PROJECT SUMMARY Stem cell therapy (SCT) has displayed immense potential in a variety of regenerative medicine applications through tissue remodeling and repair. Stem cells (SCs) not only repair injured tissue through direct differentiation towards mesenchyme lineages, but they also have a multitude of paracrine mechanisms which help in regeneration. Mesenchymal SCs (MSCs) are the most used SCs due to their easy isolation (from bone marrow or discarded adipose tissue), no ethical concerns and straightforward maintenance. However, SCT has not achieved clinical acceptance due to a variety of limiting factors such as injections to incorrect locations, lack of cell retention, poor viability of transplanted MCSs, and a high risk of protumorigenic activity. Among these a major roadblock to translation is the ability of transplanted MSCs to differentiate into cancer associated fibroblasts (CAFs). CAFs contribute to immune escape of tumors via multiple mechanisms, including secretion of multiple cytokines and chemokines and reciprocal interactions that mediate the recruitment of immune cells. Moreover, CAFs directly abrogate the function of cytotoxic lymphocytes, thus inhibiting killing of tumor cells. Clinical stem cell therapy is severely hindered by the lack of spatial and functional information about the fate of implanted cells in vivo. There is a great need for imaging tools that can provide non-invasive and longitudinal information about the location of MSCs and the differentiation of MSCs to CAFs. Present methods to determine therapeutic outcomes are all invasive end-point analyses such as histology and biomarker assays, which cause damage to regenerating tissue and do not reflect the real-time dynamics of the microenvironment. This project presents a stimuli-responsive ultrasound guided photoacoustic (US/PA) nanosensor consisting of a gold nanosphere modified with a LOXL2-responsive triblock peptide. The nanosensor is designed for US/PA monitoring of MSC location and detection of MSC differentiation to CAFs. The triblock peptide sequence is selectively cleaved by LOXL2 upon differentiation to CAF phenotypes, which triggers hydrophobic aggregation of the gold nanospheres leading to enhanced optical absorption in the first near-infrared optical window. The overall hypotheses of the project are that US/PA imaging of MSCs augmented with LOXL2-responsive plasmonic nanosensors will enable real-time spatial tracking of transplanted MSCs and allow monitoring of their differentiation to CAFs in vivo. This approach will facilitate communication between the transplanted SCs and researchers or clinicians. Successful completion of the aims will (i) showcase and validate a versatile nanosensor platform for the functional monitoring of transplanted MSCs via integration of non-invasive US/PA imaging with cell nanoengineering techniques, (ii) perform in vivo real-time US/PA guided tracking of nanosensor augmented MSCs and their differentiation into CAFs in clinica...