Bioengineered hydrogels enhance ex vivo preservation of patient-derived tumor explants for drug evaluation.

Ex vivo patient-derived tumor slices (PDTS) are currently limited by short-term viability in culture. Here, we show how bioengineered hydrogels enable the identification of key matrix parameters that significantly enhance PDTS viability compared to conventional culture systems. As demonstrated using single-cell RNA sequencing and high-dimensional flow cytometry, hydrogel-embedded PDTS tightly preserved cancer, cancer-associated fibroblast, and various immune cell populations and subpopulations in the corresponding original tumor. Cell-cell communication networks within the tumor microenvironment, including immune checkpoint ligand-receptor interactions, were also maintained. Remarkably, our results from a co-clinical trial suggest hydrogel-embedded PDTS may predict sensitivity to immune checkpoint inhibitors (ICIs) in head and neck cancer patients. Further, we show how these longer term-cultured tumor explants uniquely enable the sampling and detection of temporal evolution in molecular readouts when treated with ICIs. By preserving the compositional heterogeneity and complexity of patient tumors, hydrogel-embedded PDTS provide a valuable tool to facilitate experiments targeting the tumor microenvironment.

Making In Vitro Tumor Models Whole Again.

As a reductionist approach, patient-derived in vitro tumor models are inherently still too simplistic for personalized drug testing as they do not capture many characteristics of the tumor microenvironment (TME), such as tumor architecture and stromal heterogeneity. This is especially problematic for assessing stromal-targeting drugs such as immunotherapies in which the density and distribution of immune and other stromal cells determine drug efficacy. On the other end, in vivo models are typically costly, low-throughput, and time-consuming to establish. Ex vivo patient-derived tumor explant (PDE) cultures involve the culture of resected tumor fragments that potentially retain the intact  TME of the original tumor. Although developed decades ago, PDE cultures have not been widely adopted likely because of their low-throughput and poor long-term viability. However, with growing recognition of the importance of patient-specific TME in mediating drug response, especially in the field of immune-oncology, there is an urgent need to resurrect these holistic cultures. In this Review, the key limitations of patient-derived tumor explant cultures are outlined and technologies that have been developed or could be employed to address these limitations are discussed. Engineered holistic tumor explant cultures may truly realize the concept of personalized medicine for cancer patients.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Engineering stromal heterogeneity in cancer.

Based on our exponentially increasing knowledge of stromal heterogeneity from advances in single-cell technologies, the notion that stromal cell types exist as a spectrum of unique subpopulations that have specific functions and spatial distributions in the tumor microenvironment has significant impact on tumor modeling for drug development and personalized drug testing. In this Review, we discuss the importance of incorporating stromal heterogeneity and tumor architecture, and propose an overall approach to guide the reconstruction of stromal heterogeneity in vitro for tumor modeling. These next-generation tumor models may support the development of more precise drugs targeting specific stromal cell subpopulations, as well as enable improved recapitulation of patient tumors in vitro for personalized drug testing.

Hot or cold: Bioengineering immune contextures into in vitro patient-derived tumor models.

In the past decade, immune checkpoint inhibitors (ICI) have proven to be tremendously effective for a subset of cancer patients. However, it is difficult to predict the response of individual patients and efforts are now directed at understanding the mechanisms of ICI resistance. Current models of patient tumors poorly recapitulate the immune contexture, which describe immune parameters that are associated with patient survival. In this Review, we discuss parameters that influence the induction of different immune contextures found within tumors and how engineering strategies may be leveraged to recapitulate these contextures to develop the next generation of immune-competent patient-derived in vitro models.

Localized Delivery of Pilocarpine to Hypofunctional Salivary Glands through Electrospun Nanofiber Mats: An Ex Vivo and In Vivo Study.

Dry mouth or xerostomia is a frequent medical condition among the polymedicated elderly population. Systemic pilocarpine is included in the first line of pharmacological therapies for xerostomia. However, the efficacy of existing pilocarpine formulations is limited due to its adverse side effects and multiple daily dosages. To overcome these drawbacks, a localized formulation of pilocarpine targeting the salivary glands (SG) was developed in the current study. The proposed formulation consisted of pilocarpine-loaded Poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) nanofiber mats via an electrospinning technique. The nanofiber mats were fully characterized for their size, mesh porosity, drug encapsulation efficiency, and in vitro drug release. Mat biocompatibility and efficacy was evaluated in the SG organ ex vivo, and the expression of proliferation and pro-apoptotic markers at the cellular level was determined. In vivo short-term studies were performed to evaluate the saliva secretion after acute SG treatment with pilocarpine-loaded nanofiber mats, and after systemic pilocarpine for comparison purposes. The outcomes demonstrated that the pilocarpine-loaded mats were uniformly distributed (diameter: 384 ± 124 nm) in a highly porous mesh, and possessed a high encapsulation efficiency (~81%). Drug release studies showed an initial pilocarpine release of 26% (4.5 h), followed by a gradual increase (~46%) over 15 d. Pilocarpine-loaded nanofiber mats supported SG growth with negligible cytotoxicity and normal cellular proliferation and homeostasis. Salivary secretion was significantly increased 4.5 h after intradermal SG treatment with drug-loaded nanofibers in vivo. Overall, this study highlights the strengths of PLGA/PEG nanofiber mats for the localized daily delivery of pilocarpine and reveals its potential for future clinical translation in patients with xerostomia.

A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids.

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non-human-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7 days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E-cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (ß3-tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.

Engineering innervated secretory epithelial organoids by magnetic three-dimensional bioprinting for stimulating epithelial growth in salivary glands.

Current saliva-based stimulation therapies for radiotherapy-induced xerostomia are not fully effective due to the presence of damaged secretory epithelia and nerves in the salivary gland (SG). Hence, three-dimensional bio-engineered organoids are essential to regenerate the damaged SG. Herein, a recently validated three-dimensional (3D) biofabrication system, the magnetic 3D bioprinting (M3DB), is tested to generate innervated secretory epithelial organoids from a neural crest-derived mesenchymal stem cell, the human dental pulp stem cell (hDPSC). Cells are tagged with magnetic nanoparticles (MNP) and spatially arranged with magnet dots to generate 3D spheroids. Next, a SG epithelial differentiation stage was completed with fibroblast growth factor 10 (4-400 ng/ml) to recapitulate SG epithelial morphogenesis and neurogenesis. The SG organoids were then transplanted into ex vivo model to evaluate their epithelial growth and innervation. M3DB-formed spheroids exhibited both high cell viability rate (>90%) and stable ATP intracellular activity compared to MNP-free spheroids. After differentiation, spheroids expressed SG epithelial compartments including secretory epithelial, ductal, myoepithelial, and neuronal. Fabricated organoids also produced salivary α-amylase upon FGF10 stimulation, and intracellular calcium mobilization and trans-epithelial resistance was elicited upon neurostimulation with different neurotransmitters. After transplantation, the SG-like organoids significantly stimulated epithelial and neuronal growth in damaged SG. It is the first time bio-functional innervated SG-like organoids are bioprinted. Thus, this is an important step towards SG regeneration and the treatment of radiotherapy-induced xerostomia.

Concise Review: Salivary Gland Regeneration: Therapeutic Approaches from Stem Cells to Tissue Organoids.

The human salivary gland (SG) has an elegant architecture of epithelial acini, connecting ductal branching structures, vascular and neuronal networks that together function to produce and secrete saliva. This review focuses on the translation of cell- and tissue-based research toward therapies for patients suffering from SG hypofunction and related dry mouth syndrome (xerostomia), as a consequence of radiation therapy or systemic disease. We will broadly review the recent literature and discuss the clinical prospects of stem/progenitor cell and tissue-based therapies for SG repair and/or regeneration. Thus far, several strategies have been proposed for the purpose of restoring SG function: (1) transplanting autologous SG-derived epithelial stem/progenitor cells; (2) exploiting non-epithelial cells and/or their bioactive lysates; and (3) tissue engineering approaches using 3D (three-dimensional) biomaterials loaded with SG cells and/or bioactive cues to mimic in vivo SGs. We predict that further scientific improvement in each of these areas will translate to effective therapies toward the repair of damaged glands and the development of miniature SG organoids for the fundamental restoration of saliva secretion. Stem Cells 2017;35:97-105.

Three-Dimensional Bioprinting Nanotechnologies towards Clinical Application of Stem Cells and Their Secretome in Salivary Gland Regeneration.

Salivary gland (SG) functional damage and severe dry mouth (or xerostomia) are commonly observed in a wide range of medical conditions from autoimmune to metabolic disorders as well as after radiotherapy to treat specific head and neck cancers. No effective therapy has been developed to completely restore the SG functional damage on the long-term and reverse the poor quality of life of xerostomia patients. Cell- and secretome-based strategies are currently being tested in vitro and in vivo for the repair and/or regeneration of the damaged SG using (1) epithelial SG stem/progenitor cells from salispheres or explant cultures as well as (2) nonepithelial stem cell types and/or their bioactive secretome. These strategies will be the focus of our review. Herein, innovative 3D bioprinting nanotechnologies for the generation of organotypic cultures and SG organoids/mini-glands will also be discussed. These bioprinting technologies will allow researchers to analyze the secretome components and extracellular matrix production, as well as their biofunctional effects in 3D mini-glands ex vivo. Improving our understanding of the SG secretome is critical to develop effective secretome-based therapies towards the regeneration and/or repair of all SG compartments for proper restoration of saliva secretion and flow into the oral cavity.