Application of nucleoside analogue labelling to study the cell cycle of xenografted PDAC cell lines in the chorioallantoic membrane model

Application of nucleoside analogue labelling to study the cell cycle of xenografted PDAC cell lines in the chorioallantoic membrane model | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Application of nucleoside analogue labelling to study the cell cycle of xenografted PDAC cell lines in the chorioallantoic membrane model View ORCID Profile Robin Colenbier , Denisa Lodoaba Jurescu , View ORCID Profile Jean-Pierre Timmermans , Johannes Bogers doi: https://doi.org/10.1101/2025.09.09.675135 Robin Colenbier 1 Laboratory of Cell Biology and Histology, Faculty of Medicine and Health Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp , 2610 Antwerp, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robin Colenbier Denisa Lodoaba Jurescu 1 Laboratory of Cell Biology and Histology, Faculty of Medicine and Health Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp , 2610 Antwerp, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jean-Pierre Timmermans 1 Laboratory of Cell Biology and Histology, Faculty of Medicine and Health Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp , 2610 Antwerp, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jean-Pierre Timmermans Johannes Bogers 1 Laboratory of Cell Biology and Histology, Faculty of Medicine and Health Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp , 2610 Antwerp, Belgium 2 ElmediX NV , Esperantolaan 4, 3001 Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract The chorioallantoic membrane (CAM) model is an underutilised alternative animal model within the scope of cell cycle-related research of tumour xenografts. The usefulness of nucleoside labelling in standard rodent xenograft models is limited due to the extended labelling durations as a result of intraperitoneal route of administration. Due to its easy accessibility, the CAM allows xenograft S-phase nuclei to be labelled in as little as 30 minutes in vivo for a large number of biological replicates in parallel. We show that for the BxPC-3 and AsPC-1 cell lines, nucleoside labelling with 5-ethynyl-2’-deoxyuridine (EdU) can be multiplexed successfully with other cell-cycle markers such as cyclin B1 and Ki67, especially when combined with digital image analysis techniques. The latter also allows for accurate human versus chicken cell segmentation. Moreover, starting from embryonic day of development 14 (ED14), we observe the presence of a chicken embryonic cell type that appears to possess a high-quantity of extranuclear accumulation of EdU. Initial assessment of these cells showed that they are likely (non-proliferating) granulocytes which can be found in the embryonic liver of grafted and non-grafted embryos, as well as in xenograft sections. Importantly, these cells do not express chicken MHC II, in turn making it less likely that they represent professional antigen presenting cells. Our findings demonstrate that the CAM xenograft model can be used as a valuable tool to study the cell cycle of tumour cells in vivo . Lastly, we potentially revealed a novel biological phenomenon, namely that of extranuclear nucleoside accumulation in certain chicken embryonic cells. Download figure Open in new tab Introduction As an alternative in vivo model, the use of the chorioallantoic membrane (CAM) model in cancer research is steadily gaining popularity. This is demonstrated by the growing number of original research articles employing the CAM to evaluate cancer treatments or explore tumour biology. Despite its many advantages (e.g., low-cost, rapid turnover) the CAM assay remains underutilised in cancer research, particularly for assessing cellular proliferation and cell cycle dynamics in tumour xenografts. Furthermore, there is a scarcity of recent high-quality scientific literature regarding the use of commercial PDAC cell lines in the CAM model. 1 – 3 Consequently, the routine application of the CAM to study tumour growth and therapy responses has not yet been widely adopted. In this study, we demonstrate the feasibility of labelling actively dividing xenograft S-phase cells in the CAM using the thymidine analogues 5-bromo-2’-deoxyuridine (BrdU) and 5-ethynyl-2’-deoxyuridine (EdU). As both are considered to be golden standard methods for investigating the S-phase, their successful application in the CAM xenograft model may encourage broader use of this alternative animal model. Although already described in the context of xenografts in mice for several cancer types, these analogues are often administered intraperitoneally, which in turn leads to extended labelling durations of 3-6 hours. 4 – 6 This impedes accurate quantification of cell cycle dynamics when a high time-resolution is required. In contrast, the CAM offers easy access, allowing for tumour and embryo manipulations to be performed in a matter of seconds. This facilitates cell cycle studies in combination with exposure to compounds (such as thymidine analogues) for relatively short time-windows (i.e., < 1h). As such, the xenografted CAM model can be considered as a unique in vivo tool that enables both (immuno-) histological analyses and cell cycle analysis with improved temporal resolution compared to rodent models. Notably, another alternative animal model, namely the zebrafish xenograft is being used more frequently in recent years. These models are also characterised by a high accessibility and rapid development, making them compatible with quick experimental turnover and relatively high-throughput experiments. In turn, these could potentially also be used to study the cell cycle in xenograft contexts. However, no cell-cycle focused studies have been reported so far. 7 – 9 As in many cancer types, pancreatic ductal adenocarcinoma (PDAC) cell lines display considerable inter-cell line heterogeneity. Therefore, an appropriate biological model must accommodate a variety of cell lines. To this end, three distinct commercial PDAC cell lines (BxPC-3, AsPC-1, PANC-1) were used to induce tumours in the CAM. We demonstrate the feasibility of combining immunofluorescence (IF) microscopy techniques with S-phase labelling through BrdU/EdU detection on xenograft tissue sections. To aid in the analysis and interpretation of the acquired images, digital image processing techniques were employed to differentiate proliferating tumour xenograft cells from dividing embryonic cells. Utilising the CAM model to study the cell cycle of tumour cells provides a unique and underexplored area of research that may broaden our understanding of in vivo cancer cell cycle regulation and lead to the improved application of currently available treatment modalities or identify potential cell cycle-related vulnerabilities of cancer cells. Results 1. The CAM model can be used to reliably induce tumour formation from commercially available PDAC cell lines One of the primary advantages of the CAM model is its scalability: using a relatively small setup, a large number of biological replicates can be generated within days and analysed within the following weeks. Here, we confirm that our previously published protocol, originally optimised for the BxPC-3 cell line, can also successfully be applied to the AsPC-1 and PANC-1 PDAC cell lines. While modest success in grafting efficiencies can initially be observed when adopting the CAM model, our lab now routinely achieves tumour engraftment rates exceeding 90% and overall embryo survival rates surpassing 80% at embryonic day 10 (ED10) (three days post-grafting). Following grafting and tumour formation, the evaluated PDAC cell lines appear to retain their respective in vitro growth characteristics. BxPC-3 cells exhibit a relatively well-differentiated growth pattern in ovo , consistent with earlier observations. 10 , 11 These cells form clearly delineated tumour nodules embedded within the extracellular matrix, often merging into elongated, intercalated strands ( Figure 1 A-B ). This nodular growth pattern appears to mirror the in vitro growth pattern. AsPC-1 cells also form discrete nodules, but the presence of tubular-like structures is less evident (not shown). Additionally, these nodules generally contain fewer cells compared to those formed by BxPC-3 xenografts. In contrast, the poorly differentiated PANC-1 cells appear to be loosely interconnected and are occasionally found as strand-like organisations, often lacking clear separation from the surrounding tumour microenvironment (TME). The nuclei of PANC-1 cells are markedly polymorphic, closely reflecting their in vitro appearance ( Figure 1 C-D ). Consequently, distinguishing tumour cells from embryonic chicken cells in the CAM model can be particularly challenging in poorly differentiated cell lines, as will be discussed further. Download figure Open in new tab Figure 1. Cellular organisation of the well-differentiated PDAC cell line (BxPC-3) versus the poorly differentiated PANC-1 cell line in vitro (right panels) versus in ovo (left panels). Tissue sections are stained with secondary anti-chicken IgY antibodies (green). Nuclei are stained with Hoechst 33342 (blue). Confocal images acquired at 20x magnification. 2. The CAM model facilitates nucleoside incorporation assays in xenografted tumour cells Unlike other in vivo models, tumour cells in the CAM model can easily be exposed to (therapeutic) compounds due to the direct access to the xenograft and its vascular supply via the (extra-) embryonic membranes. This can be exploited to label dividing xenografted tumour cells: dispensing of solutions onto the CAM takes mere seconds, in turn facilitating short-term investigations. Once a solution is applied onto the CAM, compounds can be absorbed into the blood stream of the embryo and subsequently be perfused through the tumour tissue. Historically, halogenated thymidine analogues such as BrdU and IdU have been applied to investigate the S-phase in vitro as well as in vivo , predominantly in rodent models. More recently, these have been supplanted due to the emergence of (copper-based) click labelling techniques using newer thymidine analogues like EdU and F-ara-EdU. These offer superior specificity, sensitivity and multiplexing compatibility. Despite their advantages and widespread use for in vitro experiments, even these have not been adopted in the setting of the CAM model to investigate the cell cycle in xenograft cells. 2.1 BrdU labelling enables S-phase detection in xenografted PDAC tumour cells via immunohisto-fluorescence As proof-of-concept, BxPC-3 tumour-bearing embryos at various stages of development (ED10-ED14) were exposed to BrdU (up to 800 µmol/kg for labelling durations up to two hours) prior to sacrifice. No clear signs of acute embryo toxicity were observed. As shown in Figure 2 , S-phase labelling could be achieved in as little as 30 minutes of exposure to BrdU. Download figure Open in new tab Figure 2. Confocal image of a BxPC-3 xenograft cryosection labeled for 60 minutes with BrdU (800 µmol/kg) prior to sacrifice at ED13, followed by IF detection for BrdU (red), and counterstaining with secondary anti-chicken IgY antibodies (green) and Hoechst 33342 (blue). BrdU-positive xenograft nuclei (red) can be distinguished (panels A-B). Arrowheads denote BrdU-positive chicken embryonic nuclei The BrdU signal typically localizes to the nuclear periphery, a characteristic pattern resulting from the obligate double-stranded DNA denaturation step (commonly using 2M HCl) required for the detection of halogenated analogues via immunodetection. As described by Pierzyńska-Mach et al. 12 , this pattern may signify incomplete dsDNA denaturation. Nevertheless, harsher or prolonged denaturation can increase background staining and interfere with subsequent immunodetection of other markers. In extreme cases, it can lead to near-complete destruction of dsDNA helices, resulting in diminished or absent nuclear staining with standard dyes such as DAPI or Hoechst, Furthermore, (acid-based) epitope retrieval for BrdU immunodetection can be highly variable on tissue sections, leading to inconsistent results. Figure 3 illustrates the incompatibility of BrdU immunodetection protocols with subsequent IF of other epitopes in xenografted tumour cells. Here, anti-human Ki67 IF labeling was performed following a brief BrdU detection protocol (30 minutes HCl; 2M denaturation step). It is clear that homogenous nuclear staining and an intense nucleolar pattern, which are typical for Ki67, are lost following the HCl denaturation. This renders its interpretation unreliable. Additionally, performing BrdU detection (and denaturation) following IF labeling also leads to the reduction of fluorescence intensities. Download figure Open in new tab Figure 3. A-C Immunofluorescent labelling for BrdU and Ki67 following an initial 30 minutes HCl (2M) treatment on a BxPC-3 tumour cryosection. D Separate, non-contiguous tumour control sample labelled only for Ki67 without HCl pretreatment. Acquired at 40x magnification. Importantly, nuclear incorporation of BrdU is observed in both dividing human and chicken embryonic cells ( Figure 2B , arrowheads), confirming its broad application potential and high sensitivity to detect S-phase cells and absence of species-specificity. Hence, strategies are needed to avoid misidentification of embryonic cells as proliferating tumour cells. These may include human-specific IF labelling (see section 4) or nuclear feature-based classification via imaging processing software. In general, human tumour nuclei are larger while on the other hand, many chicken nuclei demonstrate intense nucleolar staining sometimes with sharply defined nuclear borders ( Figure 4 ). Download figure Open in new tab Figure 4. Gray-value representation of 60x magnification confocal microscopic sections of an AsPC-1 xenograft cryosection, stained with Hoechst 333342 depicting chicken embryonic epithelial nuclei ( EE ), nuclei of chicken embryonic blood cells ( EB ) and human AsPC-1 tumour xenograft nuclei ( T ). Mitotic figures of dividing tumour nuclei can also clearly be distinguished ( M) . Nevertheless, the nuclear staining intensities obtained via Hoechst and DAPI dyes are negatively affected in a non-linear fashion by nucleoside incorporation due to distortion of the dsDNA helix. This phenomenon occurs even in the absence of denaturation steps. Therefore, the use of these dye-dependent intensity measurements for species classification becomes challenging in the context of nucleoside labelling. Lastly, (cell-line dependent) cellular heterogeneity can further impede effortless classification, especially in dense multicellular environments, such as xenografted tumours. Consequently, additional IF labelling is required in order to segregate proliferating chicken and human cells. By replacing BrdU with EdU, reliable detection of other (cellular) markers can be performed. 2.2 5-Ethynyl-2’-deoxyuridine (EdU) labelling allows specific S-phase labelling and multiplexing with immunofluorescent labelling of cell cycle markers in xenografted tumour cells Unlike halogenated thymidine analogues, detection of EdU within tissue sections does not require dsDNA denaturation steps, making it highly compatible with IF staining, ( Figure 5 ). Moreover, BrdU signal intensities do not faithfully represent the quantity of incorporated analogue. In contrast, it is generally accepted that EdU signal intensities do closely reflect the degree of nuclear incorporation. After immunolabeling for human Ki67, we confirmed that all EdU + xenograft nuclei were also Ki67 + , confirming the specificity and compatibility of both labelling strategies in CAM xenograft tumour cells ( Figure 5 ). Additionally, nuclei that were not in S-phase at the moment of fixation (e.g., mitotic nuclei; arrowheads) lacked EdU signal. Importantly, anti-Ki67 IF shows retained perichromosomal organization in mitotic nuclei following the EdU click reaction, demonstrating the method’s suitability for studies across multiple cell cycle phases. Download figure Open in new tab Figure 5. Cryosections from an AsPC-1 xenograft (ED15) labelled with EdU (800 µmol/kg for 30 minutes) and stained for Ki67 The white dashed region outlines a Ki67-negative embryonic cell population. Arrowheads depict Ki67-positive (green), but EdU-negative mitotic nuclei, confirming the specificity of both staining techniques. The inset show a detailed view of preserved Ki67-detection. In addition to Ki67 staining, the EdU labelling in CAM xenografts can be combined with other cell cycle markers, such as cyclin B1, enabling finer resolution of the different cell cycle stages in vivo via microscopy. Combined, these markers allow in ovo segregation of proliferating tumour cells into early S-phase (ES, EdU + ), late S or early G2 (LS, EdU + CB1 + ) and G2 (EdU - CB1 + ) ( Figure 6 ) Download figure Open in new tab Figure 6. Cryosection of an AsPC-1 xenograft (ED13) IF-labelled for cyclin B1, followed by EdU detection via the click reaction showing xenograft cells at different stages of S-and G2. ES = early S-phase, LS = late S-phase, G2 = G2 phase. Image acquired at 60x magnification 2.3 Considerations for applying nucleoside labelling in the CAM model to investigate the cell cycle in xenografts As mentioned, microscopic evaluation clearly demonstrated the presence of chicken embryonic cells in close proximity to xenograft tumour cells. Additionally, nucleoside incorporation (BrdU or EdU) is not species-selective. Both observations warrant the use of techniques to distinguish human (tumour) versus embryonic chicken cells (nuclei). In essence, evaluation of the cell cycle of xenografted tumours in the CAM needs to be combined with at least one human-specific IF label to allow accurate human versus chicken segmentation of acquired images. 2.3.1 Application of both specific and non-specific immunofluorescence techniques aids tissue segmentation In order to avoid the false inclusion of (dividing) chicken embryonic cells in analyses, several experimental strategies can be employed that cooperate with digital image processing (e.g., via the QuPath software package). 13 First, distinguishing between both species can already be achieved to a large extent in a relatively simple manner via the use of anti-chicken IgY fluorophore-conjugated antibodies, which are routinely only used as secondary antibodies. In doing so, cellular as well as acellular chicken embryonic components are stained ( Figure 7 ). Download figure Open in new tab Figure 7. Comparison of the application of anti-IgY IF labelling (left, green) versus no IF labelling (right) in BxPC-3 sections at 20x magnification. Highly autofluorescent (nucleated) embryonic erythrocytes can be observed throughout the tissue (arrowheads). Nuclei are stained with Hoechst 33342. Second, nuclear features can be extracted and used to aid in the generation of a classifier model using built-in algorithms included in QuPath. This can already result in modestly accurate tissue classification for well-differentiated cell types, such as BxPC-3. ( Figure 8B ) Species-distinction can be further facilitated by combining anti-chicken IgY IF ( Figure 8C ) with additional anti-human IF, such as anti-human Ki67 ( Figure 8D ). Alternatively, one can opt to apply a simple binary classifier by manually setting a defined intensity threshold based upon visual inspection of the pixel intensity distribution in the “anti-chicken” channel ( Figure 8E ). Download figure Open in new tab Figure 8. Comparison of different classification strategies to discriminate human versus chicken cells. A . Raw confocal image of a BxPC-3 xenograft at 20x magnification. B-D . Tissue mask generation using built-in QuPath classifier models. E . (manual) Tresholding based on pixel intensity in the anti-chicken channel. The section was stained with Hoechst33342 (blue), anti-Ki67 (orange), anti-chicken IgY (green). Classifier outcomes (tumour; red versus chicken; green) are indicated by the coloured overlay in panels B-E. Human versus chicken segmentation based on the latter strategy performs well in cases where a clear distinction between tumour and chicken tissues is already present to some extent. This is often observed in xenografts of well-to moderately-differentiated cell lines (i.e. BxPC-3, AsPC-1). However, this only is reliable when staining results are also not highly variable across replicates. Nevertheless, this strategy may also induce artifacts, as non-tissue (empty) areas can be classified as tumour ( Figure 8E , Figure 9B ). In turn, a higher extent of manual curation will be warranted, which in turn can introduce additional observer bias. In contrast, for poorly-differentiated cell lines such as PANC-1, the performance of the simple thresholder is limited ( Figure 9 D-F ). Additionally, the highly variable nuclear size and intensities observed for the PANC-1 cell line may also challenge even more complex classifier models when these take nuclear features into account. Download figure Open in new tab Figure 9. A . Raw image of an AsPC-1 tumour cryosection at 60x magnification B . 2-channel classifier , C . simple thresholder . D . Raw image of a PANC-1 tumour cryosection at 20x magnification E . 2-channel classifier F . simple thresholder. Sections are labelled with anti-chicken IgY (green), anti-Ki67 (orange) and Hoechst33342 (blue). Classifier outcomes (tumour; red versus chicken; green are indicated by the coloured overlay . When human cells can be confidently separated from chicken cells and tissue, by (supervised) automated or manual annotation, individual cell detection can be performed. In this step, detection of nuclei can be performed using the built-in “cell detection” functionality. This detection can be performed for the entire image ( Figure 10 B,E ) or only for the tumour region(s) of interest ( Figure 10 C,F ). Once nuclei have been segmented, targets can be quantified in a human-specific fashion. Download figure Open in new tab Figure 10. Combination of the 2-channel classifier with the cell detection plugin from QuPath allowing separation of human from chicken embryonic nuclei A-C . BxPC-3 cryosection at 20x magnification). D-F . AsPC-1 cryosection at 40x magnification. Sections are labelled with anti-chicken IgY (green), anti-Ki67 (orange) and Hoechst33342 (blue). Bright red overlay signifies detected nuclei, yellow lines indicate the area classified as tumour. B , E : 2-channel classifier; C , F : 2-channel classifier + QuPath cell detection plugin. Not applying species-specific masks or classification strategies leads to the inclusion of a significant amount of chicken nuclei in subsequent analyses. In this example, running the cell detection algorithm for BxPC-3 cells resulted in a total of 369 detections when applied to the whole image ( Figure 10 B ), versus 256 when applied to the true tumour region only ( Figure 10 C ), which results in a 30.6% difference in detections. For the AsPC-1 cell line, a similar difference is observed: 377 detections when applied to the whole image ( Figure 10 E ) versus 249 in the tumour region ( Figure 10 F ), resulting in a 34% difference. 2.3.2 Biological causes of variability in EdU-labelling efficiency: emergence of a specific cellular but non-nuclear EdU signal When applying nucleoside labelling in the CAM to investigate the cell cycle in xenografts, the labelling solution containing the analogue should be dispensed onto the CAM as far away from the xenograft as possible. The rationale is that, in doing so, the majority of the analogue that will reach the tumour, will have passed through the embryonic cardiovascular system first. This ensures, that from the tumour cell perspective, effects of passive diffusion into the CAM (starting from the dispensing area) on the incorporation of the nucleoside analogue will be kept to a minimum. To demonstrate that the nucleoside analogues pass through the embryonic circulation, liver sections were taken from non-grafted embryos and evaluated for the presence of EdU + cells showing the clear presence of EdU + hepatoblast or hepatocyte nuclei ( Figure 11 A ). Surprisingly, from ED14 onwards, cells with cytoplasmic, granular, EdU accumulation appear. These cells were initially only sporadically observed within embryonic liver sections although they appear more frequently with increasing embryonic development stage. Eventually, these cells (further named as EdU-cyto + ) could also be observed within CAM xenografts ( Figure 11 C ). Download figure Open in new tab Figure 11. Detection of EdU within A : Embryonic liver tissue section (ED14) at 20x magnification. B: Embryonic liver tissue section (ED16) at 40x magnification. C: BxPC-3 xenograft at 40x magnification. S-phase nuclei of both embryonic liver epithelial cells ( ES ) and BxPC-3 cells ( BS ) can be found alongside chicken embryonic cells showing apparent cytoplasmic EdU inclusions (arrowheads). Nuclei are stained with Hoechst33342 (blue). Applying brightfield imaging showed that EdU indeed appeared to be localised within the cytoplasm of granulocytes ( Figure 12A ). In order to provide further insight into the identity of these apparent granulocytes, IF labelling directed to chicken MHC class II was carried out. In both liver and xenograft sections, EdU-cyto + cells were found to be negative for MHC II, confirming that these cells are likely not macrophages. On the other hand, MHC II-positive cells (Kupffer cells) with nuclear EdU incorporation are sporadically observed within liver sections ( Figure 12 C ). Download figure Open in new tab Figure 12. Illustration of nuclear and cytoplasmic EdU detection in three different tissue sections. A . Granulocytes with apparent cytoplasmic EdU (red) inclusions (arrows) are present alongside EdU-negative granulocytes in the CAM surrounding a xenograft. B . BxPC-3 xenograft (ED18) demonstrating specific nuclear EdU incorporation (red) into tumour cells ( T ) alongside cytoplasmic inclusions in non-tumour cells (arrowheads) and a single MHC II-positive cell (green). C . Embryonic liver (ED18) showing nuclear EdU incorporation into an MHC II positive cell ( MHC II ), residing in the liver parenchyma adjacent to a major blood vessel ( BV) . Images were acquired at 40x magnification. Nuclei are stained with Hoechst 33342 (blue). Discussion (Nucleoside labelling of xenografts in the CAM model is feasible and should be paired with species directed classification strategies) In this paper, we have demonstrated that our previously published protocol concerning the xenografting of the BxPC-3 cell line 10 can be applied to the AsPC-1 and PANC-1 cell lines. Additionally, the in vitro growth pattern of these cell lines appears to be well-retained during in ovo xenograft culture. Consequently, distinguishing the moderately to well-differentiated cell lines BxPC-3 and AsPC-1 from embryonic tissues and cells is relatively straightforward. In contrast, poorly differentiated cell lines, which exhibit a high degree of cellular heterogeneity, impede the clear segregation of xenografts from CAM tissues. Furthermore, we have shown that both BrdU and EdU enable efficient labelling of dividing xenograft nuclei within the CAM model. Excellent labelling can be achieved in as little as 30 minutes, which is possibly the shortest reported time interval for in vivo cell cycle labelling using nucleoside analogues. Although no obvious toxicity was observed with either compound, the toxicity profile of EdU in particular should be considered, as multiple reports have shown that prolonged exposure can lead to DNA damage accumulation and cytotoxicity. 14 – 17 We therefore recommend using the more recently developed analogue F-ara-EdU for long-term xenograft cell cycle labelling in the CAM model. While F-ara-EdU also offers high multiplexing potential and appears to have very low toxicity, it exhibits much slower incorporation kinetics into cells compared to EdU and BrdU. 18 Therefore, F-ara-EdU is not suitable for short-term cell cycle labelling procedures such as those described in this paper. Notably, no such labelling has yet been performed in the CAM model for xenografts. Nonetheless, EdU remains superior to BrdU for CAM xenografts in terms of sensitivity and compatibility with subsequent IF for cell cycle studies. This is particularly important given the non-species-specific incorporation of nucleosides into S-phase nuclei in the CAM model. We have presented simple yet effective techniques, namely the application of anti-IgY IF staining and the use of at least one anti-human IF marker in combination with built-in classifier models in the QuPath software package. When combined, these approaches provide excellent results in terms of segregating human xenograft nuclei from chicken embryonic nuclei in AsPC-1 and BxPC-3 xenografts. Although no direct comparisons were performed, this strategy appears to be robust and applicable to microscopic images acquired at 20x, 40x and 60x magnification. Importantly, this workflow allows imaging and processing of whole-tissue sections rather than imaging and quantifying select representative tissue areas – as is commonly performed. We are convinced that similar segregation strategies should be applied in any xenograft tumour model, including rodent models, to minimise observer bias in the quantification of tumour tissues. A possible limitation of this study is that the majority of examples presented were generated from BxPC-3 and AsPC-1 tissue sections (as opposed to PANC-1 tissues). Nevertheless, we are confident that these examples sufficiently demonstrate the applicability of EdU labelling in CAM xenografts. Therefore, optimisation and fine-tuning of classifier models that perform robustly across a variety of cell lines fall outside the scope of this paper. (A newly-identified biological phenomenon: non-nuclear EdU accumulation in chicken embryonic cells) In addition to the consistent labelling of S-phase cells within both embryonic liver sections and xenograft nuclei, we identified one or more embryonic cell populations that contain high amounts of extranuclear EdU. This phenomenon has not been observed with BrdU, nor was it further investigated for any of the halogenated nucleoside analogues. As such, it remains unclear whether this effect is specific to EdU. Following initial observations, several control staining procedures were performed to rule out staining-related technical artefacts. Embryonic livers from both grafted and non-grafted embryos that had not been exposed to EdU, or had been exposed to BrdU, were subjected to the EdU-detection click reaction. None of these control tissues showed nuclear or cytoplasmic staining (data not shown). Next, liver and xenograft sections from EdU-exposed embryos were incubated with an anti-BrdU antibody known to exhibit substantial cross-reactivity with DNA-incorporated EdU. 19 In both tissue types, the anti-BrdU antibody failed to reproduce the cytoplasmic signal in any embryonic cells. Therefore, it is unlikely that the cytoplasmic EdU signal represents extranuclear (fragmented) DNA. The simultaneous presence of EdU-cyto + cells alongside EdU + nuclei in both embryonic liver and xenografted tumour nuclei further rules out technical issues with the azide-EdU click reaction. It is also evident that the presence of EdU-cyto + cells is not confined to the liver, as they were also found within the CAM. These cells likely represent a ubiquitously distributed cell population present in both healthy and xenografted chicken embryos. Given that embryos were exposed to nucleoside labelling for no more than two hours, it is highly unlikely that these cells were induced by proliferation or differentiation in response to high concentrations of the analogue. Although initially observed in liver tissue sections, these cells may originate elsewhere, as other tissues have not yet been examined. Importantly, since EdU-cyto + cells are consistently present in non-grafted embryos as well, the phenomenon is not triggered by xenografting or the presence of PDAC cells. Previous studies have reported that non-dividing macrophages can accumulate and metabolise the thymidine analogue drug gemcitabine 20 , and that the embryonic chicken liver is a source of (eosinophilic) granulocytes 21 , 22 . We therefore performed an initial assessment to determine whether the EdU-cyto + cells represent (activated) antigen-presenting cells (APC). Immunofluorescence revealed that these cells are not positive for the monomorphic chicken BLA chain (MHC class II), which shows little variability across chicken immune cells. Thus, it is unlikely that the EdU-cyto + cells are classical/conventional APC. Also in the chicken (embryo), neutrophils (heterophils) are the most dominant white blood cell type. Given the high number of EdU + granulocytes observed, it is more likely that these are neutrophils rather than eosinophils. To further elucidate the identity of the EdU-cyto + cells, in-depth investigations using avian leukocyte markers are required. This may prove challenging, as only a limited number of validated characterisation markers are available for the avian immune system 23 , and even fewer are commercially accessible. Nevertheless, these findings pose an important limitation for the use of nucleoside labelling in xenografts within the CAM model during later stages of embryonic development (i.e., beyond ED14). Due to the apparent accumulation of EdU in EdU-cyto + cells, incorporation of the analogue into proliferating cells may be substantially reduced, potentially resulting in many false-negative detections of S-phase tumour cells. Assuming that the EdU-cyto + cells are indeed granulocytes, their impact on nucleoside labelling should be minimal at earlier time points, when the embryonic immune system is still very immature. To preserve the sensitivity and specificity of EdU labelling, we therefore recommend performing this assay prior to ED14 in CAM xenograft experiments. While the presence of EdU-cyto + cells presents a technical challenge for nucleoside labelling in CAM xenografts, these findings also raise several novel and important research questions: Do these cells actively accumulate EdU, and if so, what are the biological or chemical triggers? Is this phenomenon limited to the chicken embryo model, or is it also present in other in vivo models? Do EdU-cyto + cells represent a single cell type, or can multiple distinct cell types exhibit similar behaviour? Answering these questions could provide deeper insight into fundamental biological processes and the cellular handling of nucleosides and their analogues Conclusions BrdU and EdU labelling can be used to label S-phase nuclei in dividing xenograft cells within the CAM model The superiority of EdU over BrdU for short-term cell cycle labeling is maintained, as demonstrated by its multiplexing potential - a prerequisite for accurate species-specific quantification of microscopic images. We report a novel phenomenon: the apparent cytoplasmic accumulation of EdU in non-dividing chicken granulocytes. Materials and methods Cell lines and -culture The used cell lines BxPC-3 (CRL-1687), AsPC-1 (CRL-1682 and PANC-1 (CRL-1469) were obtained from ATCC. Cells were maintained and expanded at 37 °C in a humidified 5% CO 2 atmosphere in T175 culture flasks (Greiner Bio-One, Cat#660175) in Dulbecco’s modified Eagle’s medium, containing 4.5g/L glucose and supplemented with 4 mM + L-glutamine (Gibco, Cat#41965039), 1 V/V% Penicillin-Streptomycin (10,000 U/mL) (Gibco, Cat#15140122) and a final concentration of fetal bovine serum of 10 V/V% (Gibco, Cat#A5256701). Xenografting in the chorioallantoic membrane model Xenograft tumours were generated according to our previously published protocol 10 with the following modifications. All grafting was performed at ED7 using a final cell suspension volume of 50 µL rather than 100 µL. The final amount of grafted cells per embryo was 4 million for each cell line. During the preparation of the grafting cell suspension, PANC-1 cells were centrifuged at 400 G for 5 minutes rather than 250 G for 7 minutes. Nucleoside labelling of dividing cells in the CAM model 5-Bromo-2′-deoxyuridine (BrdU, Sigma-Aldrich, Cat#19-160) and 5-Ethynyl-2’-deoxyuridine (EdU, Thermofisher, Cat#466382500) was dissolved in dimethylsulfoxide (DMSO) to make a stock concentration of 100 mM and 50 mM for BrdU and EdU respectively. Subsequently, both stock solutions were sterile filtered through 0.20 µm pore size polytetrafluoroethylene (PTFE) filters (Sartorius, Cat#17575-K) and stored at -20 °C until further use. Working solutions for in ovo labelling of the cell cycle were prepared by diluting each stock solution in sterile Dulbecco’s phosphate-buffered saline (DPBS, Cat#14190169). For labelling with doses of 800 µmol/kg egg weight, a working solution of 13.33 mM (3.36 mg/mL for EdU, 4.09 mg/mL for BrdU) was prepared and warmed to 37 °C. Prior to labelling, each egg was weighed and the amount of volume to be pipetted onto each CAM was calculated based upon the following formula: . For example, an egg weighing 58.3 g would receive 159 µL of the 13.33 mM labelling solution. Then, for each egg, the appropriate amount of nucleoside-containing working solution was dispensed onto the CAM as far away from the visible tumour as possible before returning them to the egg incubator. In order to ensure low variability in labelling duration, two researchers performed the labelling in tandem, ensuring that an experimental group of 16 embryos could be labelled in less than five minutes. Tissue processing and staining Tissue collection and fixation At the end of the labelling period, eggs were placed on, and covered with ice in order to halt the cell cycle and stop nucleoside incorporation. Subsequently, embryos were sacrificed via decapitation, tumours were excised from the CAM and embryo livers removed the chicken embryo. All collected tissues were immediately rinsed three times with ice-cold PBS and fixed overnight in an ice-cold 4% formalin solution (ROTI®Histofix, Cat#3105). Tissue (pre-)processing and cryosectioning Following overnight fixation, tissues were extensively washed in PBS on a rotating shaker for 10 minute and a minimum of five changes of PBS. Tumours were further dissected with the aid of a stereomicroscope in order to remove excess CAM and blood clots. Next, tissues were pre-embedded overnight at 4 °C in a 30 W/V% sucrose solution in PBS containing 0.1 % sodium azide. The following day, pre-embedded tissues were embedded in NEG-50 cryo-embedding medium (Epredia, Cat#6502), equilibrated to-25 °C and eventually stored at – 80 °C until further use. 7 µm thick cryosections were cut from xenograft tissues, while for embryonic liver, 10 µm thick sections were cut on pre-coated glass slides (VWR, Cat# 631-0108), dried at 37 °C for 2 hours and stored at – 20 °C. Immunofluorescence labelling of cryosections Cryosections were thawed and a barrier was drawn around the tissue sections using a hydrophobic barrier pen (Sigma-Aldrich Cat#Z377821). Then, sections were rehydrated and washed in PBS for at least three washes of 10 minutes each, before being covered by a blocking and permeabilising solution in a humidified atmosphere. This solution is comprised of final concentrations of 0.1 V/V% Triton-X-100, 0.02 W/V% sodium azide, 5 V/V% horse serum (Sigma-Aldrich, Cat#H1270), 0.01 W/V% thimerosal and 0.3 W/V% bovine serum albumin (Sigma-Aldrich, Cat#A7284) in PBS at pH = 7.4. After blocking and permeabilization, tissues were washed for three times in PBS for 10 minutes. Incubation with primary antibodies was always performed prior to EdU detection via the copper-catalysed click reaction as to not interfere with epitope recognition. Primary antibodies were applied overnight at room temperature in the blocking buffer with the omission of Triton X-100, in a humidified staining tray at the following dilutions: View this table: View inline View popup Download powerpoint Table 1. List of used primary antibodies View this table: View inline View popup Download powerpoint Table 2. Secondary antibodies used for immunolabeling The following day, slides were washed three times in PBS and subsequently incubated with the appropriate secondary antibodies for one hour at room temperature in a humidified staining tray. All secondary antibodies were finally dissolved in the blocking buffer without Triton X-100 at a final concentration of 2 µg /mL together with Hoechst 33342 at a final concentration of 10 µg/mL After secondary antibody labelling tissues were washed three times in PBS and either subjected to further copper-catalysed click reaction for the detection of EdU or coverslipped in a glycerol-based anti-fading mountant (Citifluor AF1, Electron Microscopy Sciences, Cat#17970-25). Copper-catalysed click reaction In order to detect DNA-incorporated EdU, permeabilised tissues were covered and incubated at room temperature with the staining buffer comprised out of a final concentration of 2.5 µM Azide fluorophore-conjugated dye (CF®647, Biotium, Cat##92084 or Alexa Fluor™ 647 Azide, ThermoFisher, Cat#A10277). Incubation durations of 30, 60 and 120 minutes were evaluated with no discernible differences in signal-to-noise ratio nor background staining. After the click reaction, tissues were washed three times in PBS and mounted (see above). Confocal fluorescence microscopy & image processing Cryosections and in vitro cells were imaged using a Nikon Ti2 W1 spinning disk confocal microscope using the Nikon NIS-Elements software interface. Z-stack images of tissue sections were acquired and maximum-intensity projections were made before further processing for visualisation purposes through the use of the QuPath (Version 0.5.1) and Fiji software packages. Cellular classification models were built per cell line using the built-in Artificial Neural Network (ANN_MLP). In brief, a representative tissue section was manually annotated and used as input for the model. Via human-in-the-loop evaluation, parameters were adjusted until the model was able to accurately distinguish the different cell types. Declarations of interest This research is partly funded by a research grant from ElmediX NV. J.B. is the CEO and a shareholder of ElmediX NV. Acknowledgements All experimental procedures were performed in the Laboratory of Cell Biology and Histology, University of Antwerp in accordance with the Directive 2010/63/EU of the European Parliament and the Belgian Royal Decree of 29 May 2013. Funder Information Declared Elmedix NV References 1. ↵ Costanza , B. et al. Transforming growth factor beta-induced, an extracellular matrix interacting protein, enhances glycolysis and promotes pancreatic cancer cell migration . International Journal of Cancer 145 , 1570 – 1584 ( 2019 ). OpenUrl CrossRef PubMed 2. Fahmy , K. et al. Myoferlin plays a key role in VEGFA secretion and impacts tumor-associated angiogenesis in human pancreas cancer . International Journal of Cancer 138 , 652 – 663 ( 2016 ). OpenUrl PubMed 3. ↵ Harper , K. et al. The Chicken Chorioallantoic Membrane Tumor Assay as a Relevant In Vivo Model to Study the Impact of Hypoxia on Tumor Progression and Metastasis . Cancers 13 , 1093 ( 2021 ). OpenUrl PubMed 4. ↵ He , Y. et al. Quantitative Evaluation of in Vivo Target Efficacy of Anti-tumor Agents via an Immunofluorescence and EdU Labeling Strategy . Front. Pharmacol . 9 , ( 2018 ). 5. Endaya , B. B. , Lam , P. Y. P. , Meedeniya , A. C. B. & Neuzil , J. Transcriptional profiling of dividing tumor cells detects intratumor heterogeneity linked to cell proliferation in a brain tumor model . Molecular Oncology 10 , 126 – 137 ( 2016 ). OpenUrl PubMed 6. ↵ Chen , C. , Fu , Q. , Wang , L. , Tanaka , S. & Imajo , M. Establishment of a novel mouse model of colorectal cancer by orthotopic transplantation . BMC Cancer 25 , 405 ( 2025 ). OpenUrl PubMed 7. ↵ Fontana , C. M. & Van Doan , H. Zebrafish xenograft as a tool for the study of colorectal cancer: a review . Cell Death Dis 15 , 23 ( 2024 ). OpenUrl PubMed 8. Somasagara , R. R. & Leung , T. Zebrafish Xenograft Model to Study Human Cancer . Methods Mol Biol 2413 , 45 – 53 ( 2022 ). OpenUrl PubMed 9. ↵ Yao , Y. , Wang , L. & Wang , X. Modeling of Solid-Tumor Microenvironment in Zebrafish (Danio Rerio) Larvae . Adv Exp Med Biol 1219 , 413 – 428 ( 2020 ). OpenUrl PubMed 10. ↵ Colenbier , R. et al. Protocol for xenografting of BxPC-3 pancreatic tumors using a chorioallantoic membrane model . STAR Protoc 5 , 102968 ( 2024 ). OpenUrl PubMed 11. ↵ Peulen , O. et al. The Anti-Tumor Effect of HDAC Inhibition in a Human Pancreas Cancer Model Is Significantly Improved by the Simultaneous Inhibition of Cyclooxygenase 2 . PLOS ONE 8 , e75102 ( 2013 ). OpenUrl CrossRef PubMed 12. ↵ Pierzyńska-Mach , A. et al. Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy . Cell Cycle 15 , 1156 ( 2016 ). OpenUrl CrossRef PubMed 13. ↵ Bankhead , P. et al. QuPath: Open source software for digital pathology image analysis . Sci Rep 7 , 16878 ( 2017 ). OpenUrl CrossRef PubMed 14. ↵ Haskins , J. S. et al. Evaluating the Genotoxic and Cytotoxic Effects of Thymidine Analogs, 5-Ethynyl-2′-Deoxyuridine and 5-Bromo-2′-Deoxyurdine to Mammalian Cells . International Journal of Molecular Sciences 21 , 6631 ( 2020 ). OpenUrl PubMed 15. Zhao , H. et al. DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5-ethynyl-2′-deoxyuridine incorporated into DNA . Cytometry Part A 83 , 979 – 988 ( 2013 ). OpenUrl 16. Neef , A. B. & Luedtke , N. W. An Azide-Modified Nucleoside for Metabolic Labeling of DNA . ChemBioChem 15 , 789 – 793 ( 2014 ). OpenUrl CrossRef PubMed 17. ↵ Wang , L. et al. Nucleotide excision repair removes thymidine analog 5-ethynyl-2′-deoxyuridine from the mammalian genome . Proc Natl Acad Sci U S A 119 , e2210176119 ( 2022 ). OpenUrl CrossRef PubMed 18. ↵ Neef , A. B. & Luedtke , N. W. Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides . Proceedings of the National Academy of Sciences 108 , 20404 – 20409 ( 2011 ). OpenUrl Abstract / FREE Full Text 19. ↵ Liboska , R. , Ligasová , A. , Strunin , D. , Rosenberg , I. & Koberna , K. Most Anti-BrdU Antibodies React with 2′-Deoxy-5-Ethynyluridine — The Method for the Effective Suppression of This Cross-Reactivity . PLoS ONE 7 , e51679 ( 2012 ). OpenUrl CrossRef PubMed 20. ↵ Buchholz , S. M. et al. Depletion of Macrophages Improves Therapeutic Response to Gemcitabine in Murine Pancreas Cancer . Cancers (Basel) 12 , 1978 ( 2020 ). OpenUrl PubMed 21. ↵ Guedes , P. T. et al. Histological Analyses Demonstrate the Temporary Contribution of Yolk Sac, Liver, and Bone Marrow to Hematopoiesis during Chicken Development . PLOS ONE 9 , e90975 ( 2014 ). OpenUrl PubMed 22. ↵ Wong , G. K. & Cavey , M. J. Development of the liver in the chicken embryo. II. Erythropoietic and granulopoietic cells . The Anatomical Record 235 , 131 – 143 ( 1993 ). OpenUrl CrossRef PubMed 23. ↵ Härtle , S. , Sutton , K. , Vervelde , L. & Dalgaard , T. S. Delineation of chicken immune markers in the era of omics and multicolor flow cytometry . Front. Vet. Sci . 11 , ( 2024 ). View the discussion thread. Back to top Previous Next Posted September 15, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Application of nucleoside analogue labelling to study the cell cycle of xenografted PDAC cell lines in the chorioallantoic membrane model Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Application of nucleoside analogue labelling to study the cell cycle of xenografted PDAC cell lines in the chorioallantoic membrane model Robin Colenbier , Denisa Lodoaba Jurescu , Jean-Pierre Timmermans , Johannes Bogers bioRxiv 2025.09.09.675135; doi: https://doi.org/10.1101/2025.09.09.675135 Share This Article: Copy Citation Tools Application of nucleoside analogue labelling to study the cell cycle of xenografted PDAC cell lines in the chorioallantoic membrane model Robin Colenbier , Denisa Lodoaba Jurescu , Jean-Pierre Timmermans , Johannes Bogers bioRxiv 2025.09.09.675135; doi: https://doi.org/10.1101/2025.09.09.675135 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)