Mimicking the Tumor Niche: Methods for Isolation, Culture, and Characterization of Cancer Stem Cells and Multicellular Spheroids.

Cancer stem cells (CSCs) play a significant role in driving several tumor hallmarks. Their behavior and tumor progression are strictly related to the tumor microenvironment (TME). The dynamic interplay between CSCs and TME drives metastasis, chemoresistance, and disease relapse. In this chapter, we describe different techniques and protocols for isolating, culturing, and characterizing CSCs and we explain the methodology for the culture of multicellular spheroids comprising CSCs.

Treatment resistance in pancreatic and biliary tract cancer: molecular and clinical pharmacology perspectives.

INTRODUCTION: Treatment resistance poses a significant obstacle in oncology, especially in biliary tract cancer (BTC) and pancreatic cancer (PC). Current therapeutic options include chemotherapy, targeted therapy, and immunotherapy. Resistance to these treatments may arise due to diverse molecular mechanisms, such as genetic and epigenetic modifications, altered drug metabolism and efflux, and changes in the tumor microenvironment. Identifying and overcoming these mechanisms is a major focus of research: strategies being explored include combination therapies, modulation of the tumor microenvironment, and personalized approaches. AREAS COVERED: We provide a current overview and discussion of the most relevant mechanisms of resistance to chemotherapy, target therapy, and immunotherapy in both BTC and PC. Furthermore, we compare the different strategies that are being implemented to overcome these obstacles. EXPERT OPINION: So far there is no unified theory on drug resistance and progress is limited. To overcome this issue, individualized patient approaches, possibly through liquid biopsies or single-cell transcriptome studies, are suggested, along with the potential use of artificial intelligence, to guide effective treatment strategies. Furthermore, we provide insights into what we consider the most promising areas of research, and we speculate on the future of managing treatment resistance to improve patient outcomes.

Identification of novel biomarkers in the early diagnosis of malignant melanoma by untargeted liquid chromatography coupled to high-resolution mass spectrometry-based metabolomics: a pilot study.

BACKGROUND: Malignant melanoma (MM) is a highly aggressive form of skin cancer whose incidence continues to rise worldwide. If diagnosed at an early stage, it has an excellent prognosis, but mortality increases significantly at advanced stages after distant spread. Unfortunately, early detection of aggressive melanoma remains a challenge. OBJECTIVES: To identify novel blood-circulating biomarkers that may be useful in the diagnosis of MM to guide patient counselling and appropriate disease management. METHODS: In this study, 105 serum samples from 26 healthy patients and 79 with MM were analysed using an untargeted approach by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to compare the metabolomic profiles of both conditions. Resulting data were subjected to both univariate and multivariate statistical analysis to select robust biomarkers. The classification model obtained from this analysis was further validated with an independent cohort of 12 patients with stage I MM. RESULTS: We successfully identified several lipidic metabolites differentially expressed in patients with stage I MM vs. healthy controls. Three of these metabolites were used to develop a classification model, which exhibited exceptional precision (0.92) and accuracy (0.94) when validated on an independent sample. CONCLUSIONS: These results demonstrate that metabolomics using LC-HRMS is a powerful tool to identify and quantify metabolites in bodily fluids that could serve as potential early diagnostic markers for MM.

Melanoma is a type of skin cancer that can be deadly if it is not detected at an early stage. Unfortunately, the early detection of melanoma is challenging. Our team has developed a model that could be used to predict whether a person has stage I malignant melanoma based on blood serum analysis. The model was trained on data from a group of people with melanoma and it was found to be accurate in predicting melanoma at an early stage. This means that the model could be used to identify people who have skin cancer before it progresses and becomes more complicated to treat. Although the researchers recommend that further studies are conducted to validate the model in a larger population of people, this research could help with the early diagnosis of melanoma and work toward improving survival rates.

Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers.

The main aim of this study is to report basic knowledge on how a protein corona (PC) could affect or modify the way in which multifunctionalized nanoparticles interact with cells. With this purpose, we have firstly optimized the development of a target-specific nanocarrier by coupling a specific fluorescent antibody on the surface of functionalized lipid liquid nanocapsules (LLNCs). Thus, an anti-HER2-FITC antibody (αHER2) has been used, HER2 being a surface receptor that is overexpressed in several tumor cells. Subsequently, the in vitro formation of a PC has been developed using fetal bovine serum supplemented with human fibrinogen. Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), Laser Doppler Electrophoresis (LDE), and Gel Chromatography techniques have been used to assure a complete physico-chemical characterization of the nano-complexes with (LLNCs-αHER2-PC) and without (LLNCs-αHER2) the surrounding PC. In addition, cellular assays were performed to study the cellular uptake and the specific cellular-nanocarrier interactions using the SKBR3 (high expression of HER2) breast cancer cell line and human dermal fibroblasts (HDFa) (healthy cell line without expression of HER2 receptors as control), showing that the SKBR3 cell line had a higher transport rate (50-fold) than HDFa at 60 min with LLNCs-αHER2. Moreover, the SKBR3 cell line incubated with LLNCs-αHER2-PC suffered a significant reduction (40%) in the uptake. These results suggest that the formation of a PC onto LLNCs does not prevent specific cell targeting, although it does have an important influence on cell uptake.

Unraveling the Potential of miRNAs from CSCs as an Emerging Clinical Tool for Breast Cancer Diagnosis and Prognosis.

Breast cancer (BC) is the most diagnosed cancer in women and the second most common cancer globally. Significant advances in BC research have led to improved early detection and effective therapies. One of the key challenges in BC is the presence of BC stem cells (BCSCs). This small subpopulation within the tumor possesses unique characteristics, including tumor-initiating capabilities, contributes to treatment resistance, and plays a role in cancer recurrence and metastasis. In recent years, microRNAs (miRNAs) have emerged as potential regulators of BCSCs, which can modulate gene expression and influence cellular processes like BCSCs' self-renewal, differentiation, and tumor-promoting pathways. Understanding the miRNA signatures of BCSCs holds great promise for improving BC diagnosis and prognosis. By targeting BCSCs and their associated miRNAs, researchers aim to develop more effective and personalized treatment strategies that may offer better outcomes for BC patients, minimizing tumor recurrence and metastasis. In conclusion, the investigation of miRNAs as regulators of BCSCs opens new directions for advancing BC research through the use of bioinformatics and the development of innovative therapeutic approaches. This review summarizes the most recent and innovative studies and clinical trials on the role of BCSCs miRNAs as potential tools for early diagnosis, prognosis, and resistance.

Apatite-coated outer layer eggshell membrane: A novel osteoinductive biohybrid composite for guided bone/tissue regeneration.

Hybrid biomimetic materials aim to replicate the organic-inorganic constructs of mineralized tissues. During eggshell formation, the outer surface of the eggshell membrane (ESM) promotes calcium carbonate nucleation, while the inner one prevents mineralization toward the egg white and yolk. In the current study, the outer surface of the ESM acted as a heteronucleant in calcium phosphate precipitation by the vapor diffusion sitting drop method, while the inner one remained unmineralized. The aim was to fabricate a 2D biomaterial with dual functions, osteoinductive on one side and protective against cell invasion on the other side. The microstructural, physicochemical, morphological, and mechanical properties of the mineralized ESM were characterized by XRD, TGA, XPS, FTIR/Raman, HR-SEM, and mechanical testing techniques. The cytocompatibility and osteoinductive ability were assessed by biological assays of cell viability, proliferation, and osteogenic differentiation on human mesenchymal stromal cells (hMSCs). Results indicate that the outer surface of the ESM induces the heterogeneous precipitation of carbonate-apatite phase depicting biomimetic features. In addition, the apatite/ESM shows a much higher cytocompatibility than the pristine ESM and promotes the osteogenic differentiation of hMSCs more efficiently. Overall, the apatite/ESM composite exhibits compositional, crystalline, mechanical, and biological properties that resemble those of mineralized tissues, rendering it an approachable and novel material especially useful in guided tissue/bone regeneration.

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells.

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3' long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

Maslinic acid solid lipid nanoparticles as hydrophobic anticancer drug carriers: Formulation, in vitro activity and in vivo biodistribution.

Maslinic acid (MA) is a natural pentacyclic triterpenoid with inherent antitumor activity which has a very low solubility in water. MA solid lipid nanoparticles (SLNs) were prepared using Poloxamer 407 and Dicarboxylic acid-Poloxamer 407 as surfactants. Both MA SLNs are monodisperse, with sizes around 130 nm, and stable. Curcumin has been encapsulated in both types of nanoparticles without altering their colloidal properties. Moreover, SLNs greatly improve the solubility of MA and Curcumin. The cytotoxicity of MA and SLNs has been evaluated in BxPC3 human pancreatic cancer cells, MCF7 human breast cancer cells, and in a human fibroblast primary cell line. MA shows higher cytotoxic effect in BxPC3 and MCF7 cancer cells than in human primary fibroblasts. Nile Red loaded MA SLNs are quickly uptaken by BxPC3 and MCF7 cells, and show different cytoplasmic distributions depending on the cellular line. The oral or intravenous administration of MA SLNs in mice does not report any toxic effect, and the intravenous administration of fluorescent MA SLNs shows a homogeneous distribution in mice, without site-specific accumulation. Results suggest the great potential of MA SLNs as nanocarriers of anticancer drugs and as promising targeted theranostic nanodevices.

Tigecycline reduces tumorigenesis in colorectal cancer via inhibition of cell proliferation and modulation of immune response.

BACKGROUND: and Purpose: Colorectal cancer (CRC) is one of the cancers with the highest incidence in which APC gene mutations occur in almost 80% of patients. This mutation leads to ß-catenin aberrant accumulation and an uncontrolled proliferation. Apoptosis evasion, changes in the immune response and microbiota composition are also events that arise in CRC. Tetracyclines are drugs with proven antibiotic and immunomodulatory properties that have shown cytotoxic activity against different tumor cell lines. EXPERIMENTAL APPROACH: The effect of tigecycline was evaluated in vitro in HCT116 cells and in vivo in a colitis-associated colorectal cancer (CAC) murine model. 5-fluorouracil was assayed as positive control in both studies. KEY RESULTS: Tigecycline showed an antiproliferative activity targeting the Wnt/ß-catenin pathway and downregulating STAT3. Moreover, tigecycline induced apoptosis through extrinsic, intrinsic and endoplasmic reticulum pathways converging on an increase of CASP7 levels. Furthermore, tigecycline modulated the immune response in CAC, reducing the cancer-associated inflammation through downregulation of cytokines expression. Additionally, tigecycline favored the cytotoxic activity of cytotoxic T lymphocytes (CTLs), one of the main immune defenses against tumor cells. Lastly, the antibiotic reestablished the gut dysbiosis in CAC mice increasing the abundance of bacterial genera and species, such as Akkermansia and Parabacteroides distasonis, that act as protectors against tumor development. These findings resulted in a reduction of the number of tumors and an amelioration of the tumorigenesis process in CAC. CONCLUSION AND IMPLICATIONS: Tigecycline exerts a beneficial effect against CRC supporting the use of this antibiotic for the treatment of this disease.

Clinical implications of inflammation in atheroma formation and novel therapies in cardiovascular diseases.

Cardiovascular diseases (CVD) are the leading causes of death and disability in the world. Among all CVD, the most common is coronary artery disease (CAD). CAD results from the complications promoted by atherosclerosis, which is characterized by the accumulation of atherosclerotic plaques that limit and block the blood flow of the arteries involved in heart oxygenation. Atherosclerotic disease is usually treated by stents implantation and angioplasty, but these surgical interventions also favour thrombosis and restenosis which often lead to device failure. Hence, efficient and long-lasting therapeutic options that are easily accessible to patients are in high demand. Advanced technologies including nanotechnology or vascular tissue engineering may provide promising solutions for CVD. Moreover, advances in the understanding of the biological processes underlying atherosclerosis can lead to a significant improvement in the management of CVD and even to the development of novel efficient drugs. To note, over the last years, the observation that inflammation leads to atherosclerosis has gained interest providing a link between atheroma formation and oncogenesis. Here, we have focused on the description of the available therapy for atherosclerosis, including surgical treatment and experimental treatment, the mechanisms of atheroma formation, and possible novel therapeutic candidates such as the use of anti-inflammatory treatments to reduce CVD.

Eggshell Membrane as a Biomaterial for Bone Regeneration.

The physicochemical features of the avian eggshell membrane play an essential role in the process of calcium carbonate deposition during shell mineralization, giving rise to a porous mineralized tissue with remarkable mechanical properties and biological functions. The membrane could be useful by itself or as a bi-dimensional scaffold to build future bone-regenerative materials. This review focuses on the biological, physical, and mechanical properties of the eggshell membrane that could be useful for that purpose. Due to its low cost and wide availability as a waste byproduct of the egg processing industry, repurposing the eggshell membrane for bone bio-material manufacturing fulfills the principles of a circular economy. In addition, eggshell membrane particles have has the potential to be used as bio-ink for 3D printing of tailored implantable scaffolds. Herein, a literature review was conducted to ascertain the degree to which the properties of the eggshell membrane satisfy the requirements for the development of bone scaffolds. In principle, it is biocompatible and non-cytotoxic, and induces proliferation and differentiation of different cell types. Moreover, when implanted in animal models, it elicits a mild inflammatory response and displays characteristics of stability and biodegradability. Furthermore, the eggshell membrane possesses a mechanical viscoelastic behavior comparable to other collagen-based systems. Overall, the biological, physical, and mechanical features of the eggshell membrane, which can be further tuned and improved, make this natural polymer suitable as a basic component for developing new bone graft materials.

Lipid-core nanoparticles: Classification, preparation methods, routes of administration and recent advances in cancer treatment.

Nanotechnological drug delivery platforms represent a new paradigm for cancer therapeutics as they improve the pharmacokinetic profile and distribution of chemotherapeutic agents over conventional formulations. Among nanoparticles, lipid-based nanoplatforms possessing a lipid core, that is, lipid-core nanoparticles (LCNPs), have gained increasing interest due to lipid properties such as high solubilizing potential, versatility, biocompatibility, and biodegradability. However, due to the wide spectrum of morphologies and types of LCNPs, there is a lack of consensus regarding their terminology and classification. According to the current state-of-the-art in this critical review, LCNPs are defined and classified based on the state of their lipidic components in liquid lipid nanoparticles (LLNs). These include lipid nanoemulsions (LNEs) and lipid nanocapsules (LNCs), solid lipid nanoparticles (SLNs) and nanostructured lipid nanocarriers (NLCs). In addition, we present a comprehensive and comparative description of the methods employed for their preparation, routes of administration and the fundamental role of physicochemical properties of LCNPs for efficient antitumoral drug-delivery application. Market available LCNPs, clinical trials and preclinical in vivo studies of promising LCNPs as potential treatments for different cancer pathologies are summarized.

Estradiol and Estrone Have Different Biological Functions to Induce NF-κB-Driven Inflammation, EMT and Stemness in ER+ Cancer Cells.

In general, the risk of being diagnosed with cancer increases with age; however, the development of estrogen-receptor-positive (ER+) cancer types in women are more closely related to menopausal status than age. In fact, the general risk factors for cancer development, such as obesity-induced inflammation, show differences in their association with ER+ cancer risk in pre- and postmenopausal women. Here, we tested the role of the principal estrogens in the bloodstream before and after menopause, estradiol (E2) and estrone (E1), respectively, on inflammation, epithelial-to-mesenchymal transition (EMT) and cancer stem cell enrichment in the human ER+ cervical cancer cell line HeLa. Our results demonstrate that E1, contrary to E2, is pro-inflammatory, increases embryonic stem-transcription factors (ES-TFs) expression and induces EMT in ER+ HeLa cells. Moreover, we observed that high intratumoural expression levels of 17ß-Hydroxysteroid dehydrogenase (HSD17B) isoforms involved in E1 synthesis is a poor prognosis factor, while overexpression of E2-synthetizing HSD17B isoforms is associated with a better outcome, for patients diagnosed with ER+ ovarian and uterine corpus carcinomas. This work demonstrates that E1 and E2 have different biological functions in ER+ gynaecologic cancers. These results open a new line of research in the study of ER+ cancer subtypes, highlighting the potential key oncogenic role of E1 and HSD17B E1-synthesizing enzymes in the development and progression of these diseases.

Biofabrication of a tri-layered 3D-bioprinted CSC-based malignant melanoma model for personalized cancer treatment.

Conventionalin vitrocancer models do not accurately reproduce the tumor microenvironment (TME), so three-dimensional (3D)-bioprinting represents an excellent tool to overcome their limitations. Here, two multicellular tri-layered malignant melanoma (MM) models composed by cancer stem cells (CSCs) isolated from a MM established cell line or a primary-patient derived cell line, fibroblasts, mesenchymal stem cells, and endothelial cells, embedded within an agarose-collagen type I hydrogel were bioprinted. Embedded-cells showed high proliferation and metabolic activity, and actively remodeled their TME. MM hydrogels displayed similar rheological properties that skin and were able to support an early onset of vascularization. Besides, MM hydrogels displayed different response to vemurafenib compared with cell cultures, and supported tumorigenesis in murine xenotransplant achieving more mimeticin vivomodels. For the first time a tri-layered 3D-bioprinted CSC-based human MM model is developed recreating TMEin vitroandin vivoand response to treatment, being useful for precision treatment regimens against MM.

Dual Role of Fibroblasts Educated by Tumour in Cancer Behavior and Therapeutic Perspectives.

Tumours are complex systems with dynamic interactions between tumour cells, non-tumour cells, and extracellular components that comprise the tumour microenvironment (TME). The majority of TME's cells are cancer-associated fibroblasts (CAFs), which are crucial in extracellular matrix (ECM) construction, tumour metabolism, immunology, adaptive chemoresistance, and tumour cell motility. CAF subtypes have been identified based on the expression of protein markers. CAFs may act as promoters or suppressors in tumour cells depending on a variety of factors, including cancer stage. Indeed, CAFs have been shown to promote tumour growth, survival and spread, and secretome changes, but they can also slow tumourigenesis at an early stage through mechanisms that are still poorly understood. Stromal-cancer interactions are governed by a variety of soluble factors that determine the outcome of the tumourigenic process. Cancer cells release factors that enhance the ability of fibroblasts to secrete multiple tumour-promoting chemokines, acting on malignant cells to promote proliferation, migration, and invasion. This crosstalk between CAFs and tumour cells has given new prominence to the stromal cells, from being considered as mere physical support to becoming key players in the tumour process. Here, we focus on the concept of cancer as a non-healing wound and the relevance of chronic inflammation to tumour initiation. In addition, we review CAFs heterogeneous origins and markers together with the potential therapeutic implications of CAFs "re-education" and/or targeting tumour progression inhibition.

Unifying Different Cancer Theories in a Unique Tumour Model: Chronic Inflammation and Deaminases as Meeting Points.

The increase in cancer incidences shows that there is a need to better understand tumour heterogeneity to achieve efficient treatments. Interestingly, there are several common features among almost all types of cancers, with chronic inflammation induction and deaminase dysfunctions singled out. Deaminases are a family of enzymes with nucleotide-editing capacity, which are classified into two main groups: DNA-based and RNA-based. Remarkably, a close relationship between inflammation and the dysregulation of these molecules has been widely documented, which may explain the characteristic intratumor heterogeneity, both at DNA and transcriptional levels. Indeed, heterogeneity in cancer makes it difficult to establish a unique tumour progression model. Currently, there are three main cancer models-stochastic, hierarchic, and dynamic-although there is no consensus on which one better resembles cancer biology because they are usually overly simplified. Here, to accurately explain tumour progression, we propose interactions among chronic inflammation, deaminases dysregulation, intratumor genetic heterogeneity, cancer phenotypic plasticity, and even the previously proposed appearance of cancer stem-like cell populations in the edges of advanced solid tumour masses (instead of being the cells of origin of primary malignancies). The new tumour development model proposed in this study does not contradict previously accepted models and it may open up a window to interesting therapeutic approaches.

Erratum: Physiological lentiviral vectors for the generation of improved CAR-T cells.

[This corrects the article DOI: 10.1016/j.omto.2022.05.003.].

Chondro-Inductive b-TPUe-Based Functionalized Scaffolds for Application in Cartilage Tissue Engineering.

Osteoarthritis is a disease with a great socioeconomic impact and mainly affects articular cartilage, a tissue with reduced self-healing capacity. In this work, 3D printed 1,4 butanediol thermoplastic polyurethane (b-TPUe) scaffolds are functionalized and infrapatellar mesenchymal stem cells are used as the cellular source. Since b-TPUe is a biomaterial with mechanical properties similar to cartilage, but it does not provide the desired environment for cell adhesion, scaffolds are functionalized with two methods, one based on collagen type I and the other in 1-pyrenebutiric acid (PBA) as principal components. Alamar Blue and confocal assays display that PBA functionalized scaffolds support higher cell adhesion and proliferation for the first 21 days. However, collagen type I functionalization induces higher proliferation rates and similar cell viability than the PBA method. Further, both functionalization methods induce extracellular matrix synthesis, and the presence of chondrogenic markers (Sox9, Col2a, and Acan). Finally, SEM images probe that functionalized 3D printed scaffolds present much better cell/biomaterial interactions than controls and confirm early chondrogenesis. These results indicate that the two methods of functionalization in the highly hydrophobic b-TPUe enhance the cell-biomaterial interactions and the improvement in the chondro-inductive properties, which have great potential for application in cartilage tissue engineering.

Biofabrication approaches and regulatory framework of metastatic tumor-on-a-chip models for precision oncology.

The complexity of the tumor microenvironment (TME) together with the development of the metastatic process are the main reasons for the failure of conventional anticancer treatment. In recent years, there is an increasing need to advance toward advanced in vitro models of cancer mimicking TME and simulating metastasis to understand the associated mechanisms that are still unknown, and to be able to develop personalized therapy. In this review, the commonly used alternatives and latest advances in biofabrication of tumor-on-chips, which allow the generation of the most sophisticated and optimized models for recapitulating the tumor process, are presented. In addition, the advances that have allowed these new models in the area of metastasis, cancer stem cells, and angiogenesis are summarized, as well as the recent integration of multiorgan-on-a-chip systems to recapitulate natural metastasis and pharmacological screening against it. We also analyze, for the first time in the literature, the normative and regulatory framework in which these models could potentially be found, as well as the requirements and processes that must be fulfilled to be commercially implemented as in vitro study model. Moreover, we are focused on the possible regulatory pathways for their clinical application in precision medicine and decision making through the generation of personalized models with patient samples. In conclusion, this review highlights the synergistic combination of three-dimensional bioprinting systems with the novel tumor/metastasis/multiorgan-on-a-chip systems to generate models for both basic research and clinical applications to have devices useful for personalized oncology.