Student Projects 2011

Please see below for projects by SUPERS students in 2011.

Role of Pattern Recognition Receptors in Breast Cancer Metastasis

Titas Banerjee

Mentored by Dr. Andy Minn 

Hypothesis: Pattern recognition receptors (PRRs) lead to increased metastasis in breast cancer cells by enhancing inflammatory and metastasis genes.

Techniques

  • Cell Lines 231, 4175, 1833.
  • RNA Extraction.
  • Invasion Array.
  • Cell Culture.
  • Gene Silencing with siRNA.
  • Quantitative Reverse Transcriptase PCR.

Conclusions

  • PRRs play a significant role in invasion for certain metastatic breast cancer cell lines.
  • PRRs seem to regulate many known metastatic genes.

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Creating Stable Cell Lines of Inducible ATF4 and GCN2 Knockdowns

Shire Beach

Mentored by Dr. Costas Koumenis 

Goal: To create stable cell lines of inducible ATF4 and GCN2 knockdowns. These knockdowns will be used in sutdies which aim to support the hypothesis that ATF4 and GCN2 promote tumor growth.

Techniques

  • Maxi/Minipreps.
  • Tissue Culture and Transfections
  • Western Blot
  • Fluorescence Microscopy
  • FACS. 

Conclusions

The inducible knockdown constructs were transfected in to several cell lines (HT1080, B16F10) and some stable clones were obtained. Further analysis however is required to complete the goals of this project.

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Quantitative Analyses of an Orthotopic Model of GBM: Evaluation of the Bioluminescence to Tumor Volume Relationship, Radiation-Induced BBB Disruption, and Enhancement of Nanocarrier Extravasation after Radiation

Joe Benci

mentored by Dr. Jay Dorsey and Dr. Gary Kao 

Hypotheses In a mouse model of GBM, the volume of intercranial tumors determined by MRI would correlate well with the BLI data from the IVIS imaging system. Secondly, a commercially availabel infrared dye, IRDye 800CW PEG contrast agent would be an effective indicator of blood brain barrier (BBB) and blood tumor barrier (BTB) disruption by ionizing radiation in vivo. If so, this would allow for tracking the short and long-term effects of BBB and BTB disruption in response to ionizing radiation. Third, that irradiation of an orthotopic GBM tumor would lead to an increased tumor uptake of novel worm-shaped nanocarriers which were visualized and quantified by loading with a fluorescent dye encapsulation.

Techniques 

  • Stereotaxic Intercranial Injections.
  • Small Animal Magnetic Responance Imaging (MRI).
  • Bioluminescent Imaging (BLI).
  • Fluorescent Imaging. 
  • Amira. 
  • Cell Culture. 

Conclusions

  • The signal from BLI was a very accurate predictor of tumor volume up to a certain signal threshold at which the bioluminescence peaked but volumes continued to increase (likely due to a variety of factors including necrosis).
  • Increased extravasation of the IRDye was present in the brains of irradiated mice which suggested blood brain barrier disruption but more expierments are needed to confirm this hypothesis.
  • The worm-shaped nanocarrier was able to more effectively permeate the tumor tissue after irradiation and was retained there longer, suggesting this method of drug delivery, given in conjunction with ionizing radiation could be an effective method for treating glioblastoma in the clinic in the future.

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Using γH2AX to Measure the Oxygen Levels in Tumor Cells

Linda Ezidiegwu

mentored by Dr. Sydney Evans and Dr. Cameraon Koch. 

Hypothesis: The amount of oxygen present at the time of irradiation will be directly proportional to the number of γH2AX foci formed.

Techniques 

  • Cell Culture.
  • Immunohistochemistry.
  • Amido Black Assay.
  • Western Blot.

Conclusions

  • Thus far we have not been able to show that the number of γH2AX foci observed are directly proportional to O2concentration. However, an O2 effect has been established.

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The Effects of Acute Space Radiation on the Skin's Vasculature

Gabrielle James

mentored by Dr. Ann Kennedy and Dr. Jenine Sanzari. 

Hypothesis: After irradiation similar to that of a solar particle event, skin toxicity will be observed and the vasculature will be altered.

Techniques 

  • Large Animal Procedures.
  • Immunohistochemistry.
  • Microscopy.

Conclusions
The number of blood vessels in the dermis and the surface area of blood vessels decrease fourteen days after electron radiation exposure. Although statistical analyses result in no statistically significant difference between the pre-irradiated values and each time point (7, 14, and 30 days) evaluated, a trend is observed. Further experiments are necessary to confirm these findings.

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Radioprotection of Curcumin via the NRF2 Pathway

Charanya Kaushik

mentored by Dr. Stephen Tuttle 

Hypothesis: NRF2 is required for curcumin-induced radioprotection.

Techniques 

  • Cell Culture.
  • Transfection.
  • Western Blot.
  • Quantitative Reverse Transcriptase PCR.
  • Immunohistochemistry.

Conclusions
We demonstrated upregulation of inflammatory response genes (interleukin family, chemokines, tumor necrosis factor family) with radiation is reduced with curcumin diet compared to radiation alone.

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Going Big to Small: How do Macroscopic Measurements of Oxygen and Photosensitizer Concentrations in a Tumor Relate to the Actual Tumor Microenvironment?

Erin Kennedy

mentored by Dr. Theresa Busch and Dr. Shannon Gallagher-Colombo 

Goal: To determine how macroscopic spectroscopy-based measurements of oxygen and photosensitizer distributions within a tumor compare to the microscopic distribution of these parameters as determined by immunohistochemistry. We hypothesize that regional maxima and minima in oxygen and photosensitizer distributions detected by spectroscopy will be sparially representative of their actual microscopic distributions.

Techniques 

  • Cryosectioning.
  • Immunohistochemistry.
  • Fluorescence Microscopy.
  • Image Analysis Software (ImagePro, Photoshop, MATLAB) and Excel.

Conclusions

The macroscopic measurements aren't perfect, but are still helpful. All the subtle differences in photosensitizer and oxygen ditributions aren't observed, but a general idea of what is present is better than nothing. By comparing the macroscopic data to the microscopic data in the same tumor, we can determine the extent to which signals are being averaged macroscopically.

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Enzyme that Metabolizes EF5: CYPOR?

Mailene King

mentored by Dr. Sydney Evans. 

Hypothesis: Measuring CYPOR will provide the same information acquired through EF5 cube reference binding, enabling the determination of the maximum binding of EF5.

Techniques 

  • Cell Culture.
  • Immunohistochemistry.
  • Amido Black Assay.
  • Western Blot.

Conclusions

Cypor is localized in the cytoplasm. Positive correlation between CYPOR counts and EF5 adducts.

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Evaluating the Therapeutic Potential of MIBG and MABG for Pheochromocytoma

John Mikitsh

mentored by Dr. Dan Pryma, Dr. Ann-Marie Chacko, and Catherine Hou. 

Goals 

  • To radiolabel [125I]MIBG in high purity.
  • To assess the uptake of [125I]MIBG in a pheo model.
  • To assess the therapeutic efficacy of [125I]MIBG in vitro and in vivo.
  • Repeat the above with [211At]MABG as an α-therapeutic.

Techniques 

  • Radiolabeled MIBG using the ultratrace Resin and confirmed purity via TLC, Digital Autoradiography
  • and Phosphorimaging.
  • Performed cell-based radioimmuno assays with [125I/131I]MIBG and MPC 4/30/PRR cells.
  • Performed biodistributions for various studies with mice. 

Conclusions

  • [125I]MIBG can be labeled in high purity. 
  • The MPC 4/30/PRR cell line takes up [125I]MIBG, but the process needs to be optimized.
  • Ultimately, [131I]MIBG and [211At]MABG should prove to be a novel therapy for both pheochromocytoma and neuroblastoma.

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Curcumin Chemosensitization in Head and Neck Cancer Cells Sarah Porter

Sarah Porter

mentored by Dr. Stephen Tuttle. 

Hypothesis
Curcumin enhances cisplatin toxicity via covalent binding to the penultimate celenocysteine residue of thioredoxin reductase 1.

Techniques 

  • Modified MTT Assay
  • Western Blot
  • Clonogenic Assay
  • Cell Culture

Conclusions

There is data that suggests that curcumin chemosensitizes head and neck cancer cells; however, more experiments and statistical analysis must be performed.

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Improving Accuracy in Imaging and Treating Tumors

Caitlyn Riehl

mentored by Dr. Gary Kao. 

Goals and Hypotheses 

  • Use tattooing in a safe and effective manner. 
  • Utilize the imaging techniques we have to more accurately treat the mice. 
  • Ionizing radiation will cause collapse, less perfusion and shrinkage in the vasculature of tumors. 
  • U251-GFP-LUC (non-HIC) and U251-GFP-LUC-HIC cell lines, although originated from the same cell lines, have very different properties and will behave differently within the mouse model. 

 

Techniques 

  • Cell Culture.
  • Subcutaneous Tumor Injection.
  • Bioluminescent Imaging.
  • CT Scans. 
  • Fluorescent Imaging using a PEGylated Dye. 
  • Measuring Tumors using Electronic Calipers. 
  • Small Animal Tattooing. 
  • Immunohistochemistry. 
  • Use of the SARRP. 

Conclusions

  • Tattooing is a safe and durable way to identify nude mice and mark treatment sites.
  • The U251-GFP-LUC-HIC cell line has bioluminescent signal, growth rates and vascular contrast uptake patterns that seem surprisingly distinct from its parental cell line.
  • Imaging using CT contrast in mice is very possible and can help in giving more effective treatment, but needs to be explored more.
  • CD31 expression/vascular marker patterns may differ between tumors grown from different cell lines and possibly after irradiation. 
  • These techniques collectively may improve the imaging and treatment of tumors grown in mice. 

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In Vivo Profiling of Hypoxic miRNA in 9L Gliosarcoma Tumors Using the Hypoxia Marker EF5 and Laser-Capture Microdissection

James Ruggero

mentored by Dr. Amit Maity and Dr. Costas Koumenis 

Goal: Create a miRNA profile, that can be used in conjuction with a previously generated messenger RNA profile, to study the mechanisms that protect cancer cells in a hypoxic state, as well as allow them to proliferate, invade and metastasize.

Techniques 

  • Laser Capture Microdissection.
  • NanoDrop Analysis for RNA Concentration.
  • RNA Pico Card Analysis for RNA Degredation
  • In Vivo Profiling using Taqman® Array miRNA cars for real time PCR.

Conclusions

We found that a previously known miRNA in the hypoxic response, miR-210, was upregulated, confirming the utility of the Taqman® Array. Other miRNAs have been found using this array but require further analysis to evaluate their significance.

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Characterization of the Small Animal Radiation Research Platform

James Weltz

mentored by Dr. Cameron Koch and Dr. Sydney Evans

Goal: Characterize the SARRP, including accurate dosimetry, mechanical accuracy and imaging calibration.

Techniques 

  • Quantitative dosimetry using radiographic flims and ion chambers.
  • Analysis of scanner as quantitative tool. 
  • Cell culture and aseptic technique. 

Conclusions

  • Scanner is a viable quantitative tool. 
  • Film is accurate for dose calculations. 
  • Beam is not aligned with SARRP isocenter (off by order of 1mm). 

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