Fluorescence lifetime imaging microscopy of chemotherapy‐induced apoptosis resistance in a syngenic mouse tumor model

Abstract

During cancer therapy with DNA-damaging drug-agents, the development of secondary resistance to apoptosis can be observed. In the search for novel therapeutic approaches that can be used in these cases, we monitored chemotherapyinduced apoptosis resistance in a syngenic mouse tumor model. For this, syngenic murine colorectal carcinoma cells, which stably expressed a FRET-based caspase-3 activity sensor, were introduced into animals to induce peritoneal carcinomatosis or disseminated hepatic metastases. This syngenic system allowed in vitro, in vivo and ex vivo analysis of chemotherapy induced apoptosis induction by optically monitoring the caspase-3 sensor state in the tumor cells. Tumor tissue analysis of 5-FU treated mice showed the selection of 5-FU-induced apoptosis resistant tumor cells. These and chemo-naive fluorescent tumor cells could be re-isolated from treated and untreated mice and propagated in cell culture. Re-exposure to 5-FU and second line treatment modalities in this ex-vivo setting showed that 5-FU induced apoptosis resistance could be alleviated by imatinib mesylate (Gleevec). We thus show that syngenic mouse systems that stably express a FRET-based caspase-3 sensor can be employed to analyse the therapeutic efficiency of apoptosis inducing chemotherapy.

A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

“…Importantly, improving BRET sensors will require the development of brighter luminescence proteins in addition to those described above (e.g., Nanoluc; Hall et al, 2012;Stoddart et al, 2015), which are capable of energy transfer to red-shifted fluorophores. Recently, RFPs have been used in both BRET and FRET sensors in living organisms (Keese et al, 2010;Dragulescu-Andrasi et al, 2011;Bakayan et al, 2014), also for the study of a GPCR (Fruhwirth et al, 2011). A red-shifted unimolecular BRET sensor has also been successfully used to detect a cancerassociated antigen in vivo using an antibody attached to a farred fluorescent indocyanine derivative fused to Cypridina luciferase via glycol chains , illustrating the advantages of red-shifted BRET emission for in vivo detection in tissue.…”

Intravital microscopy: new insights into metastasis of tumors

“…However, as the field of FRET biosensors is still an emerging area, only a few research groups have thus far used this powerful technique in vivo. For example, FRET biosensors have been used to measure the proteolytic activity of calpain in muscles (Stockholm et al, 2005), changes in calcium concentration and myosin light-chain kinase activity in arteries (Zhang et al, 2010), and the induction of apoptosis in the skin (Bins et al, 2007), colorectal carcinomas and lymphomas (Breart et al, 2008;Keese et al, 2010). In particular, the apoptosis sensor has been of interest for tumor biology, because tumor cells have to overcome many apoptotic signals during several steps of metastasis.…”

“…2C,D) (Bins et al, 2007). Keese and colleagues used FRET IVM of this biosensor to study the induction of apoptosis resistance upon chemotherapy (Keese et al, 2007;Keese et al, 2010). Moreover, Breart and colleagues used this biosensor to study apoptosis of tumor cells by cytotoxic T lymphocytes (CTLs) and showed that a CTL kills, on average, one tumor cell every 6 hours (Breart et al, 2008).…”

Imaging molecular dynamics in vivo – from cell biology to animal models

“…In an initial study FLIM-FRET was used to image the interaction between GFP-tagged chemokine (C-X-C motif) receptor 4 (CXCR4-GFP) and red fluorescent protein (RFP)-tagged protein kinase C  (PKCA-RFP) in mammary carcinoma xenografts (Kelleher et al, 2009). In another study, FLIM was used to image a caspase-3-based FRET probe to examine the development of drug-resistance in a syngenic mouse tumor model (Keese et al, 2010). In the first case, the authors used fluorescence lifetime to demonstrate differences in receptor binding when comparing deep and shallow tumor regions.…”