A novel mouse model that closely mimics human uterine leiomyomas Michael Drosch, M.Sc., Jörn Bullerdiek, Prof. Dr., Thomas M. Zollner, M.D., Florian Prinz, M.D., Ph.D., Markus Koch, Ph.D., Nicole Schmidt, Ph.D.  Fertility and Sterility  Volume 99, Issue 3, Pages 927-935.e6 (March 2013) DOI: 10.1016/j.fertnstert.2012.11.032 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Histologic and immunohistochemical comparison of xenografts after 60 days of growth and primary tumors. (A, B) Hematoxylin and eosin (HE) staining confirms myoma-like histology with whirl-like pattern and fusiform cells. (C, D) Trichrome staining shows vast deposition of collagens (green) surrounded by muscular cells (red) in xenografts as well as primary tumors. (E, F) Abundance of smooth muscle actin (SMA) in xenografts and primary tumor confirms the smooth muscle differentiation of the generated xenografts. (G, H) Ki67 staining displays only a few positive cells in xenografts and primary tumors, indicating a less proliferative capacity. Formalin-fixed paraffin-embedded tissue, scale bar 100 μm. Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Quantification of cell proliferation in xenografts and tumor. Three different high-power fields from xenografts (60 days after transplantation) from different donors (1M02–1M05) and a representative native myoma were used for morphometric evaluation of Ki67-positive cells. Negative control was rabbit preimmune serum. Positive control was tissue from non-Hodgkin lymphoma. Data are shown as mean ± SD. Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Fluorescence in situ hybridization analysis of xenografts 60 days after inoculation showed strong hybridization signals against human centromeres (red) within the tumor, confirming human origin. Nuclear counterstain was DAPI (blue). Formalin-fixed paraffin-embedded tissue. Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Characterization of orthotopic tumors generated by intrauterine injection. Uteri were injected with 1.5 × 106 primary myoma cells under the serous membrane and excised after 4 weeks, snap frozen, and cryosectioned. (A) Fluorescence in situ hybridization identified tumorous cells positive for human centromeres (red). Red line indicates the maximum diameter of the tumor (844 μm). (B) Hybridization signals against murine centromeres (green pseudocolor) were abundant in adjacent tissue but negligible within the tumor. Nuclei were counterstained with DAPI. (C) Hematoxylin and eosin (HE) staining displayed myoma-like whirl-structure of the tumor (dashed line). (D) Immunostaining against smooth muscle actin (red) of the tumor (dashed line) and adjacent myometrium (dashed lines with arrows) confirmed smooth muscle differentiation. Magnification ×5, chromophore 3-amino-9-ethylcarbazole (AEC), formalin-fixed paraffin-embedded tissue (C and D). Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 (A) Western Blot analysis of SV40LT, p53, and smooth muscle actin (SMA) in lysates of primary cells and transformed cell lines. SV40LT and p53 were detectable in SV40ER and LM220 cells but were absent in native cells as well as carcinoma cell lines MCF7 and HeLa-Matu used as control samples. SMA was present in native and transformed cells but absent in the epithelial control cells (MCF7 and HeLa-Matu), confirming smooth muscle differentiation. (B) Soft agar assay for anchorage-independent growth. Untransduced cells do not form colonies when cultured in soft agar (top left). In turn, SV40ER-transformed cells (SV40ER) exhibit colony formation with a robust growth (top right). A proportion of cells still fail to form colonies (arrow). Immortalized cells (LM220) show fewer colonies and smaller colony size (asterisk; bottom left). The adenocarcinoma cell line A549 shows enhances colony formation as expected owing to its malignant transformation (bottom right). Photomicrographs were taken after 14 days of culture. Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Hematoxylin and eosin staining of xenografts generated by injection of the immortalized LM220 cell line shows absence of myoma-like histology, such as fiber-like patterns. In contrast, cell density is heavily reduced and there is a high proportion of cells with pycnotic nuclei (arrowheads), indicating apoptotic cells. Formalin-fixed paraffin-embedded tissue, 4 weeks after injection. Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Representative photomicrographs of xenografts derived from native uncultured primary cells after 90 days of incubation, showing the palpable tumor (left) and after excision (right). Note the sprouting of blood vessels toward the tumor. Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 4 Identification of blood vessels in xenografts by double immunostaining. Cryosections were incubated with antibodies directed against murine CD31 and smooth muscle actin (SMA). Vessels are marked by circular structures of CD31-positive cells (green) surrounded by SMA-positive cells (red). Nuclear counterstaining was with DAPI (blue). Acetone-fixed cryosection. Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 5 Gross morphology of uteri before and after injection of primary myoma cells. Cell suspension supplemented with Evans blue dye was injected underneath the subserous membrane as indicated by translucent blue coloring (left). Four weeks after injection, the injected horn (arrow) was enlarged in size compared with the unaffected horn. Fertility and Sterility 2013 99, 927-935.e6DOI: (10.1016/j.fertnstert.2012.11.032) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions