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15 pages, 2755 KiB

Open AccessArticle

Pirfenidone Reduces Epithelial–Mesenchymal Transition and Spheroid Formation in Breast Carcinoma through Targeting Cancer-Associated Fibroblasts (CAFs)

by Hamidreza Aboulkheyr Es, Thomas R Cox, Ehsan Sarafraz-Yazdi, Jean Paul Thiery and Majid Ebrahimi Warkiani

Cancers 2021, 13(20), 5118; https://doi.org/10.3390/cancers13205118 - 13 Oct 2021

Cited by 13 | Viewed by 3045

Abstract

The aim of this study was to assess the effects of pirfenidone (PFD) on promoting epithelial–mesenchymal-transition (EMT) and stemness features in breast carcinoma cells through targeting cancer-associated-fibroblasts (CAFs). Using The Cancer Genome Atlas (TCGA) database, we analyzed the association between stromal index, EMT, [...] Read more.

The aim of this study was to assess the effects of pirfenidone (PFD) on promoting epithelial–mesenchymal-transition (EMT) and stemness features in breast carcinoma cells through targeting cancer-associated-fibroblasts (CAFs). Using The Cancer Genome Atlas (TCGA) database, we analyzed the association between stromal index, EMT, and stemness-related genes across 1084 breast cancer patients, identifying positive correlation between YAP1, EMT, and stemness genes in samples with a high-stromal index. We monitored carcinoma cell invasion and spheroid formation co-cultured with CAFs in a 3D microfluidic device, followed by exposing carcinoma cells, spheroids, and CAFs with PFD. We depicted a positive association between the high-stromal index and the expression of EMT and stemness genes. High YAP1 expression in samples correlated with more advanced EMT status and stromal index. Additionally, we found that CAFs promoted spheroid formation and induced the expression of YAP1, VIM, and CD44 in spheroids. Treatment with PFD reduced carcinoma cell migration and decreased the expression of these genes at the protein level. The cytokine profiling showed significant depletion of various EMT- and stemness-regulated cytokines, particularly IL8, CCL17, and TNF-beta. These data highlight the potential application of PFD on inhibiting EMT and stemness in carcinoma cells through the targeting of critical cytokines. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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19 pages, 2959 KiB

Open AccessArticle

A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments

by Valentina Palacio-Castañeda, Simon Dumas, Philipp Albrecht, Thijmen J. Wijgers, Stéphanie Descroix and Wouter P. R. Verdurmen

Cancers 2021, 13(10), 2461; https://doi.org/10.3390/cancers13102461 - 18 May 2021

Cited by 8 | Viewed by 3004

Abstract

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin [...] Read more.

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm²·s⁻¹ and 2 µm² ·s⁻¹ for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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14 pages, 3629 KiB

Open AccessArticle

Testing Lab-on-a-Chip Technology for Culturing Human Melanoma Cells under Simulated Microgravity

by Dawid Przystupski, Agata Górska, Olga Michel, Agnieszka Podwin, Patrycja Śniadek, Radosław Łapczyński, Jolanta Saczko and Julita Kulbacka

Cancers 2021, 13(3), 402; https://doi.org/10.3390/cancers13030402 - 22 Jan 2021

Cited by 19 | Viewed by 3526

Abstract

The dynamic development of the space industry makes space flights more accessible and opens up new opportunities for biological research to better understand cell physiology under real microgravity. Whereas specialized studies in space remain out of our reach, preliminary experiments can be performed [...] Read more.

The dynamic development of the space industry makes space flights more accessible and opens up new opportunities for biological research to better understand cell physiology under real microgravity. Whereas specialized studies in space remain out of our reach, preliminary experiments can be performed on Earth under simulated microgravity (sµg). Based on this concept, we used a 3D-clinostat (3D-C) to analyze the effect of short exposure to sµg on human keratinocytes HaCaT and melanoma cells A375 cultured on all-glass Lab-on-a-Chip (LOC). Our preliminary studies included viability evaluation, mitochondrial and caspase activity, and proliferation assay, enabling us to determine the effect of sµg on human cells. By comparing the results concerning cells cultured on LOCs and standard culture dishes, we were able to confirm the biocompatibility of all-glass LOCs and their potential application in microgravity research on selected human cell lines. Our studies revealed that HaCaT and A375 cells are susceptible to simulated microgravity; however, we observed an increased caspase activity and a decrease of proliferation in cancer cells cultured on LOCs in comparison to standard cell cultures. These results are an excellent basis to conduct further research on the possible application of LOCs systems in cancer research in space. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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17 pages, 2262 KiB

Open AccessArticle

HIF2alpha-Associated Pseudohypoxia Promotes Radioresistance in Pheochromocytoma: Insights from 3D Models

by Verena Seifert, Susan Richter, Nicole Bechmann, Michael Bachmann, Christian G. Ziegler, Jens Pietzsch and Martin Ullrich

Cancers 2021, 13(3), 385; https://doi.org/10.3390/cancers13030385 - 21 Jan 2021

Cited by 12 | Viewed by 3456

Abstract

Pheochromocytomas and paragangliomas (PCCs/PGLs) are rare neuroendocrine tumors arising from chromaffin tissue located in the adrenal or ganglia of the sympathetic or parasympathetic nervous system. The treatment of non-resectable or metastatic PCCs/PGLs is still limited to palliative measures, including somatostatin type 2 receptor [...] Read more.

Pheochromocytomas and paragangliomas (PCCs/PGLs) are rare neuroendocrine tumors arising from chromaffin tissue located in the adrenal or ganglia of the sympathetic or parasympathetic nervous system. The treatment of non-resectable or metastatic PCCs/PGLs is still limited to palliative measures, including somatostatin type 2 receptor radionuclide therapy with [¹⁷⁷Lu]Lu-DOTA-TATE as one of the most effective approaches to date. Nevertheless, the metabolic and molecular determinants of radiation response in PCCs/PGLs have not yet been characterized. This study investigates the effects of hypoxia-inducible factor 2 alpha (HIF2α) on the susceptibility of PCCs/PGLs to radiation treatments using spheroids grown from genetically engineered mouse pheochromocytoma (MPC) cells. The expression of Hif2α was associated with the significantly increased resistance of MPC spheroids to external X-ray irradiation and exposure to beta particle-emitting [¹⁷⁷Lu]LuCl₃ compared to Hif2α-deficient controls. Exposure to [¹⁷⁷Lu]LuCl₃ provided an increased long-term control of MPC spheroids compared to single-dose external X-ray irradiation. This study provides the first experimental evidence that HIF2α-associated pseudohypoxia contributes to a radioresistant phenotype of PCCs/PGLs. Furthermore, the external irradiation and [¹⁷⁷Lu]LuCl₃ exposure of MPC spheroids provide surrogate models for radiation treatments to further investigate the metabolic and molecular determinants of radiation responses in PCCs/PGLs and evaluate the effects of neo-adjuvant—in particular, radiosensitizing—treatments in combination with targeted radionuclide therapies. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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16 pages, 8507 KiB

Open AccessArticle

Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip

by Min-Suk Oh, Iftikhar Ali Khawar, Dong Woo Lee, Jong Kook Park and Hyo-Jeong Kuh

Cancers 2020, 12(12), 3662; https://doi.org/10.3390/cancers12123662 - 07 Dec 2020

Cited by 7 | Viewed by 2933

Abstract

Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) represents a microtumor model that recapitulates human solid tumors in vivo, and is useful for 3D multiplex phenotypic analysis. However, the low efficiency of 3D culture and limited 3D visualization of [...] Read more.

Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) represents a microtumor model that recapitulates human solid tumors in vivo, and is useful for 3D multiplex phenotypic analysis. However, the low efficiency of 3D culture and limited 3D visualization of microtumor specimens impose technical hurdles for the evaluation of TS-based phenotypic analysis. Here, we report a 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with a tissue optical clearing (TOC) method for high-content phenotypic analysis of microtumors. To prove the utility of this method, phenotypic changes in TSs of human pancreatic cancer cells were determined by co-culture with cancer-associated fibroblasts and M2-type tumor-associated macrophages. Significant improvement was achieved in immunostaining and optical transmission in each TS as well as the entire microtumor specimen, enabling optimization in image-based analysis of the morphology, structural organization, and protein expression in cancer cells and the ECM. Changes in the invasive phenotype, including cellular morphology and expression of epithelial–mesenchymal transition-related proteins and drug-induced apoptosis under stromal cell co-culture were also successfully analyzed. Overall, our study demonstrates that a minipillar array chip combined with TOC offers a novel system for 3D culture-to-3D visualization of microtumors to facilitate high-content phenotypic analysis. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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21 pages, 2893 KiB

Open AccessArticle

Physical Cues in the Microenvironment Regulate Stemness-Dependent Homing of Breast Cancer Cells

by Hsueh-Yao Chu, Yin-Ju Chen, Chun-Jieh Hsu, Yang-Wei Liu, Jeng-Fong Chiou, Long-Sheng Lu and Fan-Gang Tseng

Cancers 2020, 12(8), 2176; https://doi.org/10.3390/cancers12082176 - 05 Aug 2020

Cited by 4 | Viewed by 3408

Abstract

Tissue-specific microenvironmental factors contribute to the targeting preferences of metastatic cancers. However, the physical attributes of the premetastatic microenvironment are not yet fully characterized. In this research, we develop a transwell-based alginate hydrogel (TAH) model to study how permeability, stiffness, and roughness of [...] Read more.

Tissue-specific microenvironmental factors contribute to the targeting preferences of metastatic cancers. However, the physical attributes of the premetastatic microenvironment are not yet fully characterized. In this research, we develop a transwell-based alginate hydrogel (TAH) model to study how permeability, stiffness, and roughness of a hanging alginate hydrogel regulate breast cancer cell homing. In this model, a layer of physically characterized alginate hydrogel is formed at the bottom of a transwell insert, which is placed into a matching culture well with an adherent monolayer of breast cancer cells. We found that breast cancer cells dissociate from the monolayer and home to the TAH for continual growth. The process is facilitated by the presence of rich serum in the upper chamber, the increased stiffness of the gel, as well as its surface roughness. This model is able to support the homing ability of MCF-7 and MDA-MB-231 cells drifting across the vertical distance in the culture medium. Cells homing to the TAH display stemness phenotype morphologically and biochemically. Taken together, these findings suggest that permeability, stiffness, and roughness are important physical factors to regulate breast cancer homing to a premetastatic microenvironment. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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Review

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25 pages, 3110 KiB

Open AccessReview

Organ-on-a-Chip Platforms for Drug Screening and Delivery in Tumor Cells: A Systematic Review

by Inês M. Gonçalves, Violeta Carvalho, Raquel O. Rodrigues, Diana Pinho, Senhorinha F. C. F. Teixeira, Ana Moita, Takeshi Hori, Hirokazu Kaji, Rui Lima and Graça Minas

Cancers 2022, 14(4), 935; https://doi.org/10.3390/cancers14040935 - 13 Feb 2022

Cited by 30 | Viewed by 8553

Abstract

The development of cancer models that rectify the simplicity of monolayer or static cell cultures physiologic microenvironment and, at the same time, replicate the human system more accurately than animal models has been a challenge in biomedical research. Organ-on-a-chip (OoC) devices are a [...] Read more.

The development of cancer models that rectify the simplicity of monolayer or static cell cultures physiologic microenvironment and, at the same time, replicate the human system more accurately than animal models has been a challenge in biomedical research. Organ-on-a-chip (OoC) devices are a solution that has been explored over the last decade. The combination of microfluidics and cell culture allows the design of a dynamic microenvironment suitable for the evaluation of treatments’ efficacy and effects, closer to the response observed in patients. This systematic review sums the studies from the last decade, where OoC with cancer cell cultures were used for drug screening assays. The studies were selected from three databases and analyzed following the research guidelines for systematic reviews proposed by PRISMA. In the selected studies, several types of cancer cells were evaluated, and the majority of treatments tested were standard chemotherapeutic drugs. Some studies reported higher drug resistance of the cultures on the OoC devices than on 2D cultures, which indicates the better resemblance to in vivo conditions of the former. Several studies also included the replication of the microvasculature or the combination of different cell cultures. The presence of vasculature can influence positively or negatively the drug efficacy since it contributes to a greater diffusion of the drug and also oxygen and nutrients. Co-cultures with liver cells contributed to the evaluation of the systemic toxicity of some drugs metabolites. Nevertheless, few studies used patient cells for the drug screening assays. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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32 pages, 3634 KiB

Open AccessReview

The Advances in Glioblastoma On-a-Chip for Therapy Approaches

by Arielly H. Alves, Mariana P. Nucci, Javier B. Mamani, Nicole M. E. Valle, Eduarda F. Ribeiro, Gabriel N. A. Rego, Fernando A. Oliveira, Matheus H. Theinel, Ricardo S. Santos and Lionel F. Gamarra

Cancers 2022, 14(4), 869; https://doi.org/10.3390/cancers14040869 - 09 Feb 2022

Cited by 8 | Viewed by 3412

Abstract

This systematic review aimed to verify the use of microfluidic devices in the process of implementing and evaluating the effectiveness of therapeutic approaches in glioblastoma on-a-chip, providing a broad view of advances to date in the use of this technology and their perspectives. [...] Read more.

This systematic review aimed to verify the use of microfluidic devices in the process of implementing and evaluating the effectiveness of therapeutic approaches in glioblastoma on-a-chip, providing a broad view of advances to date in the use of this technology and their perspectives. We searched studies with the variations of the keywords “Glioblastoma”, “microfluidic devices”, “organ-on-a-chip” and “therapy” of the last ten years in PubMed and Scopus databases. Of 446 articles identified, only 22 articles were selected for analysis according to the inclusion and exclusion criteria. The microfluidic devices were mainly produced by soft lithography technology, using the PDMS material (72%). In the microenvironment, the main extracellular matrix used was collagen type I. Most studies used U87-MG glioblastoma cells from humans and 31.8% were co-cultivated with HUVEC, hCMEC/D3, and astrocytes. Chemotherapy was the majority of therapeutic approaches, assessing mainly the cellular viability and proliferation. Furthermore, some alternative therapies were reported in a few studies (22.6%). This study identified a diversity of glioblastoma on-a-chip to assess therapeutic approaches, often using intermediate levels of complexity. The most advanced level implemented the intersection between different biological systems (liver–brain or intestine–liver–brain), BBB model, allowing in vitro studies with greater human genetic similarity, reproducibility, and low cost, in a highly customizable platform. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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21 pages, 4166 KiB

Open AccessReview

Electrochemical Sensing in 3D Cell Culture Models: New Tools for Developing Better Cancer Diagnostics and Treatments

by Micaela Oliveira, Pedro Conceição, Krishna Kant, Alar Ainla and Lorena Diéguez

Cancers 2021, 13(6), 1381; https://doi.org/10.3390/cancers13061381 - 18 Mar 2021

Cited by 17 | Viewed by 4867

Abstract

Currently, conventional pre-clinical in vitro studies are primarily based on two-dimensional (2D) cell culture models, which are usually limited in mimicking the real three-dimensional (3D) physiological conditions, cell heterogeneity, cell to cell interaction, and extracellular matrix (ECM) present in living tissues. Traditionally, animal [...] Read more.

Currently, conventional pre-clinical in vitro studies are primarily based on two-dimensional (2D) cell culture models, which are usually limited in mimicking the real three-dimensional (3D) physiological conditions, cell heterogeneity, cell to cell interaction, and extracellular matrix (ECM) present in living tissues. Traditionally, animal models are used to mimic the 3D environment of tissues and organs, but they suffer from high costs, are time consuming, bring up ethical concerns, and still present many differences when compared to the human body. The applications of microfluidic-based 3D cell culture models are advantageous and useful as they include 3D multicellular model systems (MCMS). These models have demonstrated potential to simulate the in vivo 3D microenvironment with relatively low cost and high throughput. The incorporation of monitoring capabilities in the MCMS has also been explored to evaluate in real time biophysical and chemical parameters of the system, for example temperature, oxygen, pH, and metabolites. Electrochemical sensing is considered as one of the most sensitive and commercially adapted technologies for bio-sensing applications. Amalgamation of electrochemical biosensing with cell culture in microfluidic devices with improved sensitivity and performance are the future of 3D systems. Particularly in cancer, such models with integrated sensing capabilities can be crucial to assess the multiple parameters involved in tumour formation, proliferation, and invasion. In this review, we are focusing on existing 3D cell culture systems with integrated electrochemical sensing for potential applications in cancer models to advance diagnosis and treatment. We discuss their design, sensing principle, and application in the biomedical area to understand the potential relevance of miniaturized electrochemical hybrid systems for the next generation of diagnostic platforms for precision medicine. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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21 pages, 3379 KiB

Open AccessReview

Radiobiological Studies of Microvascular Damage through In Vitro Models: A Methodological Perspective

by Luca Possenti, Laura Mecchi, Andrea Rossoni, Veronica Sangalli, Simone Bersini, Alessandro Cicchetti, Maria Laura Costantino, Christian Candrian, Chiara Arrigoni, Tiziana Rancati and Matteo Moretti

Cancers 2021, 13(5), 1182; https://doi.org/10.3390/cancers13051182 - 09 Mar 2021

Cited by 6 | Viewed by 3953

Abstract

Ionizing radiation (IR) is used in radiotherapy as a treatment to destroy cancer. Such treatment also affects other tissues, resulting in the so-called normal tissue complications. Endothelial cells (ECs) composing the microvasculature have essential roles in the microenvironment’s homeostasis (ME). Thus, detrimental effects [...] Read more.

Ionizing radiation (IR) is used in radiotherapy as a treatment to destroy cancer. Such treatment also affects other tissues, resulting in the so-called normal tissue complications. Endothelial cells (ECs) composing the microvasculature have essential roles in the microenvironment’s homeostasis (ME). Thus, detrimental effects induced by irradiation on ECs can influence both the tumor and healthy tissue. In-vitro models can be advantageous to study these phenomena. In this systematic review, we analyzed in-vitro models of ECs subjected to IR. We highlighted the critical issues involved in the production, irradiation, and analysis of such radiobiological in-vitro models to study microvascular endothelial cells damage. For each step, we analyzed common methodologies and critical points required to obtain a reliable model. We identified the generation of a 3D environment for model production and the inclusion of heterogeneous cell populations for a reliable ME recapitulation. Additionally, we highlighted how essential information on the irradiation scheme, crucial to correlate better observed in vitro effects to the clinical scenario, are often neglected in the analyzed studies, limiting the translation of achieved results. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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28 pages, 2617 KiB

Open AccessReview

Three-Dimensional Cell Culture Systems in Radiopharmaceutical Cancer Research

by Alina Doctor, Verena Seifert, Martin Ullrich, Sandra Hauser and Jens Pietzsch

Cancers 2020, 12(10), 2765; https://doi.org/10.3390/cancers12102765 - 25 Sep 2020

Cited by 33 | Viewed by 4668

Abstract

In preclinical cancer research, three-dimensional (3D) cell culture systems such as multicellular spheroids and organoids are becoming increasingly important. They provide valuable information before studies on animal models begin and, in some cases, are even suitable for reducing or replacing animal experiments. Furthermore, [...] Read more.

In preclinical cancer research, three-dimensional (3D) cell culture systems such as multicellular spheroids and organoids are becoming increasingly important. They provide valuable information before studies on animal models begin and, in some cases, are even suitable for reducing or replacing animal experiments. Furthermore, they recapitulate microtumors, metastases, and the tumor microenvironment much better than monolayer culture systems could. Three-dimensional models show higher structural complexity and diverse cell interactions while reflecting (patho)physiological phenomena such as oxygen and nutrient gradients in the course of their growth or development. These interactions and properties are of great importance for understanding the pathophysiological importance of stromal cells and the extracellular matrix for tumor progression, treatment response, or resistance mechanisms of solid tumors. Special emphasis is placed on co-cultivation with tumor-associated cells, which further increases the predictive value of 3D models, e.g., for drug development. The aim of this overview is to shed light on selected 3D models and their advantages and disadvantages, especially from the radiopharmacist’s point of view with focus on the suitability of 3D models for the radiopharmacological characterization of novel radiotracers and radiotherapeutics. Special attention is paid to pancreatic ductal adenocarcinoma (PDAC) as a predestined target for the development of new radionuclide-based theranostics. Full article

(This article belongs to the Special Issue Cancer-on-a-Chip: Applications and Challenges)

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