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  1. Article ; Online: Deciphering Stromal Changes between Metastatic and Non-metastatic Canine Mammary Carcinomas.

    Ettlin, Julia / Bauer, Alina / Opitz, Lennart / Malbon, Alexandra / Markkanen, Enni

    Journal of mammary gland biology and neoplasia

    2023  Volume 28, Issue 1, Page(s) 14

    Abstract: Cancer-associated stroma (CAS) is widely recognized to influence development and progression of epithelial tumours including breast cancer. Canine mammary tumours (CMTs) such as simple canine mammary carcinomas represent valuable models for human breast ... ...

    Abstract Cancer-associated stroma (CAS) is widely recognized to influence development and progression of epithelial tumours including breast cancer. Canine mammary tumours (CMTs) such as simple canine mammary carcinomas represent valuable models for human breast cancer also with respect to stromal reprogramming. However, it remains unclear whether and how CAS changes in metastatic tumours compared to non-metastatic ones. To characterize stromal changes between metastatic and non-metastatic CMTs and identify potential drivers of tumour progression, we analysed CAS and matched normal stroma from 16 non-metastatic and 15 metastatic CMTs by RNA-sequencing of microdissected FFPE tissue. We identified 1438 differentially regulated genes between CAS and normal stroma, supporting previous results demonstrating stromal reprogramming in CMTs to be comparable with CAS in human breast cancer and validating deregulation of pathways and genes associated with CAS. Using primary human fibroblasts activated by treatment with TGFβ, we demonstrate some of the strongest expression changes to be conserved in fibroblasts across species. Furthermore, we identify 132 differentially expressed genes between CAS from metastatic and non-metastatic tumours, with strong changes in pathways including chemotaxis, regulation of apoptosis, immune response and TGFβ signalling and validate deregulation of several targets using RT-qPCR. Finally, we identify specific upregulation of COL6A5, F5, GALNT3, CIT and MMP11 in metastatic CAS, suggesting high stromal expression of these targets to be linked to malignancy and metastasis of CMTs. In summary, our data present a resource supporting further research into stromal changes of the mammary gland in relation to metastasis with implications for both canine and human mammary cancer.
    MeSH term(s) Humans ; Animals ; Dogs ; Mammary Neoplasms, Animal/genetics ; Carcinoma ; Apoptosis ; Fibroblasts ; Transforming Growth Factor beta
    Chemical Substances Transforming Growth Factor beta
    Language English
    Publishing date 2023-07-01
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1327345-0
    ISSN 1573-7039 ; 1083-3021
    ISSN (online) 1573-7039
    ISSN 1083-3021
    DOI 10.1007/s10911-023-09542-0
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Estimating the Impact of Consecutive Blood Meals on Vector Competence of

    Veronesi, Eva / Paslaru, Anca / Ettlin, Julia / Ravasi, Damiana / Flacio, Eleonora / Tanadini, Matteo / Guidi, Valeria

    Pathogens (Basel, Switzerland)

    2023  Volume 12, Issue 6

    Abstract: The continuous expansion ... ...

    Abstract The continuous expansion of
    Language English
    Publishing date 2023-06-20
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2695572-6
    ISSN 2076-0817
    ISSN 2076-0817
    DOI 10.3390/pathogens12060849
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  3. Article ; Online: Next-generation RNA sequencing of FFPE subsections reveals highly conserved stromal reprogramming between canine and human mammary carcinoma.

    Amini, Parisa / Nassiri, Sina / Ettlin, Julia / Malbon, Alexandra / Markkanen, Enni

    Disease models & mechanisms

    2019  Volume 12, Issue 8

    Abstract: Spontaneous canine simple mammary carcinomas (mCA) are often viewed as models of human mCA. Cancer-associated stroma (CAS) is central for initiation and progression of human cancer, and is likely to play a key role in canine tumours as well. However, ... ...

    Abstract Spontaneous canine simple mammary carcinomas (mCA) are often viewed as models of human mCA. Cancer-associated stroma (CAS) is central for initiation and progression of human cancer, and is likely to play a key role in canine tumours as well. However, canine CAS lacks characterisation and it remains unclear how canine and human CAS compare. Formalin-fixed paraffin embedded (FFPE) tissue constitutes a valuable resource of patient material, but chemical crosslinking has largely precluded its analysis by next-generation RNA sequencing (RNAseq). We have recently established a protocol to isolate CAS and normal stroma from archival FFPE tumours using laser-capture microdissection followed by RNAseq. Using this approach, we have analysed stroma from 15 canine mCA. Our data reveal strong reprogramming of canine CAS. We demonstrate a high-grade molecular homology between canine and human CAS, and show that enrichment of upregulated canine CAS genes strongly correlates with the enrichment of an independently derived human stromal signature in the TCGA breast tumour dataset. Relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries. Finally, we establish the prognostic potential of the canine CAS signature in human samples, emphasising the relevance of studying canine CAS as a model of the human disease. In conclusion, we provide a proof-of-principle to analyse specific subsections of FFPE tissue by RNAseq, and compare stromal gene expression between human and canine mCA to reveal molecular drivers in CAS supporting tumour growth and malignancy.
    MeSH term(s) Animals ; Breast Neoplasms/genetics ; Dogs ; Female ; Formaldehyde ; Gene Expression Regulation, Neoplastic ; High-Throughput Nucleotide Sequencing ; Humans ; Laser Capture Microdissection ; Mammary Neoplasms, Animal/genetics ; Paraffin Embedding ; Prognosis ; Sequence Analysis, RNA ; Stromal Cells/metabolism ; Tissue Fixation ; Transcriptome/genetics
    Chemical Substances Formaldehyde (1HG84L3525)
    Language English
    Publishing date 2019-08-08
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1754-8411
    ISSN (online) 1754-8411
    DOI 10.1242/dmm.040444
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas.

    Ettlin, Julia / Clementi, Elena / Amini, Parisa / Malbon, Alexandra / Markkanen, Enni

    International journal of molecular sciences

    2017  Volume 18, Issue 5

    Abstract: Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as excellent models of human breast carcinomas. Considering the importance of CAS for human cancer, it ... ...

    Abstract Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as excellent models of human breast carcinomas. Considering the importance of CAS for human cancer, it likely plays a central role in canine tumours as well. So far, however, canine CAS lacks characterisation, and it remains unclear whether the biology between CAS from canine and human tumours is comparable. In this proof-of-principle study, using laser-capture microdissection, we isolated CAS and normal stroma from 13 formalin-fixed paraffin embedded canine simple mammary carcinomas and analysed the expression of seven known human CAS markers by RT-qPCR (Reverse Transcription quantitative PCR) and validated some targets by immunohistochemistry. We found that Col1a1 (Collagen1α1), αSMA (alpha Smooth Muscle Actin), FAP (Fibroblast activation protein), PDGFRβ (Platelet-derived growth factor receptor beta), and Caveolin-1 were significantly upregulated in canine CAS, and the expression of CXCL12 (Stromal cell derived factor 1) significantly decreased, whereas MMP2 (Matrix Metalloproteinase 1) and IL6 (Interleukin 6) did not change. Our results suggest strong similarities in CAS biology in canine and human mammary carcinomas but also reveal some differences. To the best of our knowledge, this is the first report to provide a comprehensive expression analysis of the most important CAS markers in canine simple mammary carcinomas and further supports the validity of the dog as model for human cancer.
    Language English
    Publishing date 2017-05-20
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2019364-6
    ISSN 1422-0067 ; 1422-0067 ; 1661-6596
    ISSN (online) 1422-0067
    ISSN 1422-0067 ; 1661-6596
    DOI 10.3390/ijms18051101
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.

    Amini, Parisa / Ettlin, Julia / Opitz, Lennart / Clementi, Elena / Malbon, Alexandra / Markkanen, Enni

    BMC molecular biology

    2017  Volume 18, Issue 1, Page(s) 22

    Abstract: Background: Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA ... ...

    Abstract Background: Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult.
    Methods: We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step.
    Results: We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well.
    Conclusions: We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.
    MeSH term(s) Animals ; Dogs ; Female ; Formaldehyde/chemistry ; High-Throughput Nucleotide Sequencing ; Laser Capture Microdissection ; Mammary Neoplasms, Animal/pathology ; Molecular Biology/methods ; Paraffin Embedding ; Peptide Hydrolases/metabolism ; RNA/drug effects ; RNA/isolation & purification ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA ; Sonication ; Tissue Fixation
    Chemical Substances Formaldehyde (1HG84L3525) ; RNA (63231-63-0) ; Peptide Hydrolases (EC 3.4.-)
    Language English
    Publishing date 2017-08-23
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1471-2199
    ISSN (online) 1471-2199
    DOI 10.1186/s12867-017-0099-7
    Database MEDical Literature Analysis and Retrieval System OnLINE

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