Detection of metastatic tumors after γ-irradiation using longitudinal molecular imaging and gene expression profiling of metastatic tumor nodules

Abstract

A few recent reports have indicated that metastatic growth of several human cancer cells could be promoted by radiotherapy. C6-L cells expressing the firefly luciferase (fLuc) gene were implanted subcutaneously into the right thigh of BALB/c nu/nu mice. C6-L xenograft mice were treated locally with 50-Gy γ-irradiation (γ-IR) in five 10-Gy fractions. Metastatic tumors were evaluated after γ-IR by imaging techniques. Total RNA from non-irradiated primary tumor (NRPT), γ-irradiated primary tumor (RPT), and three metastatic lung nodule was isolated and analyzed by microarray. Metastatic lung nodules were detected by BLI and PET/CT after 6-9 weeks of γ-IR in 6 (17.1%) of the 35 mice. The images clearly demonstrated high [18F]FLT and [18F]FDG uptake into metastatic lung nodules. Whole mRNA expression patterns were analyzed by microarray to elucidate the changes among NRPT, RPT and metastatic lung nodules after γ-IR. In particular, expression changes in the cancer stem cell markers were highly significant in RPT. We observed the metastatic tumors after γ-IR in a tumor-bearing animal model using molecular imaging methods and analyzed the gene expression profile to elucidate genetic changes after γ-IR.

Figure 1

Detection of metastatic tumors after γ-IR by bioluminescence imaging (BLI). (A) Schedules of γ-IR treatment and image acquisition by BLI (●) and positron emission tomography/computed tomography with [¹⁸F]FDG (■) and [¹⁸F]FLT (▲). (B) Image was acquired from 3 weeks after γ-IR, we detected metastatic tumors in chest of No. 4 and 6 mice 6 weeks after γ-IR by BLI. No. 2 mouse was confirmed by ex vivo, hut the spot was not confirm on BLI. (C) BLI images of non-IR C6-L xenografted mice. After 3–7 weeks of xenograft, no distant metastasis was detected at a secondary site in the non-IR primary tumor model. (D) fLuc gene expression in metastatic lung nodules was confirmed by RT-PCR analysis using firefly luciferase and GAPDH specific primers (fLuc, 399 bp and rat GAPDH, 595 bp as an internal control).

Figure 2

Confirmation of metastatic tumors after γ-irradiation (IR) by nuclear medicine imaging. The mice were imaged using positron emission tomography/computed tomography (PET/CT) 6 weeks after γ-IR using uptake of [¹⁸F]FDG and [¹⁸F]FLT. (A) The [¹⁸F]FLT-PET image shows no metastatic nodules from non-IR primary tumor (NRPT) at a secondary site during 6 weeks. Arrows indicate the xenografted C6-L tumor. (B) BLI, [¹⁸F]FDG and [¹⁸F]FLT PET/CT of C6-L bearing mouse (No. 4 of Fig. 1B) 6 weeks after γ-IR. The images clearly demonstrate higher uptake of [¹⁸F]FDG and [¹⁸F]FLT in four metastatic lung nodules after γ-IR (white arrow). Ex vivo indication of macroscopic observation of nodules (yellow arrows) in lung. (C) BLI and [¹⁸F]FLT PET/CT of C6-L bearing mouse (No. 6 of Fig. 1B) 6 weeks after γ-IR. [¹⁸F]FLT PET/CT and autoradiography showed high [¹⁸F]FLT uptake in metastatic nodules after γ-IR. Metastatic lung nodules were detected by [¹⁸F]FLT and autoradiography (blue arrow), and other tissue metastasis were detected in the spleen (red arrow), but not by bioluminescence.

Figure 3

Comparison of primary and metastatic lung nodules. (A) Hierarchical clustering map of 3,881 differentially expressed genes in a non-irradiated (IR) primary tumor (NRPT), γ-IR treated C6-L primary tumor (RPT), and three metastatic lung nodules: red, upregulated; green, downregulated. The significance threshold was 2-fold with a P-value <0.05. (B) Pie chart shows different biological functional terms, (C) differential gene expression patterns based on categorization of the upregulated and downregulated genes. Comparisons between the RPT vs. NRPT and metastatic lung nodules vs. RPT categories were performed.

Figure 4

Diagram showing the model presented between the epithelial mesenchymal transition (EMT) and cancer stem cell marker in γ-irradiated (IR) C6-L primary tumor (RPT) and metastatic lung nodules after 6 weeks of γ-IR. Metastatic lung nodules after γ-IR with the EMT and enhanced cancer stem cell markers, resulting in C6-L metastasis and recurrence. However, the expression of cancer stem cell marker was downregulated in metastatic lung nodules after γ-IR.