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. 2023 Jan 13;18:243–261. doi: 10.2147/IJN.S375918

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© 2023 Durand et al.

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Impact of treatments on escaped tumor cells. Fifteen U87 spheroids were seeded per well. Many of tumor cells escaped from the spheroids and adhered to the bottom of the flasks: we defined them as “escaped cells” or “invading cells”. (A) After trypsinization, recovered cells were counted and (B) cell viability was determined using the Trypan blue exclusion assay. Results are presented as mean ± SD (n ≥ 4 independent experiments). (C) The escaped cells were photographed using a transmitted light microscope (Nikon DIAPHOT 300 equipped with a Nikon Digital sight-DS-Fi1 camera) (40X magnification). These invading cells were able to form new clusters (red arrows) and clusters could be linked together by isolated cells that emitted membrane extensions. The scale bar is 100µm. (D) Histograms represent the treatment-induced mitotic catastrophe (MC). Hematoxylin and eosin staining of invading cells allowed to detect morphologically abnormal nuclei (ie micro- and multinucleation) and the MC rate (in %) was determined for each therapeutic condition as the ratio of the number of mitotic catastrophe events to the total number of cells. Results are presented as mean ± SD (n ≥ 4 independent experiments). For graphs A, B and (D) # = significant difference between RT groups versus Ctrl group and * = significant difference between “with Au@DTDTPA(Gd) nanoparticles” groups versus ‘w/o nanoparticles’ groups at p < 0.05 according to the Mann–Whitney U-test. (E) Invading cells were fixed on T15 for immunocytochemistry experiments and analyzed by confocal microscopy: visualization of F-actin was performed using Alexa Fluor™ 488-conjugated Phalloidin (green), connexin 43 was detected using a primary anti-Cx43 antibody and an Alexa Fluor 555-conjugated secondary antibody (red) while nuclei were counterstained with Hoechst 33432. Irradiated cells (RT 2×5 Gy) showed actin-rich membrane protrusions and numerous punctate Cx43 signals at these extensions. When cells were exposed to Au@DTDTPA(Gd) nanoparticles, the organization of the actin cytoskeleton was strongly modified and Cx43 immunoreactivity corresponded to diffuse intracytoplasmic staining. White arrow heads show Cx43 signals. Images on the top line: the scale bar is 150µm; Inset: the scale bar is 75µm.