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.

Translation of small-diameter tissue-engineered vascular grafts (TEVGs) for the treatment of coronary artery disease (CAD) remains an unfulfilled promise. This is largely due to the limited integration of TEVGs into the native vascular wall-a process hampered by the insufficient smooth muscle cell (SMC) infiltration and extracellular matrix deposition, and low vasoactivity. These processes can be promoted through the judicious modulation of the SMC toward a synthetic phenotype to promote remodeling and vascular integration; however, the expression of synthetic markers is often accompanied by a decrease in the expression of contractile proteins. Therefore, techniques that can precisely modulate the SMC phenotypical behavior could have the potential to advance the translation of TEVGs. In this review, we describe the phenotypic diversity of SMCs and the different environmental cues that allow the modulation of SMC gene expression. Furthermore, we describe the emerging biomaterial approaches to modulate the SMC phenotype in TEVG design and discuss the limitations of current techniques. In addition, we found that current studies in tissue engineering limit the analysis of the SMC phenotype to a few markers, which are often the characteristic of early differentiation only. This limited scope has reduced the potential of tissue engineering to modulate the SMC toward specific behaviors and applications. Therefore, we recommend using the techniques presented in this review, in addition to modern single-cell proteomics analysis techniques to comprehensively characterize the phenotypic modulation of SMCs. Expanding the holistic potential of SMC modulation presents a great opportunity to advance the translation of living conduits for CAD therapeutics.

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.

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.

Under the influence of stress and membrane damage, cells undergo immunogenic cell death (ICD), which involves the release of damage associated molecular patterns (DAMPs), natural adjuvants for enhancing an immune response. In the presence of an antigen, released DAMPs can determine the type and magnitude of the immune response, and therefore the longevity and efficacy of an antigen-specific immunity. In the last decade, the immune response effect of ICD has been shown, yet there is no tool that can induce controlled ICD with predictable results, regardless of the cell type. We designed a peptide-based tool, called [II], for controlled damage to cell membrane to induce ICD and DAMPs release. Herein we describe a series of experiments that determine that the mechanism of action of [II] includes a caspase-dependent ICD and subsequent release of immune stimulating DAMPs, on various cell types. Moreover, we tested the hypothesis that controlled DAMP release via [II] in vivo was associated with enhancement of antigen-specific adaptive immunity with influenza hemagglutinin (HA) subunit vaccine. HA and [II] showed significantly higher HA specific IgG1 and IgG2a antibodies, compared to HA-only immunized mice, while the peptide itself did not elicit antibodies. In this paper, we demonstrate the first peptide-aggregation induced immunogenic rupture (PAIIR) approach as vaccine adjuvants for increasing both humoral and cellular immunity. In consideration of its ability to enhance IgG2a responses that are associated with heterosubtypic influenza virus protection, PAIIR is a promising adjuvant to promote universal protection upon influenza HA vaccination.

Discovery of peptide domains with unique intermolecular interactions is essential for engineering peptide-based materials. Rather than attempting a brute-force approach, we instead identify a previously unexplored strategy for discovery and study of intermolecular interactions: “co-assembly of oppositely charged peptide” (CoOP), a framework that “encourages” peptide assembly by mixing two oppositely charged hexapeptides. We used an integrated computational and experimental approach, probed the free energy of association and probability of amino acid contacts during co-assembly with atomic-resolution simulations, and correlated them to the physical properties of the aggregates. We introduce CoOP with three examples: dialanine, ditryptophan, and diisoleucine. Our results indicated that the opposite charges initiate the assembly, and the subsequent stability is enhanced by the presence of an undisturbed hydrophobic core. CoOP represents a unique, simple, and elegant framework that can be used to identify the structure-property relationships of self-assembling peptide-based materials.

Inducing and maintaining a hyaline cartilage phenotype are the greatest challenge for cartilage regeneration. Synthetic chondroinductive biomaterials might be the answer to the unmet clinical need for a safe, stable, and cost-effective material capable of inducing true hyaline cartilage formation. The past decade witnessed an emergence of peptides to achieve chondrogenesis, as peptides have the advantages of versatility, high target specificity, minimized toxicity and immunogenicity, and ease of synthesis. In this study, we review peptides as the basis for creating promising synthetic chondroinductive biomaterials for in situ scaffold-based cartilage regeneration. We provide a thorough review of peptides evaluated for cartilage regeneration while distinguishing between peptides reported to induce chondrogenesis independently, and peptides reported to act in synergy with other growth factors to induce cartilage regeneration. In addition, we highlight that most peptide studies have been in vitro, and appropriate controls are not always present. A few rigorously performed in vitro studies have proceeded to in vivo studies, but the peptides in those in vivo studies were mainly introduced through systemic, subcutaneous, or intra-articular injections, with a paucity of studies employing in situ defects with appropriate controls. Clinical translation of peptides will require the evaluation of these peptides in well-controlled in vivo cartilage defect studies. In the decade ahead, we may be poised to leverage peptides to design devices that are safe, reproducible, cost-efficient, and scalable biomaterials, which are themselves chondroinductive to achieve true hyaline cartilage regeneration without the need for growth factors and other small molecules.

Peptides play an essential role in biotechnological applications as therapeutic and diagnostic agents due to their tunable activity for desired function. So far, a variety of peptide therapeutics and their peptide-based carriers have been engineered for drug delivery applications. However, in order to design and develop such systems for tissue-specific applications, the cellular microenvironment must be properly considered. Peptide-based materials have vast potential applications, particularly for cancer-related systems. Thus, in this chapter, we first focus on different design strategies and considerations for drug-release mechanisms in peptide-based materials. Then, we explain how tumor microenvironments may be distinguished from healthy tissue, including discussion of tumor-specific drug delivery strategies. Finally, the growing significance of peptide chemotherapeutics will be emphasized in terms of design concerns and current applications.

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.

Three-dimensional (3D) cell culture models are developed as a promising platform to screen anticancer therapeutics and treatments. However, current imaging techniques cannot provide 3D structures of tumor spheroids in situ. In this study, we employed label-free and noninvasive optical coherence tomography (OCT) for imaging and quantifying the 3D structures of tumor spheroids. We imaged ovarian cancer spheroids with OVCAR-8 cell line over a period of 10 days with 5,000 and 50,000 initial cell numbers. We successfully reconstructed the 3D necrotic regions via label-free intrinsic scattering attenuation contrast and evaluated the effect of Cisplatin treatment on tumor spheroids.

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.

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.

BackgroundHigh 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.MethodsWe 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.ResultsWe 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).ConclusionsThese 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.

There is a need for the development of antifouling materials to resist adsorption of biomacromolecules. Here we describe the preparation of a novel zwitterionic block copolymer with the potential to prevent or delay the formation of microbial biofilms. The block copolymer comprised a zwitterionic (hydrophilic) section of alternating glutamic acid (negatively charged) and lysine (positively charged) units and a hydrophobic polystyrene section. Cryo-TEM and dynamic-light-scattering (DLS) results showed that, on average, the block copolymer self-assembled into 7-nm-diameter micelles in aqueous solutions (0 to 100 mM NaCl, pH 6). Quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and contact angle measurements demonstrated that the block copolymer self-assembled into a brush-like monolayer on polystyrene surfaces. The brush-like monolayer produced from a 100 mg/L block copolymer solution exhibited an average distance, d, of approximately 4-8 nm between each block copolymer molecule (center to center). Once the brush-like monolayer self-assembled, it reduced EPS adsorption onto the polystyrene surface by ∼70% (mass), reduced the rate of bacterial attachment by >80%, and inhibited the development of thick biofilms. QCM-D results revealed that the EPS molecules penetrate between the chains of the brush and adsorb onto the polystyrene surface. Additionally, AFM analyses showed that the brush-like monolayer prevents the adhesion of large (> d) hydrophilic colloids onto the surface via hydration repulsion; however, molecules or colloids small enough to fit between the brush polymers (< d) were able to be adsorbed onto the surface via van der Waals interactions. Overall, we found that the penetration of extracellular organelles, as well as biopolymers through the brush, is critical for the failure of the antifouling coating, and likely could be prevented through tuning of the brush density. Stability and biofilm development testing on multiple surfaces (polypropylene, glass, and stainless steel) support practical applications of this novel block copolymer.

Nanoparticle-based therapeutics and diagnostics are commonly referred to as nanomedicine and may significantly impact the future of healthcare. However, the clinical translation of these technologies is challenging. One of these challenges is the efficient delivery of nanoparticles to specific cell populations and subcellular targets in the body to elicit desired biological and therapeutic responses. It is critical for researchers to understand the fundamental concepts of how nanoparticles interact with biological systems to predict and control in vivo nanoparticle transport for improved clinical benefit. In this overview article, we review and discuss cellular internalization pathways, summarize the field`s understanding of how nanoparticle physicochemical properties affect cellular interactions, and explore and discuss intracellular nanoparticle trafficking and kinetics. Our overview may provide a valuable resource for researchers and may inspire new studies to expand our current understanding of nanotechnology-biology interactions at cellular and subcellular levels with the goal to improve clinical translation of nanomedicines.

Therapeutic manipulation of the BCL-2 family using BH3 mimetics is an emerging paradigm in cancer treatment and immune modulation. For example, peptides mimicking the BIM BH3 helix can directly target the full complement of anti- and pro-apoptotic BCL-2 proteins to trigger apoptosis. This study has incorporated the potent BH3 α-helical death domain of BIM into peptide amphiphile (PA) nanostructures designed to facilitate cellular uptake and induce cell death. This study shows that these PA nanostructures are quickly incorporated into cells, are able to specifically bind BCL-2 proteins, are stable at physiologic temperatures and pH, and induce dose-dependent apoptosis in cells. The incorporation of a cathepsin B cleavable linker between the BIM BH3 peptide and the hydrophobic tail resulted in increased intracellular accumulation and mitochondrial co-localization of the BIM BH3 peptide while also improving BCL-2 family member binding and apoptotic reactivation. This PA platform represents a promising new strategy for intracellular therapeutic peptide delivery for the disruption of intracellular protein:protein interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The immune system plays a critical role in our health. When functioning correctly, the immune system continually protects us from the most common to the most deadly and mysterious maladies: common influenza to HIV and cancer. When functioning incorrectly, autoimmune diseases such as diabetes and multiple sclerosis ensue. Understanding how the immune system functions and learning how to rationally control it to protect against or treat disease has been a challenge historically burdened by the immunology and medical fields. Recently, exciting strides in immunological discoveries and investigational tools have allowed outsiders to enter the fold, chief among them being the biomaterials field. With biomaterials as an engineering tool, rational manipulation of the immune system at the cellular and molecular levels has expanded, shedding new light on the functioning of the immune system while also showing promising preclinical and clinical data for more effective and safer treatments. As a flourishing subset of the biomaterials field, peptides have been used as a major design component of the immune engineering strategy. Peptides, because of their chemically defined nature, engineerability, and range of complexity, provide useful functionalities when designing strategies for vaccination, cancer immunotherapy, and the treatment of autoimmune disorders. This chapter will examine the peptide-based strategies for immune modulation in detail and discuss the promise and outstanding challenges facing the field of immune engineering.