Clonal Dymanics and Phenotypic Plasticity within the Pancreatic Tumor Initiating Compartment

In pancreatic cancer and other solid tumor entities subpopulations of cancer stem cells (CSCs) or tumor initiating cells (TIC) have recently been identified. These cells were described to be tumorigenic in immunodeficient mice and give rise to the whole heterogeneity of the patient`s tumor. Besides those phenotypic markers previously associated with TIC function, little is known about pancreatic TIC. This thesis project unravels the clonal dynamics of long-term tumor growth in pancreatic cancer, and explores the phenotypic diversity within the pancreatic TIC population. Primary patient tumor samples were xenografted in immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice to remove benign human cells and receive sufficient amounts of tissue. Adherent cultures were established from xenograft tumor tissue in serum-free medium simulated with growth factors. These culture conditions allowed the enrichment of pancreatic TIC without restriction to a certain phenotype. These cultures grew as three-dimensional epithelial colonies with tight cell-cell contacts, and reliably initiated tumors in NSG mice. In order to induce TIC differentiation, culture conditions were changed to 10% FBS containing medium and withdrawal of cytokines. Subsequently, the cells lost three-dimensional growth, formed monolayers and showed irregular morphology. This was accompanied by a down-regulation of markers previously described for pancreatic TIC, stem cells of various entities or normal pancreatic progenitors. However, despite this differentiation-like phenotype, tumor initiation in serial transplantation was not substantially affected. Moreover, sorted CD133- cells formed equally efficient tumors as the CD133+ cell fraction and contained a similar proportion of CD133+ cells in vivo. In sum, these data indicate that pancreatic TIC are diverse with respect to marker expression, and exhibit an previously unknown phenotypic plasticity. To determine the clonal kinetics of individual TIC in vivo early passage serum-free cultures from 3 patients were lentivirally marked and serially transplanted over 3 generations in NSG mice. In primary mice, 0.003-0.113% of all transduced cells were detected to contribute significantly to tumor formation. However, the second and third generation tumor formation was predominantly driven by distinct TIC clones that were not detected in earlier generations, but recruited later to participate in tumor formation. Mathematical modeling indicated profound changes in the proliferation of individual TIC that produced mainly non-tumorigenic progeny with limited capacity of self-renewal. These data indicate that in pancreatic cancer long-term tumor growth is driven by the succession of transiently active TIC generating tumor tissue in temporally restricted bursts. The recruitment of inactive TIC clones to tumor formation after serial transplantation indicates a context-dependent switch between a quiescent and an active status. A clonal relation between tumorassociated fibroblast-like cells and neoplastic cells has been described in breast cancer. To investigate whether pancreatic TIC also give rise to fibroblast-like cell types xenograft tumors were analyzed in detail for their stromal compartment. Xenograft tumors contained no human stroma and attracted murine fibroblast-like cells instead. Human stroma cells were only found in xenograft tumors when these were co-transplanted with tumor cells, but engrafted with very low efficacy. Thus, due to the instability of human stroma cells in xenograft tumors grown in NSG mice, a possible clonal relation between fibroblast-like cells and neoplastic cells could not be investigated conclusively. In total, this study describes a previously unknown phenotypic and functional plasticity of pancreatic TIC. Its data show that TIC in pancreatic cancer have to be defined functionally level in vivo and that this cannot be replaced by the examination of phenotypic markers. Understanding the molecular mechanisms that regulate the activation and quiescence of pancreatic TIC might be important for future therapy approaches against pancreatic cancer

SOX2-RNAi attenuates S-phase entry and induces RhoA-dependent switch to protease-independent amoeboid migration in human glioma cells

<p>Abstract</p> <p>Background</p> <p>SOX2, a high mobility group (HMG)-box containing transcription factor, is a key regulator during development of the nervous system and a persistent marker of neural stem cells. Recent studies suggested a role of SOX2 in tumor progression. In our previous work we detected SOX2 in glioma cells and glioblastoma specimens. Herein, we aim to explore the role of SOX2 for glioma malignancy in particular its role in cell proliferation and migration.</p> <p>Methods</p> <p>Retroviral shRNA-vectors were utilized to stably knockdown SOX2 in U343-MG and U373-MG cells. The resulting phenotype was investigated by Western blot, migration/invasion assays, RhoA G-LISA, time lapse video imaging, and orthotopic xenograft experiments.</p> <p>Results</p> <p>SOX2 depletion results in pleiotropic effects including attenuated cell proliferation caused by decreased levels of cyclinD1. Also an increased TCF/LEF-signaling and concomitant decrease in Oct4 and Nestin expression was noted. Furthermore, down-regulation of focal adhesion kinase (FAK) signaling and of downstream proteins such as HEF1/NEDD9, matrix metalloproteinases pro-MMP-1 and -2 impaired invasive proteolysis-dependent migration. Yet, cells with knockdown of SOX2 switched to a RhoA-dependent amoeboid-like migration mode which could be blocked by the ROCK inhibitor Y27632 downstream of RhoA-signaling. Orthotopic xenograft experiments revealed a higher tumorigenicity of U343-MG glioma cells transduced with shRNA targeting SOX2 which was characterized by increased dissemination of glioma cells.</p> <p>Conclusion</p> <p>Our findings suggest that SOX2 plays a role in the maintenance of a less differentiated glioma cell phenotype. In addition, the results indicate a critical role of SOX2 in adhesion and migration of malignant gliomas.</p

Quantum Imaging with Incoherently Scattered Light from a Free-Electron Laser

The advent of accelerator-driven free-electron lasers (FEL) has opened new avenues for high-resolution structure determination via diffraction methods that go far beyond conventional x-ray crystallography methods. These techniques rely on coherent scattering processes that require the maintenance of first-order coherence of the radiation field throughout the imaging procedure. Here we show that higher-order degrees of coherence, displayed in the intensity correlations of incoherently scattered x-rays from an FEL, can be used to image two-dimensional objects with a spatial resolution close to or even below the Abbe limit. This constitutes a new approach towards structure determination based on incoherent processes, including Compton scattering, fluorescence emission or wavefront distortions, generally considered detrimental for imaging applications. Our method is an extension of the landmark intensity correlation measurements of Hanbury Brown and Twiss to higher than second-order paving the way towards determination of structure and dynamics of matter in regimes where coherent imaging methods have intrinsic limitations

The generality of the GUGA MRCI approach in COLUMBUS for treating complex quantum chemistry

The core part of the program system COLUMBUS allows highly efficient calculations using variational multireference (MR) methods in the framework of configuration interaction with single and double excitations (MR-CISD) and averaged quadratic coupled-cluster calculations (MR-AQCC), based on uncontracted sets of configurations and the graphical unitary group approach (GUGA). The availability of analytic MR-CISD and MR-AQCC energy gradients and analytic nonadiabatic couplings for MR-CISD enables exciting applications including, e.g., investigations of π-conjugated biradicaloid compounds, calculations of multitudes of excited states, development of diabatization procedures, and furnishing the electronic structure information for on-the-fly surface nonadiabatic dynamics. With fully variational uncontracted spin-orbit MRCI, COLUMBUS provides a unique possibility of performing high-level calculations on compounds containing heavy atoms up to lanthanides and actinides. Crucial for carrying out all of these calculations effectively is the availability of an efficient parallel code for the CI step. Configuration spaces of several billion in size now can be treated quite routinely on standard parallel computer clusters. Emerging developments in COLUMBUS, including the all configuration mean energy multiconfiguration self-consistent field method and the graphically contracted function method, promise to allow practically unlimited configuration space dimensions. Spin density based on the GUGA approach, analytic spin-orbit energy gradients, possibilities for local electron correlation MR calculations, development of general interfaces for nonadiabatic dynamics, and MRCI linear vibronic coupling models conclude this overview

The OpenMolcas Web: A Community-Driven Approach to Advancing Computational Chemistry

The developments of the open-source OpenMolcas chemistry software environment since spring 2020 are described, with a focus on novel functionalities accessible in the stable branch of the package or via interfaces with other packages. These developments span a wide range of topics in computational chemistry and are presented in thematic sections: electronic structure theory, electronic spectroscopy simulations, analytic gradients and molecular structure optimizations, ab initio molecular dynamics, and other new features. This report offers an overview of the chemical phenomena and processes OpenMolcas can address, while showing that OpenMolcas is an attractive platform for state-of-the-art atomistic computer simulations

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [&lt;1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None

Sensorineural Hearing Loss After Balloon Eustachian Tube Dilatation

Todt I, Oppel F, Sudhoff H. Sensorineural Hearing Loss After Balloon Eustachian Tube Dilatation. Frontiers in Surgery. 2021;8: 615360.Objective: Eustachian tube function is of central importance for the ventilation of the middle ear. A dysfunction can be associated with chronic otitis media, and cholesteatoma. Balloon Eustachian tube dilatation (BET) is a treatment option used to solve eustachian tube dysfunction. Although BET is widely performed, little is known about the occurrence rate of the complications associated with BET. The aim of the present study was to observe the rate of sensObjective: Eustachian tube function is of central importance for the ventilation of the middle ear. A dysfunction can be associated with chronic otitis media, and cholesteatoma. Balloon Eustachian tube dilatation (BET) is a treatment option used to solve eustachian tube dysfunction. Although BET is widely performed, little is known about the occurrence rate of the complications associated with BET. The aim of the present study was to observe the rate of sensorineural hearing loss (SNHL) after BET. Methods: We retrospectively evaluated in a chart review 1,547 patients and 2,614 procedures of BET performed in a single center between 2015 and 2019 using the Spiggle and Theis, Overath, Germany eustachian tube dilatation system. Results: We observed seven cases of SNHL after BET. In two cases, the SNHL persisted, and in five cases, the SNHL was transient. In two cases of SNHL, a simultaneous tympanoplasty was performed. The overall rate of SNHL per procedure is 0.3%. The rate of permanent SNHL is 0.08%. Conclusion: BET has a low rate of SNHL. Rapid middle ear pressure changes are assumed to cause BET-related hearing loss.orineural hearing loss (SNHL) after BET.Methods:We retrospectively evaluated in a chart review 1,547 patients and 2,614 procedures of BET performed in a single center between 2015 and 2019 using the Spiggle and Theis, Overath, Germany eustachian tube dilatation system.Results:We observed seven cases of SNHL after BET. In two cases, the SNHL persisted, and in five cases, the SNHL was transient. In two cases of SNHL, a simultaneous tympanoplasty was performed. The overall rate of SNHL per procedure is 0.3%. The rate of permanent SNHL is 0.08%. Conclusion: BET has a low rate of SNHL. Rapid middle ear pressure changes are assumed to cause BET-related hearing loss

The Potential of Tumor Debulking to Support Molecular Targeted Therapies

Oppel F, Görner M, Sudhoff H. The Potential of Tumor Debulking to Support Molecular Targeted Therapies. Frontiers in Oncology. 2020;10: 801

Specific targeting of oncogenic mutations using CRISPR-technology to destroy activated oncogenes and enhance immune response

Oppel F, Schürmann M, Sudhoff H. Specific targeting of oncogenic mutations using CRISPR-technology to destroy activated oncogenes and enhance immune response. Infinite Science Publishing. 2018;1(1): 1810046.Purpose/Objectives: In recent decades, molecular biology tools have enabled researchers to modify the genome of human cells. Today, many methods can be employed to inactivate genes, add them or change their expression. However, survival rates for patients affected by highly malignant cancer types have not improved substantially. If methodology like genome editing, which is routinely used in research laboratories, would be available to clinicians, the patients’ prognosis could be strongly improved. Materials/Methods: CRISPR-Cas9 is a method to precisely target and manipulate genomic loci and recent studies have attempted to use this method as a treatment for genetic diseases including cancer. Some of these approaches target mutant genomic sequences specifically and try to avoid affecting the respective normal loci. We combined knowledge from recent advances in CRISPR-technology with data from cancer genome sequencing studies to develop a concept for the CRISPR-mediated targeting of recurrent or individual mutations in activated oncogenes. Results: We present strategies to target mutations in activated oncogenes in a mutation-specific way using CRISPR-technology. The induction of DNA double strand breaks in key locations of oncogenic drivers is likely to abolish or reduce their oncogenic potential. CRISPR-molecules effective against highly recurrent mutations might be usable off-the-shelf for all patients with that mutation. Clinical electroporators could improve the delivery of CRISPR-molecules into tumor cells after local or systemic injection. Moreover, when combined for example with checkpoint inhibitors, CRISPR-targeting might enhance the immune-recognition of tumor cells and/or decrease the expression of ligands reducing cellular immune responses. However, considering obvious genetic risks opposing the objected benefits, this approach has to be evaluated carefully in preclinical studies. Conclusion: We believe that the mutant-specific targeting of oncogenes activated by mutation can be achieved employing the latest CRISPR-technology and that this approach could be combined with immunotherapy. Current publication: Oppel F, Schürmann M, Goon P, Albers AE, Sudhoff H. Specific Targeting of Oncogenes Using CRISPR Technology. Cancer Research. 2018 Sep 7. PubMed PMID: 30194069

Specific Targeting of Oncogenes Using CRISPR Technology

Oppel F, Schürmann M, Goon P, Albers AE, Sudhoff H. Specific Targeting of Oncogenes Using CRISPR Technology. Cancer Research. 2018;78(19):5506-5512