The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM  and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini  ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA  and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.The best results were obtained when staining 8 μm-thick sections with Cresyl violetRNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery. One of the main challenges biologists currently face is overcoming the problem of tissue heterogeneity to further understand organ function. It is crucial to distinguish which cell populations produce specific molecules or to get relevant expression profiles reflecting in vivo status.Milk is synthesized in mammary gland during lactation and though this process has been thoroughly studied, we still do not know precisely what mechanisms are involved in the intracellular transport and secretion of milk components, including supra-molecular structures, such as casein micelles ,2 which Mammary parenchyma consists of secretory alveoli organized into lobules and interconnected by a system of branching ducts separated from adipocytes by multiple layers of fibroblastic connective tissue. In the duct and alveoli, the mammary epithelium is organized into two layers, a basal layer of myoepithelial cells (MMC) and a luminal layer of MEC that secretes milk . The extet al. [Advances in molecular biology have provided new tools, including gene expression profiling, to analyze mechanisms controlling mammary gland development and differentiation ,7 and reet al. . On the et al. -11, but et al. ,13. Analet al. ,15.Therefore, to accurately and reliably follow molecular changes occurring in MEC for comparison purposes between physiologically different stages and genetically or environmentally perturbed systems, it is necessary to isolate MEC preserving biomolecule (RNA and proteins) integrity.Different techniques, such as immunomagnetic separation -19, cellin vitro. In addition, it is well-established that MEC in culture are subjected to dedifferentiation [Another means to obtain MEC homogeneous populations is from cell culture. However, one major obstacle to molecular biological studies of MEC is the lack of established cell lines that secrete, or can be induced to secrete, fat globules and milk proteins . While cntiation .et al. [Laser Capture Microdissection (LCM), first described by Emmert-Buck et al.  is now wNevertheless, LCM is an appealing technique, but it introduces additional methodological hurdles, including tissue handling  and maintenance of molecular integrity. The success of a microdissection experiment first depends upon the ability to distinguish cell types of interest from their morphological features. Immunological labelling may be required and used to assist in the identification of cells. In other words, if gene expression experiments are targeted, the challenge is to design a global protocol ensuring acceptable tissue morphology to facilitate isolation of cells while preserving accessibility and integrity of RNA, keeping in mind that this is critically tissue-dependent.Successful application of LCM in transcriptomic analyses relies upon three critical factors: good tissue morphology, capture efficiency, and maintenance of RNA molecular integrity. Effective balancing of these three factors is required to recognize regions and obtain reliable transcriptomic results. Since ruminant mammary gland is one of the richest tissues in RNAse activity, classical protocols require accommodation to preserve RNA from RNase (endogenous and exogenous) and keep it intact in captured cells. This study was carried out to address these issues with the aim of developing a convenient and reproducible protocol to isolate MECs from ruminants  lactating mammary gland, preserving tissue morphology and RNA integrity, to develop a comprehensive overview of the genome expressed in MECs in their physiological environment.Given that recent studies -38 repor® (TissuTek™) in a cryomold of 1 cm3 (Bayer™) and immediately immersed in liquid nitrogen or in SnapFrost™ system  containing cold isopentane at -80°C. Alternatively, some pieces were directly introduced in empty 1.5 ml cryotubes and immediately frozen in the same way (liquid nitrogen or SnapFrost™ system). Samples were stored at -80°C until further processing. The time delay between slaughtering and tissue freezing was less than 20 minutes.Surgical and experimental procedures were performed in compliance with the policies of INRA's Animal Care Committee. Mammary tissue was sampled from 5 goats, 2 ewes and 1 cow euthanized under safe and painless conditions, at the middle of lactation after milking and slaughtering. To preserve morphology and RNA quality we applied two different methods of freezing (liquid nitrogen or cold isopentane) immediately after collection with and without embedding medium as follows: the collected tissue was washed in cold PBS solution, 3-5 mm pieces of tissue were cut 3 and embedded in OCT®) and rinsed in three baths of distilled water before a final bath in 70% ethanol. To test whether the effect of slide temperature plays a key role in detachment of MEC from glass slides during the laser-capture process, pre-cleaned slides were chilled (on ice or at 4°C) or not (room temperature) before transfer. Manufacturers' and published protocols recommend cutting 5 to 12 μm section thicknesses. Tissue sections were 8 μm thick, a compromise to ensure an optimal RNA yield preserving morphology as well as dehydration and laser-capture process efficiency.Frozen tissue blocks were mounted on the cryostat stage  set at -20°C. Before transfer, the working environment was treated to be RNAse free and glass slides  were pre-cleaned with RNAse Zap™  or Cresyl violet® . Different protocols for dehydration and staining of frozen mammary sections were compared  after section transfer, two different methods of cold storage were tested: after cutting, the slides were immersed in cold 75% ethanol and placed at -20°C or put directly into a tube containing desiccant and stored at -80°C. To avoid RNA degradation, each step of the slide preparation process was performed as quickly as possible. Water was RNAse free. Bottles of absolute ethanol  and M-Xylene, anhydrous, >99%  were opened just before use to dehydrate a maximum number of 8 slices a day to ensure proper dehydration. Sections were stained using Histogeneed Table . They we® staining. Two chemical protectors were used: RNA later®  or RCL2® , a new fixative which preserves morphology and nucleic acid integrity. In protocols N°3 to 5, 100 μl of RCL2® or RNA later® were dropped on the tissue section just before staining, to react for 30 s and removed by tapping the slide on an absorbent paper. RNase out  or RNAsin (Promega) were prepared  and added on slice for 15 s during staining step.To preserve the quality of RNA during staining, without ruining the morphology, we tried to add RNA protectors  used commonly to block RNAse activity, before HistoGene®). Capture, which is the gentlest technique and thus maximizing biomolecules integrity, was performed under 40× or 100× magnifications using CapSure LCM macrocaps . IR Laser setting was chosen to maximize the size of the laser spot in the middle of alveoli  without contaminating the sample with non-target-tissue (MMC or interlobular stroma). Laser setting ranged between 75 to 90 mW in power, 1,300 to 3,500 μsec in duration, and 200 mV in intensity. Efficiency of microdissection was evaluated by examining the cap after capture and the tissue section remaining on the slide before and after lifting off the cap: if necessary, the non target tissue can be removed directly on the cap by lower power UV laser (2-5 mW).The LCM process was carried out using the Veritas Microdissection Arcturus system and software (Applied Biosystems2 (around 150 to 200 acini).The critical time limit for capture was estimated by examining RNA integrity and was evaluated from 30 minutes to 2 hours. The corresponding target area was between 300,000 and 600,000 μm® RNA Isolation Kit  according to the manufacturer's instruction protocol, including on-column RNase-free DNase I treatment . CapSure macrocaps with captured cells were inserted into RNase-free 500 μl microcentrifuge tube containing 25 μl of extraction buffer (XB). The tubes were inverted to allow the reaction between the buffer and the surface of the cap. RNAs were extracted from scraped sections (tissue remaining on the slide after capture) by pipetting 50 μl of XB buffer onto the remaining tissue on the glass slide and gently scraped off and transferred in RNase-free 500 μl microcentrifuge tube. RNAs from cap and section scrapes were eluted respectively with 15 μl and 30 μl of elution buffer (EB). To assess RNA quality of tissue before manipulation, one cryo-section of mammary tissue was immediately treated to extract RNA using the same protocol as that for section scrapes after LCM.Total RNA was extracted from captured cells using the PicoPurePurity, concentration and integrity of total RNA isolated in this manner were assessed using two independent techniques. RNA purity was evaluated by absorbance readings (Ratio A260/A230 and A260/A280) using the NanoDrop ND-1000 spectrophotometer . The fluorimetric method and micro-capillary electrophoresis device developed by Agilent Technologies was chosen to determine RNA concentration and quality with RNA 6000 pico LabChip Kit in the Agilent Bioanalyzer 2100 system. Quality was evaluated using the RNA Integrity Number (RIN) value introduced by Agilent .20 and random primers  using Superscript III reverse transcriptase  according to manufacturer's instructions. Then, 1 μl of RNase H  was added and incubated 20 min at 37°C to remove RNA. The obtained cDNA was stored at -20°C before qPCR.First-strand cDNA was synthesized from 5-10 ng total RNA primed with oligo(dT)CSN1S2 and LALBA transcripts, two specific markers of MEC, were quantified using qPCR (SYBR Green chemistry). Two internal control genes, S24 ribosomal protein (RPS24) and cyclophylin (PPIA) were quantified for accurate normalization of data [To estimate MEC enrichment obtained after LCM,  of data .Krt14), lymphocytes (CD3e) and macrophages (CD68). We also quantified transcripts from Fatty Acid Synthase (FASN) which is expressed in several cell types including MEC and adipocytes.Primers used were previously described ,40 and q®). Primers were designed and purchased from Eurofins Genomics (France) to amplify goat, sheep and cow genes (Table ®). First, primers efficiency was validated with a standard curve of four serial dilution points of a scraped section cDNA pool , and a no template control (NTC). qPCR amplification mixture (20 μL) contained 5 μL single strand cDNA template diluted 4 times after reverse-transcription, 10 μL 2× Power SYBR Green PCR Master Mix buffer (Applied Biosystems®) and 1.2 μL forward and reverse primers (5 μM) to reach a final primer concentration of 300 nM. After optimization of qPCR systems (efficiency -3.32 to -3.4), we used the ΔΔCt method and RQ Manager Software , as well as in the relative expression software tool , to compare expression of each gene between captured cells and its scraped tissue, following the MIQE guidelines [Comparing the structural organization of encoding genes across species  and mRNA sequences at exon-exon junctions, we identified highly conserved regions on which primer pairs were designed, using Primer Express Software, version 2.0 (Applied Biosystemsidelines .RPS24 and PPIA) was evaluated with GeNorm Visual Basic application for Microsoft Excel as described by [Reliability of reference genes , based on Pair Wise Fixed Reallocation Randomized Test© [The relative expression for each gene of interest between caps versus scraped tissues was tested for significance by a randomized test implemented in the relative expression software tool  on cell isolation and RNA yield and integrity.ca. 150 slides of tissue sections were cut from 8 mammary glands taken on three different ruminant species: goat, sheep and cow. The impact of the different steps of slide preparation was evaluated and we observed that the early steps, mainly sampling, freezing, cutting and staining of tissue, play a crucial role for a consistent success in capture of alveoli MEC from mammary sections.In this study, ® using two different frozen conditions, liquid nitrogen and cold isopentane, are shown in Figure ®/tissue/cryomold in liquid nitrogen results in loss of morphological details  obtained with or without OCTs Figure  and somes Figure . Rapid f® in liquid nitrogen  and RNA quantity. Usually, with goat and cow samples, we get 5 to 10 ng of RNA per cap . In other words, there are 3 picog RNA in 1/3 cell since we work on 8-μm tissue section thickness, and therefore one can estimate to ca. 10 picog the amount of RNA contained in one MEC. However with sheep, the amount of material we obtained was between 1.5 and 2 times higher.It is difficult to evaluate the exact number of MEC isolated after 40 to 60 min of LCM since this it depends on the number of acini present on the slide and on the MEC per acini (10 to >40). Given that the Veritas Arcturus system allows quantification of captured material via a tool estimating the total area selected before capture, we can establish a correlation between captured area  and different times of dehydration. HistoGene® stain is a special solution developed by Arcturus to stain tissues for LCM subsequently used as sources of RNA. It is a fast penetrating stain that provides good contrast by differential staining of nuclei (purple) and cytoplasm (light pink). Cresyl Violet® is a hydrophilic, basic stain that binds to negatively charged nucleic acids without water step during slide preparation to re-hydrate the tissue.The next step was to evaluate the impact of fixation, staining and dehydration on RNA integrity and yield as well as on tissue morphology. Previous studies ,38,44 ha® following the protocol recommended by Arcturus, (N°1) which easily distinguished the cytoplasm (stained in brown) and the nucleus , whereas neutral pH of Cresyl violet® solution (pH measured ~7) avoids degradation of RNA. After substitution of HistoGene® by RNase-free water (protocol N° 8), RNA degradation was reduced by less than one RIN unit. However, it was obviously not possible to recognize cells on unstained tissue sections. Given these results we decided to use the Cresyl violet® as stain for further experiments.Results are given in Table et al. [et al. have proposed to perform LCM under an argon atmosphere, thus preventing tissue rehydration to finally stabilize RNA [Dehydration is crucial to stabilize the tissue and to allow capture. All the steps following dehydration  are time consuming and it is necessary to keep tissues dehydrated to avoid degradation of RNA. A high ambient humidity could rehydrate the tissue and reactivate endogenous RNases. In addition, moisture impedes film adhesion and cell capture. For these reasons, we limit LCM to 45-60 minutes which proved to be a good compromise to overcome the variations in relative humidity. Ordway lize RNA . These a® (protocol N° 3) or RCL2®, are promising new noncrosslinking fixatives [® staining and could improve RNA quality. Actually, RNA degradation was especially reduced with RNA later® (loss of one RIN unit) whereas with RCL2® we observed a loss of 1.5 to 2.5 RIN units. However, RNA later® provokes a loss in morphology  such as RNA laterixatives , preservRNasin , a potent inhibitor of ribonucleases, was also tested prior to staining since such inhibitory activity was reported to protect RNA (included in staining solution) from degradation in LCM experiments for gene expression profiling of basal cell cancer tissues ,48. We dNevertheless, there was no considerable improvement in either quality or quantity of RNA recovered from the tissue sections after an inhibitor treatment. In conclusion, we finally decided to opt for protocol N° 7 (without any RNA protector) which is easier to handle and quicker to perform.qPCR experiments were performed on reverse transcribed RNA extracted from microdissected cells targeting specific gene transcripts to evaluate enrichment in MEC.Krt14) and MEC (LALBA and CSN1S2) was assessed after normalization, using PPAI and/or RPS24 as reference genes. FASN, a gene expressed in a large panel of tissues [LALBA (RQ mean = 1.47), CSN1S2 (RQ mean = 1.33) were significantly increased. FASN was relatively unchanged (RQ mean = 0.85), suggesting that active fatty acid synthesis which is required for energy utilization and membrane synthesis is equally expressed in MEC and in the surrounding tissue. These results attest to the enrichment in MEC after LCM even though increasing in LALBA and CSN1S2 transcripts are less striking, given the high percentage of MEC (around 80-90%) and the low number of other cell types in lactating mammary parenchyma sections.To determine potential cell contamination of the laser-captured cells by adjacent MMC or to estimate the selective capture of MEC, mRNA transcript levels of cell-specific markers were assessed by evaluating the relative expression between captured cells and their corresponding mammary tissue scrapes, after LCM. Relative quantity with specific markers for MMC  in the captured cells compared with the whole mammary tissue. A weak expression of Krt-14 was systematically observed in microdissected MEC, reflecting a slight contamination by MMC during capture. This is due to a very close proximity between MMC and MEC [CD3e (RQ mean = 0.14), a marker of lymphocytes and CD68 (RQ mean = 0.18) which suggests the putative presence of macrophages  to use to perform relative gene expression measurements made by qPCR? We chose to use isolated cells (each cap) against all scrape sections since it can be considered as a mean value of serial tissue sections.Typically, all these parameters have to be considered and the first one is usually accomplished through the use of endogenous housekeeping genes that are presumed to show stable expression levels in the samples under study. Which specific genes and how they can be measured in limited amounts of mRNA such as those extracted from microdissected cells still remains a concern. GeNorm software confirmed that LCM makes it possible to obtain highly MEC enriched material from lactating mammary tissue sections preserving RNA integrity. In addition, we provide molecular evidence for successful selectivity of the capture method despite the difficulty of disassociating luminal secretory cells (MEC) from MMC bordering the basal lamina which separates the epithelial layer from the extracellular matrix.FASN, a gene encoding the fatty acid synthase which is widely expressed in many tissues and cell types, including the alveolar secretory epithelium and adipose tissue. Relative proportions of these tissues, both involved in lipid metabolism, dramatically change during pregnancy.To accurately evaluate the expression of genes specifically expressed in MEC, such as those encoding LALBA and CSN1S2 represents a step forward in determining the transcriptional profile of a cell type and how it can be modulated by environmental factors such as feeding, stress, milking frequency, the health status or gene polymorphisms. In addition, to understand how genes expressed in several cell types are regulated, it is crucial to work on pure or at least enriched cell populations. Otherwise, expression analyses could potentially lead to artefactual results. This is well-exemplified by Capturing other cell types or stroma provides the opportunity to examine further to understand the mechanisms involved in growth regulation and morphogenesis of the mammary gland. Until now, most attention has been paid to the luminal epithelial cell which is the functionally active milk-producing cell and the most likely target cell for carcinogenesis. However attention on myoepithelial cells has begun to evolve with the recognition that these cells play an active part in branching morphogenesis and tumor suppression . HoweverqPCR: real time quantitative Polymerase Chain Reaction; MEC: Mammary Epithelials Cell; MMC: Mammary Myoepithelial Cell; RIN: RNA Integrity Number; RQ: Relative Quantification; RPS24: Ribosomal Protein S24; PPIA: cyclophylin A; Krt14: Cytokeratin 14; FASN: Fatty Acid Synthase; CSN1S2: αs2-casein; LALBA: α-lactalbumin;CB participated in study design, performed the LCM, RNA extraction and qPCR experiments, including statistical analyses, and drafted the manuscript. SM helped to perform the LCM and RNA extraction. JCH helped to perform qPCR and contributed to design qPCR systems. PD contributed to LCM protocol optimization. PM conceived the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Sarcophilus harrisii) are on the verge of extinction due to a transmissible cancer, devil facial tumour disease (DFTD). This tumour is an allograft that is transmitted between individuals without immune recognition of the tumour cells. The mechanism to explain this lack of immune recognition and acceptance is not well understood. It has been hypothesized that lack of genetic diversity at the Major Histocompatibility Complex (MHC) allowed the tumour cells to grow in genetically similar hosts without evoking an immune response to alloantigens. We conducted mixed lymphocyte reactions and skin grafts to measure functional MHC diversity in the Tasmanian devil population. The limited MHC diversity was sufficient to produce measurable mixed lymphocyte reactions. There was a wide range of responses, from low or no reaction to relatively strong responses. The highest responses occurred when lymphocytes from devils from the east of Tasmania were mixed with lymphocytes from devils from the west of Tasmania. All of the five successful skin allografts were rejected within 14 days after surgery, even though little or no MHC I and II mismatches were found. Extensive T-cell infiltration characterised the immune rejection. We conclude that Tasmanian devils are capable of allogeneic rejection. Consequently, a lack of functional allorecognition mechanisms in the devil population does not explain the transmission of a contagious cancer.Tasmanian devils ( Sarcophilus harrisii) Devil facial tumour disease (DFTD) is a transmissible Schwann cell cancer of Tasmanian devils  A predictive Acynonyx jubatus). Captive cheetahs were unable to quickly recognize alloantigens as foreign and reject the skin grafts Skin grafting between unrelated individuals has been performed to test MHC disparity in a wild mammal, the cheetah  were performed with the animals under general anaesthesia and all efforts were made to minimise pain or discomfort.All experiments describing the use of animals were undertaken with approval and under inspection of the Animal Ethics Committee of University of Tasmania, permit numbers A9491 and A11052. Captive devils were housed in groups of two or three in 100 m6 cells/mL in RPMI 1640 incomplete medium (CSL Limited 05182301) supplemented with 10% pooled devil plasma, 2 mM of glutamine (Sigma G7513) and 15 mg of gentamicin (Pfizer 61022010).Fifteen Tasmanian devils from five areas in eastern Tasmania  and nine devils from four regions in western Tasmania  were used. 2 for 144 hours. Mitogen controls were incubated under the same conditions for 96 hours. Eighteen hours prior the harvesting, cultures were pulsed with 1 µCi tritiated thymidine (Amersham Pharmacia Biotech TRA 310) to assess proliferation. Cells were harvested onto a filter paper and dried overnight at room temperature. Uptake of thymidine by proliferating cells was measured by radiation emittance using a scintillation counter . Results were obtained in counts per minute (CPM) and stimulation indices (SI) were calculated with the formula: SI = Average CPM of mixed culture/Average CPM of control cultures. Mitogen stimulated culture controls were calculated by dividing the average of incorporated thymidine in stimulated cultures by non-stimulated cultures. All SI controls were considered to be one in the MLR experiments, as no proliferation was characterised in autologous cultures. All experiments were conducted in triplicates. MLR results were considered strong when SI >10, moderate when 5<SI<10 and low or no reaction when SI<5. To compare differences in MLR stimulation indices between groups and between ‘eastern versus eastern’, ‘western versus western’ and ‘eastern versus western’, results were log transformed and analysed with nonparametric Mann Whitney test with non-Gaussian distribution.MLR experiments were conducted in U-bottomed 96 well plates (Iwaki 3870-096), mixing 100 µL of a mononuclear cell suspension from one devil with 100 µL of a mononuclear cell suspension from another devil. Background controls consisted of 200 µL of mononuclear cells from individual devils incubated alone and positive controls consisted of mononuclear cells of individual devils incubated with 50 µg/mL of Concanavalin A . Mixed lymphocyte cultures were incubated at 37°C and 5% COSeven unrelated captive devils of eastern Tasmania were used for two-way skin grafting experiments . All devils were three year old females, except TD 187, which was a three year old male. A square (3×8 cm) was drawn on the skin of the mid-dorsal region of each devil and a dermatome knife with a sterile blade was used to ‘shave’ 1 mm thick dermis and epidermis of the demarcated skin. This loose piece of skin was divided into approximately two equal pieces (approximately 3×4 cm) and one of them slid in to the cranial wound (autograft). The other piece  was placed in sterile Hartmann's solution, until the same procedure had been performed on the other devil. The allograft skin pieces were then placed in the caudal position of the wound. Surgical glue was placed on the borders of the grafts to secure the skin. A sterile dressing was applied to the surgical wound and the thoracic and chest areas were covered with cotton wool and elastic bandages under firm pressure. Bandages were changed on Day 7 and removed on Day 14. Pain relief medication (meloxicam 0.05 mg/Kg) was administered in the food for three to five days after skin graft surgery.On Days 7, 14 and 21 the grafts were visually inspected for signs of rejection. The grafts were photographed and a punch biopsy (3–4 mm) was taken from the grafts and fixed in 10% buffered formalin solution for one to four weeks. After fixation, skin biopsy punches were processed and paraffin embedded. Four to six sections (3 µm thickness) were cut onto 3-aminotriethoxysilane (Sigma A7222) coated slides for haematoxylin and eosin staining and the same number of sections was prepared for immunohistochemistry.Skin biopsies were labelled for T-cells with a rabbit anti-human CD3 antibody (Dako A0452). Isotype controls included non-specific rabbit immunoglobulin fraction (Dako X0903) and negative controls were labelled without the primary antibody . Sections of devil lymph nodes were used as positive controls.2O2 for 15 minutes and non-specific protein binding was blocked with serum-free protein block solution (Dako X0909) for 30 minutes. The antibody was diluted (1∶800) with antibody diluent and placed onto the slides for 60 minutes. Antibody binding was detected by placing a biotinylated link universal followed by streptavidin and horseradish peroxidase (30 minutes each) . A solution of diaminobenzidine (Dako K3466) was placed onto the slides for ten minutes to allow for brown colour development of positive cells and then washed with distilled water. Sections were briefly counterstained with haematoxylin for 40 seconds, dehydrated through graded alcohol solutions to xylene and coverslipped.Tissue sections were deparaffinised in xylene and rehydrated through graded alcohol solutions to water and boiled in citrate buffer solution (pH 6) in an electric pressure cooker for 10 minutes at medium heat. Slides were left to cool to 35°C and placed in PBS. Endogenous peroxidase activity was quenched by incubating sections with a solution of 3% HHistology and CD3-labelled sections were examined for signs of immunological rejection and a pathological score adapted from a previous study in human hand transplantation Taq DNA Polymerase High Fidelity Kit (Invitrogen 11304-011) was used to ensure the lowest error rate. PCR products were isolated by running 1.8% agarose TBE gels using Bioline HyperLadder IV as size marker and purified from the gel using UltraClean 15 DNA Purification Kit (MoBio 12100-300). Purified DNA fragments were cloned in a pGEM-T Easy Vector (Promega A1360) /JM109 High Efficiency Competent Cells (Promega L1001) cloning system. Twenty-four clones were picked for each sample. Plasmids were extracted using UltraClean 6 Minute Mini Plasmid Prep Kit (MoBio 12300-250) and sequenced in two directions with T7 and SP6 primers at the Australian Genome Research Facility. Two independent PCRs were performed for each individual, and only sequence variants found in more than one PCR amplification were included in the subsequent analyses to minimize nucleotide errors yielded during PCR, cloning and sequencing. The sequencing results were quality-checked in Sequencher 4.1.4 (Gene Codes) and aligned with previously identified devil MHC alleles The seven individuals used for skin graft experiments were genotyped at MHC I α chain and MHC II β chain loci through nucleotide sequencing. Genomic DNA samples were extracted from fresh or frozen whole blood using MoBio UltraClean BloodSpin Kit. The α1 domain of MHC I genes and β1 domain of MHC II genes were amplified by PCR using previously published primers and PCR conditions To confirm that the mononuclear cell suspensions were able to proliferate, all suspensions were incubated with the mitogen Con A. Each of these controls resulted in high SI, indicating that experiment conditions were optimum (data not shown). There was a wide range of MLR responses among all groups, from SI<1 to SI>100. Skin graft surgeries were performed to test whether allogeneic tissue from an unrelated devil would be immunologically accepted by the recipient devil. Five of the eight skin allografts and six of the eight autografts engrafted successfully, as determined by macroscopic and histological assessment. The remaining three allografts and two autografts showed pathological changes characteristic of mechanical trauma, resulting in failure of these tissues to engraft. The main histological alterations in these unsuccessful grafts were necrosis associated with polymorphonuclear cell infiltration, surface parakeratosis and fibrin deposition.All successful grafts appeared indistinguishable from the autografts at Day 7. Four of five engrafted allografts had macroscopic changes associated with immune rejection 14 days following the surgery. These changes consisted of scaly lesions, coagulative exudates and brown to black coloration of the allografted skin. For one allograft, these macroscopic changes were not visible until 17 days after the procedure. Microscopically, immune rejection was characterised as Grade II, III or IV rejection . Typical alterations were moderate to severe perivascular and interstitial CD3 infiltration, dermal and epidermal lymphocytic exocytosis, usually accompanied by apoptotic keratinocytes and epidermal necrosis. Spongiosis was a common finding. Most of these alterations were recognised 14 days after the surgery, and progressed to very severe rejection on Day 21. In three cases, there was total loss of the allografted skin, hence a biopsy could not be taken. MHC genotyping and MLR results for animals used for the skin graft surgeries are summarized in Mesocrycetus auratus), which was either artificially or spontaneously transmitted by social interactions The presence of a histocompatibility system should prevent the establishment of transmissible cancers in vertebrates Even though Tasmanian devils lack MHC diversity All successful allografts were acutely rejected, despite low MLR responses in three out of the four skin graft pairs and little or no mismatches in MHC I and II alleles. The rejection of the foreign tissue was usually visible macroscopically and microscopically by 14 days after the procedure. The grafts could only be assessed every seven days, as procedures with live devils require general anaesthesia. In this context, the onset of rejection occurred between seven and 14 days, which is within the normal range (8–12 days) The florid T-cell infiltration observed in the skin allografts contrasts with the absence of tumour infiltrating lymphocytes in DFTD tumours The devil population is under threat because an allogeneic tumour cell clone is being transferred between animals without immune recognition. The host is immunocompetent Table S1Skin graft scoring used for the Tasmanian devil skin grafts.(DOC)Click here for additional data file.Table S2Histopathological alterations of skin allografts of Tasmanian devils.(DOC)Click here for additional data file.


Anterograde labeling of auditory afferents showed that inhibition of Eph signaling did not misroute axons to non-auditory target regions. Similarly, we did not find vestibular axons within auditory projection regions. However, we found that pharmacologic inhibition of Eph receptors reduced the volume of the vestibular projection compartment. Inhibition of EphB signaling alone did not affect auditory or vestibular central projection volumes, but it significantly increased the area of the auditory sensory epithelium. Misexpression of EphA4 and EphB2 in VIIIth nerve axons resulted in a significant shift of dorsoventral spacing between the axon tracts, suggesting a cell-autonomous role for the partitioning of projection areas along this axis. Cochlear ganglion volumes did not differ among treatment groups, indicating the changes seen were not due to a gain or loss of cochlear ganglion cells. These results suggest that Eph-ephrin signaling does not specify auditory versus vestibular targets but rather contributes to formation of boundaries for patterning of inner ear projections in the hindbrain. Auditory and vestibular afferents enter the brainstem through the VIIIth cranial nerve and find targets in distinct brain regions. We previously reported that the axon guidance molecules EphA4 and EphB2 have largely complementary expression patterns in the developing avian VIIIth nerve. Here, we tested whether inhibition of Eph signaling alters central targeting of VIIIth nerve axons. We first identified the central compartments through which auditory and vestibular axons travel. We then manipulated Eph-ephrin signaling using pharmacological inhibition of Eph receptors and  In birds these epithelia include a single auditory organ and seven vestibular organs. The centrally projecting axons of these ganglion cells coalesce to form the VIIIth cranial (vestibulocochlear) nerve. Upon reaching the hindbrain, VIIIth nerve fibers diverge and project to distinct targets. In chickens, the primary contacts for auditory VIIIth nerve fibers are n. ngularis ,2, wherengularis ,4. In adngularis -7.in vivo and explanted neurite projections in vitro have increasingly shed light on peripheral axon guidance [As regenerative medicine advances, exciting possibilities arise to restore auditory function for deaf and hearing impaired populations -10, or vguidance ,18 but tIn this study we address the role of Eph receptor tyrosine kinases and their membrane-associated ephrin ligands, which together comprise a large family of proteins known to regulate axon guidance. Eph-ephrin signaling can be bidirectional and mediates intercellular communication that leads to attraction, repulsion or migration . Eph recPrevious studies have identified roles for Eph-ephrin signaling in the peripheral auditory and vestibular systems. Eph receptors and their ephrin ligands are expressed differentially in the inner ear of both mammals and birds during embryonic and post-natal development -29. VestIn this study we tested whether the differential expression of EphA4 and EphB2 in the VIIIth nerve components acts as an instructive cue to guide axons toward auditory versus vestibular targets. Prior to entry of VIIIth nerve central projections, late migrating neural crest cells aggregate at the peripheral side of the nerve entry point . Days laGallus domesticus) were purchased from AA Laboratories  and stored at room temperature prior to use, then placed in a rotating incubator at 39°C with a relative humidity above 70% to initiate development. All embryos were either electroporated at E2 and allowed to develop in ovo, or were transferred to a culture dish  at E3 f2HPO4, 0.4M KH2PO4, 2.3M NaCl, pH 7.4). A fine tungsten needle was used to remove overlying membranes and sterile PBS was placed over the region of interest. Either a right-sided or bilateral transfection was performed depending on the developmental stage; only the right side was used after HH stage 14 [Eggs were windowed as described previously and the embryo was visualized with a small amount of 4% India ink in phosphate buffered saline  or EphA4 (GenBank accession number D38174), or kinase inactive forms (kiEphB2 and kiEphA4), which have point mutations in the intracellular kinase domain. The pMES construct contains a chicken β-actin promoter, a CMV-IE enhancer and also encodes a GFP reporter with an internal ribosomal entry site. As a negative control, embryos were transfected with GFP alone in a pCAX vector. Transfection was assessed by microscopic examination of GFP fluorescence or anti-GFP immunolabeling in dissected brainstems and in sectioned tissue.Plasmid vectors ,41 were in vitro, corresponding with E4-E7.To inhibit forward signaling, we used soluble recombinant proteins as described previously . The rec2PO4, 20 mM NaHCO3, 3 mM HEPES, 10 mM Glucose, 2 mM CaCl2, 1.3 mM MgSO4; pH 7.0) to reveal the cochlear duct of the treated side. Small injections of rhodamine dextran amine  were delivered with a pulled glass micropipette broken to 35µm tip and attached to a Picospritzer. Injections targeted the area directly adjacent and deep to the cochlear duct where cochlear ganglion cells and their central-projecting axons are present. Samples were incubated in aCSF for 20-30 minutes and immediately fixed in 4% paraformaldehyde (PFA) in PBS.One of the goals of this study was to determine whether affecting Eph-ephrin signaling in ganglion cells would lead to mistargeting of the central auditory VIIIth nerve axons. We used a protocol we developed for injecting the basilar papilla to label the central projections of cochlear ganglion cells . Small iFollowing fixation in 4% PFA for at least 2 hours, tissue was washed in PBS for 10 minutes, then cryoprotected the tissue with overnight incubation in 30% sucrose. Tissue was embedded in OCT medium and cryosectioned at 25 µm sections in the coronal plane. Alternating series of sections were collected on chrome-alum coated glass slides then dried on a 37°C slide warmer at 37°C. Some sections were counterstained with bisbenzimide (2 µg/mL in PBS for 5 min followed by 1X PBS wash for 5 min) to label cell nuclei and facilitate identification of central auditory nuclei targets. For samples that were transfected, one series was labeled with anti-GFP immunofluorescence to enhance visualization of GFP-positive axons. All samples were labeled with anti-neurofilament immunofluorescence to view axon tracts in the hindbrain. Slides were coverslipped with Glycergel mounting medium (Dako) and stored in the dark at 4°C until analyzed. Mounted sections were enclosed with a hydrophobic Pap-pen and the slides were rinsed then incubated in blocking solution  for 1 hour at room temperature in a humid chamber. The slides were quickly washed in PBS and then incubated with the primary antibody or antibodies  in a humid chamber at room temperature overnight. Slides were then incubated with an Alexa Fluor  secondary antibody used at a 1:1000 dilution in blocking solution and incubated for 2 hours at room temperature in the dark.All samples were coded and analyzed blind to experimental conditions. The slides were viewed on a Zeiss AxioSkop-2 epifluorescence microscope and 10X objective images were taken of the hindbrain region for all sections containing the VIIIth nerve entry point. We quantified respective auditory and vestibular projection areas and dorsoventral extents of the auditory and vestibular hindbrain components as well as the right basilar papilla and cochlear ganglion areas for each image using Axiovision software to outline regions and obtain measurements based on patterns of neurofilament immunofluorescence. Volumes were subsequently calculated by summing all area measurements for each embryo and multiplying by the section thickness. Data were only collected for treated sides or control treated sides, including the right side for recombinant protein injected embryos and right, left or bilateral for transfected embryos. To estimate the surface area of the basilar papilla, we measured the segments of epithelia attached to the cochlear ganglion in each section then multiplied the sum of lengths by the section thickness. For all analyses, each embryo represented a single data point and the entire VIIIth nerve entry point region and/or the entire cochlear ganglion extent was analyzed across multiple sections.For all analyses, samples were analyzed blind to treatment group and were decoded after data collection and grouped for comparison of means. All statistical analyses were performed with JMP software using unpaired a multivariate analysis of variance (MANOVA) followed by post-hoc testing using Dunnett’s Method with a control comparison and a significance value of p < 0.05. Representative images were taken with a Zeiss Axiocam digital camera and Axiovision software. Additional image analysis, tiling and color rendering or color merging were performed using Adobe Photoshop and figures were prepared with Adobe Illustrator.We first determined the normal central trajectories of auditory and vestibular ganglion cell axons. Several complementary approaches were used, and together they revealed distinct hindbrain compartments through which auditory or vestibular axons approach their initial targets from the VIIIth nerve entry point . An overlaminaris (NL) second and third order auditory neurons, as a landmark to provide a linear demarcation between the two compartments since it is reliably situated at this transitional area . Misrouting of auditory axons into the vestibular compartment . In all of these cases, GFP labeled axons were seen only in the central vestibular projection compartments described above and not in the central auditory projections compartments . No embr6 μm3 ± 1.96 x 106 μm3 . Several factors contribute to the establishment of auditory and vestibular projection volumes. As these regions are segregated along the dorsoventral axis, we analyzed the dimensions along this axis in transfected animals. The auditory and vestibular components are segregated upon entering the hindbrain, with the vestibular VIIIth nerve projections found ventral and adjacent to a large auditory fiber tract containing projections NM to contralateral NL. We found that transfection with kiEphA4 resulted in a 15% increase in the dorsoventral extent of the auditory VIIIth nerve projection, and a 21% increase in the vestibular projection. GFP controls had a mean dorsoventral extent of 312.59 μm ± 7.40 μm (n = 23) for auditory VIIIth nerve projections and 320.04 μm ± 8.92 μm (n = 23) for vestibular VIIIth nerve projections; kiEphA4 expressing embryos had a mean dorsoventral extent of 360.90 ± 15.31 μm  and 387.06 μm ± 25.97 μm  for auditory and vestibular projections, respectively, as shown in ex ovo fusion protein injected embryos , which was significantly increased by 17% compared to IgG-Fc controls with a mean surface area of 13.34 x 104 μm2 ± 1.02 x 104 μm2 . These effects were not seen following treatment with EphA3-Fc  or EphA4-Fc . We found no effect on the basilar papilla when VIIIth nerve axons misexpressed Eph receptors (Because embryos were treated at the auditory periphery, we sought to determine whether the central effects we observed could potentially have occured secondarily to changes in the periphery. We measured the surface area of the basilar papillae and the volume of the cochlear ganglia to determine whether any central effects were correlated with peripheral changes. We found that selective inhibition of EphB signaling appeared to alter basilar papilla development . After ieceptors  and the eceptors . During The goal of this study was to determine the role of Eph-ephrin signaling in the guidance of VIIIth nerve axons to their central targets. Our results suggest that these signaling molecules do not by themselves regulate auditory vs. vestibular axon targeting. Instead, Eph family proteins appear to have more subtle roles in the designation of central projection boundaries and their extents. These hindbrain compartments for auditory and vestibular central VIIIth nerve projections may in turn contribute to axon navigation during circuit formation.Only with pharmacologic inhibition of Eph receptor signaling did we see a shift in relative amount of space within the brainstem dedicated to vestibular axons. Following treatment with EphA4-Fc, which binds strongly to ephrin-A’s and ephrin-B2  the vestIn contrast to results obtained using broad inhibition of Eph proteins, misexpression of wild type and kinase inactive forms of EphA4 and EphB2 receptors in the VIIIth nerve projections did not affect hindbrain compartment volumes. This discrepancy might reflect experimental differences in the effectiveness of treatment, as the pharmacological treatment acts on multiple targets and may reach a greater spatial extent than focal electroporation. Moreover, pharmacological treatment could result in cell-autonomous as well as non-cell-autonomous functions in central VIIIth nerve projections. The combined action of several Eph family proteins may thus be needed to determine projection volumes. KiEphA4-transfected embryos showed an expansion of the normal dorsoventral borders without a change in overall volume, suggesting that forward signaling through EphA4 influences the dimensions of the central pathway taken by the nerve projections. In embryos overexpressing the dominant negative kiEphB2, a reduction of normal spacing between the two fiber tracts was observed, suggesting that EphB2 maintains this dorsoventral spacing during hindbrain development. The separation between tracts may arise from chemorepulsive cues mediated through EphB2, which repels NM axons at the midline in chicks  and inflAlthough we did not observe mistargeting of auditory or vestibular axons to inappropriate target regions, we did observe significant changes to the central hindbrain compartments devoted to central VIIIth nerve fibers. The differential expression of EphA4 and EphB2 at the nerve entry point may thus reflect a mechanism that helps determine boundaries of fiber tracts; these tracts may facilitate axon navigation using additional guidance cues to direct axons to their hindbrain targets. Contrary to what is seen up to the nerve entry point, central vestibular projections strongly express EphA4 ,47. The The extent of the auditory sensory epithelium was increased after EphB inhibition with EphB1-Fc. Previous expression studies have shown that ephrin-B1 is highly expressed in the peripheral projections of SGN cells, whereas EphB receptors are differentially expressed in subpopulations of SGN cells and hair cells ,30. EphBPrevious studies of central auditory axon guidance have demonstrated that Eph-ephrin signaling is needed to direct axons to appropriate regions with a target ,41,43,51per se, but rather contributes to formation of boundaries for patterning of inner ear projections in the developing hindbrain. Auditory and vestibular afferents enter the brainstem through the VIIIth cranial nerve and reach their appropriate central targets. We tested the role of Eph family proteins in establishing these pathways. We inhibited Eph signaling using several approaches and examined auditory and vestibular ganglion cell projections in the brainstem. We found no evidence of mistargeting to inappropriate modality regions. We found instead that inhibition of EphA and EphB receptors reduced the volume of the putative vestibular projection compartment. Misexpression of kinase inactive forms of EphA4 and EphB2 in ganglion cells suggest that these central patterning effects are not cell-autonomous. They further show that EphA4 and EphB2 contribute to the dimensions and separation of the auditory and vestibular central projections. Central changes were not due to a gain or loss of cochlear ganglion cells. These results suggest that Eph-ephrin signaling does not specify auditory versus vestibular targets


O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly  Over 1 in 4 people are diagnosed with cancer at some point in their life PDPN is a unique transmembrane receptor that promotes tumor cell motility. PDPN expression can be induced by tumor promoters including TPA, oncogenic Ras, and Src PDPN is found at the invasive front of many tumors, which is consistent with its role in promoting malignant invasion O-glycosylated with sialic acid, α2,3 linked to galactose The extracellular domain of PDPN is highly Antibodies against PDPN can inhibit lung metastasis of transformed cells that express PDPN Unlike antibodies, lectins can survive digestive processing and retain biological activity O-linked carbohydrate chains containing sialic acid binds to PDPN to inhibit transformed cell growth and motility at submicromolar concentrations. This approach demonstrates how lectins may be used as dietary agents that target specific receptors to combat malignant cell growth.Here, we show that a lectin with affinity for O-glycosylated with sialic acid, α2,3 linked to galactose. Scholl et al have reported that PDPN associates with lectin from seeds of the legume tree Maackia amurensis (MASL) O-linked carbohydrate chains containing sialic acid We have recently reported that the Src tyrosine kinase utilizes the adaptor protein Cas/BCAR1 to augment PDPN expression in order to promote tumor cell motility As shown in We hypothesized that binding of MASL to PDPN would inhibit transformed cell migration. As shown in To verify the functional relevance of MASL targeting PDPN on cell migration, we investigated its effects on nontransformed cells transfected with PDPN or empty parental vector. Since PDPN expression is sufficient to promote cell migration As shown in Studies indicate that PDPN expression is strongly induced in about 80% of skin cancers Transwell chambers were used to further investigate the effects of MASL on melanoma cell growth and migration. As shown in We employed siRNA to verify the effects of PDPN and MASL on melanoma cell growth and migration. PDPN siRNA effectively decreased B16 Pdpn expression levels  Apoptosis usually results from the induction of caspase activity which cleaves poly(ADP-ribose) polymerase (PARP) into an 89 kD fragment in vivoin vitro to determine if this dietary lectin can survive gastrointestinal proteolysis to remain biologically active in the circulatory system. As shown in As described above, some dietary lectins can survive digestive processing to combat cancer in vivo. As shown in Since MASL, or at least its biological activity, was resistant to gastrointestinal proteolysis we examined the effects of dietary MASL on tumor cell growth in vivo. As shown in As expected, PDPN expression was evident in melanoma cells of tumors in vivoAlthough its molecular target has not been defined, Mistletoe lectin is used as an anticancer agent that can reduce tumor vascularization We examined cell lines and clinical specimens to further investigate PDPN expression and MASL sensitivity in human melanoma cells. As shown in Maackia amurensis hemagglutinin (MAH) and the 37 kD subunit is called Maackia amurensis leukoagglutinin (MAL) or Maackia amurensis mitogen (MAM) Lectins are a complex family of proteins. MASL is a tetramer formed by 32 kD and 37 kD subunits. The 32 kD subunit is called Lectins are promising anticancer agents that can be administered orally DK) below 1 nM, while antibody used for these previous studies targets a larger epitope with lower affinity  and Melanoma cell motility contributes to metastatic invasion that causes the vast majority of skin cancer deaths Dietary lectins can block the action of endogenous pro-metastatic lectins  to inhibit tumor cell growth, and can be used as medicinal cancer treatments Maackia amurensis lectin indicate that it can accurately target specific glycoproteins expressed by human cells O-linked carbohydrate chains containing sialic acid Maackia amurensis has been used as a medicinal plant for several centuries to treat ailments including cancer in parts of Asia Although some lectins may nonspecifically bind to many glycoproteins, experiments with cloned PDPN expression is relatively low in most normal cells, and found predominantly in tissues including retina, kidney podocytes, lymphatic endothelium, and lung alveolar epithelium in vivo. For example, although legumes including lentils and peanuts contain significant amounts of agglutinating lectins, their ingestion does not result in toxic platelet aggregation Maackia amurensis has been used as a medicinal plant for several centuries to treat ailments including cancer in parts of Asia Our data demonstrate that MASL targets PDPN to inhibit tumor cell growth and motility. However, MASL is bound to target other receptors containing similar sialic acid motifs. In addition, like other reagents, MASL may produce “off target” effects in a clinical setting. However, lectin pharmacology is complex and MASL may undergo enzymatic modification to form bioactive compounds Taken together, results from this study suggest that lectins exemplified by MASL could significantly expand our limited arsenal of targeted cancer treatments, particularly anticancer agents that can be administered orally. This paradigm may lead to novel skin cancer treatments, and pave the way to treat other cancers with elevated PDPN expression levels including breast Use of mice to obtain serum was approved by Committee of Animal Experiment in Nagoya University Graduate School of Medicine (Approved ID: 23132). Mouse experimental protocols were approved by the University of Medicine and Dentistry School of Osteopathic Medicine Institutional Animal Care and Use Committee (Approved ID: 10350).Mouse embryonic cells transfected with v-Src, PDPN, or empty parental vectors have been previously described Human tissue collected by the Honest Broker Program was excised and flash frozen in liquid nitrogen at the time of excision during surgery. Western blotting was performed as previously described Cells  were cultured on 35-mm poly-D-lysine–coated glass-bottomed culture dishes (MatTek P35GC-1.5-14-C) for 24 hours. MASL was conjugated to Hilyte Fluor TR (Anaspec 72052) and incubated with cells for 15 min at 37°C. Cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, washed thrice with 0.1% Tween 20 in PBS followed by 1% bovine serum albumin (BSA) in PBS for 30 minutes, incubated with PDPN antiserum (1∶100) overnight at 4°C, washed, and then labeled with goat anti-syrian hamster IgG conjugated to Alexa Fluor 488 (Molecular Probes A21110). Cells were visualized on a Zeiss Axiovert microscope as described 2, and incubated with MASL conjugated to agarose beads, or empty beads as controls (Thermo Scientific 20501), on ice for 3 hours. Beads were then washed 4 times with PBS, and eluted in SDS-PAGE sample buffer at 95°C for 5 minutes. Eluted protein was examined along with total cell lysates by Western blotting.Cells were lysed in lysis buffer  on ice for 30 min, clarified by centrifugation, diluted to 1 mg/ml in PBS supplemented with 1mM PMSF and 10 mM MgClCell monolayers were scratched and incubated with MASL for 24 hours to assess cell migration by wound healing assays, which were quantitated as the number of cells that entered an area of the wound indicated in Figure Legends as described Mice (C57BL/6) were fed with a 200 mg food pellet containing MASL to achieve doses of 0, 100, or 200 mg/kg 1 hour before blood was taken from subclavian vein. Blood was allowed to coagulate 1 hour at room temperature and clarified by centrifugation for 10 minutes at 20,000 g to obtain serum. This serum was added to cell culture medium (DMEM+10%FBS) to a final concentration of 20%. B16 cell monolayers were scratched, washed with DMEM, and then incubated with these media, or control medium not fortified with mouse serum, for 24 hours to assess their effects on cell migration by wound healing assays, which were quantitated as the number of cells that entered an area of the wound indicated in Figure Legends as described Mice (C57BL/6) were fed with a 200 mg food pellet containing MASL to achieve doses of 0 or 25 mg/kg once weekly, starting the day before subcutaneous inoculation with 100,000 B16 cells in 0.1 ml PBS. Tumor volumes were measured blindly by a trained physician with a single caliper daily. Mice were sacrificed 18 days after inoculation and dissected. Tumors were fixed in formalin, paraffin embedded, sectioned (5 microns), and processed for hematoxylin/eosin staining or immunohistochemistry to detect PDPN expression with 8.1.1 monoclonal antibody as described MASL subunits were resolved by reducing and nonreducing SDS-PAGE and stained by Coomassie dye. Bands were then extracted and digested with trypsin. Extracted protein was reduced and alkylated with iodoacetamide, and sequenced using data dependent acquisition by LC-MS/MS on a Thermo Scientific LTQ Orbitrap XL.


Burkitt lymphoma (BL) is a highly aggressive germinal center (GC) derived B cell lymphoma. Its successful treatment requires intense therapeutic protocols that include high doses of alkylating agents and central nervous system (CNS) prophylaxis . OutcomeMYC proto-oncogene at 8q24 via translocation to one of the immunoglobulin loci is typically detected in BL [A better understanding of BL pathogenesis is a prerequisite for therapeutic advance. Activation of the pos, BCL2neg, Ki67>95%) and mRNA profiles (BL gene expression signature).Based on comprehensive transcript analyses in human BL, revealing low NF-κB activity, ,4 and thThe most striking demonstration of the GC B cell origin of the BL-like tumors arising in the mutant mice was that of ongoing somatic hypermutation of the rearranged immunoglobulin variable gene segments in the tumor cells, as it is also observed in human BL. Former mouse models addressing the impact of MYC activation in B cell lymphomagenesis but nor targeting the event specifically into GC B cells, resulted in the generation of lymphomas originating from pre-GC B cells and resembling human BL only to limited extents -9.Ccnd3 [CCND3 mutations partly affecting the same codons as in the mouse tumors [TCF3/ID3 genes that result in consecutive B cell receptor upregulation and thus provide a potential mechanism for PI3K activation in human BL, which was indeed observed by both Schmitz and Sander et al. (2012).The monoclonality of the tumors arising in mice upon MYC and PI3K pathway activation in GC B cells implies, that additional, tertiary mutations are needed for tumor development. Indeed, the tumors displayed unique genomic aberrations including trisomy of chromosome 6, which comprises genomic regions commonly gained in human BL, and somatic mutations of e tumors , a findiIn summary, we hypothesized cooperation between MYC and PI3K in BL development and functionally validated this hypothesis through conditional targeted mutagenesis in the mouse. This implicates the PI3K pathway in addition to MYC as a therapeutic target in BL. Additional therapeutic targets are suggested by tertiary mutations recurrently found in the mouse tumors and human BL. Ultimately, the in vivo model offers exciting opportunities to improve BL treatment by testing novel therapeutic strategies in the context of normal physiology, including a functional immune system. A particularly attractive perspective is the study of mechanisms controlling tumor metastasis, like the dissemination of BL into the CNS.


Transcriptome analysis of bovine mammary development has provided insight into regulation of mammogenesis. However, previous studies primarily examined expression of epithelial and stromal tissues combined, and consequently did not account for tissue specific contribution to mammary development. Our objective was to identify differences in gene expression in epithelial and intralobular stromal compartments. Tissue was biopsied from non-lactating dairy cows 3 weeks prepartum, cut into explants and incubated for 2 hr with insulin and hydrocortisone. Epithelial and intralobular stromal tissues were isolated with laser capture microdissection. Global gene expression was measured with Bovine Affymetrix GeneChips, and data were preprocessed using RMA method. Moderated t-tests from gene-specific linear model analysis with cell type as a fixed effect showed more than 3,000 genes were differentially expressed between tissues . Analysis of epithelial and stromal transcriptomes using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathways Analysis (IPA) showed that epithelial and stromal cells contributed distinct molecular signatures. Epithelial signatures were enriched with gene sets for protein synthesis, metabolism and secretion. Stromal signatures were enriched with genes that encoded molecules important to signaling, extracellular matrix composition and remodeling. Transcriptome differences also showed evidence for paracrine interactions between tissues in stimulation of IGF1 signaling pathway, stromal reaction, angiogenesis, neurogenesis, and immune response. Molecular signatures point to the dynamic role the stroma plays in prepartum mammogenesis and highlight the importance of examining the roles of cell types within the mammary gland when targeting therapies and studying mechanisms that affect milk production. The bovine mammary gland undergoes morphological changes throughout the reproductive cycle. During the prepubertal/pubertal stages the gland undergoes a period of allometric growth and development characterized by the branching expansion of epithelial parenchyma and associated stroma into the fat pad. Once expansion is complete, growth and development of the gland enters a steady state period until the onset of pregnancy when growth, development and differentiation (i.e. mammogenesis) are reinitiated. Mammogenesis continues throughout the first pregnancy as epithelial structures mature and prepare for lactation. At the onset of lactation epithelial cells begin to secrete milk. Milk synthesis and secretion continue until milk removal ceases.At cessation of milk removal, or dry-off, the gland undergoes a period of rapid involution. One week following dry off there is an increase in apoptosis in alveolar structures At the systemic level mammary growth and development are regulated by hormones. Hormones either act directly to influence growth and differentiation of mammary epithelial cells or indirectly by influencing production of local signaling factors. Steroid hormone induced mammary development often occurs through indirect mechanisms and requires stromal cooperation for induction of epithelial proliferation and morphogenesis Studies conducted to characterize transcriptome changes that occur during mammary development have provided insight into key processes involved in signaling and morphogenesis of the mammary gland . Six multiparous Holstein cows from the Michigan State University Dairy Cattle Teaching and Research herd were impregnated between 90 and 105 after the onset of lactation. Cows were dried off after being milked for 310±12 days and had an average dry period length of 55±5 days. A mammary biopsy was obtained three weeks before expected calving date, during the window of expected mammogenesis 3 explants. Explants were placed on siliconized lens paper, and cultured in a humidified chamber  at 37°C for 2 h in Waymouth's media supplemented with 5 µg/ml insulin (Sigma), 0.1 µg/ml hydrocortisone (Sigma) and 50 µM 5-bromo 2-deoxyuridine  to measure rate of proliferation. Following explant culture, tissue was embedded in Tissue Tek OCT cryostat embedding compound , flash frozen in liquid nitrogen and stored at −80°C, or placed in 10% buffered formalin for 24 hr at RT. Tissues embedded in OCT were used for laser capture microdissection. Tissues fixed in buffered formalin were embedded in paraffin and used to measure rate of BrdU incorporation or to measure protein expression with immunohistochemistry.Biopsied tissue was immediately placed in cold media  and transported to the lab. Biopsied tissue was washed three times in phosphate-buffered-saline (PBS) containing; 100 U penicillin – 100 µg streptomycin per ml and 1.5 µg Amphotericin-B per ml (Invitrogen) and cut into 0.5 cmParaffin-embedded formalin fixed tissue was cut into 5 µm sections and placed on charged slides. The Zymed BrdU Staining Kit (Invitrogen) was used to detect BrdU incorporation into cells. Stained slides were examined with a Nikon Eclipse 50i light microscope . Five photomicrographs were taken per slide at 200X magnification and images were captured at a size of 2560 by 960 pixels with 32-bit per pixel depth and saved in tagged image file format (TIFF) in Image Pro Plus 5.1 . Four classes of cells were counted  using Image Pro Plus software by manually tagging stained and unstained epithelial and stromal cells in the section. Approximately 3,300 epithelial or stromal cells were counted/cow to determine percent BrdU labeled index. Data were expressed as mean percent proliferating ± standard error of the mean, and a paired student t-test was used to determine difference.Serial tissue sections (7 µm) were prepared from OCT preserved tissue with a cryostat microtome using RNase-free techniques and stored at −80°C until use (less than 8 weeks). On day of LCM, tissue sections were fixed and stained in In Situ Hybridization Pap Jars  using the Histogene Kit  following manufacturer's directions and left in xylene until LCM to ensure tissue was dehydrated.Populations of epithelial cells and intralobular stromal tissue were isolated by LCM using an Arcturus PixCell IIe LCM system (Arcturus Engineering now Molecular Devices) with CapSure LCM Transfer Film (Molecular Devices) according to manufacturer's protocol. Although intralobular stromal tissue contains a mixed population of cells that include fibroblasts, endothelial, nervous and immune cells, our goal was to enrich stromal captures with fibroblasts so during LCM care was taken to avoid blood vessels and obvious immune cells. Pictures were taken of histological sections before and after LCM to document cell types counted and tissue captured .Tissue captured onto transfer film was immediately immersed in 50 µl of isolation buffer from Acturus' Picopure kit (Molecular Devices). To obtain a minimum of 20 ng of total RNA, lysates were pooled from two slides of the same sample for epithelial captures and four slides from stromal captures. Total RNA was isolated following manufacturer's protocol for laser captured frozen tissue. Recovered RNA was analyzed using a Bioanalyzer 2100  to determine quality and quantity by use of the Picochip and multiple standards. RNA integrity number (RIN) of tissue off the slides was >8.0 for all samples.NuGen WT Ovation Pico Technology, a high-amplification method for small amounts of RNA, was used for Target preparation following manufacturer protocol to generate products for the Bovine Affymetrix GeneChip. Gene chips were hybridized and scanned using standard Affymetrix protocols. Briefly, gene chips were hybridized in an Affymetrix 640 Hybridization oven at 45°C for 16 hours with 60 rpm rotation. After hybridization, gene chips were washed according to Affymetrix protocol on a Fluidics station using non-stringent  and stringent  wash buffers. Arrays were stained using SAPE stain and antibody solutions. SAPE contains 2 ug/ul BSA, 10 ug/ml Streptavidin Phycoerythrin (SAPE) in 100 mM MES, 1M NaCl, 0.05% Tween 20 and 0.005% antifoam. The antibody solution contains: 2 mg/ml BSA, 0.1 mg/ml goat IgG, 3 ug/ml biotinylated anti-streptavidin antibodies in 100 mM MES, 1M NaCl, 0.05% Tween 20 and 0.005% antifoam. Genechips were then scanned using an Affymetrix Genearray scanner GSC3000, with 7G upgrade. The efficiency of amplification and hybridization were assessed by incorporating Affymetrix Poly-A RNA and hybridization controls with every sample.www.affymetrix.com/support). We found that epithelial and stromal samples exhibited excellent overall signals with GAPDH 3′ to 5′ ratio of <1.5, which suggests that starting material was of suitable quality for measuring gene expression www.ncbi.nlm.nih.gov/geo, accession no. GSE18768).GeneChip quality was evaluated by examining the 3′ to 5′ ratio of the GAPDH housekeeping gene, as the 3′ to 5′ ratio gives an indication of the integrity of the starting RNA  2008  of finding this ratio by chance  is also provided. This is displayed as the negative log of that P-value. Therefore, networks with scores of 2 (P<0.01) or higher have at least a 99% confidence of not being generated by random chance alone.After uploading the data into IPA, genes expressed at a greater level in epithelial and stromal transcriptomes were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. A description of the IPA networks methods can be found at Canonical pathways analysis was used to identify pathways from IPA library that were most significant to the input data set. The significance of association between the data set and the IPA curated canonical pathways is measured in two ways: 1) A ratio is calculated of the number of genes from the data set that map to the pathway divided by the total number of genes in the archived canonical pathway, and 2) Fischer's exact test is used to calculate a P-value to determine the significance of the association between the genes in the dataset and the canonical pathways.−ΔΔCTTotal RNA (640 pg) was reverse transcribed into cDNA, ligated and amplified for 8 hrs using Qiagen Quantitect Whole Transcriptome Kit following manufacturer's instructions. cDNA was used for qPCR. The qPCR analysis was performed using the StepOnePlus™ Real-Time PCR Systems , ABI TaqMan Gene Expression Master Mix and unique TaqMan® Gene Expression Assays (Applied Biosystems) specific for bovine: FN1 ; IGF1 (Bt03252280_m1); NIDI (nidogen 1/entactin Bt03272040_m1); CSN2 ; CDH1 ; PRLR , and RP18S (Bt03225196_g1) as the reference gene. The mean sample threshold cycle (CT) and mean of the reference CT for each sample were calculated from duplicate wells. The relative amounts of target gene expression for each sample were then calculated using the formula 2Immunohistochemistry was used to visualize in situ expression of MMP3 and fibronectin proteins in epithelial and stromal tissue in 5 µm sections cut from paraffin embedded sections. Prior to immunohistochemistry, slides were incubated overnight at 60°C, deparaffinized in xylene, rehydrated through a graded series of ethanol washes  and rinsed in PBS. Endogenous peroxidases were blocked for 15 minutes in 3% hydrogen peroxide in methanol. Sodium citrate antigen-retrieval was performed in a 100°C water bath. Sections were then incubated for 10 min at RT in non-immune blocking serum block , excess blocking serum was blotted off and sections were incubated with the primary mouse monoclonal anti-fibronectin  or mouse polyclonal anti-MMP-3  at RT diluted in blocking solution. Sections were washed in PBS and incubated with a broad-spectrum secondary antibody conjugated with streptavidin-peroxidase , followed by a PBS wash and 3 min incubation with 3′, 3′-diaminobenzidine chromogen substrate. All slides were counterstained with hematoxylin, dehydrated in a graded series of alcohols, cleared in xylene, and mounted with Histomount (Zymed).The relative rate of epithelial (0.34%±0.26) and stromal (0.37%±0.17) cell proliferation was not different in mammary tissue from dry cows 3 weeks prepartum. Analysis of frequency of differentially expressed genes by P-value  revealedMore than 3,000 genes (P<0.05) were differentially expressed between epithelial and stromal cell populations . FunctioEnrichment of molecular and cellular functions (P<0.005) of IPA generated gene sets were also examined. For epithelial tissue, protein synthesis  was the most highly enriched gene set ; other nMolecules tab and sorting genes by location. The majority of genes expressed at a greater level in the epithelium encoded proteins that function within the cytoplasm and nucleus . Other epithelial enriched pathways included Oxidative Phosphorylation , IGF-I Signaling Pathway  and Neuregulin Signaling . Similarly the higher expression of CDH1, PRLR and CSN2 in epithelial tissue measured with microarray analysis was validated using q-PCR, again showing greater than 150-fold expression difference between the tissues.Distribution of expression of MMP3 and fibronectin proteins that remodel and make up the extracellular matrix, respectively, was investigated using immunohistochemistry . The epiOur results demonstrate, for the first time, the unique characteristics of the transcriptomes of epithelial cells and intralobular stromal fibroblasts in mammary tissue of dry cows at three weeks prepartum. Differential gene expression between the compartments was characterized by an enrichment of genes that primarily had intracellular functions in the epithelial compartment and extracellular functions in the stromal compartment. Further, expression analysis enabled the dissecting of the tissue specific contributions for preparing the mammary gland for milk synthesis.The epithelial transcriptome was enriched with genes that clustered in ontologies and pathways characteristic of cellular turnover and development of protein and lipid synthetic machinery. Specifically, the enrichment of genes in molecular and cellular functions: cellular growth and proliferation, cell death and cell cycle indicate that at this point in mammary development the epithelial compartment is replacing old senescent cells with new cells, and cells are preparing to synthesize proteins and lipids that are necessary for lactation. Enrichment of genes in cellular assembly and organization, RNA trafficking, protein trafficking and energy metabolism molecular and cellular functions is indicative of the development of cellular infrastructure necessary to support the increased rates of protein synthesis and secretory activity needed for lactation IPA network analysis enabled the visualization of cellular distribution and queries into interactions among molecules encoded by genes that enriched epithelial or stromal transcriptomes. The top five IPA epithelial networks were composed of genes that encoded for molecules with intracellular functions. Functions of these molecules included regulators of transcription, translation and intracellular trafficking, reflective of the role of epithelial cells in the mammary and the building of cellular machinery necessary for milk synthesis Genes in stromal transcriptome enriched cellular ontologies specific for ECM and secreted molecules. Enriched cellular and molecular functions indicated that genes expressed at a greater level in the intralobular stroma encoded proteins that regulate cell-cell signaling, cellular movement, and cellular development. Stromal molecular signatures also reflected that cell turnover, proliferation and death occurred in mammary stroma of three week prepartum dry cows. Specifically, molecular and cellular functions: cellular growth and proliferation, cell death and cell cycle were enriched with genes expressed at a greater level in stromal fibroblasts.in vitro environment also removes the progesterone block, which may lead to secretory activation , ECM gene ontology and VEGF signaling pathway suggest that stromal activation is being induced during this period of mammary gland development. Stromal activation is associated with remodeling during wound healing and pathologically with cancer and fibrosis There was also transcriptome evidence for paracrine interactions between the tissues with the enrichment of the IGF1 canonical pathway and expression of IGF binding proteins. The paracrine effects of stromally derived IGF1 on epithelial cell proliferation in the bovine mammary gland is well established Examination of genes that encode proteins with plasma membrane and extracellular distributions gave insight into the interaction between the tissues and pathways that may be active during this period of mammogenesis . EpithelInterestingly although both epithelial and stromal transcriptomes showed differential contributions to the expression of proteases that remodel the ECM, only the stromal compartment showed evidence of expression of ECM molecules. These signatures were validated using immunohistochemistry. Staining showed the differential distribution of proteins that make up the extracellular matrix, fibronectin, and remodel the ECM, MMP3, as well as TGFB1 and its type II receptor (TGFBR2) .Both compartments showed unique expression of integrin receptors that serve as cell-ECM receptors and are responsible for initiating signals important to cell proliferation and differentiation. These findings suggest that stromal fibroblasts play an important role in regulating mammary function and differentiation through the generation of developmentally specific ECM molecules. The greater expression of SFRP (secreted fizzled-related proteins) in the stromal cell compartment also suggested a role for Wnts and related molecules in mediating mammogenesis during this stage of development. SFRPs have been shown to block Canonical Wnt Signaling pathways and alternatively to behave in a paracrine manner to stimulate epithelial branching Mammary epithelial and intralobular stromal cells contribute distinct molecular signatures to the mammary transcriptome in dry cows at 3 weeks prepartum. The epithelial signature was characterized by enrichment of molecules that regulate protein synthesis, metabolism and secretion. In contrast the stromal signature was primarily characterized as being enriched with genes that encode extracellular molecules important to signaling, ECM synthesis and ECM remodeling. Tissue specific signatures also provided evidence of paracrine stimulation of the IGF1 signaling pathway in the epithelial compartment from stromally produced IGF1 and stimulation of the stromal reaction from the epithelial compartment. Genes representing angiogenesis and neurogenesis were transcribed in both the epithelium and stroma. Molecular signatures point to the dynamic role the stroma plays in synthesis of signaling molecules that stimulate epithelial morphogenesis and proliferation, as well as in the synthesis of ECM conducive to milk protein synthesis. These signatures also highlight the importance of examining the roles of cells within the mammary gland when targeting therapies and studying mechanisms that impact milk production.Figure S1Experimental design flowchart shows the relation of the results reported in this manuscript to the larger study. The larger study USDA-CSREES-NRI grant The stromal effect: Remodeling and the effect of transforming growth factor- beta on the mammary gland during the dry period MICL08379. Yellow boxes highlight treatments and results that were the focus of this report.(TIF)Click here for additional data file.Figure S2Distribution of genes differentially expressed between epithelial and stromal tissue by P-value. Y-axis  = frequency (no.) of genes; x-axis =  median P-value.(TIF)Click here for additional data file.Figure S3IPA generated networks of genes expresses at a greater level in the epithelium (green). A) Network 1 Protein synthesis, cardiac dysplasia, cardiovascular disease (Score 49), B) Network 2 RNA post-transcriptional modification, cellular assembly and organization, DNA replication, recombination and repair (score 46), C) Network 3 Protein synthesis RNA post-transcriptional modification, gene expression (score 38) D) Network 4 Gene expression, cell movement, cell to cell signaling and interaction (score 38), E) Cell morphology, digestive system development and function, inflammatory response (score 36) . See below for legend.(TIF)Click here for additional data file.Figure S4IPA generated networks of genes expresses at a greater level in the stroma (red). A) Network 1 Tissue development, dermatologic disease and conditions, genetic disorders (score 51), B) Network 2 Cancer, tumor morphology, tissue morphology (score 44), C) Network 3 Cellular assembly and organization, cellular function and maintenance and cell movement (score 37) , D) Network 4 Cardiovascular system development and function, cell-to-cell signaling and interaction, connective tissue disorders (score 36), E) Network 5 Cell death, connective tissue disorders, dermatologic disease and conditions (score 36).(TIF)Click here for additional data file.Supplemental Information S1Worskeets A–Q. Lists the category of gene set ; term (i.e. specific gene ontology (GO) with GO number); count (no. of genes enriching term); % ; P-value (i.e. enrichment of gene set); genes (list of genes enriching gene set by Affymetrix ID); Bonferroni; Benjamini and FDR  for functional annotation clustering of genes expressed at a greater level in A) epithelium and B) stromal tissue. Symbol, Entrez Gene Name, Affymetrix ID, Fold Change, P-value and False Discovery Rate are listed for C) Genes in IPA generated epithelial network 1; D) Genes in IPA generated epithelial network 2; E) Genes in IPA generated epithelial network 3; F) Genes in IPA generated epithelial network 4; G) Genes in IPA generated epithelial network 5; H) Genes in IPA generated stromal network 1; I) Genes in IPA generated stromal network 2; J) Genes in IPA generated stromal network 3; K) Genes in IPA generated stromal network 4; L) Genes in IPA generated stromal network 5; M) Gene enrichment of Hepatic Fibrosis/Hepatic Stellate Cell Activation Canonical Pathway; N) Genes clustered in IGF canonical pathway; O) Gene enrichment of VEGF signaling canonical pathway. P) Genes in (XLS)Click here for additional data file.