Preoperative radiotherapy is often used to facilitate excision of soft-tissue sarcomas. We aimed define factors that affect local tumour control and patient survival.A single institution registry study of 89 patients with non-metastatic soft-tissue sarcomas having preoperative radiotherapy between 1994 and 2014. Radiologic  and histopathologic  parameters were recorded. Outcomes were the events of local recurrence, amputation, metastasis and death.Local recurrence rate was low (12%) and marginal excision gave equal local control to wide excision. Pelvic localization was associated with a higher risk for amputation. The absence of peritumoural oedema on MRI defined a subgroup of tumours with more favourable oncologic outcome. Reduction of tumour volume following radiotherapy was also associated with better patient survival. Both these radiologic parameters were associated with lower tumour grade. Tumour necrosis was not significant for patient survival. The local complication rate, mainly wound healing problems and infection, was high (40%), but did not lead to any amputation.Preoperative radiotherapy of high-risk soft-tissue sarcomas allows for good local control rate at the expense of local wound complications, which are however manageable. Marginal excision is sufficient for local control. Absence of peritumoural oedema on MRI, as well as tumour size reduction following radiotherapy are associated to superior patient survival and can be used ass early prognostic factors.The online version of this article (10.1186/s13569-018-0106-x) contains supplementary material, which is available to authorized users. Treatment of soft-tissue sarcomas is mainly surgical. Radiotherapy is indicated as an adjuvant treatment in all deep-seated tumours and in superficial tumours when a wide surgical margin is not achieved , 2. It iThere is limited amount of data regarding the outcome of surgery preceded by radiotherapy for soft-tissue sarcomas, and there is still a debate on the factors that may determine patient prognosis. Tumour necrosis may be an objective measure of the effect of preoperative radiotherapy but there is no proof of its validity as a prognostic factor . The useWe set out to investigate the outcome of patients with soft-tissue sarcomas who were treated with radiotherapy prior to surgery, and define clinical, histologic and radiologic prognostic factors associated with survival and local control of the disease in a large retrospective series.This is a single-institution registry study. Inclusion criteria for participation were the diagnosis of a soft-tissue sarcoma of the trunk or the extremities, the administration of radiotherapy treatment prior to surgery and the absence of metastases at diagnosis. Exclusion criteria were chemotherapy given in a neo-adjuvant setting and a follow-up of less than 2 years for living patients. The study confirmed to Institutional Review Board requirements. The prospective database of our department was reviewed and 121 consecutive patients with a diagnosis of soft-tissue sarcoma who had preoperative radiotherapy treatment between 1994 and 2014 were identified, out of 1005 patients who had surgery for a soft-tissue sarcoma in the same time period (12%). Of these, 89 did not have any preoperative chemotherapy  and were finally included in this study. Patient demographics and characteristics of the cohort are presented in Table Diagnosis was set in a multidisciplinary team meeting with the participation of orthopaedic surgeons, musculoskeletal radiologists, pathologists and oncologists. The decision to give preoperative radiotherapy was taken in the same meeting, with an indication to facilitate surgical resection of the tumour with an adequate surgical margin, taking into consideration the size and anatomical location of the tumour and its relationship to important structures such as the neurovascular bundle, its expected radiosensitivity and the expected morbidity related to radiotherapy and surgery. These criteria remained constant throughout the study period. Standard radiology was magnetic resonance imaging (MRI) prior to radiotherapy with another examination after given radiotherapy but prior to excision of the tumour, and a complete data set with comparable sequences prior and post radiotherapy was available for 76 patients. All MRIs were reviewed by a radiologist with many years of experience in musculoskeletal tumor imaging. Tumour dimensions (maximum dimensions in 3 axes) were measured in cm and tumour response was evaluated either as a change in tumour volume, calculated by multiplication of the maximum dimension in 3 axes, or according to the RECIST criteria using the change in the maximum diameter of the tumour, where partial response was any reduction in tumour volume ≥ 30% but with measurable tumour left, progressive disease any increase ≥ 20%, and anything else was stable disease. The degree of peritumoural oedema was subjectively evaluated in 3-grade scale  using STIR and/or T2-sequences. Chest X-ray or computed tomography was used for the detection of lung metastases. Fine-needle aspiration cytology was done for diagnosis.Radiotherapy was given as external beam photon treatment. The most common mode of radiotherapy, given in 81% of the patients, was 50 Gy given in 25 sessions of 2 Gy (5 weeks of treatment). 13% of the patients had less than 50 Gy (36–46 Gy), as a rule given in an intensity modulated treatment and 7% were treated with a dose exceeding 50 Gy (52–70 Gy). Operations were performed by consultant grade surgeons. Median time between radiotherapy and surgery was 6 weeks (range 2–28).Surgical specimens were reviewed by a dedicated musculoskeletal pathologist. The median tumour size, as measured in the excision specimen, was 11 cm. 57% of the tumours were undifferentiated pleomorfic sarcomas, 25% liposarcomas, 8% malignant peripheral nerve sheath tumours, 7% synovial sarcomas and 3% other sarcomas.Postoperative surveillance was according to the ESMO guidelines , with cl2) were used for comparisons between groups. All tests were double-sided, and a p value of ≤ 0.05 was considered significant. 95% confidence intervals are presented in brackets. The core facility of the Statistics Department of the Karolinska Institute was consulted for the analysis of the data.Statistics were done in the SPSS software  and the STATA (version 13). Survival analyses and comparisons were done using the Kaplan–Meier method and comparisons were done using log-rank test. Hazard ratios between groups were calculated using a Cox regression analysis , where possible prognostic factors were age (dichotomized around the median), gender, tumour grade (high or low), tumour volume (dichotomized around the median), surgical margin , tumour necrosis , and radiotherapy dose (dichotomized around the median). Competitive risk analysis was done using the method of Pepe and Mori. Chi square tests . We found that the tumour volume decreased after radiotherapy in 51% of the cases, increased in 40% and remained stable in 9%. Using RECIST criteria, stable disease was noted in 67% of cases, progressive disease in 18% and partial regression in 15%. Another radiologic parameter that could be evaluated with accuracy was the presence of peritumoural oedema. We observed that prior to radiotherapy 77% of the tumours had peritumoural oedema (67% moderate and 10% heavy), whilst after radiotherapy 82% of the tumours had peritumoural oedema (58% moderate and 24% heavy). The absence of peritumoural oedema, both prior to as well as after radiotherapy, was associated with reduction of tumour volume as evaluated by MRI (p = 0.005). However, there was no association of peritumoural oedema with partial regression according to RECIST criteria (not shown). Furthermore, tumour grade (p = 0.001), but not tumour volume (p = 0.897) was inversely correlated to the degree of peritumoural oedema. Likewise, tumour grade (p = 0.016), but not tumour volume (p = 0.089) was also inversely correlated to reduction in tumour volume after given radiotherapy. There was no correlation between the degree of volume change and the time period between given radiotherapy and the last MRI (data not shown).Next, the degree of tumour necrosis was quantified, based on microscopic findings after excision of the tumour, since we found post-radiotherapy MRI too unreliable regarding an accurate interpretation of tissue necrosis. In 27% of the specimens necrosis was poor, in 33% average, in 24% good and in 16% complete.Additionally, we found that the change in tumour volume had no correlation to tissue necrosis (p = 0.638). The presence or absence of peritumoural oedema prior to or after radiotherapy did not significantly correlate with the degree of tissue necrosis (p = 0.365 and p = 0.098 respectively).R0 surgical margins were achieved in 89% of the patients , whereas R1  margins were noted in 11%. No patients had R2 margins . Local recurrence was noted in 12% of the patients. A R0 surgical margin (p = 0.014) was important for local control  or the rate of wound complications. Radiotherapy dose was not associated to the rate of wound complications (p = 0.313) or local control rate (p = 0.605).There were 9 amputations . The 5 patients who underwent a primary amputation had comparable overall survival to the rest of the patients (p = 0.099). There were no amputations due to wound healing problems and infection. Limb salvage rate was 84% at 5 years and 10 years for upper extremity tumours and 89% at 5 years and 82% at 10 years for lower extremity tumours. Pelvic location was associated with a higher risk for amputation  were relatively common in this series, as they were documented in 16% of the cases. Of the 89 patients, 31 are still alive . Overall survival (OS) was 55% at 5 years and 44% at 10 years.As presented in Table We finally tested the radiologic parameters regarding their prognostic significance Table . ReductiThe decision to give preoperative radiotherapy is mainly based on the intention to downsize the tumour and make it more easily resectable. Volume reduction may result in less morbidity by sparing important anatomical structures, whereas limb-sparing surgery in cases of close proximity of the tumour to the neurovascular bundle may sometimes be feasible only when preoperative radiotherapy is successful.Our results support the notion that preoperative radiotherapy is a successful strategy in cases of high-risk tumours, such as large-volume ones and those in close proximity to the neurovascular bundle. In our cohort, average tumour size was larger than in published cohorts , 9, indiWe identified two radiologic prognostic factors that are associated with a favourable oncologic outcome, namely the absence of peritumoural oedema and the reduction of tumour volume following radiotherapy. We believe that they represent independent phenomena: The absence of oedema probably marks a more indolent biological behaviour, since there was an inverse correlation between tumour grade and the absence of oedema. Since tumour grade is, as a rule, determined with sufficient accuracy only after examination of the resection specimen, absence of peritumoural oedema can be used as an early marker to predict the oncologic outcome. Reduction of tumour volume on the other hand obviously reflects the response to treatment, although intratumoural bleeding may contribute to a stable or increasing volume and MRI sequences specific for the detection of tissue haemorrhage may be useful in this setting. Two previous studies failed to show any significance of tumour volume increase on survival , 6, and Preoperative radiotherapy was accompanied by a very high risk for local complications, often wound infections, healing problems and dehiscence, which does not depend on the time to surgery or dose. This is in line with previous publications –20 and sWe recognize the retrospective nature of this study as its main limitation. However, since our aim was not a comparison of preoperative with postoperative radiotherapy, a question which has been addressed in other studies , 24, 25,Preoperative radiotherapy allows for good local control of high-risk tumours and excellent limb salvage rates. This is at the expense of a considerable wound complication rate, which however does not pose a threat to limb survival. Simple marginal excision is safe and mutilating surgery to achieve a wide margin thus unnecessary. The absence of peritumoural oedema on MRI as well as volume reduction of the tumour after radiotherapy defines a subgroup of patients with favourable prognosis.Additional file 1: Figure S1. Local relapse rate depending on the quality of surgical margins  of patients with non-metastatic soft-tissue sarcoma of the extremities, treated with preoperative radiotherapy, calculated in a competitive risk model with death as a competing factor. Clear surgical margins are not associated to local control rate in a competitive risk model (p = 0.173).


AMP‐activated protein kinase (AMPK) is activated by environmental or nutritional stress factors, such as hypoxia, glucose deprivation, and other cell injury factors, to regulate cell energy balance and differentiation. We previously have reported that AMPKα2 deficiency resulted in the energy deprivation in tumour‐bearing liver and the enhanced‐hepatocyte death. In this study, AMPKα2 knockout mice and the liver metastasis model of colon cancer cells were used to address the role of AMPKα isoforms in tumour inflammation. First, we found that the AMPKα2 deficiency exacerbated the liver injury and recruitment of macrophages. Meanwhile, although compensatory expression of AMPKα1 was not significant after AMPKα2 knockout, AMPKα1 phosphorylation was elevated in remnant liver in AMPKα2 knockout mice, which was positively associated with the enhanced energy deprivation in the AMPKα2 deficient mice. Furthermore, the activated AMPKα1 in macrophage contributed to its polarizing to tumour‐associated phenotype. Thus, the enhanced tumour‐associated inflammation and activation of AMPKα1 in the AMPKα2 deficient mice may exacerbate the tumour development by affecting the tumour inflammatory microenvironment. Our study suggests that the two isoforms of AMPKα, AMPKα1 and AMPKα2 play different roles in controlling tumour development.Tissue damage and its associated‐inflammation act as tumour initiators or propagators.  It is not clear whether and how the energy stress in tumour microenvironment contribute to macrophage polarization. In this study, the authors found that the AMPKα2 deficiency exacerbated the liver injury and recruitment of macrophages, and elevated AMPKα1 phosphorylation in remnant liver. The activated AMPKα1 in macrophages contributed to their polarizing to tumour‐associated phenotype. The finding provides new insights that the two isoforms of AMPKα, AMPKα1 and AMPKα2 play different roles in controlling tumour development. The insufficient clearance of dead cells by phagocytes can lead to pathological inflammation,Although both tumour‐promoting and tumour‐antagonizing immune cells are present in the tumour microenvironment, cancer cells prefer to invoke the immune system facilitating the recruitment, activation, and persistence of the tumour‐promoting immune cells.Liver tissue is susceptible for tumour origination or metastasis and is abundant with AMPKα2. Our previous study has shown that the metastasis of colon cancer cells in liver resulted in hepatocyte necrosis through energy deprivation but not only because of the space conflict.22.15 SL4 cells in 100 μL of DMEM/F12 medium were intrasplenically injected into the spleen with a 26‐gauge needle after the mice were anaesthetized by intraperitoneal injection with pentobarbital (100 mg/kg). Mice were killed at the indicated day, and the blood, spleen, and liver with tumour were harvested after heart perfusion with saline. All the mice were housed in standard cages with a constant temperature range of 22 ± 2°C, 60% relative humidity and an artificial 12‐hour light/dark cycle. The study conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health , and the protocol were approved by the Institutional Animal Care and Use Committee at Capital Medical University.A liver metastasis model was carried out as described previously.2.2D‐Luciferin (15 mg/mL) was intraperitoneally injected at a dose of 10 μL/g of mouse bodyweight just before analysis. The tumour size was analysed by a Xenogen‐IVIS Lumina II device  30 minutes after injection of 2‐Deoxyglucosone 750.2.3g for 5 minutes at room temperature. The activities of AST and ALT were measured by Automatic Analyzer 7020  with the ALT, AST assay kit .Fasting blood was collected from the heart of mice and then centrifuged at the speed of 1000 2.4The composition of inflammatory cells was quantified by flow cytometry as described.2.5Livers from control mice and SL4‐injected mice were harvested and then paraffin‐sectioned and stained with haematoxylin and eosin for morphological observation. For immunohistochemistry staining, the livers sections were fixed with 4% paraformaldehyde in PBS, incubated with the primary antibodies against Mac‐2 (1:200), Ki67(1:200), CD31(1:200), and TGFβ (1:200), and then incubated with the Dako ChemMateTM EnVision System  for 30 minutes. Staining was visualized with use of diaminobenzidine and counterstaining with haematoxylin.2.6Protein was extracted using a Protein Extraction Kit containing protease inhibitor and protein phosphatase Inhibitor Cocktail. Equal amounts of protein extract (40 μg/lane) were separated using a 10% SDS‐PAGE gel. The blot was further probed with the primary antibodies anti‐GAPDH (1:1000), anti‐AMPKα1(1:1000), anti‐AMPKα2(1:1000), p‐AMPKα Thr172, and anti‐AMPKα (1:1000) overnight at 4°C and then fluorescent secondary antibodies  for 1 h at room temperature. Protein expression was analysed with the Odyssey infrared imaging system and Odyssey software and normalized to GAPDH expression.2.7Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer's protocol and 2 μg was reversed‐transcribed using the GoScript™ Reverse Transcription system . For real‐time quantitative PCR, the iQ5 system  with SYBR Green I  was used. Amplification was performed at 95°C for 5 minutes, 95°C for 45 seconds and 57°C for 45 seconds of each step for 45 cycles. The housekeeping gene GAPDH was used as a control. The primers used are shown below: Tumour necrosis factor‐α (TNF‐α): Forward, tcttctcattcctgcttgtgg; Reverse, ggtctgggccatagaactga; inducible nitric oxide synthase (iNOS): Forward, gggctgtcacggagatca; Reverse,ccatgatggtcacattctgc; arginase‐1 (Arg‐1):Forward, aaagctggtctgctggaaaa; Reverse, acagaccgtgggttcttcac.2.8As described previously,2.9P < 0.05 was considered statistically significant.The data are expressed as means ± SD. Statistical analyses involved the use of GraphPad Prism v5.00 for Windows . Comparisons among more than two groups were analysed by one‐way analysis of variance followed by the Student‐Newman‐Keuls test. 33.1+ macrophage . The decrease of blood CD4+ cells indicated more destruction of the immune system.To investigate the association between the enhanced liver injury and tumour‐induced inflammation in the AMPKα2 deficient mice, we analysed the proportions of CD3, CD33.4−/− liver‐bearing tumours. Our results of Mac‐2 immunostaining showed that there are more tumour masses scatter in livers of AMPKα2−/− mice .+ nature killer T cell recruitment in the blood  and can be phosphorylated by several upstream kinases, such as liver kinase B1 (LKB1), cAMP‐dependent kinase (PKA), and Ca and can d Figure , which s+ macrophages were increased in the AMPKα2−/− mice at day 14 post tumour implantations. And CD19+ B cells were also increased. Correspondingly, the total immune cells, ie, CD45+ cells, were increased. Thus, comparing the results of immune cell recruitment and infiltration from blood and tumours, the increased immune cells in the tumour was not only because of the change of blood cell but also the tumour microenvironment of liver tissues affected by AMPKα2 deficiency.Inflammation has recently been considered a hallmark of cancer and plays an important role in tumour initiation and progression.−/− tumour‐bearing liver.−/− mice, although the deletion of AMPKα2 resulted in decreased total AMPKα and no significant AMPKα1 compensation (Figure −/− mice than those of WT (Figure +/CD206+ macrophages (Figure AMPK has two catalytic isoforms AMPKα1 and a2, but they are tissue‐and cell‐specific expressed. Numerous studies have shown the total effect of AMPKα1 or a2 deletion on types of mouse models, however, there is a limited study to show whether AMPKα1 and a2 play different roles in the same disease conditions although they seem to be activated by the same stimuli.n Figure B. Thus, T Figure C.. It isT Figure B, C. Thes Figure G, H, I.In summary, our study showed that the two isoforms of AMPKα, AMPKα1, and AMPKα2 in different cells exploit different ways to control tumour development in the liver. Although AMPKα2 plays anti‐tumour effects, AMPKα1 in the macrophages may be activated in the tumour microenvironment and plays a pro‐tumour effect. The total AMPKα1 knockout mice or condition knockout of AMPKα1 in immune cells will help further to exploit the AMPKα1 function in tumour environment in the future studies.The authors confirm that there are no conflicts of interests.


The aim of this study was to investigate the impact of light dose rate  and associated heating on tumour response and cure.Currently delivered light dose  higher cure rate compared to light delivery alone at same irradiance and light dose. Local control and/or cures of VX2 were obtained using I-PDT with 16.5–398 mW/cm2 and ≥45 J/cm2 in 100% of the tumour.In the mouse model, cure rates of 70–90% were obtained with I-PDT using 8.4–245 mW/cm®-mediated I-PDT, a selected range of irradiance prompts effective photoreaction with tissue heating in the treatment of locally advanced mouse tumour. These irradiances were translated for effective local control of large VX2 tumours.In Photofrin We report here the results from a preclinical study that elucidates and quantifies the impact of intratumoural irradiance on tumour response and cure, toxicity, and photothermal effects in I-PDT with PhotofrinAll procedures were carried out in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Roswell Park Comprehensive Cancer Center.29 Female C3H mice, 8–12 weeks old, were obtained from the Charles River Laboratories, Frederick, MD. Animals were housed in micro isolator cages within the Cancer Center’s animal facility and provided food and water ad libitum. A suspension of 106 SCCVII cells was prepared (100 µL injection volume) and injected subcutaneously into the shoulder region of the mice. Mice were treated when SCCVII tumours reached a volume of 400–600 mm3, a size of a locally advanced murine tumour.30The SCCVII is a poorly immunogenic, syngeneic, murine tumour model that is appropriate for studying treatments for HNC.3. In this tumour model, all local recurrences after treatment were palpable within 30 days.Tumours were measured three to four times weekly, and cure was defined as complete regression of the tumour for at least 60 days. Animals were killed at 60 days after treatment or when the tumour volume reached ≥4000 mm33 A piece of VX2 tumour from a donor rabbit, of about 1 mm3, was surgically implanted into the sternomastoid muscle at the neck level of each New Zealand White (NZW) rabbit . Non-contrast-enhanced computed tomography (CT) scans were performed to assess tumour volume and determine the presence or absence of metastases. I-PDT was scheduled when tumours reached a size of 2–3 cm in their longest dimensions. Following treatment, animal health and the treatment site were monitored daily. Tumour response was determined with CT every 1–2 weeks.This is a well-accepted preclinical model to practice clinically relevant minimally invasive ablation techniques of cancerous tumours.® concentrations in excised tumour tissue by fluorescence measurements following tissue solubilisation, as previously described.34 Tumour tissue samples were collected 24 h post injection of Photofrin® in animals bearing a single tumour similar in size and location to the one treated in the study cohorts. This assay provides an average concentration of the Photofrin® in the tumour.We determined the Photofrin19 Three stained sections from each tumour were examined for morphology and pimonidazole binding at ×40 magnification with an optical microscope .Standard hematoxylin and eosin (H&E) staining was used for examining tumour tissue morphology. The Hypoxyprobe™-1 Plus Kit containing pimonidazole hydrochloride  was used to examine tumour hypoxia, following the Kit instructions. The animals were killed via cervical dislocation and tumours were excised immediately to minimise hypoxia. Positive controls (for hypoxia staining) were prepared following Busch et al.35 The model was validated by comparing the simulation results to measurements in liquid phantoms with optical properties similar to mouse tumour.35 The simulations were done for geometrical models of tumours with cylindrical diffuser and catheters. The models were reconstructed from representative magnetic resonance imaging (MRI) scans of mouse tumours and CT scans of NZW rabbit with VX2 tumour. The scans were segmented with image analysis software , as previously described.36 The tumour optical properties were measured with a non-invasive reflectance spectroscopy system . The FEM simulations were conducted by solving the light diffusion equation in 3D with Comsol 5.2 . The governing equations and model assumptions are detailed in our previous publications.36Our finite-element modeling (FEM) approach was employed to compute intratumoural light irradiance and fluence, as previously described.The laser light was delivered through 0.98 mm diameter optical fibers with a 2-cm long cylindrical diffuser  connected to 1.0 W diode-laser module that emits 630 ± 3 nm light . The laser power, emitted from the cylindrical diffuser, was measured with an integrated sphere  before and after treatment.®. The laser light was delivered through a single RD20 that was placed in a light transparent catheter . The catheter was previously inserted through the centerline of the tumour along its longest axis and horizontal to the mouse skin.C3H mice with SCCVII tumours were treated under isoflurane (3–5%) anesthesia. In one set of studies, mice were treated with a light energy of 100 J/cm that was administered with light intensities of 60, 100, 150, 200, or 400 mW/cm, with and without 5 mg/kg Photofrin37 The FEM simulations were utilised to identify light intensities for the mouse studies. The simulations suggest that two 2-cm cylindrical diffusers delivering 630-nm light at 60–150 mW/cm and 540 J/cm per fiber are required to deliver hypothetically effective intratumoural irradiance and fluence for I-PDT with Photofrin®. Mice were treated with either no drug or with Photofrin® at 3.3, 5.0, or 6.6 mg/kg that was administered via intravenous tail vein injection, 24 h prior to the light delivery. Compared to the clinical use of Photofrin®, the higher Photofrin® dose and shorter drug-light interval 24 vs. 48 h, was used to compensate for the higher metabolism of these animals in comparison to humans. A control cohort included mice (n = 21) with untreated SCCVII tumours.In subsequent studies, tumour-bearing mice were treated using two cylindrical diffusers placed within two parallel catheters that were inserted at 6 ± 1 mm apart, along the tumour centerline and horizontal to the mouse skin. In this regimen, the light fluence and irradiance were selected in order to test the hypothesis that, when accounting for light attenuation in the tumour, an effective I-PDT requires intratumoural light irradiance and fluence that are comparable to those resulting from effective EB-PDT.® was administered intravenously via a catheter in the ear vein 24 h prior to light illumination. The rabbits were treated under general anesthesia with isoflurane inhalation. Surface fiducial markers  were used for guiding the placement of the cylindrical diffuser according to the treatment plan, as detailed in Oakley et al.36 A light dosimetry system was used to measure irradiance and fluence during treatment.38 The tumour temperature was measured during treatments with optical fiber thermometers .In the NZW rabbit study, image-based pretreatment planning with our FEM simulations was conducted for each animal. The images were acquired 1–2 days prior to treatment with non-contrast-enhanced CT . A single bolus of 5 mg/kg Photofrink-space data were filtered with a two-dimensional (2D) Gaussian function to improve signal-to-noise ratio. Following 2D Fourier transforms into the spatial domain, phase images were normalised to the phantom and a 2D phase unwrapping function was applied. The temperature changes were calculated by conversion of 0.0359 radians decrease in phase values for 1 °C increase in temperature. Laser-induced irreversible thermal damage  will occur immediately when the tissue temperature exceeds 60 °C.39 Using the MRT data, we computed the volume fractions that were subjected to temperatures sufficient to induce photothermal ablation during treatment. A computer code written in MATLAB  was used to calculate the number of voxels at T > 60 °C divided by the number of voxels comprising the tumour.The temperature changes during treatment of mice were assessed with magnetic resonance thermometry (MRT). The MRT was performed using the proton resonance frequency method, in a 4.7 Tesla preclinical scanner using the ParaVision 3.0.2 MRI  with a 35 mm quadrature transceiver coil. A nuclear magnetic resonance tube filled with cottonseed oil was used as a phase normalisation reference. The MRT was performed using a 15-slice spoiled gradient echo scan . Following three baseline acquisitions scans, the interstitial light was administered, and MRT data were collected throughout the treatment and for 5 min after the laser light was off. Raw 3 or death. These end points were compared using either exact Pearson χ2 or Cox regression analysis. The exact Kruskal–Wallis rank sum test was used to compare intratumoural Photofrin® retention for different systemic doses. The percent of tumour volume at T > 60 °C was estimated assuming a nonlinear monotone-increasing exponential model and fit using a Newton-Raphson optimisation technique.40 A two-way analysis of variance was then used to compare between light-only and I-PDT with Photofrin®. All experimental conditions had sample sizes ranging from 10 to 20 animals. All tests were tested at α = 0.05 (two-sided). The general testing strategy was to first carry out the overall test at alpha, and if significant, carry out Bonferroni-adjusted pairwise comparisons. All analyses were done using SAS 9.4 . As a guide, at 80% power and n = 10 per group in a three group comparison, we could detect cure rates with roughly a 60% or larger difference between groups and hazard ratios (HRs) of roughly 4 to 1 or larger  for our time-to-event end points.The primary end points were 60-day cure or time to the tumour reaching a volume ≥4000 mm6 SCCVII cells produced tumours in 100% of mice. All untreated control tumours reached a volume of 4000 mm3 within 10–11 days of tumour cell inoculation; additionally, no palpable tumours showed spontaneous regression. Large murine tumours tend to form central necrosis and regions of tissue hypoxia, which negatively affect the response to PDT.41 Therefore, we evaluated SCCVII tumour pathology with H&E and pimonidazole staining. Histology and immunohistochemistry revealed little necrotic tissue, with minimally hypoxic regions (data not shown). The intratumoural Photofrin® concentrations in freshly excised SCCVII tumours were 2.7 ± 1.1, 3.8 ± 1.5, and 6.0 ± 1.7 mg/kg at 24 h, corresponding to the time at which I-PDT light is delivered, following intravenous administration of 3.3, 5.0, and 6.6 mg/kg, respectively.An inoculation dose of 10®. The FDA-approved light intensity of 400 mW/cm was de-escalated to 200, 150, 100, and 60 mW/cm. The corresponding intratumoural irradiances were: 9.1–979, 4.5–490, 3.4–367, 2.3–245, and 1.4–147 mW/cm2. The computed minima and maxima were at the margins and treatment fiber/tumour interface, respectively. The MRT data revealed that tumour tissue heating occurred when 100 J/cm of 630-nm light was delivered through single 2-cm cylindrical diffuser at intensities of 60–400 mW/cm  will be referred to as LITH.Having established that tissue heating is an integral feature of I-PDT, we proceeded to analyse its extent and consequences. The light-induced tissue heating  was the same (data not shown) in mice treated with either LITH or I-PDT with 5 mg/kg Photofrin®. Therefore, the analysis of relative tumour volume at T > 60 °C, following delivery of 100 J/cm 630-nm light, was combined for LITH and I-PDT. In mice treated with either 60 or 100 mW/cm, <5% of the tumour volume was subjected to T > 60 °C  following I-PDT vs. LITH, using the HR analysis, as shown in Table 2), an HR = 1.57 suggested that LITH governed tumour growth delay, and appears to have negated a possible PDT contribution. At light intensities of 200, 100, and 60 mW/cm, I-PDT appears to be somewhat, although not significantly, more effective than LITH. Only a light intensity of 150 mW/cm (3.4–367 mW/cm2) revealed a significant PDT contribution (HR = 0.04).To address the question of whether photothermal ablation or PDT governed tumour response in I-PDT, we compared tumour responses in mice treated with LITH to those treated at the same light intensity with I-PDT using 5 mg/kg Photofrin2 and fluence of 128 J/cm2 would result in an effective tumour response in mouse models.37 Using the results from those studies as a guideline, we calculated the percent tumour volume that would be illuminated at 14–75 mW/cm2 vs. light intensity in a representative geometry constructed from an MR image of an average SCCVII tumour (556 mm3); calculations were based on delivering I-PDT with two 2-cm long cylindrical-diffuser fibers spaced 6.31 ± 0.11 mm apart. The FEM was carried out using measured absorption and reduced scattering coefficients of 145 1/m and 760 1/m, respectively, which were in agreement with published data for murine tumours.42 The index of refraction was set to 1.37, and laser light was 630 nm. The 60 mW/cm appeared to be optimal when the objective was to maximise the PDT contribution by delivering 14–75 mW/cm2 to the largest portion (72%) of the tumour volume  for 60 mW/cm is shown in Fig. 2, and at light intensities >60 mW/cm, a larger part of the tumour volume will receive >75 mW/cm2.Turning the focus toward the PDT component in I-PDT, intratumoural irradiance is recognised as a major determinant of PDT effectiveness. Several studies suggest that in EB-PDT irradiance in the range of 14–75 mW/cmume Fig. . The int2, over about 9000 s), the light energy should be 540 J/cm at 60 mW/cm. In addition to 60 mW/cm, we evaluated tumour response to 100 and 150 mW/cm with 540 J/cm, which also has the advantage of shorter treatment times.Assuming that in I-PDT the intratumoural fluence should be comparable to the one delivered to the tumour surface in EB-PDT (128 J/cmn = 10 at each setting). There was a significant difference in the volume fraction of tumour >60 °C in mice treated with 5.0–147 mW/cm2  in comparison to mice treated with 8.4–245 mW/cm2 (p = 0.0020) or 12.6–367 mW/cm2 (p = 0.0007) corresponding to 100 and 150 mW/cm, respectively  higher cure rate of 70% was obtained. A 40% cure rate with LITH was observed in mice treated with 100 mW/cm and 540 J/cm  enhanced the cure rate to 90% following I-PDT in comparison to LITH, as shown in Kaplan–Meier plots  with two 2-cylindrical diffusers and 100 mW/cm and 540 J/cm, which corresponded to an intratumoural irradiance of 8.4–245 mW/cm2 and a fluence of 45–1323 J/cm2. The Photofrin® retention in VX2 tumours was 4.0 ± 0.67 mg/kg 24 h after intravenous administration of 5.0 mg/kg Photofrin®, similar retention observed in SCCVII in mice. I-PDT with 5.0 mg/kg Photofrin® was used to treat five NZW rabbits with VX2. Because the average VX2 tumour volume was >8–10 times larger than the mouse tumours and the maximum number of fibers we could implant was limited by the anatomy, relatively high light intensities (200–250 mW/cm) were required to deliver ≥8.4 mW/cm2. The average minimum of intratumoural irradiance, 20.5 ± 9.1 mW/cm2, was higher than in mice. A representative example of I-PDT that resulted in an effective response is shown in Fig. 3) was treated with 10 optically transparent closed-end brachytherapy catheters , through which the cylindrical diffuser and/or dosimetry fibers were inserted  is complicated by significant treatment-related mortality of 40 and 30% in mice treated with LITH alone and I-PDT, respectively  thermal ablation is an established treatment for cancerous tumours.39 It is typically limited to the treatment of small tumours (<3 cm) not in the vicinity of major blood vessels (that act as heat sinks) and away from nerves. High-temperature thermal ablation can cause irreversible damage to nerves. In several small studies, I-PDT has been proven safe when applied to large locally advanced tumours next to facial nerves,48 offering a potential advantage over thermal ablation.Multiple studies have demonstrated that low light irradiance and high fluence will significantly improve local tumour control with EB-PDT in preclinical and clinical settings.36 In this study, we used fibers, catheters, and CT imaging that are employed in the clinic. The impact of the Photofrin®-mediated PDT component, as compared to the thermal component, was much more pronounced in the rabbit than in the mouse model. Since the VX2 tumour is more akin to human tumours in both size and pathology, this finding is especially promising for the clinical use of I-PDT in locally advanced HNC.The FEM pretreatment planning was successfully used to translate the findings from the mouse studies to the NZW rabbit model, where the tumour size is close to human tumours. In previous publications, we demonstrated that FEM could be used to plan and guide interstitial light delivery in models mimicking human tumours.® in the treatment of patients with locally advanced HNC, utilising the guidelines in this manuscript. As a proof of concept, a treatment planning for a patient with locally advanced HNC who was amenable to receive I-PDT is shown in the 35 and used in the present preclinical study. The treatment plan suggested that 12 cylindrical-diffuser fibers with 1, 2, and 3-cm long diffusers, delivering a light intensity of 200 mW/cm per fiber, would be required to deliver an intratumoural irradiance of ≥29.4 mW/cm² and a fluence of ≥45.9 J/cm² to 100% of the tumour.We plan to follow this work with a multi-centre phase I/II clinical study to evaluate I-PDT with PhotofrinSupplementary material


Testicular germ cell tumours metastasize in a very predictable fashion involving the retroperitoneal lymph nodes first followed by either lymphatic spread via thoracic duct or hematogenous spread to distant organs like lungs, liver and brain.We encountered a case of 21-year-old gentleman with mixed germ cell tumour of testis who later presented with respiratory complaints which turned out to be a metastatic differentiated teratoma of unilateral pleura without involvement of retroperitoneal lymph nodes or any other organs.Skip Metastasis to unilateral pleura is an extremely rare entity for testicular mixed germ cell tumour and no case has been reported in the literature so far. These tumours are the most common solid tumours in young adult men between 20 and 40 years of age. More than 90% of testicular neoplasms correspond to germ cell tumours that are divided into seminomatous and nonseminomatous tumours, corresponding to the histologic subtypes of embryonal carcinoma, yolk-sac tumour, teratoma and choriocarcinoma [More than half of the germ cell tumours contain more than one cell type and are therefore known as mixed germ cell tumours .The majority of the germ cell tumours metastasize to the lymph nodes in a characteristic pattern, although choriocarcinoma preferentially has hematogenous spread. Route of spread for testicular mixed germ cell tumour depends on the histological subtypes present in the tumour . RetropeThe work has been reported in the line with the SCARE criteria .221-year-old male with history of left sided testicular swelling with increased alfa fetoprotein levels  and beta-human chorionic gonadotropin levels (66.05 mIU/ml) with normal chest radiograph, underwent left high inguinal orchidectomy in December 2016 in the outside hospital. Histopathology report of the specimen was suggestive of “Mixed seminomatous and non seminomatous germ cell tumour with yolk sac tumour (40%), mature teratoma (55%) and seminoma (5%)”. Positron emitted tomography was done 1 month later post orchidectomy suggestive of “Diffuse metabolic activity seen in the left hemi-scrotum is secondary to recent surgery. There is no metabolically active lymph node, pleural, peritoneal, omental, solid visceral, osseous lesion, ascites or pleural effusion. Isolated non metabolic nodule in the left upper lobe and left lower lobe of the lung would need serial follow up”.Patient was asymptomatic for next 9 months and didn’t come for the regular follow-ups until he developed chest pain, breathlessness and severe back pain .Fig. 1ChComputed tomography of the chest done in September 2017 suggestive of “Area of collapse-consolidation in the periphery of apical-posterior segment of left upper lobe of lung and another well-defined oval radiopacity with sharp borders is noted in superior segment of lower lobe of left lung with left hydropneumothorax- nonspecific morphology probably of benign aetiology”.Patient was managed with intercostal drainage tube insertion. Tumour markers were done showed Alpha fetoprotein levels (1606 IU/ml) and beta-human chorionic gonadotropin levels (5.40 mIU/ml). The pleural fluid sample was sent for cytology showed no malignant cells. subsequent radiographs were taken which confirmed the resolving pattern. Patient was discharged within 5 days after removal of the intercostal drainage tube.Within 2 months patient again presented with severe respiratory complaints and repeat computed tomography of chest was done in December 2017 showed “subpleural nodule in left lower lobe of lung is suggestive of metastasis. This study is negative for any significant mediastinal adenopathy, interstitial lung disease or centrilobular opacities with shallow left hydro-pneumothorax”. Patient was again managed with intercostal drainage tube and pleural fluid sent for cytology was negative for any malignant cells.Patient was again hospitalised 2 times in 15 days with similar complaints for which pleurodesis was also attempted. But in view of recurrent pleural effusions which were not responding to medical management, patient was then subjected to 4 cycles of chemotherapy with Bleomycin, Etoposide and Cisplatin for a period of 2 months. Within a week after completion of chemotherapy patient was again admitted with severe respiratory complaints. The whole-body positron emitted tomography was repeated in February 2018 showed “Appearance of the multiloculated lesion in the pleural space on the left side is a new finding compared to the prior scan and should assumed to be metastatic unless proved otherwise with fluid aspiration cytology or biopsy of the solid component in a nodule. No retroperitoneal adenopathy seen” .Fig. 2PEPatient was subjected to salvage chemotherapy with Etoposide, Carboplatin and Ifosphamide in a period of March to May 2018. Pleural fluid aspiration under sonography guidance was done which again failed to show any malignant cells. .Fig. 3Chth chemotherapy cycle done, suggestive of “Considerable increased in shift of cardio-mediastinal structures. The rest of the morphological appearance of the loculated/septated left pleural effusion remains more or less unchanged with no significant mediastinal adenopathy or pulmonary metastasis.” Mixed germ cell tumour with skip metastasis to unilateral pleura without involvement of any retroperitoneal lymph nodes or secondary to hematogenous spread involving mediastinal structures or any other distant sites is extremely rare.2)Histological subtype of metastatic lesion is different from the histological subtype of the primary tumour.3)Changing nature of histology of tumour with chemotherapy and progress of disease is known.4)Combined approach of chemotherapy and surgery is needed in case of mixed germ cell tumour depending on the histological subtype, metastasis and clinical presentation of the patient.This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.This is a case report and does not require ethics committee approval.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.1) Dr Shravani Shetye: Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Critical revision of the article, Final approval of the version to be published.2) Dr Neerajpratap Singh: Conception and design, Acquisition of data, Analysis and interpretation of data. Drafting the article, Critical revision of the article, Final approval of the version to be published.3) Manmohan Kamat: Conception and design, Acquisition of data, Analysis and interpretation of data.4) Dr Katik Nattey: Drafting the article, Critical revision of the article.5) Dr Seema Barman: Conception and design, Acquisition of dataThis is a case report and not a research study.Dr Manmohan Kamat.Not commissioned, externally peer-reviewed.None declared.


We further validated 18F-ICMT-11 specificity in a murine melanoma death-switch tumour model with conditional activation of caspase-3 induced by doxycycline.We have previously developed the caspase-based radiotracer, 18F-ICMT-11, 18F-ML-10 and 18F-FDG were assessed in B16ova and B16ovaRevC3 cells after death-switch induction.Caspase-3/7 activity and cellular uptake of 18F-ICMT-11 and 18F-ML-10 biodistribution was assessed by ex vivo gamma counting of select tissues. PET imaging was performed with 18F-ICMT-11, 18F-ML-10 and 18F-FDG. Caspase-3 activation was confirmed by immunohistochemistry.Death-switch induction was confirmed in vivo in xenograft tumours, and 18F-ICMT-11 (p < 0.001) and 18F-ML-10 (p < 0.05) and decreased 18F-FDG (p < 0.001) uptake compared with controls.Significantly increased caspase-3/7 activity was observed only in B16ovaRevC3 cells after death-switch induction, accompanied by significantly increased p < 0.01). Biodistribution studies showed significantly increased 18F-ICMT-11 tumour uptake following death-switch induction (p < 0.01), but not for 18F-ML-10. Tumour uptake of 18F-ICMT-11 was higher than that of 18F-ML-10 after death-switch induction. PET imaging studies showed that 18F-ICMT-11 can be used to detect apoptosis after death-switch induction, which was accompanied by significantly increased expression of cleaved caspase-3. 18F-FDG signal decreased in tumours after death-switch induction.B16ova and B16ovaRevC3 tumours had similar growth in vivo; however, B16ovaRevC3 growth was significantly reduced with death-switch induction  contains supplementary material, which is available to authorized users. Apoptosis, or programmed cell death, plays a crucial role during embryonic development, during differentiation, and in the maintenance of tissue homeostasis. Therefore, alterations in apoptotic signalling pathways occur in various disease pathologies; for example, excessive apoptosis is observed in ischaemia and transplant rejection , while iApoptosis can be triggered through several signalling pathways and involves a family of cysteine-dependent aspartate-directed proteases, known as caspases , 7.These are divided into two groups: initiator caspases , and effector caspases  , 10 and 18F-ICMT-11, for the in vivo assessment of tumour apoptosis and for monitoring response to cancer therapy by PET imaging, and have also shown favourable clinical dosimetry and safety profiles of 18F-ICMT-11 in a phase I trial [18F-ML-10, 18F-C-SNAT, 18F-fluorobenzyl triphenylphosphonium cation and 18F-CP18, no PET imaging agent has been approved for clinical use [We have developed and validated an isatin-based caspase-3/7-specific radiotracer,  I trial –19. Althical use –27.18F-ICMT-11 as a caspase-3-specific, non-invasive imaging radiotracer in a death-switch syngeneic murine tumour model. This tumour model was generated by transfecting the ovalbumin-expressing mouse B16 melanoma cell line (B16ova) with a tetracycline-regulated reverse transcriptional transactivator, enabling the conditional induction of constitutively active caspase-3 (B16ovaRevC3) [18F-ICMT-11 as an apoptosis radiotracer in this genetic model of conditional caspase-3 activation, which offers an experimental system devoid of confounding drug effects, and the transcriptional evolution and translation of caspase-3 activity over time after doxycycline administration. Therefore, this model is a useful tool for the validation of 18F-ICMT-11. We also evaluated another apoptosis radiotracer, 18F-ML-10 [18F-ICMT-11 along the translational pathway, and 18F-FDG to assess tumour glucose metabolism and proliferation.The present study aimed to investigate and further validate vaRevC3) . It has vaRevC3) . Thus, w8F-ML-10 , which i18F-ICMT-11 and 18F-ML-10 were performed according to previously described protocols [18F-ICMT-11 and 18F-ML-10 non-decay-corrected radio-chemical yields were 8–16% and 18–24% respectively. Radiochemical purity was > 98% at the end of synthesis for both radiotracers. 18F-FDG was obtained from Alliance Medical .Synthesis and radiolabelling of rotocols , 30. 18Fl-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin , at 37 °C in a humidified atmosphere containing 5% CO2. The medium for B16ovaRevC3 cells was further supplemented with 500 μg/ml hygromycin B (Corning).The murine melanoma B16ova and B16ovaRevC3 cell lines were kindly provided by Prof. Illidge . Both cell lines were maintained in an RPMI-1640 medium supplemented with 10% foetal calf serum, 2 mM Apoptosis was assessed in B16ova and B16ovaRevC3 cells in response to doxycycline  based on caspase activity using the Caspase-Glo-3/7 assay (Promega) according to the manufacturer’s protocol. Briefly, cells were exposed to doxycycline (0 or 2 μg/ml) for 1, 6, 24 or 48 h, and then incubated with Caspase-Glo reagent for 1 h. Luminescence was measured using a TopCount NXT microplate luminescence counter (PerkinElmer). Luminescence signal was normalized to protein content, which was determined using the BCA 96-well plate assay (Thermo Fisher Scientific). Experiments were performed in triplicate and data were expressed as fold-difference in caspase-3/7 activity (per mg of protein) from controls (mean ± SEM).5 per well) were plated into six-well plates and incubated overnight. Cells were then exposed to doxycycline  for 6 h (since caspase-3/7 activity was highest at this time point), and then incubated with 0.74 MBq/well of 18F-ICMT-11, 18F-ML-10 or 18F-FDG for 1 h at 37 °C in a humidified atmosphere containing 5% CO2. The medium was then removed, and cells were washed twice with PBS, prior to lysis in RIPA buffer (Thermo Fisher Scientific) for 15 min. Radioactivity in cell lysates was measured by gamma counting . Protein content of the lysates was determined using the Pierce BCA assay (Thermo Fisher Scientific). Experiments were performed in triplicate, using six replicates per assay, and data were expressed as percent of total decay-corrected radioactivity per milligramme protein (mean ± SEM).Cell uptake studies of the radiotracers were performed as previously described . Briefly6 tumour cells were injected subcutaneously into the neck region of the mice. Tumour dimensions were measured with callipers, and tumour growth was assessed based on tumour volume, calculated using the formula: volume = π/6 (length × width × height). Tumour growth was compared between B16ova and the genetically modified B16ovaRevC3 xenografts. To activate the death-switch in vivo, 10 days post tumour cell inoculation, doxycycline  or water (vehicle-control) was administered once per day by oral gavage for 5 days to mice bearing B16ovaRevC3 xenografts  and tumour volumes were measured for the treatment duration to show changes in tumour growth following death-switch induction. To ensure animal welfare, the weights of mice were also monitored to qualitatively assess possible adverse effects.All animal experiments were performed in accordance with the United Kingdom Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 Amendment regulations 2012 and within the published National Cancer Research Institutes Guidelines for the welfare and use of animals in cancer research . Studies18F-ICMT-11 and 18F-ML-10) was evaluated in the B16ovaRevC3 tumour model, 24 h after vehicle-control or doxycycline administration to allow activation of the death-switch, as previously reported [18F-ICMT-11 or 18F-ML-10 (1.3 ± 0.2 MBq per mouse). Mice were allocated to two groups: (1) control and (2) doxycycline (n = 6 mice per group for each radiotracer). One hour after radiotracer administration, mice were sacrificed and select tissues were excised, and radioactivity was measured by ex vivo gamma counting in these select tissues . Radiotracer biodistribution was calculated as percentage of injected dose per gram of tissue (%injected dose/g).Apoptotic radiotracer biodistribution , using parameters based on previous radiotracer distribution studies [18F-ICMT-11, 18F-ML-10 or 18F-FDG were administered and PET imaging was conducted. Mice were allocated to two groups: (1) control and (2) doxycycline (n = 6 mice per group) for each tumour model and for each radiotracer . An additional group (n = 6) for each tumour model was included to evaluate whether 18F-ICMT-11 can be used to image caspase-3 activation at an earlier time point, 6 h after doxycycline administration.PET imaging using the apoptotic radiotracers,  studies , 33, 34.Imaging was performed using the Genisys 4 Preclinical PET scanner . Based on previous studies of radiotracer distribution, data were acquired over 30–60 min in list mode and corrected for decay. 3D ML-EM (60 iterations) reconstruction was performed and presented as summed 30–60-min frames (6x5min). Image visualization was performed using the Siemens Inveon Research Workplace software as previously described .Following PET imaging, tumour tissues were collected, fixed in formalin, embedded in paraffin, and sectioned. Immunohistochemistry for cleaved caspase-3 was performed on tumour sections to evaluate apoptosis. Briefly, after antigen retrieval, sections were incubated with a primary antibody for cleaved caspase-3  overnight at 4 °C, and visualized using biotin-conjugated secondary antibodies  and 3, 3′-diaminobenzidine . Sections were then counterstained with haematoxylin and mounted with DPX (Sigma Aldrich).Image acquisition of tumour sections was performed using the AxioScan digital slide scanner (Zeiss), and QuPath  image analysis software was used for IHC quantification . Brieflyt tests, ANOVA and Tukey’s or Sidak’s multiple comparisons tests. Results were considered statistically significant at a P value of 0.05 or less.Data were plotted and analysed using Prism, version 7.0 (GraphPad Software) and expressed as mean ± standard deviation. Differences between the groups were assessed by unpaired Student’s and multiple We have outlined the overall experimental paradigm in Fig. p = 0.001 and p < 0.001, Student’s t test).We verified that caspase-3/7 induction occurs in B16ovaRevC3 cells following doxycycline exposure. Doxycycline had no significant effect on caspase-3/7 induction in B16ova cells, but a significant increase in caspase-3/7 activity was observed at 6 h (13-fold) and 24 h (3-fold) in B16ovaRevC3 cells compared with controls   and 18F-ML-10 (p < 0.05) and significantly lower uptake of 18F-FDG (p < 0.001), compared with respective controls   Fig. b.18F-ICMT-11 (p < 0.001) and 18F-ML-10 (p < 0.01), and significantly lower uptake of 18F-FDG (p < 0.05) in B16ovaRevC3 cells compared with B16ova cells (multiple t tests), Fig. 18F-ICMT-11 and decreased 18F-FDG uptake in B16ovaRevC3 cells after death-switch induction . Of interest, the uptake of 18F-ICMT-11 was 12-fold higher than that of 18F-ML-10 in untreated B16ovaRevC3 cells (p < 0.001).Significantly higher uptake of p < 0.01, multiple t tests), Fig. p < 0.001, multiple t tests), Fig. Growth of B16ova and B16ovaRevC3 tumour xenografts was evaluated in vivo. Both tumour models had similar growth kinetics over time without the administration of doxycycline . Furthermore, uptake in the small intestine could also be reflective of the prevalent physiological apoptosis of enterocytes at the villus tips and of macrophages within the intestinal lamina propria [ad libitum, it is likely that food intake prior imaging could have also additionally contributed to the intestinal radiotracer uptake. Apoptosis is also involved in normal lung structure and function, which can possibly contribute to some 18F-ICMT-11 uptake in this tissue [18F-ICMT-11 (lungblood pool).Apoptotic radiotracer biodistribution after caspase-3 activation was evaluated in the B16ovaRevC3 tumour model by ntestine . Rapid h propria , 36. Mor propria . As mices tissue . Additio18F-ICMT-11 in mice with B16ova and B16ovaRevC3 tumour xenografts at 6 and 24 h after doxycycline administration. We also conducted PET imaging using 18F-ML-10 and 18F-FDG, 24 h after vehicle-control or doxycycline administration. Representative PET images of 18F-ICMT-11 and 18F-ML-10 are displayed in Additional file 18F-FDG are displayed in Fig. We used PET imaging to assess whether caspase-3 activation can be detected using Detection of cleaved caspase-3 by immunohistochemistry was used to evaluate apoptosis in tumours following treatment with doxycycline for death-switch induction.p < 0.05, multiple t tests). Of note, after death-switch induction, a heterogeneous mix of necrosis, immune reaction, patches of tumour cells positive for cleaved caspase-3 within a dense stroma, adipose formation and vesicularization was observed within B16ovaRevC3 tumour sections  mice bearing B16ova or B16ovaRevC3 xenografts. However, following administration of doxycycline, the mean percentage (% ± SD) of cleaved caspase-3 expression in B16ovaRevC3 tumours was 23 ± 7 and 20 ± 5 at 6 and 24 h post-treatment, respectively, demonstrating a significant induction of apoptosis  higher lipophilicity and bidirectional plasma membrane permeability of 18F-ICMT-11 compared to the peptide structure of 18F-ML-10 and (b) the specificity of 18F-ICMT-11 for caspase-3/7 induction, which is the main mechanism of apoptosis induction in the death-switch model. Another consideration is that externalization of phosphatidylserine (which leads to intracellular acidification) occurs subsequent to caspase-3/7 activation [18F-ML-10 in the used model. As caspase-3/7 activation precedes phosphatidylserine exposure, imaging caspase-3/7 activity is a good strategy for the prediction of early response to treatment.By comparing reserved . There w8F-ML-10 , 40. Thetivation , which m18F-ICMT-11 and 18F-ML-18, was altered following death-switch induction in B16ovaRevC3 tumours. Based on ex vivo gamma counting, the tumour uptake of 18F-ICMT-11 was significantly higher after caspase-3 activation (death-switch induction), reflecting the specificity of the radiotracer for the target, whereas no significant change was observed for 18F-ML-10. Similar to our in vitro findings, in vivo 18F-ICMT-11 tumour distribution was significantly higher compared with 18F-ML-10, further supporting the specificity of 18F-ICMT-11 for activated caspase-3, which is the main mechanism of apoptosis induction in the death-switch model.We next investigated whether these findings can be translated in vivo. We found that B16ova and B16ovaRevC3 tumours exhibited similar growth kinetics without doxycycline administration. The administration of doxycycline significantly reduced B16ovaRevC3 tumour growth, but had no effect on parental cell line xenografts, which was in line with previous findings . Death-s18F-ML-18 after doxorubicin administration in head and neck tumour xenografts at 3 and 7 days post-treatment, but with minimal change at day 1 [18F-ML-10 uptake in nasopharyngeal carcinoma at 24 and 48 h after combined radiotherapy and cetuximab treatment, which correlated with increased TUNEL staining [18F-ML-10. Nonetheless, some of these can alter membrane permeability and integrity, and hence radiotracer distribution.Of note, a recent study demonstrated an increased uptake of at day 1 . Howeverstaining . Ionisin18F-ICMT-11 after death-switch induction was lower than that seen in vitro , which was anticipated, given that in vivo evolution of response is expected to be different from in vitro dynamics. Potential reasons for this are as follows: (a) the possibility that changes induced within the tumour microenvironment after death-switch induction could have altered the tumour uptake of the radiotracer, hence not fully reflecting the proportion of tumour cells undergoing apoptosis. For example, a heterogeneous mix of necrosis, immune reaction, patches of tumour cells undergoing apoptosis within a dense stroma, adipose formation and vesicularization was observed within these tumours after death-switch induction. It is possible that these transformations can lead to the compression of blood vessels and hence limit the amount of 18F-ICMT-11 reaching its target in tumour cells; (b) Melis et al. found that after death-switch induction, tumours undergo rapid caspase-dependent cell death, along with the release of aforementioned endogenous danger signals and quick progression towards secondary necrosis with evidence of an immune response [The fold-difference in the in vivo uptake of response , which i18F-ICMT-11 in a time-course study; however, the data obtained from these tumour models were too subjective for quantification. Nonetheless, PET imaging showed that caspase-3 activation can be detected using 18F-ICMT-11 in B16ovaRevC3 tumours, 6 and 24 h after death-switch induction, which was also accompanied by significantly increased expression of cleaved capase-3 in tumour sections, as demonstrated by IHC, confirming the induction of apoptosis in these tumours.We also investigated whether caspase-3 activation can be detected by PET imaging using 18F-FDG signal following death-switch induction, suggesting a decrease in glucose metabolism or tumour growth. A significant reduction in tumour volume was also observed, but only at 24 h, corroborating that tumour volume measurements alone are not sufficient for the prediction of response to therapy. These findings support the use of molecular imaging to predict early response to therapy since the induction of apoptosis was detected by 18F-ICMT-11 PET imaging as early as 6 h, prior a macroscopically measurable response to treatment.Interestingly, PET imaging revealed a decrease in 18F-FDG, 18F-FLT is another radiotracer used for the assessment of tumour cell metabolism; however, in contrast to 18F-FDG, 18F-FLT appears not to accumulate in inflammatory processes [18F-FDG and 18F-FLT are useful tools for the detection of changes in energy metabolism and cell proliferation, the use of these radiotracers for prediction of early response to treatment has provided variable results [18F-ICMT-11 to routine PET imaging can be beneficial in stratifying patients into responders and non-responders. Indeed, we have previously assessed 18F-ICMT-11 in numerous pre-clinical tumour models and have demonstrated added value of 18F-ICMT-11 to 18F-FDG, 18F-FLT and diffusion-weighted MRI [Beyond apoptosis imaging, the use of metabolism radiotracers to assess response to therapy is a routine clinical practice. Besides the gold-standard rocesses , 43. Whi results –48. Therhted MRI , 49.18F-ICMT-11 can detect caspase-3 activation in a doxycycline-inducible death-switch model, which further validates the specificity of the tracer without the confounding effects of anti-cancer therapeutics. These findings, along with previous evidence, verify that 18F-ICMT-11 PET imaging permits the real-time assessment of caspase-3 activation over time, and thus further support the development of this radiotracer for clinical use to monitor tumour apoptosis in response to treatment. Timing and image analysis strategies should be optimized based on the type of tumour and treatment regimen, and taking into consideration the clinical heterogeneity and dynamics of apoptosis for ultimate assessment of treatment outcome.In conclusion, we demonstrate that Additional file 1:Figure S1. PET imaging with apoptotic radiotracers. Representative PET images with (A) 18F-ICMT-11 at 6 and 24 h and (B) 18F-ML-10 at 24 h, after doxycycline administration in mice bearing B16ova and B16ovaRevC3 tumours. (PDF 560 kb)


Neovascularisation of a large tumour in the left atrium.The patient has a recent history of a transient ischaemic attack and atrial fibrillation treated with dabigatran. Recent chest radiography and spirometry test results were normal. Physical examination was unremarkable. Echocardiography revealed a large mobile mass in the left atrium attached to the atrial septum prolapsing across the mitral valve in diastole causing mitral valve obstruction Fig. a, b. PreLeft atrial myxoma is the most common primary cardiac tumour . The cli


In the Funding statement, the grant number from Vietnam National Foundation for Science and Technology Development is incorrect. The correct grant number is: 105.08–2017.11.


A novel 3D printing material based on hydroxypropyl methylcellulose (HPMC)—improved sulphoaluminate cement (SAC) for rapid 3D construction printing application is reported. The hydration heat, setting time, fluidity of paste and mortar, shape retainability, and compressive strength of extruded SAC mortar were investigated. HPMC dosage, water-to-cement (W/C) ratio, and sand-to-cement (S/C) ratio were studied as the experimental parameters. Hydration heat results reveal HPMC could delay the hydration of SAC. The initial and final setting time measured using Vicat needle would be shortened in the case of W/C ratio of 0.3 and 0.35 with HPMC dosage from 0.5% to 1.5%, W/C ratio of 0.40 with HPMC dosage of 0.5%, 0.75%, and 1.5%, and W/C ratio of 0.45 with HPMC dosage of 0.45, or be extended in the case of W/C ratio of 0.4 with HPMC dosage of 1.0% and W/C ratio of 0.45 with HPMC dosage from 0.75% to 1.5%. Fluidity measurement shows HPMC significantly improves the shape retainability. Furthermore, the addition of HPMC remarkably increased the compressive strength of extruded mortar. The results showed that HPMC could be used to prepare 3D printing SAC having satisfactory shape retainability, setting time and compressive strength. In recent years, 3D printing technology has been used for buildings and other structures, and related studies have been conducted to develop 3D concrete printing which is based on layer-by-layer printing process ,9,10,11.When the material is printed and deposited, it should maintain its extruded shape without flowing away. The application of an additive is an effective method to enhance the shape stability of printed cement-based materials. Some researchers have aimed at engineering the rheology of printed materials by modifying the mix design ,16,17 anHydroxypropyl methylcellulose (HPMC) is widely used as a thickener to modify rheological properties by saving cement-based materials from bleeding and segregation ,25,26,27Sulphoaluminate cement (SAC) exhibits fast setting and high early strength to support efficient and rapid construction; thus, SAC is advantageous to be utilized in 3D printing applications ,34,35,36In this study, a 3D printing material was developed based on SAC by incorporating HPMC. The setting time, fluidity and hydration heat of fresh SAC paste was tested by changing the water-to-cement (W/C) ratio and HPMC dosage. The fluidity of fresh SAC mortar was measured by optimizing HPMC dosage, W/C ratio, and sand-to-cement (S/C) ratio by mass. Furthermore, the early strength of the SAC mortar was tested for a series of HPMC dosages.2∙kg−1 was obtained from a commercial cement producer. The chemical composition of SAC analyzed by X-ray fluorescence (XRF) is provided in The sulphoaluminate cement (SAC) with a specific surface area of 367 mSAC pastes were prepared by mixing SAC, HPMC, and water at various W/C ratios and HPMC dosages (wt.% cement). The designed paste proportions that are shown in The SAC mortar was prepared with SAC, HPMC, sand, and water at various HPMC dosages (wt.% cement), sand-to-cement (S/C) ratio, and W/C ratio, using the mixing proportions shown in X-ray diffraction (XRD) was used to analyze the SAC clinker and hydrated SAC. Samples were grounded manually in an agate mortar to pass through a 400-mesh sieve. XRD patterns were obtained using a Bruker D8 Advance Diffractometer with Cu Kα-radiation at 2θ angles varying from 5° to 80° with an interval of 0.02°.The hydration heat was measured using an isothermal heat flow calorimeter controlled by a computer  at 25 °C. The heat release rate and the accumulative heat release of hydration during the early stage was evaluated. About 10 g of cement was weighed using electronic analytical balance with accuracy of 0.0001 g, and then was placed in a glass test tube followed by being put inside the calorimeter. After the sample equilibration finished, water was injected into cement inside the calorimeter. At the same time, the heat release started being recorded.The setting time and fluidity of fresh SAC paste and mortar were tested at room temperature (25 °C). The setting time of fresh SAC paste was determined using Vicat apparatus according to GB/T 1346–2011 (ISO 9597:2008). When the paste was close to the initial or final setting, the testing was carried out every one minuteThe fluidity of fresh SAC paste was measured according to GB/T 8077–2000. The fluidity of SAC mortar was determined using a flow table according to GB/T 2419-2005. The inner diameter of the upper and lower mouth of the mold used to measure the mortar fluidity was 70 mm and 100 mm, respectively. The frequency of the jolting apparatus was once per second, and 25 jolting were completed in 25 ± 1 s. In addition to fluidity value, the height of the mortar was recorded after removing the mold before jolting , and after jolting .The fresh mortar was extruded into cubic molds of 30 mm × 30 mm × 30 mm in size. Consequently, the molds with fresh SAC mortar were sealed with a plastic film to prevent water evaporation and then put in a standard maintenance box of cement with a temperature of 20 ± 1 °C and relative humidity of ≥90%. The SAC mortar specimens were removed carefully after 50 min and cured in the maintenance box until the intended test age was reached. Mortar specimens were cured for 1 h and 2 h. The compressive strength of SAC mortar specimens was determined using a computer-controlled compression testing machine with a loading rate in the range of 2.0–2.4 kN/s. The average compressive strength of three replicates was reported with an accuracy of 0.1 MPa.The hardened paste and mortar with and without HPMC were characterized by using scanning electron microscope (SEM)  to analyze the microstructure transformation resulted from the addition of HPMC. The hardened paste and mortar were cut into pieces which were dried in a vacuum drying oven at 30 °C for 12 h. Before the SEM test, the dried pieces were cut apart into small pieces with size of about 5 mm × 5 mm × 5 mm. For each paste and mortar, one piece was selected to be coated with gold film by gold spraying with placing the fresh section surface up. Then, the fresh section surface was observed by using SEM.The 3D printing of cement-based materials requires a reasonable setting time that is neither too short nor too long. Enough time should be provided to mixing, transporting, and printing materials in proper order to avoid extrudability loss of printing materials even becoming hardened in the tube or printing nozzle before printing. In general, the cementitious materials begin to lose their extrudability earlier than the initial setting time . Hence, The setting time of SAC paste with and without HPMC at various W/C ratios were assessed. The influence of W/C ratio and addition of HPMC on the setting time of paste is shown in The paste samples with W/C ratio of 0.30 and 0.35 displayed a constant decrease in the initial and final setting times with increasing HPMC dosage from control up to 1.5% . MoreoveIn the case of the paste with W/C ratio of 0.4, compared to the control sample, the initial setting time was reduced by 12 min, 3 min, and 5 min, respectively by adding 0.5%, 0.75%, and 1.5% HPMC. The final setting time decreased by 11 min, 5 min, and 9 min, respectively, for 0.5%, 0.75% and 1.5% HPMC dosage, while both the initial and final setting times were extended by 6 min for 1.0% HPMC dosage . In the On the basis of the numerical tool developed by Hongyan Ma et al. ,44, 2D sHPMC demonstrates can be used as a thickening admixture to increase viscoelasticity. In our tests, HPMC could shorten or extend the setting time of the freshly mixed SAC pastes with different W/C ratios and HPMC dosages measured using Vicat needle. It has been inferred that higher hydration heat release rate and higher cumulative heat evolution lead to a shorter setting time . HoweverThe results also showed that higher HPMC dosage could result in lower fluidity because 3D HPMC gel network could hold the cement particles to prevent the flow of paste. In addition, HPMC also could make the paste sticker, thus reducing fluidity. This observation is based on two reasons: the gelatinized HPMC fills the space between cement particles, and HPMC absorption of water could reduce the “virtual W/C ratio.”Both reducing W/C ratio and increasing HPMC dosage could help cement paste maintain its molded shape. The higher friction between grains results in higher yield stress and larger grain force with decreasing W/C ratio, making extrusion harder that would need more power to print materials. Thus, incorporating HPMC and increasing HPMC dosage is a better strategy to improve shape retainability of the paste with higher W/C ratio rather than reducing W/C. It also should be noted that, even at a high W/C ratio and when removing a mold after preparation, the HPMC-SAC paste could maintain its shape.The initial height, final height, and fluidity were measured according to GB/T-2419–2005 using a flow table. The initial height represents the measured height of the mortar after removing the mold before vibrating. The final height represents the measured height of the mortar after being vibrated. The initial height was used to indicate the shape retainability of the mortar (standing behavior in static condition), and the final height and fluidity were used to examine the fluid behavior during transport and extrusion-based printing. Thus, the initial height of the mortar applicable to 3D printing should be as high as possible, while the corresponding final height of the mortar should be minimized, possessing maximum desired fluidity. The higher the initial height, the higher the shape retainability. The lower final height and higher fluidity indicate that the mortar has lower yield stress, and lower extrusion force is needed to print the mortar. This approach is an easy and straightforward method to assess the standing behavior of printed materials in the static condition and their fluid behavior during transportation and printing process.The initial height, final height, and fluidity of the mortar with W/C ratio of 0.4 and various HPMC dosages were plotted against S/C ratio, as shown in In the case of the mortar with S/C ratio of 2, the initial height, final height, and fluidity of the mortar with various HPMC dosages were plotted against W/C ratio, as shown in The initial height, final height, and fluidity of the mortars with W/C ratio of 0.40 and various S/C ratios were plotted against HPMC dosage, as displayed in These results are useful to modify the SAC mortar by adjusting W/C ratio, S/C ratio, and HPMC dosage, and to select the suitable mixing proportion that has high initial height, low final height and high fluidity for 3D printing. According to The influence of the HPMC on the early strength of the extruded mortar is shown in The morphology of fresh section surface of hardened SAC paste cured for 2 h was presented in The addition of HPMC shows a delay effect on the hydration of SAC. The delay effect was enhanced due to the increasing HPMC dosage from 0.5% to 1.0% by mass of cement. While, when the HPMC dosage increased to 1.5%, the delay effect was weakened.The influence of HPMC on the setting time measured using Vicat needle depends on the W/C ratio and HPMC dosage. The HPMC composite pastes with W/C ratios of 0.30 and 0.35 have a faster setting time. While the HPMC composite pastes with W/C ratios of 0.40 and 0.45 show a decrease in the setting time with increasing HPMC dosage from 0 to 0.5%, and then an increase with increasing HPMC dosage from 0.5% to 1.0%, followed by another decrease from 1.0% to 1.5%. The reason for that is the counterbalance between the impact of hydration delay resulted from adsorption of water and gelation effect of HPMC.The addition of HPMC significantly increases the viscosity of SAC paste and mortar, presenting them with both good shape retainability in the static state and fluid behavior in the kinetic state. The effect of improving shape retainability and compressive strength is mainly attributed to the gelation of HPMC, constructing a 3D network, and this network helps SAC paste and mortar show an appealing rheological behavior that could decrease or prevent flowing.Furthermore, the addition of HPMC remarkably improves the early compressive strength of the mortar tested at 1 h and 2 h of hydration time.The addition of HPMC accelerated the growth of ettringite crystals and had no significant influence on the microstructure of SAC mortar at hydration time of 2 h.HPMC was incorporated into SAC to prepare cement-based materials that can be applied for 3D printing. The following conclusions can be drawn: