Pre- and post-surgical monitoring of experimental primary tumor growth and metastasis under neo-adjuvant treatment

Description

Methods (English)

Experimental system

Cell lines

The human LM2-4^LUC+ cells are a luciferase-expressing metastatic variant of the MDA-MB-231 breast cancer cell line derived after multiple rounds of in vivo lung metastasis selection in mice, as previously described (1). LM2-4^LUC+ were maintained in DMEM (Corning cellgro; 10-013-CV) supplemented with 5% v/v FBS (Corning cellgro; 35-010-CV), in a humidified incubator at 37oC and 5% CO2. The cell line was authenticated by STR profiling (DDC Medical, USA).

Drug and doses used

Sunitinib malate (SU11248; Sutent®©, Pfizer) is a molecular RTKI that can block angiogenesis-associated vascular endothelial growth factor receptors (VEGFRs) along with several other regulators of metastasis (2). The molecule was suspended in a vehicle formulation that contained carboxymethylcellulose sodium (USP, 0.5% w/v), NaCl (USP, 1.8% w/v), Tween-80 (NF, 0.4% w/v), benzyl alcohol (NF, 0.9% w/v), and reverse osmosis deionized water (added to final volume), which was then adjusted to pH 6. The drug was administered at 60 or 120 mg/kg/day orally by gavage as previously described (4,5). The treatment window used in all neoadjuvant studies consisted of a previously optimized 14-day period prior to surgery (4). Within this 14-day period, daily sunitinib (Su) treatment was given either at 60 mg/kg/day (for 3, 7, or 14 days followed by vehicle for 11, 7, or 0 days, respectively), or at 120 mg/kg/day for 3 days followed by 60 mg/kg/day for 0, 4, 8, or 11 days, and vehicle for 11, 7, 3, or 0 days, respectively. An example of an abbreviation in the text includes ‘Su60(14D)’, which means ‘sunitinib at 60mg/kg/day for 14 days. Schematics for all studies are shown in Table S1. Mice treated daily with vehicle for 14 days were used as controls. Detailed analysis and comparisons of these treatment regimens are described in a companion study evaluating treatment breaks on metastatic disease.

Ortho-surgical model of metastasis

Animal studies were performed in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institute of Health and according to guidelines of the Institutional Animal Care and Use Committee at Roswell Park Comprehensive Cancer Center (protocol: 1227M, PI: John M.L. Ebos).

Implantations: Experimental methodology was extended from previous work using a xenograft animal model of breast cancer spontaneous metastasis that includes orthotopic implantation followed by surgical resection of a primary tumor (termed ‘ortho-surgical’) (4). Briefly, LM2-4^LUC+ (1 x 10⁶ cells in 100μl DMEM) were orthotopically implanted into the right inguinal mammary fat pad (right flank) of 6-8-week old female SCID mice, as described previously (4,5). Primary tumor (PT)  burden was monitored with Vernier calipers using the formula width²(length x 0.5) and bioluminescence imaging (BLI) (3,4,5). Neoadjuvant treatments started 14 days before primary tumors were surgically removed at a timepoint (34-38 days post-implantation) previously optimized for maximal distant metastatic seeding but minimal localized invasion (3,4). The surgeries were planned at specific time points post-implantation to avoid invasion of primary tumor into the skin or peritoneal wall, ensuring that metastatic progression had proceed and minimizing the possibility of surgical cure (3,4). Postsurgical metastatic burden (MB) was assessed by BLI and overall survival was monitored based on signs of end stage disease as previously described(3,4).

Exclusion criteria: Two scenarios represented instances where animals were excluded from treatment studies. First, if complete removal of primary tumor was not surgically feasible because of local invasion or evidence of advanced metastatic spread (4,5). Second, if no primary or metastatic tumor was ever detected by BLI or visual assessment it was assumed there was lack of tumor-take upon implantation (3,4).

Randomization: Before treatment initiation animals were randomized by primary tumor size assessed by Vernier calipers to avoid any false results due to unequal tumor burden between groups (6).

Biomarkers

Flow cytometry

Peripheral blood was collected in tubes containing lithium heparin (BD Biosciences; 365965) by orbital bleeding one day before surgical tumor resection. Non-specific binding was blocked with normal mouse IgG (Invitrogen; 10400C) incubated with whole blood, followed by incubation with an antibody mix. After staining, cells were fixed in a lyse/fix solution (BD Biosciences; 558049), while red blood cells were lysed. Samples were analyzed with a LSR II low cytometer (Becton Dickinson), while data were acquired with FACSDiva software (Becton Dickinson) and analyzed with FCS Express 6 (DeNovo software).

Circulating tumor cells (CTC)

The antibody mix for CTC detection of human CTCs in animal models contained a rat anti-mouse CD45 (30-F11) antibody conjugated to PE (Biolegend; 103106) and mouse anti-human HLA conjugated to AlexaFluor 647 (Biolegend; 311416). CD45 staining with a rat anti-mouse CD45 conjugated to FITC (Invitrogen; MCD4501) was used to eliminate any mouse blood cells, whereas human HLA was used to identify CTC (human LM2-4^LUC+). For a positive control, LM2-4^LUC+ cells were trypsinized, washed with PBS, and stained for both CD45 and HLA. LM2-4^LUC+ were used to define the CTC gate.

Circulating myeloid-derived suppressor cells (MDSC)

The antibody mix for detection of MDSCs contained a rat anti-mouse CD45 (30-F11) antibody conjugated to PE (Biolegend; 103106), a rat anti-mouse Ly-6G/Ly-6C (Gr1) (RB6-8C5) antibody conjugated to PE-Cy7 (BD Pharmingen; 552985), and an rat anti-mouse CD11b (M1/70) antibody conjugated to eFluor450 (eBioscience; 48-0112). Mouse CD45 staining was used to select only leukocytes, and CD11b and Gr1 were used to define the granulocytic and monocytic MDSC.

Immunofluorescence

Resected tumors were frozen on dry ice in cryo-embedding compound (Ted Pella, Inc; 27300), sectioned, and fixed in a 3:1 mixture of acetone:ethanol. Non-specific binding was blocked with 2% BSA in PBS, followed by staining with antibody mix containing rabbit anti-mouse Ki67 antibody (Cell Signaling Technologies; 12202) and rat anti-mouse CD31 antibody (Dianova; DIA-310). Detection of primary antibodies was achieved using FITC conjugated goat anti-rabbit IgG (BD Pharmingen; 554020) and Cy3 conjugated goat anti-rat IgG (Invitrogen; A10522). Samples were counterstained with DAPI (Vector; H-1500) and mounted with a hard-set mounting medium for fluorescence. Random images from each section were obtained with a Zeiss AxioImager A2 epifluorescence microscope at 200x magnification, and analyzed with ImageJ. CD31+ cells (% area) and Ki67+ cells (% cells) were quantified automatically using macro functions, whereas Ki67+/CD31+ cells (proliferating endothelial cells) were quantified manually.

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[6] Suresh K. An overview of randomization techniques: An unbiased assessment of outcome in clinical research. J Hum Reprod Sci. 2011;4:8–11.