Journal: Cell reports

Article Title: Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging.

doi: 10.1016/j.celrep.2021.109689

Figure Lengend Snippet: Figure 1. Rac1 signaling is increased in MMTV-PyMT-driven metastatic breast cancer (A) Schematic of Rac1-FRET biosensor in FRET conformation upon GTP loading and Rac1 activation in cells crossed to the MMTV-PyMT-driven breast cancer model. (B) Representative images and quantification of upregulated Rac1 activity in primary MMTV-PyMT-driven mammary tumors compared to WT mammary glands (n = 7 mice per condition, 991 cells in total). (C) Spatiotemporal monitoring of live Rac1 activity in the context of the native tumor microenvironment showing the local vasculature (Qdot655) and second harmonic generation (SHG) imaging visualizing the local ECM monitored by optical window imaging in primary tumors (i). Longitudinal imaging can be achieved using these windows and further improved by image stabilization, allowing for the assessment of Rac1 activity in relation to tumor microenvironment (ii). (D) Tracking of Rac1 activity in relation to the proximity of cells to local tumor vasculature, with example excerpts showing cells proximal and distal to the local vasculature (n = 5 mice, 166 cells). (E) Upregulation of Rac1 activity at the invasive border of primary tumors as quantified through an optical window (n = 5 mice, 180 cells). Columns show averages, and error bars represent SEM; scale bars, 50 mm. Unpaired Welch’s t test, ****p < 0.0001 and *p < 0.05 (B and E); one-way ANOVA, *p < 0.05 (D).

Article Snippet: Reagent or Resource Source Identifier Antibodies Mouse monoclonal IgG1 anti-Rac1 Cytoskeleton Cat# ARC03, RRID:AB_10709099 Rabbit poly-clonal IgG anti-GFP Abcam Cat# ab290, RRID:AB_303395 Rabbit monoclonal IgG1 anti-GAPDH Cell Signaling Cat# 2118, RRID:AB_561053 Sheep-anti-Mouse IgG HRP-linked GE healthcare Cat# NA931, RRID:AB_772210 Donkey-anti-Rabbit IgG HRP-linked GE healthcare Cat# NA934, RRID:AB_772206 Rabbit monoclonal IgG1 anti-Ki67 ThermoFisher Scientific Cat# RM-9106-S1, RRID:AB_149792 Rabbit polyclonal anti-cleaved Caspase-3 Cell Signaling Cat# 9661, RRID:AB_2341188 Rabbit monoclonal anti-Cyclin B1 (EPR17060) Abcam Cat# ab181593, RRID:AB_2820245 Rabbit polyclonal anti- Cyclin A2 (C-19) Santa Cruz Biotechnology Cat# sc-596 Rabbit polyclonal anti-PAK1 Cell Signaling Cat# 2602, RRID:AB_330222 Rabbit polyclonal anti-PAK1 (Ser199/204)/PAK2 (Ser192/197) Cell Signaling Cat# 2605, RRID:AB_2160222 Mouse Monoclonal anti-beta-actin (clone AC-15) Merck (SigmaAldrich) Cat# A5441, RRID:AB_476744 Bacterial and virus strains E. coli DH5a ThermoFisher Scientific Cat# 18265017 Chemicals, peptides and recombinant proteins Qtracker 655 Vascular Label ThermoFisher Scientific Cat# Q21021MP AnnexinV-Cy5 BioVision Cat# 1013 Propidium iodide Merck, Sigma-Aldrich Cat# P4170 SalI Promega Cat# R6051 NdeI New England Biolabs Cat# R0111S Rac1 Pull-down Activation Assay Biochem Kit CytoSkeleton Inc. Cat# BK035 Experimental models: Cell lines PyMT 20065 (from mouse 20065, female) SEARCHBreast https://searchbreast.org/ MMTV-PyMT;Rac1-FRET (from mouse 8625, female) This study N/A MMTV-PyMT;Rac1-FRET;P-Rex1 ± (from mouse 138509, female) This study N/A Experimental models: Organisms/strains Rac1-FRET mice (ubiquitously expressed, ‘‘ON’’) Prof. Heidi CE Welch, Babraham Institute, UK, Johnsson et al. 2014 N/A MMTV-Her2 mice The Jackson Laboratory Stock# 002376 MMTV-PyMT mice The Jackson Laboratory Stock# 002374 K14-CreER mice The Jackson Laboratory Stock# 005107 LSL-K-Ras G12D mice The Jackson Laboratory Stock# 008179 Rac1-flox mice Prof L. Machesky, Prof O. Sansom, CRUK Beatson Institute, University of Glasgow N/A P-Rex1 KO mice Prof. Heidi CE Welch, Babraham Institute, UK, Welch et al. 2005 N/A FVB/NJ (Ausb) mice The Jackson Laboratory Stock# 001800 NOD.Cg-Prkdc < scid > IL2rg < tm1Wjl > /SzJ (Ausb) mice The Jackson Laboratory Stock# 005557 BALB/c-Fox1nu (Ausb) mice Charles River Strain# 194 (Continued on next page) Cell Reports 36, 109689, September 14, 2021 e1

Techniques: Activation Assay, Activity Assay, Imaging