Cancer Stem Cell Microenvironment Models with Biomaterial Scaffolds In Vitro

Defined by its potential for self-renewal, differentiation and tumorigenicity, cancer stem cells (CSCs) are considered responsible for drug resistance and relapse. To understand the behavior of CSC, the effects of the microenvironment in each tissue are a matter of great concerns for scientists in cancer biology. However, there are many complicated obstacles in the mimicking the microenvironment of CSCs even with current advanced technology. In this context, novel biomaterials have widely been assessed as in vitro platforms for their ability to mimic cancer microenvironment. These efforts should be successful to identify and characterize various CSCs specific in each type of cancer. Therefore, extracellular matrix scaffolds made of biomaterial will modulate the interactions and facilitate the investigation of CSC associated with biological phenomena simplifying the complexity of the microenvironment. In this review, we summarize latest advances in biomaterial scaffolds, which are exploited to mimic CSC microenvironment, and their chemical and biological requirements with discussion. The discussion includes the possible effects on both cells in tumors and microenvironment to propose what the critical factors are in controlling the CSC microenvironment focusing the future investigation. Our insights on their availability in drug screening will also follow the discussion

Promoting biological similarity by collagen microfibers in 3D colorectal cancer-stromal tissue: Replicating mechanical properties and cancer stem cell markers

Sasaki N., Asano Y., Sorayama Y., et al. Promoting biological similarity by collagen microfibers in 3D colorectal cancer-stromal tissue: Replicating mechanical properties and cancer stem cell markers. Acta Biomaterialia 185, 161 (2024); https://doi.org/10.1016/j.actbio.2024.07.001.The extracellular matrix (ECM) of cancer tissues is rich in dense collagen, contributing to the stiffening of these tissues. Increased stiffness has been reported to promote cancer cell proliferation, invasion, metastasis, and prevent drug delivery. Replicating the structure and mechanical properties of cancer tissue in vitro is essential for developing cancer treatment drugs that target these properties. In this study, we recreated specific characteristics of cancer tissue, such as collagen density and high elastic modulus, using a colorectal cancer cell line as a model. Using our original material, collagen microfibers (CMFs), and a constructed three-dimensional (3D) cancer-stromal tissue model, we successfully reproduced an ECM highly similar to in vivo conditions. Furthermore, our research demonstrated that cancer stem cell markers expressed in the 3D cancer-stromal tissue model more closely mimic in vivo conditions than traditional two-dimensional cell cultures. We also found that CMFs might affect an impact on how cancer cells express these markers. Our 3D CMF-based model holds promise for enhancing our understanding of colorectal cancer and advancing therapeutic approaches. Statement of significance: Reproducing the collagen content and stiffness of cancer tissue is crucial in comprehending the properties of cancer and advancing anticancer drug development. Nonetheless, the use of collagen as a scaffold material has posed challenges due to its poor solubility, hindering the replication of a cancer microenvironment. In this study, we have successfully recreated cancer tissue-specific characteristics such as collagen density, stiffness, and the expression of cancer stem cell markers in three-dimensional (3D) colorectal cancer stromal tissue, utilizing a proprietary material known as collagen microfiber (CMF). CMF proves to be an ideal scaffold material for replicating cancer stromal tissue, and these 3D tissues constructed with CMFs hold promise in contributing to our understanding of cancer and the development of therapeutic drugs

NOR-3, a donor of nitric oxide, increases intracellular Zn²⁺ concentration and decreases cellular thiol content: A model experiment using rat thymocytes, FluoZin-3, and 5-chloromethylfluorescein

Our previous study showed that nitroprusside, a donor of nitric oxide (NO), increased intracellular Zn2+ concentration without affecting cellular content of glutathione (GSH) although it has been proposed that the cytotoxicity of NO is resulted from its interaction with glutathione and zinc. Nitroprusside releases not only NO but also cyanides (Fe(II)CN and Fe(III)CN), CN-, Fe2+, and Fe3+. Therefore, such decomposition products may mask NO-induced action on cellular GSH content. In this study, we used NOR-3 as a donor of NO to reveal the effects of NO on intracellular Zn2+ concentration and cellular GSH content in a cytometric manner with fluorescent probes, FluoZin-3-AM and 5-chloromethylfluorescein diacetate. NOR-3 at 1-3 mM significantly increased intracellular Zn2+ concentration and decreased cellular GSH content. After the removal of extracellular Zn2+ by diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA, a chelator for Zn2+), the increase in intracellular Zn2+ concentration by NOR-3 was still observed although DTPA significantly attenuated the increase in intracellular Zn2+ concentration by NOR-3. Results suggest an involvement of both intracellular Zn2+ release and increase in membrane Zn2+ permeability. It is likely that NO induces oxidative stress, leading to an increase in intracellular Zn2+ concentration

In vitro throughput screening of anticancer drugs using patient-derived cell lines cultured on vascularized three-dimensional stromal tissues

Takahashi Y., Morimura R., Tsukamoto K., et al. In vitro throughput screening of anticancer drugs using patient-derived cell lines cultured on vascularized three-dimensional stromal tissues. Acta Biomaterialia 183, 111 (2024); https://doi.org/10.1016/j.actbio.2024.05.037.The development of high-throughput anticancer drug screening methods using patient-derived cancer cell (PDC) lines that maintain their original characteristics in an in vitro three-dimensional (3D) culture system poses a significant challenge to achieving personalized cancer medicine. Because stromal tissue plays a critical role in the composition and maintenance of the cancer microenvironment, in vitro 3D-culture using reconstructed stromal tissues has attracted considerable attention. Here, a simple and unique in vitro 3D-culture method using heparin and collagen together with fibroblasts and endothelial cells to fabricate vascularized 3D-stromal tissues for in vitro culture of PDCs is reported. Whereas co-treatment with bevacizumab, a monoclonal antibody against vascular endothelial growth factor, and 5-fluorouracil significantly reduced the survival rate of 3D-cultured PDCs to 30%, separate addition of each drug did not induce comparable strong cytotoxicity, suggesting the possibility of evaluating the combined effect of anticancer drugs and angiogenesis inhibitors. Surprisingly, drug evaluation using eight PDC lines with the 3D-culture method resulted in a drug efficacy concordance rate of 75% with clinical outcomes. The model is expected to be applicable to in vitro throughput drug screening for the development of personalized cancer medicine. Statement of significance: To replicate the cancer microenvironment, we constructed a cancer-stromal tissue model in which cancer cells are placed above and inside stromal tissue with vascular network structures derived from vascular endothelial cells in fibroblast tissue using CAViTs method. Using this method, we were able to reproduce the invasion and metastasis processes of cancer cells observed in vivo. Using patient-derived cancer cells, we assessed the possibility of evaluating the combined effect with an angiogenesis inhibitor. Further, primary cancer cells also grew on the stromal tissues with the normal medium. These data suggest that the model may be useful for new in vitro drug screening and personalized cancer medicine

がん遺伝子検査法としてのFRET-PHFA法の開発及び全自動遺伝子解析に向けた基礎評価

主1工学_物質創造工

High-throughput drug screening models of mature adipose tissues which replicate the physiology of patients’ Body Mass Index (BMI)

Obesity is a complex and incompletely understood disease, but current drug screening strategies mostly rely on immature in vitro adipose models which cannot recapitulate it properly. To address this issue, we developed a statistically validated high-throughput screening model by seeding human mature adipocytes from patients, encapsulated in physiological collagen microfibers. These drop tissues ensured the maintenance of adipocyte viability and functionality for controlling glucose and fatty acids uptake, as well as glycerol release. As such, patients’ BMI and insulin sensitivity displayed a strong inverse correlation: the healthy adipocytes were associated with the highest insulin-induced glucose uptake, while insulin resistance was confirmed in the underweight and severely obese adipocytes. Insulin sensitivity recovery was possible with two type 2 diabetes treatments, rosiglitazone and melatonin. Finally, the addition of blood vasculature to the model seemed to more accurately recapitulate the in vivo physiology, with particular respect to leptin secretion metabolism

がん遺伝子検査法としてのFRET-PHFA法の開発及び全自動遺伝子解析に向けた基礎評価

主1工学_物質創造工

Collagen Microfibers Induce Blood Capillary Orientation and Open Vascular Lumen

Achieving vascularization of engineered tissues or structures is a major challenge in the field of tissue engineering. Hitherto, studies on vascularization have demonstrated limited control of vascular network geometry, such as vasculature direction and network density. An open vascular lumen is crucial to ensure that cells survive and that metabolic activity is fully functional in large‐sized tissues. Herein, a method based on high water‐dispersible collagen microfibers (CMF) to fabricate capillary orientation‐controllable 3D tissue with an open vascular lumen using a dispensing machine is reported. A twenty micrometers‐long CMF (CMF‐20) with high dispersion property are shown to be more effective for dispensing a homogenous tissue and inducing formation of an interconnected capillary network than two hundred micrometers‐long CMF (CMF‐200). One of the advantages is the prevention of shrinkage on the z‐axis of hydrogel‐based tissue which acts as a microscaffold. The gaps between the fibers can support endothelial cell migration and maturation, thus forming a larger vascular lumen compared to CMF‐free controls. Besides, shear forces produced by the dispensing process cause the collagen microfibers to align, and these microfibers guide cell alignment by integrin‐induced adhesion. The findings based on CMF to allow blood capillary alignment and vascular lumen stabilization will be an important technology in tissue engineering

Collagen Microfibers Induce Blood Capillary Orientation and Open Vascular Lumen

Achieving vascularization of engineered tissues or structures is a major challenge in the field of tissue engineering. Hitherto, studies on vascularization have demonstrated limited control of vascular network geometry, such as vasculature direction and network density. An open vascular lumen is crucial to ensure that cells survive and that metabolic activity is fully functional in large‐sized tissues. Herein, a method based on high water‐dispersible collagen microfibers (CMF) to fabricate capillary orientation‐controllable 3D tissue with an open vascular lumen using a dispensing machine is reported. A twenty micrometers‐long CMF (CMF‐20) with high dispersion property are shown to be more effective for dispensing a homogenous tissue and inducing formation of an interconnected capillary network than two hundred micrometers‐long CMF (CMF‐200). One of the advantages is the prevention of shrinkage on the z‐axis of hydrogel‐based tissue which acts as a microscaffold. The gaps between the fibers can support endothelial cell migration and maturation, thus forming a larger vascular lumen compared to CMF‐free controls. Besides, shear forces produced by the dispensing process cause the collagen microfibers to align, and these microfibers guide cell alignment by integrin‐induced adhesion. The findings based on CMF to allow blood capillary alignment and vascular lumen stabilization will be an important technology in tissue engineering