Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

To clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03864-0.

b.The experimental units were cages of animals (3-4 animals per cage, each animal with a different mark to be followed in our Pyrat software).
Sample size 2. a. Two experimental units were allocated to each group (2 cages with 3 to 4 animals).b.In this series of experiments, it is necessary to be able to distinguish between the overall survival and sensitivity of the three groups with different implants to temozolomide.With a power = 0.8 and alpha at one-sided question = 0.05, a significant difference between the groups would be possible with an assumed therapeutic effect leading to a difference in tumor volumes of >50% with a standard deviation of 33% with a group size of n = 8.Due to technical limitations, such as implantation accuracy during surgery and histological evaluability due to the growth form of the tumor, it is recommended to use a group size of n = 10, however based on the 3R principle, we decided on n=8 to reduce the number of animals, but still be able to obtain statistically significant differences.

Inclusion and exclusion criteria
3. a.We followed the state of the animals using score sheets, which included the following criteria: ).The anesthetized mouse is positioned in the stereotactic frame in the thoracic position by hooking the mouse's incisors into the bite bar of the snout holder and tightening the nasal clamp over the snout and tightening the nasal clamp over the snout, ensuring that the mouse's head is exactly horizontal.the head of the mouse is exactly horizontal.The position of the animal is adjusted so that the tips of the ear supports are at the caudal end of the ear canal.Once the position of the skull is adjusted caudally, the bite splint is attached to the stereotaxic frame.
After adjusting the height of the ear bars, they are advanced into the caudal portion of the ear canal and secured so that the head of the mouse is exactly horizontal and immovable when touched by the is immovable when touched by the finger.After the ear rods are in place, the animal is monitored for signs of respiratory distress.The scalp was disinfected and after opening the scalp rind through a longitudinal 2-3 mm incision, the bregma was identified.The drill hole site was 3.0 mm lateral to the bregma and was first marked on the bone by a small score with a sterile cannula.A hole was then drilled through the calvaria at the marked site using a microdrill.A sterile hollow needle is then screwed into this hole exactly perpendicular to the skull calvaria of the mice.The following stereotactic coordinates are used in relation to the bregma 1 mm (anteroposterior axis), 3 mm (lateral axis), 2.5 mm (vertical axis).Through this needle, tumor cells in a total volume of 3 μl were injected into the basal ganglia of the animals over 3 minutes using a special microsyringe (model Hamilton, type 702).The needle was then slowly removed over several stages and the skin is sealed with a skin adhesive.The ear supports were loosened, the mouse removed from the stereotactic apparatus and placed on a heating pad set to 37°C until the animal regains consciousness.The animals were inspected each day and three weeks post implantation, temozolomide treatment was started.Temozolomide was purchased from Sigma-Aldrich, diluted in DMSO at a concentration of 10 mg/ml, aliquoted and stored at -80°C.For treatment, temozolomide was quickly thawed and diluted in sterile 0.9 % NaCl to a final concentration of 1 mg/ml.Mice were injected i.p. with 5 mg/kg Body weight 5 days a week for 3 weeks.

1.body weight, relative to the weight of the untreated control group
Kaplan-Meyer estimates: log-rank test (Mantel-Cox test), Graph Pad Prism 6.01 Experimental animals 8. a. Immunodeficient mice (strain NMRI-Foxn1 nu/ nu), females, 6 weeks old at the time of arrival at the TARC facility, body weight 21-27 g.After acclimatization period of 2 weeks, they were implanted at the age of 8 weeks.b.The animals were purchased from Charles River Europe and were not used in any previous procedures.Experimental procedure 9. Animal were maintained in thermostat and experiments were conducted at the Translational Animal Research Center (TARC) of the Johannes Gutenberg University Medical Center Mainz.Before implantation, mice were acclimatized for 2 weeks (see p.8), the implantations were performed.The animals were first anesthetized by i.p. injection of a ketamine/xylazine mixture in 1xPBS=phosphate salt buffer (ketamine 140 mg/xylazine 10 mg/kg body weight; application volume 0.01ml/g