Investigating Internalization, Intracellular Tracking, and Localization of NGR-Tagged Rubredoxin in HT-1080 Fibrosarcoma Cells

Date of Award

7-2025

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Bonnie Buxton, Ph.D.

Second Advisor

Francis E. Jenney, Jr., Ph.D.

Third Advisor

Lori Redmond, Ph.D.

Fourth Advisor

Kimberly Baker, Ph.D.

Abstract

Fibrosarcoma is a highly aggressive soft tissue sarcoma characterized by poor response to conventional therapies and limited treatment options. CD13, a zinc-dependent metallopeptidase overexpressed in tumor vasculature and fibrosarcoma cells, presents a promising molecular target for receptor-specific drug delivery. This study examined the internalization dynamics and intracellular localization of a NGR-tagged Rubredoxin protein (NGR-Rb) in HT-1080 fibrosarcoma cells to inform tumor-targeted therapeutic design. Rubredoxin, a thermostable iron-sulfur protein from Pyrococcus furiosus, was selected for its compact size (~6 kDa), stability under oxidative and acidic conditions, and resistance to proteolytic degradation, qualities suited for the tumor microenvironment.

To evaluate receptor-mediated uptake, four fluorescently labeled rubredoxin constructs were tested: wild-type (pFJ5.1), NGR-tagged (pFJ08), strep II–tagged (pFJ42), and dual-tagged NGR + strep II (pFJ43). HT-1080 cells were pre-stained with MitoTracker or LysoTracker and pre-treated to establish three distinct uptake conditions: physiological temperature (37°C), low temperature (4°C), and Dynasore treatment at 37°C. Following preconditioning, cells were exposed to the rubredoxin constructs for internalization analysis, which was assessed using confocal microscopy. Co-localization analysis was performed using FIJI/ImageJ, including Manders’ tM1/tM2 coefficients, Pearson’s correlation, and Costes P-values.

Western blotting confirmed robust CD13 expression in HT-1080 cells. Among the constructs, the NGR-tagged constructs (pFJ08 and pFJ43) demonstrated lysosomal localization at 15 minutes under 37°C conditions. The pFJ08 exhibited moderate co-localization (tM1 = 0.610; tM2 = 0.420), confirming that the NGR motif alone supports potential CD13-specific uptake. The pFJ43 showed stronger co-localization (tM1 = 0.786; tM2 = 0.718), though the contribution of the strep II tag remains unclear. The pFJ5.1 and pFJ42 displayed minimal uptake and diffuse cytoplasmic distribution. NGR-tagged constructs retained surface docking under inhibitory conditions, supporting potential receptor- and energy-dependent internalization.

These results identify pFJ43 as the construct with the highest lysosomal co-localization among those tested, suggesting potential as a scaffold for future CD13-targeted delivery. However, since pFJ08 also showed uptake, further studies are needed to evaluate the impact of dual-tagging. Limitations include single-sample data for each condition and time point, as well as the need to refine fluorescence quenching protocols. Future work will incorporate replicates, optimized quenching, and additional controls to strengthen mechanistic interpretation.

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