Journal: Journal for Immunotherapy of Cancer

Article Title: Novel banana lectin CAR-T cells to target pancreatic tumors and tumor-associated stroma

doi: 10.1136/jitc-2022-005891

Figure Lengend Snippet: Glycan profile of PDAC tumor cell lines and primary PSCs. (A) Summary of N-glycan analysis by MALDI-TOF mass spectrometry for CFPAC-1, Panc-1, Capan-1, and PSCs. N-glycan profiling is described in detail in the methods section. (B) Cell surface detection of high-mannose expression by biotinylated H84T BanLec. PDAC tumor cells, PSCs, and activated T cells (ATCs) were stained with 0.4 µg/mL biotinylated H84T BanLec followed by APC streptavidin. Cells were stained with streptavidin alone (2° only) for control. Surface expression was analyzed by flow cytometry and mean fluorescent intensity is represented on histogram plots. BanLec, banana lectin; PDAC, pancreatic ductal adenocarcinoma; PSC, pancreatic stellate cells.

Article Snippet: PDAC cell lines, CFPAC-1, Capan-1, and Panc-1, were obtained from ATCC (Manassas, Virginia, USA).

Techniques: Mass Spectrometry, Expressing, Staining, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Novel banana lectin CAR-T cells to target pancreatic tumors and tumor-associated stroma

doi: 10.1136/jitc-2022-005891

Figure Lengend Snippet: In vivo antitumor and anti-stromal activity of H84T CAR-T cells against three different PDACs. (A) NSG MHC KO mice were engrafted subcutaneously with 1×10 6 GFP-FfLuc labeled CFPAC-1 tumor cells and 1×10 6 PSCs. Tumors were allowed to establish for 2 weeks and then 1×10 6 NT or H84T CAR-T cells were intravenously delivered. (B) Tumor signal was quantified by bioluminescence signaling through IVIS imaging. N=4 mice per group. (C) Tumor volume was measured by the caliper and volume was calculated overtime. P value<0.001 determined by simple linear regression. (D) Residual tumors were resected on Day 36 post T-cell infusion and processed for H&E staining. Mean pixel density of H&E staining was quantified by ImageJ. Four sections per each tumor were quantified and graphed. N=3 tumors/NTR group and N=4 tumors/H84T group. P value determined by unpaired student’s t-test. (E, F) NSG MHC KO mice were engrafted subcutaneously with 2×10 6 Panc-1 tumor cells and 2×10 6 PSCs. Tumors were allowed to establish for 2 weeks and then 1×10 6 NT or H84T CAR-T cells labeled with GFP-FfLuc were delivered intravenously. T-cell signal was quantified by bioluminescence signaling through IVIS imaging (F) bioluminesence images. (G) Tumor volume was measured by the caliper and volume was calculated overtime. N=4–5 mice per group. (H, I) NSG MHC KO mice were engrafted subcutaneously with 3×10 6 GFP-FfLuc labeled Capan-1 tumor cells and 3×10 6 PSCs. Tumors were allowed to establish for 1 week and then 1×10 6 NT or H84T CAR-T cells were intravenously delivered. Tumor volume (H) was quantified by caliper measurement and tumor signal (I) was quantified by bioluminescence signaling through IVIS imaging. N=4 mice per group. (J) Residual CFPAC-1+PSC tumors from mice treated with either NT or H84T CAR T cells were stained for anti-human vimentin to detect stroma cells. Vimentin pixel count was calculated by ImageJ analysis and representative IHC images are shown for three mice in each group. CAR, chimeric antigen receptor; PDAC, FfLuc, firefly luciferase; PDAC, pancreatic ductal adenocarcinoma; PSC, pancreatic stellate cells; NT, non-transduced; IHC, immunohistochemistry; GFP, green fluorescent protein.

Article Snippet: PDAC cell lines, CFPAC-1, Capan-1, and Panc-1, were obtained from ATCC (Manassas, Virginia, USA).

Techniques: In Vivo, Activity Assay, Labeling, Imaging, Staining, Luciferase, Immunohistochemistry

Journal: Journal of Biomarkers

Article Title: Immunoreactivity of Pluripotent Markers SSEA-5 and L1CAM in Human Tumors, Teratomas, and Induced Pluripotent Stem Cells

doi: 10.1155/2013/960862

Figure Lengend Snippet: SSEA-5 and L1CAM immunoreactivity in human tumor cell lines. A variety of human tumor cell lines were immunostained with either SSEA-5 or L1CAM using diaminobenzidine reaction so that immunopositive cells could be counted in groups of 100. (a) SSEA-5 and L1CAM immunoreactivity in human tumor cells. L1CAM (yellow bars) are plotted against the left y -axis; SSEA-5 (blue bars) are plotted against the right y -axis. Capan-1 displayed the greatest number of SSEA-5+ cells, while U87MG demonstrated the highest numbers of L1CAM+ cells. For all bars not visible, the number of positive cells was less than 1%. (b) Examples of fluorescent immunoreactivity: HT-29 cells display both SSEA-5 and L1CAM immunofluorescence, with some colocalization. LNCaP cells have low levels of L1CAM immunofluorescence and no detectable SSEA-5 immunofluorescence. Microscopic fields of T98G cells are immunonegative for both L1CAM and SSEA-5.

Article Snippet: Capan-1 pancreatic adenocarcinoma cells were cultured in Iscove's Modified Dulbecco's medium (ATCC) with 20% fetal bovine serum.

Techniques: Immunofluorescence