Hypoxia-Induced FAM13A Regulates the Proliferation and Metastasis of Non-Small Cell Lung Cancer Cells

Hypoxia in non-small cell lung cancer (NSCLC) affects cancer progression, metastasis and metabolism. We previously showed that FAM13A was induced by hypoxia in NSCLC but the biological function of this gene has not been fully elucidated. This study aimed to investigate the role of hypoxia-induced FAM13A in NSCLC progression and metastasis. Lentiviral shRNAs were used for FAM13A gene silencing in NSCLC cell lines (A549, CORL-105). MTS assay, cell tracking VPD540 dye, wound healing assay, invasion assay, BrdU assay and APC Annexin V staining assays were performed to examine cell proliferation ability, migration, invasion and apoptosis rate in NSCLC cells. The results of VPD540 dye and MTS assays showed a significant reduction in cell proliferation after FAM13A knockdown in A549 cells cultured under normal and hypoxia (1% O₂) conditions (p < 0.05), while the effect of FAM13A downregulation on CORL-105 cells was observed after 96 h exposition to hypoxia. Moreover, FAM13A inhibition induced S phase cell cycle arrest in A549 cells under hypoxia conditions. Silencing of FAM13A significantly suppressed migration of A549 and CORL-105 cells in both oxygen conditions, especially after 72 and 96 h (p < 0.001 in normoxia, p < 0.01 after hypoxia). It was showed that FAM13A reduction resulted in disruption of the F-actin cytoskeleton altering A549 cell migration. Cell invasion rates were significantly decreased in A549 FAM13A depleted cells compared to controls (p < 0.05), mostly under hypoxia. FAM13A silencing had no effect on apoptosis induction in NSCLC cells. In the present study, we found that FAM13A silencing has a negative effect on proliferation, migration and invasion activity in NSCLC cells in normal and hypoxic conditions. Our data demonstrated that FAM13A depleted post-hypoxic cells have a decreased cell proliferation ability and metastatic potential, which indicates FAM13A as a potential therapeutic target in lung cancer.     

MiR-122 Induces Radiosensitization in Non-Small Cell Lung Cancer Cell Line

MiR-122 is a novel tumor suppresser and its expression induces cell cycle arrest, or apoptosis, and inhibits cell proliferation in multiple cancer cells, including non-small cell lung cancer (NSCLC) cells. Radioresistance of cancer cell leads to the major drawback of radiotherapy for NSCLC and the induction of radiosensitization could be a useful strategy to fix this problem. The present work investigates the function of miR-122 in inducing radiosensitization in A549 cell, a type of NSCLC cells. MiR-122 induces the radiosensitization of A549 cells. MiR-122 also boosts the inhibitory activity of ionizing radiation (IR) on cancer cell anchor-independent growth and invasion. Moreover, miR-122 reduced the expression of its targeted genes related to tumor-survival or cellular stress response. These results indicate that miR-122 would be a novel strategy for NSCLC radiation-therapy.     

Three-Dimensional Cell Culture: A Breakthrough in Vivo

Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model the in vivo state. As compared to the two-dimensional case, the three-dimensional (3D) cell culture allows biological cells to grow or interact with their surroundings in all three dimensions thanks to an artificial environment. Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in several studies of biological mechanisms like: cell number monitoring, viability, morphology, proliferation, differentiation, response to stimuli, migration and invasion of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug metabolism, gene expression and protein synthesis, general cell function and in vivo relevance. 3D culture models succeed thanks to technological advances, including materials science, cell biology and bioreactor design.     

Epithelial Membrane Protein 2 Suppresses Non-Small Cell Lung Cancer Cell Growth by Inhibition of MAPK Pathway

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs.     

EGFR Transgene Stimulates Spontaneous Formation of MCF7 Breast Cancer Cells Spheroids with Partly Loss of HER3 Receptor

Multicellular spheroids with 3D cell–cell interactions are a useful model to simulate the growth conditions of cancer. There is evidence that in tumor spheroids, the expression of various essential molecules is changed compared to the adherent form of cell cultures. These changes include growth factor receptors and ABC transporters and result in the enhanced invasiveness of the cells and drug resistance. It is known that breast adenocarcinoma MCF7 cells can spontaneously form 3D spheroids and such spheroids are characterized by high expression of EGFR/HER2, while the natural phenotype of MCF7 cells is EGFR^low/HER2^low. Therefore, it was interesting to reveal if high epidermal growth factor receptor (EGFR) expression is sufficient for the conversion of adherent MCF7 to spheroids. In this study, an MCF7 cell line with high expression of EGFR was engineered using the retroviral transduction method. These MCF7-EGFR cells assembled in spheroids very quickly and grew predominantly as a 3D suspension culture with no special plates, scaffolds, growth supplements, or exogenous matrixes. These spheroids were characterized by a rounded shape with a well-defined external border and 100 µM median diameter. The sphere-forming ability of MCF7-EGFR cells was up to 5 times stronger than in MCF7^wt cells. Thus, high EGFR expression was the initiation factor of conversion of adherent MCF7^wt cells to spheroids. MCF7-EGFR spheroids were enriched by the cells with a cancer stem cell (CSC) phenotype CD24^−/low/CD44⁻ in comparison with parental MCF7^wt cells and MCF7-EGFR adhesive cells. We suppose that these properties of MCF7-EGFR spheroids originate from the typical features of parental MCF7 cells. We showed the decreasing of HER3 receptors in MCF7-EGFR spheroids compared to that in MCF^wt and in adherent MCF7-EGFR cells, and the same decrease was observed in the MCF7^wt spheroids growing under the growth factors stimulation. To summarize, the expression of EGFR transgene in MCF7 cells stimulates rapid spheroids formation; these spheroids are enriched by CSC-like CD24⁻/CD44⁻ cells, they partly lose HER3 receptors, and are characterized by a lower potency in drug resistance pomp activation compared to MCF7^wt. These MCF7-EGFR spheroids are a useful cancer model for the development of anticancer drugs, including EGFR-targeted therapeutics.     

Spheroid Culture Differentially Affects Cancer Cell Sensitivity to Drugs in Melanoma and RCC Models

2D culture as a model for drug testing often turns to be clinically futile. Therefore, 3D cultures (3Ds) show potential to better model responses to drugs observed in vivo. In preliminary studies, using melanoma (B16F10) and renal (RenCa) cancer, we confirmed that 3Ds better mimics the tumor microenvironment. Here, we evaluated how the proposed 3D mode of culture affects tumor cell susceptibility to anti-cancer drugs, which have distinct mechanisms of action (everolimus, doxorubicin, cisplatin). Melanoma spheroids showed higher resistance to all used drugs, as compared to 2D. In an RCC model, such modulation was only observed for doxorubicin treatment. As drug distribution was not affected by the 3D shape, we assessed the expression of MDR1 and mTor. Upregulation of MDR1 in RCC spheroids was observed, in contrast to melanoma. In both models, mTor expression was not affected by the 3D cultures. By NGS, 10 genes related with metabolism of xenobiotics by cytochrome p450 were deregulated in renal cancer spheroids; 9 of them were later confirmed in the melanoma model. The differences between 3D models and classical 2D cultures point to the potential to uncover new non-canonical mechanisms to explain drug resistance set by the tumor in its microenvironment.     

Differential Angiogenic Potential of 3-Dimension Spheroid of HNSCC Cells in Mouse Xenograft

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.     

Advanced Glycation End-Products Enhance Lung Cancer Cell Invasion and Migration

Effects of carboxymethyllysine (CML) and pentosidine, two advanced glycation end-products (AGEs), upon invasion and migration in A549 and Calu-6 cells, two non-small cell lung cancer (NSCLC) cell lines were examined. CML or pentosidine at 1, 2, 4, 8 or 16 μmol/L were added into cells. Proliferation, invasion and migration were measured. CML or pentosidine at 4–16 μmol/L promoted invasion and migration in both cell lines, and increased the production of reactive oxygen species, tumor necrosis factor-α, interleukin-6 and transforming growth factor-β1. CML or pentosidine at 2–16 μmol/L up-regulated the protein expression of AGE receptor, p47^phox, intercellular adhesion molecule-1 and fibronectin in test NSCLC cells. Matrix metalloproteinase-2 protein expression in A549 and Calu-6 cells was increased by CML or pentosidine at 4–16 μmol/L. These two AGEs at 2–16 μmol/L enhanced nuclear factor κ-B (NF-κ B) p65 protein expression and p38 phosphorylation in A549 cells. However, CML or pentosidine at 4–16 μmol/L up-regulated NF-κB p65 and p-p38 protein expression in Calu-6 cells. These findings suggest that CML and pentosidine, by promoting the invasion, migration and production of associated factors, benefit NSCLC metastasis.     

An Evaluation of Different 3D Cultivation Models on Expression Profiles of Human Periodontal Ligament Fibroblasts with Compressive Strain

The effects of compressive strain during orthodontic treatment on gene expression profiles of periodontal ligament fibroblasts (PDLFs) have mostly been studied in 2D cell culture. However, cells behave differently in many aspects in 3D culture. Therefore, the effect of pressure application on PDLFs in different 3D structures was investigated. PDLFs were either conventionally seeded or embedded into different 3D structures (spheroids, Mebiol^® gel, 3D scaffolds) and exposed to compressive force or incubated without pressure. For one 3D scaffold (POR), we also tested the effect of different compressive forces and application times. Expression of an angiogenic gene (VEGF), a gene involved in extracellular matrix synthesis (COL1A2), inflammatory genes (IL6, PTGS2), and genes involved in bone remodelling (OPG, RANKL) were investigated by RT–qPCR. Depending on the used 3D cell culture model, we detected different effects of compressive strain on expression profiles of PDLFs. COL1A2 was downregulated in all investigated 3D culture models. Angiogenetic and proinflammatory genes were regulated differentially between models. In 3D scaffolds, regulation of bone-remodelling genes upon compressive force was contrary to that observed in 3D gels. 3D cell culture models provide better approximations to in vivo physiology, compared with conventional 2D models. However, it is crucial which 3D structures are used, as these showed diverse effects on the expression profiles of PDLFs during mechanical strain.     

miR-143 Inhibits NSCLC Cell Growth and Metastasis by Targeting Limk1

MicroRNAs (miRNAs) have essential roles in carcinogenesis and tumor progression. Here, we investigated the roles and mechanisms of miR-143 in non-small cell lung cancer (NSCLC). miR-143 was significantly decreased in NSCLC tissues and cell lines. Overexpression of miR-143 suppressed NSCLC cell proliferation, induced apoptosis, and inhibited migration and invasion in vitro. Integrated analysis identified LIM domain kinase 1 (Limk1) as a direct and functional target of miR-143. Overexpression of Limk1 attenuated the tumor suppressive effects of miR-143 in NSCLC cells. Moreover, miR-143 was inversely correlated with Limk1 expression in NSCLC tissues. Together, our results highlight the significance of miR-143 and Limk1 in the development and progression of NSCLC.     

Role of Periostin Expression in Non-Small Cell Lung Cancer: Periostin Silencing Inhibits the Migration and Invasion of Lung Cancer Cells via Regulation of MMP-2 Expression

The involvement of periostin (POSTN) in non-small-cell lung cancer (NSCLC) migration, invasion, and its underlying mechanisms has not been well established. The present study aims to determine epithelial POSTN expression in NSCLC and to assess associations with clinicopathological factors and prognosis as well as to explore the effects of POSTN knockdown on tumor microenvironment and the migration and invasion of lung cancer cells. Immunohistochemistry was used to evaluate epithelial POSTN expression in NSCLC. POSTN mRNA expression in the dissected lung cancer cells was confirmed by laser capture microdissection and real-time PCR. A549 cells were used for transfecting shRNA-POSTN lentiviral particles. Wound healing and Transwell invasion assays were used to assess the migratory and invasive abilities of A549 cells transfected with POSTN-specific short hairpin (sh)RNA. The results demonstrated significantly higher cytoplasmic POSTN expression in the whole NSCLC group compared to non-malignant lung tissue (NMLT). POSTN expression in cancer cells may be considered to be an independent prognostic factor for survival in NSCLC. POSTN knockdown significantly inhibited A549 cell migration and invasion capabilities in vitro. The activity and the expression level of matrix metalloproteinase-2 (MMP-2) were significantly decreased in A549.shRNA compared to control cells. In summary, POSTN may regulate lung cancer cell invasiveness by modulating the expression of MMP-2 and may represent a potential target for novel therapeutic intervention for NSCLC.     

Current Advances in 3D Bioprinting for Cancer Modeling and Personalized Medicine

Tumor cells evolve in a complex and heterogeneous environment composed of different cell types and an extracellular matrix. Current 2D culture methods are very limited in their ability to mimic the cancer cell environment. In recent years, various 3D models of cancer cells have been developed, notably in the form of spheroids/organoids, using scaffold or cancer-on-chip devices. However, these models have the disadvantage of not being able to precisely control the organization of multiple cell types in complex architecture and are sometimes not very reproducible in their production, and this is especially true for spheroids. Three-dimensional bioprinting can produce complex, multi-cellular, and reproducible constructs in which the matrix composition and rigidity can be adapted locally or globally to the tumor model studied. For these reasons, 3D bioprinting seems to be the technique of choice to mimic the tumor microenvironment in vivo as closely as possible. In this review, we discuss different 3D-bioprinting technologies, including bioinks and crosslinkers that can be used for in vitro cancer models and the techniques used to study cells grown in hydrogels; finally, we provide some applications of bioprinted cancer models.     

Three-Dimensional Cell Culture Models to Investigate Oral Carcinogenesis: A Scoping Review

Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve.     

Intravascular Application of Labelled Cell Spheroids: An Approach for Ischemic Peripheral Artery Disease

Mesenchymal stem cells (MSC) are known for their vascular regeneration capacity by neoangiogenesis. Even though, several delivery approaches exist, particularly in the case of intravascular delivery, only limited number of cells reach the targeted tissue and are not able to remain on site. Applicated cells exhibit poor survival accompanied with a loss of functionality. Moreover, cell application techniques lead to cell death and impede the overall MSC function and survival. 3D cell spheroids mimic the physiological microenvironment, thus, overcoming these limitations. Therefore, in this study we aimed to evaluate and assess the feasibility of 3D MSCs spheroids for endovascular application, for treatment of ischemic peripheral vascular pathologies. Multicellular 3D MSC spheroids were generated at different cell seeding densities, labelled with ultra-small particles of iron oxide (USPIO) and investigated in vitro in terms of morphology, size distribution, mechanical stability as well as ex vivo with magnetic resonance imaging (MRI) to assess their trackability and distribution. Generated 3D spheroids were stable, viable, maintained stem cell phenotype and were easily trackable and visualized via MRI. MSC 3D spheroids are suitable candidates for endovascular delivery approaches in the context of ischemic peripheral vascular pathologies.     

Anti-PD1/PD-L1 Immunotherapy for Non-Small Cell Lung Cancer with Actionable Oncogenic Driver Mutations

Anti-PD1/PD-L1 immunotherapy has emerged as a standard of care for stage III-IV non-small cell lung cancer (NSCLC) over the past decade. Patient selection is usually based on PD-L1 expression by tumor cells and/or tumor mutational burden. However, mutations in oncogenic drivers such as EGFR, ALK, BRAF, or MET modify the immune tumor microenvironment and may promote anti-PD1/PD-L1 resistance. In this review, we discuss the molecular mechanisms associated with these mutations, which shape the immune tumor microenvironment and may impede anti-PD1/PD-L1 efficacy. We provide an overview of the current clinical data on anti-PD1/PD-L1 efficacy in NSCLC with oncogenic driver mutation.     

Effect of ALDH1A1 Gene Knockout on Drug Resistance in Paclitaxel and Topotecan Resistant Human Ovarian Cancer Cell Lines in 2D and 3D Model

Ovarian cancer is the most common cause of gynecological cancer death. Cancer Stem Cells (CSCs) characterized by drug transporters and extracellular matrix (ECM) molecules expression are responsible for drug resistance development. The goal of our study was to examine the role of aldehyde dehydrogenase 1A1 (ALDH1A1) expression in paclitaxel (PAC) and topotecan (TOP) resistant ovarian cancer cell lines. In both cell lines, we knocked out the ALDH1A1 gene using the CRISPR/Cas9 technique. Additionally, we derived an ALDH1A1 positive TOP-resistant cell line with ALDH1A1 expression in all cells via clonal selection. The effect of ALDH1A1 gene knockout or clonal selection on the expression of ALDH1A1, drug transporters (P-gp and BCRP), and ECM (COL3A1) was determined by Q-PCR, Western blot and immunofluorescence. Using MTT assay, we compared drug resistance in two-dimensional (2D) and three-dimensional (3D) cell culture conditions. We did not observe any effect of ALDH1A1 gene knockout on MDR1/P-gp expression and drug resistance in the PAC-resistant cell line. The knockout of ALDH1A1 in the TOP-resistant cell line resulted in a moderate decrease of BCRP and COL3A1 expression and weakened TOP resistance. The clonal selection of ALDH1A1 cells resulted in very strong downregulation of BCPR and COL3A1 expression and overexpression of MDR1/P-gp. This finally resulted in decreased resistance to TOP but increased resistance to PAC. All spheroids were more resistant than cells growing as monolayers, but the resistance mechanism differs. The spheroids’ resistance may result from the presence of cell zones with different proliferation paces, the density of the spheroid, ECM expression, and drug capacity to diffuse into the spheroid.