Defense Date

2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Medical Physics

First Advisor

Jamal Zweit

Abstract

Cancer treatment by adoptive immune cell therapy (AIT) is a form of immunotherapy that relies on the in vitro activation and/or expansion of immune cells. In this approach, immune cells, particularly CD8+ T lymphocytes, can potentially be harvested from a tumor-bearing patient, then activated and/or expanded in vitro in the presence of cytokines and other growth factors, and then transferred back into the same patient to induce tumor regression. AIT allows the in vitro generation and activation of T-lymphocytes away from the immunosuppressive tumor microenvironment, thereby providing optimum conditions for potent anti-tumor activity. The overall objective of this study is to: a) develop multi-modality (optical- and radionuclide-based) molecular imaging approaches to study the overall kinetics of labeled adoptively transferred T- lymphocytes in vivo, b) to non-invasively image and assess in-vivo, targeting and retention of adoptively transferred labeled T-lymphocytes at the tumor site. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in vitro with Bryostatin/ Ionomycin for 18 hours, and were grown in either Interleukin-2 (IL-2) or combination of Interleukin-7 and Interleukin-15 (IL-7/IL-15) for 13 days, (cells grown in IL-2 called IL2 cells, and cells grown in IL7/15 called IL7/15 cells). In order to validate the methodology and to offer future clinical translation, both direct and indirect cell labeling methods were expanded and employed. The first method was based on direct in vitro cell labeling by lipophilic near-infrared (NIR) fluorescent probe, 1,1- dioctadecyltetramethyl indotricarbocyanine iodide, (DiR), followed by intravenous (i.v.) injection into BALB/C mice for multi-spectral fluorescence imaging (MSFI). The second method was based on indirect labeling of T- lymphocytes through transduction of a reporter gene (cell cytoplasm labeling Herpes Simplex Virus type 1- thymidine kinase (HSV-1 tk). The product of this reporter gene is an enzyme (HSV-1TK) which phosphorylates a radio labeled substrate 2-fluoro-2-deoxy-1 β- D- arabinofuranosyl-5-iodouracil ([124I]-FIAU) for Positron Emission tomography (PET) imaging. ATP based cell viability assay, flow cytometry and interferon-γ (IFN-γ) ELISA were used to investigate if there are any changes in cell viability, proliferation and function respectively, before and after direct and indirect labeling. The results showed that cell viability, proliferation, and function of labeled 4T1 specific T-lymphocytes were not affected by labeling for direct labeling methods at DiR concentration of 320µg/ml. For the indirect labeling method, the viability and proliferation results showed that cell viability decreases as multiplicity of infectious (MOI) increases. In particular, at MOI of 10 almost all cells die 3 days post transduction. At MOI of 5, cells viability was ≤ 30% and at MOI of 2 was ≤ 60%. Cell viability was 80% at MOI of 1. The results of optical imaging were as follows: when the recipient mice with established 4T1 tumors were injected with DiR labeled 4T1 specific T-lymphocytes, the 4T1 specific T-lymphocytes (IL2 cells) infused into tumor-bearing mice showed high tumor retention, which peaked 3 or 6 days post infusion depending on the tumor size and persisted at the tumor site for 3 weeks. In contrast, IL7/15 cells showed lower signal at the tumor site and this peaked on day 8. On the other case when 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes. IL2 T-lymphocytes moved out of lymphoid compartments to the site of subsequent 4T1 inoculation within two hours and peaked on day 3 and the signal persisted for 2 more weeks. In contrast with infusion of IL7/15 cells, the signal was barely detected and did not show a similar trafficking pattern as with IL2 cells. The results of the indirect labeling method, PET reporter gene (PRG) system (HSV-1tk / [124I ] FIAU ) showed that both IL2 and IL7/15 cells were successfully transduced as verified ex vivo by real time PCR and western blot. T Cells transduction efficiency was assessed from cell uptake study in comparison to stable transduced Jurkat cells which have transduction efficiency of 100 %. Both IL2 and IL7/15 cells showed lower transduction efficiency (≤ 30%) compared to Jurkat cells. Consequently, PET imaging did not show a detectable signal of transduced T cells in vivo. Biodistribution study was carried out on day 3 post [124I]-FIAU injections. Results were consistent with the optical imaging results, except for IL7/15 cells. Transduced and untransduced IL2 and IL7/15 cells were labeled with DiR and injected ( i.v.) into Balb / C mice and then imaged by both imaging modalities (MSFI and PET) at the same time. MSFI images of transduced IL2 cell showed detectable signal starting from 2 hours, peaked at 72 hours and persisted up to 2 weeks, while IL7/15 cells were detectable at the tumor site starting at 24 hours, peaked at 72 hours and persisted up to 2 weeks. By the end of this study animals were dissected and tissue activities were counted using gamma counting and expressed as % Injected dose/gram of tissue (%ID/gm). Transduced IL2 and IL7/15 cells showed higher %ID/gm than other organs at lungs, liver, spleen, tumor, lymph nodes and bone/bone marrow. IL7/15 cells compared to IL2 cells showed higher %ID/gm at same organs. Neither IL2 nor IL7/15 untransduced DiR labeled cells showed any activity at tumor site, and their activities at other organs was very low compared to transduced cells. To investigate whether labeled T-lymphocytes will localize at tumor metastases or not, and to study the difference in their migration patterns to the tumor site versus tumor metastases, 4T1 tumor cells were successfully transduced with HSV-1tk as confirmed by RT-PCR , western blot and cell uptake study. Transduced 4T1 cells were implanted in the right flank or in the mammary fat pad of the mouse. Serial PET imaging was carried out in the third and fourth week post tumor implantation to know when the tumor will metastasizes. PET imaging showed only signal at the tumor site and no metastasis were detected.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

April 2014

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