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, 10 (64), 6845-6854

A Mouse Model of Prostate Cancer Bone Metastasis in a Syngeneic Immunocompetent Host

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A Mouse Model of Prostate Cancer Bone Metastasis in a Syngeneic Immunocompetent Host

Brian W Simons et al. Oncotarget.

Abstract

We report the establishment of B6CaP, an allograft tumor line from a Hi-Myc transgenic mouse that had been backcrossed onto C57BL/6J background. This tumor line grows subcutaneously in wildtype C57BL/6J immunocompetent mice, expresses AR, and has a luminal cytokeratin profile. When digested into single cells and injected via intracardiac injection, B6CaP produces metastatic widespread metastases including frequent bone lesions. Metastatic lesions occur most often in the femur, spine, and skull, and have a mixed osteolytic/osteoblastic phenotype. B6CaP allografts are androgen dependent, and regress after castration. However, castration resistant tumors regrow after 4-6 months and can be maintained as androgen-independent clones. This is the first example of a prostate-derived tumor line that shows frequent metastasis to bone and grows in an immunocompetent host, making this model useful for studying mechanisms of bone metastasis and tumor immune response.

Keywords: bone metastasis; murine model; prostate cancer.

Conflict of interest statement

CONFLICTS OF INTEREST The authors claim no conflict of interest.

Figures

Figure 1
Figure 1. B6CaP allograft recapitulates luminal epithelial phenotype of autochthonous Myc-driven prostate cancer.
(A–D) Autochthonous tumor from a C57BL/5J Hi-Myc mouse expresses luminal cytokeratin 8 (CK8), and high levels of nuclear Androgen Receptor (AR), and MYC. (E–H) Subcutaneous allograft B6CaP expresses similar levels of CK8, AR, and MYC. (I–L) B6CaP maintains E-Cadherin expression, lacks expression of cytokeratin 14 (CK14), NKX3.1, and Synaptophysin. Insets indicate positive control stains in normal prostate (K) and pancreatic islet (L).
Figure 2
Figure 2. Immune profile of B6CaP.
(A, B) Immunohistochemistry for IBA-1 (A), CD11b (B), and F4/80 (C) indicate high levels of infiltration by macrophage/myeloid cell types. (D) Tumor infiltrating lymphocyte (TIL) populations characterized by flow cytometry of dissociated allografts (mean +/– standard deviation). (E) Immune checkpoint expression of tumor infiltrating T cells. (F) PD-L1 expression of dissociated tumor cells.
Figure 3
Figure 3. Intracardiac injection of B6CaP cells generates widespread bone and soft tissue metastases.
Representative images (H&E) of metastases in (A) femur, (B) vertebrae with spinal cord compression, (C) skull/jaw (D) lung, (E) adrenal gland, (F) abdominal lymph node, (G) seminal vesicle, (H) Eye, (I) brain, and (J) spleen. (K, L) Immunohistochemistry for pan-cytokeratin (PanCK) and AR. Bone metastases generated osteoblastic (M) and osteolytic lesions (N). Bone lesions were occasionally large and exophytic (O). After intracardiac injection, median survival was 40 days with 21% (n = 12/57) of animals surviving to the 9-week endpoint (P). Venn diagrams representing the numbers of mice with metastases to bone, lung, or other non-lung soft tissue sites, total N = 44 (Q). Venn diagram representing the locations of bone metastases to skull, spine, or limbs, total N = 34 (R).
Figure 4
Figure 4. B6CaP generates castration resistant sublines with diverse phenotypes.
B6CaP allografts partially regressed after castration and expressed low levels of cytoplasmic AR, MYC, and Ki67 but no synaptophysin (SYN) (A). B6CaP allografts partially regressed after castration, but eventually recurred as castration resistant tumors (CR). Mean time to recurrence was 140 days (B). Representative images of recurrent castrate resistant (CR) tumors (C). Increased proliferation (Ki67) and diverse expression of AR, MYC, SYN were found among the CR sublines (C). A representative slide from parental B6CaP, regressed B6CaP after castration, and each CR line was scored for expression of various markers (D).
Figure 5
Figure 5. in vivo and ex-vivo growth kinetics of B6CaP.
Growth rates of CR sublines in castrate hosts, measured as time from injection to 1.5 cm tumor size, was significantly slower than parental B6CaP cells in intact hosts (A, 42.9 vs 63.3 days, p < 0.001). Organoids retained androgen sensitivity in vitro, growing rapidly to form organoids/spheres in media containing DHT, but rarely forming small spheres without DHT (B). Three weeks after plating 105 cells as organoids, cells in media containing DHT had expanded 10-fold, but total cell number had decreased in media without DHT (C).

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