A Novel Hydrogel-Based 3D In Vitro Tumor Panel of 30 PDX Models Incorporates Tumor, Stromal and Immune Cell Compartments of the TME for the Screening of Oncology and Immuno-Therapies.

Human-relevant systems that mimic the 3D tumor microenvironment (TME), particularly the complex mechanisms of immuno-modulation in the tumor stroma, in a reproducible and scalable format are of high interest for the drug discovery industry. Here, we describe a novel 3D in vitro tumor panel comprising 30 distinct PDX models covering a range of histotypes and molecular subtypes and cocultured with fibroblasts and PBMCs in planar (flat) extracellular matrix hydrogels to reflect the three compartments of the TME-tumor, stroma, and immune cells. The panel was constructed in a 96-well plate format and assayed tumor size, tumor killing, and T-cell infiltration using high-content image analysis after 4 days of treatment. We screened the panel first against the chemotherapy drug Cisplatin to demonstrate feasibility and robustness, and subsequently assayed immuno-oncology agents Solitomab (CD3/EpCAM bispecific T-cell engager) and the immune checkpoint inhibitors (ICIs) Atezolizumab (anti-PDL1), Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA4). Solitomab displayed a strong response across many PDX models in terms of tumor reduction and killing, allowing for its subsequent use as a positive control for ICIs. Interestingly, Atezolizumab and Nivolumab demonstrated a mild response compared to Ipilimumab in a subset of models from the panel. We later determined that PBMC spatial proximity in the assay setup was important for the PD1 inhibitor, hypothesizing that both duration and concentration of antigen exposure may be critical. The described 30-model panel represents a significant advancement toward screening in vitro models of the tumor microenvironment that include tumor, fibroblast, and immune cell populations in an extracellular matrix hydrogel, with robust and standardized high content image analysis in a planar hydrogel. The platform is aimed at rapidly screening various combinations and novel agents and forming a critical conduit to the clinic, thus accelerating drug discovery for the next generation of therapeutics.

How to build a tumor: An industry perspective.

During the past 15 years, a plethora of innovative 3D in vitro systems has been developed. They offer the possibility of identifying crucial cellular and molecular contributors to the disease by permitting manipulation of each in isolation. However, improvements are needed particularly with respect to the predictivity and validity of those models. The major challenge now is to identify which assay and readout combination(s) best suits the current scientific question(s). A deep understanding of the different platforms along with their pros and cons is a prerequisite to make this decision. This review aims to give an overview of the most prominent systems with a focus on applications, translational relevance and adoption drivers from an industry perspective.

A Simple Three-dimensional Hydrogel Platform Enables Ex Vivo Cell Culture of Patient and PDX Tumors for Assaying Their Response to Clinically Relevant Therapies.

A cell culture platform that enables ex vivo tissue growth from patients or patient-derived xenograft (PDX) models and assesses sensitivity to approved therapies (e.g., temozolomide) in a clinically relevant time frame would be very useful in translational research and personalized medicine. Here, we present a novel three-dimensional (3D) ECM hydrogel system, VersaGel, for assaying ex vivo growth and therapeutic response with standard image microscopy. Specifically, multicellular spheroids deriving from either 5 patients with glioblastoma (GBM) or a renal cell carcinoma (RCC) PDX model were incorporated into VersaGel and treated with temozolomide and several other therapies, guided by the most recent advances in GBM treatment. RCC ex vivo tissue displayed invasive phenotypes in conditioned media. For the GBM patient tumor testing, all five clinical responses were predicted by the results of our 3D-temozolomide assay. In contrast, the MTT assay found no response to temozolomide regardless of the clinical outcome, and moreover, basement membrane extract failed to predict the 2 patient responders. Finally, 1 patient was tested with repurposed drugs currently being administered in GBM clinical trials. Interestingly, IC50s were lower than C max for crizotinib and chloroquine, but higher for sorafenib. In conclusion, a novel hydrogel platform, VersaGel, enables ex vivo tumor growth of patient and PDX tissue and offers insight into patient response to clinically relevant therapies. We propose a novel 3D hydrogel platform, VersaGel, to grow ex vivo tissue (patient and PDX) and assay therapeutic response using time-course image analysis.

Three-dimensional direct cell patterning in collagen hydrogels with near-infrared femtosecond laser.

We report a methodology for three-dimensional (3D) cell patterning in a hydrogel in situ. Gold nanorods within a cell-encapsulating collagen hydrogel absorb a focused near-infrared femtosecond laser beam, locally denaturing the collagen and forming channels, into which cells migrate, proliferate, and align in 3D. Importantly, pattern resolution is tunable based on writing speed and laser power, and high cell viability (>90%) is achieved using higher writing speeds and lower laser intensities. Overall, this patterning technique presents a flexible direct-write method that is applicable in tissue engineering systems where 3D alignment is critical (such as vascular, neural, cardiac, and muscle tissue).

Interplay of matrix stiffness and protein tethering in stem cell differentiation.

Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.

Light-assisted direct-write of 3D functional biomaterials.

Light-assisted 3D direct-printing of biomaterials and cellular-scaffolds has the potential to develop novel lab-on-a-chip devices (LOCs) for a variety of biomedical applications, from drug discovery and diagnostic testing to in vitro tissue engineering and regeneration. Direct-writing describes a broad family of fabrication methods that typically employ computer-controlled translational stages to manufacture structures at multi-length scales. This review focuses on light-assisted direct-write fabrication for generating 3D functional scaffolds with precise micro- and nano-architecture, using both synthetic as well as naturally derived biomaterials. Two bioprinting approaches are discussed in detail - projection printing and laser-based systems - where each method is capable of modulating multiple scaffold parameters, such as 3D architecture, mechanical properties (e.g. stiffness), Poisson's ratio, chemical gradients, biological cell distributions, and porosity. The light-assisted direct-writing techniques described in this review provide the reader with alternative approaches to fabricate 3D biomaterials for utility in LOCs.

Digital Plasmonic Patterning for Localized Tuning of Hydrogel Stiffness.

The mechanical properties of the extracellular matrix (ECM) can dictate cell fate in biological systems. In tissue engineering, varying the stiffness of hydrogels-water-swollen polymeric networks that act as ECM substrates-has previously been demonstrated to control cell migration, proliferation, and differentiation. Here, "digital plasmonic patterning" (DPP) is developed to mechanically alter a hydrogel encapsulated with gold nanorods using a near-infrared laser, according to a digital (computer-generated) pattern. DPP can provide orders of magnitude changes in stiffness, and can be tuned by laser intensity and speed of writing. In vitro cellular experiments using A7R5 smooth muscle cells confirm cell migration and alignment according to these patterns, making DPP a useful technique for mechanically patterning hydrogels for various biomedical applications.

Rapid fabrication of complex 3D extracellular microenvironments by dynamic optical projection stereolithography.

The topographic features of the extracelluar matrix (ECM) lay the foundation for cellular behavior. A novel biofabrication method using a digital-mirror device (DMD), called dynamic optical projection stereolithography (DOPsL) is demonstrated. This robust and versatile platform can generate complex biomimetic scaffolds within seconds. Such 3D scaffolds have promising potentials for studying cell interactions with microenvironments in vitro and in vivo.

Nanofiber-nanorod composites exhibiting light-induced reversible lower critical solution temperature transitions.

Stimuli-responsive materials are promising as smart materials for a range of applications. In this work, a photo-crosslinkable, thermoresponsive macromer was electrospun into fibrous scaffolds containing gold nanorods (AuNRs). The resulting fibrous nanocomposites composed of poly(N-isopropylacrylamide-co-polyethylene glycol acrylate) (PNPA) and PEGylated AuNRs were crosslinked and swollen in water. AuNRs strongly absorb in the near-infrared (NIR) region to generate heat, which triggered the fiber thermal transition upon NIR light exposure. During the thermal transition, scaffolds collapsed both macroscopically and microscopically, with individual fibers deswelling and pulling together. Exposure to a 1.1 W NIR laser decreased the diameter of swollen fibers by 34.7% from 1332 ± 193.3 to 868.9 ± 168.3 nm, and increased fiber density 116% from 209.5 ± 26.34 to 451.9 ± 23.68 fibers mm( - 1). This transition was dependent on the incorporation of the AuNRs, and was utilized to trigger the release of encapsulated proteins from the nanocomposite fiber mats. The expulsion of water from fibers upon NIR exposure caused the release rate of incorporated protein to increase greater than tenfold, from 0.038 ± 0.052 without external stimulus to 0.462 ± 0.227 µg protein/mg polymer/min with NIR exposure. These results suggest that light-responsive fibrous nanocomposites can be utilized in applications such as drug delivery.

Enhanced release of small molecules from near-infrared light responsive polymer-nanorod composites.

Stimuli-responsive materials undergo structural changes in response to an external trigger (i.e., pH, heat, or light). This process has been previously used for a range of applications in biomedicine and microdevices and has recently gained considerable attention in controlled drug release. Here, we use a near-infrared (NIR) light responsive polymer-nanorod composite whose glass transition temperature (T(g)) is in the range of body temperature to control and enhance the release of a small-molecule drug (<800 Da). In addition to increased temperature and resulting changes in molecule diffusion, the photothermal effect (conversion of NIR light to heat) adjusts the composite above the T(g). Specifically, at normal body temperature (T < T(g)), the structure is glassy and release is limited, whereas when T > T(g), the polymer is rubbery and release is enhanced. We applied this heating system to trigger release of the chemotherapeutic drug doxorubicin from both polymer films and microspheres. Multiple cycles of NIR exposure were performed and demonstrated a triggered and stepwise release behavior. Lastly, we tested the microsphere system in vitro, reporting a ∼90% reduction in the activity of T6-17 cells when the release of doxorubicin was triggered from microspheres exposed to NIR light. This overall approach can be used with numerous polymer systems to modulate molecule release toward the development of unique and clinically applicable therapies.

Novel nano-composite biomaterials that respond to light.

Composites of nanoparticles and polymers are finding wide applications to alter material properties, conductivity, and utility. Here, we show that nano-composites can be designed to heat in the presence of near infrared light. This process is useful in transitioning materials through a transition temperature for a range of applications. For example, shape-memory materials (including polymers, metals, and ceramics) are those that are processed into a temporary shape and respond to some external stimuli (e.g., temperature) to undergo a transition back to a permanent shape and may be useful in a range of applications from aerospace to fabrics, to biomedical devices and microsystem components. In this work, we formulated composites of gold nanorods (<1% by volume) and biodegradable networks, where exposure to infrared light induced heating and consequently, shape transitions. The heating is repeatable and tunable based on nanorod concentration and light intensity.