Novel target for peptide-based imaging and treatment of brain tumors

Abstract

Malignant gliomas are associated with high mortality due to infiltrative growth, recurrence, and malignant progression. Even with the most efficient therapy combinations, median survival of the glioblastoma multiforme (grade 4) patients is less than 15 months. Therefore, new treatment approaches are urgently needed. We describe here identification of a novel homing peptide that recognizes tumor vessels and invasive tumor satellites in glioblastomas. We demonstrate successful brain tumor imaging using radiolabeled peptide in whole-body SPECT/CT imaging. Peptide-targeted delivery of chemotherapeutics prolonged the lifespan of mice bearing invasive brain tumors and significantly reduced the number of tumor satellites compared with the free drug. Moreover, we identified mammary-derived growth inhibitor (MDGI/H-FABP/FABP3) as the interacting partner for our peptide on brain tumor tissue. MDGI was expressed in human brain tumor specimens in a grade-dependent manner and its expression positively correlated with the histologic grade of the tumor, suggesting MDGI as a novel marker for malignant gliomas.

Figure 1

Identification of the CooP peptide that homes to a subset of brain tumors. A, phage screen comprising two ex vivo (Ev) rounds performed by incubating phage library with i.c. HIFko tumor derived cell suspension followed by three in vivo (Iv) rounds with intravenously injected phage pool into the mice bearing intracranial HIFko tumors. Phage enrichment is shown as fold increase over the control phage. B, individual phage from the third in vivo selection round were tested for the ex vivo binding to the cell suspension derived from tumor containing brain and normal brain. Graph shows phage binding to the tumor brain relative to the normal brain. C, homing of the CooP phage to brain tumor, histologically normal brain, liver, kidney, and lung tissue compared to the control phage. Values represent a mean of three experiments. Error bars indicate ±SEM. D-F, fluorescein-conjugated CooP peptide (100 µM, 100 µl/animal) was injected into the tail vein of mice bearing intracranial tumors and allowed to circulate for 60 min followed by perfusion of the mice through the right ventricle of the heart and excision of the tumor. Staining of the tumor sections with an anti-fluorescein antibody (red in E) shows accumulation of fluorescein-conjugated CooP in HIFko clusters identified by staining of an adjacent tissue section with an antibody against the SV40 large T antibody (green in F), a marker for tumor cells. Homing of the fluorescein-conjugated CooP peptide (red) to intracranial U87MG (G) and BT4C (H) gliomas. Absence of CooP homing to subcutaneous MDA-MB-231 breast tumors (I), to subcutaneous HIFko tumors (J), and to the VEGF overexpressing i.c. tumors (K). G-K, sections were stained with an anti-PECAM1 antibody (green) to visualize tumor vasculature. D, G-K, nuclei were visualized with DAPI (blue). Magnification 200×. Images were digitally cropped in Photoshop CS6.

Figure 2

CooP peptide co-localizes with MDGI in gliomas and MDGI overexpression increases CooP homing. Fluorescein-conjugated CooP peptide (100 µM, 100 µl/animal) was injected intravenously into mice bearing intracranial HIFko (A-B) or U87MG (C-F) gliomas. CooP peptide and MDGI were visualized using antibodies against fluorescein (red in panels A, C, E-F) and MDGI (green in panels B and D). Overexpression of MDGI in the U87MG gliomas (F) increased homing of the CooP peptide compared to the control U87MG tumors (E). Nuclei were visualized with DAPI. Magnification 400×. Images were digitally cropped in Photoshop CS6.

Figure 3

MDGI is accessible via circulation in the tumor vasculature. To assess whether MDGI was accessible via circulation we injected the goat anti-MDGI (A-D) or control goat IgG (E-H) antibodies into the tail vein of intracranial U87MG tumor-bearing mice. Tumors and organs were removed one hour post-injection, prepared for histological analyses and examined for the presence of antibodies using anti-goat Alexa 594 (red in panels A-H). Blood vessels were stained with anti-PECAM1 antibody (green in panels A-H). Injected anti-MDGI antibody colocalized with blood vessels only in tumors (A) whereas no control IgG could be detected in tumors (E). No anti-MDGI antibody or control IgG was detected in other tissues examined: muscle (B and F), heart (C and G) or brain (D and H). To study the in situ expression of MDGI in tumors and other organs we stained tissue sections with the goat anti-MDGI antibody followed by the anti-goat Alexa 594 (red in I-L). Fluorescein-conjugated tomato lectin (green in I-L) was injected intravenously to visualize the vasculature. MDGI colocalized with the vascular staining only in tumors (I) but not in muscle (J), heart (K), or brain (L). Nuclei were visualized with DAPI (blue). Magnification 400×. Images were digitally cropped in Photoshop CS6.

Figure 4

Peptide-targeted SPECT/CT imaging of gliomas. A, majority of the intravenously injected peptide (20 µg in 100 µl) was rapidly excreted through the kidneys into urine. Two hours post-injection accumulation of the ¹¹¹In-conjugated CooP peptide was detectable in brain tumors (B and D) while no control peptide could be detected (C and D). E, accumulation of 5 MBq ¹¹¹In-CooP in the tumor half and the healthy half of the brain in same animals. F, biodistribution of ¹¹¹In-CooP or control peptide two hours post-injection. Error bars indicate ±SD.

Figure 5

Peptide-targeted delivery of therapeutics prolongs the life-span of mice bearing intracranial HIFko tumors. A, internalization of fluorescein-labeled CooP, CPP or Coop-CPP peptides. Fluorescence was measured at 494/518 nm after 1 h incubation of HIFko cells with 5 µM fluorescein-labeled peptides. Values represent the mean of 4 replicates in one representative experiment. Error bars indicate ±SD. B, HIFko cells were injected intracranially into nude mice. Treatment was started after tumor establishment at day 5 post-implantation (arrow). Mice were treated with intravenous injections of either saline (PBS), free Cbl or targeted drug (CooP-CPP-Cbl) every second day. Weight of the animals was monitored. Experiment was stopped when criteria for sacrification was met (>10% weight loss). Graph shows the normalized, mean body weight in each group during the experiment (±SEM). The mean weight on day 0 was set as 100%. C, brain sections of the treated mice were imaged and brain area occupied by tumor satellites was quantified from four sections/animal. Graph shows percentage of brain occupied by the tumor satellites (±SEM). D, brain sections of the treated mice were imaged and brain area occupied by the primary tumors was quantified from four sections/animal. Graph shows percentage of brain occupied by the primary tumors (±SEM). * p ≤ 0.05 (Student’s T-test).

Figure 6

MDGI is expressed in tumor cells and in blood endothelial cells, and its expression positively correlates with tumor grade in clinical human brain tumor samples. MDGI expression was studied in sections from normal brain (A), grade I (B), grade II (C), grade III (D) astrocytomas, as well as in primary (E, I-P) and secondary (F) grade IV glioblastomas, in ependymomas (G), and in medulloblastomas (H) with anti-MDGI antibodies using immunohistochemical stainings. MDGI and PECAM-1 are shown as red color and nuclei in blue. I, MDGI expression in tumor cells. J, subsequent section stained with anti-PECAM-1 antibodies. K and L, specificity of the MDGI staining was shown by using rabbit IgG staining as a control for panels I and M. M and P, MDGI expression in PECAM-1 positive endothelial cells. N, higher magnification of the boxed area in M. O, higher magnification of the boxed area in P. Q, human glioblastoma (grade IV) sections were stained with fluorescently-labeled antibodies against MDGI (red) and PECAM-1 (green). Nuclei were visualized with DAPI (blue). Insert: higher magnification of the boxed area showing that MDGI and PECAM-1 are present in the same cells. Magnification A-K 400×, M-Q 200×. Images were digitally cropped in Photoshop CS6.