Rat Prostate Tumor Cells Progress in the Bone Microenvironment to a Highly Aggressive Phenotype

Abstract

Prostate cancer generally metastasizes to bone, and most patients have tumor cells in their bone marrow already at diagnosis. Tumor cells at the metastatic site may therefore progress in parallel with those in the primary tumor. Androgen deprivation therapy is often the first-line treatment for clinically detectable prostate cancer bone metastases. Although the treatment is effective, most metastases progress to a castration-resistant and lethal state. To examine metastatic progression in the bone microenvironment, we implanted androgen-sensitive, androgen receptor-positive, and relatively slow-growing Dunning G (G) rat prostate tumor cells into the tibial bone marrow of fully immune-competent Copenhagen rats. We show that tumor establishment in the bone marrow was reduced compared with the prostate, and whereas androgen deprivation did not affect tumor establishment or growth in the bone, this was markedly reduced in the prostate. Moreover, we found that, with time, G tumor cells in the bone microenvironment progress to a more aggressive phenotype with increased growth rate, reduced androgen sensitivity, and increased metastatic capacity. Tumor cells in the bone marrow encounter lower androgen levels and a higher degree of hypoxia than at the primary site, which may cause high selective pressures and eventually contribute to the development of a new and highly aggressive tumor cell phenotype. It is therefore important to specifically study progression in bone metastases. This tumor model could be used to increase our understanding of how tumor cells adapt in the bone microenvironment and may subsequently improve therapy strategies for prostate metastases in bone.

Figure 1

Establishment of G tumors in the bone versus the prostate microenvironment. G tumor cells (2 × 10⁵) were implanted into the tibial bone marrow or ventral prostate of fully immune-competent rats. (A) Representative sections, in low and high magnifications, of G tumors in prostate at 8 weeks (encircled in black) and in bone at 8 weeks (encircled in black and with arrowheads) and 12 weeks (encircled in black). High-resolution versions of G tumors in prostate and bone slides for use with the Virtual Microscope are available as eSlide: VM02511 and as eSlide: VM02512. (B) Tumor area (mm²) in bone and prostate at 8 and 12 weeks. Values are mean ± SE; n = 6 to 10 animals in each group. *P < .05, **P < .01, ***P < .001.

Figure 2

Tumor establishment in bone and prostate of castrated and control rats. G tumor cells (2 × 10⁶) were implanted into the tibial bone marrow or prostate tissue of control or castrated rats, and tumor size (A) and tumor proliferation (BrdU) (B) were analyzed at 6 weeks. Values are mean ± SE, n = 8 to 12 animals in each group, **P < .01, ***P < .001, ns = not significant.

Figure 3

Castration treatment of established G tumors in the bone microenvironment. G tumor cells were implanted into the bone marrow (2 × 10⁵) and into the prostate tissue (2 × 10³) and grown for 8 weeks, and then the rats were either castrated or control treated. Tumor size (A), tumor proliferation (BrdU) (B), and tumor apoptosis (caspase-3) (C) were analyzed 14 days later. Values are mean ± SE; n = 7 to 8 animals in each group. *P < .05, **P < .01.

Figure 4

G tumor progression in the bone microenvironment. (A) G tumor cells (2 × 10⁵) were injected into the tibial bone marrow of castrated or control-treated rats and left for 8 or 12 weeks before tumors (n = 5 to 6 in each group) were removed, pooled, and reestablished in vitro as the following cell lines: G cells from 8-week bone tumors of control animals (G-bone-8w), G cells from 8-week bone tumors of castrated rats (G-bone-8w-cast), G cells from 12-week bone tumors of control animals (G-bone-12w), and G cells from 12-week bone tumors of castrated rats (G-bone-12w-cast). (B) Tumor cell morphology in vitro was similar to that of the original G cells. (C) Tumor cell viability measured for 7 days in vitro using an MTT assay, presented as fold change in mean absorbance (abs) ± SE compared with day 1. **P < .01 compared with the original G cell line at day 7. (D) Western blot analyses showing androgen receptor levels in the different G tumor cell lines.

Figure 5

G tumor progression in the bone microenvironment. (A) Equal numbers (2 × 10⁵ cells) from the different G tumor cell lines were reinjected into the ventral prostate of recipient rats that had been castrated or control treated, and tumor weight was measured 6 weeks later (values are mean ± SE, n = 6 to 8 in each group; *P < .05, **P < .01, ***P < .001). (B) Equal numbers of cells of each G cell line (7.5 × 10⁵) were injected into the tail vein, and tumor colonization in lungs was examined 10 weeks later (values are mean tumor weight in lungs ± SE, n = 5 to 7 in each group, **P < .01 compared with G-original cells). (C) Sections of the different G tumors in prostate. Note the changed morphology in the G-bone-cast-12w tumors, with viable tumor tissue growing around large blood vessels. High-resolution versions of G-bone-8w-cast and G-bone-12w-cast slides for use with the Virtual Microscope are available as eSlide: VM02513 and as eSlide: VM02514. (D) Representative sections, in low and high magnifications, showing lung tumor burden for the different cell lines. High-resolution versions of G-bone-8w and G-bone-12w slides for use with the Virtual Microscope are available as eSlide: VM02515 and as eSlide: VM02516.

Figure 6

G tumor cell viability in response to androgens. G tumor cell viability in vitro was measured 0, 4, and 7 days after incubation with DHT or T at different concentrations using an MTT assay. Values are mean absorbance ± SE.

Figure 7

Hypoxia in the bone marrow and prostate. Sections from bone tissue in controls and 7 days after castration, stained for hypoxia (Hypoxyprobe, brown), in low and high magnifications. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM02517.