To this end, initial conversion of silver stained BM color images into binary images identified two limitations: variable color, owing to polychromatic staining of reticulin fibers, and variable background in different sections of the same batch, limiting application of the color deconvolution method, and use of constant threshold, respectively. By blind coding image identities, to allow for threshold input (still within a narrow range), and using shape filtering to further eliminate background we were able to quantitate RF in myelofibrotic Gata-1low  and wild type (control) mice as a function of animal age. Color images spanning the whole femur BM were batch-analyzed using ImageJ software, aided by our two newly added macros. The results show heterogeneous RF density in different areas of the marrow of Gata-1low mice, with degrees of heterogeneity reduced upon aging. This method can be applied uniformly across laboratories in studies assessing RF remodeling induced by aging or other conditions in animal models.Bone marrow (BM) reticulin fibrosis (RF), revealed by silver staining of tissue sections, is associated with myeloproliferative neoplasms, while tools for quantitative assessment of reticulin deposition throughout a femur BM are still in need. Here, we present such a method, allowing  Myeloproliferative neoplasms (MPN) include polycythemia vera, essential thrombocythemia, and primary myelofibrosis (PMF), as observed in clinical and bone marrow (BM) morphologic findings . A numbeSilver impregnation to visualize subcellular structures was the keystone method used by Santiago Ramón y Cajal and Camillo Golgi, leading to their 1906 shared Nobel Prize in Physiology or Medicine. By 1937 György Gömöri consolidated the silver impregnation technique to visualize tissue reticulin fibers . Since tAssessment of BM fibrosis in mouse models has relied on using reticulin staining followed by gross evaluation of density visually or semi-quantitatively analyzed. The need for a uniform and quantitative approach across laboratories to measuring fibrotic burden is eminent when the process has to be followed as function of age, or comparatively between experimental models or conditions. Here, we describe a method of quantification of RF, overcoming two main limitations associated with image analysis: (1) variable color of the silver impregnated reticulin fibers; and (2) variable background staining. This method is not meant to replace the broadly used and accepted, though not exempt of problems and pitfalls , subjectA comprehensive study described phenotype variation in GATA-1low mice depending on genetic background . Here, wThis quantitative image analysis is the last step in a procedure involving animal selection → femur isolation → decalcification → fixation → paraffin embedding → histological sectioning → slides generation → deparaffinization → silver impregnation → image acquisition → image analysis. The silver impregnation procedure, involving around 20 steps and the use of 9 chemicals is an important source of analytical variability. However, several refinements of the protocol have produced abundant literature to troubleshoot deficient staining outcomes.Fig. S1A). Pictures, taken from each bone section  were numbered and the numbers were placed at the end of the picture name string. Thus, if 4 pictures were taken from section “a” in slide 8, they were saved under the following names: 3W30_8_a1, 3W30_8_a2, 3W30_8_a3, and 3W30_8_a4 . Hence, a large number of images, collected from multiple genotypes of animals at different ages, were unambiguously named. During image processing these annotations were coded with positive natural numbers and subsequently randomly coded with a 20 digits code as described below.Three consecutive femurs sections (5 μm each) were placed parallel to the width of the slide and referred to as “a”, “b” and “c” from the top end. Slide number, animal number, genotype and age were recorded on the slide top end (Table S1).Ten or more digital color (Red Green and Blue) RGB images (16000 × 12000 pixels) per stained and mounted bone sections were captured along the diaphysis longitudinal axis in a Nikon Eclipse 50i microscope (20 × /0.40 objective and ocular 10 × /22) with a color digital camera  controlled by the Spot Image software v5.0.37. TIFF images were saved to a dedicated folder and their filenames recorded in an Excel sheet. The original file names, organized by slide numbers within each genotype, were coded by a single number following the list order. Then the corresponding image folder was blind-coded using the broadly applicable routine (BAR v1.1.6) for ImageJ. The folder was processed in ImageJ (BAR → Snippets → Process Folder ImageJ) to generate a new folder containing image files with their names blinded to a 20 digits name . This procedure can be batch processed by running an appropriate macro, which we added to the application (Script S1).Using ImageJ platform , color ii.e., 13344 × 4920 pixels). Divide 13344/1600 = 8.34 and 4920/1200 = 4.1; (4) Fragment the large image into smaller images to facilitate image cleaning in next step: Open the whole BM image → Image → Stacks → Tools → Montage to Stacks. In the Stack Maker window, select Images per row = 8 and Images per column = 4. This mode of fragmentation generates 32 images of a manageable size, close to the size of original ones that were stitched to produce the large image; (5) Erase image artifacts in the stack using the Paintbrush Tool with a width set at around 30 pixels; (6) Get the 32 images from the stack: Image → Stacks → Stack to Images; and (7) Save the RGB images in a dedicated folder for further analysis.This procedure involves few distinct steps: (1) Overlapping pictures (1600 × 1200 pixels) along the whole diaphysis longitudinal axis length of the bone are taken and saved in a folder. This step generates over 200 RGB images; (2) Overlapping RGB images are stitched using Microsoft Image Composite Editor, generating a 500 MB image. The bone area in the RGB digital image is erased; (3) Change the image area unit from inches to pixel. Analyze → Set scale Type “pixels” in the “Unit of length” box. Take note of the width and length values shown in the upper left corner of the image (Note: In the Results table the useful columns are “Label” (file name) and “Max Thr” (threshold applied). Values recorded in “InDen” and “Raw IntDen” columns are not useful at this stage of the analysis, but are populated in step 9 below; (5) Activate “Apply” in the Threshold window to get “Convert to Mask” the thresholded image; (6) Edit → Invert the image. Required for Shape Filter processing in the next step; (7) Plugins → Shape Filter → input 70–100000 in “Area”, 15-Infinite in “Feret Diameter”, and 30-Infinite in “Circularity” options → Click OK; (8) Process → Math → Divide → Input 255 to convert from a 0–255 to a 0–1 binary image; (9) Analyze → Measure. Note: In the Results table the useful data are now in columns “Label” (the file name) and columns “InDen”/“Raw IntDen” (area of reticulin fibers); and (10) The final, processed binary image, its threshold value and its measured integrated density in the results table, are saved.Corrected RGB images are subjected to the following procedure: (1) Convert to RGB Stacks Image → Type → RGB Stacks; (2) Image → Color → Arrange Channels → Select only “2” in the “New channel order” window. Accept that channels will be reduced from 3 to 1; (3) Analyze → Set Measurements → Check only “Integrated density” (IntDen), “Limit to threshold” and “Display label” options. The “Display label” mode associates the file name with the corresponding IntDen value, critical for final data compilation and presentation; (4) Image → Adjust → Threshold → Set the upper slider to zero and adjust the lower slider till only fiber structures (highlighted in red) remain → Measure. A set is the number of images to be batch-analyzed. The number can be calculated using the following formula: 10 (images per slide) × 3 (bone sections per slide) × #genotypes × #animals/genotype × #slides/animal. Thus, when analyzing 4 genotypes by using 3 animals per genotype and 2 slides per animals, the total number of images is 720 from 24 slides. The average set size analyzed in this study was around 360 images, from 2 genotypes (WT and Gata-1 low), 3 animals/genotype and 2 slides/animal. In addition, to minimize the impact of Gömöri's silver impregnation variability on the results, all slides in a set were stained at the same time.Script S2). The first sub-macro calls for an “In” folder, containing the images to be analyzed and an empty “Out” folder were the binary, thresholded images will be stored. Then the Batch Command loops all images from the “In” folder to be thresholded one at the time with input from the user. The resulting thresholded images are stored in “Out” folder. The Results table, recording image blinded name and its corresponding threshold value remains open. When the first sub-macro is completed the second sub-macro calls for an “In” folder that is the “Out” folder from the first sub-macro, and an “Out” folder that will store the final binary images that were quantified for the area corresponding to reticulin fibers (integrated density). This sub-macro runs to completion without user intervention. The Results table, now containing all images files names with their corresponding threshold and integrated density values is transferred to an Excel sheet data calculations and statistical analysis (Table S2).The single image process analysis outline above was applied to sets of images in a batch process mode driven by a newly developed macro  in Microsoft Excel. Putative statistical differences among sections of a slide or among Gata-1 low age group  were subjected to the Tukey-Kramer test .We sought to develop a quantitative method that enables tailoring the software to analytical needs , and thuFig. S3). The polychromatic nature of RF can be directly observed by over magnification of the image . This color variability does not affect the subjective scoring evaluation to assess the progress or remission of RF, since this approach is not quantitative and it is based on the morphology and abundance of reticulin fibers, not on their color. However, color variability is a severe limitation for the application of color deconvolution in quantitative analysis.The first feature we tackled was color deconvolution of RGB images from tissues or cells, which is based on the principle that the target sub-cellular component acquires a unique color after staining. However, visual inspection of Gömöri reticulin stained images often reveals fibers with color tones varying from dark purple to dark grey. To illustrate this, ImageJ color deconvolution analysis was applied to images 64_2_30_8_b2 and 64_2_30_10_a1, number coded 665 and 668 respectively (numbering procedures are explained in Methods). The region of interest was selected using three user-defined colors taken from the color of fibers, the color of the background free of objects and the color of background elements. In image 665 reticulin fibers were deconvolved almost exclusively in color 1 channel, while in image 668 reticulin fibers were deconvolved mostly in color 2 channel, and some in color 1 channel . This calls for TH adjustment of individual images. To eliminate subjective bias in the proper assignment of threshold values, the file name (identity) of all images analyzed where assigned a random, 20 digits code name described under Materials and Methods (Table S1).Image segmentation (thresholding) in quantitative analysis of image elements (shapes) requires isolation of such elements from the background. When the background is constant for all images, a single, optimal threshold value can be applied. When BM images from Gata-1 low and wild type animals were analyzed, applying the same threshold, a suboptimal output was observed in both cases: a threshold (TH) optimal to isolate reticulin fibers (TH = 152) in the Gata-1low image renders an extremely high background in the wild type image (despite their processing and staining at the same time). Conversely, the application of a threshold value optimal for the background (TH = 95) in the wild type mouse image, underestimates the reticulin fibers content in the Gata-1low image . Background exclusion in comparative analysis is particularly important in images with low content of reticulin fibers, since the impact of the background level (noise) is inversely proportional to the reticulin level .The ImageJ Shape Filter algorithm  allows fFigure 1, the wild type genotype ages 20, 30 and 40 weeks old  displayed a very low signal regardless of the age, consistent with the lack of pathological reticulin fibers in their BM. On the other hand, in Gata-1low animals  the reticulin content progressively increased with age, with values of mean IntDen from 28.5 × 103 at 20 week-old, to 41.5 × 103 at 30 week-old and, to 93.4 × 103 at 40 week-old. The lower limit of detection corresponds to a 1.48% of reticulin fibers content with respect to the whole image area, as observed in G20 animals. Since no differences in reticulin content were observed among the three sections within each slide, regardless of the age or genotype (data not shown), the IntDen mean ± SD values were calculated from the 30 images captured from each slide. Coefficients of variation  were calculated and found to be 55%, 44% and 24% for ages 20, 30 and 40 week-old, respectively.With a controlled quantifiable method at hand, we sought to carry out BM analysis of reticulin fibers throughout the femur BM. To this end, images of Gömöri stained sections were captured longitudinally throughout the femur, either by batch analysis of around 360 images per set, or by stitching nearly 200 overlapping images. As shown in Figure 2B. This demonstrates that the high variability of the analytical data shown in Figure 1 is mainly caused by heterogeneous distribution of reticulin fiber within the BM of a femur, which subsides in older Gata-1low mice.Considering our controlled method of detection, the above high coefficients of variation are suggested to originate from heterogeneous distribution of the fibrotic tissue within the BM. To test this possibility, we obtained composite images along the whole femur, at the same level of magnification used for the acquisition of limited BM areas as described above. This approach revealed a clear heterogeneity in the distribution of reticulin fibers within the BM . Represe6 pixels corresponding to the whole femur BM area, reticulin fibers occupy an area of 2.4 × 106 pixels, which corresponds to 7.3% occupancy. A similar result was obtained by analysis of seven partial images of the same whole BM image, i.e., reticulin fibers occupy an area of 2.1 × 106 pixels, which corresponds to 6.4% occupancy . Thus, both modes of analysis render similar results.The composite analysis showed that out of 32.8 × 10via hundreds of composite images taken throughout bone marrow sections of femurs of a mouse model of myelofibrosis.Increased reticulin grade is regarded as an unfavorable prognostic factor in myelofibrotic neoplasms in human or experimental models, underscoring the need for quantitative analysis of RF progression of the disease. Computer assisted RF analysis, using color deconvolution showed a general correlation with a semi-quantitative grading methods . CurrentThe new strategy presented here requires the use of a common imaging modality, within which we have developed two macros for batch processing in the steps involving: (1) image area correction; and (2) image thresholding, shape filtering and quantification. The readiness to generate macros and adjust shape filter parameters within this mode of analysis allowed us to tailor analytical needs, thus creating a platform that might be used across laboratories.Importantly, by using the above method, we were able to determine fibrosis throughout most of the femur bone marrow, rather than in select representative sections. In the Gata-1low mice studied here, fibrosis was detected somewhat earlier and it progressed relatively slower than in Gata-1low mice on a CD1 background , 25. ThiFig. 2A), which could facilitate the diffusion of circulating fibrosis-promoting factors, such as transforming growth factor-ß [While image pixel occupancy of fibrosis in the marrow in the experimental model tested here is less than 10%, compared to total pixels recorded by the imaging modality, data showed that the range of heterogeneity of reticulin staining is high in younger mice  than in older mice . This raises the possibility that disease progression increases the percentage of areas with high fibrosis. Interestingly, whether in young or old mice, dense fibrotic patches were typically detected around sinusoids (factor-ß . The knofactor-ß  could coIn order to assess bone marrow fibrosis in humans, pathologists stain biopsy samples for reticulin and collagen fibers and grade the specimen based on a defined scale. A scale was developed by a team of international experts following evaluation of 150 paraffin-embedded biopsy sections from the posterior iliac crest of patients with bone marrow fibrosis, most of whom were diagnosed with PMF . The expvia a quantitative approach would be useful in studying the dynamics of the disease in other experimental animal models, with potential diagnostic implications to human pathology, considering the limited biopsy material typically available.Taken together, assessing the degree of fibrotic patchiness


Fitting of X-ray scattering diagrams, supported by Density Functional Theory simulations, reveals an unexpected rolling mode for these systems. The transferability of the approach opens up for improved understanding of structure–property relationships of inorganic nanotubes to the benefit of fundamental and applicative research in these systems.Determination of the atomic structure of inorganic single-walled nanotubes with complex stoichiometry remains elusive due to the too many atomic coordinates to be fitted with respect to X-ray diffractograms inherently exhibiting rather broad features. Here we introduce a methodology to reduce the number of fitted variables and enable resolution of the atomic structure for inorganic nanotubes with complex stoichiometry. We apply it to recently synthesized methylated aluminosilicate and aluminogermanate imogolite nanotubes of nominal composition (OH) Structural determination of inorganic nanotubes has lagged far behind that of their carbon-based counterparts. Here, the authors present a transferable methodology, combining wide angle X-ray scattering and computation, to quantitatively resolve the atomic structure of inorganic nanotubes with complex stoichiometry. Thanks to their one-dimensional properties and their large surface area, SWNTs are promising nano-bricks for applications in different fields, including nanoelectronics, nanofluidics, nanocatalysis, and selective molecular sieving6. Both organic and inorganic SWNTs are intensively studied for their complementary chemical and physical properties. With the discovery of single-walled carbon nanotubes (SWCNT), research in organic SWNTs grown exponentially8 providing alternatives to supramolecular self-assembly and/or polymerization of nanoscopic organic systems11. Developments in inorganic SWNTs have been slower than for SWCNTs, with substantially fewer systems available until very recently, namely BN nanotubes12, imogolite nanotubes (INTs)13, MoS2, MoO3, and SbPSx4−Sex nanotubes16. However, synthesis strategies have now emerged extending the family of inorganic SWNTs to sulfide, hydroxide, phosphate, and polyoxometalate nanotubes3. The generality of the recently proposed approach opens the way for future synthesis of a wide variety of inorganic nanotubes.Single-walled nanotubes (SWNTs) constitute an appealing class of materials in which new synthesis strategies recently emerged19. Structural resolution of more complex organic nanotubes is usually based on wide-angle X-ray scattering (WAXS) by highly oriented nanotubes fibers18 or, alternatively, oriented nanotubes in suspensions2. In ref. 20, oriented inorganic nanotubes suspensions could be obtained but with extremely low concentration. The lack of oriented samples of inorganic nanotubes, suitable for WAXS, is a major obstacle. Apart from BN nanotubes, whose structure in principle derives from that of carbon nanotubes by substituting alternatively C atoms by B and N ones, as well as the very special case of MoS2 nanotubes that assemble in a crystalline three-dimensional system, there is no unambiguous and detailed determination of the atomic structure of inorganic SWNTs.Knowledge of nanotubes’ atomic structure is crucial for comprehension of their properties. The structure of SWCNTs is currently determined with a high level of accuracy, based in particular on electron diffraction experiments coupled to the theory of diffraction from helices, as well as on a recent powder X-ray scattering study3Al2O3Si(Ge)CH3 (refs. 22). We chose them as representative examples of inorganic nanotubes of complex stoichiometry as well as for their intrinsic properties.In this article we focus on accurate determination of the atomic structure of inorganic SWNTs from powder WAXS measurements. Powder X-ray scattering method has the advantage to be a statistical method, as compared to local analysis methods such as electron diffraction, and it does not require special sample preparation. The inorganic SWNTs considered here are the newly synthesized methylated metal-oxide INTs of nominal composition (OH)3Al2O3Si(Ge)OH were discovered in soils23 and they can be easily synthesized using soft chemistry25. Their nanometric diameter is tunable at the Angstrom level, depending on the nature of synthesis precursors27. Moreover, various surface functionalizations have been achieved, either by modification of their inner surface with organic moieties22 or by grafting organic compounds on the outer part of the nanotube29. The chemical versatility of INT paves the way towards possible applications in various fields, such as stimuli-responsive materials20, molecular storage30, molecular recognition and separation31, water filtration and decontamination32 as well as catalysis33 and photocatalysis34. Furthermore, INTs are invoked in a geological context when evaluating carbon storage, metallic cations or radionuclides storage in soils37, as well as markers in the evolution of the Martian climate38.Metal-oxide INTs with composition (OH)3Al2O3Si(Ge)OH INTs are provided in numerical simulations articles40. Neither careful analysis of synchrotron Pair Distribution Function41 nor the comparison between experimental WAXS diagrams and calculated ones obtained by Tight-binding Density Functional Theory (TB-DFT) minimization42 could lead to conclusive assignment of the atomic structure of the first inorganic SWNTs to be discovered, as early as 1962, namely aluminosilicate (OH)3Al2O3Si(Ge)OH INTs. It should be underlined that WAXS diagrams for nanotubes are not formed of Bragg peaks as in crystals. They consist of a limited number of broad modulations due to the nanometric lateral extent of the nanotubes. One cannot take advantage of three-dimensional crystalline order, as was done recently by Oda and co-workers43 to solve the molecular structure of self-assembled organic nanoribbons.Quantitative interpretation of imogolite WAXS diagrams has not been achieved yet, despite intensive research and while atomic positions for (OH)44 and to a semi-empirical energy minimization. The strategy enables us to determine the atomic structure of both (OH)3Al2O3SiCH3 and (OH)3Al2O3GeCH3 nanotubes. The obtained structures are confirmed by DFT calculations.A different approach is proposed here. It is based on the reduction of the number of independent atomic positions to be fitted to WAXS diagrams, thanks to the use of helical symmetries3) with isolated (Si(Ge)O3)OH tetrahedron units connected via covalent bonding between three mutual oxygen atoms13. It can be first described with a three-dimensional (OH)3Al2O3Si(Ge)OH elementary unit arranged in a hexagonal lattice , the components of the so-called ‘chiral vector’ in the hexagonal basis  indices. Accordingly, sharp diameter distributions are reported in the literature26. Current investigations of the structure of imogolite (OH)3Al2O3Si(Ge)OH nanotubes in the literature point towards a  configuration, called ‘zig-zag’ (ZZ) by analogy with SWCNTs 21, inner hydroxyl groups are substituted by methyl groups, leading to a nominal composition (OH)3Al2O3Si(Ge)CH3. They are considered as ZZ nanotubes52, like their hydroxylated analogs. Narrow diameter distributions are reported22. Available DFT and TB-DFT results suggest the hydrogen bonding network between inner hydroxyl groups in the pristine hydroxylated INTs to be key for the energetic favorability of the ZZ structure over an armchair (AC) one53. These results prompt careful investigation of the effects of methyl-substitution of the inner hydroxyl groups on the ZZ vs. AC energy competition, which we present here.In the methylated INTs (m-INTs) discovered a few years agoQ < 1 Å−1), where INTs can be approximated as homogeneous cylinders, Amara and co-workers22 demonstrated that the inner and outer diameters of (OH)3Al2O3SixGex1−CH3 nanotubes decrease as x increases from 0 to 1. However, no information about the atomic structure of the nanotubes could be obtained at such small wave vectors. In the present study and for the first time, we have performed powder WAXS measurements over a much wider Q-range, up to 8 Å−1, for the two end-members (OH)3Al2O3SiCH3 and (OH)3Al2O3GeCH3 nanotubes, denoted SiCH3 INT and GeCH3 INT . For nanotubes of finite length, these peaks are smoothed. Specifically, in the case of methylated nanotubes, which have typical lengths of the order of 100 Å (ref. 22), these smoothing effect cannot be ignored. In this scope, the period value is not given by the position of the peak maximum but by the inflexion point of its rising edge55. To obtain inflexion point positions from the rather noisy scattering diagram, we applied a Savitzky-Golay filter with an adjustable window width as shown in Fig. l = 1, l = 2, and l = 3 planes, one obtains T = 2.45 Å for SiCH3-INTs and T = 2.48 Å for GeCH3 INTs. However, such a small period is incompatible with the structure of the primary gibbsite sheet: the Al–Al distance in gibbsite is d = 2.95 Å (ref. 56), so that the smallest period should be l = 2, 4, and 6. The period T is then found to be equal to 4.89 Å for SiCH3 INTs and 4.95 Å for GeCH3 INTs, respectively 3Al2O3GeOH nanotubes. The determination of period values around 4.9 Å for methylated INTs gives a compelling evidence about chirality modification. The ratio between the periods of normal and methylated INTs is close to 3Al2O3Si(Ge)CH3 in AC configuration , in contrast with normal (OH)3Al2O3Si(Ge)OH which present a ZZ configuration . The systematic extinction of 00l reflections for odd l values is then easily understood, since the period of an AC structure projected onto its long axis is equal to T/2  nanotube, fitted parameters can be reduced to the value of N, the positions of the atoms of the (OH)3Al2O3Si(Ge)CH3 elementary unit in Fig. 44 allow one to generate a whole nanotube structure with inner radius Ri, outer radius Re, and period T from any planar unit cell. It should be noted here that since X-rays are rather insensitive to H atoms, we considered virtual atoms at the electronic center of charge of OH and CH3 groups, with X-ray form factors equal to the weighted sum of those of their constituents. To obtain structures of physico-chemical significance, we developed an algorithm allowing us to generate a full tubular atomic structure while minimizing an energy term Egeo. The subscript ‘geo’ stands for ‘geometrical’ as minimization is made over bond lengths and angles between them. The energy is calculated via a quadratic expansion over bond lengths and angles with relevant harmonic constants and reference bond lengths and angles values taken from the literature  algorithm with optional user-defined constraints like the inner and outer radii  as well as the value of the period T determined on the WAXS diagram  peaks  and fj(Q) being the associated to scattering factors. Intensity calculations have been speed up using highly parallel calculation on GPUs59. On a regular laptop GPU (Nvidia GTX 860m), it takes about 1 h to fulfill a fitting procedure, i.e., to compute 2200 WAXS diagrams of 2048 Q-points from a structure comprising between 2840 and 5240 atoms depending on the value of N. A set of data calculated from powder X-ray scattering is created for a wide range of Ri, Re, and N values. Then, an algorithm extracts the one that matches the best the WAXS experimental data, between 1 and 8 Å−1. This procedure is based on the identification and least-square fitting of eight well-defined maxima and of a minimum of the experimental diagram. The fitting procedure is summarized in Fig. For a given set of parameters 3 imogolite nanotube is obtained with N = 9, Ri = 8.8 Å, and Re = 13.6 Å. The comparison between the calculated WAXS diagram and experiment is shown in Fig. 3 imogolite is slightly wider with  AC indices, Ri = 11.6 Å and Re = 16.2 Å. Notice that wall thicknesses are in good agreement with the thickness of a fictitious imogolite planar structure in which a gibbsite layer is coated with Si/Ge tetrahedra  and  structures of SiCH3 and GeCH3 INTs cannot be ruled out  GeCH3 nanotubes  functional and including dispersion interactions (PBE-D3). There is no significant difference between calculated WAXS diagrams for the PBE and PBE-D3 optimized structures Fig. . The besbes Fig. . These rbes Fig.  as well 3 and GeCH3 nanotubes. The DFT energy E is calculated over a periodic unit cell of the nanotube, where ZZ  and AC  nanotubes both contain 2N imogolite structural units within a period. Direct comparison of the energy per imogolite unit (E/2N) between different nanotubes structures is thus meaningful. Regardless of the inclusion (PBE-D3) or neglect (PBE) of dispersion interactions, we find the E/2N minima for SiCH3 and GeCH3 AC nanotubes to be substantially lower in energy than the corresponding minima for ZZ analogs  and GeCH3 nanotubes  explain the formation of AC nanotubes as determined experimentally –O4–Al5–O6–H7 from the nanotube cavity to its outer surface , the AC geometry results in a substantially more favorable environment for the electrons of the nanotube, contributing to lower the energy of the entire system.Extensive geometrical analysis for the nanotube structures at the DFT-optimized periodicity  group by (aprotic) phosphorous and arsenic derivatives is also computed (at TB-DFT level) to result in E/2N profiles with well-defined minima62. In addition, the E/2N profiles for ZZ and AC SiCH3 in Fig. 352, also presents minima. Altogether these results indicate that, at least for Si-based INTs, the presence of an inner H-bonding network is not actually necessary for the appearance of a well-defined E/2N minimum. The energy competition between strain of the Si3-O4 bond on the nanotube cavity, distortion of the gibbsite layer, and maximization of the H-bonding network on the outer layer  GeCH3 nanotubes  structure, PBE and PBE-D3 E/2N minima are computed for the  and  nanotubes, respectively. The disagreement for GeCH3 nanotubes is even larger with PBE and PBE-D3 computed E/2N minima for the  structure, substantially far from the experimentally fitted  value. The large deviations between experimentally fitted and energetically computed N, together with the aqueous synthesis environment for both SiCH3 and GeCH322, as well as the relatively high temperature of synthesis (90 °C), hint to a possibly over-simplistic nature of the computational models used, and to a non-negligible role for the nanotube interactions with the synthesis environment in fine-tuning the energy favored nanotube structure, an aspect previously documented experimentally for hydroxylated INTs26.In spite of the agreement between experimental and calculated WAXS diagrams for the DFT-optimized  SiCHbes Fig. , it is oE/2N differences between experimentally fitted structures and computed minima are 0.015 and 0.03 eV for SiCH3 and GeCH3, respectively. Previous (force field) molecular dynamics simulations of water confined inside hydrophobic AC CNTs in the – range  in the free energy of nano-confined water molecules as a function of the CNT radius64. Assuming, given the hydrophobic nature of both CNTs and m-INTs, that quantitatively similar changes may be present for water nano-confined inside SiCH3 and GeCH3 nanotubes, confinement of no more than 1 residual water molecule from the aqueous synthesis environment every 3–7 imogolite units could be sufficient to turn the experimentally fitted  and  structures energetically favored over the computed PBE-D3 minima . While evidently speculative, we believe this argument deserves closer investigation. Further evidence of the role of the interactions with the synthesis environment in fine-tuning the m-INTs’ structure and energy is provided by the non-negligible deviations between the experimentally fitted inner and outer radii  ZZ hydroxylated analogs, methylated INTs roll up into a  AC structure with N = 9 for SiCH3 and N = 11 for GeCH3 nanotubes. It follows that functionalization (methylation in the present case) of the INTs cavity not only offers control over the inner surface properties but it also leads to drastic structural changes such as change of the chiral vector of the nanotube. The results of the experimental WAXS fitting are supported by DFT simulations that predict AC rolling of methylated nanotubes to be energetically favored over ZZ structuring. N values corresponding to minima of DFT energies for isolated nanotubes turn out to be larger than those deduced from WAXS fitting, indirectly confirming previous experimental suggestions of the role of the synthesis medium in fine-tuning the final diameter of INTs. Furthermore, our results rule out previously proposed models which are shown to be both inconsistent with WAXS results and energetically disfavored, stressing the value of WAXS structure-determination for fundamental research in inorganic SWNTs.Whereas there has been remarkable experimental and theoretical progress in our knowledge about structure and properties of two-dimensional metal-oxide surfaces and interfaces44. Moreover, the role of the synthesis medium and the temperature, which is crucial in the synthesis of SWNTs in suspension3, is included in the WAXS fitting procedure we propose, through the choice of the internal and external radii as fitting parameters.More generally, this article introduces a singular fitting procedure to enable complete resolution of SWNTs atomic structures from WAXS diagrams. It is based, first, on the use of helical symmetries allowing one to consider the smallest unit cell and second, on semi-empirical energy minimization and ensuing reduction of the number of structural parameters to be fitted. The simple fitting approach proposed is directly applicable to the whole family of the imogolite-like metal-oxide nanotubes of geological and physico-chemical interest and whose structure is not solved precisely. It is also transferable to other large unit cell single-walled inorganic nanotubes currently synthesized and with potential applications in various fields. In the case of INTs, the unit cell is a slightly deformed hexagonal one but one should underline here that the use of helical symmetries imposes no constraint on the elementary cell3Al2SixGex1−CH3 was performed according to the procedure described by Amara et al. (2015)22. Aluminum perchlorate  was mixed in Teflon beakers with methyltriethoxysilane  or methyltriethoxygermane  for synthesizing either SiCH3 INT (x = 1) or GeCH3 INT (x = 0), respectively. The initial aluminum perchlorate concentration was set at C = 0.1 mol L−1 and the concentration of MTEG/MTES was chosen so that the ratio [Al]/([Si] or [Ge]) is equal to 2. The obtained mixtures were slowly hydrolyzed by the addition of a 0.1 mol L−1 NaOH (hydrolysis ratio [OH]/[Al] of 2), stirred overnight at room temperature, and then aged at 90 °C into an oven for 5 days. After recovering the suspensions, all samples were dialyzed against ultrapure water, using 8 kDa membranes in order to remove residual salts and alcohol in excess. Dialyses were performed until the conductivity dropped below 5 μS cm−1.The synthesis of Si or Ge nanotubes with a methylated inner cavity of nominal composition (OH)The dialyzed suspensions were dried at 60 °C during 24 h, the resulting sediment being milled in an agate mortar to obtain a fine powder. The obtained powders were held in cylindrical borosilicate glass capillaries  of 1 mm diameter that were flame-sealed.λ = 0.79176 Å was extracted from the U20 undulator beam by means of an Si (111) double monochromator. Measurements were performed using a 21 perfect crystals Si (111) multi-analyzer allowing to access to a large range of wave-vector Q: −3 Å−1) provided by Si crystals is negligible in comparison to the modulation of imogolite WAXS diagrams even for the determination of the value of the period T along the nanotube axis.The powder WAXS experiments have been carried out at the synchrotron SOLEIL  on the CRISTAL beamline. A monochromatic X-ray beam with a wavelength of 66, using the PBE67 approximation to the exchange and correlation functional. Where used, van der Waals corrections were applied according to Grimme’s DFT-D3 approach68. Core electrons were described with norm-conserving Goedecker, Teter, and Hutter pseudopotentials69. Valence electron Kohn-Sham states were expanded in terms of Gaussian functions with molecularly optimized double-ζ polarized basis sets (m-DZVP), which ensures a small basis set superposition error70. For the auxiliary basis set of plane waves a 320 Ry cutoff was used. Reciprocal space sampling was restricted to the Γ point. The adopted convergence thresholds for the geometry optimizations were 10−4 Ha Bohr−1 on the maximum atomic force, and 3 × 10−4 Ha Bohr−1 on the root mean square residual of all the atomic forces. Calculations were performed using periodic boundary conditions (>20 Å vacuum-buffer perpendicularly to the nanotube axis) with both experimentally derived and DFT-optimized values of the nanotube period along its axis. Optimized period values at PBE (PBE-D3) level are 4.87 Å (4.83 Å) for AC GeCH3 INTs, 4.72 Å (4.72 Å) for AC SiCH3 INTs, 8.50 Å (8.45 Å) for ZZ GeCH3 INTs, and 8.54 Å (8.50 Å) for ZZ GeCH3 INTs.All the DFT simulations were performed with the CP2K/Quickstep packageThe main result of this study is the WAXS diagrams’ fitting process whose details are given in Supplementary Note 3 and GeCH3 INTs are deposited at the Cambridge Crystallographic Data Centre under CCDC accession codes: 1838953, 1838955. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif [www.ccdc.cam.ac.uk/data_request/cif].The atomic structure of SiCHSupplementary InformationPeer Review File


High resolution synchrotron microtomography capable of revealing microvessels in three dimensional (3D) establishes distinct imaging markers of mouse kidney disease strongly associated to renal tubulointerstitial (TI) lesions and glomerulopathy. Two complementary mouse models of chronic kidney disease (CKD), unilateral ureteral obstruction (UUO) and focal segmental glomerulosclerosis (FSGS), were used and five candidates of unique 3D imaging markers were identified. Our characterization to differentially reflect the altered microvasculature of renal TI lesions and/or glomerulopathy demonstrated these image features can be used to differentiate the disease status and the possible cause therefore qualified as image markers. These 3D imaging markers were further correlated with the histopathology and renal microvessel-based molecular study using antibodies against vascular endothelial cells (CD31), the connective tissue growth factor or the vascular endothelial growth factor. We also found that these 3D imaging markers individually characterize the development of renal TI lesions or glomerulopathy, quantitative and integrated use of all of them provide more information for differentiating the two renal conditions. Our findings thus establish a practical strategy to characterize the CKD-associated renal injuries by the microangiography-based 3D imaging and highlight the impact of dysfunctional microvasculature as a whole on the pathogenesis of the renal lesions. To overcome this obstacle hindering earlier diagnosis and more efficient treatment of CKD8, it is imperative to develop a better understanding of the structure of the renal microvasculature highly correlated with the pathogenesis underlying CKD10. However, certain concerns regarding the procedure for diagnosing CKD including: (1) the pathophysiology-based abnormal microvasculature remains to be refined; (2) conventional pathological diagnosis depends mainly on 2D structures, but 3D imaging is recommended to profile the abnormal areas from the pathology perspective, including the fine lesions in the kidney12; and (3) there is a risk of bleeding, infection or other complications in patients after undergoing renal biopsy15; need to be addressed.Chronic kidney disease (CKD) is a worldwide health issue. In spite of the high prevalence and incidence17. Our imaging approach demonstrated previously the 3D profiling of entire microvascular networks and the neighboring fine structures in organs or tissues with micrometer resolution19. The resolution achieved by our microtomography is sufficient to resolve 1 µm test patterns with a 20x, NA = 0.4 lens.  microtomography platform based on synchrotron X-rays with greatly improved performance which is sufficient to resolve these above mentioned obstacles33. The focal segmental glomerulosclerosis (FSGS) model, concerning a specific type of glomerulopathy of which the main renal injury is typically located in the glomerulus affected36.Two complementary animal models of CKD were chosen for this study. The unilateral ureteral obstruction (UUO) model, representative of renal tubulointerstitial (TI) lesions showing dilated tubular lumens, atrophic tubular epithelial cells, leukocytic infiltration into the interstitium and extracellular matrix accumulation38. Both proteins are highly expressed in the renal lesions of CKD patients and have been shown to play a pathogenic role in this group of renal disorders. Furthermore, these two proteins are also required for the establishment of the microvasculature and the regulation of angiogenesis40.In addition, angiogenic cytokines, such as connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF), are also involved in tissue repair, and other essential biological functions, including vascular changes and fibrosis in CKDThe purpose of this study was to seek unique renal microvessel-based microscopic 3D imaging features, capable of reflecting the status of pathological lesions observed in CKD patients. Five distinct image features were successfully identified by high resolution synchrotron microtomography strongly associated with renal TI lesions and/or glomerulopathy. We further characterize these 3D image features and validated them unique imaging markers suitable for differentiating the microvascular alterations between these renal lesions in a disease course manner.Two mouse models of CKD, UUO that predominantly featured renal TI lesions and FSGS more in favor of glomerulopathy, were used to correlate the alterations in the microvasculature by simultaneous analysis with a high-resolution synchrotron-based X-ray imaging and tomography. As shown in Fig. In parallel, the severity of renal fibrosis was determined using immunohistochemistry (IHC) with collagen (Col)-III in both mouse models. As shown in Fig. The pictures in Fig. 43. In our study, a comparative analysis was performed to establish potential 3D imaging features that can be associated with individual histopathological characteristics in the two complementary mouse models of renal inflammation and fibrosis. Our extensive search of the 3D high resolution synchrotron microtomography data of normal, UUO and FSGS mouse kidneys leads to five characteristic, distinctive 3D imaging markers were identified. We label these five markers tentatively as “hedgehog-like”, “funnel-like”, “long beak-like”, “notched mushroom-like” and “earthworm-like”  theke” Fig. ; (3) theke” Fig. . Thus, tke” Fig.  and 3a.The notched mushroom-like 3D imaging marker indicated a fovea-like defect in the glomerulus (likely attributed to the random ischemia of the glomeruli affected) Fig. . The earAs shown in Fig. We further performed a detailed analysis to correlate each of these 3D imaging markers with specific histopathological features of the kidney on the interlobular arteries, arterioles, capillary network of the glomerular tufts (composed of part of arterioles and abundant capillaries) and renal interstitium in both UUO and FSGS models Figs  and 3a. In addition to determine the population of the 3D imaging markers, we also developed an automatic 3D tracing algorithm to quantify the other structure parameters in the kidney microvasculature. As shown in Fig. To dissect the mechanistic pathways underlying the changes in the microvascular networks of the kidney and the capillaries of the glomerulus in UUO and FSGS mice, we assessed the distribution and quantity of vascular endothelial cells in renal tissue sections using IHC with CD31. As shown in Fig. The grading of CD31-positive glomeruli by IHC was calculated for each group. As shown in Fig. In addition to those 3D imaging markers, tissues from the glomeruli or the renal TI lesions were collected by laser capture microdissection (LCM) to further investigate the pathogenic mechanisms. In the renal TI lesions, the renal CTGF levels were significantly increased in both the early and late stages of the disease in UUO mice compared with normal control mice Fig. . The ren46. In this study, we take advantage specifically the very high resolution and demonstrated that the performance is essential for our objective to characterize the microvasculature alternation due to CKD. We successfully identified a number of 3D imaging markers characteristic to the altered microvessels in the mouse kidneys. We also explored the potential implications of these markers in renal TI lesion and glomerulopathy using two mouse UUO and FSGS models of human CKD by comparing them with renal histopathology features and the results of renal microvessel-based molecular analysis. These 3D imaging markers were therefore firmly established capable of characterizing differential renal lesions in these distinct experimental models of CKD. Note these differential renal lesions are often observed in CKD patients and these 3D imaging markers are potentially applicable to human biopsy specimens.Synchrotron sources have been providing very bright X-ray beams for decades critical to obtain high-resolution 3D images with high throughput51. The high resolution 3D kidney microvasculature delineated by synchrotron X-ray microtomography, on the other hand, allows us to perform adequate quantitative analysis to determine valuable structural and morphological parameters. These high precision and high fidelity 3D structural parameters further enable our study of the minute modification of renal microvascularture55. As a benefit of the superior performance, for example, that the number of microvessels determined by our imaging method was significantly higher than that of lab-based micro CTs56.The key performance factor of tomography required to distinguish and characterize these image markers is the excellent 3D resolution provided by the synchrotron microtomography. The diameter of renal microvessels, such as glomerular capillaries, arterioles, and interlobular arteries, varies at a range of 40 μm to less than 2 μm, thus many small renal microvessels were not detected in previous lab-based micro CTs studiesSynchrotron microtomography results of normal control mouse kidney microvasculature, Figs Regarding the markers found in the FSGS mice, the notched mushroom-like markers indicate defects in the glomerulus and the earthworm-like markers denote deformed microvessels of varying sizes and shapes, characteristic to the FSGS model. Furthermore, these 3D imaging markers in the FSGS mice can be attributed to the sclerosis of the microvessels inside the glomerulus or those in the renal interstitial compartment Figs  and 7c. 40. Our data clearly showed that compared with normal control mice, FSGS mice showed increased levels of CTGF and VEGF in the glomeruli. Whereas, this effect was inhibited in UUO mice, which had relatively lower levels of both proteins compared with FSGS mice. The results suggest that FSGS mice may exhibit specific damage to the capillary endothelial cells of the glomeruli , and this effect may be reflected by the notched mushroom-like marker observed in the FSGS mice , Taiwan. The mice were kept in individual ventilated cages and kept at 25 ± 2 °C on a 12-hour light/dark cycle. The experimental procedures were approved by the Animal Protection Association (APA) of the Republic of China (ROC) and the NDMC Institutional Animal Care and Utilization Committee (IACUC). In addition, the number of animals used was approved by an ethical committee at the Ministry of Science and Technology of the Republic of China (MOST 103-2320-B-016-011-MY3). The experiments were performed in such a way that the number of animals used was minimized. We have confirmed that all methods were performed in accordance with the relevant guidelines and regulations.63. In brief, the mice were subjected to left ureter ligation at the junction with 6–0 silk suture under anesthesia with an intramuscular injection of Zoletil 50 . First, the back skin of the mice was shaved and the abdominal wall was cut on the back of the mice. A short suture line was dragged underneath the ureter and then tied with three knots. After the operation, a subcutaneous injection of Flunixin  was administered as an analgesic agent. The mice were sacrificed at day 7 or 14 after induction.UUO was induced in 8-week-old BALB/c male mice65. The mice were sacrificed at day 14 or 28 after induction.FSGS model was induced in 8-week-old male BALB/c mice by intravenous injection of a single dose of adriamycin PAS staining was performed for the renal pathological evaluation. Renal tissues were fixed in 10% buffered formalin immediately after removal. After approximately 24 hours, the samples were placed in an automatic tissue processor  and dehydrated. Then, the samples were embedded in paraffin using a paraffin-dispenser (Sakura Finetek). Two-micrometer-thick renal sections were cut with a sliding microtome  and were attached onto glass slides. The slides were then placed in a 75 °C incubator for 30 minutes and immersed in xylene for 10 minutes. The sections were rehydrated using a gradient alcohol series of 100%, 95%, 85%, and 70%, and the sections were finally placed in deionized water. The slides were then immersed in 0.5% periodic acid solution, placed in Schiff reagent and stained with Mayer’s hematoxylin. Masson’s trichrome staining was performed on formalin-fixed paraffin-embedded renal sections. First, the sections were stained in Weigert’s iron hematoxylin and then immersed in Biebrich scarlet-acid fuchsin solution. Next, the sections were immersed in phosphomolybdic-phosphotungstic acid solution and then transferred to aniline blue solution. Finally, the sections were immersed in acetic acid solution and dehydrated in 95% ethyl alcohol.67. In brief, the TI lesions score was semi-quantitatively determined from 0 to 4+ in an average of 5 fields per kidney section . For the glomeruli, glomerular lesions were evaluated by the percentage of glomeruli presenting sclerotic lesions, as previously described69. In brief, the glomerular lesions score was semi-quantitatively determined from 0 to 4+ in an average of 10 glomerulus per kidney section .Renal TI lesions were evaluated by the severity of the tubular dilation, interstitial fibrosis, and mononuclear cell infiltration, as previously describedFor IHC, formalin-fixed paraffin-embedded renal sections (2 μm) were stained with biotin-labeled goat anti-Col-III  or rat anti-CD31 (BD) antibodies overnight at 4 °C, washed with TBST, and incubated with a horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG or rabbit anti-rat IgG  antibody for 1 hour. The sections were then visualized with 3, 3′-diaminobenzidine (Dako) and counterstained with hematoxylin.2 per field), as previously described70. Microvessels in the sclerotic or fibrotic areas and areas closely adjacent to the tubular lumens were not included in the vessel counts. The grading of the CD31-positive glomeruli was determined as previously described, with slight modification72. In brief, 40 glomeruli were examined on each slide, and the positive staining was assigned a score ranging from 0 to 3+ ; the total grading score was calculated for each specimen using the following equation: total grading score = (% glomeruli grading negative × 0) + (% glomeruli grading 1 + × 1) + (% glomeruli grading 2 + × 2) + (% glomeruli grading 3 + × 3). The values ranged from 0 to a maximum of 300.The percentage of Col-III expression in the renal TI lesions or glomeruli was automatically quantified using the Pax-it software . To highlight the alterations in the microvessels, the distribution of CD31  in the microvessels was automatically quantified using the Pax-it software (Midwest Information Systems). After the area of the microvessels in the renal TI lesions was identified, individual microvessels were counted and presented as number of TI microvessels within a 400 × field  and then injected with a contrast agent as previously described19. In this study, barium sulfate particles were administrated to the kidney vessels via a syringe, attached to the manual pump, and injected at a rate of 1 μl/s58. Barium sulfate particles were filtered by 0.5 μm filter cup to remove aggregated barium sulfate. Then, barium sulfate particles were washed with distilled water and centrifuged at 3000 g for 60 minutes. Finally, barium sulfate particles were suspension in phosphate buffer saline (PBS) at the concentration of 30% (w/w). The filtered barium sulfate particles were suspended in the solution without aggregation and thus able to reach the smallest vessels. After fixation, the samples were washed with PBS solution for 1 hour, dehydrated with a gradient of ethanol solutions 3 times, and embedded in Embed-812  as previously described73. Finally, the samples were incubated in a 65 °C oven for 8 hours for polymerization.UUO or FSGS mice were sacrificed with an intramuscular injection of Zoletil 50 (Virbac) as previously described74. Two 550 µm silicon wafers were used to attenuate the strong X-rays to an estimated dosage of 33.9 Gy. The exposure time was 100 ms per image. The distance between sample and scintillator was 5 cm. We used a 2 × lens for overviews of the whole kidney and 5x lens to perform the local tomography; the pixel size in the final image was ~2.86 µm (2x lens) and ~1.14 µm . The resolution degraded accordingly from the best case shown in Fig. 1S obtained with a 20x lens (NA = 0.4), a reasonable trade-off for a larger field of view.The experiments were performed on the TLS 01 A beamline of the National Synchrotron Radiation Research Center . Unmonochromatized X-rays  emitted by a 4.5 tesla superconducting wavelength shifter of the 1.5 GeV electron storage ring operated at a top-up mode at a constant current of 360 mA74. All the 3D visualization tasks were processed also with the Amira 6.0. These 3D synchrotron microtomographic imaging markers were then quantitatively analyzed and presented as percentages by calculating the numbers of each imaging marker in a total of 100 randomly selected glomeruli with adjacent microvascular structures, and the imaging profiles between the two mouse models were compared.The tomographic reconstruction using filtered back projection algorithm was performed on sets of 1,000 images at equal angular spacing within 180 degrees with a code written with the IDL software. For the tracing analysis, the 3D data were first segmented based on their voxel values and then the skeleton were traced with the “centerline tree” and “auto skeleton” functions in the “skeletonization package” of the Amira 6.0 softwareEach of the five 3D synchrotron microtomographic imaging markers was rated as grade 0 to 5 scores, which depends on the degree of defects on interlobular arteries, arterioles of the hilus of the glomerulus, capillaries inside the glomerulus, and/or renal interstitial capillaries affected. The image score was then determined according to the following formula for each specimen: image score = (% “image marker grade 0” × 0) + (% “image marker grade 1” × 1) + (% “image marker grade 2” × 2) + (% “image marker grade 3” × 3) + (% “image marker grade 4” × 4) + (% “image marker grade 5” × 5). The values ranged from 0 to a maximum of 500.The tracing algorithm was based on the procedures: first, the image voxels were segmented into several connected clusters, which were defined if every voxel in the cluster was connected at least neighbor one. Second, image voxels in a connected cluster were encoded based on the idea of region-growth method. Every voxels in this connected cluster was encoded by a number representing the sequential distance of the shortest path from the origin. A codelet i was defined by voxels with coding number i−1, i, i + 1 and its center of mass was calculated based on all of these voxels. The tracing for one connected cluster was performed by simply following the codelets sequentially with increasing indices through all the encoded voxels. Finally, all of the encoded voxels were traced. Based on the rules, total vessel length, cross-sectional area of arteries, length of afferent arteriole, volume of afferent arteriole, cross-sectional area of afferent arteriole and other relevant quantitative parameters for the 3D imaging markers were then calculated.The renal tissues were embedded in OCT and immediately stored at −20 °C. The samples were placed in the pre-cooled cryostat for sectioning, and the 8-μm-thick sections were spread onto Arturus PEN membrane glass slides . The staining protocol was then performed according to the instructions that were provided with the HistoGene® LCM Frozen section staining kit (ABI). In brief, the slides were fixed in 75% ethanol for 30 seconds and washed with distilled water for 30 seconds. The HistoGene® staining solution (ABI) was then dropped onto the sections, incubated for 20 seconds, and washed with distilled water for 30 seconds. The sections were dehydrated using a gradient of alcohol solutions, 75%, 95%, and 100% ethanol, and the slides were then immersed in xylene. The desired cells or areas were selected using specialized microdissection software, ArcturusXT™ LCM System (ABI). The proteins were then purified from the samples and used for western blot analysis. Each protein sample was separated on a 10% SDS-PAGE gel and blotted onto 0.22 μm Immobilon-PSQ transfer membranes . The membranes were then incubated in blocking buffer  for 1 hour at room temperature and incubated with rabbit antibodies against CTGF, VEGF or β-actin  overnight at 4 °C. Next, the membranes were incubated with an HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz) for 1 hour at room temperature; the proteins were then detected with Chemiluminescent Reagent Plus , and images were captured on the UVP system .p value less than 0.05 was considered statistically significant.The definition of these five 3D imaging markers extracted from the microtomography image data was performed by two radiologists and two renal pathologists. Statistical analysis was performed with IBM SPSS Software 20 . The results are presented as the mean ± SEM. Comparisons among groups were performed using one-way ANOVA, with post hoc correction by Tukey’s method. A Supplementary Information


B. pilosa, shows a multi-bioactivity, including immunomodulatory and antidiabetes. Here, we investigated the anti-Listeria effect of cytopiloyne in mice by assessing mortality, clearance of L. monocytogenes, and pathology examination. The data presented herein are supplemental to our research article entitled “Cytopiloyne, a polyacetylenic glucoside from Bidens pilosa, acts as a novel anticandidial agent via regulation of macrophages” Cytopiloyne (CP), a novel polyacetylene compound extracted from  Specifications TableValue of the data•Provides a mouse animal model for assessing the antimicrobial effect of herb compounds to intracellular pathogens.•May be valuable for further studies to identify the molecular mechanism of CP involving in the intracellular survival of pathogens.•May provide a new therapeutic strategy that uses a combination treatment of antimicrobial drugs and edible immunodulatory herbs to improve the efficacy of antimicrobial drug therapy on immunosuppressed patients.1Listeria infection by assessing the survival rate  was obtained from Bioresource Collection and Research Center (Taiwan). Female 6–8 week-old C57BL/6J mice  were maintained and handled according to the guidelines of Academia Sinica Institutional Animal Care and Utilization Committee.2.2Listeria (2×106 CFU). The animals were then observed every day for determination of mortality Six groups of 6 to 8-week-old C57BL/6J mice received intraperitoneal injection of PBS (control) or PBS solution of CP  three times per week for 2 weeks. After 24 h, mice were intraperitoneally injected with 2.3Listeria (2×106 CFU). The mice were then killed at 3, 13, and 22 days  post infection. Their liver and spleen were weighed and cut into 2 parts for CFU determination and histopathological examination. For CFU count, one part of the liver and spleen were homogenized, diluted and plated on BHI agar incubated at 37 °C for 12–16 h. The rest of the organs were frozen for tissue section and then stained by H&E and Gram staining (Hucker–Conn method).As described in


Increasing evidence has demonstrated that Notch signaling is deregulated in human hematological malignancies and solid tumors. This signaling has a protumorigenic effect but may also act as a tumor suppressor. How induction of a single pathway gives rise to the opposite effects in different cell types is still unknown.This review article includes available data from peer-reviewed publications associated with the role of Notch signaling during cancer pathogenesis.Numerous reports have indicated that alterations in Notch signaling and its oncogenic activity were originally associated with the pathogenesis of T‑cell acute lymphoblastic leukemia/lymphoma (T-ALL), an aggressive hematologic tumor affecting children and adolescents. The possibility that Notch could play a significant role in human breast cancer development comes from studies on mouse mammary tumor virus-induced cancer. Numerous findings over the past several years have indicated that alterations in Notch signaling are also responsible for ovarian cancer development. Mention should also be made of the connection between expression of Notch 3 and increased resistance to chemotherapy, which remains a major obstacle to successful treatment. Notch as an oncogenic factor is also involved in the development of colon cancer, lung carcinoma and Kaposi’s sarcoma.Notch is a binary cell fate determinant and its overexpression has been described as oncogenic in a wide array of human malignancies. This finding led to interest in therapeutically targeting this pathway, especially by the use of gamma-secretase inhibitors (GSIs) blocking the cleavage of Notch receptors at the cell membrane by the inhibition of Notch intracellular domain (NICD) releasing. Preclinical cancer models have revealed that GSIs suppress the growth of cancers such as pancreatic, breast and lung cancer. It is generally accepted that Notch signaling plays a fundamental role during embryonic development that is associated with the control of cell proliferation, differentiation and apoptosis. Moreover, a great number of studies have revealed that this pathway also shows a connection to postnatal processes. Noteworthy among these are hematopoiesis, mammary gland development, maturation of gastrointestinal epithelium, immune regulation, angiogenesis and neural stem cell survival , 2.N-terminal ligand-accessible Notch extracellular domain (NECD), and a C-terminal Notch transmembrane fragment (NTM). This fragment is composed of the extracellular stub, transmembrane domain and Notch intracellular domain (NICD)  and Jagged (JAG1–2), belong to the family of the single-pass transmembrane proteins characterized by multiple epidermal growth factor (EGF)-like repeats in the extracellular region. Notch receptors are synthesized as inactive single peptide precursors. These form before reaching the plasma membrane and are proteolytically cleaved by a furin-like convertases in the trans-Golgi network. The first cleavage (S1) generates non-covalently bound heterodimers that are composed of the The significant feature of the Notch pathway is juxtacrine acting between neighboring cells. During the first step of activation, Notch ligands bind to Notch receptors on an adjacent cell. During the second step Notch receptors undergo conformational changes followed by the second cleavage (S2), catalyzed by a member of a disintegrin and metaloprotease (ADAM) family (ADAM17 or ADAM10). The third cleavage S3) is made by a presenilin-dependent γ‑secretase protease complex  consisting of presenilin 1  and anterior pharynx-defective 1 (APH1). After that, the active NICD is released into the cytoplasm and then to the nucleus, where it binds to the ubiquitous transcription factor CSL . NICD is known to convert a large co-repressor complex into a transcriptional activating complex. This complex is primarily composed of NICD, CSL, mastermind-like protein , SKIP (ski-interacting protein as a CBF1 binding protein) and p300, and in this form induces the transcription of Notch target genes. Noteworthy among these are genes encoding hairy enhancer of split (HES) family proteins, HES-related proteins (HEY), Notch-regulated ankyrin repeat protein and p21cip/waf1, cyclin D1 and 3, c‑myc and HER2 Increasing evidence has demonstrated that Notch signaling is deregulated in human hematological malignancies and solid tumors . This siNumerous reports have indicated that alterations in the Notch pathway were originally associated with the pathogenesis of T‑cell acute lymphoblastic leukemia/lymphoma (T-ALL), an aggressive hematologic tumor affecting children and adolescents. Nevertheless, this disease also occurs in adults. Patients presenting with diffuse (>25%) bone marrow infiltration are diagnosed with T‑cell acute lymphoblastic leukemia, while those with mediastinal masses and limited bone marrow involvement are diagnosed as T‑cell acute lymphoblastic lymphoma .The first evidence demonstrating that Notch might be the significant element in leukemogenesis came in the early1990s when Ellisen and coworkers characterized a rare chromosomal translocation t involving the human Notch1 gene in T‑ALL . AdditioRecent studies demonstrated a close dependency between the protumorigenic effect of Notch1 and the transcriptional regulation of cell growth and metabolism. Notch1 directly controls a great number of genes involved in anabolic pathways and also promotes cell growth through direct transcriptional upregulation of MYC . The majAmong other significant mechanisms associated with T‑cell transformation downstream of oncogenic Notch, mention should be made of the promotion of the G1-S phase. Notch1 signaling is associated with the promotion of G1-S through upregulation of CDK4 and CDK6 and downregulation of p27/KIP1 and p18/INK4C cell cycle inhibitors –27. NtziThe question arises regarding the possible relation of Notch1 activation to clinical outcome in T‑ALL. Breit et al. showed that the presence of Notch1 mutations has been significantly correlated with prednisone response, favorable minimal residual disease (MRD) kinetics and improved long-term outcome in children with T‑ALL . AnalysiIt is worth noting here the identification of a novel chalcone derivative inhibiting Notch signaling in T‑ALL. By functional and biological analysis of the most representative molecules of an in-house library of natural products, Mori et al. designed and synthesized the chalcone-derivative 8 possessing the Notch inhibitory activity at concentrations between 5–10 µm. It has been demonstrated that this inhibitor suppressed cell growth of several human T‑ALL cell lines, including DND41, KOPTK1 and TALL-1. This suppression is connected with the activation of apoptosis and increased expression of p27. Moreover, G1 cell cycle arrest has also been detected. Importantly, compound 8 inhibited Notch signaling without interfering with S2 and S3 proteolytic cleavages, depending on ADAM and GS, respectively. Nevertheless, this requires further investigation and development .Extensive research over the past half a century indicates that Notch also plays a very significant role in the pathogenesis of B‑cell malignancies. Alterations in the Notch pathway have been observed in Hodgkin’s lymphoma, Burkitt’s lymphoma, B‑cell chronic lymphatic leukemia, diffuse large B‑cell lymphoma, primary effusion lymphomas related to Kaposi’s sarcoma herpes virus infection and multiple myeloma (MM) .The possibility that Notch could play a significant role in human breast cancer development comes from studies on mouse mammary tumor virus-induced cancer. This study demonstrated that Notch4 showed an ability to transform mammary epithelium in vitro and in vivo. Moreover, an activated form of this receptor slowed ductal growth and perturbed lobular outgrowth prior to tumor formation .The majority of breast cancers express estrogen, progesterone receptors , HER2 and/or ErbB-2 . ZardawiThe presence of JAG1 in primary lesions indicated a higher risk of metastatic relapse. JAG1 expression correlated with vascular invasion and lymph node involvement . DicksonAberrant activation of the Notch signaling pathway has been associated with high grade ovarian carcinoma and carcinogenesis. Moreover, it plays a crucial role in chemoresistance in ovarian cancer patients . The higThe role of Notch1 in ovarian cancer was first described by Hopfer et al., who evaluated Notch mRNA expression in ovarian adenocarcinoma, borderline tumors and adenomas. They revealed more abundant expression of JAG2 and DLL1 in adenocarcinomas compared with adenomas. Interestingly, the stable transfection of A2780 cells with the NICD1 enhanced cell proliferation and increased colony-formation ability . The higResults of Alniaimi et al. demonstrated that Notch2 seems to be a tumour suppressor in ovarian carcinogenesis. The high expression of Notch2 mRNA has been significantly associated with poor PFS for all ovarian cancer patients, especially in grade II ovarian cancer. Interestingly, in serous and endometrioid cancer patients high expression of this mRNA was not correlated to poor PFS and OS .Min mouse model of colon cancer and suppressed the expression of Wnt target genes through epigenetic modification connected with recruitment of histone metylotransferase SET domain bifurcated 1 (SETDB1) [As revealed by studies, Notch receptors are known to be highly oncogenic in the case of colon cancer. Zhang et al. demonstrated that overexpression of Notch1 promoted proliferation, colony formation, cell cycling and tumorsphere formation of colon cancer cells in vitro. Notch1 was also responsible for the development and increased growth of implanted colon cancers in vivo . In huma(SETDB1) . Microar(SETDB1) .Notch2 expression has been reported to show a low level of expression in colon cancer. Expression of this receptor has been clearly correlated with tumor differentiation. Several studies demonstrated the negative correlation between Notch1 and Notch2. Increased expression of Notch1 or decreased expression of Notch2 is thought to be a risk factor for poor clinical outcome in colon patients , 72.In contrast, Notch3 has been shown to be highly expressed in colon cancer. Nuclear expression of Notch3 has been correlated with tumor recurrence and may serve as a novel predictive marker in recurrent stage II and III colorectal cancer . Notch3 In the context of colon cancer pathogenesis it is also worth noting microRNA. The expression patterns of these molecules help distinguish colon cancer from other diseases. Although it is known that miRNAs possess the ability to regulate gene expression by decreased translation or enhanced degradation of the target message, some studies demonstrated that Notch3 might be downregulated at both the protein and mRNA levels by miR-206 in mouse and humans. This suggests that miR-206 regulation is not restricted to the post-transcriptional level, but may also be detected at the transcriptional level . The supFor the first time Notch and its oncogenic activity in lung cancer has been discovered in a tumor-associated translocation between chromosome 15 and 19. The aberrant activation of the Notch pathway is a very characteristic feature of none-small cell lung carcinoma (NSCLC) . The higIn lung cancer cell lines, Notch3 was upregulated, which was associated with karyotipic abnormalities . In NSCLIn the context of Notch oncogenic activity, it is also worth mentioning Kaposi’s sarcoma (KS), which is a common neoplasm in HIV-1 infected individuals. Infection by KS-associated herpesvirus (KSHV) is a key factor in the development of KS , 88. IntIn summary, Notch is a binary cell fate determinant, and its overexpression has been described as oncogenic in a wide array of human malignancies Table . This fiNotch was originally characterized as an oncogene, but studies have also revealed that this pathway may have suppressive activities in some hematopoietic cells, skin and pancreatic epithelium, as well as in hepatocytes . Nicolas


However, the species specificity of non-human in-vivo models and the inadequate recapitulation of physiological conditions in-vitro are intrinsic weaknesses. Here we show that perfusion is a vital factor for engineered human tissues to recapitulate key aspects of the tumour microenvironment. Organotypic culture and human tumour explants were allowed to grow long-term (14–35 days) and phenotypic features of perfused microtumours compared with those in the static culture. Differentiation status and therapeutic responses were significantly different under perfusion, indicating a distinct biological response of cultures grown under static conditions. Furthermore, heterogeneous co-culture of tumour and endothelial cells demonstrated selective cell-killing under therapeutic perfusion versus episodic delivery. We present a perfused 3D microtumour culture platform that sustains a more physiological tissue state and increased viability for long-term analyses. This system has the potential to tackle the disadvantages inherit of conventional pharmaceutical models and is suitable for precision medicine screening of tumour explants, particularly in hard-to-treat cancer types such as brain cancer which suffer from a lack of clinical samples.Pharmaceutical research requires pre-clinical testing of new therapeutics using both  This situation is largely due to the limitations of the widely used in-vitro models and their inaccurate reflection of the clinical condition. Current in-vitro two-dimensional (2D) cell-based assays fail to recapitulate the complexity of the human tumour microenvironment, which includes three-dimensional (3D) anatomic structures of the extracellular matrix (ECM) and cellular heterogeneity reflective of the tumour niche2. In addition, tumour and supporting tissues have high plasticity under mechanical stimuli imposed by the fluidic phase of the microenvironment, a phase which includes the microcirculation of capillaries and lymph, and interstitial flow3. Interstitial flow provides mechanical clues for tissue remodelling, by manipulating the cell-ECM and intercellular interactions in a 3D in-vivo environment4. Recently, models which recapitulate the tumour microenvironment, such as 3D co-culture have been established and are beginning to be used in the re-emergence of phenotypic screening6. These models include the organotypic culture of cancer cells, integrating heterogeneous cell types and 3D scaffolds with microarchitectures in order to increase the physiological relevance of the models7. In addition, scaffold systems such as hydrogel can provide a wide range of physical and chemical properties and can therefore be optimised for various cell types8. Nevertheless, an important part of the tumour microenvironment, the flow originating from the microcirculation perfusion has been relatively ignored9. The contribution of flow can be separated into two components: shear stress and nutrient supply, however, most perfusion strategies designed to replicate the in-vivo microcirculation so far have been mainly focused on how fluid dynamics influences tumour growth or cancer cell migration8.Although average patient survival after diagnosis of cancer is increasing, some hard-to-treat cancer types such as brain tumours still suffer from the lack of effective therapeutics. Meanwhile the pharmaceutical industry is in crisis with low success rates for compounds entering the clinical phase12. Stem cell markers such as SOX2 and Nanog were found in neuroblastoma tissue11, and the expression of neuronal progenitor markers like GFAP is frequently observed in glioblastoma11. It is widely accepted that mechanical cues from the shear stress arising from blood circulation or interstitial flow are vital in tissue development and stem cell biology14, but so far there is limited information on how mechanical cues remodel the tumour microenvironment and influence self-renewal, tumour maintenance and resistance to anti-cancer therapeutics. Similarly, the role of stress as a result of nutrient depletion in the development of stem cell like traits in tumours is as yet unclear.Recently, evidence from basic and clinical oncology reveals the importance of the selection and development of cancer cell subpopulations with different pluripotency in tumour progression. A rare subpopulation of these cells has a development programme resembling that of stem cells, including self-renewable ability, promoted proliferation, and capacity for quiescence, enabling resistance to traditional chemotherapy targeted at rapidly dividing cellsIn this study, we examine the effect of continually replenishing nutrient supply on the differentiation state of 3D cultures of various tumour cells. Three representative engineered tissue models  were cultured in perfusion bioreactors to model the effect of physiological conditions on cancer cell growth and response to anti-cancer therapeutics for up to 35 days. We demonstrate that static cultures display increased indicators of cell stress and altered therapeutic responses compared to the more physiologically relevant perfused cultures.15. We have further adapted this model to allow a ‘desktop’ design for longer term longitudinal studies (<35 days) that includes of a syringe aligner for delivery of additional biochemical inputs  were observed under both conditions but only evident at day 35. Interestingly, only perfused cultures displayed expression of Tuj1 at day 14, although both cultures did so at day 35 indicating a potential shift towards differentiation into mature neurons earlier under perfusion  form endothelial like structures that can mimic angiogenesis and we find that these readily form independent of static or perfused conditions, but display slightly more stable branched structures under perfusion <14days , a marker of pluripotency and found at high levels in the nucleus of GBM cancer stem cells11, and glial fibrillary acidic protein (GFAP) which is an astrocyte marker of differentiation. Static cultures produced unusual staining for both markers, however only perfused culture demonstrated high levels of nuclear Nanog , to provide comparable nutrient supply to both the perfusion and static cultures and allow an objective comparison. The shear stress under this low perfusion rate will be limited, which can also be viewed in the velocity field simulation , to give a demonstration of how perfusion can improve the cell viability and morphological integrity over long-term culture. Morphologically, there were significant differences between static and perfused cultures. For each cell line and under each condition the perfused cultures had a greater viability and formed more defined structures.Perfusion bioreactors have been developed primarily to promote homogeneity within cell cultures in order to optimise protein production. Recently, the opportunity to adapt these technologies to culture micro-tissue and cell spheroids 25. Furthermore, cancer cells with increased pluripotent potential or ‘stem-cell’ properties are more resistant to chemotherapy, which is an emerging hypothesis to explain therapeutic resistance and cancer relapse. For instance, increased levels of Nanog can be correlated to tumour stem cells in GBM and indicate the survival and malignancy of the disease26. Moreover, SOX2 can reprogram differentiated cells into pluripotent cells in concert with other factors and is overexpressed in various cancers27. Therefore, not surprisingly, it has been suggested that targeting pluripotency transcription factors such as Nanog and SOX2 can be a promising strategy to solve the current therapeutic limitation on hard-to-treat cancer types such as GBM28. Differentiation markers such as β-tubulin III was correlated with resistance to DNA-damaging and microtubule-interrupting chemotherapy30; increased GFAP levels have been correlated with tumour volume, tumour necrosis among the GBM patients31. This is also the basis for differentiation induction therapy such as the retinoic acid treatment we mimicked in this study , extracellular matrix (Matrigel sandwich and embedded models) and supporting cells (angiogenesis). The workflow of creating microtissues in the bioreactors was optimised and standardised. The involvement of cells from human tissue will help overcome the ethical and scientific disadvantages of the existing mouse models, and provide a real tissue 3D microenvironment to preselect therapeutics for 2. For the detailed information of the medium used for each cell type please refer to Supplementary Information.Before cultured in 3D environment, all cell lines were routinely cultured as adherent cells in a humidified incubator at 37 °C with 5% CO15. Briefly, cell suspensions with 1 × 104/mL cells were added to each well, and the spheroids were allowed to form for 4 days in advance before perfusion culture was applied.The perfused 3D cancer spheroids were formed based on the protocol optimised in our lab36 and modified in our lab. Briefly, to form the bottom layer of Matrigel, 50 µL pure Matrigel was added into each well which had been pre-chilled on ice, and then the plates were incubated at 37 °C for 30 min, allowing the Matrigel to polymerise. Then for the co-culture, HUVECs were seeded onto the polymerised gel layer at the density of 1.4 × 105 cells/mL, in 250 µL endothelial basic medium  supplemented with 2% FBS  and 1% penicillin-streptomycin (PAA). After 4 hours of HUVECs seeding, OVCAR8 cells in 10% Matrigel (v/v) in 250 µL pre-chilled EBM-2 supplemented with 2% FBS and 1% penicillin-streptomycin were added onto the polymerised Matrigel at the density of 5 × 104 cells/mL. The plate was then left at 37 °C to allow the top layer of Matrigel to polymerise. The co-culture system was maintained in EBM-2 supplemented with 2% FBS and 1% penicillin-streptomycin at 37 °C, 5% CO2 for at least 24 hours before further drug testing. For the mono-culture of microtumours only, the process was the same as the procedure described in co-culture, except that the only difference was that no HUVECs were added, so cancer cells were cultured alone between the 50 µL bottom layer and 25 µL top layer of Matrigel after the polymerisation process.A modified 3D culture protocol was developed to form the Matrigel sandwich structure for both the mono-culture of microtumours and the co-culture of microtumour with angiogenesis network, based on a 3D culture protocol adapted from previous research15. Briefly, poly(dimethylsiloxane) (PDMS) polymers and crosslinking reagent  with the ratio of 10:1 were poured into a designed mould and cross-linked at 60 oC overnight. The product bioreactor, a single direction, multiple parallel perfused micro-bioreactor system is shown in Fig. The initial bioreactor was manufactured in our own lab and previously reportedFor IC50 estimation for a specific model to a certain drug, AlamarBlue  cell viability testing was conducted based on the instructions from the manufacturer. Cells were incubated with EBM-2 containing 10% AlamarBlue for 2 hours at 37 °C. The fluorescence intensity was measured at 560 nm excitation and 590 nm emission using a fluorescent micro-plate reader . The inhibition percentage of anti-cancer drug on the viability of cells was calculated as below: Percentage of Inhibition = [cell viability of untreated sample − cell viability of sample treated by anti-cancer drug]/cell viability of untreated sample × 100%. The log-dose dependent responses were then fitted into Sigmoidal functions by GraphPad Prism  to calculate IC50 estimation.2 for 30 minutes. These probes were designed for relatively long-term tracking, lasting for at least three generations of passages and therefore chosen for this study. The images were taken using a fluorescence microscope  for analysis. The details for microscope parameters are provided in the Supplementary Data.Cancer cell lines and human umbilical vein endothelial cells (HUVECs) were stained by 5 µM (in PBS) 5-chloromethylfluorescein diacetate  and (5 µM) 5-(and-6-)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine , respectively. The probes were diluted from 10 mM stock solution in DMSO into PBS and incubated with the cells at 37 °C, 5% COThe spheroids were harvested into agarose before being fixed in a 4% paraformaldehyde solution for 24 hours, they were then immersed in 70% ethanol for 24 hours. The spheroids were then embedded in paraffin to a GCP small tissue protocol  the FFPE blocks were cut at 4 um + /− 1 um using a Leica Microtome  and mounted onto SuperFrost slides (ThermoFisher Scientific Superfrost Plus). The slides were deparaffinised in formalin twice for 3 mins each, dehydrated in 100% ethanol twice for 3 mins each and then rehydrated in an ethanol gradient . The slides then underwent heat induced epitope retrieval – using a sodium citrate buffer pH 6.0 at 95 deg C for 2 mins they were then cooled on the benchtop. The slides were blocked using an endogenous peroxidise block . The antibodies were diluted in ABCAM diluent  and incubated on the samples for 24 hours at 4 °C, they were then washed in PBS and AlexaFlour-conjugated secondary antibodies were incubated for 30 mins in the dark and the slides were cleaned and mounted with a fluorescent and DAPI mountant .36. The samples were first rinsed by PBS – glycine (100 mM glycine in PBS) for three times, then blocking buffer (10% goat serum (Sigma), 1% goat F(ab′)2 anti-mouse immunoglobulin G  in staining buffer  was incubated on the samples to block the unspecific binding sites. The samples were then incubated with the desired primary antibodies at 4 °C overnight, followed by the incubation in the suitable second antibodies at room temperature for 45 minutes and finally were mounted with diaminophenylindole (DAPI) . Images were taken and processed on a confocal microscope . The details of the antibodies used in immunofluorescence are listed in the Supplementary Data.Microtumour cultured on their own or in co-culture with the vascular endothelial cells were fixed directly in the Matrigel sandwich for 15 minutes, to maintain the original 3D structures such as the tubule network formed by endothelial cells. For immunofluorescence, cells in 3D culture were stained using a protocol modified for sandwich MatrigelDue to the thickness and high cell density in the 3D structure of the Matrigel embedded model, a cell recovery protocol was applied to extract the tissue from the Matrigel hydrogel. Briefly, cultures embedded in the Matrigel were rinsed with ice-cold PBS, and the microtumours were extracted by incubation with Matrigel cell recovery solution  for 1–1.5 hour. The mixture of the Matrigel embedded culture and the recovery solution was gently shaken and checked from time to time to ensure Matrigel was fully digested. Then the extracted microtumours were separated from the solution via 150 g centrifuge, and fixed by 4% PFA for 30 minutes. The following procedure was the same as in whole-culture fixation and staining as above.Retinoic acid (10 µM) was added or perfused into the neuroblastoma microtumours in Matrigel sandwich at day 13 counted from the initial starting seeding day (as day 0), and the perfusion rate was designed as: perfusion rate (r) = medium change volume (v)/medium change time (t). For example, for culture time longer than 14 days, in static culture 1000 µL of medium will be changed every 7 days, then the perfusion rate will be: r = 1000 µL/(24 hours × 7 days) = 5.95 µL/hr. For testing time 7–14 days, in static culture 1000 µL of medium will be changed every 3 days, and this will lead to a perfusion rate r = 1000 µL/(24 hours × 3 days) = 13.8 µL/hr. For the supplementary information on ovarian cancer Matrigel sandwich model, Paclitaxel (10 µM) was applied as a classic representative for first-line anti-cancer drugs, after the microtumours were initially cultured for 3 days.Primary cells were obtained with ethical approval and informed consent from Oxford University Hospitals and all methods were carried out in accordance with relevant guidelines and regulations of the local Research Ethics Committee (REC).All experiments were repeated three times independently, with each experiment having four parallel samples. Student’s t-test was used to compare the significant difference between different culture methods. P < 0.05 was considered to be significantly different.Supplementary Information


The oxygen octahedral coupling at the La2/3Sr1/3MnO3/NdGaO3 interface induces a planar anisotropic Mn-O-Mn bond bending, which causes a significant anisotropic overlap of neighboring Mn orbitals. Due to the anisotropic double exchange interaction, it is found that the conductivity of the La2/3Sr1/3MnO3 film is enhanced when current is applied along the in-plane short crystalline axis. However, the anisotropic behavior is absent in the high temperature paramagnetic phase. Our results demonstrate anisotropic transport in the clean limit where phase separation is absent and the role of anisotropic phase percolation can be excluded.An anisotropic double exchange interaction driven giant transport anisotropy is demonstrated in a canonic double exchange system of La The double exchange (DE) mediated electron hopping6, polaron dressed by lattice distortion8 and phase separation9 are the main proposed models to explain the observed metal-to-insulator transition and colossal magnetoresistance in manganites. Among these, the DE model lies at the origin of the exhibited ferromagnetic and metallic behavior6.Among the correlated electron materials much attention has been given to the manganites due to the exhibited spin polarization and colossal magnetoresistance. Although the richness and complexity of manganites are widely believed to originate from subtle interplay between spin, charge, orbital and lattice6. In a manganite ruled by the DE electron conductivity will be strongly influenced by the transfer integrals, σ = (xϵe2/ahkT), where, ϵ is the exchange energy, x is the doping level and a is Mn-Mn distance5. Since ϵ ≈ kTC and TC is the Curie temperature, the conductivity will be proportional to the TC and σ = (xe2/ah)(Tc/T), which agrees well with the experimental observations11. The DE model hypothesizes that an anisotropic DE will lead to anisotropic resistivities (AR) as shown by theoretical simulations12. Several experimental studies have demonstrated such anisotropic behavior using elastic anisotropic strain effect in Lax5/8−PrxCa3/8MnO3 (LPCMO)13 and Pr0.65(Ca0.7Sr0.3)0.35MnO3 (PCSMO)14 thin films. Nevertheless, the giant anisotropic transport shown in LPCMO and LCSMO films, exhibiting metallic and insulating phase separation, are caused by structural distortion driven anisotropic phase nucleation and percolation13. In contrast to LPCMO and PCSMO which have sub-micrometer phase separation, La2/3Sr1/3MnO3 (LSMO) is recognized as a canonical DE system and is an ideal material for investigating anisotropic DE induced AR in the clean limit without the occurrence of the phase separation. Recently, AR was shown for a LSMO film grown on a DyScO3 (110) substrate due to a charge transfer from oxygen px to py orbitals induced by highly anisotropic tensile strain15. However, the large tensile strain in such LSMO films drives them into an insulating A-type antiferromagnetic ground state15. In this work, we present experimental evidence for anisotropic DE driven highly AR in ferromagnetic and metallic LSMO ultrathin films by employing the interfacial oxygen octahedral coupling (OOC) effect17. In contrast to anisotropic strain15, which is either so large as to destroy the ferromagnetic ground state or too small to induce anisotropic transport, the OOC is found to result in a significant anisotropic crystalline structure and therefore strong anisotropic overlap of Mn orbitals, leading to giant anisotropic transport.Furthermore, the transfer integral which depends on the overlap between oxygen 2p and Mn 3d orbitals determines the energy scale of the DE3 (NGO) (110) substrates by pulsed laser deposition17. The orthorhombic (110) substrate is equal to pseudocubic (001)pc. All films are fully in-plane strained to the NGO substrates17 and thus the lattice constant of LSMO films along [001] and [1–10] directions are 3.854 Å and 3.863 Å respectively18, leading to in-plane anisotropic strain of 0.2%. Our previous reports demonstrated a sharp interface between LSMO/NGO and uniform valence profile across the LSMO films19. Due to the interfacial OOC effect, a structure with Glazer notation of b+a−c− appears in LSMO in the region near to the interface in contrast to the strain driven a+b−c− further away from the interface17. Therefore, the bond angle (θ) along the a-axis (=[001]) θ(a) is larger than along the b−axis (=[1–10]) θ(b) in ultrathin films as schematically shown in Fig. a-axis is smaller than along the b-axis, θ(a) < θ(b). The lateral anisotropic bond angle is expected to give rise to anisotropic Mn 3d and O 2p orbital hybridization as sketched in Fig. The LSMO films were grown on NdGaOa and b measured by four-probe method are shown in Fig. Directly coupled to the strongly anisotropic crystal structure, giant transport anisotropy is observed in ultrathin LSMO films. The temperature dependent resistivities along the two orthogonal in-plane directions 17, though quantitatively different due to the fact that the van der Pauw method amplifies the anisotropic transport20. In addition, the absence of anisotropy for thicker LSMO films matches a previous study15, suggesting that the anisotropic elastic strain enforced by NGO substrate is too weak to induce AR, while the anisotropic OOC can be much more significant than strain in ultrathin films. Finally, the observed transport anisotropy should be related to the orientation of the crystal structure and is not dependent on the orientation of the step and terrace structure of the initial substrate surface. As shown in Fig. ab) of the square sample, while the resistivities and metal to insulator transition temperatures (TMIT) with currents parallel (I//) and perpendicular (I⊥) to the ab direction  will gradually dominate, the AR will become weaker for thicker LSMO films. On the other hand, one would expect a more significant anisotropic transport in the thinnest films. However, the 4 uc LSMO is highly insulating and below 200 K the resistivity is out of the measurement range, as shown in Fig. The observed enhanced conductivity along the short TA) at which the anisotropy starts to develop with the Curie temperature (TC). No sharp transition from isotropic transport to anisotropic transport occurs, therefore, the TA is estimated to be the TMIT for resistivity with the current applied along a-axis  state. These results provide experimental evidence of anisotropic double exchange interaction induced transport anisotropy as suggested by Dong et al.12.The temperature dependence of the anisotropic transport behavior was studied by comparing the critical temperature  state. To test this, we measured the magnetic field dependent transport anisotropy. As shown in Fig. TA and the electronic phase transition temperature by an Arrhenius-type plot of the resistivity as shown in Fig. ρ) vs. 1/T curve is almost linear in the measureable temperature range, but with a 9 T external magnetic field, there is a transition at around 210 K where the thermal activation energy for electronic transport with I//a-axis (I//b-axis) is reduced from 88 meV (88 meV) to 14 meV (18 meV). In contrast, the 4 uc is still highly insulating and exhibits no transition under 9 T field  substrates is a clean DE driven transport anisotropy.Finally, the 6 and 7 uc LSMO films became insulating below ~50 K due to Anderson localization effectIn conclusion, giant transport anisotropy has been induced in LSMO films by using interfacial structure engineering through the OOC effect. The OOC driven anisotropic structure leads to an anisotropic hybridization of the oxygen 2p and Mn 3d orbitals. Transport measurements show that the anisotropy develops in the FMM phase and disappears in the high temperature PM phase. Together with field dependent anisotropic behavior, our results demonstrate that the anisotropic exchange integral plays a pivotal role in anisotropic transport when double exchange dominates the conduction. Our results also suggest the vital role of Mn-O-Mn bond angle in affecting the double exchange transfer and electron conduction and provide an example to create novel properties by interfacial structure engineering.3 (NGO) (110) substrates by pulsed laser deposition, for details see reference17. The temperature dependent resistivities and magneto resistance along the two orthogonal in-plane directions a and b were measured by four-probe method in a Quantum Design physics properties measurement system (QD-PPMS).The LSMO films were grown on NdGaO