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Biobanking of patient and patient-derived xenograft ovarian tumour tissue: efficient preservation with low and high fetal calf serum based methods.

Alkema NG, Tomar T, Duiker EW, Jan Meersma G, Klip H, van der Zee AG, Wisman GB, de Jong S - Sci Rep (2015)

Bottom Line: We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%.Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues.Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.

View Article: PubMed Central - PubMed

Affiliation: University of Groningen, University Medical Centre Groningen, Department of Gynaecologic Oncology, Groningen, The Netherlands.

ABSTRACT
Using patient-derived xenografts (PDXs) for preclinical cancer research demands proper storage of tumour material to facilitate logistics and to reduce the number of animals needed. We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%. Corresponding cells from mouse replaced human tumour stromal and endothelial cells in second generation PDXs as demonstrated with mouse-specific vimentin and CD31 immunohistochemical staining. For biobanking purposes two cryopreservation methods, a fetal calf serum (FCS)-based (95%v/v) "FCS/DMSO" protocol and a low serum-based (10%v/v) "vitrification" protocol were tested. After primary cryopreservation, tumour take rates were 38% and 67% using either the vitrification or FCS/DMSO-based cryopreservation protocol, respectively. Cryopreserved tumour tissue of established PDXs achieved take rates of 67% and 94%, respectively compared to 91% using fresh PDX tumour tissue. Genotyping analysis showed that no changes in copy number alterations were introduced by any of the biobanking methods. Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues. However, FCS/DMSO-based cryopreservation is more successful. Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.

No MeSH data available.


Related in: MedlinePlus

Immunohistochemistry of patient 56 over several generations (F1 (A) and F2 (B)) and after established growth after storage using either the vitrification (Vitri) or the FCS/DMSO (FCS) protocol on either primary patient tumour tissue (F1) or tumour tissue harvested from previous generations (F2).Magnification 10× and for CD31 20×.
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f2: Immunohistochemistry of patient 56 over several generations (F1 (A) and F2 (B)) and after established growth after storage using either the vitrification (Vitri) or the FCS/DMSO (FCS) protocol on either primary patient tumour tissue (F1) or tumour tissue harvested from previous generations (F2).Magnification 10× and for CD31 20×.

Mentions: Morphology of the primary patients’ tumour and of tumours engrafted in first and second generations were compared by H&E staining (Fig. 2 and Supplementary Fig. S2–6C). Figure 2, displaying a representative series of H&E stainings for patient 56, shows that through increasing generations there was a tendency towards a more undifferentiated aspect with loss of characteristic histopathological features and increased nuclear atypia (Fig. 2A,B). Stromal infiltration was observed throughout serial transplantation. However, human stroma in the tumours was replaced by mouse stroma. This is shown by loss of human vimentin staining and gain of expression of mouse vimentin, using two vimentin antibodies raised against human and human/mouse, respectively (Fig. 2A,B). Using a monoclonal rat anti-mouse antibody for CD31, we demonstrated increased positive mouse CD31 staining of endothelial cells lining the vessel walls of PDX tumour tissue when compared to the primary tumour, suggesting replacement of human- for mouse vessels. Proliferative rate, as assessed by Ki67, remained high through generations as well as in tissue engrafted after storage for both vitrification and FCS/DMSO (Fig. 2A,B). Expression of Wilm’s Tumour (WT1), known to be primarily expressed in serous ovarian cancers, was seen in all serous patients, and absent in patient 61 (data not shown).


Biobanking of patient and patient-derived xenograft ovarian tumour tissue: efficient preservation with low and high fetal calf serum based methods.

Alkema NG, Tomar T, Duiker EW, Jan Meersma G, Klip H, van der Zee AG, Wisman GB, de Jong S - Sci Rep (2015)

Immunohistochemistry of patient 56 over several generations (F1 (A) and F2 (B)) and after established growth after storage using either the vitrification (Vitri) or the FCS/DMSO (FCS) protocol on either primary patient tumour tissue (F1) or tumour tissue harvested from previous generations (F2).Magnification 10× and for CD31 20×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594124&req=5

f2: Immunohistochemistry of patient 56 over several generations (F1 (A) and F2 (B)) and after established growth after storage using either the vitrification (Vitri) or the FCS/DMSO (FCS) protocol on either primary patient tumour tissue (F1) or tumour tissue harvested from previous generations (F2).Magnification 10× and for CD31 20×.
Mentions: Morphology of the primary patients’ tumour and of tumours engrafted in first and second generations were compared by H&E staining (Fig. 2 and Supplementary Fig. S2–6C). Figure 2, displaying a representative series of H&E stainings for patient 56, shows that through increasing generations there was a tendency towards a more undifferentiated aspect with loss of characteristic histopathological features and increased nuclear atypia (Fig. 2A,B). Stromal infiltration was observed throughout serial transplantation. However, human stroma in the tumours was replaced by mouse stroma. This is shown by loss of human vimentin staining and gain of expression of mouse vimentin, using two vimentin antibodies raised against human and human/mouse, respectively (Fig. 2A,B). Using a monoclonal rat anti-mouse antibody for CD31, we demonstrated increased positive mouse CD31 staining of endothelial cells lining the vessel walls of PDX tumour tissue when compared to the primary tumour, suggesting replacement of human- for mouse vessels. Proliferative rate, as assessed by Ki67, remained high through generations as well as in tissue engrafted after storage for both vitrification and FCS/DMSO (Fig. 2A,B). Expression of Wilm’s Tumour (WT1), known to be primarily expressed in serous ovarian cancers, was seen in all serous patients, and absent in patient 61 (data not shown).

Bottom Line: We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%.Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues.Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.

View Article: PubMed Central - PubMed

Affiliation: University of Groningen, University Medical Centre Groningen, Department of Gynaecologic Oncology, Groningen, The Netherlands.

ABSTRACT
Using patient-derived xenografts (PDXs) for preclinical cancer research demands proper storage of tumour material to facilitate logistics and to reduce the number of animals needed. We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%. Corresponding cells from mouse replaced human tumour stromal and endothelial cells in second generation PDXs as demonstrated with mouse-specific vimentin and CD31 immunohistochemical staining. For biobanking purposes two cryopreservation methods, a fetal calf serum (FCS)-based (95%v/v) "FCS/DMSO" protocol and a low serum-based (10%v/v) "vitrification" protocol were tested. After primary cryopreservation, tumour take rates were 38% and 67% using either the vitrification or FCS/DMSO-based cryopreservation protocol, respectively. Cryopreserved tumour tissue of established PDXs achieved take rates of 67% and 94%, respectively compared to 91% using fresh PDX tumour tissue. Genotyping analysis showed that no changes in copy number alterations were introduced by any of the biobanking methods. Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues. However, FCS/DMSO-based cryopreservation is more successful. Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.

No MeSH data available.


Related in: MedlinePlus