Fatty acid synthase as a feasible biomarker for triple negative breast cancer stem cell subpopulation cultured on electrospun scaffolds.

There is no targeted therapy for triple negative breast cancer (TNBC), which presents an aggressive profile and poor prognosis. Recent studies noticed the feasibility of breast cancer stem cells (BCSCs), a small population responsible for tumor initiation and relapse, to become a novel target for TNBC treatments. However, new cell culture supports need to be standardized since traditional two-dimensional (2D) surfaces do not maintain the stemness state of cells. Hence, three-dimensional (3D) scaffolds represent an alternative to study in vitro cell behavior without inducing cell differentiation. In this work, electrospun polycaprolactone scaffolds were used to enrich BCSC subpopulation of MDA-MB-231 and MDA-MB-468 TNBC cells, confirmed by the upregulation of several stemness markers and the existence of an epithelial-to-mesenchymal transition within 3D culture. Moreover, 3D-cultured cells displayed a shift from MAPK to PI3K/AKT/mTOR signaling pathways, accompanied by an enhanced EGFR and HER2 activation, especially at early cell culture times. Lastly, the fatty acid synthase (FASN), a lipogenic enzyme overexpressed in several carcinomas, was found to be hyperactivated in stemness-enriched samples. Its pharmacological inhibition led to stemness diminishment, overcoming the BCSC expansion achieved in 3D culture. Therefore, FASN may represent a novel target for BCSC niche in TNBC samples.

Polycaprolactone Electrospun Scaffolds Produce an Enrichment of Lung Cancer Stem Cells in Sensitive and Resistant EGFRm Lung Adenocarcinoma.

The establishment of a three-dimensional (3D) cell culture model for lung cancer stem cells (LCSCs) is needed because the study of these stem cells is unable to be done using flat surfaces. The study of LCSCs is fundamental due to their key role in drug resistance, tumor recurrence, and metastasis. Hence, the purpose of this work is the evaluation of polycaprolactone electrospun (PCL-ES) scaffolds for culturing LCSCs in sensitive and resistant EGFR-mutated (EGFRm) lung adenocarcinoma cell models. We performed a thermal, physical, and biological characterization of 10% and 15%-PCL-ES structures. Several genes and proteins associated with LCSC features were analyzed by RT-qPCR and Western blot. Vimentin and CD133 tumor expression were evaluated in samples from 36 patients with EGFRm non-small cell lung cancer through immunohistochemistry. Our findings revealed that PC9 and PC9-GR3 models cultured on PCL-ES scaffolds showed higher resistance to osimertinib, upregulation of ABCB1, Vimentin, Snail, Twist, Sox2, Oct-4, and CD166, downregulation of E-cadherin and CD133, and the activation of Hedgehog pathway. Additionally, we determined that the non-expression of CD133 was significantly associated with a low degree of histological differentiation, disease progression, and distant metastasis. To sum up, we confirmed PCL-ES scaffolds as a suitable 3D cell culture model for the study of the LCSC niche.

PLA Electrospun Scaffolds for Three-Dimensional Triple-Negative Breast Cancer Cell Culture.

Three-dimensional (3D) systems provide a suitable environment for cells cultured in vitro since they reproduce the physiological conditions that traditional cell culture supports lack. Electrospinning is a cost-effective technology useful to manufacture scaffolds with nanofibers that resemble the extracellular matrix that surround cells in the organism. Poly(lactic acid) (PLA) is a synthetic polymer suitable for biomedical applications. The main objective of this study is to evaluate electrospun (ES)-PLA scaffolds to be used for culturing cancer cells. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no validated targeted therapy and a high relapse rate. MDA-MB-231 TNBC cells were grown in scaffolds from two different PLA concentrations (12% and 15% w/v). The appropriateness of ES-PLA scaffolds was evaluated using a cell proliferation assay. EGFR and STAT3 gene expression and protein levels were compared in cells grown in 2D versus in 3D cultures. An increase in STAT3 activation was shown, which is related to self-renewal of cancer stem cells (CSCs). Therefore, the enrichment of the breast CSC (BCSC) population was tested using a mammosphere-forming assay and gene expression of BCSC-related stemness and epithelial-to-mesenchymal transition markers. Based on the results obtained, ES-PLA scaffolds are useful for 3D cultures in short culture periods with no BCSC-enrichment.

EGCG-Derivative G28 Shows High Efficacy Inhibiting the Mammosphere-Forming Capacity of Sensitive and Resistant TNBC Models.

Recent studies showed that Fatty Acid Synthase (FASN), a lipogenic enzyme overexpressed in several carcinomas, plays an important role in drug resistance. Furthermore, the enrichment of Breast Cancer Stem Cell (BCSC) features has been found in breast tumors that progressed after chemotherapy. Hence, we used the triple negative breast cancer (TNBC) cell line MDA-MB-231 (231) to evaluate the FASN and BCSC population role in resistance acquisition to chemotherapy. For this reason, parental cell line (231) and its derivatives resistant to doxorubicin (231DXR) and paclitaxel (231PTR) were used. The Mammosphere-Forming Assay and aldehyde dehydrogenase (ALDH) enzyme activity assay showed an increase in BCSCs in the doxorubicin-resistant model. Moreover, the expression of some transcription factors involved in epithelial-mesenchymal transition (EMT), a process that confers BCSC characteristics, was upregulated after chemotherapy treatment. FASN inhibitors C75, (-)-Epigallocatechin 3-gallate (EGCG), and its synthetic derivatives G28, G56 and G37 were used to evaluate the effect of FASN inhibition on the BCSC-enriched population in our cell lines. G28 showed a noticeable antiproliferative effect in adherent conditions and, interestingly, a high mammosphere-forming inhibition capacity in all cell models. Our preliminary results highlight the importance of studying FASN inhibitors for the treatment of TNBC patients, especially those who progress after chemotherapy.

Three-Dimensional Manufactured Supports for Breast Cancer Stem Cell Population Characterization.

Breast Cancer (BC) is the most common cancer among women and the second cause of female death for cancer. When the tumor is not correctly eradicated, there is a high relapse risk and incidence of metastasis. Breast Cancer Stem Cells (BCSCs) are responsible for initiating tumors and are resistant to current anticancer therapies being in part responsible for tumor relapse and metastasis. The study of BCSCs is limited due to their low percentage within both tumors and established cell models. Hence, three-dimensional (3D) supports are presented as an interesting tool to keep the stem-like features in 3D cell culture. In this review, several 3D culture systems are discussed. Moreover, scaffolds are presented as a tool to enrich in BCSCs in order to find new specific therapeutic strategies against this malignant subpopulation. Anticancer treatments focused on BCSCs could be useful for BC patients, with particular interest in those that progress to current therapies.

Screening of Additive Manufactured Scaffolds Designs for Triple Negative Breast Cancer 3D Cell Culture and Stem-Like Expansion.

Breast cancer stem cells (BCSCs) are tumor-initiating cells responsible for metastasis and tumor reappearance, but their research is limited by the impossibility to cultivate them in a monolayer culture. Scaffolds are three-dimensional (3D) cell culture systems which avoid problems related with culturing BCSC. However, a standardized scaffold for enhancing a BCSC population is still an open issue. The main aim of this study is to establish a suitable poly (lactic acid) (PLA) scaffold which will produce BCSC enrichment, thus allowing them to be studied. Different 3D printing parameters were analyzed using Taguchi experimental design methods. Several PLA scaffold architectures were manufactured using a Fused Filament Fabrication (FFF) 3D printer. They were then evaluated by cell proliferation assay and the configurations with the highest growth rates were subjected to BCSC quantification by ALDH activity. The design SS1 (0.2 mm layer height, 70% infill density, Zigzag infill pattern, 45° infill direction, and 100% flow) obtained the highest proliferation rate and was capable of enhancing a ALDH+ cell population compared to 2D cell culture. In conclusion, the data obtained endorse the PLA porous scaffold as useful for culturing breast cancer cells in a microenvironment similar to in vivo and increasing the numbers of BCSCs.

3D-Printed PCL/PLA Composite Stents: Towards a New Solution to Cardiovascular Problems.

Biodegradable stents (BRS) offer enormous potential but first they must meet five specific requirements: (i) their manufacturing process must be precise; (ii) degradation should have minimal toxicity; (iii) the rate of degradation should match the recovery rate of vascular tissue; (iv) ideally, they should induce rapid endothelialization to restore the functions of vascular tissue, but at the same time reduce the risk of restenosis; and (v) their mechanical behavior should comply with medical requirements, namely, the flexibility required to facilitate placement but also sufficient radial rigidity to support the vessel. Although the first three requirements have been comprehensively studied, the last two have been overlooked. One possible way of addressing these issues would be to fabricate composite stents using materials that have different mechanical, biological, or medical properties, for instance, Polylactide Acid (PLA) or Polycaprolactone (PCL). However, fashioning such stents using the traditional stent manufacturing process known as laser cutting would be impossible. Our work, therefore, aims to produce PCL/PLA composite stents using a novel 3D tubular printer based on Fused Deposition Modelling (FDM). The cell geometry (shape and area) and the materials (PCL and PLA) of the stents were analyzed and correlated with 3T3 cell proliferation, degradation rates, dynamic mechanical and radial expansion tests to determine the best parameters for a stent that will satisfy the five strict BRS requirements. Results proved that the 3D-printing process was highly suitable for producing composite stents (approximately 85⁻95% accuracy). Both PCL and PLA demonstrated their biocompatibility with PCL stents presenting an average cell proliferation of 12.46% and PLA 8.28% after only 3 days. Furthermore, the PCL/PLA composite stents demonstrated their potential in degradation, dynamic mechanical and expansion tests. Moreover, and regardless of the order of the layers, the composite stents showed (virtually) medium levels of degradation rates and mechanical modulus. Radially, they exhibited the virtues of PCL in the expansion step (elasticity) and those of PLA in the recoil step (rigidity). Results have clearly demonstrated that composite PCL/PLA stents are a highly promising solution to fulfilling the rigorous BRS requirements.

Design of a Scaffold Parameter Selection System with Additive Manufacturing for a Biomedical Cell Culture.

Open-source 3D printers mean objects can be quickly and efficiently produced. However, design and fabrication parameters need to be optimized to set up the correct printing procedure; a procedure in which the characteristics of the printing materials selected for use can also influence the process. This work focuses on optimizing the printing process of the open-source 3D extruder machine RepRap, which is used to manufacture poly(ε-caprolactone) (PCL) scaffolds for cell culture applications. PCL is a biocompatible polymer that is free of toxic dye and has been used to fabricate scaffolds, i.e., solid structures suitable for 3D cancer cell cultures. Scaffold cell culture has been described as enhancing cancer stem cell (CSC) populations related to tumor chemoresistance and/or their recurrence after chemotherapy. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. Design and fabrication parameters were first determined with SolidWorks and Slic3r software and subsequently optimized following a novel sequential flowchart. In the flowchart described here, the parameters were gradually optimized step by step, by taking several measurable variables of the resulting scaffolds into consideration to guarantee high-quality printing. Three deposition angles (45°, 60° and 90°) were fabricated and tested. MCF-7 breast carcinoma cells and NIH/3T3 murine fibroblasts were used to assess scaffold adequacy for 3D cell cultures. The 60° scaffolds were found to be suitable for the purpose. Therefore, PCL scaffolds fabricated via the flowchart optimization with a RepRap 3D printer could be used for 3D cell cultures and may boost CSCs to study new therapeutic treatments for this malignant population. Moreover, the flowchart defined here could represent a standard procedure for non-engineers (i.e., mainly physicians) when manufacturing new culture systems is required.

Electrospinning PCL Scaffolds Manufacture for Three-Dimensional Breast Cancer Cell Culture.

In vitro cell culture is traditionally performed within two-dimensional (2D) environments, providing a quick and cheap way to study cell properties in a laboratory. However, 2D systems differ from the in vivo environment and may not mimic the physiological cell behavior realistically. For instance, 2D culture models are thought to induce cancer stem cells (CSCs) differentiation, a rare cancer cell subpopulation responsible for tumor initiation and relapse. This fact hinders the development of therapeutic strategies for tumors with a high relapse percentage, such as triple negative breast cancer (TNBC). Thus, three-dimensional (3D) scaffolds have emerged as an attractive alternative to monolayer culture, simulating the extracellular matrix structure and maintaining the differentiation state of cells. In this work, scaffolds were fabricated through electrospinning different poly(ε-caprolactone)-acetone solutions. Poly(ε-caprolactone) (PCL) meshes were seeded with triple negative breast cancer (TNBC) cells and 15% PCL scaffolds displayed significantly (p < 0.05) higher cell proliferation and elongation than the other culture systems. Moreover, cells cultured on PCL scaffolds exhibited higher mammosphere forming capacity and aldehyde dehydrogenase activity than 2D-cultured cells, indicating a breast CSCs enrichment. These results prove the powerful capability of electrospinning technology in terms of poly(ε-caprolactone) nanofibers fabrication. In addition, this study has demonstrated that electrospun 15% PCL scaffolds are suitable tools to culture breast cancer cells in a more physiological way and to expand the niche of breast CSCs. In conclusion, three-dimensional cell culture using PCL scaffolds could be useful to study cancer stem cell behavior and may also trigger the development of new specific targets against such malignant subpopulation.

Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone) Scaffolds.

The cancer stem cell (CSC) population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs' phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone) (PCL), a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI) was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control). Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population.