Projects in detail

Improving the efficacy of proteasome inhibitors in the treatment of renal cell carcinoma

The pathogenesis of renal cell cancer (RCC) is in particularly driven by HIF-1α. Proteasome inhibitors (PI) specifically inhibit activation of the C-terminal transactivation domain of HIF-1α, implicating the use of PI for the treatment of RCC. The 1st generation PI bortezomib (BTZ) showed antitumor activity in individual patients with metastatic RCC a decade ago. However, in particular neurotoxicity of BTZ at the aspired dose of 1.5 mg/m2 and subsequent dose reductions in the majority of patients limited the use of BTZ in RCC patients. Concepts to improve PI-based cancer therapies have since emerged, including the more selective, irreversible 2nd generation proteasome inhibitor carfilzomib (CFZ) that lacks neurotoxicity, as well as the biology-driven concept of pre-activation of the unfolded protein response (UPR) to increase PI-mediated cytotoxicity. HIV protease inhibitors, like lopinavir, induce UPR activation in mammalian cells at therapeutic plasma levels and overcome PI resistance of multiple myeloma. Such novel options warrant re-evaluating the use of PI for RCC treatment.

 We evaluated the effects of BTZ and CFZ in combination with lopinavir on human RCC cell lines in vitro (CAKI-2, A-498, MZ-1774). Cell viability was assessed by MTS test and combination indices statistically indicating synergy between the drugs were calculated using normalized isobolograms. UPR activation was analyzed by quantitative real-time PCR of the UPR key components (i.e., BIP, XBP1, ATF4, CHOP, BCL-2, BAX). Apoptosis and reactive oxygen species (ROS) production were assessed by FACS.

 Treatment of RCC cell lines (CAKI-2, A-498 and MZ-1774) with BTZ or CFZ alone in the relevant dose range of up to 40 nM had only poor cytotoxic activity. Likewise, treatment with lopinavir alone at clinically relevant serum levels (up to 10 mM) had a moderate cytotoxic effect (40% reduction in viability). The combination of proteasome inhibitors with lopinavir showed strong synergistic cytotoxic activity across all RCC cell lines (over 90% cytotoxicity, combination index of 0.47 with BTZ and 0.05 with CFZ, values < 1 indicate drug synergy). Lopinavir in combination with CFZ resulted in an up-regulation of BIP, XBP1s, ATF4, CHOP and a significant increased BAX/BCL-2 ratio, suggesting robust UPR activation and UPR-mediated apoptotic signaling by the combination of CFZ and lopinavir. ROS production and apoptosis were significantly induced by combination therapy.

 The combination of CFZ and the approved HIV protease inhibitor lopinavir has strong synergistic cytotoxic activity against RCC cell lines in vitro at therapeutically relevant drug concentrations. This synergy is most likely explained by synergistic triggering of UPR-induced apoptotic signaling by CFZ and lopinavir. Our findings together with the most recent successful use of a combination of PI and HIV-protease inhibitors in the treatment of PI-refractory multiple myeloma in phase II suggest to test the safety and activity of CFZ combined with lopinavir in patients with metastatic RCC in a clinical trial.

Bacterial Adhesion on Soft Materials: Passive Physicochemical Interactions or Active Bacterial Mechanosensing?

The influence of mechanical stiffness of biomaterials on bacterial adhesion is only sparsely studied and the mechanism behind this influence remains unclear. Here, bacterial adhesion on polydimethylsiloxane (PDMS) samples, having four different degrees of stiffness with Young’s modulus ranging from 0.06 to 4.52 MPa, is investigated. Escherichia coli  and Pseudomonas aeruginosa are found to adhere in greater numbers on soft PDMS (7- and 27-fold increase, respectively) than on stiff PDMS, whereas Staphylococcus aureus  adheres in similar numbers on the four tested surfaces. To determine whether the observed adhesion behavior is caused by bacteria-specific mechanisms, abiotic polystyrene (PS) beads are employed as bacteria substitutes. Carboxylate-modified PS (PS-COOH) beads exhibit the same adhesion pattern as E. coli  and P. aeruginosa  with four times more adhered beads on soft PDMS than on stiff PDMS. In contrast, amine-modified PS (PS-NH2 ) beads adhere in similar numbers on all tested samples, reminiscent of S. aureus  adhesion. This work demonstrates for the first time that the intrinsic physicochemical properties associated with PDMS substrates of different stiffness strongly influence bacterial adhesion and challenge the previously reported theory on active bacterial mechanosensing, which provides new insights into the design of antifouling surfaces.

Influence of biofilms on morbidity associated with short-term indwelling ureteral stents: a prospective observational study.

Purpose: To evaluate the influence of biofilms on morbidity associated with short-term ureteral stenting using contemporary methods of biofilm examination and validated assessment of symptoms.

Methods: Patients undergoing temporary ureteral stenting for secondary ureterorenoscopy due to urinary calculi were prospectively included. The German Ureteral Stent Symptoms Questionnaire (USSQ) was used to assess stent-associated morbidity. Biofilms were removed from stents using 'pinhole extraction', a novel, validated, abrasion-based technique. Extracted biofilms were analyzed for total mass, bacterial load and mineral components. Correlation between total biofilm mass and USSQ total score was the primary outcome variable analyzed using Spearman correlation. Secondary outcomes included correlations between various biofilm characteristics and symptoms.

Results: 94 patients were included in the analysis. Extracted biofilm mass had a median of 37.0 mg (0-310.2 mg) per stent. No correlation between total biofilm mass and USSQ total score was found (Spearman r = 0.012; p = 0.911). Correlations between biofilm characteristics and morbidity were generally weak and not significant. Significant correlations could be found between biofilm mass and hematuria (r = 0.280; p = 0.007), and between the number of bacteria (qPCR) and the USSQ subscore for pain (r = 0.243; p = 0.019) and the intake of analgesics (r = 0.259; p = 0.012).

Conclusion: Based on elaborated biofilm examination methods and validated self-reported outcome measures, our findings indicate that biofilms might aggravate some lower urinary tract symptoms but are not the main trigger for stent-associated morbidity in short-term ureteral stenting.

Extraction of Biofilms From Ureteral Stents for Quantification and Cultivation-Dependent and -Independent Analyses.

Ureteral stenting is a common surgical procedure, which is associated with a high morbidity and economic burden, but the knowledge on the link between biofilms on these stents, morbidity, and the impact of the involved microbiota is still limited. This is partially due to a lack of methods that allow for a controlled extraction of the biofilms from stents. Development of an appropriate in vitro model to assess prevention of biofilm formation by antimicrobial coatings and biomaterials requires a profound understanding of the biofilm composition, including the involved microbiota. This work describes an analytical pipeline for the extraction of native biofilms from ureteral stents for both cultivation-dependent and -independent analysis, involving a novel mechanical abrasion method of passing stent samples through a tapered pinhole. The efficiency of this novel method was evaluated by quantifying the removed biofilm mass, numbers of cultivable bacteria, calcium content, and microscopic stent analysis after biofilm removal using 30 clinical stent samples. Furthermore, the extraction of in vitro formed Escherichia coli biofilms was evaluated by universal 16S quantitative PCR, a cultivation-independent method to demonstrate efficient biofilm removal by the new approach. The novel method enables effective contamination-free extraction of the biofilms formed on ureteral stents and their subsequent quantification, and it represents a useful tool for comprehensive examinations of biofilms on ureteral stents.