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Pancreatic ductal adenocarcinoma is characterised by a dense desmoplastic stroma composed of stromal cells and extracellular matrix (ECM). This barrier severely impairs drug delivery and penetration. Activated pancreatic stellate cells (PSCs) play a key role in establishing this unique pathological obstacle, but also offer a potential target for anti-tumour therapy. Here, we construct a tumour microenvironment-responsive nanosystem, based on PEGylated polyethylenimine-coated gold nanoparticles, and utilise it to co-deliver all- trans retinoic acid (ATRA, an inducer of PSC quiescence) and siRNA targeting heat shock protein 47 (HSP47, a collagen-specific molecular chaperone) to re-educate PSCs. The nanosystem simultaneously induces PSC quiescence and inhibits ECM hyperplasia, thereby promoting drug delivery to pancreatic tumours and significantly enhancing the anti-tumour efficacy of chemotherapeutics. Our combination strategy to restore homoeostatic stromal function by targeting activated PSCs represents a promising approach to improving the efficacy of chemotherapy and other therapeutic modalities in a wide range of stroma-rich tumours. Stromal-tumour interactions play an important role in pancreatic cancer progression. Here, they describe the development of a tumour microenvironment-responsive gold nanoparticle system incorporating all- trans retinoic acid (ATRA) and siRNA against heat shock protein 47 (HSP47), for use in pancreatic cancer treatment.

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID‐19 infection via proposed anti‐cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID‐19 infection. We validated the AI‐predicted biochemical inhibitory effects of baricitinib on human numb‐associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID‐19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS‐CoV‐2 viral load, inflammatory markers, and IL‐6 levels. Collectively, these data support further evaluation of the anti‐cytokine and anti‐viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID‐19 patients. Synopsis This study provides biochemical and cellular evidence confirming artificial intelligence (AI)‐predictions focused on anti‐cytokine signaling and potential anti‐viral effects for baricitinib, along with a case series, supporting its potential utility in hospitalized COVID‐19 patients. Baricitinib, an oral Janus kinase (JAK)1/JAK2 inhibitor used to treat rheumatoid arthritis, was hypothesised using AI to be useful in COVID‐19. Baricitinib‐mediated inhibition of numb associated kinases utilized by SARS‐CoV‐2 for its propagation, led to reduced viral infectivity in primary liver spheroids. Baricitinib reduces levels of cytokines implicated in COVID‐19 and inhibits their signaling. In patients with bilateral COVID‐19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS‐CoV‐2 viral load, inflammatory markers, and IL‐6 levels. Graphical Abstract This study provides biochemical and cellular evidence confirming artificial intelligence (AI)‐predictions focused on anti‐cytokine signaling and potential anti‐viral effects for baricitinib, along with a case series, supporting its potential utility in hospitalized COVID‐19 patients.

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

Cancer is a complex disease in which both genetic defects and microenvironmental components contribute to the development, progression, and metastasization of disease, representing major hurdles in the identification of more effective and safer treatment regimens for patients. Three-dimensional (3D) models are changing the paradigm of preclinical cancer research as they more closely resemble the complex tissue environment and architecture found in clinical tumors than in bidimensional (2D) cell cultures. Among 3D models, spheroids and organoids represent the most versatile and promising models in that they are capable of recapitulating the heterogeneity and pathophysiology of human cancers and of filling the gap between conventional 2D in vitro testing and animal models. Such 3D systems represent a powerful tool for studying cancer biology, enabling us to model the dynamic evolution of neoplastic disease from the early stages to metastatic dissemination and the interactions with the microenvironment. Spheroids and organoids have recently been used in the field of drug discovery and personalized medicine. The combined use of 3D models could potentially improve the robustness and reliability of preclinical research data, reducing the need for animal testing and favoring their transition to clinical practice. In this review, we summarize the recent advances in the use of these 3D systems for cancer modeling, focusing on their innovative translational applications, looking at future challenges, and comparing them with most widely used animal models.

To optimally penetrate biological hydrogels such as mucus and the tumor interstitial matrix, nanoparticles (NPs) require physicochemical properties that would typically preclude cellular uptake, resulting in inefficient drug delivery. Here, we demonstrate that (poly(lactic-co-glycolic acid) (PLGA) core)-(lipid shell) NPs with moderate rigidity display enhanced diffusivity through mucus compared with some synthetic mucus penetration particles (MPPs), achieving a mucosal and tumor penetrating capability superior to that of both their soft and hard counterparts. Orally administered semi-elastic NPs efficiently overcome multiple intestinal barriers, and result in increased bioavailability of doxorubicin (Dox) (up to 8 fold) compared to Dox solution. Molecular dynamics simulations and super-resolution microscopy reveal that the semi-elastic NPs deform into ellipsoids, which enables rotation-facilitated penetration. In contrast, rigid NPs cannot deform, and overly soft NPs are impeded by interactions with the hydrogel network. Modifying particle rigidity may improve the efficacy of NP-based drugs, and can be applicable to other barriers. Penetration of the mucus and tumor interstitial matrix is an important consideration for drug delivery devices. Here, the authors report on a study into the optimization of rigidity for the transport of nanoparticles through biological hydrogels using core-shell polymer-lipid nanoparticles.

Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.

Patient‐derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high‐throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high‐throughput screening of 2427 drugs using the cancer tissue‐originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease. We report an advanced system for the high‐throughput screening of 2427 drugs using organoids. In this system, we apply the cancer‐tissue originated spheroid (CTOS) method in an ex vivo platform from xenograft tumors, using machines to handle organoids and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.

Extrapolation of cell culture-based test results to in vivo effects is limited, as cell cultures fail to emulate organ complexity and multi-tissue crosstalk. Biology-inspired microphysiological systems provide preclinical insights into absorption, distribution, metabolism, excretion, and toxicity of substances in vitro by using human three-dimensional organotypic cultures. We co-cultured a human lung equivalent from the commercially available bronchial MucilAir culture and human liver spheroids from HepaRG cells to assess the potential toxicity of inhaled substances under conditions that permit organ crosstalk. We designed a new HUMIMIC Chip with optimized medium supply and oxygenation of the organ cultures and cultivated them on-chip for 14 days in separate culture compartments of a closed circulatory perfusion system, demonstrating the viability and homeostasis of the tissue cultures. A single-dose treatment of the hepatotoxic and carcinogenic aflatoxin B 1 impaired functionality in bronchial MucilAir tissues in monoculture but showed a protective effect when the tissues were co-cultured with liver spheroids, indicating that crosstalk can be achieved in this new human lung–liver co-culture. The setup described here may be used to determine the effects of exposure to inhaled substances on a systemic level.

Tumour hypoxia renders cancer cells resistant to cancer therapy, resulting in markedly worse clinical outcomes. To find clinical candidate compounds that reduce hypoxia in tumours, we conduct a high-throughput screen for oxygen consumption rate (OCR) reduction and identify a number of drugs with this property. For this study we focus on the anti-malarial, atovaquone. Atovaquone rapidly decreases the OCR by more than 80% in a wide range of cancer cell lines at pharmacological concentrations. In addition, atovaquone eradicates hypoxia in FaDu, HCT116 and H1299 spheroids. Similarly, it reduces hypoxia in FaDu and HCT116 xenografts in nude mice, and causes a significant tumour growth delay when combined with radiation. Atovaquone is a ubiquinone analogue, and decreases the OCR by inhibiting mitochondrial complex III. We are now undertaking clinical studies to assess whether atovaquone reduces tumour hypoxia in patients, thereby increasing the efficacy of radiotherapy. Tumour hypoxia reduces the efficacy of radiotherapy. Starting from a drug screen, here the authors demonstrate that the anti-malarial, atovaquone, reduces the oxygen consumption rate of cancer cells by inhibition of mitochondrial complex III and sensitises to radiotherapy by reducing tumour hypoxia.

Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart (“cardiac spheroids”, CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo . The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro , offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue.