Chlorinated anthracenes (Cl-Ants), persistent organic pollutants, are widely detected in the environment, posing potential lung toxicity risks due to frequent respiratory exposure. However, direct evidence and a comprehensive understanding of their toxicity mechanisms are lacking. Building on our prior findings of Cl-Ants\' immunotoxic risks, this study developed a three-dimensional coculture spheroid model mimicking the lung\'s immune microenvironment. The objective is to explore the pulmonary immunotoxicity and comprehend its mechanisms, taking into account the heightened immune reactivity and frequent lung exposure of Cl-Ants. The results demonstrated that Cl-Ants exposure led to reduced spheroid size, increased macrophage migration outward, lowered cell viability, elevated 8-OHdG levels, disturbed anti-infection balance, and altered cytokine production. Specifically, the chlorine substituent number correlates with the extent of disruption of spheroid indicators caused by Cl-Ants, with stronger immunotoxic effects observed in dichlorinated Ant compared to those in monochlorinated Ant. Furthermore, we identified critical regulatory genes associated with cell viability (ALDOC and ALDOA), bacterial response (TLR5 and MAP2K6), and GM-CSF production (CEBPB). Overall, this study offers initial in vitro evidence of low-dose Cl-PAHs\' pulmonary immunotoxicity, advancing the understanding of Cl-Ants\' structure-related toxicity and improving external toxicity assessment methods for environmental pollutants, which holds significance for future monitoring and evaluation.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, with cisplatin being a primary treatment approach. However, drug resistance and therapy failure pose a significant challenge, affecting nearly 50% of patients over time. This research had two aims: (1) to optimize a 3D cell-culture method for assessing the interplay between tumor cells and cancer-associated fibroblasts (CAFs) in vitro; and (2) to study how cisplatin impacts the Notch pathway, particularly considering the role of CAFs. Using our optimized \"3D sheet model\" approach, we tested two HNSCC cell lines with different cisplatin sensitivities and moderate, non-mutated NOTCH1 and -3 expressions. Combining cisplatin with a γ-secretase inhibitor (crenigacestat) increased sensitivity and induced cell death in the less sensitive cell line, while cisplatin alone was more effective in the moderately sensitive line and sensitivity decreased with the Notch inhibitor. Cisplatin boosted the expression of core Notch signaling proteins in 3D monocultures of both lines, which was counteracted by crenigacestat. In contrast, the presence of patient-derived CAFs mitigated effects and protected both cell lines from cisplatin toxicity. Elevated NOTCH1 and NOTCH3 protein levels were consistently correlated with reduced cisplatin sensitivity and increased cell survival. Additionally, the Notch ligand JAG2 had additional, protective effects reducing cell death from cisplatin exposure. In summary, we observed an inverse relationship between NOTCH1 and NOTCH3 levels and cisplatin responsiveness, overall protective effects by CAFs, and a potential link between JAG2 expression with tumor cell survival.

Tumor-associated macrophages are key components of the tumor microenvironment and play important roles in the progression of head and neck cancer, leading to the development of effective strategies targeting immune cells in tumors. Our study demonstrated the prognostic potential of a new scoring system (Macroscore) based on the combination of the ratio and the sum of the high and low densities of M1 (CD80+) and M2 (CD163+) macrophages in a series of head and neck cancer patients, including a training population (n = 54) and a validation population (n = 19). Interestingly, the Macroscore outperformed TNM criteria and p16 status, showing a significant association with poor patient prognosis, and demonstrated significant predictive value for overall survival. Additionally, 3D coculture spheroids were established to analyze the crosstalk between cancer cells and monocytes/macrophages. Our data revealed that cancer cells can induce monocyte differentiation into protumoral M2 macrophages, creating an immunosuppressive microenvironment. This coculture also induced the production of immunosuppressive cytokines, such as IL10 and IL8, known to promote M2 polarization. Finally, we validated the ability of the macrophage subpopulations to induce apoptosis (M1) or support proliferation (M2) of cancer cells. Overall, our research highlights the potential of the Macroscore as a valuable prognostic biomarker to enhance the clinical management of patients and underscores the relevance of a spheroid model in gaining a better understanding of the mechanisms underlying cancer cell-macrophage interactions.

UNASSIGNED: Pancreatic ductal adenocarcinoma (PDAC) represents the 4th most common cause of cancer-related deaths in Western countries. Most patients are diagnosed at advanced stages, often already with metastases. The main site of metastasis is the liver and hepatic myofibroblasts (HMF) play a pivotal role in metastatic outgrowth. Immune checkpoint inhibitors (ICI) targeting programmed death ligand 1 (PD-L1) or programmed cell death protein 1 (PD-1) improved treatment of several cancers but not of PDAC. Therefore, this study aimed to better understand the impact of HMF on PD-L1 expression and immune evasion of PDAC cells during liver metastasis.
UNASSIGNED: Formalin-fixed and paraffin embedded biopsy samples or diagnostic resection specimens from liver metastases of 15 PDAC patients were used for immunohistochemical analyses. Serial sections were stained with antibodies directed against Pan-Cytokeratin, αSMA, CD8, and PD-L1. To investigate whether the PD-1/PD-L1 axis and HMF contribute to immune escape of PDAC liver metastases, a stroma enriched 3D spheroid coculture model was established in vitro, using two different PDAC cell lines, HMF, and CD8+ T cells. Here, functional and flow cytometry analyses were conducted.
UNASSIGNED: Immunohistochemical analysis of liver tissue sections of PDAC patients revealed that HMF represent an abundant stroma population in liver metastases, with clear differences in the spatial distribution in small (1500 µm) and large (> 1500 μm) metastases. In the latter, PD-L1 expression was mainly located at the invasion front or evenly distributed, while small metastases either lacked PD-L1 expression or showed mostly weak expression in the center. Double stainings revealed that PD-L1 is predominantly expressed by stromal cells, especially HMF. Small liver metastases with no or low PD-L1 expression comprised more CD8+ T cells in the tumor center, while large metastases exhibiting stronger PD-L1 expression comprised less CD8+ T cells being mostly located at the invasion front. HMF-enriched spheroid cocultures with different ratios of PDAC cells and HMF well mimicking conditions of hepatic metastases in situ. Here, HMF impaired the release of effector molecules by CD8+ T cells and the induction of PDAC cell death, an effect that was dependent on the amount of HMF but also of PDAC cells. ICI treatment led to elevated secretion of distinct CD8+ T cell effector molecules but did not increase PDAC cell death under either spheroid condition.
UNASSIGNED: Our findings indicate a spatial reorganization of HMF, CD8+ T cells, and PD-L1 expression during progression of PDAC liver metastases. Furthermore, HMF potently impair the effector phenotype of CD8+ T cells but the PD-L1/PD-1 axis apparently plays a minor role in this scenario suggesting that immune evasion of PDAC liver metastases relies on other immunosuppressive mechanisms.

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33-955 μm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF-islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation.

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

Current treatments focused on eradicating metastatic tumors have proven unsuccessful due to cancer\'s ability to quickly undergo epithelial-to-mesenchymal transition (EMT) and metastasize to secondary sites. Using human triple negative breast cancer cells (BCCs) as a model system, this work establishes a platform for the study of aggressive cancer phenotypes by demonstrating the inhibition of human metastatic cancer cells with 3D cultured embryonic stem cells (ESCs) encapsulated in alginate microstrands (ESC-microstrands), which mimic the embryonic microenvironment and recapitulate pluripotent signaling. Coculture with ESC-microstrands significantly decreases triple negative BCC proliferation and survival and reverses abnormal cancer metabolism. In particular, coculture with ESC-microstrands markedly restricts the metastatic potential of highly aggressive cancer cells, demonstrated as decreased migration and invasion, and reversed EMT marker expression. This indicates that pluripotent signaling from 3D ESC-microstrands could restrict cancer metastasis through restriction and reversion of EMT. Furthermore, two soluble factors associated with dysregulated oncogenic signaling were identified which display altered relative mRNA expression following coculture with ESC-microstrands. Future application of this model to mechanistic studies will enable a better understanding of cancer metastasis and the discovery of therapeutic targets for metastatic diseases.

The interaction between osteogenic and angiogenic cells through a coculturing system in biocompatible materials has been considered for successfully engineering vascularized bone tissue equivalents. In this study, we developed a hydrogel-blended scaffold consisted of gelatin methacryloyl (GelMA) and alginate enriched with hydroxyapatite nanoparticles (HAP) to model an in vitro prevascularized bone construct. The hydrogel-based scaffold revealed a higher mechanical stiffness than those of pure (GelMA), alginate, and (GelMA+ HAP) hydrogels. In the present study, we generated a green fluorescent protein (GFP) knock-in umbilical vein endothelial cells (HUVECs) cell line using the CRISPR/Cas9 technology. The GFP was inserted into the human-like ROSA locus of HUVECs genome. HUVECs expressing GFP were cocultured with OB-like cells (MG-63) within three-dimensionally (3D) fabricated hydrogel to investigate the response of cocultured osteoblasts and endothelial cells in a 3D structure. Cell viability under the 3D cocultured gel was higher than the 3D monocultured. Compared to the 3D monocultured condition, the cells were aligned and developed into the vessel-like structures. During 14 days of culture periods, the cells displayed actin protrusions by the formation of spike-like filopodia in the 3D cocultured model. Angiogenic and osteogenic-related genes such as CD31, vWF, and osteocalcin showed higher expression in the cocultured versus the monocultured. These results have collectively indicated that the 3D cocultured hydrogel facilitates interaction among cells, thereby having a greater effect on angiogenic and osteogenic properties in the absence of induction media.

Mast cells (MCs) are one of the first immune cells recruited to a tumor. It is well recognized that MCs accumulate in colon cancer lesion and their density is associated with the clinical outcomes. However, the molecular mechanism of how colon cancer cells may modify MC function is still unclear. In this study, primary human MCs were generated from CD34⁺ progenitor cells and a 3D coculture model was developed to study the interplay between colon cancer cells and MCs. By comparing the transcriptomic profile of colon cancer-cocultured MCs versus control MCs, we identified a number of deregulated genes, such as MMP-2, VEGF-A, PDGF-A, COX2, NOTCH1 and ISG15, which contribute to the enrichment of cancer-related pathways. Intriguingly, pre-stimulation with a TLR2 agonist prior to colon cancer coculture induced upregulation of multiple interferon-inducible genes as well as MHC molecules in MCs. Our study provides an alternative approach to study the influence of colon cancer on MCs. The transcriptome signature of colon cancer-cocultured MCs may potentially reflect the mechanism of how colon cancer cells educate MCs to become pro-tumorigenic in the initial phase and how a subsequent inflammatory signal-e.g., TLR2 ligands-may modify their responses in the cancer milieu.