Journal: Bioactive Materials

Article Title: Tunable hybrid hydrogels with multicellular spheroids for modeling desmoplastic pancreatic cancer

doi: 10.1016/j.bioactmat.2023.02.005

Figure Lengend Snippet: Schematic of pancreatic cancer development and cancer spheroid production. A- PDAC development with cancer microenvironment alterations. B- Cancer only and Cancer + CAFs were formed into spheroids in Aggrewell400 microplates (cancer: green, CAF: orange; F-actin: Alexa Fluor 488-Phalloidin, Nucleus: DAPI). C- Spheroids were stained for live/dead dye and imaged for days 1, 3, and 7 (Green: Calcein-live, Red: Ethidium homodimer-dead, scale bar: 500 μm, insert scale bar: 100 μm). D- Size analysis of the spheroids from micrographs for days 1, 2, 3, 4, and 7 (Two-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). E− Shape analysis of the spheroids from micrographs for days 1, 2, 3, 4, and 7 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

Article Snippet: Pancreatic cancer cell line (ASPC-1) and CAF cells (ATCC #CRL-1682, ATCC #PCS-201-012) were obtained from the American Type Culture Collection (ATCC).

Techniques: Staining

Journal: Nature Communications

Article Title: The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner

doi: 10.1038/s41467-018-05793-2

Figure Lengend Snippet: rVAR2 binds specifically to a diverse repertoire of cancer cells. a Detection of cancer cells using the CytoTrack platform. Representative confocal microscopy images of indicated cell lines. Cancer cells were mixed with PBMCs in a 1:5000 ratio prior to analysis and stained with His-tagged rVAR2 in combination with anti-penta His Alexa Fluor 488 (green), an anti-CD45 Cy5 antibody (red), and DAPI (blue). Scale bars, 10 µm. b Flow cytometry measured fluorescence intensity of three breast cancer (left panel), three prostate cancer (middle panel), and three colorectal cancer (right panel) cell lines stained by His-tagged rVAR2 in combination with anti-penta His Alexa Fluor 488 ( y -axis) and a PE-conjugated anti-EpCAM antibody ( x -axis)

Article Snippet: The cells were blocked for 5 min in 10% normal donkey serum (NDS) prior to stain with PE-conjugated anti-CD45 [5B-1] antibody (Cat. No. 130-080-201, MACS Miltenyi Biotec, 1:100) for 30 min at room temperature.

Techniques: Confocal Microscopy, Staining, Flow Cytometry, Fluorescence

Journal: Nature Communications

Article Title: The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner

doi: 10.1038/s41467-018-05793-2

Figure Lengend Snippet: rVAR2-based CTC isolation using magnetic beads. a Representative confocal microscopy image of a PC3 cell captured by rVAR2-coated magnetic beads in combination with the IsoFlux™ device after spiking into 5 mL of healthy donor blood. Isolated cells were incubated with anti-cytokeratin FITC antibody (green), anti-CD45 PE antibody (red), and DAPI (blue). Scale bars, 10 µm. b A low-passage pancreatic ductal adenocarcinoma (PDAC) cell from a patient-derived xenograft captured by rVAR2-coated magnetic beads in combination with the IsoFlux™ device after spiking into 5 mL of healthy donor blood. Isolated cells were incubated with anti-cytokeratin FITC antibody (green), anti-CD45 PE antibody (red), and DAPI (blue). Scale bars, 10 µm. c CTC isolation efficiency by spike-in experiments; 100 PC3 cells or PDAC cells were spiked into 5 mL of blood and isolated by rVAR2-coated beads in combination with the IsoFlux™. The recovery was estimated by immunofluorescence staining of CK+, CD45−, DAPI+ cells (mean ± s. d., n = 3). d Recovery of PDAC cells spiked into 5 mL of blood. The number of spiked PDAC cells (10, 20, 50, 100, or 200 cells) is plotted versus the number of PDAC cells isolated by rVAR2-coated beads ( n = 5). Equation shows a linear regression model

Article Snippet: The cells were blocked for 5 min in 10% normal donkey serum (NDS) prior to stain with PE-conjugated anti-CD45 [5B-1] antibody (Cat. No. 130-080-201, MACS Miltenyi Biotec, 1:100) for 30 min at room temperature.

Techniques: Isolation, Magnetic Beads, Confocal Microscopy, Incubation, Derivative Assay, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner

doi: 10.1038/s41467-018-05793-2

Figure Lengend Snippet: rVAR2- and EpCAM-based CTC isolation and enumeration in cancer patients. a Number of CTCs isolated from 5 mL pancreatic ( n = 9), hepatocellular ( n = 4), prostate ( n = 25), and lung ( n = 6) cancer patient-derived blood using rVAR2-coated beads. CTCs were enumerated by immunofluorescence stainings and defined as CK+ CD45− DAPI+. b Representative confocal microscopy image of a circulating tumor cell isolated with rVAR2 from blood derived from one of the pancreatic cancer patients (patient 4, Table ). Isolated cells were stained with anti-cytokeratin FITC antibody (green), anti-CD45 PE antibody (red), and DAPI (blue). Scale bar, 10 µm. c Representative confocal microscopy image of circulating tumor cells isolated with rVAR2 from blood derived from one of the non-small cell lung cancer (NSCLC) patients. Isolated cells were stained with anti-cytokeratin FITC antibody (green), anti-CD45 PE antibody (red), and DAPI (blue). Scale bar, 10 µm. d Number of CK+ CD45− DAPI+ CTCs isolated using rVAR2 or anti-EpCAM antibody-coated beads from 5 mL blood from 15 of the stage II−III prostate cancer (PCa) patients ( P < 0.02, Wilcoxon test for paired data). e Number of CK+ CD45− DAPI+ CTCs isolated using rVAR2 or anti-EpCAM antibody-coated beads from 5 mL blood from six of the stage III–IV pancreatic ductal adenocarcinoma (PDAC) patients. f Number of CK+ CD45− DAPI+ CTCs isolated using rVAR2 or anti-EpCAM antibody-coated beads from 5 mL blood from four of the stage IV NSCLC patients. g Box-Whiskers plot showing post-isolation characterization of CK+ CD45− DAPI+ CTCs using EpCAM or rVAR2 stain on CTCs isolated using rVAR2 ( n = 7) or anti-EpCAM antibody-coated ( n = 7) beads, respectively. The median is presented as the center line, whiskers as min to max values, and the 25th to 75th percentiles define the box. h Number of PBMCs contaminating the isolated CTCs from patient-matched blood samples using rVAR2 or anti-EpCAM antibody-coated beads. PBMC levels were estimated by immunofluorescence stainings and defined as CK−, CD45+, DAPI+ stained cells ( P < 0.0001, Wilcoxon test for paired data) ( n = 23). i Number of anti-CK and anti-vimentin antibody stained cells in the rVAR2-based CTC enrichments from 5 mL blood samples from two of the patients with stage IV NSCLC

Article Snippet: The cells were blocked for 5 min in 10% normal donkey serum (NDS) prior to stain with PE-conjugated anti-CD45 [5B-1] antibody (Cat. No. 130-080-201, MACS Miltenyi Biotec, 1:100) for 30 min at room temperature.

Techniques: Isolation, Derivative Assay, Immunofluorescence, Confocal Microscopy, Staining

Journal: Nature Communications

Article Title: The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner

doi: 10.1038/s41467-018-05793-2

Figure Lengend Snippet: rVAR2-capture of CTCs from prostate cancer patients. a Number of CK+ CD45− DAPI+ CTCs isolated from 7.5 mL blood from four prostate cancer patients using rVAR2 or anti-EpCAM antibody-coated beads or the CellSearch® CTC platform. b CTC enumeration using rVAR2-coated beads on blood samples from prostate cancer patients with different disease stages ( n = 25) as well as from healthy controls ( n = 16) and patients with non-malignant diseases ( n = 12). ( P = 0.0001 for association between disease severity and CTC number, Kruskal–Wallis test). UTI: urinary tract infection, BPH: benign prostatic hyperplasia

Article Snippet: The cells were blocked for 5 min in 10% normal donkey serum (NDS) prior to stain with PE-conjugated anti-CD45 [5B-1] antibody (Cat. No. 130-080-201, MACS Miltenyi Biotec, 1:100) for 30 min at room temperature.

Techniques: Isolation, Infection