(18)F and (18)FDG PET imaging of osteosarcoma to non-invasively monitor in situ changes in cellular proliferation and bone differentiation upon MYC inactivation

Abstract

Osteosarcoma is one of the most common pediatric cancers. Accurate imaging of osteosarcoma is important for proper clinical staging of the disease and monitoring of the tumor's response to therapy. The MYC oncogene has been commonly implicated in the pathogenesis of human osteosarcoma. Previously, we have described a conditional transgenic mouse model of MYC-induced osteosarcoma. These tumors are highly invasive and are frequently associated with pulmonary metastases. In our model, upon MYC inactivation osteosarcomas lose their neoplastic properties, undergo proliferative arrest and differentiate into mature bone. We reasoned that we could use our model system to develop noninvasive imaging modalities to interrogate the consequences of MYC inactivation on tumor cell biology in situ. We performed positron emission tomography (PET) combining the use of both (18)F-fluorodeoxyglucose ((18)FDG) and (18)F-flouride ((18)F) to detect metabolic activity and bone mineralization/remodeling. We found that upon MYC inactivation, tumors exhibited a slight reduction in uptake of (18)FDG and a significant increase in the uptake of (18)F along with associated histological changes. Thus, these cells have apparently lost their neoplastic properties based upon both examination of their histology and biologic activity. However, these tumors continue to accumulate (18)FDG at levels significantly elevated compared to normal bone. Therefore, PET can be used to distinguish normal bone cells from tumors that have undergone differentiation upon oncogene inactivation. In addition, we found that (18)F is a highly sensitive tracer for detection of pulmonary metastasis. Collectively, we conclude that combined modality PET/CT imaging incorporating both (18)FDG and (18)F is a highly sensitive means to non-invasively measure osteosarcoma growth and the therapeutic response, as well as to detect tumor cells that have undergone differentiation upon oncogene inactivation.

Figure 1

Incidence of osteosarcomas in transgenic Eµ-tTA, Tet-o-MYC mice. When MYC is on from conception 24% (25/106) of mice will develop osteosarcomas, 38% (40/106) will develop both osteosarcomas and lymphomas, and 39% (41/106) will develop lymphomas alone. If MYC is activated when mice are weaned the incidence of osteosarcoma drops to a total of 10% (7/70), while the percentage of mice that will develop lymphoma alone rises to 90% (63/70).

Figure 2

MYC inactivation in osteosarcoma is associated with reduced cellular proliferation and bone differentiation. Tumor cells were transplanted subcutaneously into syngeneic mice. When tumors reached a diameter of 1.0 to 1.5 cm, mice were sacrificed (MYC ON) or treated with doxycycline for either 2 or 8 days (MYC OFF). Calcification begins to appear at day 2, and by day 8 the entire tumor is calcified as assayed by hematoxylin and eosin staining. Ki67 staining demonstrated that 2 days following MYC inactivation, the tumors are no longer proliferating. DAPI staining demonstrates that calcification is accompanied by a decrease in tumor cell density.

Figure 3

¹⁸F can be used in microPET to detect osteosarcoma. (A) ¹⁸F scan of a syngeneic mouse without osteosarcoma. (B) ¹⁸F scan of an Eµ-tTA, Tet-O MYC mouse detects an osteosarcoma lung metastasis (as indicated with white arrow). A CT scan confirms the calcification of the tumor mass (red arrow) T = transverse, C = coronal, S = sagittal.

Figure 4

MYC inactivation results in neither an increase nor a decrease in tumor size. An osteosarcoma cell line (1325) was injected subcutaneously into syngeneic hosts. Following MYC inactivation (Day 0) tumors stop growing, but do not reduce in size. Tumors where MYC is on continued to grow. Error bars indicate standard deviation.

Figure 5

MYC inactivation causes a slight decrease in ¹⁸FDG uptake and a significant increase in ¹⁸F uptake in osteosarcomas. An osteosarcoma cell line (1325) was injected subcutaneously into syngeneic hosts. MYC was inactivated at day 0. (A) CT scan (black and white) co-registered with an ¹⁸FDG microPET scan (color intensity indicative of tracer uptake). ¹⁸FDG was taken up in the tumor (white arrow) as well as the heart (yellow arrow) and excreted into the bladder (green arrow). (B) ¹⁸FDG uptake following MYC inactivation (Day 0) was measured. ¹⁸FDG activity spikes and then plateaus following MYC inactivation. ¹⁸FDG levels do not increase when tumors grow out in the presence of doxycycline (Day 102). In tumors where MYC was constantly on, levels of ¹⁸FDG uptake did not change significantly. (C) CT scan (black and white) co-registered with an ¹⁸F PET scan (color intensity indicative of ¹⁸F uptake). The tumor readily took up the tracer (white arrow). (D) Quantification of ¹⁸F uptake following MYC inactivation. ¹⁸F activity remains higher than in controls (MYC ON) following MYC inactivation, and then gradually decreases. At approximately 30 days following MYC inactivation, tumor uptake levels were consistent with levels prior to inactivation. ¹⁸F levels do not increase when there was evidence of new tumor growth in the presence of doxycycline (Day 102). In tumors where MYC was not inactivated, ¹⁸F uptake rises slightly and then drops as tumors become necrotic. Error bars indicate standard deviation.

Figure 6

Prolonged MYC inactivation is associated with the loss of bone formation and ¹⁸F uptake in tumors. (A) ¹⁸F and ¹⁸FDG scans of a tumor that has grown despite continuous MYC inactivation. ¹⁸F (red) and ¹⁸FDG (green) scans coregistered demonstrate that the portion of the tumor that grew while MYC was inactivated envelops the original calcified tumor. Image confirms that ¹⁸F (red) and ¹⁸FDG (green) regions do not coregister. Hematoxylin and eosin staining of two different tumors that grew despite MYC inactivation. (B and D) The original portion of the tumor contains dense patches of calcification (Black arrows) (C and E) The portion of the tumor that has grown out despite continuous MYC inactivation does not contain any osteoid. Images are from two different tumors and representative of what was observed in 5 different relapse tumors.