TDP-43 and FUS are nuclear proteins with multiple functions in mRNA processing. They play key roles in ALS  and FTD , where they are partially lost from the nucleus and aggregate in the cytoplasm of neurons and glial cells. Defects in nucleocytoplasmic transport contribute to this pathology, hence nuclear import of both proteins has been studied in detail. However, their nuclear export routes remain poorly characterized and it is unclear whether aberrant nuclear export contributes to TDP-43 or FUS pathology. Here we show that predicted nuclear export signals in TDP-43 and FUS are non-functional and that both proteins are exported independently of the export receptor CRM1/Exportin-1. Silencing of Exportin-5 or the mRNA export factor Aly/REF, as well as mutations that abrogate RNA-binding do not impair export of TDP-43 and FUS. However, artificially enlarging TDP-43 or FUS impairs their nuclear egress, suggesting that they could leave the nucleus by passive diffusion. Finally, we found that inhibition of transcription causes accelerated nuclear egress of TDP-43, suggesting that newly synthesized RNA retains TDP-43 in the nucleus, limiting its egress into the cytoplasm. Our findings implicate reduced nuclear retention as a possible factor contributing to mislocalization of TDP-43 in ALS/FTD. In FTD, a progressive degeneration of the frontal and temporal cortex leads to behavioral or language dysfunction. Eventually patients show severe cognitive impairment and die typically 7–10 years after disease onset2. ALS and FTD belong to the same disease spectrum and are thought to have a similar molecular cause, namely mislocalization and aggregation of RNA-binding proteins and, consequently, defective mRNA processing3.The RNA-binding proteins TDP-43 (TAR DNA-binding protein of 43 kDa) and FUS (Fused in sarcoma) have become infamous over the past years as being the main culprits in two fatal neurodegenerative diseases, ALS  and FTD . ALS is characterized by a progressive degeneration of motor neurons, which causes muscle weakness and eventually complete muscle paralysis. ALS patients typically die due to respiratory failure, usually 3–5 years after disease onset7. They also play a role in miRNA biogenesis and are associated with lncRNAs in paraspeckles9. A small fraction of TDP-43 and FUS is found in the cytoplasm, where they regulate stability, transport and translation of certain mRNA targets12. In post-mortem brains of ALS and FTD patients, however, the localization of TDP-43 or, less frequently, FUS is dramatically altered: TDP-43 or FUS are lost from the nucleus of many neurons and glial cells and accumulate in large cytoplasmic protein aggregates, also called inclusions15. Occasionally, cells that have lost TDP-43 or FUS from the nucleoplasm also show intranuclear TDP-43 or FUS inclusions16, although this is much more rarely seen than cytoplasmic TDP-43 or FUS inclusions. On a functional level, this is thought to cause a loss of their normal mRNA processing functions. Moreover, TDP-43 or FUS aggregates are thought to gain novel toxic functions, e.g. due to aberrant protein/RNA interactions or altered mRNP granule dynamics17.TDP-43 and FUS are ubiquitously expressed proteins that belong to the family of heterogenous nuclear ribonucleoproteins (hnRNPs). Their main site of localization is the nucleus, where they bind to gene promotors or long introns of pre-mRNAs and regulate transcription or splicing, respectively20. First, genetic mutations that alter or truncate the nuclear localization signal (NLS) of FUS and thus cause impaired nuclear import of FUS, cause familial ALS24 or motor neuron degeneration in mice27. Second, FTD patients with TDP-43 aggregates were shown to have reduced cortical levels of Exportin-2 (CAS)28. This Exportin re-exports the nuclear import receptor Importin α into the cytoplasm and therefore is required for proper Importin α/β-dependent nuclear import29. TDP-43 is imported into the nucleus by Importin α/β30, hence reduced Exportin-2 levels impair its nuclear import28. Third, the most common genetic cause of familial ALS and FTD, a hexanucleotide (GGGGCC) repeat expansion in the C9orf72 gene, is thought to functionally compromise the nuclear transport machinery, as several components involved in protein import, protein export as well as mRNA export are strong genetic modifiers of C9orf72 repeat-associated toxicity35. Consequently, enhancing nuclear import of TDP-43 and FUS could be a promising therapeutic approach, but will most likely be very hard to implement.Research over the past few years has provided strong evidence that nuclear import defects contribute to the nuclear loss and cytoplasmic accumulation of TDP-43 and FUS and to ALS and FTD pathogenesisC9orf72- and TDP-43-associated ALS and FTD: Here, specific inhibitors of the nuclear export receptor CRM1 (Exportin-1), KPT-276 and KPT-335, alleviated C9orf72 repeat-mediated neurodegeneration in the drosophila eye33 and reduced TDP-43 overexpression-induced cell death in cortical neurons36, respectively. In another study, the CRM1 inhibitors KPT-276 and KPT-350 were shown to protect against axonal damage in preclinical models of demyelination and glutamate-induced neurotoxicity37, although the underlying mechanisms are not well understood.An alternative therapeutic approach could be to curb nuclear export of TDP-43 and FUS, in order to compensate for poor nuclear import and to restore normal nuclear TDP-43 and FUS levels. Inhibition of nuclear export as a therapeutic strategy has already been tested in preclinical models of 2–3Φx2–3ΦxΦ39. In the presence of RanGTP in the nucleus, CRM1 directly binds such NESs with its cargo binding site and transports the NES-containing cargo across nuclear pore complexes (NPCs) into the cytoplasm41. Here, RanGAP promotes conversion of RanGTP into RanGDP, which leads to dissociation of the CRM1-cargo complex. Whether CRM1 also recognizes TDP-43 and FUS and mediates active nuclear export of the two proteins is unknown. So far, a CRM1-dependent NES has been predicted in the RRM2 domain of TDP-43 and mutation of key hydrophobic residues were shown to result in nuclear aggregation of TDP-4330. A leucine-rich NES has also been predicted in the RRM domain of FUS42 and deletion of this putative NES was shown to suppress toxicity associated with NLS mutant versions of FUS43. Despite these interesting observations, functionality of the predicted NESs has not been tested in bona fide nuclear export assays.CRM1 exports nuclear proteins that contain a so-called leucine-rich nuclear export signal (NES), which contains four closely spaced hydrophobic residues (Φ) and follows the consensus sequence ΦxIn this study we used the interspecies heterokaryon assay as a nuclear export assay to address whether predicted CRM1-dependent NESs in TDP-43 and FUS are functional and whether pharmacological inhibition or silencing of CRM1 blocks nuclear export of TDP-43 and FUS. As our results demonstrate CRM1-independent nuclear egress of TDP-43 and FUS, we addressed whether RNA-binding, the mRNA export machinery or the export receptor Exportin-5 are required for nuclear export of TDP-43 or FUS. As this was not the case, we engineered enlarged versions of TDP-43 and FUS to test whether nuclear egress of TDP-43 and FUS occurs by passive diffusion rather than active receptor-mediated export. Finally, using biochemical extraction and fluorescence loss in photobleaching (FLIP) experiments, we obtained support for the hypothesis that newly synthesized RNA anchors TDP-43 in the nucleus and limits its egress into the cytoplasm. Our results raise the possibility that transcriptional inhibition or defective RNA-binding may accelerate diffusion of TDP-43 out of the nucleus and thus may contribute to cytoplasmic mislocalization of TDP-43 in ALS and FTD patients.IAQSLCGEDLII) in TDP-43 and NES-289 (VQGLGENVTI) in FUS have been previously predicted as CRM1-dependent NESs30, however their activity has not been verified in bona fide nuclear export assays. NES-222 (IPKPFRAFAF) in TDP-43 and NES-301 (VADYFKQIGI) in FUS, which also fulfill the consensus criteria for recognition by CRM1, have not been predicted and studied before. To address the functionality of these predicted NESs, we introduced mutations that should abrogate CRM1-binding, by exchanging two key hydrophobic residues of one or both predicted NESs for alanine (A)  are chemically fused to generate hybrid cells, so-called interspecies heterokaryons , we identified two putative CRM1-dependent NESs in each TDP-43 (at positions 222 and 239) and FUS (at positions 289 and 301) Fig. . NES-239(A) Fig. . When ex(A) Fig. . We thenFUS Fig. . To furtone Fig. . Togethe47, impairs nuclear export of TDP-43 or FUS in the heterokaryon assay. As a positive control for LMB activity, localization of p62/SQSTM1, a known CRM1 export substrate48, was analyzed in parallel. After LMB treatment for 2 hours, p62 strongly relocalized from the cytoplasm to the nucleus , a well-known CRM1-specific inhibitoreus Fig. , demonsteus Fig. , demonst45. We reasoned that if TDP-43 and FUS would be exported via CRM1, LMB treatment or silencing of CRM1 with siRNA should shift the equilibrium towards a more nuclear localization. However, LMB treatment for 2.5 h did not alter the subcellular distribution of NLS-mutant TDP-43 or FUS , which show a partial cytosolic mislocalizationFUS Fig. . SimilarFUS Fig. , NLS-mutFUS Fig. . MoreoveFUS Fig. . Taken t49, we next tested whether Exportin-5 (XPO5) is involved in nuclear export of TDP-43 and FUS. To this end, we used siRNA to silence XPO5 in HeLa cells stably expressing NLS-mutant TDP-43 and FUS. XPO5 knockdown was efficient, as demonstrated by immunoblotting 54. In another construct, we exchanged all RGG motifs to KGG motifs (mRGG), as RGG mutations were recently shown to abrogate high affinity RNA-binding of FUS in cells55. We found that all mutant proteins were still able to shuttle from the human to the mouse nucleus of heterokaryons within 2–3 h  is fused to an NLS-containing protein-of-interest59. The GCR domain traps the fusion protein in the cytoplasm until addition of a steroid hormone, e.g. dexamethasone (DEX), triggers nuclear import; removal of the steroid hormone induces nuclear re-export  to the N-terminus of TDP-43 and FUS and thus enlarged them by 119 kDa, creating fusion proteins of 162 kDa and 172 kDa, respectively. If TDP-43 and FUS indeed exit the nucleus by passive diffusion, these large fusion proteins should leave the nucleus very slowly, as the rate of passive macromolecular diffusion through NPCs strongly decreases with increasing molecular weight61. In the absence of dexamethasone, GCR2-EGFP2-TDP-43 and -FUS were largely cytosolic, but rapidly translocated into the nucleus upon dexamethasone addition  and the bulk mRNA export machinery, we next tested the hypothesis that the two proteins exit the nucleus by passive diffusion. To test our hypothesis directly, we adapted the hormone-inducible nuclear transport assay from Love ort Fig. . We fuseion Fig. . After wiod Fig. , thus se65  and serve as a cytoplasmic reporter , the TDP-43 and FUS fusion proteins were localized in the nucleus and no relocalization and trapping in the cytoplasm by TGN-resident gM-FKBP3 was observed  fused to EYFP-NLS-FRB was readily depleted from the nucleus and co-localized with gM-FKBP3 at the TGN upon rapamycin addition  nuclear export assay, which is based on rapamycin-induced dimerization of FRB (FK506-rapamycin (FR)-binding domain) and FKBP (FK506-binding protein-12)ter Fig. . The potter Fig.  and test67.Together, our results imply that TDP-43 and FUS, at least in HeLa cells, leave the nucleus predominantly by passive diffusion, as artificial enlargement of both proteins strongly impaired their nuclear egress, which is compatible with recent models of passive diffusion through NPCs68. We therefore speculated that newly synthesized RNA could act as an anchor that retains TDP-43 and FUS in the nucleus and limits their diffusion through NPCs into the cytoplasm. To test this hypothesis, we treated HeLa cells with the transcriptional inhibitor actinomycin D (Act D) for 3 hours and examined subcellular localization and biochemical extractability of TDP-43 and FUS in comparison to untreated cells. Indeed, Act D treatment caused a slight cytosolic relocalization of TDP-43  most likely exert their promising neuroprotective effects37 independently of TDP-43 and FUS. Instead, their beneficial effects must be due to inhibiting export of other CRM1 cargoes, e.g. Tau and NRF2 . Deciphering the neuroprotective mechanism of CRM1 inhibitors in different models of neurodegeneration will be an interesting task for the future.In our study we examined how TDP-43 and FUS leave the nucleus, as the nuclear export routes of the two shuttling hnRNPs have not been characterized so far. In particular, we sought to test the hypotheses that TDP-43 and FUS are exported by the nuclear export receptor CRM1 and that inhibition of CRM1 may be a therapeutic strategy to compensate for defective nuclear import of TDP-43 and FUS. Our results clearly demonstrate that TDP-43 and FUS leave the nucleus independently of CRM1 and that pharmacologic inhibition of CRM1 is unable to restore nuclear localization of TDP-43 and FUS when their nuclear import is impaired. Our findings are in line with quantitative proteomic studies that searched for proteins that bind to CRM1 in a Ran-GTP-dependent mannerIAQSLCGEDLII)30 and FUS (NES-289 VQGLGENVTI)23 have NES activity using the heterokaryon nuclear export assay. We found that TDP-43 and FUS are able to leave the nucleus even when key hydrophobic residues in the predicted NES are exchanged for alanine. We cannot completely exclude that other functional CRM1-dependent NESs, not predicted by available NES prediction tools, are present in TDP-43 and FUS. However, we consider this highly unlikely, as we found that neither pharmacological CRM1 inhibition nor siRNA-mediated CRM1 silencing (or both combined) alter nuclear egress/localization of TDP-43 or FUS. For TDP-43, it was furthermore shown that TDP-43 localization is unaffected by CRM1 inhibition in primary rat neurons76, suggesting that our findings, at least for TDP-43, are not limited to cell lines, such as HeLa cells, but can be extended to neuronal cells.We also tested whether previously predicted leucine-rich NESs in TDP-43  domains, hence it seems possible that the point mutations/deletion alter RRM folding and impair RNA-binding of TDP-43 or FUS. Loss of RNA-binding is expected to cause nuclear aggregation of TDP-4379 and to reduce TDP-43- or FUS-associated toxicity81. In direct support of the idea that mutations in the predicted “NES-239” of TDP-43 abrogate RNA-binding, this sequence was shown to be located within the hydrophobic core of the TDP-43 RRM2 domain82 and mutations in “NES-239” were found to disrupt TDP-43’s splicing activity76. In summary, we conclude that the previously predicted and commonly annotated NESs in TDP-43 and FUS are not functional. Mutations in these motifs do not affect nuclear egress of TDP-43 and FUS and hence are not suited to experimentally trap TDP-43 or FUS in the nucleus. Instead, they most likely impair RNA-binding and RNA processing functions of TDP-43 and FUS, which should be kept in mind when interpreting data generated with such “NES” mutant versions.In light of our finding, the question arises what causes nuclear aggregation of TDP-43 when the predicted NES-239 is mutated49. Export along with mRNA appeared to be a likely scenario, as TDP-43 and FUS are well-known RNA binding proteins and FUS was found to be associated with CIP29, a component of the TREX mRNA export complex83. Overall, our data argue against a predominantly receptor-mediated nuclear export of TDP-43 and FUS, as artificial enlargement  interfered with nuclear egress of both proteins. These data support the idea that TDP-43 and FUS leave the nucleus to a large extent by passive diffusion. It is well known that passive macromolecular diffusion through NPCs decreases strongly as macromolecules increase in size. Older studies reported a size threshold of 40–60 kDa86. However, more recent studies indicate that there is no firm size threshold, but rather a soft barrier to passive diffusion that gradually intensifies with increasing size of macromolecules67. Consistent with this model, smaller molecules passively permeate NPCs within minutes, whereas larger molecules (of up to 230 kDa) were found to permeate NPCs on the time scale of hours64. Nuclear egress of TDP-43 and FUS appears to follow this model of passive diffusion: Both proteins shuttle efficiently between nuclei of heterokaryon when they carry a small epitope tag (V5-TDP-43 or HA-FUS), but poorly exit the nucleus when tagged with larger domains (>43 kDa). This suggests that TDP-43 and FUS, at least in HeLa cells, leave the nucleus by passive diffusion rather than facilitated receptor-mediated export.Besides addressing CRM1 as possible export receptor, we excluded that Exportin-5 or the mRNA export machinery solely mediate nuclear export of TDP-43 and FUS. We considered Exportin-5 as a possible candidate, as it has been genetically linked to TDP-43 toxicity87. FUS also has been suggested to self-assemble and oligomerize on chromatin-associated RNAs89. Our data imply that physiological oligomerization of TDP-43 and FUS could limit their nuclear egress, as large oligomeric TDP-43 or FUS species are expected to poorly permeate NPCs by passive diffusion. To test this hypothesis, it will be interesting to examine whether oligomerization-deficient mutants leave the nucleus at an enhanced rate, and whether enhanced nuclear egress contributes to the reported reduction in splicing activity and enhanced cytoplasmic aggregation of oligomerization-deficient TDP-4387. If so, this would underscore the proposal that stabilizing physiological TDP-43 or FUS oligomers could be an attractive therapeutic strategy to counteract cytoplasmic mislocalization and aggregation of the two proteins.The notion that TDP-43 and FUS leave the nucleus by passive diffusion implies that any molecular interactions that (i) lead to higher molecular weight complexes of TDP-43 or FUS or (ii) trap TDP-43 or FUS on nuclear structures will reduce their nuclear egress and favor a predominantly nuclear localization. In the case of TDP-43, it has been recently reported that TDP-43 forms homo-oligomers  under physiological conditions, which antagonizes its pathological aggregation3. Interestingly, a few ALS-associated mutations in TDP-43 are located in the RRM1 domain (P112H90 and D169G91) or directly adjacent to the RRM2 domain (K263E92 and K267S)93. Moreover, stress-induced acetylation of lysine residues in RRM1 and RRM2 of TDP-43, as found in ALS spinal cord, was shown to impair RNA-binding94. If mutations or acetylation cause a mild RNA-binding defect (not as severe as for the F4L mutant) and thus the mutant or acetylated protein remains soluble (not oligomerized/granular as the F4L mutant), their nuclear egress might be facilitated due to reduced retention on nuclear RNA. This speculative model would be in line with a model proposed by Ling et al., which posits that de-regulated gene expression and disrupted protein homeostasis are intimately linked3. Perturbation of RNA homeostasis may cause a vicious circle that leads to further protein and RNA homeostasis defects and drives disease progression. Loss of nuclear retention of TDP-43 on newly transcribed pre-mRNAs may be part of this detrimental cycle that eventually disturbs proper TDP-43 localization and function.For TDP-43, we furthermore found that newly synthesized RNA could retain the protein in the nucleus and limit its egress into the cytoplasm, as inhibition of transcription causes accelerated loss of TDP-43 from the nucleus. Loss of this nuclear retention mechanism could be one factor that contributes to TDP-43 mislocalization and disease progression in ALS/FTD, possibly as a consequence of the widespread RNA processing changes that occur in both diseasesFor immunocytochemistry the following antibodies were used: Alexa Fluor® 488 Donkey anti-Goat, Alexa Fluor® 488 Donkey anti-Mouse, Alexa Fluor® 488 Donkey anti-Rabbit, Alexa Fluor® 488 Donkey anti-Rat, Alexa Fluor® 555 Donkey anti-Mouse, Alexa Fluor® 555 Donkey anti-Rabbit, Alexa Fluor® 555 Donkey anti-Rat, Alexa Fluor® 647 Donkey anti-Mouse, Alexa Fluor® 647 Donkey anti-Rabbit (Invitrogen).For immunoblotting, the following antibodies were used: IRDye® 680RD Donkey anti-Mouse IgG, IRDye® 680RD Goat anti-Rat IgG, IRDye® 800CW Donkey anti-Rabbit IgG (LI-COR). For visualization of the the Dig(T)40 probe, FITC-labeled anti-Digoxigenin-AP, Fab fragments from sheep (Roche) was used.95 and pcDNA3.1-hygro(−)-HA-FUS21 as templates. TDP-43 mNES-222 and FUS mNES-289 were used as templates to generate double-mNES mutants. TDP-43 and FUS constructs carrying mutations in the NLS  as well as in the putative NESs (mNLS/double-mNES) were generated by QuikChange mutagenesis (Stratagene). Constructs encoding EGFP fused to putative NES sequences were cloned by oligonucleotide annealing and HindIII/BamHI restriction digest into the pEGFP-C1 (Clontech) mammalian expression vector. To generate GCR2-EGFP2-TDP-43 and GCR2-EGFP2-FUS constructs, TDP-43 and FUS cDNAs were cloned by enzyme restriction digest into the modified pEGFP-C1 vector containing a GCR2-EGFP2 cassette96. FUS mRRM/mZnF (F305L/K312A/K315A/K316A/F341L/F359L/F368L/D425A/N435A/F438A/W440A/R441A/N445A) and mRGG cDNAs were commercially synthesized (Genscript) and cloned by enzyme restriction digest into the pEGFP-C2 (Clontech) mammalian expression vector. Constructs encoding flag-TDP-43 and RNA-binding-deficient mutants thereof were kindly provided by Emanuele Buratti and Francisco Baralle69. EGFP-TDP-43 (F4L) construct was generated by subcloning TDP-43-4FL into the pEGFP-C3 (Clontech) mammalian expression vector. Lentiviral constructs used for generation of stable HeLa cell lines were in pCDH-Ef1-MCS-IRES-Puro (System Biosciences). Construct encoding EGFP-histone H3.3 was kindly provided by Sandra Hake97. The integrity of all constructs was verified by sequencing. Oligonucleotide sequences are available upon request.TDP-43 and FUS carrying mutations in putative nuclear export signals (NES) (mNES/double-mNES) were generated by QuikChange mutagenesis (Stratagene) using pcDNA6-TDP-43-V598. HeLa cells stably expressing TDP-43-V5, mCherry-TDP-43-dNLS and mCherry-FUS-P525L were generated by lentiviral transduction as described in99, followed by selection with puromycin . Single cell clones of mCherry-TDP-43-dNLS and mCherry-FUS-P525L-expressing lines were obtained by fluorescence activated cell sorting (FACS) and expansion of mCherry + single cell clones.HeLa cells stably expressing HA-FUS were described in2-GFP2-fusion proteins, cells were incubated for 20 min with dexamethasone .HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax (Life Technologies) supplemented with 10% (vol/vol) fetal calf serum  and Gentamycin . For the hormone-induced nuclear export assay, HeLa cells were cultured in DMEM supplemented with 10% (vol/vol) dialyzed fetal bovine serum . Transfections were carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Were indicated, leptomycin B  was added to the culture medium for the indicated time. Actinomycin D  treatment was carried out for 3 hours and sodium arsenite  treatment for 30 min. Heat shock was performed by incubating cells for 1 hour in a tissue culture incubator heated to 44 °C. To induce nuclear import of GCRHeLa cells were seeded onto coverslips in a 12-well-plate and transfected with the indicated constructs. After 5 h, medium was removed and mouse embryonic fibroblasts (MEFs) were added. 24 h post-transfection, HeLa cells were fused with MEFs by a 2 min incubation with polyethylene glycol  at room temperature. PEG was removed by washing 3 times in PBS supplemented with Glucose . Post-fusion, cells were incubated in antibiotic-free DMEM in the presence of cycloheximide  to inhibit protein synthesis and further processed by immunocytochemistry.Knockdown of Aly/REF, CRM1 and XPO5 was achieved using Dharmacon siGENOME SMARTpools composed of 4 different siRNAs. siRNA transfections were carried out with Lipofectamine 2000 at a final concentration of 25 nM siRNA. Culture medium was exchanged 5 h post-transfection and the knockdown was analyzed 72 h post-transfection by immunoblotting and immunocytochemistry.65. Briefly, HeLa cells were co-transfected for 20 hours with the plasmid pCR3-N-HA-UL10/gM-FKBP3 and either pEYFP-NLS-FRB-TDP-43 or pEYFP-NLS-FRB-FUS. As control, a plasmid encoding EYFP-NLS-FRB-pUL4 was used65. Following 10 min anisomycin (50 µM) treatment, cells were incubated for 2 h with rapamycin  or without rapamycin (−RAP) in the presence of anisomycin. Subsequently, gM was visualized by immunocytochemistry, EYFP-tagged proteins were visualized directly and nuclei were visualized by DAPI staining.The NEX-TRAP assay was performed as describedAll reagents were treated with 0.1% diethyl pyrocarbonate . Cells grown on 12 mm coverslips were fixed for 15 min at room temperature in 4% formaldehyde in PBS, permeabilized on ice for 5 min in 0.5% Triton X-100 in PBS and equilibrated on ice for 5 min with 2× SSC (Ambion) and 25% formamide in PBS. Cells were hybridized for 2 h at 37 °C with 1 ng/ml digoxygenin labeled oligo(dT)  in 2× SSC, 1 mg/ml tRNA, 0.02% bovine serum albumin (BSA), 2 mM ribonucleoside vanadyl complex , 25% formamide, 5% dextrane sulfate. Afterwards, cells were washed 2× with 2× SSC/25% formamide in PBS, followed by a washing step with 0.5× SSC in PBS and once with 0.2% Triton X-100 in PBS. Subsequently, immunocytochemistry was carried out using a FITC-coupled anti digoxygenin Fab fragment  for detection of the labeled oligonucleotide and specific antibodies for the indicated proteins, respectively.4Cl and blocked for 30 min with 0.1% saponine in PBS supplemented with 5% goat or donkey serum. Primary antibodies . If necessary for better print quality, images were processed by linear enhancement of brightness and contrast. Images presenting the NEX-TRAP results were taken using a confocal laser scanning microscope  and processed using Adobe Photoshop CS3 by linear enhancement of brightness and contrast.100. In ImageJ, ROIs corresponding to nuclei were identified by DAPI staining using the wand tool and MFI in the EGFP channel in nuclear ROI was determined. A band around the nucleus (band size = 2 µm) was set as ROI corresponding to the cytoplasm and MFI of the EGFP signal in the cytoplasmic ROI was measured. For each construct, 40 cells from one representative experiment were analyzed.For quantification of mean fluorescence intensity (MFI) in the nucleus and cytoplasm, images were imported in the public-domain software FijiHeLa cells were harvested in trypsin/EDTA (Sigma) and washed twice with ice-cold PBS. Cells were lysed in ice-cold RIPA buffer  supplemented with complete protease inhibitor cocktail (Roche). Lysates were sonicated and and protein concentrations were determined by BCA protein assay (Pierce).2, 0.1% NP-40, 10% Glycerol and complete protease inhibitor cocktail (Roche)) for 10 min on ice and centrifuged (2000 g) for 10 min at 4 °C to pellet nuclei. Nuclear proteins that are insoluble in mild cell lysis buffer remain in the pellet fraction , whereas soluble nuclear proteins are extracted into the supernatant . 4× SDS-PAGE buffer was added to the cytoplasmic fraction, the nuclear fraction was resuspended in the same volume of mild cell lysis buffer and 4× SDS-PAGE buffer was added. Equal volumes of cytoplasmic and nuclear fractions were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.HeLa cells were harvested in trypsin/EDTA (Sigma) and washed twice with ice-cold PBS. Cells were incubated in mild cell lysis buffer . The membrane was blocked in blocking buffer  and 5% milk powder) and incubated with the indicated primary antibodies  in regions-of-interest (ROI) bordering the protein bands were measured by Fiji/ImageJ software. MGVs were calculated and converted into cytoplasmic to nuclear ratios (c/n). Means of the three independent experiments were calculated and standard error of the mean indicated by error bars. The one-way ANOVA with Dunnett’s multiple comparison test Fig.  as well HeLa cells were cultured in µ-Dish 35 mm, high Glass Bottom (ibidi) and transiently transfected with EGFP-TDP-43or EGFP-histone H3.3 as a control. 24 h post-transfection cells were either left untreated or were treated with actinomycin D (5 µg/ml) for 3 hours. Images were acquired with an inverted Zeiss Axio Observer.Z1 microscope with a 63/1.4NA oil immersion lens equipped with a confocal spinning disc  and a Rapp OptoElectronic laser scanning device . Before bleaching, 5 images were taken in streaming mode with a 488 nm 50 mW SD laser; the same setting were used to acquire an image after bleaching. For bleaching, 3 circular ROIs of 6 µm each were repeatedly photobleached in the cytoplasm using a 473 nm diode laser  with full laser power with an iteration of 100 and duration of 200 ms per bleach event. An image was acquired before and after each bleach event with 15 s intervals between each bleach event.The fluorescence loss of a defined area in the nucleus of the bleached cell was measured over time, and corrected for bleaching by acquisition and background noise as follows using the Fiji/ImageJ macro “TimeSeries Analyzer”:ROI1 is defined as the average grey value of an area in the nucleus of the cells which cytoplasm was repeatedly photobleached. A corresponding area of a non-photobleached cell in the same field of view served as control for bleaching due to image acquisition and is represented by the average grey value of ROI2. ROI3 is the defined average grey value of the background. Furthermore, average grey values were normalized to the mean grey value of the 5 pre-bleach images (set to 1).Supplementary Information


EGFR mutation-positive and never smokers. For patients with EGFR mutation, anti-PD-1/PD-L1 therapy was an unfavorable factor of PFS. The grade 3 or 4 adverse events rates of anti-PD-1/PD-L1 treatment were significantly lower than that of docetaxel. Our results suggest that anti-PD-1/PD-L1 therapy significantly improves survival compared with docetaxel in patients with previously treated, PD-L1-positive, advanced NSCLC, and has a distinct safety profile from chemotherapy.Anti-PD-1/PD-L1 antibodies have been proved one of the most promising treatments against non-small cell lung cancer (NSCLC); however, whether anti-PD-1/PD-L1 antibodies can provide added benefits for pretreated patients with advanced NSCLC and which patients are most likely to benefit from anti-PD-1/PD-L1 therapy remain controversial. This meta-analysis evaluated the efficacy and safety between anti-PD-1/PD-L1 antibodies and docetaxel in previously treated, advanced NSCLC. PubMed, EMBASE and Cochrane library databases were systematically searched for eligible studies. Five studies with a total of 3,025 patients were included. Our results showed that, for all patients, anti-PD-1/PD-L1 therapy prolonged overall survival (OS)  and progression-free survival (PFS) . For patients with PD-L1 expression ≥1%, anti-PD-1/PD-L1 therapy had higher objective response rates. In subgroup analysis according to the tumor PD-L1 expression level, anti-PD-1/PD-L1 therapy was associated with longer OS and PFS in patients with high PD-L1 expression , but not in those with low expressions. In subgroup analysis of patients’ characteristics, anti-PD-1/PD-L1 antibodies showed OS benefits across most prespecified subgroups, except for patients with  One of the commonest cancers and the main cause of cancer-related death worldwide is lung cancer . Non–smaFollowing greater understanding of the multiple tumor immune escape mechanisms in recent years, programmed cell death 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) pathway inhibition, followed by immune system tumor killing effect reactivation has become a new strategy for treating cancer –7. A serThe search strategy identified 312 records that were screened for inclusion. Title review determined that 254 studies were not clinical trials, and the studies were excluded. Abstract review excluded 53 studies which did not meet the selection criteria. In total, five trials performed between the year 2012 and 2015, which included 3,025 patients, met the inclusion criteria. .The Deek’s funnel plot and Begg or Egger test demonstrated that there was no evidence of publication bias across the included studies regarding OS  was the main reason of the heterogeneity  rate, which was reported as complete response (CR) and partial response (PR) rates according to RECIST 1.1. Table ogeneity . Therefoogeneity  were sigogeneity , with anp = 0.000; fixed-effects model; Figure p = 0.000; fixed-effects model; EGFR mutation–negative status, KRAS mutation–positive status, and ALK mutation–negative status  for overall survival (OS) and progression-free survival (PFS). Compared with docetaxel, anti-PD-1/PD-L1 antibodies significantly prolonged OS . For any-grade adverse events, the frequencies of both hematologic and nonhematologic adverse events were substantially lower with anti-PD-1/PD-L1 antibodies than with docetaxel. Immune-related adverse events, including pneumonitis, have been reported in only a small percentage of patients treated with anti-PD-1/PD-L1 treatment, and these events were managed using established guidelines.This study has several limitations. One is the heterogeneity of the participant inclusion criteria, where the KEYNOTE-010 study  enrolledIn conclusion, the present meta-analysis demonstrates that anti-PD-1/PD-L1 treatment led to superior survival benefit with an improved safety profile over that for docetaxel in patients with previously treated advanced NSCLC. Patients with positive PD-L1 expression may benefit more from anti-PD-1/PD-L1 therapy. The identification of relevant biomarkers with sufficient sensitivity and specificity for predicting which patients are most likely to benefit from this therapy is needed in future studies.This meta-analysis was conducted according to the PRISMA guidelines. We systematically searched the PubMed, EMBASE and Cochrane library databases for eligible studies. The search time was from database inception to April 1, 2017. The combination of free-text words and MeSH terms were used as follows:  AND (programmed cell death 1 receptor/PD-1/programmed death-ligand 1/PD-L1) AND (nivolumab/MDX-1106/ONO-4538/BMS-936558/opdivo/atezolizumab/MPDL3280A/tecentriq/RG7446/pembrolizumab/lambrolizumab/keytruda/MK-3475) AND (docetaxel/docetaxol). Reference lists from eligible studies were also searched thoroughly for potential relevant studies.The publications identified were carefully screened. Only the most recent randomized clinical trials were included in the meta-analysis. Preclinical studies, case reports, and reviews were excluded. Two reviewers  screened all publications identified based on our inclusion criteria. In the event of disagreement between the two reviewers, we obtained and inspected the full-text article independently. In total, five studies were included in the final analysis.The inclusion criteria were: (1) randomized controlled trial; (2) patients with advanced or metastatic NSCLC after failure of previous treatments; (3) anti-PD-1/PD-L1 antibodies treatment as compared with chemotherapy; (4) published in English; (5) reported OR rate, toxicity data, or at least one form of survival data. The extracted data included: (1) study characteristics ; (2) patient characteristics ; (3) PD-1/PD-L1 therapy and docetaxel regimen; and (4) outcome measures .2 test of heterogeneity and the I2 measure of inconsistency. We considered heterogeneity present when the p-value of the Cochran Q test was <0.05 and the I2 statistic was >50%. We performed meta-regression or subgroup analyses to find the source of heterogeneity [p < 0.05 was considered significant. All statistical analyses were performed using the meta-analysis command in STATA .The methodological quality of trials was assessed by the Cochrane risk of bias tool. All the meta-analyses were performed using Review Manager 5.3 . Pooled estimates of OR rate and grade 3 or 4 treatment-related adverse events rate were computed when there was sufficient reporting of these measures. The odds ratio was used for comparing the OR rates and adverse events rates. Time-to-event outcomes (OS or PFS) were analyzed as HRs and pooled according to effect size and 95% CIs. We assessed heterogeneity in the results of the trials using the χogeneity , 27. If ogeneity , 29. A s


PD‐L1) promotes immunosuppression by binding to PD‐1 on T lymphocytes. Although tumor PD‐L1 expression is a potential predictive marker of clinical response to anti‐PD‐1/PD‐L1 therapy, little is known about its association with clinicopathological features, including prognosis, in high‐grade neuroendocrine tumors (HGNETs), including small‐cell lung carcinoma (SCLC) and large‐cell neuroendocrine carcinoma (LCNEC), of the lung. We immunohistochemically examined the membranous of expression of PD‐L1 in 115 consecutive surgical cases of lung HGNET (74 SCLC cases and 41 LCNEC cases). We examined the prognostic association of tumor PD‐L1 positivity using the log‐rank test as well as Cox proportional hazards regression models to calculate the hazard ratio (HR) for mortality. Programmed death‐ligand 1 immunostaining (at least 5% tumor cells) was observed in 25 (21%) of the 115 HGNET cases. In a univariable analysis, PD‐L1 positivity was associated with lower lung cancer‐specific  and overall  mortality. Additionally, in a multivariable analysis, PD‐L1 positivity was independently associated with lower lung cancer‐specific  and overall  mortality. Our study demonstrated the prevalence of PD‐L1 positivity in lung HGNET cases, and the independent association of tumor PD‐L1 positivity with lower mortality in lung HGNET cases. Further studies are needed to confirm our findings.Programmed death‐ligand 1 ( High‐grade neuroendocrine tumors (HGNETs), comprising small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), constitute the most aggressive lung cancer histological subtype Programmed death‐ligand 1 (PD‐L1 or CD274), an immune modulator, promotes immunosuppression by binding to programmed death‐1 (PD‐1 or PDCD1) on T lymphocytes. Emerging evidence suggests that anti‐PD‐1/PD‐L1 therapy is effective in various malignancies, including SCLC, and PD‐L1 expression in cancer cells is a potential predictive marker of the clinical response to anti‐PD‐1/PD‐L1 therapy To our knowledge, no study has examined the association of tumor PD‐L1 expression with prognosis in HGNET, although a few studies have examined PD‐L1 expression in SCLC with conflicting results We examined 115 consecutive lung HGNET cases, including 74 SCLC and 41 LCNEC cases, to assess the prognostic association of membranous PD‐L1 expression in HGNET Pathological diagnoses were made by experienced expert pulmonary pathologists (KI and YI) according to the 2015 WHO classification of lung tumors Membranous PD‐L1 expression of cancer cells was evaluated by tissue microarray immunohistochemical analysis. Using archived surgically resected specimens, which had been used for the initial pathological diagnosis of primary lung cancer, we constructed tissue microarrays as described previously μm thickness were immunostained for PD‐L1 with an anti‐PD‐L1 rabbit monoclonal antibody  using the Leica Bond III automated system . Sections were incubated at pH 9 for 20 min at 100°C. Antibody‐based PD‐L1 expression was interpreted by a pathologist (KI) who was blinded to patient details. We used the same PD‐L1‐positive/‐negative criteria as those that were previously used P < 0.0001), indicating substantial agreement.Programmed death‐ligand 1 immunostaining was conducted as previously described ™ Antibody Diluent  as nonspecific negative controls.We validated the specificity of the PD‐L1 antibody (clone: E1L3N). Because PD‐L1 is also known as B7‐H1, a B7 subfamily member, we used a B7 subfamily cell array , comprising CHO‐K1 cells overexpressing the B7 subfamily. With 10% neutral buffered formalin, we fixed CHO‐K1 cells transiently overexpressing PD‐L1 (B7‐H1), B7‐H2, B7‐DC (PD‐L2), B7‐H3, B7‐H4, B7‐1, or B7‐2, and mock‐transfected control cells and processed them to yield a formalin‐fixed paraffin‐embedded (FFPE) cell block array. Using this B7 subfamily cell array, we assessed the specificity of the PD‐L1 antibody (clone: E1L3N). Furthermore, we used sections processed with primary antibody replacement by Dako REAL2 in the air.Human SCLC cell lines, STC‐1 and Lu135, were obtained from Japanese Collection of Research Biosources . Cells were cultured in RPMI‐1640  supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% COμg protein samples were loaded into 4–12% Bis‐Tris gel . After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Invitrogen). The membrane was blocked by 5% skim milk  prior to incubation with rabbit monoclonal antibodies, anti‐PD‐L1‐antibody , and anti‐β‐actin‐antibody , for 1 h at room temperature. Immunoblots were detected using SuperSignal West Femto kit .Total protein was extracted from STC‐1 and Lu135 cell lysate. Protein concentration was determined using BCA assay reagents , and 20 β‐actin.Total RNA was extracted from SCLC cells using RNeasy Mini kit . For each sample, 100 ng of total RNA was reverse transcribed into cDNA using SuperScript III First Strand (Invitrogen) according to the manufacturer's instructions. The PD‐L1 gene was amplified with KOD FX neo  using the following primers: 5′‐ATGGTGGTGCCGACTACAAG‐3′ and 5′‐GGAATTGGTGGTGGTGGTCT‐3′. We used PCR primers 5′‐CCAACTGGGACGACATGGAG‐3′ and 5′‐TGGATGGCTACGTACATGGC‐3′ for β‐actin were as follows: 94°C for 2 min followed by 35 cycles , and a final holding temperature of 4°C. After PCR, 5 μL of each sample was electrophoresed on 2% agarose gel.The PCR conditions for PD‐L1 and P‐values were two sided, and we considered P = 0.05 or less statistically significant. The primary objective of this study is to examine the association of tumor PD‐L1 expression with prognosis in patients with lung HGNET. All the other analyses were secondary and exploratory. We interpreted P‐values very cautiously to avoid overinterpretation by considering multiple‐hypothesis testing. To investigate the associations of tumor PD‐L1 positivity with clinicopathological factors in lung HGNET cases, we performed the chi‐square or Fisher's exact test as appropriate. We used the Kaplan–Meier method and log‐rank test for survival analyses. For analyses of lung cancer‐specific mortality, we censored deaths because of other causes. We used univariable and multivariable Cox proportional hazards regression models to calculate the hazard ratio (HR) for mortality according to PD‐L1 expression status (positive vs. negative). The multivariable model initially included age (<60 vs. ≥60 years), gender , smoking history (≤20 vs. >20 pack‐years), pathological stage (p‐stage) (I vs. II–IV), neoadjuvant chemotherapy (yes vs. no), adjuvant chemotherapy (yes vs. no), histology (SCLC vs. LCNEC), and lymphovascular invasion (positive vs. negative). We created missing categories for missing cases for each variable, if applicable, and performed a backward stepwise elimination with P = 0.05 as the threshold to select variables for the final model. We confirmed the proportionality of hazards assumption using graphs of log versus log of survival time to visually assess if the lines were approximately parallel.We conducted all statistical analyses using JMP 12  and Excel 2016 . All We validated the specificity of the PD‐L1 antibody (clone: E1L3N) by using a B7 subfamily cell array, comprising CHO‐K1 cells transiently overexpressing PD‐L1 (B7‐H1), B7‐H2, B7‐DC (PD‐L2), B7‐H3, B7‐H4, B7‐1, or B7‐2, and mock‐transfected control cells. Using this cell array, we verified the specificity of the PD‐L1 antibody (clone: E1L3N) by confirming that this antibody exclusively recognized PD‐L1 (B7‐H1) among the tested B7 subfamilies Figure .We performed Western blotting and RT‐PCR analyses for PD‐L1, using the SCLC cell lines (STC‐1 and Lu135). The anti‐PD‐L1 antibody (clone: E1L3N) showed a PD‐L1 protein in Lu135, but not in STC‐1 Figure a. The siP = 0.041). However, of the 115 HGNEC patients, only 22 SCLC patients underwent neoadjuvant chemotherapy because many patients were in the early stage, and PD‐L1 positivity showed a tendency for the association of no neoadjuvant chemotherapy in SCLC (P = 0.052).Among the 115 lung HGNET cases, we observed PD‐L1 positivity (≥5% tumor cells with membranous staining) in 25 cases (21%) by immunohistochemistry  and overall  mortality  and overall  mortality (Table P = 0.0039) and overall  mortality. Among the other covariates, p‐stage II–IV was associated with higher lung cancer‐specific  and overall  mortality  for those censored. PD‐L1 positivity was associated with lower lung cancer‐specific  and overall  mortality. Multivariable analysis was not possible in LCNEC patients because of low statistical power. In univariable analysis, PD‐L1 positivity showed a tendency toward lower lung cancer‐specific  and overall  mortality.As secondary and exploratory analyses, we examined the prognostic associations of PD‐L1 positivity in SCLC and LCNEC patients using Cox regression models. The association of PD‐L1 positivity and other covariates with mortality in SCLC patients is shown in Table P = 0.022) and overall  mortality in SCLC patients, whereas it showed a tendency of lower lung cancer‐specific  and overall  mortality in LCNEC patients.Using the Kaplan–Meier method and log‐rank test, we additionally examined the prognostic associations of tumor PD‐L1 positivity in SCLC and LCNEC patients Fig. . PD‐L1 pWe examined the association of tumor PD‐L1 expression with prognosis using 115 consecutive lung HGNET cases (74 SCLC and 41 LCNEC cases). PD‐L1 expression, being involved in immune modulation, is a potential predictive marker of the clinical response to anti‐PD‐1/PD‐L1 therapy. This therapy is effective in various cancers such as SCLC Little is known about the association of tumor PD‐L1 expression with HGNET prognosis and clinicopathological features. Only Ishii et al. examined the prognostic association of tumor PD‐L1 expression in 102 SCLC cases The distinction between SCLC and LCNEC is challenging in terms of pathological diagnosis TP53, RB1, and EP300 and showed the same pattern of somatic copy number alterations Small cell lung carcinoma shares genetic alterations with LCNEC Small cell lung carcinoma has been categorized into prognostically different subtypes, although SCLC is usually considered generally as a devastating cancer. By gene expression profiling, we previously identified two groups of HGNET (better‐ and worse‐prognosis subtypes) independent of the histopathological status (SCLC vs. LCNEC) Large cell neuroendocrine carcinoma has been recently identified as a biologically heterogeneous tumor. Our gene expression profiling categorized LCNEC into two groups (better and worse prognosis) We have shown that tumor PD‐L1 positivity was independently associated with lower mortality in lung HGNET . The result in SCLC cases was similar to that obtained by Ishii et al. N = 115) was not sufficient, and the statistical power was therefore limited. Sixth, our dataset was retrospectively collected. Finally, this study may not be generalizable because we only enrolled Japanese patients in a single cancer hospital. Therefore, additional studies in other populations are needed.Our study has limitations. First, there is no standardized anti‐PD‐L1 antibody and scoring method of tumor PD‐L1 expression. For assessing PD‐L1 protein, several different clones are available. Nonetheless, we validated the specificity of a PD‐L1 antibody (clone: E1L3N) using a B7 subfamily cell array. Furthermore, we confirmed the protein and transcript expression of PD‐L1 in a SCLC cell line by conducting Western blotting and RT‐PCR analyses. Second, we utilized tissue microarrays to evaluate PD‐L1 expression in tumors. Because of intratumoral heterogeneity in lung HGNET, tumors with heterogeneous PD‐L1 expression can affect the results. This potential misclassification of tumors according to PD‐L1 expression would be randomly distributed; therefore, null results would have been obtained. Nonetheless, we have shown statistically significant associations. Furthermore, an experienced pulmonary pathologist (KI) chose each core site with a relatively large diameter (2 mm) in the most representative tumor histology. Therefore, core sites might not substantially affect the results. Third, we evaluated PD‐L1 expression only in tumor cells. PD‐L1 expression in tumor cells, tumor‐associated immune cells, or a combination of tumor and tumor‐associated immune cells might have biological or prognostic significance In conclusion, we demonstrated the prevalence and clinicopathological characteristics of tumor PD‐L1 positivity in lung HGNET  and the association of tumor PD‐L1 positivity with lower mortality in lung HGNET. This population‐based study provides useful information on PD‐L1 expression patterns in lung HGNET and may refine the role of PD‐L1 immunohistochemistry in clinical practice. Further studies with a larger sample size are warranted to confirm our findings.Y.Y. is an employee of Daiichi Sankyo Co., Ltd. M.N. received research grants from Novartis Pharma Co., Ltd., Ono Pharma Co., Ltd., Chugai Pharma Co., Ltd., Pfizer Co., Ltd., Bristol‐Myers Squibb Co., Ltd., Eli Lilly Co., Ltd., Taiho Pharma Co., Ltd., Astellas Pharma Co., Ltd., and AstraZeneca Co., Ltd., and is a consultant for Novartis Pharma Co., Ltd., Ono Pharma Co., Ltd., Chugai Pharma Co., Ltd., Eli Lilly Co., Ltd., Taiho Pharma Co., Ltd., Pfizer Co., Ltd., and Daiichi Sankyo Co., Ltd. Y.I. received research grants from Daiichi Sankyo Co., Ltd., and Chugai Pharmaceutical Co. Ltd., and is a consultant for Fujirebio Inc. All other authors declare no conflicts of interest.Figure S1. Immunohistochemical evaluation of tumor membranous PD‐L1 expression in B7 subfamily cell array. Only PD‐L1 overexpressing cells (a) showed PD‐L1 positivity. Mock‐transfected cells (b) (nonspecific negative control) and other B7 families overexpressing cells (c–h) (specific negative controls) showed PD‐L1 negativity.Figure S2. Western blot (a) and RT‐PCR (b) analyses for PD‐L1 using small cell lung carcinoma cell lines (STC‐1 and Lu135). Lu135 showed both PD‐L1 protein and transcript, whereas STC‐1 did not show PD‐L1 protein or transcript.Click here for additional data file.Table S1. PD‐L1 expression and other covariates associated with mortality* in patients with small cell lung carcinoma (SCLC).Click here for additional data file.


We observed that advanced rT classification and anemia status before salvage treatment was associated with high level of PD-L1 in recurrent NPC patients, and PD-L1 and was co-located with HIF-1α in recurrent tumors by immunofluorescence analysis. Moreover, our result suggested that PD-L1 might be a negative indicator for recurrent NPC patients as well as age, rT classification, anemia and tumor necrosis at diagnose of recurrence. Taken together, our results revealed that PD-L1 might be a potential prognostic biomarker for recurrent NPC patients, and advanced re-stage, anemia might represent as candidate biomarkers for evaluating patients’ response to anti-PD-1 / PD-L1-treatment. However, further studies are needed to clarify the underlying mechanism of hypoxia in immunosuppression process induced by PD-1 / PD-L1 axis.The expression of Programmed death-1 (PD-1) / programmed death-ligand 1 (PD-L1) has been reported to be reliable prognostic factors in various malignances including primary nasopharyngeal carcinoma (NPC). However, the exact role of PD-1/PD-L1 in recurrent NPC remains unclear. In this study, we aimed to investigate the relationship between the expression of PD-1 / PD-L1 and the clinical-pathology as well the outcomes of recurrent NPC patients (n = 132). The expression of PD-1 and PD-L1 was measured by immunohistochemistry staining. The relationship between PD-1 / PD-L1 and factors involved in clinic-pathology and outcomes of patients with NPC was assessed by correlation analysis. To further explore the association between PD-L1 and anemia, immunofluorescence analysi Nasopharyngeal carcinoma (NPC) is endemic in southern China, where annual incidence ranges from 20 to 50 cases per 100,000 . Recurreet al [et al [et al [et al [Programmed death-1 (PD-1 or CD279) is a co-receptor expressed predominantly by T cells . Programet al  reportedl [et al  delineatl [et al  has showl [et al  and Zhanl [et al . In addiTherapies targeting the PD-1 / PD-L1 pathway show promising results in the treatment of cancers through promoting antitumor T-cell activity , 21. TheIn this study, we aimed to investigate the prognostic role of PD-1 and PD-L1 in a cohort of recurrent NPC patients (n = 132). In addition, we also evaluated the correlation between PD-1 / PD-L1 expression and clinic-pathological variables as well as potential related peripheral blood biomarkers, such as hemoglobin (HB) level. Our study revealed that PD-L1 might be a potential prognostic biomarker for recurrent NPC patients, and advanced re-stage, anemia might represent as candidate biomarkers for evaluating patients’ response to anti PD-1 / PD-L1-treatment.th edition of the UICC / AJCC system, 101 patients (76.5 %) were at III-VI stage and 32 patients (24.2 %) were lymph node positive simultaneously. Pathology proven recurrent undifferentiated non-keratinizing carcinoma  and differentiated non-keratinizing carcinoma (WHO II) were diagnosed in 125 and 7 patients, respectively. The median HB level before salvage therapy was 139.0 g/l (ranges from 101.0 to 171.0 g/l) for all patients, among which 32 patients were anemic . Tumor necrosis before retreatment presented in 49 out of 132 (37.1 %) patients by pathologic diagnosis. Representative tumor necrosis with PD-L1 staining in recurrent NPC were shown in Figure All the biopsy samples of recurrent tumor n = 132) were collected. The samples were fixed by formalin and embedded into paraffin using a tissue processor. Among the 132 patients enrolled, there were 106 males and 26 females. The median time to recurrence after the initial treatment was 33.3 months (ranges from 6.6 to 190.2 months). The median age at recurrence was 46-year-old (ranges from 28 to 69 years). According to the 732 were cA total of 87 out of 132 (65.9 %) patients died during follow-ups. Of these, 35 out of 87 (40.2 %) died due to severe adverse effects, including 16 out of 35 (45.7 %) from mucosa necrosis or massive hemorrhage, 19 out of 35 (54.3 %) from other radiation-related injuries. In addition, 29 out of 87 (33.3 %) patients died due to local-regional failures and 16 out of 87 (18.4 %) due to distant failures. Other causes responsible for 2 out of 87 (2.3 %) deaths included 1 cases of cardiac disease, 1 case of digestive diseases. 5 out of 87 (5.7 %) patients died due to unknown reasons. The 3-year OS rates were 56.1 % in the 132 recurrent patients. Characteristics of the enrolled patients were summarized in Table PD-1 positive immune cells presented in 50 of 132 patients (37.9%) in a scattered manner, and PD-L1 staining was detectable in 128 tumors (97.0%), which was mainly located at the membrane or in the cytoplasm region (or both) in the tumor cells. PD-L1 was also observed to be stained in tumor-infiltration lymphocytes (TILs) dispersedly. According to ROC curve analysis for OS, the optimal cut-off value of H-score was 190  for PD-L1. The staining of PD-L1 were considered as high level (88 cases) if H-score ≥ 190. Representative staining of PD-L1 and PD-1 in recurrent NPC were shown in Figure Pre-retreatment hematologic biomarkers such as neutrophils, lymphocytes, HB and platelets were measured to identify the potential factors related to PD-1 / PD-L1. Our result showed that no significance (N. S.) between PD-1 positivity and HB level was observed in this cohort of recurrent patients Table . HoweverWe have also compared PD-1 / PD-L1 expression with numbers of neutrophils, lymphocytes, platelets and neutrophils / lymphocytes ratio before salvage therapy in this exploratory analysis, and no significantly relevant factors were identified .Hypoxia-inducible factors (HIFs) have been demonstrated to play an important role in the regulation of erythropoiesis by coordinating a series of graded hypoxic responses  and stabTo evaluate the prognostic values PD-L1 expression in recurrent NPC patients, we performed long rank tests in Kaplan-Meier survival analysis. In 132 patients with recurrent NPC, high expression of PD-L1 was correlated with significantly shorter OS  blocks from fiberscope biopsy of NPC were retrieved from the Department of Pathology. The related clinical data of these patients were retrospectively collected from our database or hospital records, including gender, age, smoking status, family history, clinical stage, pre-therapy laboratory counts of neutrophils, lymphocytes, HB and platelets as well as follow-up records. Tumor necrosis was defined as the necrosis of tumor tissue at diagnose of recurrence and assessed pathologically by two experienced pathologists independently. Anemia was defined according to World Health Organization criteria as hemoglobin < 130 g/l in men and < 120 g/l in women. All patients were followed-up for at least 3 years.A total of 179 patients with locally recurrent NPC from Sun Yat-Sen University Cancer Centre between January 2001 and March 2013 were enrolled. The cases were selected based on the following criteria: (1) local recurrence was histologically confirmed with available biopsy specimens; (2) complete clinical data; (3) non-distant metastatic simultaneously; (4) the main treatment of the initial course was radiotherapy; (5) no other malignant diseases; (6) Karnofsky score ≥ 70; (7) received salvage treatment at Sun Yat-Sen University; (8) follow-up regularly. Patients with active hepatitis, diabetes, or who failed the follow-up requirements were excluded. Therefore, we obtained the remaining 132 cases qualified for this study. All patients with recurrent NPC were re-staged according to the 7et al [et al [According to the discretion of attending oncologists with the consideration of disease condition and the preference of patients, most of these recurrent patients  were given salvage IMRT, 6 patients were treated with conventional radiotherapy, 2 with stereotactic radiotherapy, 1 with brachytherapy and 3 radiofrequency ablation, salvage endoscopic nasopharyngectomy were performed for 6 patients according to the procedure previously reported by Chen MY et al . The prel [et al . CisplatAll patients were required to be followed-up every 3-4 months in the first 3 years, every 6 months for 2 additional years, and annually after their salvage treatment. Each follow-up visit included physical examination, indirect nasopharyngoscopy, MRI of the head-and-neck regions, CT of thoracic region and ultrasound or CT of the abdomen.The samples were fixed by 4 % paraformaldehyde containing 2 % sucrose in PBS at 4 °C for overnight and embedded into paraffin using a tissue processor . FFPE sections (3 μm thick) were cut with a rotation microtome .2O2 for 10 min at room temperature. The sections were steam heated for 2.5 min in ethylene diamine tetra acetic acid buffer (PH = 8.0) to retrieve antigen. Subsequent staining was performed with a 50 min incubation period at 37°C with the monoclonal antibodies PD-1 and PD-L1 . Staining intensity was scored by the criteria: 0 as negative or trace, 1 as weak, 2 as moderate and 3 as high. The percentage of positive cells (0 - 100 %) was multiplied by the dominant intensity score of staining ranging from 0 to 3. Therefore, the overall semi-quantitative score, that is H-score, was ranged from 0 to 300 .Paraffin sections were rehydrated and steam heated to retrieve antigen under the same conditions as in IHC procedure, and blocked in normal goat serum for 30 min at room temperature. The sections were incubated with primary antibodies against PD-L1 and HIF-1α , followeOptimal cut-off point for PD-L1 H-score was determined by the area under the curve (AUC) of the receiver operating characteristic (ROC) curve at the highest positive likelihood ratio point for OS. PD-L1 expression was dichotomized into two groups (high and low), using a cut-off score of ≥ 190. OS was measured from the diagnosis of local recurrence until confirmed death or the date of last follow-up through April 2016.These statistical analyses were performed with SPSS 16.0. Measurement data were presented as mean ± standard error (S.E.M) Chi square test was used to assess the level of PD-1 and PD-L1 correlated with various clinical parameters such as age, clinical stage, anemia and tumor necrosis before re-therapy. T-test was used to evaluate the association of PD1 / PD-L1 with HB, neutrophil counts and platelet after normality test (Kolmogorov-Smirnov test). Nonparametric Mann-Whitney U test was applied to evaluate the association of PD-1 / PD-L1 with lymphocyte count and neutrophil / lymphocyte ratio. Survival analysis was depicted by Kaplan-Meier method. Univariate analysis and multivariate analysis were performed with log-rank test and Cox regression analysis, respectively. P value < 0.05 was used to denote statistical significant, and all reported P values were two sided. P values were marked as * P < 0.05, ** P <0.01, *** P < 0.001, indicating different level of significance.


In order to be compatible with the use of the PEN substrate, optimized deposition conditions of the Al2O3 film by plasma-enhanced atomic layer deposition (PE-ALD) at a low temperature (100 °C) have been developed without any relevant degradation of the final dielectric performance.Graphene is an ideal candidate for next generation applications as a transparent electrode for electronics on plastic due to its flexibility and the conservation of electrical properties upon deformation. More importantly, its field-effect tunable carrier density, high mobility and saturation velocity make it an appealing choice as a channel material for field-effect transistors (FETs) for several potential applications. As an example, properly designed and scaled graphene FETs (Gr-FETs) can be used for flexible high frequency (RF) electronics or for high sensitivity chemical sensors. Miniaturized and flexible Gr-FET sensors would be highly advantageous for current sensors technology for in vivo and in situ applications. In this paper, we report a wafer-scale processing strategy to fabricate arrays of back-gated Gr-FETs on poly (PEN) substrates. These devices present a large-area graphene channel fully exposed to the external environment, in order to be suitable for sensing applications, and the channel conductivity is efficiently modulated by a buried gate contact under a thin Al The evolution of the processing technologies for soft substrates and the discovery of new materials suitable for bending, stretching or conformably shaping , such asormation –8. These2O3 as a gate dielectric.Thanks to these properties, graphene can be considered for high frequency FETs both on conventional substrates  and on fIn addition, due to the low density of states (DOS) around the Dirac point, the carrier density of graphene is very sensitive to the adsorption of charged/polar species at or near its surface – a peculiarity particularly suitable for chemical/biological sensing. As an example, gas sensors with single molecule sensitivity have been initially demonstrated using high quality, exfoliated graphene from graphite . Conside2 (depending on the ions concentration). However, for real applications, especially for a potential totally flexible device, there is a need of a reference electrode that is an external, rigid and macroscopic element, which represents a relevant drawback. The solid IS-FET [2 or Al2O3 with film thickness in the order of 10 nm, the gate capacitance can reach hundreds or even thousands of nF/cm2, which is still reasonably high for sensing applications. It is clear that the high quality and the scaled thickness of the dielectric film fabricated below the temperature limit of the plastic support is the key point for the final devices performance.Among the various device architectures, Gr-FET-based sensors can represent a great combination between a chemical-to-electrical signal converter and an electrical signal amplifier . In partd IS-FET  (where aA low temperature (100 °C) deposition process to obtain a high quality dielectric film is essential in order to be compatible with common plastic substrates, such as poly (PET) or poly (PEN), which is also the case of our study.2O3 dielectric film with a tight control on the deposited thickness and a high level of conformal coverage. While the thickness control allows easy fabrication of a tens of nanometer thick dielectric film , the conformal coverage is essential to contain the potential local degradation of the dielectric performance. Otherwise, this degradation can take over due to the device topography and the high starting roughness of the plastic substrate.Atomic layer deposition (ALD) represents an optimal method to fabricate a good quality Al2O3 film by depositing using a plasma-enhanced ALD process and exploiting trimethylaluminium (TMA) as the metalorganic chemical precursor and O2 as the co-reagent at an optimal growth temperature of 250 °C. Nevertheless, such a temperature is still high in combination with a plastic substrate such as PEN, for which the glass transition temperature is ≈155 °C. Using ALD it is possible to grow at lower temperatures as reported in the case of plastic coating and gas diffusion barrier fabrication [ALD is essentially a low temperature process. It is possible to obtain a high quality Alrication . However2 as the oxygen source for the Al2O3 synthesis. In particular, we explored the resulting properties obtained by modifying the deposition cycle and enlarging the purging periods between the precursor exposure in order to prevent the precursor mixing and a consequent CVD growth mechanism.In our experiments we developed a 100 °C PE-ALD process using a PE ALD LL reactor by SENTECH Instruments GmbH, starting with trimethylaluminium (TMA) as a metal-organic chemical precursor and OThe developed low temperature (LT) and prolonged cycle process was analyzed on a silicon (100) reference wafer and compared to a standard temperature (ST) PE-ALD growth process. The morphology collected by tapping mode atomic force microscopy (tAFM) for the LT growth  left is 2O3. The dielectric patterning was obtained by depositing the oxide on a silicon wafer masked by a negative profile resist and then removing the resist by an ultrasonic hot chemical bath. This approach, namely the lift-off method, allows a structured layer to be defined without direct chemical/physical etching of the patterned layer. This completely avoids incomplete or over etching, resulting in a step profile which corresponds to the patterned layer thickness. It should be noted that this method is allowed only for the LT process if a standard resist for lift-off process is considered [The LT process thickness was also confirmed (43 ± 1 nm) by the local step height measure reported in nsidered . The 2502O3  of about 4.3 nm. The RMS roughness values obtained for both the LT Al2O3 , left an2O3  is defined by the Al/Al2O3 stack from one side and from the reversibly contacted mercury probe on the other side. A wafer-scale matrix of sampled positions were collected on both the ST and the LT dielectric layers. Dielectric constants of 7.94 ± 0.05 and 7.91 ± 0.05 where extracted for ST and LT dielectrics, respectively, confirming a similar dielectric quality of the two films and an extremely good homogeneity of the result on the wafer scale (standard deviation below 1%). As reported in 2 .We started with a thermally flattened PEN Teonex film purchased from Dupont Teiji Films. PEN is an analogous material to poly (PET), with superior physical and thermal stability (up to 155 °C), which are advantageous properties for flexible technology.The polymer film was reversibly bonded on a Si wafer by mechanical lamination, adopting a double face thermal release tape  and shaped by cutting along the wafer edges. This is essential for proper manageability of the substrate during fabrication and testing.The polymer film, as reported in 2O3 layer, deposited by DC-pulsed RF reactive sputter, assisted by a cooling system able to maintain the sample below 100 °C. This is a relevant precaution in order to prevent polymer degradation which may occur due to the processing, in particular, where a plasma is involved. The tAFM morphology reported in 2O3 barrier integration.The polymer film surface was coated by a 100 nm protective Al200 nm thick aluminum back gate pads have been fabricated by metal sputtering, considering the same metal deposition process previously exploited on Si during the dielectric layer testing, and patterning by a lift-off approach. The metal morphology is reported in 2O3 dielectric film was deposited by the previously analyzed LT ALD process. The surface tAFM morphology reported in A ≈30 nm thick Al2 plasma etching.Graphene transistor channels were fabricated starting from a single layer graphene film grown by CVD on large area copper foils (provided by Graphenea). The graphene membrane was transferred to a large area (100 mm diameter) of the target substrate by a PMMA-assisted wet transfer procedure and patterned by soft ORTOT, between source and drain contacts are also illustrated in Rch(Vg), the source and drain contact resistance, Rc, and the access resistance, Racc, associated with the ungated graphene access regions between the channel and the source and drain contacts (Lacc = 20 µm length per access region) are shown.After patterning the graphene channel, the source and drain contacts were fabricated by lift-off of a 30/120 nm thick Ni/Au double layer, which partially overlaps the edges of the graphene channel. A complete Gr-FET device is shown in the optical microscopy image in The total resistance resulting from the series combination of these contributions can be expressed as:2O3 dielectric deposited by ALD on the flexible substrate was characterized by metal–insulator–metal (MIM) test devices properly manufactured together with the Gr-FETs, into the same wafer. The resulting gate capacitance per unit area is Cg = 2.05 × 10−7 F/cm2. The Al2O3 film thickness of 29.9 nm is known from the growth rate previously determined on the reference substrate. The resulting dielectric constant is 6.9, which is reasonably high considering the low temperature (100 °C) adopted for the dielectric growth and the high roughness of the substrate. As a way of comparison with silicon dioxide, the resulting equivalent oxide thickness (EOT) is 16.8 nm.The capacitance of the AlSeveral arrays of independently biased back-gated Gr-FETs with different channel geometries were fabricated on the wafer scale using the above described process flow. The electrical characterization of more than 50 devices revealed a significant number of early failures. The origin of these failures is out of the scope of this paper and will be the subject of further investigations. Interestingly, working devices showed quite reproducible electrical characteristics.Id vs Vd) at incremental values of the back gate bias (Vg from 0 to 11 V) for a representative Gr-FET device with channel width W = 100 µm, channel length L = 190 µm (area = W × L = 19 × 103 µm2) and an access region length Lacc = 20 µm, defined as the distance between source (drain) and the channel region. All the output characteristics exhibit a linear (ohmic) behavior, with a gradually decreasing slope  in the considered gate bias range.Id vs Vg for a fixed drain bias (Vd = 0.1 V). It shows the typical ambipolar behavior for a graphene channel with a current minimum at the neutrality (or Dirac) point (VNP) at 9.0 V. The neutrality point is significantly shifted at high positive bias with respect to the expected ideal value calculated considering the difference between Al and neutral graphene workfunctions . This positive shift is a clear indication of graphene p-type doping, as estimated by p = Cg/q ≈ 1.2 × 1013 cm−2 [q is the electron charge. This doping can be ascribed to the effect of the chemical (PMMA) residues which normally persist after the graphene transfer. It is worth noting that due to the constrains imposed by the use of a PEN substrate, it is not possible to perform thermal annealing processes (in vacuum or Ar/H2 ambient at temperatures from 300 to 400 °C) or stronger chemical treatments typically used to remove polymeric residues after graphene transfer on standard substrates. Furthermore, a certain doping can be attributed to the electrostatic effect of fixed or trapped charges in the gate oxide when deposited at low temperature.013 cm−2 , where qIg vs Vg) collected simultaneously to the transfer characteristic measurement, showing only a negligible current flow (less than nA) in the whole back gate bias range. The transfer conductance gm of the Gr-FET reported in Id vs Vg characteristic by the formula:The insert of h = 476 cm2·V−1·s−1 and µe = 204 cm2·V−1·s−1 using the formula:From the peak transfer conductance, we calculated the hole and electron field effect mobility, µ4 µm2), the high roughness due to the plastic substrate and the unintentional doping of graphene. In particular, it is expected that the polycrystalline nature of CVD graphene has a direct effect on the current transport in a large area channel. In addition to these natural defects originating from CVD growth, the graphene membrane is subjected to significant strain if transferred to a rough surface while a certain amount of mechanical defects, such as cracks or folding is expected by the transfer procedure itself, especially onto a structured surface. Besides these macroscopic defects, nanoscale scattering mechanisms [Rc and the access resistance Racc) are still included. A more refined calculation of the graphene field effect mobility would include the elimination of the series resistance contributions in order to extract the intrinsic transfer conductance and the related mobility.These field effect mobility values are reasonably high, considering the channel area  and local back-gate on flexible PEN substrates. Particular attention was paid to the fabrication of a thin Al2O3 dielectric film at low temperature. In particular, a low temperature (100 °C) PE-ALD process was optimized and properly tested on a standard substrate (Si) in comparison with a standard PE-ALD deposition. This revealed a material with good morphology, reasonable growth per cycle and comparable dielectric performance. The optimized dielectric material deposition was exploited in order to fabricate back-gated Gr-FETs directly on a PEN substrate, with a gate oxide thickness of 30 nm. Electrical characterization of the Gr-FET devices is reported in order to evaluate key electrical parameters such as the transfer conductance, graphene doping and electron and hole mobility. The fabricated devices will represent the platform for the implementation of solid IS-FETs that can be part of a fully flexible, integrated system for sensing and signal processing.In conclusion, we reported recent advances in the fabrication of Gr-FET with large channel areas (≈10


Although the great potential of droplet based microfluidic technologies for routine applications in industry and academia has been successfully demonstrated over the past years, its inherent potential is not fully exploited till now. Especially regarding to the droplet generation reproducibility and stability, two pivotally important parameters for successful applications, there is still a need for improvement. This is even more considerable when droplets are created to investigate tissue fragments or cell cultures (e.g. suspended cells or 3D cell cultures) over days or even weeks. In this study we present microfluidic chips composed of a plasma coated polymer, which allow surfactants‐free, highly reproducible and stable droplet generation from fluids like cell culture media. We demonstrate how different microfluidic designs and different flow rates (and flow rate ratios) affect the reproducibility of the droplet generation process and display the applicability for a wide variety of bio(techno)logically relevant media. The reThis broad range of biological applications is associated with the use of highly diverse culture media possessing different properties regarding e.g. viscosity and surface energy. As culture medium components like proteins or salts significantly determine the surface energy it is obvious that the droplet generation process as well as the droplet behavior inside the micro channels highly depends on the concentration of these components. In this study we concentrate on the characterization of the droplet generation process depending on the kind of aqueous (culture) medium. The investigations focus on the droplet generation micro‐system (DMS) which is of pivotal importance for a reproducible droplet generation process and thus for future reliable assay results. Two key parameters of the DMS have to be tailored in order to achieve a high quality droplet generation: (i) provide perfect geometrical conditions for the reproducible generation of droplets from the aqueous phase and (ii) reduce the adhesion of the aqueous phase to the channel walls past the droplet generation zone.In the first part of this study the reproducibility of the droplet generation from cell culture medium as the disperse (aqueous) phase as a function of the DMS design and the applied flow rates was assessed 22.1Each DMS was assembled from two polycarbonate (PC) plates , the droplet generation micro‐system DMS Fig. A and 1B,s/s had to be introduced. This factor represents the ratio of the droplet length average sD,SP, calculated from the sample volume delivered by the syringes pump (SP) and divided by the number of generated droplets nmax, and the droplet length average sD,OD determined by the optical detection Ts/ss/s was used to calculate the length sTD,nOD of the non‐deformed droplets:In Eq. TD,nOD the exact droplet volumes were calculated. Subsequently, the volume of each individual droplet was determined employing this correction factor that is specific for each experiment. All processes including droplet generation, droplet detection and all calculations were performed using a home made C++‐procedure.From this corrected s2.22F4 as precursor 4F8, Linde, Pullach, Germany, 4.8) was used as a mixture with argon (5.0) containing 5% (v/v) c‐C4F8. The discharge was powered by a TIGRES V20‐901.Atmospheric‐pressure plasma‐activated chemical vapor deposition (PA‐CVD) of perfluorinated amorphous carbon films, then termed a‐C:F in analogy with hydrogenated “diamond‐like” carbon films, a‐C:H, was achieved using a “corona”‐activated CVD process with tetrafluoroethylene C2.32.3.1a =  0 .79 μm +/− 0.05 μm before and after the plasma coating procedure.The surface free energy (including polar and dispersive components) was determined employing the OWRK approach . Briefly, contact angles for the four fluids deionized water, formamide, ethyleneglycol (predominantly polar) and diiodomethane (disperse) were recorded using the OCA System . The investigations were performed on milled polycarbonate plates with a roughness of S2.3.22, resolution: 512 × 512 pixels).Part of the plasma film was removed from a plasma coated glass slide with a scalpel. The thickness of the film was determined at this artificially created edge employing the NanoWizard AFM  fetal calf serum and 0.01% (w/v) phenol red). All experiments were performed with PFD as continuous hydrophobic phase.Experiments with five DMS designs and various flow rate sets were performed to investigate their influence on both, the droplet volume and the droplet volume reproducibility. All deviations were evaluated using the coefficient of variation (CV). The experiments were performed using Dulbecco's Modified Eagle's Medium  of 10, 5 and 2.5 (Table 2).Prior to the experiment the DMS was intensively rinsed with PFD . The experiments were performed with PFD flow rates QTo investigate the droplet generation reproducibility in terms of the flow rate, various experiments with nine different flow rate combinations were performed. These nine experiments representing the first run started with the flow rate combination 250/25 and ended up with the flow rate combination 1000/400 (Table intra‐run‐deviation (standard deviation for each experiment of a single run),inter‐run‐deviation (standard deviation only for the 5/1 ratio experiments of the first and the second run as well as for the third and the 4th run),total inter‐run‐deviation (standard deviation for Series 1 and Series 2 experiments with the same flow rate combination).To investigate the long term behavior of the DMS the first run and the second run experiments were repeated as third and 4th run using the same DMS. The delay between Series 1 and Series 2 was ∼48 h. To evaluate the droplet generation reproducibility three deviation types were defined (Table 2.5In order to estimate the applicability of the coating for bio(techno)logically and biomedically relevant applications, various challenging aqueous media were used for droplet generation employing the 2D 90° DMS . These media comprise anticoagulated blood products , Iscove's modified dulbecco's medium . Furthermore, the surface energy decreased to 10.6 mN/m . The coated surfaces also displayed oleophobic properties (limited wettability with the not‐fluorinated tetradecane) but they highly supported the adhesion of fluorinated oils like PFD (PFD contact angle of 0°).The uncoated PC surface displayed a water contact angle of 88.3° ± 1.4° and a surface tension of 40.4 mN/m (39.6 mN/m dispersive and 0.7 mN/m polar components). After plasma coating with the precursor c‐CThe film thickness was estimated to be in the range of ∼140 nm, since AFM measurements on the identical coatings deposited on glass revealed a film thickness of 143 nm Fig. .3.23.2.1c/Qd: 500/100 and 1000/200. Being given a constant Qc and Qc/Qd ratio, the volumes of the droplets are highly dependent on the design of the DMS . This leads to the formation of a thin PFD film on the surface of the channel walls, which prevents the droplets from contacting the channel surface In the case of the junction DMS the injected PFD pushed the sample fluid against the PFD film being on the channel surface. The resulting pressure led to interactions between the involved interfaces. In the case of 90° DMS the PFD injection led to the largest pressure whereas in the case of 45° DMS there was a pressure component in z‐direction leading to a faster and more reproducible droplet generation Fig. B. These 3.3The influence of the flow rates on the droplet generation process has been described in the literature 3.3.1d a proportional increase in Qc resulted in a decrease of the droplet volume. Furthermore, a proportional increase of Qd at a constant Qc led to the formation of droplets with an increased volume. Within the investigated Qc/Qd range the droplet volume reduction with a decreasing Qc was logarithmic while there was a linear increase in the droplet volume with rising Qd values. These correlations are known from the literature and could be confirmed Given a constant Q3.3.2A strong correlation of the reproducibility of the droplet generation process with the flow rates is displayed by the experimental results. In the case of increasing the flow rate of the disperse phase the reproducibility increased. In the case of increasing the flow rate of the continuous phase the reproducibility also increased, except for the 3D DMS, which behaved reversely. For the 2D 45° DMS there was no clear correlation regarding the reproducibility of the flow rates of both phases.When the flow rate of the continuous phase was increased, the flow velocity rose and consequently the pressure increased. As a result the shear forces that were imposed onto the disperse phase also increased. As a consequence, there was a stronger constriction of the connection between the developing droplet and the disperse phase. The increased force of the continuous phase on the developing droplet obviously caused a faster  and more reproducible droplet generation.When the flow rate of the disperse phase was increased, the flow velocity rose and consequently the injection pressure, too. This also reduced the influence of the viscosity and the interfacial tension, which significantly affected the droplet generation reproducibility. In general, increasing flow rates of the continuous or the disperse phase increased the dynamics of the droplet generation and thus the reproducibility.c and for 3D DMS at low Qc. The reasons for this unusual behaviour of the 3D DMS shall be discussed following. During the droplet generation with the 3D DMS (notably at the flow rate Qc of 1000 μL/min and the Qc/Qd ratios of 5 and 10) it was observed, that the droplet diameter was smaller than the diameter of the microfluidic channel. Consequently, due to the missing stabilization that is normally exerted to the droplet by the wall of the microfluidic channel the droplet generation was inconstant (higher degree of freedom) causing a decreasing reproducibility.Comparing the results of the 2D 90° DMS Fig. A with thCompared to the 90° DMS in the 3D DMS there is a distinct downstream force component caused by the flow of the continuous phase PFD. The horizontal force component pushes the developing droplet downstream. As the vertical component of the PFD flow is smaller than in the 2D 90° DMS, the squeezing zone will be influenced by the pushed developing droplet whereas the influence of the vertical PFD force component becomes smaller. Thus, the droplet pinching–off will be more influenced by stretching the squeezing zone caused by the pushed droplet than by the squeezing effect caused by the vertical component of the PFD flow.Furthermore, in the case of 2D 90° DMS the PFD is guided from the two side channels directly into the main channel. In the case of the 3D DMS the PFD also comes from two side channels but it will be spatially distributed into an annular gap surrounding the main channel like a funnel. The abrupt junction between the side channels (arranged in 2D) and the annular gap (3D) causes undefined flow conditions leading to flow gradients with irregular flow rates. These irregular flow rates also influence the droplet generation.3.4In addition to the DMEM medium further fluidic samples Table  were subAlmost all of the investigated application relevant media could be segmented over a period of 8 h with a high quality, indicating that the DMS coating was stable over that period. However, whole blood  and human plasma slightly reduced the quality of the droplet generation process. For whole blood a perfect droplet generation was observed initially. After a few minutes the whole blood non‐cyclically interacted with the channel wall downstream the droplet generation zone. This adhesion of droplets was predominantly observed at sites at which the lowest thickness of the PFD film was expected (compare chapter 3.2). At the sites of the reduced PFD film thickness the probability of an adhesion of medium components to the channel surface obviously increases. The adhesion induced changes of the surface properties were irreversible and resulted in a decreased reproducibility of the droplet generation process.Varying the flow rates affected the droplet adhesion significantly. At flow rates of 250/25 the adhesion tendency increased while it was almost completely inhibited when the flow rates were raised to 500/100. This may be attributed to the short time period in which an individual droplet may interact with the surface.The exact reason for the high adhesion potential of blood to the channel walls could not be fully elucidated, yet. Since similar effects were observed for both anticoagulated whole blood and plasma (whole blood without cells) it is highly unlikely, that the blood cells play a major role in the changes at the interface . Consequently, it is more likely that coagulation factors interfere with the channel surface, which mask the surface and thus change its physico‐chemical properties. The adhesion would likely lead to an initiation of the coagulation cascade, which was suppressed by the added citrate in these experiments . The hypothesis that coagulation factors may interfere with the droplet generation is further supported by the observation, that media  containing serum  supported a high quality droplet generation process. However, further research on the adhesion of blood cells and coagulation factors will be performed in order to support this hypothesis.+ and OH− ions. We also suspect that the coating is irreversibly damaged since even after rinsing the system with deionized water and PFD the droplet generation process of DMEM was severely affected. The detailed elucidation of how the high H+ and OH− concentrations interfere with the coating (detachment or modification) is the subject of ongoing research.Even more dramatic, negative effects on the droplet generation process were observed when media with extreme pH values were employed. After the generation of a few initial droplets, media with a pH 10 and pH 2 could not be segmented in the DMS but only at the transition zone from the DMS to the PTFE tubing. Since this is a phenomenon usually only observed for uncoated DMS we hypothesize that the coating is not stable against high concentrations of H44F8 precursor is a perfectly suited technology for equipping the surface of microfluidic systems with the hydrophobicity that supports a stable droplet generation process. Consequently, it is possible to perform the droplet generation process without surfactants, which may negatively interfere with the bio(techno)logical applications. By choosing an appropriate DMS design and adjusting the flow rates, the droplet volumes could be tuned in a range of ∼435 nL to ∼1065 nL.A plasma coating of PC materials with the c‐COne of the most relevant parameters of the DMS was its reproducibility, which was dependent on the DMS design and the flow rates. Independent of the design all DMS revealed an excellent reproducibility for short term applications. Furthermore, a high reproducibility for long term applications was observed for high flow rates. These high flow rates of 1000 μL/min resulted in a higher dynamic of the droplet generation with a low variation in droplet volume and are thus preferable. A higher dimensionality of the PFD injection led to an improved reproducibility of the droplet generation process. Similarly, the 2D injection at an angle of 90° resulted in a superior reproducibility as compared to an injection at 45°. Consequently, the 2D 90°DMS is a promising DMS design for a reproducible droplet generation process. Further research will be focused on the development and characterization of the 3D DMS since it is highly likely, that this combination of the dimensionality and the 90° injection angle may further improve the reproducibility.c flow rate was 2000 μL/min which corresponds at a Qc/Qd ratio of 5 to a droplet generation frequency of 10 s−1. Further increasing of the Qc flow rate caused decreasing distances between the droplets which led to undesired droplet contact and, as we operate without surfactants, to fusion of droplets.The upper limit for the QThe here presented plasma coated DMS were demonstrated to be well applicable for the reproducible generation of droplets from a wide variety of bio(techno)logically relevant media. Even the droplet generation from anticoagulated blood products (like whole blood and plasma) was possible although it was less reproducible due to an increased adhesion of droplets to the channel walls in 2D DMS. However, using the 3D DMS this adhesion should be significantly reduced because of the much more pronounced PFD film between the droplets and the channel surface caused by the 3D flow‐focusing effect. Adhesion phenomena depending on the chip geometry and the used fluids will be investigated further.The results presented in this study are the basis for further development of functional chips and applications with delicate samples like suspensions of (multi)cellular systems or even tissue fragments. Both, polymer as DMS material and DMS preparation by plasma coating are convenient and cost effective. Consequently, it will be possible to offer the systems as single use devices for high‐throughput applications in industry and academia.Droplet based microfluidics has a promising application potential in the field of drug screening for personalized medicine or for pharmaceutical research, to mention only a few. Precondition for all applications is a stable and reproducible droplet generation and manipulation as well as cost‐effective equipment like microfluidic chips. In this study we present microfluidic polymer chips that are capable to generate and manipulate aqueous droplets highly reproducibly and without the use of surfactants. The droplets could be used to cultivate even mammalian cells e.g. as spheroids or tissue fragments. The chips could be manufactured by both precision manufacturing, e.g. milling, and injection molding for use as disposables. Thus, for each application the microfluidic channel structure could be milled and tested before molding a higher number of disposable chips. Each chip consists of two symmetrical chip‐halves that can be coated application‐related before assembly.The authors have declared no conflict of interest.