Abstract and poster under embargo until May 31, 2019

Al-Atrache, Zein

Appelt, Denah

Cader, Ahmad

Georges, Joseph

Ishak, Mark

Lopez, Danielle

Lynagh, Robert

Minissale, Nicholas

Osmolinski, Jonathan

Xin, Sean

Yocom, Steven S.

English

Philadelphia College of Osteopathic Medicine: DigitalCommons@PCOM

Background: Specific brain tumor diagnoses depend on histopathologicinterrogation of biopsies. Though helpful, current histopathologicaltechniques lack the speed and specificity to be effective intraoperativediagnostic tools. Here we show that in vivo imaging of rodents implantedwith human astrocytoma rapidly labeled with the physiological fluorophoresulforhodamine 101 (SR101) can immediately identify an astrocytic tumorfrom normal brain tissue.Methods: Nude rats (n= 4) were stereotactically implanted with culturedhuman astrocytoma cells unilaterally. Five-weeks post implantation, ratswere deeply anesthetized and underwent minimal bilateral craniotomies.SR101 (n=4) was applied subcortically through each craniotomy. An0.85mm diameter fiber optic imaging bundle was attached to a FluoView1000 confocal microscope, then passed through each craniotomy. Imageswere collected from the surface to a depth of five millimeters. Rodentbrains were then extracted for ex vivo imaging. Fixed tissue was imagedfollowing topical application of acridine orange (AO).Results: Areas with tumor burden were immediately identified on thehemisphere implanted with tumor cells with SR101 labeling. MinimalSR101 labeling was appreciated within hemispheres contralateral to tumorimplantation. Counterstaining with AO verified SR101 localized to areasof hypercellular astrocytoma.Conclusion: SR101 rapidly and selectively labeled regions of humanastrocytoma in rodent orthotopic xenografts. Coupled with in vivo imaging,SR101 shows utility for producing biopsy-free specific histologicaldiagnoses. Clinical application of SR101 may provide immediate andspecific intraoperative identification of astrocytic brain tumors.MethodsAbstractIntroductionIntracranial implantation: Nude rats (n=4) were anesthetized andplaced in a small animal stereotactic headframe. A small incision wasmade to expose bregma. A burr hole was made 3.5 mm lateral tobregma. Human astrocytoma cells were infused at a depth of 4.5 mmbelow the surface of the brain, the burr hole was covered with bonewax and skin incision was sutured.Craniotomy: Five-weeks following tumor implantation, animals wereanesthetized and placed in a small animal stereotactic headframe. A3mm diameter burr hole was made over the tumor implantation siteand over contralateral normal brain.Tumor labeling: To specifically label astrocytoma cells, twelvemicroliters of 50µM SR101 was injected at a depth of 3mm from thebrain surface 20 minutes prior to imaging. Acridine orange (5mM) wasapplied topically to ex vivo tissue to nonspecifically label cells in thefield of view for histological tumor verification.Imaging: We constructed our fiber optic microendoscope by couplinga 0.85mm diameter silica-based coherent fiber bundle to an OlympusFluoview 1000 confocal microscope. The pinhole was set to sample atone Airy unit and framesize set to image at Nyquist. AO and SR101were imaged sequentially with 488nm and 560nm excitation,respectively. Imaging began at the brain surface and stepwise with1mm increments to a depth of 5mm. Images were processed withlinear functions utilizing NIH ImageJ.In vivo imaging with SR101 provides rapid andspecific identification of human astrocytictumors in rodent orthotopic xenographs.SR101 is a robust live-cell imaging physiologicfluorophore routinely utilized to specificallyidentify astrocytic cells in live tissue. SR101’sstrong specificity to astrocytoma cells in freshtissue has been previously reported. Coupledwith a minimally invasive fluorescentmicroendoscope, we show SR101 may haveutility for providing immediate biopsy-free invivo histopathological diagnoses.Summary and Conclusions1. Georges, J.F., et al., Sulforhodamine 101 selectively labels humanastrocytoma cells in an animal model of glioblastoma. J ClinNeurosci, 2014. 21(5): p. 846-51.2. Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F.Sulforhodamine101 as a specific marker of astroglia in the neocortex in vivo. NatMethods 2004;1:31-37.3. Xu, C., et al., Experimental and theoretical analysis of core-to-core coupling on fiber bundle imaging. Opt Express, 2008; 16(26):21598-607.ReferencesMinimally Invasive In Vivo Real-Time Identification of Human Astrocytoma with Sulforhodamine 101Mark Ishak, DO1,2; Robert Lynagh, DO1,2; Joseph Georges, DO, PhD1,2; Hany Osman, MD3; Sean Xin, DO1,2; Zein Al-Atrache, BS1; Ahmad Cader1, MS; Danielle Lopez, BS1; Jonathan Osmolinski, MS1; Nicholas Minissale, MS1; Steven Yocom, DO1,2; and Denah Appelt, PhD 1,2Tumor Normal Brain Tumor MarginMinimally Invasive Fiber Optic MicroendoscopeEx Vivo ImagingReal-Time In Vivo ImagingNormal Brain Tumor Margin TumorFigure 1. Minimally invasive imaging. Our microendoscope constructed from a 0.85mm diameter fiber opticbundle has a smaller cross-sectional area than a Camino ICP monitor (1.30mm) and ventricular catheter(3.00mm) which are routinely placed by neurosurgeons at bedside. The microendoscope permitted immediateex vivo imaging of tissue biopsies and in vivo imaging through a miniature craniotomy.Figure 2. Ex vivo Imaging with acridine orange differentiates tumor from normal brain. Acridine orangeimmediately and nonspecifically stained cells within the field of view. Tumor regions were hypercellularcompared to normal brain. Hypercellular and hypocellar tissue regions were visualized at the tumor margin.Figure 3. In vivo Imaging with sulforhodamine 101 specifically labels astrocytoma.Normal brain lacked areas with strong SR101 labeling. Regions with strong SR101 positivity were observedthrough the craniotomy ipsilateral to tumor implantation. SR101 labeling permitted real-time in vivo visualizationof tumor margins (arrows) and regions of frank tumor. Scale bar equals 20µm.In neuro-oncology, specific tumor diagnoses are critical fordevelopment of treatment plans. Though multiple noninvasiveimaging modalities exist, such as MRI, PET and CT scan, directtumor sampling is often required for a definitive diagnosis.Sampling is typically achieved with either open biopsy orstereotactic needle biopsy. In open biopsy, a craniotomy isperformed to directly visualize and biopsy a tumor sample. Withfine needle biopsy (FNB), tumor is sampled through a small burhole. Though less invasive, FNB suffers from greater missamplingrates leading to acquisition of nondiagnositic tissue.Intraoperatively, frozen sections help determine if sampled tissuecontains tumor, necrosis, or normal brain. However, definitive tumordiagnoses require specific stains which take several days toacquire. During this time, treatment plans for patients are delayed,as different pathologies require different treatment plans. Such isthe case with astrocytoma compared to CNS lymphoma. Thesepathologies can appear similar with both noninvasive imaging andon frozen section, yet astrocytoma is best treated with surgicalresection, and lymphoma is best treated nonsurgically withchemoradiation.We propose developing a minimally invasive technique forgenerating rapid and specific in vivo intraoperativehistopathologic diagnoses of astrocytic brain tumors byutilizing a physiologic fluorophore that discriminatesastrocytoma from lymphoma, sulforhodamine 101. Thedevelopment of fiber optic fluorescent imaging devices coupled withrapid and specific fluorescent biomarkers have had widespreadutility in basic neuroscience. In this project, we model andinvestigate clinical application of these techniques on rodentorthotopic xenographs of human astrocytoma. We hypothesize invivo imaging with a miniaturized fluorescent microendoscope willprovide real-time tumor sampling and feedback which will allowimmediate intraoperative identification of these malignant braintumors.Fiber Optic Minimally Invasive Ex Vivo Imaging In Vivo ImagingResults1 Department of Neuroscience, Philadelphia College of Osteopathic Medicine; 2Department of Neurosurgery, Cooper University Hospital; 3Department of Pathology, Massachusetts General Hospital

Minimally Invasive In Vivo Real-Time Identification of Human Astrocytoma with Sulforhodamine 101

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Minimally Invasive In Vivo Real-Time Identification of Human Astrocytoma with Sulforhodamine 101

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