Mechanistic and translational studies to improve cisplatin sensitivity of testicular cancer

Testicular cancer (TC) is one of the most common solid tumors in men between 20-40 years of age. While cisplatin-based chemotherapy is highly effective in TC patients, cisplatin resistance is still an important issue and refractory testicular cancer is a significant cause of death in this relatively young age group. Therefore, we aimed to identify new therapeutic targets and treatment strategies to overcome cisplatin resistance. To assist in pre-clinical validation of novel therapeutic strategies, we established and characterized three testicular cancer patient-derived xenograft (PDX) models which we showed to faithfully reflect the patient tumor it originated from at a histological, molecular and chemosensitivity level. Furthermore, we investigated different resistance mechanisms and novel combination therapies. First, we showed that TC cell lines are characterized by a highly active PI3K/AKT/mTOR pathway, and that inhibition of this pathway, especially mTORC1/2 inhibition, is an effective therapeutic strategy to sensitize TC cell lines and TC PDX models to cisplatin treatment. Second, we investigated therapeutic strategies based on MDM2 inhibition, showing that MDM2 inhibitors sensitized TC cells to both mTORC1/2 inhibitors and cisplatin. Addition of BH3 mimetics, mimicking pro-apoptotic proteins, to these two drug combinations further enhanced treatment efficacy. Furthermore, pre-clinical efficacy of MDM2 inhibitors combined with cisplatin was demonstrated in TC mouse models. In conclusion, this thesis provides new leads for patients with refractory or cisplatin resistant testicular cancer for future combination therapies involving targeted drugs against mTORC1/2, MDM2 and the chemotherapeutic cisplatin

Die Wachstumsparameter Adiponektin und IGFBP-2: Maternale und fetale Serumkonzentration sowie mRNA-Expression in Plazenta und mononukleären Zellen als Einflussfaktoren auf fetale Wachstumsregulation

Das intrauterine Wachstum wird bestimmt durch das fetale Wachstumspotential, das maternale Nahrungsangebot sowie die plazentare Versorgung. Dieses Zusammenspiel wird durch verschiedene hormonelle Regulationsmechanismen beeinflusst, die noch nicht in ihrer Gänze erkannt sind. Adiponektin, dessen insulinsentivierenden, antiinflammatorischen und vasoprotektiven Eigenschaften insbesondere bei der Erforschung von Erkrankungen, wie Diabetes mellitus, beachtet wird, hat nach neuesten Erkenntnissen auch einen entscheidenden positiven Einfluss auf das fetale Wachstum. IGFBP-2, ist das zweithäufigste IGF-Bindungsprotein im fetalen Organismus und kann auch unabhängig von IGF wachstumshemmend wirken. Speziell für IGFBP-1 und IGFBP-3 konnte gezeigt werden, dass IGF-Bindungsproteine auf Adiponektin und dessen Wirkungen Einfluss nehmen. Arbeiten die Adiponektin und IGFBP-2 vergleichen existieren nicht. Aufgrund der konträren Stellungen, die diese beiden Proteine auf das fetale Wachstum einnehmen, soll in dieser Arbeit erstmalig das Zusammenspiel von Adiponektin und IGFBP-2 in allen drei Kompartimenten der materno-plazento-fetalen Einheit betrachtet werden. Dazu wurden maternale Serumproben (n=19), Serum aus dem Narbelschnurblut (n=20), mononukleäre Zellen (MNC) aus den entsprechenden Blutproben sowie Gewebeproben von den Plazenten (n=22) zum Zeitpunkt der Geburt gewonnen. Adiponektin korreliert dabei positiv mit fetalen Wachstum, jedoch wird es nicht in Plazenta und MNC exprimiert. Eine direkte Beeinflussung der Adiponektinwirkung durch IGFBP-2 besteht nicht. Es zeigten sich allerdings Hinweise darauf, dass IGFBP-2 im Feten negativ mit dem Metabolismus der Mutter assoziiert ist und positiv mit dem Plazentawachstum in Verbindung steht, was dafür spricht, dass IGFBP-2 ein Signalbote zwischen den Kompartimenten er materno-plazento-fetalen Einheit ist

Nucleoplasmic β-actin exists in a dynamic equilibrium between low-mobility polymeric species and rapidly diffusing populations

β-Actin, once thought to be an exclusively cytoplasmic protein, is now known to have important functions within the nucleus. Nuclear β-actin associates with and functions in chromatin remodeling complexes, ribonucleic acid polymerase complexes, and at least some ribonucleoproteins. Proteins involved in regulating actin polymerization are also found in the interphase nucleus. We define the dynamic properties of nuclear actin molecules using fluorescence recovery after photobleaching. Our results indicate that actin and actin-containing complexes are reduced in their mobility through the nucleoplasm diffusing at ∼0.5 μm2 s−1. We also observed that ∼20% of the total nuclear actin pool has properties of polymeric actin that turns over rapidly. This pool could be detected in endogenous nuclear actin by using fluorescent polymeric actin binding proteins and was sensitive to drugs that alter actin polymerization. Our results validate previous reports of polymeric forms of nuclear actin observed in fixed specimens and reveal that these polymeric forms are very dynamic

Testicular cancer: Determinants of cisplatin sensitivity and novel therapeutic opportunities:Determinants of cisplatin sensitivity and novel therapeutic opportunities

Testicular cancer (TC) is the most common solid tumor among men aged between 15 and 40 years. TCs are highly aneuploid and the 12p isochromosome is the most frequent chromosomal abnormality. The mutation rate is of TC is low, with recurrent mutations in  KIT and  KRAS observed only at low frequency in seminomas. Overall cure rates are high, even in a metastatic setting, resulting from excellent cisplatin sensitivity of TCs. Factors contributing to the observed cisplatin sensitivity include defective DNA damage repair and a hypersensitive apoptotic response to DNA damage. Nonetheless, around 10–20% of TC patients with metastatic disease cannot be cured by cisplatin-based chemotherapy. Resistance mechanisms include downregulation of OCT4 and failure to induce PUMA and NOXA, elevated levels of MDM2, and hyperactivity of the PI3K/AKT/mTOR pathway. Several pre-clinical approaches have proven successful in overcoming cisplatin resistance, including specific targeting of PARP, MDM2 or AKT/mTOR combined with cisplatin. Finally, patient-derived xenograft models hold potential for mechanistic studies and pre-clinical validation of novel therapeutic strategies in TC. While clinical trials investigating targeted drugs have been disappointing, pre-clinical successes with chemotherapy and targeted drug combinations fuel the need for further investigation in clinical setting

Dual mTORC1/2 inhibition sensitizes testicular cancer models to cisplatin treatment

Testicular cancer is the most common cancer type among young men. Despite highly effective cisplatin-based chemotherapy, around 20% of patients with metastatic disease will still die from the disease. The aim of this study was to explore the use of kinase inhibitors to sensitize testicular cancer cells to cisplatin treatment. Activation of kinases, including receptor tyrosine kinases and downstream substrates, was studied in five cisplatin-sensitive or -resistant testicular cancer cell lines using phospho-kinase arrays and Western blotting. The phospho-kinase array showed AKT and S6 to be among the top phosphorylated proteins in testicular cancer cells, which are part of the PI3K/AKT/mTORC pathway. Inhibitors of most active kinases in the PI3K/AKT/mTORC pathway were tested using apoptosis assays and survival assays. Two mTORC1/2 inhibitors, AZD8055 and MLN0128, strongly enhanced cisplatin-induced apoptosis in all tested testicular cancer cell lines. Inhibition of mTORC1/2 blocked phosphorylation of the mTORC downstream proteins S6 and 4E-BP1. Combined treatment with AZD8055 and cisplatin led to reduced clonogenic survival of testicular cancer cells. Two testicular cancer patient-derived xenografts (PDX), either from a chemosensitive or -resistant patient, were treated with cisplatin in the absence or presence of kinase inhibitor. Combined AZD8055 and cisplatin treatment resulted in effective mTORC1/2 inhibition, increased caspase-3 activity, and enhanced tumor growth inhibition. In conclusion, we identified mTORC1/2 inhibition as an effective strategy to sensitize testicular cancer cell lines and PDX models to cisplatin treatment. Our results warrant further investigation of this combination therapy in the treatment of patients with testicular cancer with high-risk relapsed or refractory disease

Establishment and characterisation of testicular cancer patient-derived xenograft models for preclinical evaluation of novel therapeutic strategies

Testicular cancer (TC) is the most common solid tumour in young men. While cisplatin-based chemotherapy is highly effective in TC patients, chemoresistance still accounts for 10% of disease-related deaths. Pre-clinical models that faithfully reflect patient tumours are needed to assist in target discovery and drug development. Tumour pieces from eight TC patients were subcutaneously implanted in NOD scid gamma (NSG) mice. Three patient-derived xenograft (PDX) models of TC, including one chemoresistant model, were established containing yolk sac tumour and teratoma components. PDX models and corresponding patient tumours were characterised by H&E, Ki-67 and cyclophilin A immunohistochemistry, showing retention of histological subtypes over several passages. Whole-exome sequencing, copy number variation analysis and RNA-sequencing was performed on these TP53 wild type PDX tumours to assess the effects of passaging, showing high concordance of molecular features between passages. Cisplatin sensitivity of PDX models corresponded with patients' response to cisplatin-based chemotherapy. MDM2 and mTORC1/2 targeted drugs showed efficacy in the cisplatin sensitive PDX models. In conclusion, we describe three PDX models faithfully reflecting chemosensitivity of TC patients. These models can be used for mechanistic studies and pre-clinical validation of novel therapeutic strategies in testicular cancer

The Association of Dexamethasone and Hydrocortisone with Cerebellar Growth in Premature Infants

Objectives: Corticosteroids are used to prevent or treat lung disease of prematurity. While neurological side effects have been reported, detailed effects on cerebellar growth are unknown. This study aimed to compare cerebellar growth in premature infants who received dexamethasone or hydrocortisone to premature infants who did not receive postnatal corticosteroids. Study Design: Retrospective case-control study in infants born at a gestational age of <29 weeks and admitted to two level 3 neonatal intensive care units. Exclusion criteria were severe congenital anomalies and cerebellar or severe supratentorial lesions. Infants were treated with dexamethasone (unit 1) or hydrocortisone (unit 2) for chronic lung disease. Controls (unit 1) did not receive postnatal corticosteroids. Sequential head circumference (HC) and ultrasound measurements of transcerebellar diameter (TCD), biparietal diameter (BPD), and corpus callosum-fastigium length (CCFL) were performed until 40 weeks' postmenstrual age (PMA). Growth was assessed using linear mixed models correcting for PMA at measurement, sex, HC z-score at birth, and a propensity score indicating illness severity. Group differences before treatment were assessed using linear regression. Results: 346 infants were included (68 dexamethasone, 37 hydrocortisone, 241 controls). Before starting corticosteroids, TCD, BPD, and HC measurements did not differ between patients and controls at a comparable PMA. After starting treatment, both types of corticosteroid had a negative association with TCD growth. BPD, CCFL, and HC growth were not negatively affected. Conclusion: Administration of dexamethasone and hydrocortisone are both associated with impaired cerebellar growth in premature infants without evident negative associations with cerebral growth

Influence of wear and overwear on surface properties of etafilcon A contact lenses and adhesion of Pseudomonas aeruginosa

PURPOSE. To determine changes in physicochemical surface properties of contact tenses (CLs) during daily wear and effects of lens wear on adhesion of a Pseudomonas aeruginosa strain from a patient with CL-related keratitis.METHODS. Ten new CL wearers used ionic, etafilcon A lenses with 58% water on both eyes for approximately 10 hours each day during 10 and 50 days. All lenses were treated daily with an appropriate lens care solution. After the CLs were worn for 10 days (first pair of lenses) and 50 days (second pair, representing overwear), hydrophobicity by water contact angles, surface roughness by atomic force microscope, elemental surface composition by x-ray photoelectron spectroscopy (XPS), and adsorbed proteins by SDS-PAGE were determined on one lens. The lens from the contralateral eye was placed in a parallel plate flow chamber for bacterial adhesion after each time interval.RESULTS. Water contact angles on lenses changed from 45degrees on unused lenses to 61degrees +/- 25degrees after 10 days of wear and changed significantly (P &lt;0.05) to 27degrees +/- 14degrees after 50 days of wear. Surface roughness increased significantly (P &lt;0.05) from 4 +/- 2 nm (unused) to 10 +/- 7 nm after 50 days of wear. These changes were accompanied by adsorption of proteinaceous material, as evidenced by XPS and SDS-PAGE, demonstrating adsorption of lysozyme, tear lipocalin, and a 30-kDa protein. Initial bacterial adhesion to worn CLs was lower than to unworn CLs. Furthermore, detachment of adhering bacteria from worn lenses was easier than from unworn lenses. The changes observed in the physicochemical surface properties of the lenses after the CLs were worn for 50 days were accompanied by reports of discomfort by 6 of the 10 new CL wearers. Multiple regression analysis revealed that the most predictive variables for an effect on initial deposition after 10 days of wear were hydrophobicity, roughness, the presence of nitrogen-rich material, including the presence of a 30-kDa protein, and the presence of oxygen-rich material-that is, the type of oxygen adsorbed (O==C or O-C). After 50 days of wear, roughness and the presence of tear lipocalin were most predictive.CONCLUSIONS. This study demonstrates that the physicochemical surface properties changed after wear and overwear, whereas overwear of the lenses decreased initial adhesion of P. aeruginosa #3 under the present experimental conditions.</p

Improved neonatal brain MRI segmentation by interpolation of motion corrupted slices

BACKGROUND AND PURPOSE: To apply and evaluate an intensity‐based interpolation technique, enabling segmentation of motion‐affected neonatal brain MRI. METHODS: Moderate‐late preterm infants were enrolled in a prospective cohort study (Brain Imaging in Moderate‐late Preterm infants “BIMP‐study”) between August 2017 and November 2019. T2‐weighted MRI was performed around term equivalent age on a 3T MRI. Scans without motion (n = 27 [24%], control group) and with moderate‐severe motion (n = 33 [29%]) were included. Motion‐affected slices were re‐estimated using intensity‐based shape‐preserving cubic spline interpolation, and automatically segmented in eight structures. Quality of interpolation and segmentation was visually assessed for errors after interpolation. Reliability was tested using interpolated control group scans (18/54 axial slices). Structural similarity index (SSIM) was used to compare T2‐weighted scans, and Sørensen‐Dice was used to compare segmentation before and after interpolation. Finally, volumes of brain structures of the control group were used assessing sensitivity (absolute mean fraction difference) and bias (confidence interval of mean difference). RESULTS: Visually, segmentation of 25 scans (22%) with motion artifacts improved with interpolation, while segmentation of eight scans (7%) with adjacent motion‐affected slices did not improve. Average SSIM was .895 and Sørensen‐Dice coefficients ranged between .87 and .97. Absolute mean fraction difference was ≤0.17 for less than or equal to five interpolated slices. Confidence intervals revealed a small bias for cortical gray matter (0.14‐3.07 cm(3)), cerebrospinal fluid (0.39‐1.65 cm(3)), deep gray matter (0.74‐1.01 cm(3)), and brainstem volumes (0.07‐0.28 cm(3)) and a negative bias in white matter volumes (–4.47 to –1.65 cm(3)). CONCLUSION: According to qualitative and quantitative assessment, intensity‐based interpolation reduced the percentage of discarded scans from 29% to 7%