In vitro studies of CS/HA and CS/HA/Si-HPMC hydrogels encapsulated in chondrocytes have shown that the proper amount of Si-HPMC increases the proliferation and deposition of the cartilage extracellular matrix. The regeneration rate of the CS/HA/Si-HPMC (3%) hydrogel reached about 79.5% at 21 days for long retention periods, indicating relatively good in vivo bone regeneration. These CS/HA/Si-HPMC hydrogels are promising candidates for tissue compatibility injectable scaffolds. The data provide proof of the principle that the resulting hydrogel has an excellent ability to repair joint cartilage using a tissue-engineered approach.An injectable hydrogel based on CS/HA/Si-HPMC composites was developed.The CS/HA/Si-HPMC hydrogel displays the tunable rheological with mechanical properties.The CS/HA/Si-HPMC hydrogel is highly porous with high swelling and degradation ratio.Increasing concentration of Si-HPMC promote an organized network in CS/HA/Si-HPMC hydrogels.Injectable CS/HA/Si-HPMC hydrogels have a high potential for cartilage tissue engineering.Cartilage damage continues to pose a threat to humans, but no treatment is currently available to fully restore cartilage function. In this study, a new class of composite hydrogels derived from water-soluble chitosan (CS)/hyaluronic acid (HA) and silanized-hydroxypropyl methylcellulose (Si-HPMC) (CS/HA/Si-HPMC) has been synthesized and tested as injectable hydrogels for cartilage tissue engineering when combined without the addition of a chemical crosslinking agent. Mechanical studies of CS/HA and CS/HA/Si-HPMC hydrogels showed that as Si-HPMC content increased, swelling rate and rheological properties were higher, compressive strength decreased and degradation was faster. Our results demonstrate that the CS and HA-based hydrogel scaffolds, especially the ones with 3.0% (w/v) Si-HPMC and 2.5/4.0% (w/v) CS/HA, have suitable physical performance and bioactive properties, thus provide a potential opportunity to be used for cartilage tissue engineering.   Cartilage is a highly specialized connective tissue that has restricted capacity for self-healing because of its compositional and structural nature, a condition that contributes to regeneration after injury  , film-forming capabilities, etc  was obtained from shrimps. HA sodium, acetic acid, sodium hydroxide, sodium ascorbate were purchased from Sigma-Aldrich. 3-Glycidoxypropyl trimethoxysilane  was purchased from Sigma-Aldrich. All other reagents are commercially available and used as received.A 0.5 g/L CS solution was prepared for the hydrogel preparation by dissolving the CS powder in 2% by volume acetic acid and stirring until the powder was completely dissolved. The beaker was covered and sealed before mixing for 1 h to achieve complete dissolution at room temperature. A stock gel of 2.5% CS in 0.1 M acetic acid was prepared and by adding 1 M NaOH dropwise, the pH was changed from 6.0 to 8.0. In the CS solution, sodium ascorbate  was applied, then the HA (5%) mixture and the CS sodium ascorbate solution were vigorously mixed with a vortex mixer. The formulation was left overnight at 4 °C resulting in complete hydration of the polymer  was dissolved for 48 h at 0.2 M NaOH with regular stirring. The solution was then steam sterilized . The solution was finally mixed with a 0.5 volume buffer of 0.26 M HEPES to allow the formation of a crosslinked hydrogel   operating at a voltage of 10 kV acceleration. The hydrogels were pre-frozen at −80 °C, then lyophilized for 24 h, cut with a knife, glued to the conductive plate, and spray gold plated.−1 within the 4000–400 cm−1 range.Using an FTIR spectrometer , the functional groups of the composite hydrogels were characterized. To create a homogeneous fine powder, the dried samples were ground with KBr and then pressed into a pellet. All samples were analyzed as a powder, thoroughly blended with KBr powder, compressed into a 1 mm clear pellet, and analyzed at room temperature in an attenuated total reflection mode with a resolution of 4 cmθ scanned angular range from 10 to 80° were collected using PANalytical X’Pert3 powder  fitted with a CuKa radiation source, actuated (40 kV and 40 mA) at a scanning speed of 6°/min. The sample was put in liquid nitrogen for 4 h before being subjected to XRD analysis and ground into a fine powder with a mortar and pestle.XRD patterns of composite hydrogel arrays in the 2The thermal stability and thermal decomposition properties of the hydrogels were evaluated by thermal gravimetric analysis . The hydrogels were instantly frozen at −80 °C, followed by lyophilization, and then ground into powder. The CS/HA and CS/HA/Si-HPMC hydrogel were heated in an aluminum pan at a heating rate of 10 °C/min from 50 to 450 °C under a nitrogen atmosphere.A tensile tester  was used to assess the tensile efficiency of the CS/HA and CS/HA/Si-HPMC composite hydrogels. A cylindrical mold with a diameter of 13 mm was used to create the hydrogel. The developed hydrogel was cut to a thickness of 1 cm and put in a Bose Electro Force . By dividing the area under the stress-strain curves, durability was measured. For each sample, the mechanical properties are based on the average value of 5 samples.All rheological experiments were performed with a rheometer  using parallel plates  at 37 °C in oscillating mode. At a frequency of 1 Hz and an elongation of 1%, the calculation is continued until the storage modulus (G′) reaches a plateau value. In a frequency sweep, samples were kept for 60 min between parallel plates, with a gap of 1 mm. At a constant voltage of 1%, the frequency sweep was carried out in the range of 0.1–100 Hz. The amplitude sweep at 1 Hz was between 0.1% and 500% elongation.The hydrogels were shaped on 24-well plates and were permitted for 24 h to crosslink. Universal Testing Systems  were used to measure the compressive modulus of hydrogels of known dimensions (height and diameter) with a 1 g cylinder load moving at 0.5 mm/s to the force (Pa) to expand according to the stretch. Since the initial surface of the cylindrical hydrogel sample was not completely smooth, between 5% and 20% elongation, the compressive modulus was measured as the curve slope.tW is the swollen state weight of the sample at equilibrium and W0 is the dry state weight of the sample. Three samples were tested using Strong swelling is one of the most beneficial properties of hydrogels. The rate of swelling of the composite CS/HA and CS/HA/Si-HPMC hydrogels was measured by the ambient temperature weighing method. The swelling behavior of the lyophilized hydrogels was observed for 3 h. In detail, pre-weighed dry CS/HA and CS/HA/Si-HPMC composite hydrogels were submerged in an aqueous solution until swelling to equilibrium. Swollen hydrogels were removed and immediately weighted with a microbalance after filter paper absorbed excess water that remained on the surfaces.in vitro degradation studies of the composite CS/HA and CS/HA/Si-HPMC hydrogels were conducted. First, 1.0 mL of each sample was put into a tube and incubated for 3 h at 37 °C to complete the gelation. Then 6.0 mL of PBS (pH∼7.4) containing the enzyme alpha-amylase was applied to the tube. Under shaking conditions (37 °C), the sample solution was maintained for another period (0–200 h). 1 mL of sample solution was taken from the tube at each interval and balanced with a fresh medium immediately. A ninhydrin assay has determined the existence of free amino acids. Finally, the solutions were measured by a Lambda 35 UV-Vis spectrophotometer  for their absorbance at 570 nm. Afterward, the samples were lyophilized and weighed (tW). The percent weight loss (W%) was used to calculate the hydrogel degradation under the following To assess weight loss due to degradation, All the experiments were performed in triplicate.2 in Dulbecco’s Modified Eagle’s Medium . In a 96 well plate containing DMEM under controlled environmental conditions (temperature 37 °C and 5% CO2 concentration), cells (1 × 106 cells/mL) were grown individually for 24 h. Then, the supernatant was replaced by 100 μL of the CS, HA, Si-HPMC, CS/HA, and CS/HA/Si-HPMC hydrogels and the CS/HA/Si-HPMC hydrogels at different concentrations (50 μg/mL at 300 µg/mL) with the plate were further incubated for 24 h under the same conditions. The supernatant was extracted and dimethyl sulfoxide (DMSO) was homogenized with the samples using a glass tissue grinder  to dissolve the crystals of formazan. The cells were incubated again for 4 h, and with 100 μL of DMSO, the formation of formazan violet was resolved. A serum-free medium acted as a monitor in this research. On a multi-well plate reader , the absorbance was read at 570 nm. As positive and negative cytotoxicity monitors, respectively, DMSO and supplemented culture medium were used. Briefly, the 105 cells were added into a 96 well plate directly to 100 μL of each hydrogel formulation. The kit was grown for 24 and 48 h in a 100 μL supplemented DMEM medium. The material was then washed in each well with PBS. After 24 and 48 h in culture, all of the medium in the wells was removed. The samples were then observed and imaged with an optical microscope.Analysis of cytotoxicity activity with 3--2,5-diphenyl-2H-tetrazolium bromide . L929 fibroblast cells  were adopted and cultured with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin  at 37 °C and 5% COn = 3) by isoflurane inhalation. To confirm the safety of the fabricated hydrogels with a 3 mm height and 6 mm diameter size was implanted into the subcutaneous parts of the Sprague–Dawley rat. The CS/HA/Si-HPMC composite hydrogel with 4 × 8 mm2 in size was implanted into the muscle pouch. After 2 and 3 weeks of implantation, three rats were sacrificed every time, and the hydrogel samples were harvested. The specimens were frozen with CryoMatrix in liquid nitrogen. The three specimens were fixed with 4% formaldehyde for 4 days, then dehydrated in 50%, 75%, and 100% ethanol and embedded with paraffin. The specimens were cut into slices of 5 μm in thickness and stained with H&E and Masson’s trichrome for observation by light microscopy .All animals were treated under the Medical Animal Care Guidelines of the Medical College of Shanghai Jiaotong University. Under anesthesia , each hydrogel (0.1 mL) loaded with 1 μg hydrogels were subcutaneously injected into the dorsal side of the mice using a 29-gauge needle. The surgery has proceeded under general anesthesia, and all attempts were made to minimize animal suffering. To evaluate the biocompatibility, control hydrogels without cells were transplanted into the back subcutaneous tissue of mice anesthetized  were randomly divided into two groups: CS/HA and CS/HA/Si-HPMC (n = 3 for each group). The cartilage was exposed  injury was created by using a 1 mm drill. Encapsulation of cells in the hydrogels method and injected in the defect area. 3 weeks after implantation, the implanted hydrogels were removed, and then fixed in 4% PFA, embedded in paraffin, and sectioned transversely into 14 μm-thickness sections. The prepared decalcified tissues were dehydrated by treating a series of ethanol molded in a paraffin block. After incubation for different periods up to 3 weeks, the cell-seeded gels were washed at room temperature with PBS and fixed for two days with 10% neutral buffered formalin. The chondrocyte-gel constructs were cultured in a chondrogenic medium for 3 weeks for in vitro cartilage regeneration and subcutaneously implanted into nude mice for further 5 or 7 weeks. All in vitro and in vivo samples (n = 3) were harvested for cartilage regeneration evaluation. The sections were stained with Safranin-O for cartilage matrix observation. Histology analysis was carried out using the areas positively stained by Safranin-O in the defects and was scored. Masson’s trichrome and hematoxylin/eosin (HE) staining were also performed for reference. The parts were eventually visualized  using an optical microscope. All reagents were purchased from Sigma-Aldrich except for hematoxylin which was obtained from Fisher Scientific, Hampton, USA.p-value was less than .05, was considered statistically significant.The mean ± standard deviation (SD) of all data was presented. By unidirectional analysis of variance (ANOVA), statistical variation was analyzed. A difference between the mean values of each category, if the To obtain the information on the microscopic structure, the hydrogels were lyophilized for morphological observation by scanning electron microscopy (SEM). The scaffolds were highly porous with a network of interconnected pores produced by freezing and lyophilization in the solvent , 1379 (amide III), and 1636 cm−1 (amide), indicating the presence of HA in the composite hydrogel. The slight shift in amide bonds can be attributed to interactions between polysaccharides, confirming complexation. Hydroxyl groups (O–H) and hydrogen bonds occurring in uncrosslinked HA and CS/HA hydrogel, respectively, are the large bands that appear at 3275 cm−1 and 3269 cm−1  and the composite hydrogel CS/HA/Si-HPMC is shown in −1 suggested the absence of an unreacted carbonyl group. The vibration of hydroxyl ions in Si-HPMC reflects the peak observed at 603 cm−1. Besides, two other bands were identified in each spectrum, 1416 cm−1 and 1319 cm−1 for Si-HPMC, which coincide with the symmetrical stretching of the carboxylate of HA and CS C–N, respectively. Due to the ionic interaction between the negatively charged HA and the positively charged CS amino group, the lack of the characteristic absorbance peak at 1172 cm−1 suggested that the CS/HA hydrogel complex had occurred. All the characteristic peaks moved between Si-HPMC and the CS/HA hydrogel composites in the IR spectra of Si-HPMC suggested that the chemical reaction took place , which is the behavior of the material and modulus of loss (G′′), indicative of viscous behavior. All groups showed that G′ was much higher than G′ without a trace of interception when the oscillating shear stress was set at 5% and the angular frequency increased from 0.1 to 100 rad/s , indicatin vitro and in vivo in an aqueous medium.The structures of the CS and HA copolymers, the hydrogels synthesized have a high-water retention capacity. There are amine and hydroxyl groups in the structure of CS and HA, which have a great ability to create hydrogen bonds. Amino groups are expected to be efficiently incorporated into the polymer network, which can increase network density in hybrid materials. The swelling ratio of the CS/HA/Si-HPMC hydrogel composite is shown in in vitro degradation test was performed and the results were shown in To examine the degradation behavior of hydrogels in a physiological state, an in vitro  staining, with the inflammatory cell nuclei stained purple and normal on fabric is pink in color  in PBS has been investigated in the present study and the findings are shown in in vivo bone regeneration. After 21 to 35 days of regeneration, bone regeneration rates for all hydrogels were above 50%. In this respect, once bone healing is complete, the prepared hydrogels can provide preserved mechanical stability and can then be progressively replaced spontaneously by new regenerative bone tissue. Although this study showed that the amount of bone regeneration of control sites was higher than nanocomposites hydrogels, the CS/HA/Si-HPMC hydrogel also showed reasonable bone in-growth and can be a good candidate to replace natural bone regeneration and to maintain space for a longer period of time. Our studies suggest that the combination of growth factors has been reported to induce rapid and successful bone-tissue regeneration. These CS/HA/Si-HPMC hydrogels are promising candidates for tissue compatibility injectable scaffolds. Based on these results, CS/HA/Si-HPMC hydrogels can provide an excellent environment for the growth and maintenance of chondrocytes. The data provide proof of the principle that the resulting hydrogel has an excellent ability to repair joint cartilage using a tissue-engineered approach. However, we could not rule out the integration between the newly formed cartilages, which merits further investigation. To understand the initial response and long-term performance of the hydrogel for cartilage regeneration, to understand in-depth the degradation of the hydrogel in vivo, and to provide quantitative biochemical estimates and thorough functional analysis of the regenerated tissues, we propose future studies in small and large animals.The In vivo quantitative biochemical evaluations of treated CS/HA and CS/HA/Si-HPMC samples were done after treatment of 35 days by using specific GAG, DNA, collagen, fibronectin, elastin, and TGF β, β1, β2 kit experimental protocols  Si-HPMC, exhibited the best comprehensive properties for bone regeneration applications. The use of CS/HA/Si-HPMC hydrogels in subcutaneous or nude mice in conjunction with chondrocytes has shown their possible use for cartilage tissue engineering. Therefore, we believe that this injectable CS/HA hydrogel loaded with Si-HPMC is a promising tissue engineering strategy and has great potential for the clinical repair of cartilage tissue.In this study, a novel water-soluble CS/HA with silanized-hydroxypropyl methylcellulose (Si-HPMC) injectable hydrogel composite was designed for tissue engineering of cartilage. Si-HPMC was homogeneously distributed into CS/HA hydrogel system and had beneficial effects on improving mechanical properties, stabilizing the network of hydrogels, and slowing down weight loss at the expense of reducing gelation time and swelling ratio. The amount of Si-HPMC(3.0%) in the CS/HA hydrogel affected the surface morphology and the pore size of the composite scaffolds. The physicochemical properties of the hydrogel network morphology, surface properties, swelling rate, degradation behavior, and mechanical, rheological, and compressive properties can be manipulated by varying the weight ratio and concentration of the CS/HA hydrogel and Si-HPMC. Besides, L929 cells were viable and proliferated well on the CS/HA/Si-HPMC hydrogel during the period of


For many tumours there is a lack of randomised data from which we can guide systemic treatments. Although gene expression profiling along with proteomics has led to advances in diagnosis, classification and prognosis, our ability to target many cancers has been further limited due to a lack of therapeutic options. The use of patient-derived xenograft (PDX) models in the setting of a rare malignancy is discussed here by Kamili et al, with the successful establishment of new model systems. This generates a demand for improving preclinical modelling. The rapidly evolving field of targeted personalised therapy is the future of oncological practice and sometimes this cannot be evaluated through traditional research methodology, such as randomised control trials (RCTs).4 Chemotherapy has often been shown to be only minimally beneficial to overall survival, and is often ineffective with intolerable side effects—though one could argue that this is the case with all treatments. The hidden costs of managing chemotherapy toxicities, with repeated admissions and discussions about side effects, are likely to be substantial. Targeted monoclonal antibodies, immune checkpoint inhibitors and CAR T-cell therapies have shown promising advances in individualised cancer treatment, however, only a few are available for standard clinical practice.4 In Phase 1 clinical trials, both response rate and progression-free survival were greatly improved with personalised oncology therapy by using biomarker selection strategies compared with those undergoing generic treatment.5 Currently, developments in the detection of cancer drug targets, comprehensive molecular profiling and personalised combined treatment regimens are all contributing to increasing availability of personalised oncology to a wider range of patients.6Despite the variability and heterogeneity of cancer types, most treatments remain ‘generic’ and usually involve chemotherapy as the mainstay.7 Currently, cell-line xenografts are the standard for preclinical research, able to create a tumour microenvironment.8 More often than not however, they do not accurately reflect the true behaviour of the host tumour and are able to adapt to in vitro growth, losing the original properties of the host tumour.7 Other models for tumour graft strategies have previously had limited success.8Patient-derived xenograft (PDX) models have been increasingly used in translational research since their development.9 They are able to accurately replicate tumour growth, diversity of tumour cells and tumour progression, including metastatic potential.10 PDX models have been shown to be used for prognostication: studies have shown that successful engraftment is associated with a poorer prognosis that can be correlated clinically.10 Another study, by exploring heterogeneous sarcoma patients with a wide range of prognoses and tumour subtypes, demonstrated that PDX models aided therapeutic decision-making in the case of a collection of disparate tumours where each one is a rare subtype.11Trials examining PDX models have shown that they can produce samples that are authentic to the host tumour.14 Children with high-risk neuroblastoma have a less than 50% chance of cure.10 This has led to recognition of an increasing need for personalised treatment for patients with high-risk neuroblastomas.15 This is especially relevant as current treatment regimens have a range of acute toxicities and long-term side effects.17 RCTs effectively test new interventions, remove allocation bias, are ethically conducted and ensure that no subject receives less-than-baseline care; however, they are expensive and can take many years to complete. RCTs are also ultimately not appropriate for the requirement for rapid developments in any field, let alone a rare cancer18—where they are less valid, almost impossible to recruit adequate numbers for and are thus performed less frequently, leading to a dearth of evidence.19 In children there is a scarcity of trials conducted, with issues relating to feasibility as well as ethics. This leads to an absence of evidence, and alternatives need to be sought.20 Existing in vivo and in vitro data will always remain the preclinical vanguards of drug development, but the clinical use of patient-derived xenografts can enhance the robustness of preclinical studies.14While neuroblastomas are the most common extracranial solid tumour in children, they are generally rare, and have a wide variety of outcomes depending on the specific, albeit variable, biology of the tumour.16 They examine different techniques including different engraftment sites and different biological sample types, such as metastatic and primary tumour samples. Previous papers have reported that PDX models are more informative than cell-line xenografts; however, they have demonstrated limited engraftment success rates, prolonged establishment of grafts and high costs.21 This paper’s key finding is that of successful engraftment via orthotopic implantation, a method leading to more rapid model development. This is in keeping with previous research in advanced sarcoma patients, which highlighted the need for time-efficient engraftment in order to see a benefit within a clinically appropriate timescale.16In this edition of the BJC, Kamili et al. investigated the reliability of establishing PDX models for high-risk neuroblastomas.16 All of the orthotopically inoculated tumours were successfully engrafted and resulted in the quickest time to engraftment compared with subcutaneously and intramuscularly inoculated tumours. Indeed, PDX models were established for 4 of 9 of patients at diagnosis and all patients tested (5/5) at relapse.16 These findings show that PDX can be established quickly, which is key in high-risk and rapidly developing tumours. Although the orthotopic model was found to be the most rapid engraftment approach, there were also subcutaneous and intramuscular xenografts that were found to be equally as representative of the donor tumour. This suggests that in future, the xenograft type can be selected depending on its clinical utility. It has also shown that PDX models can reliably be established from a diverse range of samples, depending on which is most accessible clinically.16 This is going to be especially central to rare cancers, paediatric tumours or those with a range of subtypes, though one can argue that oncology is heading in that direction already. It is also recognised that tissue samples from tumours have often been inaccessible, and this has been a limiting factor.21 This is significant for development of personalised models that can be used in future clinical trials and clinical practice. Another important aspect of this paper is the expansion of patient material for ex vivo and in vivo drug testing. Personalised PDX models would allow for prioritisation of therapeutic options and provide an evidence-based platform for decisions regarding personalised therapy.16Kamili et al. are able to address the lengthy establishment time that has been a limiting factor in previous studies.16 It emphasises the possibility that a personalised approach to cancer treatment and research can be developed with these models. With the development of reliable engraftment, this could lead to informative preclinical models for individual patients.22 It also proposes the use of xenografts to expand the current limited basis for drug testing in cancer patients, minimising the need for expensive and prolonged randomised controlled trials.9 Changes in research practice are needed to adapt to the current medical climate. Innovations, such as PDX models, have the ability to change what is considered standard practice and improve access to personalised treatment.This research explores the challenges of developing PDX models, including xenogeneic graft versus host disease and proliferation of EBV-infected cells. It does, however, propose a strategy to overcome and limit using this T-lymphocyte depletion.


Varicella zoster virus (VZV) infection commonly presents as varicella during childhood, and zoster, later in life. Here, we present a rare and interesting case of VZV infection that manifested with both cerebral and spinal infarctions and cutaneous vasculitis in the absence of a classic vesicular rash in an immunocompromised patient. Varicella zoster virus (VZV) is a human neurotropic virus that causes chickenpox (varicella) in childhood and herpes zoster (shingles) when reactivation occurs after a period of latency later in life or during decreased immunity . VasculoA 65-year-old man presented with 3 weeks of pain, numbness, and progressive weakness of the right lower back and lower extremity associated with the appearance of red lesions. He was on tacrolimus, azathioprine, and prednisone for immunosuppression due to a history of heart transplantation 3 years prior. He denied headache, neck stiffness, fever, confusion, or bowel or bladder incontinence. Physical examination was notable for the absence of a cardiac murmur, 2/5 right lower extremity weakness, and painless purpuric macules scattered along the anterior and posterior regions of his right buttock and right lower extremity in a nondermatomal distribution without vesicles .Laboratory testing was negative for human immunodeficiency virus, hepatitis, syphilis, antineutrophil cytoplasmic antibody, and cryoglobulinemia. Complement levels were normal. Blood cultures revealed no growth, and echocardiography did not reveal valvular vegetations. Pathology from a biopsy of the rash revealed an inflammatory infiltrate of lymphocytes, neutrophils, some eosinophils, and nuclear dust with extravasation of erythrocytes. This was consistent with leukocytoclastic vasculitis. Magnetic resonance imaging (MRI) with intravenous contrast of the brain and spine was notable for subacute infarcts in the bilateral cerebral hemispheres and left cerebellar hemisphere  and an eVZV vasculopathy is associated with significant morbidity and mortality when treatment is delayed. Therefore, rapid diagnosis is essential. Interestingly, our patient did not present with the classic vesicular rash of VZV but rather with cutaneous vasculitis, which is often painless, thought to be a rare manifestation of VZV infection, and reported as preceding the classic vesicular rash –5. In ouAlthough most patients with VZV vasculopathy have an abnormal MRI, the findings are not necessarily diagnostic. Additionally, a serum VZV IgG test is of little value, because it is suggestive of immunization or prior exposure to the virus and does not localize the VZV infection to the central nervous system (CNS). Therefore, confirmation of the diagnosis requires sampling of cerebrospinal fluid and detection of VZV DNA by real-time polymerase chain reaction or anti-VZV IgG antibody by enzyme immunosorbent assay , 8. BothThe presence of VZV DNA in the cerebrospinal fluid is most commonly assessed by qualitative PCR methods with a detection limit ranging from 50 to 250 copies/mL, which is similar to what was used in our case, although quantitative real-time PCR detection of VZV DNA has also been described , 10. TreIn conclusion, we present a rare case of VZV vasculopathy that manifested with both cerebral and spinal infarctions and cutaneous vasculitis without the appearance of the classic vesicular rash. We believe that this is an important case to share with the medical community given that such an atypical manifestation may result in substantial morbidity without prompt identification and treatment.


The identification and validation of strategies to individualise therapy of patients with head and neck squamous cell carcinoma require cellular and animal models that accurately represent the complex microenvironment in which human tumours grow. Recently, patient-derived xenograft and organoid models of head and neck squamous cell carcinoma have been established that recapitulate the morphology, genetics and response to therapy of the human tumours they originated from. In this review, we discuss the development of patient-derived xenograft and organoid models of head and neck squamous cell carcinoma and describe their ability to predict clinical response to therapy. We focus on the utility of these tools to enable greater precision of both approved and experimental medicines through individualised therapeutic approaches.Patient survival from head and neck squamous cell carcinoma (HNSCC), the seventh most common cause of cancer, has not markedly improved in recent years despite the approval of targeted therapies and immunotherapy agents. Precision medicine approaches that seek to individualise therapy through the use of predictive biomarkers and stratification strategies offer opportunities to improve therapeutic success in HNSCC. To enable precision medicine of HNSCC, an understanding of the microenvironment that influences tumour growth and response to therapy is required alongside research tools that recapitulate the features of human tumours. In this review, we highlight the importance of the tumour microenvironment in HNSCC, with a focus on tumour hypoxia, and discuss the fidelity of patient-derived xenograft and organoids for modelling human HNSCC and response to therapy. We describe the benefits of patient-derived models over alternative preclinical models and their limitations in clinical relevance and how these impact their utility in precision medicine in HNSCC for the discovery of new therapeutic agents, as well as predictive biomarkers to identify patients’ most likely to respond to therapy. Head and neck squamous cell carcinoma (HNSCC) describes a subset of cancers, which begin in the squamous epithelial cells lining the mucosal surfaces of the head and neck region, including the oral and nasal cavities, pharynx, and larynx. HNSCC accounts for over 90% of all head and neck cancers , which cTP53, PIK3CA, FAT1, and NOTCH1, loss of CDKN2A, and amplification of CCND1 [TP53, and likely follow an alternative tumourigenesis pathway driven by activating HRAS and inactivating CASP8 mutations as a result of ageing rather than high exposure to tobacco and alcohol [TRAF3, amplification of E2F1, and a higher rate of PIK3CA mutations than HPV-negative tumours. The overall mutational burden is lower than HPV-negative disease [TP53 and CDKN2A [TP53 and CDKN2A genetic events seen with HPV-negative disease.HNSCC carcinogenesis is driven by frequent chromosomal instability and gene mutations that differ with HPV infection status . The priof CCND1 ,10, implof CCND1 . A subgr alcohol ,12. Comm disease , with fed CDKN2A ,10, sincd CDKN2A ,15, esseLocally advanced HNSCC is treated with surgery or radiotherapy with curative intent. Frequently these modalities are supplemented with concurrent platinum-based chemotherapy  or with Precision medicine to tailor treatment plans to individual patients based on expression of predictive biomarkers is becoming more widespread in oncology as the use of diagnostics identifying treatment-sensitive patient subpopulations rises. The recent definition of a biomarker as a “defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes or responses to an exposure or intervention, including therapeutic interventions”  does lit2 of <10 mmHg [2) regions among the tumours being 29–50% [Key to enabling precision medicine in HNSCC is an understanding of the microenvironment that influences HNSCC tumour growth and response to therapy. One of the most important negative prognostic markers in HNSCC is the presence of low oxygen levels, or hypoxia. Strong evidence of the importance of hypoxia in HNSCC initially came from studies using Eppendorf oxygen electrodes to directly measure oxygen levels (pO2) in late-stage primary and nodal head and neck tumours, with patients with hypoxic tumours tending to have worse overall survival and tumour control following radiotherapy or chemoradiotherapy ,28,29,30<10 mmHg ,28,29 ang 29–50% ,28,30. Mg 29–50% .There are a number of mechanisms that underlie the poor prognosis of hypoxic tumours. One of the most-well known, and first to receive widespread attention, is the resistance of hypoxic cells to radiotherapy. Since radiation-induced DNA damage is an oxygen-dependent process, radiation doses required to kill hypoxic cells are two- to three-fold higher than those required to kill well-oxygenated cells . Knowledlow/immunehigh score had a favourable 5-year survival probability (71%) and were more likely to display an “inflamed” microenvironmental profile. In contrast, those defined as hypoxiahigh/immunelow had unfavourable 5-year survival probability (51%) and were more likely to display an “immune-desert” phenotype. Importantly, immunohistochemical staining confirmed an inverse correlation between these two features: tumours with greater CA9 staining (a marker of hypoxic tumour cells) had lower prevalence of intratumoural T cells and vice versa [Recent research also indicates that tumour hypoxia enables immune evasion by promoting and sustaining an immunosuppressive tumour microenvironment. The hypoxic niche recruits various immune cells to the tumour including macrophages, neutrophils, myeloid-derived suppressor cells, and regulatory T cells ,36,37. Hce versa . Clinicace versa . These rA range of strategies to modify hypoxia have been explored to counteract the negative consequences of tumour hypoxia to the prognosis of HNSCC treated with radiotherapy. Numerous clinical trials using nitroimidazole oxygen-mimetic radiosensitisers ,43 or su18F-labelled nitroimidazole probes [18F-misonidazole PET imaging biomarker signal [A change in approach evolved in the 1980s; rather than try to modify hypoxia within solid tumours, hypoxia-activated prodrugs (HAPs) were developed to exploit the presence of tumour hypoxia as a selective drug delivery address. HAPs feature a chemical moiety that deactivates the drug until the prodrug is selectively metabolised in low-oxygen environments by one electron reductases in an oxygen-sensitive manner ,49. Earle probes ,53. A rar signal .18F-fluoroazomycin arabinoside, FAZA) further demonstrated the prognostic potential of hypoxia biomarkers, with hypoxia-positive patients treated with chemoradiotherapy faring significantly worse than hypoxia-positive patients receiving tirapazamine plus radiotherapy [Despite this clear indication of the value of biomarker-guided hypoxia targeting, subsequent clinical trials of HAPs have not used hypoxic biomarkers to stratify patients. For example, the ensuing phase III trial of tirapazamine with chemoradiation did not select HNSCC patients based on hypoxic status and failed to demonstrate any superiority over chemoradiation alone . Analysiotherapy . The casotherapy  and patiotherapy . The mitotherapy  and the otherapy , with itotherapy ,61, undeotherapy ,63, and otherapy ,64 by elotherapy .Overall, while there is considerable evidence to justify targeting hypoxia in HNSCC, the clinical trials that have been performed to date suggest that it is imperative that future therapeutic strategies utilise prospective hypoxia biomarkers to guide their clinical development. As discussed above, the development of such therapeutic strategies also require a thorough understanding of the contribution of the different aspects of the tumour microenvironment  on treatment response, which, in turn, requires research tools that recapitulate these features of human tumours.To enable translation of precision medicine approaches for treating HNSCC into the clinic, appropriate preclinical tumour models must be employed that mirror the complex microenvironment of human tumours. Tumour xenograft models derived from human cancer cell lines are widely used to model HNSCC, with well over 300 HNSCC cell lines described in the literature, although the vast majority of these are HPV-negative . Many ofPDX models of HNSCC are generated through the direct implantation of intact tissue from human tumours into immunodeficient mice either subcutaneously or orthotopically into anatomically similar sites such as the floor of the mouth or the base of the tongue . InitialAt the histopathological level, PDX tumours accurately recapitulate the heterogeneous and complex tumour microenvironment of HNSCC with retention of cell morphology, stromal and vascular architecture, abundant regions of keratinous cells, and matched degrees of differentiation to the parent tumour ,79,81,86H1 cell adoptive transfer [The ability of PDX tumours to model patient tumour hypoxia is less established. Hypoxia is primarily determined by the tumour microvasculature , so in Ptransfer ; but ovetransfer ,101,102.transfer ,63.The majority of HNSCC PDX models generated are HPV-negative rather than HPV-positive, as HPV-negative disease represents the greater clinical need, due to its greater frequency, worse prognosis, and reduced response to therapy relative to HPV-positive disease ,23,24. HAnecdotal reports of HNSCC PDX models predicting clinical response of the donor patient have been reported in four studies. Kang et al. report two PDX models, where the response to the pan-HER inhibitor afatinib mirrored the response of the donor patient, with one patient:PDX pair showing stable disease and the other partial response . Both PDThere are various limitations associated with the use of PDX models. A long time lag typically exists between initial engraftment and establishment of PDX tumours in large cohorts of mice for evaluation of drug sensitivity, which alongside the expense of developing these models, can limit their utility. The requirement for severely immunodeficient mice to allow for successful engraftment precludes a functional immune system in the mouse host. Interactions with the immune system influence tumour cell behaviour and provide a target for immunotherapies ,109. Co-Patient-derived 3D culture models offer an alternative approach to PDX models without the cost, time or animal ethics limitations of PDX models. Multicellular spheroid models of early passage HNSCC cell lines are common ,120,121,Advances in 3D culture technology have led to the development of tissue organoids, where 3D self-organising mini-organs can be grown in culture within a basement membrane gel from either pluripotent stem cells (PSCs) or adult stem cells (ASCs) ,131,132.Cancer organoids offer promise in drug discovery as a link between 2D cell cultures and animal models for compound screening and identification of predictive biomarkers for precision medicine at a higher-throughput, with lower cost and shorter time-frames than PDX models . Across As with all preclinical models, there are limitations associated with tumour organoids. Although they accurately model epithelial cells, they lack a mesenchymal and immune component. Co-culture of tumour organoid systems with mesenchymal and/or immune cells have been reported ,149,150,The evidence outlined above demonstrates that both PDX and organoid models of HNSCC are able to recapitulate the heterogeneity of clinical HNSCC, showing histopathological, genetic, and proteomic fidelity across multiple studies ,78,79,85As previously discussed, there is anecdotal evidence that both HNSCC PDX models and organoids can predict the clinical response of the patients they originated from ,93,126, The current lack of stromal and immune components in the majority of HNSCC PDX and organoid models limits their widespread use in precision medicine. Immune checkpoint inhibitors are approved for first- and second-line therapy of HNSCC ,21,22, aPrecision medicine offers the potential in improving therapeutic success to patients with HNSCC. The development of precision approaches will require an understanding of how the tumour microenvironment influences treatment responses, the co-development of biomarkers to accompany any therapeutic agents used in these approaches, and research tools that recapitulate the complexity of human tumours. A variety of HNSCC preclinical models exist, including a large number of HNSCC cell lines, which as monolayer cultures are ideal for basic biology studies and for early evaluations of pharmacological activity. However, due to their 2D organisation and in vitro selection, these culture models often poorly model the human tumour microenvironment, lacking stromal and immune cells and physiologically relevant oxygen gradients. Newer patient-derived preclinical models including PDX and organoid models of HNSCC are better able to recapitulate the heterogeneity of clinical HNSCC, with evidence of histopathological, genetic, and proteomic fidelity across multiple studies, and anecdotal reports of accurate predictions of clinical response. Overcoming current limitations to the lack of immune and/or stromal components through humanised mice or organoid co-cultures and prospective validation of their ability to predict clinical response will further their promise and utility as clinically relevant models for precision medicine in HNSCC.


The erratum corrects a grant number listed in Acknowledgments section of the original article. J. Biomed. Opt.24(1), 017001 (2019) doi: 10.1117/1.JBO.24.1.017001] was originally published on 8 January 2019 with an erroneous grant number in the Acknowledgments section. The original grant listed was NSF PFI: BIC 11631146; the correct grant number is NSF PFI: BIC 1631146.This article [The error was corrected on 31 December 2020.


A grant number from the funder JSPS KAKENHI is missing from the Funding statement. The missing grant number is: 16H06279 (PAGS) (TT). The publisher apologizes for the error.


Sarcomatoid differentiation in renal cell carcinoma (RCC) with vena caval tumour thrombus has been shown to be associated with aggressive behaviours and poor prognosis; however, evidence of the impact of rhabdoid differentiation on prognosis is lacking. This study evaluated the impact of sarcomatoid differentiation and rhabdoid differentiation on oncological outcomes for RCC with vena caval tumour thrombus treated surgically.We retrospectively analysed patients treated surgically for RCC with vena caval tumour thrombus at our institute from Jan 2015 to Nov 2018. Prognostic variables were evaluated for associations with progression-free survival (PFS) and cancer-specific survival (CSS) by Kaplan–Meier survival analysis and log-rank test. Univariate and multivariate analyses were performed to determine independent prognostic variables.p = 0.018 and p = 0.095, respectively). The univariate and multivariate analyses both showed sarcomatoid differentiation as a significant predictor of PFS. Compared to pure RCC, patients with sarcomatoid differentiation (p = 0.002) and rhabdoid differentiation (p = 0.001) both had significantly worse CSS. The univariate analysis showed sarcomatoid differentiation, rhabdoid differentiation, metastasis and blood transfusion as significant predictors of CSS . In the multivariate analysis, sarcomatoid differentiation , rhabdoid differentiation , metastasis  and blood transfusion  all remained independent predictors of CSS.We identified 125 patients with RCC and vena caval tumour thrombus, including 17 (13.6%) with sarcomatoid differentiation alone, 8 (6.4%) with rhabdoid differentiation alone and 3 (2.4%) with both sarcomatoid and rhabdoid differentiation. Compared to pure RCC, patients with sarcomatoid differentiation but not rhabdoid differentiation have worse PFS (Sarcomatoid differentiation and rhabdoid differentiation are both independent predictors of poor prognosis in RCC with vena caval tumour thrombus treated surgically. Renal cell carcinoma (RCC) is the most common kidney tumour, comprising an estimated 2.2% of all new cancer diagnoses with 403,262 new cases and 175,098 deaths in 2018 [Rhabdoid differentiation in RCC is characterized by “sheets and clusters of variably cohesive, large epithelioid cells with vesicular nuclei, prominent nucleoli and large paranuclear intracytoplasmic inclusions” . It is pTherefore, this report describes the survival outcomes of a consecutive series of patients treated surgically for RCC with vena caval tumour thrombus and our evaluation of the impact of sarcomatoid differentiation and rhabdoid differentiation on survival outcomes.After receiving approval from the Peking University Third Hospital Medical Science Research Ethics Committee, we retrospectively analysed the data of patients treated with nephrectomy and thrombectomy for RCC with vena caval tumour thrombus at our institute from Jan 2015 to Nov 2018. Among the 131 patients pathologically diagnosed with RCC with vena caval tumour thrombus, 6 were excluded from the study: 1 with metachronous vena caval tumour thrombus, 2 with two-stage operation for RCC with vena caval tumour thrombus and 3 with incomplete follow-up data. Thus, 125 patients were included in our study. None of the patients underwent neoadjuvant therapy before surgery. Comprehensive clinical and pathological data was collected for each patient, including age, gender, tumour size, thrombus level, blood transfusion, TNM stage, histologic subtype, Fuhrman grade, tumour necrosis, sarcomatoid differentiation, rhabdoid differentiation and adjuvant target therapy of tyrosine kinase inhibitors.Tumour size was collected as the largest diameter reported in computed tomography or magnetic resonance imaging examination. The level of tumour thrombus was assigned using the Mayo classification . TNM staFirst, nephrectomy was performed following routine procedures, and lymph node dissection was performed for patients suspected to have lymph node metastasis based on enhanced CT or PET/CT results. Second, the inferior vena cava (IVC) and contralateral renal vein were isolated and blocked as follows: (a) For Mayo I tumour thrombus, the IVC tumour thrombus was squeezed back into the renal vein using the milking technique, and the IVC was partially blocked with vessel forceps. (b) For Mayo II tumour thrombus, several short hepatic veins and lumbar veins were ligated to expose the retrohepatic segment of the IVC, and the contralateral renal vein and distal and proximal IVC were blocked with rubber bands. (c) For Mayo III tumour thrombus, the liver was mobilized to expose the hepatic portal vein before blocking the IVC. (d) For Mayo IV tumour thrombus without entrance to the atrium, the milking technique and Foley catheter-assisted technique could be used to downgrade the tumour thrombus to level III. (e) For Mayo IV tumour thrombus into the atrium, thoracoabdominal midline incision and cardiopulmonary bypass were commonly necessary. Next, the junction of the renal vein and IVC was curvilinearly incised, and the tumour thrombus was pulled out once confirmed to be completely isolated. Finally, the IVC was sutured continuously after flushing the lumen with heparin saline.Follow-up was executed every 3 months for the first 2 years and semi-annually thereafter and included physical examination, laboratory tests and chest and abdomen-pelvis scans. Follow-up information was obtained through review of outpatient records and telephone calls. Progression-free survival (PFS) was calculated from the date of surgery to radiological evidence of tumour progression, death from any cause or the last follow-up. Cancer specific survival (CSS) was calculated from the date of surgery to death from RCC or the last follow-up.p < 0.05 was considered statistically significant.Normally distributed continuous variables were reported as means and standard deviations. Non-normally distributed continuous variables were reported as medians and interquartile ranges. The Student’s t test and Mann–Whitney U test were applied to compare continuous variables. The Chi-square test was applied to compare categorical variables. The Kaplan–Meier method with log-rank test was used for survival analysis and comparisons. Univariate and multivariate Cox proportional hazard models were performed to identify independent predictors associated with PFS and CSS. All statistical analyses were conducted with SPSS Statistics 22.0 . Two-tailed tests were used for all comparisons, and p = 0.060). However, RCC with sarcomatoid and/or rhabdoid differentiation more frequently had larger tumour size  and higher blood transfusion  than pure RCC. Similarly, RCC with sarcomatoid and/or rhabdoid differentiation more frequently displayed high-grade disease  and tumour necrosis .A total of 125 patients treated surgically for RCC with vena caval tumour thrombus were included in our study. Thrombus levels were Mayo I, II, III, and IV in 38, 49, 25 and 13 patients, respectively. Among those patients, 17 (13.6%) had sarcomatoid differentiation alone, 8 (6.4%) had rhabdoid differentiation alone and 3 (2.4%) had both sarcomatoid and rhabdoid differentiation. The patients’ clinicopathological demographics are outlined in Table p < 0.001) and metastasis  were significantly associated with PFS , but not RCC with rhabdoid differentiation , had significantly worse PFS , non-clear cell RCC  and sarcomatoid differentiation  were significant predictors of PFS , non-clear cell RCC  and sarcomatoid differentiation  all remained independent predictors of PFS  patients had reported disease progression with a mean PFS of 22.7 ± 1.9 months and 22 (17.6%) had died as a consequence of RCC, with a mean CSS of 33.1 ± 1.8 months. The perioperative mortality within 90 days was 10.4% (13/125). One of them died of intraoperative pulmonary embolism. Based on the Kaplan–Meier survival analysis, non-clear cell RCC  and rhabdoid differentiation  had significantly worse CSS than pure RCC . Thrombus level and histologic subtype were not associated with CSS. In the univariate analysis, sarcomatoid differentiation , rhabdoid differentiation , metastasis  and blood transfusion  were significant predictors of CSS (Table p = 0.008), rhabdoid differentiation , metastasis  and blood transfusion  all remained significant independent predictors of CSS (Table Based on the Kaplan–Meier survival analysis, RCC with sarcomatoid differentiation (mean 19.8 vs 35.3 months, RCC with sarcomatoid differentiation was first reported as a distinct histologic subtype termed sarcomatoid RCC in 1968 . SubsequRCC with sarcomatoid differentiation is classified as grade 4 by the WHO/ISUP grading system . HoweverRhabdoid differentiation, which can arise in any histologic subtype of RCC, including clear cell, papillary, chromophobe and unclassified RCC, may be a prognostic variation of RCC, similar to sarcomatoid differentiation. However, rhabdoid differentiation has not been studied as thoroughly as sarcomatoid differentiation. Gökden et al.  reportedIn contrast, a study of grade 4 RCC showed that rhabdoid differentiation alone was not associated with worse CSS . To our Furthermore, we found histologic subtype to be a significant predictor of PFS and CSS, in keeping with previous studies , 25. HowOur study has some limitations, including its retrospective nature, single-centre experience and relatively shorter follow-up compared to previous studies. Although multivariate analyses were used to identify independent predictors of PFS and CSS, it is possible that unmeasured differences existed considering the small sample of our study. In addition, we did not perform lymph node dissection routinely for all patients which may have reduced the reliability of our results regarding the prognostic impact of lymph node involvement. Finally, there was some heterogeneity in treatment after surgery which can result in different oncologic outcomes.Our study shows that sarcomatoid differentiation and rhabdoid differentiation are associated with worse CSS in patients with RCC and vena caval tumour thrombus treated surgically. Furthermore, RCC with sarcomatoid differentiation was an independent predictor for worse PFS. Blood transfusion was an important predictor of early perioperative mortality.Additional file 1: Table S1. Univariate and multivariate Cox proportional hazard regression analyses of PFS. Table S2. Univariate and multivariate logistic regression analyses of perioperative mortality within 90 days.


Osteoarthritis is a mechanical abnormality characterized by chronic joint pain associated with degeneration of the articular cartilage, synovitis, and local inflammation, leading to loss of function and pain. A connection exists between the peripheral nervous system and inflammatory joint degeneration. The process by which inflammation is influenced by the nervous system is known as neuroinflammation. One of the neuropeptides involved in peripheral neuroinflammation is nociceptin, a peptide related to the opioid class of substances. Nociceptin has both pro- and anti-inflammatory effects. Some studies show that nociceptin can be measured in synovial fluid, while other studies have not been able to detect it. The presence of nociceptin in synovial fluid could imply a molecular role for the neuropeptide in the joint, both physiologically as well as pathophysiologically. The goal of this pilot study was to determine whether nociceptin was present in the synovial fluid of osteoarthritic knees.Patients undergoing primary total knee arthroplasty were enrolled after Institutional Review Board approval was obtained. Synovial fluid was aspirated from patients’ operative knee joints and blood samples were obtained. A commercially available enzyme Immunoassay kit was used to test for nociceptin. A linear mixed-effects model was developed to account for the repeated measurements and baseline covariates. Least squares (adjusted) means were derived from the model to compare the sample types and to compare subgroups.p = 0.002 and p = 0.016 respectively). The nociceptin concentration in both plasma and synovial fluid was significantly lower in females versus males (p = 0.012).Twenty patients were included in this study. Nociceptin was detected in the synovial fluid and plasma of all patients. The mean concentration (± standard deviation) of nociceptin in synovial fluid was 28.7 ± 18.2 pg/ml. The mean concentration of nociceptin in plasma was 45.2 ± 24.3 pg/ml pre-procedure, and 40.1 ± 20.6 pg/ml post-tourniquet deflation. The nociceptin concentration in synovial fluid was significantly lower than the nociceptin concentration in plasma, both pre-procedure and post-tourniquet deflation (We demonstrated that nociceptin is present in synovial fluid and plasma of patients undergoing total knee arthroplasty. This implies a potential role for nociceptin in modulating inflammation in osteoarthritis.ClinicalTrials.gov, NCT02528916. Retrospectively registered on August 19, 2015, Osteoarthritis (OA) is a mechanical abnormality characterized by chronic joint pain associated with degeneration of the articular cartilage, synovitis, and local inflammation , leadingIt is well known that a connection exists between the peripheral nervous system and inflammatory joint degeneration . The proThe goal of this pilot study was to determine whether nociceptin was present in the synovial fluid of osteoarthritic knees. These data will be used to design a study comparing healthy subjects’ synovial fluid and plasma nociceptin concentrations to the same measurements in patients with osteoarthritis.ClinicalTrials.gov (registration number NCT02528916). EQUATOR guidelines for a cross-sectional observational study were followed. Twenty-two patients were enrolled between May and November 2014. This was a convenience sample of patients aged 18–80 years, diagnosed with osteoarthritis, undergoing primary total knee arthroplasty (TKA). Patients who were not hemodynamically stable, had undergone prior joint arthroplasty at the current surgical site, or had active infection of their knee joint, were excluded from the study. The same orthopedic surgeon performed all of the procedures. Anesthetic technique consisted of general anesthesia combined with regional anesthesia . Standard institutional anesthetic technique was followed.The study was registered retrospectively with g, 4 °C, 15 min). The plasma supernatant was aliquoted into RNAse/DNAse-free tubes and stored at − 80 °C until assayed. Synovial fluid was collected, aliquoted, and stored at − 80 °C until assayed. The concentration of nociceptin in plasma and synovial fluid was quantified by fluorescent enzyme immunoassay using the nociceptin/orphanin FQ enzyme immunoassay kit from Phoenix Pharmaceuticals, Burlingame, CA, USA.Two blood samples  and a synovial fluid sample (5 ml) were obtained from each patient. The first blood sample was collected preoperatively, during intravenous catheter placement. After induction of anesthesia, a tourniquet was applied to the operative thigh to limit blood loss and to ensure a dry operative field. The synovial fluid sample was aspirated from the knee joint after skin incision, and the second blood sample was collected 5 min after the thigh tourniquet was deflated. Blood samples were collected in BD Vacutainer K2 EDTA Plus Blood Collection Tubes  and placed on ice until processed by the addition of the protease inhibitor aprotinin and centrifugation  . Preoperp values less than 0.05 were considered statistically significant.Study data were collected and managed using REDCap (Research Electronic Data Capture) electronic data capture tools hosted at Penn State Health Milton S. Hershey Medical Center . Data weTwenty-two patients with clinically diagnosed osteoarthritis were enrolled. Technical reasons prevented us from obtaining synovial fluid samples from two patients—they were excluded from the analysis. Demographic data for the patient population are shown in Table Sixteen patients received general anesthesia plus ultrasound-guided femoral nerve blocks with a long-acting local anesthetic . Three patients underwent the procedure with continuous lumbar epidural anesthesia combined with general anesthesia . These three participants underwent bilateral total knee replacements. In these patients, synovial fluid samples were obtained from the first joint operated on only, and plasma samples were obtained after release of the first tourniquet. One female patient received subarachnoid anesthesia combined with general anesthesia, and had a femoral nerve block after the procedure. Nociceptin was detected in all plasma and synovial fluid samples (Table p = 0.002) and post-tourniquet deflation (p = 0.016). The difference in nociceptin plasma concentrations pre-procedure and post-tourniquet deflation was not statistically significant (p = 0.34).The nociceptin concentration in synovial fluid (28.7 ± 18.2 pg/ml) was significantly lower than the nociceptin concentrations in plasma, both pre-procedure (p = 0.12), body mass index (p = 0.23), preoperative pain scores (p = 0.53), or postoperative pain scores (p = 0.36). However, there was a significant correlation between nociceptin levels and gender (p = 0.012). The nociceptin concentration in plasma and synovial fluid was significantly higher in males than in females (Table There was no significant correlation between nociceptin levels and age (x p = 0.2, preoperWe demonstrated measurable levels of nociceptin in both the plasma and synovial fluid of osteoarthritis patients undergoing knee arthroplasty. The presence of this neuropeptide in synovial fluid suggests a role for this molecule either in the physiology of the joint or the pathophysiology of OA. This may have diagnostic as well as therapeutic implications for the diagnosis and management of OA, including disease modification.The detection of nociceptin in synovial fluid in our study is qualitatively similar to the results of Fiset et al. , althougThere was no control group. Our goal with this study was to determine if nociceptin was present or absent in synovial fluid, making a control group unnecessary. We did not obtain a synovial fluid sample from subjects’ contralateral knees as this was deemed an unacceptable risk to the patient.p value of 0.0008, for the difference in synovial fluid nociceptin concentration between males and females, suggests a difference that warrants further investigation. Abundant evidence indicates greater pain sensitivity among females compared with males [Given the exploratory nature of this pilot study consisting of only 20 subjects, one cannot place undue significance on results obtained when analyzing subgroups. However, the th males . Holtzmath males . Srikantth males . More reIn conclusion, nociceptin is present in the synovial fluid of patients undergoing primary knee arthroplasty. This implies a potential role for nociceptin in modulating inflammation in osteoarthritis.


Cancer cells upregulate phosphatidylserine (PS) on the cell surface as a mechanism to induce an immunosuppressive microenvironment. Development of anti-PS targeting antibodies have highlighted the ability of a PS-blockade to enhance tumour control by T cells by releasing immunosuppression. Here, C57BL/6 mice were implanted with HPV16 E7 target-expressing C3 tumours and subjected to low dose intermittent cyclophosphamide (CPA) in combination with DPX-R9F treatment targeting an E7 antigen with and without anti-PS and/or anti-PD-1 targeting antibodies. Immune responses were assessed via IFN-γ ELISPOT assay and the tumour microenvironment was further analyzed using RT-qPCR. We show that the combination of DPX-R9F and PS-targeting antibodies with and without anti-PD-1 demonstrated increased efficacy compared to untreated controls. All treatments containing DPX-R9F led to T cell activation as assessed by IFN-γ ELISPOT. Furthermore, DPX-R9F/anti-PS treatment significantly elevated cytotoxic T cells, macrophages and dendritic cells based on RT-qPCR analysis. Overall, our data indicates that anti-tumour responses are driven through a variety of immune cells within this model and highlights the need to investigate combination therapies which increase tumour immune infiltration, such as anti-phosphotidylserine.DPX is a novel delivery platform that generates targeted CD8  DPX, previously known as Depovax, is a novel formulation which enhances immune responses in humans and animals5. DPX encapsulates cancer specific peptides into a no release depot composed of an oil diluent, which provides long-lasting reservoir of antigens in vivo5. DPX formulation containing the HPV16 E7 peptide target (DPX-R9F) has demonstrated efficacy in mice bearing HPV16 E7-expressing C3 tumours, and efficacy is enhanced following PD-1 blockade, which has been shown to reinvigorate T cells, and decrease exhaustion to enhance anti-tumour responses7. Preclinical studies have demonstrated DPX formulations induce robust cytotoxic T cell (CTL) responses, and generation of tumour infiltrating CTLs8. In recent human clinical trials, DPX formulated with five survivin-specific peptides resulted in robust immune responses directed at the tumour target, survivin9. Pre-clinical and clinical data demonstrates enhanced anti-tumour responses with checkpoint inhibitors and additional adjuvants thereby expanding the clinical use of DPX-based therapies10. The combination of DPX-based formulation with checkpoint molecules and immune modulators represent different ways to target immune cells and augment their activation status7. This formulation has been demonstrated to work in combination with low dose intermittent cyclophosphamide (CPA), a treatment designated to modulate immune responses11. The sequencing of DPX-based formulation and CPA has been investigated pre-clinically, and implemented in clinical trials for its benefits as an immune modulator6. However, this treatment has yet to be investigated in combination with a specific cancer-targeted therapy as a prospective way to enhance efficacy in tumour challenge models.The ability to engage and activate T cells to tumour-associated targets has the potential to elicit strong anti-tumour responses in vivo12. Upon apoptosis, PS is ‘flipped’ to the outer leaflet of the plasma membrane, rendering it exposed to the extracellular environment12. Through a process termed efferocytosis, phagocytic cells uptake PS-expressing apoptotic membranes through a direct interaction with TIM receptors and an indirect interaction with TAM receptors13. To prevent an over-stimulated immune response to apoptotic cells, the uptake of apoptotic bodies is naturally anti-inflammatory. This process is driven through the ability of TIM and TAM receptors present on the surface of macrophages and dendritic cells to stimulate downstream expression of immune-suppressive cytokines such as transforming growth factor β (TGF-β) and interleukin-10 (IL-10)16. Within an otherwise healthy individual, this process maintains cellular homeostasis, and prevents diseases such as atherosclerosis and a variety of inflammatory diseases. However, in addition to being expressed on apoptotic cells, PS can also be abundantly expressed on malignant cells, and represents a prominent mechanism of immune evasion, which can negatively impact patient prognosis17.Phosphatidylserine (PS) is a molecule present on the inner leaflet of the plasma membrane in normal heathy cells18. Although PS expression constitutes a cellular ‘eat-me’ signal, cancerous cells often usurp the immunosuppressive capabilities of PS to enhance survival of the cells through multiple mechanisms. First, circulating PS-expressing extracellular vesicles can dampen T cell activation15. Second, phagocytic cells ingesting PS-membranes fail to become mature and functional antigen-presenting cells15. Lastly, anti-inflammatory signals such as TGF-β and IL-10 further prevent effective anti-tumour responses by other cytolytic cell types15.Within the tumour microenvironment (TME), PS becomes expressed on the outer leaflet of the plasma membrane due to rapid cell division, dysregulated membrane trafficking pathways, and upregulation of cell death associated pathways20. Moreover, when provided in combination with radiotherapy, these antibodies improved the efficacy of chemo and radiation therapy by increasing antibody-dependent cytotoxicity of PS-expressing cells. Furthermore, combination of anti-PS antibodies with anti-CTLA-4 and anti-PD-1 resulted in the increased infiltration of cytolytic T cells and reduced immune suppressive cell types such as regulatory T cells, and myeloid derived suppressor cells21. Taken together, the combination of anti-PS antibodies with current and new immunotherapies is an attractive approach to combating malignant cells.Pre-clinical studies have recently demonstrated the efficacy of targeting PS as a potential avenue to enhance current cancer immunotherapies. In vivo studies using anti-PS antibodies  demonstrated efficient targeting to malignant cells and tumour vasculature, resulting in enhanced anti-tumour responsesUsing a tumour-bearing mouse model of C3 cells expressing the HPV16 E7 antigen, we demonstrate DPX-R9F/CPA in combination with PS-targeting antibodies enhances T cells, macrophages, and dendritic cell infiltration into the tumour microenvironment providing a rationale for combining these treatments.Pathogen free, 6–8 week old female C57BL/6 mice were purchased from Charles River Labs . Mice were housed under top-filtered conditions and provided food and water ad libitum.22. The C3 cell line was maintained in IMDM supplemented with 10% fetal bovine serum, 2% penicillin–streptomycin, 50 mM mercaptoethanol and 2 mM L-Glutamine. In all tumour studies mice were implanted with 3 × 105 C3 tumour cells in the left flank on study day 0.The C3 cell line was provided by Dr. Martin Kast, and was derived from C57BL/6 embryo cells transfected with HPV16 DNA and demonstrated to express an immunodominant peptide from HPV16 E7 in the context of MHC class I on the cell surface2 x length)/2. In survival studies, mice were terminated when tumour volume reached ≤ 2000mm3, or when mice displayed signs of poor health such as dehydration, decreased activity, hunched posture, tumour ulcerations, or site of injection reactions. When endpoints were identified, mice were humanely euthanized per CCAC guidelines.Tumour growth was measured with digital calipers twice weekly, and size was calculated by the formula . For ELISPOT assays, a control irrelevant peptide was used (WT-126–134). All formulations contained a universal T helper peptide PADRE (AKXVAAWTLKAA).All peptides were synthesized by NeoMPS  with 90% purity. All studies used the peptide epitope HPV16 E77. Peptides and immunostimulant were first solubilized in buffer and mixed 10:1 (w:w) with DOPC/cholesterol mixture  to form liposomes. The mixture was then lyophilized to a dry cake which was reconstituted with Montanide ISA51 VG  immediately prior to injection. Mice were injected subcutaneously on the right flank with 50 µl of DPX-R9F containing 10 µg of R9F fused with PADRE + 20 µg of immunostimulant. For experiments with multiple injections, they were given on the same flank, but avoiding previous injection sites. In tumour challenge studies DPX was administered on study days 15 and 29. In immune monitoring studies, DPX was administered on study day 21.Peptides were formulated in DPX with a proprietary immunostimulant as previously describedCPA was reconstituted in PBS and provided in drinking water at a concentration of 0.133 mg/mL. Based on 3 mL of water/mouse/day, the CPA dose was calculated at 20 mg/kg/day. In tumour challenge studies CPA was provided for one week starting on study day 7 and then again starting on study day 22. In immune monitoring studies, CPA was provided for one week starting on study day 14.For challenge studies, monoclonal antibodies were administered on study days 15, 18 and 21, and again on study day 29, 32 and 35. For immune monitoring studies, antibodies were administered on study days 21, 24, and 27. Indicated antibodies: Anti-PS , or isotype  and anti-PD-1  were given by intraperitoneal injection.7. Single cell suspensions were prepared from splenocytes in complete RPMI media (RPMI 1640 (Gibco) + 10% FBS (Hyclone), 2% penicillin/streptomycin (Gibco), 2 mM L-Glutamine (Gibco), 50 mM mercaptoethanol (Sigma-Aldrich), and 5 mM HEPES buffer (Gibco)) and adjusted to 5 × 106 cells/mL. Peptide and splenocytes cells were then added to the IFN-γ ELISPOT plates (BD Bioscience) and incubated overnight at 37 °C in 5% CO2 and developed the following day using AEC kit (Sigma Aldrich). Spot forming units were enumerated using the ELISPOT Reader .IFN-γ ELISPOT was performed as previously describedTotal RNA from tumour tissue was isolated using RNeasy Mini Kit (QIAGEN); 4 µg aliquots were treated with DNase I (Invitrogen) and reverse transcribed using Superscript III reverse transcriptase kit (Invitrogen) and oligo d(T) primer (Invitrogen). Quantitative analysis of transcripts was performed on Rotor-Gene Q real time PCR machine using a QuantiFast SYBR Green PCR kit (QIAGEN). Data were quantified based on the standard curve method and normalized to GAPDH mRNA expression.Statistical Analysis of data was conducted with GraphPad Prism 6  software. Data were analysed by appropriate tests as indicated in the figure legends. Significant levels were denoted as: *p < 0.05, ** p < 0.01, *** p < 0.001.In tumour challenge studies mice with adverse events such as tumour ulcerations and injection site reactions which resulted in humane endpoint were excluded from the analyses presented in Fig. 6. On days 15 and 29, mice were treated with DPX formulated with R9F subcutaneously followed by antibody treatments delivered intraperitoneally on days 15, 18, 21, 29, 32, 35 with anti-PS (mch1N11) or isotype (C44), anti-PD-1 (RMP1-14), or quadruple therapy (DPX-R9F/CPA/anti-PS/anti-PD-1) , and moderately reduced tumour burden compared to other groups . Although much of the differences observed were not statistically significant over isotype controls, or DPX-R9F/CPA alone, due to the reduction in tumour burden compared to formulations lacking DPX-R9F, we sought to characterize the different immune responses generated by each of the combinations and the associated anti-tumour responses.To assess the efficacy of anti-PS targeting in combination of DPX-formulations, we utilized an established model of HPV16 E7-expressing cells injected subcutaneously into C57BL/6 mice. Starting seven days prior to DPX-R9F treatment mice were treated with 7 days of orally administered cyclophosphamide  as previously described24. Due to the unique ability of DPX to harness antigen-presenting cells and direct antigen specific T cell responses5, we sought to examine this phenomenon in combination with anti-PS antibodies as a possible mechanism of enhanced tumour control and survival. To test the ability of anti-PS treatment to affect antigen-specific responses, spleens were harvested from tumour-bearing treated mice, and subjected to an IFN-γ ELISPOT assay on study day 31 . Analysis of tumours from DPX-R9F/CPA/anti-PS treatment resulted in elevated expression of cd8α compared to untreated controls , these differences were not significant from the untreated control. Consistent with the elevated levels of cd8α mRNA, increased levels of cytotoxic markers IFN-γ (ifng) and the cytolytic granule enzyme, granzyme B (gzmb) were found in both DPX-R9F/CPA/isotype or anti-PS groups  transcripts were observed in the DPX-R9F/CPA anti-PS group, but not in treatment groups containing anti-PD-1  transcripts in the DPX-R9F/CPA/anti-PS treated group compared to untreated and isotype controls  did not result in any worse tumour burden or decreased survival  in combination with DPX-R9F/CPA exhibited some level of tumour control compared to untreated, and also resulted in differing immune responses which seemingly allowed for tumour control similar to that of the other DPX-R9F/CPA-containing groups (Fig. 7. Moreover, antigen-specific clones were found to have expanded TCR-β repertoires toward R9F, and correlated to tumour control7. Paradoxically, it remains possible that addition of anti-PD-1 to the DPX-R9F/CPA/anti-PS hindered the classical anti-tumour responses. Compared to the DPX-R9F/CPA/anti-PS treatment, the addition of PD-1 lowered overall infiltration of immunes cells determined by RT-qPCR, and had a slight reduction in ELISPOT responses. Despite these differences, the quadruple therapy did not decrease survival or tumour burden significantly from the triple therapy with anti-PS.DPX is a novel T cell activating platform which allows for active uptake of tumour-specific antigens by antigen presenting cells34. However, we are unable to observe an additive effect when including anti-PS to the therapy. In future experiments, it would be prudent to evaluate different timelines for administration of anti-PD-1 or anti-PS. Recently it has been demonstrated in both preclinical and clinical studies that timing of PD-1 may impact the generation of dysfunction T cells should sufficient priming of T cells following a vaccination of cancer peptides not take place35. In line with these recent findings, we have administered DPX and anti-PD-1 simultaneously to minimize this effect. However, combination of PD-1 with various cancer-targeting antibodies may require a further delay in anti-PD-1 administration to see maximum benefit, and greatest tumour control36. Incorporating these findings in the management of a combination antibody approach warrant further investigation to tease out how checkpoint inhibitors play a role in this novel combination therapy.We have previously demonstrated the benefit of anti-PD-1/CPA/DPX-R9F in this tumour modelIn conclusion, the addition of anti-PS to the DPX-R9F/CPA therapy in the HPV16 E7-expressing tumour model resulted in enhanced anti-tumour responses, resulting in modest levels of tumour control compared to untreated and isotype control groups. The addition of anti-PD-1 to the treatment did offer enhanced survival compared to isotype control, and was not significantly different compared to DPX-R9F/CPA/anti-PS. Whether or not APCs drive the infiltration of other leukocytes, remains an active area of research, and will require in vivo mouse models to functionally link APC chemotactic factors to lymphocyte recruitment. The anti-PS blockade is an efficient method to target the tumour microenvironment, and the results presented here warrant further investigation of tumour targeting agents with novel T cell activating therapies.All experimental protocols were approved by the university committee on laboratory animals at Dalhousie University under the guidelines of the Canadian Council on Animal Care ethical standards. All applicable international, national, and institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.


LM was detected in one-third  of randomly selected OSCC patients. The LM-positive patients had shorter overall survival (OS) compared to LM-negative group albeit not statistically significant. Highly-metastatic tumour cells formed distinct LM structures in vitro and in vivo. Importantly, the siRNA-mediated knockdown of LYVE-1 not only impaired tumour cell migration but also blunted their capacity to form LM-vessels in vitro and reduced tumour metastasis in vivo. Together, our findings uncovered, to our knowledge, a previously unknown expression and function of LYVE-1 in OSCC, whereby tumour cells could induce LM formation and promote lymphatic metastasis. Finally, more detailed studies on LM are warranted to better define this phenomenon in the future. These studies could benefit the development of targeted therapeutics for blocking tumour-related lymphangiogenesis.Lymphatic metastasis represents the main route of tumour cell dissemination in oral squamous cell carcinoma (OSCC). Yet, there are no FDA-approved therapeutics targeting cancer-related lymphangiogenesis to date. The lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1), a specific lymphatic marker, is associated with poor survival in OSCC patients. In this study, we present a potential novel mechanism of lymphatic metastasis in OSCC—lymphatic mimicry (LM), a process whereby tumour cells form cytokeratin The poor prognosis of OSCC patients is mainly attributed to the invasiveness of OSCC cells and their ability to swiftly disseminate to regional lymph nodes2. Thus, there is an urgent need to better understand the mechanisms behind OSCC metastasis, and to identify novel druggable targets that can improve the survival of OSCC patients.Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the head and neck region arising anywhere in the oral cavity. Unfortunately, despite recent advances in cancer management, the overall 5-year overall survival (OS) rate remains stagnant at around 50%4. Intratumoural vasculature has long been thought to be formed by endothelial cells alone. However, the seminal work of Maniotis and colleagues showed that aggressive uveal melanoma cells were able to acquire endothelial cell behaviour by generating de novo vessel-like networks independently of existing vascular endothelial cells5. This novel paradigm, which is termed vascular mimicry (VM), has sparked an enormous interest in the field of cancer research6. Thenceforth, myriad studies have reported intriguing aspects of VM in different types of cancer 6. Interestingly, the transcriptional signature of VM-forming tumour cells revealed remarkable phenotypic plasticity (i.e. stemness), which facilitates transdifferentiation into other cell types7. Of particular importance, current antiangiogenic therapy remains ineffective on VM, thereby paving the way for more selective and personalised approaches7. Recently, the VM channels were shown to represent a promising prognosticator and therapeutic target in head and neck squamous cell carcinomas (HNSCC)8.Vascularisation is a crucial event during tumour development and metastasis10. Furthermore, lymphatic vessel density has been shown to predict metastasis-free survival in OSCC patients better than blood microvessel density, and hence also for guiding future therapeutic approaches11. Mirroring angiogenesis, tumour cells were also shown to secrete lymphangiogenic factors that facilitate lymphangiogenesis and metastasis to sentinel lymph nodes12. Importantly, the discovery of specific markers for lymphatic endothelial cells (LEC), such as the lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1) and podoplanin (i.e. D2-40), has made it possible to distinguish between lymphatic and blood vessels13. Of note, LYVE‐1 bears a high degree of specificity for lymphatic vessels, and it has been an essential component of many important studies on tumour-induced lymphangiogenesis14. Moreover, LYVE-1 is strongly associated with nodal metastasis in OSCC, and its antibody was able to inhibit the development and progression of primary breast tumours16.Lymphatic vessels in the tumour microenvironment are the main route of dissemination in carcinomas including HNSCC, where tumour cells can preferentially metastasise to several hundred of regional lymph nodesBased on the well-investigated concept of VM, we aimed to test our hypothesis that tumour cells can attain a LEC-like phenotype and form lymphatic vessel-like structures  in OSCC tissue to facilitate tumour growth and metastasis. For this purpose, LYVE-1 was adopted in our study as a lymphatic marker using clinical samples, in vivo and in vitro experimental approaches. We also examined whether LM expresses other LEC markers such as D2-40.n = 57) using specific tumour and LEC markers . The following criteria were set to identify the LM phenomenon in tumour tissues: (1) intratumoural vessel- or capillary-like structures; (2) LM lining is positive for OSCC tumour marker (CK+) staining; and (3) positive for LEC marker (LYVE-1+) staining. Interestingly, the OSCC tissues contained vessel-like structures lined by CK+/LYVE-1+ cells as depicted in . In addition, some CK+/LYVE-1+ cells were also seen as a few “hot spots” in nests of densely packed tumour cells, where LYVE-1 immunoreactivity was observed in the tumour cell membrane and cytoplasm , normal lymphatic vessels were LYVE-1+/D2-40+  in primary OSCC tumours  into two groups based on the LM status: positive (LM+) and negative (LM−) groups. Interestingly, the estimated OS of the LM+ group (58 months) was noticeably shorter compared to the LM− group (80 months) . Yet, the correlation between LM+ status with younger age group (P = 0.016) and higher clinical T-stage (P = 0.077) represented interesting hints  and also in the low-metastatic SCC-25 cells (2 copies/μl) , we first quantified LYVE-1 mRNA in OSCC cells using the highly sensitive droplet-digital PCR (ddPCR). The ddPCR reactions with at least 13 × 10μl) Fig. . LYVE-1 μl) Fig. . We thenμl) Fig. . Additioμl) Fig. . We founμl) Fig.  or in a μl) Fig. .Fig. 2OS. To investigate the ability of HSC-3 cells to form such LM structures in vivo, cell suspension was injected into the lateral tongue border of BALB/c nude male mice (n = 18), followed by double-labelled IF on the harvested xenograft sections. Surprisingly, CK+/LYVE-1+ vessel-like structures, similar to those observed in patient samples, were detected in 8/18 (44.44%) of the xenograft sections, of which three were metastatic tumours  compared to the siControl cells .It was recently shown that endogenous LYVE-1 can facilitate transluminal cell migration21. The siLYVE-1 and siControl HSC-3 cells, labelled with CellTrace™ Far Red fluorochrome, were microinjected into the perivitelline space. On the fourth day post-injection, larvae were analysed by microscopic imaging to discern fish with a tumour metastasised outside the yolk sac region. Intriguingly, the siControl group had significantly more fish with metastasis to cloaca and tail regions compared with the siLYVE-1 group, where tumour remains largely confined within the yolk sac area   Fig. .22. Indeed, the identification of tumour cell-derived VM, as a novel model of neovascularization, has opened a new perspective in cancer research23. Increasing evidence shows that VM is strongly associated with poor survival in cancer patients, and hence represents an attractive therapeutic target24. Interestingly, in OSCC tumour tissues, we observed a phenomenon that could be similar to the VM. About one-third of randomly selected OSCC patients had vessel-like structures with surrounding CK+/LYVE-1+ cells. These structures were expressing LYVE-1 but, unlike normal LEC, they were entirely negative for D2-40, implying a key role of LYVE-1 in their development. Patients with these structures had seemingly worse survival outcome and higher clinical T-stage. However, the sample size in this report may not be large enough to reach a statistically significant difference between the LM groups. More studies with larger patient samples, across different populations, should thus be conducted to reach a resounding conclusion. Furthermore, although LYVE-1 was expressed in OSCC cell lines, only the high-metastatic cells (HSC-3) were able to form similar LYVE-1+ vessel-like structures in vitro and in vivo. Importantly, LYVE-1 knockdown in these cells has markedly blunted their ability to form such structures and reduced their migration, invasion and metastasis without a robust impact on cell viability. These findings may indicate a novel potential mechanism underlying tumour growth and metastasis in OSCC—LM  vessel-like spaces, with the absence of any specific endothelial cell markers28. However, this method has been criticised for its limited specificity. In fact, PAS staining detects extracellular matrix components, and hence PAS+ regions may also represent non-functional structures irrelevant to VM30. On the other hand, the mosaic vessel-like pattern, where tumour and endothelial cell markers are simultaneously expressed, has gained attention not only for identifying VM but also as a prognostic marker32. This mosaic pattern was long thought to be formed by a coalescence of tumour and endothelial cells during carcinogenesis 31. However, recent studies revealed that aggressive tumour cells can express genes associated with non-cancerous cell phenotype, such as endothelial cell precursors34.The VM structures are traditionally identified in tumour tissues as periodic acid-Schiff positive . Importantly, HA is synthesised by both tumour and stromal cells in OSCC, and it has been shown to regulate various cancer processes from initiation to metastasis42. Thus, it is logical to assume that LYVE-1+ OSCC cells may utilise HA/LYVE-1 interaction to facilitate oral carcinogenesis. This assumption is supported by an interesting study showing that aggressive breast cancer cells can harness their HA content to bind LYVE-1 and facilitate adhesion, hence invasion and metastasis43. Notably, the high-metastatic cells used in our study, HSC-3, were proven to be highly sensitive and responsive to HA-signalling in vitro44. Here, we have shown that HSC-3 exhibit marked suppression of tube formation, migration and invasion potential in 3D in vitro models following the siRNA-mediated knockdown of LYVE-1. More importantly, inhibition of LYVE-1 in HSC-3 cells significantly reduced their metastatic potential in zebrafish larvae. In agreement with our report, it was recently shown that anti-LYVE-1 monoclonal antibody inhibited primary tumour formation and metastasis to axillary lymph nodes in xenograft models of breast cancer16.The LYVE-1 is an essential mediator of tumour lymphangiogenesis and correlates with lymph node metastasis in HNSCC patients45. As a result, to date there are no FDA-approved therapeutics that target tumour-related lymphangiogenesis46. The de novo formation of tumour-derived mimicry channels was suggested as a plausible factor that could reduce the effects of anti-angiogenic therapy32. Thus, a better understanding of LYVE-1 function in tumour cells and its role in LM formation could benefit the development of targeted therapeutics for blocking tumour lymphangiogenesis in OSCC. Clearly, more detailed studies, including larger sample size and complete deletion models of LYVE-1, are necessary in order for such phenomenon to be better defined in the future.Finally, these pieces of evidence may bear considerable therapeutic implications. Despite the major advances in the development of new angiogenic inhibitors, the clinical success of these drugs remains, however, considerably limitedSupplementary materialsSupplementary Videos 1.Supplementary Videos 2.Supplementary Videos 3.