Radiotherapy is one of the most important therapeutic strategies for treating cancer. For decades, studies concerning the outcomes of radiotherapy mainly focused on the biological effects of radiation on tumor cells. Recently, we have increasingly recognized that the complex cellular interactions within the tumor microenvironment (TME) are closely related to treatment outcomes.As a critical component of the TME, fibroblasts participate in all stages of cancer progression. Fibroblasts are able to tolerate harsh extracellular environments, which are usually fatal to all other cells. They play pivotal roles in determining the treatment response to chemoradiotherapy. Radiotherapy activates the TME networks by inducing cycling hypoxia, modulating immune reaction, and promoting vascular regeneration, inflammation and fibrosis. While a number of studies claim that radiotherapy affects fibroblasts negatively through growth arrest and cell senescence, others argue that exposure to radiation can induce an activated phenotype in fibroblasts. These cells take an active part in constructing the tumor microenvironment by secreting cytokines and degradative enzymes. Current strategies that aim to inhibit activated fibroblasts mainly focus on four aspects: elimination, normalization, paracrine signaling blockade and extracellular matrix inhibition. This review will describe the direct cellular effects of radiotherapy on fibroblasts and the underlying genetic changes. We will also discuss the impact of fibroblasts on cancer cells during radiotherapy and the potential value of targeting fibroblasts to enhance the clinical outcome of radiotherapy.This review provides good preliminary data to elucidate the biological roles of CAFs in radiotherapy and the clinical value of targeting CAFs as a supplementary treatment to conventional radiotherapy. Further studies to validate this strategy in more physiological models may be required before clinical trial. Radiotherapy is one of the most important therapeutic strategies used for the treatment of more than two-thirds of cancer cases in western countries. In fact, radiotherapy is the curative treatment regimen for some early stage tumors  . Over thEarly studies in radiobiology mainly focused on tumor cells, overlooking the complex cellular interactions that occur within the tumor microenvironments (TME) . Cancer . Radiation induced senescence-like fibroblasts differ from replicative senescence cells in that no telomere shortening is detected [Radiotherapy leads to a series of changes in the TME networks including cycling hypoxia, immune modulation, vascular regeneration, inflammation and fibrosis . While adetected . NeverthTherapeutic resistance in cancer is defined as a process in which cancer lesions progress concurrent with or secondary to an initial response to therapeutic intervention. It is the major reason for poor prognoses of cancer. Interactions between tumor cells and stromal cells are considered to be an important cause of radiation- and chemotherapy resistance. While traditional radiobiology generally overlooks the considerable effect of radiation treatments on the TME as well as the various cytokines that the TME release as a feedback.  EvidenceStudies investigating the direct cytotoxic effects of fractionated radiotherapy on CAFs have found that fibroblasts are naturally resistant to radiation. Tommelein et al. treated primary CAFs from colorectal cancer with scheduled (fractionated dose 5 × 1.8Gy or 10 × 1.8Gy) radiotherapy which induced DNA damage, p53 activation, cell-cycle arrest in the CAFs. However, none of the regimens caused obvious cell death or changes in morphological appearances. The α–SMA expression and the CAFs’ ability to contract collagen gel was also not affected by any of the radiation regimens . In sterData concerning gene expression changes of CAFs after radiotherapy are limited. Nevertheless, altered gene expression profiles of irradiated normal fibroblasts has been explored in a number of early studies. Rodningen et al. evaluated gene expression changes in irradiated normal fibroblasts derived from skin biopsies from 30 different individuals. Fibroblasts were treated with either single dose 3.5 Gy or 10.5 Gy radiation in 3 consecutive daily fractions. Pathways that responded to radiation were associated with the following biological processes: (1) Cell cycle arrest, mainly induced by ATM and p53; (2) DNA repair, a critical process after ionizing radiation which repeated itself inside the cell; (3) ROS (reactive oxygen species) scavenging, which participates in radiation induced biological processes; (4) ECM remodeling, a process closely related to radiation induced fibrosis and fibroblast differentiation; (5) Wnt signaling, which partially regulates radiation induced matrix metalloproteinase expression; (6) IGF signaling, which regulates cell proliferation, differentiation, apoptosis and play a role in tissue homeostasis  Radiotherapy could influence the tumor promoting capability of fibroblasts in the tumor microenvironment. Grinde et al. investigated the pro-tumorgenic effect of irradiated CAFs in a murine xenograft model of lung cancer. Mice were injected with A549 lung tumor cells mixed with irradiated or control CAFs. Experimental groups were comprised of A549 cells alone, A549 co-injected with non-irradiated CAFs, A549 co-injected with single-high dose irradiated CAFs (1 × 18 Gy) and A549 co-injected with fractionated-irradiated CAFs (3× 6 Gy). The tumor promoting ability was abrogated in both groups with irradiated (3× 6 Gy or 1 × 18 Gy) CAFs (from NSCLC), suggesting that high dose fractionated RT (3× 6 Gy) or single ablative dose RT (1 × 18 Gy) may change the function of CAFs and impair their tumor promoting characteristics . What abOther studies suggest that irradiated fibroblasts may promote invasiveness and induce epithelial-to-mesenchymal transition in cancer cells Fig. . TGF-β sMatsuoka et al. explored the relationship between the proportion of CAF component and tumor chemoradiotherapy response. Paraffin fixed tumor tissue sections from 60 patients with oral squamous cell carcinoma receiving preoperative 5-Fu based chemoradiotherapy were immune stained with CAF markers to identify the CAFs component within the tumor. High CAFs component was found to be correlated with advanced pT and pN stage  and poor prognosis compared to low CAFs component group . The impCAFs have been shown to confer radiotherapy resistance to cancer cells primarily through paracrine actions of secreted growth factors. Conditioned media from cervical squamous cell carcinoma derived CAFs increased the proliferation and clonogenic survival of irradiated HeLa cells. The protective effects were enhanced by using conditioned medium from mixed culture of CAFs and HeLa cells. Growth Factor Profiling of conditioned medium showed that the CAFs secreted IGF2, insulin-like growth factor binding proteins (IGFBP)-2, − 4 and − 6 and PDGF-AA which may confer a survival signal to overcome radiation-induced cell death . CAFs haThe combination of targeting CAFs and radiotherapy has not been investigated to date. Since CAFs are naturally resistant while most tumor cells are sensitive to radiotherapy. The combination of targeting both populations may hold a promising future to radiochemotherapy. Current strategies for targeting CAFs include directly targeting and eliminating CAFs through antibody dependent recognition of specific cell surface markers, normalization of activated CAFs, targeting CAFs paracrine signaling pathways and targeting CAF-derived extracellular matrix proteins. Here, we briefly describe each of these strategies.One approach for the elimination of CAFs is through antibody targeted therapy, which requires specific surface membrane markers. As mentioned earlier, a variety of membrane proteins such as FAP, and S100A4 are upregulated in CAFs in comparison to normal fibroblasts, these proteins may serve as potential targets. Even though an individual marker may not encompass the whole CAF population, partial elimination may have the potential to hinder tumor growth or abolish CAF associated therapeutic resistance.FAP is a CAF expressed surface protein which has been studied extensively as a therapeutic target for CAF elimination. A humanized version of a murine mAb against FAP, F19  was clinically safe but showed limited activity in a phase II clinical trail of metastatic colorectal cancer. By conjugating the scFV chain of an FAP antibody to a photosensitive ferritin nanocage, the specificity of targeting FAP positive CAFs can be further enhanced . As an eS1004A is another CAF marker with potential therapeutic value. S100 belongs to one of the largest subfamilies of EF-hand calcium binding proteins, and S100A4 overexpression is strongly associated with tumor aggressiveness. S1004A is less specific than FAP as a marker for CAFs, it is expressed in macrophages as well as a fraction of epithelial cells. Monocloncal antibodies that neutralize S1004A function have been shown to reduces tumor growth and metastasis in a murine model of spontaneous breast cancer by inhibiting tumor initiation, premetastatic niche formation and by tuning the immune regulating balance between Th1 and Th2 cells . In addiMeanwhile, novel CAF markers are being discovered. Tumor endothelial marker 8  was found to be highly expressed in cancer-associated fibroblasts and other stromal cells in a variety of cancers . An ADC As CAFs can generally be described as a group of constantly active fibroblast-like cells that support tumor progression (or restrain tumors in some cases), another approach to counter its biological function is by using agents that can reverted CAFs into a quiescent state or tumor-suppressive state. In patients with pancreatic ductal adenocarcinoma (PDAC), the deficiency of fat-soluble vitamins such as vitamin A and vitamin D are commonly seen. In a mouse model of PDAC, the administration of pleiotropic agent all-trans retinoic acid (ATRA) restored the retinol levels and repressed tumor growth. CAFs isolated from these tumors were reverted into an inactivated state. The inactivated CAFs inhibited tumor growth by suppressing tumor WNT–β-catenin signalling or increasing infiltration of CD8+ T cells , 53. IntCAFs secrete a number of cytokines and growth factors to support tumor growth and regulate the malignant behavior of tumor cells. Some of these secreted proteins such as HGF and IL-6 have been studied extensively as therapeutic targets. HGF are stromal cell derived growth factors. Neutralization of HGF inhibited cancer cell invasion induced by stromal fibroblasts, suggesting a crucial role of HGF/Met signaling in the interaction between fibroblasts and tumor cells . Both mo131I-m81C6, which is an iodine-131 labelled murine mAb has been tested in phase II trials for recurrent malignant glioma patients. Compared to conventional treatment, 131I-m81C6 treatment following chemotherapy shows a survival benefit [CAF-mediated ECM remodelling causes desmoplastic reactions of tumor stoma which may promote tumor growth. Such desmoplastic tumor stroma could form a physical barrier and prevent the delivery of therapeutic agents into tumor cells, . Tenasc benefit . MMPs pl benefit . More th benefit .The profound effects of radiotherapy on fibroblasts and cross talks between fibroblasts and the TME during radiotherapy remain largely unexplored. There are certain limitations in the studies mentioned in the review that need to be improve. Firstly, effects of radiotherapy on CAFs vary among different studies, which may be due to different radiation regimens of doses and fractions that were used. Most of the aforementioned studies used a few days or weeks to explore the short-term effects of radiotherapy on CAFs and tumor cells, whereas radiotherapy has its prolonged effects on tissue, even after several years. A comparative study exploring both the short term and long term effects of radiotherapy on tumor stroma in vitro and in vivo should be conducted in the future for further clarification. Secondly, CAFs are a group of heterogeneous cells of different origins and functions. Heterogeneous functions of CAFs in tumor progression include tumor supportive and tumor suppressive actions depending on their organ of origin, tumor type and disease stage. Although the tumor-suppressive mechanisms remain less clear than the tumor-promoting mechanisms , 10. StuIn summary, the above studies provide good preliminary data to illustrate the biological roles of CAFs in cancer radiotherapy. We support the notion that targeting CAFs as a supplementary treatment to conventional radiotherapy may improve treatment outcomes significantly. Further studies to validate this strategy in more physiological models may be required before continuing to clinical trial.


Sinusoidal endothelial cells and mesenchymal CXCL12-abundant reticular cells are principal bone marrow stromal components, which critically modulate haematopoiesis at various levels, including haematopoietic stem cell maintenance. These stromal subsets are thought to be scarce and function via highly specific interactions in anatomically confined niches. Yet, knowledge on their abundance, global distribution and spatial associations remains limited. Using three-dimensional quantitative microscopy we show that sinusoidal endothelial and mesenchymal reticular subsets are remarkably more abundant than estimated by conventional flow cytometry. Moreover, both cell types assemble in topologically complex networks, associate to extracellular matrix and pervade marrow tissues. Through spatial statistical methods we challenge previous models and demonstrate that even in the absence of major specific interaction forces, virtually all tissue-resident cells are invariably in physical contact with, or close proximity to, mesenchymal reticular and sinusoidal endothelial cells. We further show that basic structural features of these stromal components are preserved during ageing. The bone marrow microenvironment modulates haematopoiesis, stem cell maintenance and differentiation. Here, the authors use 3D microscopy to map the topography of haematopoietic stem cell niche stromal components. High rates of cell production are sustained by haematopoietic stem cells (HSCs), which self-renew while generating a steady flux of multipotent progenitors (MMPs) that mature along the haematopoietic hierarchy3. Sophisticated mechanisms ensure balanced haematopoietic output and simultaneous HSC maintenance5. Fundamental regulatory cues are locally delivered by non-haematopoietic BM stromal cells, which include a plethora of increasingly defined endothelial, mesenchymal and neural cell subtypes8. A well-accepted model of the functional organization of BM posits that stromal cells exert regulation on haematopoietic cells by establishing highly specific interactions in spatially confined microanatomical units, termed niches. These entities favour maintenance or differentiation of distinct haematopoietic subtypes, of which the most widely studied are those governing HSCs10. Therefore, the detailed analysis of the structural organization and interactions established between different BM subsets through in situ microscopy can reveal essential information on the mechanisms that orchestrate haematopoiesis.Continuous de novo generation of all haematopoietic cell lineages is a major physiological process centralized in bone marrow (BM) tissues during adulthood11. BM endothelial cells are major regulators of haematopoiesis15. Genetic deletion of crucial haematopoietic factors such as the chemokine CXCL12 or stem cell factor (SCF) in endothelium results in partial to almost complete depletion of HSCs from the BM, thus proving a major functional interplay between these cell types18. Further supporting this, two- (2D) and three-dimensional (3D) microscopy studies have found that the vast majority of HSCs reside in extraluminal perisinusoidal spaces in direct contact with sinusoidal endothelial cells (SECs)21. A minor fraction of HSCs localize near arterial and arteriolar structures, which are hypothesized to constitute niches with distinct and alternative functional properties23. On the basis of spatial and functional data, SECs have also been proposed to embody multiple BM stage-specific niches involved in megakaryocyte maturation and platelet biogenesis, late B-cell development and maintenance of mature circulating B cells, among others28.Endothelial cells form the linings of BM microvascular networks, which include arterial, transitional (type H) and sinusoidal microvessels29. Mesenchymal progenitor, bone-forming and adipogenic potentials are contained in a subset of fibroblastic reticular LepR+ cells, which are most frequently observed in the vicinity of sinusoids31. The majority of LepR+ cells express substantial levels of CXCL12, exhibit the brightest green fluorescent protein (GFP) labelling in Cxcl12-Gfp knock-in reporter mice and have thus been termed CXCL12-abundant reticular cells (CARcs)34. Although other mesenchymal stromal cells, which are not equally labelled in CXCL12 reporter mice, have been described to produce CXCL1235, CARcs are essential to marrow function as they are additionally major sources of SCF and the pro-lymphoid cytokine interleukin-736. Thus, CARcs have been successively shown to fulfil principal pleiotropic roles in the regulation of HSCs, multipotent progenitors, lymphoid progenitors as well as natural killer, B and plasmacytoid dendritic cell development39. In these studies, some degree of spatial proximity was observed between all mentioned haematopoietic components and CARcs in BM sections.The mesenchymal component of BM stroma has also attracted major interest for its role in regulating haematopoiesis9. Nonetheless, it is currently not known whether these cells exert all reported functions via direct interactions in spatially restricted niches. Conclusions on CARc and SEC interactions have often been drawn merely from data generated from visual identification and manual counting of few events, within limited fields of view and randomly selected tissue regions. However, the significance of the observed spatial arrangements with respect to any proposed interacting partner has, for the most part, not been rigorously tested. Stringent spatial statistical methods to infer interactions between objects require the integration of broad contextual information, such as dimensions and geometry of the tissue volume examined and the absolute number, size and distribution of the entities analysed40. These parameters are needed to statistically discern the existence of non-random spatial dependencies indicative of cell–cell interactions within a given 3D anatomical space41.Altogether, the cardinal roles of sinusoidal microvessels and CARcs in haematopoietic regulation have been amply demonstrated42. The application of FC to study stromal cells suggests that despite their functional relevance, both SEC and CARc subsets individually contribute to a minor proportion of total BMMNCs 45. However, whether stromal cells are efficiently extracted by tissue disaggregation protocols required for FC remains to be determined. Hence, the usefulness of this technology to quantitatively and qualitatively detect pathology-related alterations in BM stromal composition is unclear. For instance, ageing is accompanied by a functional decline in haematopoiesis, which is partially due to altered HSC function46. Age-related disturbances in BM microenvironmental components are hypothesized to contribute to alterations in haematopoietic competence47 and include increased adipocyte infiltration48, perturbations of the cytokine milieu and reductions in bone volume and type H vessels15. Yet, global quantitative and spatial studies of stromal cells within intact BM landscapes and their potential changes during ageing are largely lacking.Comprehensive quantifications of BM mononuclear cell (BMMNC) content are conventionally performed via flow cytometry (FC), which is a high-throughput method for rare cell detection in heterogeneous tissues52. Here, we employed a combination of customized protocols for 3D microscopy, computational tools for image-based quantification and extensively validated spatial statistical methods to analyse the numbers, distribution and spatial coverage of CARcs and sinusoidal endothelial structures in the BM of young and aged mice.The aforementioned questions on BM structure are now becoming addressable by rapidly emerging deep-tissue imaging techniques and automatic bioimage analytical methods20. We optimized this methodology to enable the generation of high-resolution 3D reconstructions of BM volumes ranging across multiple scales, from organ-wide to subcellular levels. We have additionally developed a computational pipeline for: (i) automatic detection and segmentation of immunolabelled objects in volumetric datasets, (ii) accurate quantification of cellular populations and analysis of global and/or local spatial distributions and (iii) inference of interaction patterns between defined objects using appropriate statistical analyses.We previously employed deep-tissue imaging to study vascular microarchitecture in murine BM femoral cavitiesThe main steps of the experimental workflow for 3D quantitative microscopy (3D-QM) are summarized in Fig. + fluorescent signal of cell nuclei . Individual cells are digitally reconstructed as spheres, and continuous networks as surface meshes, which are mapped into rendered volumes , non-endothelial (CD31-) cells displaying the typical fibroblastic morphology and marker expression (CD140b) of mesenchymal reticular cells   Fig. .Fig. 23D43. To quantitatively assess this discrepancy we employed both methodologies (FC and 3D-QM) in parallel to determine the absolute number of CARcs present in either of the two femoral bones of Cxcl12-Gfp mice. FC-based quantification was performed using previously described protocols including tissue enzymatic digestion to facilitate cellular extraction57. The values obtained by FC for total BM cell content and frequencies of CARcs in BMMNCs fell within previously published values  and the BM femoral volume was calculated by image segmentation 59 and Ly6A (Sca-1)12, as determined by quantitative reverse-transcription PCR 12. In agreement with previous work and with their reported role in recruitment of circulating cells to BM, sorted SECs displayed highest levels of expression of adhesion molecules such as P-, E-selectin and VCAM-162 at messenger RNA and/or protein levels , Tek (Tie-2) and Cdh5 (VE-cadherin), further confirming their identity  subset is traceable by 3D-QM and FC63  fibresons Fig. . In a prons Fig. , ii and,ons Fig. . ECM fib21, arterial networks were significantly less abundant. Given the heterogeneous presence and longitudinal trajectories of arterial vessels, we analysed transversal slices of metaphysis and diaphysis to best capture their spatial influence  and old (20–24 months old) mice. Unexpectedly, quantification of CARc bodies revealed no major differences in old mice as compared to young counterparts Fig. . As prevsed Fig. . Of notesed Fig. . Similarsed Fig. . Furthersed Fig. . Althougsed Fig. . AltogetMulticellular organ function is the net effect of the concerted interplay of tissue constituents in space and time. Essential to our understanding of BM physiology is: (i) the identification, classification and quantification of the cellular subsets present in BM, and (ii) detailed knowledge on their structural organization into an integrated tissue architecture, which ultimately defines the nature and frequency of the interactions established. Here, we exploit the unique benefits of 3D-QM to unveil fundamental aspects of the BM microenvironment. We demonstrate that standard FC methods for cellular analyses fail to capture the vast majority of BM SECs and CARcs, which are strikingly abundant. Systematic comparison of FC with multimodal imaging data reveals that either the cellular isolation procedures or the analytical process of FC entail a disproportionate loss of stromal cells. Although unclear from our studies, inefficient isolation may be explained by the fact that stromal cells form compact, sessile networks relying in firm adherent interactions, which are likely resistant to digestion and extraction into single cell suspensions.42. However, FC relies on the premise of nearly complete recovery or homogeneous loss of distinct cell types during isolation procedures. Our results are evidently at odds with this assumption in what pertains stromal cell isolation, and further raise fundamental questions. First, whether inhomogeneities in isolation yields also apply to haematopoietic subsets needs to be thoroughly assessed, especially for cells exhibiting elongated shapes and strong adhesive phenotypes such as those of the histiocytic and antigen presenting fractions. Second, it will be crucial to understand to what extent the stromal populations extracted into suspensions and extensively studied at functional and/or transcriptomic levels ex vivo are representative of the in vivo cellular pool. Indeed, the existence of certain subgroups of stromal cells completely intolerant to isolation protocols may have been fully missed up to now. Finally, biases in cell isolation may not only arise from cell-specific but also tissue-dependent factors. In recent work, Steinert et al.68 demonstrated through meticulous image-based quantification that recovery of memory CD8+ T cells dramatically varied between organs, possibly due to the divergent biophysical properties of tissues. By providing a realistic account of memory T-cell numbers and anatomical distribution, this study led to a radical revision of prevalent models of immunological memory68. Biomechanical features of tissues may substantially change with alterations of structure, cellular and molecular composition driven by pathological stimuli. Thus, the dynamic variations in the BM stromal makeup found to occur during haematologic neoplasias, chemotherapeutic interventions or infections should be interpreted with caution until ascertained via in situ imaging71. We exploit this approach to reveal that key quantitative and spatial features of CARc networks and sinusoids are unexpectedly robust and maintained during homeostatic ageing. In contrast to the reported age-induced loss of type H vessels15, sinusoidal structures remain largely intact in the different BM regions and intrasinusoidal volumes were unchanged. Similarly, the numbers of CARcs and their spatial relationship to sinusoids do not vary significantly in aged BM. Future analyses should determine whether and to what extent the age-induced perturbations of HSC function and haematopoiesis47 relate to spatial and structural changes in BM organization.FC-based comprehensive population analyses have been historically central to the dissection of the haematopoietic hierarchyAccording to image-based estimations, and presuming only minor losses in haematopoietic cell recovery, the frequency of CARc and SEC populations is approximately 30-fold higher than assumed. Such a substantial upward revision of abundance has major implications on the perceived functional relevance of stromal cells and their proposed mode of action. Hitherto regarded as specific elements of confined niches, we demonstrate that CARcs and SECs are spatially dominant and available for physical contact with virtually all, or the majority of cellular elements in BM. Consequently, even in the absence of real interaction forces, the expected levels of association between these stromal cells and any given BM cell type will be very high. In light of our results, the significance of the reported proximities between certain haematopoietic subsets and CARcs or sinusoids could be potentially explained by pure chance. Nevertheless, as currently defined, CARcs and SECs are most likely heterogeneous mixtures of cell subtypes. A deeper understanding of such heterogeneity could help uncover zonation and labour division within these stromal pools, which would explain their multiplicity of functions, and potentially reveal the existence of highly specific stromal–haematopoietic interactions. We anticipate that 3D-QM will be pivotal in the detection of subcategories of stromal cells, based not only on phenotypic and functional criteria but also on spatial traits. Our data strongly point to the existence of two subpopulations within CARcs, based primarily on direct adjacency to sinusoidal surfaces and morphological features. Further studies should help to understand whether these two fractions represent sublineages with divergent roles.53. Multiple groups have previously investigated potential spatial dependencies  through comparison of empirically observed spatial data to that generated through simulations of randomly distributed spots in 2D and 3D microscopy images74. When performed in realistic, high-quality tissue images, the conclusions derived from these approaches with regard to the levels of random spatial associations of sinusoids and CARcs are well in line with ours21. Nonetheless, the criteria used to define randomness as well as the statistical methods for testing significant differences in spatial distributions vary largely, have not always been ideal and thus can lead to inaccurate conclusions on potential interactions. The use of the available ESD as a benchmark for complete spatial randomness avoids the need for additional a priori assumptions required to generate simulations, and provides comprehensive information on the anatomical constraints in which the observed spatial associations take place.The pipeline presented herein is widely applicable for the dissection of microanatomical features and interrogation of spatial interactions within biological tissues. The power of our approach is strongly based on the use of tissue processing methods that absolutely respect the antigenicity and structural integrity of highly sensitive subcellular structures, thereby enabling their visualization with unprecedented minute detail. We generate 3D reconstructions of intact infrastructures made of ECM fibres and CARc projections, which ultimately underlie the massive spatial influence of these cells. Furthermore, careful treatment and analysis of image datasets is essential. This includes the evaluation of our segmentation strategies through comparison to a manually annotated ground truth, or visual inspection of multiple random regions. Importantly, we propose for the first time the use of points processes, a well-established toolbox of spatial statistics, to study spatial relationships in BMIn summary, we show the power of combining quantitative imaging techniques and spatial statistics to reveal novel and key information on the cellular composition, functional interactions and homeostatic regulation of haematopoietic tissues. The datasets presented here can be further used as structural blueprints of stromal network organization and spatial coverage of the BM space in homoeostasis, which will serve to explore and identify novel phenotypes associated to haematological diseases.Cxcl12-Gfp mice have been previously described34 and Foxp3-Gfp knock-in mice63 were provided by Nicole Joller . All mice were analysed at 12–20 weeks of age. For ageing experiments, young mice were 8–12 weeks old and aged mice 20–24 months old, which were selected for displaying no apparent signs of health impairment at the moment of euthanasia. Experimental animals were not randomized and experiments were performed in a non-blinded fashion. Animals were maintained at the animal facility of the University Hospital Zürich and treated in accordance with the guidelines of the Swiss Federal Veterinary Office. Experiments and procedures were approved by the Veterinäramt des Kantons Zürich, Switzerland.C57BL/6J were purchased from Charlesriver. A detailed list of all the reagents employed in this study, including antibodies for FC and 3D-QM and the concentrations at which they were used, is provided in Supplementary Table For BM cell analyses, murine bones were first harvested and cleaned thoroughly. To ensure precise quantification, femurs were consistently dissected below the hipbone and above the kneecap. BM content was flushed directly into 5 mL of medium  using a syringe. Remaining pieces of bones were carefully minced into small-sized fragments (1 mm) and mixed with flushed BM content. Tissue suspensions were thoroughly homogenized by gentle and repeated mixing using a 1 mL pipette to facilitate dissociation of cellular aggregates. Enzymatic digestion was performed by incubation in DNAse (0.2 mg/mL) and Collagenase Type 2 (0.04 g/mL) at 37 °C for 45 min under gentle rocking. Resulting cell suspensions were then filtered through a 70 µm cell strainer, washed in phosphate-buffered saline (PBS), blocked for 15 min using TruStain fcX™ and successively stained with cocktails of fluorescently labelled antibodies for 30 min at 4 °C. Cells were then washed twice with PBS, resuspended in PBS containing 4',6-diamidino-2-phenylindole  and analysed on a LSR II Fortessa (BD Biosciences). Data analysis was performed using the FlowJo 10 software package.BM cell suspensions were prepared and immunostained as for FC analysis described above. Cellular populations of interest were sorted using a FACS Aria (BD Biosciences) equipped with 4 lasers. A total of 5000 to 10,000 cells of each endothelial subtype were directly collected in RLT lysis buffer (Qiagen). Genomic DNA (gDNA) was depleted from cellular lysates using gDNA eliminator columns (Qiagen), RNA was extracted using the RNeasy Plus Micro Kit (Qiagen) and complementary DNA (cDNA) was generated with the High-Capacity cDNA Reverse-Transcription Kit (Thermo Scientific), following the manufacturers’ instructions. Gene expression was measured with a 7500 Fast Real-Time PCR System using either TaqMan® probes or Power SYBR® Green. A full list of primer pairs employed for individual gene detection can be found in Supplementary Table 20. Mouse femurs were isolated, cleaned and immersed in PBS/2% paraformaldehyde for 6 h at 4 °C, followed by a dehydration step in 30% sucrose for 72 h at 4 °C. Femurs were then embedded in cryopreserving medium ) and snap frozen in liquid nitrogen. Bone specimens were iteratively sectioned using a cryostat until the BM cavity was fully exposed along the longitudinal axis. The OCT block containing the bone was then reversed and the procedure was repeated on the opposite face until a thick bone slice with bilaterally and evenly exposed BM content was obtained. For transversal slices, bones were manually cut after fixation with a razor blade along the transversal axis in 3–4 fragments, which were frozen separately. The procedure then continued as described above for longitudinal sections. Once BM slices were generated, the remaining OCT medium was removed by incubation and washing of the bone slices in PBS 3 times for 5 min. For immunostaining slices were incubated in blocking solution , 10% donkey serum in PBS) overnight at 4 °C. Primary antibody incubations were performed in blocking solution for 3 days at 4 °C, followed by overnight washing in PBS. Secondary antibody stainings were performed for another 3 days at 4 °C in blocking solution but in the absence of BSA to avoid cross-absorption. Immunostained thick femoral slices were successively washed in PBS overnight and incubated in RapiClear 1.52, for a minimum of 6 h, which typically increased imaging depth to 150 µm from the tissue surface without significant loss of signal intensity. The clearing protocol employed is fast, compatible with all fluorescent probes and proteins tested, and preserves the integrity of subcellular structures.Methods for 3D imaging of BM were adapted from previously published protocolsz-axis available on the Leica Application Suite X. Stitching of confocal image stacks was also performed in the dedicated tool included in the image acquisition/analysis software.For observation under the confocal microscope, BM slices were mounted on glass slides using vacuum grease. Confocal microscopy was performed with 10× (HCX PL FLUOTAR), 20× (HC PL APO CS2), 63x (HCX PL APO CS2) and 93× objectives (HC PL APO motCORR) on SP5 or SP8 Leica confocal microscopes. Low-magnification tissue-wide images were generated and ROIs were defined for subsequent detailed acquisition with 20×, 63× and 93× objectives. Image analysis was exclusively performed in high-resolution images, which met quality standards. The specific ranges for the acquisition parameters for each type of objective are provided in Supplementary Table To quantify the total BM volume in the femoral cavity µ-CT images of pre-fixed entire mouse femurs were acquired with a Bruker SkyScan 1176 in spiral scan mode and cone beam. Scans were performed with an X-ray peak voltage of 50 kV at a dynamically modulated current, typically around 270 µA. We used a 0.5 mm aluminium filter for beam energy spectrum modulation. The resulting images, reconstructed with the vendor-specific software, had an isotropic resolution of 8.67 μm/voxel. After acquisition, femurs were frozen and processed for 3D confocal imaging.Confocal image stacks were rendered into 3D volumes and analysed using Imaris v8.2 (Bitplane AG) and MATLAB software version 9.1 . The Imaris Programming Interface was used to implement all the MATLAB-generated image-processing methods described herein, as plugins for Imaris (ImarisXT). When large regions were imaged, the different tiles were stitched with the Leica Application Suite (LAS) X software (Leica microsystems). Image segmentation methods employed in this study made use of the image intensities to classify voxels and determine their correspondence to segmented BM components, which then allowed for digital reconstruction of cells or networks as objects in 3D space. Object volumes were subsequently calculated by multiplying the total number of voxels they occupied by the voxel volume calculated separately for each image stack and acquisition settings. The precise methods for segmenting individual BM components analysed in this study are described in detail below:Segmentation of DAPI fluorescent signal for determination of absolute tissue volumes: to determine tissue volumes contained in the ROIs, the outer boundaries of BM tissues were defined by image processing of the DAPI signal . The goodness of this automatic algorithm employed for CARc detection was evaluated in three manually annotated images with a total of 182 ± 3.74 annotated cells per image considered as ground truth (GT). Manual annotation was performed in Imaris by three different researchers independently to account for inter-observer variability. For this evaluation, each detected cell was assigned to its closest sphere in the GT . Detected spheres were counted as true positive (TP) when the distance between a corresponding pair was smaller than 5 µm. Otherwise it was counted as false positive (FP). All GT spheres without assigned corresponding pair were considered false negatives (FN). Precision and recall were then calculated as follows:Mean and standard deviation were calculated between the results of the different individuals. Images were discarded when the staining quality did not allow for automatic detection with the precision and recall reported here.Surface segmentation utility available in Imaris, which detects objects based on local intensities. The resulting segmentation was visually inspected to remove small individual segmented objects components of high sphericity, which were regarded as noise.Segmentation of CARc networks, ECM and arterial vessels: network-like structures were segmented using the Sinusoidal vessel segmentation: in high-resolution 3D images fluorescent signals corresponding to sinusoidal markers could only be observed along the cell surface of SECs in the endothelial walls, while the lumina and extravascular regions displayed a low to background signal intensity. In order to generate full solid reconstructions of vascular volumes we implemented methods to fill vessels as illustrated in Supplementary Fig. Segmentation of total BM volume in µ-CT-generated images: images were processed to first segment the whole femur using the same approach as described above for sinusoidal vessel segmentation. The resulting volume was subsequently thresholded to create two masks: one for the BM content and the other for the voxels corresponding to bone. The volume of these structures was directly calculated from the mask.We exclusively analysed BM slices in which integrity of the entire bone marrow cavity or at least of the complete metaphysis or diaphysis was optimal and met quality standards for spatial analysis.x), which was observed within a defined region (W) corresponding to extravascular regions of BM tissues. For this analysis, the extravascular compartment was defined as the intersection of the DAPI mask and the extravascular volume. Thus, the population of cells detected was defined as x∩W. This representation permitted the use of spatial statistic methods, which were implemented in MATLAB as plugins for Imaris (requiring ImarisXT module). The ESD in W (denoted Z) was used as a descriptor of the way in which 3D space is constrained by different cellular structures. For a given cellular structure, this function assigns each voxel (u) a distance to the closest structure as Z(u). We used the Euclidean distance, with distance zero set at every voxel that contains at least a fragment of the surface of the given structure. Voxels corresponding to the inside of the studied structures were excluded. Intravascular regions were also excluded so that only extravascular tissue regions were considered. To study the distribution of ESDs with respect to different structures, a histogram of distances for all voxels was formed and then summed in a cumulative fashion to generate the experimental CDF. The CDF of a variable defines the probability of it being smaller than or equal to the value at which it is evaluated. In this context, the ESD CDF defines the empirical proportion of volume contained within any given distance to a selected structure. CDF of ESDs to different structures are represented as filled contours in which the central solid line depicts the average probability and the upper and lower bounds of the envelope represent the standard deviation. We studied the effect of image borders in the ESD by comparing its result when confined to different boundaries. We saw the effect was negligible, probably due to the dense occupation of all the cellular structures we study herein.The representation of BM components as segmented objects allowed us to study cellular subsets as a point pattern ((xi) was not influenced by their distance to the sinusoids (Z(xi)). Under this null hypothesis, Z(xi) should be a random sample of the ESD to the sinusoids (Z(u)). Two-sample Kolmogorov–Smirnov test was used to analyse significance.To study the spatial homogeneity of CARcs with respect to sinusoids we formulated the null hypothesis that the position of the detected cells f(u) was calculated at every position u as defined in:Kσ(u) is a 3D Gaussian kernel with standard deviation σ and a diameter of 8σ for each dimension. The value of σ was calculated for each image as the average nearest neighbour distance between the represented cells. The result was the 3D probability density function of the cell distribution. To generate 2D density maps as depicted in Fig. Imaris software was used to render confocal image stacks into 3D reconstructions and visualize the results generated through computational analysis in a virtual 3D space. A customized Imaris plugin (requiring ImarisXT module) was implemented in MATLAB to easily transfer images from Imaris to MATLAB for analysis. Processed and analysed images were then returned to Imaris for visualization as 3D rendering with OpenGL. 3D volumes were represented as a Maximum Intensity Projection with a perspective of 45°. Gamma correction was applied exclusively for visualization purposes. Binary images resulting from the segmentation were represented as a surface mesh in Imaris by making use of its surface segmentation utility. To represent the distribution of cells in the form of BM tissue maps we utilized the empirical probability density, which was calculated with a custom algorithm that adapted the kernel-based density estimation method to account for the different boundaries of the BM. The tissue map Data are expressed as mean ± SEM. Mann–Whitney tests were used to assess differences between groups unless stated otherwise. Kolmogorov–Smirnov test was employed to study the homogeneity of CARcs with respect to sinusoids in Figs. https://github.com/alvgom/BM_3D-QM.The code for image analysis was implemented in Matlab as a set of user-friendly plugins for Imaris software (ImarisXT). Code and instructions for use can be found in Github All raw data are available upon reasonable request to the corresponding author.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6collagen IV CARc sinusoids


Research progress over the past 20 years has yielded several experimental Ebola virus (EBOV) vaccine candidates, which were shown to be effective in nonhuman primates when given 28 days before a lethal challenge. Of these, the vesicular stomatitis virus (VSV)-vectored vaccine against EBOV (VSV-EBOV) is unique at being able to induce rapid protection, with 100% survival achieved as soon as 7 days after EBOV challenge. mBio article, Menicucci et al. carried out a transcriptome analysis of host responses in monkeys immunized with VSV-EBOV from 28 to 3 days before challenge . It was found that surviving animals had a controlled innate immune response coupled with rapid adaptive immunity, but this was not detected in nonsurviving animals. These studies highlight the important role innate immunity plays in creating an antiviral state to restrict EBOV replication and ensuring enough time for the vaccine to induce an effective adaptive immune response.Research progress over the past 20 years has yielded several experimental Ebola virus (EBOV) vaccine candidates, which were shown to be effective in nonhuman primates when given 28 days before a lethal challenge. Of these, the vesicular stomatitis virus (VSV)-vectored vaccine against EBOV (VSV-EBOV) is unique at being able to induce rapid protection, with 100% survival achieved as soon as 7 days after EBOV challenge. In a recent  Ebola virus (EBOV), which causes sporadic and unpredictable outbreaks in Central and West Africa, is one of the deadliest pathogens known to humans. After exposure to the virus, there is an asymptomatic incubation period of 2 to 21 days before the onset of symptoms. The disease course itself is rapid and includes hypovolemic shock, hemorrhage, and multiorgan failure, with death typically occurring at 7 to 10 days after symptom onset. Case fatality rates can reach up to 90%, and there are no specific vaccines or therapeutics licensed for use against EBOV. Due to its rapid pathogenesis, fast-acting countermeasures must be deployed against EBOV infections in order to have any chance for success.Over the past 2 decades, several vaccine candidates have been tested and shown to be efficacious in nonhuman primates (NHPs) against a lethal EBOV challenge . In 2009, VSV-EBOV was administered to a researcher who had a high-risk occupational EBOV exposure in Germany, at 48 h after the incident, and in 2014 the vaccine was given to a physician working at an EBOV treatment center in Sierra Leone, at 43 h after the needlestick injury. Aside from mild symptoms, including fever at 12 h after the vaccine injection, both recipients remained healthy.Additionally, VSV-EBOV has been deployed for use on compassionate grounds during outbreak and epidemic scenarios, especially with ring vaccination of high-risk contacts to laboratory-confirmed EBOV cases, as well as contacts to these high-risk contacts. Indeed, several phase I to III clinical trials were initiated during the 2013 to 2016 EBOV epidemic in West Africa: Partnership for Research on Ebola Vaccines in Liberia I , Sierra Leone Trial to Introduce a Vaccine Against Ebola , and Ebola ça suffit! in Guinea (PACTR201503001057193). Importantly, the Ebola ça suffit! clinical trial had an estimated vaccine efficacy of 100%  play a major role in VSV-EBOV-mediated protection against EBOV infection, and past studies have highlighted the role of EBOV-specific IgG in statistically correlating with survival of EBOV disease  as well mBio. The authors undertook a thorough and detailed analysis of the transcriptome response in the whole blood of groups of 2 to 3 NHPs immunized with VSV-EBOV and then challenged with EBOV at various times after vaccination . Analysis of how gene expression changed after immunization showed the detection of 83 and 518 differentially expressed genes (DEGs) at days 3 and 7 postvaccination, but very few DEGs from day 14 onwards. The majority of DEGs from days 3 and 7 were found to be interferon-stimulated genes (ISGs) important for antiviral defense, and those exclusively identified at day 7 play a role in antiviral innate immunity, including transcription factor genes STAT1 and IRF1, inflammatory mediator genes MYD88, NFKBIA, and IL1B, as well as pathogen recognition receptor genes such as TLR2 and TLR4. Additionally, the authors also detected increased expression of genes important for antigen presentation , T-cell signaling and activation , as well as B-cell signaling, activation, proliferation, and differentiation into plasma cells  from day 7 samples. These findings are supported by the detection of elevated levels of alpha interferon (IFN-α) from NHP samples collected on days 3 and 7, as well as robust antibody responses detected from day 14 onwards. Thus, VSV-EBOV induces a robust innate immune immunity and an early adaptive response within 7 days postvaccination.Menicucci et al. addressed this knowledge gap in a study  publishen = 1]), viremic survivor (VS [n = 1]), and nonviremic survivor (S [n = 1]), thus allowing for comparison of markers between animals with different survival outcomes and potentially a gene signature determining vaccine efficacy and protection. The data showed that genes involved in innate immunity  and humoral immunity (IFLV3-19) were highly expressed in S at the day or challenge, compared to VS and VNS, but were reduced by 3 days after challenge (dpc), indicating a controlled innate immunity. ISGs  were already highly expressed in S and VS at the day of challenge, but this increase in expression was delayed in the VNS animal until 3 dpc. In contrast, genes upregulated in the VNS animal only at the day of challenge include BAK1 (involved with apoptosis), FGG (coagulation), and IL-27 (which promotes anti-inflammatory cytokine interleukin-10 [IL-10]), among others , a potential follow-up study could involve work with a nonrelated VSV vaccine (such as those against Marburg or Lassa viruses) with larger groups of NHPs, such that the role(s) of the innate immune response markers can be clearly defined and supported by statistics. This study is an important building block for the elucidation and characterization of early gene signatures contributing to survival from EBOV and will be of essential reference when designing future milestone studies regarding the mechanisms of nonspecific protection elicited by VSV-vectored vaccines.


Identification of selection signatures can provide a direct insight into the mechanism of artificial selection and allow further disclosure of the candidate genes related to the animals’ phenotypic variation. Domestication and subsequent long-time selection have resulted in extensive phenotypic changes in domestic pigs, involving a number of traits, like behavior, body composition, disease resistance, reproduction and coat color. In this study, based on genotypes obtained from PorcineSNP60 Illumina assay we attempt to detect both diversifying and within-breed selection signatures in 530 pigs belonging to four breeds: Polish Landrace, Puławska, Złotnicka White and Złotnicka Spotted, of which the last three are a subject of conservative breeding and substantially represent the native populations.ST and relative extended haplotype homozygosity (REHH) test. Breed-specific diversifying selection signals included several genes involved in processes connected with fertility, growth and metabolism which are potentially responsible for different phenotypes of the studied breeds. The diversifying selection signals also comprised PPARD gene that was previously found to have a large effect on the shape of the external ear in pigs or two genes encoding neuropeptide Y receptors (Y2 and Y5) involved in fat deposition and stress response which are important features differentiating the studied breeds. REHH statistics allowed detecting several within-breed selection signatures overlapping with genes connected with a range of functions including, among others: metabolic pathways, immune system response or implantation and development of the embryo.A two largely complementary statistical methods were used for signatures detection, including: pairwise FThe study provides many potential candidate genes with implication for traits selected in the individual breeds and gives strong basis for further studies aiming at identification of sources of variation among the studied pig breeds.The online version of this article (10.1186/s12863-018-0681-0) contains supplementary material, which is available to authorized users. Numerous studies have shown applicability of genomics in the field of quantitative genetics and identification of sources of variation of important phenotypic features such as production traits . UnfortuSimilarly, as other livestock species, pigs have been under long term selection during domestication, breeds formation and further improvement of production and functional traits. To analyze the mechanism underlying phenotypic differentiation caused by selection in pigs, the evidence of selection has been searched in genomes of various pig breeds using whole genome genotype data or high-throughput sequencing –9. The sIn the present study, we detected selection signatures at the whole genome level in three conserved pig breeds derived from the native pig populations  and a commercial Polish Landrace breed, differing in terms of production, reproduction characteristics and exterior features. Among the native breeds we included Puławska breed which is valued for good resistance to harsh environmental conditions and diseases, taste of meat (its aroma and juiciness) and usefulness for extensive breeding in ecological farms. Two other studied Złotnicka pig breeds (White and Spotted) are characterized by similar functional features but have worse productivity than Puławska breed and have more primitive character. Especially Złotnicka Spotted is being considered as a meat-lard type breed and is characterized by high subcutaneous fat content and relatively low fertility –13. The ST  [ST and REHH statistics were shown to be useful to detect selection signatures [ST is useful to detect selection signatures across breeds, represented mainly by loci that were differentially fixed in different breeds [To identify selection signatures in the analyzed breeds, we applied two different methods: first, based on Ftiation)  and aimetiation)  statistignatures  and theyt breeds .Despite the lack of typical selection in terms of production in the case of conservative breeding, the animals’ qualification into the conservation program (based on breed standard and aimed at consolidation of breed-specific traits and stabilization of exterior) may also be considered as an extensive selection and lead to similar but less pronounced changes in the frequency of alleles. The applied combination of breeds and statistical methods allowed us to search for selection signals associated with both fixed traits of individual breeds and features that are still under improvement, which may help to better understand adaptation of breeds to local environmental conditions and help to evidence processes behind good health, longevity and low environmental requirements of native pigs.n = 135), Puławska , Złotnicka White , Złotnicka Spotted  differing in terms of production, reproduction and exterior features. The animals were selected to be unrelated for at least two generations and originating from different herds. Each population sample included at least 7% of males. This was because we analyzed breeding population (reproductive studs) in which share of males is matched to the number of boars which are designed to natural matting. All animal procedures were approved by the Local Animal Care Ethics Committee No. II in Kraków - permission number 1293/2016 in accordance with EU regulations. The genomic DNA was purified using a Sherlock AX kit (A&A Biotechnology) and after quality control was genotyped with the use of the PorcineSNP60 BeadChip assay (Illumina) according to the standard Infinium Ultra protocol. The obtained genotypes were controlled for quality by evaluation of call rates and only samples with more than 97% of called genotypes were used for further analysis. Of the 61,565 assayed SNPs, a panel of 50,485 markers was further obtained by removing SNPs mapped to contigs, located on the sex chromosomes (Sscrofa10.2 genome assembly) or classified as intensity-only probes.The material of the study was genomic DNA obtained from blood or hair bulbs of 530 animals sampled from each of the four pig breeds: Polish Landrace . The signals of diversifying selection were detected using pairwise Wright’s FST [ST values obtained for pairwise comparisons at each SNP were treated according to a methodology proposed by Akey et al. [ST values were calculated (di) as:ST between breeds i and j calculated from all analyzed 43,923 SNPs. This allowed to make comparison of each breed against all other breeds under study. To account for stochasticity in locus-by-locus variation, a 10-SNP sliding window was further implemented on the obtained values. Candidate selected regions were then defined as the 99.9th percentile of the empirical distributions of window-averaged di values. The adjacent regions under selection were merged and (while searching for gene content) regions were expanded on both ends by 25 kb to detect neighboring, potentially linked genes.The initially filtered SNP set was further reduced by applying population-wide polymorphism filters. The filtering included removal of SNPs with MAF lower than 5% and SNPs with more than 20% of missing genotypes across all breeds. MAF cutoff used for SNPs filtering was applied to the whole population . This allowed to retain small proportion of SNPs that are monomorphic only in some breeds . MAF value of 0.01 was used to characterize remaining SNP polymorphisms. SNPs deviating from HWE with critical ht’s FST , the clay et al.  and furtlocus. Subsequently, a probability that two randomly selected haplotypes within a core region are identical-by-descent for the entire interval spanning the core region to a given locus was computed [P-values were ultimately obtained by a logarithmic transformation of the REHH values within these bins  and calculation of mean and standard deviation. The core haplotypes with the most extreme P-values (extended by 0.5 Mb in each direction) were filtered for frequency (> 0.25) and screened for overlapping pig ENSEMBL genes with the use of UCSC Genome Browser.The signals of positive selection within single breeds were detected using REHH statistics implemented in the Sweep v.1.1 software . First, computed , 21. Fincomputed  and calccomputed  on both The functional annotation of detected genes was performed using the KOBAS 3.0 web server  and WebGST distances created using the neighbor joining (NJ) method [The population differentiation was additionally visualized using the principal component analysis (PCA) based on SNP genotypes and a cladogram of mean pairwise F) method .ST distances were the highest between PUL and ZS (0.143 and 0.173) and the lowest level of genetic differentiation was found between PL and ZW (0.085 and 0.097) . The number of polymorphic SNPs MAF > 0.01) per breed ranged from 37,423 to 43,567 in ZS and PL breeds, respectively. The average MAF across all SNPs was the lowest in ZS (0.212) and the highest in PL (0.279). The averaged observed heterozygosity per breed ranged from 0.3 to 0.367 for the same breeds  GO biological processes there were inter alia those connected with: lipid binding, fatty acid metabolic process, cellular senescence or response to muscle stretch. When functional classification was performed for individual breeds, visible differences in the enriched GO categories were detected  and embryo implantation . The diversifying selection signals in ZW breed encompassed genes enriching very general categories of biological processes, like e.g. transcriptional repressor activity, intracellular processes or processes in nuclear lumen, however, some of the genes were connected with more specific pathways like: tryptophan metabolism  or salivary secretion . In ZS breeds, selection signals were associated with genes responsible for e.g.: neuropeptide Y receptor activity and feeding behavior , metabolic processes  and cortisol metabolic process (REST).To analyze gene content of the genome regions spanning the detected selection signals, each region was expanded by 25 kb on each end to account for potentially linked genes. This allowed for detection from 61 (ZS) to 116 (PUL) ENSEMBL genes per breed  selection signals on SSC3, SSSC7, SSC8 and SSC9 . Altogether, the regions spanned 72 different ENSEMBL genes which together did not enrich any biological processes of pathways, however, again included PPARD gene.To detect genome regions bearing variants potentially responsible for the most pronounced phenotypical differences between breeds, the pig breeds with white  and spotted  coat color patterns were compared using FC9 Table . The moslocus specific core haplotypes) consisting of minimum three and no more than 20 SNPs with average lengths ranging from 193.7 kb (PL) to 394.3 kb (ZS). For all these core haplotypes, from 37,809 (ZS) to 46,162 (PUL) EHH tests were performed – from 8.2 to 8.3 per core region, on average  to 5638 (PUL) longest non-overlapping core regions (CR – a unique genome region spanning P < 0.001) core haplotypes to 1 cM on both ends, 171 unique genes potentially being under selection were found in PL, 116 in PUL, 84 in ZW and 35 in ZS  were found in PL pigs and regulation of protein transport and localization  or muscle fiber development  in PUL pigs. In ZW breed the suggestive biological processes were involved in e.g.: metal ion homeostasis and regulation , negative regulation of innate immune response (DUSP10) and negative regulation of T cell apoptotic process (SLC46A2). In ZS breed, the most commonly represented processes included: galactosidase/galactosidase activity and lipid catabolic process .After extension of the detected significant  showed the highest genetic similarity between both Złotnicka pig breeds. Such result was expected, due to the fact that both breeds originated from the same geographic background and were created in a similar time period based on the same genetic group. The clear separation of Landrace from the Polish conserved breeds was also observed, which probably reflects its origin from German and Swedish pig breeds and its high speciation in terms of production. The applied PCA method showed the highest genetic similarity between Polish Landrace and Złotnicka White pigs which both represent white, meat-type pigs with dropping ears.In this study, by using two largely complementary methods we detected selection signatures in four pig breeds, including Landrace and three conserved native breeds representing a maternal breeding component. The native pig populations can be considered as unselected, because the conservation programs are more focused on preserving genetic diversity and breed standard than improvement of production traits. The analysis of breeds’ genetic differentiation based on a cladogram obtained with the neighbor-joining method on the averaged FST-based method, we detected large (over 1 Mb in size) selection sweeps on SSC7 (32.1–33.3 Mb) and 14 (42.2–43.7 Mb) among the top 0.1% of strongest selection signals in Polish Landrace. Several selection signals associated with these chromosomes were previously reported in other pig breeds [In the studied pig breeds, we detected several selection signals, representing genome regions that were differentially fixed in different breeds or representing within-breed selection signatures. The diversifying selection signals were detected based on the comparison of a specific breed with all other breeds under study, presuming that these signals will be characteristic for unique breed features or strongly fixed breed traits being poorly developed in the other compared breeds. This can be an explanation for the lack of selection signals being common for different breeds. With the Fg breeds , howeverg breeds , fatty ag breeds , number SC7 32.1–.3 Mb andg breeds . The aniST - based regions by additional 25 kb, which is an approximated half of mean distance between SNPs in this study. In the REHH method we extended the detected signals by 0.5 Mb in each direction. In our data, we found that core haplotype domains (their extended homozygosity) on average spans over 0.7 Mb in upstream and downstream directions from cores. We have narrowed these regions by adding 0.5 Mb to each core haplotype to account only for genes within the region of relatively high linkage with the core SNPs. Similar approach was previously proposed by Qanbari et al. [While screening detected selection signals for gene content we extended the detected regions by additional bases. Extension of the detected regions was motivated by a need to account for linkage disequilibrium and the applied sliding-window approach. While moving average smooths the data, some surrounding genes may not fall directly into the selection signals. Because of some accidental differences in allele frequencies (e.g. genetic drift), selection signal peaks can be slightly shifted in relation to real functional variant locations. To not to lose this information we extended Fi et al. . The sizST-based) selection signals detected in Polish Landrace we found several candidate genes which may be responsible for the well-developed breed characteristics like good fertility and high growth rate. One of the interesting detected genes is RASAL1 gene (RAS Protein Activator Like 1), acting as a suppressor of RAS function. The protein enhances the weak intrinsic GTPase activity of RAS proteins resulting in the inactive GDP-bound form of RAS, thereby allowing control of cellular proliferation and differentiation [HK1 (Hexokinase 1) gene which phosphorylates glucose to produce glucose-6-phosphate which is the first step in glucose metabolism pathways. Glucose metabolism is important for energy homeostasis but has also an effect on growth and meat quality traits [LC8 (LC8 dynein light chain – DYNLL1) and KHDRBS2  – related with reproduction traits. In mice, it was established that dynein regulates meiotic checkpoint during oocyte maturation [KHDRBS2 was found to be associated with the number of teats in the GWA study in Large White pigs [PRIM2 (which encodes a DNA primase – large subunit) – was previously found in Bayesian GWAS to be in a gene network associated both with the number of stillborn piglets (SB) and the number of teats (NT) [Within the strongest differential (Fntiation . A largey traits . Two othturation  and KHDRite pigs . The othats (NT) .PPARD and MAPK14 - GO:0043403) and embryo implantation  important for fertility traits. The most interesting diversifying selection candidate in this breed seems to be PPARD gene (Peroxisome proliferator-activated receptor beta/delta) located on SSC7, which was shown to be associated both with ear morphology and backfat thickness [PPARD gene that significantly reduces its transcription activity, and consequently causes enlarged external ears in pigs [Among the strongest diversifying selection signals in PUL pig breed, we detected one with length over 1 Mb on SSC7 (35.9–37.3 Mb). This region was previously described to bear several QTLs for health, meat and carcass traits and exterior . Withinhickness , 40. A p in pigs . This isTDO2, AOX1) or salivary secretion . Tryptophan (TRP), as a precursor of neurotransmitters, was shown to be involved in pale, soft, exudative (PSE) pork syndrome [In the ZW breed, large size sweeps were detected on SSC1 (230.0–232.5 Mb) and 8 (45.2–47.5 Mb). These regions were previously shown to carry mainly QTLs for meat and carcass traits (SSC1) or health (SSC8) . Withinsyndrome  and dietsyndrome . The TRPNPY1R, NPY5R), metabolic processes  and cortisol metabolic process (REST). Our interest focused especially on genes associated with neuropeptide Y (NPY) activity. NPY has been implicated in several human diseases involving fat deposition aberrations and obesity [P < 0.001) in ZS pigs (23.13 ± 3.53) than in three other analyzed breeds (in a range of 9.27 in PL to 18.03 in ZW breed) . In ZS  obesity . This ha obesity , 46 and  obesity , 48. NPY obesity  and anim obesity –52. ThesST-based comparison of white and spotted pig breeds revealed an ambiguous selection signal in SSC8 positioned in close vicinity to KIT gene – a known coat color modulator [EDNRB, KITLG or MC1R [loci affecting black spotting were also detected on SSC3 and SSC9 in exotic pig crosses [Our Fodulator . However or MC1R . Neverth or MC1R  which we crosses . This suAt least some of the detected divergent selection signals could be easily associated with genes potentially responsible for traits especially pronounced or fixed in the studied breeds and suggest the detection of genome regions connected with mechanisms underlying differentiating traits. To supplement this data with information on within-breed selection signatures, which are independent of breeds differentiation, we additionally used REHH statistics , which wP-value lower than 0.001. After extending them to 1 cM on both sides, we detected from 35 (ZS) to 171 (PL) unique genes per breed potentially being under positive selection. Several of those genes are connected with important production features like growth and fertility. Exemplarily, in PL pigs we identified 10 genes belonging to the Hox family (HOX1 to HOX9 and HOX13), which are key factors in the implantation and development of the embryo and regulate the function of the endometrium [STAT5 – responsible for the signal transduction from the estrogen receptor. The STAT5 gene itself is broadly tested for association with fertility and milk production traits [CXCL12 gene (C-X-C Motif Chemokine Ligand 12) which as a ligand for the G-protein coupled receptor plays an important role in many biological processes including embryogenesis [CXCL12 gene was identified as one of differentially expressed genes up-regulated in endometrium at 18th day of pregnancy, which can indicate its potential role in embryogenesis [To analyze in details the genomic regions with the strongest signs of selection, we took a closer look at regions bearing haplotypes with ometrium –62. In Pn traits –65. The n traits . A gene n traits  and reprn traits  was alsoogenesis . In pigsogenesis .DHCR7, CLPS, PNPLA1, PPARD, BPIFB5, CLPSL2, BPIFB1) which may be associated with former selection of this breed toward reduced fat content in carcass. We also detected genes connected with immune system processes related to resistance to bacterial  and virus  diseases, which fits to the characteristics of this native breed which is known to have a good disease resistance and adaptation to harsh environmental conditions. In Złotnicka White pigs, the strongest within-breed selection signals encompassed genes associated with inter alia the immune system processes and adaptation to environmental conditions or stress response. The first group of genes included those connected with inflammation mediated by chemokine and cytokine: GNG10, PRKCE, SOCS5 or inflammatory mediator regulation of TRP channels – MAPK10 (mitogen-activated protein kinase 10) and PRKCE (protein kinase C epsilon), which may contribute to the good health and disease resistance observed in this breed. The second group included MCFD2 gene engaged in response to a stimulus and other genes responsible for response to stress (GO:00069 50)/stress-activated MAPK cascade, like: MARC1, ERCC4, DUSP10, MAPK10.In the regions overlapping with the strongest within-breed selection signals in PUL breed as many as seven genes involved in lipid metabolism were detected (LOC102167689), membrane lipid catabolism or glucosidase/galactosidase activity (LOC102167689) which as metabolic processes are often associated with selection signals in pigs [SCLY gene encoding isoform X1 of selenocysteine lyase associated with selenium metabolism and viral mRNA translation seems to be interesting because of the fact that it was established that SCLY selenocysteine plays a key role in lipid metabolism and can be related to selenium concentration in meat [In Złotnicka Spotted breed, some predicted genes involved in lipid and carbohydrates metabolism were detected and were engaged in processes connected with: sphingolipid catabolism  and allowed us to indicate genes connected e.g. with fertility, growth and metabolism which are potentially responsible for phenotypes of the studied breeds. These selection signals also comprised PPARD gene that contributes to the shape of external ears in pigs and genes encoding neuropeptide Y receptors involved in fat deposition and stress response which are important features differentiating the studied breeds. REHH statistics pointed several within-breed selection signals overlapping with genes connected with a broad range of functions, including e.g.: metabolic pathways, immune system response or implantation and development of the embryo. The study provides many potential candidate genes responsible for traits selected in the individual breeds and gives a strong basis for further studies aimed at closing the gap between the genotype and phenotype of the studied pig breeds.Summarizing, in this study we used two different but largely complementary statistical approaches, REHH and pairwise FAdditional file 1:Breed characteristics. (DOCX 12 kb)Additional file 2:ST values (di) for all studied breeds averaged within ten-SNP sliding windows. (XLSX 4722 kb)Standardized FAdditional file 3:Genes detected within the strongest diversifying selection signals detected in the studied pig breeds. (XLSX 71 kb)Additional file 4:Summary characteristics of core regions (CR) or core haplotypes (CH) in the studied pig breeds. (XLSX 13 kb)Additional file 5:P-values for the detected core haplotypes with the highest frequency. (XLSX 730 kb)REHH Additional file 6:Genes detected within the strongest within-breed selection signals detected in the studied pig breeds. (XLSX 99 kb)Additional file 7:Comparison of selected phenotypic traits for the studied pig breeds. (DOCX 19 kb)


Contrary to the initial hypothesis, dual targeting worsened the neuroinflammatory response to intracortical probes. Therefore, probe material and CD14 deficiency were independently assessed for their effect on inflammation and neuronal density by implanting each microelectrode type in both wild-type control and -/-Cd14 mice. Histology results show that 2 weeks after implantation, targeting CD14 results in higher neuronal density and decreased glial scar around the probe, whereas the thiol-ene probe results in more microglia/macrophage activation and greater blood–brain barrier (BBB) disruption around the probe. Chronic histology demonstrate no differences in the inflammatory response at 16 weeks. Over acute time points, results also suggest immunomodulatory approaches such as targeting CD14 can be utilized to decrease inflammation to intracortical microelectrodes. The results obtained in the current study highlight the importance of not only probe material, but probe size, in regard to neuroinflammation.Intracortical microelectrodes record neuronal activity of individual neurons within the brain, which can be used to bridge communication between the biological system and computer hardware for both research and rehabilitation purposes. However, long-term consistent neural recordings are difficult to achieve, in large part due to the neuroinflammatory tissue response to the microelectrodes. Prior studies have identified many factors that may contribute to the neuroinflammatory response to intracortical microelectrodes. Unfortunately, each proposed mechanism for the prolonged neuroinflammatory response has been investigated independently, while it is clear that mechanisms can overlap and be difficult to isolate. Therefore, we aimed to determine whether the dual targeting of the innate immune response by inhibiting innate immunity pathways associated with cluster of differentiation 14 (CD14), and the mechanical mismatch could improve the neuroinflammatory response to intracortical microelectrodes. A thiol-ene probe that softens on contact with the physiological environment was used to reduce mechanical mismatch. The thiol-ene probe was both softer and larger in size than the uncoated silicon control probe.  Thus, intracortical microelectrodes are a promising technology for both basic research and the development of clinical neuroprosthetic devices.Intracortical microelectrodes allow researchers to record single-unit and multi-unit activity from individual or groups of neurons by detecting changes to the extracellular potential as a result of neurons generating action potentials . RecordeFor both clinical and research applications, intracortical microelectrodes must be able to record from single cortical neurons for long periods (months to years). Unfortunately, there are limitations to intracortical microelectrodes that impede device reliability. Many studies document the failure of intracortical microelectrodes exemplified by both decrease of signal to noise ratio and loss of number of channels detecting single units . There aInflammation ensues after the device damages tissue during implantation when blood vessels are unavoidably severed leading to blood infiltration and serum protein adsorption onto the device. Implantation results in the release of endogenous damage signals such as high mobility growth box 1 (HMGB1) and inflammatory lipids from damaged cells . Plasma In addition to the primary injurious events caused by the initial implantation, a persistent inflammatory response is present at the probe–tissue interface under chronic conditions. The pro-inflammatory microenvironment resulting from probe implantation leads to further breakdown of the blood–brain barrier (BBB) and increased vascular permeability perpetuating the inflammatory cascade .E = ∼3–6 kPa  and the brain can exacerbate inflammation . The mec∼3–6 kPa ) reduces∼3–6 kPa . As a re∼3–6 kPa .Increased BBB permeability can facilitate the infiltration of myeloid cells into the injured brain tissue. These peripheral immune cells become activated and perpetuate the inflammatory response leading to neuronal death by the probe–tissue interface . To comb-/-Cd14 mice, to reduce neuroinflammation in response to single-shank intracortical microelectrode probes.Our initial softening probes used in the Capadona Lab yielded desirable mitigation of the neuroinflammatory response to intracortical microelectrodes . However-/-Cd14 knock-out model was used to target the innate immune response while immunohistochemistry was used to evaluate the neuroinflammatory response. A neuronal nuclear protein, NeuN, was used as an immunohistochemical marker for cortical neurons around the probe . Additionally, neuronal density surrounding softening (thiol-ene) probes in e Figure . There we Figure . Howevere Figure . Altoget-/-Cd14, thiol-ene + WT, thiol-ene + -/-Cd14). Additional animals were also set up to examine more chronic (16 weeks post-implantation) time points for each of the four experimental conditions. Comparisons were made between levels of each independent variable – responses of softening thiol-ene probes versus stiff silicon probes and comparisons between -/-Cd14 and wild-type animals.To elucidate if either factor (probe stiffness or CD14 expression) drove the increased neuroinflammatory response of the combinatorial targeting, additional animals were set up as controls for each factor resulting in four different conditions  as a function of distance from the interface  was found to be greatest at the probe-tissue interface and decreased in intensity as distance from probe-tissue interface increased Figure . The onlThe current study explores how a softening thiol-ene probe and/or targeting the TLR/CD14 innate immune pathway affects inflammation and neuronal density at both 2 and 16 weeks post-implantation of intracortical microelectrode probes. Because initial results exploring synergistic effects of two different approaches resulted in increased neuroinflammation, we set out to parse out the response of each variable to gain a better understanding of each strategy alone. Our results demonstrate targeting CD14 results in higher neuronal density and decreased astroglial scarring around the probe at 2 weeks Figures , 3A,C. WPhysiological sources such as respiration and vascular pulsations result in micromotion of an intracortical probe against brain tissue resulting in strain on brain tissue which can induce tissue damage . One of Materials which are innately stiff enough to implant, but soften while residing in tissue minimize the modulus difference between probe and brain tissue. Previous literature has suggested that a softening material can compensate for increased damage footprint generated by a device with a larger cross-sectional area compared to a stiff silicon control probe . However2, which is a 4× increase over the silicon probes utilized. The bending stiffness of the probe is determined by both the Young’s modulus (E) and the probe dimensions. Although modulus of the probe material affects flexibility of the device, the probe dimensions play more of a role in reducing stiffness; bending stiffness is proportional to Et3, where t is the cross-sectional area of the probe  results in less vascular damage and tissue strain, there are other reports indicating that slower insertion speeds afford time for tissue to adapt to probe thus decreasing compressive forces  is greater than that of the uncoated silicon probe taper (0.6 mm). However, the angle of their tips are similar, at 41.6° and 47°, respectively. As such, the differences in the angle between the tips are not expected to contribute significantly to vascular damage and hence tissue response; it is unlikely to affect our study. It also should be noted that there are conflicting reports in the literature to the benefits/disadvantages of different insertion speeds. Although e forces .Together, the results found in the current study suggest that large cross-sectional area probes comprised of a minimally swelling, yet softening material cannot overcome inflammation driven by large penetration profile differences. Accordingly, the differences in the size of the electrodes is a major limitation of our study. Overall, the results presented here highlight the detriment of only considering one or two aspects of probe design and mitigation of biological response. When approaching translatable strategies to improve chronic intracortical microelectrode performance by decreasing inflammation, one needs to consider many characteristics in tandem.The current manuscript demonstrates the impact of size of the probe on the initial stages of inflammation. To reduce inflammation, the cross-sectional area of the probe should be minimized. The current study also characterizes the acute benefits of targeting CD14 and further confirms the TLR/CD14 pathway as a mechanism amenable for therapeutic targeting. In the initial weeks after probe implantation, the benefits a minimally swelling soft probe affords does not exceed the inflammation driven by a probe with 4× the cross-sectional area. When optimizing probe design for intracortical microelectrodes, many elements of the probe need to be carefully considered, especially size.6 and O2 plasma, respectively. After the 30-μm SMP layer was plasma etched down to the glass slide, the remaining silicon nitride hard mask was etched away in diluted 10:1 HF dip. A ∼3 μm SMP layer from the surface of these devices was etched in Technics RIE using O2 plasma. The devices were then released by soaking in DIW. The material is characterized by a glass transition temperature (Tg) of 52°C before, and 35 °C after softening under physiological conditions as measured by dynamic mechanical analysis (TA RSA-G2). The storage modulus E′ at 37°C (measured in tension) changes from 1.2 GPa (dry) to 35 MPa (soaked). Stainless-steel wires (∼3 mm length) were used as dummy ground and reference wires to mimic the implants involved with functional probes. Probes and wires were sterilized via a cold ethylene oxide gas cycle as previously described.Single shank, uncoated, Michigan style silicon probes  and thiol-ene based shape memory polymer (SMP) probes  were used as intracortical probes Figure . Briefly-/-Cd14 (C57/BL6 background) (strain #003726) were bred in-house. Strain of -/-Cd14 mice was verified via genotyping according to the protocols established by the vendor (Jackson Laboratories). Both male and female mice between 8 and 12 weeks of age were used for surgeries. Prior to surgery, mice were group housed with food and water ad libitium while maintained on a 12-h light/dark cycle. After surgery, mice were individually housed. All animal handling was performed in a class II sterile hood using microisolator techniques. All procedures and animal care practices comply with the protocol approved by the Case Western Reserve University Institutional Animal Care and Use Committee.C57/BL6 (strain #000664) and A total of three holes were drilled in the exposed skull using a 0.45 mm size bit (Stoetling Co.) with adequate breaks in the drilling pulses to prevent overheating of the skull . The proAt each 2 and 16 weeks post-implantation, mice were sacrificed and brain tissue was harvested. At the respective time point, mice were anesthetized with an intraperitoneal injection of ketamine/xylazine cocktail. Each animal was then transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) to fix the tissue. Mouse heads were post-fixed for an additional 2 days in 4% PFA at 4°C. After fixation, brains were extracted and immersed in 30% sucrose for at least 48 h. After dummy electrodes and wires were removed, brain tissue was cryopreserved in optimal cutting temperature compound (OCT) (Tissue-Tek). Horizontal brain tissue sections (16 μm thick) were obtained using a cryostat and stored at -80°C.® secondary antibodies (1:1000) . DAPI (Molecular Probes D3571) was incorporated in secondary antibody solution to visualize cell nuclei. Furthermore, tissue autofluorescence was reduced by incubating tissue sections with of 0.5 mM copper sulfate buffer solution for 10 min  inverted epifluorescence microscope. Fluorescent markers were imaged on single optical sections using an AxioCam MRm monochrome camera with fixed exposure times for each marker.Images of fluorescent markers were analyzed using SECOND, a custom-written MATLAB program previously used in the Capadona lab . The fluNeuronal densities at the interface were determined using AfterNeuN, another custom-written MATLAB program. Using AfterNeuN, the user manually defines the electrode implant region, any areas to be excluded from analysis, and neuronal cell bodies. The program then outputs the density of neurons at fixed radial distances from the electrode interface. Neuronal densities at uniform binned distances (50 μm bins) were then normalized to background counts from the same brain tissue slice 300–350 μm away from the interface.-/-Cd14 to silicon shank + WT were performed using unpaired t-tests. All statistical analyses assessing immunohistochemical results comparing all four conditions were performed using a general linear model with a two-way analysis of variance (ANOVA) using Minitab software with genotype (WT or -/-Cd14) and electrode material (uncoated silicon or thiol-ene) as separate factors. Results were considered significant at p < 0.05 and expressed as mean ± standard error of mean.Table HB, WV, JP, and JC contributed substantially to the conception or design of the work, analysis, and interpretation of data for the work, drafting, and revising the manuscript for important intellectual content, approved the final version to be published, and agreed to be accountable for all aspects of the work. HB, SS, XL, EM, AJS, and SL aided in the collection and analysis of histological data. AS, ME, and VD fabricated and characterized the SMP probes. All authors approved the final version to be published and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.