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Induced Myeloperoxidase Activity in Ovarian Cancer Mouse Model.Rao V. L. Papineni*, Gil Mor#, William McLaughlin, Douglas Vizard, Jennie Holmberg#, Vinicius Craveiro#, and Robert Brandau.Carestream Molecular Imaging, 4 Research Dr., Woodbridge, CT, 06525, USA and #Department of Obstetrics, Gynecology & Reproductive Sciences, Yale School of Medicine, Yale University.* rao.papineni1@carestreamhealth.com

AbstractPromotion of neutrophil-mediated inflammation at the tumor enhances the anti-tumor immune response. Cellular Myeloperoxidase (MPO) is involved in the programmed cell death in human leukemic cells. MPO is an inflammatory heme protein present in myeloid cells- neutrophils, microglia, and macrophages. Here we investigated the possibilities of nutri-pharmacological induction in MPO activity in tumor mouse model and monitor the changes non-invasively. Myeloperoxidase activity in vivo is detected by i.p injection of Luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione). Luminol a redox-sensitive compound emits blue luminescence upon exposure to oxidizing agents, and in vivo, and has been established to have unique specificity to MPO activity resultant of myeloid cell activation. We monitored real-time in vivo MPO activity in an intraperitoneal ovarian cancer tumor mouse model using tri-modal non invasive imaging. Fluorescence, luminescence, and X-ray images were obtained at different time points to determine both the progression of the intraperitoneal tumor metastasis (fluorescence) and the MPO activity (lumiscence). We show enhancement in MPO activity by administration of (i.p injection) a single dose of 0.1 ml (50% volume/volume) of nutriceuticals (BV9) using the multimodal non invasive imaging approach. The results show that basal MPO activity signal intensities were not uniform between the tumors, and in some tumors, the MPO activity was completely absent, as observed from the lack of co-registration of the fluorescence (Tumor) and the luminescence (MPO activity) signals. This indicates that a differential MPO related tumor microenvironment prevails within the mouse. BV9 elicited a robust increase in the MPO activity within 3 hrs of its administration and the luminescence signals co localized well with the fluorescence signals corresponding to that of the tumors. Further investigations assessing the role of such elicited MPO activity in ovarian cancer progression will greatly enhance the understanding of the tumor microenvironment and contribute significantly in the development of better cancer treatments.

Conclusions

Early inflammatory response during ovarian cancer tumorigenesis data show that certain sites of robust MPO activity likely prime for a rapid growth of secondary tumor at the region. Nutriceuticals like BV9 induces neutrophil activation- it is essential

to evaluate if such induction has anti-tumoral activity (therapeutic inflammation). The following non invasive methodologies can be adapted in the

development of novel therapeutics and better cancer treatments.

"Molecular Imaging - Wisdom To See For Maladies To Flee"Dr. Rao V. L. Papineni

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Dr .Rao Papineni

Disclaimer. Carestream’s pre-clinical imaging systems are not licensed to perform certain optical imaging applications that involve the in vivo imaging in mammals of (i) genetically expressed bioluminescent or fluorescent protein or (ii) conjugates of cells and light generating molecules, such applications are covered by patents owned or controlled by Caliper Life Sciences, Inc. Such patents include the following: U.S. Patents Nos. 5,650,135; 6,217,847; 7,198,774; 6,649,143; 6,939,533; 6,916,462; 6,923,951; 6,890,515; 6,908,605; 5,824,468; 6,638,752; 6,737,245 and 6,867,348; U.S. Patent Application No. 11/818,208; European Patent No. 0861093 and European Patent Application No. 991246406; Japanese Patent Nos. 3786704 and 3786903; Canadian Patent No. 2237983; Singapore Patent No. 53708; Hong Kong Patent No. 1018747; and Chinese Patent No. 951980068.

Ovarian Cancer Model and in vivo Imaging of Myeloperoxidase ActivityImaging Early Inflammation ResponseOvarian cancer stem-like cells (OCSCs) were labeled with lipophilic carbocyanine DiR fluorescent dye (Trademark of Invitrogen, NY, USA). The cells were washed thrice and were injected intra-peritoneal (i.p.) into athymic nude mice. Tumorigenesis was monitored from different angles by automatic rotation Multimodal Animal Rotation System (MARS) as shown in Figure-1. The fluorescence and x-ray images obtained at different angles are overlaid for anatomical co registration of primary and secondary tumors in the peritoneal region Figure -2.

Figure-2: Multimodal non invasive rotational analysis of ovarian cancer tumor growth. Fluorescence images from eighteen different angles overlaid over their respective x-ray images show the various locations of the primary and secondary tumors. This cancer model system was qualified for further early immune response studies.

0° 20° 40° 60° 80° 100° 120° 140° 160°

180° 200° 220° 240° 260° 280° 300° 320° 340°

Figure-1.TORSIONAL SUPPORT APPARATUS AND METHOD FOR CRANIOCAUDAL ROTATION OF ANIMALSPatent application number: 20100022866

Gilbert Feke Rao Papineni et al

The in vivo myeloperoxidase (MPO) activity during the early tumorigenesis was determined by injecting luminol substrate (i.p. 100 ml of 5 mg/ml luminol, Sigma- Aldrich MO, USA). The MPO activity responsive luminescence was imaged using the development phase imaging system with a cooled CCD Camera along with the X-ray contrast and fluorescence images of the subject.

7 Day- Post tumor

X-ray MPO Luminescence Overlay Tumor (Fluor) MPO, Tumor overlay

3 hours Post BV9

X-ray MPO Luminescence Overlay Tumor (Fluor) MPO, Tumor overlay

Control miceTumor mice

X-ray DIR MPO OverlayFluorescence Luminescence Trimodal

Induction of Neutrophil Activation

Figure-3: Myeloperoxidase activity does not co localize with all the ovarian cancer tumors. Luminescence signals that result from the myeloperoxidase (MPO) activity and viz. the activation of the neutrophils were determined and shown in the left panels (mouse with tumor) and the control mice (right panels). As seen in the encircled regions (white dotted circles), certain tumors have a putative neutrophil rich microenvironment that show MPO activity. While, certain tumors (Boxed in dotted while line) have nearly no MPO activity.

The trimodal (Luminescence (Red), fluorescence (green), and X-ray) imaging parameters are denoted below:

Iodixanol – Bladder contrast

Tumor mice

Control mice

X-ray MPO Luminescence Overlay Tumor (Fluor) MPO, Tumor overlay

X-ray MPO Luminescence Overlay Tumor (Fluor) MPO, Tumor overlay

3 hours Post BV9

Fluorescence Imaging: Tumor (green)

Exposure Type: StandardExposure Time: 60.000 sec.Exposure: 1 of 1X-binning: NoY-binning: Nof-Stop: 0.95FOV: 135.0 mmFocal Plane: 11.2 mmVertical Resolution: 300 ppiHorizontal Resolution: 300 ppiExcitation Filter Description: 550Emission Filter Description: 600

X-ray Imaging:

Exposure Type: StandardExposure Time: 2.500 min.Exposure: 1 of 1X-binning: NoY-binning: NoIllumination Source: X-Rayf-Stop: 5.60FOV: 135.0 mmFocal Plane: 11.2 mmVertical Resolution: 385 ppiHorizontal Resolution: 385 ppiIllumination Correction: YesPixel Saturation Threshold: 65535X-Ray Serial Number: 89X-Ray Energy: 17 KVPX-Ray Current: 150 uAX-Ray Filter: 0.2 mm

Figure 4: Robust increase in myeloperoxidase activity induced by BV9 nutriceutical compound. Significant increase in the luminescence signals resulting from the myeloperoxidase activity (color) were observed (Fig. 4a, b,& c). The ovarian tumor mouse model (Fig. 4b) and the control mice (Fig. 4c) show neutrophil activation within 3 hours of exposure to the BV9 nutriceutical agent.

Fig 4a

Fig 4c

Fig 4b

Mice #1 Mice #2

Is this induction of Neutrophil activation a part of the “therapeutic inflammation”.

Myeloperoxidase Activity During Early Ovarian Cancer Tumorigenesis

Presented at EB 2012

Presented at EB 2012

Presented at EB 2012

Presented at EB 2012