Cardiac surgery with cardiopulmonary bypass (CS/CPB) is associated with increased risk for postoperative complications causing substantial morbidity and mortality. To identify the molecular mechanisms underlying CS/CPB-induced pathophysiology we employed an integrative systems biology approach using the whole blood transcriptome as the sentinel organ.

Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5.

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

Whether aged hematopoietic stem and progenitor cells (HSPCs) have impaired DNA damage repair is controversial. Using a combination of DNA mutation indicator assays, we observe a 2- to 3-fold increase in the number of DNA mutations in the hematopoietic system upon aging. Young and aged hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) do not show an increase in mutation upon irradiation-induced DNA damage repair, and young and aged HSPCs respond very similarly to DNA damage with respect to cell-cycle checkpoint activation and apoptosis. Both young and aged HSPCs show impaired activation of the DNA-damage-induced G1-S checkpoint. Induction of chronic DNA double-strand breaks by zinc-finger nucleases suggests that HSPCs undergo apoptosis rather than faulty repair. These data reveal a protective mechanism in both the young and aged hematopoietic system against accumulation of mutations in response to DNA damage.

A remarkable observation emerging from recent cancer genome analyses is the identification of chromothripsis as a one-off genomic catastrophe, resulting in massive somatic DNA structural rearrangements (SRs). Largely due to lack of suitable model systems, the mechanistic basis of chromothripsis has remained elusive. We developed an integrative method termed "complex alterations after selection and transformation (CAST)," enabling efficient in vitro generation of complex DNA rearrangements including chromothripsis, using cell perturbations coupled with a strong selection barrier followed by massively parallel sequencing. We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division. Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells. Analysis of primary medulloblastoma cancer genomes verified the link between hyperploidy and chromothripsis in vivo. CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes.

Mutational activation of K-Ras is an initiating event of pancreatic ductal adenocarcinomas (PDAC) that may develop either from pancreatic intraepithelial neoplasia (PanIN) or intraductal papillary mucinous neoplasms (IPMN). Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is causally related to pancreatic carcinogenesis. Here, we deciphered the impact of COX-2, a key modulator of inflammation, in concert with active mutant K-Ras(G12D) on tumor burden and gene expression signature using compound mutant mouse lines. Concomitant activation of COX-2 and K-Ras(G12D) accelerated the progression of pancreatic intraepithelial lesions predominantly with a cystic papillary phenotype resembling human IPMN. Transcriptomes derived from laser capture microdissected preneoplastic lesions of single and compound mutants revealed a signature that was significantly enriched in Notch1 signaling components. In vitro, Notch1 signaling was COX-2-dependent. In line with these findings, human IPMN stratified into intestinal, gastric and pancreatobillary types displayed Notch1 immunosignals with high prevalence, especially in the gastric lesions. In conclusion, a yet unknown link between activated Ras, protumorigenic COX-2 and Notch1 in IPMN onset was unraveled.

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

The inhibition of angiogenesis is a promising strategy for the treatment of malignant primary and secondary tumors in addition to established therapies such as surgery, chemotherapy, and radiation. There is strong experimental evidence in primary tumors that Cyclooxygenase-2 (Cox-2) inhibition is a potent mechanism to reduce angiogenesis. For bone metastases which occur in up to 85% of the most frequent malignant primary tumors, the effects of Cox-2 inhibition on angiogenesis and tumor growth remain still unclear. Therefore, the aim of this study was to investigate the effects of Celecoxib, a selective Cox-2 inhibitor, on angiogenesis, microcirculation and growth of secondary bone tumors.

External beam radiotherapy (EBRT) with carbon ions and endoradiotherapy using radiolabeled tumor targeting agents are emerging concepts in precision cancer therapy. We report on combination effects of these two promising strategies. Tumor targeting 131I-labelled anti-EGFR-antibody (Cetuximab) was used in the prototypic EGFR-expressing A431 human squamous cell carcinoma xenograft model. A 131I-labelled melanin-binding benzamide derivative was utilized targeting B16F10 melanoma in an orthotopic syngeneic C57bl6 model. Fractionated EBRT was performed using carbon ions in direct comparison with conventional photon irradiation. Tumor uptake of 131I-Cetuximab and 131I-Benzamide was enhanced by fractionated EBRT as determined by biodistribution studies. This effect was independent of radiation quality and significant for the small molecule 131I-Benzamide, i.e., >30% more uptake in irradiated vs. non-irradiated melanoma was found (p<0.05). Compared to each monotherapy, dual combination with 131I-Cetuximab and EBRT was most effective in inhibiting A431 tumor growth. A similar trend was seen for 131I-Benzamide and EBRT in B16F10 melanoma model. Addition of 131I-Benzamide endoradiotherapy to EBRT altered expression of genes related to DNA-repair, cell cycle and cell death. In contrast, immune-response related pathways such as type 1 interferon response genes (ISG15, MX1) were predominantly upregulated after combined 131I-Cetuximab and EBRT. The beneficial effects of combined 131I-Cetuximab and EBRT was further attributed to a reduced microvascular density (CD31) and decreased proliferation index (Ki-67). Fractionated EBRT could be favorably combined with endoradiotherapy. 131I-Benzamide endoradiotherapy accelerated EBRT induced cytotoxic effects. Activation of immune-response by carbon ions markedly enhanced anti-EGFR based endoradiotherapy suggesting further evaluation of this novel and promising radioimmunotherapy concept.

Carbon ion radiotherapy (CIRT) with raster scanning technology is a promising treatment for lung cancer and thoracic malignancies. Determining normal tissue tolerance of organs at risk is of utmost importance for the success of CIRT. Here we report the relative biological effectiveness (RBE) of CIRT as a function of dose and fractionation for development of pulmonary fibrosis using well established fibrosis index (FI) model.

Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.

Glioblastoma multiforme (GBM) exhibits high resistance to the standard treatment of temozolomide (TMZ) combined with radiotherapy, due to its remarkable cell heterogeneity. Accordingly, there is a need to target alternative molecules enhancing specific GBM autocrine or paracrine mechanisms and amplifying the effect of standard treatment. Sphingosine 1-phosphate (S1P) is such a lipid target molecule with an important role in cell invasion and proliferation. Sphingosine kinase inhibitors (SKI) prevent S1P formation and induce increased production of reactive oxygen species (ROS), which may potentiate radiation cytotoxicity. We analyzed the effect of SKI singular versus combined treatments with TMZ and radiation on 2 human GBM cell lines characterized by a lack of MGMT expression and low or high expression of the anti-oxidant enzyme, glutathione peroxidase 1 (GPx1). Effects were drug concentration-, cell line-dependent and partly ROS-mediated. Clonogenic survival assay demonstrates that SKI was more effective than TMZ in increasing the sensitivity of U87 cells, which express low GPx1 amount, to a 2 Gy X-ray dose. Addition of both SKI and TMZ drastically decreased U87 cells survival compared with the combination temozolomide/radiation. SKI less effectively than TMZ sensitized LN229 cells to the 2 Gy X-ray dose. Its combination to TMZ in absence of irradiation was as efficient as TMZ combination with X-ray. We provide first evidence for SKI as an alternative or complementary treatment to TMZ, and for efficient combinations of low doses of drugs and X-ray. These may help as novel bi-modal and tri-modal therapies to contend with GBM heterogeneity.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.

The aim of this study was to analyze the robustness and diagnostic value of different compartment models for dynamic 18F-FET PET in recurrent high-grade glioma (HGG). Dynamic 18F-FET PET data of patients with recurrent WHO grade III (n:7) and WHO grade IV (n: 9) tumors undergoing re-irradiation with carbon ions were analyzed by voxelwise fitting of the time-activity curves with a simplified and an extended one-tissue compartment model (1TCM) and a two-tissue compartment model (2TCM), respectively. A simulation study was conducted to assess robustness and precision of the 2TCM. Parameter maps showed enhanced detail on tumor substructure. Neglecting the blood volume VB in the 1TCM yields insufficient results. Parameter K1 from both 1TCM and 2TCM showed correlation with overall patient survival after carbon ion irradiation (p = 0.043 and 0.036, respectively). The 2TCM yields realistic estimates for tumor blood volume, which was found to be significantly higher in WHO IV compared to WHO III (p = 0.031). Simulations on the 2TCM showed that K1 yields good accuracy and robustness while k2 showed lowest stability of all parameters. The 1TCM provides the best compromise between parameter stability and model accuracy; however application of the 2TCM is still feasible and provides a more accurate representation of tracer-kinetics at the cost of reduced robustness. Detailed tracer kinetic analysis of 18F-FET PET with compartment models holds valuable information on tumor substructures and provides additional diagnostic and prognostic value.

Pulmonary fibrosis represents a leading cause of morbidity and mortality worldwide. Therapy induced lung fibrosis constitutes a pivotal dose-limiting side effect of radiotherapy and other anticancer agents. We aimed to develop objective criteria for assessment of fibrosis and discover pathophysiological and molecular correlates of lung fibrosis as a function of fractionated whole thoracic irradiation. Dose-response series of fractionated irradiation was utilized to develop a non-invasive and quantitative measure for the degree of fibrosis - the fibrosis index (FI). The correlation of FI with histopathology, blood-gas, transcriptome and proteome responses of the lung tissue was analyzed. Macrophages infiltration and polarization was assessed by immunohistochemistry. Fibrosis development followed a slow kinetic with maximum lung fibrosis levels detected at 24-week post radiation insult. FI favorably correlated with radiation dose and surrogates of lung fibrosis i.e., enhanced pro-inflammatory response, tissue remodeling and extracellular matrix deposition. The loss of lung architecture correlated with decreased epithelial marker, loss of microvascular integrity with decreased endothelial and elevated mesenchymal markers. Lung fibrosis was further attributed to a switch of the inflammatory state toward a macrophage/T-helper cell type 2-like (M2/Th2) polarized phenotype. Together, the multiscale characterization of FI in radiation-induced lung fibrosis (RILF) model identified pathophysiological, transcriptional and proteomic correlates of fibrosis. Pathological immune response and endothelial/epithelial to mesenchymal transition were discovered as critical events governing lung tissue remodeling. FI will be instrumental for deciphering the molecular mechanisms governing lung fibrosis and discovery of novel targets for treatment of this devastating disease with an unmet medical need.

Synthetic lethality describes a genetic interaction between two perturbations, leading to cell death, whereas neither event alone has a significant effect on cell viability. This concept can be exploited to specifically target tumor cells. CRISPR viability screens have been widely employed to identify cancer vulnerabilities. However, an approach to systematically infer genetic interactions from viability screens is missing.

The occurrence and formation of genomic structural variants (SVs) is known to be influenced by the 3D chromatin architecture, but the extent and magnitude have been challenging to study. Here, we apply Hi-C to study chromatin organization before and after induction of chromothripsis in human cells. We use Hi-C to manually assemble the derivative chromosomes following the occurrence of massive complex rearrangements, which allows us to study the sources of SV formation and their consequences on gene regulation. We observe an action-reaction interplay whereby the 3D chromatin architecture directly impacts the location and formation of SVs. In turn, the SVs reshape the chromatin organization to alter the local topologies, replication timing, and gene regulation in cis We show that SVs have a strong tendency to occur between similar chromatin compartments and replication timing regions. Moreover, we find that SVs frequently occur at 3D loop anchors, that SVs can cause a switch in chromatin compartments and replication timing, and that this is a major source of SV-mediated effects on nearby gene expression changes. Finally, we provide evidence for a general mechanistic bias of the 3D chromatin on SV occurrence using data from more than 2700 patient-derived cancer genomes.

We present a protocol for the biosensor Cell-Fit-HD4D. It enables long-term monitoring and correlation of single-cell fate with subcellular-deposited energy of ionizing radiation. Cell fate tracking using widefield time-lapse microscopy is uncoupled in time from confocal ion track imaging. Registration of both image acquisition steps allows precise ion track assignment to cells and correlation with cellular readouts. For complete details on the use and execution of this protocol, please refer to Niklas et al. (2022).

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αβ sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

Helium (4He) ion beam therapy provides favorable biophysical characteristics compared to currently administered particle therapies, i.e., reduced lateral scattering and enhanced biological damage to deep-seated tumors like heavier ions, while simultaneously lessened particle fragmentation in distal healthy tissues as observed with lighter protons. Despite these biophysical advantages, raster-scanning 4He ion therapy remains poorly explored e.g., clinical translational is hampered by the lack of reliable and robust estimation of physical and radiobiological uncertainties. Therefore, prior to the upcoming 4He ion therapy program at the Heidelberg Ion-beam Therapy Center (HIT), we aimed to characterize the biophysical phenomena of 4He ion beams and various aspects of the associated models for clinical integration.