Journal: Science Advances

Article Title: Contrasting temporal dynamics of fluorescence and photoacoustic signals from Cetuximab-IRDye800 conjugate in EGFR-overexpressing tumors

doi: 10.1126/sciadv.adv4639

Figure Lengend Snippet: ( A ) Schematic representation of the synthesis of Cetuximab-IRDye800 conjugate. The N -hydroxysuccinimide (NHS) ester of the dye was reacted with the anti-EGFR antibody, Cetuximab, in predefined ratios to develop the conjugate, followed by purification through gel filtration chromatography. “ x ” represents the respective number of dye molecules per antibody. ( B ) Ultraviolet-visible (UV-Vis) absorption spectra of IRDye800 (magenta) and Cetuximab-IRDye800 (cyan) in phosphate-buffered saline (PBS). The spectrum for Cetuximab-IRDye800 shows characteristic absorption peaks for both IRDye800 and Cetuximab along with a peak at 703 nm characteristic of H-aggregate formation. ( C ) SDS-PAGE image of Cetuximab-IRDye800 showing binding of the dye to both the heavy and light chains of the Cetuximab antibody. Free dye, pseudocolored in blue (IRDye800) band, is shown as a reference. HC, heavy chain; LC, light chain. ( D ) Table showing the conjugation ratio of IRDye800 per mole Cetuximab in a reaction to generate Cetuximab-IRDye800 with varying IRDye800 ratios, as mentioned in the sample name. The conjugation ratios for IRDye800 are listed along with the conjugation efficiency. Data are presented as mean ± SD. n ≥ 2 per sample.

Article Snippet: The human anti-EGFR antibody (Cetuximab, ERBITUX from Eli Lilly) was obtained from Premium Health Services (Columbia, MD, USA).

Techniques: Purification, Filtration, Chromatography, Saline, SDS Page, Binding Assay, Conjugation Assay

Journal: Science Advances

Article Title: Contrasting temporal dynamics of fluorescence and photoacoustic signals from Cetuximab-IRDye800 conjugate in EGFR-overexpressing tumors

doi: 10.1126/sciadv.adv4639

Figure Lengend Snippet: ( A ) Hematoxylin and eosin (H&E) and corresponding immunofluorescence image of tumor cross section obtained from a mouse tumor 6 hours after Cetuximab-IRDye-AF injection. ( B ) H&E and corresponding immunofluorescence image of tumor cross section obtained from a mouse tumor 24 hours after Cetuximab-IRDye-AF injection [scale bars, 2.5 mm and 100 μm (inset)]. ( C to G ) Quantification of Cetuximab-IRDye-AF distribution heterogeneity parameters including mean Cetuximab-IRDye-AF intensity (C), %Cetuximab-IRDye-AF coverage area (D), Cetuximab-IRDye-AF–to–CD31 ratio to quantify the amount of Cetuximab-IRDye-AF per blood vessel (E), distance of Cetuximab-IRDye-AF from nearest CD31 + blood vessel (F), and Cetuximab-IRDye-AF–to–EGFR ratio to quantify number of EGFR bound by Cetuximab-IRDye-AF (G). Data are presented as mean ± SD ( n ≥ 10 tumor sections per group from more than five mice each for the 6 and 24 hours and two mice from the untreated group), analyzed using Kruskal-Wallis test, followed by post hoc pairwise comparisons using Dunn’s test, except for Cetuximab-IRDye-AF distance to CD31 (F), which is analyzed using Welch’s t test. P values are provided for each graph.

Article Snippet: The human anti-EGFR antibody (Cetuximab, ERBITUX from Eli Lilly) was obtained from Premium Health Services (Columbia, MD, USA).

Techniques: Immunofluorescence, Injection

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A) Cell lines were incubated with anti-EGFR-PE and the PE fluorescence was measured by flow cytometry. The median fluorescence intensity for the respective quadrant was used to calculate relative levels of EGFR expression on cell surface compared to the A431 cell line. Proteomic data was calculated from mass spectrometer peptide counts. (B) Relative levels of EGFR expression on cell surface of several CRC cell lines and breast cancer cell lines, BT-20 and MDA-MB-468, compared to the A431 cell line.

Article Snippet: An analogue of the anti-EGFR affibody [ ], Z EGFR 1907’ -Cys was expressed in E.coli BL21 (DE3) and purified on a Ni-NTA column from lysates as described in the methods section.

Techniques: Incubation, Fluorescence, Flow Cytometry, Expressing, Mass Spectrometry