Drosophila model of tumourigenesis, we demonstrate a role for the AMP Defensin in the control of tumour progression. Our results reveal that Tumour Necrosis Factor mediates exposure of phosphatidylserine (PS), which makes tumour cells selectively sensitive to the action of Defensin remotely secreted from tracheal and fat tissues. Defensin binds tumour cells in PS-enriched areas, provoking cell death and tumour regression. Altogether, our results provide the first in vivo demonstration for a role of an endogenous AMP as an anti-cancer agent, as well as a mechanism that explains tumour cell sensitivity to the action of AMPs.Antimicrobial peptides (AMPs) are small cationic molecules best known as mediators of the innate defence against microbial infection. While in vitro and ex vivo evidence suggest AMPs’ capacity to kill cancer cells, in vivo demonstration of an anti-tumour role of endogenous AMPs is lacking. Using a  Animals have a natural defence system – the immune system – that is needed to fight off disease-causing microbes, known as pathogens. One way the immune system attacks pathogens is by producing small microbe-killing molecules called antimicrobial peptides. These antimicrobial peptides carry a positive charge, which allows them to interact with and disrupt the negatively charged cell surfaces of many microbes. Healthy animal cells do not have these negatively charged components on their cell surface, which means they are invisible to antimicrobial peptides. Studies have reported that antimicrobial peptides can attack cancer cells grown in a dish. However, it was unclear whether they could fight cancer cells in a live animal.Parvy et al. have now addressed this issue by studying tumours in the larvae of fruit flies. Flies with tumours made an antimicrobial peptide called Defensin, which normally helps to fight infections. When Parvy et al. deleted the gene coding for Defensin, less tumour cells were dying and the tumours became bigger. This result indicated that Defensin was protecting the fruit flies from tumours. Examining the tumours under the microscope showed that Defensin protein interacted with the membranes of tumour cells. Defensin was not, however, interacting with healthy cells.Further analysis revealed that a negatively charged component of cell membranes called phosphatidylserine, which normally faces the inside of healthy cells, is exposed to the outer surface of tumour cells. This negatively charged molecule renders cancer cells visible to positively charged Defensin. Importantly, the exposure of the phosphatidylserine is mediated by the fly equivalent of a protein called Tumour Necrosis Factor, a key player in cancer biology. Defensin binding to tumour cells leads to their death.These experiments in the fruit fly highlight key molecular mechanisms that allow antimicrobial peptides to fight cancer cells in a living organism. Because human tumour cells can also expose phosphatidylserine, these latest findings may open up the possibility of a new kind of anti-cancer therapy for patients. Drosophila, where the single TNF homolog Eiger (Egr), produced by tumour-associated macrophages (TAMs), has been shown to drive cell death of neoplastic tumours, generated in larval imaginal discs by the loss of apico-basal complex components such as Disc large 1, Scribble or Lethal giant larvae (disc large 1 Drosophila mutant larvae (40.2dlg hereafter referred to as dlg), activate Toll pathway in the Drosophila fat body in an Egr-dependent manner, and this immune activation is necessary for TNF-dependent tumour cell death  or dlg mutant larvae (defensin in fat bodies of dlg larvae compared to wild-type ones (drosomycin and attacin A), even though data were highly variable amongst biological replicates, while other AMPs were not transcriptionally regulated   also displayed increased defensin expression, confirming def gene induction as a consequence of dlg-driven epithelial transformation rather than whole body dl loss  . Interesopin A1) , and deler types . This prmicrobes . Moreove dl loss . We concdefensin (def) gene using the CRISPR/Cas9 system (sk3def and sk4def)    (Hanson  defsk4) . Survivabacteria   . Further animals . Additioal discs , while idefensin expression by RT-qPCR in various immune tissues of tumour bearing animals. We observed that the fat body, homologue to the mammalian liver and adipose tissue, and the trachea, a network of tubes transporting oxygen to cells that resembles the mammalian vasculature, were the main sources of defensin in these animals . We noticed leaky HA protein expression in the tracheal system , which resulted in significant upregulation of Defensin in the fat body  tagged form of Defensin dlg;imd (E20)dlg;rel   or the g;relE20) . Consistterparts . Altogetdefensin expression and tumour phenotype in dlg mutant larvae where imd expression had been knocked down specifically within tracheal cells . In this setting, defensin expression was significantly reduced in the whole larvae  is a negatively charged phospholipid, normally restricted to the inner leaflet of the cell membrane. However, PS can be exposed on the outer leaflet for example in apoptotic cells, which tags these cells for phagocytosis , and observed a strong decrease in cell surface-exposed PS compared to control tumours   . This in tissues , Def-HA  animals . However tumours .dlg;def and dlg;imd mutants , another member of the same apico-basal complex as dlg, showed significant cell surface PS exposure, which was Egr dependent , allowed to establish a role for the AMP in long-term memory  by tumour cells . ImportaDrosophila. Our data point to a conserved mechanism of tumour control by AMPs, a potent arm of the innate immune system. Importantly, we identify cellular features within tumours, which may be predictive of their sensitivity to be targeted by AMPs. This study provides a new paradigm to decipher the molecular mechanisms influencing anti-tumoural functions of an AMP, which may extend to other non-canonical roles of AMPs, such as in ageing, long-term memory and wound healing. Moreover, together with the new genetic tools allowing targeting of all Drosophila AMPs  on agar/grape juice plates  supplemented with yeast paste. Plates were kept at 25°C until hatching. 200 larvae hatched within a 4 hr time window were collected and transferred to rearing bottles containing 45 mL of fly food to avoid any developmental delay and/or starvation effects due to overcrowding conditions. The tumour phenotype was analysed close to the normal developmental time, i.e. 7 days after hatching. Tumours were dissected and stained for further analysis (see below).sk3def and sk4def mutants were generated using CRISPR/cas9 technology . Male flies (5–7 days old) were pricked in the thorax with a needle dipped in a concentrated pellet of fresh overnight bacterial culture. Infected flies were kept at 29°C. Listeria innocua was cultured in Brain heart infusion medium at 37°C and used at optical density of 200 nm (OD600). Erwinia carotovora carotovora 15 was cultured in Luria-Bertani broth at 29°C and used at OD600 200. Experiments were repeated three times independently and one representative experiment is shown.Survival of Tissues were dissected in Phosphate Buffer Saline (PBS) and fixed for 20 min in 4% formaldehyde. Fat body samples were fixed for 30 min using the same protocol. Tissues were washed three times in PBS containing 1% of Triton-X100 (PBT) and incubated overnight at 4°C with primary antibodies. Tissues were washed five times in PBT and incubated for 2 hr at RT with secondary antibodies and 4’,6-Diamidine-2′-phenylindole dihydrochloride (DAPI). After three washes in PBT, tissues were mounted in Vectashield using Secured-Seal spacers (Thermo Fisher Scientific).Confocal images were captured using a Zeiss 710 or Zeiss 880 with Airyscan confocal microscope and processed with Fiji 2.0.0 or Adobe Photoshop C.S5.1.dlg;def double mutant rescue experiments . All qPCR experiments were carried out on three to seven independent biological replicates (see figure legends for details) and, larvae were sampled from the same bottle used to analyse the tumour phenotype. GraphPad Prism7 software was used for graphical representation and statistical analysis. Primer targets and sequences are presented in key resources table.Total RNA from 7 to 10 whole larvae or tissues collected from 10 to 20 larvae  was extracted using TRIzol according to the manufacturer’s instructions. RNA was treated with Turbo DNA free Kit and RNA concentration and quality were monitored using a Nanodrop (Thermo Fisher Scientific). The same amount of total RNA (1 μg for whole larvae and fat body or 100 ng for trachea) was used to perform the three independent reverse-transcriptions using the High-Capacity cDNA Reverse Transcription Kit. cDNAs were pooled and qPCR was performed using PerfeCTa SYBR green following the manufacturer’s instructions. cDNA amplification was monitored with Applied Biosystems 7500 fast instruments. Serial 10-fold dilutions of an external standard were used to produce a standard curve, and RNA samples were included to control for the absence of DNA contamination. sk3def and sk4def mutants, genomic DNA was extracted from single flies by grinding whole animals with a 200 μL pipette tip containing 50 μL of squishing buffer  followed by incubation at 37°C for 30 min. Proteinase K was heat-inactivated at 95°C for 2 min. 1 μL of genomic DNA was used for PCR amplification using PfuUltra II Fusion DNA Polymerase and Eppendorf Mastercycler Ep Gradient Thermal Cycler.To verify the sequence of Tumours were dissected in PBS and incubated in Annexin V binding buffer containing 5% of Alexa Fluor (488 nm or 568 nm) conjugated Annexin V for 10 min. Tissues were washed quickly in Annexin V binding buffer and fixed in 4% formaldehyde for 20 min. Immunostaining, mounting and imaging were then carried out as described above.We used Fiji 2.0.0 software to quantify Annexin V staining at the tumour surface. Maximal Z-projection of whole tumours were generated and the Colour Threshold tool was then used to detect the red pixels that represented Annexin V staining at the surface of tumours. The hue slider was set to include only the red signal, and the brightness slider was adjusted to exclude any background signal. This area was then selected and measured in pixels.Drosophila hemolymph using two complementary approaches, namely Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) for molecular mass determination and trypsin digestion followed by nanoLiquid Chromatography/Electrospray Ionisation tandem Mass Spectroscopy (nanoLC/ESI MS/MS) for molecular integrity confirmation. Those analyses confirmed the similar mass between natural and synthetic Defensin (respectively provided by Bulet EIRL and Genepep) as well the presence of disulphide bonds within synthetic Def, demonstrating it is the mature peptide (Data not shown).The mature Defensin peptide was synthesised (over 90% purity) and the purity controlled by Genepep using Ultra Performance Liquid Chromatography/Mass Spectrometry. We subsequently compared the integrity of synthetic Defensin with authentic Defensin purified from 2 anaesthesia using Nanoject II (Drummond) and glass capillaries 3.5 inch (Drummond). Larvae were injected with 69 nL of PBS containing 7.5 nmol of synthetic Defensin or 69 nL of PBS only as control. Larvae were then gently transferred into agar/grape juice plates and kept at 25°C. Tumours were dissected 4 hr after injection and stained for visualisation of nuclei and tumour cell death as describe above.For injection experiments, larvae were collected, washed three times in cold sterile PBS and injected on a fly pad upon COAll data are presented as mean ± SD and n values are indicated in the figure legends. Statistical analyses were carried out using GraphPad Prism7 software and only significant differences are indicated in dot-plots. Survival upon infection was analysed using Log-rank test. qPCR data were analysed using unpaired t-test with a two-tailed p value. Tumour volume and tumour cell death comparing two samples were analysed using Mann-Whitney test with a two-tailed p value and the one comparing three genotypes were analysed using One-way ANOVA followed by Turkey’s multiple comparisons test. Tumour volume from injected larvae was analysed using Two-way ANOVA followed by Turkey’s multiple comparisons test and only significant statistical differences between ctrl and Defensin injected larvae for each genotype tested are indicated.http://dx.doi.org/10.5525/gla.researchdata.834.All raw data is available through  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Drosophila" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Utpal Banerjee as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Georg Halder (Reviewer #3).Thank you for submitting your article "The antimicrobial peptide Defensin cooperates with Tumour Necrosis Factor to drive tumour cell death in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Drosophila cancer model. They report that defensin is secreted by the fat body and trachea in response to Toll and Imd signalling and that defensin kills tumour cells by binding to cell surface phosphatidylserine, which become exposed in response to TNF signalling. This is novel and, if true, may provide the foundation of new approaches to understanding cell death and growth control in tumours.This is an interesting manuscript about a topic of wide significance. The authors investigate the role of the antimicrobial peptide defensin in killing tumour cells in a eLife.For a rather radical proposal, the data need to be solid. In general, the results are convincing and carefully controlled but here are a few that are very important to the model but are not as convincing (or at least not as clearly presented) as most of the others. The reviewers' consensus is that the following issues need to be addressed before this paper could be accepted by dlg mutant animals. This figure is hard to interpret. Tumour tissue seems to be marked by anti-Dcp1, a cell death marker, but it's not clear whether this marks the whole tumour. Moreover, the HA-defensin staining is very limited and does not obviously colocalise with Dcp1 or anything else. The idea that defensin binds to tumour tissue is central to the model 4E does not strongly support the idea. To be fair, the other data in Figure 4 are clearer and more convincing, but 4E is a problem. It would be better to show an image of the whole disc first so that we can appreciate the morphology and also how the staining looks over an entire tumour and not only in a small selected region. It would also be good to show the individual channels of 4G and to comment on the amount of non-overlap that is present.1) In Figure 4E they claim to show that humoral defensin is binding to 'transformed imaginal discs' in 2) Aspects of Figure 7, which is central to the conclusion that defensin binds to exposed PS on tumour cells, are also of concern. Panels A purport to show that defensin colocalises with annexin V, a marker of exposed PS, but the images do not show that convincingly. It is clear in A' and A' that there is plenty of non-overlap between the green annexin and red defensin. Much red defensin appears to label annexin-negative tissue. Without clearer data, preferably quantified, it is hard to support the conclusion.egr mutants, which is a secreted protein that activates JNK signalling, it is not clear whether the requirement for JNK activation is in tumour cells and/or other cells. JNK could just as well act non-autonomously and this experiment would distinguish whether their model is correct or not. To further investigate this, the authors should test the effect of defensin overexpression on the efficacy of JNK induced cell death. This might best be done in clones with weak JNK activation, e.g. puckered hypomorphic clones. However, a scenario of double overexpression of defensin with a construct to activate JNK may be sufficient.3) The model that JNK activation is a prerequisite for defensin mediated tumour cell killing is of particular interest, as AMPs lack strong specificity towards killing cancer cells. However, since the experiments were done in 4) It is notable that the data in Figure 7C shows that the statistical confidence of the differences is rather low.5) Both the written description and the diagrams in Figures 1 and 7 of the complex signalling between tumour cells, haemocytes, FB and trachea, involving TNF, Toll, Imd, and defensin, are complex and hard to follow. Of course this is partly because biology is complicated, but a better job could be done. It is important that readers do understand both the anatomical and the signalling aspects of the model. […] For a rather radical proposal, the data need to be solid. In general, the results are convincing and carefully controlled but here are a few that are very important to the model but are not as convincing (or at least not as clearly presented) as most of the others. The reviewers' consensus is that the following issues need to be addressed before this paper could be accepted by eLife.1) In Figure 4E they claim to show that humoral defensin is binding to 'transformed imaginal discs' in dlg mutant animals. This figure is hard to interpret. Tumour tissue seems to be marked by anti-Dcp1, a cell death marker, but it's not clear whether this marks the whole tumour. Moreover, the HA-defensin staining is very limited and does not obviously colocalise with Dcp1 or anything else. The idea that defensin binds to tumour tissue is central to the model 4E does not strongly support the idea. To be fair, the other data in Figure 4 are clearer and more convincing, but 4E is a problem. It would be better to show an image of the whole disc first so that we can appreciate the morphology and also how the staining looks over an entire tumour and not only in a small selected region. It would also be good to show the individual channels of 4G and to comment on the amount of non-overlap that is present.+ve areas on the tumours . For organisational purposes, pictures depicting basal UAS-Def-HA expression patterns and following fat body overexpression in wild type discs  are now in a new Figure 4—figure supplement 1.The goal of Figure 4E was to unambiguously show that none-tumour produced Def (fat body overexpressed Def-HA) binds to tumours but not wild type tissues . As the reviewer pointed out, the assessment of endogenous Def in tumours does supports the selective binding of the AMP to the tumours . However, the reviewer is correct in that old Figure 4E was not clear enough to make the case for Def tumour binding and/or Dcp-1 co-localization. One of the issues is that, in addition to the tumour itself, tumour associated tissues, which are negative for Dcp-1 but produce Defensin (fat body and trachea) are almost always captured in these images. Def expression in such accessory tissues is obviously more robust in the context of Def-HA overexpression . We now provide new and significantly improved images of a whole tumour and zoomed-in areas of tumour tissue, including individual channels . For completeness, we also present quantifications, which confirm enrichment of endogenous Def in Dcp-140.2;egr3/3dlg animals . 40.2;egr3/3dlg tumours don’t expose PS and are refractive to Def killing action . Consistently, they present no detectable staining for Def-HA .Furthermore to unambiguously demonstrate the specificity of ectopically produced Def to bind to tumours exposing PS, we overexpressed Def-HA in the fat body of 2) Aspects of Figure 7, which is central to the conclusion that defensin binds to exposed PS on tumour cells, are also of concern. Panels A purport to show that defensin colocalises with annexin V, a marker of exposed PS, but the images do not show that convincingly. It is clear in A' and A' that there is plenty of non-overlap between the green annexin and red defensin. Much red defensin appears to label annexin-negative tissue. Without clearer data, preferably quantified, it is hard to support the conclusion.+ve versus AnnexinV-ve tumour areas. Moreover, a new experiment showing that Def-HA can’t associate to 40.2;egr3/3dlg tumours also support this conclusion .As for the previous point we now present quantification of Def and AnnexinVco-localization . We also present individual channels for a more clear visualization of Annexin V and Def staining . As noted by the reviewer, there are tumour areas stained with Def and not AnnexinV and vice versa. However, our data from images and quantifications clearly shows an enrichment of Def within AnnexinV3) The model that JNK activation is a prerequisite for defensin mediated tumour cell killing is of particular interest, as AMPs lack strong specificity towards killing cancer cells. However, since the experiments were done in egr mutants, which is a secreted protein that activates JNK signalling, it is not clear whether the requirement for JNK activation is in tumour cells and/or other cells. JNK could just as well act non-autonomously and this experiment would distinguish whether their model is correct or not. To further investigate this, the authors should test the effect of defensin overexpression on the efficacy of JNK induced cell death. This might best be done in clones with weak JNK activation, e.g. puckered hypomorphic clones. However, a scenario of double overexpression of defensin with a construct to activate JNK may be sufficient.This is indeed a very interesting question. However, we would like to point that, our data definitely suggests that Egr and PS exposure, which is dependent on Egr, are a pre-requisite for Def killing of tumour cells. Even though Egr does activate JNK signaling, it may also be doing more than that in these tumours. In order to best address the reviewer’s question following his/her line of thought and with the tools that were available to us to perform experiments within the timeframe of this revision we did the following:wtengrailed-gal4 (eng>hep) and analysed wing discs. This condition, led to mild apoptosis in the wing pouch but was not sufficient to drive detectable PS exposure , we overexpressed wild type JNK. Here, we used exposure . Therefoexposure .”. We overexpressed activated hemipterous using actrotund-gal4 (rn>hep) as acteng>hep led to embryonic lethality. However, actrn>hepdiscs presented a complete apoptosis of the wing pouch, and consistently extremely rare adult escapers were devoid of wings  It is notable that the data in Figure 7C shows that the statistical confidence of the differences is rather low.def expression levels within the dlg animals. Moreover, we have done ANOVA to compare multiple samples in old Figure 7C , which is more stringent than T-test used in other cases in the manuscript where we compared only two sets of RT-QPCR data . Therefore, even * is meaningful. Importantly, we do present several lines of evidence on the functional importance of Egr in the system.This is a fair point. Reasons for this are likely due to the variability in 5) Both the written description and the diagrams in Figures 1 and 7 of the complex signalling between tumour cells, haemocytes, FB and trachea, involving TNF, Toll, Imd, and defensin, are complex and hard to follow. Of course this is partly because biology is complicated, but a better job could be done. It is important that readers do understand both the anatomical and the signalling aspects of the model.We have now simplified the diagrams  and text to focus on the key points addressed in this manuscript.


Oncolytic virotherapy is thought to result in direct virus-induced lytic tumour killing and simultaneous activation of innate and tumour-specific adaptive immune responses. Using a chimeric vesicular stomatitis virus variant VSV-GP, we addressed the direct oncolytic effects and the role of anti-tumour immune induction in the syngeneic mouse lung cancer model LLC1.−/−). Therapeutic efficacy of VSV-GP was assessed in vivo in syngeneic C57BL/6 and athymic nude mice bearing subcutaneous tumours. VSV-GP treatment effects were analysed using bioluminescent imaging (BLI), immunohistochemistry, ELISpot, flow cytometry, multiplex ELISA and Nanostring® assays.To study a tumour system with limited antiviral effects, we generated interferon receptor-deficient cells (LLC1-IFNAR1+ T cells and surviving mice showed little protection from tumour re-challenge, indicating limited therapeutic contribution by the activated immune system.Interferon insensitivity correlated with VSV-GP replication and therapeutic outcome. BLI revealed tumour-to-tumour spread of viral progeny in bilateral tumours. Histological and gene expression analysis confirmed widespread and rapid infection and cell killing within the tumour with activation of innate and adaptive immune-response markers. However, treatment outcome was increased in the absence of CD8These studies present a case for a predominantly lytic treatment effect of VSV-GP in a syngeneic mouse lung cancer model. There is an initial tumour-selective viral replication and tumour cell killing, which subsequently leads to activation of innate and adaptive immunity. The activation of anti-tumour immunity holds the potential to induce long-lasting tumour remissions and immunological memory formation.6The development of oncolytic virotherapy has gained significant momentum in recent years with the clinical approval of the first oncolytic virus  in the western hemisphere9 VSV belongs to the family of Rhabdoviridae10 and its rapid replication cycle and wide host cell range make for a promising therapeutic agent.11 Tumour selectivity of VSV is predominantly based on defects in the antiviral defence capabilities of malignant cells,8 a feature commonly seen in many human malignancies.13 VSV-GP is a chimeric VSV variant with its glycoprotein (G) replaced by the lymphocytic choriomeningitis virus (LCMV) derived glycoprotein (GP). This results in the abrogation of VSV’s neurotoxicity without sacrificing its oncolytic potential as shown in a variety of different preclinical tumour models.17 In addition, pre-existing immunity is absent in the general population and induction of a neutralising antibody response is reduced,18 making systemic delivery possible. The clinical proof-of-concept of successful targeting disseminated lesions after intravenous injection was recently shown with another oncolytic virus.19Variants of vesicular stomatitis virus (VSV) have been thoroughly investigated for their oncolytic potential.21 In immuno-compromised hosts with human tumour xenografts the treatment outcome is directly linked to the lytic activity within the tumour with only innate immune components responding to the viral challenge.22 Conversely, immune-competent models are paramount to assess the immune response in both trajectories—the potential anti-tumour immune activation as well as its virus-countering effects.23 VSV-based virotherapy has been preclinically tested on numerous xenograft and syngeneic models,24 with the latter displaying an effective induction of anti-tumour immunity in a number of VSV-treated cancer models.27 So far, most of these studies in immune-competent hosts point towards a significant immune-response contribution to the overall therapeutic effect of VSV treatment.The overall therapeutic effect of virotherapy is shaped by the interaction between virus, tumour cells and the immune system.For this study we generated a mouse Lewis lung cancer LLC1 made highly permissive for VSV-GP via type 1 interferon receptor knockout. We could demonstrate that resistance to type 1 IFN-mediated antiviral protection in vitro translates to an increased and prolonged intratumoural virus activity in vivo, which resulted in complete remission of established subcutaneous tumours both in immune-competent and -deficient hosts. However, despite significant activation of innate and adaptive immune responses by VSV-GP, their contribution to the overall therapeutic effect was ineffectual in this particular tumour setting. We believe this model can inform on studies of potentially rare clinical instances in which a tumour response to virotherapy is lytic-dominant with little contribution of anti-tumour immunity.2. VSV-GP, VSV-GP-GFP, VSV-GP-Luciferase and VSV-GP-ΔG have been described previously.17 Viruses were propagated and titred on BHK-21 cells (ATCC).LLC1 cells were obtained from ATCC  and maintained in high glucose DMEM  supplemented with 10% heat inactivated FCS, 4mM L-Glutamine , 100 units/ml penicillin and 0.1 mg/ml streptomycin (Gibco) at 37 °C/5% COIfnar1 gene (NM_010508.2) were purchased from GeneCopoeia . The following pairwise target sites were chosen: (1) L: TCCTGAGAATATAGACGTC – R: TGCTCCACTTTAGGGTGTA; (2) L: TGCCTGAATGTCAACATAC – R: TGTGTCCAGTAAAGAGAAT; (3) L: TCTTCGTGGAATGAGGTTG – R: TGGCGGCTTCTTACCTGTG. LLC1 cells were transfected with three TALEN pairs (0.6 µg each) using Trans-IT  following manufacturer’s instructions.Plasmids encoding Transcription Activator-Like Effector Nucleases (TALEN) sequences targeting the murine heterodimeric interferon type I receptor complex Cell suspensions were stained for flow cytometry analysis using the following specific antibodies: anti-IFNAR1 mouse ; APC-conjugated anti-mouse-IgG1 from goat  and sorted into single cell clones using FACS Aria . For quantification of cell surface IFNAR1, selected clones were incubated using the same antibodies and analysed using FACS Canto II (BD Biosciences).4 cells/well were seeded in 24-well plates and treated with 500 U/mL of universal IFN-α A/D  for 16 h or left untreated. Cultures were infected with VSV-GP-GFP at MOI 0.1 for 24 h before assessing fluorescence expression.5 × 104 cells/well were plated in 96-well plates and treated with universal IFN-α A/D (PBL) at different concentrations for 16 h prior virus infection with various VSV-GP concentrations. Seventy-two hours later MTT viability assay was performed as described previously.172 × 101nu/nu mice or C57BL/6JRj mice weighing 16–20 g were obtained from Janvier . A health status certificate was supplied with every mouse delivery. Tumours were implanted by subcutaneous injection of 100 µl of 1 × 106 LLC1-IFNAR1−/− or 5 × 105 LLC1 cells in the flank of syngeneic C57BL/6JRj or athymic Rj:NMRI-Foxn1nu/nu mice. Tumour size was measured twice a week with a calliper and volume was calculated using the formula: length × width² × 0.4. Treatment commenced when tumours reached a size of 0.05 to 0.07 cm3. PBS-based solutions containing 108 TCID50 of virus were used for intratumoural (30 µl) or intravenous (100 µl) injection. Mice were sacrificed when tumour size reached 0.8 cm³ or tumours showed signs of ulcerations. Animals were euthanised by CO2 asphyxiation and cervical dislocation or via short-term isoflurane anaesthesia followed by cervical dislocation. For bilateral tumours 3 × 105 LLC1-IFNAR1−/− cells were injected into the flanks of C57BL/6JRj and Rj:NMRI-Foxn1nu/nu mice. For luciferase imaging, Lumina system was used  as described in ref. 16 For CD8+ T cell depletion, mice were intraperitoneally injected with 100 µg anti-mouse CD8  or anti-IgG2b-a-kIH  antibody. Depletion was repeated on days 0, 2, 6 and 10 post virus treatment. Mice were treated intravenously with 108 TCID50 VSV-GP on days 0, 4 and 8. CD8+ T cells depletion was confirmed by staining against CD3 , CD8  and CD43 . The studies were designed in compliance with ARRIVE guidelines. Detailed additional information is provided in supplementary methods . Six to eight-week-old female athymic Rj:NMRI-Foxn Methods .−/− tumours were processed using the mouse tumour dissociation kit  and the GentleMACS dissociator (Miltenyi Biotec) according to manufacturer’s instructions. Single cell suspension was filtered through 70 µm cell strainer, washed with medium and centrifuged for 10 min at 1600rpm. Approximately 5 × 107 cells were layered on top of Lympholyte-M solution . After density gradient centrifugation, cells were removed from the interphase and washed using PBS. FC receptors were blocked by incubating cells for 20 min at 4 °C with FACS buffer containing FCR block CD16/32 (BD Pharmingen) and rat and hamster serum . Cells were subsequently stained for flow cytometry as described below.Splenocytes were isolated via 40 µm cell strainer. Erythrocytes were lysed, and PBS-washed cells were resuspended in appropriate buffer for flow cytometry or IFNγ ELISpot. LLC1-IFNAR1Cells were stained with the H-2Kb VSV-NP-PE-tetramer (Biomedica) followed by staining for surface markers using the following antibodies: CD45.2-PerCP-Cy5.5 , CD90.2-AF488 , CD8-BV510 , CD14-APC-Cy7 , CD19-APC-Cy7  and CD4-APC-Cy7 . Non-viable cells were stained using LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Thermofisher). Samples were analysed using FACS Canto II (BD Biosciences) and data analysis was performed using the FlowJo software .6 splenocytes were incubated overnight at 37 °C with 5 × 104 LLC1-IFNAR1−/− cells or 10 µg/mL mSurvivin peptides  or VSV-NP peptide  in IFNγ capture antibody pre-coated plates. Development of spots was performed as advised by the manufacturer and enumerated using the ImmunoSpot S6 reader .IFNγ secretion of splenocytes was investigated using the IFNγ ELISpotPLUS kit . Briefly, 2.5 × 10Resected tumours were cut in two halves; one was stored in RNAlater  at 4 °C for NanoString analysis (see below), one was fixed in 4% paraformaldehyde prior to paraffin embedding (FFPE). Three-micron tissue sections were de-waxed with xylene, rehydrated in a graded ethanol series and blocked with 3% hydrogen peroxide. Antigen retrieval was performed for all primary antibodies except VSV-N (no pre-treatment) by heating the sections in Tris-EDTA buffer  for 20 min. Sections were incubated for 1 h at room temperature with the following primary antibodies: VSV-N , cleaved Caspase 3 , CD8a , CD4  or PD-L1 . A biotinylated or HRP-conjugated antibody followed by DAB or Refine Red chromogen was used for detection. Haematoxylin  was applied for counterstaining. Staining was performed on the automated Leica IHC Bond-III™ platform (Leica Biosystems). Microscopy was conducted with a Zeiss AxioImager M2 microscope , slide scans were obtained using a Zeiss AxioScan Z1 scanner. Density of IHC stain positive cells was quantified with the image analysis software Halo 2.1  using the Cytonuclear IHC analysis module.29 Heatmaps of NanoString data were generated using TreeView.30Tumours were homogenised with the SpeedMill PLUS  and RNA was extracted using Phenol:Chloroform:Isoamyl Alcohol (25:24:1)  and MagMAX-96 Total RNA Isolation Kit (Thermo Fisher) following manufacturer’s instructions. Extracted RNA was analysed for differential expression by means of the nCounter PanCancer Immune Profiling Panel and the nCounter FLEX Analysis System . Profiled data were pre-processed following the manufacturer’s recommendations.g for 20 min. Lysates were analysed using the Procartaplex Cytokine & Chemokine 26-Plex Mouse Panel 1 on a Luminex MAGPIX fluorescence imager (Thermo Fisher Scientific) and LEGENDplexTM Mouse Anti-Virus Response Panel (13-plex) (BioLegend) followed by flow cytometry analysis. Cytokine concentrations were normalised to total protein concentration in the lysate measured using the Pierce BCA protein assay (Thermo Fisher Scientific).Resected tumours were snap frozen and homogenised using the SpeedMill PLUS  in 500 µl Procartaplex cell lysis buffer (Thermo Fisher) per 100 mg tissue. Homogenate was centrifuged at 6000 × p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.GraphPad Prism software  was used for statistical analysis. ANOVA test was applied to assess significance levels. Kaplan–Meier survival curves were compared using the Log-rank (Mantel-Cox) test. Data are presented as mean ± SEM or SD as noted. Statistically significant differences were encoded as follows: *32 interferon insensitivity is often lacking in murine models of cancer.33 To generate a tumour system with limited antiviral effect of type I interferon signalling that more closely mimics the condition found in many human cancers, the subunit 1 of the heterodimeric interferon type I receptor complex (IFNAR1) was knocked-out in murine LLC1 lung cancer cells. Consequently, surface expression of IFNAR1 was absent in LLC1-IFNAR1−/− cells compared to parental LLC1 cells   increased from 9 days post treatment in PBS-treated mice to 26 days in VSV-GP i.t. and 37 days in VSV-GP i.v. treated mice. The VSV-GP treatment effect showed a dose dependency in both time-to-progression as well as in overall survival , tumours treated with 107 TCID50 VSV-GP relapsed earlier (day 10–20) while a low dose of 106 TCID50 showed only a partial response and rapid relapse. Median survival increased from 18 days (106 TCID50), to 25 (107 TCID50) and 42 days (108 TCID50 days), respectively, compared to control at 11 days. To address if virus replication is required for the therapeutic effect we treated LLC1-IFNAR1−/− tumours with a replication-deficient VSV-GP variant (VSV-ΔG-GP) via intratumoural or systemic application. These tumours were completely resistant to replication-incompetent VSV-ΔG-GP therapy with no objective response or survival benefit compared to control  Fig. . Althougval Fig. . Comparerol Fig. .Fig. 2Th−/− tumours. As in immune-competent hosts, subcutaneous LLC1 tumours grown in athymic NMRI-nu mice were resistant to intratumoural VSV-GP treatment  of the CD8+ cells .To test whether a CD8lls Fig. . Intraveion Fig. . Triple −/− tumours by VSV-GP therapy  were re-challenged via contralateral subcutaneous injection of parental or IFNAR1-deficient LLC1 cells and monitored for tumour outgrowth. Naïve, age-matched animals developed LLC1 wt or IFNAR1−/− tumours within 8 days. In LLC1-IFNAR1−/− tumour-cured mice, challenge with parental LLC1 tumours resulted in tumour growth equal to naïve mice . As expected, a strong IFNγ response resulted from recognition of the immunodominant epitope of VSV-GP (VSV-NP52–59)39 by the splenocytes of VSV-GP treated mice, which was absent in mock treated or tumour-free naïve mice . The same dynamic pattern was observed in athymic nude mice .We next used a VSV-GP variant expressing firefly luciferase (VSV-GP-Luc) to monitor intratumoural virus activity depending on tumour permissiveness (IFNAR1 wt or deficient) and adaptive immune status (syngeneic C57BL/6J mice vs. athymic nude mice). In immune-competent syngeneic hosts, intratumoural injection of 10ays Fig.  indicatiice Fig. . Howeverurs Fig.  compareddel Fig. . We nextdel Fig. . Remarkadel Fig. .Fig. 4In−/− tumours were resected three and seven days post intravenous VSV-GP treatment. Immunohistochemical analysis confirmed extensive virus infiltration of and replication in tumour tissue and increased induction of apoptosis compared to mock treated tumours , RANTES (CCL5), and IP-10 (CXCL10) and anti-inflammatory cytokines IL-4, IL-10 and IL-13 3 and 7 days post treatment Fig. . Althoug+ cells, cytotoxic cells and T cells, was strongly upregulated over time after VSV-GP administration  and additionally suggests a pronounced immune cell activation. A more comprehensive analysis of the immune signature revealed a differentiated immune response over time in the treated tumours. Despite the knockout of the interferon receptor in these tumours, a strong innate immune response could be observed, increasing up to day 7 post infection . The adaptive immune signature also presented a differentiated upregulation that progressed at least up to day 7 post infection  insertion of a reporter gene (CT26LacZ) resulted in a derivative highly permissive for vaccinia, VSV, and other viruses.33 In contrast to our system, no single gene defect explains the reduced antiviral phenotype in CT26LacZ cells but rather the downregulation of a pool of genes, which are crucial for innate antiviral responses.Many oncolytic viruses display a particular sensitivity towards innate antiviral immunity41 with increased immunogenicity of CT26LacZ cells due to the insertion of a foreign immunodominant antigen (LacZ). An important finding from our study is that despite the VSV-GP treatment-related proinflammatory shift in the tumour microenvironment, the immune system does not appear to mount an effective anti-tumour response. In contrast to the CT26LacZ model, protection of mice with long-term remission after VSV-GP treatment of LLC1-IFNAR1−/− tumours against a tumour re-challenge was minimal. Clinical trials have demonstrated that T cell infiltration and PD-L1 upregulation correlate with an increased response probability to immunotherapy.51 Here, we demonstrate that the viral targeting and lytic processing of LLC1-IFNAR1−/− tumours results in a strong upregulation of immune activation markers such as CD8+ T cells and PD-L1 expression. Moreover, immune-focused transcriptome analysis revealed a significant induction of gene signatures for both innate and adaptive immune responses. Yet, the treatment efficacy of VSV-GP on LLC1-IFNAR1−/− tumours does not diminish in the absence of a functional T cell response. When analysing the specificity of CD8+ T cells, we monitored a sizeable antiviral T cell response yet failed to detect a response against multiple epitopes of survivin, an LLC-associated antigen.35 Even more so, up to 50% of the tumour infiltrating lymphocytes were reactive to a single immune-dominant viral epitope. Compared to TILs, the frequency of antiviral T cells among splenocytes was significantly lower, highlighting the antiviral nature of the strong intratumoural immune activation. It has been well recognised that the immune response triggered by an oncolytic virus can exert effects in both directions: anti-tumour and anti-virus.21 The dominant trajectory of this response depends on a number of factors, such as the immunogenicity of the virus, the (neo-) antigenic composition and antigen presentation machinery of the tumour cell, the release of soluble danger signals and cytokines, and the tumour microenvironment.More importantly, in contrast to LLC1 tumours, CT26 are considered highly immunogenic,+ T cell responses against virally encoded as well as tumour-related antigens in tumour models.9 This immune activation is essential for the VSV treatment effect in certain models, such as B16, as evidenced by therapy failure after CD8+ or NK cell depletion.52 Interestingly, the same authors report that enhancing effector T cell activity by Treg depletion can actually reduce the anti-tumour response by unleashing forces for faster virus clearance, highlighting the delicate balance between antiviral and anti-tumour immunity. Our finding that absence of CD8+ T cells does not decrease the VSV-GP treatment effect on LLC1-IFNAR1−/− tumours runs opposite to the dynamic in B16 tumours. Yet, similar mechanisms might be involved—the difference being that the immune balance is shifted toward antiviral rather than anti-tumour CD8+ T-cell activation in the LLC1-IFNAR1−/− model.As for the immunogenicity of VSV variants, it is well established that VSV induces strong CD853 Cancer vaccine approaches against survivin have been described,36 though due to its self-antigen nature, responses are of low affinity. Other cancer vaccine studies largely reverted to the model antigen chicken ovalbumin (OVA) expressing LLC-OVA tumour cells.54 It is conceivable that the lack of immune-dominant antigens in LLC1 tumours further drives the shift of the VSV-GP treatment-induced CD8+ T cell response towards a predominant antiviral rather than anti-tumour immunity.The LLC1 tumour has been well established as a poorly immunogenic tumour with downregulated MHC presentation and an immunosuppressive mMDSC-rich tumour microenvironment.55 In contrast, we detected a significant induction of predominantly inflammatory cytokines IFN-γ, IL-17a, IL-22, MP-1α, RANTES and IP10. This is in line with reports of VSV treatment on B16 tumours with rapid induction of cytokines with anti-tumour activity.25 However, in our setting with LLC1-IFNAR1−/− tumours these proinflammatory signals are likely facilitators of the dominating antiviral immune response.LLC1 tumours showed a downregulation of inflammatory cytokines such as IL-12, IL-5, IL-10 and TNF-α after a cell-based Reovirus vaccine treatment.−/− tumours is predominantly due to the direct oncolytic activity is further supported by the correlation in magnitude and duration between tumour response and intratumoural virus activity, reported via bioluminescence imaging. In non-responsive LLC1 wt tumours, virus activity could only be detected for the first 24 h post injection of VSV-GP, followed by a sharp drop. Of note, the continued bioluminescence signal in the responsive LLC1-IFNAR1−/− tumours was increased in athymic nude mice compared to C57BL/6J. Although the decreased magnitude of luciferase signal in immune-competent black mice compared to athymic nude mice could in principle be partially explained by differences in skin absorption,56 the kinetic of the luciferase signal was profoundly prolonged in athymic nude mice. This further supports the view that antiviral T cells can actively curb VSV-GP virotherapy.That the treatment effect of VSV-GP on LLC1-IFNAR157 In syngeneic hosts, the systemic effect on non-injected tumour lesions has predominantly been described as and attributed to immune-mediated anamnestic effects.59The bioluminescence approach also revealed that VSV-GP can travel via secondary viremia to distant non-injected tumour sites in a syngeneic tumour model with intact immune surveillance. Previously, tumour-to-tumour spread has preclinically been shown predominantly in the xenograft setting using immune-compromised hosts.61 Naturally, the therapeutic effect in these immune-driven therapeutic responses is often diminished in athymic mice, even though the virus replicates to higher levels.62 A treatment effect that heavily depends on the lytic interaction between virus and tumour is rather the exception in syngeneic tumour models. In our setting, we see a complete abrogation of both efficacy as well as the proinflammatory intratumoural cytokine activation when we apply replication-incompetent VSV-ΔG-GP directly into the tumour, further highlighting the dominance of the lytic effect. We previously demonstrated the strong oncolytic potential of VSV-GP in immune-deficient xenograft models using a number of different human cancer models16 and show here, for the first time, a strong lysis-dominant treatment effect in an immune-competent host. This disconnect between the clearly oncolysis-driven therapeutic effect and the apparently non-contributing immune activation may be relevant for tumours with a very limited repertoire of tumour-associated antigens in which immune-activating interventions may show limited promise. We are aware of a number of model-related limitations and future studies will have to corroborate the relevance of the proposed shift from anti-tumour to antiviral activities. These could include for example testing other tumour derivations such as the well-studied B16 with IFNAR1−/− knockout or the use of oncolytic viruses other than VSV-GP. Together, our results suggest that successful oncolysis does not necessarily induce anti-tumour immunity in select tumour models, despite robust immune activation. An impaired antiviral condition of the target tumour cells provides the permissive environment for this extended lytic activity and this study might have ramifications for rare instances of tumours responding to virotherapy with a lytic pattern and no significant anti-tumour immunity.For virotherapy settings in cases with induction of strong anti-tumour immunity, continued viral replication and spread is often found to be dispensable.Supplementary Material


The purpose of this study is to design a backpack to push the lumbar region forward and confirm the change in the sagittal plane of the spine using radiography when wearing the backpack to present an effective backpack wearing method that can help spinal alignment. Place the question addressed in a broad context and highlight the purpose of the study. A total of 14 adult volunteers participated in the study. The study was carried out on the subjects without carrying a backpack, with a general backpack, and with a backpack designed to push the lumbar region forward. We investigated cervical, thoracic, lumbar, and sacral alignment under these three conditions. Lumbar lordosis showed a significant decrease in the state of wearing a general backpack compared to the case without a backpack, and a significant increase in the state of wearing a backpack designed to push the lumbar region forward rather than a general backpack. In addition, the sacral slope was significantly increased when carrying the backpack designed to push the lumbar region forward, compared to carrying the general backpack. There was a significant correlation between the sacral and lumbar alignment change when wearing the backpack compared to the state without a backpack. The results of this study indicate that wearing a backpack designed to push the lumbar region forward may contribute to the recovery of lumbar lordosis that is reduced when wearing a general backpack. This may be due to an increase in the sacral slope corresponding to the inferior angle of lumbar spine. Backpack is a piece of commonly used carrying equipment, and many studies have recently reported that it may adversely affect the spine . In addiSpinal deformity does not occur in the sagittal plane or frontal plane alone, but rather in a complex form. Most of the major causes of spinal diseases are closely related to the shape of the sagittal plane as well as the alignment of the frontal plane . Much reThere are many factors related to sagittal plane alignment of the spine when the posture deformity is examined. A previous study reports that as the Internet usage rate increased, people spend a lot of time in front of the computer, which caused lumbar lordosis to disappear, and the restoration of lumbar lordosis is important in the restoration of the balance in the sagittal plane of the spine , and it Backpacks commonly used by students and adults have also been reported to contribute to the increase in lumbar and cervical deformity mentioned above. As a result, it has been reported that adults feel a sense of heaviness, fatigue, and even lower back pain due to backpacks . The bacThe purpose of this study is to design a backpack to restore the decrease in lumbar lordosis which negatively affects the spinal balance and confirm the change in the sagittal plane of the spine using radiography to present an effective backpack wearing method that can be beneficial to spinal alignment. A total of 14 adult volunteers participated in the study. Subjects were between 23 and 28 years of age and were fully informed about the study and gave their consent to participate in the study. This study was approved by Gimcheon University Bioethics Committee (GU-201709-HRa-06-02). Subjects were excluded from the study if they had a medical history of spine such as chronic back pain, deformity, spondylolysis, spondylolisthesis, scoliosis, and Scheuermann’s kyphosis.The subjects participated in the study without carrying a backpack, carrying a general backpack, and carrying a backpack designed to push the lumbar region forward. The backpack is a general two strap backpack, 300 mm wide, 450 mm tall, and 100 mm in length front and back, with a Velcro attached to the back of the backpack, where the Styrofoam designed in this study can be attached and detached. The Styrofoam is 300 mm wide, 120 mm long, and 36 mm high, with rounded parts that contact the back to smoothly apply the force pushing the lumbar region forward. If Styrofoam is not attached, it is a general backpack, and if a Styrofoam is attached, it is a backpack designed to push the lumbar region forward. The highest part of the Styrofoam reached the L2 site, and was attached to the Velcro of the back plate.In the previous study, the weight of a backpack was mainly studied in the range of 5%, 10%, and 15% of the weight of the subjects, and it was found that the weight of a backpack more than 10% of the subject’s weight affected the subject’s spine . TherefoRadiographic images were obtained in all three states, without carrying the backpack, with the general backpack, and with the backpack designed to push the lumbar region forward. In order to acquire the lateral projection radiograph of the whole spine of the subject, the teleradiograph method was used by digital X-ray radiography . For the test method, the centerline of the X-ray is positioned between thoracic spine seven–10 according to the subject’s height, and the two images of X-ray in upper and lower directions were stitched to express the whole image of the spine. To minimize distortion of the image caused by X-ray diverging, the focus to detector distance (FID) was set to 90 inches and the external auricular meatus and femur heads were used as reference points in the x-ray field size to include the whole spine of the subject. In order for the weight of the backpack used in this study to be evenly distributed throughout the spine, the subject should stand with his feet spread shoulder-width wide, and the subject was instructed to perform weight-bearing, keeping the knees unbent. In order to minimize the overlap of the shoulder and humerus with the thoracic spine column in the image, the angle between the humerus long axis and the coronal plane forms about 30 degrees by placing the subject’s hand on the support positioned at the shoulder height.Spinal alignment indicators were measured and analyzed using 3D radiography measurement software  based on radiographic images taken in three conditions: Without a backpack, with wearing a general backpack, and with wearing a backpack designed to push the lumbar region forward.Experienced radiologists with more than two years of clinical experience measured cervical, thoracic, lumbar, and sacral alignment. In order to examine the cervical alignment, the lower cervical lordosis, that is, the angle formed by the two tangent lines drawn at the C2 posterior corner and C7 posterior corner were measured. Since thoracic kyphosis angle is less visible because the one–four thoracic spine is hidden from the shoulder, the angle between the fifth thoracic vertebrae superior border and the 12th thoracic vertebrae inferior border was measured. The lumbar lordosis angle was measured between the first lumbar vertebrae superior border and the first sacral vertebrae superior border. The sacral slope angle is also the angle formed between the superior endplate of first sacrum and the horizontal line .Statistical analysis of all collected data was done using the SPSS version 20.0 statistics program for Windows . The general characteristics of the subjects were presented as mean and standard deviation of all variables measured using descriptive statistics. Repeated measures ANOVA was used to compare each spinal alignment in three states. In addition, in order to investigate the correlation between the spinal alignment without wearing a backpack and with wearing a general backpack, and also the correlation between the alignment without wearing a backpack and with wearing a backpack designed to push the lumbar region forward, the bivariate coefficient of correlation was calculated. For the test of the statistical significance, the significance level α was set to 0.05 for all analyses.The subjects were 14 male adults with 24.43 ± 1.50 of age, and their height was 173.64 ± 5.69 cm, and their weight was 73.44 ± 7.92 kg.Radiographs were analyzed to compare the lower cervical alignment in all three conditions: Without the backpack, the general backpack, and the backpack designed to push the lumbar region forward. Repeated measures ANOVA results showed no significant differences between all three conditions .Thoracic kyphosis was measured in the three conditions, and repeated measures ANOVA results showed no significant differences between them .Lumbar lordosis was measured in three conditions and repeated measures ANOVA results showed statistically significant differences. There was a significant decrease in the state of wearing a general backpack compared to the state without carrying a backpack, and a significant increase in the state of wearing a backpack designed to push the lumbar region forward compared to the state of wearing a general backpack .The sacral slope was measured in the three conditions and the repeated measures ANOVA results showed statistically significant differences. The sacral slope increased significantly with the backpack designed to push the lumbar region forward, rather than the case of carrying a general backpack and without a backpack .In this study, the analysis of correlation of the spinal alignment changes between the condition without a backpack and the condition with carrying a general backpack showed a significant correlation in sacral alignment change and lumbar alignment change. Also, the analysis of correlation of the spinal alignment changes between the condition without a backpack and the condition with carrying a backpack designed to push the lumbar region forward showed a significant correlation in sacral alignment change and lumbar alignment change .Recently, the number of patients with spinal alignment deformity is increasing, and it is known that a backpack, the article-carrying equipment commonly used by adults, contributes to the spinal alignment deformity. In particular, it reduces the normal curve of lumbar, which also affects the overall spinal alignment . TherefoPrevious studies have used nonradiating methods to examine spinal alignment. To measure the frontal spinal curve, a scoliometer was used and for the lateral curves, that is, for the measurement of thoracic kyphosis and lumbar lordosis, kyphometer was utilized , or to iIn a previous study, the experiment was conducted with the weight of a backpack at 10% of body weight of the subject to determine the effect of backpack on spinal alignment . The weiFor the measurement of lumbar lordosis, the region between the first lumbar spine superior border and the first sacral spine superior border was measured. In a previous study, a mean lumbar lordosis of a typical Korean was known to be about 49 degrees , and thiThe sacral slope angle showed a significant increase in the state of wearing the backpack designed to push the lumbar region forward than wearing the general backpack. This means that the backpack designed to push the lumbar region forward contributed to the restoration of lumbar lordosis, but this was mainly done at the L5-S1 level. Sacral slope can be thought of as the inferior angle of the lumbar spine because the lower arc of lumbar lordosis is geometrically identical to the sacral slope . As we gThoracic kyphosis is measured between the superior border of the fifth thoracic vertebrae and the inferior border of the 12th thoracic vertebrae, and because the one–four thoracic spine is hidden from the shoulder, radiographic images are based on the fifth thoracic vertebrae. In general, the normal range of thoracic kyphosis is known to be 20–40 degrees. In the results of this study, the kyphosis angle was measured within the range indicated in the previous study. Previous studies have reported that cervical lordosis and lumbar lordosis affected thoracic kyphosis for balance . There aWearing a backpack tends to increase head-on-neck extension, that is, upper cervical lordosis. This is because wearing a backpack causes lumbar flexion and the tilting of the body forward to move the center of gravity (COG) into the BOS, and upper cervical lordosis increases in order to keep the eyes’ view of the subject forward . Upper cIn this study, as a result of analyzing the correlation of spinal alignment change between the state without a backpack and the state with wearing a general backpack, there was a significant correlation between sacral alignment change and lumbar alignment change. Also, as a result of analyzing the correlation of spinal alignment change between the state without a backpack and the state with wearing a backpack designed push the lumbar region forward, there was also a significant correlation between sacral alignment change and lumbar alignment change. In a previous study, sacral slope and lumbar lordosis were reported to be closely associated . The resThe balance of pelvic and lumbar region has a strong correlation with pain, dysfunction, deformity and degenerative diseases ,25, and In conclusion, the results of this study suggest that wearing a backpack designed to push the lumbar region forward may contribute to the restoration of lumbar lordosis which is reduced when a general backpack is worn. It is thought that this change in lumbar alignment may be due to the change in the sacral slope.


In Vivo Resistance to Ceftolozane/Tazobactam in Pseudomonas aeruginosa Arising by AmpC- and Non-AmpC-Mediated Pathways” [In the article titled “athways” , a grantWe thank Morouge Alramadhan for technical support. We also thank the Universidad El Bosque for financial support for whole genome sequencing (Grant no. PCI-2017-9447). This work was supported by the National Institutes of Health (Grant nos. K08-AI113317 (TTT) and K24-AI121296 (CAA)), the Millennium Science Initiative of the Ministry of Economy, Development and Tourism, Government of Chile (JMM), and the Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT), FONDECYT 1171805, Ministry of Education, Government of Chile (JMM).


Cancers of Unknown Primary (CUP) comprise a heterogeneous clinical entity of confirmed metastatic cancer where the primary site of origin is undetectable. It has a poor prognosis with limited treatment options. CUP is historically under-researched; however, understanding its biology has the potential to not only improve treatment and survival by implementation of biomarkers for patient management, but also to greatly contribute to our understanding of carcinogenesis and metastasis across all cancer types. Here we review the current advances in CUP research and explore the debated hypotheses underlying its biology. The evolution of molecular profiling and tissue-of-origin classifiers have the potential to transform the diagnosis, classification and therapeutic management of patients with CUP but robust evidence to support widespread use is lacking. Precision medicine has transformed treatment strategy in known tumour types; in CUP, however, there remains a clinical need for a better understanding of molecular characteristics to establish the potential role of novel or existing therapeutics. The emergence of liquid biopsies as a source of predictive and prognostic biomarkers within known tumour types is gaining rapid ground and this review explores the potential utility of liquid biopsies in CUP. Cancer of Unknown Primary (CUP) is a rare heterogeneous clinical syndrome of metastatic cancers for which the primary site of origin is elusive. It is an under-researched entity, where little is understood of its biology and debate remains as to its true definition and classification. Two predominant theories exist: that CUP tumours are metastatic tumours that have arisen from a small undetectable or regressed primary lesion; or that CUP is a single metastatic entity, with no primary tumour that exists. If the metastatic tumours originated from a primary tumour, be it regressed or dormant, then CUP tumours may be more biologically similar or molecularly traceable to the primary, and therefore may respond to treatments aimed at the primary. This makes CUP a syndrome of distinct tumour types that require site-specific therapy. However, if CUP is a single cancer entity it is hypothesised that a shared molecular signature may exist to explain the aggressive phenotype. This view suggests treating tumours based on primary tumour type will not improve outcomes.1, we will explore how novel trial design and personalised medicine could improve treatment strategies for patients with CUP. We will highlight the rapidly evolving field of liquid biopsies for diagnosis and monitoring and the potential applications for them in CUP.Current treatments for CUP depend on individual clinical presentation; however, most patients have poor prognosis disease with no consensus of ‘optimal’ chemotherapy, lack of robust prognostic and predictive biomarkers, and no access to targeted or immune-therapies. Research within CUP is hampered by the heterogeneity of the condition and there have been few advancements in treatments when compared to other metastatic cancers such as lung. In addition to the recent review of current advances in molecular characterisation and tissue-of-origin (TOO) research within CUP2 Incidence has been decreasing since the early 1990s, which likely reflects improvements in primary cancer site determination. Despite this improvement CUP remains the fifth leading cause of cancer death in the UK, carrying a poor prognosis with a median survival of 6-16 months.3 Patients with CUP have disseminated cancer at presentation where the originating site of the tumour is not readily detectable; this presentation is initially defined as malignancy of unknown origin (MUO). In the UK, MUO represents ~15% of all new cancer diagnoses.4 Patients with MUO undergo appropriate radiological investigations, tumour marker assessment and biopsy, determined by clinical presentation, in search of the cancer’s TOO. For the majority (two thirds) of patients presenting with MUO, these investigations will reveal or be suggestive of the tumour’s primary TOO. For the remaining patients a diagnosis of confirmed CUP is made.5CUP make up 3–5% of total cancer diagnoses worldwide.5 Current European and US guidelines recommend a meticulous, step-wise histopathological approach including assessing tissue morphology and appropriate application of immunohistochemistry (IHC) to not only confirm malignancy, but exclude highly treatable non-carcinomas  (reviewed in5). By definition CUP tumours are therefore carcinomas of unknown primary, with other cancer types excluded , diagnosis is challenging.ded Fig. . The mos6 Patients with unfavourable CUP subtype (~80%) are more likely to have visceral disease, high tumour burden and exhibit poor response to standard chemotherapy. Prognosis is much shorter (6-12 months) than the favourable subtype and, for patients who do not undergo treatment, life expectancy can be as little as 4 weeks.7 Traditionally, patients within the unfavourable group have been treated as a single cancer entity due to shared clinical features, namely a short clinical history, atypical metastatic distribution, aggressive disease and an undetectable primary tumour. Patients with a good performance status and normal Lactate Dehydrogenase (LDH) can be considered for palliative chemotherapy. There is a lack of international consensus guiding optimal chemotherapy selection, but standard treatment for the unfavourable subtype is currently doublet chemotherapy, usually containing platinum and/or taxane, with response rates of 25-40% and median overall survival of 9 months. Multiple phase II studies have found no superior combination cytotoxics, and there is no standard second line treatment.8Patients with confirmed CUP are sub-classified into two subgroups based on histopathological and clinical features: favourable and unfavourable see Fig. . PatientLittle is understood of the disease pathogenesis and the biology explaining the CUP phenotype is debated. Two predominant hypotheses exist: 1) CUP tumours arise from a small, dormant or regressed primary tumour that is undetectable and are therefore a group of distinct primaries with an undetectable primary lesion. 2) No primary tumour exists or will exist, and ‘true’ CUP tumours arise independently to a tumour mass and are biologically distinct from other metastatic tumours.11 It is notable, however, that despite improvements in imaging and histopathological diagnosis, up to 50% of patients with CUP do not have a primary tumour detected at autopsy. Additionally, in cases of occult breast cancer, which represent 0.3–1% of all breast cancer cases, frequently no primary breast lesion is found at diagnostic imaging or pathological review of mastectomy specimens.12The biology underlying early dissemination from a small/regressed primary lesion is not easily explained and research is hampered by the paradox of a lack of primary tissue. Interestingly, in around one quarter of patients with CUP, the primary tumour lesion becomes clinically or radiologically detectable later in the patient’s lifetime. Furthermore, autopsy studies have identified a primary tumour lesion in 50-80% of patients with previously diagnosed CUP; these are predominantly small primary lesions of the pancreas, lung or colon.16Theories of cancer dissemination are debated around known tumour types. The traditional view of metastatic progression is a stepwise process of progressive molecular aberrations initiating carcinogenesis, progressing to autonomous proliferation and invasion with subsequent tumour cell spread at a late stage of tumour growth (termed linear progression). Subsequent metastases are molecularly similar to their primary tumour and metastatic potential appears to be linked to primary tumour size. However, this view has been challenged by pre-clinical and clinical data in many types of cancers including melanoma, breast, pancreatic and small cell lung cancer.17 Breast cancer tumours are felt to have early metastatic potential and disseminated cancer disseminated tumour cells (DTCs) are found in the bone marrow of patients with localised breast cancer; often remaining dormant for many years.19 Mouse models of melanoma have also demonstrated cancer cell dissemination as an early step in primary tumour development, with DTC dormancy being maintained by immune cells during immunosurveillance.20 In other cancer types, dissemination is early and rapid. In mouse models of pancreatic cancer, tumour cells disseminate early and metastatic tumour formation can precede the formation of the primary pancreatic tumour.16 Parallel progression better describes how a small, undetectable primary tumour could lead to aggressive and widespread metastasis, such as that found in patients with CUP.Parallel progression describes dissemination of primary tumour cells as an early event where subsequent clonal evolution of the metastasis is distinctly different to that of the primary tumour.21 It is hypothesised that a complex interplay between the tumour microenvironment, as well as immune, genetic and epigenetic features may make the primary lesion regress or remain dormant,22 although early invasive and metastatic behaviour is justifiably assumed, uncoupling proliferation from dissemination.Spontaneous tumour regression has been reported in several tumour types but is best described in cases of melanoma, where 5% of metastatic cases present with an occult primary.23 A further study, employing genome wide transcriptome analysis, compared 41 CUP tumours with a molecularly predicted TOO to 186 metastatic lesions from 10 known tumour types. The authors describe CUP tumours as more distinct from their predicted primary tumour than the metastatic lesions from their confirmed primary tumour types. CUP tumour transcripts were enriched for DNA damage and repair genes, lacked tumour protein expression and showed more chromosomal instability, when compared to metastases of known tumour types.24 The authors suggest this supports the hypothesis of parallel progression in these cases but it may also be a reflection of the aggressive and advanced nature of the disease. Although these studies suggest some commonality in the biology of CUP they include small numbers of patients. Conversely, in 40 patients with favourable CUP, subtype microRNA signatures were compared to analogous known tumour type and showed no significant differences in expression, suggesting at least within the favourable subtype CUP tumours are biologically similar to their analogous primary.25Finding a unifying molecular ‘CUP signature’ to explain the CUP phenotype remains elusive. Researchers have found considerable mutational heterogeneity between patient’s tumours. A handful of recent studies have elucidated common molecular pathways that are interrupted and may explain the aggressive and early dissemination pattern of CUP tumours. In 17 relapsed CUP tumours aberrations that affected cell cycle arrest, combined with either epigenetic deregulation or cell signalling activation, namely PI3K and MAPK signal transduction pathway alterations, were found consistently across the tumours analysed and the authors proposed a role for novel combinational therapies.26 Further molecular characterisation of larger numbers of CUP tumours would increase our understanding of the heterogeneity of the syndrome and determine if molecular CUP subtypes exist that may respond better to existing or novel treatments. Meanwhile, predicting TOO remains valuable to identify those patients that may benefit from alternative treatments.In the absence of a unique biology that unifies CUP tumours as a single entity, it is most likely that the CUP syndrome is a limitation of primary tumour determination. There remains an unexplained biology behind primary tumour regression, early dissemination, and aggressive metastatic phenotype. It is generally believed that CUP tumours, if a primary tumour of origin can be predicted, will behave and respond to treatments similarly to the predicted tumour type.Although debated, TOO classifiers are seen as a gateway to improving survival and therapeutic options in patients with CUP. Cancer treatment decisions are currently based on histological classification; without a TOO, treatment options are limited and access to newer therapies is difficult. There is an assumption that administering therapeutics based on the predicted primary tumour site will improve response rates and survival in patients with CUP.29 Many of these TOO classifiers have been successfully applied to CUP tumours to aid diagnosis  in poorly differentiated cancers and those requiring a second round of IHC.42 This suggests that in the more difficult cases, as observed in CUP, gene expression profiling could be useful.Molecular methods applied to tumour tissue are able to predict the TOO, using gene expression profiling, gene microarrays, microRNA and DNA methylation analysis. Analysing known primaries generates a tumour-type gene signature database for classifying cancers, and this can predict the primary origin of known tumour types with accuracies of 80-95%.is Table .30–39 Ho44 This is due to a lack of randomised, prospective data demonstrating improved clinical outcomes when treatments are chosen based on the predicted TOO. Only a handful of studies  of cases the gene expression profiling predicted a tumour type that was likely to respond to platinum-doublet chemotherapy, including NSCLC, bladder, breast and ovarian cancer. The other half of the patients were predicted tumour types where empiric platinum-doublet chemotherapy would not be standard of care, including colon, pancreatic and hepatocellular carcinoma. Patients who were predicted to have platinum-responsive tumour types had significantly longer progression-free survival (PFS) and overall survival (OS), compared to those tumour types where platinum chemotherapy is not standard of care  and OS median 17.8 vs. 8.3 months, P = 0.0052; HR 0.37 (95% CI: 0.18–0.76)).38A recent but small prospective phase II trial enrolled 46 patients with confirmed CUP treated with carboplatin, paclitaxel and everolimus. Thirty-eight of these patients were successfully molecularly profiled with a 2000-gene expression microarray. In 50% .39Finally, a recent large but retrospective study predicted the TOO based on DNA methylation profiles of tumour tissue with 99·6% specificity (95% CI 99·5–99·7) and 97·7% sensitivity (96·1–99·2). Of 216 tumours assayed, 31 patients had therapy directed to the predicted TOO. An improved OS of 13.6 months was observed in these patients, compared to 6 months in the 61 patients who received empiric therapy , and without gene expression profiling they would not have been classified as CUP colorectal-subtype. ESMO CUP guidelines include both histopathologically and molecularly confirmed CRC within this favourable clinical subtype, despite not routinely recommending the molecular profiling of CUP tumours.43Importantly, these gene-expression profiling studies have aided the identification of a favourable colorectal-subtype of CUP. Two retrospective studies Table  revealed39 Importantly, these patients could have EGFR and ALK fusion protein and PDL1 testing, where administration of appropriate targeted therapy significantly improves OS.50The evidence base is currently not robust enough to recommend routine molecular TOO predictions for diagnosis in CUP. These studies do highlight a role for identifying subtypes of CUP that may gain clinical improvement with site-directed therapy, as demonstrated for the colorectal subtype of CUP. In addition, a significant proportion of patients with adenocarcinoma CUP are predicted to have lung cancer as their primary tumour type.51 Increasingly, patients are treated with a precision oncology approach; therapy selection is based on molecular characteristics of the tumour and targeted to the biology of the disease process. As our molecular understanding of cancers improves, for example from comprehensive datasets such as TCGA, common molecular characteristics of cancer are discovered and new classifications are born.52 It is predicted 1 in 10 cancer patients might be classified (and possibly treated) differently using molecular classifications of cancer.53 Modern trial designs now reflect the known molecular heterogeneity within cancer types and so-called ‘basket trials’ include patients based on shared molecular drivers irrespective of their tumour type. Patients with CUP could gain huge benefits from such an approach and are the epitome of requiring personalised treatments. To date, no molecular sub-classification for CUP tumours exists and only a limited number of CUP tumours have been molecularly characterised to elucidate a role for targeted therapies.There is a movement away from traditional cancer classification by tissue and organ type alone, with more cancers now being sub-classified not only by histological subtype but also by their molecular characteristics.The discovery of multiple driver mutations has paved the way for novel targeted therapeutics across multiple tumour types. Many of these molecular drivers are not tumour-type specific and are common across several tumour types. Therapies targeting these driver mutations have had a significant impact on response rates and survival in previously poor-prognosis metastatic tumours.59 Significant mutational heterogeneity exists, and mutations found in CUP are found amongst many different cancer types. For example, KRAS mutations are found in 30-50% of colorectal cancers and up to 25% of lung cancers.62 Studies investigating the presence of driver mutations and molecular aberrations in CUP vary widely in their conclusions regarding whether changes are ‘potentially druggable’ (ranging from 15-96%). Only one phase I trial to date has reported early clinical follow-up data of 11 patients with CUP that have been matched to therapies based on molecular aberrations; four of those patients treated had stable disease and one a mixed response.23 Within the literature there are numerous case reports of patients with CUP being treated successfully with targeted therapies  but poor in CRC (0%) and cholangiocarcinoma (12%), despite the majority of cases having the same V600E somatic BRAF mutation. These results exemplify the challenges of predicting response to targeted therapy based on single driver mutations alone.70 A recent study evaluating genetic aberrations in CUP highlighted that the common pathways interrupted may require combinatorial therapies for clinical responses, and this should be considered when designing trials for targeted drugs.23The prognostic or predictive significance of driver mutations are tumour-type specific and therefore their value in CUP is uncertain without a TOO. For example, it is well documented that BRAF mutations are predictive of response to BRAF inhibitor monotherapy in melanoma, but not in colorectal carcinoma.Immunotherapies are emerging as important therapeutics in several cancer types, with promising response rates and in some cases long-term durable responses in the metastatic setting. It would be reasonable to expect that a subset of patients with CUP may respond to immunotherapy. Markers of responsiveness to immunotherapy are under intense research and debate. Those currently under investigation in known tumour types include immune biomarker expression, high tumour mutational burden (TMB), and microsatellite instability (MSI) and mismatch repair deficiency (dMMR).68 Recently published works have demonstrated the efficacy of pembrolizumab in dMMR and MSI-high tumours irrespective of tumour cell origin;72 consequently, pembrolizumab is the first drug to gain pan-cancer FDA approval for use in any MSI-high/dMMR solid tumour. The frequency of high TMB, PDL1 expression and MSI-high amongst CUP tumours has been evaluated by a few studies, which suggest one or more of these biomarkers may be present in up to 28% of cases.73 Determining a potential immunotherapy-responsive subgroup of CUP is hampered by a lack of validated predictive biomarkers and further research is desperately needed. The opening of the Roche Foundation Medicine Phase II global CUP trial (NCT03498521) will make some progress in evaluating the efficacy of novel molecularly-guided therapies, including immunotherapy, in patients with unfavourable CUP and may aid biomarker discovery.There are two case reports describing responses to immunotherapy in patients with CUP; one patient with high expression levels of programmed cell death ligand-1 (PDL1), and the other had confirmed dMMR . This makes direct comparisons of this treatment with site-specific chemotherapy difficult, due to significant treatment overlap.74 There is a significant degree of both inter- and intra-tumour heterogeneity within known tumour types, as well as clonal and sub-clonal variations between metastatic sites in the same patient.75 Tissue biopsies taken from single metastatic sites are less likely to determine the tumour origin by IHC than biopsies taken from the primary tumour,76 and are also unlikely to capture the heterogeneity of the cancer burden, which has direct implications for treatment response and resistance. Tumour biopsies are invasive, sometimes anatomically difficult to obtain and the procedure is not without risk of harm to the patient; they are therefore usually performed only once - at the point of diagnosis. However, our emerging understanding of treatment resistance mechanisms, especially with targeted agents, has meant it is increasingly important for repeated evaluation of the tumour at progression to better understand tumour evolution and guide appropriate therapy.A tumour biopsy, usually of the primary tumour, is currently the ‘gold standard’ for tumour evaluation and diagnosis, and thus determination of initial treatment. Unfortunately there are inherent limitations in both acquisition and interpretation of tumour biopsies, together with uncertainty over whether a single tumour biopsy, which is often very small, may be representative of the entire tumour landscape.77 Liquid biopsies , tumour microRNAs (miRNAs) and platelet-derived tumour mRNA, as well as analysis of DNA, RNAs and protein expression from individual circulating tumour cells (CTCs) .A wealth of recent advances in liquid biopsies have shown them to be clinically useful for tumour biomarker detection in known tumour types; for example, blood-based EGFR mutation testing in NSCLC is used within the clinic to detect circulating tumour DNA (ctDNA) harbouring common EGFR mutations that predict response/resistance to EGFR inhibitors.68 This indicates the feasibility of using ctDNA to not only profile tumour but also to track resistance mechanisms whilst on therapy. In known tumour types we know that ctDNA can be used to detect specific cancer mutations and track response and resistance to therapies and predict relapse prior to radiological progression.85 For patients with CUP, there is a clinical need for early non-invasive indicators of treatment response or resistance, such that futile treatments can be halted and detrimental side effects can be avoided.To date, only one group has used patient cfDNA to profile the mutational landscape of CUP tumours. Evaluating 442 patients with targeted sequencing of 54-70 genes, they found 80% of patients harboured at least one genetic alteration. The mutational profile was comparable to studies profiling CUP tumour tissue, and demonstrated heterogeneity similar to that seen across all tumour types. In one patient, serial on-treatment ctDNA samples demonstrated dynamic changes in the mutation profile that were consistent with response and then resistance to therapy.86 It is therefore apparent that mutational profiling alone can only stratify small numbers of patients to possible therapeutic targets, and only a proportion of these patients will gain benefit from these treatments. Epigenetic alterations, such as DNA methylation, histone modification and microRNA-mediated gene regulation, assert transcriptional control and regulate gene expression without genetic modification. Emerging evidence suggests these alterations may provide a more useful analysis of DNA and overcome the uncertainty of mutational analysis, as they better reflect gene expression and therefore function. It is thought that epigenetic modifications play a key role in tumour initiation and cancer predisposition, and epigenetic changes are tumour and tissue-type specific.87 Epigenetic biomarkers are already in use in the clinical setting and potentially useful in predicting TOO from CUP tumour tissue.39 Several groups have demonstrated that epigenetic changes in ctDNA can be a useful biomarker for cancer prognosis, prediction of treatment response and post-operative detection of minimal residual disease (reviewed in ref. 88).One limitation of cfDNA mutational analysis is determining the biological significance and pathogenicity of mutation results, given that mutations can be found in non-malignant/healthy individuals.74 Notably, the majority of CTCs are undergoing some stage of apoptosis in the bloodstream, which contributes to ctDNA levels.90 The exact mechanisms underlying haematogenous dissemination of CTCs from the primary and/or metastatic tumour are not fully understood, but this may be a passive process of tumour shedding or through active intravasation, including epithelial-to-mesenchymal transition (EMT), cell-to-cell cooperation and vasculogenic mimicry.92 Epithelial cells undergoing EMT lose their characteristic cell-to-cell interactions, become mobile and gain invasive properties,93 and CTCs that have undergone EMT may reverse this process during the process of metastasis formation, though only a very small proportion of CTCs subsequently propagate a distant metastasis.74CTCs are primary tumour or metastatic cells that have entered the peripheral circulation as singular cells or as cell clusters that are termed circulating tumour microemboli (CTM).94 but they are found in many cancer types. The number of CTCs present varies between cancer type and patient, and their observed prevalence also depends on the technology platform used for their detection. CTCs are promising as a liquid biopsy and various clinical studies have confirmed the negative prognostic significance of CTCs on survival in numerous tumour types, most notably in breast, colorectal and prostate cancer (reviewed in95).CTCs are rarely found in healthy normal volunteers or in patients with benign disease,97 The most recent publication evaluated 10 CUP patients and found CTCs in 50% of cases (median CTC count: 31). CTCs were more frequent in treatment-naive patients and in one patient CTC levels decreased with treatment.96 Two of the studies used the CellSearch® platform; this is the only FDA-approved CTC technology that detects EpCam-positive CTCs. It therefore fails to detect CTCs that have down-regulated this epithelial marker, for example during invasion whilst undergoing epithelial-to-mesenchymal transition. New marker-independent approaches, such as the RareCyte and HDSCA which were developed on the principle that ‘no cell is left behind’, should improve CTC assay sensitivity because all nucleated cells in the sample are captured onto slides, allowing staining with a flexible panel of markers and physical picking of single cells for molecular characterisation.98 Application of these approaches to CUP should improve the recovery and detection of CUP CTCs that may have lost their epithelial markers by nature of the disease.Patients with CUP often have a high tumour burden and aggressive clinical course. It would be reasonable to expect that these patients may have a relatively high frequency of CTCs, reflecting their metastatic capability, as well as high ctDNA levels, reflecting rapid tumour cell proliferation and death. A handful of studies have evaluated CTCs in a small number of patients with CUP Table .68,94,96100 Small-cell lung cancer CTC-derived explants faithfully reflected tumour responses to chemotherapy, indicating that these may be ideal models for understanding tumour resistance mechanisms.101 Therefore, these CTC-derived explant (CDX) models may allow for novel drug testing and discovery,99 and may serve as an informative way to investigate tumour heterogeneity. If successfully applied to CUP, this could reveal insights into the biology and heterogeneity of CUP tumours and allow for rational drug testing to improve therapeutic options for patients with CUP. Of note, a recent publication described a similar approach using tumour tissue-derived cell culture and patient-derived explants (PDX) from a patient with neuroendocrine CUP. Successful pathway analysis and drug trial analysis in these models identified AKT pathway activation and response to an AKT inhibitor in the absence of actionable mutations.102Isolation of CTCs has the further advantage of allowing functional analysis of the single tumour cell, including whole-genome sequencing, gene expression profiling, epigenetic studies, and proteomics. In vivo models from cultured CTCs have been established from patients with advanced cancer (lung and melanoma) and exhibiting high CTC counts.109 Many of these studies have focussed on early detection of disease in search of a non-invasive cancer screening tool. However, we know that ctDNA mutation detection is more sensitive in advanced tumours,110 reflecting higher tumour burden and increased circulating genetic material from the tumour. The TOO studies report sensitivities of 60-98% dependent on tumour type and molecular approach.107 These studies demonstrate that liquid biopsy molecular characterisation is feasible, especially in the metastatic setting. Predicting the TOO via molecular profiling is a debated topic within CUP; these methods have yet to be applied to CUP tumours and will likely be more challenging. CUP tumours are by definition tumours without histological definition and therefore more difficult to molecularly classify. In addition, multiple metastatic deposits commonly observed in patients with CUP are likely to increase ctDNA heterogeneity. CUP tumours may have lost obvious ‘molecular signposts’ to the TOO, which may make CTC phenotyping unlikely to be helpful. Although research in this area is very exciting, utility of any of these methods remains to be seen with regards to determining the TOO in patients with CUP.A single blood test that can detect and diagnose the cancer type is seen as the ‘holy grail’ of cancer diagnostics, and rapid advancements in the field of liquid biopsies is bringing this closer to reality. Numerous recent proof-of-concept studies in common tumour types claim sensitive and specific cancer detection with liquid biopsies Table . MethodsThe limited studies evaluating ctDNA and CTCs in CUP demonstrate its feasibility, but only with further research will we establish if it is clinically useful and applicable. TOO classifiers using liquid biopsies show exciting potential for diagnosis within known tumour types but have yet to be applied to patients with CUP. It may be invaluable in streamlining the diagnostic pathway for patients with CUP and MUO, as well as the rapid diagnosis of potentially curable cancers where diagnosis is time critical, for example germ cell carcinoma and lymphoma. The most useful liquid biopsies will most likely be multi-analyte; not only predicting TOO, but also providing better prognostication, detecting actionable mutations and markers of response to chemotherapy and/or immunotherapy, and enabling early response or resistance monitoring Fig. .Fig. 3PrCUP remains an unmet clinical and research need. A lack of validated prognostic and predictive biomarkers means the scientific rationale for existing and novel therapy selection is lacking, with therapeutics in clinical development remaining out of reach. A small proportion of patients within the favourable subset respond well to treatments of their analogous tumour type, and achieve meaningful improved survival. However, the majority of patients comprise the unfavourable subtype, and are currently treated with combination chemotherapy based on clinical and radiological characteristics and limited histopathological information. This ‘one-size-fits-all’ approach does not reflect the heterogeneity of these tumours, and emphasises the need for better treatment stratification.Within CUP, as well as other tumour types, there remains an important need to find novel biomarkers, ideally liquid-based, that better predict response to chemotherapy, immunotherapy and targeted therapy. There is also a need for robust evidence before implementation of liquid biopsies in the clinical management of CUP, and it is therefore necessary for well-designed and adequately powered clinical trials to prospectively incorporate liquid biomarker discovery into the trial design (Supplementary Box Supplementary Table S1 - Summary of published case studies of patients with CUP treated with targeted and novel therapiesSupplementary Box 1