Cytotoxic and molecular differences of anticancer agents on 2D and 3D cell culture.

BACKGROUND: Cancer and multidrug resistance are regarded as concerns related to poor health outcomes. It was found that the monolayer of 2D cancer cell cultures lacks many important features compared to Multicellular Tumor Spheroids (MCTS) or 3D cell cultures which instead have the ability to mimic more closely the in vivo tumor microenvironment. This study aimed to produce 3D cell cultures from different cancer cell lines and to examine the cytotoxic activity of anticancer medications on both 2D and 3D systems, as well as to detect alterations in the expression of certain genes levels. METHOD: 3D cell culture was produced using 3D microtissue molds. The cytotoxic activities of colchicine, cisplatin, doxorubicin, and paclitaxel were tested on 2D and 3D cell culture systems obtained from different cell lines (A549, H1299, MCF-7, and DU-145). IC50 values were determined by MTT assay. In addition, gene expression levels of PIK3CA, AKT1, and PTEN were evaluated by qPCR. RESULTS: Similar cytotoxic activities were observed on both 3D and 2D cell cultures, however, higher concentrations of anticancer medications were needed for the 3D system. For instance, paclitaxel showed an IC50 of 6.234 µM and of 13.87 µM on 2D and 3D H1299 cell cultures, respectively. Gene expression of PIK3CA in H1299 cells also showed a higher fold change in 3D cell culture compared to 2D system upon treatment with doxorubicin. CONCLUSION: When compared to 2D cell cultures, the behavior of cells in the 3D system showed to be more resistant to anticancer treatments. Due to their shape, growth pattern, hypoxic core features, interaction between cells, biomarkers synthesis, and resistance to treatment penetration, the MCTS have the advantage of better simulating the in vivo tumor conditions. As a result, it is reasonable to conclude that 3D cell cultures may be a more promising model than the traditional 2D system, offering a better understanding of the in vivo molecular changes in response to different potential treatments and multidrug resistance development.

Rationally designed ß-cyclodextrin-crosslinked polyacrylamide hydrogels for cell spheroid formation and 3D tumor model construction.

In vitro tumor models are essential for understanding tumor behavior and evaluating tumor biological properties. Hydrogels that can mimic the tumor extracellular matrix have become popular for creating 3D in vitro tumor models. However, designing biocompatible hydrogels with appropriate chemical and physical properties for constructing tumor models is still a challenge. In this study, we synthesized a series of ß-cyclodextrin (ß-CD)-crosslinked polyacrylamide hydrogels with different ß-CD densities and mechanical properties and evaluated their potential for use in 3D in vitro tumor model construction, including cell capture and spheroid formation. By utilizing a combination of ß-CD-methacrylate (CD-MA) and a small amount of N,N'-methylene bisacrylamide (BIS) as hydrogel crosslinkers and optimizing the CD-MA/BIS ratio, the hydrogels performed excellently for tumor cell 3D culture and spheroid formation. Notably, when we co-cultured L929 fibroblasts with HeLa tumor cells on the hydrogel surface, co-cultured spheroids were formed, showing that the hydrogel can mimic the complexity of the tumor extracellular matrix. This comprehensive investigation of the relationship between hydrogel mechanical properties and biocompatibility provides important insights for hydrogel-based in vitro tumor modeling and advances our understanding of the mechanisms underlying tumor growth and progression.

The transcriptional landscape of cancer stem-like cell functionality in breast cancer.

BACKGROUND: Cancer stem-like cells (CSCs) have been extensively researched as the primary drivers of therapy resistance and tumor relapse in patients with breast cancer. However, due to lack of specific molecular markers, increased phenotypic plasticity and no clear clinicopathological features, the assessment of CSCs presence and functionality in solid tumors is challenging. While several potential markers, such as CD24/CD44, have been proposed, the extent to which they truly represent the stem cell potential of tumors or merely provide static snapshots is still a subject of controversy. Recent studies have highlighted the crucial role of the tumor microenvironment (TME) in influencing the CSC phenotype in breast cancer. The interplay between the tumor and TME induces significant changes in the cancer cell phenotype, leading to the acquisition of CSC characteristics, therapeutic resistance, and metastatic spread. Simultaneously, CSCs actively shape their microenvironment by evading immune surveillance and attracting stromal cells that support tumor progression. METHODS: In this study, we associated in vitro mammosphere formation assays with bulk tumor microarray profiling and deconvolution algorithms to map CSC functionality and the microenvironmental landscape in a large cohort of 125 breast tumors. RESULTS: We found that the TME score was a significant factor associated with CSC functionality. CSC-rich tumors were characterized by an immune-suppressed TME, while tumors devoid of CSC potential exhibited high immune infiltration and activation of pathways involved in the immune response. Gene expression analysis revealed IFNG, CXCR5, CD40LG, TBX21 and IL2RG to be associated with the CSC phenotype and also displayed prognostic value for patients with breast cancer. CONCLUSION: These results suggest that the characterization of CSCs content and functionality in tumors can be used as an attractive strategy to fine-tune treatments and guide clinical decisions to improve patients therapy response.

EMMPRIN promotes spheroid organization and metastatic formation: comparison between monolayers and spheroids of CT26 colon carcinoma cells.

Background: In vitro studies often use two-dimensional (2D) monolayers, but 3D cell organization, such as in spheroids, better mimics the complexity of solid tumors. To metastasize, cancer cells undergo the process of epithelial-to-mesenchymal transition (EMT) to become more invasive and pro-angiogenic, with expression of both epithelial and mesenchymal markers. Aims: We asked whether EMMPRIN/CD147 contributes to the formation of the 3D spheroid structure, and whether spheroids, which are often used to study proliferation and drug resistance, could better model the EMT process and the metastatic properties of cells, and improve our understanding of the role of EMMPRIN in them. Methods: We used the parental mouse CT26 colon carcinoma (CT26-WT) cells, and infected them with a lentivirus vector to knock down EMMPRIN expression (CT26-KD cells), or with an empty lentivirus vector (CT26-NC) that served as a negative control. In some cases, we repeated the experiments with the 4T1 or LLC cell lines. We compared the magnitude of change between CT26-KD and CT26-WT/NC cells in different metastatic properties in cells seeded as monolayers or as spheroids formed by the scaffold-free liquid overlay method. Results: We show that reduced EMMPRIN expression changed the morphology of cells and their spatial organization in both 2D and 3D models. The 3D models more clearly demonstrated how reduced EMMPRIN expression inhibited proliferation and the angiogenic potential, while it enhanced drug resistance, invasiveness, and EMT status, and moreover it enhanced cell dormancy and prevented CT26-KD cells from forming metastatic-like lesions when seeded on basement membrane extract (BME). Most interestingly, this approach enabled us to identify that EMMPRIN and miR-146a-5p form a negative feedback loop, thus identifying a key mechanism for EMMPRIN activities. These results underline EMMPRIN role as a gatekeeper that prevents dormancy, and suggest that EMMPRIN links EMT characteristics to the process of spheroid formation. Conclusions: Thus, 3D models can help identify mechanisms by which EMMPRIN facilitates tumor and metastasis progression, which might render EMMPRIN as a promising target for anti-metastatic tumor therapy.

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.

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 .

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.

Construction of An Orthotopic Xenograft Model of Non-small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities.

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model. Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease's progression and facilitate the development of potential anticancer drugs.

Evaluating Nanoparticles Penetration by a New Microfluidic Hydrogel-Based Approach.

Reliable predictions for the route and accumulation of nanotherapeutics in vivo are limited by the huge gap between the 2D in vitro assays used for drug screening and the 3D physiological in vivo environment. While developing a standard 3D in vitro model for screening nanotherapeutics remains challenging, multi-cellular tumor spheroids (MCTS) are a promising in vitro model for such screening. Here, we present a straightforward and flexible 3D-model microsystem made out of agarose-based micro-wells, which enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging, including live high-resolution optical microscopy, can be done in situ without manipulating spheroids.

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.

Assessing the distribution of cancer stem cells in tumorspheres.

Cancer Stem Cells presumably drive tumor growth and resistance to conventional cancer treatments. From a previous computational model, we inferred that these cells are not uniformly distributed in the bulk of a tumorsphere. To confirm this result, we cultivated tumorspheres enriched in stem cells, and performed immunofluorescent detection of the stemness marker SOX2 using confocal microscopy. In this article, we present an image processing method that reconstructs the amount and location of the Cancer Stem Cells in the spheroids. Its advantage is the use of a statistical criterion to classify the cells in Stem and Differentiated, instead of setting an arbitrary threshold. Moreover, the analysis of the experimental images presented in this work agrees with the results from our computational models, thus enforcing the notion that the distribution of Cancer Stem Cells in a tumorsphere is non-homogeneous. Additionally, the method presented here provides a useful tool for analyzing any image in which different kinds of cells are stained with different markers.

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.

Uniform sized cancer spheroids production using hydrogel-based droplet microfluidics: a review.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

Pentoxifylline and Norcantharidin Modify p62 Expression in 2D and 3D Cultures of B16F1 Cells.

Three-dimensional cell cultures have improved the evaluation of drugs for cancer therapy, due to their high similarity to solid tumors. In melanoma, autophagy appears to show a dual role depending on the progression of the disease. p62 protein has been proposed for the evaluation of autophagic flux since its expression is an indicator of the state of autophagy. Pentoxifylline (PTX) and Norcantharidin (NCTD) are drugs that have been shown to possess anticancer effects. In this work, we used B16F1 mouse melanoma cells in two-dimensional (2D) monolayer cultures and three-dimensional (3D) spheroids to test the effect of PTX and NCTD over the p62 expression. We analyzed the effect on p62 expression through Western blot and immunofluorescence assays. Our results indicate that PTX decreases p62 expression in both cell culture models, while Norcantharidin increases its expression in 3D cultures at 24 h. Therefore, these drugs could have a potential therapeutic use for the regulation of autophagy in melanoma, depending on the state of evolution of the disease.

Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an "off-the-shelf" bioartificial liver system.

The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.

A Pillar/Perfusion Plate Enhances Cell Growth, Reproducibility, Throughput, and User Friendliness in Dynamic 3D Cell Culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from a necrotic core due to limited nutrient and oxygen diffusion and waste removal and have a limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids were loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow velocity was maintained within perfusion wells and the pillar plate was separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in a dynamic 3D cell culture.

Biofabrication of 3D adipose tissue via assembly of composite stem cell spheroids containing adipo-inductive dual-signal delivery nanofibers.

Reconstruction of large 3D tissues based on assembly of micro-sized multi-cellular spheroids has gained attention in tissue engineering. However, formation of 3D adipose tissue from spheroids has been challenging due to the limited adhesion capability and restricted cell mobility of adipocytes in culture media. In this study, we addressed this problem by developing adipo-inductive nanofibers enabling dual delivery of indomethacin and insulin. These nanofibers were introduced into composite spheroids comprising human adipose-derived stem cells (hADSCs). This approach led to a significant enhancement in the formation of uniform lipid droplets, as evidenced by the significantly increased Oil red O-stained area in spheroids incorporating indomethacin and insulin dual delivery nanofibers (56.9 ± 4.6%) compared to the control (15.6 ± 3.5%) with significantly greater gene expression associated with adipogenesis (C/EBPA, PPARG, FABP4, and adiponectin) of hADSCs. Furthermore, we investigated the influence of culture media on the migration and merging of spheroids and observed significant decrease in migration and merging of spheroids in adipogenic differentiation media. Conversely, the presence of adipo-inductive nanofibers promoted spheroid fusion, allowing the formation of macroscopic 3D adipose tissue in the absence of adipogenic supplements while facilitating homogeneous adipogenesis of hADSCs. The approach described here holds promise for the generation of 3D adipose tissue constructs by scaffold-free assembly of stem cell spheroids with potential applications in clinical and organ models.

Patient-derived follicular lymphoma spheroids recapitulate lymph node signaling and immune profile uncovering galectin-9 as a novel immunotherapeutic target.

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.