Abstract
Abstract The ability to observe changes in molecular behavior during cancer cell invasion in vivo remains a major challenge to our understanding of the metastatic process. Here, we demonstrate for the first time, an analysis of RhoA activity at a subcellular level using FLIM-FRET (fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer) imaging in a live animal model of pancreatic cancer. In invasive mouse pancreatic ductal adenocarcinoma (PDAC) cells driven by mutant p53 (p53R172H), we observed a discrete fraction of high RhoA activity at both the leading edge and rear of cells in vivo which was absent in two-dimensional in vitro cultures. Notably, this pool of active RhoA was absent in noninvasive p53fl knockout PDAC cells, correlating with their poor invasive potential in vivo. We used dasatanib, a clinically approved anti-invasive agent that is active in this model, to illustrate the functional importance of spatially regulated RhoA. Dasatanib inhibited the activity of RhoA at the poles of p53R172H cells in vivo and this effect was independent of basal RhoA activity within the cell body. Taken together, quantitative in vivo fluorescence lifetime imaging illustrated that RhoA is not only necessary for invasion, but also that subcellular spatial regulation of RhoA activity, as opposed to its global activity, is likely to govern invasion efficiency in vivo. Our findings reveal the utility of FLIM-FRET in analyzing dynamic biomarkers during drug treatment in living animals, and they also show how discrete intracellular molecular pools might be differentially manipulated by future anti-invasive therapies. Cancer Res; 71(3); 747–57. ©2011 AACR.
🔬 Techniques
💻 Software
✨ Fluorophores
🧪 Sample Preparation
🏭 Microscope Brands
💻 Software Details
💾 Data Repositories
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📋 Methods
Cell culture Primary mouse
PDACs were derived from tumors harvested from Pdx1-Cre-GFP, LSL-KRas G12D/+ , LSL-Trp53 R172H/+ mice and Pdx1-Cre-GFP, LSL-KRas G12D/+ , Trp53 LoxP/+ mice ( 23 ). Cell lines were tested 1 month before experiments and authenticated by morphology, growth curve analysis, and Mycoplasma detection in accordance with the ATCC cell line verification test recommendations and cultured as described in detail in SI Materials and Methods . Drug treatment in vitro and in vivo Dasatinib (a kind gift from Bristol Myers Squibb) was administered daily by oral gavage in 80 mmol/L citrate buffer [10 mg/kg] or 100 nM in vitro ( 33 ). ROCK inhibitor Y27632 (Calbiochem) was used at 10 μM and cell permeable C3 Transferase (Cytoskeleton) was used at 0.125 μg/ml.
Plasmids
For details of the modified GFP-RFP Raichu-RhoA reporter see SI Materials and Methods .
Organotypic invasion assay
Organotypic cultures were set up as described ( 32 ).
RhoA activity assay
GTP loading of RhoA was determined using a RhoA activation kit as described in manufacturers protocol (cytoskeleton).
Imaging
All imaging was performed on a Nikon Eclipse TE2000-U inverted microscope with a LaVision Biotec Trim-scope scan head. See SI Materials and Methods .
Fluorescence life-time imaging of RhoA
FRET reporter in vitro and in vivo For detailed procedure see SI Materials and Methods .
Data Analysis
Data was analyzed using the built-in TCSPC fluorescence lifetime analysis functionality of ImSpectorPro (LaVison Biotec, Germany). See details in SI Materials and Methods .
Supplementary Material 1 2 3 4 5 6 7
📊 Figures
Figure 1
Fluorescence life-time imaging of RhoA activity within a migrating pancreatic tumor cell population during wound healing. A, Schematic of the adapted GFP-RFP Raichu-RhoA reporter adapted from Yoshizak...
Figure 2
RhoA activity is required for PDAC invasion. A,B H&E-stained sections of p53 fl and p53 R172H cells on organotypic matrix. C, Quantification of p53 fl and p53 R172H PDAC cell invasion u00b1 Y27632 or ...
Figure 3
Spatial regulation of RhoA activity during invasion. A-C, Representative fluorescence images of p53 fl and p53 R172H cells expressing the Raichu-RhoA reporter (green) on the matrix surface or during i...
Figure 4
Spatial regulation of RhoA activity in live animals. A,B Representative in vivo fluorescence images of p53 fl and p53 R172H PDAC cells expressing the Raichu-RhoA reporter (green) with corresponding in...
Figure 5
Dasatinib inhibits mutant p53-driven PDAC cell invasion. A,B H&E-stained sections and quantification of mutant p53 R172H or p53 R175H -driven PDAC cell invasion u00b1 dasatinib in the organotypic matr...
Figure 6
Spatial regulation of RhoA activity in invasive p53 R172H PDAC cells upon dasatinib treatment in vivo . A and C, Representative in vivo fluorescence images of mutant p53 R172H PDAC cells expressing th...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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