Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer

FACS-sorted BMRCs/CTCs were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 and stained for immunofluorescence (IF) using selected primary and secondary antibodies, according to published procedures [21]. Alexafluor (AF) 594 (red) and AF-488 (green) tagged secondary antibodies against mouse and rabbit primary antibodies were used for IF. Bright-field and fluorescent microscopic images were captured using Zeiss Axio Observer microscope Z1 (Carl Zeiss, Jena, Germany) and data were analyzed by ZEN2 software (Carl Zeiss). For DEPArray, FACS-isolated cells were subsequently washed with SB115 buffer, loaded into the DEPArray cartridge, and imaged with 10× objective (Menarini Silicon Biosystems, Inc.). Data were analyzed using the custom Fixed_Low_Density program of the DEPArray v3.0 platform. CTCs were visualized by DAPI nuclear staining, Ki67, and other markers as listed. DEPArray positive/negative IF values were determined by CellBrowser^TM software, which calculate multiple parameters (background, mean fluorescence, etc.) during cell detection. Intensity threshold settings are then automatically applied to captured images, equalizing fluorescence levels in all images, but without altering primary fluorescence intensity values. Proliferative status of CTCs was evaluated by mean fluorescence intensity using FlowJo ver 10 software. Imaging parameters were established using control cells, and all subsequent images were captured using the same settings.

For immunohistochemistry (IHC), harvested tissue was fixed and stained. Sources of antibodies were: HLA-ABC (#565292) and mouse anti-cleaved PARP (Asp214) (#552596) were received from BD Biosciences San Jose, CA, USA; GCDFP-15 mammaglobin cocktail (#906H-08) from Sigma, St. Louis, MO, USA; Rabbit anti-RBL2 (#ab76234) from Abcam, Cambridge, MA, USA; Rat anti-Ki67 (#TA801577) from Origene, Rockville, MD, USA; Rabbit anti-pNDRG1 (#5482) from Cell Signaling Technology; and mouse anti-p70s6K1 (#MABS82) from EMD Millipore, Burlington, MA, USA; Anti-mitochondrial antibody (#MSM1-739-P), CD44 (#960-MSM2-P), PanCK (#MSM2-371-P), and Muc1 (#4582-MSM18-P) antibodies from Neobiotechnologies, Union City, CA, USA; and CD298 (#GTX114272) from Genetex, Irvine, CA, USA. All histochemical and antibody staining was performed by the HMRI Research Pathology Core. IHC on mouse tissue using antibodies of mouse origin were performed using M.O.M. elite peroxidase kit; dual IHCs were performed using ImmPRESS Duet Double Staining HRP/AP Polymer Kit, and triple IHCs were performed by multiplexing with ImmPRESS-AP Anti-Rat IgG, Mouse Adsorbed Polymer Detection Kit (Vector Labs, Burlingame, CA, USA). Slides were imaged using an EVOS XL Cell Imaging System (ThermoFisher Scientific) and quantified using ImageJ software by the Pathology Core facility at Houston Methodist Hospital (Houston, TX, USA) [19,20,21,22].