The Impact of Mitochondria in Ovarian Cancer Cell Metabolism, Proliferation, and Metastasis.

Mitochondrial dysfunctions are significantly implicated in cancer initiation, progression, and metastasis, which have been shown for several cancers including ovarian cancer.An increase in mitochondrial dysfunction is also associated with drug resistance along with cancer progression, which in part is related to its specific microenvironment that is characterized by ascites, low glucose levels, and hypoxia that causes ovarian cancer cells to switch to mitochondrial respiration to enable their survival. Peritoneal ascitic fluid accumulation is a specific feature of ovarian cancer, and it is a major cause of its metastatic spread that also presents challenges for effective treatment. Among the treatment difficulties for ovarian cancer is the mutation rate and frequency of mtDNA in ovarian cancer tissue that can affect the efficiency of chemotherapeutic drugs. The varied and multiple mutations of different types enable metabolic reprogramming, cancer cell proliferation, and drug resistance.New specific information on mechanisms underlying several of the mitochondrial dysfunctions has led to proposing various mitochondrial determinants as targets for ovarian cancer therapy, which include targeting specific mitochondrial proteins and phosphoproteins as well as reactive oxygen species (ROS) that accumulate abnormally in cancer cells. Because of the genetically and histologically heterogeneous nature of the disease, combination therapy approaches will be necessary to combat the disease and achieve progress in effective treatment of ovarian cancer. This chapter will address (1) mitochondrial vulnerabilities underlying dysfunction and disease; (2) mitochondrial dysfunction in ovarian cancer; (3) present treatment difficulties for ovarian cancer and new potential treatment strategies to target ovarian cancer mitochondrial metabolism; and (4) biobehavioral factors influencing ovarian cancer development.

Cancer Spheroids Embedded in Tissue-Engineered Skin Substitutes: A New Method to Study Tumorigenicity In Vivo.

Tumorigenic assays are used during a clinical translation to detect the transformation potential of cell-based therapies. One of these in vivo assays is based on the separate injection of each cell type to be used in the clinical trial. However, the injection method requires many animals and several months to obtain useful results. In previous studies, we showed the potential of tissue-engineered skin substitutes (TESs) as a model for normal skin in which cancer cells can be included in vitro. Herein, we showed a new method to study tumorigenicity, using cancer spheroids that were embedded in TESs (cTES) and grafted onto athymic mice, and compared it with the commonly used cell injection assay. Tumors developed in both models, cancer cell injection and cTES grafting, but metastases were not detected at the time of sacrifice. Interestingly, the rate of tumor development was faster in cTESs than with the injection method. In conclusion, grafting TESs is a sensitive method to detect tumor cell growth with and could be developed as an alternative test for tumorigenicity.

Chondroitin Sulfate Proteoglycan 4 Provides New Treatment Approach to Preventing Peritoneal Dissemination in Ovarian Cancer.

Most epithelial ovarian cancer (EOC) patients are diagnosed with peritoneal dissemination. Cellular interactions are an important aspect of EOC cells when they detach from the primary site of the ovary. However, the mechanism remains underexplored. Our study aimed to reveal the role of chondroitin sulfate proteoglycan 4 (CSPG4) in EOC with a major focus on cell-cell interactions. We examined the expression of CSPG4 in clinical samples and cell lines of EOC. The proliferation, migration, and invasion abilities of the CSPG4 knockdown cells were assessed. We also assessed the role of CSPG4 in spheroid formation and peritoneal metastasis in an in vivo model using sh-CSPG4 EOC cell lines. Of the clinical samples, 23 (44.2%) samples expressed CSPG4. CSPG4 was associated with a worse prognosis in patients with advanced EOC. Among the EOC cell lines, aggressive cell lines, including ES2, expressed CSPG4. When CSPG4 was knocked down using siRNA or shRNA, the cell proliferation, migration, and invasion abilities were significantly decreased compared to the control cells. Proteomic analyses showed changes in the expression of proteins related to the cell movement pathways. Spheroid formation was significantly inhibited when CSPG4 was inhibited. The number of nodules and the tumor burden of the omentum were significantly decreased in the sh-CSPG4 mouse models. In the peritoneal wash fluid from mice injected with sh-CSPG4 EOC cells, significantly fewer spheroids were present. Reduced CSPG4 expression was observed in lymphoid enhancer-binding factor 1-inhibited cells. CSPG4 is associated with aggressive features of EOC and poor prognosis. CSPG4 could be a new treatment target for blocking peritoneal metastasis by inhibiting spheroid formation.

A method for reproducible high-resolution imaging of 3D cancer cell spheroids.

Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

Hybrid Nanoparticle-Assisted Chemo-Photothermal Therapy and Photoacoustic Imaging in a Three-Dimensional Breast Cancer Cell Model.

Bioinspired nanoparticles have recently been gaining attention as promising multifunctional nanoplatforms for therapeutic applications in cancer, including breast cancer. Here, the efficiency of the chemo-photothermal and photoacoustic properties of hybrid albumin-modified nanoparticles (HSA-NPs) loaded with doxorubicin was evaluated in a three-dimensional breast cancer cell model. The HSA-NPs showed a higher uptake and deeper penetration into breast cancer spheroids than healthy breast cell 3D cultures. Confocal microscopy revealed that, in tumour spheroids incubated with doxorubicin-loaded NPs for 16 h, doxorubicin was mainly localised in the cytoplasm, while a strong signal was detectable at the nuclear level after 24 h, suggesting a time-dependent uptake. To evaluate the cytotoxicity of doxorubicin-loaded NPs, tumour spheroids were treated for up to 96 h with increasing concentrations of NPs, showing marked toxicity only at the highest concentration of doxorubicin. When doxorubicin administration was combined with laser photothermal irradiation, enhanced cytotoxicity was observed at lower concentrations and incubation times. Finally, the photoacoustic properties of doxorubicin-loaded NPs were evaluated in tumour spheroids, showing a detectable signal increasing with NP concentration. Overall, our data show that the combined effect of chemo-photothermal therapy results in a shorter exposure time to doxorubicin and a lower drug dose. Furthermore, owing to the photoacoustic properties of the NPs, this nanoplatform may represent a good candidate for theranostic applications.

Effects of Nanosecond Pulsed Electric Field (nsPEF) on a Multicellular Spheroid Tumor Model: Influence of Pulse Duration, Pulse Repetition Rate, Absorbed Energy, and Temperature.

Cellular response upon nsPEF exposure depends on different parameters, such as pulse number and duration, the intensity of the electric field, pulse repetition rate (PRR), pulsing buffer composition, absorbed energy, and local temperature increase. Therefore, a deep insight into the impact of such parameters on cellular response is paramount to adaptively optimize nsPEF treatment. Herein, we examined the effects of nsPEF ≤ 10 ns on long-term cellular viability and growth as a function of pulse duration (2-10 ns), PRR (20 and 200 Hz), cumulative time duration (1-5 µs), and absorbed electrical energy density (up to 81 mJ/mm3 in sucrose-containing low-conductivity buffer and up to 700 mJ/mm3 in high-conductivity HBSS buffer). Our results show that the effectiveness of nsPEFs in ablating 3D-grown cancer cells depends on the medium to which the cells are exposed and the PRR. When a medium with low-conductivity is used, the pulses do not result in cell ablation. Conversely, when the same pulse parameters are applied in a high-conductivity HBSS buffer and high PRRs are applied, the local temperature rises and yields either cell sensitization to nsPEFs or thermal damage.

INSIDIA 2.0 High-Throughput Analysis of 3D Cancer Models: Multiparametric Quantification of Graphene Quantum Dots Photothermal Therapy for Glioblastoma and Pancreatic Cancer.

Cancer spheroids are in vitro 3D models that became crucial in nanomaterials science thanks to the possibility of performing high throughput screening of nanoparticles and combined nanoparticle-drug therapies on in vitro models. However, most of the current spheroid analysis methods involve manual steps. This is a time-consuming process and is extremely liable to the variability of individual operators. For this reason, rapid, user-friendly, ready-to-use, high-throughput image analysis software is necessary. In this work, we report the INSIDIA 2.0 macro, which offers researchers high-throughput and high content quantitative analysis of in vitro 3D cancer cell spheroids and allows advanced parametrization of the expanding and invading cancer cellular mass. INSIDIA has been implemented to provide in-depth morphologic analysis and has been used for the analysis of the effect of graphene quantum dots photothermal therapy on glioblastoma (U87) and pancreatic cancer (PANC-1) spheroids. Thanks to INSIDIA 2.0 analysis, two types of effects have been observed: In U87 spheroids, death is accompanied by a decrease in area of the entire spheroid, with a decrease in entropy due to the generation of a high uniform density spheroid core. On the other hand, PANC-1 spheroids' death caused by nanoparticle photothermal disruption is accompanied with an overall increase in area and entropy due to the progressive loss of integrity and increase in variability of spheroid texture. We have summarized these effects in a quantitative parameter of spheroid disruption demonstrating that INSIDIA 2.0 multiparametric analysis can be used to quantify cell death in a non-invasive, fast, and high-throughput fashion.

Thermomagnetic Resonance Effect of the Extremely Low Frequency Electromagnetic Field on Three-Dimensional Cancer Models.

In our recent studies, we have developed a thermodynamic biochemical model able to select the resonant frequency of an extremely low frequency electromagnetic field (ELF-EMF) specifically affecting different types of cancer, and we have demonstrated its effects in vitro. In this work, we investigate the cellular response to the ELF electromagnetic wave in three-dimensional (3D) culture models, which mimic the features of tumors in vivo. Cell membrane was modelled as a resistor-capacitor circuit and the specific thermal resonant frequency was calculated and tested on two-dimensional (2D) and three-dimensional (3D) cell cultures of human pancreatic cancer, glioblastoma and breast cancer. Cell proliferation and the transcription of respiratory chain and adenosine triphosphate synthase subunits, as well as uncoupling proteins, were assessed. For the first time, we demonstrate that an ELF-EMF hampers growth and potentiates both the coupled and uncoupled respiration of all analyzed models. Interestingly, the metabolic shift was evident even in the 3D aggregates, making this approach particularly valuable and promising for future application in vivo, in aggressive cancer tissues characterized by resistance to treatments.

In Ovarian Cancer Multicellular Spheroids, Platelet Releasate Promotes Growth, Expansion of ALDH+ and CD133+ Cancer Stem Cells, and Protection against the Cytotoxic Effects of Cisplatin, Carboplatin and Paclitaxel.

A high platelet count is associated with a poor prognosis in ovarian cancer (OvCa). Despite good clinical responses with platinating agents in combination with taxanes, numerous OvCa patients relapse due to chemotherapy resistance. Here, we report that treatment of OvCa cells A2780, OVCAR5 and MDAH with releasate from activated platelets (PR) promoted multicellular tumor spheroid (MCTS) formation. These OvCa-MCTSs had increased percentages of CD133+ and aldehyde dehydrogenase (ALDH)+ cells, bona fide markers of OvCa cancer stem cells (CSCs). PR increased OVCAR5- and MDAH-MCTS viability and decreased the cytotoxic and pro-apoptotic effects of paclitaxel, cisplatin and carboplatin. PR increased the volume of spontaneously formed OVCAR8-MCTSs and counteracted their size reduction due to cisplatin, carboplatin and paclitaxel treatment. PR promoted the survival of ALDH+ and CD133+ OvCa cells during cisplatin, carboplatin and paclitaxel treatment. In conclusion, molecules and growth factors released by activated platelets (EGF, PDGF, TGF-ß, IGF and CCL5) may protect tumor cells from chemotherapy by promoting the expansion of ALDH+ and CD133+ OvCa-CSCs, favoring drug resistance and tumor relapse.

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate.

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-ß2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.

Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) for Monitoring of Drug Response in Primary Cancer Spheroids.

Malignant ascites is a fluid, which builds up in the abdomen and contains cancer cells in the form of single cells or multicellular clusters called spheroids. Malignant ascites has been observed in patients suffering from ovarian, cervical, gastric, colorectal, pancreatic, endometrial, or primary liver cancer. The spheroids are believed to play a major role in chemo resistance and metastasis of the cancer. To ease the discomfort of patients, malignant ascites (MA) is often drained from the abdomen using a procedure called paracentesis. MA retrieved via this minimal invasive procedure is a great source for cancer spheroids, which can be used for testing chemotherapeutic drugs and drug combinations. Herein, the existing workflow is adapted to make concurrent monitoring of drug accumulation, drug response, and drug metabolites feasible using primary spheroids or spheroids grown without a scaffolding matrix. To achieve this, those spheroids are embedded in matrigel, before fixing them with formalin. This makes it possible to process, store, and ship samples at room temperature. This new approach might be used to choose the best targeted therapy for each patient and thereby facilitate personalized medicine.

Supramolecular Nanofibrillar Thermoreversible Hydrogel for Growth and Release of Cancer Spheroids.

Growth of three-dimensional cancer spheroids (CSs) in man-made hydrogels mimicking natural extracellular matrix is an important and challenging task. Herein, we report on a supramolecular temperature-responsive hydrogel designed for the growth and subsequent release of CSs. A filamentous hydrogel was formed at 37 °C from an aqueous suspension of cellulose nanocrystals surface-functionalized with temperature-responsive polymer molecules. The encapsulation of cells in the hydrogel enabled effective growth of CSs with dimensions determined by the concentration of cellulose nanocrystals in the hydrogel. On demand release of CSs without loss of cell viability and spheroid integrity was achieved upon hydrogel cooling. The tumorigenic properties of the released CSs were examined by encapsulating and re-growing them in fibrin hydrogel. The results in this work can be used in fundamental cancer research and in cancer drug screening.

Targeting of Micelles and Liposomes Loaded with the Pro-Apoptotic Drug, NCL-240, into NCI/ADR-RES Cells in a 3D Spheroid Model.

PURPOSE: To develop transferrin (Tf)-targeted delivery systems for the pro-apoptotic drug, NCL-240, and to evaluate the efficacy of this delivery system in ovarian cancer NCI/ADR-RES cells, grown in vitro in a 3D spheroid model. METHODS: Tf-targeted PEG-PE-based micellar and ePC/CHOL-based liposomal delivery systems for NCL-240 were prepared. NCI/ADR-RES cells were used to generate spheroids by a non-adhesive liquid overlay technique. Spheroid growth and development were monitored by size (diameter) analysis and H&E staining. The targeted formulations were compared to untargeted ones in terms of their degree of spheroid association and penetration. A cell viability analysis with NCL-240-loaded micelles and liposomes was performed to assess the effectiveness of Tf-targeting. RESULTS: Tf-targeted polymeric micelles and Tf-targeted liposomes loaded with NCL-240 were prepared. NCI/ADR-RES cells generated spheroids that demonstrated the presence of a distinct necrotic core along with proliferating cells in the spheroid periphery, partly mimicking in vivo tumors. The Tf-targeted micelles and liposomes had a deeper spheroid penetration as compared to the untargeted delivery systems. Cell viability studies using the spheroid model demonstrated that Tf-mediated targeting markedly improved the cytotoxicity profile of NCL-240. CONCLUSION: Transferrin targeting enhanced delivery and effectiveness of micelles and liposomes loaded with NCL-240 against NCI/ADR-RES cancer cells in a 3D spheroid model.

Three-dimensional perfused tumour spheroid model for anti-cancer drug screening.

OBJECTIVE: To build an in vitro-perfused, three-dimensional (3D) spheroid model based on the TissueFlex system for anti-cancer drug efficacy testing in order to mimic avascular micro-tissues with inherent O2, nutrient and metabolite gradients, and to provide a more accurate prediction of drug toxicity and efficacy than traditional in vitro tumour models in conventional static culture well plates. RESULTS: The perfused cancer spheroid model showed higher cell viability and increased diameter of spheroids over a relatively long culture period (17 days). Three anti-cancer drugs with different cytotoxic mechanisms were tested. In perfusion, lower cytotoxicity was observed for traditional cytotoxic drug 5-fluorouracil and microtubule-interfering, paclitaxel, showed greater interruption of spheroid integrity. For the hypoxic-dependent drug, tirapazamine, there was no significant difference observed between static and perfusion cultures. CONCLUSION: The perfusion culture provides a better homeostasis for cancer cell growth in a more controllable working platform for long-term drug testing.

Multidrug resistance characterization in multicellular tumour spheroids from two human lung cancer cell lines.

BACKGROUND: Most of the knowledge about the mechanisms of multidrug resistance in lung cancer has been achieved through the use of cell lines isolated from tumours cultivated either in suspensions of isolated cells or in monolayers and following exposition to different cytostatic agents. However, tumour cell lines growing as multicellular tumour spheroids (MTS) frequently develop multicellular resistance in a drug-independent form. The aim of this study was to characterize the phenotypic and functional differences between two human NSCLC cell lines (INER-37 and INER-51) grown as traditional monolayer cultures versus as MTS. METHODS: After 72 hours treatment with anticancer drugs, chemosensitivity in monolayers and tumour spheroids cultures was assessed using MTT assay. Reverse transcription-polymerase chain reaction was employed to detect the mRNAs of multidrug resistance-related genes. The expression of P-gp was analyzed by immunohistochemical staining and cell cycle profiles were analyzed using FACS. RESULTS: The results indicate that when grown as MTS each lung cancer cell line had different morphologies as well as and abrogation of cell proliferation with decrease of the G2/M phase. Also, MTS acquired multicellular resistance to several chemotherapeutic agents in only a few days of culture which were accomplished by significant changes in the expression of MDR-related genes. CONCLUSION: Overall, the MTS culture changed the cellular response to drugs nevertheless each of the cell lines studied seems to implement different mechanisms to acquire multicellular resistance.

Comparison between polypyridyl and cyclometalated ruthenium(II) complexes: anticancer activities against 2D and 3D cancer models.

The aim of this study was to illustrate the dramatically different anticancer activities between coordinatively saturated polypyridyl (1 a-4 a) and cyclometalated (1 b-4 b) ruthenium(II) complexes. The cyclometalated complexes 1 b-4 b function as DNA transcription inhibitors, exhibiting switch-on cytotoxicity against a 2D cancer cell monolayer, whereas the polypyridyl complexes 1 a-4 a are relatively inactive. Moreover, complexes 1 b-4 b exhibit excellent cytotoxicity against 3D multicellular tumor spheroids (MCTSs), which serve as an intermediate model between in vitro 2D cell monolayers and in vivo 3D solid tumors. The hydrophobicity, efficient cell uptake, and nucleus targeting ability, as well as the high DNA binding affinity of complexes 1 b-4 b, likely contribute to their enhanced anticancer activity. We surmise that cyclometalation could be a universal approach to significantly enhance the anticancer activity of substituted polypyridyl Ru(II) complexes. We also suggest that 3D MCTSs may be a more practical platform for anticancer drug screening than 2D cancer monolayer approaches.

Suspension Culture System for Isolating Cancer Spheroids using Enzymatically Synthesized Cellulose Oligomers.

Isolating cancer cells from tissues and providing an appropriate culture environment are important for a better understanding of cancer behavior. Although various three-dimensional (3D) cell culture systems have been developed, techniques for collecting high-purity spheroids without strong stimulation are required. Herein, we report a 3D cell culture system for the isolation of cancer spheroids using enzymatically synthesized cellulose oligomers (COs) and demonstrate that this system isolates only cancer spheroids under coculture conditions with normal cells. CO suspensions in a serum-containing cell culture medium were prepared to suspend cells without settling. High-purity cancer spheroids could be separated by filtration without strong stimulation because the COs exhibited antibiofouling properties and a viscosity comparable to that of the culture medium. When human hepatocellular carcinoma (HepG2) cells, a model for cancer cells, were cultured in the CO suspensions, they proliferated clonally and efficiently with time. In addition, only developed cancer spheroids from HepG2 cells were collected in the presence of normal cells by using a mesh filter with an appropriate pore size. These results indicate that this approach has potential applications in basic cancer research and cancer drug screening.

Evaluation of the cytotoxic and immunomodulatory effects of sonodynamic therapy in human pancreatic cancer spheroids.

Sonodynamic therapy (SDT) exploits the energy generated by ultrasound (US) to activate sound-sensitive drugs (sonosensitizers), leading to the generation of reactive oxygen species (ROS) and cancer cell death. Two-dimensional (2D) and three-dimensional (3D) cultures of human pancreatic cancer BxPC-3 cells were chosen as the models with which to investigate the therapeutic effects of the US-activated sonosensitizer IR-780 as pancreatic cancer is still one of the most lethal types of cancer. The effects of SDT, including ROS production, cancer cell death and immunogenic cell death (ICD), were extensively investigated. When subjected to US, IR-780 triggered significant ROS production and caused cancer cell death after 24 h (p ≤ 0.01). Additionally, the activation of dendritic cells (DCs) led to an effective immune response against the cancer cells undergoing SDT-induced death. BxPC-3 spheroids were developed and studied extensively to validate the findings observed in 2D BxPC-3 cell cultures. An analysis of the pancreatic cancer spheroid section revealed significant SDT-induced cancer cell death after 48 h after the treatment (p ≤ 0.01), with this being accompanied by the presence of SDT-induced damage-associated molecular patterns (DAMPs), such as calreticulin (CRT) and high mobility group box 1 (HMGB1). In conclusion, the data obtained demonstrates the anticancer efficacy of SDT and its immunomodulatory potential via action as an ICD-inducer.

Comparing in vitro cytotoxic drug sensitivity in colon and pancreatic cancer using 2D and 3D cell models: Contrasting viability and growth inhibition in clinically relevant dose and repeated drug cycles.

BACKGROUND: In vitro drug screening that is more translatable to the in vivo tumor environment can reduce both time and cost of cancer drug development. Here we address some of the shortcomings in screening and show how treatment with 5-fluorouracil (5-FU) in 2D and 3D culture models of colorectal cancer (CRC) and pancreatic ductal adenocarcinomas (PDAC) give different responses regarding growth inhibition. METHODS: The sensitivity of the cell lines at clinically relevant 5-FU concentrations was monitored over 4 days of treatment in both 2D and 3D cultures for CRC (SW948 and HCT116) and PDAC (Panc-1 and MIA-Pa-Ca-2) cell lines. The 3D cultures were maintained beyond this point to enable a second treatment cycle at Day 14, following the timeline of a standard clinical 5-FU regimen. RESULTS: Evaluation after one cycle did not reveal significant growth inhibition in any of the CRC or PDAC 2D models. By the end of the second cycle of treatment the CRC spheroids reached 50% inhibition at clinically achievable concentrations in the 3D model, but not in the 2D model. The PDAC models were not sensitive to clinical doses even after two cycles. High content viability metrics point to even lower response in the resistant PDAC models. CONCLUSION: This study reveals the limitations of testing drugs in 2D cancer models and short exposure in 3D models, and the importance of using appropriate growth inhibition analysis. We found that screening with longer exposure and several cycles of treatment in 3D models suggests a more reliable way to assess drug sensitivity.

Synergistic eradicating impact of 5-fluouracil with FeO nanoparticles-diethyldithiocarbamate in colon cancer spheroids.

Background: Cancer stem cells' (CSCs) resistance to 5-fluorouracil (Fu), which is the main obstacle in treating colon cancer (CC), can be overcome by ferroptosis. The latter, herein, can be triggered by FeO nanoparticles (inducer of iron accumulation) and diethyldithiocarbamate-inhibited glutathione system and aldehyde dehydrogenase (ALDH1A1-maintained stemness, therapeutic resistance and metastasis). Materials & methods: Nanocomplex of FeO nanoparticles and diethyldithiocarbamate (FD) was used in combination with Fu to investigate its potential synergistic anti-CSC influence using CC spheroid models. Results: In Fu + FD-treated spheroids, the strongest growth inhibition, the highest cell death percentage, and the lowest CD133+-CSCs percentage and stemness gene expressions (e.g., drug efflux transporter), and the strongest antimetastatic effect were recorded with high synergistic indexes. Conclusion: Fu + FD represents effective combination therapy for chemoresistant CC cells.

[Box: see text].