Application of a Patient Derived Xenograft Model for Predicative Study of Uterine Fibroid Disease

Abstract

Human uterine fibroids, benign tumors derived from the smooth muscle layers of the uterus, impose a major health burden to up to 50% of premenopausal women in their daily life. To improve our understanding of this disease, we developed and characterized a patient-derived xenograft model by subcutaneous transplantation of pieces of human uterine fibroid tissue into three different strains of severe combined immunodeficient mice. Engrafted uterine fibroid tissue preserved the classical morphology with interwoven bundles of smooth muscle cells and an abundant deposition of collagenous matrix, similar to uterine fibroids in situ. The grafts expressed both estrogen receptor 1 and progesterone receptor. Additionally, both receptors were up-regulated by estrogen treatment. Growth of the fibroid grafts was dependent on 17β-estradiol and progesterone supplementation at levels similar to women with the disease and was studied for up to 60 days at maximum. Co-treatment with the antiprogestin mifepristone reduced graft growth (four independent donors, p<0.0001 two-sided t-test), as did treatment with the mTOR inhibitor rapamycin (three independent donors, p<0.0001 two-sided t-test). This in vivo animal model preserves the main histological and functional characteristics of human uterine fibroids, is amenable to intervention by pharmacological treatment, and can thus serve as an adequate model for the development of novel therapies.

Conflict of interest statement

Competing Interests: MF, NS, IG, ALF and MK were full-time employees of Bayer Pharma AG Research at the time this research has been conducted. Work presented in this publication is not patented, nor does it relate to commercial products of Bayer Pharma AG being in development or marketed. This commercial affiliation does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. The other authors disclosed no potential conflicts of interest.

Figures

Fig 1. 17β Estradiol and Progesterone support growth of fibroid xenografts.

CB17-SCID mice were ovariectomized, and either not treated or supplemented with either 17β Estradiol (0.1mg/60d release pellets) alone, or 17β Estradiol in combination with progesterone (25mg/60d release pellets). Per mouse, four small grafts of each fibroid and myometrial tissue from one donor was transplanted. Between four to six mice were transplanted per donor and treatment group in this experiment (for details see S1 Table)The weight of the grafted fibroid tissue pieces at grafting is indicated with the dotted line. Mice were sacrificed after 60d, and grafts were weighted. Symbols indicate individual graft weights, together with group means ± s.e.m.

Fig 2. Immunohistochemistry of Estradiol receptor alpha, Progesterone receptor, and Ki67 in human fibroid grafts.

Uterine fibroid and myometrial grafts from the mouse xenograft experiment shown in Fig 1 were stained for ERα, PR and Ki67.

Fig 3. Optimization of 17β Estradiol and Progesterone dose supplement for uterine fibroid xenograft growth.

Ovariectomized SCID outbred mice were either not treated or supplemented with Progesterone pellets as indicated, and standard 17β Estradiol pellets (0.05mg/90d release, panel A); or 17β Estradiol pellets as indicated, and standard Progesterone pellets (25mg/60d release, panel B). Small pieces of fibroid tissue where grafted at an average weight indicated with the dotted line. For this experiment, 4 to 5 mice per donor with eight grafts each where used per treatment group. Mice were sacrificed after 60d, and grafts were weighted. Symbols indicate individual graft weights, together with group means ± s.d. Significance of difference between treatment and control groups was evaluated a two-sided t-test with Dunnett´s correction for multiple testing.

Fig 4. Fibroid grafts retain characteristics of human uterine fibroids.

Fibroid xenografts transplanted with approx. 10mg and grown for 60d in SCID mice supplemented with 17β Estradiol and Progesterone to 31mg were stained for BrdU incorporation (left, see S1 Fig for higher resolution), Desmin (middle) and collagen (extracellular matrix) (right). Scale bar = 500μm.

Fig 5. Growth kinetics of human fibroid xenografts in CB17 SCID mice.

17β Estradiol and Progesterone supplemented SCID mice were grafted with small (A) or large (B) grafts with fibroid tissue from four different patients. The dashed and dotted lines indicate graft weights of small and large grafts at transplantation on day 0, respectively. Mice were killed after 15, 30 and 45 days (A) or 45 days (B) and grafts were removed and weighted. Each individual graft weight is shown, together with group means ± s.d. Tissue from donors with the same numbers in panel A and B are identical. For statistical analysis, in (A) log transformed group means d15 to d30, and d30 to d45 were compared. In (B), 1graft weights were normalized to the respective xenograft transplantation weight of 10mg for small and 40mg for large grafts, and log transformed normalized data were compared to each other. Significance of difference between treatment and control groups was evaluated using a two-sided t-test.

Fig 6. Rapamycin and Mifepristone inhibit growth of fibroid xenografts in SCID mice.

E2 and P supplemented SCID-beige mice were grafted with large grafts (for Rapamycin) and small grafts (for Mifepristone, SCID-beige) from fibroid tissue from three and four different patients, respectively. The dashed line indicates the average weight of the grafts at day 0. (A) Mice were treated with Mifepristone from day 0 using 10mg/60d release pellets; this is approx. 6.5mg/kg/d. (B) Mice were treated p.o. with either 15mg/kg/d Rapamycin in 2.5% PEG400 in H2O or vehicle alone from d4 of the experiment. Mice were killed after 42–45 days and grafts were removed and weighted. Each symbol indicates the weight of an individual graft, together with group means ± s.d. Significance of difference between treatment and control groups was evaluated using a two-sided t-test.