Intravital FLIM-FRET Imaging Reveals Dasatinib-Induced Spatial Control of Src in Pancreatic Cancer

Abstract

Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, timedependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation. Cancer Res; 73(15); 4674-86. Ó2013 AACR.

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

“…However, we emphasize that this approach should be generalizable to other FRET-based biosensors, once the reporter module is modified to include mTurquoise and suitable acceptor. For example, we have found very similar results using the optimized calcium biosensor probe, Twitch2b 45 . Thus, with minimal effort the approach described here could be applied to many other biosensor probes by exchanging the earlier, more photo-labile, variants of the cyan FPs.…”

Fast bi-exponential fluorescence lifetime imaging analysis methods

“…As lifetime characteristics of fluorophores are usually sensitive to their environment, FLIM can be a powerful tool to monitor physiological parameters such as pH, O 2 ,Ca 2 , or image cellular protein-protein interactions that link to diseases or tumors [2][3][4].…”

“…In this Letter we will only discuss nonfitting time-domain methods applicable to highly parallel single-photon avalanche diode (SPAD) arrays [5][6][7]. Time-domain FLIM usually employs time-gated cameras [6,[8][9][10] or time-correlated single-photon counting (TCSPC) modules [1,3]. Only the former technique can provide high-speed acquisition.…”

Intravital microscopy of cancer: New insights into the spatiotemporal dynamics of the disease

“…In a multi-modal imaging study, pancreatic adenocarcinoma cell response to the anti-invasive drug dasatinib has been mapped and revealed the benefits of dual targeting in this disease (25). Similarly, intravital imaging has been used to monitor the intracellular pharmacokinetics of PARP-1 inhibitors and of microtubule inhibitors and underlined the heterogeneity of tumor response to chemotherapy (26,27).…”

“…* Mapping of tumor tracks, architecture, and response to drug treatments has been achieved via intravital microscopy and has enabled the identification of chemoresistant cells that may repopulate the tumor (5,18,25,26,27). We, therefore, envisage that intravital imaging will increase our understanding of the mechanisms of chemoresistance, which remains a key challenge for cancer research.…”