Visualizing Extracellular Vesicles and Their Function in 3D Tumor Microenvironment Models

Extracellular vesicles (EVs) are cell-derived nanostructures that mediate intercellular communication by delivering complex signals in normal tissues and cancer. The cellular coordination required for tumor development and maintenance is mediated, in part, through EV transport of molecular cargo to resident and distant cells. Most studies on EV-mediated signaling have been performed in two-dimensional (2D) monolayer cell cultures, largely because of their simplicity and high-throughput screening capacity. Three-dimensional (3D) cell cultures can be used to study cell-to-cell and cell-to-matrix interactions, enabling the study of EV-mediated cellular communication. 3D cultures may best model the role of EVs in formation of the tumor microenvironment (TME) and cancer cell-stromal interactions that sustain tumor growth. In this review, we discuss EV biology in 3D culture correlates of the TME. This includes EV communication between cell types of the TME, differences in EV biogenesis and signaling associated with differing scaffold choices and in scaffold-free 3D cultures and cultivation of the premetastatic niche. An understanding of EV biogenesis and signaling within a 3D TME will improve culture correlates of oncogenesis, enable molecular control of the TME and aid development of drug delivery tools based on EV-mediated signaling.     

Patient-Derived Organoids as a Model for Cancer Drug Discovery

In the search for the ideal model of tumours, the use of three-dimensional in vitro models is advancing rapidly. These are intended to mimic the in vivo properties of the tumours which affect cancer development, progression and drug sensitivity, and take into account cell–cell interactions, adhesion and invasiveness. Importantly, it is hoped that successful recapitulation of the structure and function of the tissue will predict patient response, permitting the development of personalized therapy in a timely manner applicable to the clinic. Furthermore, the use of co-culture systems will allow the role of the tumour microenvironment and tissue–tissue interactions to be taken into account and should lead to more accurate predictions of tumour development and responses to drugs. In this review, the relative merits and limitations of patient-derived organoids will be discussed compared to other in vitro and ex vivo cancer models. We will focus on their use as models for drug testing and personalized therapy and how these may be improved. Developments in technology will also be considered, including the use of microfluidics, 3D bioprinting, cryopreservation and circulating tumour cell-derived organoids. These have the potential to enhance the consistency, accessibility and availability of these models.     

Polymeric Hydrogels for In Vitro 3D Ovarian Cancer Modeling

Ovarian cancer (OC) grows and interacts constantly with a complex microenvironment, in which immune cells, fibroblasts, blood vessels, signal molecules and the extracellular matrix (ECM) coexist. This heterogeneous environment provides structural and biochemical support to the surrounding cells and undergoes constant and dynamic remodeling that actively promotes tumor initiation, progression, and metastasis. Despite the fact that traditional 2D cell culture systems have led to relevant medical advances in cancer research, 3D cell culture models could open new possibilities for the development of an in vitro tumor microenvironment more closely reproducing that observed in vivo. The implementation of materials science and technology into cancer research has enabled significant progress in the study of cancer progression and drug screening, through the development of polymeric scaffold-based 3D models closely recapitulating the physiopathological features of native tumor tissue. This article provides an overview of state-of-the-art in vitro tumor models with a particular focus on 3D OC cell culture in pre-clinical studies. The most representative OC models described in the literature are presented with a focus on hydrogel-based scaffolds, which guarantee soft tissue-like physical properties as well as a suitable 3D microenvironment for cell growth. Hydrogel-forming polymers of either natural or synthetic origin investigated in this context are described by highlighting their source of extraction, physical-chemical properties, and application for 3D ovarian cancer cell culture.     

3D Printed Solutions for Spheroid Engineering and Cancer Research

In multicellular organisms, cells are organized in a 3-dimensional framework and this is essential for organogenesis and tissue morphogenesis. Systems to recapitulate 3D cell growth are therefore vital for understanding development and cancer biology. Cells organized in 3D environments can evolve certain phenotypic traits valuable to physiologically relevant models that cannot be accessed in 2D culture. Cellular spheroids constitute an important aspect of in vitro tumor biology and they are usually prepared using the hanging drop method. Here a 3D printed approach is demonstrated to fabricate bespoke hanging drop devices for the culture of tumor cells. The design attributes of the hanging drop device take into account the need for high-throughput, high efficacy in spheroid formation, and automation. Specifically, in this study, custom-fit, modularized hanging drop devices comprising of inserts (Q-serts) were designed and fabricated using fused filament deposition (FFD). The utility of the Q-serts in the engineering of unicellular and multicellular spheroids-synthetic tumor microenvironment mimics (STEMs)—was established using human (cancer) cells. The culture of spheroids was automated using a pipetting robot and bioprinted using a custom bioink based on carboxylated agarose to simulate a tumor microenvironment (TME). The spheroids were characterized using light microscopy and histology. They showed good morphological and structural integrity and had high viability throughout the entire workflow. The systems and workflow presented here represent a user-focused 3D printing-driven spheroid culture platform which can be reliably reproduced in any research environment and scaled to- and on-demand. The standardization of spheroid preparation, handling, and culture should eliminate user-dependent variables, and have a positive impact on translational research to enable direct comparison of scientific findings.     

Hijacked Immune Cells in the Tumor Microenvironment: Molecular Mechanisms of Immunosuppression and Cues to Improve T Cell-Based Immunotherapy of Solid Tumors

The understanding of the tumor microenvironment (TME) has been expanding in recent years in the context of interactions among different cell types, through direct cell–cell communication as well as through soluble factors. It has become evident that the development of a successful antitumor response depends on several TME factors. In this context, the number, type, and subsets of immune cells, as well as the functionality, memory, and exhaustion state of leukocytes are key factors of the TME. Both the presence and functionality of immune cells, in particular T cells, are regulated by cellular and soluble factors of the TME. In this regard, one fundamental reason for failure of antitumor responses is hijacked immune cells, which contribute to the immunosuppressive TME in multiple ways. Specifically, reactive oxygen species (ROS), metabolites, and anti-inflammatory cytokines have central roles in generating an immunosuppressive TME. In this review, we focused on recent developments in the immune cell constituents of the TME, and the micromilieu control of antitumor responses. Furthermore, we highlighted the current challenges of T cell-based immunotherapies and potential future strategies to consider for strengthening their effectiveness.     

The Apoptosis Paradox in Cancer

Cancer growth represents a dysregulated imbalance between cell gain and cell loss, where the rate of proliferating mutant tumour cells exceeds the rate of those that die. Apoptosis, the most renowned form of programmed cell death, operates as a key physiological mechanism that limits cell population expansion, either to maintain tissue homeostasis or to remove potentially harmful cells, such as those that have sustained DNA damage. Paradoxically, high-grade cancers are generally associated with high constitutive levels of apoptosis. In cancer, cell-autonomous apoptosis constitutes a common tumour suppressor mechanism, a property which is exploited in cancer therapy. By contrast, limited apoptosis in the tumour-cell population also has the potential to promote cell survival and resistance to therapy by conditioning the tumour microenvironment (TME)—including phagocytes and viable tumour cells—and engendering pro-oncogenic effects. Notably, the constitutive apoptosis-mediated activation of cells of the innate immune system can help orchestrate a pro-oncogenic TME and may also effect evasion of cancer treatment. Here, we present an overview of the implications of cell death programmes in tumour biology, with particular focus on apoptosis as a process with “double-edged” consequences: on the one hand, being tumour suppressive through deletion of malignant or pre-malignant cells, while, on the other, being tumour progressive through stimulation of reparatory and regenerative responses in the TME.     

In Vitro Models of Biological Barriers for Nanomedical Research

Nanoconstructs developed for biomedical purposes must overcome diverse biological barriers before reaching the target where playing their therapeutic or diagnostic function. In vivo models are very complex and unsuitable to distinguish the roles plaid by the multiple biological barriers on nanoparticle biodistribution and effect; in addition, they are costly, time-consuming and subject to strict ethical regulation. For these reasons, simplified in vitro models are preferred, at least for the earlier phases of the nanoconstruct development. Many in vitro models have therefore been set up. Each model has its own pros and cons: conventional 2D cell cultures are simple and cost-effective, but the information remains limited to single cells; cell monolayers allow the formation of cell–cell junctions and the assessment of nanoparticle translocation across structured barriers but they lack three-dimensionality; 3D cell culture systems are more appropriate to test in vitro nanoparticle biodistribution but they are static; finally, bioreactors and microfluidic devices can mimicking the physiological flow occurring in vivo thus providing in vitro biological barrier models suitable to reliably assess nanoparticles relocation. In this evolving context, the present review provides an overview of the most representative and performing in vitro models of biological barriers set up for nanomedical research.     

Chemokine-Cytokine Networks in the Head and Neck Tumor Microenvironment

Head and neck squamous cell carcinomas (HNSCCs) are aggressive diseases with a dismal patient prognosis. Despite significant advances in treatment modalities, the five-year survival rate in patients with HNSCC has improved marginally and therefore warrants a comprehensive understanding of the HNSCC biology. Alterations in the cellular and non-cellular components of the HNSCC tumor micro-environment (TME) play a critical role in regulating many hallmarks of cancer development including evasion of apoptosis, activation of invasion, metastasis, angiogenesis, response to therapy, immune escape mechanisms, deregulation of energetics, and therefore the development of an overall aggressive HNSCC phenotype. Cytokines and chemokines are small secretory proteins produced by neoplastic or stromal cells, controlling complex and dynamic cell–cell interactions in the TME to regulate many cancer hallmarks. This review summarizes the current understanding of the complex cytokine/chemokine networks in the HNSCC TME, their role in activating diverse signaling pathways and promoting tumor progression, metastasis, and therapeutic resistance development.     

3D Bioprinting of Gelatin–Xanthan Gum Composite Hydrogels for Growth of Human Skin Cells

In recent years, bioprinting has attracted much attention as a potential tool for generating complex 3D biological constructs capable of mimicking the native tissue microenvironment and promoting physiologically relevant cell–cell and cell–matrix interactions. The aim of the present study was to develop a crosslinked 3D printable hydrogel based on biocompatible natural polymers, gelatin and xanthan gum at different percentages to be used both as a scaffold for cell growth and as a wound dressing. The CellInk Inkredible 3D printer was used for the 3D printing of hydrogels, and a glutaraldehyde solution was tested for the crosslinking process. We were able to obtain two kinds of printable hydrogels with different porosity, swelling and degradation time. Subsequently, the printed hydrogels were characterized from the point of view of biocompatibility. Our results showed that gelatin/xanthan-gum bioprinted hydrogels were biocompatible materials, as they allowed both human keratinocyte and fibroblast in vitro growth for 14 days. These two bioprintable hydrogels could be also used as a helpful dressing material.     

Ptk7 Is Dynamically Localized at Neural Crest Cell–Cell Contact Sites and Functions in Contact Inhibition of Locomotion

Neural crest (NC) cells are highly migratory cells that contribute to various vertebrate tissues, and whose migratory behaviors resemble cancer cell migration and invasion. Information exchange via dynamic NC cell–cell contact is one mechanism by which the directionality of migrating NC cells is controlled. One transmembrane protein that is most likely involved in this process is protein tyrosine kinase 7 (PTK7), an evolutionary conserved Wnt co-receptor that is expressed in cranial NC cells and several tumor cells. In Xenopus, Ptk7 is required for NC migration. In this study, we show that the Ptk7 protein is dynamically localized at cell–cell contact zones of migrating Xenopus NC cells and required for contact inhibition of locomotion (CIL). Using deletion constructs of Ptk7, we determined that the extracellular immunoglobulin domains of Ptk7 are important for its transient accumulation and that they mediate homophilic binding. Conversely, we found that ectopic expression of Ptk7 in non-NC cells was able to prevent NC cell invasion. However, deletion of the extracellular domains of Ptk7 abolished this effect. Thus, Ptk7 is sufficient at protecting non-NC tissue from NC cell invasion, suggesting a common role of PTK7 in contact inhibition, cell invasion, and tissue integrity.     

The Ephb2 Receptor Uses Homotypic,Head-to-Tail Interactions within Its Ectodomain as an Autoinhibitory Control Mechanism

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, as well as mediate many other cell–cell communication events. Their dysfunctional signaling has been shown to lead to various diseases, including cancer. The Ephs and ephrins both localize to the plasma membrane and, upon cell–cell contact, form extensive signaling assemblies at the contact sites. The Ephs and the ephrins are divided into A and B subclasses based on their sequence conservation and affinities for each other. The molecular details of Eph–ephrin recognition have been previously revealed and it has been documented that ephrin binding induces higher-order Eph assemblies, which are essential for full biological activity, via multiple, distinct Eph–Eph interfaces. One Eph–Eph interface type is characterized by a homotypic, head-to-tail interaction between the ligand-binding and the fibronectin domains of two adjacent Eph molecules. While the previous Eph ectodomain structural studies were focused on A class receptors, we now report the crystal structure of the full ectodomain of EphB2, revealing distinct and unique head-to-tail receptor–receptor interactions. The EphB2 structure and structure-based mutagenesis document that EphB2 uses the head-to-tail interactions as a novel autoinhibitory control mechanism for regulating downstream signaling and that these interactions can be modulated by posttranslational modifications.     

The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction

Background: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. Methods: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell–cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. Results: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell–cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. Conclusion: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations.     

Tumor Extracellular Matrix Stiffness Promptly Modulates the Phenotype and Gene Expression of Infiltrating T Lymphocytes

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4⁺ and CD8⁺ lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host’s immune system. The presence or absence, in particular, of cytotoxic CD8⁺ cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4⁺ and CD8⁺ cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.     

Comprehensive Integrated Single-Cell Whole Transcriptome Analysis Revealed the p-EMT Tumor Cells—CAFs Communication in Oral Squamous Cell Carcinoma

Cancer-associated fibroblasts (CAFs) and partial epithelial–mesenchymal transition (p-EMT) tumor cells are closed together and contribute to the tumor progression of oral squamous cell carcinoma (OSCC). In the present study, we deeply analyzed and integrated OSCC single-cell RNA sequencing datasets to define OSCC CAFs and p-EMT subpopulations. We highlighted the cell–cell interaction network of CAFs and p-EMT tumor cells and suggested biomarkers for the diagnosis and prognosis of OSCC during the metastasis condition. The analysis discovered four subtypes of CAFs: one p-EMT tumor cell population, and cycling tumor cells as well as TNFSF12-TNFRSF25/TNFRSF12A interactions between CAFs and p-EMT tumor cells during tumor metastasis. This suggests the prediction of therapeutically targetable checkpoint receptor–ligand interactions between CAFs and p-EMT tumor cells in OSCC regarding the metastasis status.     

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.     

An Emerging Role for Calcium Signaling in Cancer-Associated Fibroblasts

Tumors exist in a complex milieu where interaction with their associated microenvironment significantly contributes to disease progression. Cancer-associated fibroblasts (CAFs) are the primary component of the tumor microenvironment and participate in complex bidirectional communication with tumor cells. CAFs support the development of various hallmarks of cancer through diverse processes, including direct cell–cell contact, paracrine signaling, and remodeling and deposition of the extracellular matrix. Calcium signaling is a key second messenger in intra- and inter-cellular signaling pathways that contributes to cancer progression; however, the links between calcium signaling and CAFs are less well-explored. In this review, we put into context the role of calcium signaling in interactions between cancer cells and CAFs, with a focus on migration, proliferation, chemoresistance, and genetic instability.     

The iNKT Cell–Macrophage Axis in Homeostasis and Disease

Invariant natural killer T (iNKT) cells are CD1d-restricted, lipid-reactive T cells that exhibit preponderant immunomodulatory properties. The ultimate protective or deleterious functions displayed by iNKT cells in tissues are known to be partially shaped by the interactions they establish with other immune cells. In particular, the iNKT cell–macrophage crosstalk has gained growing interest over the past two decades. Accumulating evidence has highlighted that this immune axis plays central roles not only in maintaining homeostasis but also during the development of several pathologies. Hence, this review summarizes the reported features of the iNKT cell–macrophage axis in health and disease. We discuss the pathophysiological significance of this interplay and provide an overview of how both cells communicate with each other to regulate disease onset and progression in the context of infection, obesity, sterile inflammation, cancer and autoimmunity.     

Single-Cell RNA Sequencing Analysis for Oncogenic Mechanisms Underlying Oral Squamous Cell Carcinoma Carcinogenesis with Candida albicans Infection

Oral squamous cell carcinoma (OSCC) carcinogenesis involves heterogeneous tumor cells, and the tumor microenvironment (TME) is highly complex with many different cell types. Cancer cell–TME interactions are crucial in OSCC progression. Candida albicans (C. albicans)—frequently pre-sent in the oral potentially malignant disorder (OPMD) lesions and OSCC tissues—promotes malignant transformation. The aim of the study is to verify the mechanisms underlying OSCC car-cinogenesis with C. albicans infection and identify the biomarker for the early detection of OSCC and as the treatment target. The single-cell RNA sequencing analysis (scRNA-seq) was performed to explore the cell subtypes in normal oral mucosa, OPMD, and OSCC tissues. The cell composi-tion changes and oncogenic mechanisms underlying OSCC carcinogenesis with C. albicans infec-tion were investigated. Gene Set Variation Analysis (GSVA) was used to survey the mechanisms underlying OSCC carcinogenesis with and without C. albicans infection. The results revealed spe-cific cell clusters contributing to OSCC carcinogenesis with and without C. albicans infection. The major mechanisms involved in OSCC carcinogenesis without C. albicans infection are the IL2/STAT5, TNFα/NFκB, and TGFβ signaling pathways, whereas those involved in OSCC carcinogenesis with C. albicans infection are the KRAS signaling pathway and E2F target down-stream genes. Finally, stratifin (SFN) was validated to be a specific biomarker of OSCC with C. albicans infection. Thus, the detailed mechanism underlying OSCC carcinogenesis with C. albicans infection was determined and identified the treatment biomarker with potential precision medicine applications.     

Extracellular Vesicles and Their Role in the Spatial and Temporal Expansion of Tumor–Immune Interactions

Extracellular vesicles (EVs) serve as trafficking vehicles and intercellular communication tools. Their cargo molecules directly reflect characteristics of their parental cell. This includes information on cell identity and specific cellular conditions, ranging from normal to pathological states. In cancer, the content of EVs derived from tumor cells is altered and can induce oncogenic reprogramming of target cells. As a result, tumor-derived EVs compromise antitumor immunity and promote cancer progression and spreading. However, this pro-oncogenic phenotype is constantly being challenged by EVs derived from the local tumor microenvironment and from remote sources. Here, we summarize the role of EVs in the tumor–immune cross-talk that includes, but is not limited to, immune cells in the tumor microenvironment. We discuss the potential of remotely released EVs from the microbiome and during physical activity to shape the tumor–immune cross-talk, directly or indirectly, and confer antitumor activity. We further discuss the role of proinflammatory EVs in the temporal development of the tumor–immune interactions and their potential use for cancer diagnostics.     

Impairment of Hypoxia-Induced CA IX by Beta-Blocker Propranolol—Impact on Progression and Metastatic Potential of Colorectal Cancer Cells

The coexistence of cancer and other concomitant diseases is very frequent and has substantial implications for treatment decisions and outcomes. Beta-blockers, agents that block the beta-adrenergic receptors, have been related also to cancers. In the model of multicellular spheroids formed by colorectal cancer cells we described a crosstalk between beta-blockade by propranolol and tumour microenvironment. Non-selective beta-blocker propranolol decreased ability of tumour cells to adapt to hypoxia by reducing levels of HIF1α and carbonic anhydrase IX in 3D spheroids. We indicated a double action of propranolol in the tumour microenvironment by inhibiting the stability of HIF1α, thus mediating decrease of CA IX expression and, at the same time, by its possible effect on CA IX activity by decreasing the activity of protein kinase A (PKA). Moreover, the inhibition of β-adrenoreceptors by propranolol enhanced apoptosis, decreased number of mitochondria and lowered the amount of proteins involved in oxidative phosphorylation (V-ATP5A, IV-COX2, III-UQCRC2, II-SDHB, I-NDUFB8). Propranolol reduced metastatic potential, viability and proliferation of colorectal cancer cells cultivated in multicellular spheroids. To choose the right treatment strategy, it is extremely important to know how the treatment of concomitant diseases affects the superior microenvironment that is directly related to the efficiency of anti-cancer therapy