Optical coherence tomography for multicellular tumor spheroid category recognition and drug screening classification via multi-spatial-superficial-parameter and machine learning.

Optical coherence tomography (OCT) is an ideal imaging technique for noninvasive and longitudinal monitoring of multicellular tumor spheroids (MCTS). However, the internal structure features within MCTS from OCT images are still not fully utilized. In this study, we developed cross-statistical, cross-screening, and composite-hyperparameter feature processing methods in conjunction with 12 machine learning models to assess changes within the MCTS internal structure. Our results indicated that the effective features combined with supervised learning models successfully classify OVCAR-8 MCTS culturing with 5,000 and 50,000 cell numbers, MCTS with pancreatic tumor cells (Panc02-H7) culturing with the ratio of 0%, 33%, 50%, and 67% of fibroblasts, and OVCAR-4 MCTS treated by 2-methoxyestradiol, AZD1208, and R-ketorolac with concentrations of 1, 10, and 25 µM. This approach holds promise for obtaining multi-dimensional physiological and functional evaluations for using OCT and MCTS in anticancer studies.

Maximum-a-posteriori Estimation of Retinal Oxygen Tension Based on Photon Counting Statistics.

The regularization of retinal oxygen tension estimation was previously proposed with an assumption that phosphorescence intensity images are corrupted by additive Gaussian noise. Based on this assumption, a regularized least-squares estimate has been shown to be better than a conventional least-squares estimation. However, this assumption is inconsistent with the acquisition process of phosphorescence intensity images acquired using an intensified charge-coupled device camera. Almost the entire acquisition process is governed by the natural aspects of photons. Therefore, a method based on photon counting statistics is more appropriate. In this study, we propose a regularized oxygen tension estimation method based on photon counting statistics and a phosphorescence lifetime imaging model.

Peptide aggregation-induced immunogenic cell death in a breast cancer spheroid model.

Utilizing multicellular aggregates (spheroids) for in vitro cancer research offers a physiologically relevant model that closely mirrors the intricate tumor microenvironment, capturing properties of solid tumors such as cell interactions and drug resistance. In this research, we investigated the Peptide-Aggregation Induced Immunogenic Response (PAIIR), an innovative method employing engineered peptides we designed specifically to induce immunogenic cell death (ICD). We contrasted PAIIR-induced ICD with standard ICD and non-ICD inducer chemotherapeutics within the context of three-dimensional breast cancer tumor spheroids. Our findings reveal that PAIIR outperforms traditional chemotherapeutics in its efficacy to stimulate ICD. This is marked by the release of key damage-associated molecular patterns (DAMPs), which bolster the phagocytic clearance of dying cancer cells by dendritic cells (DCs) and, in turn, activate powerful anti-tumor immune responses. Additionally, we observed that PAIIR results in elevated dendritic cell activation and increased antitumor cytokine presence. This study not only showcases the utility of tumor spheroids for efficient high-throughput screening but also emphasizes PAIIR's potential as a formidable immunotherapeutic strategy against breast cancer, setting the stage for deeper exploration and potential clinical implementation.

Peptide Aggregation Induced Immunogenic Rupture (PAIIR).

Immunogenic cell death (ICD) arises when cells are under stress, and their membranes are damaged. They release damage-associated molecular patterns (DAMPs) that stimulate and drive the type and magnitude of the immune response. In the presence of an antigen, DAMPs ride the longevity and efficacy of antigen-specific immunity. Yet, no tool can induce the controlled ICD with predictable results. A peptide-based tool, [II], is designed that aggregates in the cell and causes cell membrane damage, generates ICD and DAMPs release on various cell types, and hence can act as an adjuvant. An influenza vaccine is prepared by combining [II] with influenza hemagglutinin (HA) subunit antigens. The results show that [II] induced significantly higher HA-specific immunoglobulin G1 (IgG1) and IgG2a antibodies than HA-only immunized mice, while the peptide itself did not elicit antibodies. This paper demonstrates the first peptide-aggregation induced immunogenic rupture (PAIIR) approach as a vaccine adjuvant. PAIIR is a promising adjuvant with a high potential to promote universal protection upon influenza HA vaccination.

Controllable membrane damage by tunable peptide aggregation with albumin.

Aggregation of otherwise soluble proteins into amyloid structures is a hallmark of many disorders, such as Alzheimer's and Parkinson's disease. There is an increasing evidence that the small aggregations, instead of ordered fibrillar aggregates, are the main structures causing toxicity. However, the studies on the small aggregation phase are limited due to the variety of structures and the complexity of the physiological environment. Here, we showed an engineered co-assembling oppositely charged amyloid-like peptide pair ([II]) as a simple tool to establish methodologies to study the mechanism and kinetics of aggregation and relate its aggregation to toxicity. The toxicity mechanism of [II] is through cell membrane damage and stress, shown with YAP and eIF2α, as in the amyloid protein-initiated diseases. Albumin is demonstrated as an extrinsic and physiologically relevant molecule in controlling the aggregation lag time and toxicity of [II]. This study represents a molecular engineering strategy to create simplistic molecular tools for establishing methodologies to study the aggregation process and kinetics of amyloid-like proteins in various conditions. Understanding the nature of protein aggregation kinetics and linking them to their biological functions through engineered peptides paves the way for future designs and drug development applications.

Synthesis of Niobium-Alumina Composite Aggregates and Their Application in Coarse-Grained Refractory Ceramic-Metal Castables.

Niobium-alumina aggregate fractions with particle sizes up to 3150 µm were produced by crushing pre-synthesised fine-grained composites. Phase separation with niobium enrichment in the aggregate class 45-500 µm was revealed by XRD/Rietveld analysis. To reduce the amount of carbon-based impurities, no organic additives were used for the castable mixtures, which resulted in water demands of approximately 27 vol.% for the fine- and coarse-grained castables. As a consequence, open porosities of 18% and 30% were determined for the fine- and coarse-grained composites, respectively. Due to increased porosity, the modulus of rupture at room temperature decreased from 52 MPa for the fine-grained composite to 11 MPa for the coarse-grained one. However, even the compressive yield strength decreased from 49 MPa to 18 MPa at 1300 °C for the fine-grained to the coarse-grained composite, the latter showed still plasticity with a strain up to 5%. The electrical conductivity of fine-grained composite samples was in the range between 40 and 60 S/cm, which is fifteen magnitudes above the values of pure corundum.

Characterization and quantification of necrotic tissues and morphology in multicellular ovarian cancer tumor spheroids using optical coherence tomography.

The three-dimensional (3D) tumor spheroid model is a critical tool for high-throughput ovarian cancer research and anticancer drug development in vitro. However, the 3D structure prevents high-resolution imaging of the inner side of the spheroids. We aim to visualize and characterize 3D morphological and physiological information of the contact multicellular ovarian tumor spheroids growing over time. We intend to further evaluate the distinctive evolutions of the tumor spheroid and necrotic tissue volumes in different cell numbers and determine the most appropriate mathematical model for fitting the growth of tumor spheroids and necrotic tissues. A label-free and noninvasive swept-source optical coherence tomography (SS-OCT) imaging platform was applied to obtain two-dimensional (2D) and 3D morphologies of ovarian tumor spheroids over 18 days. Ovarian tumor spheroids of two different initial cell numbers (5,000- and 50,000- cells) were cultured and imaged (each day) over the time of growth in 18 days. Four mathematical models (Exponential-Linear, Gompertz, logistic, and Boltzmann) were employed to describe the growth kinetics of the tumor spheroids volume and necrotic tissues. Ovarian tumor spheroids have different growth curves with different initial cell numbers and their growths contain different stages with various growth rates over 18 days. The volumes of 50,000-cells spheroids and the corresponding necrotic tissues are larger than that of the 5,000-cells spheroids. The formation of necrotic tissue in 5,000-cells numbers is slower than that in the 50,000-cells ones. Moreover, the Boltzmann model exhibits the best fitting performance for the growth of tumor spheroids and necrotic tissues. Optical coherence tomography (OCT) can serve as a promising imaging modality to visualize and characterize morphological and physiological features of multicellular ovarian tumor spheroids. The Boltzmann model integrating with 3D OCT data of ovarian tumor spheroids provides great potential for high-throughput cancer research in vitro and aiding in drug development.

Small Molecule Targeting of Oxysterol-Binding Protein (OSBP)-Related Protein 4 and OSBP Inhibits Ovarian Cancer Cell Proliferation in Monolayer and Spheroid Cell Models.

The development of precision drugs for the selective treatment of ovarian cancer will require targeting proliferative factors selectively expressed in ovarian tumors or targeting unique physiological microenvironments specific for ovarian tumors. Here, we report that oxysterol-binding protein (OSBP)-related protein 4 (ORP4) is a potential druggable precision target in ovarian cancer cells. ORP4 has limited expression in normal tissues and was recently recognized to be a cancer-specific driver of cellular proliferation, including in patient-isolated leukemias. We demonstrate that ORP4 is strongly expressed in a panel of ovarian cancer cell lines. The antiproliferative natural product compound OSW-1 targets ORP4 and OSBP. Our results demonstrate that the OSW-1 compound has high antiproliferative potency in both monolayer and three-dimensional ovarian cancer spheroid models, especially compared to the standard-of-care agents cisplatin and paclitaxel. OSW-1 compound treatment induces a loss of ORP4 expression after 48 h, which is coincident with the cytotoxic effects of OSW-1. The absence of extracellular lipids markedly potentiated the cytotoxicity of OSW-1, which was reversed by addition of extracellular free cholesterol. OSBP, but not ORP4, is reported to transport cholesterol and other lipids between organelles. Our results indicate that the targeting of ORP4 is responsible for the antiproliferative activity of the OSW-1 compound, but that in the absence of exogenously supplied cholesterol, which might be similar to the in vivo ovarian cancer microenvironment, possible OSW-1 targeting of OSBP further potentiates the anticancer activity of the compound. Overall, ORP4 and potentially OSBP are revealed as potential druggable targets for the development of novel treatments for ovarian cancer.

Natural and Synthetic Biomaterials for Engineering Multicellular Tumor Spheroids.

The lack of in vitro models that represent the native tumor microenvironment is a significant challenge for cancer research. Two-dimensional (2D) monolayer culture has long been the standard for in vitro cell-based studies. However, differences between 2D culture and the in vivo environment have led to poor translation of cancer research from in vitro to in vivo models, slowing the progress of the field. Recent advances in three-dimensional (3D) culture have improved the ability of in vitro culture to replicate in vivo conditions. Although 3D cultures still cannot achieve the complexity of the in vivo environment, they can still better replicate the cell-cell and cell-matrix interactions of solid tumors. Multicellular tumor spheroids (MCTS) are three-dimensional (3D) clusters of cells with tumor-like features such as oxygen gradients and drug resistance, and represent an important translational tool for cancer research. Accordingly, natural and synthetic polymers, including collagen, hyaluronic acid, Matrigel®, polyethylene glycol (PEG), alginate and chitosan, have been used to form and study MCTS for improved clinical translatability. This review evaluates the current state of biomaterial-based MCTS formation, including advantages and disadvantages of the different biomaterials and their recent applications to the field of cancer research, with a focus on the past five years.

The effects of size and shape of the ovarian cancer spheroids on the drug resistance and migration.

BACKGROUND: High fatality in ovarian cancer is attributed to metastasis, propagated by the release of multi-cellular aggregates/spheroids into the peritoneal cavity and their subsequent mesothelial invasion of peritoneal organs. Spheroids are therefore a common and clinically relevant in vitro model for ovarian cancer research. Spheroids in patients vary significantly in size and shape and display enhanced resistance to anti-cancer drugs compared to monolayers. However, there is no consensus on how spheroid size and shape affect drug resistance. Moreover, existing data regarding the influence of epithelial-to-mesenchymal transition (EMT) profile on spheroid shape and migration is inconclusive. METHODS: We formed spheroids with OVCAR-3 and OVCAR-8 cells, chosen for their established genetic similarity to the patient tumor samples. We monitored their morphology using confocal microscope with dipping objective and fluorescent microscope. We characterized important EMT biomarkers; E-cadherin, Vimentin and Slug through western blotting in monolayers and spheroids. We treated these spheroids with Taxol and Cisplatin and investigated their migratory profile based on their morphology. RESULTS: We report two distinct multicellular structures: loose aggregates (OVCAR-3) and compact spheroids (OVCAR-8). We attribute these different morphologies to the expression of the EMT biomarkers, and their changes upon spheroid formation. Importantly, we did not observe a difference in resistance to the anti-cancer drugs as a function of spheroid size and shape. However, migration capacity of compact spheroid (OVCAR-8) was 15-fold higher compared to that of loose aggregates (OVCAR-3). CONCLUSIONS: These results highlight the importance of spheroid size and shape on anti-cancer drug resistance and migration profiles. The results of this study can, therefore, help to elucidate general rules for ovarian cancer studies based on 3D samples.

Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation.

Neutrophils are the most abundant type of white blood cells. Upon stimulation, they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) is part of immune defense mechanisms but also plays an important role in many chronic and inflammatory diseases such as atherosclerosis, rheumatoid arthritis, diabetes, and cancer. For this reason, much effort has been invested into understanding biochemical signaling pathways in NETosis. However, the impact of the mechanical micro-environment and adhesion on NETosis is not well-understood. Here, we studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Young's modulus E) within the physiologically relevant range from 1 to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Neutrophils were cultured on these substrates and stimulated with potent inducers of NETosis: phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. Furthermore, inhibition of phosphatidylinositide 3 kinase (PI3K), which is involved in adhesion signaling, completely abolished LPS-induced NETosis but only slightly decreased PMA-induced NETosis. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.

Chromatin swelling drives neutrophil extracellular trap release.

Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.

Tenascin-C derived signaling induces neuronal differentiation in a three-dimensional peptide nanofiber gel.

The development of new biomaterials mimicking the neuronal extracellular matrix (ECM) requires signals for the induction of neuronal differentiation and regeneration. In addition to the biological and chemical cues, the physical properties of the ECM should also be considered while designing regenerative materials for nervous tissue. In this study, we investigated the influence of the microenvironment on tenascin-C signaling using 2D surfaces and 3D scaffolds generated by a peptide amphiphile nanofiber gel with a tenascin-C derived peptide epitope (VFDNFVLK). While tenascin-C mimetic PA nanofibers significantly increased the length and number of neurites produced by PC12 cells on 2D cell culture, more extensive neurite outgrowth was observed in the 3D gel environment. PC12 cells encapsulated within the 3D tenascin-C mimetic peptide nanofiber gel also exhibited significantly increased expression of neural markers compared to the cells on 2D surfaces. Our results emphasize the synergistic effects of the 3D conformation of peptide nanofibers along with the tenascin-C signaling and growth factors on the neuronal differentiation of PC12 cells, which may further provide more tissue-like morphology for therapeutic applications.

Three-Dimensional Laminin Mimetic Peptide Nanofiber Gels for In Vitro Neural Differentiation.

The extracellular matrix (ECM) provides biochemical signals and structural support for cells, and its functional imitation is a fundamental aspect of biomaterial design for regenerative medicine applications. The stimulation of neural differentiation by a laminin protein-derived epitope in two-dimensional (2D) and three-dimensional (3D) environments is investigated. The 3D gel system is found to be superior to its 2D counterpart for the induction of neural differentiation, even in the absence of a crucial biological inducer in nerve growth factor (NGF). In addition, cells cultured in 3D gels exhibits a spherical morphology that is consistent with their form under in vivo conditions. Overall, the present study underlines the impact of bioactivity, dimension, and NGF addition, as well as the cooperative effects thereof, on the neural differentiation of PC-12 cells. These results underline the significance of 3D culture systems in the development of scaffolds that closely replicate in vivo environments for the formation of cellular organoid models in vitro.

Semiautomated registration of pre- and intraoperative CT for image-guided percutaneous liver tumor ablation interventions.

PURPOSE: In CT-guided liver tumor ablation interventions, registration of a preoperative contrast-enhanced CT image to the intraoperative CT image is hypothesized to improve guidance. This is a highly challenging registration task due to differences in patient poses and large deformations, and therefore high registration errors are expected. In this study, our objective is to develop a method that enables users to locally improve the registration where the registration fails, with minimal user interaction. METHODS: The method is based on a conventional nonrigid intensity-based registration framework, extended with a novel point-to-surface penalty. The point-to-surface penalty serves to improve the alignment of the liver boundary, while requiring minimal user interaction during the intervention: annotating some points on the liver surface at those regions where the conventional registration seems inaccurate. RESULTS: The method is evaluated on 18 clinical datasets. It improves registration accuracy compared with the conventional nonrigid registration in terms of average surface distance (from 2.75 to 2.05 mm) and target registration error (from 6.92 to 5.8 mm). CONCLUSIONS: In this study, we introduce a semiautomated registration algorithm that improves the accuracy of image registration.

Antigenic GM3 Lactone Mimetic Molecule Integrated Mannosylated Glycopeptide Nanofibers for the Activation and Maturation of Dendritic Cells.

The ability of dendritic cells to coordinate innate and adaptive immune responses makes them essential targets for vaccination strategies. Presentation of specific antigens by dendritic cells is required for the activation of the immune system against many pathogens and tumors, and nanoscale materials can be functionalized for active targeting of dendritic cells. In this work, we integrated an immunogenic, carbohydrate melanoma-associated antigen-mimetic GM3-lactone molecule into mannosylated peptide amphiphile nanofibers to target dendritic cells through DC-SIGN receptor. Based on morphological and functional analyses, when dendritic cells were treated with peptide nanofiber carriers, they showed significant increase in antigen internalization and a corresponding increase in the surface expression of the activation and maturation markers CD86, CD83 and HLA-DR, in addition to exhibiting a general morphology consistent with dendritic cell maturation. These results indicate that mannosylated peptide amphiphile nanofiber carriers are promising candidates to target dendritic cells for antigen delivery.

A Modular Antigen Presenting Peptide/Oligonucleotide Nanostructure Platform for Inducing Potent Immune Response.

The design and development of vaccines, which can induce cellular immunity, particularly CD8+ T cells hold great importance since these cells play crucial roles against cancers and viral infections. Covalent conjugation of antigen and adjuvant molecules has been used for successful promotion of immunogenicity in subunit vaccines; however, the stimulation of the CD8+ T-cell responses by this approach has so far been limited. This study demonstrates a modular system based on noncovalent attachment of biotinylated antigen to a hybrid nanofiber system consisting of biotinylated self-assembling peptide and CpG oligodeoxynucleotides (ODN) molecules, via biotin-streptavidin interaction. These peptide/oligonucleotide hybrid nanosystems are capable of bypassing prior limitations related with inactivated or live-attenuated virus vaccines and achieve exceptionally high CD8+ T-cell responses. The nanostructures are found to trigger strong IgG response and effectively modulate cross-presentation of their antigen "cargo" through close proximity between the antigen and peptide/ODN adjuvant system. In addition, the biotinylated peptide nanofiber system is able to enhance antigen uptake and induce the maturation of antigen-presenting cells. Due to its versatility, biocompatibility, and biodegradability with a broad variety of streptavidin-linked antigens, the nanosystem shown here can be utilized as an efficient strategy for new vaccine development.

Local delivery of doxorubicin through supramolecular peptide amphiphile nanofiber gels.

Peptide amphiphiles (PAs) self-assemble into supramolecular nanofiber gels that provide a suitable environment for encapsulation of both hydrophobic and hydrophilic molecules. The PA gels have significant advantages for controlled delivery applications due to their high capacity to retain water, biocompatibility, and biodegradability. In this study, we demonstrate injectable supramolecular PA nanofiber gels for drug delivery applications. Doxorubicin (Dox), as a widely used chemotherapeutic drug for breast cancer treatment, was encapsulated within the PA gels prepared at different concentrations. Physical and chemical properties of the gels were characterized, and slow release of the Dox molecules through the supramolecular PA nanofiber gels was studied. In addition, the diffusion constants of the drug molecules within the PA nanofiber gels were estimated using fluorescence recovery after the photobleaching (FRAP) method. The PA nanofiber gels did not show any cytotoxicity and the encapsulation strategy enhanced the activity of drug molecules on cellular viability through prolonged release compared to direct administration under in vitro conditions. Moreover, the local in vivo injection of the Dox encapsulated PA nanofiber gels (Dox/PA) to the tumor site demonstrated the lowest tumor growth rate compared to the direct Dox injection and increased the apoptotic cells within the tumor tissue for local drug release through the PA nanofiber gels under in vivo conditions.

Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres.

Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane.

A fast regularized least-squares method for retinal vascular oxygen tension estimation using a phosphorescence lifetime imaging model.

BACKGROUND: Monitoring retinal oxygenation is of primary importance in detecting the presence of some common eye diseases. To improve the estimation of oxygen tension in retinal vessels, regularized least-squares (RLS) method was shown to be very effective compared with the conventional least-squares (LS) estimation. In this study, we propose an accelerated RLS estimation method for the problem of assessing the oxygenation of retinal vessels from phosphorescence lifetime images. METHODS: In the previous work, gradient descent algorithms were used to find the minimum of the RLS cost function. This approach is computationally expensive, especially when the oxygen tension map is large. In this study, using a closed-form solution of the RLS estimation and some inherent properties of the problem at hand, the RLS process is reduced to the weighted averaging of the LS estimates. This decreases the computational complexity of the RLS estimation considerably without sacrificing its performance. RESULTS: Performance analyses are conducted using both real and simulated data sets. In terms of computational complexity, the proposed RLS estimation method is significantly better than RLS methods that use gradient descent algorithms to find the minimum of the cost function. Additionally, there is no significant difference between the estimates acquired by the proposed and conventional RLS estimation methods. CONCLUSION: The proposed RLS estimation method for computing the retinal oxygen tension is computationally efficient, and produces estimates with negligible difference from those obtained by iterative RLS methods. Further, the results of this study can be applied to other lifetime imaging problems that have similar properties.