When the same combination therapy was used in the EGFR-negative cancer cell line MCF-7, only around 10% of cancer cells underwent apoptosis

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When the same combination therapy was used in the EGFR-negative cancer cell line MCF-7, only around 10% of cancer cells underwent apoptosis. anti-EGFR functionalisation significantly increased the association of tAuNRs with each EGFR-positive cancer cell. Considering this, the MTT assay showed that photothermal therapy (PTT) significantly increased cancer cell death ( 97%) in head and neck cancer cell line Fluorocurarine chloride Fluorocurarine chloride CAL-27 using tAuNRs but not uAuNRs, apoptosis being the major mechanism of cell death. This successful targeting and therapeutic outcome highlight the future use of tAuNRs for molecular photoacoustic imaging or tumour treatment through plasmonic photothermal therapy. for 5 min. Then, 1 106 cells in 100 L FACS buffer (10% FBS in PBS) were blocked Fluorocurarine chloride with 5 L Human TruStain FcX (#422301, BioLegend, San Diego, CA, USA) for 10 min on ice. The cells were incubated with either 0.05 g of recombinant monoclonal human EGFR (Research Grade Cetuximab Biosimilar, #FAB9577B, R&D Systems, Minneapolis, MN, USA) or of mouse IgG1 isotype (#IC002B, R&D Systems) biotinylated antibodies for 30 min on ice. Following washing, streptavidinCFITC secondary antibody (#F0030, R&D systems) was added at a concentration of 10 L/106 cells (10 g/mL) and incubated with the cells for 30 min on ice in a dark place. Cells were washed and then analysed on an Attune Flow Cytometer (Applied Biosystems, Temecula, CA, USA) and the results were analysed using the Attune software (Applied Biosystems, Life Technologies, Waltham, MA, USA). The percentage of EGFR-positive cells, mean and median fluorescent intensities were determined for all the four cancer cell types stained using the anti-EGFR antibody compared with the isotype control. 2.3. Immunofluorescence Imaging (IF) Cells were plated onto glass coverslips and allowed to grow to 70% confluence. Cells were washed with PBS and fixed in 4% PFA for 10 min at RT. The wells were further washed twice in PBS for 5 min each time and blocked in 10% (= 3, the dotted lines represent 95% CI. 3.3. In Vitro Targeting Efficiency To identify if there were any differences in cellular tAuNRs uptake in EGFR-positive KYSE-30 and CAL-27 cancer cells, the number of AuNRs aggregates associated with each Rabbit Polyclonal to SGCA cell following co-incubation was quantified based on small dots of clearly visible optical scatters from such AuNRs. Dark-field images indicated a higher AuNRs optical scattering in tAuNRs compared with uAuNRs (Physique 4A). A two-tailed nonparametric MannCWhitney 0.0001) between targeted and untargeted KYSE-30, indicating that antibody functionalisation increased the number of tAuNRs aggregates with optical scatter per cell (Physique 5A). The same was observed for the second EGFR-positive cancer cell line CAL-27 with a significantly higher ( 0.0001) number of AuNRs aggregates per cell in the targeted than in the untargeted AuNRs (Figure 5B). Open in a separate window Physique 4 Images showing different intensities of AuNRs signals in EGFR-positive and unfavorable cancer cell lines. Dark-field, DAPI and Texas Red images of (A) EGFR-positive cancer cell line KYSE-30 and CAL-27 and (B) EGFR-negative cancer cell lines Hep G2 and MCF-7 were taken for both Fluorocurarine chloride tAuNRs and uAuNRs groups. All dark-field (where bright yellow-to-orange colour aggregates represent AuNRs), DAPI and Texas Red (MemBrite fix) images were captured using an inverted Nikon microscope with a uniform setting throughout. Scale bar, 200 m. Open in a separate window Physique 5 Targeting increased the number of AuNRs in cancer cells. (ACD) Dot plots show a qualitative quantification of AuNRs aggregates with a strong optical scatter from aggregates imaged and counted per cell. MannCWhitney test showed that there was a significantly higher number of AuNRs aggregates counted in tAuNRs than in uAuNRs in: Fluorocurarine chloride (A) KYSE-30 ( 0.0001); (B).