To describe the management of patients with occult anterior uveal melanomas presenting with extrascleral extension.Retrospective case series including five patients with small pigmented nodular mass on the episclera. Each lesion was documented by slit-lamp photography and measured with high-frequency ultrasound imaging (ultrasound biomicroscopy). Diagnosis of uveal melanoma was confirmed by biopsy with lamellar sclerectomy. Immediate scleral patch graft repair was performed. Later, each tumour was treated with palladium-103 ophthalmic plaque brachytherapy. The mean plaque diameter was 12 mm . A mean apex prescription dose of 87 Gy  to a tumour depth of 2 mm from the inner sclera delivered over 7 continuous days. The main outcome measures were best-corrected visual acuity, changes in tumour and scleral characteristics and complications.During each surgery, residual tumour was visualised within an emissary passageway at the deep plane of scleral resection. At a mean of 80 months  follow-up, no patients experienced graft infection, scleromalacia or rejection. Biopsy was required to establish the diagnosis, transillumination failed, and therefore ultrasound measurements were used to determine the plaque size required to treat the relatively occult intraocular component. Despite these challenges, there were no cases of local tumour recurrence, secondary enucleation or metastatic disease. Attributed to cataract surgery, visual acuities improved in three patients and two were stable.Extrascleral uveal melanoma extension can occur with undetectable, occult intraocular tumours. In these cases, plaque radiation effectively induced local tumour control, preserved vision and prevented metastasis. Anterior extrascleral melanoma extension has been shown to occur in association with an easily recognisable underlying tumour.This study shows it can occur with an occult uveal melanoma, is diagnosed by scleral tumortumour biopsy and is treatable with palladium-103 plaque radiation therapy.This knowledge will help physicians with early intervention, thereby, saving the sight, eye and life of patients with occult uveal melanoma presenting with extrascleral extension. In addition, the literature and staging according to the 8th edition American Joint Committee on Cancer (AJCC) suggests that the presence of extrascleral extension (ESE) ≥5 mm increases the risks of metastasis and death.Uveal melanoma is the most common primary intraocular malignancy and represents 5% of all melanomas diagnosed in the USA.Extraocular extension can be primary due to tumour growth or secondary to prior surgery, inflammation, laser or radiation damage.Most uveal melanomas can be diagnosed based on clinical presentation and imaging.Herein, we describe the diagnosis of and plaque treatment for a clinical case series of small extrascleral uveal melanomas with occult intraocular tumours.This is a retrospective, interventional, non-comparative case series of five patients with uveal melanoma presenting with ESE. All patient records were available for review. Data recorded at initial visit included the patient’s medical history, clinical findings and ancillary tests including ultrasonography (B-scan), ultrasound biomicroscopy (UBM), optical coherence tomography (OCT), photography  and fundus fluorescein angiography, where indicated. In addition, patient’s systemic evaluation, operative records, histopathological results, follow-up period, complications and final visual outcomes were recorded. All cases were performed by a single surgeon (PTF). Main outcomes were defined as best-corrected visual acuity, surgical complications, local control and metastatic disease.All patients underwent tumour and partial thickness scleral diagnostic biopsy. A #57 Bard-Parker blade was used to create a rectangular box, allowing at least 2 mm margins around the edges of the extrascleral tumour nodule . Then a Histopathological features of all cases were analysed for cell type, presence of melanin, nuclear grade, tumour-infiltrating lymphocytes and necrosis. The patients were informed about the pathology result and the risks associated with untreated residual AJCC T1 intraocular melanomas. Therefore, an initial whole-body positron emission tomography-CT (PET/CT) staging was performed.103Pd and 125I radioactive plaque placement for uveal melanoma as covering the tumour plus a 2–3 mm margin of normal appearing tissue.The 2014 American Brachytherapy Society (ABS) consensus guidelines defined normal 103Pd plaque or 125I had been used. Additional plaque parameters included: plaque size, type of plaque, apical tumour dose and hours of irradiation. Critical ocular structures were defined as the tumour apex, subjacent sclera, lens, fovea, optic disc and opposite eye wall.For the purposes of this study, we calculated and compared the intraocular radiation dose distribution of these patients as if both Ophthalmic examinations were performed every 4–6 months. This included but was not limited to evaluation of the tumour bed documented by slit-lamp and gonio-photography as well as UBM. The posterior segments were evaluated with extended indirect ophthalmoscopy, fundus photography and low-frequency ultrasound imaging. Fluorescein angiography and OCT were performed every 6 months to monitor for radiation vasculopathy. At each visit, test results were compared with the patient’s prior test-examinations for evidence of change. Post-treatment systemic surveillance involved abdominal radiographic imaging every 6 months for the first 5 years and then yearly.Over 36 years of clinical practice, only five patients presented with this unusual form of extrascleral uveal melanoma extension. Their mean age was 52 years . The mean follow-up duration at last visit was 80 months . One patient had a history of breast cancer and one cutaneous basal cell carcinoma. All the tumours were pigmented. Two lesions were temporal and three were nasal .Pretreatment tumour characteristics of the ESE are found in Of interest, the mean pretreatment largest basal intraocular tumour dimensions as measured by UBM were 3.6 mm  and the mean pretreatment tumour height was only 1.5 mm . UBM of the tumour for case #3 showed 1.4 mm thickness  which reAccording to the 8th edition AJCC staging system for uveal melanoma, the category of anatomic extent for all 5 (100%) tumours was T1cN0M0 (stage IIA) in that extraocular extension was ≤5 mm in all patients.A histopathological diagnosis of uveal melanoma was established in each case. Histopathological evaluation of the melanomas revealed that 40% (n=2/5) contained epithelioid cells and 60% (n=3/5) were mixed cell tumours. The mean tumour depth on the histopathological specimen was 0.6 mm . The mean largest histopathological basal tumour dimension was 2 mm . The channels between the ESE and the uvea were not seen on histopathology. As was thought due to the typically diagonal path of emissary vessels through the sclera.Before resection, the mean uveal melanoma depth  beneath the biopsy site by UBM was 1.5 mm . In consideration of their small size, we used a minimum axial intraocular radiation prescription distance of 2.0 mm (from inner sclera) in all cases. The tumour’s basal dimensions were based on the UBM appearance of the uveal shadows noted on transillumination prior to biopsy. Mean plaque diameter was 12 mm . Due to the presence of pigmented tumour against the pigmented ora serrata, intraoperative tumour transillumination was not positive, hence larger plaque sizes, with resultant larger margins were used (as to cover any occult tumour component).103Pd and 125I) intraocular dosimetry was performed using the radionuclides available in our centre.103Pd in every case.Then, according to current practice and as reflected in the ABS consensus guidelines, when and extraocular nodule is small, globe sparing plaque irradiation was performed.103Pd tumour dose of 87 Gy  was delivered. The mean dose to sclera was 155.8 Gy . The mean radiation doses to lens, optic nerve and fovea were 28.6 Gy , 2.2 Gy  and 1.9 Gy , respectively . Myopia was present in 80% (n=4/5) of patients. The post-treatment vision improved to a mean of 20/16  at a mean follow-up of 80 months attributed to three patients having interim cataract surgery during the follow-up period.All patients were phakic at the time of treatment. Three (60%) developed radiation-induced cataract and underwent cataract surgery.In this series, local tumour control (no recurrence) was achieved in 100% patients. Eye retention was seen in all patients. No patient developed metastasis at the last follow-up.This series analysed the results of five cases of ESE with occult subjacent uveal melanomas. Biopsy was required to establish the diagnosis. Scleral grafts were placed for tectonic ocular stability, to prevent treatment-induced scleral thinning and for cosmesis. All patients were at risk for scleral thinning as biopsy involved a lamellar scleral dissection followed by episcleral plaque radiation therapy. Treatment resulted in vision retention, local cancer control and lack of metastatic disease. Cataract formation and vision retention were related to the anterior ocular locations of the tumours and thus the radiation source. This resulted in high mean doses to the lens (28.6 Gy) and low doses to the macular fovea (1.9 Gy) as well as the optic nerve (2.2 Gy). We attribute the high rates of local control and lack of metastases to the small tumour sizes (cT1 category by AJCC staging) as well as the use of relatively large treatment margins.et al used 125I for 19 patients with uveal melanoma with ESE, where tumour recurrence rates and survival rates were not adversely affected.et al also used 125I in a single patient resulting in local control for 2.5 years.et al used plaque brachytherapy in eight patients with extrascleral tumour thicknesses varying from 1 to 10 mm. During 10 years follow-up, they noted one local recurrence and three patients with metastasis.106Ru in a single patient with ciliary body melanoma and ESE who showed no local recurrence at 30 months after treatment.There exist multiple reports of management of small uveal melanomas with ESE .30–32 BeIn contrast to those studies, our Medline and PubMed searches using the terms: extrascleral, uveal melanoma, diagnostic biopsy and brachytherapy, revealed no cases of epibulbar ESE with occult subjacent uveal melanomas and thus no consensus on their treatment. Contrary to reports that suggest ESE is usually present with the presence of a large uveal melanoma, our series demonstrated that a small, and thus benign looking pigmented epibulbar tumour could be a potentially life-threatening condition.et al suggested that small tumours with extraocular extension may have originated from melanocytes which are present in the natural emissary channels along arteries, veins and nerves.Uveal melanomas are known to invade sclera through different intrascleral routes.However, the lack of metastatic disease at a mean follow-up of 80 months in our series supports that tumour size (AJCC T-size) at presentation is a significant factor for the prediction of metastasis.The tumour’s basal dimensions were based on the UBM and subjective appearance of the uveal shadows. In this series, UBM was required to make the diagnosis due to the small ‘occult’ intraocular components. Tumour thickness could only be determined by UBM. Both outpatient clinical and intraoperative transillumination were unreliable in these cases, mostly due to the presence of pigmented tumour that lacks contrast with the pigmented ciliary body band. Therefore, relatively large plaque sizes were used so as to cover any missing occult tumour component.Presence of myopia in 80% patients, in this series, suggests that there could be a component of scleral thinning induced by myopia associated with ESE. However, as the literature reveals no such correlation, this finding might require further research.103Pd plaque study.The mean radiation dose to lens was 28.6 Gy, which is similar to the dose causing cataract in our prior The limitations of this study are its retrospective nature, small sample size, variable follow-up and lack of control group. However, this observational case series offers unique information including our surgical approach in consideration of variable and somewhat uncertain basal tumour diameters.103Pd plaque radiation therapy. Our patient’s clinical courses were found to be uncomplicated in that scleral grafting was performed at the time of biopsy, and subsequent 103Pd brachytherapy controlled the occult uveal melanomas.In summary, herein is described a case series of small uveal melanomas presenting with ESE, treated by excisional biopsy combined with scleral grafting and staged postoperative A search of the MEDLINE database was conducted using the keywords extrascleral, uveal melanoma, diagnostic biopsy and brachytherapy. Additional references were culled from the bibliographies of these references. These references were evaluated for their pertinence to the topic, with special consideration given to papers that detailed clinical outcomes of extrascleral tumours.


Meningoencephalomyelitis and visceral dissemination infection are rare but life-threatening complications of either the primary infection or reactivation of varicella-zoster virus (VZV) in immunocompromised patients. To date, few studies have reported the co-existence of VZV meningoencephalomyelitis and the visceral dissemination of VZV infection.A 23-year-old male was diagnosed with lupus nephritis class III and was being treated with oral prednisone and tacrolimus. The patient exhibited herpes zoster 21-day after the initiation of therapy and experienced unbearable abdominal pain and generalized seizures 11 days after the onset of a zoster rash. Magnetic resonance imaging showed progressive lesions in the cerebrum, brainstem, and cerebellum, as well as meningeal thickening and thoracic myelitis. Computed tomography showed pulmonary interstitial infiltration, partial intestinal dilatation, and effusion. Metagenomic next-generation sequencing revealed 198,269 and 152,222 VZV-specific reads in the cerebrospinal fluid and bronchoalveolar lavage fluid, respectively.Based on the clinical and genetic findings, this patient was finally diagnosed with VZV meningoencephalomyelitis and visceral disseminated VZV infection.The patient received intravenous acyclovir (0.5 g every 8 hours) combined with plasma exchange and intravenous immunoglobulin. Treatment against secondary bacterial and fungal infections, organ support therapy and rehabilitation training were given simultaneously.The patient’s peripheral muscle strength did not improve and repeated metagenomic next-generation sequencing showed the persistence of VZV-specific reads in the cerebrospinal fluid. The patient finally abandoned therapy due to financial constraints at the 1-month follow-up.Patients with autoimmune diseases receiving immunosuppressive therapy should be warned about the possibility of developing serious neurological infections and visceral disseminated VZV infections as side effects. Early diagnosis and the early initiation of intravenous acyclovir therapy are important for such cases. Patients suffering from meningoencephalomyelitis usually have a worse prognosis with a multitude of neurological sequelae.–11 Visceral disseminated VZV infection is another rare but life-threatening complication of VZV infection.,13 Visceral organ involvement may present as severe abdominal pain, hepatitis, pancreatitis, pneumonia, and other symptoms.,14–16 Herein, we report a rare case of severe VZV meningoencephalomyelitis with visceral disseminated VZV infection in a patient with lupus nephritis (LN) by metagenomic next-generation sequencing (mNGS).CNS infection caused by VZV typically manifests as vasculopathy, encephalitis, cerebellitis, meningitis, and myelitis, with or without rash.A 23-year-old male was first admitted to the hospital on April 26th, 2022, due to having developed a malar rash and experiencing swelling of the lower extremities for 1 week. Physical examination revealed facial butterfly erythema, enlarged lymph nodes, periungual vasculitis-like rash, bilateral lower extremity edema, and normal blood pressure. Auxiliary examination  analysis showed a white blood cell count of 78 × 106 cells/L (84.6% neutrophils), glucose level of 9.5 mmol/L, and protein level of 5.03 g/L. No significant pathogens were isolated in the CSF culture. The CSF sample and bronchoalveolar lavage fluid (BALF) sample were sent for mNGS analysis to identify the responsible pathogen on June 1st, 2022. Written informed consent was provided by the patient’s parents.The patient was readmitted to the hospital on May 28th, 2022, presenting with a painful rash with blisters on the left thoracodorsal area for 1 week. Physical examination conducted on the second admission showed a large band-like rash with vesicles in the left thoracic and lumbar dermatomes   sequences were detected in the CSF sample (42 sequence reads) . The results obtained by mNGS conducted on the CSF sample detected 2677 unique deoxyribonucleic acid (DNA) reads of VZV and had a genomic coverage of 64.34%. Only 1 CMV-specific read was detected in the CSF sample . Furthermore, the patient still complained of experiencing abdominal pain, while abdominal CT showed partial intestinal dilatation and intestinal effusion. A lumbar puncture was re-performed to observe cytologic and protein changes in the CSF and to allow for mNGS testing on June 17th, 2022. The analysis of the CSF showed a decreased number of white blood cells and decreased protein levels  were negative both in the serum and CSF. The detection of identical oligoclonal immunoglobulin G bands in the serum and CSF confirmed the disruption of the blood-CSF barrier. A third lumbar puncture was performed on July 8th, 2022. The CSF analysis showed that the white blood cell count decreased to 8 × 1019% Fig. D. As the,17 Cell-mediated immunity plays an important role in the prevention of VZV reactivation in adults. Patients affected by autoimmune diseases like SLE have a significantly increased risk of VZV reactivation. Treatment with glucocorticoids and immunosuppressants may further increase this risk.,19 Moreover, immunocompromised individuals are more likely to present rare but potentially lethal manifestations, such as meningoencephalomyelitis and disseminated visceral involvement. VZV meningoencephalomyelitis has been reported to be associated with the direct viral invasion of the spinal cord and retrograde viral transport to the brain.,22 Meanwhile, the spread of VZV from the dorsal root ganglion toward the viscera may be related to visceral disseminated VZV infection.,23 In the present case, the patient received oral prednisone and tacrolimus from the time of being diagnosed with LN class III. To avoid relapse, the doses of the steroids and immunosuppressants were not reduced immediately when the patient achieved complete remission of proteinuria within a week. Although the time interval between the initiation of immunosuppressive therapy and the onset of the zoster rash was only 21 days, the induction therapy caused a marked decrease in the lymphocyte counts of this patient. Notably, lymphocytopenia (<1000 cells/μL) is considered an independent risk factor for the development of severe infections in patients with SLE.VZV is a highly infectious herpesvirus that causes varicella during primary infection and HZ during reactivation. Regrettably, the application of the quantitative polymerase chain reaction to VZV DNA in the CSF was not performed during the patient’s disease course. We only speculate that our patient may have had a high viral load of VZV in the CSF at the time of the first presentation, as there were nearly 2 hundred thousand VZV-specific reads detected by mNGS in the CSF. In addition, this number of VZV-specific reads decreased after continuous antiviral treatment, which was consistent with the changes observed in the biochemical indexes of the CSF after therapy.Our patient originally suffered from a zoster rash 1 week before the onset of abdominal pain and generalized seizures. The association between the neurological symptoms and HZ was properly established by mNGS to identify specific VZV sequences in the CSF. Although trace CMV-specific reads were also detected in the CSF, they almost turned negative after 14 days of ganciclovir treatment. In contrast, the patient had persistent VZV-specific sequences in the CSF along with progressively intracranial lesions and thoracic spine myelitis on MRI. Therefore, the diagnosis of meningoencephalomyelitis caused by VZV was considered. Moreover, the large number of VZV-specific reads detected by mNGS in the BALF provided crucial evidence for the disseminated seeding of the lung, leading to the inference that the unexplained abdominal pain was caused by disseminated VZV seeding the intestine. Studies have reported that high viral loads of VZV are associated with a higher incidence of severe intracranial lesions and more frequent neurological sequelae. Several studies have recommended intravenous acyclovir at a dose of 10 to 15 mg/kg 3 times daily for 14 to 21 days in such cases.,28 It has also been suggested that treatment be discontinued when the CSF tests negative for VZV DNA. Although our patient was treated with intravenous acyclovir for 55 days in conjunction with plasma exchange and intravenous immunoglobulin, VZV-specific sequences were still detected in the CSF. The persistent presence of VZV-specific reads in the CSF may also be associated with the poor prognosis of this patient.Intravenous acyclovir has been widely accepted as the primary therapy for disseminated VZV infection and severe VZV CNS infection. The early use of antiviral drugs at the onset of initial symptoms is recommended to improve prognosis. The patient started antiviral therapy eleven days after the onset of the zoster rash, and this may be partly responsible for their poor prognosis. The optimum dose and duration of antiviral therapy remain unknown.In conclusion, we have reported a rare case of VZV-induced severe meningoencephalomyelitis and disseminated visceral infection in a patient with LN treated with tacrolimus and high doses of glucocorticoids. Patients with autoimmune diseases receiving immunosuppressive therapy should be warned about the increased risk of develop VZV infection and reactivation. mNGS is essential for the early diagnosis of insidious neurological and visceral disseminated VZV infections in immunocompromised patients, and the early initiation of intravenous acyclovir therapy is important for such cases.Conceptualization: Tian Tao, Jun Chen.Data curation: Kunlan Long, Lijia Zhi, Yuexian Ma, Hong Yan.Formal analysis: Song Zhang, Shuqin Liu.Methodology: Lizeyu LV, Yue Xu.Project administration: Liangbin Zhao, Peiyang Gao.Software: Ling Wu.Writing - original draft: Tian Tao, Jun Chen.Writing - review & editing: Liangbin Zhao, Peiyang Gao.


This study aims to analyse lung tumour motion and to investigate the correlation between the internal tumour motion acquired from four-dimensional computed tomography (4DCT) and the motion of an external surrogate.A data set of 363 4DCT images was analysed. Tumours were classified based on their anatomical lobes. The recorded gross tumour volume (GTV) information included the centroid GTV motion in the superior–inferior, anteroposterior and left-right directions, and in three-dimensions (3D). For the internal/external correlation, the RPM surrogate breathing signals of 260 patients were analysed via an in-house script. The external motion was correlated with the 3D centroid motion, and the maximum tumour motion via Spearman’s correlation. The effect of tumour volume on the amount of motion was evaluated.r = 0.21) and moderate in the middle (r = 0.51) and the lower (r = 0.52) lobes. There was no obvious difference in the correlation coefficients between the maximum tumour displacement and the centroid motion. No correlation was found between the tumour volume and the magnitude of motion.The greatest 3D tumour amplitude was found for tumours located in the lower part of the lung, with a maximum of 26.7 mm. The Spearman’s correlation of the internal 3D motion was weak in the upper (Our results suggest that tumour location can be a good predictor of its motion. However, tumour size is a poor predictor of the motion.This knowledge of the distribution of tumour motion throughout the thoracic regions will be valuable to research groups investigating the refinement of motion management strategies. Furthermore, the motion-encompassing approach can be performed by acquiring slow CT and inhalation/exhalation BH CT. The gating technique is another way of managing respiratory motion and refers to the process of tracking the respiratory cycle in order to deliver the radiation dose to a certain portion. Gating the radiation beam can be achieved by tracking fiducial markers (FMs) as internal surrogates, or based on the use of external surrogates. Finally, four-dimensional CT (4DCT) images can be acquired to account for respiratory motion.The accuracy of radiation dose delivery is compromised by organ motion for tumours in the thoracic cavity. To accommodate tumour motion, wide treatment margins are required. However, increased treatment toxicity is associated with widened margins. Numerous motion management techniques aimed at minimising the extent of the motion, generating a spatiotemporal data set of the patient’s anatomical structures. Stereotactic ablative radiotherapy (SABR) is commonly used for early-stage non-small cell lung cancer (NSCLC), with targets of a maximum diameter less than 5 cm. Larger maximum target diameters have also been reported, however, there are insufficient data on the safety and efficacy for SABR in this setting at present. SABR has also been used for intra- and extracranial metastases to achieve long-term local control. SABR treatment for early stage NSCLC has been shown to offer superior treatment outcomes with high local control rates and manageable toxicities both as single- and multifraction regimens compared with conventional radiotherapy.4DCT has the potential to account for respiratory motion during the entire treatment process, starting from the image acquisition to the treatment delivery. It allows the patient’s internal organ motion to be quantified as a function of the respiratory cycle by acquiring oversampled data for each acquired slice, However, for lung SABR, the internal target volume (ITV) is created to account for the movement of the GTV during breathing  with reduced margins, in order to minimise the volume of tissues exposed to the higher radiation dose. The clinical target volume (CTV), a margin introduced to account for the microscopic expansion of the disease in conventional treatments, is not typically used in SABR.reathing .8 in which an external marker is also used to monitor patient breathing during the image acquisition for the radiotherapy planning scan. The accuracy of the correlation between the external marker, which is assumed to be directly related to the diaphragm motion, and the internal tumour position is a crucial question if the tumour motion is to be tracked indirectly.One of the most widely adopted approaches for taking respiratory motion into account for thoracic SABR treatments is 4DCT, However, there is a paucity of data on the correlation between the external surrogate and the internal motion defined using 4DCT for a large cohort of patients. Willmann et al investigated the internal/external correlation using a fiducial marker and external surrogate in 4DCT and found that fiducial markers provided better internal motion prediction compared to the external surrogate method.The correlation between the motion of the diaphragm and external surrogate have been intensively studied using fluoroscopy, based on the assumption of a direct correlation between the tumour and motion of the abdominal surrogate.The present study was conceived to examine respiratory-induced lung tumour motion and its correlation with abdominal movement as monitored by the external marker. To this end, a novel method to analyse the RPM external marker signal was developed and applied to test the correlation of tumour motion across a range of patient and tumour characteristics.363 lung cancer patients who underwent SABR treatment between July 2013 and September 2020 at the Northern Ireland Cancer Centre (NICC) were selected for this retrospective study. The candidates underwent free-breathing (FB) 4DCT as a part of their treatment with no additional selection criteria. Patients with multiple masses contoured as a single structure were excluded from this study. The use of the data within this study population was approved by the Belfast Health and Social Care Trust (BHSCT) with research governance permission number 19067CMcG-SS, REC Ref 20/HSC/0002.Free-breathing 4DCT data sets were generated using the GE large-bore Optima 580 and Discovery 590 RT  machines. The scanners were coupled with the Varian RPM v. 1.6  for monitoring patient breathing. The RPM system uses an infrared tracking camera that captures the reflected signal in the anteroposterior (AP) direction from a lightweight marker block placed on the patient’s abdomen. The CT reconstruction software retrospectively uses the RPM signal to rearrange the breathing sequence of the acquired volumetric data.i.e. the planning target volume.Scanners acquired 4DCT with the cine acquisition protocol for all patients. With the aid of the Advantage 4D workstation , the phase binning approach was applied to bin the slices into 10 breathing phases, with the 0 and 50% phases corresponding to the end-inhalation and end-exhalation phases, respectively. Patients were imaged with a 4DCT protocol of 120 KV, 250 mA, 0.5 s tube rotation time, and slice thickness of 2.5 mm. The 4DCT data sets were imported into the Varian Eclipse treatment planning system (TPS) v. 16.01 . The tumour segmentations, were completed on the phases 0% , 50% , and the maximum intensity projection image (GTV-MIP). From these, a composite ITV structure was generated, reflecting a motion-compensated GTV. Dose prescriptions were made to the ITV plus a further 5 mm margin,  Tumour classification based on the attachment to rigid structures was performed by a clinical oncologist. Given that tumour motion is secondary to the unrestricted inflation of lung, tumours deemed to be possibly invading into the chest wall were defined as ‘attached’, and labelled ‘free’ if not, so that restricted motion could be accounted for.Tumours were classified, based on their location in the pulmonary zone, into upper, middle and lower, by dividing the lung into three regions of approximately equal height in the sagittal plane. The tumours were also classified based on the anatomical lung lobes, by direct visualisation of the lung fissures. ie. right upper, right middle, right lower lobes and the left upper and lower lobes. No left and right lung tumour grouping was made, as it was previously shown that there is no significant difference in the mean motion amplitude between left and right lungs.0%-50% motion) was calculated using equation 1, along with the motion in the superior-inferior (SI), AP, and left-right (LR) directions (The coordinates of the centre of mass at phases 0% (GTV0%) and 50% (GTV50%) were recorded .A. The crections .B.In this equation,  and  are the coordinates of the centre of the tumour in phase 0% and phase 50%, respectively. A patient’s breathing characteristics can influence the position of the end-inhalation and end-exhalation. Indeed, phase 0% is not always the phase of maximum inhalation, and similarly, phase 50% is not always the phase of maximum exhalation for some patients. To account for this variation, the full extent of the tumour motion was accounted for by introducing an ITV type motion approach integrating the GTV-MIP with the GTV0% and GTV50% to generate the ITV structure. In some cases, the ITV is reviewed against the GTV from all phases to make sure it encompasses the whole tumour extent.0%-50% motion) and SI measurements to account for the maximum tumour motion in this study. The latter recalculated motion is referred to, in this work, as the ‘3D maximum motion (3Dm), and the SI maximum motion (SIm).The external respiratory motion analysis was based on the RPM external marker. This allows the analysis of a large number of breathing signals which also require no further manual processing. Out of the 363 patients, 260 patient breathing files were accessible for the analysis. Each breathing trace  was analysed by means of a customised MATLAB  script to define the external breathing motion (median amplitude in the AP direction) which could then be correlated with the internal motion measured from the 4DCT.A summary of the key elements of the script are summarised. The patients’ breathing periods and amplitudes were measured from the RPM signal. For the amplitude measurements, the breathing signals from the beginning of the X-ray irradiation (first BeamOn) until the last BeamOff were considered as illustrated in m, and the SIm motion via Spearman’s correlation for each anatomical lobe. The correlation using the entire RPM signal from the beginning (C1) to the end of the scan (C19) is referred to as the ‘RPM signal’. The correlation with the section of the signal corresponding to the tumour sampling area C9 to C12 is referred to as the ‘Tumour RPM’. The relationship between the tumour volume, analysed in the exhalation phase, and the magnitude of motion (3D0%-50% and SI) was also evaluated.The correlation between the internal tumour motion, acquired from 4DCT, and the external surrogate motion was investigated by considering the entire X-ray BeamOn signal as illustrated in The relative position of the analysed tumours within the lung was acquired using the average intensity projection (AIP) 4DCT data set. The GTV50% relative position in the SI direction was measured as the ratio of the distance from the apex of the lung to the centre of the GTV structure and the distance from the apex of the lung to the diaphragm passing through the GTV. These distances were measured using the frontal view of the AIP data set. The relative position in the AP direction was the ratio of the distance from the posterior lung boundary to the centre of the GTV and the total distance in the AP direction from the posterior lung boundary. The LR relative position is the ratio of the distance from the centre of the vertebra to the centre of the GTV and the total distance in the LR direction from the centre of the vertebra to the distal edge of the lung contour. The relative positions in the AP and LR directions were measured using the transverse plane.p < 0.05). The null hypothesis was set such that the distribution of motion is the same across all groups.Statistical analysis was performed using SPSS v. 27 . The Spearman’s correlation was used to assess the internal/external correlation, and the Pearson correlation was used to assess the linearity between the tumour volume and motion. Correlation between the motion of the centre of mass in the 3D, SI, AP, and LR directions across lobes was examined by Kruskal–Wallis with Bonferroni adjustment and Mann–Whitney tests , the median and the range are reported throughout the paper.A total of 363 tumours were analysed and found to have a median GTV0% volume of 5.3 cc (range: 0.2–66.2 cc), median GTV50% of 5.3 cc (range: 0.2–65.5 cc), and median ITV of 8.5 cc (range: 0.6–142.7 cc). More than half (61.7%) of the analysed tumours were located in the upper lobe of the lung, 6.6% in the middle lobe and 31.7% in the lower lobe. The normality of the distribution of the 3D0%-50% motion amplitude) was 4.2 mm (range: 0.1–26.7 mm) across all tumours. 16.5% of the analysed tumours had a motion greater than 10 mm, only 3% had a motion amplitude greater than or equal to 20 mm. The median amplitude was 3.0 mm (range: 0–26.5 mm), 1.5 mm (range: 0–13.4 mm), and 1.0 mm (range: 0–8.1 mm) in the SI, AP, and LR directions, respectively. The Kruskal–Wallis test indicated that there is a significant difference in the 3D0%-50% motion across the three categories of anatomical lobes (p < 0.001) but there is no difference in the 3D0%-50% motion between the upper and middle lobe (p = 0.79).0%-50%) in the upper, middle and lower lobes was 3.4 mm, 4.0 mm, and 9.9 mm as presented in p < 0.001), but not between the upper and middle lobe, p = 0.24. However, there is no significant difference in the AP and LR motion across anatomical lobes (p = 0.53) and (p = 0.12), respectively.In 0%-50% motion, was classified into three groups, motion <5 mm, motion ≥5 mm and motion ≥10 mm.The histogram in r = 0.95, and the Mann–Whitney analysis indicated no significant difference between the two amplitudes (p = 0.92). The Spearman’s correlation between the internal 3D0%-50% motion was weak when all 260 tumours were considered . However, differences in the strength of the correlation were apparent for different lung lobes , as illustrated in The RPM breathing signal is a 1D signal acquired in the AP direction from the top of the patient’s abdomen. The Spearman’s correlation between the external marker amplitude from the ‘RPM signal’ and the amplitude at tumour sampling area ‘Tumour RPM’ showed a strong correlation r = 0.19), and moderate in the middle (r = 0.41), and lower lobe (r = 0.56). The correlation, when the breathing trace was restricted to when the tumour was being scanned ‘Tumour RPM’, showed a similar trend with no difference in correlation coefficient. The comparison with the tumour maximum motion 3Dm and SIm did show similar correlation coefficients for the upper and lower lobes, but not in the middle lobe . As the motion in the SI direction is dominant, the effect of the tumour volume was examined in the SI direction but no linear relationship was found . The classification of tumours based on the attachment to rigid structures indicated that only three tumours were possibly attached (3D0%-50% motion range: 2.9 mm), and due to this limited number of attached tumours no statistical analysis was performed.The dependence of the 3D tumour motion on the GTV volume showed no linear relationship  was determined using 4DCT scans. A total of 24 patients were used to build a correlative model, 11 of which were included in the internal/external assessment. The internal tumour motion in the SI and AP direction was assessed against the AP motion of the external marker block. The results illustrated the best association between the AP motion of the external marker and the SI motion of the GTV . This work also studied the correlation between the internal tumour motion and the motion of implanted transponders in the SI, AP and LR directions, and concluded that the transponders better reflected tumour motion prediction than the external marker block.A recent study by Willmann et al assessed the internal/external correlation by building a population correlative model using a linear mixed model. In their work, 101 radical tumours (tumour volume ranges between less than 10 and more than 100 cc) were used to measure the motion of lung tumours in 4DCT and to investigate the correlation between patient and tumour characteristics  and motion amplitudes. Their results showed that radical tumours located in the lower lobe of the lung had higher motion amplitudes in the SI direction compared with the other lobes (p < 0.001). The AP and mediolateral motion amplitudes did not differ between lobes . In addition, the amount and direction of the motion of tumours was not predictable by their individual characteristics. The research presented in this manuscript confirms these findings in a larger cohort, albeit with tumours smaller in size.Another internal tumour motion-based study in a large number of radically treated tumours was published in 2015 by Tan et al. This trend is not always the case however, as upper lobe tumours might have an unexpectedly higher motion amplitude. Hoisak et al, reported the largest motion for a tumour in the upper right lobe of the lung of 27.6 mm in a small cohort of mixed early and locally advanced lung cancers. Our findings demonstrate that tumour location in the vicinity of the diaphragm is not necessarily a good predictor of increased motion as we also report an upper lobe tumour with motion amplitude of 20.3 mm  Despite the general trend of tumour motion , In this work, the analysis of 363 SABR tumours showed that tumours in the upper lobe of the lung typically have the least amount of motion compared with middle and lower lobe tumours. Tumours closer to the diaphragm showed the highest amount of motion amplitude, as expected. Moreover, for the majority of the cases, the tumour motion was predominantly in the SI direction. These findings are in accordance with previously published work. is presented in p < 0.001). Similar p values were presented for the tumour motion in the AP and LR directions. Higher mean and median tumour motion in the SI, AP, and LR were reported by Knybel et al, and Liang et al, where the measurements were based on the motion of real-time tumour data.A comparison between the amount of centre of mass tumour motion of this study and selected previously published studiesSupplementary Table 1.Click here for additional data file.p < 0.0001), which is comparable to the data presented in this study.The accuracy of gated and tracked radiotherapy treatments that can be based on the use of an external surrogate can be affected by the degree of correlation between the movement of the external marker and the movement of the patient’s internal anatomy. In such a scenario, it is important to ensure there is spatial and temporal correlation between the moving tumour and the external surrogate for an adequate period of time. In the present analysis, the correlation was assessed between the median amplitude measured from the RPM external marker signal and the internal 3D tumour motion. Willmann et al compared the tumour motion in the SI and AP directions with the motion of the external reflecting marker block structure imaged during the 4DCT (motion between the phases 0 and 50%) using a linear mixed model. The best association was found between the AP motion of the external marker structure and SI motion of the GTV  and the maximum motion using the ITV motion approach . There was no difference in the correlations obtained with these different approaches.Our analysis included the measurements of tumour motion using the tumour centroid motion  and the attachment degree to a rigid structure (p = 0.008) (n = 44). Similarly, Wu et al reported lower motion amplitudes for tumours attached to fixed structures and tumours with large volumes (n = 42). As SABR is considered only for early-stage targets (T1N0 and T2N0), the vast majority of the targets considered in this study were not invasive in rigid structures (n = 360), which mitigates the need for tumour attachment analysis.The effect of tumour attachments to rigid structures in non-SABR targets have shown a trend in some studies, but this was found to be non-linear. An early study by Seppenwoolde et al has demonstrated that free lower lobe tumours tend to have more motion compared with attached tumours or tumours in the upper lobe.One of the limitations of this work is the use of breathing files generated by the RPM signals during the 4DCT acquisition, which might be relatively short compared with the nominal SABR treatment delivery time. Although the RPM signals are for treatment planning purposes, significant deviation from intrafraction motion may or may not occur. In comparison with other studies that considered different means of tumour motion quantification and internal/external correlation, this work can give an insight into the behaviour of SABR targets during irradiation prospectively. The results presented in this work can aid future auto-segmentation tools and enlighten other research areas where motion management is in its infancy, such as MRI planning for early-stage lung cancer.This study provides a retrospective analysis of tumour motion in lung SABR patients. To our knowledge, this is the largest sample of SABR patient data analysed for both tumour respiratory-induced motion and internal/external correlation using the widely accessible RPM external surrogate. Our findings are consistent with previous publications but the present patient sample was at least two and a half times greater than previous reports. In addition, this study has analysed how internal motion correlates with the motion of an external marker and has shown that there is a good correlation between the two when the tumour is located in the lower part of the lung.


This study investigated how personal characteristics such as generalized trust, self-consciousness and friendship, and desire for self-presentation are related to the subjective well-being of university students who use Twitter in Japan, including the effects of their online communication skills. We conducted a survey in May 2021 with Twitter users and analyzed their log data between January 2019 and June 2021. The log data of 501 Twitter users, including the number of public tweets, retweets, and emotional expressions among different patterns of social media  and academic standings, were analyzed using ANOVA and stepwise regression analyses. The results showed that the number of tweets and retweets, with and without photos/videos, increased in 2020 and 2021 compared to 2019, and the ratio of positive sentences remained almost the same for the two-and-a-half-year period of this study. However, the proportion of negative sentences increased slightly. It is clear that the factors which affected the university students’ subjective well-being differed depending on the respective patterns of social media use. Since January 2020 and the beginning of the COVID-19 pandemic, many people have experienced changes in their lives. Since then, many cities and countries have experienced lockdowns, and people were asked to physically distance themselves from each other during the quarantine and lockdown arrangements. One of the most common ways to connect with family members and friends was through the Internet, mainly via social media, to make phone calls, send text messages, and share posts with others. However, connecting with friends and relatives through social media could not fully compensate for the loss of face-to-face interaction. Previous research has indicated that people’s well-being has been affected significantly, and how they connect has changed   5] conduTo investigate the effects of the COVID-19 pandemic on young people’s online communication behaviors and subjective well-being, we used Twitter as the major tool to probe into this issue. Using sentiment analysis, we examined how different combinations of social media can affect subjective well-being. Previous studies also indicate that self-consciousness, and friendship and self-presentation  16] are are16] aFrom May 10 to 22, 2021, we conducted an online survey with university students in the Kanto region of Japan. A total of 1,694 students submitted their responses, and 1,681 were analyzed, as 13 were incomplete. However, only 577 participants had public tweets and/or retweets between January 2019 and June 2021. Therefore, in this study, we conducted the analysis of the data obtained from the 577 participants.In the survey, their personal characteristics, including generalized trust toward others (α = 0.80), self-consciousness and friendship, desire for self-presentation and admiration, usage of various social media, online communication skills (α = 0.80), and subjective well-being (α = 0.86), were measured. Ye  conducteWe collected and analyzed the log data using the Twitter API. The number of tweets, number of retweets, number of tweets with photos/videos, and number of retweets of tweets with photos/videos were calculated as features. Terms including “コロナ” , “corona,” and “covid” —hereinafter “COVID-19”—were set as keywords directly related to COVID-19, and the number of tweets and retweets including these keywords were calculated and analyzed.Sentiment analysis was performed using a neural network model implemented using the flairNLP library . The neuTable st year during the pandemic), and from January to June 2021 (2nd year during the pandemic). We used data corresponding to the first half of the year for analysis. We matched this to the usage pattern of students as the academic year started in the first half of the year, and university students started to develop their social networks, especially first-year students. This arrangement also allowed us to match our data collection period  with the tweets and retweets they posted. At this stage, we removed data from 8 participants’ data as their tweet/retweet records were insufficient for us to conduct further analysis. We considered participants to be users of a particular social media platform if they spent at least 20% of their social media time on that particular platform. Of the 15 possible combinations of usage patterns, we observed nine patterns that met the following requirements: (1) LINE only (n = 5); (2) Twitter only (n = 70); (3) Instagram only (n = 2); (4) LINE and Twitter (n = 149); (5) LINE and Instagram (n = 11); (6) Twitter and Instagram (n = 17); (7) LINE, Twitter, and Instagram (n = 282); (8) LINE, Instagram, and Facebook (n = 1); and (9) all four social media platforms (n = 32). For further analysis, we only included Patterns 2 (n = 70), 4 (n = 149), and 7 (n = 282), as they are the only three patterns that account for more than 10% of the participants. The results are presented in Table The collected data were compared separately in 2019 (before the pandemic), 2020  and the number of tweets and retweets with photos/videos  increased from 2019 to 2021 in overall results. Therefore, there was a growing trend in the number of tweets posted and retweets (including photos/videos) from 2019 to 2021, reflecting an increase in Twitter usage. With regard to the number of tweets about COVID-19, there was a slight increase in the overall result . There were few retweets about COVID-19. Regarding the ratio of positive and negative sentences on Twitter, we noted an increase in negative sentences  and a decrease in positive sentences  in this period. Figure As shown by Ye , the posWe further analyzed the data according to the academic standing of the participants (based on their academic standing in 2020–21). As shown in Table p < 0.05), rejection avoidance factor , praise acquisition factor , online communication skills , Twitter usage period , number of Twitter accounts , number of Twitter accounts followed , number of Twitter account followers , and subjective well-being  based on ANOVA and post-hoc tests.Ye  clarifieWe further conducted a stepwise multiple regression using subjective well-being as the dependent variable for demographic attributes, factors related to personal characteristics, and Twitter usage Tables  and 4 asWe also compared the standardized coefficients between the patterns, and the results are presented in Table In this study, we collected personal data from university students enrolled in the Kanto region of Japan through a survey. In addition, we collected their log data (public posts) on Twitter. Then we analyzed the relationships between their social media use patterns, emotional expressions on Twitter, and subjective well-being using these variables.We analyzed whether emotional expression in tweets and retweets posted by university students on Twitter changed since the beginning of the COVID-19 pandemic. Additionally, we analyzed the results using social media patterns and found that the number of tweets and retweets (including tweets and retweets with photos and videos) increased from 2019 to 2021 for all major patterns. This result is probably related to the fact that about one-third of the participants were freshmen who used Twitter to build new interpersonal relationships in April 2021 when they started their university lives . In 2019In general, the effects of generalized trust, self-consciousness and friendship, and self-presentation on subjective well-being echoed previous findings. These results included: (i) a high level of generalized trust (except for Pattern 7), self-establishment (except for Pattern 2), and praise acquisition from others improved their levels of subjective well-being, and (ii) a high level of self-appeal (for Pattern 2), self-indetermination, and self-independency (the latter two for Pattern 7) reduced their levels of subjective well-being.Regarding emotional expressions, we first analyzed the proportion of positive and negative sentences on Twitter. We noted that the proportion of positive sentences remained almost unchanged for 2.5 years, whereas the proportion of negative sentences increased slightly Tables  during tLooking at the findings through the lens of academic standings, we noted that while first-year students had a higher ratio of positive sentences most of the time, the percentage of positive sentences saw a decrease in 2021. All three other groups also either had a decrease or flatted out from 2020 to 2021. The findings of the first-year students relate to their academic journey, as these participants were in high school in 2019 and 2020, and entered university in 2021. The reduction in the ratio of positive sentences could be due to the change in their living environment (as they would relocate from their hometown to the university and faced changes in their lives) and their attitude toward COVID-19. Indeed, our ANOVA results showed that the differences in the ratios in 2021 among the four groups were statistically insignificant.However, there was an interesting observation regarding the ratio of negative sentences. While there was an increase in the ratio of negative sentences in 2020 for second-, third- and fourth-year students, there was a decreasing trend for first-year students. For first-year students (who were high school students in 2019 and 2020), their lives were probably not significantly adversely affected by COVID-19 in 2020. They might even have fresh experience in participating in the coursework through various types of online courses. Therefore, these new exposures might make them feel adventurous and have fewer complaints. However, second-year students might have initially experienced a lot of pressure in 2019 (as they were facing university admission examinations when the news about COVID-19 broke out in December 2019) and had a sharp increase in the ratio of negative sentences. These students probably had a more difficult time during their campus life compared to other grades, as all classes in the Spring semester were conducted online, and most classes were still conducted online in the Autumn semester. All extracurricular activities were suspended during that period, which meant that they had few opportunities to communicate with their classmates in person to reduce their anxiety and stress. As a result, we still observed an increase in the ratio of negative sentences in 2021.We also noted that for Twitter-only users (Pattern 2), subjective well-being decreased if they spent too much time on the Internet via their smartphones. An effect of the ratio of positive sentences was observed for users who used Twitter, LINE, and Instagram (Pattern 7). The ratio of positive sentences on Twitter was positively related to subjective well-being, which may indicate that users with higher levels of subjective well-being are more likely to use more positive sentences. As Ye  clarifieTo conclude, our findings show that emotional expressions that affect subjective well-being differed depending on the pattern of use of other social media, even if all participants used Twitter.The finding of this study shows the relationships between the subjective well-being of university students in Japan and their social media use patterns. Therefore, we suggest that public health authorities should consider taking our findings in developing programs help young adults who face stress and other mental health issues due to the COVID-19 crisis  [19], suThis paper presents valuable information on how university students’ emotional expressions on Twitter were related to their subjective well-being in Japan. Through the results of regression analyses, we found that subjective well-being was affected by the time spent on the Internet through smartphones (for Pattern 2), the percentage of positive sentences (for Pattern 7), and the number of Twitter accounts .This study has some limitations. Even though 1,681 social media users participated in the survey, only 577 of them had public tweets and retweets. Additionally, among the final 501 responses, we could only analyze three of the 15 possible combinations of social media usage patterns. Furthermore, the number of tweets and retweets we collected from the respondents’ public records was insufficient to analyze their behavior further quarterly. To better understand how social media usage patterns relate to users’ subjective well-being, it would be necessary to recruit more respondents of different ages with more public tweets/retweets to analyze in more detail.


Dear Editor,With great interest we read the recent article by Fujii et al. , which bFirstly, the findings diverge from the outcomes published in the study conducted by Spiegel et al., where no discernible correlation between major adverse kidney events at 30 days (MAKE30) and IRVF patterns was established . The autSecondly, and arguably of utmost significance, Fujii et al. omitted the consideration of fluid responsiveness at the timepoint of IRVF-assessment and nevertheless conclude that their findings deliver a potential rationale to guide post-resuscitation fluid therapy and removal in sepsis. They arbitrarily defined the initial resuscitation phase as the first 24 h hours after sepsis onset. Strong prerequisites for the right time to start fluid withdrawal have been proposed based on simple physiological principles: Beside adequate tissue perfusion, patients must not be preload responsive, because if a patient is on the steep part of the Frank–Starling curve, fluid withdrawal can lead to a decrease in cardiac output . The fulIn summary, this study provides only a preliminary exploration into the complex cardio-pulmo-renal interactions it seeks to investigate, especially in the context of fluid responsiveness and venous congestion. It illustrates that assessment of fluid status remains challenging and will likely never be based on a single parameter. Ultrasonography represents an attractive tool in assessing fluid status due to its non-invasiveness as well as rapid and near universal availability in the ICU setting , 2, 5. HTherefore, these findings need replication in adequately powered studies with increased sample size and thoughtful covariate selection for appropriate adjustment of statistical models to finally gain robust and generalizable results that may become routine part of clinical care.


With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons. In vitro human iPSC-derived models that mimic VZV’s entry route into the nervous system via axon termini, and additionally allow interaction with immune cell populations, can progress our understanding of innate immune cell responses to neuronal VZV infection and replication. Here we demonstrate that hiPSC-macrophages, even though unable to control an ongoing VZV infection in hiPSC-neurons on their own, are – in contrast to hiPSC-neurons – not ignorant towards VZV challenge. Therefore, we believe our established bi-partite hiPSC-derived neuro-immune co-culture model for VZV infection to be highly suitable for furthers studies aiming to link innate and adaptive immunity towards VZV using even more advanced iPSC-derived neuro-immune co-culture models.VZV is a neurotropic herpesvirus that naturally infects over 95% of the human population, resulting in a large burden of disease. + T-cells are of central importance during the subsequent adaptive immune response (Varicella-zoster virus (VZV) or human herpesvirus 3 (HHV-3) is a highly species-specific human neurotropic alpha herpesvirus. Upon initial infection - typically during childhood years - the virus enters the host via the upper respiratory mucosal epithelium, which serves as entry point for the initial VZV spread to local lymphoid tissues. Here, VZV infects T-cells, which then act as vehicle to spread the virus via the blood to the skin, leading to the initial distinct VZV pathology known as varicella or chickenpox . This firesponse –6. Althoresponse . Furtherresponse .In vitro, the scarce availability and inherent variability of primary human neural tissue have been hampering progress in VZV research. In the last decade, neuronal in vitro models derived from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) have become important platforms in the study of VZV neurobiology   was diffier work . In brie5 cells/ml in StemSpan SFEMII medium (STEMCELL Technologies) containing 100 ng/ml SCF Immunotools), 100 ng/ml TPO (PeproTech), 100 ng/ml Flt-3l (Immunotools), 50 ng/ml M-CSF (Immunotools) and 20 ng/ml GM-CSF (Immunotools) to allow expansion of the cells. After 2 days of expansion (day 14), cells were passaged at a concentration of 3-5 x 105 cells/ml and allowed to grow for another 3 days. At day 17, expanded hiPSC-HPC were harvested and resuspended at a concentration of 0.5 x 106 cells/ml in X-Vivo medium (Lonza) supplemented with 100 ng/ml M-CSF (Immunotools) to direct further differentiation into hiPSC-macrophages. Brightfield images of hiPSC-macrophages were obtained using a Fluovert Leitz microscope equipped with an Olympus UC30 camera. Finally, at day 24, hiPSC-macrophages were harvested and subjected to a CD14 selection step using anti-human CD14 magnetic beads (Miltenyi Biotec) for downstream experiments. Note that no selection step was performed for the characterization of the cells with mass cytometry. Purity was additionally confirmed via flow cytometry (NovoCyte Quanteon™). Cultures were maintained at 37°C and 5% CO2 throughout the entire protocol.hiPSC – the original hiPSC line from which hiPSC-NSC were derived  – were f® Gold antifade reagent (ThermoFisher). The following antibody combinations were used: mouse anti-β-III-tubulin (Tuj1)  and rabbit anti-GFAP  in combination with the respective secondary antibodies, goat anti-mouse AF555  and goat anti-rabbit FITC , guinea pig anti-NeuN  in combination with donkey anti-guinea pig Cy3 , and chicken anti-peripherin  in combination with goat anti-rabbit FITC . Immunofluorescent microscopic images of hiPSC-neurons were obtained using a PerkinElmer UltraVIEW Vox spinning disk confocal system.Immunostaining of hiPSC-neurons was performed as previously described . In brie6 cells were aliquoted to staining tubes. First, dead cells were stained with Cell-ID™ Cisplatin  for 5 min at RT and quenched with Maxpar® Cell Staining Buffer  supplemented with ethylenediaminetetraacetic acid . Next, Fc receptor block  was performed for 10 min at RT, and antibody cocktail of metal-conjugated antibodies (Standard BioTools) (® Fix and Perm buffer (Standard BioTools) overnight at 4°C. The next day samples were transferred to -80°C and stored until the acquisition. For mass cytometry analysis, samples were shipped on dry ice to the core facility in Genyo , thawed, and washed with Maxpar® Cell Acquisition Solution . Cells were acquired on a mass cytometer  at an event rate of 400 cells/s together with EQ Four Element Calibration Beads (Standard BioTools). Raw data were normalized using CytoQP function bead_normalize , counted and 2 x 10ioTools) Table 1 ormalize . FlowJo + hiPSC-macrophages were transfected with poly(dA:dT)  using Lipofectamine 3000 Reagent (Invitrogen) or were treated with Lipofectamine 3000 Reagent alone, according to the manufacturer’s instructions. Unstimulated hiPSC-macrophages were used as a control. After 24h of incubation, supernatants were collected and a LEGENDplex (BioLegend) assay was performed according to manufacturer’s instructions to determine the level of TNF-α, IFN-λ1, IFN-α2, IFN-β and IL-6 as part of the Human Anti-Virus Response Panel. Data were acquired using the NovoCyte Quanteon™ flow cytometer and subsequently analyzed by means of the accompanying LEGENDplex data analysis software (BioLegend) according to manufacturer’s instructions.CD14This study makes use of the recombinant VZV-eGFP/ORF23 strain , propagam/v) sucrose, 0.08% (m/v) glutamate, 8% iFBS) by scraping the cells off the culture flasks. The cells underwent two cycles of freezing in liquid nitrogen and thawing in a 37°C water bath, followed by sonication for 15 s at 20% amplitude . The resulting VZV cell lysates (further named as CF VZV-eGFP/ORF23) were pooled and stored at -80°C. The absence of intact cells was confirmed by culturing the VZV lysate in DMEM/F12 + 10% iFBS (Gibco™) for seven days. Titers of CF VZV-eGFP/ORF23 were determined by an infection foci assay, as described above.Cell-free (CF) VZV was obtained following lysing VZV-eGFP/ORF23-infected ARPE-19 cells, according to the protocol of Sloutskin et al.  with minS. aureus Bioparticles™ (Invitrogen) according to the manufacturer’s instructions. In brief, pHrodo bioparticles™ were added to hiPSC-macrophages at a final concentration of 0.1 mg/ml and incubated for 2h at 37°C. hiPSC-macrophages were subsequently harvested and washed with PBS pH 7.4. A live/dead staining was performed by incubating the samples with 7-AAD  for 10 min at RT, prior to their acquisition by flow cytometry (NovoCyte Quanteon™). Cells-only and beads-only samples served as negative controls. Second, the ability of CD14+ hiPSC-macrophages to phagocytose VZV was assessed by inoculation of the cultures with CF VZV-eGFP/ORF23. More specifically, hiPSC-macrophages were inoculated with CF VZV-eGFP/ORF23 at a multiplicity of infection (MOI) of 0.00149. Similarly, control lysate [CF control (ARPE-19)], derived from non-infected ARPE-19 cells according to the same procedure, was added to hiPSC-macrophages. At 2 and 48 hours post-inoculation, hiPSC-macrophages were harvested and washed with wash buffer (Sheath (BD), 0.1% BSA, 0.05% NaN3). A live/dead staining was performed by incubating the samples with 7-AAD  for 10 min at RT, prior to their acquisition by flow cytometry (NovoCyte Quanteon™). FlowJo software v10.0 was used for subsequent data analysis.First, the phagocytic capacity of CD14+ hiPSC-macrophages was assessed using pHrodo Green 5 U/ml, STEMCELL Technologies) for 24h (at day 21) before inoculation and were treated for the entire duration of the experiment for seven days. Furthermore, 28-day old hiPSC-neurons were transfected with pdAT  using Lipofectamine 3000 Reagent (Invitrogen) or were treated with Lipofectamine 3000 Reagent alone for 24h. Unstimulated hiPSC-neurons were used as control. For all different stimulations, hiPSC-neurons were harvested at day 29 and stored in DNA/RNA shield (Zymo Research) at -80°C for downstream RNA isolation and subsequent RT-qPCR and RNA sequencing analyses.22-day old hiPSC-neurons were inoculated with CF VZV-eGFP/ORF23 (MOI = 0.00026) or CF control (ARPE-19) for 72h and further cultured for 4 days. For IFN-α2 treatment, cultures were pre-treated with IFN-α2  treatment for 48h. Furthermore, CD14+ hiPSC-macrophages were transfected with pdAT  using Lipofectamine 3000 Reagent (Invitrogen) or were treated with Lipofectamine 3000 Reagent alone for 24h. Unstimulated hiPSC-macrophages were used as control. At the end of the 48 or 24h incubation period, respectively, cells were harvested and stored in DNA/RNA shield (Zymo Research) at -80°C for downstream RNA isolation and subsequent RT-qPCR and RNA sequencing analyses.CD14+ hiPSC-macrophages were stimulated with CF VZV-eGFP/ORF23 (MOI = 0.00033 or 0.00021) or CF control (ARPE-19), with or without IFN-α2 (1.2 x 1020 primers (Invitrogen™).RNA extraction and cDNA synthesis of samples stored in DNA/RNA shield were performed as described previously . In brie® 480 System , using following thermal cycling conditions: 1 cycle of 2 min at 95°C , followed by 40 cycles of 5 s at 95°C (denaturation) and 20 s at 61°C . No reverse transcriptase controls (NRT) were run for each sample. Relative quantification to hGAPDH was performed using the Pfaffl Method  were added to the neurons in the somal compartment for 3 days, prior to the inoculation of the neuronal axons with 1.5 x 104 PFU ARPE-19-associated VZV-eGFP/ORF23. At 72 hpi, the medium was changed. Brightfield and immunofluorescent microscopic images were taken at 3 and 7 dpi. For imaging hiPSC-neurons (with hiPSC-macrophages) in XonaChips™, images of complete axonal and somal compartments were obtained by the stitching of multiple 20x images in brightfield and eGFP, acquired by an inverted fluorescence microscope equipped with a motorized stage  and ZEN blue software (Zeiss). Fiji image analysis freeware was used for image processing and analysis (http://fiji.sc). For the quantification of VZV-eGFP/ORF23 foci in the neuronal cultures, images were cut into two, dividing the somal and axonal compartments. By means of manual intensity thresholding, the number of VZV-eGFP/ORF23 foci could be retrieved, as previously described  compartmentalized neuronal model separating axons from neuronal cell soma to allow axonal VZV infection, as established previously . In briehttps://nanostring.com/products/ncounter-assays-panels/immunology/host-response/). Further pathway and gene set enrichment analysis was performed using the topGO and clusterProfiler R packages.RNA quality was verified using the TapestationTM . RNA sequencing was performed as described in Bartholomeus et al. . In brieTo evaluate whether VZV viral transcripts were captured within the RNA-sequencing the VIRTUS pipeline was used . First, ® Pro Version 16. A p-value < 0.05 was considered statistically significant. All data were modelled using a linear mixed-effects model, accounting for the repeated measures, i.e. independent experiments and/or repeated measurements for each observation. The conditions on normality of residuals and homoscedasticity for applying linear mixed-effects models were primarily checked to be in an acceptable range to perform this type of analysis. For RT-qPCR data of OAS1 mRNA expression by hiPSC-macrophages and for the quantification of VZV-eGFP/ORF23 foci in the axonal compartment, log-transformed data were used. For the quantification of VZV-eGFP/ORF23 foci in the somal compartment (x), log(x+1)-transformation was used, considering zero values in the original dataset. Post-hoc analyses for linear mixed-effects models were carried out with Tukey HSD correction for multiple comparisons.Graphs representing quantitative data were obtained using GraphPad Prism v.8.2.1 software. Statistical analyses were carried out using the statistical software JMPhiPSC-neurons were generated from a previously established hiPSC-derived neural stem cell line (hiPSC-NSC) , wherebyin vitro counterpart  as well as the immune responsiveness (pdAT stimulation) of our cultured hiPSC-macrophages, but at the same time suggests the high immune-evasive properties of VZV and/or the low innate immune reactivity of macrophages towards VZV.To demonstrate innate immune responsiveness of the cultured hiPSC-macrophages, we first lipofected hiPSC-macrophages with pdAT, a well-known synthetic DNA analogue that activates the type I and III IFN-signalling pathway. This resulted in significant production of high levels of anti-viral cytokines, including TNF-α, IFN-λ1, IFN-α2, IFN-β and IL-6, by hiPSC-macrophages  unstimulated hiPSC-macrophages , (ii) lipofectamine control for hiPSC-macrophages , (iii) hiPSC-macrophages after lipofection with pdAT , (iv) hiPSC-macrophages challenged with CF control (ARPE-19) lysate  and (v) hiPSC-macrophages challenged with CF VZV-eGFP/ORF23 lysate . Conditions (i), (ii) and (iii) refer to the experiments presented in Given this extensive RNA-Seq dataset, a heat map Figure 7Lastly we performed an additional VIRTUS bioinformatics analysis  on our Rin vitro stem cell-derived neuronal models are of great interest for studying neuron-pathogen interactions  can be found here: GSE228390 (GEO).Conceptualization: PP, BO, PD, TB-H, EB, and MB; methodology: PP, BO, PD, KL, PM, AB, MA-R, and CM; investigation: TB-H with help of EB, JG, PM, MB, JS, TC, HR, LD, JS, EB, AB, and PR; formal analysis: TB-H, JG, and EB; writing—original draft: EB, JG, and PP; writing—review and editing: PP, BO, PD, PM, AB, ML, CS-D, CM, and KL; funding acquisition: PP, BO, PD, MA-R, CM, and KL; resources: ML and CS-D; supervision: PP, BO, and PD. All authors contributed to the article and approved the submitted version.


Trigeminal ganglia (TG) neurons are an important site of lifelong latent varicella-zoster virus (VZV) infection. Although VZV-specific T-cells are considered pivotal to control virus reactivation, their protective role at the site of latency remains uncharacterized.Paired blood and TG specimens were obtained from ten latent VZV-infected adults, of which nine were co-infected with herpes simplex virus type 1 (HSV-1). Short-term TG-derived T-cell lines (TG-TCL), generated by mitogenic stimulation of TG-derived T-cells, were probed for HSV-1- and VZV-specific T-cells using flow cytometry. We also performed VZV proteome-wide screening of TG-TCL to determine the fine antigenic specificity of VZV reactive T-cells. Finally, the relationship between T-cells and latent HSV-1 and VZV infections in TG was analyzed by reverse transcription quantitative PCR (RT-qPCR) and in situ analysis for T-cell proteins and latent viral transcripts.VZV proteome-wide analysis of ten TG-TCL identified two VZV antigens recognized by CD8 T-cells in two separate subjects. The first was an HSV-1/VZV cross-reactive CD8 T-cell epitope, whereas the second TG harbored CD8 T-cells reactive with VZV specifically and not the homologous peptide in HSV-1. In silico analysis showed that HSV-1/VZV cross reactivity of TG-derived CD8 T-cells reactive with ten previously identified HSV-1 epitopes was unlikely, suggesting that HSV-1/VZV cross-reactive T-cells are not a common feature in dually infected TG. Finally, no association was detected between T-cell infiltration and VZV latency transcript abundance in TG by RT-qPCR or in situ analyses.The low presence of VZV- compared to HSV-1-specific CD8 T-cells in human TG suggests that VZV reactive CD8 T-cells play a limited role in maintaining VZV latency.The online version contains supplementary material available at 10.1186/s12974-023-02820-y. Most adults worldwide are infected with the neurotropic human alphaherpesvirus varicella-zoster virus (VZV), which causes chickenpox during primary infection and herpes zoster (HZ) in one-third of non-vaccinated individuals upon reactivation of latent VZV. Following primary infection, VZV establishes lifelong latency in neurons of trigeminal ganglia (TG) and dorsal root ganglia (DRG) along the entire neuraxis . FurtherRM) to control HSV reactivation .DNA and RNA extraction from human TG samples and subsequent cDNA synthesis were performed as described previously , 17. ReaT [GAPDH . In addiGeneious Prime version 2022.2.2 (Dotmatics) and the Geneious alignment algorithm with default parameters were used to align viral sequences. The predicted binding rank for minimal HSV epitopes and their VZV homologous peptides of similar length to relevant HLA-I alleles was analyzed using the netMHCpan4.1 algorithm with default parameters accessed at IEDB [VLT RNA expression by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively, as described previously [VLT [POLR2A , and the bacterial gene dapB  using the RNAScope 2.5 HD Assay (Advanced Cell Diagnostics). RNA integrity in tissue sections and specificity of the RNAScope Assay was demonstrated by robust POLR2A staining and absence of dapB ISH signal in all sections analyzed . Data were analyzed using two-tailed Spearman exact test where appropriate. Results were considered significant at Heparinized blood and TG were obtained during autopsy from ten individuals living in The Netherlands with an average ± standard deviation (SD) post-mortem interval of 6 ± 1.5 h. The cohort included 2 men and 8 women, the average age was 80 ± 11 years, and the majority had an underlying neurodegenerative disease, such as Alzheimer’s disease or multiple sclerosis. The cause of death was not related to acute herpesvirus infections  neurons in human and mouse ganglia [VLT expression in consecutive TG sections (n = 3 per donor) from five VZV-infected subjects by IHC and ISH . Second, we have previously observed that the level of CD8α transcripts—a measure for T-cell infiltration/retention—correlates with HSV-1 genome load in human TG, indicating that the extent of T-cell infiltration into the TG correlates with HSV-1 viral load [CD3D T-cell transcripts and VZV DNA or viral RNAs selectively expressed during VZV latency (VLT and the VLT-ORF63 fusion transcript) [VLT, or VZV VLT-ORF63 RNA levels and CD3D transcript abundance, suggesting that T-cell infiltration and/or retention in the human TG does not correlate with VZV viral load  as well as latent infection . Whereas VZV-specific T-cells are known to be protective against HZ, their localization and functional roles at the sites of viral latency remain unknown. In the present report, we assessed the abundance of VZV reactive T-cells in human TGs by determining the reactivity of T-cells derived from latently VZV-infected TG of ten elderly adults. Our data show that VZV reactive CD8 T-cells are present in only a small subset of latently VZV-infected human TG and that they are much less frequently observed than HSV-1-specific T-cells.We used two complementary APC platforms to detect virus-specific T-cells in human TG-TCL. First, we cocultured the TG-TCL with autologous B-LCL as APC and observed robust frequencies of HSV-1 reactive T-cells, especially CD8 T-cells, in 3 of 4 TG-TCL (75%). This is in line with the selective retention of HSV-1-specific T-cells in human TG latently infected with HSV-1 that we have previously observed . In contOf the two VZV epitopes we identified, the epitope in ORF29 was found to be cross-reactive with the homologous HSV-1/2 proteins UL29. This is in line with a previous study in which we also identified abundant cross-reactive T-cells targeting ORF29/UL29 in blood-derived T-cells . In factTo determine how widespread the cross-reactivity of HSV-1 reactive T-cells with VZV antigens is, we performed an in silico analysis of 10 previously reported HSV-1 minimal epitopes . Whereas111–121 epitope found here was also identified as a CD8 T-cell epitope in skin-derived TCL from the site of a healed HZ lesion [The other identified epitope that was specific to VZV mapped to ORF9. We recently reported that VZV ORF9 was highly immunoprevalent in human skin- and blood-derived TCL being frequently recognized by CD8 T-cells in these tissues . In factZ lesion . This suVLT expression in TG , and therefore, potentially experienced waning VZV-specific T-cell immunity. VZV-specific memory T-cells are maintained at a low frequency in skin and blood throughout life, but decline in number and/or functional capacity with older age leading to increased risk of developing HZ , 38–41. In conclusion, the current study shows that VZV-specific CD8 T-cells are scarce in latently VZV-infected human TG, suggesting that the protective role of VZV reactive CD8 T-cells against herpes zoster is of limited importance at the site of latency.Additional file 1: Figure S1. Control stainings for the in situ hybridization assays. Staining for the human gene POLR2A and the bacterial gene dapB were performed as positive and negative controls, respectively, for the in situ hybridization assays shown in main, Fig. Additional file 2: Figure S2. VZV antigen-specific responses of additional human TG-derived CD8+ T-cells. TG-derived T-cell lines of donor TG01–TG03 and TG05–TG10 were incubated with Cos-7 cells transfected with vectors encoding the indicated subject-specific HLA class I allele together with the individual VZV open reading frames. Empty vector and phytohemagglutinin were used as negative and positive controls, respectively. Levels of secreted IFNγ were determined by ELISA. Data are presented as the individual and mean OD450 values of two independent replicates. Horizontal dashed lines indicate the threshold of T-cell response set at twice the value of the empty vector control. Viral ORF nomenclature according to VZV reference strain Dumas.Additional file 3: Table S1. General characteristics and HLA genotype of study subjects used for T-cell analysis.Additional file 4: Table S2. General characteristics of the study subjects used for histological analysis in main, Fig. Additional file 5: Table S3. Primers used to generate the VZV ORF29 truncated fragments.Additional file 6: Table S4. Homology of HSV-1 CD8 T-cell epitope-containing regions with orthologous VZV proteins.


HG received a research fellowship, grant number: 3029440/2019-7, from Conselho Nacional de Desenvolvimento Científico e Tecnológico (https://www.gov.br/cnpq/pt-br). MF received a scholarship, grant number: 2017/03367-3, from Fundação de Amparo à Pesquisa do Estado de São Paulo (https://fapesp.br/). RTVP received a scholarship, grant numbers: 88882.376665/2019-01 and 88887.689454/2022-00, from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (https://www.gov.br/capes/pt-br).There are errors in the Funding section. The correct Funding statement is: This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, grant number: 2021/12535-2 to MCAS (The publisher apologizes for the error.


Acinetobacter baumannii (ACB), P. aeruginosa (PSA) and Enterobacterales are needed. Sulbactam-durlobactam (SUL-DUR) has potent, selective activity against ACB. Cefepime (FEP) is a common first-line therapy for hospital/ventilator associated pneumonia caused by non-ACB Gram-negative pathogens, but resistance to FEP is increasing. This study investigated the in vitro synergy of SUL-DUR + FEP against relevant pathogens.Due to the incidence of polymicrobial nosocomial infections, effective therapies with activity against ACB, n=4; PSA, n=4; E. coli, n=3; K. pneumoniae, n=3). One wild type K. pneumoniae isolate was included. Antibiotic concentrations simulated the free-steady state average concentration of clinically administered doses in patients .Static time-kills were performed in duplicate against fourteen FEP-resistant isolates (10 reduction in CFU/mL relative to 0h control). SUL-DUR plus FEP showed synergy  against one ACB isolate with an elevated MIC to SUL-DUR (32 mg/L). SUL-DUR plus FEP showed synergy vs. all PSA isolates . Against Enterobacterales, combination therapy was indifferent due to significant kill from SUL-DUR  with ∼ 4 log10 kill relative to 0h. SUL-DUR plus FEP was synergistic  vs. one E. coli isolate with an elevated MIC to SUL-DUR ( >128 mg/L). SUL-DUR plus FEP showed synergy against one of the K. pneumoniae with 2.97 log10 CFU/mL reduction relative to SUL-DUR and an additive effect against the other two K. pneumoniae tested . No antagonism was observed in any isolates.SUL-DUR alone showed significant activity against ACB consistent with the MIC for isolates at 8 mg/L and lower (range: 0.91-2.14 login vivo to verify these results are warranted.Synergy and no antagonism was observed with the combination of SUL-DUR and FEP. Further pharmacokinetic/pharmacodynamic studies conducted David P. Nicolau, PharmD, Allergan: Advisor/Consultant|Allergan: Grant/Research Support|Cepheid: Advisor/Consultant|Cepheid: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Tetraphase: Advisor/Consultant|Tetraphase: Grant/Research Support|Venatorx: Advisor/Consultant|Venatorx: Grant/Research Support|Wockhardt: Advisor/Consultant|Wockhardt: Grant/Research Support Christian M. Gill, PharmD, Cepheid: Grant/Research Support|Entasis therapeutics: Grant/Research Support|Everest Medicines: Grant/Research Support|Shionogi: Grant/Research Support


Recurrence and metastasis of bladder cancer are major factors affecting patient prognosis. Endoscopic cryoablation achieved a better clinical outcome among clinical patients and could be synergistic with ICIs. Thus, this study aimed to evaluate the immunological mechanism of cryoablation for bladder cancer to reveal the therapeutic mechanism.We systematically reviewed the clinical prognosis of patients underwent cryoablation at Huashan Hospital in these first‐in‐human studies (ChiCTR‐INR‐17013060). Murine models were constructed to explore cryoablation‐induced tumour‐specific immunity, which was further confirmed by primary bladder tumour organoids and autologous lymphocytes cocultured system.Cryoablation improved progression‐free survival and recurrence‐free survival respectively. Assessment of murine models after cryoablation confirmed microenvironment remodelling and tumour‐specific T cells expansion. Enhanced antitumour effects were found after coculture of organoids with autologous lymphocytes collected from post‐cryoablation. We also demonstrated cryoablation‐induced tumour elimination required IFNGR expression on tumour cells. In addition, a long‐lasting antitumour memory response is achieved by cryoablation and could be enhanced after combination with ICIs.This study revealed endoscopic cryoablation is an efficient and safe therapy for bladder tumour treatment. The tumour‐specific immune responses induced by cryoablation could reduce tumour recurrence and metastasis. Endoscopic cryoablation eliminated bladder cancer cells and released tumour antigens to induce abscopal effect. Cryoablation remodulated microenvironment immune landscape and induced tumour‐specific T‐cell responses. Tumour‐specific immunity induced by cryoablation was depended on tumour expression of IFNGR. Cryoablation plus ICIs promoted effective long‐term antitumour immunity. The prediction that T cells could develop more potent antitumour effector functions due to cryoablation was validated using in vitro and in vivo experimental models, including organoids and adoptive cellular immunotherapy (ACT). In particular, we observed a markedly elevated antitumour effect of T cells in peripheral blood after cryoablation compared to pre‐cryoablation T cells when cocultured with autologous primary cancer organoids. Moreover, a higher level of IFN‐γ was detected in supernatants collected from the coculture system after cryoablation. When IFNGR was knocked out, the specific antitumour immunity induced by cryoablation disappeared. We hypothesised that cryoablation‐induced antitumour immunity depends on IFN‐γ signalling. This phenomenon was verified in tumour microarrays (TMAs), as patients with low IFNGR expression are prone to relapse. Finally, the synergistic effect of combined cryoablation and anti‐PD‐1 treatment was demonstrated.The present study revealed the dual function of cryoablation in eliminating orthotopic tumour cells and inducing changes in systemic immunologic memory and other immunological effects, which prevent the progression and recurrence of the tumour in patients with bladder cancer.22.1Tissue samples from two cohorts of patients with bladder cancer were collected at Huashan Hospital. Patients in cohort 1 were enrolled in a randomised clinical trial that evaluated the safety and feasibility of EBCA in patients with NMIBC or T2a MIBC (registration number: ChiCTR‐INR‐17013060). Tissue specimens from these patients were collected during diagnostic transurethral resection before endoscopic cryoablation. Tissue detachment from TMAs and immunohistochemistry (IHC) staining analysis were performed as previously described.2.25 cells/mouse) into the right armpit of each mouse. After 14 days, the tumour was frozen using a specially designed cryoablation unit (Patent No. 201510398917.6) for two cycles, with each cycle involving 40‐second freezing and 90‐s thawing. No treatment was administered to mice in the blank group. The control group underwent surgical resection of the primary tumour. The mice were anesthetised with isoflurane, and strict aseptic conditions were maintained during surgery. The surgical sites were sterilised twice a day after surgery, and the bedding was changed daily to prevent infection. On the second day after surgery, MB49 cells (5 × 105 cells/mouse) were reimplanted in the contralateral armpit. The size and volume of the secondary tumours were observed and growth curves were plotted. The experimental endpoint was defined as death or a secondary tumour volume ≥ 2000 mm3.All animal studies were approved by the Ethics Committee and performed in accordance with ethical standards. Male wild‐type C57 BL/6J  between 6 and 8 weeks of age were used. A tumour‐bearing mouse model was constructed by subcutaneous injection of the mouse bladder cancer carcinoma cell line MB49 (5 × 102.32 humidified incubator. Luciferase‐expressing (MB49‐luc) was generated by transfection with lentiviral vectors  encoding for firefly luciferase. All cells were tested for Mycoplasma  and retested prior to use.MB49 murine urothelial tumour cell was obtained from Millipore Corporation  and B16F10 melanoma cell line was purchased from ATCC. MB49 and B16F10 cells were placed in complete DMEM  and RPMI‐1640 medium  medium containing 10% fetal bovine serum , 1% penicillin/streptomycin at 37°C in a 5% CO2.4Fastq files generated from RNA‐sequencing were processed to quantitate clonotypes using MiXCR software,2.52O2/MeOH to inhibit endogenous peroxidase. To block nonspecific binding sites, the slides were implanted with a blocking buffer, including 10% horse serum. The slides were then exposed to primary antibodies in a wet chamber at 4°C for 16 h and treated with a biotin‐labelled goat anti‐mouse/rabbit IgG secondary antibody. The excess antibody was removed by washing thrice with PBS and visualised using the DAB Substrate Kit until the desired brown colour was achieved. Slides were scanned using Pannoramic SCAN II.IHC staining was performed using dewaxed tissue slides. After hydration, tissue slides were heated in citrate buffer  to extract antigens and 3% H2.6g for 10 min. After resuspension, red blood cells were lysed using the Red Blood Cell Lysis Buffer . To investigate tumour‐related effector molecules, 1 × 106 cells were incubated in RPMI‐1640 with 5% FBS and Cell Stimulation Cocktail  at 37°C. After 4 h, the cells were harvested and analysed further. Information regarding the antibodies used for flow cytometric analysis is listed in Table The mice were anaesthetised and euthanised at predetermined time points. For the analysis of lymphocyte subsets and function in the tumour microenvironment, the secondary tumours were isolated, weighed and treated with RPMI‐1640, 5% FBS, collagenase IA , and DNase I  at 37°C for 45 min, while splenic lymphocytes were extracted by grinding. Subsequently, the digested and splenic lymphocytes were filtered through 70 µm cell strainers and centrifuged at 300 2.72 for 45 min. Isolated tissues were centrifuged at 350 g for 5 min at room temperature. The supernatant was discarded, and the precipitate was resuspended in 60% Matrigel matrix . Following this, 250 µL of the mixture was added to each well of a six‐well plate and incubated at 37°C with 5% CO2 for 30 min. Once the Matrigel solidified, 2 mL of organoid medium was added to each well and changed regularly every 3 or 4 days. Tumour organoids with good activity after three to six generations were isolated from Matrigel, dissociated into single cells, and cocultured in vitro with autologous T cells collected before and after cryoablation at a 1:10 ratio. T‐cell separation was performed as previously described.The protocol for culturing organoids in vitro has been reported previously.2.8+ T lymphocytes and CD8+ T lymphocytes were depleted by intraperitoneal injection of 200 µg neutralising antibody  on the day of cryoablation and every 3 days from the second day after cryoablation. The depletion efficacy was verified by flow cytometry. Anti‐mouse PD‐1  was injected into rechallenged mice after cryoablation or surgical resection of primary tumours every 3 days for a total of five times.CD42.95 cells/well. RPMI 1640 (Gibco) was added with anti‐mouse CD3/CD28 (5 µg/mL) and IL‐2 (2000 IU/mL) for cell culture and expansion. MB49‐Luc cells were subcutaneously injected (3 × 106 cells/mouse) into the right armpit of 6‐week‐old male NPSG mice . When a small nodule could be palpated under the skin about 3 days after implantation, activated T cells (5 × 106 cells) were injected via the tail vein into the mice for ACT. On days 3, 6, 9, 14 and 18, the mice were imaged using intravital imaging. The efficacy of ACT was evaluated by comparing the volume and fluorescence intensity of the tumours.The spleens of mice after cryoablation or tumour resection were collected, ground and filtered through a 70 µm cell strainer. T cells were purified using the Pan T‐cell isolation kit (Miltenyi Biotec) before being seeded into six‐well plates at 1 × 102.10Knockout of the IFNGR gene in the MB49 cell lines via the CRISPR/Cas9‐gDNA system was accomplished by Genomeditech . The gRNA sequence for IFNGR is TCGCGCAGGAATGGGCCCGC. The IFNGR knockout cell line was selected by flow cytometry analysis, which based on functional testing by adding recombinant mouse IFN‐γ   at a concentration of 100ng/mL for 48 h.2.11−ΔΔCT method was used to calculate different expression of RNAs. The experiments were repeated three times. All primers used were listed in Table Total RNAs were extracted from tissues by using Trizol  following the manufacturer's instruction as previously described.2.12t‐tests. One‐way or two‐way ANOVA tests were used to determine p value. The data were analysed using Student's t‐test when normally distributed, or using nonparametric tests if not. Statistical significance was set at p < .05.All statistical analyses were performed using GraphPad Prism 9 . Survival analysis was performed using the Kaplan–Meier method, and survival differences were compared using the log‐rank test. For sequencing analysis, comparisons between two groups were performed using unpaired Student's 33.1p = .0006), and a 2‐year recurrence‐free survival (RFS) of 81.6% and 60.0% , respectively . The study met its primary outcome with a local control rate of 91.5% in the cryoablation group compared to 76.5% in the TURBT group. Of these patients, 88 were screened for eligibility at the hospital, and 68 patients completed the trial Table . The triy Figure  and D. Ty Figure . The cely Figure . The tumy Figure . Kaplan–y Figure . We alsoy Figure . These r3.2+ and CD8+ T cells into the tumours. In particular, the number and proportion of CD4+ T lymphocyte infiltration increased significantly  are a group of T cells residing in the tumour microenvironment with both effector and memory T‐cell characteristics that perform both immunosurveillance and antitumour functions. In our study, elevated expression of CD103+CD69+ Trm cells was observed after rechallenge in the cryoablation group  in the group that received a combination of cryoablation and tislelizumab compared with those who underwent cryoablation alone  was found to be associated with a higher risk of recurrence after cryoablation. IFNGR knocked out could render cryoablation‐induced tumour‐specific T cells ineffective in eliminating tumours. In addition, whether tumour intrinsic factors may induce recurrence and metastasis after cryoablation also deserves further investigation in our follow‐up study.5Endoscopic cryoablation eliminates tumour cells and releases tumour antigens, promoting long‐lasting tumour‐specific immunity to inhibit bladder tumour recurrence. Combining endoscopic cryoablation with ICIs offers new treatment options for patients with advanced bladder cancer.The authors have declared that no conflict of interest exists.Supporting InformationClick here for additional data file.