Inhibition of angiopoietin-2 in LuCaP 23.1 prostate cancer tumors decreases tumor growth and viability

Abstract

Background: Angiopoietin-2 is expressed in prostate cancer (PCa) bone, liver, and lymph node metastases, whereas, its competitor angiopoietin-1 has limited expression in these tissues. Therefore, we hypothesized that the inhibition of angiopoietin-2 activity in PCa will impede angiogenesis, tumor growth, and alter bone response in vivo.

Methods: To test our hypothesis we used L1-10, a peptide-Fc fusion that inhibits interactions between angiopoietin-2 and its receptor tie2. We blocked angiopoietin-2 activity using L1-10 in established subcutaneous and intra-tibial LuCaP 23.1 xenografts. We then determined the effect of L1-10 on survival, tumor growth, serum PSA, proliferation, microvessel density, and angiogenesis-associated gene expression in subcutaneous tumors. We also determined serum PSA, tumor area, and bone response in intra-tibial tumors.

Results: The administration of L1-10 decreased tumor volume and serum PSA, and increased survival in SCID mice bearing subcutaneous LuCaP 23.1 tumors. Histomorphometric analysis, showed a further significant decrease in tumor epithelial area within the L1-10 treated LuCaP 23.1 subcutaneous tumors (P=0.0063). There was also a significant decrease in cell proliferation (P=0.012), microvessel density (P=0.012), and a significant increase in ANGPT-2 and HIF-1α mRNA expression (P≤0.05) associated with L1-10 treatment. Alternatively, in LuCaP 23.1 intra-tibial tumors L1-10 treatment did not significantly change serum PSA, tumor area or bone response.

Conclusions: Our results demonstrate that inhibiting angiopoietin-2 activity impedes angiogenesis and growth of LuCaP 23.1 PCa xenografts. Based on these data, we hypothesize that angiopoietin-2 inhibition in combination with other therapies may represent a potential therapy for patients with metastatic disease.

Figure 1. L1–10 decreases tumor volume and serum PSA while improving survival in subcutaneous LuCaP 23.1 tumor xenografts

(A) Serum PSA levels in control and L1–10 treated animals bearing LuCaP 23.1 subcutaneous tumors. (B) Subcutaneous LuCaP 23.1 tumor volume in control and L1–10 treated animals. (C) Survival in SCID mice with subcutaneous LuCaP 23.1 tumors. (D) Weight of subcutaneous LuCaP 23.1 tumors in control and L1–10 treated animals. For all experiments: control (n=9) and L1–10 treated (n=9) animals were sacrificed when tumors reached 1g in size. Results are plotted as mean ± SEM. * indicates significance (p<0.05).

Figure 2. Histomorphometric analysis reveals a decrease in tumor area and an increase in necrotic regions of L1–10 treated LuCaP 23.1 subcutaneous xenografts

(A) Histomorphometric analysis of necrotic area as a % of tumor area in control and L1–10 treated LuCaP 23.1 subcutaneous tumors. (B) Histomorphometric analysis of tumor area in control and L1–10 treated LuCaP 23.1 subcutaneous tumors * (p=0.0063). (C) Representative images of control and L1–10 treated LuCaP 23.1 subcutaneous tumors. * Highlights necrotic regions. For all experiments: control (n=9) and L1–10 treated (n=9). Results are plotted as mean ± SEM.

Figure 3. L1–10 decreased epithelial cell proliferation in LuCaP 23.1 subcutaneous xeonografts

A graph of % BrdU positive cells in control and L1–10 treated LuCaP 23.1 subcutaneous tumors * (p=0.012). Arrows highlight BrdU positive nuclei. Control (n=9) and L1–10 treated (n=9). Results are plotted as mean ± SEM.

Figure 4. L1–10 decreased microvessel density in LuCaP 23.1 subcutaneous xeonografts and alters angiogenesis-associated gene expression

(A) Microvessel density in control and L1–10 treated LuCaP 23.1 subcutaneous tumors * (p=0.012). Arrows indicate CD34 positive vessels in representative tumor samples. (B) Angiogenesis-associated gene expression (human epithelial and mouse stromal) in control and L1–10 treated LuCaP 23.1 subcutaneous tumors * (p≤0.05). mRNA expression was calculated relative to RPL13a mRNA expression and then normalized to angiopoietin-1 mRNA expression. For all experiments: control (n=9) and L1–10 treated (n=9). Results are plotted as mean ± SEM.

Figure 5. No difference in α smooth muscle actin (αSMA) was observed in the vessels of control and L1–10 treated LuCaP 23.1 subcutaneous xeonografts

Representative images of the immunohistochemical co-localization of CD34 (green), αSMA (red), control Rat IgG, and MOPC-21 antibodies in L1–10 (n=4) and control (n=4) treated LuCaP 23.1 subcutaneous tumors.

Figure 6. Tumor growth and bone response are altered in L1–10 treated LuCaP 23.1 intra-tibial tumors

(A) Serum PSA levels in animals bearing LuCaP 23.1 intra-tibial tumors in control and L1–10 treated animals. (B) Bone mineral density (BMD) of LuCaP 23.1 control (left tibia) and tumored (right tibia) of control and L1–10 treated tumors. (C) Radiographs of the right tumored tibiae of a representative animal from the control and L1–10 treated group after three and six weeks. (D) Mineralized tibiae were harvested 6 weeks after tumor injection and embedded in methyl methacrylate. Five micron sections were stained with Goldner's trichrome stain. In tumor-bearing animals the tumor replaced the marrow and promoted trabecular bone growth. Arrows highlight osteoblastic bone. (E) Bone histomorphometry of Goldner’s trichrome stained tumored tibiae. For all experiments: control (n=8) and L1–10 (n=9). Results are plotted as mean ± SEM. * indicates significance (p<0.05).