Use of nanobody 99mTc-D10 of a size as small as 15.5 kDa enables detection of tumours by single photon emission computed tomography (SPECT) imaging already 45 min post intravenous administration with high tumour uptake (>3% ID/g) in small MDA-MB-468 and A431 tumours, with tumour volumes of 52.5 mm3 ± 21.2 and 26.6 mm3 ± 16.7, respectively. Fast blood clearance with a serum half-life of 4.9 min resulted in high in vivo contrast and ex vivo tumour to blood and tissue ratios. In contrast, no accumulation of 99mTc-D10 in MDA-MB-231 tumours characterized by a very low expression of EGFR was observed. Here we present specific and high contrast in vivo visualization of small human tumours overexpressing EGFR by preclinical multi-pinhole SPECT shortly after administration of anti-EGFR nanobody 99mTc-D10.The detection of tumours in an early phase of tumour development in combination with the knowledge of expression of tumour markers such as epidermal growth factor receptor (EGFR) is an important prerequisite for clinical decisions. In this study we applied the anti-EGFR nanobody  Besides accurate information of tumour load and spread, the retrieval of the expression of biomarkers on the tumour cell surface at the earliest time point is a prerequisite for a successful targeted therapeutic approach. In order to acquire information on expression of tumour associated proteins E.coli, that is considered to be an economic, fast and straightforward production method with high yields. They can easily be modified with various tags e.g. hexahistidine (6 × His), myc or a free cysteine that allows a site-specific labelling for biomedical imaging.A requirement for probes to be used for tumour imaging is their fast and specific accumulation in the tumour and as little as possible uptake in healthy tissue thus generating a high contrast within the tumour shortly after probe administration. In order to achieve a fast removal from the blood pool the ideal imaging probe should be as small as possible31199mTc-tricarbonyl species [99mTc(CO)3]+ to target the EGFR that is frequently overexpressed in a variety of tumoursIn this study, we applied and radiolabelled the recently developed anti-EGFR nanobody D10 with 3 in nude mice using [99mTc(CO)3]+ labelled anti-EGFR nanobody 99mTc-D10 by multi-pinhole SPECT 45 min after intravenous administration.Here we present non-invasive, specific and high contrast visualization of very small EGFR overexpressing human tumours with sizes ranging from 10 to 100 mmE.coli cultures and purified via the 6 × His tags by metal affinity chromatography. For radiolabelling, the 6 × His tags of the nanobodies were site-specifically labelled with [99mTc(CO)3]+ with a specific activity of 183 MBq/nmol ± 35 and 182 MBq/nmol ± 51 and a radiochemical yield of 48.8% ± 7.0 and 58.2% ± 6.7 for the anti-EGFR nanobody 99mTc-D10 (N = 12) and for the control nanobody 99mTc-F5 (N = 10), respectively. After purification, radiochemical purities, i.e. the amount of protein-bound activity, with values of 97.7% ± 1.2 (99mTc-D10) and 98.2% ± 1.1 (99mTc-F5) were determined by instant thin layer chromatography (ITLC). In comparison to the nanobodies, the anti-EGFR immunoglobulin G1 (IgG1) Cetuximab was modified with HyNic prior radiolabelling due to a missing 6 × His tag. Labelling of Cetuximab with 99mTc-pertechnetate and tricine as coligand resulted in a specific activity of 1700 MBq/nmol ± 105 with a yield of 44.1 ± 10.1% and a purity of 97.3%  between the radiolabelled 99mTc-D10 and 99mTc-F5 (red line) and unlabelled (blue line) nanobodies D10 and F5 could be detected indicating that 99mTc-D10 still bound the same amount of EGFR on cells as D10  ruling out any unspecific binding of nanobodies to the investigated tumour cells  ratios of 36.2 ± 20.9 in 5 out of 6 animals . Therefore, tumours with volumes below 8 mm3 cannot be detected by the here used SPECT system. This explains the absent signals within the tumour of approximately 7 mm3 in the SPECT scans of one out of six mice. Although this very small tumour was not visualized by in vivo SPECT due to the limited resolution, a distinct uptake of up to 2.18% ID/g of 99mTc-D10 was proven by ex vivo biodistribution.A resolution of more than 2 mm was determined for the multi-pinhole collimators used here by SPECT scans with a Jaszczak phantom corresponding to a volume of at least 8 mmin vivo tumour detection with anti-EGFR nanobody 99mTc-D10 was compared to the IgG199mTc-Cetuximab that was used to visualize A431 tumour lesions with comparable mean tumour volumes of 40 mm3  45 min post injection. In vivo SPECT scans showed no uptake of 99mTc-Cetuximab in the small tumour lesions at the early time point. It was not possible to define the tumour at the early time point due to the high background in any of the scans performed 45 min post injection. High uptakes were found in all organs and large blood vessels like the inferior vena cava and the external iliac veins  were used for in vivo tumour detection with anti-EGFR nanobody 99mTc-D10. All MDA-MB-468 tumours were detected reliably by in vivo SPECT scans 45 min after administration of 17 pmol anti-EGFR nanobody 99mTc-D10 as shown by signals present in the tumour and very low background signals resulting in a tumour to tissue  ratio of 42.8 ± 27.0 . The lack of 99mTc-D10 to accumulate within the tumour indicated no unspecific binding or tumour uptake.MDA-MB-231 tumours with a very low expression of EGFR showed no detectable uptake of the anti-EGFR nanobody 9mTc-D10  in vivo.vo SPECT  nor ex vanalyses , Table 23 was investigated by single plane illumination microscopy (SPIM) 45 min after intravenous administration of anti-EGFR nanobody AF488-D10. This allows the analysis of whole tumour lesions and provides the possibility to three-dimensionally acquire the tumour tissue penetration of nanobody D10 labelled to AlexaFluor 488 (AF488-D10) on a cellular resolutionTo assess the distribution of anti-EGFR nanobody D10 through the tumour tissue, a small high EGFR expressing A431 tumour with a volume of 6.6 mmA movie of the whole z-stack is provided in the 99mTc-D10 is especially suitable to detect small tumour lesions expressing EGFR with high specificity and high contrast 45 min after administration. These features are mandatory for tracers to enable the detection of tumours with small sizes at earliest time points to improve the overall prognosis for the patient. In vivo SPECT scans were performed already after 45 min in tumour lesions expressing high levels of EGFR with an excellent contrast with low background signals resulting in a tumour to tissue ratio ranging from 11 to up to 80. This early detection was feasible due to the fast blood clearance with a half life in blood of only 4.9 min and a very low plateau of 0.26% ID/g ± 0.24 of the remaining activity in the blood pool.In this study we demonstrate that the use of anti-EGFR nanobody 99mTc-D10 is suitable to visualize small tumour lesions with weights below 100 mg corresponding to tumour volumes below 100 mm3. It is known that most tumours originate as small avascular structures and have to establish their own tumour derived blood vessels to grow beyond a few millimetres1515161718192099mTc-D10 can be explained by the small size of the nanobody enabling a good tissue penetration. Recent studies have shown that smaller antibodies are characterized by a better penetration through the tissue1099mTc-D10 is quickly removed from the tissue as well as from the blood resulted in this study in a high tumour contrast with high in vivo tumour to tissue and ex vivo tumour to muscle ratios.Here, we show that the anti-EGFR nanobody 3 with tumour weights far beyond 100 mg3423in vivo as well as ex vivo for the published anti-EGFR nanobodies3499mTc-D10, we found lower background activities in the blood (ex vivo) as well as in the tissue (in vivo) compared to the reported anti-EGFR nanobodies343) of tumours used in these studies, leading to better vascularisation of the tumour with more fenestrated blood vessels33 were shown to contain many small and mostly immature blood vessel that were homogenously distributed throughout the tumouret al. reported an inverse relationship between tumour uptake and tumour weight with EGFR targeting in A431 tumours with different anti-EGFR nanobodies3. This effect can be explained on one hand by intratumoural heterogeneity and on the other hand by the diameter of the blood vessels that becomes narrower from the periphery to the centre and might reduce the amount of nanobody reaching the centreNanobodies are subject of investigation for several years and many studies were published targeting EGFR or HER2 overexpressing tumours, however mainly tumours with diameters of approximately 0.5–1 cm3199mTc-Cetuximab led to a significantly lower tumour to blood ratio compared to nanobody 99mTc-D10, despite a more than 10 fold higher affinity towards EGFR (0.5 nM)ex vivo tumour to blood ratio was significantly impaired (P < 0.001) for 99mTc-Cetuximab after 25 h compared to 99mTc-D10 after 100 min. Targeting of EGFR with 99mTc-Cetuximab in our setting resulted in a relatively low overall A431 tumour uptake of 6.5% ID/g compared to tumour uptakes of up to 20% ID/g observed by others2099mTc-Cetuximab we observed a long serum half-life (approx. 3 h in this murine model) which can be explained by the relatively large size of the IgG1, 152 kDa, and its removal from the body via hepatic excretion, together with the recycling process in the cells of the liver afforded by the binding of its Fc portion to the FcRn receptor2799mTc-D10 was observed in our study the anti-EGFR nanobody 99mTc-D10 is predominantly removed by renal excretion. High uptakes in the kidneys have previously been reported for many other nanobodies3410232429303463334The full IgGet al. recently reported that the removal of the C-terminal His tag from an anti-PSMA nanobody also markedly reduced its renal uptake and could be combined with gelofusine and lysine to decrease the renal uptake below 4% ID/g at 3 h post injectionThe renal uptake is mediated by the Low Density Lipoprotein receptor-related protein 2  in the proximal tubular cells36233334in vivo SPECT with 99mTc-D10 tumours could be clearly visualized with a very low background resulting in high tumour to tissue ratios.For a successful tumour visualization, high contrast is needed to define the tumour from the surrounding tissue. Here, by the use of ex vivo biodistribution analysis. The uptake of 99mTc-D10 in the muscle of A431 tumour bearing mice was very low with 0.14% ID/g ± 0.1, however, with a distinctive variance. This variance is even more pronounced after calculation of the ratio. For 99mTc-Cetuximab, this effect was less marked, since the muscle uptake was 3-fold higher, and varied to a lesser extent with values of 0.43% ID/g ± 0.08 compared to 99mTc-D10. The lower variation in this experimental set up can be explained by the late time point of the in vivo SPECT scan at 24 h post 99mTc-Cetuximab injection. The variation of the ratios did not interfere with interpretation and analysis of images since sufficient contrast between tumour and muscle was provided.The variations of tumour to tissue ratios are large compared to the data of 99mTc-D10 to EGFR expressing tumour cells since no tumour uptake ex vivo and consequently no tumour uptake in vivo could be observed in the low EGFR expressing MDA-MB-231 tumour model.Our results also illustrate specific tumour detection of nanobody 99mTc-D10 represents a versatile tool for the specific detection of small EGFR overexpressing tumour lesions and the assessment of EGFR expression in tumours by in vivo SPECT. Of crucial importance, we show the possibility to acquire high contrast images with a high tumour to background ratio shortly after probe administration, i.e. allowing imaging at the same day. Therefore, anti-EGFR nanobody 99mTc-D10 might be suitable in a clinical setting as non-invasive diagnostic tracer to not only detect small tumours but also to obtain information on the expression level of EGFR at the time of diagnosis and during disease progression.Our study shows that E. coli BL21-DE3 were grown overnight in 400 ml 2 YT medium. Nanobodies were harvested by lysis of the bacteria with BugBugster extraction reagent  supplemented with Lysozyme and Benzonase, and purified by IMAC .Selection and screening of the anti-EGFR nanobody D10 was published recently2 in a humidified atmosphere and detached with 0.05% trypsin/EDTA  at a subconfluent stage.Human mammary carcinoma cell lines MDA-MB-231 and MDA-MB-468 and human epidermoid carcinoma cell line A431  were cultured in high glucose (4.5 g/l) DMEM with Glutamax  supplemented with 10% fetal calf serum . All cells were grown in monolayer at 37 °C, 5% COTumour cells were seeded on 6 cm petri dishes and allowed to grow to a subconfluent stage, washed with cold phosphate buffered saline (PBS) and lysed with radioimmunoprecipitation assay (RIPA) buffer . For Western blot analysis, 25 μg total lysate were separated by sodiumdodecylsulfate polyacrylamide gel electrophoresis  and blotted on nitrocellulose membrane. Primary antibodies targeting EGFR (#2232) and Actin (#MAB1501) were purchased from Cell Signaling  and MerckMillipore , respectively. HRP-labelled secondary anti-mouse (#NA931VS) and anti-rabbit (#NA934VS) antibodies were purchased from Amersham . Proteins were detected by brief incubation with the ECL detection kit (Amersham) and by acquisition on a ChemiDoc XRS system .99mTc(CO)3]+) species via their C-terminal hexahistidine tags (6 × His). The CRS Kit  was used to produce the [99mTc(CO)3]+ intermediate out of 2400–4000 MBq of 99mTc pertechnetate in 1 ml saline. After boiling for 20 min at 100 °C, 500 μl of [99mTc(CO)3]+ were neutralized with 1 M HCl to pH 7.5 and incubated with 50 μg nanobody (1 mg/ml) for 90 min at 56 °C. The radiolabeld nanobodies were purified from unbound activity on Amicon Ultra centrifugal filters .The nanobodies were labelled with technetium-99m tricarbonyl , the free ε-amino groups were modified with 6-hydrazinonicotinamide (HyNic). 10 nmol antibody were incubated with an 30 molar excess of succinimidyl 6-hydraziniumnicotinate hydrochlorate (SHNH)  for 2 h at ambient temperature in a 100 mM phosphate buffer (pH 7.4). 20 μl of the antibody-HyNic conjugate (2 mg/ml) in 100 mM phosphate buffer (pH 6.0) were incubated with 500 μl 99mTc pertechnetate (1000–2000 MBq), SnCl2  and tricine  as coligand for 20 min at ambient temperature. The radiolabelled Cetuximab was purified from unbound activity on Amicon Ultra centrifugal filters . The radiochemical yield and purity of the radiolabelling procedures were determined by instant thin layer chromatography on iTLC-SG strips  and 1% HCl in methanol as mobile phase.For radiolablelling of the full IgG99mTc-D10 and 99mTc-F5 and IgG199mTc-Cetuximab were labelled with 99mTc as described above. 2–3 μg of the radiolabelled proteins were mixed with mouse serum and incubated at 37 °C. Samples were taken at 0, 30, 60, 120 and 240 min and analyzed by thin layer chromatography using iTLC-SG strips and 1% HCl in methanol as mobile phase.Nanobodies The anti-EGFR nanobody D10 was produced with a free terminal cysteine as described previously6 cells were blocked in 2% bovine serum albumin (BSA)  in PBS and incubated with 100 μl nanobody or antibody diluted in 2% BSA (10 μg/ml) for 30 min on ice. The nanobodies were detected by consecutive incubation with an anti-myc antibody  (clone 9E10) and goat-anti-mouse-PE   for 30 min on ice. Fluorescence intensities were measured by flow cytometry on a FACSAria cell sorter with FACSDiva software . Data were analyzed using FlowJo software .Binding capacities of the labelled and unlabelled nanobodies were assessed by flow cytometry. Cells were detached by brief incubation with trypsin/EDTA, washed and counted. 1 × 106 in 100 μl PBS) were injected subcutaneously in the right flank of 8–12 weeks old male athymic nude mice (NMRI-Foxn1)  under control of 2% isoflurane. Cells of the human mammary carcinoma cell lines MDA-MB-231 (1 × 106 in 20 μl PBS) and MDA-MB-468 (2 × 106 in 20 μl PBS) were implanted orthotopically in the fat pad of right abdominal mammary gland of 8–12 weeks old female athymic nude mice (NMRI-Foxn1)  under the control of a general Ketamine/Rompun anaesthesia as described previously39All animal experiments were carried out in accordance with the German animal welfare law and were approved by local authorities (Lower Saxony State Office for Consumer Protection and Food safety–LAVES). Cells of the human epidermoid carcinoma cell line A431 (1.5 × 10in vivo by contrast-enhanced computed tomography (CT) scans under 1% isoflurane anaesthesia. 100 μl Ultravist370  were injected intravenously and two 360° scans were carried out on a low-dose in vivo small animal CT  with the following parameter: 90 kV, 200 μA and a field of view of 40  and 73 mm . Image reconstruction was performed using a standard filtered backprojection algorithm implemented in the vendors software resulting in data sets with a voxel size of either ~80 × 80 × 80 μm3 or 140 × 140 × 140 μm3.All tumour volumes were determined 99mTc-F5 and 99mTc-D10  in the tail vein. Mice that received radiolabelled 99mTc-Cetuximab  were started 45 min and 24 h post intravenous injection. Images were acquired over 360° in 10 projections of 300 sec each into 256 × 256 matrices, resulting in a total scan time of 50.6 min. SPECT scans with the anti-EGFR nanobody 99mTc-D10 and the control nanobody 99mTc-F5 were performed in the same animals using identical scan protocols. 99mTc-D10 was injected intravenously at least 72 h after administration of the control nanobody 99mTc-F5.One to two days after CT imaging, SPECT was performed on a triple-head clinical gamma camera  equipped with multi-pinhole collimators with 6 pinholes and a field of view of 50 × 60 mm  on tumour bearing mice under 1% isoflurane anaesthesia. SPECT scans of mice that received radiolabelled nanobodies were started 45 min post intravenous injection of 4 and scanned with the in vivo scan protocol as described above.Phantom scans were conducted with a custom made Jaszczak phantom with rod diameters ranging from 1.0 to 2.0 mm. The phantom was filled with approximately 2 ml [99mTc]NaTcO3. Since CT and SPECT scans were performed on separate systems on different days, each mouse was imaged in the same animal holders, which included 6 holes each filled with 99mTc-pertechnetate (<30 kBq) that were used for manual alignment.Image reconstruction was performed using an ordered-subset expectation maximization algorithm implemented in the HiSPECT software  resulting in data sets with a voxel size of 600 × 600 × 600 μmReconstructed CT and SPECT data sets were quantified and analyzed for determination of tumour volume and tumour uptake using Scry v. 5.0 .in vivo tumour to tissue ratio. Tumour and tissue uptake were expressed as percent of injected dose per cubic centimeter (% ID/cm3).SPECT data were decay corrected and normalized. All tumour volumes were segmented from contrast enhanced CT and SPECT images by a region growing algorithm. For reference a region with the same size like the tumour containing tissue only has been segmented on the contra lateral side and was used for determination of Following SPECT scans, mice were sacrificed by cervical dislocation under isoflurane anesthesia. The organs were dissected, blotted dry and weighed. Remaining activity in organs and tumours was measured in a gamma counter , decay corrected and expressed as percent injected dose per gram (% ID/g). The total injected dose of each mouse was determined by measuring the activity in the syringes before and after injection.The anti-EGFR nanobody D10 was labelled site-specifically with a AlexaFluor 488-maleimido dye on the free cysteine. 14 pmol of the anti-EGFR nanobody AF488-D10 were injected in the tail vein of a xenografted A431 cancer xenografted nude mouse. The mouse was sacrificed 45 min post injection and the tumour and the spleen were dissected, fixed in 4% paraformaldehyde (PFA)  and sectioned coronally in cold PBS. Slices were collected in PBS in 6 well plates and removed individually for clearing. For SPIM analysis tumour tissue was optically cleared at room temperature with organic solvents using an altered 3DISCO protocol as described previouslyWe acquired z-stack measurements with 5 μm step size and a total range of 200 μm from the whole tumour material. In addition, we imaged single pictures from the tumour in a tumour depth of 175 μm and generated tagged image file format (tiff) or we converted the z-stack in an Audio Video Interleave (AVI) movie format. For AVIs of whole tumour z-stacks we used 40 pictures and 20 frames/second and a x-resolution of 720 and y-resolution of 576.ex vivo and in vivo comparison. Unless otherwise stated all values were calculated as mean ± standard deviation (SD).Statistical analysis was performed using GraphPad Prism v. 6.01 for Windows . Unpaired two-tailed t-Tests were used to determine the significant difference between the groups in How to cite this article: Krüwel, T. et al.In vivo detection of small tumour lesions by multi-pinhole SPECT applying a 99mTc-labelled nanobody targeting the Epidermal Growth Factor Receptor. Sci. Rep.6, 21834; doi: 10.1038/srep21834 (2016).


Onychomatricoma is a rare and benign tumour of the nail matrix but originates rarely from the ventral portion of the proximal nail fold. This tumour is characterised by fingerlike projections that invade the nail plate. This lesion, of unknown aetiology, is typically asymptomatic with slow progression. Localisation on the finger is the most frequently described. We report the case of a 68-year-old woman who has an onychomatricoma in an unusual location, the fifth toe of the left foot. Due to its clinical appearance, the tumour can be confused with and treated as onychomycosis. However, if it is resistant to an oral antifungal well behaved treatment, one must consider onychomatricoma diagnosis. A 68-year-old woman went to a dermatologist for an opinion regarding the thickening and yellowing of the nail plate on the fifth toe of the left foot, which had appeared gradually over the past 2 years. She had no history of prior traumatism, and the lesion was pain-free. Clinical examination showed a thickening of the nail plate, a xanthonychia, and distal splinter haemorrhages of the nail plate on the 5th toe . DermoscOnychomatricoma (OM) is a rare and benign fibroepithelial tumour of the nail matrix. It was first described in 1992 by Baran and Kint. They have reported three cases of filamentous funnel-shaped tumours in the nail matrix . The sexIt is a rare tumour for which nail plate modification originates in the matrix , 8. OccaOnychomatricoma is a rare benign tumour of the nail matrix commonly occurring on the fingernails. This case is of interest due to its unusual location on the fifth toenail.


However, it is not yet known how these cells contribute to tumour vascularization. In the present study, we investigated the role of infiltrating CD11b+ myeloid cells in the vascularization and recurrence of oral squamous cell carcinoma (OSCC). In a xenograft mouse model, local irradiation caused vascular damage and hypoxia in the tumour and increased infiltration of CD11b+ myeloid cells. These infiltrating cells showed characteristics of M2 macrophages (M2Mφs) and are associated with the promotion of vascularization. M2Mφs promoted tumour progression in recurrence after irradiation compared to non-irradiated tumours. In addition, we found that CD11b+ myeloid cells, as well as CD206+ M2Mφs, are increased during recurrence after radiotherapy in human OSCC specimens. Our findings may lead to the development of potential clinical biomarkers or treatment targets in irradiated OSCC patients.Despite the fact that radiation is one of the standard therapies in the treatment of patients with oral cancer, tumours can recur even in the early stages of the disease, negatively impacting prognosis and quality of life. We previously found that CD11b Surgery is standard therapy for the treatment of patients with oral cancer, even at advanced stages. However, chemo- and radiotherapy also play major roles in the treatment of advanced OSCC to avoid functional disorders and cosmetic disturbances. Despite advances in treatment modalities, tumours may recur within the irradiated field, leading to a poor prognosis. Thus, improving local control of the primary tumour with radiotherapy would increase the cure rate of oral cancer. To that end, it is necessary to understand how tumour vasculature can be restored after irradiation, given that a local dose should eliminate existing tumour endothelial cells. Since Folkman proposed that tumours cannot grow beyond 2 to 3 mm in size without forming new blood vessels2et al. also demonstrated in lung and breast tumour models that host-derived BMDC infiltration in tumours was stimulated by local irradiation and facilitates tumour recurrence through paracrine effects on irradiated tumour vasculature+ BMDCs, but not EPCs, make significant contributions to facilitate tumour regrowth after irradiation.We have previously shown in an intracranial GBM xenograft model that irradiation induces recruitment of BMDCs into tumours, restoring radiation-damaged vasculature by vasculogenesis and thus allowing the growth of surviving tumour cells+ myeloid cells recruited into tumours are thought to subsequently differentiate into macrophages. Macrophages surrounding the tumour are referred to as tumour-associated macrophages (TAMs), which are believed to act as key regulators of tumour angiogenesis, migration, metastasis, and treatment resistance91012131415+ myeloid cells recruited into irradiated tumours may be differentiated into a proangiogenic phenotype of macrophages. Therefore, our research aimed to investigate whether CD11b+ myeloid cells are recruited into oral cancer after irradiation and how they are differentiated into macrophages in relation to tumour relapse, which remains largely uncharacterized.CD11b+ cells with that of non-irradiated control tumours. We found that recurrences contained significantly higher numbers of CD11b+ cells  of tumour-bearing mice in various cancers19mal mice . These d+ cells affected cancer cell biology in vitro or tumour growth in vivo. To test this, OSC-19-luc cells were co-cultured with BMDCs of normal control mice or tumour-bearing mice, and the proliferation of OSC-19-luc cells was assessed by the IVIS system. The results showed no significant difference between the control and tumour-bearing BMDCs with respect to OSC-19 cell proliferation, suggesting that in spite of different CD11b positive rates, BMDCs have no effect on OSC-19 cell proliferation . TEMs have been reported to be highly proangiogenic and to interact with the formation of tumour blood vessels+VEGFR+ circulating precursor cells that contribute to the structural luminal surface of capillaries were not detectable. These results indicate that recruited CD11b+ cells are highly related to the macrophage lineage  is a crucial factor in the induction of BMDC influx. Various animal studies showed that HIF-1α regulates many chemokines that may induce recruitment of BMDCs into tumours, including chemokine ligand 2 (CCL2 or MCP1)30315+ cells into the tumour.We have shown that irradiation induces recruitment of CD11b+ monocyte precursors were increased in the BM, spleen, and PB of tumour-bearing mice in colon adenocarcinoma, mammary adenocarcinoma, and lymphoma. Moreover, those immature myeloid cells are also further produced in the PB of patients with cancer19+ cells in the PB and BM of OSC-19 tumour-bearing mice, suggesting that BMDCs can be altered by tumours, likely secretory cytokines and chemokines.Previous studies reported that CD11b+ myeloid cells. Subclass analyses of these cells showed high co-expression of F4/80, indicating that they are of the macrophage lineage. TEMs, which have shown the proangiogenic activity of myeloid cells in tumours, were small in number, which is consistent with a previous reportet al. demonstrated in mouse xenografts that conditional depletion of TEMs prevented angiogenesis and delayed tumour growth. We also reported that approximately half of the CD11b+ myeloid cells recruited into the irradiated GBM were TEMs5+Gr-1+myeloid cells, known as myeloid-derived suppressor cells (MDSCs), that have been reported accumulate in several tumours, suppress tumour immunity and thus promote tumour growth+F4/80+ cells. Moreover, Ahn et al. suggested that CD11b-neutralizing antibodies enhanced tumour responsiveness to radiation in transplanted tumours, whereas depletion of Gr-1+ cells did not show the same effect. Although the possible involvement of CD11b+Gr-1-Ly-6Chi monocytic MDSCs in tumour relapse after radiation may not be excluded, CD11b+ monocytic cells, but not CD11b+ neutrophils or granulocytes, seem to be more critical as a proangiogenic populationBMDC accumulation in OSC-19 tumours after local irradiation was largely composed of CD11bin vitro, while they promoted tumour growth and regrowth in vivo. This effect was more significant and lasted much longer in tumours injected at pre-IR sites. CD31 endothelial cells in the tumours of non-IR sites showed that OSC-19 tumours injected with M2Mφs formed more blood vessels. These results suggested that the effect of M2Mφs on neovascularization acts in more ischaemic conditions and contributes to relapse in OSCC tumours. Indeed, in conditions such as wound and ischaemic heart disease models, polarization of the M2 phenotype is promoted in infiltrating macrophages. Oxygen deprivation functionally links to progressive induction of proangiogenic M2MφsM2Mφs have been reported to have protumoural functions, including functions in angiogenesis and alterations in invasiveness. In our study, M2Mφs had no effect on OSC-19 cell proliferation We have previously reported in a GBM model that tumour vascularization mainly relies on angiogenesis in normal conditions; however, when local irradiation inhibits angiogenesis and induces hypoxia, vasculogenesis predominates. In this study, CD31 endothelial cells decreased to 36% of control levels by 2 weeks after 12 Gy irradiation so that angiogenesis was inhibited by irradiation in an OSCC model. Because BMDCs of tumour-bearing mice also promoted tumour growth in non-IR sites, it is possible that angiogenesis was also stimulated. However, the effect of tumour-bearing BMDCs was limited to the initial stage of non-IR tumours and was more significant in pre-IR sites; therefore, BMDCs more likely contribute to vasculogenesis in irradiated tumours. Proangiogenic M2Mφs are reported to secrete several angiogenic factors, such as VEGF, IL-8 (CXCL8), bFGF (basic fibroblast growth factor), TP (thymidine phosphorylase), and MMP 11+ myeloid cells in recurrence also has clinical implications. Evidence has accumulated suggesting that recruitment of myeloid cells into tumours is correlated with clinical outcomes accompanied by treatment. A previous study reported that macrophage density positively correlated with microvessel counts and negatively correlated with patient relapse-free survival in lung canceret al. also reported that CD163+ macrophage expression predicts an unfavourable clinical outcome in HNSCC after definitive chemoradiotherapy+ M2Mφs was also increased in recurrences of OSCC specimens after radiotherapy. These results suggest that increased myeloid cells or TAMs in tumours or PB may be independent prognostic markers. Local and distant recurrence occurs in approximately 30% of OSCC. Hence, it is crucial to identify novel biomarkers that predict patients at high risk of relapse.The observation of increased CD11bet al. developed a unique peptide called M2pep that preferentially binds to M2Mφs, including TAMsPseudomonas exotoxin (PE) directly killed IL-13Rα2 positive cellsAlthough several TAM-targeting cancer therapies have been explored, whole TAM depletion may also affect monocyte/macrophage-mediated host defence. Therefore, a M2Mφs-specific targeting agent would need to exert high anti-tumour effects with radiation while minimizing toxicity+ myeloid cells into OSCC and that these are polarized towards M2Mφs shortly before tumours start to regrow. M2Mφs rather than CD11+ myeloid cells may contribute to neovasculogenesis and relapse in OSCC after radiation.In summary, our data suggest that local irradiation induces recruitment of CD11bOSC-19 and HSC-3 cell lines (human squamous cell carcinoma of the tongue) were purchased from the Japanese Collection of Research Bioresources. YCU-OR891 cells  were obtained from Dr. Oridate of Yokohama City University. OSC-19-luc cells were retrovirally transduced with the luciferase gene, as previously describedFemale 4–7-week-old BALB/c nude mice weighting 14–18 g were purchased from SLC Japan. All studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee at Yokohama City University.4Cl solution. BMDCs of OSC-19 tumour-bearing mice were obtained from female BALB/c nude mice previously implanted with OSC-19 cells.BMDCs were harvested by flushing the femurs and tibias of female BALB/c nude mice with RPMI1640 Glutamax (Thermo Fisher Scientific) supplemented with 10% FBS using a 25G needle and a syringe. BMDCs were passed through a 70 μm cell strainer, and red blood cells were removed by NHFor BMDC differentiation into BM-derived macrophages (BM-Mφs), BMDCs were cultured for an additional 7 days in the same medium supplement with 10 ng/ml recombinant macrophage colony-stimulating factor (M-CSF) (Peprotech). The efficiency of differentiation was confirmed by FACS analysis for CD11b and F4/80 antigen surface expression . To induTMIII Reverse Transcriptase (Thermo Fisher Scientific). PCR with TaKaRa Ex Taq (Takara) was performed with primers (Sigma Genosys), as shown in Supplementary Table S141424344Total RNA was isolated using RNeasy, and reverse transcription was performed using SuperScriptFor two co-culture studies, OSC-19-luc cells were plated in 24-well plates with or without BMDCs of normal control mice or OSC-19 tumour-bearing mice and with or without BM-derived M1Mφs (BM-M1Mφs) or BM-derived M2Mφs (BM-M2Mφs) at indicated ratios. Then, proliferation of OSC-19-luc cells was assessed via the enzymatic activities of luciferase using the IVIS system  on the indicated date.6 cells) were subcutaneously (s.c.) implanted on the back of a female BALB/c nude mouse. Tumour volume (V) was calculated with callipers as V = L × W2 × 0.5 . When the tumour volume reached approximately 200 mm3, 12 Gy of local irradiation was administered (HITACH X-ray MBR-1520-4) using a specialized lead jig. For the pre-IR model, 12 Gy of irradiation was performed before tumour implantation. For the two co-injection studies, OSC-19 cells with or without BMDCs of normal control mice or OSC-19 tumour-bearing mice were s.c. injected into non-irradiated (non-IR) or pre-IR sites. For the other experiment, OSC-19 cells were injected with or without BM-M1Mφs or BM-M2Mφs.OSC-19 cells  according to the manufacturer’s instructions. All sections were mounted in ProLong® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and imaged with Leica DMI 4000B fluorescent microscopy (Leica Microsystems Inc.). Isotype-matched IgG was used as a negative control.Tumour samples were fixed by 4% paraformaldehyde cardiac perfusion and then embedded in OCT compound (Sakura Finetek). Frozen sections (5–10 μm) were incubated with the following primary anti-mouse antibodies overnight at 4 °C: CD31 (BD Bioscience), CD11b (BioLegend), Tie-2, F4/80, Gr-1 (eBioscience), VEGFR2 (R&D Systems), CD68 (Serotec), CD206 (Santa Cruz) and iNOS (Abcam). They were then incubated with secondary antibodies for 30 min at room temperature, including Alexa Flour 488, 546, Streptavidin labeled with Alexa Flour 488, 546 and 647 (Thermo Fisher Scientific). Hypoxic areas were detected using pimonidazole hydrochloride staining  and CD68 (Dako) overnight at 4 °C. The reaction was visualized with the Vector Stain ABC kit and ImPACT DAB Substrate (Vector Laboratories). Sections were counterstained with haematoxylin and viewed with an OLYMPUS BH2 microscope (Olympus).6 cells) and injected into the flank of female BALB/c nude mice. Matrigel with bFGF served as a control. One week after inoculation, Matrigel plugs were surgically removed, and frozen sections were analysed by immunofluorescence staining for the presence of CD31 endothelial cells.Matrigel  supplemented with 75 ng of basic fibroblast growth factor (bFGF) (Peprotech) was mixed with BM-M2Mφs  and CD11b-PE antibodies (BD Pharmingen), followed by Streptavidin-APC antibody (eBioscience). All samples were pre-incubated with Fc-block (BioLegend) prior to staining. Analysis was performed using MoFlo® Astrios (Beckman Coulter) and Summit Software ver. 4.3 (Beckman Coulter).Mouse whole blood was collected with 1.5% EDTA-2Na used as the anticoagulation agent, and red blood cells were eliminated by a lysing buffer (BD Pharmingen). Leukocyte cells and BMDCs of normal control mice or OSC-19 tumour-bearing mice were stained with CD11b-PE antibody (BD Pharmingen). For IL-13Rα2 and CD11b double staining, OSC-19 tumours grown at non-IR sites or pre-IR sites in nude mice were minced and enzymatically digested with 3 mg/ml Type VI collagenase and 2 U/ml hyaluronidase (SIGMA). They were further treated with 0.1 mg/ml DNase I (Wako) and 0.09% NHt-test was used (GraphPad Prism software version 5.02). P values (exact significance) less than 0.05 were considered statistically significant.Statistical analyses for multiple-group comparison were analysed by one-way ANOVA. For two-group comparisons, Student’s How to cite this article: Okubo, M. et al. M2-polarized macrophages contribute to neovasculogenesis, leading to relapse of oral cancer following radiation. Sci. Rep.6, 27548; doi: 10.1038/srep27548 (2016).


We propose an experimental and theoretical framework for the study of capillary filling at the micro-scale. Our methodology enables us to control the fluid flow regime so that we can characterise properties of Newtonian fluids such as their viscosity. In particular, we study a viscous, non-inertial, non-Washburn regime in which the position of the fluid front increases linearly with time for the whole duration of the experiment. The operating shear-rate range of our apparatus extends over nearly two orders of magnitude. Further, we analyse the advancement of a fluid front within a microcapillary in a system of two immiscible Newtonian liquids. We observe a non-Washburn regime in which the front can accelerate or decelerate depending on the viscosity contrast between the two liquids. We then propose a theoretical model which enables us to study and explain both non-Washburn regimes. Furthermore, our theoretical model allows us to put forward ways to control the emergence of these regimes by means of geometrical parameters of the experimental set-up. Our methodology allows us to design and calibrate a micro-viscosimetre which works at constant pressure. The study of fluid flow at the micro- and nano-scale , 2 has bh ∼ tν where h is the position of the meniscus and the exponent ν = 1/2 [Besides potential technological applications, there are basic questions that remain to be understood, in particular those related to the dynamics of the moving contact line , 14–17.  ν = 1/2 .−5 s, and prior to the Washburn quasi-steady regime, a transient, viscous regime ensues such that h ∼ t, i.e. the front propagates at constant speed. We further propose a method to experimentally control the cross-over time between our transient and the Washburn regimes so that we can make the duration of the transient regime to last for an arbitrarily long time, in particular, until the microchannel is completely full.Our aim in this paper is to propose a method of control of fluid flow in a microchannel. This enables us to analyse a flow regimes different from that studied by Washburn. Within these regimes, we show that we can characterise rheological properties, e.g. viscosity, of the fluid which is particularly suitable for potential automatisation. In a typical situation, where a single fluid is pushed through a micro-channel, we show that a set-up see Figs  & 2 is pμPIVs).The fact that within the transient regime the front is moving with constant velocity enables us to simplify the experimental measure of the viscosity. It can be therefore used to propose a new type micro-rheometer based on the measurement of the position of the front in contrast to methods based on tracer particles such as micro particle image velocimeters , on a glass substrate according to Replica Moulding and Soft Lithography microfabrication methods [s and 0.008 s depending of the chosen frame rate. The 10× objective allows us a 2 mm view of the channel. Photographs of the experimental setup are shown in H = 0.420 to 0.050 m and connected to a microtube of biocompatible material with uniform internal circular cross-section of diameter d = 0.254 mm and length lt = 0.430 m  is confined at the bottom of the tube and the plasma on the top. The plasma is extracted using an sterile pipette, under the extraction hood to avoid contamination of the sample.We study the behavior of three fluids of different viscosities and densities: water, ethylene glycol at two different volume concentrations and blood plasma. Plasma was obtained from human blood samples from two anonymous donors randomly selected. Blood samples were delivered for our experiments from the blood bank of the Hematology Department of the Clinical Hospital of Barcelona, in tubes of 10 or 5 η1, (a glycerol solution at 19% of volume concentration). We then study the properties of the dynamics of the fluid front of a second fluid of unknown viscosity, η2, which is being pushed into the microchannel by a column of glycerol solution. We have used two Newtonian fluids to measure their viscosity using our microcapillary device, namely, water and ethylene glycol at 44% volume concentration. We have used glycerol at this dilution because its density, ρ, is then practically identical to ethylene glycol at 44% volume concentration.In a second set of experiments, we consider a two-fluid system where we fill the reservoir with a liquid of known viscosity, We study fluid flow inside a capillary microchannel using a pressure-driven flow using the experimental set-up described in the Materials section. In this method, a pressure difference is set and the velocity of the fluid-air interface inside the microchannel is measured.Our experiments consist of measuring the average position of the front as the fluid fills the capillary.h(t), as a function of time at a column height of H = 0.290 m. At this pressure the channel is filled in less than 50 seconds. We observe that, for all three liquids, the mean position of the front behaves asν ≃ 1, consistent with a constant front velocity. Note that this behaviour is different from the predicted by Washburn’s law. According to Washburn’s result, the front velocity should be expected to decay as t−1/2.lc = 4 cm) is 8% for water, 11% for ethylene glycol, and 13% for blood plasma (see According to asma see .h(t) vs time). By measuring the position of the front, h(t), at different times , we can calculate the associated velocity as the time derivative of h(t). The average velocity is then obtained as the by taking the mean of all the “instantaneous” values over the length of the microchannel.In m and n to physical properties of the fluid and geometrical parameters of our experimental setup. The quantity n corresponds exactly to the capillary pressure associated to the curvature of the front. Regarding the parameter m, the theory developed in the Methods section enables us to relate m with the viscosity of the liquid. m and n obtained form linear fitting of the data shown in We now proceed to measure the velocity of the fluid front as the height of the liquid column, i.e. the pressure, varies. Our results are shown in H, which takes an average of t = 600 s. For each height, H, we obtain a velocity profile of the moving fluid front with a dispersion non superior to the 1.82% for water, 1.77% for ethylene glycol at 44% and 2.60% for blood plasma.Within the pressure range in which our experiments take place, the constant velocity regime is preserved until the whole capillary is filled for each value of h. In H = 200 mm, its velocity increases. By contrast, if the experiment is done with a single-fluid system (ethylene glycol at 44%), we do not observe such an acceleration. Rather, consistent with our results of our one-fluid systems, its front velocity remains constant as it fills the capillary.Our experiments with the two-fluid system consist of measuring the velocity of advancement of the front of the fluid being pushed, which fills the capillary, This result has non-trivial implications in the way we measure the viscosity of the fluid which fills the capillary. The method developed for measuring the viscosity in a single-fluid system is based on the fact that the advancement of the front occurs at constant velocity. Since this is no longer true for the two-fluid system, we must develop an alternative method.In order to explain the experimental results reported in the Results section, we propose a general mathematical model which accounts for the possibility of dealing with two fluids with positive viscosity contrast.b is the height of the micro-channel. We write the velocity of the fluid front inside the microchannel as:h(t) is the position of the front at time t and η2 is the viscosity to be determined. ΔP(t) is the total pressure drop:Phyd = ρgH, Pcap is the capillary pressure, and, last, the pressure drop generated by the resistance of the microtube connecting the fluid container to the microchannel, ΔPR(t), which is now time-dependent, due to the presence of a second fluid of known viscosity, η1, which progresively fills the microtube:l1(t) and l2(t) are such that l1(t) + l2(t) = lt at all time, where lt is the length of the microtube feeding the capillary , we have assumed that the microtube connecting the container and the microchannel has a circular cross-section and that the flow inside the microtube obeys Poiseuille’s law.We assume Darcy’s law with a permeability of a microrectangular cell, lary see . FurtherTaking into account that, due to mass conservation, η1 = η2 ≡ η. In this case The general equation h(t) increases, this approximation becomes less accurate, which implies that this linear regime is valid for either short microchannels or early times. Similarly, the approximation may fail for microchannels with small gap height b. This phenomenon is illustrated in b = 150 μm and b = 300 μm), the velocity stays approximately constant as the front advances through the capillary, in agreement with our approximation b of the channel, we expect that the approximation leading to b = 50 μm, where the front velocity is no longer constant as the front advances through the capillary.h(t):h(t) ≈ t1/2. In particular, in In the general case, when the above assumption does not apply, integration of From m in terms of the geometrical parameters of our setup and on physical properties of the fluid, in particular its viscosity. Using Eqs 10), we osing Eqs  and (10)n = Pcap, so that the capillary pressure associated to the curvature of the front can also be estimated from our experiments. In the case of water, our experimental results show that the capillary pressure is estimated to be Pcap = 184Pa. This result can be verified by using the definition of the capillary pressure in terms of the curvature radii of the front:τ is the surface tension, and θ is the contact angle of the fluid with the channel wall. By analysing experimental images of the fronts, we estimate the value of the contact angle, θ = 108 degree. Then, provided the values of the surface tension for water, τ = 0.0728Nm−1, and those of the parameters b = 300 μm and w = 1 mm, we estimate the capillary pressure for water to be Pcap = 194.97 Pa. This value is feasible and compatible with our experimental results shown in Comparing Eqs  and 10)10), n = Our method provides an effective description in which the pressure drop induced by gravity (hydrostatic pressure) is dominant over all other effects, including capillary ones. Our observations , then H, implies that, h , according to:C is given by:E = 3π2r6/2b4w2 and D = 2b3wlt/3πr4. Using the definition hr(t) and a is a positive constant. Using the fact that η2 by measuring the front velocity at two different points of the microchannel. Consider two positions on the microchannel, h1 and h2, and the associated  velocities, hr (η2:η2 in terms of the (known) viscosity of the pushing fluid, the geometric characteristics of the experimental setup and experimental measurements of We now describe a procedure to measure the viscosity of the fluid that fills the capillary, based on the experimental determination of the velocity and position of its front within the microchannel. We start by defining the quantity, on of hr , we have1Eη1η2-1,E = 3π2rh˙1-h2h˙2From Eqsη2 should be independent of both the height of the liquid column, H, and the height of the capillary, b. To check that our method satisfies these consistency criteria, we have performed measurements for different values of H and b. In order to proceed further, we first fix b and we study the behaviour of η2 as H varies. H. We further compare the viscosities obtained from the two-fluid model η2 is independent of the height of the liquid column, H. We also observe an excellent agreement between the estimates produced by our two methods.For this method of measuring the unknown viscosity of the capillary-filling Newtonian liquid to be consistent, the viscosity η2 that are independent of b, we now proceed to study the behaviour of the viscosity η2 as b varies. Results are shown in b. We also compare the estimates obtained with our two-fluid method to those obtained with the single-fluid method. We observe that both methods consistently predict a value of the viscosity which is independent of b. We further show that our estimates of the viscosities for water and ethylene glycol agree with those in the literature.In order to check that our method is consistent and therefore produces measurements of −1), the cone-plate rheometre produces spurious results, as the viscosity is not a constant but it depends on the shear rate. This spurious effects are not present in our microfluidic device which produces the right result with a constant viscosity.We have found evidence that our method provides more reliable measures of the viscosity in a wider range of shear rates than a standard, commercial cone-plate rheometre. We present results in h(t) ≈ t. This regime is a viscous, transient regime which eventually relaxes onto the well-known Washburn regime where h(t) ≈ t1/2. Note that this transient regime is different from the inertial regime, which is characterised by much shorter time-scales of the order of 10−5 seconds, whereas our linear regime extends to times of the order of minutes. In fact, from the experimental point of view, it is straightforward to control the duration of the linear regime by, for example, changing the geometrical parameters of the device. Furthermore, in addtion to the value of the exponent ν to be different from that prescribed by Washburn’s law, the values of the velocity may differ greatly from those expected from Washburn’s prediction, as they depend on the geometrical parameters of the experimental setup. Also, the fact that the velocity of advancement of the front is constant allows for a much simplified method to measure the viscosity and the consequent construction of a viscosimeter.We have proposed an experimental and theoretical framework to study capillary filling at the micro-scale. Our methodology enables us to control the fluid flow regime in such a way as to allows us to characterise the properties of the fluid such as the viscosity. In particular, we have been able to characterise a fluid flow regime where the front position b, although other ways are possible such as varying the diameter of the biocompatible microtube connecting the reservoir to the microchannel.It is also worth mentioning that our linear regime appears at constant inlet pressure conditions due to the geometry of the experimental set up, rather than by controlling the flow rate and letting the pressure to adapt. We have shown that a possible way to control whether we are within the linear regime is to modify the height of the microchannel, We have further presented an experimental and theoretical methodology that allows us to determine the viscosity of a fluid in a systems of two immiscible liquids by analysing the propagation of the front within a microcapillary. This methodology has the advantage of allowing for the use of very small samples of potentially expensive or rare liquids, as we do not need to fill the device with such liquid. By using such a device and the associated theoretical model, we have been able to explore a non-Washburn regime in which the fluid front within the capillary accelerates or decelerates depending on the viscosity contrast between both liquids: if the fluid that fills the capillary is more viscous than the liquid that pushes it into the capillary, then the front accelerates, and viceversa.H, or the value of the capillary gap, b. Using this method, we obtain results consistent with those reported in the literature.Under these conditions we cannot use the methodology proposed in one-fluid systems to measure viscosities of Newtonian fluids, so we have devised a new method. This new method is based on, for a fixed height of the liquid column, measuring the front velocity at two different positions within the microcapillary. We have checked that the viscosity so obtained does not exhibit spurious dependencies on experimental factors such as the height of the liquid column, The flow regime studied in this paper, in which we can observe both acceleration and decelaration of the fluid front, may induce to misinterpret the data regarding the properties of the fluid flow regime being observed. The theoretical approach that we have devised in this paper allows us to reliably measure the viscosity of Newtonian fluids regardless of the fluid regime introduced by our experimental setup. This theoretical framework is the stepping stone towards studying more complex fluids such as whole blood and other non-Newtonian fluids, as it allows to separate spurious dependencies produced by experimental artifacts from genuine non-Newtonian effects.ad hoc corrections, which are necessary when using other types of viscometers.This methodology has allowed us to design and calibrate a micro-viscosimeter to determine the viscosity with measurements over a wide range of shear rate, extending about two orders of magnitude. Our device is capable of obtaining the correct value of the viscosity of Newtonian fluids for low values of the shear rate without the need of Finally, we have shown evidence that our method is more reliable than a standard, commercial cone-plate rheometer for small shear rates, as shown in


In the Funding section, a grant number from the funder National Institute of Neurological Disorders and Stroke (NINDS) is missing. The additional grant number is: #1R56NS091616-01.


The aim of the current special issue was to bring together articles on various aspects of tumour modelling focusing on treatment response and prediction of clinical outcome based on functional imaging techniques.Together with technological and radiobiological advances, tumour modelling is an emerging area of oncology, which plays a key role in predicting treatment outcome in cancer patients. Despite the complexity of tumour biology and its microenvironment, computational and mathematical models of virtual cancer behaviour and response to treatment are successfully developed and employed for clinical research. A mathematical  model with optimal control for tumour-immune response with chemoimmunotherapy is presented by F. A. Rihan et al. in their research paper that describes the interaction of tumour cells and immune response cells with external therapy. In their model, the authors propose a numerical technique to solve the optimal control problem and identify the best strategy to combine chemotherapy and immunotherapy in order to minimise tumour load while maximising the strength of the immune system.Current diagnostic and imaging methods provide in-depth metabolic information that can be employed for tumour modelling. Latest technical and molecular advances in the field of nuclear medicine offer new possibilities in functional imaging, overcoming some of the confines imposed by previous diagnostic techniques. Positron emission tomography (PET) is the most advanced technology designed to provide metabolic information of disease, treatment monitoring, and also evaluation of treatment outcome. Together with other quantitative imaging techniques, such as dynamic contrast-enhanced MRI, PET is a promising diagnostic tool assisting in patient stratification for specific therapies, evaluation of drug efficacy, assessment of chemo- and radiotherapy outcome, and prediction of survival. in silico models of tumour growth, development, and behaviour during treatment. Tumour-related parameters such as cell proliferation, hypoxia, angiogenesis, and pH as well as PET/CT-specific biophysical parameters by means of SUV  and Hounsfield units are revisited in the context of computational modelling of complex processed involving tumour kinetics and treatment outcome.This research idea is underlined in the paper by M. Jennings et al., which focuses on PET-specific parameters and radiotracers in theoretical tumour modelling. The work reviews the use of PET/CT information applied to in silico models that aim to simulate or interpret the acquired image. The paper discusses two main streams of PET tracer modelling: (1) models of specific tracer/oxygen dynamics with the aim of simulating PET images leading to results that are in close agreement with real PET images and (2) utilisation of PET data within a separate tumour model with the aim of creating a more specific model that is predictive of response to treatment. The authors have collated analytical compartment models of tracer pharmacokinetics, stochastic models of treatment response using probability distribution functions, and reports on model predictions within clinical radiotherapy planning systems of dose distribution. A pertinent conclusion of the review is that the role of computer models based on functional imaging techniques in understanding patient-specific tumour behaviour is effusively justified.While several PET radiotracers have been developed for clinical use to target specific tumour properties, FDG (fluorodeoxyglucose) continues to be the most commonly used radionuclide in functional imaging as it offers unique information for tumour detection, staging, target definition, and response monitoring during radio- and chemotherapy. In this special issue, J. Jeong and J. O. Deasy have modelled the relationship between FGD uptake and tumour radioresistance as a function of the tumour microenvironment. The mechanistic model has considered cellular status to be dictated by glucose and oxygen content, showing that cells in the intermediate stress state (that receive glucose but not oxygen) present an increased avidity of FDG when compared to well-oxygenated cells. The role of hypoxia and the current status of hypoxia imaging including PET-specific markers are discussed by L. G. Marcu et al. in their review paper written in the context of head and neck cancer. Hypoxia-specific PET markers have been implemented in several clinical trials to quantify hypoxic tumour subvolumes for dose painting and personalised treatment planning. Tracer pharmacokinetics and PET-derived functional parameters serve as important input data forThe challenge of hypoxia in cancer management is further analysed by M. M. Iglesias et al. in a research article looking at the multimodality functional imaging in radiotherapy planning and the relationship between dynamic contrast-enhanced (DCE) MRI, diffusion-weighted MRI, and FDG-PET in order to develop predictive individualised models of tumour response to radiotherapy in head and neck cancer patients. For an optimal biologically guided radiotherapy, to obtain relevant datasets on tumour hypoxia and cellular density, there is a need to understand the correlations and interactions between various functional imaging modalities. Parameters such as SUV (PET), Hounsfield units, dose, ADC (apparent diffusion coefficient) maps (MRI), and contrast exchange coefficients (DCE-MRI) have been recorded for each patient from the study and the relationship between parameters analysed. The authors concluded that the above functional parameters based on different image datasets are valuable in describing in a complex manner tumour oxygenation and vascularisation, cell density, and tumour malignity offering, therefore, treatment personalisation and optimisation.A central motif of today's cancer management is personalised treatment planning and delivery. While this trend is featured in all papers of the current special issue, a more focused view upon treatment individualisation via multidimensional radiotherapy is presented in the work of I. Toma-Dasu and A. Dasu. According to the authors, the key approach to maximise the individualisation of treatment is by combining multiparameter information from imaging with predictive information from biopsies and molecular analyses and also by monitoring tumour response to treatment. The emphasis of the paper is on biologically adapted radiation therapy (BIOART), which is based on both pretreatment conditions and intrinsic responsiveness assessed using functional imaging techniques. The authors suggest that the BIOART concept should replace the simple dose painting approach for a more optimal management of radioresistant subvolumes within the BTV . Functional imaging is shown to have the potential to provide a paradigm shift in treatment planning and optimisation that extends beyond target definition.In silico modelling in cancer research was and continues to be a very important tool of treatment simulation and optimisation, contributing to a more personalised medicine for an improved patient outcome. The use of computational models for treatment assessment and outcome prediction is fast growing and the power of in silico models as preclinical tools becomes acknowledged. Therefore, multidisciplinary research leading to predictive models of tumour response to treatment using functional imaging techniques needs to be encouraged to enable further developments in the diagnostic and treatment of cancer.Loredana G. MarcuLoredana G. MarcuEva BezakEva BezakIuliana Toma-DasuIuliana Toma-DasuAlexandru DasuAlexandru Dasu


The correct assessment of volume status and prediction of response to filling fluid challenge is of crucial importance in the patient with septic shock. A patient is a fluid responder if there is an increase in cardiac index (CI) >15% in response to the fluid challenge. Identifying which patient will be a fluid responder is crucial. The aim of this prospective study is to evaluate which patient is a fluid responder in septic shock using dynamics echocardiography with the distensibility index of the inferior vena cava (DIIVC), which has been shown to be predictive of an increase in CI [In a period of 1 year, 42 adult patients were admitted to the ICU in septic shock. All patients were treated according to the guidelines of the Surviving Sepsis Campaign International Guidelines for Management of Severe Sepsis and Septic Shock 2012 [Sixteen patients (38%) had a change in DIIVC <18%, on average 11 ± 5%, and when subjected to fluid challenge did not have an increase in CI >15%, average 8 ± 3%, not proving to be a fluid responder. Twenty- six patients (62%) had a change in DIIVC >18%, on average 45 ± 22%, and when subjected to fluid challenge had an increase of the CI >15%, on average 31 ± 8%, proving to be a fluid responder.We have shown that the DIIVC can predict the response of patients to fluid challenge regardless of the values of CVP and then distinguish fluid responders from nonresponder patients. This can help us in the choice of treatment for patients in septic shock.


This work deals with the flow and heat transfer in upper-convected Maxwell fluid above an exponentially stretching surface. Cattaneo-Christov heat flux model is employed for the formulation of the energy equation. This model can predict the effects of thermal relaxation time on the boundary layer. Similarity approach is utilized to normalize the governing boundary layer equations. Local similarity solutions are achieved by shooting approach together with fourth-fifth-order Runge-Kutta integration technique and Newton’s method. Our computations reveal that fluid temperature has inverse relationship with the thermal relaxation time. Further the fluid velocity is a decreasing function of the fluid relaxation time. A comparison of Fourier’s law and the Cattaneo-Christov’s law is also presented. Present attempt even in the case of Newtonian fluid is not yet available in the literature. Heat transfer phenomenon is involved in wide ranging industrial and engineering processes including nuclear reactor cooling, space cooling, energy production, biomedical applications such as magnetic drug targeting, heat conduction in tissues etc. and many others. Heat conduction law proposed by Fourier  has beenThe purpose of this paper is to study the boundary layer flow of upper-convected Maxwell (UCM) fluid induced by exponentially stretching sheet using Cattaneo-Christov heat flux model. Maxwell fluid is a popular viscoelastic fluid that can give the influence of fluid relaxation time. On the other hand, the study of viscous flow and heat transfer above stretching surfaces has been widely addressed research area due to its abundant applications in chemical and manufacturing processes including polymer extrusion, continuous casting of metals, extrusion of copper wires, die forging, paper production and several others. Several interesting boundary layer flow problems involving the stretching surfaces have been addressed in recent years –14. The y = 0. The sheet is stretched in its own plane with the velocity T0 denotes the heating/cooling reference temperature. This is reasonable since in extrusion process, the material properties and in particular the elasticity of the extruded sheet is being pulled out by a constant force. Invoking the boundary layer approximations, the equations governing the two-dimensional flow and heat transfer of incompressible UCM fluid are expressed as below:u and v denote the velocity components along the x—and y—directions respectively, v is the kinematic viscosity, λ1 is the fluid relaxation time, T is the local fluid temperature and q is the heat flux which satisfies the following relationship [λ2 is the relaxation time for heat flux, V is the velocity vector and k is the thermal conductivity. Eliminating q from Eqs (α (= k / ρcp) is the thermal diffusivity. The boundary conditions are imposed as below:Consider the steady two dimensional incompressible flow of upper-convected Maxwell (UCM) fluid over an elastic sheet located at T0eAx/2L  is consi∂y)=−∇.q,where u ∂y)=−∇.q,where u )q)=−k∇T,in which∂y)=−∇.q,where u Using the following similarity transformations η=U02νLePr = v / α is the Prandtl number.Eq  is ident1 = 0, the case of Newtonian fluid is obtained. Further Λ2 = 0 corresponds to the case of classical Fourier’s heat conduction law.It is important to point out through the Eqs –10) tha tha10) tst order ODEs by making a substitution  = . This yields the following:We employ the shooting method with fifth order Runge-Kutta procedure for the numerical solution of the present problem. First of all we reduce the Eqs  and 9)  9) and bu1 = f”(0) and u2 = θ’(0). The values of these conditions are then iteratively estimated through Newton’s method such that solutions satisfy the boundary conditions at infinity (given in −5.given in  with theθ’(0) is directly proportional to the dimensionless relaxation time Λ1. However it appears to decrease upon increasing the fluid relaxation time. Notably, the value of θ’(0) is negative when A = −1.5 revealing the reverse flow near the wall which will be explained later. There is a significant growth in the wall temperature gradient θ’(0). When A is incremented. When A enlarges, this leads to larger a surface temperature and hence larger heat transfer rate from the sheet.Physical interpretation to the behavior of the embedded parameters is assigned in this section. In 1 may be regarded as increase in fluid viscosity. This increased viscosity opposes the fluid motion and consequently the velocity decreases. It is also clear that velocity profiles are tilted towards the stretching wall when Λ1 is increased which means that boundary layer thickness is an increasing function of Λ1. The obtained results are in accordance with the results of Han et al. [n et al.  in whichPr on the thermal boundary layer with and without the consideration of thermal relaxation time. The behavior of Pr on θ is qualitatively similar in both the cases i.e. the temperature and thermal boundary layer thickness both are found to decrease upon increasing Pr. Notably the variation in temperature θ is similar in magnitude in both Fourier and Cattaneo-Christov heat flux models. Physically, the Prandtl number Pr is inversely related with the thermal diffusivity α. As Pr enlarges, one anticipates less thermal effect to penetrate into the fluid. Due to this reason the thermal boundary layer becomes thinner when Pr is increased. The thinner thermal boundary layer leads to a steeper temperature profile indicating larger wall slope of temperature function.A on the temperature profile is sketched. This Figdepicts an interesting phenomenon of “Sparrow-Gregg hill (SGH)” for negative temperature exponent A in which temperature θ first approaches to a maximum and then exponentially descends to zero when η is increased. This means that for some negative A, one expects reverse heat flow in the vicinity of the sheet. This result is consistent with the findings of Magyari and Keller [A, we observe a sharp growth in wall slope of temperature function.In d Keller  for the 1 on thermal boundary layer. Stronger viscous force associated with the larger Λ1 resists the flow and enhances the temperature. This leads to the conclusion that temperature in viscoelastic fluid is greater than the viscous fluid. In 2. on the temperature distribution is sketched. We observe that temperature θ has inverse relationship with the thermal relaxation time. We also notice that temperature θ approaches the free stream condition at shorter distances above the sheet for bigger Λ2. Notably, the variation in temperature θ with thermal relaxation time is of similar magnitude in Newtonian and Maxwell fluids.2 at different values of Λ1. θ’(0) linearly increases with an increment in Λ2 whereas it appears to decrease when Λ1 is increased. θ’(0) against the Prandtl number with the variations in Λ1 and Λ2. This Fig is complementing the numerical results of θ’(0) given in θ’(0) are nearly a straight line revealing that heat transfer rate grows linearly when Pr is augmented. We observe that θ’(0) approaches to zero for vanishing Prandtl number.Hydrodynamic boundary layer is thinner in viscoelastic fluid when compared with the viscous fluid.2.Temperature and thermal boundary layer thickness are decreasing functions of relaxation time ΛA.Interesting Sparrow-Gregg Hills (SGH) for the temperature distribution exist for negative temperature exponent The behaviors of parameters in Cattaneo-Christov model are qualitatively similar to those in Fourier’s heat conduction law.1 = 0.The present consideration for the Newtonian fluid case can be recovered by choosing ΛCattaneo-Christov heat flux model is used to describe the heat transfer in viscoelastic flow induced by an exponentially stretching sheet. The major points of this study may be summarized as under:


In doing this, a mechanism has been established to fulfill the FDA regulatory requirements for translating promising PET imaging probes from preclinical research into human clinical trials in an efficient and cost-effective manner.We have developed an efficient, streamlined, cost-effective approach to obtain Investigational New Drug (IND) approvals from the Food and Drug Administration (FDA) for positron emission tomography (PET) imaging probes . The required application and supporting data for the INDs were collected in a collaborative effort involving appropriate scientific disciplines. This path to INDs was successfully used to translate three [The online version of this article (doi:10.1007/s11307-014-0735-2) contains supplementary material, which is available to authorized users. Thus, it is reasonable to surmise that there is a critical but unmet need to identify an efficient and cost-effective methodology of translating promising PET probes from the preclinical to the clinical domain and obtaining first-in-human data.positron emission tomography (PET) is an exceptionally sensitive and noninvasive imaging modality that produces a quantitative, three-dimensional image of biological and pharmacological processes occurring in the body. This study’s approach involves administration of PET imaging probes and detection of the resulting 511-MeV gamma rays, due to positron decay, with a PET scanner. Over the past three decades, more than 2,000 PET probes have been developed fluoro-arabinofuranosylcytosine (FAC) analogs within a 1-year time span, as described below.This collaborative approach has been successfully used to obtain three INDs for the [18 F] fluoro-β-d-arabinofuranosyl)cytosine (d-[18 F] FAC), 1-(2′-deoxy-2′-[18 F] fluoro-β-l-arabinofuranosyl)cytosine (l-[18 F] FAC), and 1-(2′-deoxy-2′-[18 F] fluoro-β-l-arabinofuranosyl)-5-methylcytosine (l-[18 F] FMAC), have been designed to closely resemble the structure of the natural dCK substrate, deoxycytidine, and the nucleoside pro-drug gemcitabine fluoro-d-glucose (FDG) for assay of transport and phosphorylation of glucose [ in mice  provide  glucose .in vivo assay of dCK enzyme activity with PET in cancer patients correlated with the in vitro assays of dCK expression and enzyme activity from surgically excised cancer tissues.We utilized the IND approach for investigating the three FAC analogs in human subjects to (a) assess the safety profile of the FAC probes in human subjects and (b) determine the bio-distribution of the three FAC analogs in cancer patients and evaluate whether the d-[18 F] FAC, l-[18 F] FAC, and l-[18 F] FMAC, a 14-day preclinical safety pharmacology and toxicology study was conducted in Sprague-Dawley rats by UCLA DLAM and the Rodent Physiology Core under GLP conditions  postinjection in groups 1–4, and the late effects of the administered probes were monitored on day (+14) post injection in groups 5–8. Rats in groups 1 and 5 were administered the carrier solution as the control group and those in groups 2 and 6, groups 3 and 7, and groups 4 and 8 were administered with the test probes Following completion of preclinical studies for the FAC analogs, a pre-IND meeting was scheduled with the FDA to discuss the best approach for submission of the three INDs, one for each FAC probe. Based on the data package submitted and guidance on preclinical studies required to satisfy microdose administration criteria , the FDAd-[18 F] FAC, l-[18 F] FAC, and l-[18 F] FMAC). Upon completion of the 30-day reviews which responded to questions raised by the FDA, UCLA was granted permission to proceed with the phase 1 clinical studies of 20–30 human subjects for each probe.INDs, in traditional 11-part paper format, per regulations in 21 CFR 312.23, were submitted to the FDA for each FAC analog  preclinical safety pharmacology and toxicology; (2) preclinical imaging and dosimetry; (3) chemistry, controls, and manufacturing; (4) clinical study design; and (5) an overall IND submission strategy to satisfy the phase 1 IND requirements in an open and cooperative manner with the FDA. Using the FAC analog PET probes developed at UCLA as a case study, we have demonstrated that this overall phase 1 IND approach can successfully be applied to obtain FDA approval of phase 1 INDs in an academic site efficiently and at a reasonable cost.Although the protocol developed herein has proved successful, we recommend requesting a pre-IND meeting with the FDA prior to initiating preclinical safety pharmacology and toxicology studies in rodents. We surmise that if we had engaged the FDA team earlier and made this request prior to initiating these studies, safety pharmacology studies may have been waived or a limited number of studies would have satisfied the FDA, due to the microdose, tracer levels of PET probes administered according to the eIND guidance document. As such, it is prudent to initiate a pre-IND FDA meeting early in the study development process, which may result in additional cost and time savings for the IND submission. Based on experience gained from the FAC IND project, an optimized project timeline to IND for PET probes is provided here Fig. .Fig. 5.IIt is the goal of the DMMP at UCLA to continue working closely with the FDA to fulfill the regulatory requirements for PET imaging probes. New requirements outlined in the FDA Safety and Innovation Act (FDASIA) move towards electronic submission of IND applications in Electronic Common Technical Document (eCTD) format. We hope the FDA will make special accommodations for academic sites regarding PET probes in tracer doses by waiving the requirement for submission of eCTD INDs or providing additional tools to academic sites that reduce the cost and time of IND submissions while still maintaining the quality and safety standards relevant to PET probes.in vivo biology of disease in patients, (2) aid in the drug discovery and development processes, and (3) patient stratification for appropriate drug(s) and assessing therapeutic responses.It is critical for academic sites to have a streamlined and cost-effective approach to translate PET imaging probes from preclinical research to clinical investigations and investigate the degree to which preclinical findings for these probes apply to humans. This will allow for the (1) development and introduction of new molecular imaging diagnostics of disease that serve as an array of new assays to investigate the ESM 1(PDF 4693 kb)Below is the link to the electronic supplementary material.


Effects of M2 tumour-associated macrophages on the pathogenesis of diffuse large B cell lymphoma (DLBCL) are still controversial. Our data showed that the number of CD163-positive M2 macrophages correlated negatively with DLBCL prognosis. Macrophage depletion by clodronate liposomes significantly suppressed tumour growth in a xenograft mouse model of DLBCL using OCI-Ly3 cells. Moreover, M2 polarization of macrophages induced legumain expression in U937 cells. Exogenous legumain promoted degradation of fibronectin and collagen I, which was abolished by administration of a legumain inhibitor RR-11a. Overexpression of legumain in Raw 264.7 cells also induced tube formation of endothelial cells in matrigel. In the xenograft mouse model of DLBCL, decreased fibronectin and collagen I, as well as increased legumain expression and angiogenesis were found at the late stage tumours compared with early stage tumours. Co-localization of legumain and fibronectin was observed in the extracellular matrix of tumour tissues. Administration of the legumain inhibitor to the xenograft DLBCL model suppressed tumour growth, angiogenesis and collagen deposition compared with the control. Taken together, our results suggest that M2 tumour-associated macrophages affect degradation of the extracellular matrix and angiogenesis via overexpression of legumain, and therefore play an active role in the progression of DLBCL. Diffuse large B cell lymphoma (DLBCL) is the most frequent Non-Hodgkin’s lymphoma, accounting for 30–40% of newly diagnosed lymphomas245et al. suggested that CD68-positive TAMs have no prognostic value in DLBCLSeveral clinical data suggest that a high density of tumour-associated macrophages (TAMs), especially M2 TAMs can predict adverse outcomes in many kinds of cancers7Legumain is an asparaginyl endopeptidase that is classified as a member of the C13 family of cysteine proteases1417Here, we provide evidence that a high percentage of M2 TAMs predicts poor outcomes in DLBCL patients. M2 TAMs in the lymphoma microenvironment overexpressed and secreted legumain. In addition, legumain was involved in angiogenesis and tumour progression via extracellular matrix (ECM) remodelling. Therefore, we have identified a novel therapeutic target as well as a diagnostic index for the treatment of DLBCL.Whether a higher ratio of CD163- or CD68-positive macrophages can serve as a predictive index for a poorer prognosis of DLBCL is still controversial. Here, we collected clinical data from 139 DLBCL patients with various stages of disease and calculated their international prognostic index (IPI). In the tumour samples, immunohistochemical staining of CD163 was performed to visualize the CD163-positive macrophages. Our data showed various ratios of CD163-positive macrophages in DLBCL tumour tissues. The mean ratio of CD163-positive M2 macrophages was 13.23% . Flow cytometry analysis suggested that the ratio of CD206-positive M2 TAMs increased with tumour progression . Clodrolip (25 mg/kg), RR-11a (0.2 mg/kg), or the vehicle only (control liposome) was used to treat mice. As shown in As the largest cell population in the tumour microenvironment, the effects of macrophages on tumour pathogenesis have been widely studied in various tumour models. Several lines of evidence support that a high ratio of M2 polarized macrophages (M2 TAMs) leads to a poor outcome of DLBCL. Marchesi and colleagues suggested high M2 TAM levels at diagnosis as an unfavourable prognostic factor in patients with DLBCL21In vitro, experiments showed that conditioned medium from M2 TAMs induced cleavage of ECM and formation of vessel tubes. Moreover, administration of the legumain inhibitor indicated that these effects were mediated by legumain. At this time, it is still hard to distinguish the major source of legumain in the tumour microenvironment since both tumour cells themselves and tumour associated macrophage have been proven to overexpress this protein. Lin et al. revealed that stress stimuli induced TRAF6 ubiquitination of pro-legumain by K63 polyubiquitin thus enhanced both the intracellular stability and secretion of legumain in the tumour cellsECM remodelling, including ECM cleavage, modulating angiogenesis and immune responses provides essential signals for tumour progressionin vitro. It has been reported that pericellular proteases contribute to cleavage of the ECM and the release of angiogenic factors such as vascular endothelial growth factor. Meanwhile, the degradation products might also directly promote angiogenesis.Studies on cathepsins show that they either degrade ECM proteins via being secreted into the extracellular area or internalize ECM components such as collagen through endocytosis and degrade them in lysosomes29Considering the critical role of TAMs, especial M2 TAMs in the progression of tumours, diverse strategies aimed to delete M2 TAMs have been designed and achieved encouraging results on limiting tumour progressionIn this study, we identified the M2 macrophage marker CD163 as a potential prognostic index for DLBCL. Moreover, legumain secreted by M2 TAMs contributes to cleavage of the ECM as well as angiogenesis in tumour microenvironments. Administration of a legumain inhibitor or deletion of M2 TAMs limits tumour expansion, ECM cleavage and angiogenesis. Therefore, our study suggests a novel potential method to optimize the treatment of DLBCL.Human DLBCL cell line OCI-Ly3 was kindly provided by Jun Chen  and cultured in RPMI 1640 supplemented with 20% fetal bovine serum (FBS). The human monocytic cell line U937, human umbilical vein endothelial cells  and the mouse monocytic cell line Raw 264.7 were kind gifts from Dr. Ralph Reisfeld . U937 and Raw 264.7 cells were cultured in RPIM 1640 supplemented with 10% FBS. HUVECs were cultured in EGM-2 endothelial cell medium . Lentivirus production was performed as described previously8 cells/ml) was subcutaneously injected at the forth rib of mice. For tumour staging, mice were divided randomly into two groups (n = 5) and sacrificed when the lymphoma volume reached 800 mm3 (early stage) or 1,500 mm3 (late stage). To analyze the effect of M2 TAMs and legumain on tumour progression, mice were divided randomly into three groups (n = 5) and intravenously injected with PBS (control) or clodronate liposomes (25 mg/kg) every 4 days ; or intraperitoneally injected with legumain inhibitor RR-11a (0.2 mg/kg) every day for 12 days. Tumour growth curves were recorded and the mice were sacrificed at the end of the experiments. Primary tumours were collected for tissue sectioning and homogenate preparation.Six- to eight-week-old male NOD/SCID mice were purchased from Beijing HFK Bio-Technology  and housed at the Animal Centre of Collagen of Life Science, Nankai University. All animal experiments were approved by the Institutional Animal Care and Use Committee at Nankai Univeristy, and were performed in accordance with relevant guidelines and regulations. To establish the animal model, 100 μl of OCI-Ly3 cells  for 1 h, washed with acidified water, dehydrated, and mounted in a resinous medium. Images were captured by a BX53 research microscope .To isolate murine peritoneal macrophages, inject 1 ml of 3% Brewer thioglycollate medium into the peritoneal cavity of each mouse. 4 days later, the mice were injected with 5 ml of ice cold PBS (with 3% FBS) into the peritoneal cavity. Then the peritoneal fluid was withdrawn slowly and centrifuged at 1500 rpm for 8 min, the cells were resuspended in RPMI 1640 supplemented with 10% FBS. To stimulate M1/M2 polarization in macrophages, 100 ng/ml lipopolysaccharide  or 20 ng/ml mouse interleukin 4  were added to the macrophages and incubated for 24 h or 96 h at 37 °C.OCI-Ly3 xenograft tumour tissues were collected, digested, and filtered to prepare mono-cellular suspensions. To quantify the ratio of CD206-positive TAMs in primary tumour cells, cells were washed twice with PBS containing 1% FBS, dispersed cells were incubated with PE/Cy5 anti-mouse F4/80  and FITC anti-mouse CD206 (Biolegend) antibody for 15 min on ice. The resulting suspensions were immediately analysed using a digital BD Caliber flow cytometer .OCI-Ly3 xenograft tumour tissues were ground and filtered to prepare tumour homogenates. U937 monocytic cells were treated with the tumour homogenates. RNAs from lymphoma tissues. U937 cells and primary macrophages were isolated using Trizol Reagent  and reverse transcribed to cDNA with a reverse transcription system . Then, we conducted semi-quantitative PCR by agarose gel electrophoresis. The primers used were as follows : h-legumain, ggcacagatgtctatcagggag and ctcatcatagtaacaggcgtaggac; h-CD163, cgagttaacgccagtaagg and gaacatgtcacgccagc; h-IL-10, ggttgccaagccttgtctga and agggagttcacatgcgcct; h-MMP9, atgcgtggagagtcgaaatc and tacacgcgagtgaaggtgag; h-GAPDH, gcaccgtcaaggctgagaac and tggtgaagacgccagtgga; m-IL-1β, atccagcttcaaatctcgc and atctcggagcctgtagtgc; m-iNOS, gaaacgcttcacttccaatg and aatccacaactcgctccaa; m-IL-6, ttgccttcttgggactgat and ttgccattgcacaactctt; m-CXCL-9, tcatcttcctggagcagtg and ttcttcacatttgccgagt; m-Arg-1, cagtctggcagttggaagc and ttggcagatatgcagggag; m-IL-10, caacatactgctaaccgactc and catggccttgtagacacct; m-legumain, cgccgtgctgagaggtgacg and gactccttggggttggccgc; m-F4/80, cgtgctggagcaagcgacca and tctccccggtcacagtgcca; m-CD206, acgcagtggttggcagtggg and ttgccaggtccccaccctcc; m-TNF-α, cctcttctcattcctgcttg and cacttggtggtttgctacg; m-CCL2, cctgctgttcacagttgcc and tggacccattccttcttgg; m-GAPDH, tgcccagaacatcatccct and gaagtcgcaggagacaacc.The detailed procedures of immunohistochemical and immunofluorescence analyses have been described previously2 for 30 min to solidify the matrigel. Once the matrigel had set, 200 μl Raw 264.7 cell-derived conditioned medium was mixed with 50 μl resuspended HUVEC cells (approximately 2 × 104 cells) and then added to the plates. After 6 h, the wells were washed with PBS and the tube network was imaged using the IX51 research microscope.HUVECs were used in the tube formation assay. Briefly, matrigel was coated onto 48-wells plates and then incubated at 37 °C with 5% COFibronectin and collagen I were coated to the bottom of 96-well plates and treated with or without legumain for 2 h. Then, protein samples were collected and subjected to western blotting with antibodies against fibronectin (Santa Cruz Biotechnology) and collagen I (Abcam). To detect proteins extracted from cells, equivalent amounts of total protein from each lysate were loaded for western blotting to analyze the target protein level with the indicated antibody including anti-legumain and anti-β-actin (Santa Cruz Biotechnology) antibodies. An ECL chemiluminescence kit  was used to detect the proteins.GraphPad Software  was used for data analysis. The statistical significance among groups was determined by a t-test and two-tailed analysis of variance. A P-value of less than 0.05 was regarded as statistically significant.How to cite this article: Shen, L. et al. M2 tumour-associated macrophages contribute to tumour progression via legumain remodelling the extracellular matrix in diffuse large B cell lymphoma. Sci. Rep.6, 30347; doi: 10.1038/srep30347 (2016).