Abstract 628: A method to examine spatial relationships between tumor cells and vasculature using a mouse orthotopic PDX glioblastoma model
Authors: Nagaraja Tavarekere N. , Keenan Kelly , Irtenkauf Susan , Hasselbach Laura , Panda Swayamprava , Cabral Glauber , Ewing James R. , Mikkelsen Tom , deCarvalho. Ana 
Published: July 2016

Abstract
Introduction: Glioblastoma progression requires angiogenesis and/or vascular co-option. A major pathway for its infiltration is the perivascular space. We present herein a method to visualize glioblastoma cells with respect to tumor-associated vasculature within and beyond the tumor mass.
Experimental procedures: A neurosphere cell line, HF3253, developed from cultures of a human glioblastoma biopsy sample was labeled with PKH26, a red fluorescent dye, using a commercially available kit (Sigma-Aldrich). The labeled cells were implanted into the right striatum in immunocompromised nude mice (n = 5) and allowed to grow for 2-4 weeks. Terminally (one mouse at 2, two at 3 and the remaining two at 4 weeks), the mice were intravenously injected with fluorescent isothiocyanate (FITC) dextran of 70 kDa molecular weight. The brains were removed after 5 min of dextran circulation, immersion fixed in 10% neutral buffered formalin for 2 days and then sectioned into 100 μm thick coronal slices using a vibratome. Slices were stained with the nuclear counterstain 4’,6-diamidino-2-phenylindole (DAPI) to visualize native mouse brain cells at 405 nm in an Olympus FV1200 laser scanning confocal microscope. Relative distributions of the dextran and PKH26 fluorochromes were also examined using the same microscope. FITC-dextran (green fluorochrome)-labeled microvessels and PKH26-labeled tumor cells (red fluorochrome) were excited by a laser beam at 488 and 568 nm, respectively, and emissions were detected with a photomultiplier tube through 522 and 585 nm emission filters, respectively.
Results: The normal vasculature on the contralateral side appeared well perfused, filled with the green dextran fluorescence. Intricate, fine networks of microvessels were present. The ipsilateral hemisphere showed bright red spots of tumor cells in the striatum in closely distributed groups among the blue DAPI-stained mouse cells. Compared to contralateral side, the vasculature associated with such groups was dilated and irregular, with fewer interconnections. Interestingly, these irregular networks were also observed away from the main tumor cell mass in 3- and 4-week-old tumors, suggesting probable future infiltrative pathways. Red-labeled single cells and groups of tumor cells could be seen along vascular branches penetrating brain tissue adjacent to the tumor. The strong fluorescence of the tumor cell tracer PKH26 persisted over the 4 week experimental period, suggesting its utility in imaging tumor infiltration in this model.
Conclusions: This technique generated high resolution images of tumor cell distribution and associated microvasculature in an orthotopic glioblastoma model. With further quantification, it can help in establishing infiltrative patterns of different PDX tumor models, and may also be useful to measure discrete responses to therapies that affect tumor cells and/or vasculature.
Citation Format: Tavarekere N. Nagaraja, Kelly Keenan, Susan Irtenkauf, Laura Hasselbach, Swayamprava Panda, Glauber Cabral, James R. Ewing, Tom Mikkelsen, Ana deCarvalho. A method to examine spatial relationships between tumor cells and vasculature using a mouse orthotopic PDX glioblastoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 628.