Biological characterization of preclinical Bioluminescent Osteosarcoma Orthotopic Mouse (BOOM) model: A multi-modality approach

Abstract

Osteosarcoma (OS) is a bone malignancy that affects children and adolescents. It is a highly aggressive tumor and typically metastasizes to lungs. Despite aggressive chemotherapy and surgical treatments, the current 5 year survival rate is 60-70%. Clinically relevant models are needed to understand OS pathobiology, metastatic progression from bones to lungs, and ultimately, to develop more efficacious treatment strategies and improve survival rates in OS patients with metastasis. The main goal of this study was to develop and characterize an in vivo OS model that will allow non-invasive tracking of tumor progression in real time, and aid in studying OS pathobiology, and screening of potential therapeutic agents against OS. In this study, we have used a multi-modality approach using bioluminescent imaging, electron microscopy, micro-computed tomography, and histopathology to develop and characterize a preclinical Bioluminescent Osteosarcoma Orthotopic Mouse (BOOM) model, using 143B human OS cell line. The results of this study clearly demonstrate that the BOOM model represents the clinical disease as evidenced by a spectrum of changes associated with tumor establishment, progression and metastasis, and detection of known OS biomarkers in the primary and metastatic tumor tissue. Key novel findings of this study include: (a) multimodality approach for extensive characterization of the BOOM model using 143B human OS cell line; (b) evidence of renal metastasis in OS orthotopic model using 143B cells; (c) evidence of Runx2 expression in the metastatic lung tissue; and (d) evidence of the presence of extracellular membrane vesicles and myofibroblasts in the BOOM model.

Keywords: Bioluminescent; Extra-cellular membrane vesicles; Myofibroblasts; Orthotopic; Osteosarcoma; Preclinical.

Fig. 1

Bioluminescent imaging of intra-tibial growth of 143B-luc cells. Mice were injected with 143B-luc cells intra-tibially and tumor growth was monitored weekly by luciferase imaging (A). Within 2 weeks, mice developed spontaneous metastasis to lungs (A). A representative image of ex-vivo bioluminescent imaging of lung metastatic lesions is shown (B). Kinetics of 143B-luc generated tumor in real time is shown in C. Detection of bioluminescent signal in 3D views (coronal, transaxial and saggital) is shown in D.

Fig. 2

Photomicrograph illustration showing the presence of mCherry positive 143B-luc cells (as indicated by an arrow) in the bone (primary tumor site) (A) and lung (metastatic site) tissue (B).

Fig. 3

Hematoxylin and Eosin staining showing primary tumor in the tibia (A and B) and OS metastatic lesions in lungs (C and D) and kidney (E and F). The inset shows the presence of osteoid (pink color) in the tumor. The reader is recommended to zoom to 200% to view tumor cells in different mitotic stages. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Graphic illustrations showing tumor burden profile in the BOOM model from two rounds of experiments. Bone, lung and kidney sections were examined histogically for the detection of tumors (A). Number of metastatic lesions per lung or kidney sections were also examined (B).

Fig. 5

Photomicrograph illustration showing the presence of osteoid (pink color) in the primary tumor as detected by Goldner staining. The osteoid (as indicated by an arrow) is deposited by rapidly proliferating osteosarcoma cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Photomicrograph illustration showing Ki-67 (a proliferation marker) immunostaining in actively dividing osteosarcoma cells in tibia (A), lungs (B) and kidneys (c). Arrows indicate mitotic figures where Ki-67 is maximal.

Fig. 7

Photomicrograph illustration showing ezrin immunostaining in osteosarcoma tumor tissue in tibia and lungs. Panels A and B are treated with anti-ezrin primary antibody. Brown staining indicates ezrin expression (also indicated by arrows). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Photomicrograph illustration showing immunlocalization and expression of Runx2 in osteosarcoma tissue microarray, 143B human OS cells and tumor tissue of the BOOM model. Panels A (10×), B (40×), C and D demonstrate positive staining in osteoblastic osteosarcoma core sample of bone cancer tissue microarrays, 143B human OS cell line in vitro, and in the tumor tissue isolated from the BOOM model, respectively. Panel E (40X) shows positive immunostaining for Runx2 in metastatic OS lesions in the lung tissue.

Fig. 9

Photomicrograph illustration showing the presence of myofibroblast like cells in the primary tumor tissue of the BOOM model as identified by light microscopy (Panel A) and α-SMA (Panel B), and desmin (Panel C) immunostaining.

Fig. 10

Transmission Electron Micrograph composite showing representative images of myofibroblasts and EMVs in the primary tissue of the BOOM model. The ultrastructural features that are characteristic of myofibroblasts include presence of RER, peripheral distribution of filaments, and a prominent fibronexus junction (Panel A). Panel B shows representative images (B1) of EMVs and multivesicular body (B2) in the tumor tissue of the BOOM model.

Fig. 11

Photomicrograph illustration showing representative 3D reconstructions of μCT scans of control (non-tumor-bearing) vs. orthograft tumor (tumor-bearing). Osteoblastic (OBL)/osteolytic (OLL) lesions are indicated by an arrow (Panel A). The lower panel (B) shows representative 3D μCT–generated sections of tibias showing increased cortical thickening and destruction of cortical bone in tumor-bearing mice versus control.

Fig. 12

Graphic illustration comparing cortical bone parameters i.e. cortical fraction (BV/TV) (A), cortical bone mineral density (B), cortical thickness (C), and polar moment of inertia (pMOI) (D) between control (non-tumor-bearing) and tumor-bearing bone (n≥3).