Wnt/β-catenin  signalling pathway activity is necessary and used independently several times for specification of vegetal fate and endoderm, gut differentiation, maintenance of epithelium in adult intestine and the development of gut-derived organs in various vertebrate and non-vertebrate organisms. However, its conservation in later stages of digestive tract development still remains questionable due to the lack of detailed data, mainly from Spiralia.Pdu-Tcf gene, a Tcf/LEF orthologue and a component of Wnt/β-catenin pathway from Platynereis dumerilii, a spiralian, marine annelid worm. Pdu-Tcf undergoes extensive alternative splicing in the C-terminal region of the gene generating as many as eight mRNA isoforms some of which differ in the presence or absence of a C-clamp domain which suggests a distinct DNA binding activity of individual protein variants. Pdu-Tcf is broadly expressed throughout development which is indicative of many functions. One of the most prominent domains that exhibits rather strong Pdu-Tcf expression is in the putative precursors of endodermal gut cells which are detected after 72 h post-fertilization (hpf). At day 5 post-fertilization (dpf), Pdu-Tcf is expressed in the hindgut and pharynx (foregut), whereas at 7 dpf stage, it is strongly transcribed in the now-cellularized midgut for the first time. In order to gain insight into the role of Wnt/β-catenin signalling, we disrupted its activity using pharmacological inhibitors between day 5 and 7 of development. The inhibition of Wnt/β-catenin signalling led to the loss of midgut marker genes Subtilisin-1, Subtilisin-2, α-Amylase and Otx along with a drop in β-catenin protein levels, Axin expression in the gut and nearly the complete loss of proliferative activity throughout the body of larva. At the same time, a hindgut marker gene Legumain was expanded to the midgut compartment under the same conditions.Here we characterize the Platynereis.Our findings suggest that high Wnt/β-catenin signalling in the midgut might be necessary for proper differentiation of the endoderm to an epithelium capable of secreting digestive enzymes. Together, our data provide evidence for the role of Wnt/β-catenin signalling in gut differentiation in  Nematostella vectensis . After the addition of fresh hybridization buffer, larvae from one sample were divided into several groups for staining of different genes and pre-hybrized for 2–4 h at 63 °C in a thermal block with a cover. After replacement of the pre-hybridization solution with 50 µl of digoxigenin-labelled RNA probes, 2 ng/µl in hybridization buffer (denatured previously for 10 min at 90 °C), the samples were hybridized approximately for 16–18 h overnight in a thermal block at 63 °C.The following day, probes were collected for reuse and replaced with 250 µl of plain hybridization buffer. After the larvae settled to the bottom of the tubes (10–15 min), another 15-min wash with 250 µl of Hyb-Mix or 0.5 ml of 2× SSCT/50% formamide (Sigma) followed. Subsequently, the samples were washed twice in 2× SSCT/50 formamide for 30 min, twice in 2× SSCT for 15 min and twice in 0.2× SSCT for 30 min. All solutions used from hybridization up to this step were prepared using DEPC-treated autoclaved deionized water and pre-heated to the hybridization temperature of 63 °C The samples were kept in a thermal block at the hybridization temperature throughout this portion of the experiment (even during exchange of solutions). After last SSCT wash, the samples were transferred back to room temperature and washed twice with RT/cold 1× PTw and blocked in 2% w/V Boehringer-Mannheim Blocking Reagent (Roche 11 096 176 001) in maleic acid buffer  for 1 h at RT, slowly rocking. The larvae were then incubated with anti-digoxigenin-AP, Fab fragments from sheep  and monoclonal anti-acetylated tubulin  at 4 °C overnight (the latter was only applied for fluorescent in situ hybridization) while shaking on a nutating mixer.On the third day, the samples were washed three times with PTw (5–10 min each) and four times in PTw (30 min each). After the last wash, the larvae were stored overnight at 4 °C in PTw. The next morning, the samples were briefly (5–10 min) washed again with PTw.® Blue Alkaline Phosphatase Substrate (Vector Laboratories SK-5300) solution which was prepared according to the manufacturer’s instructions with 100 mM Tris–Cl, pH 8.5, 0.2% Tween20. A signal was developed in the dark at room temperature while slowly rocking.In the case of fluorescent in situ hybridization, the larvae were washed twice with 100 mM Tris–Cl, pH 8.5, 0.2% Tween20, filtered through a 0.22-µm syringe filters (Merck Millipore), and stained on 4-well or 24-well plate in a Vector2  followed by two washes with the same buffer supplemented with 50 mM MgCl2. All solutions were filtered through a 0.22-µm syringe filter (Merck Millipore). The staining was done in the AP staining solution with MgCl2, 1.65 µl NBT (Roche 11 383 213 001) and 1.65 µl BCIP (Roche 11 383 221 001) per ml. Stock solutions of NBT and BCIP were first centrifuged for 2 min at 14,100 rcf to pellet the precipitated material. A signal was developed in the dark, overnight at 4 °C.For bright-field microscopy, after washing the larvae in PTw, we washed them twice (10 min each wash) in alkaline phosphatase (AP) staining solution without MgClIn both cases, the larvae were checked regularly for a developing signal. Staining was stopped by transferring them back to microtubes and washed five times (about 10 min) in PTw.® 555 goat anti-mouse IgG (H + L)  secondary antibody. After three washes  in PTw, the larvae were gradually transferred through 10-min washes in a dilution series of 33, 66 and 97% (two times) to 97% 2,2′-thiodiethanol  in PTw and stored at 4 °C in the dark for up to several weeks.For fluorescent in situ, larvae were blocked in a Blocking 1 solution  consisti2 to sea water with worms to the final concentration of 50 mM, which is substantially less than previously used by others [Adult worms were subjected to starvation for 2 days in order to empty their digestive tracts prior to dissection. They were then immobilized by the addition of 1 M MgCly others , 40, 60 ® Blue followed by staining with DAPI together with Alexa Fluor® 555 goat anti-mouse IgG (H + L)  secondary antibody. Stained samples were transferred to and stored in 97% TDE.Whole mount in situ hybridization was done using the same protocol that was used for larvae with following modifications (as described in ): proteiPdu-Tcf were washed 1× with distilled water, 1× in 70% EtOH for 30 s and 100% ethanol for 1 min. After the ethanol had been removed, it was replaced with 400 µl of Spurr low viscosity embedding resin  and incubated for at least 20 min while gently rocking. They were then placed into moulds filled with Spurr resin and left for 2 h at room temperature. The larvae were positioned and oriented within the moulds and placed at 72 °C overnight. Blocks were then sectioned to 4-µm thin sections.Larvae stained with NBT/BCIP for ® EdU Alexa Fluor® 594 Imaging Kit  according to the manufacturer’s instructions, followed by DAPI staining of nuclei overnight. Finally, the larvae were transferred gradually to 97% TDE mounting medium for confocal fluorescence microscopy.Chemical treatment with Wnt/β-catenin pathway activator or inhibitors was done from 5 dpf as described. At 6 dpf, 5-ethynyl-2′-deoxyuridine was added to the water to a 20 µM final concentration so it could be incorporated into the DNA of replicating cells until 7 dpf when the larvae would be fixed by 4% PFA/PTw and stored in 100% methanol. Proliferating cells were detected using Click-iTLarvae treated with Wnt/β-catenin pathway activator or inhibitors between days 5 and 7 of development were fixed and stored in 100% methanol. Dead or dying cells were detected using Click-iT™ TUNEL Alexa Fluor™ 488 Imaging Kit  and nuclei counterstained with Hoechst 33342 following the kit protocol. After counterstaining, the larvae were gradually transferred to 97% TDE in PTw.P. dumerilii were washed thoroughly with half a litre of filtered natural sea water (FNSW), treated with 0.1 mg/ml proteinase K in FNSW for 25 s to permeabilize the eggshell and then rinsed quickly with half a litre of FNSW. The zygotes were microinjected with the mixture of 0.4 µg/µl SuperTOPFlash-tdTomato (courtesy of Vladimir Korinek) and 1:5 Fast Green FCF dye. SuperTOPFlash-tdTomato carries the gene for tdTomato fluorescent protein under a promoter with 8 Tcf/LEF binding sites, which makes it a reporter that is responsive to Wnt/β-catenin signalling [Fertilized eggs of gnalling .z-stacks with a z-step of 0.42- and 0.42-µm pixel size resulting in cubic voxels. The appropriate wavelengths for excitation (633 or 635 µm for Vector Blue) and emission detection (720–800 µm for Vector Blue) and z-compensation of laser intensity and detector gain to compensate for signal loss with increasing depth in the sample were used. EdU and TUNEL stainings were imaged in Fusion software with 0.45 µm voxel size by Zyla 4.2 PLUS sCMOS camera (Andor) and the Dragonfly 503 spinning-disc confocal system (Andor) mounted on a Leica DMi8 core with HC PL APO 40×/1.30 OIL CS objective lens.Stained larvae were placed on a glass slide with three layers of Scotch tape (approximate thickness of each layer was 50 µm) as spacers in 80 µl of 97% TDE as a mounting medium which has the same refractive index as glass and low photobleaching . Immunofz-stacks. Maximum z-projections  or 3D reconstructions (for in situ hybridization) were done with FIJI software (in the latter case using its 3D Viewer plug-in). Images were cropped and resized in FastStone Image Browser, and the figures were assembled in Adobe Illutrator CS4. Proliferating cells marked by EdU incorporation and dead/dying cells marked by TUNEL staining were counted manually on maximal projections of whole z-stacks using the Cell Counter plug-in in FIJI. The differences between treatments were evaluated by a Student’s t-test.Brightness and contrast were adjusted linearly and uniformly in the same way for all Bright-field images and composite images of bright field and fluorescence were taken on a Nikon Diaphot 300 inverted microscope with DIC optics by Canon EOS1100D camera and utilizing the Canon EOS Utility software Remote Shooting function. We took a bright-field image from every individual after in situ hybridization and assigned them to categories according to the expression in the gut to quantify the effect of chemical treatment.Platynereis dumerilii. Phylogenetic analysis  variant on mixed stage cDNA which was used as a template Fig. a. We obsPlatynereis dumerilii has only one Tcf gene and a diversity of isoforms is produced by alternative mRNA splicing.Moreover, the end of C-clamp (−) Tcf is encoded by an intron of the same gene, directly following the basic tail and containing a cryptic termination codon. When the splice site is skipped and the intron retained, this stop codon prematurely terminates translation, resulting in a shorter protein that does not contain a C-clamp but does have the both β-catenin binding and HMG DNA binding domain, forming a potentially functional transcription factor of yet another isoform (here named X7). Although we observed C-clamp (−) C terminus in combination with only one HMG variant, we cannot rule out that it can also combine with the another one to making a putative isoform, X8. In summary, it appears that, like most protostomes, Pdu-Tcf and Wnt/β-catenin signalling’s role in Platynereis development, we performed whole mount in situ hybridization with an antisense digoxigenin-labelled probe that was complementary to the N-terminal part of Pdu-Tcf mRNA, which is common to and should hence detect all isoforms. We investigated Pdu-Tcf expression at 24, 48 and 72 hpf and 5 and 7 dpf larvae as well as adult worms.In order to get an overall view of the Pdu-Tcf was expressed at quite low levels broadly in both episphere  and hyposphere , except the area around the forming stomodaeum/blastoporus. In the episphere, it was detected slightly more laterally, while it was more abundant in ventrolateral region (neuroectoderm) of the hyposphere.At 24 hpf  added tPdu-Tcf in amputated tails of adult worms , which is incorporated into replicating DNA in vivo, to the water containing 6 dpf larvae. EdU contains an ethynyl group that after activation is later labelled with a fluorescent dye to visualize proliferating cells [Wnt/β-catenin signalling in general induces cell proliferation , and in ng cells .Platynereis at 7 dpf, most of the proliferating cells were located in the frontal part of the head and in the proliferative zone between the last segment and pygidium, surrounding the hindgut , at the base of second and third pair of parapodia, the jaws and on the foregut/midgut and midgut/hindgut borders. Only few proliferating cells were seen on the ventral side of midgut.In gut Fig. a. Some cOverall, there were significantly less proliferating cells throughout the body of IWR-1-endo treated larvae, whereas the number of proliferating cells in the presence of Wnt activator CHIR99021 was not changed Fig. c. Thus, We also checked for cell death by TUNEL staining of fragmented DNA. There were significantly more dead or dying cells in the larvae treated with CHIR99021 or IWR-1-endo. However, this test does not discriminate between apoptosis and other forms of cell death. Since most dead cells are present on the surface of the body, which is in direct contact with the environment, and the dead cells are seen in the presence of either activator or inhibitor, the cell death cannot account for the observed changes in gene expression inside the gut which are different for each chemical. Moreover, expression of some gut marker genes shows that a morphologically normal gut was present in all experimental groups.Platynereis gut consists of three major parts, a foregut, a midgut and a hindgut. To visualize the effect of chemical manipulation of the Wnt/β-catenin pathway on gut differentiation, we used the expression of several digestive enzymes that have been previously described to be specific to different Platynereis gut compartments [Pdu-Subtilisin-1 and Pdu-Subtilisin-2, a polysaccharide digesting enzyme Pdu-α-Amylase and a precursor of an intracellular digestive protease Pdu-Legumain.artments : extracePdu-Subtilisin-1 showed only faint expression in the hindgut and was primarily expressed in the midgut  were expressed at lower levels or diminished in the midgut upon Wnt/β-catenin inhibition  the leech Hellobdela triseralis [Platynereis, Pdu-Nk2.1 , sea uriewed in , ascidiaiewed in , Drosophosophila  and otheosophila , or it cosophila . Pdu-Cdxtynereis , 56 and apitella . We usedgut Fig. a, close ted Fig. . The chaTcf gene repertoire in invertebrates (both deuterostomes and protostomes) [Tcf gene, and their repertoire of isoforms has been underappreciated until recently, although their presence in other organisms as Drosophila has also been mentioned. Thus, it is not clear whether complex regulation by Tcf proteins is limited only to organisms that possess multiple Tcf/LEF genes or whether it is also present in Protostomia with only single Tcf gene, and if yes, how it is achieved. Here, we have shown that a large diversity of Tcf isoforms is present in a spiralian which can potentially compensate for the lack of gene diversity in invertebrates. According to our BLAST search, Tcf isoforms have been already found in the sequencing of many invertebrate genomes. From vertebrates, the top hits usually belonged to various isoforms of the Tcf7l2 (Tcf4) gene. Since the duplications of Tcf genes occurred only after the divergence of Platynereis and vertebrate lineages, this does not reflect closer homology than with other vertebrate Tcf genes, but it could rather point out to closest functional similarity. It is interesting that Tcf4 is able to both activate [Tcf1 and Tcf3 are considered to be purely an activator or an inhibitor of gene expression, respectively [It was previously pointed out that a great difference exists between the diversity and specialized functions of vertebrate Tcf proteins with their many isoforms in contrast to the poor ostomes) , 21. Theactivate  and repractivate  transcriactivate  and it iactivate , while Tectively . We hypoPlatynereis in an increasingly greater depth, most recently up to the midtrochophore stage. Expression profiling based on single-cell RNA seq, back-mapping to the larval body based on a reference gene expression atlas and subsequent clustering according to the typical expression fingerprints of major organ systems [Hnf4 and symmetrically positioned along the ventral midline on the level between second and third segment. Since Pdu-Tcf is expressed broadly in putative endodermal and mesodermal cells in the body of the 48 hpf larva, it is probable that it is expressed in Hnf4-positive cells as well. Moreover, Pdu-Tcf is present in internal clusters of cells at 72 hpf reminiscent of Hnf4+ putative gut precursor cells and might thus represent the developing gut. Hence, it is possible that Pdu-Tcf is expressed in the gut from the earliest stages of its development.The conservation of Wnt/β-catenin signalling’s role in digestive tract development still remains questionable due to a lack of detailed data from mostly invertebrate phyla, namely the group Spiralia, which do not contain any of the “classical” model organisms. Cell lineage tracing and fate mapping have been done several times in  systems  revealedPdu-Tcf transcripts can be detected in the hindgut and two paired domains (one of them comprising the jaws) in the foregut, but only slightly in the midgut of a 5 dpf larva. This may possibly be due to the fact that the midgut at this stage consists primarily of large not yet cellularized macromeres. Interestingly, this pattern corresponds to the expression of Tcf4 and Lef1 in the developing chicken gut, where they are necessary for formation of gizzard microvilli and differentiation of hindgut cells, respectively. However, in the cellularized gut of the 7 dpf larva, Tcf is already strongly transcribed in the midgut but less in hindgut evoking more the expression of Tcf4 in both developing and adult mammalian gut [Pdu-Tcf is expressed in the developing jaws, it is of note that the chitinous mandibles of treated larvae are smaller and less developed. Together with our finding that cell proliferation in the gut (and throughout the whole body) is severely reduced or abolished in larvae treated with a strong Wnt inhibitor, these results suggest that Wnt/β-catenin signalling is necessary for midgut cellularization which requires cell division, reminiscent of the Tcf4 role in the maintenance of mammalian adult gut epithelium [Later in development, lian gut , 85. Notithelium .Pdu-Nk2.1 shows that the midgut cells of larvae, whose Wnt/β-catenin signalling was inhibited, retained endodermal characteristics. Yet, the midgut marker genes Pdu-Subtilisin-1, Pdu-Subtilisin-2, Pdu-α-Amylase and Pdu-Otx, which are under normal circumstances expressed at high levels in the midgut but absent from the hindgut at 7 dpf, are lost upon pharmacological inhibition of Wnt/β-catenin pathway. At the same time, the hindgut marker gene legumain protease precursor expands from hindgut to midgut. Taken together, these two pieces of evidence suggest that midgut cells lose their ability to express digestive enzymes and obtain (or retain) hindgut-like characteristics, when Wnt/β-catenin is inhibited. All changes in gene expression are summarized in the scheme illustrated in Fig. Expression of endodermal marker gene Pdu-Subtilisin-1 and Pdu-Legumain are expressed in both the midgut and the hindgut, whereas we found that Pdu-Subtilisin-1 was predominantly a midgut gene and Pdu-Legumain was expressed strongly in the hindgut but only very weakly in the midgut . The likely explanation for this discrepancy comes from the fact that Williams and colleagues described the expression on 6 dpf stage, when cellularization of the gut is still occurring and the differentiation of the gut has not been completed. Initially, the midgut and hindgut cells are more alike, and only at 7 dpf stage, the expression patterns become more resolved with most of the digestive enzymes expressed in the broad midgut, while the narrow hindgut is dedicated more to defecation than digestion.There is small, but a notable difference between the expression patterns of digestive enzymes reported in our paper and in previous work conducted by Jékely group  which rePdu-Cdx can also be considered a hindgut gene, but unlike Pdu-Legumain, we saw quite the opposite effect on its expression, which was downregulated upon Wnt inhibition and upregulated in the midgut in the presence of a Wnt activator. Pdu-Cdx is a direct target that is activated by Wnt/β-catenin signalling [Cdx. The transcription of Cdx genes is fully triggered only by Tcf proteins that have a functional, intact C-clamp [Pdu-Tcf isoform is expressed in the gut, the involvement of a C-clamp (−) isoform might explain why Pdu-Cdx transcription is not activated in the midgut under normal circumstances, with the exception of the ventral floor and both foregut and hindgut borders that are closest to Wnt sources.gnalling . On the  C-clamp . AlthougPdu-Axin, but not dramatically, although reported to be the case at 55 dpf [Axin genes and only Axin2, but not Axin, has been shown to be regulated by Wnt/β-catenin signalling [Platynereis has most probably only one axin gene, whose responsiveness to Wnt/β-catenin signalling might be time and tissue dependent. Immunostaining against β-catenin clearly shows that the stronger inhibitor IWR-1-endo indeed reliably downregulates Wnt/β-catenin signalling and β-catenin staining and the effects on gene expression shared with IWR-1-endo suggest that the other inhibitor, JW55, works as well but produce milder phenotypes at the same concentration. The reliability of the activator CHIR99021 remains questionable since it always produced mild or no effects at this stage (except the missing Pdu-Otx-positive cells of the anus) although it works very well on younger larvae . The absence of strong phenotypic and gene expression differences in the presence of the activator can also suggest that the Wnt/β-catenin signalling has a permissive instead of an instructive role in gut development. In such a scenario, the pathway’s activity is required, but not sufficient for the gut endoderm to acquire midgut fate and gene expression fingerprint. Therefore, other transcriptions factors and signalling pathways would have to be involved to pattern the developing gut as has been shown for Hox genes [The observation that chemical manipulation of the Wnt/β-catenin pathway from 5 to 7 dpf does affect the expression of the Wnt target gene t 55 dpf , which mgnalling  even thognalling . On the ox genes , Hedgehoox genes , 89, BMPox genes  in otherPdu-Tcf itself might be a target of Wnt/β-catenin pathway. Upon Wnt activation, it is strongly transcribed in both the midgut and the hindgut, whereas the expression is lower after inhibition. This points to a positive feedback loop to reinforce Pdu-Tcf expression in the gut, where high amounts might be necessary. Positive autoregulation has been described before for LEF1 in colon cancer [Xenopus midbrain [Pdu-Tcf expression in the presence of Wnt/β-catenin inhibitors in the rest of the body, e.g. brain ganglia. This could be explained by the presence of a negative feedback loop to maintain steady and localized Pdu-Tcf expression. Such dual regulation in two different tissues, if confirmed, could be achieved by the action of tissue-specific sets of transcriptional cofactors. Further investigation will be needed to verify these results and ascertain the real situation about Pdu-Tcf autoregulation.Based on preliminary results, it seems that n cancer , XTcf-4 midbrain  and zebrmidbrain . It is amidbrain , 96, butmidbrain . InteresPdu-Tcf alone. Our in situ hybridization stainings show that it is expressed in the hindgut and some parts of foregut at 5 dpf, whereas it is present in both midgut and less in hindgut later at 7 dpf. The same is true for Pdu-Axin at 7 dpf. If we take a look at immunofluorescence staining of β-catenin as a proxy of Wnt/β-catenin pathway activity, we see it is present in high amounts in the midgut but not in the hindgut. This difference might be the results of the combinatorial action of factors involved in Wnt/β-catenin signalling cascade or in the modulation of Wnt signal, e.g. sources of Wnt proteins, expression of Frizzled receptors or soluble Wnt inhibitory proteins. When the Wnt/β-catenin pathway is inhibited, β-catenin diminishes from the midgut, whereas no change occurs in the hindgut, since β-catenin levels there are naturally low. As a result, the gene expression regulated by Wnt signalling via β-catenin in both parts of the gut exhibits hindgut-like characteristics.The differences between the midgut and the hindgut and the observed midgut to hindgut transition caused by inhibition of Wnt signalling Fig.  probablyTcf homologue in the marine polychaete annelid Platynereis dumerilii. It produces an array of mRNA variants via alternative splicing with a potentially different DNA binding capacity and function. Pdu-Tcf and several other components of Wnt/β-catenin signalling pathway  are present in the developing gut of Platynereis and inhibition of Wnt/β-catenin signalling causes the midgut to obtain a hindgut-like expression of digestive enzymes and leads to a loss in cell proliferation. Taken together, Platynereis appears to be a useful model to investigate the roles of Wnt/β-catenin signalling in organ development in a relatively simple system and to find features of gut differentiation and maintenance that have been conserved in Bilateria.In this paper, we described a single


Cohen's d statistic assumes that average values are good estimators of the position of a distribution of numbers and also assumes Gaussian (or bell-shaped) underlying data distributions. In this paper, we present an alternative evaluative statistic that can quantify differences between two data distributions in a manner that is similar to traditional effect size calculations; however, the proposed approach avoids making assumptions regarding the shape of the underlying data distribution. The proposed sorting statistic is compared with Cohen's d statistic and is demonstrated to be capable of identifying feature measurements of potential interest for which Cohen's d statistic implies the measurement would be of little use. This proposed sorting statistic has been evaluated on a large clinical autism dataset from Boston Children's Hospital, Harvard Medical School, demonstrating that it can potentially play a constructive role in future healthcare technologies.Effect size refers to the assessment of the extent of differences between two groups of samples on a single measurement. Assessing effect size in medical research is typically accomplished with Cohen's  The proposed sorting statistic is defined inx is equal to the median of y, then , as the numerator. The denominator represents a theoretical maximum possible distance between the positional estimates of the two distributions, if we accept the number of measurements provided in each distribution as fixed and the values of those measurements as fixed but allow class membership to vary. This normalization procedure forces the resultant sorting statistic to take on values ranging from 0 to 1, with high values representing pairs of distributions that are more dissimilar to each other and values near 0 representing pairs of distributions that are nearly identical. Note that if the median of  y, then  returns d statistic was performed, along with a correlation analysis comparing the proposed sorting statistic with the negatively valued Cohen's d statistic. All statistical analyses were performed with Matlab .A correlation analysis comparing the relationship between the proposed sorting statistic and the positively valued Cohen's d statistic and the proposed sorting statistic. A plot of the relationship between Cohen's d statistic and the proposed sorting statistic across all measurements is provided in Each of the 4788 measurements acquired using FreeSurfer was compared on a group-wise basis  using both Cohen's d statistic  as well as the strong negative correlation between the proposed sorting statistic and the negatively valued Cohen's d statistic . The strong correlations form a clear V-like pattern, the arms of the “V” representing a general agreement between the two measurements in assessing differences between the autistic and healthy populations, whereby increases in our proposed sorting statistic are strongly associated with Cohen's d statistic moving further away from zero. The region in between the two arms of the “V”  are highly overlapping and as you can see from their histograms  above about 0.1 may have potential in helping characterize autism as there are many more autistic samples with elevated GCSTS values relative to the healthy participants, despite the fact that low GCSTS values (~0.05) fall in a region of high overlap between our two groups of interest.Note the strong positive correlation between the proposed sorting statistic and the positively valued Cohen's l sulcus  whose Costograms ; these ad statistic , as well as highly negatively correlated with negative Cohen's d values . Measurements with an elevated sorting statistic and low Cohen's d . Although the distributions in Figures Gaussian distributions are extremely common in real-world data analysis, which has supported the widespread use of Cohen's d statistic because of assumptions made about the underlying data distribution. This helps demonstrate the proposed sorting statistical metric's potential to assist in the identification of feature measurements that may contribute to the accurate prediction of disease status in the context of a multivariate machine learning technology responsible for combining a series of measurements  to form a final prediction. Future work will assess this proposed sorting statistic's potential to play a role in assessing an individual measurement's potential to contribute to multivariate machine learning technologies for health care.The superior temporal region Figures  and 3 isd statistic of 0.2 corresponds to a small effect size, 0.5 to a medium effect size, and 0.8 to a large effect size. Based on our autism data and referring to a visual inspection of Ds = 0.2), medium effect sizes (Ds = 0.45), and large effect sizes (Ds = 0.7). Note that these values are very close to the equivalent values for Cohen's d statistic, facilitating interpretation of the results of the proposed sorting statistic. It should also be noted that because of the sorting procedure, there is increased computational requirements for the proposed sorting statistic relative to computing Cohen's d statistic.Cohen suggested that a It should also be noted that the proposed sorting statistic has potential towards being applied to sortable categorical variables, a data type for which standard mathematical analysis is not normally possible. To support assessment of group-wise differences in sortable categorical variables, the equation would need to be modified to replace the difference (minus operation) with a distance metric compatible with sortable categorical variables .


Previous research usually focused on single protein or gene in tumor development, actually highly heterogeneous nature and different signaling pathways largely contribute to tumor progression and tumor patients’ outcomes. Therefore, using combinatorial biomarkers to evaluate the prognostic features and guide management is gradually accepted and urgently needed. β-catenin is a well-known crucial factor in astrocytoma progression and it is involved in aquaporin1 (AQP1) mediated cell migration. In this study, we revealed the function of AQP1 in astrocytoma progression and provided the first clinical evidence that AQP1 expression was positively correlated with β-catenin. Furthermore, we proved the functional role of AQP1/β-catenin pathway in astrocytoma progression. More importantly, we discovered that combination of AQP1 and β-catenin expression was an independent prognosis factor for astrocytoma patients and it was a better survival predictor than either AQP1 or β-catenin alone. In conclusion, our study provided a novel more precise prognostication for predicting astrocytoma prognosis based on combinatorial analysis of AQP1 and β-catenin expression. Astrocytomas are the most common primary intracranial tumors in adults . Despitein vitro cell migration/invasion [Aberrant activation of Wnt/β-catenin signaling has been described in a range of malignancies including astrocytoma , 10. As invasion , 14.in vitro studies [Recently, β-catenin was revealed to be involved in aquaporin 1 (AQP1) mediated cell migration by  studies –17. AQP1 studies , 18. HowIn the present study, we explored the relationship between AQP1 and β-catenin by clinical and cellular analyses and demonstrated for the first time that AQP1 expression was positively correlated with β-catenin in astrocytoma specimens. We also discovered the functional role of AQP1/β-catenin pathway in astrocytoma progression. More importantly, we discovered that combination of AQP1 and β-catenin expression was an independent prognosis factor for astrocytoma patients and it was a better survival predictor than either AQP1 or β-catenin alone. Patients with AQP1 low/β-catenin low had the best outcome while the survival of the other three subgroups  was similar. Either high expression of AQP1 or β-catenin indicated a poor outcome. In conclusion, our study provided a novel more precise prognostication for predicting astrocytoma prognosis based on combinatorial analysis of AQP1 and β-catenin expression.P = 0.003, Figure P = 0.133, Table In the current study, we examined the expression of β-catenin in a cohort of 160 astrocytoma specimens , including 45 astrocytoma cases and 6 normal temporal lobe samples. The validation data confirmed that it was up-regulated in astrocytoma compared with normal tissues . The exogenous expression of AQP1 was confirmed by Western blot and kidney tissue from mouse was used as a positive control . Patients with AQP1 low/β-catenin low exhibited better outcome compared with other 3 groups Figure , while oP = 0.048; PFS: P = 0.015) independent of age, tumor size and histological grade.Therefore, combination of AQP1 and β-catenin expression had a better prognostic value and it was further confirmed by multivariate analysis. As shown in Table In our study, endogenous AQP1 expression could not be detected in LN229 cell line by Western blot. Only endogenous AQP1 expression in astrocytoma tissues could be found by immunohistochemistry analysis and Western blot. Previous study have pointed out that glioma cells were isolated and grown as cell lines would lose AQP1 expression such as U87 , STTG-1  and D54 (WHO grade IV) [In the present study, children patients were included in the cohort. According to WHO Classification of Tumors of the Central Nervous System 2016 (Revised 4th edition) and related literature, for diffuse midline glioma, H3 K27-mutant diffuse midline glioma predominates in children but can also be seen in adults. The finding of H3 K27-matation confers a worse prognosis than that of wildtype cases , 24. TheAccumulation of cytoplasmic β-catenin is an indispensable step for its translocation to nucleus . In thiset al. reported that the prognosis of patients with low expression of β-catenin tended to be better compared with patients with high expression of β-catenin by detecting 63 samples of astrocytoma [Altered expression and/or sub-localization of β-catenin have been reported to be correlated with poor survival in patients with craniopharyngioma, colorectal cancer, and breast carcinoma –30. Howerocytoma . While irocytoma . In thisIn conclusion, combined AQP1 and β-catenin expression may be used as a powerful predictor of survival of astrocytoma patients. It showed greater prognostic value than single marker and could be more reliable for clinical application.This study was reviewed and approved by Institutional Ethics Committee of Tianjin Medical University Cancer Institute & Hospital, and all the patients signed an informed consent for participation of the study and the use of their biological tissues prior to surgery. All experiments were performed in accordance with relevant guidelines and regulations of Institutional Ethics Committee of Tianjin Medical University Cancer Institute & Hospital.Paraffin-embedded specimens from 160 astrocytoma patients diagnosed between 1999 and 2013 were randomly selected. All patients had undergone surgical resection at the Tianjin Medical University Cancer Institute & Hospital and no patients had received radiotherapy or chemotherapy prior to resection. Two pathologists independently reviewed all histological slides and graded each astrocytoma according to 2007 World Health Organization criteria . PatientIn brief, sections (5 μm thick) were dewaxed, hydrated, and heated for antigen retrieval. They were blocked with hydrogen peroxide and normal goat serum, and subsequently incubated overnight with rabbit anti-human polyclonal antibody against AQP1  or mouse anti-human polyclonal antibody against β-catenin . All sections were stained with 3,3′-diaminobenzidinetetra-hydrochloride (DAB).Stained tissue sections were reviewed by two pathologists in a blinded manner based on a double scoring system (staining intensity multiplied by staining area). Staining intensity for AQP1 and β-catenin was scored as follows . The positive staining area of AQP1 was scored as follows: 0 (0%–25%); 1 (26%–50%); 2 (51%–75%); 3 (76%–100%), producing a total scores range of 0 to 9. The positive staining area of β-catenin was scored as 0–100, producing a total score range of 0 to 300. In our present study, low and high expression of AQP1 was regarded as score 0–4 and score 5–9, respectively. Low and high expression of β-catenin was regarded as score 0–99 and score 100–300, respectively.2 incubator at 37°C. Cells had been tested and authenticated by DNA (STR) profiling, work performed by Beijing Microread Genetics Co., Ltd. .LN229 human glioblastoma cell line was obtained from the American Type Culture Collection  and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in a 5% COhttp://www.addgene.org/). The sequences of the inserts were 100% correct. Lentiviruses were produced by co-transfection of lentiviral plasmid, packing plasmids ΔR and pVSVg into HEK-293T cells. After transfection, supernatant was collected and virus was used to infect LN229 cells. Lentivirus-infected cells were screened by 1 μg/ml puromycin for 2 weeks to establish stably cell clones (named AQP1/LN229) and verified by western blot analysis. GFP-labeled AQP1-overexpressed cells were named AQP1/LN229 and 3×Flag labeled AQP1-overexpressed cells were named 3×Flag-AQP1/LN229, respectively.Full length of AQP1 was amplified by PCR using primers for human AQP1 . AQP1 with a green fluorescent protein (GFP) label or 3×Flag label was cloned into pCDH-CMV-MCS-EF1-Puro lentiviral vector (5 different β-catenin specific RNA interference sequences were applied (#1-#5). The sequences were following: (#1: 5′-gctccttctctgagtggtaaa-3′); (#2: 5′-aggtgctatctgtctgctcta-3′); (#3: 5′-ttgttatcagaggactaaata-3′); (#4: 5′-gttctccgaacgtgtcacgt-3′); (#5: 5′-gcttggaatgagactgctgat-3′). Lentiviruses were produced by co-transfection of lentiviral vector and packing plasmids ΔR and pVSVg into HEK-293T cells. Supernatant was collected and virus was used to infect cultured AQP1 over-expressing LN229 cells. Expression of β-catenin was verified by western blot analysis and these cells were named AQP1-shβ-catenin/LN229.Cells were seeded in 35-mm dishes 24 hours before they were fixed. In AQP1/LN229 cells, AQP1 localization was directly analyzed by GFP fluorescence. In 3×Flag-AQP1/LN229 cells, AQP1 was stained with Alexa fluor 546-conjugated goat anti-rabbit IgG  after incubated with primary AQP1 antibody at 4°C overnight. β-catenin was stained with Alexa fluor 594-conjugated goat anti-mouse IgG  in AQP1/LN229 cells and Alexa fluor 488-conjugated goat anti-mouse IgG  in 3×Flag-AQP1/LN229 cells after incubated with primary β-catenin antibody at 4°C overnight. DAPI  was used to stain nuclei. Images were acquired using a fluorescence microscope (magnifications 200×).Astrocytoma tissues were washed three times with ice-cold PBS and then re-suspended with CO-IP lysis buffer (pH 7.4). Tissue lysates were gently rotated at 4°C overnight followed by centrifuging at 12,000g for 5 minutes and the pellet was discarded. Nonspecific protein was removed by adding Protein A  and gently rotated at 4°C for 1 hour. Then the mixture was centrifuged and the supernatant was divided into three groups with following different antibodies treatments. The precipitates were washed three times with CO-IP lysis buffer, re-suspended in sample buffer and boiled for 5 minutes for western blot examination.Cells (3×Flag-AQP1/LN229 and 3×Flag-vector/LN229) were washed three times with ice-cold PBS and then re-suspended with CO-IP lysis buffer (pH 7.4). Cell lysates were gently rotated at 4°C overnight followed by centrifuging at 12,000g for 10 minutes and the pellet was discarded. The supernatant was immunopurified with anti-Flag M2 affinity gel  and eluted with Flag peptides. The eluates were re-suspended in sample buffer and boiled for 5 minutes for Western blot examination.Frozen astrocytoma specimens of Figure Cells were seeded in 35-mm dishes and cultured for 72 hours. Following treatment with BrdU  for 48 hours, cells were incubated with mouse anti-BrdU antibody , followed by incubation with Alexa Fluor 594-conjugated secondary antibody  for 1 hour in the dark. Finally, cells were stained with DAPI (1 µg/ml) and BrdU-DAPI double-positive cells were regarded as BrdU-positive cells.Cells were plated and incubated in 24-well plates. At the end of the incubation period, viable cells were quantified using 3--2, 5-diphenyltetrazolium bromide (MTT). Briefly, 50 μl of the MTT stock solution (5 mg/ml) was added to each well. After 4 hours of incubation, medium was removed and the converted dye was solubilized with dimethyl sulfoxide. The absorbance of the converted dye was measured at a wave length of 570 nm.Briefly, stably transfected cells (AQP1/LN229 or vector/LN229) were seeded in complete medium in six-well culture plates containing a top layer of 0.35% agar and a bottom layer of 0.6% agar. The plates were then incubated at 37°C for 4 weeks and later stained with 0.005% crystal violet for 1 hour. Colonies larger than 50 µm were scored and photographed at 200× magnifications using an Olympus microscope .5 cells/well in 200 μl of serum-free DMEM containing 0.1% bovine serum albumin (BSA). 600 μl medium (serum-free DMEM containing 0.1% BSA) with 50 ng/ml recombinant human epithelial growth factor  were added to the lower chambers. The cells were allowed to migrate for 24 hours at 37°C. Afterward, cells at the upper layer of the membrane were scraped and cells at the lower layer were stained with Giemsa solution and photographed under a microscope. The number of cells was quantified in randomly selected fields.Migration assays were performed using 24-well transwell migration chambers  with polyethylene membranes (8 μm pore size). The upper chambers were seeded with 1.0×10in vitro. Briefly, transwell inserts for 24-well plates were coated with prediluted matrigel. 1.0 × 105 cells/well were loaded into the upper compartment of the chambers, and medium (serum-free DMEM containing 0.1% BSA) with 50 ng/ml EGF was added to the lower chambers. After 24 hours of incubation, the invading cells were fixed, stained, counted and photographed under a microscope in five pre-determined fields at 200× magnifications.Boyden chamber invasion assays were performed to measure cell invasion 2 test were performed for group comparisons and correlations between two variables were evaluated by Spearman’s Rank-Correlation test. Overall survival (OS) and progression-free survival (PFS) rates were estimated using the Kaplan-Meier method, and the log-rank test was applied to compute P-values. The Cox proportional hazards regression model was performed toward the identification of relevant prognostic factors. For analysis of in vitro cellular experiments, statistical significance for comparisons between groups was determined using a two-tailed Student’s t-test. All data was presented as mean ± standard deviation. Three independent experiments were performed. A two-sided P<0.05 was considered statistically significant in all analyses.The SPSS 17.0 software package was used for statistical analysis. Mann-Whitney U test, ANOVA test and χ


Fibrosis is characterized by fibroblast proliferation and fibroblast differentiation into myofibroblasts, which generate a relaxation-free contraction mechanism associated with excessive collagen synthesis in the extracellular matrix, which promotes irreversible tissue retraction evolving towards fibrosis. From a thermodynamic point of view, the mechanisms leading to fibrosis are irreversible processes that can occur through changing the entropy production rate. The thermodynamic behaviors of metabolic enzymes involved in fibrosis are modified by the dysregulation of both transforming growth factor β (TGF-β) signaling and the canonical WNT/β-catenin pathway, leading to aerobic glycolysis, called the Warburg effect. Molecular signaling pathways leading to fibrosis are considered dissipative structures that exchange energy or matter with their environment far from the thermodynamic equilibrium. The myofibroblastic cells arise from exergonic processes by switching the core metabolism from oxidative phosphorylation to glycolysis, which generates energy and reprograms cellular energy metabolism to induce the process of myofibroblast differentiation. Circadian rhythms are far-from-equilibrium thermodynamic processes. They directly participate in regulating the TGF-β and WNT/β-catenin pathways involved in energetic dysregulation and enabling fibrosis. The present review focusses on the thermodynamic implications of the reprogramming of cellular energy metabolism, leading to fibroblast differentiation into myofibroblasts through the positive interplay between TGF-β and WNT/β-catenin pathways underlying in fibrosis. Fibrosis is an irreversible and non-physiological scarring process associated with inflammation and increased extracellular matrix (ECM) deposition contributing to tissue damage. Fibrosis is characterized by fibroblast proliferation, fibroblast differentiation into myofibroblasts, and synthesis of ECM including collagen and proteoglycans. Several causes are known for fibrosis, such as infection, alcohol, drug, genetic alterations, radiation, or environmental factors . FibrosiFrom a thermodynamic point of view, myofibroblastic cells arise from exergonic processes and generate heat within their surroundings. The mechanisms leading to the development of fibrosis are similar to many irreversible processes that can occur due to a change in the entropy production rate ,11,12,13Several molecular mechanisms can induce and develop the fibrotic process. In fibrosis, thermodynamic behaviors of metabolic enzymes are modified by the dysregulation of both transforming growth factor β (TGF-β) signaling and the canonical WNT/β-catenin pathway ,15.Upregulation of TGF-β1 activates lactate dehydrogenase (LDH) expression, leading to lactate production, and induces fibroblast differentiation into myofibroblast and excessive collagen deposition ,17. MyofUpregulation of the canonical WNT/β-catenin pathway activates pyruvate dehydrogenase kinase-1 (PDK-1), which decreases pyruvate dehydrogenase complex (PDH) activity. Upregulation of canonical WNT/β-catenin pathway also stimulates Lactate Dehydrogenase A (LDH-A) and monocarboxylate lactate transporter-1 (MCT-1) . Thus, WTGF-β1 and WNT/β-catenin pathways leading to fibrosis can be considered dissipative structures that exchange energy or matter with their environment . These pWe focus this review on the thermodynamic implications in the reprogramming of cellular energy metabolism, enabling fibroblast differentiation into myofibroblasts through the positive interplay of the molecular signaling pathways TGF-β and WNT/β-catenin underlying the fibrotic process.Myofibroblasts are contractile non-muscle cells that exhibit bundles of actin filament containing α-SMA. Myofibroblasts are connected by α-SMA peripheral focal adhesions and gap junctions in the granulation tissue . FibroblDifferentiation of fibroblasts into myofibroblasts requires the participation of physical and chemical factors such as an increase in tissue stiffness ,27 and tThe molecular motor of myofibroblasts is the non-muscle myosin IIA (NMIIA) . NMIIA iMyosin filaments link actin filaments in thick contractile actomyosin bundles like stress fibers. The NMIIA molecules assemble into bipolar filaments. A tilted state of the myosin head enables a conformational change that moves actin filaments in an anti-parallel manner. The actin–myosin cross-bridge (CB) cycle of NMIIA is overall like smooth and striated muscle myosin. An ATP molecule binds the NMIIA-ATPase site on the head of the myosin, which enables the dissociation of actin from the NMIIA head, and the ATP hydrolysis. Subsequently, NMIIA binds to actin. The power stroke then occurs with a tilt of the NMIIA head, generating a CB single force with a displacement of few nanometers, and then releasing ADP from the acto-NMIIA complex, as the final stage of a CB cycle. A new CB cycle starts when a new ATP molecule dissociates actin from the myosin head.From a thermodynamic point of view, contractile tissues are open biological systems not in equilibrium state through the exchanges of energy and matter with their surroundings, and can operate either close to or far away from thermodynamic equilibrium ,33. In tHowever, the singularity of the motor molecular NMIIA that characterizes contractile non-muscular myofibroblast cells is its dramatically slow cycle of contractile kinetics compared to that of myosin of contractile muscle cells ,19. IndeIn the fibrosis process, myofibroblasts generate a relaxation-free contraction mechanism associated with excessive collagen synthesis in the ECM that promotes irreversible tissue retraction, evolving towards fibrosis and finally apoptosis of myofibroblasts ,34,35,36The mechanisms leading to fibrosis are like many irreversible processes that can occur due to a change in the entropy production rate. This rate represents a thermodynamic quantity, such as heat production, Gibbs energy, or ionic conductance, and quantifies these irreversible processes ,11,12,13The molecular signaling pathways leading to the development of fibrosis are considered as dissipative structures that exchange energy or matter with their environment. They operate as far-from-equilibrium open thermodynamic systems under non-linear regime evolving to non-stationary states. Circadian rhythms are also dissipative structures that operate as far-from-equilibrium thermodynamics ,12. MoreThe production ,40,41 anSeveral studies have shown that reactive oxygen species (ROS) have a major role in the fibrosis process. ROS production has been observed in several fibrotic tissues such as renal fibrosis and fibrotic myocardial infarction ,54,55,56ROS production is enhanced by TGF-β1 activation ,71,72,73Several studies have shown that tumor suppressor p53 is a critical factor in the fibrotic process . MoreoveROS production and inflammation act in a vicious circle in which inflammation stimulates ROS, which in turn enhance inflammation through activation of the NF-κB pathway ,95,96. IThe canonical WNT/β-catenin pathway is upregulated in fibrotic tissues such as the liver, skin, lungs, kidneys, and heart ,102,103.WNT extracellular ligands bind Frizzled (FZD) receptors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP 5/6), and disheveled (DSH), which results in β-catenin accumulation and then nuclear β-catenin translocation to bind with the T-cell factor/lymphoid enhancer factor (TCF/LEF) . TCF/LEFDownregulation of the WNT/β-catenin pathway leads to the absence of binding between the extracellular WNT ligands and the FZD/LRP 5/6 complex. Thus, adenomatous polyposis coli (APC), AXIN and glycogen synthase kinase-3β (GSK-3β) form a β-catenin destruction complex and mediate the proteasomal β-catenin degradation . GSK-3β NF-κB signaling, a key mediator of inflammation ,111,112,The phosphatidylinositol 3-kinase/serine/threonine kinase (protein kinase B)/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is involved in cell growth, cell proliferation, protein synthesis, and energetic metabolism ,131,132.WNT3a, a canonical WNT ligand, activates TGF-β1 expression via β-catenin-dependent Smad2 activation, leading to the differentiation of fibroblasts into myofibroblasts . ConversMoreover, activation of TGF-β also stimulates the WNT pathway by inhibiting dickkopf-related protein 1 (DKK1) expression, an inhibitor of WNT/β-catenin pathway . After TUpon WNT ligand activation, AXIN forms a complex with Smad7 and the E3 ubiquitin ligase Arkadia to promote the degradation of Smad7 , which iYAP and TAZ  mechanical signaling drives persistent fibroblast activation and sustainable fibrogenesis. Elevated YAP and TAZ levels are observed in fibrosis . YAP andStimulation by WNT3a inhibits the destruction complex because YAP/TAZ dissociates from the β-catenin destruction complex and then allows β-catenin nuclear translocation. TAZ binds DSH and dephosphorylates it upon stimulation with WNT3a. Then TAZ dissociates DSH from the destruction complex . Indeed,TAZ is also a downstream mediator of WNT signaling, independent of Hippo signaling . The actWNT/β-catenin signaling induces β-catenin and TAZ accumulation and activation of the TAZ-dependent gene responses .The non-Smad pathways such as MAPK, TGF-β activated kinase (TAK1), JNK, or PI3K/Akt are also involved in interactions between TGF-β1 and canonical WNT/β-catenin pathways ,165. PhoThe Warburg effect is involved in various tumor and non-tumor diseases  and occuAlthough aerobic glycolysis produces fewer ATP molecules than mitochondrial oxidative phosphorylation, glycolysis is much faster than oxidative phosphorylation . ConsequAerobic glycolysis generates the energy and metabolites involved in the regulation of cellular functions, including proliferation, extracellular matrix production, autophagy, and apoptosis .In fibrosis, aerobic glycolysis provides the necessary ATP, amino acids, and nucleotides for ECM synthesis , and is From an energy metabolism perspective, the TGF-β1 and WNT/β-catenin pathways are central regulators of glycolytic energy metabolism in fibrosis.Glycolysis is induced in a hypoxia-independent manner by (1) the WNT/β-catenin pathway activation via direct activation of WNT target genes, PDK1 and MCT-1, and via the stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) ,175,176;The TGF-β1 and WNT/β-catenin pathways activate each other in a positive feedback loop through (1) Smad signaling pathway via Smad2/3, Smad2/3/NOX4/ROS, and the Smad2/3-4 complex/YAP/TAZ, dependent on Hippo signaling; and (2) non-Smad signaling pathways such as MAPK, TAK1, JNK, and PI3K/Akt signaling .HIF-1α stabilization is sustained in a hypoxia-independent manner through the molecular signaling pathways, which consist of (1) WNT/β-catenin target genes, c-Myc and cyclin D1; (2) WNT/β-catenin-induced PI3K/Akt; (3) TGF-β1-induced PI3K/Akt; (4) WNT/β-catenin-induced TAZ independent of the Hippo signaling; PKM2/β-catenin complex-induced c-Myc; and (5) the positive interplay between the TGF-β1 and WNT/β-catenin pathways.HIF-1α is a heterodimeric nuclear transcription factor composed of two subunits: HIF-1alpha and HIF-1β. HIF-1α stabilization results in its nuclear translocation, dimerization with HIF-1β, and the transcription of genes encoding glycolysis enzymes . HIF-1α Aberrant HIF signaling after HIF-1α stabilization induces energy metabolism reprogramming in the context of fibrosis by increasing glycolysis energy metabolism and decreasing the entry of glucose-derived carbons into the TCA cycle, reducing the mitochondrial oxidative phosphorylation energy metabolism ,179. HIFHIFs reprograms glucose and glutamine metabolism to promote the Warburg effect. HIF-1α stabilization increases glycolysis by (1) overexpression of glucose transporters (Glut) mediating increased glucose uptake and (2) activation of glycolytic enzymes such as Glut, HK2, PKM2, LDH-A, and PDK1 ,182,183.PI3K/Akt signaling is activated by cytosolic β-catenin accumulation ,146. ActGlut-1 and Glut-3 are essential for the insulin-sensitive homeostasis of glucose transport . After gd-fructose-6-phosphate to β-d-fructose-1,6-biphosphate and stimulates aerobic glycolysis during fibroblast differentiation into myofibroblasts to generate sufficient energy and stabilize HIF-1α levels [The allosteric enzyme phosphofructokinase (PFK) catalyzes the conversion from β-α levels . This coα levels . In addiα levels ,194.The shunting of glucose carbons far from the TCA cycle results in glutamine metabolism reprogramming. The WNT target gene c-Myc stimulates glutaminolysis and drives glutamine uptake into the cytosol and the mitochondria . c-Myc-aUpregulation of glycolysis, despite an overall decrease in mitochondrial respiration, results in increased levels of the TCA cycle intermediate succinate . The dicTGF-β also induces HIF-1α stabilization and LDH5 expression via TGF-β-induced HIF-1α overexpression, which promotes glycolysis energy metabolism and lactate production through activating HIF-1α-regulated glycolytic enzymes in idiopathic pulmonary fibrosis . TGF-β iHypoxia can also be critically involved in fibrosis, and stabilizes HIF-1α by inhibiting HIF prolyl-hydroxylase, which depends on oxygen as a cofactor for its enzyme activity. Thus, hypoxia causes lowered HIF prolyl-hydroxylase activity, leading to HIF-1α stabilization . Thus, tNumerous biological systems exhibit oscillatory behaviors such as glycolysis ,212, or The numerous transcription factors, responsible for the regulation of circadian rhythms, are circadian locomotor output cycles kaput (Clock), brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 , Period 1 (Per1), Period 2 (Per2), Period 3 (Per3), and Cryptochrome (Cry 1 and Cry 2) ,215. TheThe Clock/Bmal1 heterodimer also activates the transcription of retinoic acid-related orphan nuclear receptors, Rev-Erbs, and retinoid-related orphan receptors (RORs). Bmal1 transcription can be activated by RORs via a positive feedback loop, whereas they can be repressed by Rev-Erbs via a negative feedback loop .The imbalance of circadian rhythms is directly related to a negative feedback loop performed by a protein on its own gene expression ,220. CirCircadian rhythms regulate numerous physiological and metabolic functions, such as heart rate, blood pressure, body temperature, sleep–awake, and feeding patterns . SimilarmPer2−/− mouse models develop severe liver fibrosis [The fibrotic process is associated with irregular circadian phases ,224,225.fibrosis . Per2 exfibrosis . Diminutfibrosis ,228. Bmafibrosis . ROS havfibrosis . Howeverfibrosis , which pfibrosis . High blfibrosis ,234.ROR expression is increased during fibroblast differentiation into myofibroblasts . ROR actBmal1 has direct transcriptional control on components of TGF-β signaling . Smad7 aN-acetyltryptamine), naturally secreted by the pineal gland [Melatonin  the WNT/β-catenin pathway activation via direct activation of WNT target genes, PDK1 and MCT-1 and via the stabilization of the hypoxia-inducible factor 1 alpha (HIF-1α), and (2) the TGF-β1 pathway activation via the HIF-1α stabilization. TGF-β1 and WNT/β-catenin pathways activate each other in a positive feedback loop through (1) the Smad signaling pathway via Smad2/3, Smad2/3/NOX4/ROS, Smad2/3-4 complex/YAP/TAZ dependent of the Hippo signaling, and (2) the non-Smad signaling pathways such as PI3K/Akt signaling.HIF-1α stabilization is sustained in a hypoxia-independent manner through molecular pathways: (1) WNT/β-catenin target genes, c-Myc and cyclin D1; (2) WNT/β-catenin-induced PI3K/Akt; (3) TGF-β1-induced PI3K/Akt; (4) WNT/β-catenin-induced TAZ independent of the Hippo signaling; PKM2/β-catenin complex-induced c-Myc; and (5) the positive interplay between the molecular pathways TGF-β1 and WNT/β-catenin. Aberrant HIF signaling with HIF-1α stabilization induces energy metabolism reprogramming in the context of fibrosis by increasing glycolysis energy metabolism and decreasing the entry of glucose-derived carbons into the TCA cycle, thereby reducing mitochondrial oxidative phosphorylation energy metabolism. HIF-1α is a central regulator of the glycolysis energy metabolism involved in organ fibrosis, indicating a similarity between inflammation and cancer.The molecular mechanisms of the fibrotic process, including the TGF-β1 and WNT/β-catenin pathways, can be considered dissipative structures that exchange energy or matter with their environment. They are open systems working far from the thermodynamic equilibrium and operate under a non-linear regime evolving towards non-stationary states. Indeed, circadian rhythms, which are also far-from-equilibrium open thermodynamic systems, directly drive the fibrotic process by regulating the molecular pathways TGF-β1 and WNT/β-catenin involved in the reprogramming of cellular energy metabolism, enabling the differentiation of fibroblasts into myofibroblasts and leading to the fibrotic process.


N = 1643) review was undertaken to explore the relationship between these ten molecular subtypes and traditional clinicopathological features. IntClust subtypes were significantly associated with histological type, tumour grade, receptor status, and lymphocytic infiltration (p < 0.0001). Lymph node status and Nottingham Prognostic Index [NPI] categories were also significantly associated with IntClust subtype. IntClust 3 was enriched for tubular and lobular carcinomas, the latter largely accounting for the association with CDH1 mutations in this cluster. Mucinous carcinomas were not present in IntClusts 5 or 10, but did not show an association with any of the remaining IntClusts. In contrast, medullary-like cancers were associated with IntClust 10 (15/26). Hormone receptor-positive tumours were scattered across all IntClusts. IntClust 5 was dominated by HER2 positivity (127/151), including both hormone receptor-positive (60/72) and hormone receptor-negative tumours (67/77). Triple-negative tumours comprised the majority of IntClust 10 (132/159) and around a quarter of IntClust 4 (52/217). Whilst the ten IntClust subtypes of breast cancer show characteristic patterns of association with traditional clinicopathological variables, no IntClust can be adequately identified by these variables alone. Hence, the addition of genomic stratification has the potential to enhance the biological relevance of the current clinical evaluation and facilitate genome-guided therapeutic strategies.The integration of genomic and transcriptomic profiles of 2000 breast tumours from the METABRIC [Molecular Taxonomy of Breast Cancer International Consortium] cohort revealed ten subtypes, termed integrative clusters (IntClust/s), characterised by distinct genomic drivers. Central histopathology ( Molecular subtypes of breast cancer, defined by genetic alterations and expression patterns, tracks with traditional classification parameters. A team including histopathologists from the University of Cambridge, UK, and the University of Nottingham, UK, correlated the 10 subgroups of breast cancer previously discovered by analysing the genomic and transcriptomic architecture of 2000 tumours to standard diagnostic indicators routinely assessed by pathologists. Considering 1643 samples from these cases with available histopathological review data, the researchers showed that the novel subtypes were significantly associated with tumour type, tumour grade, receptor status, lymph node status, the extent of infiltration of anti-tumour immune cells, and the score of a commonly applied clinical assessment tool called the Nottingham Prognostic Index. However, these traditional variables alone could not fully demarcate the molecularly defined subgroups, suggesting that a combined approach holds potential value for evaluating patients and guiding therapeutic decision-making. Approaches for effective and systematic genomic stratification of BCs are urgently required in order to facilitate therapeutic strategies.The molecular heterogeneity of breast cancer (BC) is well-recognised.3 mostly reflect tumour classification based on these markers alongside proliferation, with luminal subtypes showing ER and/or PR expression, a HER2-positive subtype, and basal-like BCs being usually negative for all three receptors (triple-negative). This is now reflected in the most recent TNM prognostic stage grouping, which incorporates anatomic stage, grade, ER/ PR and HER2 receptor status and Oncotype Dx recurrence score [8th edition AJCC cancer staging manual].5The traditional classification of BC has utilised tumour morphology and assessment of oestrogen receptor [ER], progesterone receptor [PR] and human epidermal growth factor receptor 2 (HER2) expression. Expression signatures7 The METABRIC  study identified ten subtypes of BC termed integrative clusters (IntClust), by joint analysis of copy number and expression data to detect the cis genomics.8 These ten subtypes show characteristic copy number aberrations (CNAs), and importantly are associated with distinct patterns of survival and response to neoadjuvant chemotherapy.9 IntClusts 3, 4, 7 and 8 have the best prognosis, IntClusts 1, 6 and 9 have an intermediate prognosis, and IntClusts 2, 5 and 10 a poor prognosis.10 IntClust 4 comprises a mixture of ER-positive and -negative tumours and is characterised by a relative paucity of CNAs and a gene expression signature reflecting immune activation. The majority of ER-positive and HER2-negative tumours are distributed within 8 IntClusts , but have variable degrees of genomic instability and distinct CNAs. For example, IntClust 3 has low genomic instability and a high frequency of PIK3CA mutations, IntClust 6 has amplification of 8p12 with upregulation of ZNF703, a common Luminal B BC oncogene11 and IntClust 2 has high genomic instability and amplification of 11q13/14. IntClust 10, composed of tumours with a high rate of TP53 mutations and 5q deletion, has a very poor prognosis in the short term, but patients surviving beyond 6 years following treatment have an excellent long-term outcome. IntClust 5, associated with HER2 amplification, has the worst prognosis in this cohort of patients derived from the pre-trastuzumab era.Next-generation sequencing has further refined the molecular profiles.The tumours in the original METABRIC cohort were collected between 1977–2005 from five centres in the UK and Canada. The original annotation of these tumours was based on the primary pathology reports, with obvious differences in terminology for the classification of histological tumour types over time and between the five contributing centres. Hence, the relationship between the IntClust subgroups and traditional clinicopathological factors has not been systematically investigated to date. Here, we have addressed this shortcoming by conducting detailed central review of the tumour pathology of the majority of cases comprising the original METABRIC study and have tested for associations with IntClust subtype.A total of 1643 cases (83%) from the METABRIC cohort were available for central pathology review. The key clinicopathological features of these cases are provided in Supplementary Table p < 0.000001; for significant Chi-square residuals see Fig CDH1 mutations. On closer analysis, this is largely related to the lobular carcinomas within this IntClust; as would be expected, none of the tubular carcinomas in IntClust 3 harboured CDH1 mutations (OR 0.38 for tubular vs. 20.42 for lobular BC). Mixed tumours were also increased in IntClust 8. Medullary carcinomas were associated with IntClust 10 , which did not contain any tubular, lobular or mucinous carcinomas. Mucinous carcinomas were distributed in IntClusts 3, 4, 7 and 8, but did not show an association with any one cluster.The IntClusts showed significant associations with tumour type (p = 0.02). Looking at other variables, lesion size was not correlated with differential distribution of lobular BC across IntClusts (p = 0.46).Of 114 lobular carcinomas, 91 were in good prognosis IntClusts (80%), ten were in intermediate prognosis IntClusts (9%), and 13 were in poor prognosis IntClusts 2 and 5 . 23 lobular carcinomas were of the solid/pleomorphic varieties, traditionally associated with worse clinical outcome. However, 13 of these fell in the good prognosis IntClusts (57%), and conversely, only 5/13 lobular carcinomas in IntClust 5 and 2 were of solid/pleomorphic subtype Table . While hDuctal NST tumours with apocrine differentiation fell in all IntClusts except IntClust 2, with the largest proportion in IntClust 10  Table . The disp < 0.000001; Fig. p < 0.000001) of grade: tubule formation, nuclear pleomorphism and mitotic count  predominantly fell in IntClusts 3, 4 and 8 (Suppl Table p < 0.001). Two-hundred and thirty of 1637 cases showed high levels of lymphocytic infiltration, and they were predominantly in IntClust 4 (n = 57) and 10 (n = 64) , representing 20% and 35% of cancers in these IntClusts, respectively . Strengths of association of ER and HER2 within IntClusts are depicted in the residual Chi-Square analysis in Fig. Hormone receptor and HER2 status across the entire cohort is summarised in Supplementary Table ER + HER2- tumours were associated with IntClusts 3, 6, 7 and 8. ER + PR + cases formed the majority in IntClusts 2 (73%), 3 (64%), 6 (60%), 7 (69%), 8 (75%) and 9 (51%), while ER + PR- was the predominant fraction in poor prognosis IntClust 1 (47%).HER2 status was available from IHC supplemented by copy number analysis where required for 1611 cases  and formed the largest proportion of cases within this group ; of the remainder, 23% (52/227) group in IntClust 4 and form 24% of this cluster (52/217).The remaining IntClusts except IntClust 5 showed a negative association with ER−HER2- status.p < 0.001). IntClusts 1, 5, 6 and 9 showed a greater frequency of lymph node positivity compared to other IntClusts , 384 cases (49%) were in good prognosis IntClusts, and only 216 (28%) were in the poor prognosis IntClusts. Lymph node negative cases were mostly  distributed in good prognosis IntClusts, but 138 (16%) were in intermediate prognosis and 184 (21%) were in poor prognosis IntClusts.A significant association was observed between lymph node status and IntClust (p < 0.001) with the NPI status . Heavier lymphocytic infiltration was observed in ER − (43%) vs. ER + cancers (12%) (p < 0.001). ER + cancers were evenly spread across good and poor NPI categories , whereas ER − cancers were skewed towards poorer NPI prognostic categories (79%) (p = 0.0004).Seventy-five cases were ER − (27%) and 204 were ER + (73%); ER status was unavailable in 1 case. The ER + tumours were of lower grade (17% grade 1 and 15% grade 3), compared with ER − tumours ; 2 (LumB 48%); 3 (LumA 69%); 4 (LumA 34%); 5 (Her2 55%); 6 (LumB 49%); 7 (LumA 66%); 8 (LumA 67%); 9 (LumB 48%) and 10  , 2 (66%), 6 (71%), 9 (67%) while ER + /Her2-ve, low proliferation BCs predominated in IntClusts 3 (70%), 4 (45%), 7 (46%) and 8 (51%). Eighty-three percent of IntClust 5 was Her2 + while 75% of IntClust 10 was ER − /HER2-ve  and this may skew the distribution of pathological and prognostic variables within the IntClusts. Histopathological parameter modelling of prognostic effects within the IntClusts, though attempted, was underpowered in sub-categories to draw definite conclusions. Addressing the prognostic and predictive effects of histological features within the IntClusts will require analyses on larger data sets from multiple sources, with detailed pathological and clinical annotation.Our study has several limitations. Materials were from archival samples at multiple institutions and pre-dated the trastuzumab era. There is a bias towards banking of large cancers in the historical tumour bank collections that comprise the METABRIC cohort , 4 fell in IntClust 8  and 6 belonged to IntClust 4 . This illustrates that a histologically distinct special type BC can have diverse genomic drivers.Tubular carcinomas, a well-recognised special type of BC, showed the least variability and were distributed in the good prognosis IntClusts consistent with the excellent behaviour of tubular carcinomas documented in other series.CDH1 mutations, and this is secondary to the association with lobular carcinoma. Although the majority of lobular carcinomas belong to the good prognosis IntClusts, a significant number were found in the intermediate and poor prognosis IntClusts. This distribution was not explained by pleomorphic or solid subtype, but showed an association with grade 3 lobular tumours. This supports the findings of Rakha et al.,15 who found an association with worse prognosis for grade 3 lobular carcinomas, but not the pleomorphic subtype on its own. Of interest, the two largest groups of lobular carcinomas were present in IntClust 3 and 4; both defined by low genomic instability. The former is associated with frequent PIK3CA mutations, while the latter is associated with immune response. Immune related and hormone-related subtypes of lobular carcinomas have been recently described in the TCGA (The Cancer Genome Atlas)16 and RATHER cohorts.17 This may offer novel treatment options for lobular BC dependent upon the molecular profile.Other typically ER + histological types, including lobular and mucinous cancers, showed a more variable distribution across IntClusts. IntClust 3 is enriched for 18Mucinous tumours were relatively evenly distributed across all IntClusts except IntClusts 5 and 10. This finding highlights the heterogeneity in underlying genomic drivers of mucinous tumours despite their distinctive morphological appearance and typical ER + HER2− phenotype. Mucinous carcinomas less frequently harboured 1q and 16p gain and 16q and 22q loss compared to other histotypes in this series, as described by others.19 Here, most triple-negative and basal-like cancers including medullary-like tumours belonged to IntClust 10, which is associated with the greatest genetic instability, harbouring characteristic cis-acting alterations  associated with impaired DNA damage repair and cell-cycle checkpoint regulation. However, a significant proportion also fell in IntClust 4 characterised by low genomic instability and prominent lymphocytic infiltration. A CD8 + lymphocyte infiltrate is a good prognostic feature in basal-like cancers21 and further evidence is emerging that lymphocytic infiltration may risk stratify patients.22 It has become apparent that a subset of ER − breast tumours, including those that belong to IntClusts 4 and 10, are associated with a significant immune response conferring better outcome.The prognostic relevance of medullary-like BC remains controversial.23 fell within good and intermediate prognosis IntClusts. Despite their association with lymphovascular invasion and suggested poorer outcome in some series,24 micropapillary carcinomas fell across the good prognosis IntClusts 3, 4 and 7 with two in IntClust 9 defined by high-genetic instability, 20q amplification and intermediate prognosis. The two Adenoid cystic carcinomas both fell into IntClust 4, consistent with their known indolent behaviour despite their basal-like features, and have recently been shown to harbour a characteristic MYB-NFIB gene translocation with low background genetic instability.26Some special type tumours were present in relatively small numbers in the series precluding ability to draw definitive conclusions. Papillary carcinomas, which are a mixture of Luminal A and B types,27 Interestingly, mixed-NST and special type tumours predominantly fell in the good prognosis ER + IntClusts 3 and 8, possibly reflecting more indolent underlying molecular biology related to the special type component.Ductal NST carcinomas lacking the characteristics of a special histological type form a morphologically and prognostically heterogeneous group, so it is unsurprising that they are distributed across all IntClusts. Two thirds of the NST carcinomas showing apocrine differentiation fell in the intermediate and poor IntClusts, with only 10% in IntClust 5 consistent with reported HER2 positivity rates for these tumours.28 Although there was an association between grade and IntClust, tumours of grades 1–3 fell across all IntClusts except 10. Grade 1 tumours were more predictive of IntClust subtype, whereas grade 3 tumours were found in all IntClusts including those associated with good clinical outcome. Hence, tumour grade has limited utility in predicting the underlying genomic drivers of an individual cancer. Grade 3 cancers in intermediate and poorer prognostic IntClusts had a higher proportion of BC-specific deaths  than grade 3 cancers in good prognosis IntClusts . Hence the genomic heterogeneity may explain differential rates of BC-specific death within grade 3 BC.Grade is a routinely assessed traditional pathological prognostic factor, which shows strong association with patient outcome.p = 0.001), as well as ER + (p = 0.02) but not ER-ve subgroups (p = 0.89). A recent study of the molecular portraits of BC including the METABRIC cohort29 indirectly surmised that differentially expressed genes associated with poor tubular differentiation differed from signatures of either marked nuclear pleomorphism or higher mitotic counts, which were both enriched for proliferation genes. Given the sporadic distribution across IntClusts for BCs with poor tubular differentiation, it is unsurprising that these are not enriched for a particular gene-set, and the tubular differentiation component of grade may be a product of several biologically complex events.Looking at the individual components of grade, poor tubular differentiation was the only component that was significantly associated with BC-specific deaths in the whole cohort  and HER2 copy number data (HER2 positivity defined as copy number > 6). Of these, 59 cases (92%) appear to have false-negative immunohistochemical staining, with a HER2 copy number ranging from 6.02 to 51.2 (mean 12.9). False-negative immunohistochemistry results may be explained by inadequate fixation in this historical series of tumours, and by sampling errors in TMAs related to intralesional heterogeneity. Fourteen false-negative cases fell within IntClust 5 (mean HER2 copy number 22.4), strengthening the association between this IntClust and HER2 amplification. An analysis of the NOAH study found that HER2-enriched tumours showed a higher pathological complete response rate following trastuzumab therapy compared with non-HER2-enriched clinically HER2 + tumours.33 Patients in the METABRIC cohort had not received trastuzumab, and it remains to be demonstrated if the IntClust 5 group respond better to HER2-targeted agents.HER2 + tumours predominate in IntClust 5 regardless of ER status, but they also occur in other groups. The diversity of clinically HER2 + tumours has been reported previously; using PAM50 only 64% are assigned to the HER2-enriched group, the remainder distributing across all intrinsic subtype categories.34 The copy number high Luminal A sub-class has been shown to have a poorer prognosis compared to the low copy number variety.34 This category harbours 8q gain, 5q loss and 20q gain and parallels the intermediate and poor prognosis IntClusts in the METABRIC landscape. Given the ability of METABRIC IntClusts to provide prognostic information, it is unsurprising that the IntClusts associate strongly with parameters such as grade and lymph node status and to the traditional NPI.35 However, positive lymph node status or high NPI can occur even in the good prognosis IntClusts. IntClust subtype provides a basis for the behavioural diversity in historical prognostic models, and integration of genomic data with clinicopathological factors offers superior prognostic models to either variable alone.37Pathway analysis within the IntClusts can help us understand why some tumours with distinctive morphology behave aberrantly in terms of prognosis. Molecular interrogation of different histological subtypes of BC for such new drivers will help further unravel links with microscopy. For example, the molecular profile of ER-positive luminal A BCs has been investigated from six different data sets including the TCGA and METABRIC.In summary, we have conducted a central pathology review of the largest case series of BC with matched genomic data. While the genome-driven IntClusts are significantly associated with traditional clinicopathological variables, IntClust subtype could not be reliably predicted based on current histological parameters alone. Adoption of genome-guided therapeutic strategies first requires classification of tumours into homogenous groups in terms of genomic alterations. IntClust subtypes are classified on this basis; therefore, a test for prospective assignment of IntClust subtype in the clinical setting, especially for predictive analyses, would be useful.8 IntClusts 3, 4, 7 and 8 are defined as the good prognosis groups, IntClust 1, 6 and 9 as intermediate prognosis groups, and IntClust 2, 5 and 10 as poor prognosis groups.10All patient samples were acquired with appropriate consent from respective institutional review boards . DNA and RNA isolation from samples was followed by hybridisation to the Affymetrix SNP 6.0 and Illumina HT-12 v3 platforms for profiling. Full details of genomic and transcriptomic profiling and CNA/ variation analyses and allocation to integrative clusters are described in Curtis et al.n = 1980) only if histology slides were available for central pathology review along with matched clinical and expression analyses data. Any cases with no tumour slides available or non-invasive disease only were excluded from analysis.Cases were included from the METABRIC cohort , lymphocytic infiltration, presence of lymphovascular invasion, and presence of in situ disease (DCIS and LCIS) and precursor lesions. For purposes of analyses, tumour types were grouped as follows: tubular , lobular , mucinous, medullary-like, NST, mixed-NST and special type, and other rare types .A representative H&E section from each case was reviewed by a specialist breast pathologist . The following variables were assessed: histological tumour type,n = 405) were reviewed by two or more of the study histopathologists. Any discrepancies in histological type and grade between the central reviewers were resolved by consensus review of scanned images. The concordance between the specialist breast pathologists was excellent (kappa statistic 0.9) for all variables assessed.At least 25% of the cases  or ‘high’ (moderate to severe).40 Micro-arrays were stained for ER, PR and HER2 using routine immunohistochemistry protocols summarised in Supplementary Table 41 The proportion of cells staining positively (Allred score) was used to determine ER and PR status: 0 = 0, < 1% = 1, 1%–9% = 2, 10%–32% = 3, 33–66% = 4, > 66% = 5.42 Any score of 2 or above was deemed positive. For cases with missing IHC data (due to missing/unavailable cores), the ER, PR status was supplemented from the histopathology reports at the time of diagnosis. HER2 was scored as per UK guidelines.43 A 2 + score or missing HER2 status was supplemented by results from original histopathology reports, if available, or from METABRIC HER2 gene copy number analysis ( > 6.0 copies deemed positive).44Tissue micro-arrays were constructed from the BC cases as per standard protocol.p-value < 0.01) and, therefore, the distribution of tumour grade does not explain properly the distribution of histological types in each IntClust.In order to determine whether clinicopathological variables could predict molecular subtype, ten multivariable logistic regression models were fit for each of the ten IntClust groups, with the dependent variable as one IntClust vs. the remaining nine clusters. Clinical and pathological variables  were taken as predictors of each IntClust. The predicted probability of a tumour belonging to each of the ten clusters was derived based on these models. Final IntClust subtyping was based on the highest assigned probability and these assignments compared to gold-standard subtyping based on genomic data. Also, to evaluate whether grade or receptor status explains the differences in histological types within IntClusts, a log-linear model of conditional independence:  and a more general model that also includes the association between IntClusters and Histology:  were fitted. The two models were compared with the Deviance test, showing that the more general model produced a better fit to the data , may be downloaded by requesting the METABRIC Data Access Committee. Gene expression data, copy number data from the original METABRIC publication can be found on the freely available cBioPortal. Published IDs for the cases included in the study have been provided in a supplementary file .Supplementary File 1Supplementary File 2Supplementary File 3Supplementary File 4Supplementary File 5


HIV leads to a spectrum of neurologic diseases collectively termed HIV‐associated neurocognitive disorders (HAND). Drug abuse, particularly methamphetamine (meth), is a frequently abused psychostimulant among HIV+ individuals and its abuse exacerbates HAND. The mechanism by which HIV and meth lead to brain cell dysregulation is not entirely clear. In this study, we evaluated the impact of HIV and meth on astrocyte senescence using in vitro and several animal models. Astrocytes constitute up to 50% of brain cells and play a pivotal role in marinating brain homeostasis. We show here that HIV and meth induce significant senescence of primary human fetal astrocytes, as evaluated by induction of senescence markers , senescence‐associated morphologic changes, and cell cycle arrest. HIV‐ and meth‐mediated astrocyte senescence was also demonstrated in three small animal models . Senescent astrocytes in turn mediated neuronal toxicity. Further, we show that β‐catenin, a pro‐survival/proliferation transcriptional co‐activator, is downregulated by HIV and meth in human astrocytes and this downregulation promotes astrocyte senescence while induction of β‐catenin blocks HIV‐ and meth‐mediated astrocyte senescence. These studies, for the first time, demonstrate that HIV and meth induce astrocyte senescence and implicate the β‐catenin pathway as potential therapeutic target to overcome astrocyte senescence.Emerging evidence suggests that cell senescence plays an important role in aging‐associated diseases including neurodegenerative diseases.  In particular, HIV‐infected individuals are at an increased risk of neurocognitive and motor impairments  activity and p16Campisi, . p16INK4et al., et al., et al., et al., et al., β‐Catenin is a central mediator of the Wnt/β‐catenin pathway. The Wnt/β‐catenin signaling pathway is vital to various functions in the CNS including neurogenesis, neurotransmitter release, induction of long‐term potentiation and depolarization resulting in increased synaptic strengths, and memory consolidation ; at day 6, HFAs were stained for SA‐βGal. Meth significantly induced SA‐βGal expression even at the lower meth dose of 10 μm  for 5 days, and at day 6 after first meth treatment, SA‐βGal expression was measured. At lower doses of daily meth treatments (10–30 μm), meth did not enhance SA‐βGal expression above what was observed with equivalent single‐dose treatment , meth enhanced SA‐βGal expression in comparison with the single‐dose treatment, as a daily 100 μm meth induced Sa‐βGal at 8% higher than 100 μm single meth dose (17% vs. 25%), a daily 300 μm meth 9% higher than 300 μm single meth dose (31% vs. 22%). Meth also increased the expression of another marker of cell senescence, p161NK4A, as evaluated by Western blot  in comparison with vehicle‐treated cells, while induced cells in G1 phase by 16%  and cell cycle arrest.We first examined whether meth induces senescence of HFAs. human fetal astrocytes were treated with a single dose of meth 0–1000 μ000 μm; aBaL  or mock infected. VSVG‐HIV induced SA‐βGal in HFAs by fivefold (50% vs. 10%) in comparison with VSVG alone  that may be driving astrocyte senescence in HIV infection and meth treatments of astrocytes, we evaluated the role of β‐catenin in these responses. We previously demonstrated that β‐catenin signaling is robustly expressed in astrocytes  were transiently transfected with a Wnt/β‐catenin signaling reporter plasmid, TOPflash. TOPflash consists of several TCF/LEF DNA binding sites linked to firefly luciferase. TCF/LEF and β‐catenin bind to TCF/LEF DNA sequences to regulate gene expression. Meth at 300 μet al., m), which induces β‐catenin expression by inhibiting GSK3β, a component of the β‐catenin destruction complex. Treatment of HIV‐infected HFAs with LiCl rescued SA‐βGal expression to baseline , leading to inflammation and neurotoxicity. To explore the consequence of astrocyte senescence on neurons, conditioned media (CM) from senescent astrocytes were added to neurons. Specifically, human LUHMES cells were differentiated into dopaminergic neurons in vivo, we used IL‐2rγc‐/‐ (NSG) mice reconstituted with human PBLs (huPBLs) and infected with HIVBaL by i.p, as described , which is characterized by elevated levels of potent inflammatory cytokines ; adult male HIV‐1 transgenic Fischer F344 rats (HIV‐1 Tg) and control Fischer F344 rats (non‐Tg) at 3–4 weeks of age from Harlan Laboratory , and adult male Sprague Dawley rats (180e275 g) from Harlan. The animals were housed under pathogen‐free conditions in accordance with the Institutional Animal Care and Use Committee  at Rush University Medical Center and University of Toledo, and the ethical guidelines for care of laboratory animals at the National Institutes of Health.INK4A antibody , GAPDH antibody , antineurofilament heavy chain . Click‐iT Plus EdU Alexa Fluor Flow Cytometry Assay kit (C10646) was from Invitrogen .Methamphetamine (M8750), LiCl, BIO, and propidium iodide were purchased from Sigma . Access to meth was approved by state and federal regulations. Senescence‐associated β‐galactosidase  kit and anti‐GFAP G5 antibody were purchased from Cell Signaling Inc . Other antibodies include Iba1 antibody , active β‐catenin antibody , total β‐catenin antibody , p16m) or vehicle control (1xDPBS). Meth was added once or daily at specified dose for 5 days, as indicated.HFAs  were cultured in complete astrocyte medium according to the supplier's protocol. Early passages (p2‐p4) of HFAs were used for all experiments. For meth treatment of HFAs, the cells were seeded in 8‐well cell culture slide chambers . Media were changed the next day, and the cells were treated with meth  were produced in 293T cells by cotransfection of VSVG expression plasmid and HIVBaL plasmid in 293T cells using the calcium precipitation method. The HIV‐1BaL plasmid and VSV‐G expression plasmid were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. HFAs were infected with VSVG‐HIV at 10 ng or 20 ng of p24 overnight. Next day, cells were washed extensively with 1xDPBS and cultured in fresh astrocyte medium.Vesicular stomatitis virus (VSV)‐G glycoprotein BaL plasmid or control plasmid (pcDNA3) using Lipofectamine 2000 (Invitrogen). For experiments where HFAs were HIV‐infected and meth‐treated, meth was added to the cells during infection and thereafter at the specified concentrations.Human fetal astrocytes (HFAs) were transiently transfected with pHIV‐1et al., m was used to augment β‐catenin activities as described   plus 0.15% Tween‐20 (Thermo Scientific) for 1 h at room temperature. Membranes were probed with primary antibodies with indicated dilutions in SuperBlock TW20 (0.12%). Primary antibodies used were as follows: rabbit anti‐p16INK4A (1:500), mouse monoclonal anti‐active β‐catenin antibody (1:1000), rabbit anti‐β‐catenin antibody (1:50000), anti‐GFAP (1:2000), Iba1 (1:500), anti‐synaptophysin (1:2000), anti‐NF‐H (1:2000), anti‐GAPDH (1:50000). Membranes, after overnight incubation with indicated dilutions of primary antibodies in SuperBlock TW20 (0.12%), were washed three times for 45 min with TBS‐T and incubated with a 1:5000 dilution of anti‐mouse IgG or anti‐rabbit IgG‐HRP secondary antibody  in SuperBlock T20 (0.1%) for 1 h at room temperature. Membranes were again washed three times for 45 min with TBS‐T and exposed to SuperSignal Femto ECL substrate reagent (Thermos Scientific) for p16INK4A and active β‐catenin, and SuperSignal Pico ECL substrate reagent for total β‐catenin and GAPDH. Films were exposed and developed on a Konica SLX‐101A auto processor. Band densitometry was quantitated using ImageJ software , using GAPDH as loading controls.HFAs and hippocampus tissues from human PBMC‐reconstituted NSG mice infected with HIV (see below) were lysed in 1xRIPA buffer in the presence of protease inhibitors (Sigma). 10 μg of cell lysates was loaded onto a 10% SDS‐PAGE for detection of β‐catenin and 20 μg lysates for p16et al., β‐catenin‐dependent transcription activities were analyzed using a T‐cell factor/lymphoid enhancer (TOPflash) reporter . Control cell samples for flow compensation analysis were without either EdU labeling, propidium iodide, or both. All data were analyzed in FlowJo software.We adapted a cell cycle analysis using EdU Click‐It Plus Flow Cytometry kit  in combination with propidium iodide. Briefly, HFAs were treated with 10 μet al., 7 human PBMCs by intraperitoneal injection (i.p.). One to two weeks after reconstitution, mice were bled by retro‐orbital perfusion and extent of reconstitution was determined by flow cytometry. Reconstituted mice were infected with 104 TCID50/mouse of HIVBaL i.p.These experiments were performed as described  and 80 kunitz units per mL DNase‐1 (Invitrogen) in Ca2+/Mg2+‐free PIPES cysteine buffer (Sigma) for 1 h at 37°. After 1 h, an additional 25 kunitz units per mL DNase‐1 was added for an additional 25 min. Digestion was then massed through a 70‐μm strainer with 1% BSA in DMEM and then overlayed onto a layer of isotonic 90% Percoll  and spun at 200 g at 4 °C for 10 min. The myelin and Percoll layers were collected and myelin was removed using the Myelin Removal Bead Kit  according to the manufacturer's instructions.Mice were anesthetized by inhalation of isoflurane and then perfused with 30–50 mL of ice‐cold PBS. Brains were collected and the hippocampus was lysed in 1xRIPA buffer for Western blotting analysis. Astrocytes were isolated as described (Lovatt ° and then washed two times in PBS. Cells were fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) for 30 min at 4°. Cells were washed with Perm/Wash (BD Biosciences) according to the manufacturer's instructions. Cells were then stained with rat anti‐GFAP‐Alexa Fluor 488 and rabbit anti‐p16INK4A  in BD Perm/Wash for 30 min at 4°. Cells were washed two times with BD Perm/Wash and then stained with an anti‐rabbit secondary antibody for the anti‐p16INK4A conjugated to Alexa Fluor 647 for 30 min at 4 °C. Cells were washed two times with BD Perm/Wash and then one additional time with PBS before being run on a BD Fortessa Flow Cytometer (BD Biosciences). Astrocytes were identified as CD45loCD11bnegGFAP+ and 2000 live astrocytes were collected. Flow cytometry was analyzed with FlowJo Analysis Software .Single cell suspensions of cells isolated from mouse brain were stained with LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen) according to the manufacturer's instructions. Cells were washed with PBS and stained for CD45‐PE‐Cy7 and CD11b‐PE  for 30 min at 4−1, i.p., every 2 h × 4) or saline control  as described  at 45 °C for 2 h. SA‐β‐Gal staining of these tissues was performed according to the manufacture's protocol .P values less than 0.05 were considered significant. All error bars are representative of standard deviation within the sample group. Experiments were repeated at least three times.Comparisons between groups depending on experiment were analyzed using unpaired Student's t‐test, where appropriate. This work was supported by R01 DA 033966 (LA), R01 NS060632 (LA), R01 NS084817 (XH), and DA007606 (BY). National Institutes of Health .None declared.