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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

Copy number analysis of ovarian cancer patient tumours and their matched PDX tumours using genome-wide SNP array.(A) CNA plots represented the copy number alterations between the primary tumour of patient 56, PDX tumour after first engraftment (F1) and PDX tumour after 3rd engraftment (F3). Genomic gain is indicated in blue and genomic loss is indicated in red over all chromosomes. In the upper CNA plot, the average genomic alteration of all three samples is presented in a similar manner (blue: amplification and red: loss). Below each CNA plot of each sample, the bar with colors represents the allelic events (yellow for loss of heterozygosity (LOH); purple for allelic imbalance). (B) Quantitative CNA concordance analysis of tumours of patients and their corresponding PDXs by hierarchical clustering. (C) Quantitative CNA concordance analysis of engrafted tumours of patient 56 after preservation using both methods compared to freshly propagated tumours by hierarchical clustering. Note that the scale bar of the Pearson Value is different for (B and C).
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f4: Copy number analysis of ovarian cancer patient tumours and their matched PDX tumours using genome-wide SNP array.(A) CNA plots represented the copy number alterations between the primary tumour of patient 56, PDX tumour after first engraftment (F1) and PDX tumour after 3rd engraftment (F3). Genomic gain is indicated in blue and genomic loss is indicated in red over all chromosomes. In the upper CNA plot, the average genomic alteration of all three samples is presented in a similar manner (blue: amplification and red: loss). Below each CNA plot of each sample, the bar with colors represents the allelic events (yellow for loss of heterozygosity (LOH); purple for allelic imbalance). (B) Quantitative CNA concordance analysis of tumours of patients and their corresponding PDXs by hierarchical clustering. (C) Quantitative CNA concordance analysis of engrafted tumours of patient 56 after preservation using both methods compared to freshly propagated tumours by hierarchical clustering. Note that the scale bar of the Pearson Value is different for (B and C).

Mentions: We performed a genome-wide single nucleotide polymorphism (SNP) microarray on tumour material from five independent patients (30, 36, 37, 56 and 84) and their corresponding PDX tumours of different generations (F1, F2 and F3). Besides these samples, bio-banked tumours of PDX 56 using both freezing methods were also included for genotyping analysis. After pre-processing and quality control, resulting data were used to calculate copy number alterations (CNAs) across the entire human genome and were compared among different samples. Four samples from PDX 30 and 84 did not pass quality control and were not included for subsequent analysis. The pattern of CNAs was compared between the primary tumour and different generations of PDX tumours. Grafted tumours maintained the CNA pattern of the parental patient tumour (Fig. 4A and Supplementary Fig. S7). We observed more accumulation of deletion events in the genome of PDX tumours, which seemed to be enhancements of existing genomic aberrations of the primary tumour specimen (Fig. 4A). This could be due to the influence of enrichment of human tumour cells after implantation since mouse stroma replaced the human stroma as aforementioned.


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)

Copy number analysis of ovarian cancer patient tumours and their matched PDX tumours using genome-wide SNP array.(A) CNA plots represented the copy number alterations between the primary tumour of patient 56, PDX tumour after first engraftment (F1) and PDX tumour after 3rd engraftment (F3). Genomic gain is indicated in blue and genomic loss is indicated in red over all chromosomes. In the upper CNA plot, the average genomic alteration of all three samples is presented in a similar manner (blue: amplification and red: loss). Below each CNA plot of each sample, the bar with colors represents the allelic events (yellow for loss of heterozygosity (LOH); purple for allelic imbalance). (B) Quantitative CNA concordance analysis of tumours of patients and their corresponding PDXs by hierarchical clustering. (C) Quantitative CNA concordance analysis of engrafted tumours of patient 56 after preservation using both methods compared to freshly propagated tumours by hierarchical clustering. Note that the scale bar of the Pearson Value is different for (B and C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Copy number analysis of ovarian cancer patient tumours and their matched PDX tumours using genome-wide SNP array.(A) CNA plots represented the copy number alterations between the primary tumour of patient 56, PDX tumour after first engraftment (F1) and PDX tumour after 3rd engraftment (F3). Genomic gain is indicated in blue and genomic loss is indicated in red over all chromosomes. In the upper CNA plot, the average genomic alteration of all three samples is presented in a similar manner (blue: amplification and red: loss). Below each CNA plot of each sample, the bar with colors represents the allelic events (yellow for loss of heterozygosity (LOH); purple for allelic imbalance). (B) Quantitative CNA concordance analysis of tumours of patients and their corresponding PDXs by hierarchical clustering. (C) Quantitative CNA concordance analysis of engrafted tumours of patient 56 after preservation using both methods compared to freshly propagated tumours by hierarchical clustering. Note that the scale bar of the Pearson Value is different for (B and C).
Mentions: We performed a genome-wide single nucleotide polymorphism (SNP) microarray on tumour material from five independent patients (30, 36, 37, 56 and 84) and their corresponding PDX tumours of different generations (F1, F2 and F3). Besides these samples, bio-banked tumours of PDX 56 using both freezing methods were also included for genotyping analysis. After pre-processing and quality control, resulting data were used to calculate copy number alterations (CNAs) across the entire human genome and were compared among different samples. Four samples from PDX 30 and 84 did not pass quality control and were not included for subsequent analysis. The pattern of CNAs was compared between the primary tumour and different generations of PDX tumours. Grafted tumours maintained the CNA pattern of the parental patient tumour (Fig. 4A and Supplementary Fig. S7). We observed more accumulation of deletion events in the genome of PDX tumours, which seemed to be enhancements of existing genomic aberrations of the primary tumour specimen (Fig. 4A). This could be due to the influence of enrichment of human tumour cells after implantation since mouse stroma replaced the human stroma as aforementioned.

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