Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that drive GBM growth and invasion, few studies have targeted these fuels for therapeutic management. The glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), was administered together with a calorically restricted ketogenic diet (KD-R) to treat late-stage orthotopic growth in two syngeneic GBM mouse models: VM-M3 and CT-2A. DON targets glutaminolysis, while the KD-R reduces glucose and, simultaneously, elevates neuroprotective and non-fermentable ketone bodies. The diet/drug therapeutic strategy killed tumour cells while reversing disease symptoms, and improving overall mouse survival. The therapeutic strategy also reduces edema, hemorrhage, and inflammation. Moreover, the KD-R diet facilitated DON delivery to the brain and allowed a lower dosage to achieve therapeutic effect. The findings support the importance of glucose and glutamine in driving GBM growth and provide a therapeutic strategy for non-toxic metabolic management. Purna Mukherjee et al demonstrate that administering a glutamine analogue together with a calorie-restricted ketogenic diet can reduce brain tumour growth and inflammation, while enhancing survival, in two syngeneic orthotopic mouse models. A defining characteristic of GBM is the secondary structures of Scherer, which includes diffuse parenchymal growth and invasion over the subpial surface, along white matter tracks, and through the Virchow-Robin space2. The highly invasive nature of GBM makes most current therapies ineffective. Most patients die from intracranial pressure due to inflammation, edema, and distal tumour cell invasion. Hence, the proliferative and invasive nature of GBM is the major hindrance for effective therapeutic intervention.Glioblastoma (GBM) has the highest mortality rate among primary brain tumours in adults and remains largely unmanageable with a life expectancy of about 6–24 months following diagnosis. Despite decades of research, little progress has been made in improving GBM survival5. Also recognized are abnormalities in the number, structure, and function of mitochondria in GBM tumour tissue10. In addition, recent studies show abnormalities in GBM mitochondrial-associated membranes, which are also critical for normal mitochondrial function11. Moreover, the content and composition of cardiolipin, the signature lipid of the mitochondrial inner membrane that regulates oxidative phosphorylation (OxPhos), shows deviations from the normal in five different murine GBM models12. As mitochondrial function and efficiency is dependent on structure13, OxPhos is expected to be lower in GBM than in normal brain tissue. Indeed, significant evidence shows that OxPhos is defective in human and murine GBM14.GBM contains a range of morphologically diverse neoplastic cell types that express glial, stem cell, and myeloid/mesenchymal markers19. Although glucose is the primary metabolic fuel for neurons and glia, normal brain cells can transition to ketone bodies (primarily β-hydroxybutyrate) for energy under hypoglycemic conditions20. In contrast, GBM cells lack metabolic flexibility and are susceptible to death in response to reduced glucose bioavailability21. Although brain glutamine levels are tightly regulated via the glutamine-glutamate cycle, the bioavailability of glutamine increases following surgery, radiation, and chemotherapy, which fuels tumour growth22. If glucose and/or glutamine remain available, the tumour cells will utilize the fuel and grow, making long-term management difficult.Regardless of GBM cell type, glucose and glutamine are required for growth and invasion through glycolysis and glutaminolysis, respectively23. The primary ketone bodies, β-hydroxybutyrate and acetoacetate, are also neuroprotective and non-fermentable25. We found previously that the KD-R is a non-toxic anti-inflammatory approach to reduce pathology in experimental human and mouse brain tumours23. The neoplastic GBM cells can also synthesize high-energy phosphates through mitochondrial substrate level phosphorylation supported by glutaminolysis26. In addition, glutamine provides a continuous source of nitrogen for de novo synthesis of nucleotides and proteins in cancer cells27. The extent of glucose and glutamine metabolism in tumour cells depends not only on the tumour cell type, but also on the location and microenvironment.The calorically restricted ketogenic diet (KD-R) is a low carbohydrate, high fat diet, that reduces blood glucose and increases blood ketone bodies to therapeutic levels, while simultaneously inducing anti-inflammatory effects29. Therapeutic success depends on modifications in dosage, scheduling, and timing in order to enhance efficacy while reducing toxicity6. 6-diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist that was initially isolated from a Streptomyces broth in 1956. DON blocks multiple glutaminases, thus restricting key metabolites needed for glutaminolysis and the synthesis of nucleotides and proteins30. The anti-tumour efficacy of DON was confirmed in different tumour models, and in humans with various cancers30.Recently, the efficacy of simultaneous inhibition of both glycolysis and glutaminolysis has been demonstrated in experimental cancer models and in a human GBM case report2. In addition to the VM-M3 tumour, we also evaluated the effects of the KD-R and DON combination on the growth of the CT-2A brain tumour. The highly angiogenic CT-2A tumour was produced from 20-methancolanthrene in the cerebral cortex of a C57BL/6J mouse, and has several characteristics in common with the neoplastic neural stem cells found in human GBM32. Abnormalities in the electron transport chain were found in both tumours12. Our data show that the combination of KD-R and DON administration cause massive tumour cell death in both of these preclinical GBM mouse models and highlights the importance of targeting glycolysis and glutaminolysis simultaneously for the metabolic management of GBM.In the present study, we show a unique therapeutic response from the simultaneous targeting of glucose and glutamine using a KD-R and DON, respectively, in syngeneic orthotopic mouse models of GBM. The VM-M3 GBM tumour arose in the cerebral cortex of a mouse from the VM/Dk inbred strain, which has a high incidence of spontaneous brain tumours relative to other strains. The mesenchymal VM-M3 tumour cells invade distally throughout the brain using the secondary structures of Scherer2. Additional results were obtained in C57BL/6J inbred mice bearing the syngeneic CT-2A high-grade stem cell glioma32. DON, at the concentrations used in this study, had no toxic effects in non-tumour-bearing VM/Dk mice, as was shown previously for C57BL/6J mice33.The aim of this research was to determine if the simultaneous targeting of glucose and glutamine, while under therapeutic ketosis, could manage late-stage growth and enhance survival without toxicity in two different syngeneic mouse models of GBM. We administered DON 7 days after tumour implantation, which is considered late for the VM-M3/Fluc tumour, as neurological signs and symptoms first appear at 10 to 12 days post implantation with morbidity occurring in 100% of the tumour-bearing mice by 14–18 daysTo observe the effect of the KD-R and DON on the VM-M3 tumour model, we conducted three consecutive experiments. Experiment 1 included four groups  with lower DON dosages (0.1 and 0.5 mg/kg). Experiment 2 involved both in vivo and ex vivo imaging of two study groups (KD-R and KD-R + DON) following the administration of a higher dose of DON (1.0 mg/kg). Experiment 3, a survival study, also used the higher DON dose (1.0 mg/kg) and included the four groups from Experiment 1   that were treated with a higher dose of DON (1.0 mg/kg). Due to the observed synergy between the KD-R and DON in experiment 1, only the KD-R group was selected for further experiments. All mice in this experiment expressed tumour burden by day 7. Individual brain bioluminescent values are shown in Fig. 35. In other words, the ketone levels are higher and the glucose levels are lower under KD-R feeding than under SD-R feeding despite similar caloric restriction and reduced body weight. A KD-UR was also not included in this study, as consumption of ketogenic diets in unrestricted amounts can cause insulin insensitivity and weight gain, as we previously described36.A standard diet restricted (SD-R) group or a ketogenic diet unrestricted (KD-UR) group were not included in this study. We previously showed that a KD-R is more effective at reaching the target level of blood glucose and ketones than is a SD-RThe VM mice from experiment 2 were sacrificed after in vivo imaging, and their brains were immediately resected and individually imaged ex vivo for total bioluminescence as described in the Methods. Ex vivo imaging provides a more accurate assessment of tumour progression than in vivo brain imaging due to the direct contact of the d-luciferin with the brain tissue and the removal of the blood–brain barrier as a limiting factor. The individual ex vivo bioluminescent values for the DON-treated mice were below the baseline value indicating a reduction in tumour cell viability Fig. . These vHistological evaluation of brain tumour tissue from mice with the VM-M3 tumour is shown in Fig. 2. VM-M3 cells were observed invading from the core tumour into the normal appearing brain  has been linked to the therapeutic efficacy of the ketogenic diet (KD) for brain cancerent Fig. .37. The KD-R reduced the expression of Iba-1 as shown in both immunohistochemistry and western blot  and reduced TNF-α and Iba-1 levels, suggesting reduced inflammation as compared to the SD-UR control brains. The ability of DON treatment to arrest tumour growth and promote survival late in disease progression suggests a potentially direct role for glutamine metabolism in VM-M3/Fluc growth and invasion. This finding is consistent with the importance of glutamine in the metabolism of mesenchymal cells from which the VM-M3 tumour is derived42. Although DON treatment alone was effective in reducing tumour growth and invasion in the control SD-UR mice, a synergistic effect was observed when DON was administered together with the KD-R, providing evidence for a diet/drug interaction. The findings also support the press-pulse therapeutic strategy for the metabolic management of cancer with the KD-R serving as a press therapy and DON serving as a pulse therapy6.This study documents the therapeutic action of glucose and glutamine targeting in the VM-M3 and CT-2A syngeneic mouse models of GBM. It is known that calorie restriction, ketogenic diets, and DON can reduce metabolites in the glycolytic and glutaminolysis pathways, respectively43. Although the mechanism remains to be determined, these findings suggest that the KD has potential as a facilitator of drug delivery to the brain for a broad range of neuro-pathological conditions. It is also important to mention that our previous findings clearly demonstrated that caloric restriction can maintain the blood vessel integrity in the mouse CT-2A and the human U-87 GBM and thus reduce the leakiness of the neovasculature44. Interestingly, a recent observation by Gordon et al. showed that DON treatment promotes the recovery of blood–brain barrier integrity in the mouse model of cerebral malaria33. Further studies are needed to clarify the mechanism by which a KD-R facilitates drug delivery to the brain.Interestingly, the level of DON in the brain tumour tissue was higher under KD-R feeding than under SD-UR feeding suggesting that the KD-R facilitates DON delivery to the brain. This is significant because it allowed for a lower therapeutic dose of DON when administered under a KD-R regimen. A similar observation was made previously in showing that a KD-R facilitated the delivery of the iminosugar, N-butyldeoxynojirimycin, across the blood–brain barrier in mice with Sandhoff disease30. No noticeable toxicity was seen in the DON-treated mice until the end of the study when mild muscle loss was observed in some of the mice. It is proposed that issues with DON toxicity arise when there is an appreciable competition for glutamine between host and tumour. There is also increasing evidence supporting the protective role of glutamine supplementation for cancer patients45. Since tumour consumption of glutamine is dissipative, glutamine supplementation might not increase tumour growth46. However, additional preclinical studies will be needed to determine if glutamine supplementation could be used together with DON. It is our view that strategies for managing toxicity of a therapeutically effective drug would be as important as developing a new drug. Our results indicate that the KD-R enhances the therapeutic action of DON, thus reducing dosage and toxicity.DON has shown therapeutic efficacy in human cancers of the blood, colon, and lung, but issues of toxicity were noted in some cases32, whereas the VM-M3 tumour shares several characteristics with the invasive mesenchymal cell found in most GBM2. Hence, our findings suggest that the KD-R + DON therapeutic strategy could potentially target the two most common neoplastic cell types found in human GBM3. Our recent human GBM case report provides support for our prediction15.The therapeutic action of the diet/drug effect seen in the VM-M3 tumour was also seen in the CT-2A neural stem cell tumour. As observed from histological analysis, the KD-R + DON treatment caused massive mitotic arrest or catastrophe in the CT-2A brain tumour cells. The results show that therapeutic efficacy against orthotopic brain tumour growth was greater using the diet/drug combination than in using either the KD-R or DON alone. The CT-2A tumour shares several characteristics with glioma stem cells49. We previously showed that calorie restriction and restricted ketogenic diets target the IGF-1, PI3K, AKT, and Hif-1α signaling pathways in the CT-2A tumour50. The down-regulation of these pathways would reduce angiogenesis, inflammation, and mTOR signaling, while enhancing tumour cell apoptosis52. As glucose is the fuel for glycolysis, the pentose phosphate pathway, and serine biosynthesis, the KD-R should reduce multiple growth metabolites, tumour cell glutathione levels, and nucleotide synthesis. Furthermore, the KD-R should also reduce one-carbon metabolism especially in glioma cells lacking OxPhos capacity53. Hence, the glucose-restricting action of the KD-R will target multiple signaling pathways linked to glioma growth and progression.Several mechanisms could underlie the therapeutic action of the diet/drug therapy used to treat the VM-M3 and CT-2A tumours. The KD-R will simultaneously target the glycolytic and pentose phosphate pathways, which are upregulated in GBM54. Ketone bodies increase the redox span of the CoQ couple, thus reducing oxidative stress in normal brain cells54. These ketone bodies are also neuroprotective and have beneficial therapeutic value for various diseases55. As ketone bodies are non-fermentable and require efficient OxPhos for generating ATP54, the VM-M3 and CT-2A cells will be unable to metabolize these metabolites for energy due to insufficient OxPhos25. Ketone bodies cannot be used as an alternative energy fuel in cells with defective mitochondria25. Previous studies demonstrated that glioma cells cannot use ketone bodies and that ketone bodies do not stimulate tumour growth57. Hence, the KD-R has a dual function in (1) targeting glucose-dependent signaling pathways that drive tumour growth, and (2) in providing an alternative metabolic fuel to normal brain cells under glucose restriction.In addition to restricting glucose availability, the KD-R will also elevate β-hydroxybutyrate and acetoacetate, the major circulating ketone bodies produced in the liver, from the active metabolism of medium chain triglycerides59. α-KG is a precursor for lipid synthesis through reductive carboxylation and is the substrate for ATP synthesis through the succinate-CoA ligase reaction in the TCA cycle under hypoxia60. As mentioned previously, however, glutamine targeting is more challenging than is glucose targeting due to the importance of glutamine for the immune system and the gut61. Consequently, glutamine targeting must be done strategically to avoid damage to those systems that are needed for normal physiological function15. It is for this reason that we administered DON to the tumour-bearing mice at low doses and transiently. We administered DON 7 days after tumour implantation, which is considered late for the VM-M3/Fluc tumour because neurological signs and symptoms start to appear 10–12 days post implantation followed by 100% death in 14–18 days. The late administration of DON was chosen to ensure adequately high ketone levels were achieved in order to facilitate neuroprotection and to reduce tumour inflammation. Secondly, the late treatment could serve as a comparable model when treating late-stage GBM patients. A 60–70% reduction in tumour growth was observed when DON was administered at 0.5 mg/kg once or twice per week. In comparison, a near complete resolution of the brain tumour was achieved when 1.0 mg/kg of DON was administered with the same schedule, as seen with bioluminescent imaging. These results are significant because no other clinical or pre-clinical study using DON considered a KD-R as a method for protecting the host from toxicity. Case reports of GBM and other metastatic cancers show the significance of the KD-R when combined with other therapies63.While the KD-R will reduce metabolites through glycolysis and the pentose phosphate pathway, DON will block glutaminolysis, thus depriving the tumour cells of the amide nitrogen needed for nucleotide and protein synthesis, and at the same time, will deplete the glutamate needed for synthesis of α-ketoglutarate (α-KG)30. Although it is known that DON targets glutaminolysis in tissues58 and that reduced levels of circulating glucose will reduce glycolysis65, we were unable to find consistent brain metabolite changes in pathways of glycolysis and glutaminolysis in a preliminary metabolomic analysis of KD-R + DON-treated VM-M3 mouse brains. Due to the confounding variables in the in vivo environment , we believe that an in vitro analysis in defined media could provide a clearer picture of the changes in metabolites of the glucose and glutamine pathways due to DON or other drug treatments in the VM-M3 and CT-2A cells. It will also be important to determine if other glutaminolysis inhibitors, e.g., epigallocatechin-3-gallate (EGCG), bis-2- ethyl sulfide (BPTEs), and CB-83966, can match the therapeutic action of DON against brain cancer when used together with the KD-R. Pretreatment with a ketogenic diet before implementing the standard of care is suggested in order to protect normal brain cells from treatment toxicities22.Our findings support previous suggestions that reduced glutaminolysis would be a key therapeutic action of DON against the growth of glutamine-dependent tumours, including GBM69. Previous studies from Bachoo and colleagues suggest minimal 13C-glutamine anaplerosis in GBM implying that glutamine restriction in this context may not be therapeutic68. As DON inhibits multiple glutaminases, it is possible that DON does not directly impact glutamine anaplerosis in vivo, but acts on other related pathways and systems33. We recently described how glutamine is the only amino acid not requiring an energy investment to generate ATP through mitochondria substrate level phosphorylation (mSLP)25. Tardito et al. found that under glutamine starvation, intracellular oleate was unaffected, and glucose-dependent glutamate production increased, implying that the contribution of glutamine to growth is largely independent of anaplerosis69. Further, Oizel et al. identified two clusters of GBM that are glutaminehigh and glutaminelow based on glutamine utilization16. These investigators also mentioned that the most aggressive GBM contained mesenchymal cells that utilized the highest levels of glutamine. The VM-M3 cells are highly invasive and of mesenchymal origin37. Nevertheless, we use caution in the interpretation of our data, as the metabolic effects of DON’s action in killing the GBM cells were inferred rather than measured directly.It is important to mention that our findings in the VM-M3 and CT-2A syngeneic GBM models might be considered contradictory to some previous studies in GBM patients and in experimental systems15. Because of the high mortality following a GBM diagnosis, our findings should have clinical implications especially in light of our recent case report15. Furthermore, our studies reveal a potential role for the ketogenic diet in facilitating drug delivery to brain tumours.In the present study, we demonstrate the benefits of targeting glutamine under a KD-R for managing experimental GBM. The influence of targeting both glucose and glutamine on the metabolic pathways involved with GBM growth is shown in Fig. 28. Male and female mice between 8–10 weeks of age were used for all studies. All animal procedures and protocols were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee at Boston College under assurance number A3905–01.Mice of the VM/Dk (VM) strain were obtained as a gift from H. Fraser . The C57BL/6J (B6) mice were obtained originally from the Jackson Laboratory, Bar Harbor, ME. All mice used in this study were housed and bred in the Boston College Animal Care Facility using IACUC approved husbandry conditions37. The VM-M3 tumour manifests all of the invasive characteristics seen in human GBM2. The CT-2A tumour was originally produced from implantation of 20-methylcholanthrene into the cerebral cortex of a C57BL/6J mouse and was broadly classified as a poorly differentiated highly malignant anaplastic astrocytoma and a cell line was produced from this tumour31. Both cell lines are mycoplasma free and we use MycoZap (Lonza) kit to eradicate mycoplasma. More recent studies have classified the CT-2A tumour as a neural stem cell tumour32. The VM-M3 and the CT-2A cell lines were transduced with a lentivirus vector (gift from Miguel Sena-Esteves) containing the firefly luciferase gene under control of the cytomegalovirus promoter (VM-M3/Fluc) to produce VM-M3/Fluc and CT-2A/Fluc cell lines37. This transduction allows the cells to be tracked in the brain using bioluminescent imaging.The VM-M3 tumour used in this study arose spontaneously in the cerebrum of an adult male mouse of the VM/Dk inbred strain. A cell line was prepared from the VM-M3 tumour2. Briefly mice were anaesthetized with isoflurane (5% in oxygen). The tops of the heads were disinfected with ethanol and a small incision was made in the scalp over the midline. A 3 mm3 burr hole was made in the skull over the right parietal region behind the coronal suture and lateral to the sagittal suture. Small (1 mm3) tumour fragments were implanted approximately 1.5–2.0 mm deep into the cortical region using a trocar, as previously described. The skin flaps were closed with 7 mm reflex clips. The mice were placed in a warm room (24 °C) until they were fully recovered. The procedure confirms 100% recovery within a few hours of implantation. The GBM tumour cells are highly invasive regardless of implantation method, and all tumour-implanted mice reach morbidity at approximately 12–15 days.Tumour implantation was performed using our standard protocol70. Mice were individually housed beginning at the initiation of the 15 h fast. Food intake for the KD-R mice was measured to be between 1–3 g per day to maintain a 15–18% body weight reduction in each mouse. Mice were weighed daily to ensure weight maintenance  prior to initiation of the study. Tumour fragments were implanted on day zero. Upon implantation of the tumour, mice remained on the SD-UR until a 15-h fast was initiated 72 h after implantation. Following the fast, mice were introduced to the Ketogenic diet restricted (KD-R), or remained on the SD-UR, depending on the study group. The KD-R has a caloric density of 7.12 Kcal/g. The percent nutritional breakdown of KetoGEN  is as follows: 2.1% of Cal from carbohydrates, 8.7% of Cal from protein, and 89.2% of Cal from fat (Supplementary Table Mice receiving the SD-UR were given the standard chow diet (Lab Diet) ad libitum for the duration of the study. For those mice that received DON injections, a fresh stock was prepared and diluted to an appropriate concentration in PBS and was administered intraperitoneally (i.p.). The DON stock solution in PBS was stored at −20 °C for the duration of the study and mice received 200 μl injections of 0.1–1.0 mg/kg. Mice in the DON survival study received DON at a dosage that was appropriate for the individual mouse response. Some doses were skipped if the mice appeared lethargic or if body weight loss exceeded 1.5 g from the previous day. Studies were terminated at the time of morbidity for the control SD-UR group.37. Briefly, for in vivo imaging, mice received an i.p. injection of d-lucifierin (50 mg/kg) in PBS and Isofluorane (5% in oxygen). Imaging times ranged from 1 to 5 min, depending on the time point. For ex vivo imaging, brains were removed and imaged in 0.3 mg d-luciferin in PBS. The IVIS Lumina cooled CCD camera system was used for light acquisition. Data acquisition and analysis was performed with Living Image software .The Xenogen IVIS system is used to record the bioluminescent signal from the labeled tumours as we previously describedBrain tumour samples were fixed in 10% neutral buffered formalin (Sigma) and embedded in paraffin. The brain tumour samples were sectioned at 5 μm, were stained with haematoxylin and eosin (H&E) at the Harvard University Rodent Histopathology Core Facility , and were examined by light microscopy using either a Zeiss Axioplan 2 or Nikon SMZ1500 light microscope. Images were acquired using SPOT Imaging Solutions  cameras and software. All histological sections were evaluated at the Harvard University Rodent Histopathology Core Facility.For immunohistochemistry, the tissue sections from untreated and treated tumour-bearing mice were deparaffinized, rehydrated, and washed. The tissue sections were then heat treated (95 °C) in antigen unmasking solution  for 30 min. Tissue sections were blocked in goat serum (1:10 in PBS) for one hour at room temperature, treated with Ki-67 primary antibody  overnight at 4 °C followed by Alexafluor conjugated anti-rat secondary antibody at 1:100 dilution. Nuclei were stained using NucBlue (Invitrogen). Images were captured by the EVOS FL Cell Imaging System fluorescent microscope. For Iba-1, tissue sections were blocked with Iba-1 primary antibody  overnight at 4 °C followed by biotinylated anti-rabbit secondary at 1:2000 dilution. Avidin-biotin reaction was complete by incubating the tissues in ABC reagent (Vector Lab) for 30 min followed by DAB substrate reaction. Methyl green was used for staining the nucleus.2EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L NaPPi,1 mmol/L a-glycerophosphate, 1 mmol/L Na3PO4, 1 Ag/mL leupeptin, and 1 mmol/L phenylmethylsufonyl fluoride.Frozen tumour and normal brain tissues were homogenized in ice-cold lysis buffer containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl,1 mmol/L Naxg for 20 min. Supernatants were collected and protein concentrations were estimated using the Bio-Rad detergent-compatible protein assay.Lysates were transferred to 1.7 mL Eppendorf tubes, mixed on a rocker for 1 h at 4 °C, and then centrifuged at 8100Approximately 100 μg of total protein from each tissue sample was denatured with SDS-PAGE sample buffer  and was resolved by SDS-PAGE on 4 to 12% Bis-Tris gels (Invitrogen). Proteins were transferred to a polyvinylidene difluoride immobilon TM-P membrane (Millipore) overnight at 4 °C and blocked in either 5% nonfat powdered milk or 5% bovine serum albumin in TBS with Tween 20 (pH 7.6) for 1–3 h at room temperature. Membranes were probed with primary antibodies  overnight at 4 °C with gentle shaking. The blots were then incubated with the appropriate secondary antibody (anti-rabbit) for 1 h at room temperature and bands were visualized with enhanced chemiluminescence. Each membrane was stripped and reprobed for β-actin as an internal loading control and the ratio of the Iba-1 to β-actin was analyzed by scanning densitometry (FluorChem 8900 Software).xg for 5 min to precipitate proteins. A 200 μl aliquot of the supernatant was transferred to a 1.5 ml tube and incubated at 60 °C for 30 min in a shaking water bath to perform the DON derivatization reaction. For the brain tissue, 5 μl of n-butanol containing 3 N HCl was added per milligram tissue. The tissue sample was then homogenized with a glass pestle and was then vortexed and centrifuged, and prepared similarly to the plasma samples. Standard solutions were prepared by serial dilution to generate concentrations from 10 to 100 μM. DON was added to untreated mouse plasma or brain tissue to generate an internal standard curve. A high resolution LC-MS QToF method (Agilent 6550) was used to analyze DON in the second procedure  was used to analyze DON in the first procedure Fig. . Mice weure Fig. . Mice we36.All mice were fasted for 2 h before blood collection to stabilize blood glucose levels. Blood glucose and ketone levels were measured using the Keto-Mojo monitoring system . Whole blood from the tail was placed onto the glucose or ketone strip. The keto-mojo meter was used to determine the mmol levels of glucose and β-hydroxybutyrate in the blood. The GKI was determined as we previously describedVM-M3 brain tumour tissue and normal brain tissue from the VM/Dk mice were homogenized and processed following the Quantikine Mouse TNF-α Immunoassay protocol (R&D systems). TNF-α levels were measured in triplicate and adjusted with the protein level of each sample. The limit of detection is <10 pg/ml Tumour necrosis factor (TNF).t-test to perform a two-sided pairwise comparison among the groups (SPSS 14.0). In each figure, error bars are mean ± SEM and n is the number of individual mice analyzed. The Survival studies were plotted on a Kaplan–Meir curve using Graph Pad Prizm software and significance was determined using the log-rank test.Body weight, food intake, tumour growth, and plasma metabolite levels were analyzed using the one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test or by a Student’s Further information on research design is available in the supplementary fileReporting SummaryDescription of Supplementary DataSupplementary Data 1


Acute kidney injury (AKI) is independently associated with morbidity and mortality in critically ill neonates, children, adolescents, and young adults. AKI occurs commonly in this population, and the vast majority of published studies utilize only a serum creatinine based criteria for AKI diagnosis and staging. While urine output criteria have been a part of all AKI systematic and multidimensional AKI definitions for the past 15 years, oliguria based on these definitions is difficult to extract from the electronic health record. This manuscript reviews the published data regarding the impact of oliguria on patient outcomes, and the contribution of oliguria to % fluid overload and resultant changes in serum creatinine based epidemiology. The aim of this manuscript is to demonstrate that oliguria is an incredibly valuable biomarker for the management of patients with, or at-risk for, AKI. Acute kidney injury (AKI) occurs commonly in patients admitted to intensive care units and is independently associated with morbidity and mortality across the entire spectrum of patient age, from neonates to elderly adults –3. WhileThe aims of this manuscript are to review the published data regarding the impact of oliguria on patient outcomes, and the contribution of oliguria to fluid overload and resultant changes in serum creatinine based epidemiology. After this review, it will become clear that oliguria is an invaluable biomarker in the management of the patient with, or at-risk for AKI.Current diagnostic and staging criteria based on the recommendations of the Kidney Disease Improving Global Outcomes (KDIGO) Work Group for AKI are listed in The multi-national prospective Assessment of Worldwide Acute kidney injury, Renal angina and Epidemiology (AWARE) study enrolled nearly 5,000 children to evaluate potential associations between KDIGO Stage 2 or 3 AKI and outcomes in critically ill children . Twenty-Multiple studies have demonstrated the association between ICU fluid accumulation and morbidity and mortality in children. These studies were the subject of a comprehensive systematic review and meta-analysis by Alobaidi et al. and an extensive review is beyond the scope of this paper . In gene2) patient with septic shock. We will assume the patient receives 60 ml/kg of crystalloid  for fluid resuscitation and then is put on a fixed rate of fluid of 1,600 ml/day . In the following case, we can assess the resultant fluid accumulation status based on a standard volume of fluid resuscitation, required daily intake, and oliguria based on KDIGO staging. The case involves a 30 kg , which randomized 1,000 adult patients to a liberal vs. conservative fluid provision strategy after resuscitation . They foIn summary, most published studies in the AKI field rely on serum creatinine based definitions, due to their ease of data extraction from the electronic health record as well as the technical challenges of urine collection in neonates, infants and toddlers without an indwelling bladder catheter . Yet, AKSG wrote the article and is responsible for all of the content.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Modelling of cell flow for biomedical applications relies in many cases on the correct description of fluid-structure interaction between the cell membrane and the surrounding fluid. We analyse the coupling of the lattice-Boltzmann method for the fluid and the spring network model for the cells. We investigate the bare friction parameter of fluid-structure interaction that is mediated via dissipative coupling. Such coupling mimics the no-slip boundary condition at the interface between the fluid and object. It is an alternative method to the immersed boundary method. Here, the fluid-structure coupling is provided by forces penalising local differences between velocities of the object's boundaries and the surrounding fluid. The method includes a phenomenological friction coefficient that determines the strength of the coupling. This work aims at determination of proper values of such friction coefficient. We derive an explicit formula for computation of this coefficient depending on the mesh density assuming a reference friction is known. We validate this formula on spherical and ellipsoidal objects. We also provide sensitivity analysis of the formula on all parameters entering the model. We conclude that such formula may be used also for objects with irregular shapes provided that the triangular mesh covering the object's surface is in some sense uniform. Our findings are justified by two computational experiments where we simulate motion of a red blood cell in a capillary and in a shear flow. Both experiments confirm our results presented in this work. Deformation of elastic membranes due to an external shear flow or interactions with other such objects is an important problem in basic research as well as in biomedical applications. One of the most pronounced examples is the red blood cell (RBC) with its membrane composed of a lipid bilayer and a cytoskeleton. This membrane behaves as a viscoelastic material with property of area-conservation as described by Skalak in . ElasticMost membrane models are either derived from continuum laws or based on spring networks. Here, we focus on the latter due to their simplicity and similarity to the cytoskeleton. The membrane itself is usually described by a triangular mesh of interconnected points with elastic forces between them defining the elasticity .The flow of the fluid in which the cells are immersed is usually computed from governing equations for fluid dynamics, for example, the Navier–Stokes equations for incompressible flow . RecentlX is set to be the velocity of the surrounding fluid u. Because of the fixed fluid discretisation, the fluid velocity at the position X is obtained from convolution with suitably chosen δ functionThe crucial element in such models of the cell flow is the coupling of the membrane mesh and the underlying velocity field of the fluid. An efficient way of imposing these boundary conditions is via immersed boundary method (IBM), first introduced by Peskin ; for theδ function is a challenging task.The concrete form of the delta function influences numerical properties of the method. Yang et al.  use a smThe IBM does not account for the mass of the boundary. The mesh points are massless and in situations when mass of the membrane does play a role, the use of the IBM is limited. A variant of IBM has been introduced by Kim and Peskin , where tIn this article, we elaborate a different approach of the coupling between the membrane mesh and the fluid using dissipative force coupling. This so-called force-coupling algorithm was first introduced by Ahlrichs and Dunwegin  and lateSince its inception, the force-coupling algorithm has seen several improvements in terms of accuracy and flexibility. Ladd et al.  have devThis approach introduces a friction coefficient that needs to be properly calibrated. In the detailed analysis of force-coupling algorithm , 22, FisWe studied this method in ; howeverξ.The coupling force is scaled by a prefactor called friction coefficient denoted as Similar methods for modelling deformable objects have been proposed. Reasor et al.  introducIn the present work, we focus on proper determination of the friction coefficient. It is a purely phenomenological term, and we determine its value in such a way that the movement of objects corresponds to reality. As a ground truth, we take a physically relevant experiment, and we determine the friction coefficient for one specific triangulation of this object. This calibrated value will serve as a reference starting point for any other mesh with different mesh density or size of the object. We derive the explicit formula for computation of friction coefficient based on this reference value.The paper is organised as follows. In Our computational model consists of three parts: solver for the fluid, mechanical model based on spring networks for the cells, and coupling between the fluid and the cell.ni defined for each lattice point x, discrete velocity vector ei, and time t. We use the D3Q19 version of the lattice-Boltzmann method . The governing LB equations areδt is the time step and Δi denotes the collision operator that accounts for the difference between pre- and postcollision states and satisfies the constraints of mass and momentum conservation. In the lattice-Boltzmann method, the fluid flow needs to be evaluated with a half-step correction of the local force in order to be consistent with the Navier–Stokes equation. Therefore, the external forces can be incorporated by half-step Verlet algorithm. We refer to works of Ahlrichs and Dellar [u and the density of the fluid ρ are evaluated fromThis method describes the fluid dynamics and is based on fictive particles. These particles propagate and collide over a fixed three-dimensional discrete lattice. The unknown variable is the particle density function d Dellar  for detad Dellar . The velks is the stretching stiffness coefficient, κ is a nonlinear function mimicking neo-Hookean behaviour of cell's membrane, l is the current length of the edge between those two points, and l0 is the length in a cell's relaxed state. In the model we use for computations, there are all together five elastic coefficients: ks for the shear stretching, kb for the bending rigidity, kal for local area expansion, kag for preservation of total surface, and kv for preservation of total volume of the cells or other objects. Further details on formulas for each such elastic moduli are presented in [Cell's membrane is covered by mesh points, linked together into a triangular mesh. Elastic properties of the cell membrane are represented with different types of force-like bonds between neighbouring mesh points. To take the mechanoelastic properties of the immersed objects into account, geometrical entities in this mesh  are used to model stretching, bending, stiffness, and other properties of the membrane. One such example is the stretching force between two neighbouring mesh points defined asented in . These cented in .f exerted on mesh points. This force causes motion of the mesh point according to the Newton equation of motion.m is the mass of the mesh point. The source of f is either from the abovementioned elastomechanical properties of the immersed object or from the fluid-structure interaction.The sum of all such elastic forces defines force v of the mesh point and the fluid velocity u at the same position:ξ is a friction coefficient. F enters   5) descrξ⟶∞, the no-slip condition should be preserved.Friction coefficient does not correspond to any physical quantity and is purely phenomenological. It enforces the no-slip condition, and in the limit ξ.In numerical computations, however, we need to use finite value. This value is dependent on different features. In the next sections, we determine the correct value of To determine the friction coefficient, we need to compare our computational approach with analytical results. We decided to set as a reference the movement of a solid spheroidal object  in a fluid. There are theoretical computations that give us exact solutions for the velocity of such sphere. We can then compare them with computed results using our model. This way, we can inversely get the correct value for the friction coefficient.It is of course a priori not clear whether a hollow membrane consisting of interlinked mesh points can be compared with a rigid sphere since dissipation in the inside fluid may affect the friction. The presented simulations however show a good resemblance of the reality.m is the mass and v is the velocity of the object. We focus on the flow with low Reynolds number, and for these, the drag force of the fluid on the objects is given by Stokes law asr is the radius of the object, v is its velocity relative to the surrounding fluid, and ν is the dynamic viscosity of the fluid. Theory assumes a solid rigid object immersed in an unbounded fluid. Actually, our model assumes elastic objects; however, by setting the elastic coefficients high, we can model solid objects as well. We have discussed the question of domain boundaries in [μm, simulation box with dimensions 200  μm is suitable.The motion of solid objects immersed in the fluid is described by Newton's second law of motion:aries in , and as aries in . With diBased on this theoretical knowledge, we design two different experiments.F0 is applied on the sphere , we adopt the value of ξ and try again. By this inverse process, we will be able to determine correct values of ξ. At the beginning, we chose the values of ξ with regular incrementation; then, for finer calibration, we used simple bisection/step-doubling method.At this point, we can perform computer simulations of both experiments to find a proper value of ξ, we need to perform full 3D simulation. Therefore, we first calibrate ξref for a reference sphere and afterwards we derive a formula for direct computation of ξ for an arbitrary sphere based on the value ξref. For the whole calibration process, we used a simulation software ESPResSo [This process is quite computationally demanding. To evaluate both experiments for one single value of ESPResSo , releaseμm with 393 mesh points as a reference sphere. We use the following elastic parameters: ks=1.0, kb=0.5, kal=0.2, kag=5.0, and kv=10.0. Such high values ensure that the sphere is not deformed and behaves like a rigid one. The mass of individual mesh points is set to 0.25 pg.From our previous experiences with similar computations, we decided to pick the sphere with radius 4  −3 and dynamic viscosity to 1.5375  mPa · s, which are values of blood plasma. The spatial step of the lattice-Boltzmann grid equals 1.0  μm. All simulations were performed in a cubic simulation box with edge 200  μm [μs.We set density of the fluid to 1025  kg · m 200  μm . The timF0=0.4  nN. From  with radius. Our proposition is to use the following expression for computation of friction coefficient for nonspherical objects with Note that for spheres, the newly proposed relation is consistent with . The forTo verify the hypotheses, we use extended theoretical results concerning movement of rotationally symmetric ellipsoids in a fluid. Such ellipsoids are created from a sphere by prolonging (prolate ellipsoids) or shortening (oblate ellipsoids) of the sphere along one axis .ν is the dynamic viscosity of the fluid, v is the relative velocity of the ellipsoid to the fluid, a is the radius of circular cross section of the ellipsoid, and K is the shape factor. K depends on the ratio a/b and on the flow direction. The concrete values of K for different cases are taken from [The relation  that is ken from  and showv∞ for the terminal velocity experiment and FA for the balancing force experiment read asUsing , we can v∞ and FA in both experiments.These expressions give us expected values of K was computed using expressions from Now, we test our hypothesis. We choose 6 different ellipsoids (three of them are prolate and three are oblate). Each ellipsoid is triangulated using open source software GMSH . The triF0 and v0 were identical to those from the calibration of the reference sphere in Each ellipsoid is put in two different flows, one in an axial direction and one in a transversal direction. This results in 12 different scenarios. Each of these scenarios served as a starting point for both computational experiments, one for terminal velocity and one for balancing force. Altogether, we have thus computed 24 simulations. In these simulations, the size of the simulation box, elastic coefficients, and values for v∞ and simulated v∞. Analogous statement holds for the expected balancing force FA and simulated FA.Assuming that the hypothesis is correct, we should obtain the same values of expected Using the corresponding friction coefficient, we have performed simulations for all 24 scenarios and we collected the computed values of terminal velocities and balancing forces, respectively. The actual results are presented in The Δ-columns contain relative differences. In the table, we can see that the simulated quantities  are fairly close to the expected values. The relative difference is always under 5%.−3, and their respective viscosities are presented in μm. The relation (Increasing viscosity means that with given velocity gradient, the shear stress increases. The friction coefficient is responsible for transfer of forces between the fluid and immersed objects, and thus, it is natural to expect dependence of the calibrated friction on viscosity. Our auxiliary simulations revealed that indeed the friction coefficient for the reference sphere is different for various viscosities. Therefore, we performed the sphere calibration from relation  remains To demonstrate the effect of different friction coefficients on real flow of cells, we performed a simulation of flow of a red blood cell exposed to Poiseuille flow in a tube with diameter comparable with the size of the cell. The parabolic profile of the fluid velocity means slower fluid velocities close to the tube wall and large velocity around the axis of the channel. The red blood cell when exposed to such flow deforms to a so-called parachute shape , 38, 39.r=5  μm and length 50  μm. We consider fluid with density ρ=1000  kg · m−3 and viscosity 1.5  mPa · s flowing in a tube with volumetric flow rate 0.054  μl · s−1. The elasticity of red blood cells is determined by its elastic coefficients. We take the following values:We set up a simulation in a tube with diameter orted in . Data frorted in  have beeorted in , 41. TheInitial spatial orientation of the cell is transversal with respect to the axial direction. In When a different value of friction coefficient was used in the same experiment, the shape of the red blood cell was not affected. This seems to be in contradiction with results from previous sections where we claim that friction coefficient must be properly set depending on the mesh density and that we cannot choose its value arbitrarily. There is, however, a crucial difference between the terminal velocity and the balancing force experiments and the flow in an empty channel: in the empty channel, there is no external force exerted on the flowing object, and the object is being drifted by the fluid freely. There is thus much less transfer of force needed between the object and fluid compared with experiments, where external forces act against the movement of the object.In situations, however, where the object does not flow freely in the flow, the friction coefficient must be set properly as demonstrated by the following example. We designed another test, where the cell is squeezed between two obstacles. Here, the obstacles substitute external forces by their influence on the cell. In Different shape is clearly visible at time instance 2  ms. With higher value of friction, the shape is more prolonged in axial direction than with lower value. This can be explained by larger transfer of force to the cell membrane. Near obstacles, the flow is almost zero due to no-slip condition, while in between the obstacles, the flow is fast. With larger friction, fluid near the obstacles decelerates the cell and in the middle it accelerates the membrane, causing more prolonged shape of the cell compared with the case with lower friction.Delay is pronounced later, at 3.5ms and 5ms. This can be explained by the fact that when passing the obstacles, the no-slip condition causes more effective deceleration of the cell for higher friction, and thus for larger force transfer between static fluid and moving membrane.Objects immersed in a shear flow exhibit complex behaviour. Movement of rigid ellipsoidal particles in a shear flow has been studied in . The casξok is the correct value computed from (ξ1=0.5ξok and ξ2=1.5ξok. The aim is to verify whether computations using ξok give results corresponding to biological data, while computations using ξ1 or ξ2 give results diverging from the data.Using these data, we will validate relation . We perfted from , while tShear flow can be induced between two parallel surfaces that move relative to one another. In practice, this means either one of them is stationary or the other moving or two plates moving with the same velocity in opposite directions, as depicted in v0 and −v0 can be computed from h being the distance between the walls. We will use a triangular mesh covering the surface of the cell with 393 mesh points and with surface area of approximately 142  μm2. The model requires proper parameters so that the simulated cell has elasticity of a real red blood cell. The following values of the elastic parameters similar to those from [The shear rate ose from  have beeμm, h=20  μm enclosing the cell located in its center. We use the following fluid properties to simulate suspending medium used in experiments from [ρ=1045  kg/m3, ν=10.7  mPa · s. The desired shear rate in the range from 0 to 200  s−1 can be generated by velocities ranging from 0 to 0.002  m/s. To obtain correct ξref, we extrapolate the values in ξref=9.57 for ν=10.7  mPa · s. Finally, using (We used a cubical computational domain with dimensions 20 × 20 × 20  nts from , 44: ρ=1y, using , we can μm and the lattice grid of 2  μm.In all the experiments, we use time step of 0.1  ξok just slightly overshoot data from experiments. Clearly, the results for ξ1 overshoot the experimental data significantly. The results for ξ2 overshoot the experimental data in the range L2 error is 4.01 for ξok, 4.21 for ξ2, and 9.11 fro ξ1. This shows that the case for ξok best fits the experimental data.Computational results are summarized in We analysed fluid-structure interaction that is based on a dissipative force between the fluid and structure. This interaction is mediated via friction coefficient between the mesh points and the fluid grid. In this work, we answered the question of proper value of the friction coefficient. The analysis was gradually established by first calibrating the reference sphere, generalisation to arbitrary sphere, proposal for arbitrary shape, validation of proposed hypothesis for ellipsoidal objects, and demonstration of validity for nonconcave objects, namely, for red blood cells.Our study relies on explicit solutions to terminal velocity and balancing force experiments. Unfortunately, for asymmetrical or biconcave shapes, we are not aware of any such explicit solutions, and our study could not be extended to, for example, concave objects.Nevertheless, we expect the relation  to be suIn case of red blood cells, their biconcave shape resembles ellipsoids quite well. The validity of the proposed results for red blood cells has been demonstrated in


Various mechanisms such as second primary lesion, tumour seeding or lymphogenous and haematogenous metastasis could be proposed to explain the nature of dual malignant lesions. We report the case of a glottic laryngeal carcinoma combined with a secondary endotracheal tumour. Following the imaging modalities, the patient underwent total laryngectomy and wide excision of the trachea. Histopathology ultimately established that the tracheal lesion was a metastatic tumour secondary to regional lymphatic spread of the glottic tumour. To our knowledge, there is no previous report in the English literature concerning tracheal lymphogenous metastatic involvement in the context of laryngeal malignancy. Paradoxical lymphatic spread must always remain an issue of head and neck oncology. Laryngeal cancer commonly metastasizes via the cervical lymph nodes to distant sites. Haematogenous spread occurs less frequently, accounting for only about 10% of all distant metastases. The most common site for laryngeal squamous cell carcinoma metastasis is the lungs, followed by the liver, bones and central nervous system [We report the case of a glottic laryngeal carcinoma with solitary endotracheal metastasis.A 60-year-old male was referred to our department, complaining about gradually worsening hoarseness, during the last 8 month period. Occasional dysphagia and foreign-body sensation were also reported upon referral. The patient was a heavy smoker for more than 20 years, reporting an average of 20 cigarettes per day. Alcohol was also a factor, and although no real alcohol abuse or indulgence was noted, the patient was a rather frequent user.Medical history only revealed arterial hypertension under treatment with beta blockers. Haematological and biochemical tests did not show any significant abnormalities.Physical examination included a full head and neck examination, complemented with flexible fiberoptic laryngoscopy. Typical ear, nose and throat examination did not reveal any abnormal findings and neck palpation was negative. However, fiberoptic laryngoscopy revealed a lesion affecting both vocal cords and anterior commissure, while vocal cord mobility appeared impaired. On these grounds, a cervicothoracic and upper abdomen computed tomography (CT) scan with intravenous gadolinium was decided and the patient was scheduled for direct microlaryngoscopy and biopsy of the lesion under general anaesthesia.Imaging confirmed the laryngeal lesion, yet it also indicated a second lesion about 2 cm below the inferior end of the primary one, arising somewhere between the first and second tracheal ring. Intermediate tissue appeared grossly normal . No signOn the other hand, histopathological examination after biopsy of the lesion under general anaesthesia confirmed the diagnosis of squamous cell carcinoma. The lesion was carefully mapped and proved to be a glottic carcinoma affecting the anterior commissure and appearing in strong correlation with the thyroid cartilage. The lesion infiltrated the left and the first tertile of the right vocal cord. No subglottic extension was noted. In this context, the patient was informed and consent for radical surgical therapy was obtained.The patient underwent total laryngectomy and wide excision of the trachea which included the second tumour within safe limits . The proThe final pathological report was of crucial importance in our case. First of all, the surgical margins of resection were found to be free of disease. Second, histological sections from the tumour of the glottis showed the characteristic morphology of squamous cell carcinoma. Cancer cells were large in size and polygonal in shape with eosinophilic cytoplasm and nuclei with moderate variation in size and shape. There were a moderate number of mitoses and keratinisation could be focally observed. Cancer cells showed an infiltrative pattern consisting mainly of nests and trabeculae that invaded the vocalis muscle in both the vocal cords. The perichondrium of thyroid cartilage was focally invaded by cancer cells. Histological sections from the tumour of the trachea showed morphological features identical to those of the tumour of the glottis. An upward infiltrating pattern could be noticed. Moreover, a comparative immunohistochemical study of the two tumours showed strong positivity of cancer cells in stains for keratins AE1/AE3 and 34βΕ12 and moderate positivity in stains for CK5/6, CK8/18 and epithelial membrane antigen. Immunohistochemistry for D2-40 antigen (podoplanin) illustrated the positivity of the lymphatic endothelium. Immunohistochemical stains for other vascular endothelia (CD31 and CD34 antigens) were also performed, and were negative. In the region between the two tumours, many lymphatics containing neoplastic emboli could be observed . FinallyVarious mechanisms could be proposed to explain the nature of such a rare case of dual lesions.The case of a simultaneous second primary tracheal tumour must be accounted for. First of all, in order to clarify terminology, we use the established term ‘simultaneous’ to refer to primary tumours diagnosed at the same time, whereas ‘synchronous’ refers to lesions detected within a 6-month  or 1 yeaMulticentric carcinomas of the aerodigestive tract are not rare. The literature suggests that the patients with primary squamous cell carcinoma in the upper aerodigestive tract run an increased risk of developing a second tumour . The conMoreover, the possibility of neoplastic spread by tumour cell seeding would be an alternative worth taking into consideration. Neoplastic seeding is a well-documented concept of mechanical tumour disruption and consequent cell implantation into adjacent or distant traumatised tissues. Although it remains a rare mechanism of tumour dissemination, yet it is accepted as an explanation for unusual patterns of tumour recurrence. Tumour implantation into tracheotomy and percutaneous endoscopic gastrostomy sites has been postulated , 7 in paFinally, the possibility of a metastatic endoluminal tracheal tumour was considered. Lymphogenous and haematogenous metastasis of laryngeal cancer are not rare. The lymphatic metastatic pattern of laryngeal squamous cell carcinoma is well known. Nodal metastasis is mainly concentrated in cervical regional lymph nodes, particularly Robin’s levels IIa and III while metastases outside of this level are rather unusual . AdvanceThe histopathology report shed light on these key questions, although an effort to make a molecular comparison of the two tumours was unsuccessful, due to insufficient DNA preservation. The identical morphological and immunohistochemical features of the two tumours and the upward infiltrating pattern of the tracheal tumour, combined with the striking finding of neoplastic emboli in intermediate lymphatics were not decisive about the nature of the lesions, however, established that against all odds the tracheal lesion was most probably a metastatic tumour secondary to regional lymphatic spread of the tumour of the glottis. Since the tracheal lumen has not been reported as a possible metastatic site for glottic carcinomas, this unique case underlines the need for all head and neck surgeons to remain vigilant for paradoxical lymphatic tumour spread.We report a unique case of endoluminal tracheal metastasis of a glottic carcinoma, secondary to regional lymphatic spread. To our knowledge, there is no previous report in the English literature concerning tracheal metastatic involvement in the context of laryngeal malignancy. Paradoxical lymphatic spread must always remain an issue within the field of head and neck oncology.The author(s) declare that they have no conflict of interest.


Re is between 0.002 and 0.01, while it is enhanced when Re is between 0.1 and 0.2 compared with that at a constant flow rate. In the viscoelastic fluid, on the other hand, the time pulsing does not change the mixing degree when the Weissenberg number WiWi = 50.Fluid mixing plays an essential role in many microfluidic applications. Here, we compare the mixing in time pulsing flows for both a Newtonian fluid and a viscoelastic fluid at different pulsing frequencies. In general, the mixing degree in the viscoelastic fluid is higher than that in the Newtonian fluid. Particularly, the mixing in Newtonian fluid with time pulsing is decreased when the Reynolds number  In the swift development of the micro-fabrication process, microfluidic technology has been widely recognized in many practical applications such as medical diagnostics , biologiRe less than 150 [Various approaches have been developed to improve the microfluidic mixing, and are usually categorized into active methods and passive methods ,13,14. Fthan 150 . In contthan 150 , ultrasothan 150 , electrothan 150 ,24 and mthan 150 .Besides the reliance on external fields, the flow is also controlled by introducing time pulsing to the flow ,27,28. UDespite the fact that much research has focused on the time pulsing mixing, this study mainly concentrates on the mixing in a Newtonian fluid. In fact, the rheological properties of the fluid also have a high impact on the mixing efficiency. Recently, viscoelastic fluid is proposed to improve the mixing efficiency in the passive microfluidic channels ,33,34.In the viscoelastic fluid, elastic stress only decays after a characteristic time due to its elasticity. For its viscoelastic nature, the viscoelastic fluid can realize chaotic vortices even at a low Reynolds number condition ,35. In tIA and IB of the T-junction. The fluid on one side contains a small amount of fluorescent dye for experimental view. The two fluids are confluent at the center of the inlet channel and flow into a narrow channel before finally entering into an expanded mixing channel. The fluorescent color in the mixing channel is monitored to determine the mixing efficiency at the flow distance, S0, from the inlet channel. The length and width of the inlet channels are L1 = 2 mm and W1 = 100 µm, respectively. The confluence channel has dimensions of L2 = 600 µm and W2 = 50 µm, and the mixing channel has dimensions of L3 = 2 mm and W3 = 600 µm. The mixer structure has a depth of h = 100 µm, and the outlet is set at the end of the mixing channel.The T-junction microfluidic mixer is designed as shown in The T-junction is fabricated using standard soft lithography processes. The mold of the design is first fabricated by spinning coat, exposure and development of a 100 µm thick SU8 photoresist on a silicon wafer. Then, the liquid polydimethylsiloxane (PDMS) consisting of pre-polymer (base) and cross-linker (curing agent) with a weight ratio of 10:1 is poured onto the SU8 mold. The PDMS becomes solid after 4 h of heating in the 65 °C oven. The solid PDMS with the mixer pattern is bonded to a glass slide after plasma treatment, and the microfluidic channel is formed. The completed T-junction mixer chip is shown in In the experiment, we studied both the Newtonian fluid mixing and viscoelastic fluid mixing. In the Newtonian fluid, the response of a simple shear has a linear relationship between the applied shear stress and the rate of shear, i.e., iffusion .The viscoelastic fluid usually contains polymer chains, which can be stretched by the stress from the fluid and relax to an equilibrium state when the stress is released. Therefore, significant elasticity is manifested, and the flow behaviors are enriched. The elasticity can be characterized using the Weissenberg number, defined as ng flows . Such inng flows ,39,40. Cng flows .λ of about 0.12 s.Here, the expanded T-junction mixer introduces the stress to the polymer in the viscoelastic fluid. For the mixing comparison, the Newtonian fluid, 75% glycerol is used, which has a viscosity of 25 mPa·s. For the viscoelastic fluid, 100 ppm polyacrylamide (PAM) with a molecular weight of 18 million is used, which has a similar viscosity (27 mPa·s) with that of glycerol at the shear rate of 100 1/s and relaxation time Ci at every pixel i. The grey value depends on the dye concentration and can be used to derive the mixing efficiency. The flow rate A while at IB, it is a constant. The flow rates are expressed as: f is the time pulsing frequency. The flow rate at IA oscillates between 0 and 2f and a duty cycle of 50%. The time pulsing signal will disturb the flow and further affect the mixing between fluid A and fluid B.In the mixing analysis, the same types of fluids are injected from the two inlets of the T-junction mixer, which are named fluid A and fluid B, respectively. Rhodamine B is diluted in fluid A as fluorescent dye with a concentration of 10 µg/mL. The dye is excited by a 475 nm laser, and the fluorescent color in the channel is recorded as the grey value S0 from the inlet channel at a specific recorded time jt is expressed as:iC is the recorded grey value at the pixel i on the cross line of the channel with the total pixel number of P. C0 is the mean value of the recorded grey values within the mixing channel. It can be seen that S0 in an adequately long recorded time range t0 as a collection of the fluid flowing through S0 at every recorded time ti. Therefore, the averaged mixing degree can be expressed as: N is the number of recorded frames for the concentration profile. We can also see that As the injected flow rate is time-dependent, both the instantaneous mixing degree Re ranges from 0.002 to 0.2. The color in the flow map indicates the normalized concentration profile of the fluorescent dye. The red color presents a fluorescent dye concentration close to 1, while the blue color presents a concentration close to 0. At S0 from the inlet channel. Here, we derive the S1 = 1000 µm and ending at a distance S2 = 2000 µm from the inlet channel, as shown in We first investigate the mixing in the T-junction mixer when glycerol is injected into both inlets at the same constant flow rate. The fluorescent concentration profile in the mixing channel is illustrated in f = 0.1 Hz, 0.2 Hz, and 1 Hz as expressed in Equation (1a) while keeping a constant flow rate of f = 0. The time-averaged mixing degree S1 to S2 is plotted in f = 0 and gradually decreases as f increases to 1 Hz. A similar trend is also observed for the case when Then, we modulate fluid A at a time pulsing frequency f = 0 is the lowest compared to that at non-zero pulsing frequencies. This indicates that the disturbance starts to contribute to the diffusion.In a higher flow rate of f = 0.1 Hz and f = 0.2 Hz, while it remains about the same for f = 0 and f = 1 Hz. This phenomenon contradicts with the previous analysis that the mixing should be lower at a higher flow rate because of the shorter diffusion time. Here, we conclude that the impact of the intrinsic diffusion change can be neglected at this high flow rate. Instead, the disturbance contributes more significantly to the diffusion, especially at f = 0.1 Hz and f = 0.2 Hz, which makes the mixing enhanced compared to that at constant injected flow. As f = 0. It is also noticed that at a fixed flow rate, the f. Therefore, a stronger fluid oscillation in the channel can be induced.As S2 = 2000 µm for 20 s, as shown in f = 0, which is due to the high intrinsic diffusion. At flow rate f = 0. This confirms the contribution of diffusion disturbance to the mixing. At the high flow rate of 500 µL/h, the f = 0.1 Hz and 0.2 Hz, which is much higher than that at f = 0.To further understand the fluid oscillation, we analyze the instantaneous mixing degree f = 0.1 Hz . As shown in Re = 0.002 and Wi = 1 in the micro-mixer. In the Newtonian fluid, compared to that at the constant flow rate, the mixing with time pulsing is decreased when Re is between 0.002 and 0.01, while increased when Re is between 0.1 and 0.2. In the viscoelastic fluid, the time pulsing does not change the mixing degree when WiWi = 50. The improved mixing in viscoelastic fluid stems from the strong fluid oscillation in the micro-mixer.In conclusion: The time pulsed mixing in both the Newtonian fluid and viscoelastic fluid is experimentally investigated in a T-junction micro-mixer. The mixing efficiency of the fluid is compared for different pulsing frequencies and flow rates. In general, the mixing degree in the viscoelastic fluid is higher than that in Newtonian fluid and reaches 0.82 at


The CXCL12/CXCR4 chemokine pathway is involved in cervical cancer pathogenesis and radiation treatment (RT) response. We previously reported that radiochemotherapy (RTCT) and concurrent administration of the CXCR4 inhibitor plerixafor improved primary tumour response. The aims of this study were to determine optimal sequencing of RTCT and plerixafor, the mechanisms responsible for improved response and the effect of plerixafor on late intestinal toxicity.Orthotopic cervical cancer xenografts were treated with RTCT (30 Gy in 2 Gy fractions and cisplatin) with or without concurrent, adjuvant or continuous plerixafor. The endpoints were growth delay and molecular and immune cell changes at the end of treatment. Late intestinal toxicity was assessed by histologic examination of the rectum 90 days after a single 20 Gy fraction.RTCT increased CXCL12/CXCR4 signalling and the intratumoral accumulation of myeloid cells; the addition of plerixafor mitigated these effects. All of the RTCT and plerixafor arms showed prolonged tumour growth delay compared to RTCT alone, with the adjuvant arm showing the greatest improvement. Plerixafor also reduced late intestinal toxicity.Adding Plerixafor to RTCT blunts treatment-induced increases in CXCL12/CXCR4 signalling, improves primary tumour response and reduces intestinal side effects. This combination warrants testing in future clinical trials. When tumours progress locally after RTCT or when metastases develop, treatment options are often limited and ineffective. There is an important need for new therapeutic approaches to overcome radiation treatment resistance, prevent metastases and further improve cure rates.Cervical cancer is the fourth leading cause of cancer death in women worldwide despite improvements in cervical screening and human papilloma virus (HPV) vaccination over the past decades.3 It has been implicated in HPV infection and cervical carcinogenesis, malignant progression, the development of metastases and RT response.3 Our group previously reported that concurrent treatment of cervical cancer patient-derived xenografts (PDXs) with RTCT and the CXCR4 inhibitor plerixafor (AMD3100) produced substantial tumour growth delay and reduced lymph node metastases without increasing early (acute) intestinal toxicity compared to RTCT alone.4 This report builds on these findings by investigating different ways of sequencing RTCT and plerixafor for optimal efficacy, and the mechanisms responsible for improved treatment response as a foundation for future phase I/II clinical trials. We also evaluated long-term tumour control with the combination of RTCT and plerixafor, and the effect of plerixafor on late intestinal toxicity, the most common serious side effect of RTCT after treatment of cervical cancer.Recently, the CXCL12/CXCR4 pathway has emerged as being of particular interest and relevance in cervical cancer as outlined in a recent review.5 were bred, housed and treated in accordance with protocols that conform to the Canadian Council on Animal Care.Six- to 8-week-old female NOD-Rag1nullIL2rgnull (NRG) female mice (impaired adaptive immunity but intact chemokine pathways and myeloid cell immunity) and C57BL/6 mice (fully immunocompetent)7 The radiation treatment growth delay experiments were done using OCICx 20 to maintain continuity with our previous studies.4 This PDX has been extensively characterised by our group.8 The tumour eradication experiments were done using OCICx 3. This PDX was selected because it has a radiation dose-response characteristic (data not shown) that results in a small proportion of tumour ‘cures’ with RTCT alone (50 Gy + concurrent cisplatin), making it better suited for evaluating long-term disease eradication with the addition of plerixafor. In general, these PDX models have been shown to mirror the clinical and biological behaviour of cervical cancer in patients, including the development of lymph node metastases, and respond similarly to RTCT.8The two PDX models used for these experiments were developed from clinical cervical cancer biopsies at the Princess Margaret Cancer Centre and grown orthotopically in the cervixes of mice as previously described.All of the imaging and RT experiments were performed using a dedicated 225 kVp small animal irradiator and integrated cone-beam CT imager  with the mice anaesthetised and immobilised in a lucite jig. Following implantation, the size of the cervical PDXs was monitored weekly using CT imaging. Tumour-bearing mice were randomly assigned to control or experimental groups when the tumours reached a size of 5–7 mm.10 cisplatin alone is not standard-of-care treatment for newly diagnosed patients with potentially curable cervical and was therefore beyond the scope of this study.CT-guided, fractionated RT regimens reflective of clinical practice were used for the tumour growth delay studies. RT was planned using pre-treatment CT images to identify the cervical tumour volume. Customised treatment plans were developed using circular collimators 8 mm in diameter and multiple beams with roughly equal angular distribution around the tumour isocenter. The RT dose was 30 or 50 Gy in 2 Gy fractions at a dose rate of 3 Gy/minute, delivered Monday to Friday for 3 or 5 weeks. Cisplatin 4 mg/kg was administered intraperitoneally (ip) one day each week during RT. Plerixafor  5 mg/kg/day was administered by continuous subcutaneous (sc) infusion using implanted osmotic pumps  concurrently with RTCT for 3 or 5 weeks, adjuvantly  or continuously . Combinations of cisplatin alone and plerixafor were not investigated. While previous reports have suggested that plerixafor may enhance the effectiveness of chemotherapy,x, y, z) based on an ellipsoid model of volume. Two observers independently estimated tumour size from the CT images. Disagreements were resolved by joint review of the images.Mice were followed after treatment with serial CT imaging to monitor tumour regrowth and were euthanised when the tumours regrew to a size of 1–1.5 cm. The tumour volume was calculated from the largest diameter in each dimensional plane  was used to evaluate phosphorylated CXCR4 (pCXCR4), phosphorylated AKT (pAKT), phosphorylated ERK (pERK), CD31 (microvessels) and PD-L1 levels. Myeloid cell surface markers were also assessed, including CD11b (immune cells), Ly6G (granulocytic myeloid-derived suppressor cells—MDSCs) and F4/80 (macrophages). The hypoxia marker EF5 (2-(2-Nitro-1H-imidazole-1-yl)-N-acetamide), a gift from Dr C. Koch (University of Pennsylvania), was injected intraperitoneally at a dose of 10 mg/kg 3 hours before euthanising the mice. The IHC antibodies and dilutions are summarised in Supplemental Table Since CXCL12 is a secreted molecule, gene rather than protein expression was assessed using reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from frozen tissue using the Qiagen RNeasy Mini Total RNA Extraction kit . Total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase (Life Technologies). RT-PCR was performed using SYBR Green PCR Master Mix (Life Technologies) with CXCL12 specific primers. The 2-ΔΔ Ct method was used to calculate relative CXCL12 expression, using the L32, HSP90 and YWAZ housekeeping genes as endogenous controls for normalisation. The CXCL12 and housekeeping gene primer sequences are provided in Supplemental Table To evaluate the general tolerability of high-dose abdominal RT, C57BL/6 non-tumour-bearing mice received whole abdominal RT to a dose of 50 Gy in 2 Gy daily fractions over 5 weeks, using a single 3 × 4 cm treatment field with or without concurrent weekly cisplatin (4 mg/kg) and concurrent plerixafor (5 mg/kg/day). The mice were euthanised 22–29 weeks after completing treatment. The intestines were removed, stained with haematoxylin and eosin (H&E) and examined by a small animal pathologist for histologic evidence of radiation injury.11 The rectum was also examined using IHC for changes in CXCR4 expression and T-cell  and myeloid cell  accumulation.In a separate series of experiments, C57BL/6 mice were anaesthetised and a small polyethylene catheter  was inserted 10 mm into the rectum. CT imaging of the catheter was performed to localise the isocenter of a cylindrical radiation treatment volume approximately 5 mm in diameter and 5 mm in length. A single 20 Gy radiation fraction delivered to this volume using a 360° rotational arc. Cisplatin 4 mg/kg was administered as a single ip dose two hours before irradiation. Plerixafor 5 mg/kg/day was administered sc for 30 days beginning 3 days before irradiation. The mice were euthanised 30 days or 90 days after irradiation. The distal colon and rectum were excised, sectioned longitudinally, stained with H&E and examined by a small animal pathologist for histologic evidence of radiation injury using a previously described radiation injury score (RIS), which is summarised in Supplemental Table 3.p < 0.05 were considered statistically different.For the tumour growth delay studies, the times for the individual tumours to regrow to the size at the start of treatment were calculated using linear interpolation between the points closest to the regrowth time, and the median regrowth time was then calculated for each treatment group. The Mann-Whitney test was used to compare tumour regrowth times and IHC expression levels between two treatment groups, and one-way ANOVA with the Newman-Keuls test was used for comparisons among multiple treatment groups. Results with 4 We have since further validated this result in separate experiments using the OCICx 20 PDX model. The pooled results of three concurrent OCICx 20 experiments are shown in Fig. p = 0.02). For perspective, the improvement in tumour response with the addition of concurrent plerixafor to RTCT was greater than the improvement with the addition of concurrent cisplatin to RT alone  .We previously reported that the combination of RTCT and concurrent plerixafor for 3 weeks was associated with longer tumour growth delay compared to RTCT alone.p = 0.006 and 0.003, respectively). RTCT and adjuvant plerixafor for 3 weeks significantly delayed tumour regrowth compared to RTCT and concurrent plerixafor for 3 weeks (p = 0.018). There was a trend towards delayed tumour regrowth with adjuvant plerixafor for 3 weeks compared to continuous plerixafor for 6 weeks (3 weeks during RTCT and 3 weeks after RTCT) (median regrowth time 27.5 weeks), although this did not reach statistical significance (p = 0.10). There was no difference in tumour control between 3 and 6 weeks of adjuvant plerixafor (p > 0.9).The optimal sequencing of RTCT and plerixafor  was evaluated according to the schema outlined in Fig. While tumour growth delay is a relevant and commonly used pre-clinical study endpoint, it does not necessarily correlate with long-term tumour control and cure because of different radiation treatment sensitivities and response kinetics between clonogenic cells (that must be eradicated to achieve cure) and other cell populations. To determine if the combination of RTCT and concurrent plerixafor improved long-term tumour control compared to RTCT alone, we increased the dose of RT (50 Gy in 2 Gy daily fractions over 5 weeks) and concurrent cisplatin (4 mg/kg ip weekly for 5 weeks) and used a different orthotopic cervical cancer PDX model OCICx 3) to ‘calibrate’ the experiment and ensure that RTCT alone was on the cusp of eradicating the tumours. Long-term control was defined as the absence of tumour re-growth using CT imaging for at least 20 weeks after RTCT and absence of tumour cells in the cervix, uterus or bladder histologically at the time of euthanasia. As shown in Fig.  to ‘caliTo investigate treatment-induced changes in tumour CXCL12/CXCR4 signalling and the immune cell microenvironment, mice bearing the OCICx 20 model were treated with RT or RTCT for 3 weeks (30 Gy) and concurrent or adjuvant plerixafor for 3 weeks. The mice were euthanised at the end of treatment and the primary tumours were excised and analysed using RT-PCR and IHC. The complete dataset for the concurrent plerixafor experiment is shown in Supplemental Fig. RTCT alone resulted in increased CXCL12 gene expression. There were significant RTCT-induced increases in pCXCR4, pAKT (assessed in the concurrent experiment only) and pERK protein expression, suggesting CXCR4-dependent activation of downstream cellular pathways. RTCT also increased tumour accumulation of myeloid cell populations, including Ly6G + MDSCs and F4/80 + macrophages, as well as upregulation of PD-L1 expression by tumour cells. There were no changes in the tumour microvasculature (CD31) or tumour hypoxia (EF5) with treatment.The addition of either concurrent or adjuvant plerixafor abrogated the RTCT-related increases in CXCL12/CXCR4 signalling and myeloid cell accumulation. CXCL12 gene expression, as well as pCXCR4, pAKT, pERK, Ly6G and F4/80 protein expression, returned to near-control levels.The C57BL/6 mice tolerated whole abdominal RT to a dose of 50 Gy in 2 Gy daily fractions, with or without concurrent weekly cisplatin and concurrent plerixafor, with no change in behaviour or weight (data not shown). There was no histologic evidence of intestinal radiation injury in any of the treatment groups 22–29 weeks after completing treatment (data not shown).11To further investigate sub-acute and late intestinal side effects, a single, targeted RT dose of 20 Gy was administered to the rectum, with or without cisplatin and plerixafor, and the mice were euthanised 30 or 90 days later. A previously described radiation injury score (RIS) based on the seven histologic features listed in Supplemental Table p = 0.001 relative to RTCT) indicating radiation protection or mitigation of radiation injury by plerixafor. High (grade 3) levels of rectal vascular sclerosis, wall fibrosis or colitis cystica profunda were seen in 60% of mice in the RT group, 70% of mice in the RTCT group and in none of the mice in the RTCT + plerixafor group.Focal histologic changes consistent with rectal mucosal injury were seen in the majority of mice 30 days after RT. The 30-day RIS was zero in the control arm and uniformly higher in the RT, RTCT and RTCT + plerixafor arms with mean values of 5.4, 4.8, 5.2, respectively, as shown in Fig. To investigate the mechanism by which plerixafor prevents or mitigates rectal radiation injury, we evaluated rectal CXCR4 levels and immune cell accumulation 90 days after treatment. This experiment was also done twice, and the pooled results are shown in Fig. 12 High pre-treatment tumour CXCL12 or CXCR4 expression is associated with early recurrence and death after potentially curative RT in patients with cervical cancer or other malignancies.12 In addition, we found that RT dramatically increases tumour CXCL12 expression  and activation of stimulator of interferon genes (STING).18 Interactions between hypoxia and this delayed DNA damage response will be investigated in future experiments to develop biomarkers for future clinical trials and understand when blood and tumour samples should optimally be acquired relative to the start of treatment.The CXCL12/CXCR4 chemokine pathway plays a central role in immune and tumour cell trafficking and has been implicated in tumour development and progression, the development of metastases and RT response.4 and provides further evidence that the combination of RTCT and concurrent plerixafor improves tumour response compared to RTCT alone  risk of late bone marrow aplasia among cervical cancer patients in future clinical trials, plerixafor should be started towards the end of RTCT after the last dose of concurrent cisplatin.The optimal timing and duration of CXCR4 inhibition relative to RTCT are key considerations in clinical trial design. RTCT and concurrent plerixafor was chosen for our initial experimentslts Fig.  indicatelts Fig. . This im12 that could influence RT sensitivity.20 However, the results of our timing and duration studies imply that plerixafor is modulating pathways that influence tumour recovery towards the end of and/or early after a course of fractionated RT, rather than intrinsic cellular sensitivity directly. This is consistent with other reports that have shown myeloid cell accumulation during and after RT to strongly influence treatment response.22 We identified an increase in Ly6G + MDSCs and F4/80 + macrophages with RT or RTCT alone and a reduction to baseline levels with the addition of either concurrent or adjuvant plerixafor  side effects. The results provide a strong rationale for future clinical trials of RTCT and plerixafor or another CXCL12/CXCR4 inhibitor aimed at improving the efficacy of frontline curative therapy for patients with cervical cancer and possibly other malignancies.In conclusion, this study demonstrates that RTCT increases CXCL12/CXCR4 signalling in cervical cancer, leading to intratumoral accumulation of MDSCs and other myeloid cell populations that may contribute to disease recurrence. Furthermore, it provides the strongest possible evidence using clinically relevant tumour models and treatment protocolsLecavalier and Chaudary, Cervix Plerixafor Supplemental Material


Mediastinal pure choriocarcinomas are exceedingly rare representations of germ cell tumours and are associated with a poor prognosis. To date, fewer than 20 cases have been reported. This current report describes an elderly patient who developed a large rapidly growing mediastinal tumour. Unfortunately, the patient expired before a definitive diagnosis could be reached. An autopsy revealed that the histomorphological features of the tumour showed two distinct tumour cell populations (syncytio- and cytotrophoblasts), and the diagnosis of choriocarcinoma was made. Immunohistochemical analysis showed a characteristic staining pattern in agreement with published studies. Here, we report a case of primary mediastinal choriocarcinoma in an elderly male with concurrent metastasizing prostate adenocarcinoma treated with long-term goserelin deposits, which, as we speculate, could have induced the choriocarcinoma. Choriocarcinoma is a rare malignant tumour that most commonly develops from the chorionic part of the placenta. In males, it is most commonly seen in the testes as part of a mixed germ cell tumour in young males. Extragonadal occurrence is rare—preferentially in axial structures, such as the mediastinum, retroperitoneum, and brain. The prognosis of mediastinal choriocarcinoma is usually poor, which underlines the necessity of rapid diagnosis .We report here a rare case of primary mediastinal choriocarcinoma in an elderly patient with concurrent goserelin-treated metastasized prostate adenocarcinoma.A 71-year-old man presented with a 5-week history of severe back pain. Five years earlier, the patient was diagnosed with metastatic prostate adenocarcinoma (Gleason score 4+4) and was treated with gonadotropin releasing hormone (GNRH) agonist implants . After 9 months of treatment, prostate specific antigen (PSA) values returned to normal levels.Upon admission, bone scintigraphy showed no bone metastases but a potential compression fracture of the L2 vertebra. CT-scan confirmed a lesion in L2 and revealed a 5-cm spherical tumour located in the anterior mediastinum and multiple lung metastases. The periphery of the mediastinal tumour had high levels of Fludeoxyglucose metabolism as seen on PET-CT. The lung metastases and L2 were also PET-positive. Needle biopsy from the mediastinal tumour showed necrosis but no tumour cells. Decompressive laminectomy was performed, and suspected tumour tissue was sent for histological examination. Unfortunately, the patient expired before any conclusive diagnosis could be reached and therapy instituted.Autopsy revealed a 5.5 cm spherical tumour in the mediastinum adherent to but not invading the left lung. The lung parenchyma bilaterally contained multiple suspected metastases. The prostate was slightly enlarged but showed no macroscopical signs of adenocarcinoma. Both testes were atrophic and without signs of focal lesions.The extensive microscopic examination of the mediastinal tumour revealed widespread necrosis and peripheral areas with two distinct tumour cell populations (syncytio- and cytotrophoblasts) . MorpholOn the basis of the findings above, we concluded that the tumour was a primary mediastinal choriocarcinoma.The origin of extragonadal choriocarcinomas has been debated . They haDiagnosis of extragonadal choriocarcinomas can be challenging due to samples comprising predominantly necrosis but only small amounts of tumour cells suitable for diagnosis. Immunohistochemical markers can often promote correct diagnosis.Our findings are similar to previously published immunohistochemical studies of choriocarcinomas. The tumour was classically positive for hCG. The germ cell markers SALL4 and glypican 3 exhibited a characteristic binary staining pattern  .  in vitro study found inhibitory properties of endogenous GNRH in a single choriocarcinoma cell line [At time of diagnosis, the patient was 71 years old. This differs significantly from the typical epidemiology of mediastinal choriocarcinomas, which occurs primarily in younger patients . On the ell line . Howeverell line .Insights into the potential carcinogenic properties of goserelin and knowledge of expression of GNRH receptors may be utilized in the diagnosis and treatment of choriocarcinomas.Extragonadal choriocarcinomas are extremely rare germ cell tumours, and diagnosis requires advanced immunohistochemical analysis. The prognosis is usually poor, which underlines the necessity of rapid diagnosis in order to institute treatment before progression hinders treatment. In this case report, we speculate that long-term treatment with goserelin may have stimulated the development of the choriocarcinoma.