Granulomatous dermatitis (GD) is the most common among a variety of skin reactions that may occur in the varicella‐zoster virus (VZV) reactivation area. It is thought that the formation of granulomas may be the result of a delayed hypersensitivity reaction to viral envelope glycoproteins. Immune checkpoint inhibitors (ICIs), such as nivolumab stimulate T cells and promote hypersensitivity reactions, leading to the formation of granulomas in VZV wrapping proteins, thus triggering VZV‐GD. Few cases of the use of ICIs in patients diagnosed with VZV‐GD have been reported in the literature. Here, we report the clinical case of a patient with metastatic lung cancer which was treated with nivolumab who subsequently developed VZV‐GD. Accurate clinical diagnosis and prompt treatment with antiviral agents have resulted in a complete resolution of the clinical picture.Treatment with ICIs may result in VZV reactivation.Accurate differential diagnosis and early treatment led to the resolution of VZV‐GD.Few cases of ICI and VZV reactivation have been reported in the literature.Full and timely resolution of VZV‐GD allowed the continuation of ICI treatment. Varicella‐zoster virus (VZV‐GD) is a cutaneous reaction that can appear in the area in which a reactivation of the VZV takes place. It may occur during treatment with ICIs but very few cases are described in the literature.A differential diagnosis of dermatological adverse events (dAEs) related to treatment with ICIs should be carried out. dAEs occur in 34%–45% of patients treated with ICIs.Here, we report the clinical case of a patient with metastatic lung cancer which was treated with nivolumab who subsequently developed VZV‐GD. Accurate clinical diagnosis and prompt treatment with antiviral agents have resulted in a complete resolution of the clinical picture.A 65‐year‐old woman presented to the clinic, following the appearance of supraclavicular lymphadenopathy with a diameter of 20 mm. A biopsy was performed, with a subsequent diagnosis of lung adenocarcinoma with mutation Exon 19 EGFR, ALK and ROS not rearranged, PDL‐1 negative.Positron emission tomography (PET) and computed tomography (CT) scans were performed which indicated multiple mediastinal lymphadenopathy. Treatment with Afatinib was initiated and the disease was subsequently controlled for seven months. Following progression of the disease, no T790M mutation was detected in the circulating DNA, or after a new biopsy of the lesion.The patient then commenced chemotherapy with six cycles of pemetrexed and cisplatin and whilst complete metabolic remission of a very short duration was achieved, it was followed by a rapidly evolving relapse. A PET/CT scan showed diffuse adenopathies, right adrenal loggia nodularity (SUV 12.1) right iliac adenopathies and cruralw inguin (SUV 13.7). The patient commenced treatment with nivolumab and achieved a complete response which was documented by PET scan. After six months of treatment, there was widespread erythema evident at the level of the humeral‐scapula articulation, with severe itching and pain. Subsequently, 2–3 days later, maculae and papules appeared which evolved into vesicles and then pustules. The area was affected throughout by severe itching and pain.Treatment with nivolumab was subsequently temporarily discontinued. The patient commenced treatment with valaciclovir, 1000 mg three times a day for seven days in addition to fusidic acid cream which was applied twice a day to the damaged skin.Dermatological diagnosis was a grade 3 dAE due to VZV‐GD, with interesting scapular and supraclavicular cutaneous areas Figs a and 1b.A clinical reassessment after three weeks from the diagnosis of the infection documented a good resolution of the clinical picture with improvement in the cutaneous erythema, the rash had dried with the formation of crusts and almost complete disappearance of the symptoms of itching.At the time of writing this report, the patient is continuing treatment with nivolumab with excellent disease control.The primary infection of VZV is chickenpox, manifested by viremia with a diffuse rash and seeding of multiple sensory ganglia where the virus establishes life‐long latency.There are more than one million cases of HZ in the United States each year with an annual rate of three to four cases per 1000 people. Most cases of HZ can be diagnosed clinically.Nivolumab is a human immunoglobulin monoclonal antibody, which binds to programmed death‐1 receptor (PD‐1) and blocks its interaction with the PD‐L1 and the PD‐L2. Interaction of the PD‐1 with the PD‐L1 and PD‐L2 ligands, which are expressed by the antigen‐presenting cells, cancer cell or by other cells in tumor microenvironment, involves inhibition of T cell proliferation and cytokine secretion. Nivolumab strengthens T cell responses, including antitumor responses.Most frequent adverse events during treatment with ICIs are dAEs, which may range from vesicular, maculopapular, follicular, papular to exfoliative lesions or lupus‐like reactions.According to the European Society for Medical Oncology guidelines on the management of dAEs, the first requirement is to rule out any other etiology of the skin problem Table . 8 In ouNo authors report any conflict of interest.


Tumour acidosis is considered to play a central role in promoting cancer invasion and migration, but few studies have investigated in vivo how tumour pH correlates with cancer invasion. This study aims to determine in vivo whether tumour acidity is associated with cancer metastatic potential.Breast cancer cell lines with different metastatic potentials have been characterised for several markers of aggressiveness and invasiveness. Murine tumour models have been developed and assessed for lung metastases and tumour acidosis has been assessed in vivo by a magnetic resonance imaging-based chemical exchange saturation transfer (CEST) pH imaging approach.+CD24−), highlighting their propensity to migrate and invade, coinciding with the measurement obtained by in vitro assays. MRI-CEST pH imaging successfully discriminated the more aggressive 4T1 and TS/A tumours that displayed a more acidic pH. Moreover, the observed higher tumour acidity was significantly correlated with an increased number of lung metastases.The higher metastatic potential of 4T1 and TS/A primary tumours, in comparison to the less aggressive TUBO and BALB-neuT ones, was confirmed by the highest expression of cancer cell stem markers (CD44The findings of this study indicate that the extracellular acidification is associated with the metastatic potential. Unfortunately, by the time many breast tumours are detected with such techniques, metastasis may have already begun and remains unnoticed on imaging. Even at the time of initial cancer surgery, metastasis may have already occurred in many patients who eventually die of breast cancer.2 Once the tumour has spread, fewer curative treatment options are available to reduce cancer mortality upon metastasis.3 Predicting the tendency of a primary tumour to form secondary metastatic lesions, or its metastatic potential, remains a major challenge in cancer research. In fact, knowledge of tumour metastatic potential may help physicians to select the adequate treatment based on tumour aggressiveness while avoiding unnecessary side effects on patients.4 Established breast cancer prognostic markers are based on size and grade of the primary tumour and on the presence of lymph-node metastasis.5 However, definitive diagnosis and treatment planning are currently based on biopsy samples that can assess only limited portions of the tumour tissue with limited success in assessing its heterogeneity. Therefore, histological typing is only a weak prognostic marker of metastasis risk.Breast cancer is the second leading cause of cancer-related death in women worldwide. In these patients, metastases at distant sites and not the primary tumours are still the main cause of death.7 Among the several factors, the compensatory hypoxia-induced shift to anaerobic glycolysis for energy production can lead to a significant increase in production and release of acidic metabolites into the extracellular matrix (ECM). As a consequence, the progressive reduction of extracellular pH (pHe) within tumours, known as acidosis, is another feature of the tumour microenvironment.8 Tumour acidity has shown to affect most of the steps in the metastatic cascade, either directly or indirectly. In fact, acidic microenvironment is closely associated with increased mutagenesis, sustained proliferation and drug resistance, leading to more aggressive behaviour and metastasis.11Indeed, tumour aggressiveness is associated with many processes, including hypoxia, altered glucose metabolism, angiogenesis and inflammation that determine the heterogeneity of tumour microenvironment.12 hypoxia,13 redox status,14 metabolites and several other targets of the tumour microenvironment.19To date, the various steps of the metastatic cascade have been investigated by imaging-based approaches, looking for relationships between cancer aggressiveness and tumour vascularisation,20 Recently, MRI-based approaches exploiting the pH-sensitive chemical exchange-dependent saturation transfer (CEST) technique have been successfully investigated and validated for measuring tumour pH.21 Among these approaches, MRI-CEST pH mapping exploiting x-ray contrast agents have been shown to provide accurate pH measurements with high spatial resolution.24 Such improvements allowed for the first time to investigate the relationship between tumour metabolism and acidosis, providing evidence of their relationship in a breast murine model.25 In addition, the capability to quantify the heterogeneity of tumour pHe has been exploited for assessing the early therapeutic effect of drugs targeting tumour metabolism and for monitoring the onset of resistance.26 Other studies have explored tumour pH imaging for evaluating treatment response to other drugs affecting various pathways of tumour metabolism.29 Moreover, these molecules have also been validated for characterising tumour perfusion properties.31 Notably, this approach, owing to the clinical safety of the iodinated contrast agents, has already been translated to the clinic providing tumour pHe maps in breast and ovarian cancer patients.32 Therefore, MRI-CEST pH imaging holds promise to quantitatively assess tumour pHe with high accuracy and spatial resolution and our hypothesis is that this approach can provide reliable information on the role of tumour acidosis in cancer progression and metastasis.On the other hand, very few studies have investigated tumour acidosis as a possible marker of the metastatic potential. One reason relies in the poor spatial resolution, long acquisition times, limited accuracy and reduced clinical translatability of the proposed approaches for measuring pH.In this study, we investigated several murine breast cancer models from low to highly metastatic that showed different cancer aggressiveness and invasiveness both in vitro and in vivo. Using MRI-CEST pH imaging, we observed that more aggressive cancer murine models showed increased tumour acidity in comparison to less aggressive ones.33 and were grown in Dulbecco’s modified Eagle’s medium supplemented with 20% foetal bovine serum (FBS) and 100 U/mL penicillin and 100 µg/mL streptomycin (Pen/Strep); 4T1 and TS/A cells were grown in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL Pen/Strep and 2 mM l-glutamine. TUBO is considered to be a non-metastatic cell line; triple-negative breast cancer 4T1 cells, instead, are a highly metastatic cell line derived from a spontaneous BALB/c mammary tumour34 and HER2+ TS/A cells are a metastasising cell line derived from a spontaneous BALB/c mammary tumour.35 Cells were not authenticated but tested as mycoplasma-free and passaged in our laboratory for <2 months after their resuscitation.TUBO cells were derived from a lobular carcinoma arising spontaneously in a BALB-neuT mousel-glutamine and with 25 mmol/L of PIPES and HEPES to adjust pH to 6.8.36 We maintained tumour cells in this medium for 8 to 10 weeks until they completely recovered and grew at the same rate as parental cells.4T1 chronically exposed to acidic pH (pH 6.8) were maintained in RPMI-1640 supplemented with 10% FBS, 100 U/mL Pen/Strep and 2 mM 37 and protein contents were quantified by BCA assay according to the manufacturer’s instructions (Thermo Fisher 23221). For western blotting, 20 µg were loaded per well on 7.5% precast TGX gel (Bio-Rad 4561026). Proteins were then transferred on PVDF membrane (Bio-Rad 10026933). The primary antibodies used for western blotting were as follows: CA IX , NHE1  and vinculin . Secondary antibodies were purchased from Sigma . Clarity-ECL substrate was used to reveal bands (Bio-Rad 1705060) and protein expression was finally determined by quantification of band intensity using ImageLab (Bio-Rad). Real-time PCR method is described in Protein extraction was performed as previously described,4 TUBO, TS/A or 4T1 cells were seeded into 96-well plates and incubated overnight to allow adherence. The following day, the pHe was measured using a pH-sensitive fluorescent probes , as described in Supplementary Material.38A total of 5 × 10For migration and invasion assays, cells were plated and carried out and analysed as described in Supplementary Material.39 were bred at the Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy. Mice were maintained in specific pathogen-free conditions  and treated in accordance with University Ethical Committee and European guidelines under directive 2010/63. During housing, animals were monitored twice daily for health status. BALB/c mice  were inoculated subcutaneously (s.c.) with TUBO , 4T1  and TS/A  cells on both flanks. BALB-neuT mice (n = 8) were selected for the study from 25 weeks of age (mean weight 25 g). Altogether, pHe maps were measured for 22, 24, 16 and 16 4T1, TS/A, TUBO and BALB-neuT tumours, respectively, and tumour volumes ranged between 100 and 500 mm3.BALB/c mice were purchased from Charles River Laboratories , while BALB-neuT mice4 4T1 pH 6.8 and 3 × 104 4T1 cells subcutaneously injected, n = 10 for each group) 6 days following tumour inoculation were divided into two groups: one (control) was provided with drinking water and the other (bicarbonate) was provided with 200 mmol/L NaHCO3 ad libitum, which continued for the duration of the experiment. 4T1 pH 6.8 and 4T1 tumour-bearing mice were imaged for tumour pH measurement 7 days after treatment and mice group were treated until tumour volumes reached 400 mm3.4T1 pH 6.8 and 4T1 tumour-bearing animals  and tiletamine/zolazepam 20 mg/kg  and breath rate was monitored by an air pillow placed below the animal  during all the experiment. MRI-CEST pH mapping was performed upon intravenous injection of ca. 270 µL of iopamidol  into the tail vein through a 27-gauge needle catheter. MRI-CEST images were acquired on a Bruker Avance 7 T MRI scanner using a 30-mm insert coil. A detailed description of the acquisition protocols is provided in Supplementary Material.26 Tumour pixels were clustered into three groups: group I for pixels displaying pHe values >7.0, group II for pixels displaying pHe values >6.7 and <7.0 and group III for pixels displaying pHe values <6.7. The percentage of pixels of each group was multiplied by a factor between 1 and 3, to obtain the acidity score, in accordance with the equation:The acidity score was calculated to assess the heterogeneous pHe distribution within the tumour regions.The acidity score ranges from 1 (less acidic) to 3 (more acidic), defining tumour regions with different acidosis levels.To collect tumours, mice were sacrificed by cervical dislocation right after the end of the acquisition.5 cells were incubated with 100 μM 2-NBDG  amino]-d-glucose, Focus Biomolecules, Plymouth, USA) dissolved in glucose-free medium for 10 min, and their fluorescence was measured by flow cytometry as described in Supplementary Material.For flow cytometry-based glucose uptake assays, 4 × 10Moreover, cells and fresh primary tumours were stained for membrane antigens. The following antibodies were used: Alexa Fluor647-conjugated anti-stem cell antigen-1 (Sca1), phycoerythrin (PE)-conjugated anti-CD44 and PE/Cy7-conjugated anti-CD24 . All samples were collected and analysed using a CyAn ADP Flow Cytometer and Summit 4.3 software (DakoCytomation) as described in detail in Supplementary Material.Fixed cells and frozen tumour samples were stained for GLUT1. See Supplementary Material for further details.3 (ca. 10 mm diameter) (n = 5 mice for each breast cancer murine model). Immediately afterwards, mice were euthanised, and resected lungs were fixed in phosphate-buffered 4% paraformaldehyde and paraffin-embedded. Histological sections were cut from each lobe at 100 μm intervals and stained with haematoxylin and eosin. Metastases were counted with a Nikon SMZ1000 stereomicroscope  and ImageJ software .40 Groups of five or more tumour cells were scored as a metastasis. Moreover, the percentage of the area occupied by metastases was calculated by measuring the area of metastasis in respect to the total lung area by using ImageJ.Spontaneous pulmonary metastases were studied by histological examination in tumour-bearing mice when the primary tumours had grown to a volume of ca. 400 mmP value < 0.05 was considered statistically significant.Statistical analysis was performed by using the GraphPad Prism 7 software . Data are presented as mean ± SD, unless otherwise stated. One-way analysis of variance (ANOVA) and Bonferroni’s multiple comparison test were used to test for statistically significant differences between the measurements of parameters among the four groups of breast cancers. The Pearson’s product–moment correlation test was used to search for correlation between parameters. For all tests, a We evaluated the invasiveness and migration capability of some murine breast cancer cell lines and whether their aggressiveness was related to specific molecular markers.P < 0.01, Fig. In vitro cell migration studies showed that TUBO cells were slower to close the wound compared to 4T1 and TS/A cells Fig. . 4T1 cel40 and of CD44+CD24− cells41 was low in TUBO, but intermediate in 4T1 and high in TS/A cells, indicating that higher levels of CSCs were correlated with the migration and invasion capabilities of breast cancer cells. Moreover, the expression of CD44, a cell surface marker whose activation by its ligand hyaluronan induces cytoskeletal changes, promotes cell proliferation and survival and enhances cellular motility, thus contributing to the metastatic process,42 progressively increased from TUBO to 4T1 and TS/A cells , a cell population characterised by self-renewal and tumour-forming abilities, thereby promoting tumour growth and metastases formation. As shown in Fig. lls Fig. . CollectHigh metabolic rates in cancer cells determine the net production of protons that are secreted for maintaining the intracellular pH homeostasis. Therefore, a complex interplay between glycolytic activity, glucose transporters and acid–base transporters results in the net extracellular acidification that favours cell migration and invasion. By means of a fluorescent glucose analogue  compared to the other breast cancer models used . The corresponding mean acidity score values .Altered tumour pHe is known to promote several aspects of tumour progression and invasion. We therefore next asked whether the level of tumour acidification may correlate with the formation of lung metastases. MRI-CEST pHe maps were acquired in vivo in tumour-bearing mice upon intravenous administration of iopamidol, a pH-responsive agent. All the investigated murine breast cancer models showed a marked extracellular acidification, with mean pHe values of 6.84 ± 0.03, 6.96 ± 0.03, 6.79 ± 0.02, and 6.8 ± 0.03 for TUBO, BALB-neuT, 4T1, and TS/A, respectively Fig. . Signifiues Fig.  of the T36 We maintained tumour cells in this medium for 10 weeks until they completely recovered and grew at the same rate as parental cells , suggesting that cells adapted to an acidic pHe may have a partial CSC phenotype.Aiming to further study whether acidity may be an aetiological factor in promoting metastasis, we adapted 4T1 cancer cells to a chronic acidic environment, by culturing them in a medium buffered at pH 6.8.P = 0.03). Conversely, a higher level of both MCT1 and MCT4 mRNA was expressed by 4T1 cells conditioned at pH 6.8 .Then, we examined in the 4T1 and the 4T1 cultured at pH 6.8 cells the expression of NHE1, CA9 and MCTs transporters by means of quantitative PCR (qPCR)  defective and chaotic vasculature formation, (ii) proton accumulation from several pathways including ATP hydrolysis, pentose phosphate and serine besides glycolysis, (iii) increased expression of proton transporters such as H+-ATPases, Na+/H+ exchangers, Na+/HCO3− co-transporters51 and monocarboxylate transporters and (iv) lactate produced by cells other than tumour cells that sustains the tumour growth and proliferation.52 The key role played by tumour acidosis in cancer aggressiveness is further validated by the ability to induce on tumour-associated macrophages and fibroblasts the secretion of cathepsins that degrade the ECM and cell-adhesion molecules promoting invasiveness.54 In addition, it has been shown that buffering the acidic tumour microenvironment reduces metastasis formation.45 Therefore, acidosis may be a superior biomarker for understanding the complex role of the tumour microenvironment in cancer progression.This study aims to validate tumour pHe as an innovative biomarker of metastatic potential in several breast cancer murine models. We have exploited in vivo tumour pHe measurements to characterise three transplantable breast cancer mouse and one transgenic breast tumour models, of which two are aggressive (highly metastatic: 4T1 and TS/A), and two are indolent (poorly metastatic: TUBO and BALB-NeuT). A recognised hallmark of cancer commonly associated to cancer aggressiveness is the abnormally high rate of glycolysis and of lactate production, that is the end product of fermentative glucose metabolism. Therefore, high tumour lactate levels have been correlated with metastasis likelihood in several cancers,25 This approach showed a marked acidic microenvironment for two well-established human breast tumour models, MCF-7 and MDA-MB-231 cells, consistent with a upregulated glycolytic metabolism.55 Herein, by mapping in vivo tumour pHe, we show that the more aggressive breast tumours displayed a more pronounced tumour acidic microenvironment, whereas the less aggressive tumours showed a relatively less acidic environment. Although the average tumour acidosis measurements did not provide a clear differentiation between the less and more aggressive tumours models, the exploitation of the acidity score index, which considers the heterogeneity of the tumour acidosis in the whole tumour, allowed to clearly distinguish the two different tumour types.Only recently, the development of MRI-CEST based pH imaging has provided a powerful tool for a comprehensive in vivo investigation of tumour acidosis.56 However, although the two tumour models showed a different metastatic potential, in vivo optical images did not provide any clear discrimination, likely due to the low pH sensitivity of the probe and to the inherently limited tissue depth penetration of the technique. Recently, ex vivo analysis by exploiting a fluorescent pH-responsive probes showed that tumour acidic regions overlap with highly proliferative and invasive regions at the tumour–stroma interface, likely promoting tumour invasion.57 Positron emission tomography-based tracers targeting tumour acidosis58 or for imaging tumour hypoxia or for targeting CXCR4 chemokine receptor involved in metastasis59 have been proposed with promising clinical translatability, despite radiation exposure and limited spatial resolution hamper accurate heterogeneity measurements. Tumour vessel permeability has been investigated on xenograft models of metastatic and non-metastatic breast and prostate tumour using MRI albumin-GdDTPA contrast agent, providing evidence that an adequate vascularisation and/or permeability is needed for metastasis to occur.12 Overall, these studies highlight the importance of MRI-based methods that can provide sufficient spatial resolution, depth penetration and pH accuracy for non-invasively assessing tumour pHe and for accurate quantification of its heterogeneity. Despite the higher accuracy in assessing extracellular tumour pH for MRI-based methods that require the injection of a pH-responsive probe, the low extravasation of the agent in poorly vascularised or necrotic tumour areas can hinder the pH measurement in these regions. Anyway, in the investigated tumour murine models, the high accumulation of the pH-responsive probe in the whole tumours (as indicated by the high coverage of coloured pixels in the tumour regions) resulted in pH measurements representative of the whole tumour. Moreover, the large heterogeneity that we observed in tumour pHe values among the investigated tumour murine models is in agreement with the range of pHe values reported by others using MRI-based approaches and confirmed by invasive microelectrode measurements.63Several imaging-based studies have investigated different aspects of the tumour microenvironment as potential reporters of the metastatic potential. A fluorescent pH-activatable probe for optical imaging was exploited for assessing differences in acidic microenvironment in two human hepatic xenografts (HepG2 and HCCLM3).64 Our in vivo data support this hypothesis of a causative link between tumour acidosis and metastatic potential, but it is still not possible to draw a strong conclusion. Therefore, new studies will be required to investigate whether the reduction of tumour acidosis will hamper and delay the development of metastases. Furthermore, since metabolic pathways essential for tumour growth are being explored as novel targets to anti-cancer drug development, accurate in vivo pH imaging may be a promising tool for monitoring and predicting the therapeutic response.65Importantly, the relationship between tumour acidosis and metastatic propensity is a valuable clinical issue, since death rate in breast cancer patients is heavily dependent on metastases. In addition to the strong correlation observed between the acidity score and the number of lung metastases, we further investigated whether a causative link exists between tumour acidosis and invasiveness. First, the acclimation of the 4T1 tumour cells to low pH promoted CSC markers and increased expression levels of MCT1/4 proton transporters that resulted, when inoculated in mice, in an increased tumour acidic microenvironment and in a two-fold increase of lung metastases. Second, prolonged bicarbonate treatment was not able to induce any change in tumour acidosis and this was accompanied by similar numbers of lung metastases. Although bicarbonate treatment was initially proposed to reduce the incidence of metastases by inhibiting tumour acidity, several studies have shown contradictory results with a strong dependence on the investigated tumour types.In conclusion, we demonstrate that more aggressive breast cancer cell lines present more acidic tumour pHe that results in increased lung metastases. Our data suggest that MRI-CEST imaging of tumour pHe may be a valuable tool for predicting the metastatic potential of primary tumours. However, additional studies are necessary to verify the generalisability of these findings in other cancer types. These results may open new interest in assessing the role of tumour acidosis in several aspects of tumour progression and response to therapy.Supplementary Materials


ICD-11 criteria one needs to experience persistent and pervasive longing for the deceased and/or persistent and pervasive cognitive preoccupation with the deceased, combined with any of 10 additional grief reactions assumed indicative of intense emotional pain for at least six months after bereavement. Contrary to the 5th revision of the Diagnostical and Statistical Manual of Mental Disorders . That iICD-11 and their comparability against external standards when varying the number of additional criteria ICD-11 an the ICG , 7, yet and PCBD . MoreoveICD-11 to establish the robustness of clinically-relevant findings  and the variety of other aspects relevant to the validity of a diagnosis [for reviews:  Veni grant [Grant ID: 016.veni195.113]. The funder did not play a role in the study design, the writing of the report or in the decision to submit the article for publication. RR was supported by a grant of Deutsche Forschungsgemeinschaft [Grant ID: RO2042/7-1]. The funder did not play a role in the study design, the writing of the report or the decision to submit the article for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vestibular schwannoma (VS) is a Schwann cell-derived tumour arising from the vestibulocochlear nerve. Although benign, it represents a threat to intracranial structures due to mass effect and carries a small risk of malignant transformation. VS therefore represents an important healthcare burden. We review the literature regarding pathogenesis, risk factors, and diagnosis of VS. The current and future potential management strategies are also discussed. A narrative review of all relevant papers known to the authors was conducted. The majority of VS remain clinically stable and do not require interventional procedures. Nevertheless, various surgical techniques exist for removing VS, the most common of which are translabyrinthine and retrosigmoid approaches. Due to surgical risks such as hearing loss, facial nerve dysfunction, post-operative headache, and cerebrospinal fluid leakage, a "watch and rescan" approach is adopted for most patients. Radiotherapy is a useful alternative and has been shown to have a similar response for growth restriction. Due to the heterogeneous nature of VS, there is a lack of consensus regarding management of tumours that are too large for conservative management but too small to indicate surgery. Emerging biologic therapies, such as Bevacizumab, Everolimus, and Lapatinib, as well as anti-inflammatories like aspirin are promising potential treatments; however, long-term evidence of their efficacy is required. The knowledge base regarding VS continues to improve. With increased understanding of the pathogenesis of these tumors, we believe future work should focus on pharmacologic intervention. Biologic therapies aimed toward improved patient outcomes are particularly promising. Vestibular schwannoma (VS), also called acoustic neuroma, is a benign Schwann cell-derived tumour arising from the vestibulocochlear nerve. These tumors represent 85% of intracranial growths arising at the cerebellopontine angle . The KooAlthough benign, VS represents a risk to various intracranial structures due to mass effect. The most common symptoms include progressive hearing loss and tinnitus which are reported in over 60% of patients. Larger tumours can cause hydrocephalus and brainstem compression leading to symptoms such as facial paraesthesia, vertigo, and headache .VS accounts for approximately 8% of all intracranial tumours with an incidence of 10.4 per million per year . The majThis article aims to provide an up to date review of our understanding of the pathogenesis and diagnosis of VS with a focus on current management strategies. Emerging treatment options are also discussed and explored.The majority of VS grow from the inferior vestibular nerve with rare cases arising from the superior vestibular, or cochlear portion of the nerve . The hisNF2, a tumour suppressor gene on chromosome 22, play a vital role in the development of both sporadic and NF2-related disease [NF2 protein product, Merlin (schwannomin), leads to deregulation of various intracellular signalling pathways such as Rac1, Ras, PAK1, and mTORC1. Inactivation of other tumour suppressor genes including LZTR1, SMARCB1, and COQ6 are also linked to schwannoma development  to the cerbellopontine angle in children has been linked to a higher incidence of VS after a latency period of 20–55 years . Other sMultiple epidemiological studies failed to establish a significant link between mobile phone exposure and VS –25. HoweVS is often diagnosed due to otological or neurological symptoms. Otological symptoms include progressive sensorineural hearing loss, unilateral tinnitus, and vertigo. These are more common than the neurological symptoms such as trigeminal and facial nerve impairment, headache, and hydrocephalus amongst the general population , 30.Up to 20% of patients presenting to Ear, Nose, and Throat (ENT) clinics have symptoms suggesting a lesion at the cerebellopontine angle . TherefoAll patients presenting with unilateral tinnitus or sensorineural hearing loss require MRI as the gold standard investigation , 33. ForVarious approaches exist to treat patients with VS. These include observation, termed a ‘watch and rescan’ approach, surgical removal, and radiotherapy. The main aim of interventional treatment is to remove or debulk the tumour to prevent mass effect .Observation is an accepted treatment option due to the slow growth rate of VS. A systematic review of 41 papers showed a mean tumour growth rate of 1–2 mm/year with up to 75% of tumours showing no further growth . An obseAlthough observation can increase the risk of tumour progression and mass effect, it seems a safe approach due to the minimal growth rate. Furthermore, delaying surgical intervention appears to have no increased risk in mortality . Since gA range of surgical methods for VS removal are discussed in the literature and mainly pertain to large tumours. The approaches include: middle fossa (MF), extended middle fossa, transotic, and endoscopic resection. The translabyrinthine (TL) and retrosigmoid (RS) approaches are the most commonly used in the United Kingdom (UK) and are further discussed below . The benThe TL approach refers to a retroauricular incision with posterior extension toward the mastoid tip. This provides quick access to the internal acoustic canal, facial nerve, and tumour. A major disadvantage is poor preservation of hearing, limiting the use of the TL approach to those with minimal or absent preoperative hearing.With the RS approach, a suboccipital incision exposes the transverse and sigmoidal sinuses, and subsequent cerebellar manipulation exposes the internal acoustic canal. Although this method is superior in preserving hearing, there is an increased risk of facial and cochlear nerve damage . Of courIn a systematic review of 35 studies (n = 5064), it was reported that facial nerve dysfunction was less likely to occur with the RS versus TL approach for tumours greater than 1.5 cm . EvidencMortality and tumour recurrence did not differ between surgical approaches with mortality rates as low as 0.2% . Non-speThere are three forms of radiotherapy treatment for VS management: stereotactic radiosurgery (SRS), fractionated stereotactic radiotherapy (FSRT), and proton beam therapy. SRS and FRST are most commonly used, as there is limited availability of proton beam therapy and inadequate evidence of its efficacy . The maiVarious advantages and disadvantages apply to SRS and FRST. SRS uses Gamma Knife technology to expose the tumour to a singular dose of radiation and is less applicable to large lesions  . FRST reA recent systematic review comparing FRST and SRS reported similar rates of tumour control with an average of 4.8% and 5% of patients undergoing treatment requiring rescue therapy, respectively. Facial and trigeminal nerve deterioration was less for SRS. These comparisons were based on limited evidence; however, and without randomised controlled trials (RCTs). Furthermore, only two studies on FRST were evaluated limiting the validity of estimates regarding this treatment. More studies are required to confidently compare these two therapies .Ranges of controlled studies demonstrate comparable progression-free survival rates and side effects, such as nerve palsies and hearing deterioration, between radiotherapy and microsurgery –61. A reThe evidence presented is inconclusive regarding the best treatment options for all categories of VS. While most small tumours are managed conservatively and larger tumours with surgery and/or radiotherapy, there is a grey area surrounding best management options for tumours falling between categories . More roAs knowledge surrounding the molecular pathology of VS improves, targeted biologic therapies are emerging at the forefront of treatment. Bevacizumab, Everolimus, and Lapatinib are potential options for treating VS. Furthermore, a link between tumour growth and non-steroidal anti-inflammatory medications such as aspirin has been recognised.Bevacizumab is a monoclonal antibody and vascular endothelial growth factor (VEGF) inhibitor. VEGF is a key mediator of angiogenesis and subsequently aids tumour growth . PlotkinSince then, more promising data has arisen. In a systematic review on the safety and efficacy of Bevacizumab, 41% of patients receiving treatment had tumour regression, 20% experienced hearing improvement, and 69% had no further hearing deterioration , 71.Furthermore, various side effects have been reported in a dose-dependent relationship with Bevacizumab including hypertension, proteinuria, and infection , 72. AltEverolimus is an mTOR complex 1 (mTORC1) inhibitor. mTORC1 activation has been implicated in tumour growth as a result of merlin deficiency. Furthermore, inhibition of mTORC1 has demonstrated antiangiogenic properties , 75. In Lapatinib is an EGFR/ErbB2 inhibitor with demonstrated promise for tumor growth inhibition during in vitro trials . The CNSStudies also suggest that aspirin may be beneficial to delay tumour growth through its anti-cyclooxygenase 2 (COX-2) effect. A proliferative effect of COX-2 expression in 30 tumours was observed . AnotherMore recent evidence is not as promising. In a 2019 study, tissue samples of 1048 tumours were analysed. It was discovered that while COX-2 expression increased with tumour proliferation, the use of acetylsalicylic acid did not alter COX-2 expression . This stVS is a clinically important disease with an evolving knowledge base. There is now a vast scope of literature pertaining to risk factors, diagnosis, and treatment. With improved understanding of the pathogenesis, future work should focus on biologic interventions due to the current risks and lack of high quality RCTs of different surgical and radiologic treatment options.


This article introduces the Twitter Parliamentarian Database (TPD), a multi-source and manually validated database of parliamentarians on Twitter. The TPD includes parliamentarians from all European Free Trade Association countries where over 45% of parliamentarians are on Twitter as well as a selection of English-speaking countries. The database is designed to move beyond the one-off nature of most Twitter-based research and in the direction of systematic and rigorous comparative and transnational analysis. The TPD incorporates, in addition to data collected through Twitter’s streaming API and governmental websites, data from the Manifesto Project Database; the Electoral System Design Database; the ParlGov database; and the Chapel Hill Expert Survey. By compiling these different data sources it becomes possible to compare different countries, political parties, political party families, and different kinds of democracies. To illustrate the opportunities for comparative and transnational analysis that the TPD opens up, we ask: What are the differences between countries in parliamentarian Twitter interactions? How do political parties differ in their use of hashtags and what is their common ground? What is the structure of interaction between parliamentarians in the transnational debate? Alongside some interesting similarities, we find striking cross-party and particularly cross-national differences in how parliamentarians engage in politics on the social media platform. While many authors have argued that social media data have the potential to revolutionize social science research , 2, schoWe contribute to this endeavor by presenting a database of parliamentarians on Twitter. The Twitter Parliamentarian Database (TPD) contains all the tweets of members of 27 parliaments . The database has been painstakingly and systematically validated to address issues of reliability and validity characteristic of much of the existing research on Twitter politician communication. To address issues of data availability, the TPD incorporates, in addition to data collected through Twitter’s streaming API and governmental websites, data from the Manifesto Project Database on the parliamentarians’ political parties ; the ChaWhat are the differences between countries in parliamentarian Twitter interactions? (2) We look at differences between how different parties label political issues, asking: how do political parties differ in their use of hashtags and what is their common ground? (3) We study the structure of mentions between parliamentarians internationally, asking: what is the structure of interaction between parliamentarians in the transnational debate? We do not here aim for definitive answers, but rather use the analyses to illustrate the affordances of the database in relation to research questions that were previously difficult to address. Through including a variety of analyses we demonstrate the different functionalities and areas of research that the TPD is able to touch upon, especially analyses of communication patterns between and within a large number of countries, as well as the content of the communication. While the affordances of Twitter are the same across all the countries, we find cross-party and particularly cross-national differences in how parliamentarians engage in politics on the social media platform.In this paper, we carry out a tentative analysis using the data in order to demonstrate the potential of the database. While the TPD allows for a wide range of analyses, we focus our demonstration on comparative and transnational research, as the database fills a considerable research gap in these fields resulting from the lack of large-scale data sources. To illustrate the capacity of the database to answer questions pertaining to this research, we carry out three illustrative and exploratory studies. (1) We look at parliamentarian’s Twitter use across nations, with a focus on coalitions and divisions through who retweets whom, asking: non plus ultra for social scientific research ).Focusing on parliamentarians limits the scope of research, but the advantage is that the TPD attempts to resolve several important issues regarding the delineation of the population and sampling in a way that is straightforward, transparent, and verifiable. An additional advantage is that parliamentarians are public figures, which significantly reduces ethical issues regarding privacy protection and increases possibilities to match data obtained from Twitter with data from other sources. In an effort to capitalize on these advantages, the database presented in this article extends earlier work by: including a much greater number of countries; complementing Twitter data with data drawn from other sources; and developing elaborate procedures to maintain data accuracy. The TPD data enable a broad range of Twitter research e.g. , 46–50).–50.46–50Several research domains can reached by the TPD through linking it to other databases, such as; the Electoral System Design Database (ESDD), the Manifesto Project, the Chapel Hill Expert Survey (CHES) and ParlGov—meaning that the TPD can extend existing comparative work to Twitter, to examine differences in for example the ways parliamentarians express themselves and engage with one another, within nations, within parties as well as transnationally. To illustrate the TPD’s potential, we focus on three domains of comparative research as a demonstration of how the database could begin to answer questions in these areas.We use several methodologies to illustrate the TPD’s potential for cross-national and comparative research. First, we can use the TPD to investigate national differences in the way parliamentarians use Twitter. Political science literature has a long-standing tradition of comparing political culture and political debate among various nations –55, showThe limitations of exclusively following parliamentarians on Twitter should be acknowledged. First, as mentioned, the focus on parliamentarians limits the scope of the research, since parliamentarian tweets only constitute a subset of political discussions on Twitter. This limits usefulness in relation to, for instance, campaigning research, as the TPD only gathers data on parliamentarians who are already in office. It should also be noted that in some cases, parliamentarians may choose not to individually interact with constituents but rather present themselves through the party account. Further, the timeliness of updating the database following elections is largely dependent on the updating of official government websites, which may not occur in some countries until coalitions are formed. In rare cases, this may be several months. While the TPD has a limited scope outside of incumbent parliamentarian communication, it may serve as a starting point for questions about other political debates, by using various techniques to expand from parliamentarian users to other parts of Twitter.In selecting countries, we aim to contribute to the large and growing body of comparative and transnational analysis. We included all member states and candidate member states of the European Free Trade Association (EFTA) where over 45% of parliamentarians are on Twitter . In addiparliamentarians in the TPD, we consulted official government websites and retrieved all the identities of incumbent members. From these websites, we also collected the parliamentarians’ party affiliations and when available, data on regions and constituencies. When the official government websites did not provide the party information for parliamentarians , we used Google searches to establish party affiliation. For those websites where English was not available, it was double checked with translation programs that the list we obtained from these websites is the most current and up-to-date list of incumbent members. Identifying parliamentarians on Twitter is occasionally challenging due to e.g. common names and parody accounts, so we followed a protocol to identify and verify Twitter accounts, which involved comparing pictures with those on the government website, examining the number of followers, scrutinizing the lists of followers, and reading the content of the tweets. If the tweets were not in English, they were translated through Google Translate.To identify the Due to the data protection regulation, we only include parliamentarians that are in public service during the time of data collection. Hence, persons who are not currently in parliament but are campaigning to be elected are excluded, as are those who served in one legislative period but were not re-elected in the next. If an account was set to private, it was not included. Inactive accounts, defined as accounts that have not made public tweets after 2014, were also excluded. For the year of 2018, the Twitter Parliamentarian Database captured 6,281,684 tweets from 6,437 parliamentarians active on Twitter out of a possible 8,098, meaning that 79.6% of parliamentarians had an active Twitter account.The tweets of the parliamentarians are collected using Twitter’s streaming API, including their mentions, retweets, and hashtags. As mentioned with regards to prior research, the streaming API can encounter certain limitations due to its rate limit, which, when used to follow a certain query , may be reached much faster than following a certain user, who may only tweet a couple of times per day. Hence, following certain user  results in the streaming API gathering almost all tweets of that user as the amount of tweets per minute rarely exceeds the rate limit imposed by the API.The database is updated following elections. Therefore there may be several electoral periods for some countries, if they had an election during the time of data collection. For example, if persons A and B are elected in 2015, and there is an election in 2018, where person B is reelected but person A is not, there will be data for person B across both periods, but only for person A until the election in 2018. Data collection for a parliamentarian starts from after they are elected, hence their tweet ids before the election date are not included. The Twitter accounts of members of new parliaments are checked for several months following elections, since we discovered that it takes time for new parliamentarians to find their footing on Twitter, and that some parliamentarians may later create new accounts for their parliamentary service, separate from their personal or campaign accounts. The database includes data from as early as May 2017 for some countries, and has been continually updated since, capturing many interesting political events such as the span of Donald Trump’s presidency, the Catalonia referendum, the Brexit debate. The user ids from the database can be used to retrieve older tweets from parliamentarians across the electoral periods where members were collected, or be used to gather further data. Hence, any researcher can use the Twitter ids currently provided in the TPD as a starting point for their own research to update a country of interest following an election.115th U.S. Congress Tweet Ids dataset ),A simple but powerful way of operationalizing the level of cohesion within a country is to compare the fraction of retweets made to members of external parties. Countries whose parliamentarians frequently use retweets to endorse members of other parties can be assumed to have more amicable between-party relations compared to countries whose parties mostly retweet internally. However, this quantitative approach has certain limitations, as it fails to capture, for instance, divisions involving multiple parties in coalitions or situations in which a fraction of a party is strongly divided from other parties. As there are many forms that divisions can take in these networks, we turn to a more qualitative approach to network analysis: Visual Network Analysis . This hiTo look beyond descriptive metrics for examining the differences in parliamentarian twitter interactions, for this preliminary demonstration we focus on the networks with 30 or more nodes and filter the networks by their giant component. We plot the networks using the ForceAtlas2 visualization algorithm, which uses a number of properties to structure the networks in such a way that highly connected nodes tend to be closer to each other, and less connected nodes further away from each other . In Fig Using this qualitative method identify four distinct types of political network structures see . Type 1 We can furthermore use additional measures to compare the categories of networks identified using VNA, to demonstrate that the networks are not only visually distinct. χ2 and Cramer’s V measures, wherein Cramer’s V show the strength of that relationship, thereby indicating how ‘neatly’ the networks cluster based on party: fragemented networks are expected to have a stronger relationship between party-cluster membership, whereas a weak relationship would indicate more overall cohesion in the network. For brevity, we only report the Cramer’s V value in χ2 results indicated a significant relationship between party and cluster membership (p < 0.00). We see that type 1 networks have much stronger relationships between party and cluster membership than type 4 networks. Therefore, it is clear that type 4 networks generally retweet across party lines.We also look at the relationship between cluster and party membership through All in all, visual analysis and basic network measures can be combined to interpret the types of network structures that emerge from the data. The measures provided are by no means exhaustive and may differ depending on the research question. Using the information from the ESDD , we can Textual data from Twitter provides an avenue into seeing how parliamentarians navigate different issues and engage in discursive conflict. Despite this potential, there has been limited research on content of parliamentarians’ tweets, and none that has focused on international comparison . The TPDThe use of labels in political discourse reveal the different ways that opposing political parties discuss the same issues. This is apparent through brief exposure to political debate: while one party may speak of “tax reform,” the other focuses on “tax relief.” Using different labels for the same issue indicates how central labelling is to politics . The wayhow do political parties differ in their use of hashtags and what is their common ground?Analyzing labels used on Twitter is made easier by Twitter providing affordances for explicitly labeling tweets, in the form of hashtags. While hashtags serve many purposes, broadly speaking they can be used to index conversations , convey To take a first step toward a comparison of how different parties label issues, we develop a simple computational method that captures which hashtags are partisan and which are shared. To study this, we look at the two largest parties in all countries, in terms of the number of active users on Twitter (note that these are not necessarily the parties that have the most seats in parliament). We count each hashtag used by these parliamentarians and then normalize according to the total number of hashtags used by the party. If a hashtag is used by both parties, the overlapping part of the use is seen as the intersection between the parties. As in a Venn diagram, the intersection of the sets are the common hashtags, and the non-intersecting parts are hashtags characteristic of the party. The relative size of the intersection thereby gives a measure of the similarity in hashtag use between the two parties, which corresponds to the Jaccard similarity measure . Since dA first striking takeaway from this A first thing to note is that the most common intersecting hashtags for Canada and Australia are “cdnpoli” and “auspol” respectively, although they are not used with the same relative frequency by each party. These are general country-level hashtags for marking the nation which the discussion concerns, and are broadly employed for political debate also outside of professional politicians We focus here on the US for a deeper look into the ways parties make use of hashtags, and the relation to how political issues are labelled. Looking at The most important intersecting hashtags found in Other important intersecting hashtags point to common ground values, to which both parties are happy to profess their support. These include “veterans,” “neverforget,” and “stem”. Similar common ground is found in national emergencies, such as the hurricane striking Puerto Rico. However, even here the language differs somewhat between the parties: Republicans are more likely to refer to the event as “hurricaneirma”, while Democrats speak of its impact on “PuertoRico”. DACA —an Obama-era executive action, now turned Trump-vetoed bill with broad bipartisan support among voters—is an important hashtag in the intersection, but is driven in particular by Democratic politicians, who also employ the related hashtags “dreamact”, “dreamers”, “protectdreamers”. There is, perhaps somewhat tellingly, broad bipartisan use of the hashtag “mepolitics,” denoting a criticism of the country’s polarized political discourse.As we have demonstrated, we see that politicians use hashtags to express partisan claims. While there is some common ground (for instance when it comes to the importance of caring for veterans), politicians generally use hashtags in outspokenly partisan ways: they use specific hashtags to push different kinds of issues on the agenda or to express a partisan take on the same issue. This case has served to illustrate the range of possible research opened by the TPD in examining language and discourse of politicians on Twitter.what is the structure of interaction between parliamentarians in the transnational debate?The TPD not only allows for cross-national comparative research but also for research on international and transnational politics, by enabling analysis of how parliamentarians of different countries communicate with one another –89. ThroWe begin by examining the network of mentions between individual parliamentarians, filtered by its giant component see . In mostTo arrive to a more precise measure for quantifying the level of external communication between countries, we look at the fraction of external tweets, network diameter, average path length and modularity. The modularity indicates the strength of the division of the network. The network diameter measures the longest distance between any two nodes in the network, whereas average path length counts the average graph distance between all pairs of nodes. These measures help to understand the structural connectivity amongst individual national parliamentarians on a European level. Aggregating on country level, we examine centrality with PageRank. The network diameter is 18 and the average path length is 5.8, indicating that there is quite some distance between distant nodes in this network. The network has a modularity of 0.678, suggesting that the network is relatively divisible into separate clusters. All these numbers suggest that parliamentarians communicate mostly with their compatriots, resulting in a very sparse and highly divided international communication network.Although international communication accounts for a low proportion of the total communication , in absolute terms it is still substantial cross-national traffic . Looking closer at these mentions gives insight in patterns of communication across EFTA countries. We examine the fraction of external mentions per country, to determine the proportion of mentions that are dedicated to international debate . When loTo further examine the extent of the transnational debate, Figs In summary, the results of our demonstration suggest that debates among national parliamentarians remain, by and large, contained within national boundaries. The TPD not only makes it possible to study the degree of transnational communication, but also to examine the position of countries, parties, or individual parliamentarians within that debate and the role of different issues. While our exploratory analysis has shown which countries are more central to transnational debates within Europe, the TPD makes it possible to study transnational politics on Twitter at a grater scale and in more detail than data sources have previously allowed for.This paper presents a database that responds to the methodological issues regarding delineation, sampling and validation methods commonly used in Twitter research, using a variety of data sources along side manually validated Twitter information. The opportunities of this database for comparative and transnational research were illustrated through three tentative studies, looking at 1) national differences between parliamentarians’ Twitter use, 2) differences between political parties’ hashtag use and 3) the structure of transnational debates.Through our exploratory analyses, we discover similarities in the ways which politicians use Twitter across countries. Overall when communicating with other politicians, they prefer to use mentions rather than retweets. There are also many differences: some countries have a very active, thriving Twitter culture in which all functionalities are used, others refrain from mentioning and retweeting, and yet others may not use the platform much at all. Apart from documenting differences in how Twitter is used, the database can also provide an empirical foundation for research into long-standing questions in political science. To illustrate the database as a tool for studying these questions, we used the structure of retweet networks as a proxy for coalitions and divisions in parliamentarian Twitter networks. Using Visual Network Analysis, we could distinguish four different kinds of retweet networks: bipolar, fringe party and cohesive. These structures show whether or not there is a lot of endorsement across party lines, or if the political culture seems more strictly partisan. Indeed, when looking at clustering measures, we see that networks that appear more divided have a higher clustering coefficient. We find that majoritarian systems have less external retweets and higher clustering coefficients, and most commonly resulted in bipolar structures. On the other hand, proportional systems are the only systems that resulted in cohesive networks. This suggests that there is a correspondence between the nature of political system and patterns of political communication but further investigation is necessary to arrive at robust conclusions.Comparisons between parties are made through using hashtags as a conduit for issue labelling. We compare countries with majoritarian systems, as they have two large opposing parties that comprise the bulk of the politicians’ Twitter activity. We find that there are very few hashtags that are shared between the opposing parties, and, in line with existing literature , 96, 97,The TPD does not only allow comparisons of countries and parties but also the study of transnational communication. By way of illustration, we studied cross-European parliamentarian communication. We found that cross-national mentions constitute only a tiny portion of the total politician Twitter use, which confirmed by the fraction of external mentions per country. Therefore there is very little international interaction amongst national parliamentarians. Additionally, through PageRank measures we can see that Germany and the United Kingdom take central positions in these debates. Hence, we have demonstrated that the TPD is able to explore the degree in which cross-European communication exists for parliamentarians on Twitter, along with the position of the countries, parties or individual parliamentarians across a number of different issues with more detail than previous studies.This paper has therefore demonstrated that the TPD is a powerful database for carrying out research on parliamentarians’ use of Twitter, in particular for cross-country comparative and transnational research, which has thus far struggled with data availability. All in all, our database addresses some of the current methodological issues with Twitter research and provides a starting point for studying communication, contention and cooperation not only within countries, but also comparatively across borders. As far as we are aware, the TPD is the most comprehensive database of parliamentarians on Twitter that exists, and it is able to provide a framework for more standardized comparative methodology in politician Twitter research.S1 File(PDF)Click here for additional data file.S1 FigShows the individual country retweet networks for 2018. The nodes are colored by party and the network is visualized with the ForceAtlas2 algorithm, with each node sized by in-degree (1-10) and scaling set to 1.(TIF)Click here for additional data file.S1 TableThe legislative period id is an arbitrary id number that distinguishes each legisla-tive period.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableHigh values indicate that a term occurs more frequently than chance amongst one party.(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Several studies have revealed that vasopressor may be more appropriate for treating intraoperative hypotension and preventing hypervolemia. This study compared the effects of vasopressor infusion and fluid supplementation on intestinal microcirculation during treating intraoperative hypotension. Thirty-two rats were randomly divided into the following four groups: Light Anesthesia group ; Deep Anesthesia group ; Fluid DA group (1.5–1.8% isoflurane and fluid supplementation); and Norepinephrine DA group (1.5–1.8% isoflurane and norepinephrine infusion). At 240 min, perfused small vessel density (PSVD) of the mucosa did not differ significantly between the Fluid DA and Norepinephrine DA groups , and tissue oxygen saturation of the mucosa was lower in the Fluid DA groups than in the Norepinephrine DA groups . At 240 min, TSVD and PSVD of the seromuscular layer were higher in the Norepinephrine DA group than in the Fluid DA group. Fluid administration was higher in the Fluid DA group than in the Norepinephrine DA group . Our results showed that norepinephrine can resuscitate intraoperative hypotension related microcirculatory alteration and avoid fluid overload. The common causes of perioperative hypoperfusion are general anesthesia-induced hypotension, blood loss, and fluid loss. Intraoperative administration of crystalloids is commonly used to treat perioperative hypoperfusion. However, research has proved that fluid loading has no influence on anesthesia-induced hypotension2, and excessive fluid supplementation may cause hypervolemia-related glycocalyx impairment3, tissue edema, respiratory distress syndrome4, and abdominal compartment syndrome5. Postoperative fluid overload has been demonstrated to be strongly correlated with mortality6. Several studies have shown that vasopressors may be more appropriate for treating anesthesia-induced hypotension and prevent hypervolemia8. Furthermore, microcirculatory dysfunction can occur in the absence of arterial hypotension12. Intestinal microcirculatory dysfunction can lead to the disruption of the intestinal mucosal barrier and the development of multiple organ dysfunction syndrome13. To the best of our knowledge, no study has compared the treatment effects of fluid supplementation and vasopressor infusion on intestinal microcirculation. The primary aim of this study was to compare the treatment effects of fluid supplementation and vasopressor infusion on intestinal microcirculation during intraoperative anesthesia-related hypotension in an open abdominal surgery rat model  mcg in the Norepinephrine DA group. Mean arterial pressure (MAP) and the heart rate are given in Fig. P = 0.788]. At 240 min, MAP did not significantly differ between the Fluid DA and Norepinephrine DA groups .The mean body weights and standard deviation of the rats were 266 (8) g in the light anesthesia (LA) group, 265 (21) g in the deep anesthesia (DA) group, 253 (23) g in the Fluid DA group, and 266 (13) g in the Norepinephrine DA group. According to the treatment protocol, the mean total fluid supplementation and standard deviation for 240 min was 8 μl/g in the LA and DA groups. Fluid supplementation was higher in the Fluid DA group than in the Norepinephrine DA group . Results of other exploratory variables are as follows. In repeated measures analysis, PSVD of the mucosa was lower in the DA group than in the LA and Norepinephrine DA groups  vs 22.3 (4.2) mm/mm2, P = 0.001]. At 240 min, TSVD and PVSD of the mucosa did not differ significantly between the DA and Fluid DA groups . At 240 min, TSVD of the mucosa did not differ significantly between the Fluid DA and Norepinephrine DA groups . In repeated measures analysis, StO2 of the mucosa was lower in the DA and Fluid DA groups than in the LA and Norepinephrine DA groups  of the mucosa was lower in the Fluid DA groups than in the Norepinephrine DA groups .The microcirculation images of the mucosa and seromuscular layer at baseline and 240 min are shown in Fig. 2, P = 0.05; PSVD: 28.3 (3.8) vs 23.2 (4.2) mm/mm2, P = 0.039]. In repeated measures analysis, StO2 of the seromuscular layer was lower in the DA group than in the LA groups  % vs 64 (7) %, P = 0.045], but StO2 of the seromuscular layer did not significantly differ among the DA, Fluid DA, and Norepinephrine DA groups.In repeated measures analysis, TSVD and PSVD of the seromuscular layer did not differ significantly among the four groups Fig. . At 240 Our study did not show a significant difference of PSVD of the intestinal mucosa at 240 min between norepinephrine infusion and fluid administration. From the results of comparisons of other exploratory variables, we found that norepinephrine improve tissue oxygenation of the intestinal mucosa and the microcirculation of the terminal seromuscular layer with less amount of fluid when compared with fluid administration.14, in which low to moderate doses of norepinephrine were found to preserve hemodynamic stability without any major concerns related to splanchnic organ perfusion and oxygenation. Norepinephrine actively expel blood from the splanchnic capacitance vessels, causing a rapid increase in the venous return. Pathophysiologic mechanisms of anesthesia-related hypotension include vasodilation, impairment of sympathetic nervous system, and compromised baroreflex regulation15. Norepinephrine can attenuate vasodilation and decrease the requirement of fluid supplement. Our results support that the adequate use of vasopressor can significantly decrease the amount of fluid administered. Over-administration of saline can lead to hyperchloremic metabolic acidosis and can damage the endothelial glycocalyx16. In addition, administrating a large volume of crystalloids to maintain normovolemia can reduced capillary perfusion and tissue oxygen partial pressure17. Aggressive fluid therapy leads to increased cardiac filling pressures, which results in the release of natriuretic peptides, and these peptides markedly disrupt the glycocalyx structure and function, leading to increased vascular leakage18. Moreover, the resuscitation effect of fluid supplementation may be limited or not persistent. Norberg et al. reported that a bolus of 25 mL/kg saline administered to healthy volunteers during isoflurane anesthesia had no effect on decreased blood pressure2.Our results that norepinephrine infusion resulted in better intestinal microcirculation than fluid supplementation may indicate that norepinephrine can be safely used to restore intraoperative anesthesia-related hypotension. This finding is supported by the animal study of Hiltebrand et al.19. Mucosal microcirculatory dysfunction may account for the development of intestinal edema, stress ulcer bleeding, and bacterial translocation. Conversely, microcirculatory dysfunction was more severe in the seromuscular layer than in the mucosa during fluid supplementation for intraoperative hypotension. We suggested that overloaded fluid may result in more tissue edema in the seromuscular layer than in the mucosa. Hydrostatic edema has been reported to activate the signal transduction pathway that results in intestinal contractile dysfunction20. Microcirculatory dysfunction in the seromuscular layer may account for ileus.A relevant concern is that difference in microcirculatory alterations between the mucosa and seromuscular layer. In the present study, compared with seromuscular layer, microcirculation was more severely affected in the mucosa during intraoperative hypotension. In a septic shock pig model, Hiltebrand et al. suggested that decreased capillary density may cause intestinal mucosal acidosis21. If hemodynamic coherence exists, correcting systemic hemodynamic variables is able to correct microcirculatory perfusion and oxygen delivery21. However, many studies have described conditions of a loss of hemodynamic coherence between macrocirculation and microcirculation23. We then did a post-hoc investigation of the correlation among heart rate, MAP, TSVD, PSVD, and StO2 at 240 min. We found that MAP was weakly correlated with PSVD  and StO2 . Further studies are warranted to investigate the coherence of microcirculation variables and other systemic hemodynamic variables  or laboratory data (eg. lactate level).Coherence of the macrocirculation and the microcirculation is the correlation between systemic hemodynamic variables and microcirculatory variables2. Second, the anesthetized rats were under spontaneous breathing. Mechanical ventilation with considerably positive end-expiratory pressure can lead to splanchnic hypoperfusion and marked decreases in hepatic, portal venous and mesenteric blood flow24. Third, our model was an open abdominal surgery model. Further studies in rat model with an intact abdominal wall, the effect of increase intra-abdominal pressure resulted from excessive intestinal edema should be considered. Fourth, as our previous description of the hyperchloremic metabolic acidosis after over-administration of saline, it is warranted for further studies to investigate the treatment effect of a balanced fluid on microcirculation.This study has several limitations. First, in the Fluid DA group, the MAP goal could not be maintained even with aggressive volume resuscitation. This observation is compatible with the result of Norberg et al.In conclusion, norepinephrine can resuscitate intraoperative hypotension related microcirculatory alteration and avoid fluid overload. Moreover, overload fluid may impair microcirculation in the intestinal seromuscular layer. We suggest that the resuscitating effects on microcirculation should be considered in further studies of investigating perioperative hypotensive resuscitation.In this study, 32 male Wistar rats  were used, and this study was approved by the Animal Care and Use Committee of the Laboratory Animal Center of the College of Medicine, National Taiwan University, Taipei, Taiwan (No. 20130287). The rats were maintained on a 12-h light–dark cycle with free access to water and food. The rats were handled according to the Guidelines for the Treatment of Laboratory Animals of the Institute Animal Care and Use Committee of National Taiwan University.The 32 rats were randomly assigned to four groups (8 rats in each group) Fig. . In the 25. Atropine sulfate (0.05 mg/kg in 0.9% sodium chloride) was administered subcutaneously to reduce respiratory tract secretions and block vagal reflexes elicited by the manipulation of the intestinal viscera. Arterial blood pressure and the heart rate were continually monitored via the right common carotid artery catheter and recorded at baseline, 60 min, 120 min, 180 min, and 240 min. The rectal temperature was continuously monitored. A 3 cm long midline laparotomy was performed to exteriorize a segment of terminal ileum which was located at 6 to 10 cm proximal to the ileocecal valve. The mucosa and seromuscular layer of the terminal ileum were prepared for microcirculation examination as described in our previous study26. Euthanasia was performed by exsanguination cardiac arrest under anesthesia after finishing all examinations.Anesthesia, tracheostomy, and right common carotid artery and external jugular vein cannulation were performed as described in our previous studyAfter a 15-min stabilization period, baseline microcirculation examinations for the mucosa and seromuscular layer was performed with a sidestream dark-field video microscope  and a tissue oxygen monitor . The time after the examination was set as 0 min, and the rats received treatment according to their grouping. Intestinal microcirculation was examined again at 120 min and 240 min.28. At each time point, three continuous image sequences (10 s) were digitally stored for each measured site, and the data of the three images were averaged for obtaining the required statistics. Analyses were performed by a single investigator who was blinded to grouping. At each time point, StO2 was measured at three points of the intestinal mucosa and seromuscular layer, and the data of these points were averaged for statistical calculation.Intestinal microcirculation parameters, including TSVD (less than 20 μm) and PSVD were analyzed using semi-automated analysis software  according to the guidelines of microcirculation analysis29, this study (n = 8 rats per group) was powered to detect a 10% difference of PSVD of the mucosa between the two groups at 240 min, with an α level of 0.05 (single-tailed) and a β level of 0.2 (80% power), assuming a control PSVD of 29 (2.3) mm/mm2.Primary outcome in this study was defined as the difference of PSVD of the mucosa between the Fluid DA and Norepinephrine DA group. Based on our experience30, and appropriate statistical analysis was used according to the normality of data. Primary outcome was examined by the Mann–Whitney U-test or the t-test according to the normality of PSVD. A P value of < 0.05 was considered significant. Other exploratory variables were examined as follows. Differences in means over time among the four groups were analyzed using general linear model repeated measures ANOVA . Differences in means at 240 min among the four groups were analyzed using one-way ANOVA, followed by the Tukey test for post hoc analysis. The P values for comparisons of exploratory data were not adjusted.Data analysis was performed using statistical software . Data normality was examined by W/S test


Immunogenic cell death (ICD) and tumour-infiltrating T lymphocytes are severely weakened by elevated reactive oxygen species (ROS) in the tumour microenvironment. It is therefore of critical importance to modulate the level of extracellular ROS for the reversal of immunosuppressive environment. Here, we present a tumour extracellular matrix (ECM) targeting ROS nanoscavenger masked by pH sensitive covalently crosslinked polyethylene glycol. The nanoscavenger anchors on the ECM to sweep away the ROS from tumour microenvironment to relieve the immunosuppressive ICD elicited by specific chemotherapy and prolong the survival of T cells for personalized cancer immunotherapy. In a breast cancer model, elimination of the ROS in tumour microenvironment elicited antitumour immunity and increased infiltration of T lymphocytes, resulting in highly potent antitumour effect. The study highlights a strategy to enhance the efficacy of cancer immunotherapy by scavenging extracellular ROS using advanced nanomaterials. Reactive oxygen species in the tumour microenvironment can have an immunosuppressive effect. Here, the authors devise a nanoparticle that anchors to the extracellular matrix within tumours and scavenges reactive oxygen species, resulting in an enhanced immune response within the tumour. Recent studies revealed that immunogenic cell death (ICD) elicited by specific chemotherapy or radiotherapy makes the dead cell corpses ‘visible’ to dendritic cells (DCs) that present antigens to T cells with specific antitumour immune responses, which then control residual tumour cells9. However, ICD induced immune response can be severely weakened and even abolished by elevated reactive oxygen species (ROS) in the tumour microenvironment (TME)11. In the meantime, T cells become dysfunctional after reaching the tumour site16. Therefore, how to modulate the level of extracellular ROS is utterly important to reverse the immunosuppressive nature of the TME.Cancer immunotherapies that stimulate the inherent immunological systems of the body to recognize, attack, and eradicate tumour cells have demonstrated varying degrees of success21. Under the potent ICD-inducing therapies, damage-associated molecular patterns (DAMPs) are secreted from dying cells or exposed on the outer layer of the cell membrane to facilitate immune responses. Release of adjuvant high mobility group protein B1 (HMGB1) as “find me” signals is essential to induce DC maturation24. However, HMGB1 is often oxidized by ROS in the TME and its stimulatory activity is thus neutralized11. Although there have been strategies reported to enhance tumour cell death in an immunogenic way by increasing the amount of released HMGB1 into the TME, the fate of released HMGB1 has received limited attention25. Therefore, how to keep the stimulatory activity of HMGB1 in the TME is a major challenge. Despite the potential, the antitumour immunity triggered by ICD is also limited, which is mainly due to the dysfunctional T cells in the TME. Elevated extracellular ROS controls immune regulation and reduces the proliferation and antitumour function of T cells29. Therefore, strategies to extend the survival of T cells and recover immune functions by scavenging ROS in the TME are urgently needed.ICD provides dying cancer cells with stimuli to elicit immune responses as a tumour vaccineECM-NS) modified with ECM targeting peptide. Dual-benzaldehyde terminated polyethylene glycol as a caging polymer was introduced to construct the crosslinked “stealth” delivery system (PEG-TECM-NS) with pH sensitive imine bonds. This intelligent nanoscavenger can sweep away the ROS from the TME to relieve the immunosuppressive ICD elicited by oleandrin (OLE) anticancer drug and prolong the survival of T cells for “personalized” cancer immunotherapy   was prepared by automated solid-phase peptide synthesis at a 100 µmol scale on a rink amide resin using standard Fmoc-based protocols30  was conjugated onto the surface of PEI-PPS nanoparticles through an amidation reaction, termed as TECM-NS  were prepared and crosslinked by the PEGylation of CHO-PEG-CHO via pH sensitive benzoic imine bond on the surface of TECM-NS. The coating efficiency of PEG on the surface of nanoparticles was about 100%   incubation  (PPS) was synthesized via anionic polymerization of propylene sulfide using 3-mercaptopropionic acid as an initiator  Fig. . TEM reszer Fig. . As showPEG Fig. . BecauseECM-NS were evaluated. The characteristics of OLE loaded PEG-TECM-NS were summarized . The significantly increased OLE release from PEG-TECM-NS at pH 6.8 with 10 mM H2O2 . PEG was also coated on the surface of TECM-C18 (PEG-TECM-C18). Parallel studies showed that PEG-TECM-C18 was incapable of scavenging ROS . The supernatant was freeze dried and the PEG content was measured by 1H NMR  using biotinylated nanoparticles and substrate coated with rat tail collagen type I  is a sensitive technique that measures the heat of reaction of two aqueous solutions when one is titrated against the other35. As shown in Fig. ECM-NS and TECM-NS in the heterogeneous environment of 4T1 murine mammary tumour. After 4T1 tumour pieces (1 cm3) were incubated with TECM-NS and PEG-TECM-NS at pH 7.4 and pH 6.8, respectively, confocal laser scanning microscopy (CLSM) images of the sectioned slices from the 4T1 tumour were examined. The complete overlap of TECM-NS or PEG-TECM-NS signal at pH 6.8 with anti-collagen antibody further confirmed the collagen specificity of TECM-NS  as “eat me” signal. Therefore, the ability of PEG-TECM-NS/OLE to induce ICD was determined by examining the HMGB1 release and CRT exposure by immunofluorescence staining. As shown in Fig. ECM-NS/OLE at pH 6.8 with 100 µM H2O2 induced more extracellular release of HMGB1 than that of PEG-TECM-NS/OLE without pretreatment. Meanwhile, pretreatment of PEG-TECM-NS/OLE at pH 6.8 with 100 µM H2O2 had the similar ability to induce extracellular release of HMGB1 to that of free OLE. These results indicated that PEGylated TECM-NS/OLE with crosslinked structure could restrain drug leakage in physiological environment. The combination of the pH sensitive cleavage of the benzoic imine with de-shieldable PEG and ROS mediated hydrophilic transition of thioether induced disruption of nanoparticles could rapidly trigger drug release. Furthermore, HMGB1 release also confirmed that free OLE and PEG-TECM-NS/OLE could efficiently induce immunogenicity of the tumour cells. Released HMGB1 in supernatant of 4T1 cells was measured by ELISA  via ICD. 4T1 tumour cells were treated with free OLE, PEG-TECM-NS/OLE, and PEG-TECM-NS/OLE pretreated at pH 6.8 with 100 µM H2O2 for 12 h. Then, Bone marrow dendritic cells (BMDCs) were co-cultured with pretreated 4T1 tumour cells for another 24 h. The frequency of CD80+CD86+ mature BMDCs after co-culture with 4T1 tumour cells pretreated with free OLE was significantly higher that of the control group, indicating that ICD from dying 4T1 cells elicited by OLE can induce DC maturation and immune response  and tumour necrosis factor α (TNF-α) as indicators of DC activation elicited by ICD from dying tumour cells were determined by ELISA  were higher than those of the control group, confirming that OLE could induce strong antitumour immunity. PEG-TECM-NS/OLE with pretreatment induced higher level of IL-12p40 and TNF-α than PEG-TECM-NS/OLE without pretreatment, suggesting that the release of OLE was significantly accelerated at acid environment in the presence of H2O2 due to the pH-induced PEG de-shielding and ROS induced hydrophilic transition of thioether, resulting in the disruption of nanoparticles. Mice were immunized intravenously with the tumour lyste three times. Lymph nodes were then harvested to measure the percentages of CD80+CD86+ DCs. The results verified that PEG-TECM-NS/OLE pretreated with pH 6.8 buffer and 100 µM H2O2 could induce the maturation of DCs  and then incubated with or without 100 µM H2O2 or PEG-TECM-NS. T-cell proliferation was measured by carboxyfluorescein (CFSE) dilution , the suppressive effect of H2O2 on T-cell proliferation of was partially abrogated, showing that PEG-TECM-NS can scavenge extracellular ROS to extend the survival of T cells and recover immune responses. We also found H2O2 concentration dependent inhibition of T-cell proliferation  and ICG-labelled PEG-TECM-NS was detected by photoacoustic imaging  and analysed by ELISA. As shown in Fig. ECM-NS/OLE. The result indicated that PEG-TECM-NS/OLE can elicit effective ICD. As shown in Fig. ECM-NS/OLE was obviously elevated, which agreed well with the result in Fig. ECM-NS/OLE markedly reduced the number of Ki67 proliferating tumour cells and increased the number of dUTP nick-end labelling (TUNEL)-positive tumour cells . After stirring for 24 h, the mixture was dialyzed. Then the aqueous solution of CHO-PEG-CHO was added to TECM-NS solution to coat PEG on the surface of TECM-NS through benzoic bonds.PEI-PPS was prepared by a solvent displacement method. Typically, the solution of dimethyl sulfoxide (DMSO) containing 10 mg PEI-PPS polymer was added dropwise to water. The mixture was dialyzed to remove DMSO at room temperature. Then T−1 in 20 mM acetic acid) in TBS  to rows A–D on a 96-well plate . The plates were incubated overnight at 4 °C on a rotating table, then inverted, emptied, and washed with TBS (3 × 300 µL). All the wells (i.e. rows A–H) were then incubated with 100 µL of TBS containing 5% BSA for 2 h at 37 °C, and subsequently washed with 3 × 300 µL of TBS containing 0.1% Tween-20. The plates were incubated with the PEG-TECM-NS for 3 h. The fluorescence was measured.Collagen-coated plates were prepared by adding 50 µL of a 1:100 dilution of rat tail collagen type I (2.78 mg mLECM-NS pretreated with pH 6.8 buffer and 10 mM H2O2 after centrifugation were added and incubated with cells. Then 4T1 cells were stained with Annexin V-FITC (5 µL for one sample)/7-AAD (5 µL for one sample)  for 30 min at room temperature and then added 500 µL binding buffer to analyze by a flow cytometry.4T1 Cells were seeded in six well plates. The supernatant of PEG-TAll animal experiments were performed under a National Institutes of Health Animal Care and Use Committee (NIHACUC) approved protocol. BALB/C mice  were subcutaneously implanted with 1 × 106 4T1 cells. 4T1 cells (ATCC) were authenticated for mycoplasma.−1) and IL-4 (10 ng mL−1) at 37 °C for 7 days to acquire immature DC. 4T1 cells were pretreated with free OLE, PEG-TECM-NS/OLE, and PEG-TECM-NS/OLE pretreated with pH 6.8 buffer and 100 µM H2O2. Then, immature BMDCs (1 × 106) were co-cultured with 4T1 cells (1 × 105) for 24 h. After various treatments, DCs stained with antibodies  for 30 min at 4 °C and measured by a flow cytometer. Anti-CD11c-APC-Cy7, anti-CD80-FITC, and anti-CD86-PE were diluted according to the manufacturer’s direction (1:500).BMDCs were isolated from the femur of BALB/C mice. The bone marrow cells were harvested and treated with ACK lysis buffer for 5 min. The cells were washed twice with PBS. The bone marrow was cultured in medium with GM-CSF  and H2O2 were added. After 3 days, the CFSE signal of gated splenocytes was analyzed by a flow cytometer.Splenocytes were isolated and incubated for 4 h. We collected the cells and labelled with CFSE (1 mM) for 5 min. Then, 0.6 mg mL−1) with 0.2 mg mL−1 DNase I for 1 h at 37 °C. Suspensions were filtered and the single cells were stained with fluorescently labelled antibodies . Anti-CD4-PE-Cy5, anti-CD8-PE-Cy7, and anti-CD25-Alexa Fluor ®700, anti-CD62L-APC, anti-CD44-PerCP, anti-Foxp3-PE, and anti-IFN-γ-PercCP/Cy5.5 were diluted according to the manufacturer’s direction (1:500).The harvested tumours were explanted and cut into small pieces. Then, the small pieces were immersed in 5 mL collagenase IV (1 mg mLECM-NS with crosslinked structure (DFO-PEG-TECM-NS). As a control, we also prepared N-PEG-TECM-NS without crosslinked structure DFO-N-PEG-TECM-NS. Zr89-DFO-PEG-TECM-NS, and Zr89-DFO-N-PEG-TECM-NS were intravenously injected into the 4T1 tumour-bearing mice. An Inveon small-animal PET scanner  was used to acquire whole-body PET images at predetermined time points after injection.Firstly, we prepared DFO modified PEG-TECM-NS with crosslinked structure (DOTA-PEG-TECM-NS). As a control, we also prepared N-PEG-TECM-NS without crosslinked structure DOTA-N-PEG-TECM-NS. Gd3+-DOTA-PEG-TECM-NS and Gd3+-DOT -N-PEG-TECM-NS were intravenously injected into the 4T1 tumour-bearing mice. A high magnetic field micro-MR scanner  was used for the scanning at indicated time points after injection.Firstly, we prepared DOTA modified PEG-TECM-C18/OEL, and PEG-TECM-NS/OLE . Then the tumour volume was calculated using the formula: tumour volume (mm3) = (length × width2) × 1/2.BALB/C mice were inoculated with 4T1 tumour cells. The mice were randomly grouped and treated with PBS, free OLE, PEG-T6) was injected into the caecum wall. Then the incision was sutured. The mice were randomly grouped and treated with PBS, free OLE, PEG-TECM-C18/OEL, and PEG-TECM-NS/OLE . Mice were sacrificed by cervical dislocation after treatment. Spleen, tumour, and main organs were collected for immunofluorescence and flow cytometry analysis.Mice were anesthetized with isoflurane. A small incision was made to exteriorize the caecum. CT26 cell suspension  from. The significant differences between two groups were evaluated by Student’s unpaired Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary