For the purpose of understanding, the governing system of partial differential equations for synovial fluid flow velocity and temperature distribution in the knee joint has been successfully solved for the first time. Therefore, such an article is shedding light on the convective diffusion of the viscous flow along the articular surfaces of the joints through the introduction of power-law fluids with different features of permeability, and stagnation point flow along a magnetic field. Henceforth, the frictional energy causes the knee joint’s temperature to increase. By way of filtration, heated synovial fluid reaches the articular cartilage and provides heat to the bone and cartilage. The lubricant in the joint cavity is properly mixed with this cooled fluid. A rectangular region flow and diffusion model is used to define the issue, thermal diffusion and flow inside the intra-articular gap, as well as flow and thermal diffusion within the porous matrix covering the approaching bones at the joint. Using the similarity solution approach, the linked mixed boundary value problem is addressed. The fluid has been shown to resist moving into or out of the cartilage in certain sick and/or aging synovial joints, causing the temperature to increase. By changing the values of the parameters from their usual levels, it is observed that the temperature did increase in aged and sick joints which impact cartilage and/or synovial fluid degradation. Plane Couette flow is the steady flow between two infinite parallel plates moving in opposite directions at a constant speed1 and pipe Poiseuille flow is when a constant pressure gradient in the axial direction drives a steady flow in an infinitely long circular pipe. The Navier-Stokes equations are a set of equations that describe both flows. These flows are easy to understand which are examples of shear flows and the steady analytical solution is known in both cases2. Extensional viscosity and viscoelastic properties help preserve fluid film conditions in these circumstances. Oscillatory movements with a small amplitude as those seen in running and leaping, may benefit greatly from the presence of viscoelasticity3.The flow properties of synovial fluid are crucial to understanding the many kinds of lubrication that occur in human knee joints . In the context of lubrication, visco-elastic and viscous flow properties are most often the ones that are investigated as part of the research process. At a constant shear rate (steady shear flow), such as Couette or pipe Poiseuille flows, 1. In fluids with higher complexity, such as synovial fluid, the viscosity does indeed exhibit variations in response to changes in shear rate , demonstrate a propensity to undergo mechanical interlocking or entanglement. The phenomenon of entanglement occurs due to the elongated, chain-like structures exhibited by molecules such as HA. In situations where the fluid encounters low shear rates, there is ample opportunity for the molecular chains to engage and interweave, resulting in the creation of a network-like configuration that effectively impedes the flow. The phenomenon of entanglement leads to a significant rise in the perceived viscosity of the fluid in question under the given circumstances. In essence, the variation in viscosity of synovial fluid with shear rate can be attributed to the interaction between molecular entanglement at low shear rates and shear-induced disentanglement at higher shear rates. The rheological behavior of a substance plays a crucial role in facilitating joint lubrication and preserving joint health1. It offers a mathematical representation of viscosity as follows:c fluids consistency is thought of as a measurement of how long it takes for the fluid to relax and d is the rate index, which quantifies the dependency of shear-thinning viscosity on shear rate5.The Cross model is a frequently employed model for describing shear-thinning behavior in synovial fluid7. Their findings which are shown below in Table 9. It is believed that the molecular weight and concentration of Hyaluronic Acid (HA) are the factors that decide the flow properties of SF in health and illness, the amount of HA which is contained in synovial fluid might change. HA is a kind of glycosaminoglycan that is found in high concentrations in connective tissue12. The d-glucuronic acid and n-acetyl-glucosamine units which make up the HA polymer’s repeat unit are alternated with one another, see Fig. 14. The HA molecule has been the subject of a significant amount of study because of its peculiar physical characteristics, despite the fact that its structure seems to be rather straightforward14.The shear viscosity which is steady and other relevant metrics have been recorded several times in a variety of patient groups. Rainer et al. conducted one of the most extensive research up to this point in which they analyzed more than two hundred diseased and postmortem samples however they did not describe the sizes of the individual groups16. The intrinsic viscosity was the most accurate approach after fitting the data to the Mark–Houwink equation which is as follows:16.Since HA is an unbranched polymer, its molecular weight, and concentration may be used to describe the dissolved solution, see Fig. 16. In addition to the molecular weight and concentration of HA, the rheological behavior is typically compatible with theoretical expectations for polymer solutions20. The HA network is a shear-thinning, visco-elastic liquid when it has a molecular weight and concentration that are physiologically relevant. Therefore, the HA concentration could be able to explain a significant portion of the observed diversity in flow characteristics. However, this has not yet been shown explicitly21. The fact that healthy SF contains a larger viscosity, more HA, and a higher molecular weight than osteoarthritis (OA) or rheumatoid arthritis (RA) synovial fluid does provide evidence for the existence of a link between the two conditions21. If protein contributes to viscosity then the consequence of this would be to reduce the disparities that exist between the groups.However, the detrimental impact that polydispersity has on the sensitivity of centrifugation it was decided that intrinsic viscosity would be a better measurement technique to utilize. Since, the viscous and viscoelastic characteristics of HA have been described under a broad variety of different environmental circumstances23. Last but not least consider the diverse array of factors that may lead to osteoarthritis (OA). It is possible that various disease etiologies might result in various SF compositions. According to the findings of some preliminary research, the primary components of joint fluid seem to be the same as those of SF, protein, HA, and phospholipids27. For the measurement of the concentration of protein, HA, and phospholipids straightforward and dependable procedures have been developed, see Table 30.However, there might be additional factors contributing to this fluctuation. For instance, the vast majority of studies did not take into account, the environment of the joint may have been altered by pharmaceutical therapies. According to the findings of Dahl et al., the concentration of HA is unaffected, despite the possibility that other therapies might have an effect. For instance, viscosupplementation is an example of a pharmaceutical intervention that has the potential to alter the HA content of joint fluid30. Due to the fact that several organizations have utilized varying ranges of shear rates, it is not possible to compute some parameters of the Cross model. One of the drawbacks of the research conducted by Rainer and colleagues is that they only looked at postmortem samples from healthy joints. Their findings probably underestimate the viscosity of synovial fluid seen in joints that are really healthy since SF may become more watery when a person passes away32. In order to address this issue, we provide an idea of the use of the power law model in conjunction with the stagnation point flow in order to investigate the temperature and flow behavior of synovial fluid in the knee joint under a variety of different parametric conditions. Later on, the flow at the stagnation point permits us to deliver nanofluid drugs into the SF of the knee joint. The major focus of this research is on the heat transfer and flow structure of pseudo-plastic non-Newtonian fluids flowing over a permeable flat plate in the direction of the base fluid. Energy, continuity, and momentum equations are used to describe the subject under investigation. The similarity solution is used to simplify these equations into two nonlinear ordinary differential equations (ODEs)48. The resultant ODEs then solved numerically using the software package.Comparing the different studies proves to be a challenging task due to the utilisation of distinct rheological models by various researchers. Dahl et al. (1985), Rainer (1996), and Seller et al. (1971) have conducted comprehensive studies and employed appropriate models for their researchIn section "14, together with Davies15 conducted research on the viscosity of human SF samples in 1966, and found that the fluids associated with RA were predominantly Newtonian but the fluids associated with other disease states displayed shear-thinning and had a greater viscosity. The experiment utilized the cone-on-cone geometry with a minimal volume of SF (0.3 ml). Whereas shear-thinning was seen in bovine SF during the same year as Davies et al.15 in which fluid originates from the different joints. A cone and plate rheometer were used throughout all of these tests and investigations. In 1968, Palfrey et al.16 published findings that were comparable using an oscillating apparatus. However, they discovered that oscillatory studies were more difficult to interpret than steady-shear tests. In 1968, Ferguson et al.17 conducted an experiment in which they compared the SF that was extracted from diseased patients’ left and right knees. The authors of this research demonstrated an intriguing finding in that they revealed the viscosity of SF did not seem to alter for the same patient during the course of the day.Dintenfass18 conducted an experiment in which they assessed the viscosity of 80 samples of diseased human SF using three different shear rates. In 1978, Cooke et al.22 conducted an experiment in which shear rates ranging from 0.1 to 1000 were used to determine the constant shear viscosity of a selection of healthy and sick human fluids. He examined one postmortem sample that was considered to be normal as well as a combined sample of another two samples. He carried out each and every one of his tests at a temperature of 21 degrees Celsius in which SF was collected from 59 individuals suffering from a variety of arthritic diseases and analyzed23. They made their measurements at 37 degrees Celsius using cone and plate geometries however they did not provide the shear range across which they tested. It would seem that the maximum shear rate that they used was 150 n which is equal to n derived from the power law model, the samples into mechanical and inflamed fluids. n was found to be less than 0.85 from regular SF28. These were taken from individuals who had a kind of arthritis that was not associated with any shifts in their SF. On the other hand, fluids classified as inflammatory were much less viscous and had a lower degree of shear-thinning . The behavior of visco-elastic materials under enormous deformations that may be significant in vivo is difficult to investigate using computer methods because of the complexity involved. The behavior of a viscoelastic material, on the other hand, is independent of the amplitude of the motion it is subjected to for movements with sufficiently small amplitudes and it can be defined by a few parameters57. Therefore, the most obvious manifestation of elastic behavior in liquids is seen in motions with low amplitudes and high velocities. This circumstance when it occurs in a human joint, is analogous to activities like running or leaping (3 Hz). Other movements such as standing might have substantially longer characteristic periods than walking which happens at a frequency closer to one hertz (1 Hz). Some people feel that the viscoelastic qualities of SF help to sustain and preserve joint tissue even when the joint is subjected to high-velocity motion57.Researchers have started to define complicated fluids in the linear viscoelastic range even if meaningful motion may occur at greater deformations which is important. The physical entanglement that occurs among long molecules is one of the sources of viscoelastic motion in liquids. The network does not get disentangled even when it is exposed to deformation of a modest amplitude particularly when this deformation occurs across brief time periods. Instead, the chains are stretched and compressed which results in the storage of energy in a manner similar to that of a solid subjected to elastic deformation. However, molecular disentanglement is triggered by processes that include larger movements and longer time periods58. The link between temperature and viscosity in joint fluid was observed across the range of 25–4063. In line with the finding, Webb et al.28 observed within the same body of research that investigated the piezo viscous effect in which viscosity dropped by a factor of 2.5 to 5 times between the temperatures of 5 and 35 degrees Celsius. The majority of researchers working in this area have examined the rheology of SF at temperatures that are somewhat close to 65. In addition, it was difficult to determine elastic characteristics at higher temperatures, most likely because of the temporal temperature superposition effects. As a result, it has been determined that evaluating these qualities at a temperature of 55.The temperature has an effect on the viscosity of SF which was something that was not really touched on in the previous discussion. The rheological behavior of various polymer solutions is seen to be dependent on temperature in the same way as it is dependent on time. This observation is supported by some molecular evidenceThe flow in the boundary layer is two-dimensional and in a steady state, the flat plate is unmoving and porous. Both the velocity and temperature of the free stream are equal to entclass1pt{minimax and y directions respectively. The power-law number is indicated by the letter n in the exponent, which is connected to the base fluid. Both Newtonian fluids and pseudo-plastic fluids have n values that are either 1 or between 0 and 1, depending on the kind of fluids. Additionally, the value of n for dilatant fluids is greater than 1. The mass transfer velocity, denoted here by 68. The dimensional governing equations for the boundary layer may be stated as follows;54, the thermo-physical properties are as follows:In the governing system, We provide the following similarity transformations to facilitate the mathematical analysis of our research.mentclasspt{minimaThe quantities of physical interest in this work are the local Skin friction coefficient We explore the flow of both Newtonian and non-Newtonian fluids that could be utilized to model the synovial fluid flow. The SF flow equations are not amenable to easy mathematical investigation since the real geometry of the area where the flow occurs is fairly complicated. Thus, analyzing the flow in a simple geometry can help to explain the model’s efficacy and assess the SF models. Therefore, the complex interaction between the permeability of cartilage, the stagnation point, and the magnetic field as it relates to temperature distribution in a synovial joint has been analyzed using a more straightforward mathematical model. The heating caused by friction might potentially have an effect on the physical qualities of the articular surface as well as the properties of the synovial fluid.c are constant numbers. Hence, the utilization of suction or impermeable surfaces leads to an augmentation in the quantity of both Newtonian and Non-Newtonian fluids, resulting in an elevation of fluid velocity. However, the converse effect occurs during injection phenomena. Also, the boundary layer thickness is decreased by utilizing Newtonian fluid for suction and an impermeable surface, but the velocity gradient is unaffected while Non-Newtonian fluids have a greater velocity gradient across the surface during injection, and such difference is accentuated by increasing the fluid concentration. The impact that permeability has on the velocity profile of both Newtonian and non-Newtonian fluids is seen in the Figs. The impact of the Newtonian and Non-Newtonian fluids for the three different 25. Nevertheless, during injection, the boundary layer area expands, which is an indicator that injection generates greater fluid diffusion and extends the boundary layer. A shift in the velocity profile close to the walls is produced when n is varied from its default value. The shear-thinning and chemically thickening viscosity are to blame for this peculiar behavior seen by the velocity profile. It is important to note that although the velocity profile for 69. The flow changes gradually while maintaining a nearly constant viscosity. The rise in the concentration of HA has the greatest effect on the domain’s center, and as a consequence, the viscosity around the center of the domain has grown quite high, whilst it is relatively low near the domain’s boundaries69.Suction has the effect of slowing down the expansion of the boundary layer, which corresponds with the observation, that the velocity slows down in direct proportion to the suction parameter70.Temperature profiles for an impermeable surface, suction, and injection are shown in Figs. 69. Thus, high temperature near the surface of the articular cartilage drops as it moves toward the contact between the bone and cartilage. This is because of the fact that friction causes a localized increase in temperature at the cartilage surface, therefore the temperature of the fluid likewise rises18. In the process of inhibition, the fluid is absorbed into the cartilage due to heat therefore, cools down as moves closer to the bone cartilage interface. According to Figs. In every instance, the thickness of the boundary layer is reduced when a non-Newtonian fluid is used. Physically, a rise in temperature of the synovial fluid because of the viscous nature of the joint tissues, causes a portion of the input mechanical energy to be dissipated as heatHence, the Impact of different permeability on non-dimensionalized velocity and temperature profiles along flow characteristics of joints over a surface with various fluids can be seen in Figs. 71. Therefore, the velocity of the synovial fluid increases as the parameter M rises for both Newtonian and non-Newtonian fluids in the case of knee joints. When there is a greater distance between the bones of a joint, the axial velocity slows down. As the power-law index is increased, there is a corresponding rise in the mean concentration distribution72.Figures M in the presence of varied free stream conditions henceforth the interaction between the applied magnetic field and the fluid particles, the temperature distribution improves with higher levels of the magnetic field for both the fluids72. The thickness of the thermal boundary layer grows in proportion to the magnetic field in the synovial fluid scenario for the Newtonian and non-Newtonian fluids. It has been noticed that the passage of fluid into or out of the cartilage is impeded in some diseases and/or aged synovial joints, leading to an increase in temperature because of the fact that the temperature rises49.Figures 5. These numbers allude to the effects of aging as well as disorders that might impact the degradation of synovial fluid as well as cartilage. Even if they are not very noteworthy, increases in temperature result in a reduction in the viscosity of the synovial fluid5. It is projected that the global average temperature would increase by little more than 1 degree Celsius, and there would be some regionally heightened temperature gradients. Generally, the temperature varies greatly depending on the articulate gap such that the synovial fluid closure to the articular cartilage5. Thus, the peak temperature is roughly the same as the temperature of the cartilage surface. Therefore, an increase in knee contact forces causes a higher amount of heat to be generated. Hence, a heavier patient will have more knee contact during a given activity, then it stands to reason that would create more heat, and lead to rising temperature both within and around the knee joint15.When the values of the parameters are changed from their usual levels, a spike in temperature is observed in aged and sick joints70. When the stretching velocity of the surface is greater than the free stream velocity, an inverted boundary layer structure is created71 such BL creation does not occur when 71.The velocity and temperature profiles for a number of different At the stagnation point, the kinetic energy term in the Bernoulli equation is nullified as a result of the absence of velocity. The dominance of static pressure and internal energy results in the fluid particle’s total energy. As the fluid departs from the stagnation point, it undergoes a redistribution of energy among kinetic energy, potential energy, and internal energy. This redistribution is influenced by the flow conditions and the presence of gravitational forces. These forces are particularly relevant in the context of the thin layer of fluid adjacent to a solid surface, where viscous effects play a significant role, see Figs. In the vicinity of the stagnation point, the boundary layer tends to exhibit a notably reduced thickness. The presence of a thin boundary layer has a direct impact on the velocity and pressure distributions surrounding the stagnation point. Consequently, these distributions have an influence on the energy equation. Understanding the redistribution of energy in a fluid as it moves along a surface requires a comprehensive analysis of the pressure and velocity gradients within the boundary layer.The energy equation is a useful tool for analyzing the heat transfer process between a solid surface and a fluid medium. The rate of heat transfer and the development of the thermal boundary layer can be influenced by the temperature distribution within the boundary layer. This temperature distribution is dependent on the energy equation.a is provided in Figs. a results in a lower non-dimensional temperature. These Figures depict results for the suction case to both Newtonian and non-Newtonian fluids, therefore the effect is applicable to all types of fluids. Increasing the value of the convective parameter results in an increase in the dimensionless surface temperature 71. An increase in the convective parameter leads to a decrease in the thermal resistance of the hot surface, which causes an increase in the dimensionless surface temperature. In addition, it is important to observe that as parameter a gets closer and closer to infinity, the The temperature profile for various values of a convective parameter The influence of various power-law fluids on the Nusselt number for impermeable surfaces, suction, and injection is seen in Fig. Figure 55.The boundary layer thickness is decreased by utilizing Newtonian fluid for suction and an impermeable surface, but the velocity gradient is unaffected. Non-Newtonian fluids have a greater velocity gradient across the surface during injection, and such difference is accentuated by increasing the fluid concentrationThe dimensionless temperature decreases initially for the suction case, which involves Newtonian to non-Newtonian fluids, and for the impermeable surface case, the temperature eventually rises.Physically, a rise in temperature of the synovial fluid because of the viscous nature of the joint tissues, which causes a portion of the input mechanical energy to be dissipated as heat.59.The synovial fluid’s composition and structure in which HA makes the fluid non-Newtonian, and its concentration affects synovial fluid responsiveness71.The rate of friction does not rely on the velocity of the SF. Though at high velocity or load, surface heating and viscoelastic processes might influence the tribology of the interface, the rate of motion does not directly impact the energy wasted per unit sliding distance under boundary lubrication conditionsThe use of a non-Newtonian fluid with a large volume fraction allowed for the decrease of the non-dimensional temperature on the impermeable surface. In the case of suction, the volume fraction does not have a significant impact on the surface temperature; however, using a non-Newtonian fluid . A fluid whose characteristics alter when it interacts with a chemical agent whose mass fraction is considerably less than that of the fluid and whose diffusion velocity is much slower than that of the fluid, as a result, the diffusion is carried by the fluid at a speed close to that of the fluid itself. The findings are relevant not only to the flow of synovial fluid but also to the chemical interaction of HA with polymeric fluids. The flow of a shear-thinning, chemically-reacting fluid that may be utilized to represent the flow of synovial fluid is a major focus of study in this article. The most important results of this study are as follows:


Non-gestational choriocarcinoma (NGC) is a rare subtype of malignant germ cell tumour and there is no consensus on its treatment. The lack of suitable preclinical models for NGC is a challenge in drug discovery research. Patient-derived xenograft (PDX) models recapitulate the tumour microenvironment of the original cancer tissue. Therefore, they have received considerable attention for studies on rare cancer. Here, we aimed to establish a PDX model from a patient with recurrent NGC.-Prkdcscid1l2rgtm1Wjl/SzJ) and maintained for more than three in vivo passages. Subsequently, we evaluated the molecular characteristics of the PDX model using immunohistochemistry, polymerase chain reaction, and RNA sequencing. Moreover, the PDX tumours were transplanted into BALB/c nude mice, and we evaluated their sensitivity for cisplatin and methotrexate.Fresh NGC tumour tissue was immediately transplanted into a severely immune-deficient mouse . Finally, we demonstrated that the PDX tumour was sensitive to cisplatin and methotrexate; therefore, its clinical response to the agents was similar to that of the primary tumour.The PDX tumour maintained the morphological features of NGC. Moreover, Immunohistochemistry revealed that the human chorionic gonadotropin, cytokeratin 7, and EpCAM expression levels were similar to those in the primary tumour. Furthermore, serum human chorionic gonadotropin levels were elevated in both the primary tumour and the PDX models. Additionally, using PCR analysis with species-specific primers, we confirmed that the PDX tumour contained human genes and was derived from human tissue. Moreover, the gene expression profile of the NGC was compared with that of epithelial ovarian cancer samples and cell lines, and 568 dysregulated genes in the NGC were extracted. The expression of the dysregulated genes in PDX was significantly correlated with that in the primary tumour (RWe successfully established a PDX model of NGC, to the best of our knowledge, for the first time. The established PDX retained the molecular and transcriptome characteristics of the primary tumour and can be used to predict drug effects. It may facilitate further research and the development of novel therapeutic agents for NGC.The online version contains supplementary material available at 10.1186/s12885-023-11626-3. Choriocarcinoma is a malignant tumour that forms when trophoblastic cells, a part of the placenta, proliferate abnormally and become tumour-like . ChoriocHuman chorionic gonadotropin (hCG), which is secreted by trophoblasts, is a useful biomarker for diagnosis and monitoring the treatment response. In addition to pathological findings, a history of sexual intercourse and pregnancy is critical information that could facilitate distinguishing between GC and NGC. The standard treatment for NGC has not been established; however, surgical removal of the tumour and systemic chemotherapy are effective treatment options . ParticuPatient-derived xenograft (PDX) models are created by transplanting the patient’s tumour tissue into immune-deficient mice. PDX tumours retain the properties of primary patient tumours, such as glandular, vascular, and stromal structures; cellular complexity; and cytogenetic features –12. In aIn the present study, a PDX model of NGC was established successfully for the first time, to the best of our knowledge. Additionally, the histological, molecular, and pharmacological characteristics of the PDX model were investigated. The PDX model could facilitate the elucidation of the molecular background of NGC and the development of novel therapeutic strategies.One patient with a gynaecological malignancy was included. To establish the PDX models, a residual tumour was obtained from the left ovary during a fourth surgical procedure, and clinical data of the patient were collected. The study protocol was approved by the ethics committee of the Nagoya University Graduate School of Medicine in Japan , and written informed consent was obtained from the patient. We also confirmed that the consent to publish was obtained from the study participant.-PrkdcscidIl2rgtm1Wjl/SzJ) and BALB/c nude mice were purchased from Oriental Bio Service, Inc. . The mice were maintained in a pathogen-free, temperature-controlled environment with a 12 h light/dark cycle, and NSG mice were fed autoclaved water and irradiated food. The mice were carefully monitored, and tumour volumes were calculated using the following formula: volume = length × width × width × 1/2. NSG mice were used for the PDX model establishment. BALB/c nude mice were used in experiments to observe how the established PDX model responded to the drugs. The humane endpoint criteria required euthanasia if the tumour length diameter was > 2 cm, weight loss in a few days was > 20%, or self-mutilation occurred. All animal experiments were approved by the Animal Care and Use Committee of the Nagoya University Graduate School of Medicine .Four-week-old female NSG , 4.0 mg/kg of midazolam , and 5.0 mg/kg of butorphanol . When the tumour length diameter reached approximately 2 cm, the tumours were harvested and passed. The generation harbouring the patient-derived material was termed P1, with subsequent generations numbered consecutively . The PDX model was considered established after the P3 generation, and the tumours were cryopreserved using liquid nitrogen in CELLBANKER .To establish the PDX model, a residual tumour was obtained from the left ovary during a fourth surgical procedure. The tumour was washed with PBS and sectioned into approximately 8 mm3. The mice received intraperitoneal administrations of either cisplatin  or PBS four times every 2 days. Similarly, for the second assay, 10 mice were subcutaneously transplanted with PDX tumours (P7 generation) and randomly assigned to two groups. Thereafter, they received intraperitoneal administrations of either methotrexate  or PBS four times every 3 days. All mice were sacrificed when the tumour volume was > 1500 mm3.For the drug sensitivity assay, 10 BALB/c nude mice were subcutaneously transplanted with PDX tumours (P6 generation). From the viewpoint of animal welfare, the number of experimental mice was set to the minimum effective number. They were then randomly divided into two groups when the tumour volume reached approximately 100 mm2O2 in methanol for 10 min (for CK7 and EpCAM) or 0.03% H2O2 in methanol for 10 min (for hCG). Subsequently, the sections were blocked with appropriate serum using the SAB‑PO (R) kit , according to the manufacturer’s protocol (for CK7 and EpCAM), or 1% BSA in PBS (for hCG). Thereafter, the sections were incubated with the following primary antibodies at 4 °C for 12–16 h: anti-hCG Ab , anti-CK7 Ab , and anti-EpCAM Ab . The CK7- and EpCAM-treated sections were incubated with appropriate secondary antibody for 10 min, followed by peroxidase-labelled streptavidin for 5 min using the SAB‑PO (R) kit; the hCG-treated sections were incubated with the secondary antibody conjugated to horseradish peroxidase-labelled polymer  for 60 min. After incubation, the sections were stained with the DAB working reagent for 4–15 min. Finally, the sections were counterstained with haematoxylin, dehydrated, and mounted. All images were acquired using a Zeiss Axio Imager A1 microscope .The PDX tumour tissues were fixed in 4% paraformaldehyde for 12–16 h and embedded in paraffin. Formalin-fixed and paraffin-embedded tissues were sliced at a thickness of 4 μm using a microtome. Sections were deparaffinized and rehydrated in xylene and a gradient series of ethanol. For antigen retrieval, the sections were heated in Tris/EDTA buffer (pH 9.0) for 20 min at 90 °C (for cytokeratin 7 (CK7) and EpCAM) in a microwave. Antigen retrieval was not performed for hCG. Thereafter, to block endogenous peroxidase, the sections were incubated with 0.3% Hg for 10 min at 15–25 °C. Plasma samples were analysed using enzyme immunoassays (CLEIA) with α-hCG and β-hCG-CTP monoclonal antibodies .Blood samples were collected by cardiac puncture in the mice at the time of their sacrifice and centrifuged at 2,000 ×NHDF-Neo  cells were purchased from Lonza . This cell line was isolated from neonatal foreskin tissue. These cells were cultured in DMEM medium  supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) and incubated at 37 °C and 5% CO2. Cells tested negative for mycoplasma contamination.gDNA was extracted from the P3 tumour tissues of the PDX model, the primary tumour tissues, NHDF cells, and normal tissues of BALB/c nude mice using the QIAamp® DNA Mini kit , according to the manufacturer’s protocol. The extracted gDNA was quantified using a NanoDrop 1000 spectrophotometer . Thereafter, 100 ng of gDNA was amplified using Blend Taq® -Plus , and PCR was performed using a 2720 Thermal Cycler (Thermo Fisher Scientific) with the following protocol: 1 cycle at 94 °C for 2 min, followed by 28 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The PCR products were separated using 1.5% agarose gel electrophoresis with 3 µL of a TrackIt 100-base pair DNA ladder . The amplified fragments were observed by exposing the gel to UV light.http://pachterlab.github.io/kallisto/). The data were summarized using the tximport package (ver. 1.18.0) of R software (ver. 4.1.2) and RStudio .Total RNA was extracted from primary patient and PDX tumour tissues (P3) using the miRNeasy Mini Kit (Qiagen). RNA sequencing was performed by Rarevariant, Inc. , and the sequencing data were used to quantify the expression level of each gene with Kallisto  datasets for the comparison, including GSE121103, which contains the RNA expression data of clinical samples of epithelial ovarian cancers (EOCs). In addition, GSE192446, GSE190792, GSE131753, GSE43362, and GSE38541 are the gene expression profiles of germ cell tumour cell lines. GSE192446 contains data from our previous study [2 fold change > 1. Following comparison of the EOC clinical sample and germ cell tumour cell line data, DEGs in NGC were extracted. Subsequently, Gene Ontology (GO) analysis for the DEGs was performed using an online database, DAVID (https://david.ncifcrf.gov/).Heatmap and hierarchical clustering analyses were performed using the heatmap.2 function of the gplots package (ver. 3.1.0). The genes with low read counts  were excluded. Moreover, considering several transcripts encode the same gene, only the transcript with the highest expression was used for the heatmap analysis. Differentially expressed genes (DEGs) were identified with an absolute logt test. Data are presented as the mean ± standard deviation (SD). P < 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism v.9.4 . Statistical comparisons between two groups were performed using the Welch’s An 18-year-old woman  was admitted to our hospital with severe abdominal pain and bloating. She had no history of sexual intercourse. Computed tomography (CT) revealed a huge right ovarian tumour (approximately 18 cm × 12 cm × 23 cm) . Subsequently, she received further chemotherapy (three cycles of MEA), resulting in negative hCG levels. Nevertheless, the patient experienced re-recurrence at the left fallopian tube approximately six months after the last treatment. Due to the strong desire for fertility-sparing, a left salpingectomy (third surgery) was planned after one cycle of MEA therapy. Fortunately, intraoperative findings suggested the tumour was localized to the left fallopian tube, and the pathological examination confirmed the negative margin. As a result, the left ovary was preserved, and three cycles of MEA therapy were administered. However, after approximately another six months, elevated hCG levels were observed, and a recurrent lesion was found in the left ovary. The patient still maintained a strong desire for fertility-sparing; therefore, a left ovarian cystectomy was performed (fourth surgery) without adjuvant chemotherapy, and the tumour was used for the analysis Fig. F–H. A reA fresh tumour sample was obtained from the patient during the fourth surgery and immediately transplanted into the NSG mouse model. This sample was histologically confirmed to comprise pure choriocarcinoma. The tumours became palpable 42 days after implantation and were transplanted into second-generation mice 76 days after implantation. Subsequently, the PDX model was established through serial passages. The tumour-bearing periods of the P2 and P3 generations were 78 and 61 days, respectively. No metastasis to lungs or other organs was observed in all mouse generations. Moreover, we confirmed that the PDX tumours (P4) were tumorigenic in NSG mice after cryopreservation. The success rate after the transplant was 100%. Furthermore, PDX tumours were engrafted even in BALB/c nude mice. All mice used for PDX establishment did not show weight loss more than 20%.Haematoxylin & eosin (H&E) staining showed a substantial proliferation of cells with atypical nuclei of varying sizes in all PDX tumour passages, and tumour cells were arranged in biphasic proliferation of cytotrophoblastic cells rimmed by syncytiotrophoblast cells. The H&E staining findings revealed that primary morphology and tissue architecture were preserved in all PDX tumour passages Fig. A. AdditiThereafter, we evaluated the serum hCG level of the mice, as this was the biomarker examined in the patient Fig. E. The seGAPDH, as shown in Fig. GAPDH was observed  using RNA-seq and multiple GEO datasets. Heatmap analysis showed that the gene expression profiles of PDX were more similar to those of the primary tumour than those of EOC tissue. Fig. A. SimilaFinally, to evaluate the responsiveness of the PDX model to CDDP and MTX , we analysed their effects on tumorigenicity in nude mice. We found that treatment with CDDP significantly reduced the tumour volume compared to that of the control P < 0.05, Fig. , Fig. 5AIn the present study, a PDX model from a patient with NGC was established successfully. A previous report has demonstrated a mini-PDX model for NGC . The PDXThe clinical course showed that the patient had refractory cancer, which repeatedly relapsed despite several chemotherapeutic regimens and multiple surgeries. The aggressiveness of the tumour may be associated with our successful establishment of a PDX model. In a previous study, patients whose cancers failed to engraft into nude mice had an 81% reduction in the risk of death from pancreatic cancer . In geneWhen comparing histological and gene expression profiles, we found a high degree of similarity between the PDX model and primary tumour. Similar to GC, NGC reveals the abnormal proliferation of atypical trophoblasts histologically and presents a two-cell pattern organized into two types of trophoblastic cells: syncytiotrophoblastic and cytotrophoblastic. Trimorphic proliferation was observed in certain cases, including in intermediate trophoblasts. These structures are mixed and proliferate in full or sheet-like forms, infiltrate, and proliferate in the surrounding tissues and blood vessels. They are often associated with haemorrhage and necrosis. In addition, the chorionic villi are absent , 27. H&EFurthermore, as hCG is secreted into the blood, serum hCG levels are extremely useful biomarkers for periodic monitoring to evaluate a therapeutic response . Our PDXFurthermore, the heatmap analysis of RNA-seq revealed the similarity of gene expression profiles between PDX and primary tumours. These results are consistent with previous studies comparing PDX and corresponding patient tumours in breast and colon cancer , 31.In PDX, human lymphoma and mouse tumours can develop spontaneously –34. TherGiven that the response to drugs is important for evaluating the clinical predictive value of any PDX model, we validated whether the results of drug intervention using our PDX model were comparable to the clinical outcomes of NGC. The most commonly used chemotherapy for NGC is a platinum-based regimen, including BEP and TIP therapies, which is based on a treatment for MGCT , 6. MoreThis study had some limitations. First, only one PDX model was established. However, NGC is a very rare cancer, and it occurs in only three cases in our institution in the past 10 years. Therefore, it needs too much time to establish several PDX models, and we have to keep trying to establish several PDX models of NGC. Second, the RNA-seq data used to evaluate gene expression may not be optimal. Hence, the subsequent pathway analysis might have limited significance. Third, the genetic characteristics of NGC were not assessed. Therefore, to further research about the molecular background of NGC using our PDX model, whole genome sequencing (WGS) is essential for evaluating the genetic similarity between the PDX model established and the original tumour. Fourth, the sensitivity to CDDP and MTX was evaluated. In the present study, to evaluate whether the NGC-derived PDX model reflects the properties of both MGCT and GCN, CDDP was selected as a key drug for MGCT and MTX, as an important drug for GTN. However, to further clarify the drug sensitivity of the PDX model, the effects of other drugs, such as etoposide and bleomycin, should be assessed in future.A PDX model of NGC was established and a high degree of similarity between the PDX and primary tumours was demonstrated. The results suggested that the PDX model represents the histopathological and gene expression characteristics of primary tumours. Therefore, the model could provide a promising platform for novel therapeutic strategies for NGC. Further studies are required to improve the prognosis of patients with NGC.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4


Cancer metastasis, the process by which tumour cells spread throughout the body and form secondary tumours at distant sites, is the leading cause of cancer-related deaths. The metastatic cascade is a highly complex process encompassing initial dissemination from the primary tumour, travel through the blood stream or lymphatic system, and the colonisation of distant organs. However, the factors enabling cells to survive this stressful process and adapt to new microenvironments are not fully characterised. Drosophila have proven a powerful system in which to study this process, despite important caveats such as their open circulatory system and lack of adaptive immune system. Historically, larvae have been used to model cancer due to the presence of pools of proliferating cells in which tumours can be induced, and transplanting these larval tumours into adult hosts has enabled tumour growth to be monitored over longer periods. More recently, thanks largely to the discovery that there are stem cells in the adult midgut, adult models have been developed. We focus this review on the development of different Drosophila models of metastasis and how they have contributed to our understanding of important factors determining metastatic potential, including signalling pathways, the immune system and the microenvironment. Drosophila melanogaster (Drosophila) have been widely used to study the molecular and genetic underpinnings of human cancer . F. F39]. FSerial transplants, where tumours are repeatedly harvested and retransplanted into new adult hosts, have allowed the study of primary tumour growth and metastasis over an even longer period B. Caussilgl and brat were sufficient to drive metastasis in the adult host . T. T41]. Tult host ,76. Brieult host ,76,79,80and head ,76. By elgl and brat tumours had a similar rate of metastasis in the first instance, continuous passaging of the tumour cells into new hosts increased the rate of metastasis in lgl but not brat mutants . A. ADrosoptastases , whereastastases . Understtastases .As with transplant assays, adult models of metastasis allow the development of a tumour to be studied over a longer period, better recapitulating human disease. In contrast to transplant experiments, generating clones in situ using MARCM in adult flies circumvents practical issues with microinjection, making such models far more amenable to high-throughput screening. In addition, a secondary tumour forming in the adult tumour model cannot be an artefact of the injection and importantly, it allows many stages of metastasis, including the very first and last, to be followed in a single adult fly.Drosophila models are ideally placed to address this, and they have already yielded significant findings implicating EMT, dysregulated cell signalling and different aspects of the microenvironment in metastasis.Since the earliest discoveries of cancer-related genes in larvae, the development of rudimentary transplantation experiments demonstrating the differences between benign and malignant neoplasms and, more recently, the development of adult tumour models, research using fruit flies has been instrumental in enhancing our understanding of factors underlying cancer. An important focus of future cancer research is the metastatic cascade. Metastasis is the biggest cause of cancer-related death, but the mechanisms remain unclear. Drosophila metastasis model has advantages and disadvantages, and each is ideally suited to investigating distinct aspects of metastasis using different techniques. Although larvae have large populations of mitotic cells amenable to induction of tumour growth, they are limited in the time the tumours can grow and are thus unsuitable for long term studies of tumour behaviour. This can be overcome by transplanting larval tumours into adult hosts and monitoring their growth. The main disadvantage of this method is that the primary tumour did not grow in the adult host, meaning you are missing the initial key steps in the metastatic cascade, as well as inducing possible artefacts through injection. More recently, adult models have been developed based on the finding of mitotic intestinal stem cells in the adult gut. These models are advantageous because you can model the entire metastatic cascade and monitor growth and behaviour over long periods. However, all Drosophila models are based on the expression of markers such as GFP, RFP and luciferase. It is possible that some of these markers may affect cell behaviour, survival and response to the immune system. Therefore, it may be an important future endeavour to establish endogenous markers of tumorigenesis to study how tumours behave in the absence of these ectopic factors.Each type of Drosophila has limitations but offers also important advantages that can be exploited. There are now a great variety of metastasis models, from the genetically induced to transplantation experiments, that are continuing to contribute to our understanding of how cells become metastatic. Moreover, the great amenability to genetic manipulation that Drosophila offers will help to dissect how gene expression is able to modulate the extraordinary cell plasticity shown by tumour cells and how it is required for their adaptation to new microenvironments.In summary, as a model for metastasis


Further, when IgM was positive, the value of the orbicularis oculi muscle in the ENoG test results was significantly higher (p < 0.05), and symptoms of tinnitus and hyperacusis occurred more frequently (p < 0.05). The initial and final HB grades were significantly higher in IgG-positive patients than in IgG-negative patients (p < 0.05). When IgG was positive, the values of nasalis and oris muscles in the ENoG test results were significantly higher (p < 0.05), and symptoms of dizziness occurred more frequently (p < 0.05). This study confirmed that the more active the immunological action of the VZV in the body, the greater the damage to the facial and vestibulocochlear nerves, which are associated with the degree of facial paralysis and the accompanying otologic symptoms.Ramsay Hunt syndrome (RHS) has a poor prognosis because of varicella-zoster virus (VZV) infection. This is most closely related to severe inflammation in the geniculate ganglion of the facial nerve due to VZV infection or reactivation. This study investigated whether there were differences in the prognosis and accompanying symptoms of facial paralysis based on the presence or absence of VZV IgM and IgG antibodies. This study was conducted as a retrospective chart analysis of 105 patients with RHS who were admitted to our hospital between 2015 and 2021. The House–Brackmann (HB) grade and electroneurography (ENoG) was used to evaluate the degree of facial paralysis. Patients’ subjective symptoms were evaluated by dividing them into dizziness, tinnitus, hyperacusis, and hearing loss. No difference was observed in the initial HB grade with or without IgM; however, the final HB grade was significantly higher in IgM-positive patients than in IgM-negative patients ( Varicella-zoster virus (VZV), also known as the chickenpox–shingles virus, is one of the most common causes of infection in the central nervous system. VZV infection can lead to encephalitis, meningitis, Ramsay Hunt syndrome (RHS), stroke, and other neurological complications. In particular, when VZV infects the seventh cranial nerve, which controls the facial muscles, it can cause peripheral facial paralysis along with symptoms such as tinnitus, pain and blisters around the ear, and dizziness. Despite its infrequency, RHS presents a significant challenge for clinicians due to its potential for severe complications and the need for early diagnosis and prompt management . RHS accIn addition to serological tests that use enzyme-linked immunosorbent assay (ELISA) to detect VZV infection, polymerase chain reaction has recently been introduced as a method for detecting VZV infection in saliva, blood, or nervous tissue samples, and several studies have reported a relationship between VZV infection and RHS ,5,6.Specifically, in patients with RHS, VZV DNA was found not only in the external ear and oral vesicle, but also in the middle-ear mucosa, facial nerve sheath, and cerebrospinal fluid . FurtherThis study aimed to investigate whether there were differences in the prognosis of facial paralysis and accompanying symptoms based on the presence or absence of VZV IgM and IgG antibodies, which are responsible for the body’s immune response to VZV. To our knowledge, no previous research has been conducted on this topic, and we believe that this study will provide insights into predicting the prognosis and symptoms of facial paralysis in RHS patients.In this study, we retrospectively analyzed the medical records of 105 patients with RHS who were admitted to the Department of Otorhinolaryngology at Kyung Hee University Hospital  and underwent high-dose steroid therapy from 2015 to 2022 (8 years). The diagnostic criteria for RHS were as follows: (1) acute or subacute peripheral facial palsy; (2) vesicular rash in the pattern of the external ear, external auditory canal, or oropharynx; and (3) absence of any brainstem or cerebellar lesions on magnetic resonance imaging . PatientWe used the House–Brackmann (HB) grading system to assess the degree of facial palsy at admission and six months after onset . The HB High-dose steroids were administered to patients who met the inclusion criteria. High-dose steroid therapy involved the use of 5 mg of steroids per kg of body weight for the first 4 days, followed by a reduction over 6–8 days . High-dose steroids were used with caution even in normal patients without underlying diseases because they can cause metabolic effects and an increased vascular pressure as well as worsen underlying conditions such as hypertension or diabetes. In particular, for patients with diabetes whose blood sugar control was poor, the endocrinology department was consulted, and they recommended using oral antidiabetic drugs or insulin during the high-dose steroid therapy. Moreover, for patients with hypertension, when blood pressure was not continuously controlled while taking oral corticosteroids, an oral antihypertensive agent was additionally taken by referring to the cardiology department. Additionally, all patients were administered famciclovir 750 mg orally once daily for one week, and the use of eye drops, ointments, and eye patches during sleep was recommended to protect the cornea .Blood samples were collected from the patients upon admission and stored in a freezer at −20 °C. VZV IgM and IgG antibodies were detected using ELISA. IgM antibodies were measured using a solid-phase enzyme immunoassay (EIA), and IgG antibodies were measured using the captured EIA. Results were classified as negative when the values were <0.90, intermediate when between 0.90 and 1.10, and positive when >1.10. Patients with intermediate results were excluded from the study [To accurately evaluate the axonal degeneration of the facial nerve, the prognosis of facial paralysis, electroneurography (ENoG) was performed between 5 and 14 days after the onset of RHS ,12. The For measurement, surface electrodes were attached to the upper and lower lateral sides of the eyebrow, nasolabial fold, and upper lip, and the ground electrode was attached to both wrists. The nerve stimulator was positioned at the stylomastoid foramen and started with a low current of 10 mA, gradually increasing the intensity of stimulation by 10 mA up to a maximum current of 80 mA. The maximum electrical stimulation applied to the muscle was 1 Hz AC for 0.2 ms, and all tests were conducted using the DS7A model  ,15. The t-test was used. Multiple regression tests were used to examine the impact of VZV IgM and IgG on each dependent variable, with adjustments for sex and age. SPSS statistical software was used for the statistical analysis, with a significance level of p < 0.05.For categorical data, such as sex, direction of facial paralysis, HB grade, medical history, and symptoms reported by the patients, chi-square and multiple logistic regression tests were used. For the normality test for continuous data, the Kolmogorov–Smirnov test was conducted, such as for the ENoG results. Then, to analyze the difference between the mean, an independent Of the 105 patients with RHS, 38 were IgM-positive and 71 were IgM-negative, whereas 91 were IgG-positive and 18 were IgG-negative. The results are summarized in The mean age of IgG-positive patients was 50.73 ± 15.80 years, while that of IgG-negative patients was 52.77 ± 17.87 years, indicating no statistically significant difference between the two groups. In the IgG-positive group, 35 were men and 56 were women, while in the IgG-negative group, 3 were men and 15 were women. No significant difference was observed in terms of sex between the two groups. In the IgG-positive group, 16 patients had hypertension and 10 had diabetes, whereas there were no patients with hypertension or diabetes in the IgG-negative group, indicating no statistically significant difference between the two groups. In addition, among IgG-positive patients, 49 had dizziness, 26 had tinnitus, 39 had hyperacusis, and 21 had hearing loss; among IgG-negative patients, 14 had dizziness, 3 had tinnitus, 12 had hyperacusis, and 7 had hearing loss. No statistically significant differences were observed in coexisting symptoms between the IgG-positive and -negative groups .p = 0.553; final HB grade, p < 0.0001) ( 0.0001) . Next, wp = 0.001; final HB grade, p = 0.030). The Kolmogorov–Smirnov test was performed to test the normality of the ENoG result values. As a result of the analysis, it was confirmed that the value of the ENoG satisfied normality (p > 0.05). The ENoG results for the four facial muscles based on the IgM and IgG levels are shown in p = 0.018). Similarly, the mean ENoG values for the four facial muscles were higher in the IgG-positive group than in the IgG-negative group. The average ENoG values for nasalis, orbicularis oris, and the four facial muscles were significantly higher in the IgG-positive group than in the IgG-negative group . To evaluate the effects of IgM and IgG on neurological symptoms, a multiple logistic regression analysis was performed (The initial and final HB grades based on IgG levels are shown in erformed . In the erformed .In this study, we confirmed the correlation between VZV IgM and IgG antibodies collected from the blood of RHS patients and the prognosis of facial paralysis and related symptoms. Based on the results of the initial and final HB grading systems and ENoG, we concluded that the higher the activity of VZV IgM and IgG, which play an important immunological role in VZV, the worse the prognosis of facial paralysis. In addition to facial paralysis symptoms, we also found that the higher the activity of VZV IgM and IgG, the higher the frequency of accompanying symptoms.In the context of VZV infection, VZV Ig refers to the specific antibodies produced by the immune system in response to the presence of the varicella-zoster virus. These antibodies, including IgM and IgG, play a critical role in detecting and neutralizing the virus, aiding in the diagnosis and management of VZV-related diseases. Several studies have been conducted so far regarding the long-term prognosis of facial paralysis and accompanying symptoms in patients with RHS. According to one previous study, the prognosis of facial paralysis was worse in patients with RHS, who were old, had hypertension or diabetes mellitus as an underlying disease, and had dizziness as a comorbidity . In anotVZV IgM and IgG are synthesized in the body in response to VZV infection, which causes vesicular rashes on the skin; synthesis begins in the body between two and five days, reaching a maximum level between two and three weeks . VZV IgMVZV IgG is the most abundant immunoglobulin in the body and is responsible for long-lasting immune effects, comprising approximately 80% of the total immunoglobulin. Additionally, VZV IgG can be distributed throughout the body, not just in the blood and lymphatic fluid. According to Charlotte et al., VZV IgG is expressed in approximately 80% of patients with acute VZV infection . In the When comparing the characteristics and differences between VZV IgM and IgG, a notable feature of this study is that in cases where VZV IgG is positive, the severity and prognosis of facial paralysis are worse than in cases where VZV IgM, which is known to play an important role in acute immune response, is positive. This is because VZV IgM is synthesized faster in the body during the acute phase and reaches its maximum level faster than VZV IgG, which acts over a longer period of six months or more, with a greater difference in immune activity at a ratio of 1:8. Therefore, VZV IgG is more actively involved in immune activity and yields statistically significant results in terms of the initial HB grade and mean ENoG values. However, because the sample size of the study population with RHS was limited to 105 individuals, further research is necessary.Furthermore, based on our results, there was also a correlation between VZV IgM and IgG and otologic symptoms. When VZV IgM is positive, symptoms of tinnitus and auditory hypersensitivity are relatively more common, whereas when VZV IgG is positive, symptoms of dizziness are more common. This suggests that an increase in VZV IgM and IgG, which act on the vestibulocochlear nerve, is more likely to result in otologic symptoms. However, it is difficult to explain why VZV IgM and IgG cause different otologic symptoms in the positive cases. Further molecular biological research can confirm whether VZV IgM and IgG specifically act on the cochlear and vestibular nerves, respectively, and this topic can be discussed further.In this study, we evaluated the effects of VZV IgM and IgG antibodies collected from the blood of patients with RHS on the severity of early facial paralysis, the prognosis of facial paralysis after treatment, and accompanying otologic symptoms. We found that the prognosis of facial paralysis was worse when each serological marker was positive, as confirmed by the HB grade and ENoG results for the four facial muscles. In addition, we found more cases of tinnitus and dizziness in the positive serological marker group than in the negative serological marker group. The results of this study confirmed a correlation between the immunological action of VZV in the body and damage to the facial and vestibulocochlear nerves, which has an effect on the severity of facial paralysis and its accompanying otologic symptoms. As a limitation of this study, when VZV IgM and IgG antibodies were tested at our hospital, the results were only negative or positive, not quantitative values. The higher the quantitative level of VZV IgM and IgG antibodies, the poorer the prognosis of facial paralysis and the more otologic symptoms may occur; thus, additional research is needed. Moreover, our institution conducted tests for VZV IgM and IgG antibodies once at the time of admission, but these results may vary depending on when the patient was infected with the varicella-zoster virus. In addition, VZV IgM and IgG antibodies in the body become negative over time after infection with VZV, which can affect the prognosis of RHS. It is considered a limitation that this study could not conduct such an analysis due to the retrospective nature of the study. In a future study, additional tests for VZV IgM and IgG antibodies need to be performed, not just one test at the time of admission.


The guideline-recommended tissue particle count of 10 was not achieved in 208 patients (82.2%) and the required tumour-bearing tissue particle count of 5 was not achieved in 133 patients (52.6%). Tissue particle count, tumour-infiltrated area and tumour cell count were only weakly correlated. Most cases featured an infiltrated area ≥ 4.5 mm2 . Cases with more tissue particles showed only a moderate increase in infiltrated area and tumour cells compared to cases with fewer particles. Biopsies are often used to determine predictive biomarkers, particularly Her2/neu and PD-L1. Diagnostic standards to ensure representative material have been suggested in guidelines to reduce false-negative predictions. However, the real-world practice seems to substantially deviate from recommended standards. To the best of our knowledge, this is the first systematic study describing the relationships between endoscopic tissue fragment number, actual infiltrated tumour area and carcinoma cell number. The data question the tissue particle number as a quality assessment parameter. We advocate histopathological reports indicating on which basis statements on therapy-relevant biomarkers were made. Digital pathology has the potential to objectively quantify the tissue for documentation, quality assessment and future clinical studies.Guidelines regulate how many (tumour-bearing) tissue particles should be sampled during gastric cancer biopsy to obtain representative results in predictive biomarker testing. Little is known about how well these guidelines are applied, how the number of tissue particles correlates with the actual tumour-infiltrated area and how many absolute tumour cells are captured. The study included endoscopic biopsies of untreated carcinomas of the upper gastrointestinal (GI)-tract during the 2016–2020 review period. Archival (H&E)-stained histological sections were digitised and the tumour areas were manually annotated. The tumour-bearing tissue area and absolute carcinoma cell count per case were determined by image analysis and compared with a reference primary surgical specimen. Biopsies from 253 patients were analysed. The following mean values were determined: (a) tumour tissue particle number: 6.5  = 3.33), (b) number of tumour-bearing tissue particles: 4.7 , (c) tumour-infiltrated area: 7.5 mm Gastric carcinomas can be divided into two anatomical regions: (a) proximal carcinoma of the cardia region, which may include adenocarcinomas of the gastroesophageal junction and (b) distal gastric carcinoma of the antrum region. The relevance of these tumour localisations are subject to significant geographic differences:in Eastern Europe, parts of Asia, South America: 80% distal (antrum)7.in Northern Europe and USA: 60% proximal (cardia/fundus)Endoscopically-obtained tumour biopsies confirm the histological tumour identity but are also used to determine therapy-relevant biomarkers. In the case of inoperable patients or metastases that are difficult to access, these biopsies are the only tumour cell source available. Currently, clinically relevant predictive biomarkers in upper gastrointestinal tract carcinomas are Her2/neu and PD-L1. Future indications for targeted therapy are likely to expand the biomarker portfolio, possibly including Claudin 18.2 and FGFR2http://www.krebsdaten.de/magenkrebs). The proportion of cardiac carcinomas has been increasing in recent years.In absolute numbers, an incidence of about 14,000 new cases per year is found in Germany . A German expert opinion on Her2/neu testing states more precisely that "…for reliable diagnosis of Her2/neu status the specific number of particles is less relevant than the quality of biopsies and the number of actual tumour-containing particles obtained" and calls for five tumour-containing particles from different tumour areas to avoid false-negative results due to the high intra-tumoral heterogeneity of Her2/neu11. Her2/neu stratification followed the known criteria of the TOGA trail. An adenocarcinoma of the upper GI tract is considered Her2/neu positive if (a) at least five contiguous carcinoma cells are Her2/neu positive on endoscopic biopsies or (b) at least 10% of the tumour is Her2/neu positive on surgical specimens12. PD-L1 is also a therapeutically relevant biomarker that is determined immunohistochemically on tumour tissue of carcinomas of the upper GI tract. The so-called combined positive score (CPS) is used for this purpose, which considers positivity on both tumour cells and certain mononuclear inflammatory cells (such as macrophages). As with Her2/neu, the endoscopically obtained tissue particles are often used for determination2.Studies have determined how many endoscopic tissue particles are required to measure these biomarkers with high sensitivityIn this study, we addressed the following questions: (A) how well are the requirements of the national S3 guideline are respected by gastroenterologists, oncologists and pathologists? (B) How well does the number of tissue particles correlate with the absolute area infiltrated by tumour and the absolute tumour cell count? Background: currently, there is no definition of what constitutes a “tumour-bearing biopsy particle”. Tumour-bearing particles could consist of only a few single tumour cells or be infiltrated entirely with tumour cells. The number of tumour-bearing particles would be the same, but the absolute area invaded by tumour cells and the actual tumour cell count would differ significantly in these two scenarios. With this premise, tumour area and absolute tumour cell count might be more objective and meaningful representative metrics than tumour-bearing particle count. (C) How many carcinoma cells are obtained by biopsies? (D) How many carcinoma cells make up a primarily operated adenocarcinoma of the oesophagus?To answer these questions, (H&E)-stained histological sections were digitised. The number of tissue particles was determined and tumour-bearing area and absolute carcinoma cell count per case were assessed by image analysis.To the best of our knowledge, this is the first such work on carcinomas of the oesophagus and stomach.We searched the pathology medical record system for all endoscopic tumour biopsies obtained from primary carcinomas of the oesophagus and stomach over a 6-year period 2016–2020). The biopsies were submitted by one university hospital , nine peripheral hospitals and five gastroenterologists . All cases in which the primary slides were not suitable for digitisation due to scratches or other artifacts were re-cut and re-stained from the original paraffin blocks (2 μm sections), with subsequent digitisation. Examples of histology and tissue particle numbers are shown in the supplementary data . QuPath is a commonly used open-source software for digital pathology image analysis2). For tumour cell detection, we initially tested two tools: a native cell detection tool from QuPath and a QuPath-implementation of StarDist algorithm14. The former provided visually better carcinoma cell detection  and allowed better flexibility concerning the parameter selection and was used for all further experiments. The main parameter change for cell detection tool defaults was minimum area of tumour nuclei, which was initially set at 25 µm2. Previously annotated tumour-bearing regions were processed by a cell detection tool to calculate the absolute tumour cell numbers , allowing the adequate separation of tumour cells from other cell populations in virtually all cases . The absolute tissue area interspersed by tumour cells per case could thus be precisely quantified mm. For tumA representative case of the tubular  differentiated adenocarcinoma of the oesophagus treated with primary surgery was selected. The specimen was routinely processed according to the institute standards and the tumour was completely submitted for paraffin embedding (n blocks = 29) The largest tumour dimension was 6.7 cm. One representative paraffin block was completely cut in 50 μm steps (thickness 2 µm) yielding 50 histological sections. Since an average tumour cell in this case had a diameter of 25–30 μm, the stepwise sequence of 50 μm ensured that a new tumour cell layer was present on each new H&E step section. The tumour area of the remaining paraffin blocks was used to calculate the absolute tumour cell numbers considering three dimensions of the respective tissue fragments. All H&E-stained step sections (29 + 50 = 79) were digitised and tumour cell number calculated using QuPath, as detailed above.Data were recorded in Microsoft Excel (Version 2016). Statistical analyses were performed using R statistical programming language (version 4.1.2) including the packages ggplot2 and patchwork. For statistical testing, Student's T-test was used. Alpha was set to 5% and corrected for the multiple comparisons problem with the Bonferroni method (α/[number of tested hypotheses]). For subgroup analyses, the respective subgroup was tested against the rest of the collective.Patients gave their written consent to usage of their tumor specimens. The objective of the project is primarily in the field of diagnostics and quality assurance, an approval was obtained from the University of Cologne Ethics Committee. An approval was obtained from the University of Cologne Ethics Committee (reference number: 20-1583 and 10-242).All authors confirm that methods used were carried out in accordance with relevant guidelines and regulations. The experimental protocols were approved by the licensing committees. We confirm that informed consent was obtained from all subjects and/or their legal guardians.In the considered period of 2016–2020, 253 patient cases with endoscopically obtained biopsies of primary carcinomas of the upper gastrointestinal tract were available. These included 230 oesophageal carcinomas, 19 gastric carcinomas and 4 carcinomas spanning both regions. Histologically, 171 (67.6%) were adenocarcinomas including 143 tubular-intestinal carcinomas, 9 poorly cohesive carcinomas including signet ring cell carcinoma, 16 well differentiated carcinomas and 3 tumours showing mucinous differentiation, while 77 were squamous cell carcinomas (30.4%) and 5 were other subtypes (2%). Most patients were male (76.7%) , the university hospital  or by general physician practices   = 3.33]  ; squamous cell carcinoma: 6.9 particles/per case   (p = 0.508).2 was achieved in 61.7% of cases  of ≥ 4.5 mmThe mean number of tumour-bearing tissue particles was 4.68 per case  Fig. B. Again,The required number of tumour-bearing particles, n ≥ 5, was not achieved in 133 of 253 cases (52.6%).Cases with n ≥ 10 particles and n ≥ 8 are enriched for n ≥ 5 tumour-bearing particles, 39/45, 86.7% and 68/78, 86.0%.On average, 13,492 carcinoma cells were detected per case . The cell number was only weakly correlated with the total tissue particle count (r = 0.20) and the tumour-bearing tissue particle count (r = 0.38) . The tiA was substantially correlated with the tumour cell number (r = 0.71). Tissue particle count (r = 0.28) and tumour-bearing particle count (r = 0.46) showed low correlations.The average tiA per case was 7.46 mm2 . The mean number of tumour cells per tumour-bearing particle was 3,033 .The mean area per tissue particle was 1.29 mm2 ; squamous cell carcinoma mean tiA = 8.95 mm2  (p = 0.06191).Subgroup-analysis by histotype showed a small but not significant difference: adenocarcinoma mean tiA = 6.85 mm2 (p < 0.001) and 10.54 vs. 6.8 mm2 (p = 0.001). However, cut-off analysis showed that cases with fewer particles had a relatively large tiA and tcN . Subgroup-analyses of submitting institutions and histotypes did not show any relevant differences. Cut-off analysis showed that 17.8% of cases (45/253) contained ≥ 10 tissue particles and 85.8% contained ≥ 4 particles (217/253). A tiA ≥ 4.5 mm2 was achieved by 61.7% of all cases (156/253).In the present study, we measured the tissue particle number, tiA and tumour cell number (tcN) in a cohort of n = 253 tumour-biopsies of the upper GI-tract by digital image-analysis. Both tiA and tcN were only weakly correlated to the number tissue particles and the number of tumour-bearing particles. The particles showed a broad spectrum of different sizes , < 10 tissue particles were taken, while < 5 tumour-bearing particles were detected in 133 patients (52.6%). Thus, the reasons are speculative: (a) ignorance of the S3 guideline, (b) a fear of adverse-effects, especially bleeding and (c) estimation to have identified enough tumour-bearing tissue. It must be noted that this is a study of a metropolitan region in Western Germany and can in no way claim to be representative of the whole country. A multicentre study of different regions and federal states would be necessary and, in our opinion, would also be of interest.The employed digital-image analysis to quantify tiA and tcN is easy to apply. The QuPath software is open-source and free of charge. It is compatible with all common histological whole-slide scanners. We suggest that the quantitation of tissue particle numbers and tumour cell content should be included in studies of histological biomarkers to verify representativeness. Still, the procedure has yet to be optimised and validated. However, we have deliberately chosen the freely available software QuPath, since all working groups worldwide are given the opportunity to carry out similar investigations.Overall, 83% of the investigated adenocarcinomas showed tubular  tumour differentiation, while 4% corresponded to poorly cohesive carcinoma including signet ring cell carcinoma. Different subtypes of carcinoma are found in different parts of the world due to the varying risk constellations. In the East-Asian region, the proportion of Helicobacter-related and distally localised gastric carcinomas is much higher than in Western Europe, where this tumour type has been declining for years. This is also accompanied by the high proportion of tubular-intestinal differentiated tumours in Western Europe. This explains the high proportion of adenocarcinomas of the oesophagus and junction-tumours in our collective.There were no significant differences between the submitting institutions if grouped into university hospital, primary care hospitals and general physicians. Still, there was obvious variation between the cases  with no or minimally acceptable levels of contamination through other cells. However, individual determination of the optimal software settings for tumour cell nuclei detection might be necessary, which in our case was between 25 and 40 μm2. Thus, the biopsy in this case accounted for 0.01%.During the course of manuscript development, the question arose how many tumour cells constitute an adenocarcinoma of the oesophagus (EAC) not treated with neoadjuvant therapy. We were able to show that a 6.7 cm large EAC consists of just over 100 million tumour cells . We also received biopsy-material from the same patient preoperatively. Three tissue particles contained a total of 11,009 carcinoma cells, which infiltrated an area of 7.67 mmThis is the first work to systematically capture the relationships between endoscopically obtained tissue particle number, actual infiltrated tumour area and absolute carcinoma cell count. By considering absolute carcinoma cell count or tumour area, future clinical studies will be more standardised and comparable. We advocate that histopathological reports should indicate on which basis (tissue particle number or carcinoma cell number or tumour area) statements on therapy-relevant biomarkers are made. In the case of the negative detection of a predictive biomarker, it is highly relevant whether this statement is based on sufficiently representative tumour material.Supplementary Legends.Supplementary Figure 1.Supplementary Figure 2.Supplementary Table 1.Supplementary Table 2.


Cancer of unknown primary (CUP) encloses a group of heterogeneous tumours, the primary sites for which cannot be identified at the time of diagnosis, despite extensive investigations. CUP has always posed major challenges both in its diagnosis and management, leading to the hypothesis that it is rather a distinct entity with specific genetic and phenotypic aberrations, considering the regression or dormancy of the primary tumour; the development of early, uncommon systemic metastases; and the resistance to therapy. Patients with CUP account for 1–3% of all human malignancies and can be categorised into two prognostic subsets according to their clinicopathologic characteristics at presentation. The diagnosis of CUP mainly depends on the standard evaluation comprising a thorough medical history; complete physical examination; histopathologic morphology and algorithmic immunohistochemistry assessment; and CT scan of the chest, abdomen, and pelvis. However, physicians and patients do not fare well with these criteria and often perform additional time-consuming evaluations to identify the primary tumour site to guide treatment decisions. The development of molecularly guided diagnostic strategies has emerged to complement traditional procedures but has been disappointing thus far. In this review, we present the latest data on CUP regarding the biology, molecular profiling, classification, diagnostic workup, and treatment. Cancer of unknown primary origin (CUP) is a diverse category of cancers with varying clinical and histological characteristics for which no original tumour site has been identified despite a thorough diagnostic workup . It is tRecently, a consensus was reached on the first diagnostic layer for CUP. History and physical examination; full blood count; along with serum markers, a CT scan of the chest/abdomen/pelvis, and a biopsy of the most accessible lesion, followed by immunohistochemical testing should be the starting point. On the other hand, the symptom-guided magnetic resonance imaging (MRI) or ultrasound, a positron emission tomography/computerised tomography (PET/CT) scan, targeted gene panels, immunohistochemical markers, and whole genome sequencing remain debatable . With reFrom the epidemiological perspective, around 8600 new CUPs are diagnosed each year in the United Kingdom, making this the fifteenth most prevalent cancer . Since tAny degree of smoking raises the chance of CUP with respiratory system metastases to 4.9%. In a study, smoking was strongly linked to an increased risk of CUP, with a relative risk of 3.66% for ongoing, heavy smokers (>25 cigarettes/day) compared to never smokers (standardised for other risk factors), and a relative risk of 5.12% for patients with CUP who died within less 12 months since the diagnosis . Other rGenerally, the ongoing improved knowledge of the biology of several cancers has enabled a more-accurate classification, diagnosis, and prognosis, as well as providing guidance in the tailoring of specific therapies. However, in CUP, the data are not very mature. The early dissemination of tumour cells implies subsequent independent progression of the primary tumour and metastases, under the selection pressure of the immune system. The clinical observation of the high systemic relapse rate in CUP patients with localised disease treated with curative intention with surgery and/or radiotherapy supports that model. Still, molecular platforms represent a key component of the diagnostic work-up and clinical management . Many tumours have an abnormal number of chromosomes, a condition known as aneuploidy. Chromosomal instability (CIN), a mechanism of continual chromosomal missegregation, is a common cause of aneuploidy . Oncogenn = 92), followed by intermediate and low AS  and high AS . The high-intermediate AS groups lacked an enriched genetic alteration, and the presence of a TP53 or KRAS mutation did not correlate with a high AS. This differs from what was previously known about these mutations in aneuploid cancers. The researchers concluded that CUP patients present individual gene alterations implicated in immune evasion and resistance to ICI, but further clinical investigations are needed to clarify the interplay between CIN, point mutations, and the immune system. CIN is a known driver of early dissemination and aggressive behaviour of CUP. In a recently published study, researchers investigated the genomic information of CUP samples analysed using a hybridisation-capture-based next-generation sequencing (NGS) assay in 410 cancer-associated genes . CUP samPI3K, Ras, p53, PTEN, Rb, and p16INK4a are among the oncogenes and tumour suppressors that are often mutated in cancerous cells  fluoro-2-deoxy-d-glucose (FDG) PET/CT, but still the detection rate is around 40% [68Ga-DOTA-NOC receptor PET/CT is recommended for the identification of primary neuroendocrine tumours, along with their metastases [Image-assisted technologies has revolutionised the diagnosis of CUP. CT and conventional MRI have both been used to locate lesions, considering the clinical manifestation of CUP. The diagnostic accuracy of CT scans is around 55% (36–74%), mainly in pancreatic, colorectal, and lung cancer, while MRI has a sensitivity of 70% in detecting primary breast cancers . Howeveround 40% . The mostastases . MammogrEndoscopy should be directed towards investigating specific symptoms and signs or when specific histopathological findings are available. Fiberoptic bronchoscopy is reasonable for patients with respiratory symptoms and/or expression of CK7 and TTF1, whereas colonoscopy should be requested for those with abdominal symptoms or occult blood in the stool and/or expression of CK7, CK20, and CDX2. The sensitivity and specificity of the endoscopies are generally low.In almost 70% of CUP patients, more than one marker can be concomitantly elevated in a non-specific way. Routine request of cancer antigen 125 (CA 125), cancer antigen 15.3 (CA 15-3), carbohydrate antigen 19-9 (CA 19-9), and carcinoembryonic antigen (CEA) is not recommended due to lack of prognostic and/or predictive value . HoweverVIM is an early detection biomarker [Improvements in nucleic acid sequencing technologies have enabled the detection of low quantities of tumour genetic material within the blood and show the potential to be both sensitive and specific to an individual’s tumour. These blood-based biomarkers include cfDNA, tumour microRNAs (miRNAs), and platelet-derived tumour mRNA, as well as analysis of DNA, RNAs, and protein expression from individual circulating tumour cells. Within this context, the use of liquid biopsies reduces the need for intrusive diagnostic biopsies and provides enough material to perform the diagnostic procedures. For instance, even though the presence of aberrant hypermethylation of tumour-suppressor genes in serum DNA has been detectable before the millennium, more sensitive and quantitative techniques for analysis of DNA methylation are required to expedite its incorporation in the clinical setting . In diffiomarker ,63. Overiomarker . Molecular profiling technologies including microarray-based gene expression profiling, reverse transcriptase polymerase chain reaction, RNA sequencing, somatic gene mutation profiling with NGS, and DNA methylation profiling were used to define the primary culprit among patients with CUP. However, the implementation of tissue-of-origin classifiers in CUP is limited due to the absence of primary tumour. Some studies were conducted to validate predictions of the primary origin, on the basis of autopsy data, latent primary emergence, or IHC. Gene expression profiling has been directly compared to IHC, within known metastatic tumour types. Accuracy of gene expression profiling was 89%, compared with 83% for IHC when only one round of IHC determined the diagnosis, whereas in poorly differentiated cancers, such as CUP, the percentages were 83% and 67%, respectively . NeverthPlexinB2 (PlxnB2) is a semaphorin receptor implicated in the regulation of cancer cell proliferation, invasiveness, and metastatic spreading ,69,70,71Traditionally, CUP patients who are classified into one of the favourable subsets are treated according to their corresponding primary guidelines for metastatic disease. CUP patients with poorly differentiated carcinoma with midline distribution  should be managed like poor prognosis germ cell tumours with platinum-based combination chemotherapy. More than 50% response has been reported, with 15–25% complete responders and 10–15% long-term disease-free survivors. Women with papillary adenocarcinoma of the peritoneal cavity are optimally treated as FIGO stage III ovarian cancer. The recommended strategy includes aggressive surgical cytoreduction, followed by platinum-based postoperative chemotherapy. The median response rate is 80%, whilst 30–40% of the patients are complete responders. Similarly to FIGO stage III ovarian cancer patients, the median survival is 36 months ,77. For p = 0.069) compared with empiric therapy [The treatment of patients with unfavourable CUP subsets is usually empirical chemotherapy, consisting of either taxanes or platinum-based regimens, on the basis of randomised trials showing dismal survival improvements . The bio therapy .CUPISCO NCT03498521) is an ongoing prospective, phase II, randomised study designed to elucidate the potential benefit of treatment following genomic profiling, as compared to standard chemotherapy of CUP patients . The stu21 is an Within this context, immunotherapies have the potential to improve outcomes in this population, due to the PD-L1 expression and high tumour mutational burden in 22.5% and 11.8% of CUP patients, respectively . OverallInitially, some anecdotal cases showed clinical activity of immune checkpoint inhibitors in CUP, irrespective of the presumed tissue of origin . ThroughCUP is a heterogeneous group of metastatic tumours with a distinct natural history that mainly depends on clinicopathological criteria. While favourable groups are treated according to their corresponding primary tumour, unfavourable groups are treated with empirical chemotherapy, usually having a dismal prognosis. Several tissue-of-origin classifiers have been developed, collecting evidence that supported their translational potential in the clinical management of CUP patients. Several studies focused on genomic analysis of ctDNA and included some CUP cases among other tumour types, showing high sensitivity rates in the identification of oncogenic and actionable alterations in CUP. Small non-coding RNAs and epigenetic modifications are particularly appealing. Such biomarkers could potentially endorse the access to more specific therapies. The knowledge of the primary site remains fundamental because specific driver mutations could be predictive of responses in some tumour types but not in others. Immunotherapy is emerging as a potentially winning therapeutic strategy in several cancer types. Reasonably, it has gained interest even in the subset of CUP patients. Liquid biopsy could help in unveiling druggable alterations using a non-invasive approach. Therefore, molecular diagnostics, combined with genetic profiling, might become the standard of care for future CUP management. There is a significant limitation of the research on therapeutic strategies in CUP; this is the non-inclusion of many patients within the expanded favourable CUP subsets in the randomised trials who may be treated by their oncologists according to a potential primary tumour. New comprehensive clinical trial designs have been proposed to overcome the methodological issues encountered in CUP research implementing the latest diagnostics and therapeutic advances of CUP research.


As an effective tool for minimising costly trial-and-error design methods, it expedites translation into clinical practice to improve therapeutic outcomes and limit adverse effects for cancer patients.One of the main challenges in improving the efficacy of conventional chemotherapeutic drugs is that they do not reach the cancer cells at sufficiently high doses while at the same time affecting healthy tissue and causing significant side effects and suffering in cancer patients. To overcome this deficiency, magnetic nanoparticles as transporter systems have emerged as a promising approach to achieve more specific tumour targeting. Drug-loaded magnetic nanoparticles can be directed to the target tissue by applying an external magnetic field. However, the magnetic forces exerted on the nanoparticles fall off rapidly with distance, making the tumour targeting challenging, even more so in the presence of flowing blood or interstitial fluid. We therefore present a computational model of the capturing of magnetic nanoparticles in a test setup: our model includes the flow around the tumour, the magnetic forces that guide the nanoparticles, and the transport within the tumour. We show how a model for the transport of magnetic nanoparticles in an external magnetic field can be integrated with a multiphase tumour model based on the theory of porous media. Our approach based on the underlying physical mechanisms can provide crucial insights into mechanisms that cannot be studied conclusively in experimental research alone. Such a computational model enables an efficient and systematic exploration of the nanoparticle design space, first in a controlled test setup and then in more complex  While chemotherapy is one of the most common treatments for cancer, it comes with a significant drawback: the administered drugs do not reach the cancer cells at sufficiently high doses while at the same time attacking healthy tissues. This causes significant side effects and suffering in cancer patients. Magnetic nanoparticles have emerged as a promising approach to overcome this deficiency by achieving more specific tumour targeting. Chemical functionalisation of the nanoparticle surface allows for attaching a chemotherapeutic agent, and an external magnetic field directs the nanoparticles to the target tissue , the particles form a single magnetic domain. Therefore, they become highly magnetic in the presence of an external magnetic field but revert to a non-magnetic state when the field is removed, called superparamagnetism  in a flow chamber. Among the various types of magnetic nanoparticles, SPIONs are the most extensively investigated because of their biocompatibility  challenges: The computational setup is depicted in Fig. We decompose this system into two distinct but fully coupled regions, which we model as a multiphase porous medium: the region of the free fluid The magnetic nanoparticles are dispersed in the fluid and transported by its flow. Additionally, a cylindrical magnet is positioned below the flow chamber and exerts a magnetic force on the nanoparticles. Many publications  or the culture medium (in the flow chamber). The ECM is a meshlike structure with voids where the cells are attached or migrate and where the fluid flows. We model the tumour cells as a highly viscous fluid (rather than a solid), as most tumour-growth models do  and the interstitial fluid , respectively. As an adequate assumption in this case, we assume that the ECM does not deform in this study, i.e., the porosity is constant in space and time The voids in the ECM constitute the pore space, and the ratio of the volume of the pore space to the total volume is the porosity The fluid phases share the pore space of the ECM, and the fraction occupied by each fluid phase is the saturation k is the intrinsic permeability of the ECM. The permeability describes how easily a specific fluid flows through the ECM. Since the culture medium and the tumour cells share the same pore space, they interact: the flow of one phase impedes the flow of the other. The relative permeability thus is a function of the corresponding saturation The two fluid phases are governed by the mass balance equationIn this contribution, we employ our previously developed tumour-growth model  nanoparticles are subjected to the external magnetic field To evaluate Eq. d Olbert  and Cacid Olbert  derived The magnetic field Numerical Recipes  an an13) anIn our case, the convective terms dominate the Smoluchowski advection–diffusion equation, which causes numerical instabilities when using the standard Galerkin procedure. To overcome this issue, we use the streamline-upwind Petrov–Galerkin (SUPG) method \document in Eqs. , respectTable -1.We first analyse the fluid flow around and through the tumour spheroid for the four different configurations. Figure We employ a one-domain approach due to its simplicity while retaining essential physics. If more complex flow patterns around the tumour spheroid are of interest, such as transitional flow with vortices or even turbulent flow, one can solve the Navier–Stokes equations in t et al. , Coroneot et al.  or Ager t et al. .We now investigate the distribution of the nanoparticles for the four different configurations. For all configurations, the nanoparticles are injected in the upper half of the inflow boundary with a mass fraction of The resulting magnetic flux density Figure The results in Fig. We here only consider the force exerted by the external magnetic field. However, the magnetised nanoparticles also exert forces on each other when they are close. According to Furlani and Ng , Keavenyz-axis) as we do here. Our approach enables arbitrary length and diameter of the magnet and an arbitrary position of the magnet, thus allowing for a more realistic and flexible simulation of the experimental setup.We here employ an analytical expression for the magnetic force of a cylindrical magnet of finite length. Furlani and Ng , 2007 anin vivo scenarios and clinical practice. Accordingly, Stillman et al. (in silco models should include. The approach we present here is readily extendable to the vascular version of our tumour-growth model, which includes the vasculature, angiogenesis, and the surrounding host tissue (Kremheller et al. Finally, we only study a simplified model in the experimental test setup and the computational model: we consider the tumour spheroid with the ECM and the flow in the flow chamber, but such a setup does not include the blood vessels and the surrounding tissue, as we did in Wirthl et al.  for a din et al.  argued tin vitro and in silico, forms the basis for further studies in more complex scenarios, e.g., in a vascular in vivo model.Motivated by a recent experimental test setup (Behr et al. in silico model will enable a fast and systematic exploration of the nanoparticle design space, which is impossible in experimental research alone: this reduces the number of experiments required to the most promising candidates, bypassing costly and time-consuming trial-and-error design methods (Karolak et al. Developing a comprehensive