthesis of magneto-electric (ME) core–shell cobalt ferrite-barium titanate nanorods (CFO@BTO NRs) to achieve “on demand” drug release in vitro. Physical characterizations confirmed the formation of pure CFO@BTO NRs with appropriate magnetic and ferroelectric response, favorable for an externally controlled drug delivery system. Functionalization of NRs with doxorubicin (DOX) and methotrexate (MTX) achieved up to 98% drug release in 20 minutes, under a 4 mT magnetic field (MF). We observed strong MF and dose dependent cytotoxic response in HepG2 and HT144 cells and 3D spheroid models (p < 0.05). Cytotoxicity was characterized by enhanced oxidative stress, causing p53 mediated cell cycle arrest, DNA damage and cellular apoptosis via downregulation of Bcl-2 expression. In addition, MF and dose dependent inhibition of Multidrug Resistance (MDR) pump activity was also observed (p < 0.05) indicating effectivity in chemo-resistant cancers. Hence, CFO@BTO NRs represent an efficient carrier system for controlled drug delivery in cancer nanotherapeutics, where higher drug uptake is a prerequisite for effective treatment.

Assessment of a 3D neural spheroid model to detect pharmaceutical-induced neurotoxicity_suppl2

Assessment of a 3D neural spheroid model to detect pharmaceutical-induced neurotoxicity_suppl2

Efficiency of GrowDex&lt;sup&gt;®&lt;/sup&gt; nanofibrillar cellulosic hydrogel when generating homotypic and heterotypic 3D tumor spheroids

&lt;abstract&gt; &lt;p&gt;In recent times, homotypic and heterotypic 3D tumor spheroid (HTS) models have been receiving increasing attention and come to be widely employed in preclinical studies. The present study is focused on the generation of homotypic (A549 and MDA-MB-231, separately) and heterotypic (A549 + NIH/3T3; MDA-MB-231 + NIH/3T3) 3D tumor spheroids by using GrowDex&lt;sup&gt;®&lt;/sup&gt; nanofibrillar cellulosic (NFC) hydrogel as the scaffold. Light microscopic observations and F-actin staining confirmed the formation of spheroids. The proliferation efficiency indicated an expansion of cell population and an increase in spheroid size over time. The distribution, interaction pattern and influence of fibroblasts on the epithelial cell types were observed in terms of the size and shape of the HTS against homo-spheroids. An interesting observation was that, with an increase in the size of HTSs, many more fibroblast cells were found to occupy the core region, which, perhaps, was due to the faster growth of tumor cells over normal cells. Thus, normal and tumor cells, especially with origins from two different species, can be cultured together in 3D format, and this can potentially enhance our knowledge of tumor microenvironments and cell-cell interaction. These spheroids could be used to improve microphysiological systems for drug discovery and to better understand the tumor microenvironment.&lt;/p&gt; &lt;/abstract&gt;

Three-dimensional spheroids of choroid-retinal vascular endothelial cells as an in-vitro model for diabetic retinopathy: Proof-of-concept investigation

Diabetic retinopathy (DR) is a primary microvascular complication of diabetes mellitus and a vision-threatening condition. Vascular endothelial growth factor (VEGF) induces neovascularization and causes metabolic damage to the retinal and choroidal vasculature in diabetic patients. Existing drug screening models and treatment strategies for DR need to be refined through the establishment of relevant pre-clinical models, which may enable development of effective and safe therapies. The present study discusses the development of an in-vitro three-dimensional (3D) spheroid model, using RF/6A choroid-retinal vascular endothelial cells, to closely mimic the in-vivo disease condition. Compact, reproducibly-sized, viable and proliferating RF/6A spheroids were fabricated, as confirmed by microscopy, live/dead assay, cell proliferation assay and histological staining. In-vitro angiogenesis was studied by evaluating individual effects of VEGF and an anti-VEGF monoclonal antibody, Bevacizumab, and their combination on cellular proliferation and 3D endothelial sprout formation. VEGF stimulated angiogenic sprouting while Bevacizumab demonstrated a dose-dependent anti-angiogenic effect, as determined from the cellular proliferation observed and extent and length of sprouting. These investigations validated the potential of RF/6A spheroids in providing an alternative-to-animal, pathophysiologically-relevant model to facilitate pre-clinical and biomedical research related to DR.