Chemically and mechanically defined hyaluronan hydrogels emulate the extracellular matrix for unbiased in vivo and in vitro organoid formation and drug testing in cancer

Finding a material that emulates the in vivo microenvironment to allow complex cell phenotypes in vitro and without biasing cell fate in vivo remains a significant challenge. Hyaluronan is over-expressed in breast cancer and in several other tumours. Mammary epithelial cells invade tissues through both active degradation of the matrix and ameboid-like mechanisms. Thus, we synthesized a defined, biomimetic hydrogel composed of hyaluronan (HA) and matrix metalloproteinase-cleavable (MMPx) crosslinker and used oxime crosslinking to ensure tunability of the resulting HA-MMPx hydrogel. This strategy allowed us to identify the optimal matrix to study the growth and polarization of healthy and diseased mammary epithelial cancer cells first in vitro and then in vivo. We then extended the platform to study nine different cancer types in vitro. We demonstrate that primary, patient-derived breast cancer cells from biopsies established organoids within HA-MMPx and, relative to Matrigel®, had different growth rates and responses to drugs, underscoring the importance of the extracellular environment to cell fate. We established patient-derived xenografts (PDX) using HA-MMPx in SCID mice and showed superior reproducibility compared to Matrigel®. Fascinatingly, PDX grown in HA-MMPx did not induce resident murine macrophage polarization whereas those grown in Matrigel® showed an increase in the proportion of alternatively activated cells, indicating that Matrigel® itself skewed macrophage polarization. Importantly, HA-MMPx did not bias the immune cell response in vivo and supported a diverse range of organoid phenotypes in vitro.     

In vitro Toxicity Testing of Nanoparticles in 3D Cell Culture

Common 2D cell cultures do not adequately represent the functions of 3D tissues that have extensive cell–cell and cell–matrix interactions, as well as markedly different diffusion/transport conditions. Hence, testing cytotoxicity in 2D cultures may not accurately reflect the actual toxicity of nanoparticles (NPs) and other nanostructures in the body. To obtain more adequate and detailed information about NP–tissue interactions, we here introduce a 3D‐spheroid‐culture‐based NP toxicology testing system. Hydrogel inverted colloidal crystal (ICC) scaffolds are used to create a physiologically relevant and standardized 3D liver tissue spheroid model for in vitro assay application. Toxicity of CdTe and Au NPs are tested in both 2D and 3D spheroid cultures. The results reveal that NP toxic effects are significantly reduced in the spheroid culture when compared to the 2D culture data. Tissue‐like morphology and phenotypic change are identified to be the major factors in diminishing toxicity. Acting as an intermediate stage bridging in vitro 2D and in vivo, our in vitro 3D cell‐culture model would extend current cellular level cytotoxicity to the tissue level, thereby improving the predictive power of in vitro NP toxicology.     

Facile One Step Formation and Screening of Tumor Spheroids Using Droplet‐Microarray Platform

Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo “microenvironment” compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and resistance testing. Established and commonly used platforms for routine screenings of cell spheroids, based on microtiter plates of 96‐ and 384‐well formats, require relatively large numbers of cells and compounds, and often lead to the formation of multiple spheroids per well. In this study, an application of the Droplet Microarray platform, based on hydrophilic–superhydrophobic patterning, in combination with the method of hanging droplet, is demonstrated for the formation of highly miniaturized single‐spheroid‐microarrays. Formation of spheroids from several commonly used cancer cell lines in 100 nL droplets starting with as few as 150 cells per spheroid within 24–48 h is demonstrated. Established methodology carries a potential to be adopted for routine workflows of high‐throughput compound screening in 3D cancer spheroids or microtumors, which is crucial for the fields of fundamental cancer research, drug discovery, and precision medicine.     

A Spontaneous 3D Bone‐On‐a‐Chip for Bone Metastasis Study of Breast Cancer Cells

Bone metastasis occurs at ≈70% frequency in metastatic breast cancer. The mechanisms used by tumors to hijack the skeleton, promote bone metastases, and confer therapeutic resistance are poorly understood. This has led to the development of various bone models to investigate the interactions between cancer cells and host bone marrow cells and related physiological changes. However, it is challenging to perform bone studies due to the difficulty in periodic sampling. Herein, a bone‐on‐a‐chip (BC) is reported for spontaneous growth of a 3D, mineralized, collagenous bone tissue. Mature osteoblastic tissue of up to 85 µm thickness containing heavily mineralized collagen fibers naturally formed in 720 h without the aid of differentiation agents. Moreover, co‐culture of metastatic breast cancer cells is examined with osteoblastic tissues. The new bone‐on‐a‐chip design not only increases experimental throughput by miniaturization, but also maximizes the chances of cancer cell interaction with bone matrix of a concentrated surface area and facilitates easy, frequent observation. As a result, unique hallmarks of breast cancer bone colonization, previously confirmed only in vivo, are observed. The spontaneous 3D BC keeps the promise as a physiologically relevant model for the in vitro study of breast cancer bone metastasis.     

Multicellular Tumor Spheroids (MCTS) as a 3D In Vitro Evaluation Tool of Nanoparticles

Multicellular tumor spheroid models (MCTS) are often coined as 3D in vitro models that can mimic the microenvironment of tissues. MCTS have gained increasing interest in the nano‐biotechnology field as they can provide easily accessible information on the performance of nanoparticles without using animal models. Considering that many countries have put restrictions on animals testing, which will only tighten in the future as seen by the recent developments in the Netherlands, 3D models will become an even more valuable tool. Here, an overview on MCTS is provided, focusing on their use in cancer research as most nanoparticles are tested in MCTS for treatment of primary tumors. Thereafter, various types of nanoparticles—from self‐assembled block copolymers to inorganic nanoparticles, are discussed. A range of physicochemical parameters including the size, shape, surface chemistry, ligands attachment, stability, and stiffness are found to influence nanoparticles in MCTS. Some of these studies are complemented by animal studies confirming that lessons from MCTS can in part predict the behaviour in vivo. In summary, MCTS are suitable models to gain additional information on nanoparticles. While not being able to replace in vivo studies, they can bridge the gap between traditional 2D in vitro studies and in vivo models.     

Biomimicry 3D Gastrointestinal Spheroid Platform for the Assessment of Toxicity and Inflammatory Effects of Zinc Oxide Nanoparticles

Our current mechanistic understanding on the effects of engineered nanoparticles (NPs) on cellular physiology is derived mainly from 2D cell culture studies. However, conventional monolayer cell culture may not accurately model the mass transfer gradient that is expected in 3D tissue physiology and thus may lead to artifactual experimental conclusions. Herein, using a micropatterned agarose hydrogel platform, the effects of ZnO NPs (25 nm) on 3D colon cell spheroids of well‐defined sizes are examined. The findings show that cell dimensionality plays a critical role in governing the spatiotemporal cellular outcomes like inflammatory response and cytotoxicity in response to ZnO NPs treatment. More importantly, ZnO NPs can induce different modes of cell death in 2D and 3D cell culture systems. Interestingly, the outer few layers of cells in 3D model could only protect the inner core of cells for a limited time and periodically slough off from the spheroids surface. These findings suggest that toxicological conclusions made from 2D cell models might overestimate the toxicity of ZnO NPs. This 3D cell spheroid model can serve as a reproducible platform to better reflect the actual cell response to NPs and to study a more realistic mechanism of nanoparticle‐induced toxicity.     

A Self‐Assembled DNA Origami‐Gold Nanorod Complex for Cancer Theranostics

A self‐assembled DNA origami (DO)‐gold nanorod (GNR) complex, which is a dual‐functional nanotheranostics constructed by decorating GNRs onto the surface of DNA origami, is demonstrated. After 24 h incubation of two structured DO‐GNR complexes with human MCF7 breast cancer cells, significant enhancement of cell uptake is achieved compared to bare GNRs by two‐photon luminescence imaging. Particularly, the triangle shaped DO‐GNR complex exhibits optimal cellular accumulation. Compared to GNRs, improved photothermolysis against tumor cells is accomplished for the triangle DO‐GNR complex by two‐photon laser or NIR laser irradiation. Moreover, the DO‐GNR complex exhibits enhanced antitumor efficacy compared with bare GNRs in nude mice bearing breast tumor xenografts. The results demonstrate that the DO‐GNR complex can achieve optimal two‐photon cell imaging and photothermal effect, suggesting a promising candidate for cancer diagnosis and therapy both in vitro and in vivo.     

Bioprinting-Based High-Throughput Fabrication of Three-Dimensional MCF-7 Human Breast Cancer Cellular Spheroids

Cellular spheroids serving as three-dimensional(3D) in vitro tissue models have attracted increasing interest for pathological study and drug-screening applications. Various methods, including microwells in particular, have been developed for engineering cellular spheroids. However, these methods usually suffer from either destructive molding operations or cell loss and non-uniform cell distribution among the wells due to two-step molding and cell seeding. We have developed a facile method that utilizes cellembedded hydrogel arrays as templates for concave well fabrication and in situ MCF-7 cellular spheroid formation on a chip. A custom-built bioprinting system was applied for the fabrication of sacrificial gelatin arrays and sequentially concave wells in a high-throughput, flexible, and controlled manner. The ability to achieve in situ cell seeding for cellular spheroid construction was demonstrated with the advantage of uniform cell seeding and the potential for programmed fabrication of tissue models on chips. The developed method holds great potential for applications in tissue engineering, regenerative medicine, and drug screening.     

Superporous Poly(ethylene glycol) Diacrylate Cryogel with a Defined Elastic Modulus for Prostate Cancer Cell Research

The physical and mechanical properties of the tumor microenvironment are crucial for the growth, differentiation and migration of cancer cells. However, such microenvironment is not found in the geometric constraints of 2D cell culture systems used in many cancer studies. Prostate cancer research, in particular, suffers from the lack of suitable in vitro models. Here a 3D superporous scaffold is described with thick pore walls in a mechanically stable and robust architecture to support prostate tumor growth. This scaffold is generated from the cryogelation of poly(ethylene glycol) diacrylate to produce a defined elastic modulus for prostate tumor growth. Lymph node carcinoma of the prostate (LNCaP) cells show a linear growth over 21 d as multicellular tumor spheroids in such a scaffold with points of attachments to the walls of the scaffold. These LNCaP cells respond to the growth promoting effects of androgens and demonstrate a characteristic cytoplasmic‐nuclear translocation of the androgen receptor and androgen‐dependent gene expression. Compared to 2D cell culture, the expression or androgen response of prostate cancer specific genes is greatly enhanced in the LNCaP cells in this system. This scaffold is therefore a powerful tool for prostate cancer studies with unique advantages over 2D cell culture systems.     

三维成球培养优化间充质干细胞的研究进展

间充质干细胞(mesenchymal stem cells,MSCs)源于发育早期的中胚层,因其来源广泛、具有多向分化潜能、低免疫原性和自我更新能力,在组织工程和再生医学应用中显示出巨大的潜力,也是当前基础研究和临床研究中应用最多的一类干细胞。然而,间充质干细胞的临床应用面临许多挑战,比如治疗所需细胞数量巨大,细胞质量存在异质性,细胞体内移植后存活率低,以及二维(two-dimensional,2D)贴壁培养导致间充质干细胞特征衰减等。三维(three-dimensional,3D)成球培养可以更好地模拟体内微环境,且大量的研究证明,3D成球培养增强了间充质干细胞的细胞存活和因子分泌能力,促进了干细胞特征维持、细胞迁移和血管生成,在临床医学领域具有广阔的应用前景。基于此,综述了体外3D成球培养的方法、3D成球培养优化的间充质干细胞的生物学特性及应用,并对3D成球培养未来的研究方向进行展望。     

Thermoplasmonic‐Triggered Release of Loads from DNA‐Modified Hydrogel Microcapsules Functionalized with Au Nanoparticles or Au Nanorods

Microcapsules consisting of hydrogel shells cross‐linked by glucosamine–boronate ester complexes and duplex nucleic acids, loaded with dyes or drugs and functionalized with Au nanoparticles (Au NPs) or Au nanorods (Au NRs), are developed. Irradiation of Au NPs or Au NRs results in the thermoplasmonic heating of the microcapsules, and the dissociation of the nucleic acid cross‐linkers. The separation of duplex nucleic acid cross‐linkers leads to low‐stiffness hydrogel shells, allowing the release of loads. Switching off the light‐induced plasmonic heating results in the regeneration of stiff hydrogel shells protecting the microcapsules, leading to the blockage of release processes. The thermoplasmonic release of tetramethylrhodamine‐dextran, Texas Red‐dextran, doxorubicin‐dextran (DOX‐D), or camptothecin‐carboxymethylcellulose (CPT‐CMC) from the microcapsules is introduced. By loading the microcapsules with two different drugs (DOX‐D and CPT‐CMC), the light‐controlled dose release is demonstrated. Cellular experiments show efficient permeation of Au NPs/DOX‐D or Au NRs/DOX‐D microcapsules into MDA‐MB‐231 cancer cells and inefficient uptake by MCF‐10A epithelial breast cells. Cytotoxicity experiments reveal selective thermoplasmon‐induced cytotoxicity of the microcapsules toward MDA‐MB‐231 cancer cells as compared to MCF‐10A cells. Also, selective cytotoxicity towards MDA‐MB‐231 cancer cells upon irradiation of the Au NPs‐ and Au NRs‐functionalized microcapsules at λ = 532 or 910 nm is demonstrated.     

Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High‐Content Drug Screening

Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung‐mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.     

Efficient One‐Step Production of Microencapsulated Hepatocyte Spheroids with Enhanced Functions

Hepatocyte spheroids microencapsulated in hydrogels can contribute to liver research in various capacities. The conventional approach of microencapsulating spheroids produces a variable number of spheroids per microgel and requires an extra step of spheroid loading into the gel. Here, a microfluidics technology bypassing the step of spheroid loading and controlling the spheroid characteristics is reported. Double‐emulsion droplets are used to generate microencapsulated homotypic or heterotypic hepatocyte spheroids (all as single spheroids <200 μm in diameter) with enhanced functions in 4 h. The composition of the microgel is tunable as demonstrated by improved hepatocyte functions during 24 d culture (albumin secretion, urea secretion, and cytochrome P450 activity) when alginate‐collagen composite hydrogel is used instead of alginate. Hepatocyte spheroids in alginate‐collagen also perform better than hepatocytes cultured in collagen‐sandwich configuration. Moreover, hepatocyte functions are significantly enhanced when hepatocytes and endothelial progenitor cells (used as a novel supporting cell source) are co‐cultured to form composite spheroids at an optimal ratio of 5:1, which could be further boosted when encapsulated in alginate‐collagen. This microencapsulated‐spheroid formation technology with high yield, versatility, and uniformity is envisioned to be an enabling technology for liver tissue engineering as well as biomanufacturing.     

Hyaluronidase-triggered anticancer drug and siRNA delivery from cascaded targeting nanoparticles for drug-resistant breast cancer therapy

Drug resistance renders standard chemotherapy ineffective in the treatment of connective tissue growth factor (CTGF)-overexpressing breast cancer.By co-embedding the breast tumor cell-penetrating peptide (PEGA-pVEC) and hyaluronic add (HA) as a targeting media,novel cascaded targeting nanoparticles (HACT NPs) were created on a rattle mesoporous silica (rmSiO2) scaffold for the pinpoint delivery of siRNAs along with an anticancer drug,aiming at overcoming the drug resistance of CTGF-overexpressing breast cancer in vivo.The targeting nanoparticles selectively accumulated in the vasculature under the guidance of the PEGA-pVEC peptide,cascaded by receptor-mediated endocytosis with the aid of another targeting agent,HA,presenting a greater in vivo tumor targeting ability than single targeting ligand vectors.In addition,an HA shell prevented the leakage of therapeutic drugs during the cargo transport process,until the hyaluronidase (HAase)-triggered degradation upon lysosomes entering,guaranteeing a controllable drug release inside the target cells.When the protective shell disintegrates,the released siRNA took charge to silence the gene associated with drug resistance,CTGF,thus facilitating doxorubicin-induced apoptosis.The cascaded targeting media (PEGA-pVEC and HA) advances precision-guided therapy in vivo,while the encapsulation of siRNAs into a chemotherapy drug delivery system provides an effident strategy for the treatment of drug resistance cancers.     

Multiscale Modulation of Nanocrystalline Cellulose Hydrogel via Nanocarbon Hybridization for 3D Neuronal Bilayer Formation

Bacterial biopolymers have drawn much attention owing to their unconventional three‐dimensional structures and interesting functions, which are closely integrated with bacterial physiology. The nongenetic modulation of bacterial (Acetobacter xylinum) cellulose synthesis via nanocarbon hybridization, and its application to the emulation of layered neuronal tissue, is reported. The controlled dispersion of graphene oxide (GO) nanoflakes into bacterial cellulose (BC) culture media not only induces structural changes within a crystalline cellulose nanofibril, but also modulates their 3D collective association, leading to substantial reduction in Young's modulus (≈50%) and clear definition of water–hydrogel interfaces. Furthermore, real‐time investigation of 3D neuronal networks constructed in this GO‐incorporated BC hydrogel with broken chiral nematic ordering revealed the vertical locomotion of growth cones, the accelerated neurite outgrowth (≈100 µm per day) with reduced backward travel length, and the efficient formation of synaptic connectivity with distinct axonal bifurcation abundancy at the ≈750 µm outgrowth from a cell body. In comparison with the pristine BC, GO‐BC supports the formation of well‐defined neuronal bilayer networks with flattened interfacial profiles and vertical axonal outgrowth, apparently emulating the neuronal development in vivo. We envisioned that our findings may contribute to various applications of engineered BC hydrogel to fundamental neurobiology studies and neural engineering.     

Hyaluronic acid-bound letrozole nanoparticles restore sensitivity to letrozole-resistant xenograft tumors in mice

Letrozole is a potent aromatase inhibitor and superior to other defined selective estrogen receptor modulators such as tamoxifen in treating hormone-responsive postmenopausal breast cancer patients. Patients who receive this drug may become insensitive to the effects of estrogen deprivation induced by letrozole. Letrozole has known side effects on bone metabolism due to systemic ablation of estrogen production. The purpose of this study was to examine the therapeutic efficacy of hyaluronic acid-bound letrozole nanoparticles (HA-Letr-NPs) in restoring sensitivity to letrozole-resistant (LTLT-Ca) cells. To target letrozole to LTLT-Ca cells, hyaluronic acid-bound letrozole nanoparticles were prepared by nanoprecipitation using biodegradable PLGA-PEG co-polymer. Binding specificity of HA to CD44 on the cell surface was analyzed in vitro using FITC-CD44 Ab and CD44 siRNA by flow cytometry. Effects on in vitro cytotoxicity and aromatase enzymatic activity of HA-Letr-NPs were performed in MCF-7 breast cancer cells, MCF-7 cells over-expressing aromatase (MCF-7/Aro), and LTLT-Ca cells resistant to letrozole. Preclinical efficacy of HA-Letr-NPs was examined in mice using LTLT-Ca xenograft tumors. HA-Letr-NPs were restricted to a maximum size of 100 nm. The in vitro drug release assay showed that the highest released concentration of letrozole occurred after 23 hours at 37 degrees C in phosphate-buffered saline. HA-Letr-NPs on MCF-7/Aro and LTLT-Ca cells showed an IC50 of 2 microM and 5 microM, respectively. HA-Letr-NPs were more efficacious in inhibiting tumor growth, reducing in vitro cellular and in vivo tumor aromatase enzyme activity more than the corresponding Letr-NPs or letrozole. HA-Letr-NPs restored and maintained a prolonged sensitivity and targeted delivery of letrozole in letrozole-resistant tumors in vivo.     

Hydrogen Peroxide Responsive Iron–Based Nanoplatform for Multimodal Imaging–Guided Cancer Therapy

Cancer multimodal phototherapy triggered by hydrogen peroxide has attracted widespread attention as a dominating strategy to increase phototherapeutic efficiency. Herein, a hydrogen peroxide responsive iron oxide nanoplatform, with the diameter of about 50 nm, is fabricated to intracellularly trigger the Fenton reaction and achieve synergistic photodynamic therapy and photothermal therapy. The nanoplatform based on iron oxide nanoparticles is decorated with indocyanine green (ICG, photosensitizer) and hyaluronic acid (HA, targeting molecular) through electrostatic interaction, thus the as‐prepared nanoplatform (IONPs‐ICG‐HA) exhibits excellent active targeting ability and biocompatibility. More importantly, it can effectively utilize the intratumoral overproduced hydrogen peroxide to generate reactive oxygen species for cancer cell killing via intracellular Fenton reactions. In vitro and in vivo experiments reveal that the IONPs‐ICG‐HA nanocomposites realize effective photoacoustic/photothermal/fluorescence imaging–guided phototherapy, leading to promising hydrogen peroxide responsive cancer theranostics.     

3D Jet Writing: Functional Microtissues Based on Tessellated Scaffold Architectures

The advent of adaptive manufacturing techniques supports the vision of cell‐instructive materials that mimic biological tissues. 3D jet writing, a modified electrospinning process reported herein, yields 3D structures with unprecedented precision and resolution offering customizable pore geometries and scalability to over tens of centimeters. These scaffolds support the 3D expansion and differentiation of human mesenchymal stem cells in vitro. Implantation of these constructs leads to the healing of critical bone defects in vivo without exogenous growth factors. When applied as a metastatic target site in mice, circulating cancer cells home in to the osteogenic environment simulated on 3D jet writing scaffolds, despite implantation in an anatomically abnormal site. Through 3D jet writing, the formation of tessellated microtissues is demonstrated, which serve as a versatile 3D cell culture platform in a range of biomedical applications including regenerative medicine, cancer biology, and stem cell biotechnology.     

A 3D In Vitro Cancer Model as a Platform for Nanoparticle Uptake and Imaging Investigations

In order to maximize the potential of nanoparticles (NPs) in cancer imaging and therapy, their mechanisms of interaction with host tissue need to be fully understood. NP uptake is known to be dramatically influenced by the tumor microenvironment, and an imaging platform that could replicate in vivo cellular conditions would make big strides in NP uptake studies. Here, a novel NP uptake platform consisting of a tissue‐engineered 3D in vitro cancer model (tumoroid), which mimics the microarchitecture of a solid cancer mass and stroma, is presented. As the tumoroid exhibits fundamental characteristics of solid cancer tissue and its cellular and biochemical parameters are controllable, it provides a real alternative to animal models. Furthermore, an X‐ray fluorescence imaging system is developed to demonstrate 3D imaging of GNPs and to determine uptake efficiency within the tumoroid. This platform has implications for optimizing the targeted delivery of NPs to cells to benefit cancer diagnostics and therapy.