Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects
Pritha Ray1, Abhijit De, Manishkumar Patel
1Molecular Imaging Program at Stanford, Departments of Radiology and Bioengineering, Bio-X Program, School of Medicine, Stanford University, California 94305-5427, USA.
Purpose:
Capsase-3 plays an important role in chemotherapy-induced apoptosis in many cancers. Herein, we applied a multimodality reporter vector to monitor caspase-3 activation indirectly in live cells and tumors of living animals undergoing apoptosis.
Experimental Design:
A fusion protein (MTF) was constructed by combining three different reporter proteins, red fluorescent protein (mRFP1), firefly luciferase (FL), and HSV1-sr39 truncated thymidine kinase (TK), linked through a caspase-3 recognizable polypeptide linker. After cleavage by caspase-3, a significant gain in mRFP1, FL, and TK activity are observed by fluorescence-activated cell sorting and enzyme-based assays. A melanoma cell line (B16F10-mtf-hrl) stably expressing mtf (to measure caspase-3 activation) and hrl-IRES-gfp (to determine the decrease in a number of viable cells) vectors was generated to measure two independent molecular events upon treatment.
Results:
Upon induction with 8 mumol/L staurosporine, the fusion protein showed a 2.8-fold increase in FL (P = 0.03), a 1.5-fold increase in TK (P = not significant), and a 2-fold increase in mRFP1 (P = 0.05) activity in 293T cells. Bioluminescence and micropositron emission tomography imaging of the apoptotic B16F10-mtf-hrl tumors showed a 2-fold higher FL activity (897 versus 416) and a 2-fold higher TK activity (10.3 versus 3.87) than control tumors when normalized with RL activity. Using a similar normalization approach, the time kinetics of caspase-3 activation by two protein kinase-C inhibitors was noninvasively monitored in living mice.
Conclusion:
This multimodality caspase sensor vector could effectively and noninvasively monitor caspase-3 activation from single live cells to a multicellular tumor environment and, thus, would be a valuable tool for drug screening in preclinical models and future patient cell based therapy.
Insights
Researchers developed a novel reporter vector to noninvasively monitor caspase-3 activation, a key indicator of chemotherapy-induced apoptosis. This tool aids in tracking cancer cell death in real-time for drug screening.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Caspase-3 is crucial for chemotherapy-induced apoptosis in various cancers.
- Monitoring caspase-3 activation is vital for assessing cancer treatment efficacy.
Purpose of the Study:
- To develop and validate a multimodality reporter vector for indirect monitoring of caspase-3 activation in live cells and tumors.
- To assess the vector's utility in tracking apoptosis in preclinical cancer models.
Main Methods:
- A fusion protein (MTF) was engineered, combining mRFP1, firefly luciferase (FL), and HSV1-sr39 truncated thymidine kinase (TK) via a caspase-3-cleavable linker.
- A melanoma cell line (B16F10-mtf-hrl) was established to express the MTF reporter and a viability marker (hrl-IRES-gfp).
- Caspase-3 activation was measured by increased reporter activity (mRFP1, FL, TK) using fluorescence-activated cell sorting and enzyme assays; tumor imaging utilized bioluminescence and micro-PET.
Main Results:
- In 293T cells, staurosporine induced significant increases in FL (2.8-fold) and mRFP1 (2-fold) activity.
- In vivo, B16F10-mtf-hrl tumors showed a 2-fold increase in FL and TK activity compared to controls.
- The system successfully monitored caspase-3 activation kinetics noninvasively in mice treated with protein kinase-C inhibitors.
Conclusions:
- The multimodality caspase sensor vector effectively monitors caspase-3 activation from single cells to tumors.
- This tool is valuable for noninvasive drug screening in preclinical models.
- It holds potential for future applications in patient cell-based therapy monitoring.
