Radium-223 Inhibits Osseous Prostate Cancer Growth by Dual Targeting of Cancer Cells and Bone Microenvironment in Mouse Models

Abstract

Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models.Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12-17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology.Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects.Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335-46. ©2017 AACR.

Figure 1. Radium-223 suppresses LNCaP and LuCaP 58 prostate cancer growth in bone in mouse models

A, Serum PSA levels in mice bearing LNCaP tumors, measured biweekly during dosing (mean ± SD, n = 10–14, p = 0.02771). B, Serum PSA levels in mice bearing LuCaP 58 PDX tumors, measured biweekly during dosing (mean ± SD, n = 16–18, p = 0.00191). C, Tumor area in mice bearing LNCaP tumors (n = 12–13, p = 0.00928). D, Tumor area in mice bearing LuCaP 58 tumors (n = 11, p = 0.09817). E, Total tissue area in mice bearing LNCaP tumors (n = 7–10, p = 0.91945). F, Total tissue area in mice bearing LuCaP 58 tumors (n = 11, p = 0.0697). G, The length of tumor-bone interface in mice bearing LNCaP tumors (n = 6–10, p = 0.00122). H, The length of tumor-bone interface in mice bearing LuCaP 58 tumors (n = 8–11, p = 0.00014). In box plots, horizontal lines show 5^th, 25^th, 50^th, 75^th and 95^th centiles and crosses indicate mean values. ns = not significant,* p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2. Radium-223 inhibits tumor-induced osteoblastic reaction resulting from LNCaP and LuCaP 58 prostate cancer growth in bone in mice

A–B, Representative micro-CT reconstructions of healthy and tumor-bearing tibias in (A) LNCaP and (B) LuCaP 58 models imaged from the medial side (measurement area starting 0.5 mm below the growth plate). Respective sagittal sections are shown on the right. Bone volume of tibias in mice bearing C, LNCaP (n = 12–13, p = 0.00450) or D, LuCaP 58 (n = 8–10, p < 0.001) tumors. In box plots, horizontal lines show 5^th, 25^th, 50^th, 75^th and 95^th centiles and crosses indicate mean values. Representative radiographs of healthy and tumor-bearing tibias in (E) LNCaP and (F) LuCaP 58 models. Red line delineates the osteoblastic/osteolytic/mixed lesion area. Osteoblastic/mixed lesion area measured biweekly during dosing in mice bearing (G) LNCaP tumors (mean ± SD, n = 13, p = 0.00366) and (H) LuCaP 58 tumors (mean ± SD, n = 11–17, p = 0.01702). I, PINP levels in mice bearing LNCaP tumors were measured biweekly (mean ± SD, n = 13, p<0.001). J, PINP levels in mice bearing LuCaP 58 tumors were measured biweekly (mean ± SD, n = 11–15, p = 0.00548). * p<0.05, ** p<0.01, *** p<0.001.

Figure 3. Radium-223 suppresses pathological bone formation in LNCaP and LuCaP 58 models of prostate cancer growth in mice

A, Representative MGT staining of bone architecture and tumor area in LNCaP and LuCaP 58 tumor-bearing mice treated with vehicle or Radium-223 (300 kBq/kg, i.v.). Turquoise indicates bone, pale pink tumor and dark pink bone marrow. Scale bar = 1 mm; 25x magnification. B, Total bone area in mice bearing LNCaP tumors (n = 12–13, p = 0.00059). C, Total bone area in mice bearing LuCaP 58 tumors (n = 11, p = 0.05883). D, Trabecular bone area relative to bone marrow area in mice bearing LNCaP tumors (n = 12–13, p = 0.43710). E, Trabecular bone area relative to bone marrow area in mice bearing LuCaP 58 tumors. (n = 11, p = 0.0212). F, The number of osteoblasts relative to bone surface in mice bearing LNCaP tumors (n = 12–13, p = 0.00127). G, The number of osteoblasts relative to bone surface in mice bearing LuCaP 58 tumors (n = 11, p = 0.0014). H, The number of osteoclasts relative to tumor-bone interface (TBI) in mice bearing LNCaP tumors (n = 6–10, p = 0.01207). I, The number of osteoclasts relative to TBI in mice bearing LuCaP 58 tumors (p = 8–11, 0.19644). In box plots, horizontal lines show 5^th, 25^th, 50^th, 75^th and 95^th centiles and crosses indicate mean values. ns = not significant,* p<0.05, ** p<0.01, *** p<0.001.

Figure 4. The effect of radium-223 treatment on visceral metastases in LuCaP 58 prostate cancer PDX model in mice

The number of mice with visceral metastases in mice treated with 28 mmol/L sodium citrate as vehicle or with radium-223 (300 kBq/kg, i.v.). The bars represent all mice, mice with smaller (PSA < 5 ng/mL) and mice with larger (PSA > 5 ng/mL) amount of tumor growth. The numbers above the bars represent the number of mice with visceral metastasis per the number of mice in a respective group.

Figure 5. Radium-223 is deposited in the intratumoral bone matrix and induces DNA double-strand breaks in tumor cells, osteoblasts and osteoclasts in LuCaP 58 mouse model of prostate cancer growth in bone

A, Autoradiography analysis of undecalcified tissue sections was used to define the localization of radium-223 particles (black dots) in osteoblastic/mixed bone metastasis. Analysis was done at 24 h after single intravenous administration of radium-223 (300 kBq/kg). Green arrows indicate radium-223 deposition in the proximity of osteoblasts and blue arrows point radium-223 deposits within the tumor bed. Scale bar = 100 μm; 200x magnification. B, Total activity of healthy and contralateral tumor-bearing tibias of mice treated with single dose of radium-223 measured with a gamma counter (cpm, counts per minute; n = 11–12). C, Relative serum PSA 72 h after radium-223 dosing (% of value on day 0; mean ± SD, n = 11–12, p = 0.04926). D, Relative necrotic tumor area in animals sacrificed 24, 48, or 72 h after a single dose of radium-223 and 72 h after vehicle dosing (n = 4–5, p = 0.27134). E, γ-H2AX positive tumor cells (n/microscope field). The animals were sacrificed 24, 48, or 72 h after a single radium-223 dose and 72 h after vehicle dosing (n = 4–5; p = 0.00297, 0.03665 and 0.00714 for 24h, 48h and 72 h, respectively. F, γ-H2AX positive osteoblasts (% of all osteoblasts). The animals were sacrificed 24, 48, or 72 h after a single radium-223 dose and 72 h after vehicle dosing (n = 3–5; p = 0.03148 for 72h). G, γ-H2AX positive osteoclasts (% of all osteoclasts). The animals were sacrificed 24, 48, or 72 h after a single radium-223 dose and 72 h after vehicle dosing (n = 4–5; p = 0.00714 for 72h). In box plots, horizontal lines show 5^th, 25^th, 50^th, 75^th and 95^th centiles and crosses indicate mean values. ns = not significant,* p<0.05, ** p<0.01, *** p<0.001.

Figure 6. Radium-223 therapy exhibits a dual targeting mode-of-action that destroys tumor cells and inhibits tumor-induced pathological bone reaction

A, Tumor-induced dysregulation of bone remodeling is a key driver in prostate cancer-derived bone metastases. Prostate cancer bone metastases are commonly associated with abnormal osteoblastic bone growth. Prostate cancer cells release paracrine factors that affect both osteoblastic and osteoclastic activity and stimulate abnormal bone remodeling. Osteoblasts, in turn, secrete factors that promote progression of the prostate cancer metastases and stimulate osteoclast activity. The growth factors released from bone by activated osteoclasts further stimulate tumor growth. This complex vicious cycle of prostate cancer bone metastasis results in increased tumor growth and altered bone structure stability resulting eventually in bone destruction. RANKL, receptor activator of nuclear factor kappa-B ligand. B, Radium-223 is a targeted α-therapy with a dual targeting mode-of-action. Radium-223 inhibits the vicious cycle of prostate cancer bone metastases by inducing tightly localized cytotoxic effects through double-stranded DNA breaks in osteoblasts, osteoclasts and prostate cancer cells.