1.18 Automated Classification of Glioblastoma Margins in Label-Free SRS Microscopy Images

Posted on December 22, 2014

S. B. Lewis2, M. Ji3, X. S. Xie3, D. A. Orringer1  3Harvard University,Department Of Chemistry And Chemical Biology,Cambridge, MA, USA 1University Of Michigan Health System,Neurosurgery,Ann Arbor, MI, USA 2University Of Michigan Medical School,Ann Arbor, MI, USA

Introduction: Glioblastoma (GBM) is the most common and most aggressive form of intrinsic intracranial malignancy. Its surgical treatment is complicated by its capacity to infiltrate the surrounding parenchyma, with biopsies of ostensibly-normal tumor margin revealing large numbers of tumor cells after H&E staining. Stimulated Raman spectroscopy (SRS) microscopy is a label-free technique which can deliniate fine structure in fresh tissue, including nuclei and axons, using relative abundances of lipid and protein as contrast. Here we have derrved a program which automatically quantifies the cellularity, axonal density, and overall lipid:protein ratio, and classifies the sample as normal brain, infiltrating tumor or dense tumor. 

Methods: Two normal and two mouse xenograft models of GBM were imaged with SRS microscopy, at 2930 and 2845 cm-1 (the CH3 and CH2 Raman peaks, respectively). 1682 regions of interest were collected from these four brains and processed in Matlab using the Image Processing Toolbox. Briefly, nuclei were detected in the CH3 channel using the H-maxima transform and classified according to their compactness and intensity. Axons (in the CH2 channel) were hilighted via convolution of the image with the Sobel edge kernel and solidified via image closure; thresholding of the result was conducted via Otsu's method and candidate blobs were sorted by eccentricity and Euler number. Discriminant analysis was then used to classify each region of interest as "normal," "marginal" or "dense core". 

Results: The model differentiated normal tissue from dense core with 100% sensitivity and 100% specificity. When challenged to distinguish between normal tissue, marginal samples and dense core, abnormal tissue was detected with and 95.02% sensitivity 100% specificity. 

Conclusion: This model holds promise for the rapid and automated differentiation of normal and tumorous tissue during resection of malignant brain neoplasms, even when such tissue is not distinguishable by eye. This distinction is apparent without labels, staining, or fixation. Furthermore, aspects of both this technology and this algorithm may be applicable to the microscale segmentation of many tumors whose margins are unclear, or whose complete resection is imperative. 

 

1.19 ALDH marks a population of canine cancer stem cells which are preferentially targeted by dog NK cells

Posted on December 22, 2014

R. J. Canter5,6, E. Ames6, S. Mac6, S. Grossenbacher6, M. Kent3, W. Culp3, M. Chen4, W. J. Murphy6  3UC Davis School Of Veterinary Medicine,Surgical And Radiological Sciences,Davis, CA, USA 4University Of California – Davis,Pathology And Laboratory Medicine,Sacramento, CA, USA 5University Of California – Davis,Surgery/Surgical Oncology,Sacramento, CA, USA 6University Of California – Davis,Laboratory Of Cancer Immunology,Sacramento, CA, USA

Introduction: Aldehyde dehydrogenase (ALDH) is a common cancer stem cell (CSC) marker in diverse solid human tumors. We hypothesized that ALDHbright cells would demonstrate the CSC phenotype in dog soft tissue sarcomas (STS) and that these dog CSCs could be preferentially targeted by dog NK cells.  

Methods: ALDHbright cell populations from canine tumor lines and fresh canine primary STS were evaluated for long term colony outgrowth and their ability to form tumors in NOD-SCID IL2 receptor gamma chain null (NSG). STS tissue was obtained from primary dog STS samples and canine patient-derived xenografts (PDX) and evaluated by immunohistochemy (IHC) and flow cytometry for CSC markers including CD24, CD44, and ALDH.  Stained slides were reviewed by a blinded pathologist and scored for percentage and intensity of ALDH-positive cells. Flow cytometry was performed using a BD Fortessa cell sorter (BD Biosciences), and cell viability was analyzed using 7-Aminoactinomycin (7-AAD). Dog NK cells were isolated from leukocyte filters obtained from healthy donors at the School of Veterinary Medicine. NK cytotoxicity was assessed by chromium release and flow cytometry. Parametric and non-parametric statistical tests were performed as appropriate.
 

Results: ALDHbright canine tumor cells displayed properties of CSCs, including selective tumor formation in NSG mice after cell sorting into ALDHbright and ALDHdim populations and long term colony outgrowth in methylcellulose. Using positive selection with magnetic beads, we observed that canine NK cells are responsive to human cytokines, including IL-2, IL-12, and IL-18 with a 3 – 10-fold expansion in NK cells over 14 days. Ex vivo activated dog NK cells demonstrated 35 – 45% cytotoxicity and 57 – 62% cytotoxicity against dissociated dog STS tumors at effector-to-target ratios of 10:1 and 20:1, respectively. IHC staining of dog PDX specimens showed a marked reduction in ALDH score (P<0.05) after intra-tumoral injection of allogeneic dog NK cells compared to controls.

Conclusion: ALDHbright cells exhibit CSC properties in dog STS, and dog NK cells appear to possess an intrinsic ability to recognize and target them. Dog STS appear to be a valuable model to facilitate clinical translation of NK immunotherapy and targeting of CSCs.

1.20 Gene silencing of SphK1 with nanoparticles as an innovative approach against cancer progression

Posted on December 22, 2014

I. Woelfel1, K. P. Terracina3, S. Lima5, C. Oyeniran5, J. Newton5, H. Aoki3, D. Avni5, P. Mukhopadhyay3, N. Hait5, A. Raza3, X. Wu4, H. Yamamoto4, S. Spiegel5, K. Takabe2,3,5  1Virginia Commonwealth University,School Of Medicine,Richmond, VA, USA 2VCU Massey Cancer Center,Richmond, VA, USA 3Virginia Commonwealth University,Department Of Surgery,Richmond, VA, USA 4Osaka University,Suita, Osaka, Japan 5Virginia Commonwealth University,Department Of Biochemistry And Molecular Biology,Richmond, VA, USA

Introduction:  

Gene therapy as an effective treatment modality for cancer has been sought for decades. Its full therapeutic potential has not been realized due to many barriers, including efficiency and cost. Small interfering RNA (siRNA) has emerged as a successful technology for specifically silencing gene expression in vitro. However, due to the small size and delicate nature of these molecules, an effective delivery system that allows these molecules to reach cancer and thus be applicable in patient treatment is necessary. Recent innovations in nanoparticle technology have enabled the development of a new delivery system: super carbonate apatite (sCA). sCA system is extremely inexpensive, and has been reported to deliver genes specifically to cancer due to the characteristic size and leakiness of peritumoral vessels. Sphingosine-1-phosphate, generated by Sphingosine kinase 1 (SphK1), has been established as a key lipid signaling molecule in cancer progression and is integral to cell survival, proliferation, migration, angiogenesis, and lymphangiogenesis. We investigated the ability of this new nanoparticle delivery system (sCA) with SphK1 siRNA to effectively knockdown SphK1 and suppress its physiological functions in vitro.

Methods:

A carbonate apatite inorganic nanoparticle delivery solution was created using CaCl2 and NaCO3 ions. 4T1 murine breast cancer cells were treated with 2 ug/mL of SphK1 siRNA, Non-targeting siRNA or a control containing no siRNA using the inorganic solution with a 4-hour serum free incubation time. At 4 hours 1 mL of 10% FBS media was added. 24 hours after the initial application of inorganic solution the old media was removed and 2 mL of DMEM with 10% FBS was added.  mRNA was harvested at the 48 hour time for qPCR. Cell survival was measured using WST-8 assay. To study the effects of silencing on cell migration, a point scratch assay was conducted using established technique with images collected at 0, 15 and 24 hours. Analysis of the images was conducted using Image J. 

Results:

We successfully introduced SphK1 siRNA into the 4T1 cells using sCA system, demonstrated through qPCR with a statistically significant reduction of SphK1 message production (67%, p value 0.008). We found that sCA transfection of SphK1 siRNA significantly decreased 4T1 cell survival compared to vector treated cells. We also observed faster closure of scratch area in cells treated with a non-targeting vector, as opposed to cells treated with SphK1 siRNA. 

Conclusion:

We have shown that our new inorganic nanoparticle delivery system, sCA, was successful in gene silencing of SphK1 in vitro and suppressed 4T1 cell proliferation and migration. Considering the advantage of sCA in gene delivery to cancer, we have high expectations regarding the application of this approach in vivo, which has the potential to transform the treatment frontier and to positively impact patient outcomes in the future. 

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