Comprehensive DSRCT multi-omics analyses unveil CACNA2D2 as a diagnostic hallmark and super-enhancer-driven EWSR1::WT1 signature gene

IF 20.1 1区 医学 Q1 ONCOLOGY
Florian Henning Geyer, Alina Ritter, Seneca Kinn-Gurzo, Tobias Faehling, Jing Li, Armin Jarosch, Carine Ngo, Endrit Vinca, Karim Aljakouch, Azhar Orynbek, Shunya Ohmura, Thomas Kirchner, Roland Imle, Laura Romero-Pérez, Juan Díaz-Martín, Stefanie Bertram, Enrique de Álava, Clémence Henon, Sophie Postel-Vilnay, Ana Banito, Martin Sill, Yvonne Versleijen-Jonkers, Benjamin Friedrich Berthold Mayer, Martin Ebinger, Monika Sparber-Sauer, Sabine Stegmaier, Daniel Baumhoer, Wolfgang Hartmann, Jeroen Krijgsveld, David Horst, Olivier Delattre, Patrick Joseph Grohar, Thomas Georg Phillip Grünewald, Florencia Cidre-Aranaz
{"title":"Comprehensive DSRCT multi-omics analyses unveil CACNA2D2 as a diagnostic hallmark and super-enhancer-driven EWSR1::WT1 signature gene","authors":"Florian Henning Geyer,&nbsp;Alina Ritter,&nbsp;Seneca Kinn-Gurzo,&nbsp;Tobias Faehling,&nbsp;Jing Li,&nbsp;Armin Jarosch,&nbsp;Carine Ngo,&nbsp;Endrit Vinca,&nbsp;Karim Aljakouch,&nbsp;Azhar Orynbek,&nbsp;Shunya Ohmura,&nbsp;Thomas Kirchner,&nbsp;Roland Imle,&nbsp;Laura Romero-Pérez,&nbsp;Juan Díaz-Martín,&nbsp;Stefanie Bertram,&nbsp;Enrique de Álava,&nbsp;Clémence Henon,&nbsp;Sophie Postel-Vilnay,&nbsp;Ana Banito,&nbsp;Martin Sill,&nbsp;Yvonne Versleijen-Jonkers,&nbsp;Benjamin Friedrich Berthold Mayer,&nbsp;Martin Ebinger,&nbsp;Monika Sparber-Sauer,&nbsp;Sabine Stegmaier,&nbsp;Daniel Baumhoer,&nbsp;Wolfgang Hartmann,&nbsp;Jeroen Krijgsveld,&nbsp;David Horst,&nbsp;Olivier Delattre,&nbsp;Patrick Joseph Grohar,&nbsp;Thomas Georg Phillip Grünewald,&nbsp;Florencia Cidre-Aranaz","doi":"10.1002/cac2.70015","DOIUrl":null,"url":null,"abstract":"<p>Desmoplastic small round cell tumor (DSRCT) is an aggressive cancer that predominantly affects adolescents and young adults, typically developing at sites lined by mesothelium [<span>1, 2</span>]. DSRCT is genetically defined by a chromosomal translocation that fuses the N-terminus of EWS RNA binding protein 1 (<i>EWSR1</i>) to the C-terminus of Wilms tumor protein (<i>WT1)</i>, forming EWSR1::WT1 [<span>3</span>]. This fusion encodes a potent transcription factor and is the only known driver of oncogenic transformation in DSRCT [<span>4</span>]. The lack of a comprehensive understanding of DSRCT biology parallels its dismal survival rate (5%-20%) [<span>1</span>]. These challenges are exacerbated by the absence of clinical trials, the limited systematic collection and analysis of DSRCT biomaterial [<span>1</span>], and the notable lack of specific diagnostic markers, necessitating resource-intensive molecular testing for an accurate diagnosis.</p><p>Here we first focused on identifying promising candidates for validation as single, fast, and reliable diagnostic DSRCT markers. For this, we performed differential gene expression (DEG) analysis on datasets comprising patient samples from 32 DSRCT and 20 morphological mimics, identifying 23 genes overexpressed in DSRCT (log<sub>2</sub> fold change (log<sub>2</sub>FC) &gt; 2.5; adjusted <i>P</i>-value (<i>Padj)</i> &lt; 0.01; Figure 1A, Supplementary Figure S1A). Secondly, we analyzed EWSR1::WT1 binding sites derived from chromatin immunoprecipitation followed by sequencing (ChIP-seq) data [<span>5</span>] obtained from the JN-DSRCT-1 cell line, identifying 2,065 genomic loci likely regulated by EWSR1::WT1 (Figure 1A). Third, we established JN-DSRCT-1 and SK-DSRCT2 cell lines expressing doxycycline (DOX)-inducible short hairpin RNA (shRNA)-mediated EWSR1::WT1 knockdown (KD) (Supplementary Figure S1B). Differential protein expression (DEP) analysis of these cells identified 104 proteins consistently regulated across both cell lines (log<sub>2</sub>FC &gt; 1.0 and <i>Padj</i> &lt; 0.01; Figure 1A, Supplementary Table S1). The intersection of these analyses revealed calcium voltage-gated channel auxiliary subunit alpha2delta 2 (CACNA2D2) and IQ motif containing G (IQCG) as potential DSRCT biomarkers (Figure 1A). <i>CACNA2D2</i> was selected for validation due to its significantly higher expression in DSRCTs compared to <i>IQCG</i> (<i>P</i> &lt; 0.001; Figure 1A). Indeed, DSRCT exhibited the highest expression of <i>CACNA2D2</i> among all studied morphological mimics and normal tissues (<i>P &lt;</i> 0.001; Supplementary Figures S1C-D). Further ChIP-seq data and motif analyses of EWSR1::WT1 binding coordinates and histone marks in JN-DSRCT-1 and four DSRCT patient samples [<span>5, 6</span>] suggested a direct regulatory role of EWSR1::WT1 through an enhancer interaction at the <i>CACNA2D2</i> locus (Figure 1B). Notably, KD of EWSR1::WT1 in JN-DSRCT-1 resulted in a loss of the EWSR1::WT1 signal and Histone H3 lysine 27 acetylation (H3K27ac) enhancer marks at the <i>CACNA2D2</i> locus (Figure 1B). Additionally, chromatin interaction data [<span>6</span>] revealed 19 loops connecting the EWSR1::WT1 binding site to the transcription start site of <i>CACNA2D2</i>, which were depleted upon KD of <i>EWSR1::WT1</i> (Figure 1C). Super enhancer (SE) analysis further demonstrated that the EWSR1::WT1-bound enhancer exhibited a characteristic SE H3K27ac profile in JN-DSRCT-1, which was lost upon EWSR1::WT1 KD (Figure 1D, Supplementary Table S2).</p><p>Post-transcriptional and post-translational KD of EWSR1::WT1 in three DSRCT cell line models expressing different EWSR1::WT1 isoforms (Supplementary Figure S2A) resulted in a significant reduction in CACNA2D2 expression (Figures 1E–F, Supplementary Figure S1B, Supplementary Figures S2B–F). Additionally, ChIP-seq data derived from MeT-5A mesothelial cells [<span>6</span>] – the potential cell of origin of DSRCT [<span>7, 8</span>] – ectopically expressing different EWSR1::WT1 isoforms (-KTS, +KTS, or -KTS/+KTS) suggested direct regulation, as evidenced by the co-occurrence of H3K27ac signals and signals for V5- or HA-tagged EWSR1::WT1 isoforms at the <i>CACNA2D2</i> enhancer region (Supplementary Figure S2G). Notably, MeT-5A cells transfected with a control vector showed no substantial signal at this locus (Supplementary Figure S2G). Publicly available RNA-sequencing (RNA-seq) data from MeT-5A cells [<span>6</span>] expressing different EWSR1::WT1 isoforms showed that <i>CACNA2D2</i> was differentially expressed in the presence of EWSR1::WT1 (4.1 ≤ log<sub>2</sub>FC ≤ 5.9, <i>Padj</i> &lt; 0.001) (Supplementary Figure S2H). Finally, quantitative polymerase chain reaction (qPCR) analysis of MeT-5A cells stably expressing a DOX-inducible ectopic EWSR1::WT1 expression cassette confirmed that upon EWSR1::WT1 induction, <i>CACNA2D2</i> was significantly and highly overexpressed (Supplementary Figure S2I). Taken together, these results emphasize that EWSR1::WT1 is sufficient to drive <i>CACNA2D2</i> expression. SE analysis of MeT-5A-derived data strikingly showed that the <i>CACNA2D2</i> enhancer bound by EWSR1::WT1 became a SE upon ectopic expression of EWSR1::WT1<sup>− KTS + KTS</sup> (Supplementary Figure S2J).</p><p>To explore whether <i>CACNA2D2</i> could serve as a surrogate indicator of oncogenic <i>EWSR1::WT1</i> transformation, we defined a <i>CACNA2D2</i> gene set and gene signature by performing a correlation analysis of gene expression data from 32 DSRCT patient samples (Supplementary Figure S3A, Supplementary Tables S3-S4). Next, an EWSR1::WT1 signature was computed by performing a combined DEG analysis of newly generated in vivo and in vitro [<span>4</span>] material derived from three DSRCT cell lines upon <i>EWSR1::WT1</i> KD (Supplementary Figure S3A, Supplementary Table S4). Notably, fast gene set enrichment analysis (fGSEA) of the <i>CACNA2D2</i> gene set demonstrated a highly significant (<i>Padj</i> &lt; 0.001) and strong positive enrichment for the EWSR1::WT1 signature (normalized enrichment score, NES<sub>EWSR1::WT1</sub> = 3.6). Moreover, single sample gene set enrichment analysis (ssGSEA) of expression data from 32 DSRCT patient samples confirmed that the EWSR1::WT1 signature significantly correlated with that of CACNA2D2 (<i>r</i> = 0.75), highlighting a transcriptional interconnection between <i>CACNA2D2</i> and <i>EWSR1::WT1 in situ</i> (Figure 1G). These observations were further supported by single-cell (sc)-derived signatures from orthotopically-generated tumors using two DSRCT cell lines with DOX-inducible KD of EWSR1::WT1 at primary (<i>n</i> = 221) and metastatic (<i>n</i> = 221) locations (Figure 1G, Supplementary Table S4). Indeed, ssGSEA of our single-cell data showed highly significant correlation between the NES of our generated EWSR1::WT1 and CACNA2D2 signatures (Figure 1G), regardless of tumor location, implying that CACNA2D2-associated genes are also characteristic features of metastasized DSRCT cells (Supplementary Figure S3B).</p><p>To delineate the specificity of the interaction between <i>CACNA2D2</i> and <i>EWSR1::WT1</i> in DSRCT, we performed ssGSEA using our EWSR1::WT1 and CACNA2D2 signatures on expression data from 20 DSRCT morphological mimics (Figure 1H). Here, non-DSRCT cancer entities showed significantly lower NES and correlation strength for all signatures compared to DSRCT (Supplementary Figures S3C-D). These results further emphasized the high specificity of the CACNA2D2 and EWSR1::WT1 interplay in DSRCT. Moreover, both bulk- and sc-derived CACNA2D2 signatures precisely distinguished DSRCT cell clusters from non-tumor cells in single-cell RNA-sequencing (scRNA-seq) data from four DSRCT patients (<i>n</i> = 11 samples) [<span>9</span>] (Figure 1I, Supplementary Figure S3E). Concordantly, all predicted normal cell types within these tumors exhibited low enrichment of both CACNA2D2 signatures (Supplementary Figures S3F-G).</p><p>Further, dimensional reduction of <i>CACNA2D2</i>-associated CpG sites in 24 DSRCT patient samples, compared with 192 samples from 13 morphological mimics [<span>10</span>] revealed distinct clustering of all DSRCT samples, which was unique to <i>CACNA2D2</i> compared to other described EWSR1::WT1-regulated genes or <i>IQCG</i> (Figure 1A, Supplementary Figures S4A-B). Additionally, these <i>CACNA2D2</i>-associated CpG sites exhibited significant (<i>P</i> &lt; 0.001) and specific hypomethylation in DSRCT patient samples, collectively suggesting that the <i>CACNA2D2</i>-associated methylation signature is a distinct and specific feature of DSRCT (Supplementary Figure S4C).</p><p>To assess the diagnostic utility of CACNA2D2, we optimized a staining protocol for DSRCT cell line xenografts, achieving consistent and robust membranous or cytoplasmatic staining, even uncovering micrometastases (Figure 1J, Supplementary Figure S4D).</p><p>Finally, we assembled the largest collection of fresh-frozen and paraffin-embedded DSRCT patient samples analyzed to date (<i>n</i> = 61), comprising primary, metastatic, and post-treatment samples, and supplemented it with 249 patient samples from 18 different DSRCT morphological mimics (Supplementary Table S5). CACNA2D2 immunoreactivity was evaluated using a modified Immune Reactive Score (IRS) (Supplementary Material and Methods). Excitingly, DSRCT tumor sections exhibited the highest IRS for CACNA2D2 (IRS<sub>mean</sub> = 10.5, 6 ≤ IRS<sub>DSRCT</sub> ≤ 12, <i>P</i> &lt; 0.001) (Supplementary Figure S4E-F), with specificity reaching 98% when applying a cutoff of IRS &gt; 1 (Figure 1K-M, Supplementary Figure S4E). Indeed, even samples derived from CIC- and BCOR-rearranged sarcomas, as well as fusion-positive alveolar rhabdomyosarcomas, showed negligible mean protein expression compared to DSRCT (IRS<sub>CIC</sub> = 0.21, IRS<sub>BCOR</sub> = 0, IRS<sub>fp-ARMS</sub> = 0.56). Furthermore, 100% sensitivity was achieved when applying an IRS cutoff of ≤ 6, implying that DSRCT samples consistently displayed strong staining for CACNA2D2 (Figure 1M). Thus, we recommend a single CACNA2D2 staining for clinically and histologically compatible DSRCT differential diagnosis. If IRS<sub>CACNA2D2</sub> ≤ 1, the diagnosis should be reconsidered or re-evaluated using molecular diagnostic procedures (such as fluorescence in situ hybridization, qRT-PCR, or next-generation sequencing), if available (Figure 1N). Conversely, if IRS<sub>CACNA2D2</sub> &gt; 1, a diagnosis of DSRCT may be established. Also, CACNA2D2 staining may be used to rule out DSRCT within the broad spectrum of small-round-blue-cell tumors, potentially offering extensive diagnostic utility.</p><p>Finally, the high, specific, and homogenous membranous expression of CACNA2D2 in DSRCT, combined with the highly specific antibody described here, makes CACNA2D2 an ideal candidate for targeted therapeutic approaches, including drug delivery using antibody-drug conjugates or CAR-T cell therapy. Future studies should investigate the precise role of CACNA2D2 in DSRCT biology, with a focus on its potential contributions in tumor cell fitness, differentiation, and tumorigenic potential.</p><p>In conclusion, here we developed an extensive toolset for DSRCT research (Supplementary Figure S4G), a validated blueprint for how such resources could be harnessed in other cancer entities, and identified CACNA2D2 as a singular, powerful DSRCT biomarker.</p><p>Florian Henning Geyer, Florencia Cidre-Aranaz, and Thomas Georg Phillip Grünewald conceived the study. Florian Henning Geyer and Florencia Cidre-Aranaz wrote the paper and drafted all figures and tables. Florian Henning Geyer carried out all in vitro and in vivo experiments and performed all bioinformatic and statistical analyses. Florian Henning Geyer, Alina Ritter, and Thomas Georg Phillip Grünewald performed immunohistochemical evaluation and scoring of tumor samples and TMAs. Florencia Cidre-Aranaz, Roland Imle, and Ana Banito performed and/or coordinated in vivo experiments. Olivier Delattre provided microarray expression data. Seneca Kinn-Gurzo performed in vitro experiments on BER cell lines. Tobias Faehling and Clémence Henon performed single-cell bioinformatic analyses. Karim Aljakouch and Azhar Orynbek performed MassSpec and analyzed MassSpec data. Alina Ritter, Jing Li, Endrit Vinca, Laura Romero-Perez, Martin Sill, and Shunya Ohmura contributed to experimental procedures. Wolfgang Hartmann and Benjamin Friedrich Berthold Mayer provided clinical and/or histological guidance. Enrique De Álava, Juan Díaz-Martín, Stefanie Bertram, Sophie Postel-Vilnay, Martin Ebinger, Monika Sparber-Sauer, Daniel Baumhoer, Carine Ngo, David Horst, Yvonne Versleijen-Jonkers, Armin Jarosch, Sabine Stegmaier, and Thomas Kirchner provided clinical samples. Patrick Joseph Grohar, Thomas Georg Phillip Grünewald, and Jeroen Krijgsveld provided laboratory infrastructure. Florencia Cidre-Aranaz and Thomas Georg Phillip Grünewald supervised the study and data analysis. All authors read and approved the final manuscript.</p><p>The authors declare no competing interests.</p><p>The laboratory of Thomas Georg Phillip Grünewald is supported by grants from the Matthias-Lackas Foundation, the Dr. Leopold und Carmen Ellinger Foundation, the European Research Council (ERC CoG 2023 #101122595), the Deutsche Forschungsgemeinschaft (DFG 458891500), the German Cancer Aid (DKH-70112257, DKH-7011411, DKH-70114278, DKH-70115315), the Dr. Rolf M. Schwiete foundation, the SMARCB1 association, the Ministry of Education and Research (BMBF; SMART-CARE and HEROES-AYA), and the Barbara and Wilfried Mohr foundation. The research team of Florencia Cidre-Aranaz was supported by the German Cancer Aid (DHK-70114111), and the Dr. Rolf M. Schwiete Stiftung (2020-028 and 2022-31). In addition, this work was delivered as part of the PROTECT team supported by the Cancer Grand Challenges partnership funded by Cancer Research UK, the National Cancer Institute, the Scientific Foundation of the Spanish Association Against Cancer And KiKa (Children Cancer Free Foundation). Florian Henning Geyer, Tobias Faehling, Endrit Vinca, and Alina Ritter were supported by the German Academic Scholarship Foundation. In addition, Endrit Vinca was supported by scholarships from the Heinrich F.C. Behr foundation and the Rudolf and Brigitte Zenner foundation, Tobias Faehling by the Heinrich F.C. Behr foundation, and Florian Henning Geyer and Alina Ritter are supported by the German Cancer Aid through the ‘Mildred-Scheel-Doctoral Program’ (DKH-70114866). This project is co-funded by the European Union (ERC, CANCER-HARAKIRI, 101122595). All views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.</p><p>In vivo experiments were approved by the government of North Baden and conducted in accordance with ARRIVE guidelines and recommendations of the European Community (86/609/EEC) and UKCCCR (guidelines for the welfare and use of animals in cancer research). Open slides or tissue-microarrays from human formalin-fixed, paraffin-embedded or cryopreserved tissue samples were retrieved from the archives of the Institute of Pathology of the LMU Munich, the Charité Berlin, The Biobank of the Hospital Universitario Virgen del Rocío of Seville, the Hospital Gustave Roussy (Villejuif), the Bone Tumor Reference Center at the University of Basel, the University of Essen, the Cooperative Weichteilsarkom Studiengruppe (CWS) study center, the Klinikum Stuttgart (ethics committee from the Medical Faculty of the Eberhard-Karls University and University Hospital of Tübingen, approval no. 207/2022BO2), the Radboud University Medical Center, the Pathology Institute of the LMU Munich (approval no. 550-16 UE), and the University of Heidelberg (approval no. S-211/2021).</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":"45 6","pages":"702-708"},"PeriodicalIF":20.1000,"publicationDate":"2025-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.70015","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.70015","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Desmoplastic small round cell tumor (DSRCT) is an aggressive cancer that predominantly affects adolescents and young adults, typically developing at sites lined by mesothelium [1, 2]. DSRCT is genetically defined by a chromosomal translocation that fuses the N-terminus of EWS RNA binding protein 1 (EWSR1) to the C-terminus of Wilms tumor protein (WT1), forming EWSR1::WT1 [3]. This fusion encodes a potent transcription factor and is the only known driver of oncogenic transformation in DSRCT [4]. The lack of a comprehensive understanding of DSRCT biology parallels its dismal survival rate (5%-20%) [1]. These challenges are exacerbated by the absence of clinical trials, the limited systematic collection and analysis of DSRCT biomaterial [1], and the notable lack of specific diagnostic markers, necessitating resource-intensive molecular testing for an accurate diagnosis.

Here we first focused on identifying promising candidates for validation as single, fast, and reliable diagnostic DSRCT markers. For this, we performed differential gene expression (DEG) analysis on datasets comprising patient samples from 32 DSRCT and 20 morphological mimics, identifying 23 genes overexpressed in DSRCT (log2 fold change (log2FC) > 2.5; adjusted P-value (Padj) < 0.01; Figure 1A, Supplementary Figure S1A). Secondly, we analyzed EWSR1::WT1 binding sites derived from chromatin immunoprecipitation followed by sequencing (ChIP-seq) data [5] obtained from the JN-DSRCT-1 cell line, identifying 2,065 genomic loci likely regulated by EWSR1::WT1 (Figure 1A). Third, we established JN-DSRCT-1 and SK-DSRCT2 cell lines expressing doxycycline (DOX)-inducible short hairpin RNA (shRNA)-mediated EWSR1::WT1 knockdown (KD) (Supplementary Figure S1B). Differential protein expression (DEP) analysis of these cells identified 104 proteins consistently regulated across both cell lines (log2FC > 1.0 and Padj < 0.01; Figure 1A, Supplementary Table S1). The intersection of these analyses revealed calcium voltage-gated channel auxiliary subunit alpha2delta 2 (CACNA2D2) and IQ motif containing G (IQCG) as potential DSRCT biomarkers (Figure 1A). CACNA2D2 was selected for validation due to its significantly higher expression in DSRCTs compared to IQCG (P < 0.001; Figure 1A). Indeed, DSRCT exhibited the highest expression of CACNA2D2 among all studied morphological mimics and normal tissues (P < 0.001; Supplementary Figures S1C-D). Further ChIP-seq data and motif analyses of EWSR1::WT1 binding coordinates and histone marks in JN-DSRCT-1 and four DSRCT patient samples [5, 6] suggested a direct regulatory role of EWSR1::WT1 through an enhancer interaction at the CACNA2D2 locus (Figure 1B). Notably, KD of EWSR1::WT1 in JN-DSRCT-1 resulted in a loss of the EWSR1::WT1 signal and Histone H3 lysine 27 acetylation (H3K27ac) enhancer marks at the CACNA2D2 locus (Figure 1B). Additionally, chromatin interaction data [6] revealed 19 loops connecting the EWSR1::WT1 binding site to the transcription start site of CACNA2D2, which were depleted upon KD of EWSR1::WT1 (Figure 1C). Super enhancer (SE) analysis further demonstrated that the EWSR1::WT1-bound enhancer exhibited a characteristic SE H3K27ac profile in JN-DSRCT-1, which was lost upon EWSR1::WT1 KD (Figure 1D, Supplementary Table S2).

Post-transcriptional and post-translational KD of EWSR1::WT1 in three DSRCT cell line models expressing different EWSR1::WT1 isoforms (Supplementary Figure S2A) resulted in a significant reduction in CACNA2D2 expression (Figures 1E–F, Supplementary Figure S1B, Supplementary Figures S2B–F). Additionally, ChIP-seq data derived from MeT-5A mesothelial cells [6] – the potential cell of origin of DSRCT [7, 8] – ectopically expressing different EWSR1::WT1 isoforms (-KTS, +KTS, or -KTS/+KTS) suggested direct regulation, as evidenced by the co-occurrence of H3K27ac signals and signals for V5- or HA-tagged EWSR1::WT1 isoforms at the CACNA2D2 enhancer region (Supplementary Figure S2G). Notably, MeT-5A cells transfected with a control vector showed no substantial signal at this locus (Supplementary Figure S2G). Publicly available RNA-sequencing (RNA-seq) data from MeT-5A cells [6] expressing different EWSR1::WT1 isoforms showed that CACNA2D2 was differentially expressed in the presence of EWSR1::WT1 (4.1 ≤ log2FC ≤ 5.9, Padj < 0.001) (Supplementary Figure S2H). Finally, quantitative polymerase chain reaction (qPCR) analysis of MeT-5A cells stably expressing a DOX-inducible ectopic EWSR1::WT1 expression cassette confirmed that upon EWSR1::WT1 induction, CACNA2D2 was significantly and highly overexpressed (Supplementary Figure S2I). Taken together, these results emphasize that EWSR1::WT1 is sufficient to drive CACNA2D2 expression. SE analysis of MeT-5A-derived data strikingly showed that the CACNA2D2 enhancer bound by EWSR1::WT1 became a SE upon ectopic expression of EWSR1::WT1− KTS + KTS (Supplementary Figure S2J).

To explore whether CACNA2D2 could serve as a surrogate indicator of oncogenic EWSR1::WT1 transformation, we defined a CACNA2D2 gene set and gene signature by performing a correlation analysis of gene expression data from 32 DSRCT patient samples (Supplementary Figure S3A, Supplementary Tables S3-S4). Next, an EWSR1::WT1 signature was computed by performing a combined DEG analysis of newly generated in vivo and in vitro [4] material derived from three DSRCT cell lines upon EWSR1::WT1 KD (Supplementary Figure S3A, Supplementary Table S4). Notably, fast gene set enrichment analysis (fGSEA) of the CACNA2D2 gene set demonstrated a highly significant (Padj < 0.001) and strong positive enrichment for the EWSR1::WT1 signature (normalized enrichment score, NESEWSR1::WT1 = 3.6). Moreover, single sample gene set enrichment analysis (ssGSEA) of expression data from 32 DSRCT patient samples confirmed that the EWSR1::WT1 signature significantly correlated with that of CACNA2D2 (r = 0.75), highlighting a transcriptional interconnection between CACNA2D2 and EWSR1::WT1 in situ (Figure 1G). These observations were further supported by single-cell (sc)-derived signatures from orthotopically-generated tumors using two DSRCT cell lines with DOX-inducible KD of EWSR1::WT1 at primary (n = 221) and metastatic (n = 221) locations (Figure 1G, Supplementary Table S4). Indeed, ssGSEA of our single-cell data showed highly significant correlation between the NES of our generated EWSR1::WT1 and CACNA2D2 signatures (Figure 1G), regardless of tumor location, implying that CACNA2D2-associated genes are also characteristic features of metastasized DSRCT cells (Supplementary Figure S3B).

To delineate the specificity of the interaction between CACNA2D2 and EWSR1::WT1 in DSRCT, we performed ssGSEA using our EWSR1::WT1 and CACNA2D2 signatures on expression data from 20 DSRCT morphological mimics (Figure 1H). Here, non-DSRCT cancer entities showed significantly lower NES and correlation strength for all signatures compared to DSRCT (Supplementary Figures S3C-D). These results further emphasized the high specificity of the CACNA2D2 and EWSR1::WT1 interplay in DSRCT. Moreover, both bulk- and sc-derived CACNA2D2 signatures precisely distinguished DSRCT cell clusters from non-tumor cells in single-cell RNA-sequencing (scRNA-seq) data from four DSRCT patients (n = 11 samples) [9] (Figure 1I, Supplementary Figure S3E). Concordantly, all predicted normal cell types within these tumors exhibited low enrichment of both CACNA2D2 signatures (Supplementary Figures S3F-G).

Further, dimensional reduction of CACNA2D2-associated CpG sites in 24 DSRCT patient samples, compared with 192 samples from 13 morphological mimics [10] revealed distinct clustering of all DSRCT samples, which was unique to CACNA2D2 compared to other described EWSR1::WT1-regulated genes or IQCG (Figure 1A, Supplementary Figures S4A-B). Additionally, these CACNA2D2-associated CpG sites exhibited significant (P < 0.001) and specific hypomethylation in DSRCT patient samples, collectively suggesting that the CACNA2D2-associated methylation signature is a distinct and specific feature of DSRCT (Supplementary Figure S4C).

To assess the diagnostic utility of CACNA2D2, we optimized a staining protocol for DSRCT cell line xenografts, achieving consistent and robust membranous or cytoplasmatic staining, even uncovering micrometastases (Figure 1J, Supplementary Figure S4D).

Finally, we assembled the largest collection of fresh-frozen and paraffin-embedded DSRCT patient samples analyzed to date (n = 61), comprising primary, metastatic, and post-treatment samples, and supplemented it with 249 patient samples from 18 different DSRCT morphological mimics (Supplementary Table S5). CACNA2D2 immunoreactivity was evaluated using a modified Immune Reactive Score (IRS) (Supplementary Material and Methods). Excitingly, DSRCT tumor sections exhibited the highest IRS for CACNA2D2 (IRSmean = 10.5, 6 ≤ IRSDSRCT ≤ 12, P < 0.001) (Supplementary Figure S4E-F), with specificity reaching 98% when applying a cutoff of IRS > 1 (Figure 1K-M, Supplementary Figure S4E). Indeed, even samples derived from CIC- and BCOR-rearranged sarcomas, as well as fusion-positive alveolar rhabdomyosarcomas, showed negligible mean protein expression compared to DSRCT (IRSCIC = 0.21, IRSBCOR = 0, IRSfp-ARMS = 0.56). Furthermore, 100% sensitivity was achieved when applying an IRS cutoff of ≤ 6, implying that DSRCT samples consistently displayed strong staining for CACNA2D2 (Figure 1M). Thus, we recommend a single CACNA2D2 staining for clinically and histologically compatible DSRCT differential diagnosis. If IRSCACNA2D2 ≤ 1, the diagnosis should be reconsidered or re-evaluated using molecular diagnostic procedures (such as fluorescence in situ hybridization, qRT-PCR, or next-generation sequencing), if available (Figure 1N). Conversely, if IRSCACNA2D2 > 1, a diagnosis of DSRCT may be established. Also, CACNA2D2 staining may be used to rule out DSRCT within the broad spectrum of small-round-blue-cell tumors, potentially offering extensive diagnostic utility.

Finally, the high, specific, and homogenous membranous expression of CACNA2D2 in DSRCT, combined with the highly specific antibody described here, makes CACNA2D2 an ideal candidate for targeted therapeutic approaches, including drug delivery using antibody-drug conjugates or CAR-T cell therapy. Future studies should investigate the precise role of CACNA2D2 in DSRCT biology, with a focus on its potential contributions in tumor cell fitness, differentiation, and tumorigenic potential.

In conclusion, here we developed an extensive toolset for DSRCT research (Supplementary Figure S4G), a validated blueprint for how such resources could be harnessed in other cancer entities, and identified CACNA2D2 as a singular, powerful DSRCT biomarker.

Florian Henning Geyer, Florencia Cidre-Aranaz, and Thomas Georg Phillip Grünewald conceived the study. Florian Henning Geyer and Florencia Cidre-Aranaz wrote the paper and drafted all figures and tables. Florian Henning Geyer carried out all in vitro and in vivo experiments and performed all bioinformatic and statistical analyses. Florian Henning Geyer, Alina Ritter, and Thomas Georg Phillip Grünewald performed immunohistochemical evaluation and scoring of tumor samples and TMAs. Florencia Cidre-Aranaz, Roland Imle, and Ana Banito performed and/or coordinated in vivo experiments. Olivier Delattre provided microarray expression data. Seneca Kinn-Gurzo performed in vitro experiments on BER cell lines. Tobias Faehling and Clémence Henon performed single-cell bioinformatic analyses. Karim Aljakouch and Azhar Orynbek performed MassSpec and analyzed MassSpec data. Alina Ritter, Jing Li, Endrit Vinca, Laura Romero-Perez, Martin Sill, and Shunya Ohmura contributed to experimental procedures. Wolfgang Hartmann and Benjamin Friedrich Berthold Mayer provided clinical and/or histological guidance. Enrique De Álava, Juan Díaz-Martín, Stefanie Bertram, Sophie Postel-Vilnay, Martin Ebinger, Monika Sparber-Sauer, Daniel Baumhoer, Carine Ngo, David Horst, Yvonne Versleijen-Jonkers, Armin Jarosch, Sabine Stegmaier, and Thomas Kirchner provided clinical samples. Patrick Joseph Grohar, Thomas Georg Phillip Grünewald, and Jeroen Krijgsveld provided laboratory infrastructure. Florencia Cidre-Aranaz and Thomas Georg Phillip Grünewald supervised the study and data analysis. All authors read and approved the final manuscript.

The authors declare no competing interests.

The laboratory of Thomas Georg Phillip Grünewald is supported by grants from the Matthias-Lackas Foundation, the Dr. Leopold und Carmen Ellinger Foundation, the European Research Council (ERC CoG 2023 #101122595), the Deutsche Forschungsgemeinschaft (DFG 458891500), the German Cancer Aid (DKH-70112257, DKH-7011411, DKH-70114278, DKH-70115315), the Dr. Rolf M. Schwiete foundation, the SMARCB1 association, the Ministry of Education and Research (BMBF; SMART-CARE and HEROES-AYA), and the Barbara and Wilfried Mohr foundation. The research team of Florencia Cidre-Aranaz was supported by the German Cancer Aid (DHK-70114111), and the Dr. Rolf M. Schwiete Stiftung (2020-028 and 2022-31). In addition, this work was delivered as part of the PROTECT team supported by the Cancer Grand Challenges partnership funded by Cancer Research UK, the National Cancer Institute, the Scientific Foundation of the Spanish Association Against Cancer And KiKa (Children Cancer Free Foundation). Florian Henning Geyer, Tobias Faehling, Endrit Vinca, and Alina Ritter were supported by the German Academic Scholarship Foundation. In addition, Endrit Vinca was supported by scholarships from the Heinrich F.C. Behr foundation and the Rudolf and Brigitte Zenner foundation, Tobias Faehling by the Heinrich F.C. Behr foundation, and Florian Henning Geyer and Alina Ritter are supported by the German Cancer Aid through the ‘Mildred-Scheel-Doctoral Program’ (DKH-70114866). This project is co-funded by the European Union (ERC, CANCER-HARAKIRI, 101122595). All views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.

In vivo experiments were approved by the government of North Baden and conducted in accordance with ARRIVE guidelines and recommendations of the European Community (86/609/EEC) and UKCCCR (guidelines for the welfare and use of animals in cancer research). Open slides or tissue-microarrays from human formalin-fixed, paraffin-embedded or cryopreserved tissue samples were retrieved from the archives of the Institute of Pathology of the LMU Munich, the Charité Berlin, The Biobank of the Hospital Universitario Virgen del Rocío of Seville, the Hospital Gustave Roussy (Villejuif), the Bone Tumor Reference Center at the University of Basel, the University of Essen, the Cooperative Weichteilsarkom Studiengruppe (CWS) study center, the Klinikum Stuttgart (ethics committee from the Medical Faculty of the Eberhard-Karls University and University Hospital of Tübingen, approval no. 207/2022BO2), the Radboud University Medical Center, the Pathology Institute of the LMU Munich (approval no. 550-16 UE), and the University of Heidelberg (approval no. S-211/2021).

Abstract Image

全面的 DSRCT 多组学分析揭示了 CACNA2D2 作为诊断标志和超级增强子驱动的 EWSR1::WT1 特征基因。
结缔组织增生小圆细胞瘤(DSRCT)是一种侵袭性癌症,主要影响青少年和年轻人,通常发生在间皮层排列的部位[1,2]。DSRCT的遗传定义是染色体易位,将EWS RNA结合蛋白1 (EWSR1)的n端与Wilms肿瘤蛋白(WT1)的c端融合,形成EWSR1::WT1[3]。这种融合编码一种有效的转录因子,是DSRCT[4]中唯一已知的致癌转化驱动因素。缺乏对DSRCT生物学的全面了解与其令人沮丧的生存率(5%-20%)相似。由于缺乏临床试验,DSRCT生物材料[1]的系统收集和分析有限,以及明显缺乏特定的诊断标记,需要资源密集的分子检测来进行准确诊断,这些挑战加剧了。在这里,我们首先专注于确定有希望的候选物,作为单一、快速、可靠的诊断性DSRCT标记物进行验证。为此,我们对来自32例DSRCT和20例形态模拟患者样本的数据集进行了差异基因表达(DEG)分析,鉴定出23个基因在DSRCT中过表达(log2倍变化(log2FC) &gt;2.5;调整p值(Padj) &lt;0.01;图1A,补充图S1A)。其次,我们分析了来自染色质免疫沉淀的EWSR1::WT1结合位点,随后从JN-DSRCT-1细胞系获得测序(ChIP-seq)数据[5],确定了2065个可能由EWSR1::WT1调控的基因组位点(图1A)。第三,我们建立了表达多西环素(DOX)诱导的短发卡RNA (shRNA)介导的EWSR1::WT1敲低(KD)的JN-DSRCT-1和SK-DSRCT2细胞系(Supplementary Figure S1B)。这些细胞的差异蛋白表达(DEP)分析鉴定出104种蛋白在两种细胞系中一致受到调节(log2FC &gt;1.0和Padj &lt;0.01;图1A,补充表S1)。这些分析的交集揭示了钙电压门控通道辅助亚基alpha2delta 2 (CACNA2D2)和含有IQ基序G (IQCG)作为潜在的DSRCT生物标志物(图1A)。之所以选择CACNA2D2进行验证,是因为它在dsrct中的表达明显高于IQCG (P &lt;0.001;图1 a)。事实上,DSRCT在所有形态学模拟和正常组织中CACNA2D2的表达最高(P &lt;0.001;补充数据S1C-D)。进一步的ChIP-seq数据和对JN-DSRCT-1和4个DSRCT患者样本中EWSR1::WT1结合坐标和组蛋白标记的基序分析[5,6]表明EWSR1::WT1通过CACNA2D2位点的增强子相互作用直接调控(图1B)。值得注意的是,JN-DSRCT-1中EWSR1::WT1的KD导致了CACNA2D2位点EWSR1::WT1信号和组蛋白H3赖氨酸27乙酰化(H3K27ac)增强子标记的缺失(图1B)。此外,染色质相互作用数据[6]显示了连接EWSR1::WT1结合位点和CACNA2D2转录起始位点的19个环,这些环在EWSR1::WT1 KD时被耗尽(图1C)。超级增强子(SE)分析进一步表明,EWSR1::WT1结合的增强子在JN-DSRCT-1中表现出典型的SE H3K27ac谱,在EWSR1::WT1 KD上缺失(图1D,补充表S2)。在表达不同EWSR1::WT1亚型的三种DSRCT细胞系模型中,EWSR1::WT1的转录后和翻译后KD (Supplementary Figure S2A)导致CACNA2D2表达显著降低(图1E-F, Supplementary Figure S1B, Supplementary Figure S2B-F)。此外,来自MeT-5A间皮细胞[6]的ChIP-seq数据- DSRCT的潜在起源细胞[7,8]-异位表达不同的EWSR1::WT1亚型(-KTS, +KTS或-KTS/+KTS)表明直接调控,证明H3K27ac信号和V5或ha标记的EWSR1::WT1亚型在CACNA2D2增强子区域共出现(Supplementary Figure S2G)。值得注意的是,用对照载体转染的MeT-5A细胞在该位点未显示明显的信号(补充图S2G)。来自表达不同EWSR1::WT1亚型的MeT-5A细胞[6]的公开rna测序(RNA-seq)数据显示,在EWSR1::WT1存在时,CACNA2D2存在差异表达(4.1≤log2FC≤5.9,Padj &lt;0.001)(补充图S2H)。最后,对稳定表达dox诱导的异位EWSR1::WT1表达盒的MeT-5A细胞进行定量聚合酶链反应(qPCR)分析,证实EWSR1::WT1诱导后,CACNA2D2显著高过表达(Supplementary Figure S2I)。综上所述,这些结果强调EWSR1::WT1足以驱动CACNA2D2的表达。对met - 5a衍生数据的SE分析显示,EWSR1::WT1结合的CACNA2D2增强子在异位表达EWSR1::WT1−KTS + KTS时成为SE (Supplementary Figure S2J)。 为了探讨CACNA2D2是否可以作为致癌EWSR1::WT1转化的替代指标,我们通过对32例DSRCT患者样本的基因表达数据进行相关性分析,定义了CACNA2D2基因集和基因标记(补充图S3A,补充表S3-S4)。接下来,通过对来自三个DSRCT细胞系的EWSR1::WT1 KD新生成的体内和体外[4]材料进行联合DEG分析,计算EWSR1::WT1特征(补充图S3A,补充表S4)。值得注意的是,CACNA2D2基因集的快速基因集富集分析(fGSEA)显示了高度显著的(Padj &lt;EWSR1::WT1特征呈强正富集(归一化富集分数,NESEWSR1::WT1 = 3.6)。此外,对32例DSRCT患者样本表达数据的单样本基因集富集分析(ssGSEA)证实,EWSR1::WT1与CACNA2D2的特征显著相关(r = 0.75),突出了CACNA2D2与EWSR1::WT1之间的原位转录互连(图1G)。这些观察结果进一步得到了原位生成肿瘤的单细胞(sc)来源特征的支持,使用两个DSRCT细胞系,在原发(n = 221)和转移(n = 221)位置具有dox诱导的EWSR1::WT1 KD(图1G,补充表S4)。事实上,我们的单细胞数据的ssGSEA显示,无论肿瘤位置如何,我们生成的EWSR1::WT1和CACNA2D2特征的NES之间存在高度显著的相关性(图1G),这意味着CACNA2D2相关基因也是转移性DSRCT细胞的特征(补充图S3B)。为了描述CACNA2D2和EWSR1::WT1在DSRCT中相互作用的特异性,我们使用我们的EWSR1::WT1和CACNA2D2签名对来自20个DSRCT形态模拟的表达数据进行了ssGSEA(图1H)。在这里,与DSRCT相比,非DSRCT癌症实体的所有特征的网元和相关强度都明显较低(补充图S3C-D)。这些结果进一步强调了CACNA2D2和EWSR1::WT1相互作用在DSRCT中的高特异性。此外,在来自4名DSRCT患者(n = 11个样本)的单细胞rna测序(scRNA-seq)数据中,大量和sc来源的CACNA2D2特征都能精确区分DSRCT细胞簇和非肿瘤细胞(图1I,补充图S3E)。与此一致的是,这些肿瘤中所有预测的正常细胞类型都表现出CACNA2D2两种特征的低富集(补充图sgf - g)。此外,与来自13个形态模拟[10]的192个样本相比,24个DSRCT患者样本中CACNA2D2相关CpG位点的降维显示,所有DSRCT样本都有明显的聚类,与其他描述的EWSR1:: wt1调节基因或IQCG相比,这是CACNA2D2所独有的(图1A,补充图S4A-B)。此外,这些cacna2d2相关的CpG位点表现出显著的(P &lt;0.001)和DSRCT患者样本中的特异性低甲基化,共同表明cacna2d2相关的甲基化特征是DSRCT的独特和特异性特征(补充图S4C)。为了评估CACNA2D2的诊断效用,我们优化了DSRCT细胞系异种移植的染色方案,实现了一致且坚固的膜或细胞质染色,甚至发现了微转移(图1J,补充图S4D)。最后,我们收集了迄今为止分析的最大的新鲜冷冻和石蜡包埋的DSRCT患者样本(n = 61),包括原发、转移和治疗后样本,并补充了来自18种不同DSRCT形态模拟的249例患者样本(补充表S5)。采用改进的免疫反应性评分(IRS)评价CACNA2D2的免疫反应性(补充材料和方法)。令人兴奋的是,DSRCT肿瘤切片显示CACNA2D2的IRS最高(IRSmean = 10.5, 6≤IRSDSRCT≤12,P &lt;0.001)(补充图S4E-F),当应用IRS &gt的截止值时,特异性达到98%;1(图k - m,补充图S4E)。事实上,即使是来自CIC和bcor重排肉瘤的样本,以及融合阳性的肺泡横纹肌肉瘤,与DSRCT相比,平均蛋白表达也可以忽略不计(IRSCIC = 0.21, IRSBCOR = 0, IRSfp-ARMS = 0.56)。此外,当IRS截止值≤6时,灵敏度达到100%,这意味着DSRCT样品始终显示CACNA2D2的强染色(图1M)。因此,我们推荐单一的CACNA2D2染色作为临床和组织学相容的DSRCT鉴别诊断。如果IRSCACNA2D2≤1,则应使用分子诊断程序(如荧光原位杂交、qRT-PCR或下一代测序)重新考虑或重新评估诊断(图1N)。反之,如果IRSCACNA2D2 &gt;1、可以建立DSRCT的诊断。 此外,CACNA2D2染色可用于排除广谱小圆蓝细胞肿瘤的DSRCT,具有潜在的广泛诊断价值。最后,CACNA2D2在DSRCT中的高特异性和均匀性膜表达,结合本文描述的高度特异性抗体,使CACNA2D2成为靶向治疗方法的理想候选者,包括使用抗体-药物偶联物或CAR-T细胞疗法给药。未来的研究应探讨CACNA2D2在DSRCT生物学中的确切作用,重点关注其在肿瘤细胞适应性、分化和致瘤潜能方面的潜在贡献。总之,我们为DSRCT研究开发了一个广泛的工具集(补充图S4G),为如何在其他癌症实体中利用这些资源提供了一个经过验证的蓝图,并确定了CACNA2D2是一个单一的、强大的DSRCT生物标志物。Florian Henning Geyer, Florencia Cidre-Aranaz和Thomas Georg Phillip grnewald构思了这项研究。Florian Henning Geyer和Florencia Cidre-Aranaz撰写了论文并起草了所有的数据和表格。Florian Henning Geyer进行了所有体外和体内实验,并进行了所有生物信息学和统计分析。Florian Henning Geyer、Alina Ritter和Thomas Georg Phillip grnewald对肿瘤样本和tma进行免疫组化评价和评分。Florencia Cidre-Aranaz, Roland Imle和Ana Banito进行和/或协调体内实验。Olivier Delattre提供了微阵列表达数据。Seneca Kinn-Gurzo对BER细胞系进行了体外实验。Tobias Faehling和clacimence Henon进行了单细胞生物信息学分析。Karim Aljakouch和Azhar Orynbek进行了MassSpec测试并分析了MassSpec数据。Alina Ritter, Jing Li, Endrit Vinca, Laura Romero-Perez, Martin Sill和Shunya Ohmura为实验程序做出了贡献。Wolfgang Hartmann和Benjamin Friedrich Berthold Mayer提供临床和/或组织学指导。Enrique De Álava、Juan Díaz-Martín、Stefanie Bertram、Sophie Postel-Vilnay、Martin Ebinger、Monika Sparber-Sauer、Daniel Baumhoer、Carine Ngo、David Horst、Yvonne versleijenjonkers、Armin Jarosch、Sabine Stegmaier和Thomas Kirchner提供了临床样本。Patrick Joseph Grohar、Thomas Georg Phillip gr<s:1> newald和Jeroen Krijgsveld提供了实验室基础设施。Florencia Cidre-Aranaz和Thomas Georg Phillip grnewald监督了这项研究和数据分析。所有作者都阅读并批准了最终的手稿。作者声明没有利益冲突。Thomas Georg Phillip grnewald的实验室得到了Matthias-Lackas基金会、Leopold博士和Carmen Ellinger基金会、欧洲研究理事会(ERC CoG 2023 #101122595)、德国研究协会(DFG 458891500)、德国癌症援助(DKH-70112257、DKH-7011411、DKH-70114278、DKH-70115315)、Rolf M. Schwiete博士基金会、SMARCB1协会、教育和研究部(BMBF;SMART-CARE和HEROES-AYA),以及芭芭拉和威尔弗里德·莫尔基金会。Florencia Cidre-Aranaz的研究团队得到了德国癌症援助(DHK-70114111)和Dr. Rolf M. Schwiete Stiftung(2020-028和2022-31)的支持。此外,这项工作是由英国癌症研究中心、国家癌症研究所、西班牙抗癌协会科学基金会和KiKa(儿童无癌基金会)资助的癌症大挑战合作伙伴关系所支持的PROTECT团队的一部分。Florian Henning Geyer, Tobias Faehling, Endrit Vinca和Alina Ritter得到了德国学术奖学金基金会的支持。此外,Endrit Vinca获得了Heinrich F.C. Behr基金会和Rudolf and Brigitte Zenner基金会的奖学金,Tobias Faehling获得了Heinrich F.C. Behr基金会的奖学金,Florian Henning Geyer和Alina Ritter通过“mildred - scheel -博士项目”(dhh -70114866)得到了德国癌症援助组织的支持。该项目由欧盟(ERC, CANCER-HARAKIRI, 101122595)共同资助。然而,所表达的所有观点和意见仅代表作者的观点和意见,并不一定反映欧盟或欧洲研究理事会的观点和意见。欧盟和授权机构都不能对此负责。体内实验由北巴登政府批准,并按照欧洲共同体(86/609/EEC)和UKCCCR(癌症研究动物福利和使用指南)的指导方针和建议进行。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信