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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

Establishment of the ovarian cancer PDX model.(A) Making a single cut in the neck, two pieces were subcutaneously transferred to and implanted on either side of the flank of 6–12 weeks old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumours were measured once or twice a week and after reaching appropriate size, tumours were harvested for either direct propagation into a further generation or for storage. (B) Tumour growth of fresh implanted tumour tissue from patient 56 and further propagation of the tumour (green line) into the second generation (red lines). (C) Tumour growth of stored and subsequently thawed and re-implanted tumour tissue from patient 56. Tumour tissue was either directly frozen after patients primary surgery (F1) using either the vitrification (green line) or FCS/DMSO (black line) protocol. After establishment of a PDX, tumour tissue was harvested from the mouse (F2) and frozen using either the vitrification (red line) or FCS/DMSO (blue line) protocol.
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f1: Establishment of the ovarian cancer PDX model.(A) Making a single cut in the neck, two pieces were subcutaneously transferred to and implanted on either side of the flank of 6–12 weeks old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumours were measured once or twice a week and after reaching appropriate size, tumours were harvested for either direct propagation into a further generation or for storage. (B) Tumour growth of fresh implanted tumour tissue from patient 56 and further propagation of the tumour (green line) into the second generation (red lines). (C) Tumour growth of stored and subsequently thawed and re-implanted tumour tissue from patient 56. Tumour tissue was either directly frozen after patients primary surgery (F1) using either the vitrification (green line) or FCS/DMSO (black line) protocol. After establishment of a PDX, tumour tissue was harvested from the mouse (F2) and frozen using either the vitrification (red line) or FCS/DMSO (blue line) protocol.

Mentions: For proper cross-verification of ovarian cancer patients, an experienced gynaecologic-oncological pathologist reviewed histological slides of the tumour to reconfirm the diagnosis. Pathological examination diagnosed high-grade serous adenocarcinoma for 31 cases, endometrioid carcinoma for 7 cases, clear cell carcinoma for 3 cases, mucinous carcinoma in 1 case and a mixed phenotype tumour for 3 cases (Table 1). Median latency time, defined as time from implantation till first tumour growth was observed, was 43 days but varied between histology subtypes (Table 1). After reaching a size of at least 1 cm3, tumours were harvested and serially transplanted in mice to establish further generations, as well as stored using the vitrification and/or FCS/DMSO method (Fig. 1A). After successful establishment in first generation (F1), all tumours showed successful engraftment in further generations.


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)

Establishment of the ovarian cancer PDX model.(A) Making a single cut in the neck, two pieces were subcutaneously transferred to and implanted on either side of the flank of 6–12 weeks old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumours were measured once or twice a week and after reaching appropriate size, tumours were harvested for either direct propagation into a further generation or for storage. (B) Tumour growth of fresh implanted tumour tissue from patient 56 and further propagation of the tumour (green line) into the second generation (red lines). (C) Tumour growth of stored and subsequently thawed and re-implanted tumour tissue from patient 56. Tumour tissue was either directly frozen after patients primary surgery (F1) using either the vitrification (green line) or FCS/DMSO (black line) protocol. After establishment of a PDX, tumour tissue was harvested from the mouse (F2) and frozen using either the vitrification (red line) or FCS/DMSO (blue line) protocol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Establishment of the ovarian cancer PDX model.(A) Making a single cut in the neck, two pieces were subcutaneously transferred to and implanted on either side of the flank of 6–12 weeks old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumours were measured once or twice a week and after reaching appropriate size, tumours were harvested for either direct propagation into a further generation or for storage. (B) Tumour growth of fresh implanted tumour tissue from patient 56 and further propagation of the tumour (green line) into the second generation (red lines). (C) Tumour growth of stored and subsequently thawed and re-implanted tumour tissue from patient 56. Tumour tissue was either directly frozen after patients primary surgery (F1) using either the vitrification (green line) or FCS/DMSO (black line) protocol. After establishment of a PDX, tumour tissue was harvested from the mouse (F2) and frozen using either the vitrification (red line) or FCS/DMSO (blue line) protocol.
Mentions: For proper cross-verification of ovarian cancer patients, an experienced gynaecologic-oncological pathologist reviewed histological slides of the tumour to reconfirm the diagnosis. Pathological examination diagnosed high-grade serous adenocarcinoma for 31 cases, endometrioid carcinoma for 7 cases, clear cell carcinoma for 3 cases, mucinous carcinoma in 1 case and a mixed phenotype tumour for 3 cases (Table 1). Median latency time, defined as time from implantation till first tumour growth was observed, was 43 days but varied between histology subtypes (Table 1). After reaching a size of at least 1 cm3, tumours were harvested and serially transplanted in mice to establish further generations, as well as stored using the vitrification and/or FCS/DMSO method (Fig. 1A). After successful establishment in first generation (F1), all tumours showed successful engraftment in further generations.

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