Cancer remains one of the leading causes of death worldwide. The advancements in 3D tumour models provide in vitro test-beds to study cancer growth, metastasis and response to therapy. We conducted this systematic review on existing experimental studies in order to identify and summarize key biomimetic tumour microenvironmental features which affect aspects of cancer biology. The review noted the significance of in vitro hypoxia and 3D tumour models on epithelial to mesenchymal transition, drug resistance, invasion and migration of cancer cells. We highlight the importance of various experimental parameters used in these studies and their subsequent effects on cancer cell behaviour.The development of biomimetic, human tissue models is recognized as being an important step for transitioning in vitro research findings to the native in vivo response. Oftentimes, 2D models lack the necessary complexity to truly recapitulate cellular responses. The introduction of physiological features into 3D models informs us of how each component feature alters specific cellular response. We conducted a systematic review of research papers where the focus was the introduction of key biomimetic features into in vitro models of cancer, including 3D culture and hypoxia. We analysed outcomes from these and compiled our findings into distinct groupings to ascertain which biomimetic parameters correlated with specific responses. We found a number of biomimetic features which primed cancer cells to respond in a manner which matched in vivo response. Cancer remains one of the leading causes of death despite notable advances in modern medicine . In vitrSeveral previous literature reviews of experimental studies involving 2D and 3D in vitro tumour models, the microenvironmental features of different tumour models and drug responses have drawn conclusions that 3D in vitro tumour models mimic physiological characteristics and display biomimetic response to chemotherapy ,10,11,12In this review we have systematically analysed the effect of the addition of biomimetic features, including physiological hypoxia and 3D culture, on cancer cell behaviour. We have focused in particular on cancer cell growth, invasion, EMT and response to therapeutic interventions within biomimetic 3D cultures.The systematic review was conducted using a number of search engines including “Medline“, “Embase“ and “Web of Science“ with the same initial search terms: “cancer“ and “hypoxia“, then narrowed down further using terms including “2D“ and “3D“ for all databases used. The databases above were all accessed on 28 May 2020. All publications in English language until 28 May 2020 were included for screening. Spelling variations and similar search terms were included for thorough inclusion of existing publications .Inclusion and exclusion criteria were decided and a total of 475 citations were identified through the initial search, excluding duplications, non-full text and non-eligible publications according to the exclusion criteria, 141 publications were deemed eligible for systematic qualitative analysis .Lists of materials, methods, outcomes and conclusions from each publication were recorded via an Excel spreadsheet. To minimize outcome reporting bias, data collection was conducted by two independent investigators, with a third investigator comparing the two data sets for discrepancies and any identified were further analysed by a fourth investigator before final outcome from the specific publication was recorded for the qualitative analysis.2 or partial pressure of oxygen in the overlaying media. The pO2 within 3D cultures can drop to 0% causing cell migration away from the core of 3D cultures where this is measured directly within 3D tissue models [Pathological hypoxia plays an established role in tumour growth, invasion and migration, involving mechanisms such as EMT, angiogenesis and glucose metabolism. Tumour masses of over 500 micrometer diameters display gradients of oxygen concentration, affecting aspects of tumour growth as described above [e models .2 probes to measure the media and or 3D tumour models for dissolved oxygen; stains such as pimonidazole [In this review a total of 51 publications ,62,63,64nidazole  as a tumnidazole . The hyp2 of arterial blood is a measure of the effectiveness of transfer of oxygen from the atmosphere (air) by the lungs. Once dissolved, this oxygen is measured using mmHg units, and 7.8 mmHg is roughly equivalent to 1% atmospheric oxygen. The majority of studies in this review have not directly measured hypoxia in 3D cultures, but instead utilised hypoxia chambers to control or “set“ the free atmospheric oxygen. Many researchers will rely upon the equilibrium achieved between free atmospheric oxygen of the chamber and dissolved oxygen in the culture media, and thus this was difficult to verify for each study. There are issues around how quickly this process occurs, especially given aspects such as: (i) the constant temperature of the culture; (ii) the opening and closure of hypoxia chambers, which can cause fluctuations in oxygen tension; (iii) an understanding of the oxygen diffusion coefficient of the 3D scaffold materials used in 3D culture and; (iv) the multiple monitoring of the media pO2 and the 3D culture pO2, which should preferably be done in the core and at the surface [When directly measuring oxygen in tissues, this is done so by measuring the partial pressure of oxygen (mmHg) and therefore reported levels of tissue oxygenation use this measure. The pO surface .2, is distinct from atmospheric oxygen concentration, which is 21% in air [Tissue normoxia, measured as pO% in air . Existin% in air . Taking % in air , one wou3D tumour models are considered to better recapitulate the in vivo tumour microenvironment compared to standard 2D cultures. In this review we have identified a total of 37 publications ,96,97,98These studies used a variety of measurements to determine the extent of biomimicry within in vitro cultures, using a range of criteria including: Genetic expression of angiogenic and matrix remodelling genes, protein synthesis of angiogenic, invasive and matrix remodelling molecules,; morphological basis involving regulators such as expression of hypoxia induced factors, proangiogenic factors, EMT markers, and factors associated with tumour cell stemness; increased IL-8 expression; measured increases in growth of cancer and invasion.3D culture and the presence of hypoxia, measured by the various methods outlined in EMT is a process identified as one of the key features in tumour invasion and metastasis. We identified a total of 19 publications ,106,107 We identified a total of 42 publications ,122,123 Overall, 85% of experimental outcomes showed 3D tumour cultures displayed increased resistance to anticancer therapies and 15% showed increased sensitivity in 3D when compared with 2D cultures. Of those which showed increased sensitivity in 3D, the therapies tested were predominantly proapoptotic agents, novel targeted therapy, hypoxia activated drugs, radiotherapy or natural compounds .Furthermore, we investigated the specific response from each cancer type to multiple therapeutic interventions. We categorized the cell lines into 11 organ systems. All cancer types showed a trend of overall increased resistance in 3D compared to 2D in vitro tumour cultures. Most cancer types showed similar responses ranging from 72% to 88% of studies displaying enhanced resistance to chemotherapy in 3D. Studies involving urological and head & neck cancers reported 100% chemo resistant outcome in 3D, whereas prostate cancers and sarcomas showed a significantly lower proportion for chemo resistant outcomes in 3D compared to other cancer types at 57% and 56% prospectively .We identified 42 publications from our cohort that specifically studied chemotherapy resistance in 3D cultures, and similar numbers of studies used in scaffold-based cultures and non-scaffold-based cultures (53% vs. 47%). On further investigation, the studies where scaffold-based 3D cultures were used, a much greater percentage of studies used native materials (72%) compared to non-native materials (28%). Similar to the cohort of studies concerning biomimicry of tumour culture, collagen and alginate appear to be the most frequently used materials for native and non-native scaffolds. This noteworthy finding could be due to native ECM proteins being deemed more biomimetic as a 3D scaffold, with materials such as collagen used extensively. Collagen type I is found in the dense fibrotic tissue and the tumour microenvironment, and by using this material, studies report the use of native materials to be more consistent between in vitro and in vivo findings .When describing cancer progression and metastasis, invasion and migration often go hand in hand and are described as parts of a process facilitating cancer spread. We investigated the evidence within the scope of this review for the definition and measurable markers used for invasion and migration, and whether these were distinct to one or the other process. 16 publications ,112,124 Migration imaging assays describe the process as the movement of tumour cells from one position to another, for example across porous membranes, movement within the tumour spheroids or out into the surrounding matrix in an in vitro setting. The morphology of cancer invasion is described in a more aggressive manner, with terms such as invasive, spiky projections and invadopodias into the surrounding ECM. Fibronectin coding genes were found to be up-regulated in studies describing cancer cell migration and various EMT markers such as vimentin, E-cadherin, MMP2 and MMP9, which were all associated with and used to measure cellular invasion. Only invasion is described in an in vivo setting, which has been duly noted in multiple publications. Florczyk et al. described the two cellular processes as, “invasion in vivo and migration in vitro by effective degradation of ECM” . Therefo2, and is distinct from ambient atmospheric oxygen, which is 21%. Tissue normoxia, or physiological hypoxia, ranges between 3–7%, thus when conducting comparisons of hypoxic vs normoxic cultures in cancer studies, a more appropriate choice for hypoxia level should be no higher than 2% oxygen and normoxia control samples should be conducted within the “normal” physiological oxygen levels to truly represent the in vivo tumour and stromal microenvironments [It is important to consider the average range of oxygen concentration in physiologically and pathologically hypoxic tissues when conducting in vitro tumour cultures. Normal tissue oxygen saturation is based on pOronments .To highlight the significance of ranges of hypoxia, over a fifth 22%) of studies included in this review explored tumour hypoxia using 3% oxygen or higher for the hypoxic samples, and furthermore 5% of studies % of stud,47,51 usApart from the level of hypoxia involved in these in vitro studies, the length of exposure also plays a vital role for the biomimicry of these tumour models. There is evidence to suggest that acute hypoxia is more likely to contribute to the malignant progression of tumour cells when compared with chronic hypoxia, meaning that an exposure period of 72 h or less used by the majority (71%) of tumour models included in this review is a sufficient period for hypoxic exposure for malignant progression of most cancer cells. HIF1’s activation has been shown to upregulate EMT within 18hrs of exposure to 1% oxygen and upregulation of angiogenesis related markers can be seen within 2 to 4 h . One stuIn this review we can deduce that 3D tumour models have shown to be a better representation of the in vivo tumour microenvironment regardless if they are scaffold based or rely upon cell-generated ECM deposition to develop 3D matrix, when compared to 2D cultures. Multicellular spheroids and ECM/scaffold-based tumour cultures have similarities in their shared three-dimensional nature in terms of presence of hypoxic regions and their effects on tumour growth and metastasis. Both scaffold and non-scaffold-based tumour cultures demonstrate that tumour cells play a significant role in the secretion of various ECM proteins such as collagen and fibronectin. There is significant data to suggest that non scaffold based in vitro cancer cell cultures, with an absence of extracellular ECM have a less significant effect on the biomimetic properties of the tumour models in terms of hypoxia driven gene expressions, cell ECM interaction driven growth and invasion, and malignant progression. The role of ECM in facilitating tumour invasion and migration is still not fully understood, however evidence has shown that ECM remodelling and infiltration of ECM is strongly primed by various soluble factors by the infiltrating tumour cells . This woHowever, when we analysed the studies exclusively exploring the role of 3D tumour models on EMT, the majority of studies involved scaffold based cultures in comparison with studies looking at biomimicry as a generic theme. ECM components such as collagens, fibronectins, and hyaluronic acid have been shown to play a crucial regulatory role in the EMT signalling pathways. Studies have shown growth factors such as TGF-B, which is present in or sequestered by ECM components, via the TBG-B-ECM axis increases expression of pathways for EMT execution in a dependent manner. Overall, ECM not only provides a structurally supportive role in cancer growth and invasion, but also ECM derived signals provide critical anchor points involved in EMT during the tumorigenesis process and the fine-tuned interaction between the ECM and tumour cells have a vital role in pathogenesis of cancer . TherefoCSCs have been isolated and characterised from numerous types of solid cancers such as breast, colorectal, and hepatocellular carcinomas. Their role in tumorigenesis and metastasis has been described in previous literature and their self-renewal, replicative and differentiative abilities via common signalling pathways have been hypothesised to be the basis for metastatic tumour growth . This reThe preference of native material over non-native material for scaffold based in vitro tumour models in drug resistance studies is the logical approach since the purpose of these experiments are for prediction of in vivo therapy response in an in vitro laboratory setting. Therefore, in order to recapitulate the natural in vivo tumour microenvironment, the choice of natively occurring scaffold material is an obvious preference.Response to all therapy classes conveyed a trend of increased resistance to therapy in 3D in vitro tumour cultures compared to 2D cultures. Furthermore, five out of six of the chemotherapy classes expressed congruent response of increased resistance to therapy in all experimental setups in 3D compared to 2D. Pro-apoptotic agents stood out showing almost even-handed response. We uncovered that 9/10 experimental setups expressing sensitivity to proapoptotic agents used the drug triapizamine. Tirapazamine, a hypoxia- activated agent, showed increased sensitivity in 3D tumour in all the studies which involved the drug. Due to the presence of intrinsic hypoxic cores in 3D tumour constructs, tirapazamine targets hypoxic regions of the tumour, therefore is highly sensitive in such tumour cultures compared to 2D tumour cultures which lack hypoxic regions . The tarThe proportional increase in chemosensitivity of prostate cancer and sarcomas can be accounted for by the multiple specific enzymes targeting novel chemotherapy agents tested in each of the publications compared to other cancer types studied in other publications. When sarcoma cell lines responses to novel targeted therapy DZNep compared to a classical chemotherapy agent cisplatin, all cell lines displayed chemosensitivity to DZNep and resistance to cisplatin . The limThe increased drug resistance in 3D tumour cultures compared to 2D culture systems is not only owing to its oxygen diffusion gradient. The presence of hypoxia is a well-known factor responsible for drug resistance via several difference mechanisms such as hypoxia induced G0 cell cycle arrest . The incNature Research stating invasion as “the ability of cells to become motile and to navigate through the ECM within a tissue or to infiltrate neighboring tissues“ [Cell migration in the non-malignant setting usually refers to the orchestrated movement of cells in a particular direction to a specific location . Cell intissues“ . Neverthtissues“ . TherefoIt is crucial to recognise possible reporting biases which may have resulted during the course of this comprehensive review. Publication, time lag and language biases were noted since this review deemed only full text publications up until 28 May 2020 in English language eligible for analysis. Outcome reporting bias was also taken into account and two independent investigators were involved in the data collection process separately, without influence from external parties. Nevertheless, taking into account of possible biases, this report should still show a relative comprehensive analysis of the topic due to the inclusive search and analysis methods.To conclude, this comprehensive systematic review has analysed in depth the published outcomes in existing literature of the role of 3D cell culture techniques and the corresponding physiological hypoxia on cancer cell behaviour, in particular focusing on growth, invasion, EMT and response to therapeutic interventions. Hypoxia level and duration is a crucial consideration when partaking in in vitro tumour hypoxia studies and must reflect the tissue oxygen concentrations. Whether to use scaffold-based tumour models should be carefully considered when exploring the effects of 3D tumour models on EMT and furthermore the choice of scaffolds in drug resistance studies highlights the importance for in vitro tumour models to recapitulate the in vivo tumour microenvironment. Migration and invasion are distinct but related processes both play crucial roles in cancer progression and metastasis. The development of and optimisation of in vitro 3D tumour models using a variety of microenvironments is essential in the study of cancer growth and progression, and advances in cancer treatments.


Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1.Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1.+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy.3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD83 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response. RT to the primary tumour in low-burden metastatic PCa may also provide benefit7 through RT-induced anti-tumour immune responses enhanced with iRT. Whilst iRT may improve tumour control, the evidence from pre-clinical PCa models investigating RT combined with adjuncts14 frequently use RT doses considerably higher than in clinical practice, such as 10, 20, or 25 Gy fractions. Standard clinical RT delivers 2 Gy fractions over 6 weeks to a dose of 74–78 Gy. Recent developments have seen moderate hypofractionation with 62 Gy in 20 fractions over 4 weeks, and ultrahypofractionation with 36.25 Gy in 5 fractions over 1–2 weeks, with no increase in acute toxicity.15 Although these studies suggest a change in practice with increased dose per fraction, and reduced overall numbers of fractions, the dose per fraction in clinical practice remains lower than those in pre-clinical iRT PCa experiments.Radiotherapy (RT) combined with androgen deprivation therapy (ADT) is a standard-of-care therapy for high risk localised or locally advanced prostate cancer (PCa).16 immunological effects19 and anti-tumour immune responses.23 Despite the observation of pro-inflammatory tumour effects following RT, the immunosuppressive tumour immune microenvironment (TIME) of pre-clinical models includes abundant tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). These myeloid-derived cells may impede RT-induced anti-tumour responses25 and promote tumour progression through growth factors, cytokines, chemokines and reactive oxygen species, increasing survival and growth of cancer cells. Although RT is a potential immunostimulatory anti-PCa therapy, with opportunities for combination with immunotherapy, it may be difficult to harness immune-mediated anti-tumour responses in PCa based on its moderate mutational burden and disappointing immunotherapy clinical trial results.28 However, the first FDA-approved immunotherapy, albeit without RT, for a solid-organ cancer was Sipuleucel-T in PCa,31 and immunomodulation may be effective if appropriately combined with RT. A clinical study combining anti-CTLA4 with a 8 Gy fraction to PCa bony metastases demonstrated a non-significant improvement in overall survival,33 and a trial investigating the efficacy of anti-PD-1 and RT in high-risk and oligometastatic PCa is underway.34Anti-tumour RT effects include DNA breaks causing apoptosis,To test the hypothesis that RT fractions used in the clinic induce PCa TIME effects, flank tumour allografts in immunocompetent mice were treated with RT and effects of RT investigated as a primary objective using tumour growth delay, flow cytometry (FACS) and NanoString immune gene expression changes. Effects of iRT with concomitant anti-PD-L1 were investigated as a secondary objective, testing the hypothesis that iRT increases anti-tumour effects.transgenic adenocarcinoma mouse prostate) model in C57BL/6 mice harbouring a construct comprising the minimal rat probasin promoter driving prostate-specific epithelial expression of SV40 large T antigen.36 MyC-CaP cells were derived from a c-myc transgenic mouse with prostate cancer.37 Both TRAMP-C1 and MyC-CaP cells express androgen receptor,38 however, TRAMP-C1 are androgen-independent whilst MyC-CaP cells are androgen-sensitive. TRAMP-C1 and MyC-CaP cells are useful for immunocompetent subcutaneous tumour models investigating therapies requiring an immune system prior to clinical trials. TRAMP-C1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 0.005 mg/ml bovine insulin, 10 nM dehydroisoandrosterone, 5% Nu-Serum IV, 5% foetal bovine serum (FBS), 1% penicillin and 100 μg/ml streptomycin. MyC-CaP cells were cultured in DMEM with 10% FBS, 1% penicillin and 100 μg/ml streptomycin. Cells were maintained mycoplasma free (LookOut® Mycoplasma PCR Detection Kit) at 37 °C and 5% CO2.TRAMP-C1 (ATCC® CRL-2730™) and MyC-CaP (ATCC® CRL-3255™) cells were purchased from American Type Culture Collection (ATCC®). TRAMP-C1 cells were derived from the TRAMP (b) and PD-L1 expression, and changes induced by γIFN. To test whether RT induces MHC-I and PD-L1 expression, TRAMP-C1 cells were treated with 2, 6 and 10 Gy RT using a Caesium irradiator at 1.46 Gy/min ± 1.0 ng/mL γIFN. After 72 h incubation cells were detached using Accutase solution, washed with PBS and stained with Pe-Cy7 H-2Kb , BV421-CD274  and LIVE/DEAD™ Fixable Green Dead Cell Stain Kit  or Propidium Iodide in 100 μl FACS buffer (PBS with 2% FBS). For western blot analysis, whole-cell extracts were prepared from PBS-washed cells using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using Pierce™ Rapid Gold BCA Protein Assay Kit (Thermoscientific) using a bovine serum albumin standard. Proteins were reduced at 95 oC with Laemmli gel loading buffer containing 2-mercaptoethanol, separated on 4–15% polyacrylamide gels containing SDS and transferred to PVDF membranes. Anti-PD-L1  and vinculin  primary antibodies were used, and membranes incubated with 1:5000 HRP conjugated secondary antibodies  and visualised with ECL detection reagent .40,000 TRAMP-C1 or MyC-CaP cells were plated in duplicate in 6-well plates and treated with 0, 0.1, 1.0 or 10 ng/mL mouse recombinant murine gamma interferon (γIFN) (Invitrogen PMC4034) for FACS quantification of MHC-I complex  were housed in groups of six to limit fighting, in a pathogen-free facility with 12-h light cycles, in individually ventilated cages on woodchip bedding, with access to water and food ad libitum, at 22 °C (range 21–24 °C) and 50% humidity (range 35–75%), with environmental enrichment and bedding material, and monitored for body weight changes twice weekly. TRAMP-C1 (2 × 106) or MyC-CaP (1.0 × 106) cells in PBS and 1:1 high concentration phenol red-free Matrigel (Corning) were injected into the flank of mice under isofluorane inhalational anaesthesia on a heat mat to aid recovery. Tumours were measured pre- and post-treatment using digital callipers three times weekly (tumour volume = π/6 × length × width × height). When tumours reached 100 mm3 mice were assigned to treatment groups on a ‘first come, first allocated’ basis using a randomly generated treatment list . RT was delivered in the afternoon under isofluorane inhalational anaesthesia with physiological monitoring  on a heat mat using a Gulmay 320 irradiator . 10 mg/kg anti-PD-L139  or isotype control  in PBS was administered by intra-peritoneal injection on days 1 (i.e. with the first 5 Gy RT fraction at 100 mm3 tumour size), 4, 7, and 10.39 All procedures were conducted in the home cage except tumour cell injection, RT and recovery from anaesthetic. All mice were culled by a schedule one procedure at end-point in the morning using pentobarbitone injection overdose followed by cervical dislocation according to institutional guidelines.Animal procedures were performed according to UK Animal law (Scientific Procedures Act 1986) and ARRIVE guidelines, with local ethics and Home Office approval. Naive male 6–8 week old immunocompetent C57BL/6 and FVB mice , 5 U/mL DNAse I (195 U/mL)  in Hanks Balanced Salt Solution and passed through a 70 µm nylon cell strainer. Red blood cells were removed using lysis buffer . Cells were incubated for 5 min at room temperature with purified anti-mouse CD16/CD32 Fc block  before the addition of cell surface antibodies. An eBiosciences FOXP3 intracellular staining kit  was used, and antibodies used for immune cell staining Table . Data weTM analysis software v2.5 for quality control and normalisation of gene transcripts using NanoString analysis guidelines and housekeeping genes.Total RNA was extracted from four 10 µm sections of formalin-fixed paraffin-embedded tissue samples using an RNeasy FFPE kit . Samples were analysed using the nCounter® mouse PanCancer Immune Profiling Panel, and data acquired with the nCounter® SPRINT profiler. Data were imported into nSolver3 to reach end-point size of 400 mm3 compared against control untreated tumours, a single mouse being considered an experimental unit. T-tests were performed for FACS data. All results are mean ± standard error of the mean, p < 0.05 being statistically significant.Statistical analysis was performed using GraphPad Prism 8 . For in vitro work ordinary one-way ANOVA tests were performed with Dunnett’s or Tukey’s post hoc adjustment for multiple comparisons. For in vivo tumour growth delay experiments, ordinary one-way ANOVA was performed using Tukey’s test for multiple comparisons following Brown-Forsythe’s test for equality of the means. Tumour growth delay was defined as a significant increase in time (days) for a tumour treated at 100 mmTo select PCa cells for iRT experiments in a syngeneic immunocompetent in vivo model, TRAMP-C1 and MyC-CaP cells were chosen for in vitro and in vivo analysis. As RT induces a γIFN response cells were initially treated with γIFN, and effects on MHC-I and PD-L1 analysed with FACS. In vitro treatment of TRAMP-C1 with γIFN increased MHC-I expression in a dose-dependent manner at 72 h. This effect was not observed in MyC-CaP cells, where baseline MHC-I expression remained low with increasing γIFN Fig. . IncreasTo assess the suitability of TRAMP-C1 for iRT experiments combining anti-PD-L1 and RT, MHC-I and PD-L1 expression was investigated in vitro following RT. MHC-I expression was increased in TRAMP-C1 at 72 h following 6Gy RT with 1 ng/mL γIFN, whereas expression of control transferrin receptor protein remained unchanged Fig. . A non-s3  in RT-treated tumours compared to control untreated tumours. A significantly reduced proportion of CD11b+Gr1+ expressing MDSCs was observed in 3 × 5 Gy RT-treated tumours compared to untreated controls  cells remained unchanged in 3 × 5 Gy RT-treated tumours compared to untreated control tumours , CD11b+Ly6C-Ly6G+ (p = 0.057) cells, and CD11b+Ly6G+ neutrophils (p = 0.070) in radio-recurrent tumours versus controls   Fig. . Correspols Fig. , even th+ cells, however a significant reduction in the proportion of CD45+CD3+CD4+ T-cells, and a non-significant reduction in the proportion of CD45+CD3+CD8+ T-cells (p = 0.08) was seen in treated versus untreated tumours  was seen in 3 × 5 Gy RT-treated tumours. The TAM cell population in MyC-CaP was M2-polarised in 3 × 5 Gy RT-treated and untreated control tumours. A non-significant increase in iNOS-expressing TAMs following 3 × 5 Gy RT in MyC-CaP was observed (data not shown), consistent with findings in TRAMP-C1. At the tumour regrowth end-point in MyC-CaP, TIME FACS analysis demonstrated no significant differences in the proportion of myeloid immune cell subtypes in 3 × 5 Gy RT-treated versus control tumours  or later time-points. It is possible that there is a relatively narrow therapeutic temporal window of opportunity for immunotherapy post-RT to be effective in highly aggressive immunosuppressive tumour models such as the autochthonous TRAMP model and our TRAMP-C1 flank allograft models, albeit these studies used different RT doses and fractionation. The TIME results from our experiments suggest that despite RT, the CD8+ T-cells which play important roles in the anti-tumour effects of ablative RT58 remain at low levels following 3 × 5 Gy RT, despite iRT with anti-PD-L1. This observation suggests that therapeutic strategies combining RT with T-cell-based therapies such as immune checkpoint blockade may not be clinically effective without first finding strategies to enhance T-cell immunosurveillance. Monocytes, in particular, play important roles in the immune evasion/tolerance of PCa,59 and it may be that immunotherapy directed at inhibiting TAMs or MDSCs recruitment to the TIME in combination with RT are needed to improve disease control in PCa.Previous experiments in autochthonous TRAMP models developing spontaneous PCa suggest that neither RT  nor immunotherapy alone (in the form of a tumour vaccine) can prime an anti-tumour immune response in animals with evolving tumours, but that iRT could result in anti-tumour T cell activation, although this effect was dependent on the relative timing of RT and immunotherapy.3 tumours, which we found to be 6–8 weeks in the conditions tested, led to changes in the immunogenicity of these tumours, perhaps with the development of immune tolerance in the animals, such that iRT was less effective. A variation in the efficacy of immunomodulation, dependent upon the timing of initiation of treatment following tumour implantation, and consistent with a decay of the initial immune response following tumour implantation has been reported.60 However, failure of delayed immunomodulation may be due to accumulation of a greater tumour burden or acquisition of an immunosuppressed TIME. RT may need to be continued for a greater number of fractions than 3 × 5 Gy to enhance effects of anti-PD-L1. This warrants further analysis, provided animals tolerate a higher number of RT fractions over several weeks, and given that RT is administered under general anaesthetic. An additional limitation of these experiments is the absence of concomitant ADT, given that this is administered with 2 Gy RT fractions as standard-of-care in patients. However, adding ADT would have introduced a third variable (in addition to RT and anti-PD-L1), and the TRAMP-C1 model is androgen independent. Moreover, recent clinical trials such as PACE (Prostate Advances in Comparative Evidence)15 are investigating hypofractionated RT without ADT versus conventional RT plus ADT. It may be the case that ADT plus RT leads to enhanced anti-tumour responses through addition of anti-PD-L1, and this possibility warrants investigation in future studies.These experiments have several limitations. Flank tumour allografts were used, rather than orthotopic tumours, or de novo PCa arising within transgenic mice, which might better recapitulate the TIME of spontaneously arising PCa. Standard implanted tumour models may not be ideal for iRT studies as they may fail to properly model the physiological setting in which tolerogenic tumours arise over extended time periods. However, the flank allograft tumour approach within immunocompetent syngeneic mice was necessary to deliver iRT with follow-up and measurement of tumour growth. It is possible that the length of time between subcutaneous injection of TRAMP-C1 cells and formation of 100 mm3 If iRT was successful then this may permit a reduction in the necessary RT dose for effective treatment, and could have clinical benefit for patients as RT reduces quality of life due to side effects.6 Immunotherapy is an attractive option combined with RT given recent clinical trial results where RT to the primary tumour in oligometastatic disease led to clinical benefit,7 suggesting possible RT-induced immunological responses which may be enhanced with iRT. However, for iRT to be successfully used in the clinic, studies need to be performed in pre-clinical models and early phase clinical trials to determine the optimal timing, sequence of delivery, and type of immunomodulation used in iRT.The possibility for an iRT approach to be beneficial in patients with PCa is an important idea to test given that the 5-year biochemical relapse rate for high-risk patients is 32–70%.In conclusion, 3 × 5 Gy hypofractionated RT resulted in a tumour growth delay in MyC-CaP and TRAMP-C1 PCa models. 3 × 5 Gy resulted in an initial increase in TAMs and DCs, and upregulation of PD-1/PD-L1, in the TRAMP-C1 TIME, however, anti-PD-L1 combined with RT in the conditions tested did not increase tumour growth delay. These results suggest that adjuncts beyond immunomodulation, such as perhaps combination with PCa focal ablation therapies or other cancer cell-targeting treatments, which warrants evaluation in future studies, are necessary to improve the anti-tumour response from RT fraction sizes used in the clinic.Supplementary figure legendsFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8Figure S9Figure S10Figure S11Table S1Table S2Table S3


Small cell lung cancer (SCLC) is characterised by high relapse rates. Tumour-initiating cells (TICs) are responsible for drug resistance and recurrence of cancer. Rovalpituzumab tesirine (Rova-T), a potent humanised antibody–drug conjugate, selectively targets delta-like protein 3, which is highly expressed in SCLC TICs. The experimental drug CBL0137 (CBL) inhibits the histone chaperone FACT (facilitates chromatin transcription), which is required for the expression of transcription factors that are essential for TIC maintenance. Rova-T and CBL each target SCLC TICs as single agents. However, acquired or intrinsic resistance to single agents is a major problem in cancer. Therefore, we investigated the potential effect of combining Rova-T and CBL in SCLC to eradicate TICs more effectively. Our preclinical studies report a novel and highly translatable therapeutic strategy of dual targeting TICs using Rova-T in combination with CBL to potentially increase survival of SCLC patients. Tumour-initiating cells (TICs) within most solid tumours, including SCLC,5 Rova-T is considered to be the first biomarker-directed treatment for SCLC and is particularly effective against TICs.5Rovalpituzumab tesirine (Rova-T) is an antibody–drug conjugate (ADC) that comprises a humanised anti-delta-like protein 3 (DLL3) monoclonal antibody attached to a DNA-damaging pyrrolobenzodiazepine toxin.7 Recently, we reported that CBL as a single agent preferentially targets TICs in SCLC and has potent anti-cancer activity against SCLCs when combined with cisplatin.8CBL0137 (CBL) targets FACT (facilitates chromatin transcription), a histone chaperone that is expressed at high levels in tumours and is required for the expression of transcription factors that are essential for TIC maintenance.Thus, the TIC-targeting mechanisms of CBL and Rova-T are entirely different, targeting two different proteins, FACT and DLL3, respectively, that are highly expressed in SCLC TICs. Here, we investigated the therapeutic efficacy of these drugs in combination using both in vitro and patient-derived xenograft (PDX) models of SCLC. PDXs are well recognised as predictors of human cancer biology and patient response to treatment. The objective of these preclinical studies was to report a novel and highly translatable therapeutic strategy of dual targeting TICs that can potentially increase survival of SCLC patients.high (TIC) and CD133low (non-TIC) cells were generated from NCI-H82 (H82) and NCI-H526 (H526) as described previously.8 The PDX tumour (JHU-LX102), derived from a chemotherapy-naive SCLC patient, was a generous gift from Dr. Charles M. Rudin . NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice, commonly known as the NOD scid IL-2 receptor gamma knockout (NSG) mice, were inoculated in both flanks with 50,000 tumour cells in each and randomised into cohorts of four animals per arm with mean starting tumour volumes of 100 mm3. Mice were treated with vehicle control for CBL + IgG control, Rova-T alone, CBL alone or the combination of Rova-T with CBL.8 Tumour volumes were measured three times a week until vehicle-treated mice reached ~1200 mm3, at which time all mice were euthanised using a gradient controlled CO2 inhalation, followed by cervical dislocation, and the tumours removed. In a second experiment, tumour-bearing mice were treated with the vehicle controls and the drugs as above, but now each group was treated until tumours reached the maximum size of ~1200 mm3. For in vivo limiting dilution assays, cell suspensions from residual tumours harvested from the tumorigenicity studies were inoculated subcutaneously in limiting dilutions (103–105) into naive NSG mice.9 The TIC frequency (TIF) was calculated using the ELDA software (http://bioinf.wehi.edu.au/software/elda/).10The CD1335 We observed a higher expression of DLL3 in TICs of H82 and H526 compared to non-TICs  after day 55 in the combination group , CBL alone (P < 0.05) or vehicle control (P < 0.05)  Fig. . We obse05) Fig. . We coul05) Fig. . Rova-T 05) Fig. , indicat5 and CBL8 as single agents reduce tumour-initiating capacity in vivo4 or in vitro.8 To determine whether Rova-T combined with CBL can reduce tumour recurrence by targeting TICs, we performed in vivo limiting dilution assays on residual tumours. Tumours derived from control mice were shown to have a TIF of 1:1424, which was reduced to 1:1961 and 1:2598 in CBL or Rova-T-treated mice, respectively. A substantial reduction in the TIF to 1:11,871 was observed in tumours derived from mice treated with the combination of Rova-T and CBL , and it has not yet exhibited dose-limiting toxicity. Therefore, using CBL in combination with Rova-T may add therapeutic value to disappointing recent results with Rova-T alone in SCLC,Supplementary material


Furthermore, the HPMC/T2/HA/hLF 1-11fd matrix allowed for the release of the drug in a simulated physiological environment, linked to an optimal hydration time, and the peptide antimicrobial activity was preserved for up to 15 months of storage, a very promising result considering the chemical liability of proteinaceous material. For its properties, the freeze-dried matrix developed in this study could be a good platform for the delivery of antimicrobial peptides in the precorneal area to treat infectious phenomena of the ocular surface.Infectious ocular keratitis is the leading cause of blindness worldwide. Bacterial resistance to classical pharmacological treatments raised the interest of researchers towards antimicrobial peptide (AMP)-based therapy. hLF 1-11, a synthetic antimicrobial peptide derived from the N-terminus of human lactoferrin, proved effective against different bacteria and yeast but, like all proteinaceous materials, it is unstable from chemical, physical, and biological points of view. In this study, new freeze-dried solid matrices containing mucoadhesive polymers were prepared and characterized in terms of rheology, hydration time, bioadhesion, drug content, and in vitro release. The formulation HPMC/T2/HA/hLF 1-11 Infectious keratitis is the leading cause of blindness worldwide. Delayed or inadequate treatment of this infection can lead to corneal perforation, which can evolve into endophthalmitis and complete vision loss. The WHO has estimated that corneal ulcers are responsible for 1.5–2.0 million new cases of monocular blindness every year ,4,5. TheCandida albicans, multidrug-resistant Staphylococcus aureus, and Acinetobacter baumannii strains  were calculated for the weights of the matrices belonging to two different batches for each developed formulation . CV rang−1, taking into account the weaker interactions of the macromolecules when the fluid is at rest, such as during the formation of adhesive bonds [To understand the influence of the freeze-drying conditions on the stability of the final product, the rheological characteristics were checked before and after lyophilization. All formulations showed pseudoplastic behavior and ve bonds .disp starting dispersion showed a viscosity of 227.7 ± 11.82 mPa*s. The presence of high molecular weight HA (1000–1800 kDa) produced a predictable increase in viscosity: HPMC/HA/VAisp gave the highest viscosity value (528.3 mPa*s) [disp, 321.2 mPa*s). After freeze-drying, the viscosity of the dispersions obtained from the reconstituted matrices tended to decrease, but only in the case of HPMC/HA/VAfd was the difference statistically significant . This behavior could be attributable to a partial depolymerization of HA during the lyophilization process [fd), directing the choice towards this formulation.The HPMC/VA3 mPa*s) . The add process . This phThe role of the lyophilization process on the stability of drugs was verified by determining the VA content of the final matrices. Drug recovery values (%), summarized in fd and HPMC/T2/VAfd, respectively. On the other hand, matrices HPMC/VAfd (WST: 12 min) and HPMC/HA/VAfd (WST: 14 min) needed 10 times longer than trehalose-based matrices to rehydrate. The replacement of mannitol with trehalose, highly soluble in water  appeareat 20 °C . Moreoveat 20 °C . Clearlyat 20 °C .2, for HPMC/VAfd was 327.0 ± 10.67 erg/cm2 and tended to increase when a part of the HPMC was replaced with HA (HPMC/HA/VAfd = 378.7 ± 21.56 erg/cm2), confirming the data in the literature about the mucoadhesive properties of hyaluronic acid, even if the differences were not statistically significant (p > 0.05). Moreover, these W values were statistically different from those obtained by putting into contact the two surfaces of mucin to determine mucin–mucin interactions (cohesion work: 244.0 ± 14.65 erg/cm2), demonstrating that the polymer/mucin adhesion and not the mucin/mucin cohesion was measured. Therefore, lower values than 244.0 ± 14.65 erg/cm2 would be found if the interaction/interpenetration between mucin and polymer molecules did not happen.Tests were performed by re-dissolving the matrices with a volume of 30 μL of water. The work of adhesion, erg/cmpow and HApow polymers showed a broad endothermic peak (more intense for HApow). In the case of HA, next to the transition at 172.54 °C ascribable to the dehydration process, there appeared an exothermal transition at 223.78 °C for the degradation phenomenon [Influence of the lyophilization process on the components of the formulations under study was verified by DSC analysis. The thermal transitions are graphically reported in enomenon . DSC of enomenon .fd), the lyophilization process appeared to modify the amorphous state, a typical feature of HPMC, since another thermal peak appeared at 145.28 °C for HPMCfd. The replacement of a part of HPMC with HA gave similar results for both the physical mixture and freeze-dried matrix.An interaction between HPMC and mannitol seemed to exist, since a widened peak to 158.36 °C, and a new endothermic transition onset at 208.35 °C were observed when the HPMC physical mixture was evaluated. In the case of freeze-died matrices (HPMCphmix), a transition peak at 93.31 °C appeared. Thermogram of pure trehalose revealed two peaks at 102.21 °C (sharp) and at 183.14 °C (wider); the first peak is attributable to the melting of the crystalline part of the dehydrated trehalose, while the latter peak can be associated with the melting of its anhydrous counterpart, which corresponds to the β-form of anhydrous trehalose [When part of mannitol had been replaced with trehalose in the physical mixture , while the introduction of trehalose appeared to modify the peak at 147.44 °C. The change in the thermal profile could suggest a close interaction among all components of the formulation.HPMC/T2/HAfd, 79.11% ± 17.02% for HPMC/HA/VAfd, 59.79% ± 1.51% for HPMC/T2/VAfd, and 59.54% ± 0.94% for HPMC/T2/HA/VAfd. Trehalose-containing matrices initially appeared to have a greater release, probably due to faster hydration as demonstrated by the value of WST ; afterwards they showed a slower release.The matrices under study were subjected to in vitro release studies using vertical diffusion cells (VDC); the amount of released drug through the period of the experiment (4 h) was determined by RP-HPLC. All the formulations showed the same drug-release profile  swelling, and consequent drug dissolution . When inFrom the preliminary studies described above and carried out with the model drug, the HPMC/T2/HA formulation appeared to be the most promising for the delivery of the antimicrobial peptide hLF 1-11 in terms of matrix chemico-physical characteristics, mucoadhesion, ability to preserve the integrity of the drug during the freeze-drying process, and good drug-release profile.fd) had a greater peptide recovery (86.74% ± 1.83 %) than the formulation containing only the bulking agent and polymers (HPMC/HA/hLF1-11fd: 68.38% ± 4.43 %), as previously found for the VA-loaded matrix and in a previous work for freeze-dried matrices containing antibodies [® 20 water solution to avoid unspecific surface adsorption by the peptide, given that it has been reported that Tween® 20 appears to absorb reversibly on very hydrophobic surfaces and to form a removable layer [HPMC/T2/HA/hLF 1-11 matrices were prepared by freeze drying. We evaluated the matrix integrity/alteration visually and by monitoring the changes in peptide content over different storage times by HPLC. The results were compared with the same formulation without trehalose (HPMC/HA/hLF 1-11) used as a reference to confirm its cryoprotectant activity towards the target drug. A good cake quality was obtained, with no fractures or cake collapses, as also seen with vancomycin. As shown in tibodies . The redtibodies , and thile layer .Moreover, a long-term drug stability study was performed on the selected formulation HPMC/T2/HA/hLF 1-11 by monitoring the change in drug recovery up to 6 months. The analysis of drugs in the samples was performed by RP-HPLC, which allowed for the detection of eventual impurities due to the degradation phenomenon. As shown in S. epidermidis was 1 μM, showing potency and bactericidal properties of the peptide.MIC and MBC of hLF 1-11 against hLF1-11 and the release of hLF 1-11 from the HPMC/T2/HA/hLF1-11fd matrix were assessed by an S. epidermidis ATCC 35,184 killing assay. As shown in S. epidermidis, which was comparable to hLF 1-11 solutions. Notably, a volume of 20 µL of the suspended matrix exerted the same antibacterial activity of a 6.4 µM solution of hLF 1-11, which resulted in a statistically significant 4-log reduction of cell viability. These results indicate that, when released from the matrix, hLF1-11 induces a dose-dependent reduction of S. epidermidis viability, demonstrating that the matrix maintains the antimicrobial properties of the peptide unaltered. This result appears relevant, since degradation of peptides can occur during release from delivery systems. When the solid hLF 1-11 is released from the matrix in biological fluid, it absorbs water. The polymeric matrix produces a viscous gel through which the peptide has to diffuse, but aggregation phenomena inside the device can also happen, affecting peptide release and bioavailability. Similar results were obtained using formulations prepared 1, 2, 6, and 15 months in advance.The inhibitory activity of Therefore, these results highlight the matrix’ capacity to release the peptide while preserving its antimicrobial activity, which is also maintained during a long-term storage of 15 months, underlying the ability of the matrix to maintain the original structure of the hLF 1-11 peptide.fd was also tested for preliminary in vitro release studies. In HPMC/T2/HA/hLF 1-11The research in the field of antimicrobial peptides and novel approaches for the treatment of infections has mainly focused on identifying more powerful and selective AMPs. However, a deeper knowledge on the delivery of these molecules, considering the problems associated with an efficient and safe release of AMPs, is essential. In fact, with an appropriate drug-delivery system, it is possible to obtain a reduction of the chemical or biological degradation of the AMPs, either in the formulation or after administration, and decrease adverse side effects and control the AMPs’ release rate.Different vehicles have been developed for the administration of peptides; newer ones are systems that exploit metallic or polymer nanoparticles or microgels/nanogels or surfactant self-assembling systems to control peptide exposure and release as well as to protect incorporated peptides from degradation. Although there are good opportunities for success, numerous issues still remain poorly understood, especially regarding the factors that control the load of the peptide into and the release from the various drug-delivery systems .fd was selected for the delivery of the hLF 1-11 since it showed good drug recovery and no chemical degradation for up to 6 months (long-term stability). Furthermore, the HPMC/T2/HA/hLF 1-11fd matrix allowed for the release of the drug in a simulated physiological environment, linked to an optimal hydration time, and the peptide antimicrobial activity was preserved for up to 15 months of storage. This is a very promising result considering the chemical liability of proteinaceous material. For its properties, the freeze-dried matrix developed in this study could be a good platform for the delivery of antimicrobial peptides in the precorneal area to treat infectious phenomena of the ocular surface. These polymers were chosen on the basis of the results obtained by Burgalassi et al. 2018 [Since an optimal vehicle does not seem to have been found yet, in this study, new solid matrices containing mucoadhesive polymers obtained by freeze-drying were prepared and characterized. The formulation HPMC/T2/HA/hLF 1-11al. 2018  who had Moreover, the freeze-drying technology allowed us to obtain a peptide-loaded solid device, overcoming, for example, the alteration of the proteinaceous material following the use of other matrix manufacturing processes, such as the solvent casting, where high temperature or mechanical impacts could destroy the peptide structure.Emerging technologies represent an opportunity to regulate peptide release and minimize the need for repeated administrations. This research opens new perspectives for ocular topical administration of antimicrobial peptides, providing the basis for technological and biopharmaceutical optimization of the developed formulation.The formulation could be improved to provide a combined therapy, releasing more than one drug at the same time. In addition, its texture and design could be modified to facilitate administration with the aid of a device, since freeze-dried materials are fragile and may be damaged during manipulated by the patient. Combination therapy might represent a promising option, as synergistic effects would allow for the use of lower doses of both compounds, thus reducing the frequency of emergent drug-resistant strains. Further studies will be performed to evaluate whether the previously described synergistic effects obtained by combining hLF 1-11 with various antibiotics  or antif


The tripartite mission of land-grant universities  continues to produce astounding numbers of college graduates, inventions, and discoveries.Land-grant universities wishing to assert the benefits of a college degree must become more efficient, increase teaching excellence, engage with community stakeholders, conduct research that matters, describe how activities impact local needs, and refocus on affordability and accessibility.Additional public investment in animal science research, teaching, and extension activities are urgently needed.Animal scientists should consider how connected they are to the mission of land-grant universities that speaks directly to the 21st century needs of its partners and stakeholders.The year 2012 marked the sesquicentennial celebration of the Morrill Act of 1862 , the conDuring the same year that the nation was celebrating the sesquicentennial anniversary of the Morrill Land-Grant Act, the American Society of Animal Science (ASAS) implemented Innovate 2012 , an initAnimal Frontiers, the official journal of ASAS. This literature has included an examination of data pertaining to students who have attended land-grant university animal science programs  of the land-grant university, In 2011, then Nokia CEO Stephen Elop sent to his employees what has come to be known as his “burning platform” memo . In thisHere, we see a CEO signaling that his company was ablaze with challenges that required a course of action on the part of his employees that went far beyond what would be considered typical and usual. So, one must ask, are land-grant universities similarly standing on a burning platform at this moment in history? In response to this decided decline in public support, This essay closes with a question: If animal scientists took a good look around right now, would they come to find themselves standing on a burning platform as well? Even a cursory examination of the goals and objectives related to the Innovate 2012 initiative would seem to provide at least some clues to that effect. Perhaps more substantially, the call made by


Osteosarcoma (OS) originates in the skeletal system and has a rising global incidence. Long Non-coding RNAs (lncRNAs) are key regulators of human cancers development and progression. However, their roles in the development of OS are not well understood. This research aimed to investigate the effect of a long non-coding RNA (lncRNA), MRUL, on OS and revealed its potential molecular mechanisms. The bioinformatics analysis demonstrated that lncRNA MRUL was involved in regulating nucleic acid-templated transcription, cellular macromolecule biosynthetic process, immune response, and inflammatory response. In this work, the expression of lncRNA MRUL was detected by quantitative real-time polymerase chain reaction (qRT-RCR) in both cancer tissues and cell lines. We found that lncRNA MRUL was up-regulated in cancer tissues and cell lines. Functional experiments showed that knockdown of lncRNA MRUL inhibited OS cell proliferation, and metastasis. At the same time, we found that lncRNA MRUL interacted with miR-125a-5p to suppress FUT4 expression. Moreover, inhibition of miR-125a-5p abrogated the biological roles of lncRNA MRUL knockdown on OS cell proliferation, migration, and invasion. In conclusion, these results demonstrated that OS-upregulated lncRNA MRUL promoted cell proliferation, and metastasis via negatively regulating miR-125a-5p, and imply that lncRNA MRUL may be a potential biomarker for OS. Long non-coding RNAs are linear RNAs that contain over 200 nucleotides in length . It accoMicroRNA (miRNA) is a class of single-strand non-coding RNAs . MicroRNIn vitro experiments in cell lines of OS, lncRNA UCA1 knockdown inhibited cell proliferation, invasion and migration, and accelerated cell apoptosis, while lncRNA-UCA1 overexpression had the opposite effects  was used to evaluated the PPI information of LncRNA MRUL co-expressing genes w. Cytoscape software (version 3.6.1) was used to constructed the PPI networks.2.The normal human bone cell line hFOB 1.19, the OS cell lines SW1353, U2OS, and MG-63 were from ATCC. We cultured cells in DMEM , supplemented them with 10% FBS  and maintained them in a humidified incubator at 37°C with 5% COMRUL siRNA and Negative siRNA were synthesized by the Shanghai GenePharma Co. OS cells were transfected with siRNAs by Lipofectamine 2000 . MRUL siRNA-1 5′–GGCCUUUGUUUGCAGUUUATT–3′; MRUL siRNA-2: 5′–UUUCUACUGUUACUGUGUCTT–3′; Negative siRNA: 5′–UUCUCCGAACGUGUCACGUTT–3′.We follow the cell fractionation assay according to the previous report .TM luciferase reporter vector at the XhoI and NotI sites. The mutant sequence of lncRNA MRUL or FUT4 at the 3′UTR was also built into the vector.In order to build luciferase reporter vectors, the whole lncRNA MRUL or the 3′UTR fragment of FUT4 that contains the expected latent binding sites, were cloned into the pmiR-RB-REPORTAs for luciferase activity assay, we applied Lipofectamine 2000 (Invitrogen) to co-transfect each construct with marked miRNAs (RiboBio) in OS cells for 48h. They were performed with the Dual-Luciferase Reporter Assay System with the instruction of the manufacturer. Moreover, we quantified the Luminescent signals by BioTek Synergy HTX multi-mode reader, and presented the luciferase activities by relative hRluc/hluc ratio.TM 6 Flex manufactured by Thermo Fisher Scientific (United States). Furthermore, Sangon  was used to construct all the primer sequences. MRUL(F): 5′–ACCCACAGACAACTGTGGACCC–3′, MRUL(R): 5′–GCCGCCCCTATTGTTGCCCA–3′; GAPDH (F): 5′–ACCCAGAAGACTGTGGAGG–3′, GAPDH (R): 5′–TTCTAGACGGCAGGTCAGGT–3′. The reference gene for lncRNA and mRNA was chosen as GAPDH. The internal control for miRNA was set as U6. We also applied the 2–ΔΔCt method to quantify the gene expression.We extracted total RNA from tissues and cells with TRIzol reagent . For lncRNA and mRNA, the reverse transcription of total RNA to cDNA was performed by Reverse Transcriptase . Then we conducted qPCR with a SYBR Green PCR Kit . The fluorescence quantitative PCR instrument was QuantStudio3) in 96-well plates and treated them with 10 μl CCK-8 solution for 2 h, and then analyzed the spectrophotometrically at 450 nM by automatic microplate reader .We assessed OS cells proliferative ability with the CCK-8 assay according to the manufacturer’s instruction. Next, we plated SW1353 and U2OS cells , nucleic acid-templated transcription (GO:1903506), transcription (GO:0006355), gene expression (GO:0010468), histone H3-K4 methylation (GO:0051568) . And lncKEGG pathway analysis revealed that lncRNA MRUL positively co-expressing genes were involved in regulating Lysine degradation, TGF-β signaling pathway, signaling pathways regulating pluripotency of stem cells, Fanconi anemia pathway, Taste transduction, Spliceosome, RNA degradation, Homologous recombination, mRNA surveillance pathway, other glycan degradation, and Cell cycle . LncRNA In order to reveal the interaction among lncRNA MRUL positively and negatively correlated genes, we constructed PPI networks using STRING database. As present in By qRT-PCR, lncRNA MRUL expression in OS tissues was significantly higher than in normal tissues . BesidesTo explore the impact of lncRNA MRUL on the biological function of OS cells, we use siRNAs (siMRULs)  to silenWe had constructed lncRNA MRUL knockdown cell lines by transfecting cells with the siMRULs and detected the efficiency by the transwell experiments. It could be seen that the migration and invasion capabilities of these lncRNA MRUL knockdown SW1353 , 8A,B anIt was found that the expression of target genes was regulated by a growing number of lncRNA via sponging miRNA. The bioinformatics analysis showed that miR-125a-5p could be sponged by lncRNA MRUL. Besides, lncRNA MRUL knockdown was found to significantly upregulate the expression of miR-125a-5p and down-regulate FUT4 in SW1353 and U2OS cells with qRT-PCR . On the In vitro experiments, siMRULs were used to silence its expression in SW1353 and U2OS cell lines. The results indicated that lncRNA MRUL knockdown reduced the proliferation ability of SW1353 and U2OS cells, and inhibited the migration and invasion ability of OS cells in vitro. To investigate the regulatory mechanism of lncRNA MRUL in OS, we found that lncRNA MRUL promoted the progress of OS cells by regulating the miR-125a-5p/FUT4 axis.This present study explored the potential functions of lncRNA MRUL in OS using bioinformatics analysis and functional validation. The bioinformatics analysis demonstrated that lncRNA MRUL was involved in regulating cellular macromolecule biosynthetic process, nucleic acid-templated transcription, immune response, and inflammatory response. Through qRT-PCR, the expression of lncRNA MRUL in cancer tissues was found to be significantly more than that in normal tissues. In recent studies, it is shown that lncRNAs are involved in multiple processes in cancer progression. They can interact with DNA, RNA and protein molecules, as well as complexes of DNA, RNA and proteins to enhance or inhibit the functions of these molecules and play a significant role in gene expression, which enhances the ability of cancer cells to differentiate, grow and metastasize . LncRNAsThere is growing evidence that dysregulation of microRNAs (miRNAs) are involved in the progression of cancer. By analyzing the ectopic expression and target sites and of HCC cells, it was found that miR-125b and miR-125a-5p inhibited the expression of cyclin D1 and SIRT7, but induced p21WAF1/Cip1−dependent G1 cell cycle arrest . Sirtuinin vitro assays. Therefore, the in vivo assays should also be conducted to further validate our findings.Several limitations also existed in this study. First, despite the expression levels of lncRNA MRUL were detected in a small sample size OS samples, the correlation between the expression of MRUL, miR-125a-5p, and FUT4 and clinical parameters should be further validated using a bigger sample size, which could be helpful for us to understand the functional importance of this axis. Second, the findings of this study were concluded with In conclusion, we demonstrated that the downregulation of lncRNA MRUL significantly up-regulated the expression of miR-125a-5p but suppressed the corresponding target gene FUT4, and inhibited the occurrence and invasion of OS. The results showed that lncRNA MRUL could be a promising target to treat the disease of OS.All datasets generated for this study are included in the article/SL and DM designed the research. CT and XS performed the research and wrote the manuscript. KH and HZ helped in the analysis of the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To the Editor:The optimal approach to fluid resuscitation for patients with sepsis is uncertain. Data from sub-Saharan Africa have indicated potential for harm from aggressive treatment with intravenous fluids , 2. NeveThe role of lung ultrasonography for guiding fluid management for patients with sepsis is undefined. Sonographic B-lines correlate with measurements of extravascular lung water –5. IncreWe recruited a cohort of 70 patients who presented to the Queen Elizabeth Central Hospital in Blantyre, Malawi, with suspected sepsis. The aims of the study were to estimate the effect of intravenous fluid treatment on lung ultrasound findings and to examine the relationship between lung ultrasound findings and the development of hypoxemia. Some results were reported previously in an abstract .a.m. and 4 p.m. Suspected sepsis was defined as an abnormal temperature measurement (≥38.0°C or <36.0°C) or symptoms of fevers, chills, or rigors in the preceding week, and evidence of hemodynamic instability . Written informed consent was provided by participants or their surrogates. Prisoners were excluded, as were any patients who had received more than 2 L intravenous fluid prior to screening. Ethical approval was granted by the Malawi College of Medicine (P.04/18/2385) and by the University of California San Francisco (227029).Participants were a convenience sample of adults over 18 years old who presented to the Queen Elizabeth Central Hospital with suspected sepsis during weekdays, recruited between 7 Eight-zone lung ultrasonography was performed by an experienced operator (R.J.W. or P.D.) using a Philips S4-1 Lumify phased array transducer at enrollment, and again at 3, 6, 24, 48, and 72 hours. While participants were supine, 4-second video clips were recorded at each quadrant of the hemithorax for a total of eight clips per exam. Clips were reviewed offline by an experienced reader (R.J.W.) at a later time, and a B-line score was generated for each exam. B-lines are hyperechoic artifacts that extend from the pleura through the depth of the image. The maximal number of B-lines visualized in a single frame for each clip was summed to generate a B-line score .Vital signs, including pulse oximetry on room air, were measured at each time point. The volume of intravenous fluid given between each time point was recorded. Clinical decisions regarding intravenous fluids were made by each participant’s clinical team strictly without input from research personnel.To account for repeated measures, mixed-effects regression was used to model the relationship between the volume of intravenous fluid treatment and the B-line score at any time point, and between the B-line score and oxygen saturation. To test whether the baseline B-line score predicted impending hypoxemia, analysis was restricted to the subset of participants who were normoxemic—had an oxygen saturation of 90% or greater while breathing room air—at baseline. Simple logistic regression was used to model the baseline B-line score as the predictor and the development of hypoxemia—any measurement of oxygen saturation <90% while breathing room air—in the first 72 hours of hospitalization as the outcome. To assess the performance of the baseline B-line score for predicting impending hypoxemia, the area under the receiver operating curve was calculated, with 1,000 bootstrap replications to obtain bias-corrected 95% confidence intervals (CIs).Clinical characteristics for the 70 participants enrolled in the study from July 18, 2018, through October 26, 2018, are shown in P < 0.001).The volume of intravenous fluid treatment was significantly associated with the number of B-lines detected. Within the first 72 hours of hospitalization, every additional liter of intravenous fluid treatment was associated with approximately one additional B-line .The number of B-lines detected was significantly associated with oxygen saturation. For every additional B-line detected, a participant’s oxygen saturation was decreased by 0.24%  times the odds of developing hypoxemia within the subsequent 72 hours. The area under the receiver operating characteristic curve (Among the 39 participants who were normoxemic on presentation, the baseline B-line score predicted the development of hypoxemia within 72 hours of enrollment. Every additional B-line detected at baseline was associated with 1.5 (95% CI, 1.1–2.0; ic curve  was 0.86In this study, we found a significant association between the volume of intravenous fluid received and the number of B-lines subsequently detected with lung ultrasonography. The number of B-lines detected is significantly associated with a concurrent measurement of oxygen saturation. Lastly, the baseline lung ultrasound exam might identify normoxemic patients who are at increased risk of developing hypoxemia within the first 72 hours of hospitalization. Because this is an observational study, these findings suggest, but do not establish, a potential role for using lung ultrasonography to assess fluid tolerance in patients with sepsis.An important strength of this study is that it was performed in a hospital in Malawi, where there is a paucity of—and, perhaps, the greatest need for—data on fluid resuscitation in patients with suspected sepsis. A limitation of this study is the small sample size. A further limitation of this study is that the ultrasound exams were scored by a single experienced reader who had access to the full data set and, thus, was not formally blinded to data on intravenous fluid volume or oxygen saturation.Despite these limitations, we believe the results of this study attest to the relationship between intravenous fluid treatment, sonographic B-lines, and clinical outcomes, such as impaired oxygenation. Testing an ultrasound-guided fluid resuscitation strategy in sepsis warrants further investigation.


We first analyzed the radioactive uptake of positron emission tomography (PET) tracers in BAT of CT26 xenograft mice with 18F-fluorodeoxyglucose ([18F]FDG) and [18F]DPA714. We also studied the BAT uptake of [18F]DPA714 in CT26, A549 and LLC tumor models. The dynamic distribution of [18F]FDG is quite variable among animals, even in mice of the same tumor model . Contrarily, [18F]DPA714 produced high-quality and stable BAT imaging in different tumor models and different animals of the same model. Interestingly, %ID/g-mean of [18F]DPA714 in BAT was significantly higher on day 26 than that on day 7 in CT26 xenograft model. Taken together, these results strongly indicate the potential feasibility of [18F]DPA714 PET imaging in investigating BAT and energy metabolism during tumor progression in preclinical and clinical study.Brown adipose tissue (BAT) is important in monitoring energy homeostasis and cancer cachexia. Different from white adipose tissue, BAT is characterized by the presence of a large number of mitochondria in adipocytes. Translocator protein 18 kDa (TSPO), a critical transporter, is expressed in the outer membrane of mitochondria. We speculated that [ Numerou18F-fluorodeoxyglucose ([18F]FDG) positron emission tomography (PET) is the main approach to monitor BAT activity, and it is based on the high expression of glucose transporter in BAT [18F]FDG PET are quite discrepant and vulnerable to environmental influences DPA [18F]DPA714, which is derived from the nature ligand of TSPO and has been used in clinical trials, is a highly TSPO specific and practically mature PET tracer [18F]FDG and [18F]DPA714 as PET tracers in the imaging of BAT has not been reported.Translocator protein 18 kDa (TSPO), expressed in the outer membrane of mitochondria, is a reliable inflammatory biomarker and related to many human diseases, including metastatic cancer, inflammatory and neurological diseases (such as Alzheimer’s disease and Parkinson’s disease) . An impo[18F]DPA ,20. [18FT tracer . However18F]FDG and [18F]DPA714 in BAT of the same tumour model. Then, we investigated the performance of [18F]DPA714 in the BAT of different tumour-bearing mice that may result in cachexia. We also studied the uptake of [18F]DPA714 in BAT at different stages of the tumour model.In this study, we first compared the PET imaging effects and applicability of FDG was performed as classic studies [18F]DPA714 was similar to the reported protocol in literature DPA714 as the standard reference, and collect the target peak. The final radiochemical purities of products [18F]FDG and [18F]DPA714 were both >95%.The commercial  studies . The radterature . Simply,18F]FDG were conducted at 45 min after injection, and the microPET scans (15 min per mouse) were performed at 60 min  after [18F]DPA714 administration. After scanning, data reconstruction  was handled by the Inveon Acquisition Workplace (IAW) software (Siemens Inveon). The regions of interest (ROIs) were delineated in the reconstructed images by manual selection of the statistical investigator who was blind to experimental design and grouping. PMOD software (PMOD Technologies Ltd) was used for analysing the data of radioactive uptake (%ID/g) in the ROIs after reconstruction and quantification.All tumour-bearing mice were scanned with microPET . During the scan, the mice were anesthetized continuously by inhalation of 2% isoflurane. All animals were given (100 ± 10) μCi of radiolabeled tracer by tail vein injection. Static whole-body microPET scans (10 min per mouse) for FDG PET imaging is a classic imaging method to detect the BAT activity. In this research, taking [18F]FDG imaging as control, we comparatively studied the differences between [18F]DPA714 and [18F]FDG PET imaging for the monitoring of BAT activity in the same tumour-bearing mouse model. When the tumour volumes of CT26 model increased to about 500 mm3, [18F]FDG scanning was performed on the first day and [18F]DPA714 scanning was performed on the third day. As shown in 18F]FDG in BAT of the scapular region in the CT26 tumour-bearing animals varied dramatically. Of the sixteen mice scanned , seven had significant BAT uptake in the scapular region , while the BAT uptake of [18F]FDG in the other nine animals was not obvious . Moreover, in accordance with previous studies, our PET imaging showed that [18F]FDG was mainly distributed in the heart, liver, brain and tumour. The data summarized from the results of CT26 tumour uptake of [18F]FDG are shown in S Table 1 in the Supplemental Information.[18F]DPA714 in mice were significantly different from that of [18F]FDG. In the same CT26 xenograft model mice, the BAT uptake of [18F]DPA714  in the scapular region was significantly higher than that of [18F]FDG . The uptake values of [18F]DPA714 (%ID/g-mean) at different time points in six organs (including tumour) in CT26 tumour-bearing mice were calculated and presented in supplemental S 18F]DPA714 in tumour tissue was relatively lower at all the time points.Interestingly, as shown in 18F]DPA714 in different kinds of tumour-bearing models. Considering the representativeness and differences of species and location, we chose CT26, A549 and LLC tumour models (n = 6 mice for each model group) for the study of the usability characteristics of [18F]DPA714 as a PET tracer for BAT monitoring. The average tumour volume of each group was (500 ± 100) mm3 at the scanning time point. As shown in 18F]DPA714 in the scapular region was all relatively high in the three tumour-bearing models .Then, in order to study the stability and durability in application of the tracer in BAT imaging, we investigated the variation of the uptake of [18F]DPA714 tracer in CT26 xenograft mice. We performed PET scans at day 7 and day 26 after tumour cell injection by [18F]DPA714 tracing. Similar to the previous findings, %ID/g-mean and %ID/g-max of the radioactive uptake of [18F]DPA714 in the scapular region were both the highest among all the organs (or ROIs)   ,c). For 18F]DPA714 for the monitoring of BAT in the CT26 colorectal cancer model, compared with the commonly used PET tracer [18F]FDG. As a specific ligand of TSPO protein, [18F]DPA714 showed a more stable and sensitive imaging of the BAT (which is rich in mitochondria and TSPO) than [18F]FDG in CT26 xenograft mice. Then, [18F]DPA714 PET also showed good quality imaging for BAT in the A549 and LLC tumour models. Furthermore, with the development of tumour, uptake of [18F]DPA714 by BAT in the late stage was significantly higher than that in early stage.In this study, we firstly evaluated the imaging efficacy of [18F]FDG PET imaging is the gold standard for the study of BAT activity in vivo [18F]FDG in BAT is relatively little. The imaging results of [18F]FDG PET depend on the imaging conditions and the stimulated activation level of BAT. The BAT [18F]FDG uptake can not only be affected by fasting status, diabetes, muscle activity and drugs (such as β-receptor blockers), but also related to the population characteristics , temperature and seasons FEPPA, can be used for the imaging of BAT and to detect the browning of WAT [18F]DPA714, a mitochondrial TSPO-based imaging tracer.A large number of fat droplets and high concentration of mitochondria are typical characteristics of BAT. At present, most of the researches on BAT are focused on uncoupling proteins 1 (UCP1), expressed on mitochondrial inner membrane . In factg of WAT ,25. HoweNowadays, most of the studies on BAT have paid their attention to energy expenditure, obesity, insulin resistance and other energy metabolic disorders ,26,27. S18F]DPA714 in BAT in the late stage of tumour was significantly higher than that in the early stage in the same tumour model. In patients with malignant tumours, the long-term presence of tumour tissue will cause chronic inflammation of the body. Inflammatory factors such as IL-6 and PTHrP, secreted by tumour tissues and mainly released by stimulating sympathetic nervous system and norepinephrine, act on β3-adrenergic receptor in adipose tissues and further activate the expression of related transcription factors and thermogenes such as UCP1, and induce the browning of WAT DPA714 is an ideal molecular imaging tracer to study the effect of tumour on the BAT activity and WAT browning. The safety of this tracer has been confirmed in several clinical subjects, and we also studied and confirmed that [18F]DPA714 can be used as a PET imaging tool for early warning and intervention efficacy evaluation of cachexia. We will further research the role of BAT in different tumour progression with this PET imaging tracer in the future preclinical and clinical studies.In conclusion, [Click here for additional data file.


Additional modification of glycogen polymers with poly led to a reduction in the elimination rate and lower uptake in internal organs . Our results highlight the potential of multimodal glycogen-based nanopolymers as a carrier for drug delivery systems in tumour diagnosis and treatment.As a natural polysaccharide polymer, glycogen possesses suitable properties for use as a nanoparticle carrier in cancer theranostics. Not only it is inherently biocompatible, it can also be easily chemically modified with various moieties. Synthetic glycogen conjugates can passively accumulate in tumours due to enhanced permeability of tumour vessels and limited lymphatic drainage (the EPR effect). For this study, we developed and examined a glycogen-based carrier containing a gadolinium chelate and near-infrared fluorescent dye. Our aim was to monitor biodistribution and accumulation in tumour-bearing rats using magnetic resonance and fluorescence imaging. Our data clearly show that these conjugates possess suitable imaging and tumour-targeting properties, and are safe under both  Therefore, a safe drug delivery system capable of accumulating and providing controlled drug release at the tumour site is highly desirable. Nanoparticles represent a versatile carrier for drug delivery due to their ability to passively accumulate in solid tumours. This occurs via the enhanced permeability and retention (EPR) effect, a relatively universal phenomenon whereby nano-sized structures (up to approx. 200 nm in size) become entrapped in tumour tissue due to either leaky endothelia or aberrant tumour vessel architecture. Restricted or even completely absent lymphatic drainage in tumours limits the release and passive accumulation of compounds3. Nanotherapeutic carrier can accumulate in tumours via the EPR effect without any targeting ligand6. Their size above the renal threshold (molecular weight > ca 45 kDa/hydrodynamic diameter > 8 nm) leads to prolonged circulation in the bloodstream increasing the probability of accumulation at the target site3. Importantly, polymers can control the release of drugs, being composed of a structure responsive to external stimuli, such as pH changes in acidic cancer-cell environments7, the presence of enzymes, and redox potential9. Thus to minimise the drug release in the bloodstream and the cytotoxic effect on healthy cells12.The majority of anticancer drugs are highly toxic and adversely affect healthy cells due to a lack of specific targeting13. However, this can be mitigated by surface modification: for instance, grafting nanoparticles with poly(ethylene oxide) (PEO)  – PEG) is a well-established procedure for reducing nanoparticle toxicity, prolonging circulation in the bloodstream, and minimising interactions with the immune system14. On the other hand, PEO also has the potential to be both immunogenic and antigenic and degrading to produce reactive oxygen species16. Therefore, alternative polymers such as poly(2-oxazolines) (POx) are currently the subject of extensive testing18. POx are significantly more stable compared to PEO20, retain their non-fouling (protein-repellent) potential, have low unspecific organ deposition and do not exhibit immunogenicity22. This so-called “stealth” effect leads to the prolonged circulation of coated nanoparticles in the blood and increases the likelihood of accumulation in tumour tissue via the EPR effect22.One limitation of nanoparticles is that they tend to opsonise, clearing quickly through the reticuloendothelial system in the liver and spleen23, synthetic polymers27 and carbon nanotubes28, natural carbohydrate polymers such as polysaccharides are versatile and unique carriers due to their high biocompatibility, biodegradability, and availability32. Polysaccharides can also be easily modified to control drug release while incorporating both diagnostic and therapeutic entities. Polysaccharide surfaces can also be tailored with ligands for more specific targeted delivery, e.g. using folate to bind to overexpressed folate receptors on cancer cells33.In addition to other tried-and-tested drug delivery systems such as proteins34, dextran36, chitosan38, pectin42, starch43 or hyaluronic acid45. However, only a few studies have explored the potential of glycogen46. Glycogen is a natural hyperbranched polymer of suitable size for the EPR effect  and above the renal threshold, it is degraded into D-glucose by intracellular enzymes and further metabolized by normal physiological glycolysis46. Moreover, it is biocompatible, biodegradable47, widely available as a natural renewable resource 49, and can be easily modified with drugs or diagnostic moieties, what makes glycogen ideally suited for use in the construction of a multimodal drug delivery system50.Most studies of polysaccharide drug delivery systems use alginatein vivo conditions on an animal tumor model of hepatocellular carcinoma (HUH7 human cancer cell line). Glycogen was modified with two imaging moieties: a gadolinium chelate for magnetic resonance (MR) and a near-infrared fluorescent dye for fluorescence imaging. A multimodal agent was deemed most favourable given each method provides a different set of information on biodistribution, accumulation, and clearance of probes under in vivo conditions. In order to enhance tumour-targeting properties and eliminate any possible adverse effects on other organs, the polymers were modified with POx. Using tumour-bearing rats, we evaluated imaging properties, biodistribution, safety, in vivo accumulation, and clearance. To the best of our knowledge, this is the first demonstration of imaging potential of these contrast agents on in vivo animal model.In the absence of evidence to indicate glycogen is efficient as a nanocarrier in a clinically relevant model such as a tumour-bearing animal, we performed a detailed characterisation of a modified glycogen carrier under 1-weighted MR signals and corresponding contrast-to-noise ratio (CNR) values were linearly dependent on concentration of Gd3+ in the conjugates  was higher compared to the GM (4.2 ± 0.07 mM−1s−1) and GOX (3.0 ± 0.16 mM−1s−1) probes  emitted no fluorescence signal after excitation  and glycogen-based conjugates without POx (GG). A commercially available MR contrast agent gadoterate meglumine (GM) served as a control. MR properties of the conjugates were examined by MR imaging at 4.7 T and relaxometry at 0.5 T. Ttes Fig. , with thbes Fig. . Relaxivs−1 Fig. . The higion Fig. . The fluin vivo imaging , we carried out histological analysis of tumour tissues. Histology excluded the presence of fibrosis and steatosis, only necrosis was found in tumour centres.After intravenous administration of conjugates, we assessed biodistribution and accumulation of the glycogen-based probes (GG and GOX) and control agent (GM) using MR and fluorescence ing Fig. . The MR ing Fig. . The higing Fig. . The GOXing Fig. . The higing Fig. ; the CNRing Fig. . The siging Fig. . To confin vivo imaging  Fig. . The higDue to the attenuation of fluorescence in deeper tissues, probe uptake in the kidneys was analysed by MRI only. Signals from the GM control and modified glycogen-based GOX conjugate decreased to initial values 2 h after injection. The GG signal increased above the pre-injection value throughout the whole examination even at the lowest probe concentration Fig.  p < 0.0. The sigin vivo imaging, ex vivo fluorescence analysis of the internal organs  was performed at two time points: day 2 and day 7.Fluorescence imaging is limited by its attenuation and refraction of optical signals in biological tissues, particularly in deeper structures. Therefore, to assess probe accumulation more precisely and to confirm the results of in vivo MRI data, ex vivo analysis revealed GG continuously accumulated in tumours, with the highest uptake on day 7 following probe injection . However, it should be noted that control rats without tumours also exhibited higher AST values. Therefore, the increase might be connected to the specific rat strain used. Importantly, no difference was found between biochemical levels of all measured compounds in rats injected with glycogen-based probes and in those injected with commercial GM probes  on MR images. Notably, glycogen concentrations of synthesised nanoparticles can even be increased where required. Although POx can enhance tumour targeting by slowing biodegradation and blood circulation time, our POx-modified polymers exhibited lower MR relaxivity, decreasing imaging effectiveness as a result. This effect may have been due either to the more restricted availability of water molecules in gadolinium chelates, or to the higher conformational mobility of gadolinium chelates at the end of POx chains compared to chelates directly attached to the glycogen core.To prove the visualisation ability and sensitivity of probes using MR and optical imaging methods, we examined solutions of different conjugate concentrations. MR imaging, MR relaxometry, and fluorescence imaging proved suitably sensitive to glycogen nanoparticles. Boasting high MR relaxivity, good contrast on Tin vivo multimodal imaging results clearly confirmed the accumulation of glycogen-based conjugates in tumour tissue, displaying sustained MR contrast enhancement. Contrast in glycogen-based conjugates was higher than that observed in the commercial MR contrast agent, the plausible explanation being continuous accumulation due to the EPR effect. The difference between the commercial GM agent and the glycogen-based conjugates was highest on day 3 after i.v. injection. Glycogen-based probe accumulation peaked on day 2 in the case of GG (concentration of 0.04 mmol Gd3+/kg), confirming it the carrier most suitable for drug delivery to tumours. It should be noted that tumours were monitored only until day 7, at which point massive tumour necrosis occurred (confirmed by histology), a finding that obviously compromises the validity of our MR data. Probe accumulation in tumours was also confirmed by in vivo and ex vivo fluorescence imaging. We observed a rapid decline in the in vivo fluorescence signal within the first days after administration. Previous animal model studies to have used other imaging probes report the dissociation of MR and fluorescence signals, a similar effect possibly caused by lysosomal degradation of fluorescent dyes52.To evaluate drug delivery, polymer probes were tested for biodistribution, accumulation, and clearance in hepatocellular xenograft tumours using an animal cancer model. Our in vivo MR data show that throughout the entire examination POx conjugates issued the lower MR signals, a result more attributable to lower MR relaxivity (3.4-times lower compared to GG) than to lower accumulation in tumours considering the corresponding fluorescence signals revealed no significant differences between the probes with and without POx. However, non-POx probes exhibited slightly higher fluorescence signals (for both in vivo and ex vivo), accumulating in greater amounts as a result.Our 2. To that end, the use of beneficial non-biofouling (protein-repellent) compounds such as POx and PEO14 is recommended. Confirming the stealth effect of POx, our model demonstrates that conjugates modified with POx slowed the elimination rate and reduced the uptake of POx-modified conjugates in rat livers, spleens and kidneys compared to the unmodified version . We observed significantly lower in vivo MRI and fluorescence signals from POx-modified conjugates in the liver and kidneys throughout the whole examination, a finding supported by our ex vivo analysis of fluorescence signals from internal organs. Based on our results comparing tumours with other organs, POx not only caused internal organs to be protected against probe uptake but, in contrast to our expectations, inhibited accumulation in target tumour tissue.All drug delivery systems need to minimise the toxic effect of the drug incorporated while mitigating effects on non-targeted tissue3+ probe concentrations were much lower (up to 0.04 mmol Gd3+/kg) compared to standard doses routinely administered in human medicine (approximately 0.1 mmol Gd3+/kg)53. Therefore, we assume any adverse effect of Gd3+ was minimal. Moreover, in our model, we found no pathological changes in organs, confirmed by histological and biochemical analysis of blood proteins. Given our glycogen-based conjugates were uptaken by cells via endocytosis and intracellularly degraded into D-glucose units46, we assume they degraded to form water and carbon dioxide in tumour tissue. Similar to commercial clinically approved Gd-based MR contrast agents, we hypothesise the released chelate (DOTA) and fluorescent dye were then cleared from the body by the kidneys or liver. Nevertheless, it should be noted that these conjugates can carry other imaging moieties, thus impacting on sensitive radionuclides and certain applications such as 19F MR probes.The merits of any drug delivery system are largely dependent on its ability to ensure efficient clearance from the body. In our study, both MR and fluorescence imaging revealed that probes were eliminated from the organism mainly through the kidneys, similar to the preferred elimination route of MR chelates such as DOTA. As predicted, polymer modification with oxazolines reduced renal clearance. Elevated MR signals in the kidneys of rats administered polymers without POx persisted even 7 days after application, raising safety concerns and underscoring the importance of oxazoline modification. However, it should be noted that GdThe most important factor to consider when designing any drug delivery system is toxicity risk. Our biochemistry and immunohistochemistry results confirm the non-toxicity and biocompatibility of the glycogen-based carriers tested. Incubated cells exposed to probes showed comparable viability and proliferation to controls. Moreover, no prominent changes in blood serum protein levels were detected after intravenous probe administration in rats. Importantly, histology examination revealed no pathological changes in internal organs  after administration.54, thus providing safe and effective anticancer treatment for specific types of tumours.A key advantage of our drug delivery model for future applications is that it can be tailored to release drugs at particular sites of interest and/or under specific biological conditions, e.g. pH changes in solid tumoursin vitro and in vivo results highlight the potential of glycogen-based probes as carriers for drug delivery systems in tumour diagnosis and treatment.In this study, we demonstrate that our novel glycogen-based compounds are suitable for multimodal imaging of solid tumours, being of the requisite biological properties for tumour targeting. Biocompatible and safe, these compounds can be easily modified to control the biological fates of these conjugates. Although oxazoline modification prevents uptake of probes in internal organs, it also inhibits accumulation in target tumour tissue. Both our 46.Glycogen (type II from oysters) was used for nanoparticle synthesis. Two polymer conjugate variants were prepared: glycogen-based conjugates modified with POx (GOX) and glycogen-based conjugates without POx (GG). Glycogen conjugates grafted and non-grafted with GOX were synthesised as previously describedMR imaging was performed on a 4.7 T scanner  using a resonator coil with an internal diameter of 7 cm . MR images were processed using ImageJ software . All optical images (fluorescence) were acquired on an IVIS Lumina XR imager  and processed using Living Image software .1) of conjugates was assessed by measuring T1 relaxation times at various probe concentrations (0.01–0.36 mM Gd3+) on a 0.5 T relaxometer  using a saturation recovery sequence .We examined the MR and optical properties of GG, GOX, and GM, a commercial MR contrast agent used as a control. MR relaxivity (r1-weighted MR images of conjugates in tubes were acquired using a Rapid Acquisition with Refocused Echoes (RARE) sequence (repetition time (TR) = 125 ms, echo time (TE) – 11.6 ms, spatial resolution 0.2 × 0.2 × 1.5 mm3, scan time 6 min 24 s). A tube filled with water served as a reference. Regions of interest (ROI) of the same size were drawn around each sample in MR images, with the CNR between the reference and sample of choice calculated for each agent concentration.T2/sr]/(μW/cm2)). Fluorescence images were overlaid on photographs to localise the optical signal.Fluorescence images of the same samples were acquired during a 2-second exposure using aperture (f/stop) 4 and binning 4. Fluorescence excitation was set at 745 nm and emission at 810–875 nm. ROIs were drawn around each tube, with the emitted optical signal expressed as the radiance efficiency  in accordance with the European Communities Council Directive (2010/63/EU). Animals were kept in ventilated cages under a 12-h light cycle and given free access to food and water.6) above the right hind leg. During implantation, animals were anaesthetised by isoflurane inhalation . Prior to implantation, cells were cultured for 2 weeks in a DMEM culture medium supplemented with 10% foetal bovine serum (FBS), 5% L-glutamine, and a 5% penicillin/streptomycin solution .Tumours were induced in immunodeficient RNU nude rats  by subcutaneous injection of HUH7 cells .Animals (n = 48) were divided into 6 experimental groups according to the type of conjugate administered and based on two concentrations: GG 0.02 mmol Gdin vivo imaging experiments . Body temperature was maintained using a heating system, with breathing monitored throughout.Animals were kept under general inhalation anaesthesia for the duration of all 1-weighted MR images of tumours, livers and kidneys were acquired before and after intravenous administration of GG, GOX and GM at two concentrations (0.02/0.04 mmol Gd3+/kg). MR imaging was performed before, immediately after, and then 10 min, 2 h, 1, 2, 3 and 7 days after conjugate administration.Three weeks after tumour induction, T3, 8 acquisitions, scan time 4 min 16 s; gradient echo sequence for the liver and kidneys: TR = 72 ms, TE = 4.6 ms, spatial resolution 0.25 × 0.25 × 1.50 mm3, 12 acquisitions, scan time 3 min 41 s. CNR values of the tumour, liver and kidneys in relation to muscle tissue were calculated at each time point from ROIs manually outlined around each tissue type. CNR maps were calculated based on pixel-wise processing of MR images using a custom-written script in Matlab .MRI examination parameters were as follows: spin-echo sequence for tumour imaging with TR = 125 ms, TE = 11.6 ms, spatial resolution 0.25 × 0.25 × 1.50 mm2/sr]/(μW/cm2)) was calculated from each ROI outlined around the liver and tumour tissues.After MRI, near-infrared fluorescence images  of rat tumours and livers were acquired based on a 30-second exposure time, aperture 4 (f/stop) and binning 4. Fluorescence images were overlaid on photographs for anatomic localisation of the optical signal. The average fluorescence efficiency  and internal organs removed for histological analysis. Tumours, livers, kidneys and spleens were placed in a 4% formaldehyde solution overnight (pH 7.4) at 4 °C and then embedded in paraffin blocks. Paraffin samples were cut into 4-μm tissue sections and routinely stained with haematoxylin and eosin. Each organ was examined for the presence of pathological changes by an experienced pathologist.Statistical analysis was performed using GraphPad Prism 6.02 . A standard two-tailed Student’s t-test was used to compare differences between groups. Comparisons of three or more groups were performed by analysis of variance (one-way ANOVA), with the significance level set at p < 0.05. For mean values and standard deviations, see graphs.Supplementary information.