City 5.0: Citizen involvement in the design of future cities.

A citizen-centric view is key to channeling technological affordances into the development of future cities in which improvements are made with the quality of citizens' life in mind. This paper proposes City 5.0 as a new citizen-centric design paradigm for future cities, in which cities can be seen as markets connecting service providers with citizens as consumers. City 5.0 is dedicated to eliminating restrictions that citizens face when utilizing city services. Our design paradigm focuses on smart consumption and extends the technology-centric concept of smart city with a stronger view on citizens' roadblocks to service usage. Through a series of design workshops, we conceptualized the City 5.0 paradigm and formalized it in a semi-formal model. The applicability of the model is demonstrated using the case of a telemedical service offered by a Spanish public healthcare service provider. The usefulness of the model is validated by qualitative interviews with public organizations involved in the development of technology-based city solutions. Our contribution lies in the advancement of citizen-centric analysis and the development of city solutions for both academic and professional communities.

Irradiated Triple-Negative Breast Cancer Co-Culture Produces a Less Oncogenic Extracellular Matrix.

Triple-negative breast cancer is the most common and most deadly cancer among women. Radiation is a mainstay of treatment, administered after surgery, and used in the hope that any remaining cancer cells will be destroyed. While the cancer cell response is normally the focus of radiation therapy, little is known about the tumor microenvironment response after irradiation. It is widely reported that increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. Aside from the classical fibrotic response, ratios of collagen isoforms have not been studied in a radiated tumor microenvironment. Here, we created one healthy co-culture of stromal fibroblasts and adipose-derived stem cells, and one triple-negative breast cancer co-culture, made of stromal fibroblasts, adipose derived stem cells, and triple-negative breast cancer cells. After irradiation, growth and decellularization of co-cultures, we reseeded the breast cancer cells for 24 h and analyzed the samples using mass spectrometry. Proteomic analysis revealed that collagen VI, a highly oncogenic collagen isoform linked to breast cancer, was decreased in the irradiated cancer co-culture. This indicates that the anti-cancer impact of radiation may be not only cell ablative, but also influential in creating a less oncogenic microenvironment.

Activation of PPARα by Fenofibrate Attenuates the Effect of Local Heart High Dose Irradiation on the Mouse Cardiac Proteome.

Radiation-induced cardiovascular disease is associated with metabolic remodeling in the heart, mainly due to the inactivation of the transcription factor peroxisome proliferator-activated receptor alpha (PPARα), thereby inhibiting lipid metabolic enzymes. The objective of the present study was to investigate the potential protective effect of fenofibrate, a known agonist of PPARα on radiation-induced cardiac toxicity. To this end, we compared, for the first time, the cardiac proteome of fenofibrate- and placebo-treated mice 20 weeks after local heart irradiation (16 Gy) using label-free proteomics. The observations were further validated using immunoblotting, enzyme activity assays, and ELISA. The analysis showed that fenofibrate restored signalling pathways that were negatively affected by irradiation, including lipid metabolism, mitochondrial respiratory chain, redox response, tissue homeostasis, endothelial NO signalling and the inflammatory status. The results presented here indicate that PPARα activation by fenofibrate attenuates the cardiac proteome alterations induced by irradiation. These findings suggest a potential benefit of fenofibrate administration in the prevention of cardiovascular diseases, following radiation exposure.

In vitro cellular and proteome assays identify Wnt pathway and CDKN2A-regulated senescence affected in mesenchymal stem cells from mice after a chronic LD gamma irradiation in utero.

Reliable data on the effects of chronic prenatal exposure to low dose (LD) of ionizing radiation in humans are missing. There are concerns about adverse long-term effects that may persist throughout postnatal life of the offspring. Due to their slow cell cycle kinetics and life-long residence time in the organism, mesenchymal stem cells (MSCs) are more susceptible to low level genotoxic stress caused by extrinsic multiple LD events. The aim of this study was to investigate the effect of chronic, prenatal LD gamma irradiation to the biology of MSCs later in life. C3H mice were exposed in utero to chronic prenatal irradiation of 10 mGy/day over a period of 3 weeks. Two years later, MSCs were isolated from the bone marrow and analyzed in vitro for their radiosensitivity, for cellular senescence and for DNA double-strand break recognition after a second acute gamma-irradiation. In addition to these cellular assays, changes in protein expression were measured using HPLC-MS/MS and dysregulated molecular signaling pathways identified using bioinformatics. We observed radiation-induced proteomic changes in MSCs from the offspring of in utero irradiated mice (leading to ~ 9.4% of all detected proteins being either up- or downregulated) as compared to non-irradiated controls. The proteomic changes map to regulation pathways involved in the extracellular matrix, the response to oxidative stress, and the Wnt signaling pathway. In addition, chronic prenatal LD irradiation lead to an increased rate of in vitro radiation-induced senescence later in life and to an increased number of residual DNA double-strand breaks after 4 Gy irradiation, indicating a remarkable interaction of in vivo radiation in combination with a second acute dose of in vitro radiation. This study provides the first insight into a molecular mechanism of persistent MSC damage response by ionizing radiation exposure during prenatal time and will help to predict therapeutic safety and efficacy with respect to a clinical application of stem cells.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq unravels tumour heterogeneity plus M2-like tumour-associated macrophage infiltration and aggressiveness in TNBC.

Triple-negative breast cancer (TNBC) is characterized by a more aggressive clinical course with extensive inter- and intra-tumour heterogeneity. Combination of single-cell and bulk tissue transcriptome profiling allows the characterization of tumour heterogeneity and identifies the association of the immune landscape with clinical outcomes. We identified inter- and intra-tumour heterogeneity at a single-cell resolution. Tumour cells shared a high correlation amongst stemness, angiogenesis, and EMT in TNBC. A subset of cells with concurrent high EMT, stemness and angiogenesis was identified at the single-cell level. Amongst tumour-infiltrating immune cells, M2-like tumour-associated macrophages (TAMs) made up the majority of macrophages and displayed immunosuppressive characteristics. CIBERSORT was applied to estimate the abundance of M2-like TAM in bulk tissue transcriptome file from The Cancer Genome Atlas (TCGA). M2-like TAMs were associated with unfavourable prognosis in TNBC patients. A TAM-related gene signature serves as a promising marker for predicting prognosis and response to immunotherapy. Two commonly used machine learning methods, random forest and SVM, were applied to find the genes that were mostly associated with M2-like TAM densities in the gene signature. A neural network-based deep learning framework based on the TAM-related gene signature exhibits high accuracy in predicting the immunotherapy response.

A Five-Year report on the conception and establishment of the MSc Radiation Biology at the Technical University of Munich.

PURPOSE: The MSc Radiation Biology course is a highly interdisciplinary degree program placing radiation biology at the interface between biology, medicine, and physics, as well as their associated technologies. The goal was to establish an internationally acknowledged program with diverse and heterogeneous student cohorts, who benefit from each other academically as well as culturally. We have completed a Five-Year evaluation of the program to assess our qualification profile and the further direction we want to take. MATERIALS AND METHODS: We evaluated the student cohort's data from the last 5 years regarding gender, age, and nationality as well as the highest degree before applying and career path after graduation. RESULTS: Data shows a great diversity regarding nationalty as well as undergraduate background. Cohort sizes could be increased and future prospects mainly aimed to a PhD. Measures after regular quality meetings and students' feedback led to improving the curriculum and workload, teacher's training, and changes to examination regulations. CONCLUSIONS: After 5 years, statistics show that our expectations have been met exceedingly. All graduates had excellent career opportunities reflecting the necessity of this MSc and its topics. We are continuously working on improving the program and adapting the curriculum to the requirements in radiation sciences. The future vision includes an expansion of the program as well as undergraduate education opportunities in this field.

Extended in vitro culture of primary human mesenchymal stem cells downregulates Brca1-related genes and impairs DNA double-strand break recognition.

Mesenchymal stem cells (MSCs) are multilineage adult stem cells with considerable potential for cell-based regenerative therapies. In vitro expansion changes their epigenetic and cellular properties, with a poorly understood impact on DNA damage response (DDR) and genome stability. We report here results of a transcriptome-based pathway analysis of in vitro-expanded human bone marrow-derived mesenchymal stem cell (hBM-MSCs), supplemented with cellular assays focusing on DNA double-strand break (DSB) repair. Gene pathways affected by in vitro aging were mapped using gene ontology, KEGG, and GSEA, and were found to involve DNA repair, homologous recombination (HR), cell cycle control, and chromosomal replication. Assays for the recognition (γ-H2AX + 53BP1 foci) and repair (pBRCA1 + Î³-H2AX foci) of X-ray-induced DNA DSBs in hBM-MSCs show that over a period of 8 weeks of in vitro aging (i.e., about 10 doubling times), cells exhibit a reduced DDR and a higher fraction of residual DNA damage. Furthermore, a distinct subpopulation of cells with impaired DNA DSB recognition was observed. Several genes that participate in DNA repair by HR (e.g., Rad51, Rad54, BRCA1) show a 2.3- to fourfold reduction of their mRNA expression by qRT-PCR. We conclude that the in vitro expansion of hMSCs can lead to aging-related impairment of the recognition and repair of DNA breaks.

Oncogenic Linear Collagen VI of Invasive Breast Cancer Is Induced by CCL5.

The triple-negative breast tumor boundary is made of aligned, linear collagen. The pro-oncogenic impact of linear collagen is well established; however, its mechanism of formation is unknown. An in vitro analogue of the tumor border is created by a co-culture of MDA-MB-231 cells, adipose derived stem cells, and dermal fibroblasts. Decellularization of this co-culture after seven days reveals an extracellular matrix that is linear in fashion, high in pro-oncogenic collagen type VI, and able to promote invasion of reseeded cells. Further investigation revealed linear collagen VI is produced by fibroblasts in response to a paracrine co-culture of adipose derived stem cells and MDA-MB-231, which together secrete high levels of the chemokine CCL5. The addition of monoclonal antibody against CCL5 to the co-culture results in an unorganized matrix with dramatically decreased collagen VI. Importantly, reseeded cells do not exhibit pro-oncogenic behavior. These data illustrate a cellular mechanism, which creates linear extracellular matrix (ECM) in vitro, and highlight a potential role of CCL5 for building striated tumor collagen in vivo.

A novel epigenetic signature for overall survival prediction in patients with breast cancer.

BACKGROUND: Breast cancer is the most common malignancy in female patients worldwide. Because of its heterogeneity in terms of prognosis and therapeutic response, biomarkers with the potential to predict survival or assist in making treatment decisions in breast cancer patients are essential for an individualised therapy. Epigenetic alterations in the genome of the cancer cells, such as changes in DNA methylation pattern, could be a novel marker with an important role in the initiation and progression of breast cancer. METHOD: DNA methylation and RNA-seq datasets from The Cancer Genome Atlas (TCGA) were analysed using the Least Absolute Shrinkage and Selection Operator (LASSO) Cox model. Applying gene ontology (GO) and single sample gene set enrichment analysis (ssGSEA) an epigenetic signature associated with the survival of breast cancer patients was constructed that yields the best discrimination between tumour and normal breast tissue. A predictive nomogram was built for the optimal strategy to distinguish between high- and low-risk cases. RESULTS: The combination of mRNA-expression and of DNA methylation datasets yielded a 13-gene epigenetic signature that identified subset of breast cancer patients with low overall survival. This high-risk group of tumor cases was marked by upregulation of known cancer-related pathways (e.g. mTOR signalling). Subgroup analysis indicated that this epigenetic signature could distinguish high and low-risk patients also in different molecular or histological tumour subtypes (by Her2-, EGFR- or ER expression or different tumour grades). Using Gene Expression Omnibus (GEO) the 13-gene signature was confirmed in four external breast cancer cohorts. CONCLUSION: An epigenetic signature was discovered that effectively stratifies breast cancer patients into low and high-risk groups. Since its efficiency appears independent of other known classifiers (such as staging, histology, metastasis status, receptor status), it has a high potential to further improve likely individualised therapy in breast cancer.

Long-term culture of mesenchymal stem cells impairs ATM-dependent recognition of DNA breaks and increases genetic instability.

BACKGROUND: Mesenchymal stem cells (MSCs) are attracting increasing interest for cell-based therapies, making use of both their immuno-modulating and regenerative potential. For such therapeutic applications, a massive in vitro expansion of donor cells is usually necessary to furnish sufficient material for transplantation. It is not established to what extent the long-term genomic stability and potency of MSCs can be compromised as a result of this rapid ex vivo expansion. In this study, we investigated the DNA damage response and chromosomal stability (indicated by micronuclei induction) after sub-lethal doses of gamma irradiation in murine MSCs at different stages of their in vitro expansion. METHODS: Bone-marrow-derived tri-potent MSCs were explanted from 3-month-old female FVB/N mice and expanded in vitro for up to 12 weeks. DNA damage response and repair kinetics after gamma irradiation were quantified by the induction of γH2AX/53BP1 DSB repair foci. Micronuclei were counted in post-mitotic, binucleated cells using an automated image analyzer Metafer4. Involvement of DNA damage response pathways was tested using chemical ATM and DNA-PK inhibitors. RESULTS: Murine bone-marrow-derived MSCs in long-term expansion culture gradually lose their ability to recognize endogenous and radiation-induced DNA double-strand breaks. This impaired DNA damage response, indicated by a decrease in the number of γH2AX/53BP1 DSB repair foci, was associated with reduced ATM dependency of foci formation, a slower DNA repair kinetics, and an increased number of residual DNA double-strand breaks 7 h post irradiation. In parallel with this impaired efficiency of DNA break recognition and repair in older MSCs, chromosomal instability after mitosis increased significantly as shown by a higher number of micronuclei, both spontaneously and induced by γ-irradiation. Multifactorial regression analysis demonstrates that in vitro aging reduced DNA damage recognition in MSCs after irradiation by a multiplicative interaction with dose (p < 0.0001), whereas the increased frequency of micronuclei was caused by an additive interaction between in vitro aging and radiation dose. CONCLUSION: The detrimental impact of long-term in vitro expansion on DNA damage response of MSCs warrants a regular monitoring of this process during the ex vivo growth of these cells to improve therapeutic safety and efficiency.

Factors affecting the performance of public out-patient services.

Objective The delivery of public out-patient services is an essential part of complex healthcare systems, but the contribution of public out-patient services is often ill defined and poorly evaluated. The aim of this study was to identify and better understand those factors that may affect the performance of out-patient services to provide health service managers, clinicians and executives with a conceptual framework for future decision-making processes. Methods The present qualitative research involved five exploratory case studies. These case studies were conducted across two specialties at hospitals in the Metro North Hospital and Health Service in Queensland. Data were obtained from 38 interviews and 15 focus groups, and were analysed to identify common themes. Further analysis helped identify the most significant factors and build a conceptual framework for understanding the relationships between those factors and their effect on performance. Results Across both specialties there were 10 factors (scheduling, performance, service framework, categorisation or prioritisation of patients, internal and external stakeholders, resources, service demand, culture, system challenges and medical stakeholders) identified that may affect the performance of out-patient services. These factors were condensed into five core domains: culture, stakeholders, resources, demand and system reform. Conclusion Strategies to address the five core domains identified may provide a framework for sustainable improvement in the delivery of out-patient services. What is known about the topic? The provision of specialist out-patient services is an essential element of health service delivery. Access to specialist services in the public sector is challenging because of the escalating demand associated with an increasing and aging demographic. The factors that may affect the delivery of out-patient services need to be addressed for long-term sustainable improvement. What does this paper add? This paper provides a conceptual framework grounded in rigorous qualitative data analysis for understanding the internal and external factors that affect waiting times for specialist out-patient services. The results of this qualitative research indicate that there are five core domains that may influence waiting times in the public out-patient setting. When these domains are addressed at the strategic, tactical and operational levels, they have the potential to provide significant improvement in the delivery of out-patient services. What are the implications for practitioners? This paper guides the attention of relevant stakeholders towards the five core domains identified (culture, stakeholders, resources, demand and system reform) that influence the performance of waiting times at the operational, tactical and strategic levels within the public hospital setting.

NHEJ Contributes to the Fast Repair of Radiation-induced DNA Double-strand Breaks at Late Prophase I Telomeres.

Exposure of cells to ionizing radiation induces DNA double-strand breaks. To repair double-strand breaks correctly, cells must distinguish between the ends of chromosomes (telomeres) and DNA double-strand breaks within chromosomes. Double-strand breaks in telomeric DNA may lead to telomere shortening and mutagenesis. Eukaryotic cells repair double-strand breaks primarily by two mechanisms: error-free homologous recombination and error-prone nonhomologous end joining, of which homologous recombination is used in early meiotic prophase I to create recombined haploid gametes by two meiotic cell divisions lacking an intervening S-phase. Genotoxic exposures put meiosis at risk to transmit mutations, and ionizing radiation is known to induce large double-strand break-marking phospho (gamma)-H2AX foci along the cores and ends of mouse meiotic chromosomes. However, it remained unclear through which repair pathway the ionizing radiation-induced telomeric double-strand breaks are repaired in late prophase I spermatocytes. Using male wild-type and nonhomologous end joining-deficient (severe combined immunodeficient) mice, this study investigated the kinetics of in vivo double-strand break formation and repair at telomeres of late prophase I chromosomes up to 12 h after 0.5 Gy of whole-body gamma irradiation. Late pachytene and diplotene spermatocytes revealed overlapping gamma-H2AX and telomere repeat signal foci, indicating telomeric DNA damage. The comparison of double-strand break repair rates at telomeres and internal prophase chromosome sites revealed a more rapid double-strand break repair at wild-type telomeres during the first hour after irradiation. Increased double-strand break foci numbers at nonhomologous end joining-deficient telomeres and chromosomes and a slowed repair rate in this DNA-dependent protein kinase catalytic subunit mutant suggest that the fast repair of double-strand breaks in telomeric DNA repeats during late prophase I is largely mediated by canonical nonhomologous end joining.

Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

Time to wait: a systematic review of strategies that affect out-patient waiting times.

Objective Out-patient waiting times pose a significant challenge for public patients in need of specialist evaluation and intervention. The aim of the present study was to identify and categorise effective strategies to reduce waiting times for specialist out-patient services with a focus on the Australian healthcare system. Methods A systematic review of major health databases was conducted using the key terms 'outpatient*' AND 'waiting time', 'process*' AND 'improvement in outpatient clinics'. Identified articles were assessed for their relevance by sequential review of the title, abstract and full text. References of the selected manuscripts were scanned for additional relevant articles. Selected articles were evaluated for consistent and emerging themes. Results In all, 152 articles were screened, of which 38 were included in the present review. Numerous strategies identified in the articles were consolidated into 26 consistent approaches. Three overarching themes were identified as significantly affecting waiting times: resource realignment, operational efficiency and process improvement. Conclusions Strategies to align resources, increase operational efficiency and improve processes provide a comprehensive approach that may reduce out-patient waiting times. What is known about the topic? Out-patient waiting times are a challenge in most countries that seek to provide universal access to health care for all citizens. Although there has been extensive research in this area, many patients still experience extensive delays accessing specialist care, particularly in the public health sector. The multiple factors that contribute to bottlenecks and inefficiencies in the referral process and affect patient waiting times are often poorly understood. What does this paper add? This paper reviews the published healthcare literature to identify strategies that affect specialist out-patient waiting times for patients. The findings suggest that there are numerous operational strategies that affect waiting times. These strategies may be categorised into three overarching themes (resource alignment, operational efficiencies and out-patient processes) that, when actioned in a coordinated approach, have the potential to significantly reduce out-patient waiting times. What are the implications for practitioners? This paper identifies evidence-based strategies for aligning resources, improving operational efficiency and streamlining processes, which may provide improvements to specialist out-patient waiting times for patients. Addressing the identified organisational, person-related, cultural and attitudinal factors will assist health system managers and health practitioners target the most appropriate improvement activities to reduce waiting times.

DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts.

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G1-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.

p53-Dependent Senescence in Mesenchymal Stem Cells under Chronic Normoxia Is Potentiated by Low-Dose γ-Irradiation.

Mesenchymal stem cells (MSCs) are a source of adult multipotent cells important in tissue regeneration. Murine MSCs are known to proliferate poorly in vitro under normoxia. The aim of this study is to analyze the interaction of nonphysiological high oxygen and low-dose γ-irradiation onto growth, senescence, and DNA damage. Tri-potent bone marrow-derived MSCs from p53 wildtype and p53-/- mice were cultured under either 21% or 2% O2. Long-term observations revealed a decreasing ability of wildtype mMSCs to proliferate and form colonies under extended culture in normoxia. This was accompanied by increased senescence under normoxia but not associated with telomere shortening. After low-dose γ-irradiation, the normoxic wildtype cells further increased the level of senescence. The number of radiation-induced γH2AX DNA repair foci was higher in mMSCs kept under normoxia but not in p53-/- cells. P53-deficient MSCs additionally showed higher clonogeneity, lower senescence levels, and fewer γH2AX repair foci per cell as compared to their p53 wildtype counterparts irrespective of oxygen levels. These results reveal that oxygen levels together with γ-irradiation and p53 status are interconnected factors modulating growth capacity of BM MSCs in long-term culture. These efforts help to better understand and optimize handling of MSCs prior to their therapeutic use.

Three-dimensional microtissues essentially contribute to preclinical validations of therapeutic targets in breast cancer.

A 3D microtissues using T47D and JIMT-1 cells were generated to analyze tissue-like response of breast cancer cells after combined human epidermal growth factor receptor 2 (HER2)-targeted treatment and radiation. Following lentiviral knockdown of HER2, we compared growth rate alterations using 2D monolayers, 3D microtissues, and mouse xenografts. Additionally, to model combined therapeutic strategies, we treated HER2-depleted T47D cells and 3D microtissues using trastuzumab (anti-HER2 antibody) in combination with irradiation. Comparison of HER2 knockdown with corresponding controls revealed growth impairment due to HER2 knockdown in T47D 2D monolayers, 3D microtissues, and xenografts (after 2, 12, and ≥40 days, respectively). In contrast, HER2 knockdown was less effective in inhibiting growth of trastuzumab-resistant JIMT-1 cells in vitro and in vivo. Combined administration of trastuzumab and radiation treatment was also analyzed using T47D 3D microtissues. Administration of both, radiation (5 Gy) and trastuzumab, significantly enhanced the growth inhibiting effect in 3D microtissues. To improve the predictive power of potential drugs--as single agents or in combination--here, we show that regarding tumor growth analyses, 3D microtissues are highly comparable to outcomes derived from xenografts. Considering increased limitations for animal experiments on the one hand and strong need of novel drugs on the other hand, it is indispensable to include highly reproducible 3D microtissue platform in preclinical analyses to validate more accurately the capacity of future drug-combined radiotherapy.

Short and long-term phototoxicity in cells expressing genetic reporters under nanosecond laser exposure.

Nanosecond-duration laser pulses are exploited in a plethora of therapeutic and diagnostic applications, such as optoacoustic imaging. However, phototoxicity effects of pulsed radiation in living cells, in particular those expressing genetic reporters, are not well understood. We established a three-dimensional fluorescent protein expressing cellular model in order to reliably investigate the extent and major exposure parameters responsible for both photobleaching and phototoxicity under pulsed laser exposure, unveiling a variety of possible effects on living cells, from reversible photobleaching to cytotoxicity and cell death. Significant losses of fluorescence levels were identified when exposing the cells to illumination conditions considered safe under common standards for skin exposure in diagnostic imaging applications. Thus, the use of photolabile fluorescent proteins and their in vivo exposure parameters have to be designed carefully for all applications using pulsed nanosecond radiation. In particular, loss of signal due to bleaching may significantly alter signals in longitudinal measurements, making data quantification challenging.