sj-mp4-1-lan-10.1177_00236772231169344 - Supplemental material for Urinary bladder catheterisation of female pigs: Influence of bladder content and <i>Escherichia coli</i> urinary tract infection on procedural outcome

Supplemental material, sj-mp4-1-lan-10.1177_00236772231169344 for Urinary bladder catheterisation of female pigs: Influence of bladder content and Escherichia coli urinary tract infection on procedural outcome by Kristian Stærk, Louise Langhorn, Bo Halle and Thomas Emil Andersen in Laboratory Animals</p

Additional file 3: Figure S3. of Effects of the lysosomal destabilizing drug siramesine on glioblastoma in vitro and in vivo

Patient-derived spheroids exposed to siramesine. The glioblastoma stem cell-like containing spheroid (GSS) cultures T78, T86 and T87 were exposed to siramesine (0-15 μM) for 24 h. Light microscopy imaging showed that the spheroids started to disintegrate already at 5–10 μM. Scalebar 100 μm. (TIF 2610 kb

Additional file 4: Figure S4. of Effects of the lysosomal destabilizing drug siramesine on glioblastoma in vitro and in vivo

Propidium iodide uptake in patient-derived spheroids. In order to evaluate the expected diffusion of siramesine through the membranes used for culturing of spheroid-brain slice co-cultures, spheroids alone were placed directly upon these membranes and exposed to medium with 20 μM siramesine. The medium was present below the membranes similar to the procedure when culturing brain slice cultures. (A) Propidium iodide (PI) uptake on day 6 was detected as both red (arrows) and yellow (arrowheads) fluorescence in both T78 and T86 spheroids compared to control cultures. (B) Measuring PI uptake by using a software classifier identifying red and yellow staining per total area, a significant PI uptake was clearly seen, especially on day 6. Scalebar 100 μm. Data are displayed as mean values ± SEM, and **P < 0.01, ***P < 0.001 were assessed by one-way ANOVA. AU, arbitrary units. (TIF 7565 kb

Ki-67 labeling index and scoring of stem cell markers in cultured spheroids, in vivo xenografts and implanted spheroids.

<p>Ki-67 LI (A) was measured in cultured spheroids, in vivo xenografts and implanted spheroids using ki-67 stained histological sections. A semi-quantitative scoring was performed for CD133 (B) nestin (C) and podoplanin (D). Data are shown as means ± SEM, n = 4–9 for cultured spheroids, n = 2–12 for in vivo xenografts, n = 5–58 for implanted spheroids, and statistical significance *P<0.05, **P<0.01, ***P<0.001 was investigated using ANOVA with Bonferroni correction for comparison of all groups.</p

Quantitation of area and invasion in confocal images.

<p>The spheroid area was measured in the confocal images on day 0, 3 and 6 (T78: n = 48, T86: n = 73, T87: n = 21, U87: n = 21) (A) as well as the invasion area outside the spheroid (B). Data are shown as means ± SEM, and statistical significance *P<0.05, **P<0.01, ***P<0.001 was investigated using ANOVA with Bonferroni correction for comparison with day 0.</p

Characterization of GSS cultures.

<p>The three GSS cultures were cultured in serum-free medium as spheroids, which upon trypsination to single cells developed new spheroids (A-C). Cells were seeded and the cell number estimated at day 1–5 (n = 3) (D-F). Differentiation assays were performed showing expression of the astrocytic marker GFAP (G-I) and the neuronal marker MAP2 (J-L), here illustrated for T78. The MGMT status for each culture was determined by PCR and T78 and T87 were found to be methylated whereas T86 was unmethylated (M). All three GSS cultures were derived from IDH1-negative tumors representing primary glioblastomas (M). Scalebar 100 μm (A-C) and 50 μm (J-L).</p

Invasion in the in vitro invasion model.

<p>Thin 3 μm sections of spheroids implanted into brain slice cultures were stained by immunostained using anti-human vimentin (A, C, E, G) and CD56 (B, D, F, H) antibodies. Co-cultures with both U87 spheroids (A, B), T78 (C, D), T86 (E, F) and T87 spheroids (G, H) were stained. Spheroid area (I), invasion area (J) and longest invasion distance (K) were measured on the immunostained sections (T78: n = 147, T86: n = 98, T87: n = 35, U87: n = 36). Data are shown as means ± SEM, and statistical significance */$P<0.05, **/$ $P<0.01, ***/$ $$ P<0.001 was investigated using ANOVA with Bonferroni correction for comparison of all groups. $ is comparison of the GSS cultures to U87. Scalebar 100 μm (A-H).</p

Confocal images of spheroid invasion.

<p>Confocal z-stacks were recorded on day 0, one hour after implantation of DiI-labeled spheroids into the brain slice cultures and after 3 and 6 days. The z-stacks were superimposed into one image representing the entire spheroid. The glioma cell lines U87 showed no particular invasion of cells into the brain slice cultures (A-C), whereas the three GSS cultures all showed pronounced invasion of tumor cells into the brain tissue (D-L). Scalebar 100 μm.</p