We and others have previously shown that ADGRF1, an adhesion G protein-coupled receptor, is overexpressed and associated with poor survival in many cancers, including human epidermal growth factor receptor-2 (HER2) breast cancer (BC). Also, we have reported the tumour-promoting function of ADGRF1 using preclinical models of HER2+ BC. In this study, we investigated the effect of ADGRF1 overexpression in an orthotopic in vivo model as well as downstream signalling of ADGRF1 in HER2+ BC.

We utilized a doxycycline (Dox)-induced ADGRF1 overexpression system in HER2+ BC cell lines and performed various in vitro and in vivo studies. Following ADGRF1 overexpression in the presence/absence of Matrigel, laminin-111 or collagen-IV, we performed the mammosphere assay to assess the tumorigenicity of breast epithelial cells, as well as cAMP/IP1 assays and RNA-sequencing, to understand the receptor function and pharmacology. We conducted cross-linking-aided immunoprecipitation and mass spectrometry to confirm the physical interaction between ADGRF1 and the extracellular matrix proteins present in Matrigel.

We found that ADGRF1 switched from a tumour-promoting to tumour-suppressive function upon interaction with laminin-111. Interaction of ADGRF1 with laminin-111 resulted in inhibition of Gαs coupling and STAT3 phosphorylation, induction of senescence, increase in HER2 expression, and improvement of sensitivity to anti-HER2 drugs in HER2+ BC.

ADGRF1 switches from a tumour-promoting to tumour-suppressive function upon interaction with laminin-111, leading to improvements in sensitivity to anti-HER2 drugs. Leveraging ADGRF1 interactions with laminin-111 may allow the design of novel therapies against ADGRF1 in HER2+ BC.

TGF-β ligands suppress growth yet can paradoxically and potently promote cancer invasion and metastasis depending on downstream pathway mutational context, such as loss of Mothers against decapentaplegic homolog 4 (Smad4). Here, we characterised phenotypes and associated gene expression signatures in conditional murine intestinal adenoma with and without Smad4. Conditional Lgr5-CreERT2 activation in Apcfl/flSmad4fl/fl mice resulted in homozygote floxed alleles (ApcΔ/ΔSmad4Δ/Δ) and adenoma formation. The adenoma phenotype was discordant, with reduced small intestinal adenoma burden yet development of large non-metastatic caecal adenoma with nuclear localisation of phospho-Smad2/3. Derived ApcΔ/ΔSmad4Δ/Δ adenoma organoids resisted TGF-β1 dose dependent growth arrest and cell death (IC50 534 pM) compared to ApcΔ/ΔSmad4+/+ (IC50 24 pM). TGF-β1 (390 pM) altered adenoma bulk mRNA expression most significantly for Id1low and Spp1high in ApcΔ/ΔSmad4Δ/Δ. Single cell RNAseq of caecal adenoma identified expansion of Lgr5low, Pak3high and Id1low progenitor populations in ApcΔ/ΔSmad4Δ/Δ. Of the 76 Smad4 and TGF-β1 dependent genes identified in Apcfl/flSmad4fl/fl adenoma organoids, only 7 human equivalent genes were differentially expressed in SMAD4 mutated colorectal cancer (TCGA cohorts), including ID1low. SMAD4low, ID1low SPP1high and PAK3high all correlated with poorer survival. Murine adenoma identified Smad4 dependent gene expression signatures that require further evaluation as functional biomarker classifiers of SMAD4 mutated cancer subtypes.

Cellular biomechanics plays a critical role in cancer metastasis and tumor progression. Existing studies on cancer cell biomechanics are mostly conducted in flat 2D conditions, where cells' behavior can differ considerably from those in 3D physiological environments. Despite great advances in developing 3D in vitro models, probing cellular elasticity in 3D conditions remains a major challenge for existing technologies. In this work, optical Brillouin microscopy is utilized to longitudinally acquire mechanical images of growing cancerous spheroids over the period of 8 days. The dense mechanical mapping from Brillouin microscopy enables us to extract spatially resolved and temporally evolving mechanical features that were previously inaccessible. Using an established machine learning algorithm, it is demonstrated that incorporating these extracted mechanical features significantly improves the classification accuracy of cancer cells, from 74% to 95%. Building on this finding, a deep learning pipeline capable of accurately differentiating cancerous spheroids from normal ones solely using Brillouin images have been developed, suggesting the mechanical features of cancer cells can potentially serve as a new biomarker in cancer classification and detection.

Platinum(II) complexes such as cisplatin, among a few others, are well-known anticancer metal-based drugs approved for clinical use. In spite of their wide acceptance, the respective chemotherapy is associated with severe side effects and the ability of tumors to quickly develop resistance. To overcome these drawbacks, the novel strategy is considered, which is based on the use of platinum complexes with bioactive ligands attached to act in synergy with platinum and to further improve its pharmacological properties. Among the recently introduced multiaction prodrugs is the Pt(IV) complex with two lonidamine ligands, the latter selectively inhibiting hexokinase and, thus, glycolysis in cancer cells. While platinum-based multiaction prodrugs exhibit increased levels of activity toward cancer cells and, thus, are considered potent to overcome the resistance to cisplatin, there is a crucial need to uncover their mechanism of action by revealing all possibly affected processes and targets across the whole cellular proteome. These are challenging tasks in proteomics requiring high-throughput analysis of hundreds of samples for just a single drug-to-proteome system. In this work, we performed these analyses for 8-azaguanine and the experimental Pt(IV)-lonidamine complex applied to ovarian cancer cell line A2780 employing both mechanism- and compound-centric ultrafast chemical proteomics approaches. These approaches were based on protein expression analysis and thermal proteome profiling, respectively. Data obtained for the Pt(IV)-lonidamine complex revealed regulation of proteins involved in the glucose metabolic process associated with lonidamine, further supporting the multiaction mechanism of this prodrug action.

For many years, the gold standard in the study of malignant tumors has been the in vitro culture of tumor cells, in vivo xenografts or genetically modified animal models. Meanwhile, three-dimensional cell models (3D cultures) have been added to the arsenal of modern biomedical research. 3D cultures reproduce tissue-specific features of tissue topology. This makes them relevant tissue models in terms of cell differentiation, metabolism and the development of drug resistance. Such models are already being used by many research groups for both basic and translational research, and may substantially reduce the number of animal studies, for example in the field of oncological research. In the current literature, 3D cultures are classified according to the technique of their formation (with or without a scaffold), cultivation conditions (static or dynamic), as well as their cellular organization and function. In terms of cellular organization, 3D cultures are divided into "spheroid models", "organoids", "organs-on-a-chip" and "microtissues". Each of these models has its own unique features, which should be taken into account when using a particular model in an experiment. The simplest 3D cultures are spheroid models which are floating spherical cell aggregates. An organoid is a more complex 3D model, in which a self-organizing 3D structure is formed from stem cells (SCs) capable of self-renewal and differentiation within the model. Organ-on-a-chip models are chips of microfluidic systems that simulate dynamic physical and biological processes found in organs and tissues in vitro. By combining different cell types into a single structure, spheroids and organoids can act as a basis for the formation of a microtissue - a hybrid 3D model imitating a specific tissue phenotype and containing tissue-specific extracellular matrix (ECM) components. This review presents a brief history of 3D cell culture. It describes the main characteristics and perspectives of the use of "spheroid models", "organoids", "organ-on-a-chip" models and "microtissues" in immune oncology research of solid tumors.

The development of more physiologically relevant cancer models has led to the increased adoption of three-dimensional (3D) cell culture systems, such as tumor spheroids, which more accurately replicate the complex tumor microenvironment compared to traditional two-dimensional (2D) cultures. This study utilizes the hanging drop technique to generate melanoma spheroids from A375 and SK-MEL28 cell lines, including both parental and drug-resistant variants. These spheroids were embedded in Matrigel and treated with vemurafenib, cilengitide, or their combination to evaluate the effects on invasion. The combination therapy significantly reduced invasion, particularly in resistant SK-MEL28 spheroids. These findings underscore the importance of 3D spheroid models in studying drug efficacy and resistance mechanisms in cancer.

Metal nanoparticles are commonly found in our daily lives and pose great risks to people's health. Therefore, it is crucial to establish a research model for the toxic effects of metal nanoparticles. In recent decades, three-dimensional (3D) cell models have attracted increasing interest in the fields of cell barriers, nanotoxicology, and drug screening, as they have significant advantages over two-dimensional (2D) cell models in accurately simulating in vivo behavior of human cells. The accurate spatiotemporal reaction characteristics achieved through the diffusion effect of metal nanoparticles in Matrigel scaffolds are of great importance in nanotoxicology. However, traditional impedance sensors face challenges in performing spatiotemporal dynamic impedance monitoring and evaluating the toxic impact of metal nanoparticles on 3D cells. Here, we propose an impedance sensor that integrates a plug-in vertical electrode array (PVEA) chip with a multi-channel detection system. This sensor can dynamically record 3D cell impedance in the vertical direction, which is consistent with the temporal and spatial progression of metal nanoparticle penetration, and also closely related to the spatiotemporal activity of cells influenced by metal nanoparticles. This method can detect subtle changes in impedance signals at different positions caused by the diffusion of metal nanoparticles, and has high application value in Nanotoxicology evaluation. This universal, high-throughput 3D cell impedance sensor has great potential in toxicity detection and drug screening.

The multifactorial nature of cancer requires treatment that involves simultaneous targeting of associated overexpressed proteins and cell signaling pathways, possibly leading to synergistic effects. Herein, we present a systematic study that involves the simultaneous inhibition of human topoisomerases (hTopos) and histone deacetylases (HDACs) by multitargeted quinoline-bridged hydroxamic acid derivatives. These compounds were rationally designed considering pharmacophoric features and catalytic sites of the cross-talk proteins, synthesized, and assessed for their anticancer potential. Our findings revealed that the compound 5c significantly produced anticancer effects in vitro and in vivo by reducing the tumor growth and its size in the A549 cell-induced lung cancer xenograft model through multiple mechanisms, primarily by multi-inhibition of hTopoI/II and HDACs, especially HDAC1 via atypical binding. The present paper discusses detailed mechanistic biological investigations, structure-activity effects supported by computational docking studies, and DMPK studies and provides future scope for lead optimization and modification.

3D segmentation of biological structures is critical in biomedical imaging, offering significant insights into structures and functions. This paper introduces a novel segmentation of biological images that couples Multi-Aperture representation with Transformers for 3D (MAT3D) segmentation. Our method integrates the global context-awareness of Transformer networks with the local feature extraction capabilities of Convolutional Neural Networks (CNNs), providing a comprehensive solution for accurately delineating complex biological structures. First, we evaluated the performance of the proposed technique on two public clinical datasets of ACDC and Synapse multi-organ segmentation, rendering superior Dice scores of 93.34±0.05 and 89.73±0.04, respectively, with fewer parameters compared to the published literature. Next, we assessed the performance of our technique on an organoid dataset comprising four breast cancer subtypes. The proposed method achieved a Dice 95.12±0.02 and a PQ score of 97.01±0.01, respectively. MAT3D also significantly reduces the parameters to 40 million. The code is available on https://github.com/sohaibcs1/MAT3D.

Profiling of Patient-Derived organoids is necessary for drug screening and precision medicine. This step requires accurate segmentation of three-dimensional cellular structures followed by protein readouts. While fully Convolutional Neural Networks are widely used in medical image segmentation, they struggle to capture long-range dependencies necessary for accurate segmentation. On the other hand, transformer models have shown promise in capturing long-range dependencies and self-similarities. Motivated by this, we present 3D-Organoid-SwinNet, a unique segmentation model explicitly designed for organoid semantic segmentation. We evaluated the performance of our technique using an Organoid dataset from four breast cancer subtypes. We demonstrated consistent top-tier performance in both the validation and testing phases, achieving a Dice score of 94.91 while reducing the number of parameters to 21 million. Our findings indicate that the proposed model offers a foundation for transformer-based models designed for high-content profiling of organoid models.

Current in vitro models for gastric cancer research, such as 2D cell cultures and organoid systems, often fail to replicate the complex extracellular matrix (ECM) found in vivo. For the first time, this study utilizes a gelatin methacryloyl (GelMA) hydrogel, a biomimetic ECM-like material, in 3D bioprinting to construct a physiologically relevant gastric cancer model. GelMA's tunable mechanical properties allow for the precise manipulation of cellular behavior within physiological ranges. Genetic and phenotypic analyses indicate that the 3D bioprinted GelMA (3Db) model accurately mimics the clinical tumor characteristics and reproduces key cancer hallmarks, such as cell proliferation, invasion, migration, angiogenesis, and the Warburg effect. Comparisons of gene expression and drug responses between the 3Db model and patient-derived xenograft models, both constructed from primary gastric cancer cells, validate the model's clinical relevance. The ability of the 3Db model to closely simulate in vivo conditions highlights its crucial role in identifying treatment targets and predicting patient-specific responses, showcasing its potential in high-throughput drug screening and clinical applications. This study is the first to report the pivotal role of GelMA-based 3D bioprinting in advancing gastric cancer research and regenerative medicine.

Breast cancer is among the most prevalent malignant tumors globally, with carboplatin serving as a standard treatment option. However, resistance often compromises its efficacy. DNA damage repair (DDR) pathways are crucial in determining responses to treatment and are also associated with immune infiltration. This study aimed to identify the DDR genes involved in carboplatin resistance and to elucidate their effects on prognosis, immune infiltration, and drug sensitivity in breast cancer patients.

A 3D-culture model resistant to carboplatin was constructed and sequenced. Co-expressed DDR genes were analyzed to develop a predictive model. Immune infiltration analysis tools were employed to assess the immune microenvironment of patients with varying expression levels of these risk genes. Additionally, drug sensitivity predictions were made to evaluate the efficacy of other DNA damage-related drugs across different risk groups. Molecular assays were performed to investigate the role of the key gene TONSL in breast cancer.

By integrating data from public database, we established a prognostic signature comprising thirteen DDR genes. Our analysis indicated that this model is associated with immune infiltration patterns in breast cancer patients, particularly concerning CD8+ T cells and NK cells. Additionally, it demonstrated a significant correlation with sensitivity to other DDR-related drugs, suggesting its potential as a biomarker for treatment efficacy. Compared to the control group, TONSL-knockdown cell lines exhibited a diminished response to DNA-damaging agents, marked by a notable increase in DNA damage levels and enhanced drug sensitivity. Furthermore, single-cell analysis revealed elevated TONSL expression in dendritic and epithelial cells, particularly in triple-negative breast cancers.

Carboplatin resistance-related DDR genes are associated with prognosis, immune infiltration, and drug sensitivity in breast cancer patients. TONSL may serve as a potential therapeutic target for breast cancer, particularly in triple-negative breast cancer, indicating new treatment strategies for these patients.

Oncological diseases consistently occupy leading positions among the most life-threatening diseases, including in highly developed countries. At the same time, the second most common cause of cancer death is colorectal cancer. The current level of research shows that the development of effective therapy, in this case, requires a new grade of understanding processes during the emergence and development of a tumor. In particular, the concept of cancer stem cells that ensure the survival of chemoresistant cells capable of giving rise to new tumors is becoming widespread. To provide adequate conditions that reproduce natural processes typical for tumor development, approaches based on increasingly complex cellular systems are being improved. This review discusses the main strategies that allow for the study of the properties of tumor cells with an emphasis on colorectal cancer stem cells. The features of working with tumor cells and the advantages and disadvantages of 2D and 3D culture systems are considered.

The physical microenvironment plays a crucial role in tumor development, progression, metastasis and treatment. Recently, we proposed four physical hallmarks of cancer, with distinct origins and consequences, to characterize abnormalities in the physical tumor microenvironment: (1) elevated compressive-tensile solid stresses, (2) elevated interstitial fluid pressure and the resulting interstitial fluid flow, (3) altered material properties (for example, increased tissue stiffness) and (4) altered physical micro-architecture. As this emerging field of physical oncology is being advanced by tumor biologists, cell and developmental biologists, engineers, physicists and oncologists, there is a critical need for model systems and measurement tools to mechanistically probe these physical hallmarks. Here, after briefly defining these physical hallmarks, we discuss the tools and model systems available for probing each hallmark in vitro, ex vivo, in vivo and in clinical settings. We finally review the unmet needs for mechanistic probing of the physical hallmarks of tumors and discuss the challenges and unanswered questions associated with each hallmark.

Pathobiology of asthma and chronic obstructive pulmonary disease (COPD) is associated with changes among respiratory epithelium structure and function. Increased levels of PM2.5 from urban particulate matter (UPM) are correlated with enlarged rate of asthma and COPD morbidity as well as acute disease exacerbation. It has been suggested that pre-existing pulmonary obstructive diseases predispose epithelium for different biological response than in healthy airways. The aim of this study was to assess the impact of PM2.5 on the biological response of healthy as well as asthma and COPD respiratory epithelium using 3D/spheroid culture model.

The spheroids from 5 healthy controls, 8 asthma patients, and 8 COPD patients were exposed to 100 µg/ml of PM2.5 for 24 h.

The common pattern for healthy asthma and COPD epithelium inflammatory response to PM2.5 stimulation include the increase in IL-1β, IL-6, IL-8 mRNA expression, and secretion of IL-6. Asthmatic spheroids produced higher amount of TNF-α and IL-8, whereas COPD spheroids expressed increased mRNA level of MUC5AC and decreased level of MMP7. PM2.5 treatment induced changes in AHR and TLR4 expression on secretory epithelium in COPD.

The response of airway epithelium to air pollution is different in healthy people than in obstructive lung disease patients. The impairment of airway epithelium in asthma and COPD changes their response to toxic environmental stimuli. This physiological dysfunction might be associated with diseases exacerbation of obstructive lung diseases.

3-Dimensional (3D) cultures of cancer cell lines exhibit unique morphologies, a feature that can be utilized for understanding several aspects of solid tumors. This study aims to investigate the morphology and morphometrics of agarose hydrogel-induced 3D aggregates of SiHa cell line. Floating 3D aggregates of SiHa cells were obtained using liquid overlay technique on 1 % agarose hydrogel. The aggregates were monitored daily for its progressive growth and morphology, and documented. The morphometric analysis of the multicellular 3D spheroids were performed on cultures in the late exponential/log phase (the 93rd hour in culture). The morphology exhibited by the 3D aggregates at this stage was "grape-like". Three morphometric parameters viz. average area, average number, and average roundness of aggregates, were determined and was found that the compactness of the aggregates was the maximum on Day 5 post-seeding. Additionally, the aggregates exhibited multi-acinar structures and extracellular matrix, characteristic of tumour progression. Establishing the morphological parameters of SiHa is an approach to better understand the characteristics of 3D aggregates. Such analysis has a direct bearing on translational benefits towards solid tumour research.

Triple-negative breast cancer (TNBC) is highly aggressive and lacks targeted therapies, posing a major challenge in oncology. Traditional two-dimensional (2D) cell cultures fail to capture the tumor microenvironment's complexity, whereas three-dimensional (3D) cultures provide a more accurate model of tumor biology. We developed an advanced 3D culture system for TNBC cell lines BT-20 and MDA-MB-231, enhancing the hanging-drop method with Matrigel to restore essential extracellular matrix interactions. Confocal imaging showed MDA-MB-231 cells forming clusters typical of aggressive carcinoma, while BT-20 cells organized into duct-like structures. Molecular analysis of PI3K and β-catenin target genes revealed distinct expression patterns, with PI3K overexpressed and β-catenin downregulated in 3D cultures. Moreover, β-catenin distribution in the 3D cell culture closely resembles its pattern in tissue. These findings underscore the value of 3D models in understanding TNBC progression and in supporting the exploration of novel therapeutic strategies.

LAG3 plays a regulatory role in immunity and emerged as an inhibitory immune checkpoint molecule comparable to PD-L1 and CTLA-4 and a potential target for enhancing anti-cancer immune responses. We generated 3D cancer cultures as a model to identify novel molecular biomarkers for the selection of patients suitable for α-LAG3 treatment and simultaneously the possibility to perform an early diagnosis due to its higher presence in breast cancer, also to achieve a theragnostic approach. Our data confirm the extreme dysregulation of LAG3 in breast cancer with significantly higher expression in tumor tissue specimens, compared to non-cancerous tissue controls. LAG3 blockade inhibited proliferation of in vitro and ex vivo 3D human organoids and immune micro-environment through both a decrease of PD-L1, TIM-3 and CTLA4 expression and an increased production of several pro-inflammatory cytokines (IFNγ, IL-12, IL-6, IL-1β, TNFα) and EMT markers. These effects trigger a more permissive anti-tumor immune reaction, recruiting immune cells to the tumor sites, boosting the anti-tumor response. LAG3 acts as an immunosuppressive molecule in breast cancer, inhibiting T CD8 + cell proliferation and cytokine production by T cells. We proposed the modulation of a novel checkpoint molecule, such as LAG3, as potential biomarkers associated to a rapid diagnosis.

Mouse gastrulation entails concomitant changes in cell fate, tissue shape, and embryo size. The use of a reproducible in vitro system is crucial for dissecting the mechanisms that coordinate these events. Here, we present a protocol for generating a 3D culture of epiblast stem cells (3D EpiSCs), which grow as epithelial spheroids mimicking key features of the gastrulating mouse embryonic epiblast. We describe steps for spheroid formation, growth, and passaging, followed by imaging or further downstream analyses. For complete details on the use and execution of this protocol, please refer to Sato et al.1.

Organelle-targeted photosensitizers (PSs) offer valuable tools for improving photodynamic therapy (PDT), yet systematic studies on how different organelles influence phototherapeutic outcomes are limited. In particular, the connection between organelle targeting and various modes of programmed cell death remains unclear. In this study, we developed a series of PSs using the Coumarin-Quinazolinone (CQ) scaffold, each designed to target different organelles, including the mitochondria, endoplasmic reticulum (ER), lysosome, and nucleolus. Our results show that their PDT performance is highly dependent on their localization, with phototoxic index (PI) ranging from 2 to 245. Notably, the mitochondria-targeted CQ-Mito and ER-targeted CQ-ER exhibited profound phototherapeutic performances, with PI of 167 and 245 respectively. Our further study reveals that CQ-Mito causes cell death by both apoptosis and ferroptosis, while CQ-ER primarily triggers ferroptosis. This study not only provides new agents for PDT but also offers insights into how organelle targeting influences cell death mechanisms, which can shed light on the design of PSs for controlled cell death.

Cancer is one of the most common and fatal diseases worldwide and kills millions of people every year. Cancer drug resistance, lack of efficacy, and safety are significant problems in cancer patients. A multiorgan-on-a-chip (MOC) device consisting of breast and liver compartments was designed with AutoCAD software. The MOC molds were printed by a Formlabs Form 2 3D printer. MDA-MB-231, HepG2, and MCF-10 A cells were used for the MOC experiments. The cell lines were cultured at 37 °C with 5% CO2, and cell viability was assessed via Alamar blue dye to generate pharmacodynamics (PD) data. Drug concentrations from the cell culture media were analyzed via Agilent 1260 Infinity II HPLC with a Waters Symmetry C18 column and used to generate pharmacokinetics (PK) data. The PK and PD data were modeled and simulated by Monolix and Simulix software, respectively. The safety and efficacy of drug dosing regimens were compared, and the best dosing regimens were selected. This research designed and fabricated a unique MOC consisting of liver and breast compartments that overcomes the need for sealing or assembling. It was used for PK-PD modeling and simulations, and its functionality was proven experimentally. The new MOC will be helpful in preclinical trials to evaluate the efficacy and safety of drugs.

Canine mammary gland tumour (CMT) is the most common spontaneous tumour in intact female dogs and often exhibits metastases. Auranofin (AF) is a gold complex used for treating rheumatism. The excellent anti-tumour ability of AF has been demonstrated in various types of human and canine tumours. In this study, five CMT cell lines (CIPp, CMT-7364, CHMp, CIPm and CTBp) and three CMT primary cells (G7894, L1883 and L6783) were used to explore the anti-tumour effect of AF on CMT. Two CMT cell lines (CIPp and CMT-7364) were used to search the underlying mechanism of the effect of AF on CMT. The results showed that AF inhibited the growth, migration, invasion, and colony formation abilities of CMT cells. Additionally, the growth of CMT in a 3D cell culture model was effectively suppressed by AF. Furthermore, AF induced cell apoptosis of CMT cells via the PI3K/AKT pathway. In conclusion, AF effectively induces CMT apoptosis by regulating the PI3K/AKT pathway, indicating that AF should be explored as a potential CMT treatment in future studies.

The use of conventional two-dimensional (2D) culture of the porcine intestinal epithelial cell (IEC) line IPEC-J2 in animal nutrition research has the disadvantage that IEC function is studied under unphysiological conditions, which limits the ability of transferring knowledge to the in vivo-situation. Thus, the aim of the present study was to establish a more convincing and meaningful three-dimensional (3D) culture of IPEC-J2 cells, which allows to study cell function in a more tissue-like environment, and to compare the effect of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) on ER stress indicators and the expression of tight junction proteins (TJP), inflammatory and apoptosis-related genes and the modulatory role of 1,25-dihydroxy-vitamin D3 (1,25D3) on these parameters in 2D and 3D cultures of IPEC-J2 cells.

A published protocol for 3D culture of Caco-2 cells was successfully adopted to IPEC-J2 cells as evident from fully differentiated 3D IPEC-J2 spheroids showing the characteristic spherical architecture with a single layer of IPEC-J2 cells surrounding a central lumen. Treatment of 2D IPEC-J2 cells and 3D IPEC-J2 spheroids with TM for 24 h markedly increased mRNA and/or protein levels of the ER stress target genes, heat shock protein family A (Hsp70) member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3), whereas co-treatment with TM and 1,25D3 did not mitigate TM-induced ER stress in IPEC-J2 cells in the 2D and the 3D cell culture. In contrast, TM-induced expression of pro-inflammatory [interleukin-6 (IL6), IL8] and pro-apoptotic genes [BCL2 associated X, apoptosis regulator (BAX), caspase 3 (CASP3), CASP8] and genes encoding TJP [TJP1, claudin 1 (CLDN1), CLDN3, occludin (OCLN), cadherin 1 (CDH1), junctional adhesion molecule 1 (JAM1)] was reduced by co-treatment with TM and 1,25D3 in 3D IPEC-J2 spheroids but not in the 2D cell culture.

The effect of 1,25D3 in the IPEC-J2 cell culture is dependent on the culture model applied. While 1,25D3 does not inhibit TM-induced expression of genes involved in inflammation, apoptosis and TJP in conventional 2D cultures of IPEC-J2 cells, TM-induced expression of these genes is abrogated by 1,25D3 in the more meaningful 3D IPEC-J2 cell culture model.

This review centers on the synthesis and characterization of both natural and synthetic hydrogels, highlighting their diverse applications across various fields. We will delve into the evolution of hydrogels, focusing on the importance of polysaccharide-based and synthetic variants, which have been particularly chosen for 3D spheroid development in cancer research and drug delivery. A detailed background on the research and specific methodologies, including the in-situ free radical polymerization used for synthesizing these hydrogels, will be extensively discussed. Additionally, the review will explore various applications of these hydrogels, such as their self-healing properties, swelling ratios, pH responsiveness, and cell viability. A comprehensive literature review will support this investigation. Ultimately, this review aims to clearly outline the objectives and significance of hydrogel synthesis and their applications.

Background/objectives: The functional activity of a certain tumor determines the effectiveness of primary NK cells and NK-92 cell line-based cancer therapy; their therapeutic effectiveness against different tumors can vary. This work provides a direct simultaneous comparison of the cytotoxic effects of in vitro-activated peripheral NK (pNK) cells and NK-92 cells in spheroid models of BT-474, MCF7 and SKOV-3 carcinomas and uncovers the reasons for the differential effectiveness of NK cells against tumors. Methods: Tumor spheroids of similar size and shape, obtained from agarose molds, were incubated with NK-92 or pNK cells for 24 h. Tumor cell death was detected using flow cytometry or confocal microscopy. Cytokine production, granzyme B levels and NK cell degranulation analyses were performed, along with pNK and target-cell phenotypic characterization. Results: While NK-92 and pNK cells lysed BT-474 spheroids with comparably low efficiency, pNK cells were more capable of eliminating MCF7 and SKOV-3 spheroids than NK-92 cells were. The results of the functional and phenotypic analyses strongly support the participation of the NKG2D-NKG2DL pathway in pNK cell activation induced by the most sensitive cytotoxic attack on SKOV-3 spheroids, whereas the CX3CR1-CX3CL1 axis appears to be involved in the pNK reaction against MCF-7 spheroids. Conclusions: We provide a new approach for the preliminary identification of the most promising NK cell receptors that can alter the effectiveness of cancer therapy depending on the specific tumor type. Using this approach, NK-92 cells or pNK subsets can be selected for further accumulation and/or genetic modification to improve specificity and reactivity.

The secretion rate of albumin is a key indicator of function in liver tissue models used for hepatotoxicity and pharmacokinetic testing. However, it is not generally clear how to determine molecular secretion rates from measurements of the molecular concentration in supernatant media. Here, we develop computational and analytical models of molecular transport in an experimental system that enable determination of albumin secretion rates based on measurements of albumin concentration in supernatant media. The experimental system is a 3D-bioprinted human liver tissue construct embedded in a 3D culture environment made from packed microgel particles swollen in liquid growth media. The mathematical models reveal that the range of albumin synthesis rates necessary to match experimentally measured albumin concentrations corresponds to reaction-limited conditions, where a steady state of albumin spatial distribution is rapidly reached between media exchanges. Our results show that temporally resolved synthesis rates can be inferred from serial concentration measurements of collected supernatant media. This link is critical to confidently assessing in vitro tissue performance in applications where critical quality attributes must be quantified, like in drug development and screening.

Here, we designed, synthesized and characterized three new cyclometalated Ru(II) complexes, [Ru(phen)2(1-(4-Ph-Ph)-IQ)]+ (phen = 1,10-phenanthroline, IQ = isoquinoline, RuIQ9), [Ru(phen)2(1-(4-Ph-Ph)-7-OCH3-IQ)]+ (RuIQ10), and [Ru(phen)2(1-(4-Ph-Ph)-6,7-(OCH3)2-IQ)]+ (RuIQ11). The cytotoxicity experiments conducted on both 2D and 3D multicellular tumor spheroids (MCTSs) indicated that complexes RuIQ9-11 exhibited notably higher cytotoxicity against A549 and A549/DDP cells when compared to the ligands and precursor compounds as well as clinical cisplatin. Moreover, the Ru(II) complexes displayed low toxicity when tested on normal HBE cells in vitro and exposed to zebrafish embryos in vivo. In addition, complexes RuIQ9-11 could inhibit A549 and A549/DDP cell migration and proliferation by causing cell cycle arrest, mitochondrial dysfunction, and elevating ROS levels to induce apoptosis in these cells. Mechanistic studies revealed that RuIQ9-11 could suppress the expression of Nrf2 and its downstream antioxidant protein HO-1 by inhibiting Nrf2 gene transcription in drug-resistant A549/DDP cells. Simultaneously, they inhibited the expression of efflux proteins MRP1 and p-gp in drug-resistant cells, ensuring the accumulation of the complexes within the cells. This led to an increase in intracellular ROS levels in drug-resistant cells, ultimately causing damage and cell death, thus overcoming cisplatin resistance. More importantly, RuIQ11 could effectively inhibit the migration and proliferation of drug-resistant cells within zebrafish, addressing the issue of cisplatin resistance. Accordingly, the prepared Ru(II) complexes possess significant potential for development as highly effective and low-toxicity lung cancer therapeutic agents to overcome cisplatin resistance.

The extracellular matrix (ECM) governs a wide spectrum of cellular fate processes, with a particular emphasis on anoikis, an integrin-dependent form of cell death. Currently, anoikis is defined as an intrinsic apoptosis. In contrast to traditional apoptosis and necroptosis, integrin correlates ECM signaling with intracellular signaling cascades, describing the full process of anoikis. However, anoikis is frequently overlooked in physiological and pathological processes as well as traditional in vitro research models. In this review, we summarized the role of anoikis in physiological and pathological processes, spanning embryonic development, organ development, tissue repair, inflammatory responses, cardiovascular diseases, tumor metastasis, and so on. Similarly, in the realm of stem cell research focused on the functional evolution of cells, anoikis offers a potential solution to various challenges, including in vitro cell culture models, stem cell therapy, cell transplantation, and engineering applications, which are largely based on the regulation of cell fate by anoikis. More importantly, the regulatory mechanisms of anoikis based on molecular processes and ECM signaling will provide new strategies for therapeutic interventions (drug therapy and cell-based therapy) in disease. In summary, this review provides a systematic elaboration of anoikis, thus shedding light on its future research.

Photodynamic therapy delivers more targeted cell killing than classical chemotherapy. It uses light-absorbing compounds, photosensitizers (PSs), to generate lethal reactive oxygen species (ROS) at sites of localized irradiation. Transition metal complexes are attractive PSs due to their photostability, visible-light absorption, and high ROS yields. Here, we introduce a low-molecular weight, photostable iridium complex, [Ir(thpy)2(benz)]Cl, 1, that localizes to the Golgi apparatus, mitochondria, and endoplasmic reticulum, absorbs visible light, phosphoresces strongly, generates 1O2 with 43% yield, and undergoes cellular elimination after 24 h. 1 shows low dark toxicity and under remarkably low doses (3 min, 20-30 mJ s-1 cm-2) of 405 or 455 nm light, it causes killing of bladder (EJ), malignant melanoma (A375), and oropharyngeal (OPSCC72) cancer cells, with high phototoxic indices > 100-378. 1 is also an efficient PS in 3D melanoma spheroids, with repeated short-time irradiation causing cumulative killing.

Upregulation of CXC motif chemokine 10 (CXCL10) in melanoma patients has been found to be associated with melanoma progression. However, the role of endogenous CXCL10 from the host in melanoma tumor growth remains unclear. In the present study, we found that host-derived endogenous CXCL10 production was dramatically augmented during subcutaneous B16F10 melanoma tumor growth and that host ablation of CXCL10 in Cxcl10-/- mice showed a decrease in both angiogenesis and tumor growth of B16F10 melanoma in vivo. Several signaling pathways involved in production of pro-angiogenic factors and tumor growth were activated by CXCL10 in B16F10 melanoma cells. CXCL10 increased expression of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor subunit-B (PDGF-B), fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and angiopoietin 2 (Angpt2), in B16F10 melanoma cells, resulting in enhanced tube formation and proliferation of human umbilical vein endothelial cells in vitro. In addition, CXCL10 directly enhanced B16F10 melanoma tumor growth in an in vitro three-dimensional cell culture system. Together, our findings reveal that amplified host-derived endogenous CXCL10 is critical for B16F10 melanoma angiogenesis and tumor growth. Therefore, CXCL10 might represent a therapeutic target for melanoma.

A better understanding of ductal carcinoma in situ (DCIS) is urgently needed to identify these preinvasive lesions as distinct clinical entities. Semaphorin 3F (SEMA3F) is a soluble axonal guidance molecule, and its coreceptors Neuropilin 1 (NRP1) and NRP2 are strongly expressed in invasive epithelial BC cells.

We utilized two cell line models to represent the progression from a healthy state to the mild-aggressive or ductal carcinoma in situ (DCIS) stage and, ultimately, to invasive cell lines. Additionally, we employed in vivo models and conducted analyses on patient databases to ensure the translational relevance of our results.

We revealed SEMA3F as a promoter of invasion during the DCIS-to-invasive ductal carcinoma transition in breast cancer (BC) through the action of NRP1 and NRP2. In epithelial cells, SEMA3F activates epithelialmesenchymal transition, whereas it promotes extracellular matrix degradation and basal membrane and myoepithelial cell layer breakdown.

Together with our patient database data, these proof-of-concept results reveal new SEMA3F-mediated mechanisms occurring in the most common preinvasive BC lesion, DCIS, and represent potent and direct activation of its transition to invasion. Moreover, and of clinical and therapeutic relevance, the effects of SEMA3F can be blocked directly through its coreceptors, thus preventing invasion and keeping DCIS lesions in the preinvasive state.

Cytochrome P450 3A5 (CYP3A5) has been proposed as a predictor of therapy response in subtypes of pancreatic ductal adenocarcinoma cancer (PDAC). To validate CYP3A5 as a therapeutic target, we developed a high-content image organoid-based screen to quantify the phenotypic responses to the selective inhibition of CYP3A5 enzymatic activity by clobetasol propionate (CBZ), using a cohort of PDAC-derived organoids (PDACOs). The chemoresistance of PDACOs to a panel of standard-of-care drugs, alone or in combination with CBZ, was investigated. PDACO pharmaco-profiling revealed CBZ to have anti-cancer activity that was dependent on the CYP3A5 level. In addition, CBZ restored chemo-vulnerability to cisplatin in a subset of PDACOs. A correlative proteomic analysis established that CBZ caused the suppression of multiple cancer pathways sustained by or associated with a mutant form of p53. Limiting the active pool of CYP3A5 enables targeted and personalized therapy to suppress pro-oncogenic mechanisms that fuel chemoresistance in some PDAC tumors.

Stable isotope-resolved metabolomics comprises a critical set of technologies that can be applied to a wide variety of systems, from isolated cells to whole organisms, to define metabolic pathway usage and responses to perturbations such as drugs or mutations, as well as providing the basis for flux analysis. As the diversity of stable isotope-enriched compounds is very high, and with newer approaches to multiplexing, the coverage of metabolism is now very extensive. However, as the complexity of the model increases, including more kinds of interacting cell types and interorgan communication, the analytical complexity also increases. Further, as studies move further into spatially resolved biology, new technical problems have to be overcome owing to the small number of analytes present in the confines of a single cell or cell compartment. Here, we review the overall goals and solutions made possible by stable isotope tracing and their applications to models of increasing complexity. Finally, we discuss progress and outstanding difficulties in high-resolution spatially resolved tracer-based metabolic studies.

Efforts to find compounds selectively affecting cancer cells while sparing normal ones have continued to grow. Nitric oxide (NO) is critical in physiology and pathology, including cancer. It influences cellular processes like proliferation, apoptosis, and angiogenesis. The intricate interaction of NO with cancer cells offers innovative treatment possibilities, but its effects can vary by concentration and site. Ruthenium complexes capable of releasing NO upon stimulation show for this purpose. These versatile compounds can also enhance photodynamic therapy (PDT), a light-activated approach, which induces cellular damage. Ruthenium-based photosensitizers (PSs), delivering NO and producing reactive oxygen species (ROS), offer a novel strategy for improved cancer treatments. In this study, a nitro-ruthenium porphyrin conjugate: {TPyP[Ru(NO2)(bpy)2]4}(PF6)4, designated RuNO2TPyP, which releases NO upon irradiation, was investigated for its effects on lung cells (non-tumor MRC-5 and tumor A549) in 2D and 3D cell cultures. The findings suggest that this complex has potential for PDT treatment in lung cancer, as it exhibits photocytotoxicity at low concentrations without causing cytotoxicity to normal lung cells. Moreover, treatment of cells with RuNO2TPyP followed by light irradiation (4 J cm-2) can induce apoptosis, generate ROS, promote intracellular NO formation, and has anti-migratory effects. Additionally, the complex can modify tumor cell structures and induce photocytotoxicity and apoptosis in a 3D culture. These outcomes are attributed to the internalization of the complex and its subsequent activation upon light irradiation, resulting in NO release and singlet oxygen production.

Supramolecular delivery systems with the prolonged circulation, the potential for diverse functionalization, and few toxin-related limitations have been extensively studied. For the present study, we constructed a linear polyglycerol-shelled polymersome attached with the anti-HER-2-antibody trastuzumab. We then covalently loaded the anticancer drug DM1 in the polymersome via dynamic disulfide bonding. The resulted trastuzumab-polymersome-DM1 (Tra-PS-DM1) exhibits a mean size of 95.3 nm and remarkable drug loading efficiency % of 99.3%. In addition to its superior stability, we observed the rapid release of DM1 in a controlled manner under reductive conditions. Compared to the native polymersomes, Tra-PS-DM1 has shown greatly improved cellular uptake and significantly reduced IC50 up to 17-fold among HER-2-positive cancer cells. Moreover, Tra-PS-DM1 demonstrated superb growth inhibition of HER-2-positive tumoroids; specifically, BT474 tumoroids shrunk up to 62% after 12 h treatment. With exceptional stability and targetability, the PG-shelled Tra-PS-DM1 appears as an attractive approach for HER-2-positive tumor treatment.

Cellular biomechanics plays critical roles in cancer metastasis and tumor progression. Existing studies on cancer cell biomechanics are mostly conducted in flat 2D conditions, where cells' behavior can differ considerably from those in 3D physiological environments. Despite great advances in developing 3D in vitro models, probing cellular elasticity in 3D conditions remains a major challenge for existing technologies. In this work, we utilize optical Brillouin microscopy to longitudinally acquire mechanical images of growing cancerous spheroids over the period of eight days. The dense mechanical mapping from Brillouin microscopy enables us to extract spatially resolved and temporally evolving mechanical features that were previously inaccessible. Using an established machine learning algorithm, we demonstrate that incorporating these extracted mechanical features significantly improves the classification accuracy of cancer cells, from 74% to 95%. Building on this finding, we have developed a deep learning pipeline capable of accurately differentiating cancerous spheroids from normal ones solely using Brillouin images, suggesting the mechanical features of cancer cells could potentially serve as a new biomarker in cancer classification and detection.

Breast cancer is a relevant cause of mortality in women and its triple-negative subtype (TNBC) is usually associated with poor prognosis. During tumor progression to metastasis, angiogenesis is triggered by the sprouting of endothelial cells from pre-existing vessels by a dynamic chain of events including VE-cadherin downregulation, actin protrusion, and integrin-mediated adhesion, allowing for migration and proliferation. The binding of tumoral and tumor-associated stromal cells with the extracellular matrix through integrins mediates angiogenic processes and certain integrin subtypes, such as the αvβ3 integrin, are upregulated in hypoxic TNBC models. Integrin αvβ3 inhibition by the high-affinity binding disintegrin DisBa-01 was previously demonstrated to induce anti-tumoral and anti-angiogenic responses in traditional 2D cell assays. Here, we investigate the effects of integrin αvβ3 blockage in endothelial and TNBC cells by DisBa-01 in 3D cultures under two oxygen conditions (1% and 20%). 3D cultures created using non-adhesive micromolds with Matrigel were submitted to migration assay in Boyden chambers and fluorescence analysis. DisBa-01 inhibited cell migration in normoxia and hypoxia in both MDA-MB-231 and HUVEC spheroids. Protein levels of integrin αvβ3 were overexpressed in HUVEC spheroids compared to MDA-MB-231 spheroids. In HUVEC 3D cultures, sprouting assays in collagen type I were decreased in normoxia upon DisBa-01 treatment, and VE-cadherin levels were diminished in HUVEC spheroids in hypoxia and upon DisBa-01 treatment. In conclusion, the blockage of integrin αvβ3 by DisBa-01 inhibits cell migration in 3D culture and interferes with tumor-derived responses in different oxygen settings, implicating its crucial role in angiogenesis and tumor progression.

ADGRF5 (GPR116) has been identified as a facilitator of breast cancer cell migration and metastasis, yet the underlying mechanisms remain largely elusive. Our current study reveals that the absence of ADGRF5 in breast cancer cells impairs extracellular matrix (ECM)-associated cell motility and impedes in vivo tumor growth. This correlates with heightened expression of matrix metalloproteinase 8 (MMP8), a well-characterized antitumorigenic MMP, and a shift in the polarization of tumor-associated neutrophils (TANs) towards the antitumor N1 phenotype in the tumor microenvironment (TME). Mechanistically, ADGRF5 inhibits ERK1/2 activity by enhancing RhoA activation, leading to decreased phosphorylation of C/EBPβ at Thr235, hindering its nuclear translocation and subsequent activation. Crucially, two C/EBPβ binding motifs essential for MMP8 transcription are identified within its promoter region. Consequently, ADGRF5 silencing fosters MMP8 expression and CXCL8 secretion, attracting increased infiltration of TANs; simultaneously, MMP8 plays a role in decorin cleavage, which leads to trapped-inactivation of TGF-β in the TME, thereby polarizing TANs towards the antitumor N1 neutrophil phenotype and mitigating TGF-β-enhanced cell motility in breast cancer. Our findings reveal a novel connection between ADGRF5, an adhesion G protein-coupled receptor, and the orchestration of the TME, which dictates malignancy progression. Overall, the data underscore ADGRF5 as a promising therapeutic target for breast cancer intervention.

Metastasis is one of the defining features of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor prognosis. In this study, the palmitoyl transferase ZDHHC20 was identified in an in vivo short hairpin RNA (shRNA) screen as critical for metastatic outgrowth, with no effect on proliferation and migration in vitro or primary PDAC growth in mice. This phenotype is abrogated in immunocompromised animals and animals with depleted natural killer (NK) cells, indicating that ZDHHC20 affects the interaction of tumor cells and the innate immune system. Using a chemical genetics platform for ZDHHC20-specific substrate profiling, a number of substrates of this enzyme were identified. These results describe a role for palmitoylation in enabling distant metastasis that could not have been detected using in vitro screening approaches and identify potential effectors through which ZDHHC20 promotes metastasis of PDAC.