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. 2017 Jan 12;2(1):e89760.
doi: 10.1172/jci.insight.89760.

A patient-derived-xenograft platform to study BRCA-deficient ovarian cancers

Erin George  1 , Hyoung Kim  1 , Clemens Krepler  2 , Brandon Wenz  3 , Mehran Makvandi  4 , Janos L Tanyi  1 , Eric Brown  5 , Rugang Zhang  2 , Patricia Brafford  2 , Stephanie Jean  1 , Robert H Mach  4 , Yiling Lu  6 , Gordon B Mills  6 , Meenhard Herlyn  2 , Mark Morgan  1 , Xiaochen Zhang  1 , Robert Soslow  7 , Ronny Drapkin  1 , Neil Johnson  8 , Ying Zheng  9 , George Cotsarelis  9 , Katherine L Nathanson  3 , Fiona Simpkins  1   2
Affiliations

A patient-derived-xenograft platform to study BRCA-deficient ovarian cancers

Erin George et al. JCI Insight. .

Abstract

Approximately 50% of high-grade serous ovarian cancers (HGSOCs) have defects in genes involved in homologous recombination (HR) (i.e., BRCA1/2). Preclinical models to optimize therapeutic strategies for HR-deficient (HRD) HGSOC are lacking. We developed a preclinical platform for HRD HGSOCs that includes primary tumor cultures, patient-derived xenografts (PDXs), and molecular imaging. Models were characterized by immunohistochemistry, targeted sequencing, and reverse-phase protein array analysis. We also tested PDX tumor response to PARP, CHK1, and ATR inhibitors. Fourteen orthotopic HGSOC PDX models with BRCA mutations (BRCAMUT) were established with a 93% success rate. The orthotopic PDX model emulates the natural progression of HGSOC, including development of a primary ovarian tumor and metastasis to abdominal viscera. PDX response to standard chemotherapy correlated to that demonstrated in the patient. Pathogenic mutations and HGSOC markers were preserved after multiple mouse passages, indicating retention of underlying molecular mechanisms of carcinogenesis. A BRCA2MUT PDX with high p-CHK1 demonstrated a similar delay of tumor growth in response to PARP, CHK1, and ATR inhibitors. A poly (ADP-ribose) polymerase (PARP) inhibitor radiotracer correlated with PARP1 activity and showed response to PARP inhibition in the BRCA2MUT PDX model. In summary, the orthotopic HGSOC PDX represents a robust and reliable model to optimize therapeutic strategies for BRCAMUT HGSOC.

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Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. A potentially novel BRCAMUT ovarian patient-derived-xenograft (PDX) platform.
(A) Patient tumors removed during surgery are transplanted onto the fallopian tube fimbria/ovaries of NSG mice for PDX development and grown in tissue culture. Mouse tumors are then used to establish a live tumor bank or expanded for preclinical trials. Models can also be used for functional biomarker studies. Targeted genomic sequencing and reverse phase protein array (RPPA) analysis is performed on all patient and PDX samples. Primary tumor cultures may be used for in vitro 2D and 3D drug screening. (B) Representative BRCAMUT PDX (c.8945delAA; WO-2-1) 10 weeks after transplantation showing primary tumor replacing host ovary (upper panel), diaphragmatic metastasis (middle panel), and primary ovarian tumor and normal mouse ovary (lower panel). (C) PDX tumor size by palpation over time for select PDX models comparing BRCAMUT (WO-2-1, WO-3-1, WO-10-1) with BRCAWT (WO-4-1, WO-12-1, WO-13-1) HGSOC over 3 mouse passages (MPs). BRCAMUT PDXs demonstrated slower growth rates compared with BRCAWT at MP1 (0.11 mm/day vs. 0.29 mm/day; P < 0.001). However, in subsequent passages MP2 and MP3, growth rates were similar (0.19 mm/day for BRCAMUT vs. 0.25 mm/day for BRCAWT; P = 0.08 for MP2, 0.177 mm/day for BRCAMUT vs. 0.18 mm/day for BRCAWT; P = 0.9 for MP3). Dots represent mean of determinations with SEM. A linear mixed-effects model was used to compare growth rates/day between groups. Individual mouse data shown in dot plots in Supplemental Figure 4. (D) Example of preclinical platform using a BRCA2MUT (c.8945delAA) PDX model (WO-2). The patient was diagnosed with ovarian cancer in 2012 and had primary cytoreductive surgery (1° CRS) followed by standard chemotherapy (carboplatin and paclitaxel) and had a complete remission (CR). Tumor obtained at secondary CRS for recurrent disease in January 2014 was used for PDX generation. PDX tumors grew in 12 to 16 weeks to approximately 1,000 mm3. Tumors were harvested and expanded for a preclinical trial. Mice were randomized to control or carboplatin 50 mg/kg weekly and tumor growth as measured by weekly ultrasound. All mice showed CR. Similarly, the patient’s tumor also responded to a platinum-based chemotherapy regimen as illustrated in the graph of her serum CA-125. The box-and-whisker plots show the median, with boxes extending from the 25th to 75th percentile and the whiskers extending from minimum to maximum values of the dataset. Control, n = 9 mice; carboplatin, n = 6 mice.
Figure 2
Figure 2. Ovarian cancer epithelial markers and morphologic characteristics in parent tumors are preserved over multiple mouse passages.
(A) BRCAMUT patient-derived-xenograft (PDX) models (WO-2, WO-3) and 2 BRCAWT PDX models (WO-4, WO-20) were evaluated by H&E and immunohistochemistry for epithelial ovarian cancer markers. PAX8 (paired box 8, nuclear stain), CK7 (cytokeratin 7, cytoplasmic stain), and ER (estrogen receptor, nuclear stain) in parent tumor and matched PDXs up to mouse passage 3 (MP1–MP3) are shown. Magnification is ×10 for large panels and ×100 for inserts. (B) Five matched BRCAMUT patient/PDXs and 5 matched BRCAWT patient /PDXs were reviewed for SET (solid, pseudoendometrioid, and translational cell carcinoma-like) morphology in a blinded fashion. WO-4 (BRCAWT), WO-2-1 (BRCA2MUT), and WO-3-1 (BRCA1MUT) representative parent and PDX tumors of MP1, MP2, and MP3 H&E sections shown demonstrating SET criteria in BRCAMUT and not in BRCAWT tumors. Parent WO-2-1 tumor showed micropapillary features, while the MP1–MP3 tumors showed both micropapillary and solid architecture. Magnification is ×10 for all panels.
Figure 3
Figure 3. Illustration of the BRCA1 and BRCA2 genes, including exons, introns, and functional domains noting location of BRCA mutations in the patient-derived-xenograft (PDX) models.
RING, really interesting new gene; PALB2, partner and localizer of BRCA2; BRCT, BRCA1 C-terminal; OB, oligonucleotide/oligosaccharide-binding domain.
Figure 4
Figure 4. Genomic profiles are preserved from parent tumors to PDX tumors.
Targeted sequencing of full genes or all exons was performed using a 157-targeted-gene panel on both BRCAMUT and BRCAWT patient tumors with matched patient-derived xenografts (PDXs) highlighting deleterious, likely deleterious, and variants of unknown significance (VUS).
Figure 5
Figure 5. Targeting the ATR-CHK1 axis in BRCA1/2MUT cell models.
(A) Viability of established ovarian cancer cells after treatment with 1 μM PARPi (AZD2281), 1 μM CHK1i (MK8776), 1 μM ATRi (AZD6738), and 30 μM carboplatin was assessed with an MTT assay. Cell models included PEO1 (BRCA2MUT), JHOS4 (BRCA1MUT), and the homologous recombination–proficient cells, PEO4 (BRCA2REV). Cells were incubated in their respective drug concentrations for 5 days. PARPi was significantly more cytotoxic in BRCAMUT relative to BRCA2REV cells (32.9% ± 0.7% cell viability for BRCA2MUT cells and 42.3% ± 2.0% for BRCA1MUT cells vs. 76.7% ± 2.0% for BRCA2REV cells; P < 0.00001 and P = 0.0002 for BRCA2 vs. BRCAREV and BRCA1 vs. BRCAREV, respectively). Carboplatin treatment appeared to have a similar effect to that of PARPi in all lines: BRCA2MUT (41.2% ± 3.9% cell viability; P = 0.07 carboplatin compared with PARPi); BRCA1MUT (55.0% ± 5.2% viability; P = 0.0003 carboplatin vs. PARPi); and BRCA2REV (96.3% ± 4.6% viability; P = 0.01 carboplatin vs. PARPi). Only the BRCA2MUT cell model was sensitive to 1 μM CHK1i (56.6% ± 1.1% cell viability). Both BRCAMUT and BRCA2REV cells were sensitive to ATRi monotherapy (8.6% ± 0.2%, 38.9% ± 1.4%, and 26.3% ± 0.5% cell viability for BRCA2MUT, BRCA1MUT, and BRCA2REV cells, respectively; P < 0.001 for all lines ATRi vs. carboplatin treatment). The box-and-whisker plots show the median, with boxes extending from the 25th to 75th percentile and the whiskers extending from minimum and maximum values of the dataset (n = 5 per group). Two-way ANOVA was performed before the Tukey’s honestly significant difference test to determine whether there was an overall difference between the groups. The data shown are a single representative experiment with 5 determinations. Three independent experiments were performed. (B) Colony forming assay was performed with 1 μM PARPi, 1 μM CHK1i, 1 μM ATRi, and 30 μM carboplatin treatment in BRCA2MUT (PEO1) and BRCA2REV (PEO4) cell models. Cells were incubated in their respective drug concentrations for 13 days. Colony formation was decreased in BRCA2MUT compared with BRCA2REV cells with PARPi (3.3% ± 1.3% vs. 116.8% ± 5.2%; P < 0.00001), CHK1i (28.6% ± 3.1% vs. 94.1% ± 6.0%; P = 0.0001), and carboplatin (3.9% ± 1.2% vs. 43.3% ± 2.8%; P < 0.00001). All values are percentage colony formation relative to control. Both cell models showed decreased colony formation with ATRi (6.4% ± 1.6% for BRCA2MUT vs. 10.9% ± 1.4% for BRCA2REV; P = 0.08). (C) BRCA2MUT (PEO1) and BRCA2REV (PEO4) cells were treated with 1 μM PARPi, 1 μM CHK1i, and 1 μM ATRi and lysates were collected after 24 hours of treatment. Western blot for the indicated total and phosphoproteins is shown.
Figure 6
Figure 6. Targeting the ATR-CHK1 axis in a BRCA2MUT PDX model.
(A) Reverse phase protein array (RPPA) analysis for 308 total and phosphoproteins in patient tumors and their corresponding PDXs were analyzed. Two proteins (p-CHK1 on Ser345 and total CHK1) were selected and plotted on a log2-fold scale. WO-2-1 (BRCA2MUT c.8945delAA) demonstrated elevated p-CHK1 relative to other tumors. (B) PDX tumors with high and low p-CHK1 by RPPA were compared by Western blot for p-CHK1 and total CHK1. (C and D) WO-2-1 PDXs were randomized into the following groups: vehicle control; PARPi (AZD2281) 100 mg/kg by oral gavage daily; CHK1i (MK8776) 50 mg/kg i.p. every 3 days; ATRi (AZD6738) 50 mg/kg daily; and carboplatin 50 mg/kg weekly. Tumor volume was measured by weekly ultrasound. There was a significant decrease in all treatment groups relative to control (P < 0.0001) and a significant difference relative to carboplatin treatment (P = 0.0001 for PARPi, P = 0.0072 for CHK1i, and P < 0.00001 for ATRi). ANOVA analysis was conducted to evaluate differences among means. Tukey’s honestly significant difference (HSD) test was used for all pairwise mean comparisons. PARPi treatment decreased average tumor volume by 35% over treatment duration with 68.8% (n = 11 of 16) and 31.2% (n = 5 of 16) showing partial remission (PR) and stable disease (SD), respectively, by RECIST 1.1 (37). CHK1i treatment resulted in a 25% (n = 2 of 8) SD rate and 75% (n = 6 of 8) with PD (progression of disease). ATRi treatment resulted in a 22.2% (n = 2 of 9) SD rate, and 77.8% (n = 7 of 9) with PD in average tumor volume, respectively. Carboplatin treatment resulted in 100% CR. Each symbol represents mean of determinations with SEM. Control, n = 7; PARPi n = 16; carboplatin, n = 5; ATRi, n = 9; CHK1i, n = 6 (see Supplemental Figure 9 for individual mouse response). (E) Lysates from PDX tumors after 2 weeks of treatment were immunoblotted for the indicated proteins. (F) PET imaging of PARP-1 with [18F]FTT and PET/CT fused images are shown in untreated and PARPi-treated mouse. WO-2-1 (BRCA2MUT c.8945delAA) PDX received oral doses of PARPi (AZD2281) 50 mg/kg every day for 1 month and on the day of PET imaging prior to the imaging study. White arrows point to tumors. (G) Digital autoradiography (left panel) of tumor sections and PARP-1 immunofluorescence (IF; right panel) of adjacent tumor sections comparing control with PARPi-treated tumor sections (n = 2) is shown. Representation of 1 of 2 independent experiments is shown. (H) Correlative data shown for PET, autoradiography (Rad), and IF modalities for determining PARP-1 expression. Graphical representation of 3 independent experiments and associated results are shown. Four regions of interest were drawn in each dataset. The box-and-whisker plot shows the median, with boxes extending from the 25th to 75th percentile and the whiskers extending from minimum to maximum values of the dataset (n = 4).
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