Aims Extracorporeal photopheresis (ECP) is a UVA-based phototherapy of whole blood and well established as a first line or combination therapy for the treatment of cutaneous T-cell lymphoma, systemic sclerosis, graft-versus-host disease and is used to control organ transplant rejection. While the proapoptotic activity on activated T-cells is evident, the clinical efficacy of this treatment also appears to be based on other yet unknown mechanisms. In this study, we aimed to identify novel mechanisms of ECP regardless of the patient’s background situation. Main methods To better understand the immediate consequences of ECP, we analyzed blood plasma of patients with different ECP indications immediately before and after treatment with regard to proteins and lipid mediators. Key findings While proteome profiling identified substantial inter-individual differences in the protein composition, no significant alteration was detectable upon treatment. In contrast, several fatty acids and lipid mediators were found to be significantly altered by ECP. Remarkably, upregulated lipid mediators including polyunsaturated fatty acids, 12-HEPE and 13-OxoODE have been described to be anti-inflammatory, while the downregulated molecules sphingosine-1-phosphate (S1P) and stearic acid are potent pro-inflammatory mediators. A selective sphingosine-1-phosphate-1 receptor (S1P1) modulator AUY954, which decreases S1P1 and experimentally reduces transplant rejection in vivo, showed greater anti-proliferative activity in human lung fibroblasts from COPD patients compared to normal lung fibroblasts, confirming that this pathway may be important in ECP and its mode of action. Significance and outlook In conclusion, we suggest that the ECP-induced changes in lipid mediators may contribute to the remarkable anti-inflammatory effects of the treatment. Depending on their lipid status, patients may benefit from novel treatment regimens combining ECP with lipid modulators. This could be used for the prevention of transplant organ rejection, the treatment of acute or chronic GvHD or transplant organ rejection and the long-term treatment of various skin diseases. This study uncovers novel mechanisms of ECP, that can be used to establish clinically relevant lipid profiles of patients to support patient stratification, predictive or prognostic purposes and thus personalized medical care in the framework of PPPM practice. A combination with S1P modulators may therefore have beneficial effects.

Here, we performed photo-SAR studies in a series of photoswitchable tubulin inhibiting antimitotics, and discovered that fluorescent antenna motifs permit their single-photon photoisomerisation in biological conditions at up to >600 nm; we were also able to visualise the temporal onset of their long-term photopharmacological effects, using large-field-of-view microscopy. Previously, azobenzene analogues of the tubulin polymerisation inhibitor combretastatin A4 (PSTs) had been developed to optically control microtubule dynamics in living systems, with subsecond response time and single-cell spatial precision, by reversible in situ photoswitching of their bioactivity with near-UV/visible light. First-generation PSTs were sufficiently potent and photoswitchable for use in live cells and embryos. However, the link between their seconds-scale and hours-scale bioactivity remained untested. Here, we now used tandem photoswitching/microscopy to reveal the timing of onset of their long-term bulk cytostatic effects. Since the scope for modifications to tune their photo-structure-activity-relationship or expand PST function had been unknown, we then synthesised a panel of novel PSTs exploring structural variations that tune photoresponse wavelengths and lipophilicity, identifying promising blue-shifted analogues that are better-compatible with GFP/YFP imaging. Taken together, these results can guide new design and applications for photoswitchable microtubule inhibitors. Finally, we also identified tolerated sites for linkers to attach functional cargos, and tested them with fluorescent "antennas" as reporters. Serendipitously, we found that antennas can greatly enhance long-wavelength single-photon photoisomerisation, by a hitherto un-explored mechanism. This final result will drive progress towards near-quantitative long-wavelength photoswitching of photopharmaceuticals in living systems, with minimal molecular redesign and general application scope.

Advanced breast cancer, as well as ineffective treatments leading to surviving cancer cells, can result in the dissemination of these malignant cells from the primary tumor to distant organs. Recent research has shown that microRNA 200c (miR-200c) can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear whether miR-200c expression alone is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. The ex vivo analysis of metastatic sites in a multitude of organs, including lung, liver, brain, and spleen, revealed a dramatically reduced metastatic burden in mice with miR-200c-expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR-200c on collective- and single-cell migration in vitro, utilizing MDA-MB 231 and MCF7 cell systems with genetically modified miR-200c expression. Analysis of collective-cell migration revealed confluence-dependent motility of cells with altered miR-200c expression. Additionally, scratch assays showed an enhanced predisposition of miR-200c-negative cells to leave cell clusters. The in-between stage of collective- and single-cell migration was validated using transwell assays, which showed reduced migration of miR-200c-positive cells. Finally, to measure migration at the single-cell level, a novel assay on dumbbell-shaped micropatterns was performed, which revealed that miR-200c critically determines confined cell motility. All of these results demonstrate that sole expression of miR-200c impedes metastasis formation in vivo and migration in vitro and highlights miR-200c as a metastasis suppressor in breast cancer.

Mass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence the variation of perturbation profiles using the two colon carcinoma cell lines SW480 and HCT116. These memory effects are largely driven by differences in growth states that persist into the perturbation experiment. In SW480 cells, memory effects combined with moderate treatment effects amplify the variation in multiple omics levels, including eicosadomics, proteomics, and phosphoproteomics. With stronger treatment effects, the memory effect was less pronounced, as demonstrated in HCT116 cells. Subculture homogeneity was controlled by real-time monitoring of cell growth. Controlled homogeneous subculture resulted in a perturbation network of 321 causal conjectures based on combined proteomic and phosphoproteomic data, compared to only 58 causal conjectures without controlling subculture homogeneity in SW480 cells. Some cellular responses and regulatory events were identified that extend the mode of action of arsenic trioxide (ATO) only when accounting for these memory effects. Controlled prior subculture led to the finding of a synergistic combination treatment of ATO with the thioredoxin reductase 1 inhibitor auranofin, which may prove useful in the management of NRF2-mediated resistance mechanisms.

Proteome characterization of Vemurafenib-sensitive and-resistant melanoma cells V0 and V1 of nuclear fraction. Left: Hierarchical clustering of Z-scored expression values for significantly changed proteins, revealing differences between V0 and V1. Left: Profiles of the five main clusters. Two clusters with the highest difference are selected and the significant regulated groups are depicted. Red: upregulated in V1, green: downregulated in V1.

Comparison of protein expression in A375 and M24met by 2D-gel electrophoresis. Corresponding proteins which are assigned in the 2-D gel in Figure 2. Accession numbers are from the Uniprot database. Vol indicates the volume identified by 2D-gel electrophoresis n.d =not determined

AbstractAdvanced breast cancer as well as insufficient treatment can lead to the dissemination of malignant cells from the primary tumor to distant organs. Recent research has shown that miR-200c can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear, whether sole miR-200c expression is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. Theex vivoanalysis of metastatic sites in a multitude of organs including lung, liver, brain, and spleen has revealed a dramatically reduced metastatic burden of mice with miR-200c expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR-200c on collective and single cell migrationin vitro, utilizing MDA-MB 231 and MCF7 cell systems with genetically modified miR-200c expression. Analysis of collective cell migration has resulted in confluence dependent motility of cells with altered miR-200c expression. Additionally, scratch assays have shown enhanced predisposition of miR-200c negative cells to leave cell clusters. The in-between stage of collective and single cell migration was validated using transwell assays, which have displayed reduced migration of miR-200c positive cells. Finally, to measure migration on single cell level, a novel assay on dumbbell shaped micropatterns was performed, which revealed that miR-200c critically determines confined cell motility. All of these results demonstrate that exclusive expression of miR-200c impedes metastasis formationin vivoand migrationin vitroand highlight miR-200c as metastatic suppressor in breast cancer.

Activity of vemurafenib and M8 against A375, M24met and V0, V0.1, V1, V10 (Proliferation assay).*Paulitschke et al. 2010

An overview of the applied workflow is depected in Figure S4. All used cell systems and applied techniques are depicted. In addition to that the read out of this study design is visualized,

Mass spectrometry-based omics technologies are increasingly used to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by the effect size and the variation of each molecular parameter. While the former is largely determined by the biological system, the latter can be tuned by the experimental workflow. Here, we unequivocally show that memory effects originating from subculture of colon carcinoma cells before treating with arsenic trioxide exacerbate the variation of multiple omics levels, including eicosadomics, proteomics and phosphoproteomics, without necessarily impacting on effect size. Real-time monitoring of individual samples enables control over subculture homogeneity and improves the median variation >2-fold across omics levels. This considerably facilitated mode of action deconvolution and resulted in a bilevel perturbation network of 321 causal conjectures. Controlling memory effects from subculture revealed key signaling cascades and transcriptional regulatory events that extend the molecular understanding of arsenic trioxide in solid tumors. ### Competing Interest Statement Philipp Paulitschke is the founder and CEO of PHIO scien-tific GmbH. All other authors declare no competing finan-cial interests.

The legend of the supplemetary figures is given.

Morphologic characterization of A375 and M24met melanoma cells. (a) Microscopic analyses; the arrows indicate a round cell of A375 and a tube-like structure formed by M24met. Immunohistochemical staining of A375 (b) and M24met (c) with S100, HMB45, Nestin, and p75NTR. (d) FACS analysis of A375, M24met and solvent control with CD90, FE, CD45, CD31, CD34 and CD40.

Advanced breast cancer as well as insufficient treatment can lead to the dissemination of malignant cells from the primary tumor to distant organs. Recent research has shown that miR-200c can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear, whether sole miR-200c expression is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. The ex vivo analysis of metastatic sites in a multitude of organs including lung, liver, brain, and spleen has revealed a dramatically reduced metastatic burden of mice with miR-200c expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR-200c on collective and single cell migration in vitro, utilizing MDA-MB 231 and MCF7 cell systems with genetically modified miR-200c expression. Analysis of collective cell migration has resulted in confluence dependent motility of cells with altered miR-200c expression. Additionally, scratch assays have shown enhanced predisposition of miR-200c negative cells to leave cell clusters. The in-between stage of collective and single cell migration was validated using transwell assays, which have displayed reduced migration of miR-200c positive cells. Finally, to measure migration on single cell level, a novel assay on dumbbell shaped micropatterns was performed, which revealed that miR-200c critically determines confined cell motility. All of these results demonstrate that exclusive expression of miR-200c impedes metastasis formation in vivo and migration in vitro and highlight miR-200c as metastatic suppressor in breast cancer.

Inflammatory profile in A375 and M24met melanoma cells. Distribution of inflammatory proteins in A375 and M24met (a). M8 downregulates proteins involved in inflammation, as exemplified by the inflammatory proteins lysyl oxidase homolog 2, plasminogen activator inhibitor 1 (PAI-1), ADAMTS-1, pentraxin, granulins, that are upregulated in M24met (b,c). Correlation of the candidates with tumor microenvironment and IL-1beta and VEGF response (d).

Acquired drug resistance constitutes a serious obstacle to the successful therapy of cancer. In the process of therapy resistance, microRNAs can play important roles. In order to combat resistance formation and to improve the efficacy of chemotherapeutics, the mechanisms of the multifaceted hsa-miR-200c on drug resistance were elucidated. Upon knockout of hsa-miR-200c in breast carcinoma cells, a proteomic approach identified altered expression of glutathione S-transferases (GSTs) when cells were treated with the chemotherapeutic drug doxorubicin. In different hsa-miR-200c expression systems, such as knockout, inducible sponge and inducible overexpression, the differential expression of all members of the GST family was evaluated. Expression of hsa-miR-200c in cancer cells led to the repression of a multitude of these GSTs and as consequence, enhanced drug-induced tumor cell death which was evaluated for two chemotherapeutic drugs. Additionally, the influence of hsa-miR-200c on the glutathione pathway, which is part of the phase II detoxification mechanism, was investigated. Finally, the long-term effects of hsa-miR-200c on drug efficacy were studied in vitro and in vivo. Upon doxycycline induction of hsa-miR-200c, MDA-MB 231 xenograft mouse models revealed a strongly reduced tumor growth and an enhanced treatment response to doxorubicin. A combined treatment of these tumors with hsa-miR-200c and doxorubicin resulted in complete regression of the tumor in 60% of the animals. These results identify hsa-miR-200c as an important player regulating the cellular phase II detoxification, thus sensitizing cancer cells not expressing this microRNA to chemotherapeutics and reversing drug resistance through suppression of GSTs.

In recent years, nanomechanics has evolved into a mature field, and it has now reached a stage which enables the fabrication and study of ever more elaborate devices. This has led to the emergence of arrays of coupled nanomechanical resonators as a promising field of research serving as model systems to study collective dynamical phenomena such as synchronization or topological transport. From a general point of view, the arrays investigated so far can be effectively treated as scalar fields on a lattice. Moving to a scenario where the vector character of the fields becomes important would unlock a whole host of conceptually interesting additional phenomena, including the physics of polarization patterns in wave fields and their associated topology. Here we introduce a new platform, a two-dimensional array of coupled nanomechanical pillar resonators, whose orthogonal vibration directions encode a mechanical polarization degree of freedom. We demonstrate direct optical imaging of the collective dynamics, enabling us to analyze the emerging polarization patterns, follow their evolution with drive frequency, and identify topological polarization singularities.

MAPK inhibitors (MAPKi) show outstanding clinical response rates in melanoma patients harbouring BRAF mutations, but resistance is common. The ability of melanoma cells to switch from melanocytic to mesenchymal phenotypes appears to be associated with therapeutic resistance. High‐throughput, subcellular proteome analyses and RNAseq on two panels of primary melanoma cells that were either sensitive or resistant to MAPKi revealed that only 15 proteins were sufficient to distinguish between these phenotypes. The two proteins with the highest discriminatory power were PTRF and IGFBP7, which were both highly upregulated in the mesenchymal‐resistant cells. Proteomic analysis of CRISPR/Cas‐derived PTRF knockouts revealed targets involved in lysosomal activation, endocytosis, pH regulation, EMT, TGFβ signalling and cell migration and adhesion, as well as a significantly reduced invasive index and ability to form spheres in 3D culture. Overexpression of PTRF led to MAPKi resistance, increased cell adhesion and sphere formation. In addition, immunohistochemistry of patient samples showed that PTRF expression levels were a significant biomarker of poor progression‐free survival, and IGFBP7 levels in patient sera were shown to be higher after relapse. Therapy resistance towards BRAF inhibition and relapse in skin cancer are frequently associated with cellular phenotype switching, but the molecular control of this plasticity and discriminating markers remain unclear. Multidimensional expression profiling of resistant and sensitive primary melanoma reveals functional biomarkers and potential targets associated with poor progression‐free survival. Comparative proteome and transcriptome profiling of primary skin cancers identifies biomarkers associated with therapy resistance and relapse.

Semiconductur nano- and micropillars represent a promising platform for hybrid nanodevices. Their ability to couple to a broad variety of nanomechanical, acoustic, charge, spin, excitonic, polaritonic, or electromagnetic excitations is utilized in fields as diverse as force sensing or optoelectronics. In order to fully exploit the potential of these versatile systems e.g. for metamaterials, synchronization or topologically protected devices an intrinsic coupling mechanism between individual pillars needs to be established. This can be accomplished by taking advantage of the strain field induced by the flexural modes of the pillars. Here, we demonstrate strain-induced, strong coupling between two adjacent nanomechanical pillar resonators. Both mode hybridization and the formation of an avoided level crossing in the response of the nanopillar pair are experimentally observed. The described coupling mechanism is readily scalable, enabling hybrid nanomechanical resonator networks for the investigation of a broad range of collective dynamical phenomena.