Comparison of 5-aminolevulinic acid and MMP-14 targeted peptide probes in preclinical models of GBM

Abstract

Rationale: Developing novel pre-operative and intraoperative imaging approaches for glioblastoma multiforme (GBM) could aid therapeutic intervention while sparing healthy normal brain, which remains a significant clinical challenge. 5-aminolevulinic acid (5-ALA) is the only intraoperative imaging agent approved to aid the resection of GBM. Matrix metalloproteinase 14 (MMP14), which is overexpressed in GBM, is an attractive target for preoperative and intraoperative imaging of GBM. Prior studies have shown the feasibility of near-infrared fluorescence (NIRF) imaging and positron emission tomography (PET) imaging of GBM xenografts in mice using MMP-14 targeted peptide probes. The present studies assessed the tumor-specific localization and contrast of these MMP-14 targeted peptides relative to 5-ALA in GBM models. Methods: Fluorescence and PET imaging was performed after i.v. injection of 5-ALA and the MMP-14 targeted peptide probes (non-labeled or radiolabeled with ⁶⁴Cu) in mice bearing human GBM orthotopic xenografts (U87, D54). Imaging signals were correlated to MMP-14 expression determined by immunofluorescence. Tumor-to-normal brain ratio (TBR) and Dice similarity coefficient (DSC) relative to tumor defined by ex vivo pathology or in vivo magnetic resonance imaging were determined for each imaging agent. Results: NIRF signals from the MMP-14 targeted peptide probes showed comparable TBR (p < 0.05) but significantly higher DSC (p < 0.05) relative to 5-ALA. NIRF signals from the peptide probes significantly correlated with MMP-14 expression (p < 0.05). MMP-14 binding peptide labeled with ⁶⁴Cu showed moderate DSC (0.45) while PET signals significantly correlated (p < 0.05) with NIRF signals from a co-injected MMP-14 substrate peptide. NIRF and PET signals localized in residual tumor regions in the resection cavity during in situ resection. Conclusions: MMP-14 targeted peptides showed favorable TBR and higher tumor localization than 5-ALA in GBM orthotopic models. Further development of MMP-14 targeted peptide probes could lead to improved pre-operative and intraoperative management of GBM.

Keywords: 5-ALA; GBM; MMP-14; NIRF; PET.

Figure 1

NIRF signal from MMP-14 targeted peptide probes localizes in macroscopic and microscopic GBM orthotopic xenografts and correlates with histological MMP-14 expression. A, representative images of fixed brain slices from GBM-bearing mice dosed i.v. with the substrate-binding peptide and 5-ALA (top panels) or the substrate peptide and 5-ALA (bottom panels); corresponding brightfield and NIRF panels are from the same 1 mm brain slice, which was also used to obtain histologic sections for the corresponding H+E and MMP-14 immunofluorescence (yellow; blue, nuclei stained by DAPI) panels. White dotted line indicates tumor region. B-C, least-squares fit of linear regression between histological MMP-14 immunofluorescence MFI and NIRF signal MFI in 1 mm brain slices from GBM-bearing mice dosed i.v. with the substrate-binding peptide (B) or the substrate peptide (C); dotted lines show the 95% confidence interval of the correlation. D, brightfield and NIRF images of a 1 mm fixed brain slice from a mouse dosed i.v. with the substrate peptide, with white box indicating the region of implantation and the corresponding zoomed in areas from histology sections stained with H+E or MMP-14 immunofluorescence (4x original magnification). MFI, mean fluorescence intensity; a.u., arbitrary units.

Figure 2

PpIX fluorescence from 5-ALA and NIRF from MMP-14 targeted peptide probes provide comparable tumor-to-normal brain ratios in GBM orthotopic xenografts. A, representative images (brightfield, PpIX fluorescence, NIRF) of 1 mm fixed brain slices from GBM-bearing mice dosed i.v. with 5-ALA and the substrate-binding peptide (top panels) or 5-ALA and the substrate peptide (bottom panels); corresponding panels are from the same 1 mm brain slice. White dotted line indicates tumor region. B, mean ± SD tumor-to-normal brain ratio (TBR) of mean fluorescence intensity for each imaging agent quantified in 1 mm fixed brain slices; significance determined by ANOVA (ns, not significant).

Figure 3

NIRF from MMP-14 targeted peptide probes exhibit significantly greater tumor localization relative to PpIX fluorescence from 5-ALA in GBM orthotopic xenografts. A, representative images of fixed brain slices from GBM-bearing mice dosed i.v. with 5-ALA and the substrate-binding peptide (top panels, U87 xenograft) or 5-ALA and the substrate peptide (bottom panels, D54 xenograft); corresponding brightfield, PpIX, and NIRF panels are from the same 1 mm brain slice, which was also used to obtain a histologic section for the corresponding H+E panel. White dotted line indicates tumor region. B, mean ± SD signal-to-normal brain ratio (SBR) of PpIX fluorescence quantified in intra-tumor and extra-tumor regions of fixed brain slices from GBM-bearing mice (significance determined by unpaired t-test; **p < 0.01). C, representative H+E image of a brain section from a U87 GBM-bearing mouse dosed i.v. with 5-ALA and the substrate-binding peptide; overlaid lines indicate registered regions of tumor (red, H+E) or fluorescence signals (blue, NIRF low resolution Pearl system; green, NIRF high resolution Odyssey scanner; black, PpIX fluorescence) determined in the 1 mm fixed brain slice from which the histology section was obtained. D, mean ± SD 2-dimensional Dice similarity coefficient of each imaging agent relative to tumor region defined by H+E in fixed brain slices from GBM-bearing mice (significance determined by ANOVA followed by Dunn's multiple comparisons test; *, p < 0.05; ***, p < 0.001; ns, not significant).

Figure 4

A cocktail of MMP-14 targeted peptide probes results in tumor localization and correlation of PET and NIRF signals in GBM orthotopic xenografts. A, representative images of segmented, registered brain regions from in vivo T₂-weighted MRI and PET imaging of a GBM-bearing mouse dosed i.v. with [⁶⁴Cu]Cu-binding peptide, substrate peptide, and 5-ALA; NIRF panel is from a 1 mm fixed brain slice imaged ex vivo. B, mean ± SD SUV_mean in tumor and contralateral normal brain regions of mice dosed i.v. with [⁶⁴Cu]Cu-binding peptide; significance determined by paired t-test (**, p < 0.01). C, least-squares fit of linear regression between in vivo [⁶⁴Cu]Cu-binding peptide SUV_mean and ex vivo substrate peptide NIRF MFI in fixed brain slices; dotted lines show the 95% confidence interval of the correlation. a.u., arbitrary units.

Figure 5

PET signals from radiolabeled MMP-14 targeted peptide probes spatially localize within MRI-defined tumor regions in GBM orthotopic xenografts. A-B, representative images of in vivo T₂-weighted MRI, PET/CT, and segmented, registered brain regions from MRI and PET images of GBM-bearing mice dosed i.v. with [⁶⁴Cu]Cu-substrate-binding peptide (A) or [⁶⁴Cu]Cu-binding peptide (B). C, mean ± SD 3-dimensional Dice similarity coefficient of each radiolabeled peptide relative to tumor region defined by T₂-weighted MRI.

Figure 6

MMP-14 substrate-binding peptide shows NIRF signal in residual tumor during mock resection of a GBM orthotopic xenograft. A, pre-resection in vivo T₂-weighted MRI of a of GBM-bearing mouse used for mock resection. B, NIRF/brightfield overlay images of the GBM-bearing mouse head during in situ resection after opening the skull cap prior to removing tumor (Pre-resection) and after resection of the superior xenograft portion (1^st resection). C, NIRF/brightfield overlay images of the extracted whole brain with resected xenograft portions after 1^st and 2^nd resections of xenograft portions containing NIRF signal. D, NIRF/brightfield overlay image of fixed tissue (top panel) and H+E stained section indicating pathological tumor (bottom panel) from the 2^nd resection containing the inferior portion of the GBM orthotopic xenograft. Arrowhead and asterisk across panels indicate superior and inferior portions, respectively, of the GBM orthotopic xenograft.

Figure 7

Mid-resection PET signals from radiolabeled MMP-14 targeted peptide probes were consistent with resected and residual intracranial tumor regions of GBM orthotopic xenografts. A, D, representative in vivo PET/CT, PET/CT with overlaid T₂-weighted MRI segmented brain region (PET/CT/MRI), and PET/CT/MRI with overlaid tumor VOI (red) pre-resection images from GBM-bearing mice dosed i.v. with [⁶⁴Cu]Cu-substrate-binding peptide (A) or [⁶⁴Cu]Cu-binding peptide (D). B, E, representative in situ PET/CT and PET/CT with overlaid tumor VOI (red) post-resection images from GBM-bearing mice dosed i.v. with [⁶⁴Cu]Cu-substrate-binding peptide (B) or [⁶⁴Cu]Cu-binding peptide (E). C, F, mean ± SD percent PET activity in intracranial tumor VOI, contralateral normal brain VOI, and resected tissue VOI at respective pre-resection, mid-resection, and post-resection in situ PET/CT imaging sessions of GBM-bearing mice dosed i.v. with [⁶⁴Cu]Cu-substrate-binding peptide (C, n = 2 mice/group) or [⁶⁴Cu]Cu-binding peptide (F, n = 2 mice/group). VOI, volume of interest.