The activity of pyrazolo[4,3-e][1,2,4]triazine and pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulphonamide derivatives in monolayer and spheroid breast cancer cell cultures.

In the last decade, an increasing interest in compounds containing pyrazolo[4,3-e][1,2,4]triazine moiety is observed. Therefore, the aim of the research was to synthesise a novel sulphonyl pyrazolo[4,3-e][1,2,4]triazines (2a, 2b) and pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulphonamide derivatives (3a, 3b) to assess their anticancer activity. The MTT assay showed that 2a, 2b, 3a, 3b have stronger cytotoxic activity than cisplatin in both breast cancer cells (MCF-7 and MDA-MB-231) and exhibited weaker effect on normal breast cells (MCF-10A). The obtained results showed that the most active compound 3b increased apoptosis via caspase 9, caspase 8, and caspase 3/7. It is worth to note that compound 3b suppressed NF-κB expression and promoted p53, Bax, and ROS which play important role in activation of apoptosis. Moreover, our results confirmed that compound 3b triggers autophagy through increased formation of autophagosomes, expression of beclin-1 and mTOR inhibition. Thus, our study defines a possible mechanism underlying 3b-induced anti-cancer activity against breast cancer cell lines.

Actively targeted photodynamic therapy in multicellular colorectal cancer spheroids via functionalised gold nanoparticles.

Photodynamic therapy (PDT) holds great potential to overcome limitations associated with common colorectal cancer (CRC) treatment approaches. Targeted photosensitiser (PS) delivery systems using nanoparticles (NPs) with targeting moieties are continually being designed, which are aimed at enhancing PS efficacy in CRC PDT. However, the optimisation of targeted PS delivery systems in most, in vitro PDT studies has been conducted on two dimensional (2D) monolayers cell cultures. In our present study, we developed a nano PS delivery system for in vitro cultured human colorectal three-dimensional multicellular spheroids (3D MCTS). PEGylated gold nanoparticles (PEG-AuNPs) were prepared and attached to ZnPcS4PS and further functionalised with specific CRC targeting anti-Guanylate Cyclase monoclonal antibodies(mAb). The ZnPcS4-AuNP-Anti-GCC Ab (BNC) nanoconjugates were successfully synthesised and their photodynamic effect investigated following exposure to laser irradiation and demonstrated enhanced anticancer effects in Caco-2 cells cultivated as 3D MCTS spheroids. Our findings suggest that targeted BNC nanoconjugates can improve the efficacy of PDT and highlight the potential of 3D MCTS tumour model for evaluating of targeted PDT.

Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro.

Statins, the drugs used for the treatment of hypercholesterolemia, have come into the spotlight not only as chemoadjuvants, but also as potential stem cell modulators in the context of regenerative therapy. In our study, we compared the in vitro effects of all clinically used statins on the viability of human pancreatic cancer (MiaPaCa-2) cells, non-cancerous human embryonic kidney (HEK 293) cells and adipose-derived mesenchymal stem cells (ADMSC). Additionally, the effect of statins on viability of MiaPaCa-2 and ADMSC cells spheroids was tested. Furthermore, we performed a microarray analysis on ADMSCs treated with individual statins (12 µM) and compared the importance of the effects of statins on gene expression between stem cells and pancreatic cancer cells. Concentrations of statins that significantly affected cancer cells viability (< 40 µM) did not affect stem cells viability after 24 h. Moreover, statins that didn´t affect viability of cancer cells grown in a monolayer, induce the disintegration of cancer cell spheroids. The effect of statins on gene expression was significantly less pronounced in stem cells compared to pancreatic cancer cells. In conclusion, the low efficacy of statins on non-tumor and stem cells at concentrations sufficient for cancer cells growth inhibition, support their applicability in chemoadjuvant tumor therapy.

In Situ Self-Assembling Liver Spheroids with Synthetic Nanoscaffolds for Preclinical Drug Screening Applications.

Drug-induced liver injury (DILI) is one of the most common reasons for acute liver failure and a major reason for the withdrawal of medications from the market. There is a growing need for advanced in vitro liver models that can effectively recapitulate hepatic function, offering a robust platform for preclinical drug screening applications. Here, we explore the potential of self-assembling liver spheroids in the presence of electrospun and cryomilled poly(caprolactone) (PCL) nanoscaffolds for use as a new preclinical drug screening tool. This study investigated the extent to which nanoscaffold concentration may have on spheroid size and viability and liver-specific biofunctionality. The efficacy of our model was further validated using a comprehensive dose-dependent acetaminophen toxicity protocol. Our findings show the strong potential of PCL-based nanoscaffolds to facilitate in situ self-assembly of liver spheroids with sizes under 350 µm. The presence of the PCL-based nanoscaffolds (0.005 and 0.01% w/v) improved spheroid viability and the secretion of critical liver-specific biomarkers, namely, albumin and urea. Liver spheroids with nanoscaffolds showed improved drug-metabolizing enzyme activity and greater sensitivity to acetaminophen compared to two-dimensional monolayer cultures and scaffold-free liver spheroids. These promising findings highlight the potential of our nanoscaffold-based liver spheroids as an in vitro liver model for drug-induced hepatotoxicity and drug screening.

Impact of the thyroid hormone T3 and its nuclear receptor TRα1 on colon cancer stem cell phenotypes and response to chemotherapies.

Colorectal cancers (CRCs) are highly heterogeneous and show a hierarchical organization, with cancer stem cells (CSCs) responsible for tumor development, maintenance, and drug resistance. Our previous studies showed the importance of thyroid hormone-dependent signaling on intestinal tumor development and progression through action on stem cells. These results have a translational value, given that the thyroid hormone nuclear receptor TRα1 is upregulated in human CRCs, including in the molecular subtypes associated with CSC features. We used an established spheroid model generated from the human colon adenocarcinoma cell line Caco2 to study the effects of T3 and TRα1 on spheroid formation, growth, and response to conventional chemotherapies. Our results show that T3 treatment and/or increased TRα1 expression in spheroids impaired the response to FOLFIRI and conferred a survival advantage. This was achieved by stimulating drug detoxification pathways and increasing ALDH1A1-expressing cells, including CSCs, within spheroids. These results suggest that clinical evaluation of the thyroid axis and assessing TRα1 levels in CRCs could help to select optimal therapeutic regimens for patients with CRC. Proposed mechanism of action of T3/TRα1 in colon cancer spheroids. In the control condition, TRα1 participates in maintaining homeostatic cell conditions. The presence of T3 in the culture medium activates TRα1 action on target genes, including the drug efflux pumps ABCG2 and ABCB1. In the case of chemotherapy FOLFIRI, the increased expression of ABC transcripts and proteins induced by T3 treatment is responsible for the augmented efflux of 5-FU and Irinotecan from the cancer cells. Taken together, these mechanisms contribute to the decreased efficacy of the chemotherapy and allow cells to escape the treatment. Created with BioRender.com .

Characterization of the effects of thymol derivatives on colorectal cancer spheroids.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies with a high mortality rate. In the last few years, attention has been focused on substances of natural origin with anticancer activity. One such substance is thymol and its derivatives, which have been shown to have an antitumor effect also against CRC cells. In our study, we focused on determining the biological and antibacterial effects of thymol and thymol derivatives. Analyses were performed on a 3D model of human colon carcinoma cell lines (HCT-116 and HT-29) - spheroids. The cytotoxic (MTT assay) and genotoxic effect (comet assay) of thymol and derivatives: acetic acid thymol ester and thymol ß-D-glucoside were determined. ROS levels (ROS-Glo™ H2O2 Assay) and total antioxidant status (Randox TAS Assay) were also monitored. Last but not least, we also detected the effect of the derivatives using a disk diffusion assay and determined the number of colonies on the plates on selected bacteria such as Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Lactobacillus brevis, Lactobacillus pentosus and Weizmannia coagulans. The derivatives did not show a significant inhibitory effect on the growth of LAB bacteria (lactic acid bacteria) in contrast to thymol. Overall, thymol derivatives are cytotoxic, genotoxic and increase ROS levels. Among the derivatives tested, acetic acid thymol ester (IC50 ~ 0.2 µg/ml) was more effective. The second derivative tested (thymol ß-D-glucoside) was effective at higher concentrations than thymol. Our research confirmed that thymol derivatives have a toxic effect on the 3D model of intestinal tumor cells, while they do not have a toxic effect on selected intestinal bacteria. Thus, they could bring new significance to the prevention or treatment of CRC.

Apoptotic effect of 5-fluorouracil-doxorubicin combination on colorectal cancer cell monolayers and spheroids.

BACKGROUND: Drug combination studies help to improve new treatment approaches for colon cancer. Tumor spheroids (3D) are better models than traditional 2-dimensional cultures (2D) to evaluate cellular responses to chemotherapy drugs. The cultivation of cancer cells in 2D and 3D cultures affects the apoptotic process, which is a major factor influencing the response of cancer cells to chemotherapeutic drugs. In this study, the antiproliferative effects of 5-fluorouracil (5-FU) and doxorubicin (DOX) were investigated separately and in combination using 2D and 3D cell culture models on two different colon cancer cell lines, HT-29 (apoptosis-resistant cells) and Caco-2 2 (apoptosis-susceptible cells). METHODS: The effect of the drugs on the proliferation of both colon cancer cells was determined by performing an MTT assay in 2D culture. The apoptotic effect of 5-FU and DOX, both as single agents and in combination, was assessed in 2D and 3D cultures through quantitative real-time polymerase chain reaction analysis. The expression of apoptotic genes, such as caspases, p53, Bax, and Bcl-2, was quantified. RESULTS: It was found that the mRNA expression of proapoptotic genes was significantly upregulated, whereas the mRNA expression of the antiapoptotic Bcl-2 gene was significantly downregulated in both colon cancer models treated with 5-FU, DOX, and 5-FU + DOX. CONCLUSION: The results indicated that the 5-FU + DOX combination therapy induces apoptosis and renders 5-FU and DOX more effective at lower concentrations compared to their alone use. This study reveals promising results in reducing the potential side effects of treatment by enabling the use of lower drug doses.

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Polymeric nanofiber leveraged co-delivery of anti-stromal PAK1 inhibitor and paclitaxel enhances therapeutic effects in stroma-rich 3D spheroid models.

The role of tumor stroma in solid tumors has been widely recognized in cancer progression, metastasis and chemoresistance. Cancer-associated fibroblasts (CAFs) play a crucial role in matrix remodeling and promoting cancer cell stemness and resistance via reciprocal crosstalk. Residual tumor tissue after surgical removal as well as unresectable tumors face therapeutic challenges to achieve curable outcome. In this study, we propose to develop a dual delivery approach by combining p21-activated kinase 1 (PAK1) inhibitor (FRAX597) to inhibit tumor stroma and chemotherapeutic agent paclitaxel (PTX) to kill cancer cells using electrospun nanofibers. First, the role of the PAK1 pathway was established in CAF differentiation, migration and contraction using relevant in vitro models. Second, polycaprolactone polymer-based nanofibers were fabricated using a uniaxial electrospinning technique to incorporate FRAX597 and/or PTX, which showed a uniform texture and a prolonged release of both drugs for 16 days. To test nanofibers, stroma-rich 3D heterospheroid models were set up which showed high resistance to PTX nanofibers compared to stroma-free homospheroids. Interestingly, nanofibers containing PTX and FRAX597 showed strong anti-tumor effects on heterospheroids by reducing the growth and viability by > 90 % compared to either of single drug-loaded nanofibers. These effects were reflected by reduced intra-spheroidal expression levels of collagen 1 and α-smooth muscle actin (α-SMA). Overall, this study provides a new therapeutic strategy to inhibit the tumor stroma using PAK1 inhibitor and thereby enhance the efficacy of chemotherapy using nanofibers as a local delivery system for unresectable or residual tumor. Use of 3D models to evaluate nanofibers highlights these models as advanced in vitro tools to study the effect of controlled release local drug delivery systems before animal studies.

Transcriptomic profiling analysis of the effect of palmitic acid on 3D spheroids of ß-like cells derived from induced pluripotent stem cells.

Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic ß-cell function and mass. In ß-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in ß-cell dysfunction and mass. However, there is a lack of human relevant in vitro model to identify the underlying mechanism through which palmitate induces ß-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of ß-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in ß-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at  0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at  0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment.

Minocycline and photodynamic priming significantly improve chemotherapy efficacy in heterotypic spheroids of pancreatic ductal adenocarcinoma.

The prognosis for patients with advanced-stage pancreatic ductal adenocarcinoma (PDAC) remains dismal. It is generally accepted that combination cancer therapies offer the most promise, such as Folforinox, despite their associated high toxicity. This study addresses the issue of chemoresistance by introducing a complementary dual priming approach to attenuate the DNA repair mechanism and to improve the efficacy of a type 1 topoisomerase (Top1) inhibitor. The result is a regimen that integrates drug-repurposing and nanotechnology using 3 clinically relevant FDA-approved agents (1) Top1 inhibitor (irinotecan) at subcytotoxic doses (2) benzoporphyrin derivative (BPD) as a photoactive molecule for photodynamic priming (PDP) to improve the delivery of irinotecan within the cancer cell and (3) minocycline priming (MNP) to modulate DNA repair enzyme Tdp1 (tyrosyl-DNA phosphodiesterase) activity. We demonstrate in heterotypic 3D cancer models that incorporate cancer cells and pancreatic cancer-associated fibroblasts that simultaneous targeting of Tdp1 and Top1 were significantly more effective by employing MNP and photoactivatable multi-inhibitor liposomes encapsulating BPD and irinotecan compared to monotherapies or a cocktail of dual or triple-agents. These data are encouraging and warrant further work in appropriate animal models to evolve improved therapeutic regimens.

Exploration of isoxazole analogs: Synthesis, COX inhibition, anticancer screening, 3D multicellular tumor spheroids, and molecular modeling.

In this study, a new series of Isoxazole-carboxamide derivatives were synthesized and characterized via HRMS, 1H-, 13CAPT-NMR, and MicroED. The findings revealed that nearly all of the synthesized derivatives exhibited potent inhibitory activities against both COX enzymes, with IC50 values ranging from 4.1 nM to 3.87 µM. Specifically, MYM1 demonstrated the highest efficacy among the compounds tested against the COX-1, displaying an IC50 value of 4.1 nM. The results showed that 5 compounds possess high COX-2 isozyme inhibitory effects with IC50 value in range 0.24-1.30 µM with COX-2 selectivity indexes (2.51-6.13), among these compounds MYM4 has the lowest IC50 value against COX-2, with selectivity index around 4. Intriguingly, this compound displayed significant antiproliferative effects against CaCo-2, Hep3B, and HeLa cancer cell lines, with IC50 values of 10.22, 4.84, and 1.57 µM, respectively, which was nearly comparable to that of doxorubicin. Compound MYM4 showed low cytotoxic activities on normal cell lines LX-2 and Hek293t with IC50 values 20.01 and 216.97 µM respectively, with safer values than doxorubicin. Furthermore, compound MYM4 was able to induce the apoptosis, suppress the colonization of both HeLa and HepG2 cells. Additionally, the induction of Reactive oxygen species (ROS) production could be the mechanism underlying the apoptotic effect and the cytotoxic activity of the compound. In the 3D multicellular tumor spheroid model, results revealed that MYM4 compound hampered the spheroid formation capacity of Hep3B and HeLa cancer cells. Moreover, the molecular docking of MYM4 compound revealed a high affinity for the COX2 enzyme, with energy scores (S) -7.45 kcal/mol, which were comparable to celecoxib (S) -8.40 kcal/mol. Collectively, these findings position MYM4 as a promising pharmacological candidate as COX inhibitor and anticancer agent.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Development and in-depth characterization of BRAFi-resistant melanoma cell lines in vitro and in vivo.

Regardless of the clinical response and improved patient survival observed following treatment with BRAFi like Vemurafenib (Vem), rapid development of resistance still remains as a major obstacle in melanoma therapy. In this context, we developed and characterized two acquired Vem-resistant melanoma cell lines, A375V and SK-MEL-28V, and an intrinsically Vem-resistant cell line, RPMI-7951. Altered morphology and growth rate of the resistant cell lines displayed spindle-shaped cells with filopodia formation and enhanced proliferation rate as compared to parental cells. Further in vitro characterization in 2D models confirmed the emergence of a resistant phenotype in melanoma cells. To mimic the in vivo tumor microenvironment, spheroids were developed for both parental and resistant cell lines to recognize materialization of invadopodia structures demonstrating elevated invasiveness and proliferation of resistant cells-based spheroids, especially A375V. Importantly, we validated A375V cell line in vivo to prove its tumorigenicity and drug resistance in tumor xenograft model. Taken together, our established clinically relevant Vem-resistant tumor model could be beneficial to elucidate drug resistance mechanisms, screen and identify novel anticancer therapies to overcome BRAFi resistance in melanoma.

Colon cancer inhibitory properties of Caulerpa lentillifera polysaccharide and its molecular mechanisms based on three-dimensional cell culture model.

Caulerpa lentillifera is rich in polysaccharides, and its polysaccharides show a significant effect in different biological activities including anti-cancer activity. As an edible algae-derived polysaccharide, exploring the role of colon cancer can better develop the application from a dietary therapy perspective. However, more in-depth studies of C. lentillifera polysaccharide on anti-colon cancer activity and mechanism are needed. In this study, we found that Caulerpa lentillifera polysaccharides (CLP) showed potential anti-colon cancer effect on human colon cancer cell HT29 in monolayer (IC50 = 1.954 mg/mL) and spheroid (IC50 = 0.402 mg/mL). Transcriptomics and metabolomics analyses revealed that CLP had an inhibitory effect on HT29 3D spheroid cells by activating aminoacyl-tRNA biosynthesis as well as arginine and proline metabolism pathways. Furthermore, the anti-colon cancer effects of CLP were confirmed through other human colon cancer cell HCT116 and LoVo in monolayer cells (IC50 = 1.890 mg/mL and 1.437 mg/mL, respectively) and 3D spheroid cells (IC50 = 0.344 mg/mL and 0.975 mg/mL, respectively), and three patient-derived organoids with IC50 values of 6.333-8.780 mg/mL. This study provided basic data for the potential application of CLP in adjuvant therapeutic food for colon cancer on multiple levels, while further investigation of detailed mechanism in vivo was still required.

Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients.

Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O2-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed "MMIR" (multimodal infrared), incorporate an NIR O2-sensitive metalloporphyrin (PtTPTBPF) and deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing for oxygen gradient quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated the nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest, and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroid size, and formation method influenced spheroid oxygenation. Some spheroids produced from HCT116 and dental pulp stem cells exhibited "inverted" oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered "normal" gradient of hypoxia toward the core caused by diffusion. Further microscopy analysis of spheroids with an "inverted" gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H indicated the formation of a glycolytic core and localization of OxPhos-active cells at the periphery. Collectively, we demonstrate a strong potential of NIR-emitting ratiometric nanosensors for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models.

Identification of a set of genes potentially responsible for resistance to ferroptosis in lung adenocarcinoma cancer stem cells.

Scientific literature supports the evidence that cancer stem cells (CSCs) retain inside low reactive oxygen species (ROS) levels and are, therefore, less susceptible to cell death, including ferroptosis, a type of cell death dependent on iron-driven lipid peroxidation. A collection of lung adenocarcinoma (LUAD) primary cell lines derived from malignant pleural effusions (MPEs) of patients was used to obtain 3D spheroids enriched for stem-like properties. We observed that the ferroptosis inducer RSL3 triggered lipid peroxidation and cell death in LUAD cells when grown in 2D conditions; however, when grown in 3D conditions, all cell lines underwent a phenotypic switch, exhibiting substantial resistance to RSL3 and, therefore, protection against ferroptotic cell death. Interestingly, this phenomenon was reversed by disrupting 3D cells and growing them back in adherence, supporting the idea of CSCs plasticity, which holds that cancer cells have the dynamic ability to transition between a CSC state and a non-CSC state. Molecular analyses showed that ferroptosis resistance in 3D spheroids correlated with an increased expression of antioxidant genes and high levels of proteins involved in iron storage and export, indicating protection against oxidative stress and low availability of iron for the initiation of ferroptosis. Moreover, transcriptomic analyses highlighted a novel subset of genes commonly modulated in 3D spheroids and potentially capable of driving ferroptosis protection in LUAD-CSCs, thus allowing to better understand the mechanisms of CSC-mediated drug resistance in tumors.

"A lactose-modified chitosan accelerates chondrogenic differentiation in mesenchymal stem cells spheroids".

Spheroids derived from human mesenchymal stem cells (hMSCs) are of limited use for cartilage regeneration, as the viability of the cells progressively decreases during the period required for chondrogenic differentiation (21 days). In this work, spheroids based on hMSCs and a lactose-modified chitosan (CTL) were formed by seeding cells onto an air-dried coating of CTL. The polymer coating can inhibit cell adhesion and it is simultaneously incorporated into spheroid structure. CTL-spheroids were characterized from a morphological and biological perspective, and their properties were compared with those of spheroids obtained by seeding the cells onto a non-adherent surface (agar gel). Compared to the latter, smaller and more viable spheroids form in the presence of CTL as early as 4 days of culture. At this time point, analysis of stem cells differentiation in spheroids showed a remarkable increase in collagen type-2 (COL2A1) gene expression (~700-fold compared to day 0), whereas only a 2-fold increase was observed in the control spheroids at day 21. These results were confirmed by histological and transmission electron microscopy (TEM) analyses, which showed that in CTL-spheroids an early deposition of collagen with a banding structure already occurred at day 7. Overall, these results support the use of CTL-spheroids as a novel system for cartilage regeneration, characterized by increased cell viability and differentiation capacity within a short time-frame. This will pave the way for approaches aimed at increasing the success rate of procedures and reducing the time required for tissue regeneration.

A vascularized breast cancer spheroid platform for the ranked evaluation of tumor microenvironment-targeted drugs by light sheet fluorescence microscopy.

Targeting the supportive tumor microenvironment (TME) is an approach of high interest in cancer drug development. However, assessing TME-targeted drug candidates presents a unique set of challenges. We develop a comprehensive screening platform that allows monitoring, quantifying, and ranking drug-induced effects in self-organizing, vascularized tumor spheroids (VTSs). The confrontation of four human-derived cell populations makes it possible to recreate and study complex changes in TME composition and cell-cell interaction. The platform is modular and adaptable for tumor entity or genetic manipulation. Treatment effects are recorded by light sheet fluorescence microscopy and translated by an advanced image analysis routine in processable multi-parametric datasets. The system proved to be robust, with strong interassay reliability. We demonstrate the platform's utility for evaluating TME-targeted antifibrotic and antiangiogenic drugs side-by-side. The platform's output enabled the differential evaluation of even closely related drug candidates according to projected therapeutic needs.

Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.