Mesenchymal Stem/Stromal Cells under Stress Increase Osteosarcoma Migration and Apoptosis Resistance via Extracellular Vesicle Mediated Communication - Fig 6

(A) A simplified interaction schema between EV-derived miRNAs and changes in gene expression in osteosarcoma cells (B) Metabolic pathways involved in osteosarcoma metastasis highlighting the lactate importer MCT1 (SLC16A1) and oxidative phosphorylation pathways (ATP5B). (C) Cell migration pathway in osteosarcoma associated with exposure to EV miRNAs emphasizing the interaction between the focal adhesion (PTK2) and oxidative stress pathways (NFE2L2).</p

Mesenchymal Stem/Stromal Cells under Stress Increase Osteosarcoma Migration and Apoptosis Resistance via Extracellular Vesicle Mediated Communication - Fig 4

(A) Schematic procedure for determining microRNAs and genes associated with changes in OS cells after treating with to EVs from MSCs (4day EVs) and SD-MSCs (12day EVs). (B) Quantitative RT-PCR validation of changes in metastasis and metabolism associated genes after OS incubation with MSCs and SD-MSC-EVs. (C) Quantitative RT-PCR showing changes in expression of shortlisted miRNA after MSC-EVs treatment.</p

Effects of EVs and conditioned media from SD-MSCs on OS wound healing.

<p><b>(A)</b> KHOS cell monolayer was scratched with a p200 micropipette tip, incubated with CCM or SD-MSC conditioned media, and imaged every 6 hrs, after replacing the media with serum free media, SD-MSC conditioned media or EV free media. <b>(B)</b> Scratched KHOS cell monolayer treated with EVs from SD-MSCs for either 4 days or 12 days and imaged every 6 hrs after replacing the media with SDM + EVs. Wound closure area was determined by ImageJ software analysis. Data presented as the means of three independent measurements. * P< 0.05, ** P< 0.01, *** P< 0.005 compared to untreated controls.</p

miRNA transferred by EVs regulate PTK2 and expression.

<p><b>(A)</b> Quantitative RT-PCR showing changes in expression of four miRNA after MSC-EVs treatment. <b>(B)</b> Quantitative RT-PCR expression of downstream target PTK2/FAK. <b>(C)</b> Quantitative RT-PCR showing downregulation of FAK1repressor LKB1.</p

Comparative analysis of OS survival in the presence of mesenchymal stem cells (MSCs) or serum-deprived MSCs (SD-MSCs).

<p><b>(A)</b> KRSOS cells grown with either complete culture media (CCM) or media without serum (SDM) in the presence of transwell inserts with donor-matched MSCs or SD-MSCs. <b>(B)</b> KHOS grown in either CCM or serum-free media in the presence of inserts containing either MSC or serum-deprived MSC cells. Data presented as means ± SD, Columns, mean of three independent experiments; bars, standard deviation (SD).</p

Apoptosis analysis of OS cells incubated with conditioned media from serum-deprived MSCs.

<p><b>(A and B)</b> DNA quantification of KRSOS and KHOS osteosarcoma cells treated with 0.1 μM doxorubicin for 24 h in the presence of complete culture media (control), SD-MSC media (CM) or EVs from SD-MSCs (EVs). <b>(C)</b> OS cells treated with doxorubicin in the presence of either SDM, CCM. Caspase activity measured as an increase in the amount of fluorogenic DEVD₂, a caspase 3 substrate. The data presented as the means ± SD of 3 independent experiments, *P < 0.05. <b>(D)</b> Western blot of cleaved caspase-3 in KRSOS and KHOS treatment with doxorubicin in the presence of SD-MSC EVs.</p

DataSheet_1_Ex Vivo Modeling of Human Neuroendocrine Tumors in Tissue Surrogates.docx

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community’s ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo.</p