Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model

Abstract

A new transplantable ovarian tumor model is presented using a novel folate receptor (FR) positive, murine ovarian cancer cell line that emulates the human disease and induces widespread intraperitoneal (i.p.) tumors in immunocompetent mice within 4-8 weeks of implantation. Tumor development was monitored using a new positron emission tomography (PET) FR-targeting reporter with PET/computerized tomography (PET/CT) and fluorescence molecular tomography (FMT) using a commercial FR-targeting reporter. Conventional structural magnetic resonance imaging (MRI) was also performed. Adult female C57BL/6 mice were injected i.p. with 6 × 10(6) MKP-L FR+ cells. Imaging was performed weekly beginning 2 weeks after tumor induction. The albumin-binding, FR-targeting ligand cm09 was radiolabeled with the positron emitter (68)Ga and used to image the tumors with a small animal PET/CT. The FR-reporter FolateRSense 680 (PerkinElmer) was used for FMT and flow cytometry. Preclinical MRI (7 T) without FR-targeting was compared with the PET and FMT molecular imaging. Tumors were visible by all three imaging modalities. PET/CT had the highest imaging sensitivity at 3-3.5 h postadministration (mean %IA/g mean > 6) and visualized tumors earlier than the other two modalities with lower kidney uptake (mean %IA/g mean < 17) than previously reported FR-targeting agents in late stage disease. FMT showed relatively low FR-targeted agent in the bladder and kidneys, but yielded the lowest anatomical image resolution. MRI produced the highest resolution images, but it was difficult to distinguish tumors from abdominal organs during early progression since a FR-targeting MRI reporter was not used. Nevertheless, there was good correlation of imaging biomarkers between the three modalities. Tumors in the mouse ovarian cancer model could be detected using FR-targeted imaging as early as 2 weeks post i.p. injection of tumor cells. An imaging protocol should combine one or more of the modalities, e.g., PET/CT or PET/MRI for optimal tumor detection and delineation from surrounding tissues.

Keywords: 68Ga; FolateRSense; MRI; PET/CT; cm09; fluorescence molecular tomography; folate receptor targeting; immunocompetent ovarian cancer mouse model.

Figure 1

Folate receptor expression in MKP-L cells in vitro and in vivo. (A) FR protein expression in MKP-L cells detected by Western blot using rabbit anti-FR polyclonal antibody. Low FR expressing IG10 murine ovarian cancer cells were used as controls. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels were used as loading controls. (B) FR protein expression in IG10 (left two histograms) and MKP-L (right two histograms), detected with flow cytometry using fluorescent FR680. Cells not exposed to FR680 (autofluorescence) were used as negative controls. The gate for FR positive events was set outside of the control histogram. Background staining (0.1 and 0.3%) and the percentage of FR+ IG10 and MKP-L cells are shown (13.6 and 90.3%, respectively). The same FR680 concentration was used for both cell lines. (C) MKP-L tumor histology. Female mice were injected i.p. with 6 × 10⁶ cells; tumors were isolated at necropsy, formalin-fixed, and paraffin embedded. Five micron sections were prepared for histology. Left panel: HE staining of a MKP-L tumor section (10×). Image shown is representative of tumors from three different mice, with at least two tumor sites sampled. Second and third panels (from left to right): IHC staining for FR (10× and 40× objectives, respectively), using anti-FR antibody. FR positive cells are shown in brown (S: stroma, T: tumor). Right panel: IHC image of one section stained with isotype control antibody. Scale bars: 100 μm.

Figure 2

Internalization of ⁶⁸Ga-cm09 in MKP-L cells (n = 3). All data are normalized for protein content. By 2 h, the amount of ⁶⁸Ga-cm09 internalized reached 4587.94 ± 550.08 fmol/mg protein (A), whereas the amount of ⁶⁸Ga-cm09 bound to the cell surface reached 19415.90 ± 2543.50 fmol/mg protein (B). Specific receptor-mediated internalization and surface bound fraction was blocked by folic acid in the medium (p = 0.011 and p = 0.001, respectively). Error bars represent standard deviations.

Figure 3

Detection of i.p. MKP-L tumors by ⁶⁸Ga-cm09 PET/CT in late stage disease in a control mouse vs 5 tumor-bearing mice (T# = tumor (and tumor index); K = kidneys), showing the various levels of disease (numbers of tumors, presence or not of ascites). All images were obtained 3–3.5 h after injection of the radiotracer. Images presented in %IA/g. Individual images are scaled based on the highest activity tumor. (A) Control mouse (Mouse 12); (B) Mouse 8, 5 weeks after MKP-L cell injection (T1 = 7.6 %IA/g, T2 = 7.7 % IA/g, K = 12.3 %IA/g); (C) Mouse 10, 7 weeks after MKP-L cell injection (T1 = 13.9 %IA/g, kidneys, K = 12.6 %IA/g); (D) Mouse 5, 6 weeks after MKP-L cell injection (T1 = 13.9 %IA/g, K = 12.5 %IA/g); (E) Mouse 1, 5 weeks after MKP-L cell injection (T1 = 15.1 %IA/g, T2 = 13.5 %IA/g, T3 = 12.1 %IA/g, K = 10.4 %IA/g); (F) Mouse 3, 5 weeks after MKP-L cell injection with ascites (tumors were dispersed throughout peritoneum and were not assigned indices, ascites activity = 6.9 %IA/g, K = 14.1 %IA/g). (G) Tumor to muscle ratios of ⁶⁸Ga-cm09 over time based on image ROI analysis in mice that had observable tumors at each weekly imaging session. In week 2, number of mice (N_M) = 4, number of tumors (N_T) = 3; in week 3, N_M = 4, N_T = 7; in week 4, N_M = 4, N_T = 7; in week 5, N_M = 7, N_T = 21; in week 6, N_M = 7, N_T = 24; in week 7, N_M = 3, N_T = 12. (H) Biodistribution and (I) PET/CT data in the same MKP-L-bearing mice (n = 3) injected with ⁶⁸Ga-cm09. The biodistribution study at 4 h postinjection (p.i.) was performed after PET/CT imaging (3 h). The PET color scales are in units of %IA/g. Error bars represent standard deviations.

Figure 4

(A) Small animal PET/CT maximum intensity projection images of MKP-L tumor bearing mouse (Mouse 15) at 3 h post injection of ⁶⁸Ga-cm09 (T# = tumor (and tumor index). PET/CT scan of nonblocked (left) and preblocked (right) of the same mouse indicates specific tumor uptake of ⁶⁸Ga-cm09. Uptake in tissues is presented in units of %IA/g. The tail signal in the preblocked mouse is due to extravasation that occurred during the initial attempt of the tail vein injection. (B) PET/CT uptake at 3 h p.i. of ⁶⁸Ga-cm09 in the nonblocked and blocked MKP-L tumor-bearing mouse (p = 0.001). (C) PET/CT organ or tumor to muscle uptake ratios between nonblocked and blocked mouse (p = 0.002). Error bars represent standard deviations.

Figure 5

Coronal and dissection images of a C57BL/6 mouse (Mouse 1) sacrificed 5 weeks after MKP-L injection showing ascites and widespread i.p. tumors. Images in the top row are the most dorsal and include stomach and kidneys, middle row images are medial, and bottom row images are the most ventral and include the bladder. (A,E,H) T₂-weighted MR images. (B,F,I) PET/CT images. (C,G,J) FMT images. (D) Gross dissection of i.p. tumors. Yellow arrows indicate tumors and/or tracer uptake outside of the kidneys and bladder. The PET color scales are in units of %IA/g.

Figure 6

Coronal images of a C57BL/6 mouse (Mouse 7) 2–7 weeks after MKP-L injection before the development of ascites. (A,D,G,J,M,P) T₂-weighted MR images. (B,E,H,K,N,Q) PET/CT images. (C,F,I,L,O,R) FMT images. Yellow arrows indicate tumors and/or tracer uptake outside of the kidneys and bladder. The PET color scales are in units of %IA/g.

Figure 7

FMT imaging of MKP-L tumors. (A–F) FMT images of a tumor-bearing mouse (Mouse 10) injected with 2 nmol FR680 at 2–7 weeks post-tumor implantation show the development of tumors in the i.p. cavity. (G) FMT images of a nontumor-bearing mouse injected with 2 nmol FR680. (H) Tumor volumes in Mouse 10 over time. Blue bars represent total tumor volume in the i.p. cavity, red bars represent tumor volume in the left side of the i.p. cavity, and green bars represent tumor volume in the right side of the i.p. cavity. (I) Tumor to muscle ratios of quantified FR680 fluorescence signal in the i.p. cavity over time. Error bars represent standard error.

Figure 8

MRI images of a periadrenal tumor at 6 weeks (Mouse 10) after induction taken with different imaging sequences. (A) T₂-weighted turbo spin echo (TSE), (B) T₁-weighted volume interpolated gradient echo (VIBE), which makes intestinal tissue appear bright white, (C) T₁-weighted TSE, and (D) T₁-weighted TSE after the administration of a gadolinium-based contrast agent. The tumor area is outlined in white. (E–H) Magnified images showing the periadrenal tumors and surrounding adrenal and intestinal tissue.