Non-metastatic Cell Line Research Articles

SLAMF1/CD150 expression and topology in prostate and breast cancer cell lines.

SLAMF1/CD150receptor is aberrantly expressed in malignant hematopoietic cells compared to ubiquitous expression in their normal analogues. However, the data about CD150expression and function outside the hematopoietic system arelimited. The aim of this pilot study was to examine the profile of mRNA expression of CD150isoforms and the protein topology in highly and low malignant breast (BC) and prostate cancer (PC) cell lines. The study was performed on BCT47D, MDA-MB-231, ВСС/Р and BC/ML cell lines and PC LNCap, Du-145and PC-3cell lines. The quantitative polymerase chain reaction was applied for study of CD150isoforms mRNA expression and flow cytometry was used for determination of protein localization. Analyzed BC cell lines did not express CD150on the cell surface membrane (csCD150-), but more than 45% of cells were positive for CD150cytoplasmic reaction (cyCD150+). The cyCD150expression level in T47D cells of luminal BC subtype was higher than in basal BC cell lines MDA-MB-231, ВСС/Р and BC/ML. The PC cell lines expressed CD150both on the cell surface and in cytoplasm. The highest number of csCD150+ and cyCD150+ cells was revealed in less aggressive androgen responsive, non-metastatic LNCap cell line. All studied BC and PC cell lines expressed mRNA of canonical transmembrane mCD150and novel nCD150isoforms but with different pattern of prevalence. Soluble CD150isoform was revealed at the low level only in BCC/P BC cell line and LNCap, PC-3PC cell lines. We have shown that BC and PC cell lines differentially expressed multifunctional receptor CD150at the mRNA and protein levels that may indicate its association with the degree of their malignancy.

Comparison of FTIR spectra of different breast cell lines to detect spectral biomarkers of pathology

Many efforts have been made for several years to make Fourier Transform Infrared (FTIR) micro-spectroscopy usable in the cytological diagnostic. In fact, some events arising when cells undergo a disease, as alterations in the relative content of biochemical cellular components and variations of the proteins secondary structure, lead to modifications of the FTIR spectra, as changes of relative intensity and/or spectral shifts of specific peaks. In this study, we compare FTIR spectra measured for three different breast cell lines: the non-malignant MCF10A cell line, the malignant non-metastatic MCF7 cell line and the metastatic MDA cell line. The major differences of average absorption spectra involve a blue-shift of amide I and II bands and a larger relative intensity of nucleic acid bands in MCF7 spectra with respect to the other ones. Curve fitting also provided important information about spectral markers able to discriminate the three cell lines. In fact, the ratio of sum of the areas of peaks at 2852 and 2922 cm−1 with respect to the sum of the areas of peaks at 2873 and 2958 cm−1 is able to differentiate the non-malignant, malignant and metastatic cells among them. These results suggest that FTIR micro-spectroscopy may be a potentially useful tool for breast cancer diagnosis at cytological level.

Discrimination of Different Breast Cell Lines on Glass Substrate by Means of Fourier Transform Infrared Spectroscopy.

Fourier transform infrared (FTIR) micro-spectroscopy has been attracting the interest of many cytologists and histopathologists for several years. This is related to the possibility of FTIR translation in the clinical diagnostic field. In fact, FTIR spectra are able to detect changes in biochemical cellular components occurring when the cells pass to a pathological state. Recently, this interest has increased because it has been shown that FTIR spectra carried out just in the high wavenumber spectral range (2500–4000 cm−1), where information mainly relating to lipids and proteins can be obtained, are able to discriminate cell lines related to different tissues. This possibility allows to perform IR absorption measurements of cellular samples deposited onto microscopy glass slides (widely used in the medical environment) which are transparent to IR radiation only for wavenumber values larger than 2000 cm−1. For these reasons, we show that FTIR spectra in the 2800–3000 cm−1 spectral range can discriminate three different cell lines from breast tissue: a non-malignant cell line (MCF10A), a non-metastatic adenocarcinoma cell line (MCF7) and a metastatic adenocarcinoma cell line (MDA). All the cells were grown onto glass slides. The spectra were discriminated by means of a principal component analysis, according to the PC1 component, whose values have the opposite sign in the pairwise score plots. This result supports the wide studies that are being carried out to promote the translation of the FTIR technique in medical practice, as a complementary diagnostic tool.

Virtual Screening for Biomimetic Anti-Cancer Peptides from Cordyceps militaris Putative Pepsinized Peptidome and Validation on Colon Cancer Cell Line.

Colorectal cancer is one of the leading causes of cancer-related death in Thailand and many other countries. The standard practice for curing this cancer is surgery with an adjuvant chemotherapy treatment. However, the unfavorable side effects of chemotherapeutic drugs are undeniable. Recently, protein hydrolysates and anticancer peptides have become popular alternative options for colon cancer treatment. Therefore, we aimed to screen and select the anticancer peptide candidates from the in silico pepsin hydrolysate of a Cordyceps militaris (CM) proteome using machine-learning-based prediction servers for anticancer prediction, i.e., AntiCP, iACP, and MLACP. The selected CM-anticancer peptide candidates could be an alternative treatment or co-treatment agent for colorectal cancer, reducing the use of chemotherapeutic drugs. To ensure the anticancer properties, an in vitro assay was performed with “CM-biomimetic peptides” on the non-metastatic colon cancer cell line (HT-29). According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results from peptide candidate treatments at 0–400 µM, the IC50 doses of the CM-biomimetic peptide with no toxic and cancer-cell-penetrating ability, original C. militaris biomimetic peptide (C-ori), against the HT-29 cell line were 114.9 µM at 72 hours. The effects of C-ori compared to the doxorubicin, a conventional chemotherapeutic drug for colon cancer treatment, and the combination effects of both the CM-anticancer peptide and doxorubicin were observed. The results showed that C-ori increased the overall efficiency in the combination treatment with doxorubicin. According to the acridine orange/propidium iodine (AO/PI) staining assay, C-ori can induce apoptosis in HT-29 cells significantly, confirmed by chromatin condensation, membrane blebbing, apoptotic bodies, and late apoptosis which were observed under a fluorescence microscope.

GPCR48 promotes invasion and metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma cells

Objective: To observe the G protein-coupled receptor 48 (GPCR48) expression in hepatocellular carcinoma (HCC) cell lines with different metastatic potential and its characteristics effect on the invasion and metastasis of Huh7 hepatoma cells via epithelial-mesenchymal transition (EMT). Methods: Western blot was used to detect the protein expression level of GPCR48 in HCC cells with different metastatic potential. The lentivirus vector expressing GPCR48 gene was constructed. GPCR48 was overexpressed in Huh7 hepatoma cells. The GPCR48 overexpression level was detected by real-time PCR and Western blot. Transwell invasion and migration assay was used to detect the Huh7 hepatoma cells invasion and migration ability in the Control, Mock and GPCR48 overexpression group. Real-time PCR and Western blot were used to detect Huh7 hepatoma cells mRNA and protein expression levels of the EMT related markers (E-cadherin, N-cadherin, vimentin, and γ catenin) in the Control, Mock and GPCR48 overexpression groups, respectively. Analysis of variance was used to compare the differences between data sets. Results: GPCR48 protein expression level in metastatic HCC cell lines was significantly higher than non-metastatic HCC cell lines (P < 0.05). The lentivirus vector expressing the GPCR48 gene had effectively transfected the Huh7 hepatoma cells and stably expressed the GPCR48mRNA and protein. Compared with the Mock and the Control group, Huh7 hepatoma cells invasion and migration ability in the GPCR48 overexpression group was significantly enhanced (F≥5.54, P < 0.05), and the mRNA and protein expression levels of epithelial phenotypic markers E-cadherin and γ-catenin were decreased (P < 0.05). The mRNA and protein expression levels of the mesenchymal phenotypic markers N-cadherin and Vimentin were increased (P < 0.05), indicating that EMT changes occurred in Huh7 hepatoma cells had overexpressed GPCR48. Conclusion: GPCR48 expression level is positively correlated with the metastatic potential of HCC cells. GPCR48 overexpression can down-regulate the expression of epithelial phenotypic markers and up-regulate the expression of mesenchymal phenotypic markers, and induce EMT changes in HCC cells, thus promoting HCC cells invasion and migration.

Microarray analysis reveals ONC201 mediated differential mechanisms of CHOP gene regulation in metastatic and nonmetastatic colorectal cancer cells

The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

Assessment of TSPAN Expression Profile and Their Role in the VSCC Prognosis.

The role and prognostic value of tetraspanins (TSPANs) in vulvar squamous cell carcinoma (VSCC) remain poorly understood. We sought to primarily determine, at both the molecular and tissue level, the expression profile of the TSPANs CD9, CD63, CD81, and CD82 in archived VSCC samples (n = 117) and further investigate their clinical relevance as prognostic markers. Our studies led us to identify CD63 as the most highly expressed TSPAN, at the gene and protein levels. Multicomparison studies also revealed that the expression of CD9 was associated with tumor size, whereas CD63 upregulation was associated with histological diagnosis and vascular invasion. Moreover, low expression of CD81 and CD82 was associated with worse prognosis. To determine the role of TSPANs in VSCC at the cellular level, we assessed the mRNA levels of CD63 and CD82 in established metastatic (SW962) and non-metastatic (SW954) VSCC human cell lines. CD82 was found to be downregulated in SW962 cells, thus supporting its metastasis suppressor role. However, CD63 was significantly upregulated in both cell lines. Silencing of CD63 by siRNA led to a significant decrease in proliferation of both SW954 and SW962. Furthermore, in SW962 particularly, CD63-siRNA also remarkably inhibited cell migration. Altogether, our data suggest that the differential expression of TSPANs represents an important feature for prognosis of VSCC patients and indicates that CD63 and CD82 are likely potential therapeutic targets in VSCC.

The Influence of Cold Atmospheric Pressure Plasma-Treated Media on the Cell Viability, Motility, and Induction of Apoptosis in Human Non-Metastatic (MCF7) and Metastatic (MDA-MB-231) Breast Cancer Cell Lines.

Breast cancer remains the most common type of cancer, occurring in middle-aged women, and often leads to patients’ death. In this work, we applied a cold atmospheric pressure plasma (CAPP)-based reaction-discharge system, one that is unique in its class, for the production of CAPP-activated media (DMEM and Opti-MEM); it is intended for further uses in breast cancer treatment. To reach this aim, different volumes of DMEM or Opti-MEM were treated by CAPP. Prepared media were exposed to the CAPP treatment at seven different time intervals and examined in respect of their impact on cell viability and motility, and the induction of the apoptosis in human non-metastatic (MCF7) and metastatic (MDA-MB-231) breast cancer cell lines. As a control, the influence of CAPP-activated media on the viability and motility, and the type of the cell death of the non-cancerous human normal MCF10A cell line, was estimated. Additionally, qualitative and quantitative analyses of the reactive oxygen and nitrogen species (RONS), generated during the CAPP operation in contact with analyzed media, were performed. Based on the conducted research, it was found that 180 s (media activation time by CAPP) should be considered as the minimal toxic dose, which significantly decreases the cell viability and the migration of MDA-MB-231 cells, and also disturbs life processes of MCF7 cells. Finally, CAPP-activated media led to the apoptosis of analyzed cell lines, especially of the metastatic MDA-MB-231 cell line. Therefore, the application of the CAPP system may be potentially applied as a therapeutic strategy for the management of highly metastatic human breast cancer.

Alcoholic extract of Tarantula cubensis (Theranekron®) induce autophagy on gastric cancer cells

Aim: To evaluate the effects of theranekron in respect of autophagy on gastric cancer that is the fifth leading cancer type worldwide. Metods: In the present study, metastatic AGS and non-metastatic MKN-45 human gastric cell lines were used together with HEK-293 non-cancer cells as controls. Cytotoxic effect of theranekron besides appropriate treatment time was investigated through cell proliferation by using Cell Proliferation assay Kit (MTT) using different concentrations of the drug. The autophagic effect of the drug was determined using the LC3-GFP translocation assay and western blot analysis. All experiments were performed also using the ethanol since Tarantula cubensis spider was processed and diluted in 60% alcohol to generate as a drug. Results: MTT assay results demonstrated that the half maximal inhibitory concentration of theranekron was ~100 μM, its effect was found to be significant at 6 hrs, and theranekron decreased the cell viability in all cell lines without specificity in respect to the increasing concentrations. Additionally, a significantly increased GFP accumulation was detected in the autophagosomes of the cells treated with theranekron compared to non-treated cells, indicating the presence of autophagy. Conclusion: These findings were confirmed by LC3-I to LC3-II conversion with the western blot analysis. The data of ethanol experiments; however, demonstrated that ethanol also induced a cytotoxic effect and autophagic cell death. Our results suggested that theranekron results in cell death and stimulate autophagy process, but it is not specific for cancer cells since it represented similar results on non-cancer control cells. Moreover, the effect of theranekron on cell death might mostly occur through alcohol in which it is extracted.

Prognostic Implications of the Complement Protein C1Q and Its Correlation with Immune Infiltrates in Osteosarcoma

BackgroundOsteosarcoma (OS) is the most widespread bone tumour among childhood cancers, and distant metastasis is the dominant factor in poor prognosis for patients with OS. Therefore, it is necessary to identify new prognostic biomarkers for identifying patients with aggressive disease.MethodsTwo OS datasets (GSE21257 and GSE33383) were downloaded from the Gene Expression Omnibus (GEO) and subsequently subjected to weighted gene co-expression network analysis (WGCNA) and differential gene expression analysis (DGE) to screen candidate genes. A prognostic model was constructed using OS data derived from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program to further screen key genes and perform gene ontology (GO) analysis. The prognostic values of key genes were assessed using the Kaplan–Meier (KM) plotter. The GEO dataset was used for immune infiltration analysis and association analysis of key genes. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to validate the expression levels of potentially crucial genes in OS cell lines.ResultsIn the present study, we found 114 genes with a highly significant correlation in the module and 44 downregulated genes; 25 candidate genes overlapped in the two parts of the genes. Among these, three key genes, C1QA, C1QB, and C1QC, were the most significant hub genes, which had the highest node degrees, were clustered into one group, and implicated in most significant biological processes (regulation of immune effector process). Moreover, these three key genes were negatively associated with the prognosis of OS and positively associated with three immune cells (follicular helper T cells, memory B cells, and CD8 T cells). Additionally, compared to non-metastatic OS cell lines, the expression of three key genes was significantly downregulated in metastatic OS cell lines.ConclusionOur results revealed that three key genes (C1QA, C1QB, and C1QC) were implicated in tumour immune infiltration and may be promising biomarkers for predicting metastasis and prognosis of patients with OS.

Abstract PS19-25: Metastatic TNBC is highly associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism programmation

Abstract Background: Triple negative breast cancer (TNBC) is characterized by heterogeneous metastatic and metabolic properties, which difficult the diagnosis and development of specific treatment. Therefore, the understanding of the mitochondria´s role in the different grades of TNBC could be relevant for the design of novel specific TNBC treatments. This research aimed to explore the mitochondria morphology and gene expression of proteins related to oxidative and non-oxidative metabolism in metastatic and non-metastatic TNBC cell lines. Methods: We performed a gene expression analysis of mitochondrial biogenesis, EMT and principal metabolism-related genes (Integrated DNA Technologies, CA, USA) in three breast cancer cell lines HCC-1395, MDA-MB-231 and MCF-12A. After the gene expression qPCR data mining, in sílico analisis were performed using confocal microscopy (Leica Microsystems,WZL, DE) in the cell lines where mitochondrial distribution, morphology, function and ROS production were measured. Results: The metastatic TNBC-cell line showed a preference for fatty acid biosynthesis and glycolytic metabolism since overexpression of genes related to both pathways was observed. These metabolism features were accompanied by a fused mitochondrial morphology and lower mitochondrial activity since was observed less mitochondrial density accompanied by the downregulated expression of biogenesis-related genes such as PGC1α. In contrast, the non-metastatic TNBC-cell line presented a hyperfragmented mitochondria, a stress associated mitochondrial morphology accompanied by upregulated expression of mitochondrial biogenesis-related genes, both characteristics related to the higher ROS production observed in this cell line. Conclusions: Metastatic TNBC was characterized by a mitochondrial function and morphology similar to control ranges with a metabolic gene program directed to glycolysis and FA usage. While mitochondria of the non-metastatic TNBC cell line was characterized by a higher density and potential, related to an increase of mitochondrial biogenesis, which in turn was associated with higher levels of ROS and with the consequence of a hyperfragmented mitochondrial morphology. These characteristics can provide a better understanding of the metastasis observed in TNBC and contribute to the development of a specific and personalized TNBC therapy. Key words: Triple Negative Breast Cancer, Mitochondria, Cell-metabolism. Citation Format: Claudia D Aguayo-Millán, Perla Perez-Trevino, Sandra Santuario-Facio, Alberto Camacho, Noemí Garcia, Rocio Ortiz-Lopez. Metastatic TNBC is highly associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism programmation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-25.

Concentration-Dependent Pro- and Antitumor Activities of Quercetin in Human Melanoma Spheroids: Comparative Analysis of 2D and 3D Cell Culture Models.

Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (>12.5 µM) or low (<6.3 µM) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively. Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (>0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.

MBRS-28. EXOSOMES DRIVE MEDULLOBLASTOMA METASTASIS IN A MMP2 AND EMMPRIN DEPENDENT MANNER

INTRODUCTIONRecurrent/metastatic medulloblastoma (MB) is a devastating disease with an abysmal prognosis of less than 10% 5-year survival. The secretion of extracellular vesicles (EVs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. The most investigated EV’s are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. Matrix metalloproteinases (MMPs) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. We hypothesise that exosomal MMP2 and its inducer EMMPRIN could enhance metastasis of MB.METHODSProliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. Gelatin zymography and western blotting were performed to confirm MMP2 functional activity in cell lines and exosomes. Nanoscale flow cytometry was used to measure surface exosomal EMMPRIN levels. Exosomal MMP2 and EMMPRIN were modulated at the RNA level.RESULTSNumber of exosomes is directly related to the migratory behaviour of parental MB cell lines (p<0.01). Notably, functional exosomal MMP2 and EMMPRIN levels also correlate with this. Furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair by ~3.8-fold (p<0.01). Exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells.CONCLUSIONTogether this data suggests that exosomal MMP2 and EMMPRIN may promote medulloblastoma metastasis and supports analysis of exosomal MMP2 and EMMPRIN levels in patient cerebral spinal fluid samples.

MiRNA‑490‑3p promotes the metastatic progression of invasive ductal carcinoma.

MicroRNA (miRNA/mir)-490-3p has been defined as a tumor suppressor in different types of cancer, including breast cancer. However, miR-490-3p has been shown to function as a tumor suppressor and promoter in a context-dependent manner in hepatocellular and lung cancer. Contrary to previous studies, the present study revealed that miR-490-3p expression was significantly higher in invasive ductal carcinoma (IDC) tissue specimens, the most common form of breast cancer, compared to tumor-adjacent normal tissue specimens (n=20). Its expression was also higher in the more metastatic breast cancer cell line, MDA-MB-231, compared to the non-metastatic breast cancer cell line, MCF7, and the moderately metastatic breast cancer cell line, MDA-MB-468. The expression of miR-490-3p was induced following transforming growth factor (TGF)-β-induced epithelial-to-mesenchymal transition (EMT) in MCF10A cells. Gain-and loss-of-function assays revealed that the expression of miR-490-3p regulated the proliferation, colony formation, EMT, migration and invasion in vitro, but not the apoptosis of MDA-MB-468 and MDA-MB-231 cells. The knockdown of miR-490-3p expression in MDA-MB-231 cells inhibited experimental metastasis in a tumor xenograft assay. As in lung cancer, miR-490-3p was found to target and downregulate the expression of the tumor suppressor RNA binding protein poly r(C) binding protein 1 (PCBP1). PCBP1 protein and miR-490-3p expression inversely correlated in patients with ductal carcinoma in situ (DCIS; n=10; no nodal involvement) and IDC (n=10; different stages of metastatic progression) with a significantly higher miR-490-3p expression in patients with IDC compared to those with DCIS. The expression of miR-490-3p was negatively associated with both overall and disease-free survival in the patients with breast cancer included in the present study. On the whole, the results confirm a pro-metastatic role of miR-490-3p in IDC, establishing it as a biomarker for disease progression in these patients.

&lt;p&gt;Knockdown of Long Non-Coding RNA LOC100132707 Inhibits the Migration of Uveal Melanoma Cells via Silencing JAK2&lt;/p&gt;

Background/ObjectiveAlthough lots of long non-coding RNAs (lncRNAs) have been demonstrated to be involved in carcinogenesis, the functions of numerous of lncRNAs remain unknown. Bioinformatics online database showed that lncRNA LOC100132707 was highly expressed in metastatic melanoma tissues, and its expression predicted a lower overall survival rate in melanoma patients. However, LOC100132707 function in uveal melanoma (UM) progression still remains unclear. In the present study, we aimed to elucidate the role and molecular mechanisms underlying LOC100132707 in UM.MethodsRT-PCR was used to detect the levels of LOC100132707 in UM cells. Cell migration, invasion and tumorigenesis were tested by using the transwell chamber assay and in vivo assay.ResultsLOC100132707 expression in metastatic UM cell line MM28 was significantly higher than that of the non-metastatic UM cell lines, MP38, MP46 and MP65, as well as the expressions of LOC100132707-related genes, including XRN1, PARP14, JAK2, DDX60, BUB1 and SAMD9L. LOC100132707 downregulation significantly repressed cell migration and invasion abilities, whereas overexpressing JAK2 rescued these effects. Consistently, upregulation of LOC100132707 induced significant increases in cell migration and invasion abilities via upregulating JAK2. In addition, silencing of LOC100132707 significantly repressed the in vivo tumor formation ability in UM cells.ConclusionThis study reveals that silence of LOC100132707 represses the migration of UM via downregulating JAK2. The LOC100132707/JAK2 axis might serve as a potent target for the prevention and treatment of UM metastasis.

D283 Med, a Cell Line Derived from Peritoneal Metastatic Medulloblastoma: A Good Choice for Missing Protein Discovery.

Since the Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2010, many techniques have been adopted for the discovery of missing proteins (MPs). Because of these efforts, only 1481 MPs remained as of July 2020; however, by relying only on technique optimization, researchers have reached a bottleneck in MP discovery. Protein expression is tissue- or cell-type-dependent. The tissues of the human testis and brain have been reported to harbor a large number of tissue-specific genes and proteins; however, few studies have been performed on human brain tissue or cells to identify MPs. Herein a metastatic cell line derived from brain cancer, D283 Med, was used to search for MPs. With a traditional and simple shotgun workflow to separate the peptides into 20 fractions, 12 MPs containing at least two unique non-nested peptides (amino acid length ≥9) were identified in this cell line with a protein false discovery rate of <1%. Following the same experimental protocol, only one MP was found in a nonmetastatic brain cancer cell line, U-118 MG. Furthermore, 12 MPs were verified as having two non-nested unique peptides by matching them with corresponding chemically synthesized peptides through parallel reaction monitoring. These results clearly demonstrate that the appropriate selection of experimental materials, either tissues or cell lines, is imperative for MP discovery. The data obtained in this study are available via ProteomeXchange (PXD021482) and PeptideAtlas (PASS01627).

Abstract PO-035: A novel bioengineered model of osteosarcoma recapitulates critical tumor phenotypes within a native bone microenvironment

Abstract Traditional in-vitro models of solid bone tumors used in basic and preclinical research are unable to faithfully recapitulate human physiology. Specifically, monolayer cultures of osteosarcoma fail to recapitulate various features of 3D tumors, and they lack the native bone milieu, where tumor growth, metastasis, and response to therapy are critically dependent on cancer cell interactions with the bone matrix, supporting cells, and secreted regulatory factors. Based on our previous success in developing a biomimetic tissue model of Ewing sarcoma, we extended our approach towards bioengineering a novel human 3D tumor model of osteosarcoma within a bone microenvironment that can overcome some of these limitations. Briefly, osteosarcoma cell lines were used to generate tumor-like aggregates which were subsequently introduced into, and cultured within, engineered bone tissue scaffolds. Our bioengineered osteosarcoma model was able to recapitulate several aspects of native tumors and their microenvironment missing in monolayer cultures. Significantly higher expression of cancer-related genes (as seen in patients), re-activation of the hypoxic and glycolytic tumor phenotype, re-activation of hypoxia-mediated expression of vascular endothelial growth factor VEGF-α (a critical component of tumor angiogenesis), induction of vasculogenic mimicry, more physiologically relevant growth rates, and co-culture with adjacent functional osteoblasts within a demineralized native bone scaffold were all achieved. Additionally, we were able to capture a degree of both intra-tumor heterogeneity, with the presence of a necrotic core and variability in proliferation across the tumor aggregates, as well as inter-tumor heterogeneity, with notable differences observed between our models generated from metastatic and non-metastatic osteosarcoma cell lines. Finally, the osteosarcoma cell lines were transduced with a GFP-luciferase reporter, allowing us to visualize the engineered tumors in situ within the bone scaffolds, and to evaluate tumor cell responses to various canonical and experimental drug treatments. Notably, unlike monolayers, our bioengineered osteosarcoma model could be cultured stably for several weeks. This allowed us to apply a clinical treatment regimen cycle of linsitinib, an experimental drug in an ongoing phase II clinical trial for Ewing sarcoma, in also being effective at preventing the growth of non-metastatic osteosarcoma bioengineered tumors. Overall, we were able to successfully generate a new, more native-like 3D bioengineered model of osteosarcoma with significant advantages over traditional in-vitro culture that has the potential to explore novel questions regarding tumor biology, the tumor microenvironment, and translational applications. Citation Format: Alan Chramiec, Alessandro Marturano-Kruik, Keith Yeager, Luke Hao, Roberta Lock, Gordana Vunjak-Novakovic. A novel bioengineered model of osteosarcoma recapitulates critical tumor phenotypes within a native bone microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-035.

Plasma Treatment Limits Human Melanoma Spheroid Growth and Metastasis Independent of the Ambient Gas Composition

Simple SummaryDespite recent advances in therapeutic options, melanoma remains a deadly disease with a poor prognosis. Physical gas plasma has been proposed as a promising technology for the treatment of melanoma. This study aimed to develop and investigate a convenient test system based on three-dimensional cell cultures (spheroids) of two melanoma cell lines in response to physical gas plasma. The experimental approach combined high-content imaging technology and different gas plasma treatment modalities (direct and indirect, gas compositions). Our results revealed that plasma treatment was toxic for both cell lines predominantly dependent on the treatment time. Furthermore, we addressed the question of safety and morphological changes in response to physical gas plasma exposure and found no support for metastatic progression. Treatment with physical gas plasma effectively limited the growth of human 3D melanoma spheroids and provided a versatile test system for more in vivo-like tumor tissue.Melanoma skin cancer is still a deadly disease despite recent advances in therapy. Previous studies have suggested medical plasma technology as a promising modality for melanoma treatment. However, the efficacy of plasmas operated under different ambient air conditions and the comparison of direct and indirect plasma treatments are mostly unexplored for this tumor entity. Moreover, exactly how plasma treatment affects melanoma metastasis has still not been explained. Using 3D tumor spheroid models and high-content imaging technology, we addressed these questions by utilizing one metastatic and one non-metastatic human melanoma cell line targeted with an argon plasma jet. Plasma treatment was toxic in both cell lines. Modulating the oxygen and nitrogen ambient air composition (100/0, 75/25, 50/50, 25/75, and 0/100) gave similar toxicity and reduced the spheroid growth for all conditions. This was the case for both direct and indirect treatments, with the former showing a treatment time-dependent response while the latter resulted in cytotoxicity with the longest treatment time investigated. Live-cell imaging of in-gel cultured spheroids indicated that plasma treatment did not enhance metastasis, and flow cytometry showed a significant modulation of S100A4 but not in any of the five other metastasis-related markers (β-catenin, E-cadherin, LEF1, SLUG, and ZEB1) investigated.

Silibinin releasing mesoporous bioactive glass nanoparticles with potential for breast cancer therapy

Mesoporous materials have attracted extensive consideration in the field of drug delivery. The targeted release of anti-neoplastic agents depends on the carrier used to transport such drugs in the body. In this study, mesoporous bioactive glass nanoparticles (MBGN) of an average size of 120 ± 10 nm were used to load and release silibinin, which is a natural flavonolignan used as anticancer agent and tumor suppressor. Mesoporous bioactive glass nanoparticles were characterized in terms of morphology and release potential after being loaded with silibinin. The loading and release amounts of silibinin were quantified using UV-VIS spectrophotometer at λmax = 280 nm in PBS buffer solution (pH 7.4). It was found that MBGN have a maximum loading efficiency of 61% against optimal silibinin concentration (i.e. 40 μg/ml silibinin in PBS buffer solution (pH 7.4)). The drug was released in a sustained manner from loaded mesoporous bioactive glass nanoparticles and a release of 18 μg/ml silibinin was measured during the first 24 h post-drug loading. Further, the chemotherapeutic effect of silibinin was tested on metastatic MDA-MB-231 breast cells and the non-metastatic breast cell line MCF-10A at two different concentrations (9.0 μg/ml and 18 μg/ml). As our results show, silibinin exhibits a chemotherapeutic potential towards MDA-MB-231 cell line, which is a much lower concentration compared to values reported in literature. According to our knowledge, this is the first study elucidating the use of MBGN as a carrier for silibinin, as well as testing its lethal effect in contact with breast neoplastic cells. Taken collectively, the results of the present study suggest that silibinin-loaded MBGN can be applied as an effective drug delivery system to cause a chemo preventive response which has the potential to be beneficial for the treatment of breast cancer.

Abstract 2714: The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models

Abstract Introduction: Progression into metastatic disease is a critical step in most malignancies. Cancer cells may enter circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. CSCs are known to form cell clusters in vitro (spheroids/tumorspheres). We reported that tumorspheres also circulate in blood in vivo. We found that they carry cancer stem cells and strongly correlate with metastatic disease. We collected tumorspheres from blood by filtration. This project aims to test the metastatic potential of tumorspheres by developing an assay using in vivo circulation models. The goal is to establish a novel prognostic test for metastatic predisposition in cancer patients. Methods: To develop an in vivo assay for metastasis we use the chorioallantoic circulation in the chicken embryo, with several advantages: easy access to circulation, lack of immune response, rapid turnaround time (5 to 10 days) and inexpensive and simple technique. For clinical applications we introduced three technical innovations: (i) to make fertilized chicken embryos easily available we worked out a method to store viable embryos beyond 30 days; (ii) we developed a new coverglass chamber technique for micromanipulations; and (iii) for microscopic analysis new over-drained chorioallantois preparations can accommodate high resolution objectives. To test tumorspheres in embryonic circulation we used both highly metastatic (e.g. MDA-MB-436) and non-metastatic (e.g. MCF7) cell lines with GFP label, DiI membrane dye and CFDA-SA dye. For molecular studies DNA was extracted 5 days after cancer spheroid implantation and used qPCR to detect human sequences. Results: In traditional storage few embryos survive beyond 6 days. As fertilized eggs carry pluripotent stem cells, we optimized temperature, gas and humidity combinations to match known stem cell optimums. By suppressing metabolism, blocking embryo adhesion and controlling gas and humidity exchange we extended viable embryo storage beyond 30 days. We modified the traditional window method by forming visually clear microscopic coverglass chambers, and the new over-draining approach lowered the chorioallantois membranes and allowed high resolution imaging. The results showed invasive and hematogenic dissemination of the cancer spheroids. Molecular studies of human markers detected metastatic progression and generated quantitative data. Conclusions: We optimized the in vivo embryonic metastasis assay. We show that it has the potential for a sensitive and quantitative method to diagnose metastatic predisposition in cancer patients. The molecular approach (DNA extraction, PCR markers) is easily up-scalable, the instrumentation is available in most hospitals and offers cost effective and critical early diagnosis of cancer progression. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Kathleen Marinelli, Christine Kyritsis, Viktoria Denes. The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2714.