Understanding and Modeling Metastasis Biology to Improve Therapeutic Strategies for Combating Osteosarcoma Progression

Abstract

Osteosarcoma is a malignant primary tumor of bone, arising from transformed progenitor cells with osteoblastic differentiation and osteoid production. While categorized as a rare tumor, most patients diagnosed with osteosarcoma are adolescents in their second decade of life and underscores the potential for life changing consequences in this vulnerable population. In the setting of localized disease, conventional treatment for osteosarcoma affords a cure rate approaching 70%; however, survival for patients suffering from metastatic disease remain disappointing with only 20% of individuals being alive past 5 years post-diagnosis. In patients with incurable disease, pulmonary metastases remain the leading cause for osteosarcoma-associated mortality; yet identifying new strategies for combating metastatic progression remains at a scientific and clinical impasse, with no significant advancements for the past four decades. While there is resonating clinical urgency for newer and more effective treatment options for managing osteosarcoma metastases, the discovery of druggable targets and development of innovative therapies for inhibiting metastatic progression will require a deeper and more detailed understanding of osteosarcoma metastasis biology. Toward the goal of illuminating the processes involved in cancer metastasis, a convergent science approach inclusive of diverse disciplines spanning the biology and physical science domains can offer novel and synergistic perspectives, inventive, and sophisticated model systems, and disruptive experimental approaches that can accelerate the discovery and characterization of key processes operative during metastatic progression. Through the lens of trans-disciplinary research, the field of comparative oncology is uniquely positioned to advance new discoveries in metastasis biology toward impactful clinical translation through the inclusion of pet dogs diagnosed with metastatic osteosarcoma. Given the spontaneous course of osteosarcoma development in the context of real-time tumor microenvironmental cues and immune mechanisms, pet dogs are distinctively valuable in translational modeling given their faithful recapitulation of metastatic disease progression as occurs in humans. Pet dogs can be leveraged for the exploration of novel therapies that exploit tumor cell vulnerabilities, perturb local microenvironmental cues, and amplify immunologic recognition. In this capacity, pet dogs can serve as valuable corroborative models for realizing the science and best clinical practices necessary for understanding and combating osteosarcoma metastases.

Keywords: canine cancer; comparative oncology; experimental models; metastasis biology; translational therapeutics.

Figure 1

The metastatic cascade in osteosarcoma. (A) Primary OS tumor, usually in the long bones. (B) Tumor cells acquire an invasive phenotype and migrate away from the primary tumor and invade into surrounding tissues (step 1). Tumor cells interact with the basement membrane and endothelial cells to intravasate into the blood microvasculature (step 2) and travel in the circulation (step 3). (C) Upon arrival at the secondary site (lung), tumor cells arrest via size restriction or adhesion interactions with the pulmonary microvascular endothelial cells (step 4). (D) Once tumor cells extravasate out of the blood vessels, they must be able to adapt and survive in the lung microenvironment (step 5). At this vulnerable stage, tumor cells can undergo a number of fates which include- enter cellular dormancy, die off, or if the stresses of the lung microenvironment can be successfully managed, tumor cells can proliferate into multi-cellular micrometastases (step 6). Micrometastases can enter into a state of “angiogenic dormancy” and remain the same size, or regress if cell death is greater than proliferation, or recruit local blood vessels and form a vascularized secondary tumor (step 7).

Figure 2

Imaging models to study the metastatic cascade in cancer. (A) The CAM model (whole mount image) showing the chick embryo and highly vascularized CAM. A small renal cell carcinoma (RCC) explant can be seen growing in the dashed white box. Zoomed image of a different established GFP-expressing RCC tumor where the entire tumor, associated vasculature, and corresponding fluorescence image (below) are shown (Image courtesy of Matthew Lowerison and Pengfei Song, UIUC). (B) High magnification, single cell imaging of a GFP-labeled prostatic carcinoma cell (PC) migrating through capillary plexus (labeled with rhodamine-lectin) and forming invadopodia (yellow arrowheads) into the lumen of 2 adjacent capillaries in the CAM model (Image courtesy of Fabrice Lucien and Yohan Kim, Mayo Clinic). (C) The PuMA is an ex vivo lung explant model where tumor cells in viable lung tissue is maintained in cell culture. The lung slices are kept at an air-liquid interface on top of a gelatin sponge. (D) Shows the lung parenchyma (stained red with DAR4M) and eGFP-expressing MG63 OS cells (OS) interacting with vessel-like structures (v). Scalebar = 100 μm. See Lizardo and Sorensen (88) for methods. (E) The WHRIL model allows for the direct visualize of lung tissue through a window in the mouse chest cavity (89) as shown with the dashed white circle. (F) Fluorescent micrograph showing the lung microvasculature (v) (labeled red with tetramethylrhodamine) and GFP-expressing breast cancer cell (BC). Blue fibers represent second harmonic imaging of connective tissue (c) fibers. Scalebar = 15 μm (Images courtesy of David R. Entenberg, Albert Einstein College of Medicine). UIUC, University of Illinois at Urbana-Champaign.

Figure 3

(A) Syngeneic orthotopic mouse model of primary bone OS (K7M3) with concurrent spontaneous pulmonary metastases development visualized by bioluminescent imaging, (B) with corollary histology of established pulmonary metastatic lesions and (C) micro CT images of the OS primary lesion showing profound osteolysis and contralateral unaffected tibia. (D) Bioluminescent imaging of an experimental metastases model in athymic nude mice following tail vein injection with the Abrams (canine OS) luciferase cell line demonstrating correlation between luminescent signal and (E) gross macroscopic tumor burdens.

Figure 4

(A) Glioblastoma spheroids in high throughput high-density hanging drop culture on a microchip. Method allows for the rapid screening of novel therapeutic agents in cancer cells growing in 3D whereby diffusional gradients and cell-cell interactions are more accurately recapitulated than 2D cell culture conditions (monolayer). Green dye (Celltox™ Promega) shows cell death after 24 h of culture (Image courtesy of Anurup Ganguli and Rashid Bashir, UIUC). (B) Relative size of hydrogel scaffolds for the study of (C) 3D glioblastoma spheroids by confocal fluorescent microscopy and associated (D) homotypic (cell-cell) and heterotypic (cell-ECM) interactions by scanning electron microscopy (Images courtesy of Emily Chen and Brendan Harley, UIUC). UIUC, University of Illinois at Urbana-Champaign.

Figure 5

Radiologic assessment and relevance of comparable anatomic size and metabolic activity of OS tumors arising in pet dogs. (A) Early detection of emerging pulmonary metastatic lesion (red arrowhead) with (B) subsequent rapid macroscopic growth (red arrowhead) over a period of 8 weeks documented by serial CT imaging. Metabolic activity of (C) primary bone OS (Image courtesy of Kim Selting, UIUC) and (D) pulmonary metastases (Image courtesy of Lynn Griffin, Colorado State University) using PET/CT imaging in pet dogs with OS. UIUC, University of Illinois at Urbana-Champaign.