AZ12756122, a novel fatty acid synthase inhibitor, decreases resistance features in EGFR-TKI resistant EGFR-mutated NSCLC cell models.

Different EGFR tyrosine kinase inhibitors (TKIs) have been developed for the treatment of non-small cell lung cancer (NSCLC) patients harboring sensitizing mutations in the EGFR gene. Apart from acquired secondary mutations, multiple resistance mechanisms have been reported, such as the overexpression of fatty acid synthase (FASN), a multi-functional enzyme essential for the de novo lipogenesis, or the increase of cancer stem cells, a small subpopulation within the tumor responsible for relapse, metastasis, and resistance to therapies. Hence, the purpose of this work is to evaluate the novel FASN inhibitor AZ12756122, both alone and in combination with gefitinib and osimertinib, in EGFR-mutated (EGFRm) lung adenocarcinoma cell models sensitive and resistant to EGFR-TKIs. The molecular effect of AZ12756122 (alone and in combination with EGFR-TKI) on FASN, EGFR/STAT3, Akt/mTOR, and MAPK signaling pathways was analyzed using RT-qPCR and Western blot. FASN expression was also evaluated in samples from patients with EGFRm NSCLC through immunohistochemistry. Our findings revealed that AZ12756122 caused cytotoxic effects inducing apoptosis, downregulated FASN expression and activity, decreased the activation of EGFR and Akt/mTOR pathway, and reduced cancer stem-like cells. Furthermore, the combination of AZ12756122 and osimertinib sensitized cells to EGFR-TKI, showing a synergistic effect that resulted in a reduction in the activation of EGFR, Akt/mTOR, and MAPK signaling pathways. Our study also showed that FASN+ EGFRm NSCLC patients exhibited a longer mPFS in patients who responded to EGFR-TKI treatment. In conclusion, FASN inhibition should be further studied for the treatment, alone or in combination with EGFR-TKIs, for EGFRm NSCLC patients.

The solvent chosen for the manufacturing of electrospun polycaprolactone scaffolds influences cell behavior of lung cancer cells.

The development of a trustworthy in vitro lung cancer model is essential to better understand the illness, find novel biomarkers, and establish new treatments. Polycaprolactone (PCL) electrospun nanofibers are a cost-effective and ECM-like approach for 3D cell culture. However, the solvent used to prepare the polymer solution has a significant impact on the fiber morphology and, consequently, on the cell behavior. Hence, the present study evaluated the effect of the solvent employed in the manufacturing on the physical properties of 15%-PCL electrospun scaffolds and consequently, on cell behavior of NCI-H1975 lung adenocarcinoma cells. Five solvents mixtures (acetic acid, acetic acid-formic acid (3:1, v/v), acetone, chloroform-ethanol (7:3, v/v), and chloroform-dichloromethane (7:3, v/v)) were tested. The highest cell viability ([Formula: see text] = 33.4%) was found for cells cultured on chloroform-ethanol (7:3) PCL scaffolds. Chloroform-dichloromethane (7:3) PCL scaffolds exhibited a roughness that enhanced the quality of electrospun filament, in terms of cell viability. Our findings highlighted the influence of the solvent on fiber morphology and protein adsorption capacity of nanofilaments. Consequently, these features directly affected cell attachment, morphology, and viability.

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.

Fatty Acid Synthase Inhibitor G28 Shows Anticancer Activity in EGFR Tyrosine Kinase Inhibitor Resistant Lung Adenocarcinoma Models.

Epidermal growth factor receptor (EGFR) tyrosine kinases inhibitors (TKIs) are effective therapies for non-small cell lung cancer (NSCLC) patients whose tumors harbor an EGFR activating mutation. However, this treatment is not curative due to primary and secondary resistance such as T790M mutation in exon 20. Recently, activation of transducer and activator of transcription 3 (STAT3) in NSCLC appeared as an alternative resistance mechanism allowing cancer cells to elude the EGFR signaling. Overexpression of fatty acid synthase (FASN), a multifunctional enzyme essential for endogenous lipogenesis, has been related to resistance and the regulation of the EGFR/Jak2/STAT signaling pathways. Using EGFR mutated (EGFRm) NSCLC sensitive and EGFR TKIs' resistant models (Gefitinib Resistant, GR) we studied the role of the natural polyphenolic anti-FASN compound (-)-epigallocatechin-3-gallate (EGCG), and its derivative G28 to overcome EGFR TKIs' resistance. We show that G28's cytotoxicity is independent of TKIs' resistance mechanisms displaying synergistic effects in combination with gefitinib and osimertinib in the resistant T790M negative (T790M-) model and showing a reduction of activated EGFR and STAT3 in T790M positive (T790M+) models. Our results provide the bases for further investigation of G28 in combination with TKIs to overcome the EGFR TKI resistance in NSCLC.

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.

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.