Nondestructive, quantitative viability analysis of 3D tissue cultures using machine learning image segmentation.

Ascertaining the collective viability of cells in different cell culture conditions has typically relied on averaging colorimetric indicators and is often reported out in simple binary readouts. Recent research has combined viability assessment techniques with image-based deep-learning models to automate the characterization of cellular properties. However, further development of viability measurements to assess the continuity of possible cellular states and responses to perturbation across cell culture conditions is needed. In this work, we demonstrate an image processing algorithm for quantifying features associated with cellular viability in 3D cultures without the need for assay-based indicators. We show that our algorithm performs similarly to a pair of human experts in whole-well images over a range of days and culture matrix compositions. To demonstrate potential utility, we perform a longitudinal study investigating the impact of a known therapeutic on pancreatic cancer spheroids. Using images taken with a high content imaging system, the algorithm successfully tracks viability at the individual spheroid and whole-well level. The method we propose reduces analysis time by 97% in comparison with the experts. Because the method is independent of the microscope or imaging system used, this approach lays the foundation for accelerating progress in and for improving the robustness and reproducibility of 3D culture analysis across biological and clinical research.

Glioblastoma Spheroid Invasion through Soft, Brain-Like Matrices Depends on Hyaluronic Acid-CD44 Interactions.

Increased secretion of hyaluronic acid (HA), a glycosaminoglycan abundant in the brain extracellular matrix (ECM), correlates with worse clinical outcomes for glioblastoma (GBM) patients. GBM cells aggressively invade the brain parenchyma while encountering spatiotemporal changes in their local ECM, including HA concentration. To investigate how varying HA concentrations affect GBM invasion, patient-derived GBM cells are cultured within a soft, 3D matrix in which HA concentration is precisely varied and cell migration observed. Data demonstrate that HA concentration can determine the invasive activity of patient-derived GBM cells in a biphasic and highly sensitive manner, where the absolute concentration of HA at which cell migration peaked is specific to each patient-derived line. Furthermore, evidence that this response relies on phosphorylated ezrin, which interacts with the intracellular domain of HA-engaged CD44 to effectively link the actin cytoskeleton to the local ECM is provided. Overall, this study highlights CD44-HA binding as a major mediator of GBM cell migration that acts independently of integrins and focal adhesion complexes and suggests that targeting HA-CD44-ezrin interactions represents a promising therapeutic strategy to prevent tumor cell invasion in the brain.

Cancer-Associated Fibroblasts Confer Gemcitabine Resistance to Pancreatic Cancer Cells through PTEN-Targeting miRNAs in Exosomes.

Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related death in the United States. Even though the poor prognosis of PDAC is often attributed to late diagnosis, patients with an early diagnosis who undergo tumor resection and adjuvant chemotherapy still show tumor recurrence, highlighting a need to develop therapies which can overcome chemoresistance. Chemoresistance has been linked to the high expression of microRNAs (miRs), such as miR-21, within tumor cells. Tumor cells can collect miRs through the uptake of miR-containing lipid extracellular vesicles called exosomes. These exosomes are secreted in high numbers from cancer-associated fibroblasts (CAFs) within the tumor microenvironment during gemcitabine treatment and can contribute to cell proliferation and chemoresistance. Here, we show a novel mechanism in which CAF-derived exosomes may promote proliferation and chemoresistance, in part, through suppression of the tumor suppressor PTEN. We identified five microRNAs: miR-21, miR-181a, miR-221, miR-222, and miR-92a, that significantly increased in number within the CAF exosomes secreted during gemcitabine treatment which target PTEN. Furthermore, we found that CAF exosomes suppressed PTEN expression in vitro and that treatment with the exosome inhibitor GW4869 blocked PTEN suppression in vivo. Collectively, these findings highlight a mechanism through which the PTEN expression loss, often seen in PDAC, may be attained and lend support to investigations into the use of exosome inhibitors as potential therapeutics to improve the effectiveness of chemotherapy.

Matrix stiffness mediates pancreatic cancer chemoresistance through induction of exosome hypersecretion in a cancer associated fibroblasts-tumor organoid biomimetic model.

In pancreatic ductal adenocarcinoma (PDAC), the abundant stromal cells which comprise the tumor microenvironment constitute more than 90% of the primary tumor bulk. Moreover, this desmoplastic environment has been found to be three times stiffer than normal pancreas tissue. Despite the importance of studying the desmoplastic environment of PDAC, there is still a lack of models designed to adequately recapitulate this complex stiff microenvironment, a critical hallmark of the disease that has been shown to induce chemoresistance. Here, we present a bio-mimetic, 3-dimensional co-culture system that integrates tumor organoids and host-matching stromal cancer associated-fibroblasts (CAFs) that recapitulates the complex, fibrotic matrix of PDAC using advanced biomaterials. With this model, we show that matrix-activated CAFs are able to "re-engineer" the fibrotic environment into a significantly stiffer environment through lysyl-oxidase dependent crosslinking. Moreover, we show that culture of CAFs in this model leads to an increase of exosomes; extracellular vesicles known to promote chemoresistance. Finally, using previously identified exosome inhibitors, climbazole and imipramine, we demonstrate how abrogation of exosome hypersecretion can reduce matrix stiffness-induced chemoresistance. These data highlight the importance of the development of new models that recapitulate not only the cellular composition found in PDAC tumors, but also the biophysical stresses, like stiffness, that the cells are exposed to in order to identify therapies that can overcome this critical feature which can contribute to the chemoresistance observed in patients. We believe that the 3D bio-mimetic model we have developed will be a valuable tool for the development, testing, and optimization of "mechano-medicines" designed to target the biophysical forces that lead to tumor growth and chemoresistance.

Extracellular Matrix Proteins Confer Cell Adhesion-Mediated Drug Resistance Through Integrin α v in Glioblastoma Cells.

Chemotherapy resistance to glioblastoma (GBM) remains an obstacle that is difficult to overcome, leading to poor prognosis of GBM patients. Many previous studies have focused on resistance mechanisms intrinsic to cancer cells; the microenvironment surrounding tumor cells has been found more recently to have significant impacts on the response to chemotherapeutic agents. Extracellular matrix (ECM) proteins may confer cell adhesion-mediated drug resistance (CAMDR). Here, expression of the ECM proteins laminin, vitronectin, and fibronectin was assessed in clinical GBM tumors using immunohistochemistry. Then, patient-derived GBM cells grown in monolayers on precoated laminin, vitronectin, or fibronectin substrates were treated with cilengitide, an integrin inhibitor, and/or carmustine, an alkylating chemotherapy. Cell adhesion and viability were quantified. Transcription factor (TF) activities were assessed over time using a bioluminescent assay in which GBM cells were transduced with lentiviruses containing consensus binding sites for specific TFs linked to expression a firefly luciferase reporter. Apoptosis, mediated by p53, was analyzed by Western blotting and immunocytofluorescence. Integrin α v activation of the FAK/paxillin/AKT signaling pathway and effects on expression of the proliferative marker Ki67 were investigated. To assess effects of integrin α v activation of AKT and ERK pathways, which are typically deregulated in GBM, and expression of epidermal growth factor receptor (EGFR), which is amplified and/or mutated in many GBM tumors, shRNA knockdown was used. Laminin, vitronectin, and fibronectin were abundant in clinical GBM tumors and promoted CAMDR in GBM cells cultured on precoated substrates. Cilengitide treatment induced cell detachment, which was most pronounced for cells cultured on vitronectin. Cilengitide treatment increased cytotoxicity of carmustine, reversing CAMDR. ECM adhesion increased activity of NFκB and decreased that of p53, leading to suppression of p53-mediated apoptosis and upregulation of multidrug resistance gene 1 (MDR1; also known as ABCB1 or P-glycoprotein). Expression of Ki67 was correlative with activation of the integrin α v -mediated FAK/paxillin/AKT signaling pathway. EGFR expression increased with integrin α v knockdown GBM cells and may represent a compensatory survival mechanism. These results indicate that ECM proteins confer CAMDR through integrin α v in GBM cells.

Injectable, macroporous scaffolds for delivery of therapeutic genes to the injured spinal cord.

Biomaterials are being developed as therapeutics for spinal cord injury (SCI) that can stabilize and bridge acute lesions and mediate the delivery of transgenes, providing a localized and sustained reservoir of regenerative factors. For clinical use, direct injection of biomaterial scaffolds is preferred to enable conformation to unique lesions and minimize tissue damage. While an interconnected network of cell-sized macropores is necessary for rapid host cell infiltration into-and thus integration of host tissue with-implanted scaffolds, injectable biomaterials have generally suffered from a lack of control over the macrostructure. As genetic vectors have short lifetimes in vivo, rapid host cell infiltration into scaffolds is a prerequisite for efficient biomaterial-mediated delivery of transgenes. We present scaffolds that can be injected and assembled in situ from hyaluronic acid (HA)-based, spherical microparticles to form scaffolds with a network of macropores (∼10 µm). The results demonstrate that addition of regularly sized macropores to traditional hydrogel scaffolds, which have nanopores (∼10 nm), significantly increases the expression of locally delivered transgene to the spinal cord after a thoracic injury. Maximal cell and axon infiltration into scaffolds was observed in scaffolds with more regularly sized macropores. The delivery of lentiviral vectors encoding the brain-derived neurotrophic factor (BDNF), but not neurotrophin-3, from these scaffolds further increased total numbers and myelination of infiltrating axons. Modest improvements to the hindlimb function were observed with BDNF delivery. The results demonstrate the utility of macroporous and injectable HA scaffolds as a platform for localized gene therapies after SCI.

Bioengineered scaffolds for 3D culture demonstrate extracellular matrix-mediated mechanisms of chemotherapy resistance in glioblastoma.

Originating in the brain, glioblastoma (GBM) is a highly lethal and virtually incurable cancer, in large part because it readily develops resistance to treatments. While numerous studies have investigated mechanisms enabling GBM cells to evade chemotherapy-induced apoptosis, few have addressed how their surrounding extracellular matrix (ECM) acts to promote their survival. Here, we employed a biomaterial-based, 3D culture platform to investigate systematically how interactions between patient-derived GBM cells and the brain ECM promote resistance to alkylating chemotherapies - including temozolomide, which is used routinely in clinical practice. Scaffolds for 3D culture were fabricated from hyaluronic acid (HA) - a major structural and bioactive component of the brain ECM - and functionalized with the RGD (arginine-glycine-aspartic acid) tripeptide to provide sites for integrin engagement. Data demonstrate that cooperative engagement of CD44, through HA, and integrin αV, through RGD, facilitates resistance to alkylating chemotherapies through co-activation of Src, which inhibited downstream expression of BCL-2 family pro-apoptotic factors. In sum, a bioengineered, 3D culture platform was used to gain new mechanistic insights into how ECM in the brain tumor microenvironment promotes resistance to chemotherapy and suggests potential avenues for the development of novel, matrix-targeted combination therapies designed to suppress chemotherapy resistance in GBM.

Injectable, Hyaluronic Acid-Based Scaffolds with Macroporous Architecture for Gene Delivery.

INTRODUCTION: Biomaterials can provide localized reservoirs for controlled release of therapeutic biomolecules and drugs for applications in tissue engineering and regenerative medicine. As carriers of gene-based therapies, biomaterial scaffolds can improve efficiency and delivery-site localization of transgene expression. Controlled delivery of gene therapy vectors from scaffolds requires cell-scale macropores to facilitate rapid host cell infiltration. Recently, advanced methods have been developed to form injectable scaffolds containing cell-scale macropores. However, relative efficacy of in vivo gene delivery from scaffolds formulated using these general approaches has not been previously investigated. Using two of these methods, we fabricated scaffolds based on hyaluronic acid (HA) and compared how their unique, macroporous architectures affected their respective abilities to deliver transgenes via lentiviral vectors in vivo. METHODS: Three types of scaffolds-nanoporous HA hydrogels (NP-HA), annealed HA microparticles (HA-MP) and nanoporous HA hydrogels containing protease-degradable poly(ethylene glycol) (PEG) microparticles as sacrificial porogens (PEG-MP)-were loaded with lentiviral particles encoding reporter transgenes and injected into mouse mammary fat. Scaffolds were evaluated for their ability to induce rapid infiltration of host cells and subsequent transgene expression. RESULTS: Cell densities in scaffolds, distances into which cells penetrated scaffolds, and transgene expression levels significantly increased with delivery from HA-MP, compared to NP-HA and PEG-MP, scaffolds. Nearly 8-fold greater cell densities and up to 16-fold greater transgene expression levels were found in HA-MP, over NP-HA, scaffolds. Cell profiling revealed that within HA-MP scaffolds, macrophages (F4/80+), fibroblasts (ERTR7+) and endothelial cells (CD31+) were each present and expressed delivered transgene. CONCLUSIONS: Results demonstrate that injectable scaffolds containing cell-scale macropores in an open, interconnected architecture support rapid host cell infiltration to improve efficiency of biomaterial-mediated gene delivery.

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

In situ coherent diffractive imaging.

Coherent diffractive imaging (CDI) has been widely applied in the physical and biological sciences using synchrotron radiation, X-ray free-electron laser, high harmonic generation, electrons, and optical lasers. One of CDI's important applications is to probe dynamic phenomena with high spatiotemporal resolution. Here, we report the development of a general in situ CDI method for real-time imaging of dynamic processes in solution. By introducing a time-invariant overlapping region as real-space constraint, we simultaneously reconstructed a time series of complex exit wave of dynamic processes with robust and fast convergence. We validated this method using optical laser experiments and numerical simulations with coherent X-rays. Our numerical simulations further indicated that in situ CDI can potentially reduce radiation dose by more than an order of magnitude relative to conventional CDI. With further development, we envision in situ CDI could be applied to probe a range of dynamic phenomena in the future.

Brain-Mimetic 3D Culture Platforms Allow Investigation of Cooperative Effects of Extracellular Matrix Features on Therapeutic Resistance in Glioblastoma.

Glioblastoma (GBM) tumors exhibit potentially actionable genetic alterations against which targeted therapies have been effective in treatment of other cancers. However, these therapies have largely failed in GBM patients. A notable example is kinase inhibitors of EGFR, which display poor clinical efficacy despite overexpression and/or mutation of EGFR in >50% of GBM. In addressing this issue, preclinical models may be limited by the inability to accurately replicate pathophysiologic interactions of GBM cells with unique aspects of the brain extracellular matrix (ECM), which is relatively enriched in hyaluronic acid (HA) and flexible. In this study, we present a brain-mimetic biomaterial ECM platform for 3D culturing of patient-derived GBM cells, with improved pathophysiologic properties as an experimental model. Compared with orthotopic xenograft assays, the novel biomaterial cultures we developed better preserved the physiology and kinetics of acquired resistance to the EGFR inhibition than gliomasphere cultures. Orthogonal modulation of both HA content and mechanical properties of biomaterial scaffolds was required to achieve this result. Overall, our findings show how specific interactions between GBM cell receptors and scaffold components contribute significantly to resistance to the cytotoxic effects of EGFR inhibition.Significance: Three-dimensional culture scaffolds of glioblastoma provide a better physiological representation over current methods of patient-derived cell culture and xenograft models. Cancer Res; 78(5); 1358-70. ©2017 AACR.

Integrating the glioblastoma microenvironment into engineered experimental models.

Glioblastoma (GBM) is the most lethal cancer originating in the brain. Its high mortality rate has been attributed to therapeutic resistance and rapid, diffuse invasion - both of which are strongly influenced by the unique microenvironment. Thus, there is a need to develop new models that mimic individual microenvironmental features and are able to provide clinically relevant data. Current understanding of the effects of the microenvironment on GBM progression, established experimental models of GBM and recent developments using bioengineered microenvironments as ex vivo experimental platforms that mimic the biochemical and physical properties of GBM tumors are discussed.

G-quadruplex conformation and dynamics are determined by loop length and sequence.

The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components) length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, N-methyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and how the GQ conformation determines the small molecule and protein binding propensity.