Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis

BC PDX mouse models were established in Dr. Michael Lewis’ laboratory at the Lester and Sue Smith Breast Center in Baylor College of Medicine. Methods used to establish these PDX models have been recently reported [26]. Mice transplanted with tumors from passages 2 through 11 were used for our studies. Animal care for the mice bearing the BC PDX tumors, as well as age- and gender-matched control mice, was in accordance with the NIH Guide for the Care and Use of Experimental Animals with approval from the Baylor College of Medicine Institutional Animal Care and Use Committee.

In brief, mammary fat pad epithelium was surgically cleared from 3- to 4-week-old SCID/Beige female mice. Subsequently, fresh breast tumor fragments collected directly from patients were orthotopically transplanted into the cleared mammary fat pads. When tumor size reached 1,000 mm³, mice were killed, and tumors were re-transplanted into additional mice up to 11 passages. Importantly, the primary and serially passaged PDXs have shown genomic, proteomic, phenotypic, and histologic consistency with the tumor of origin, and they are also genetically and proteomically stable across multiple transplant generations [26].

After anesthesia, peripheral blood (500 to 700 μl) was collected from the vena cava inferior of each animal, which was then killed by cervical dislocation. Tibias, femurs, and hip-bones were collected, and bone marrow was flushed from the bones by using phosphate-buffered saline (PBS) supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA). Whole blood was processed within 1 hour of collection to lyse red blood cells (RBCs) by incubation with ammonium chloride (StemCell Technologies, Vancouver, BC, Canada) per manufacturer’s protocol.

RBC-depleted cells and bone marrow cells were then washed twice with PBS at room temperature, pelleted, and fixed with 10% neutral buffered formalin for 3 hours at room temperature. Cell pellets were embedded in paraffin and cut in consecutive sections of 5-μm thickness. Five consecutive sections of every 15 sections were stained by using immunohistochemistry (IHC) with anti-human pan-cytokeratin (clone AE1/AE3 against cytokeratins 1–8, 10, 13–16, and 19; Source: Dako, Carpinteria, CA, USA) and nuclear counterstain (hematoxylin). CTCs and BM-DTCs were identified as cytoplasmic human pan-cytokeratin-positive and nuclear counterstain-positive cells. A CTC or BM-DTC cluster was defined as a group of two or more CTCs or BM-DTCs, respectively. At least two PDX-bearing mice were tested per line (range, 2 to 7), and in total, five age-matched non-tumor-bearing female mice were used as controls. Lung metastases (LMs) were identified by IHC in these PDX lines and were reported in the previous study [26].

The following nine of 18 screened lines had published Affymetrix gene expression data (GEO:GSE46106), and at least three mice screened for CTCs (BCM-3107, BCM-3204, BCM-3561, BCM-3613, BCM-3887, BCM-3963, BCM-4272, BCM-4664, BCM-4888) [26]. Of these nine lines, CTC clusters were present in three lines and LM were detected in six lines (Table 1). To identify differential genetic signatures corresponding to CTC clusters and to LM, analysis of variance followed by t-tests were performed by applying linear modeling to our microarray experiments by using the Linear Models for Microarray Data (LIMMA) method, and subsequent manual curation was used, as described previously [27]. A genetic profile overlapping these two genetic signatures was derived and interrogated for prediction of distant metastasis-free survival in publicly available datasets [28].

The number of CTCs was reported per 20,000 nucleated cells (≈20 μl of blood). This was based on our initial testing of >20 representative mice from nine PDX models showing that the average number of RBC-depleted nucleated cells was 20,000 per 20 μl of blood collected. The DTC count was reported as number of DTCs per 2 million bone marrow cells. In addition, the CTC and BM-DTC detection rates were calculated per each PDX line as the ratio of the number of mice with one or more CTCs and DTCs, respectively, and the number of mice tested. Lung metastatic rate was previously reported as the ratio of the mice affected by metastases and the number of mice tested per each line [26]. The association between the presence of CTCs and BM-DTCs was evaluated within the same mice by using Fisher’s Exact test and Cochran-Mantel-Haenszel test to adjust for different PDX lines. The data are presented in a collapsed 2 × 2 contingency table without different strata representing different PDX lines. The association between the ability to produce CTCs or CTC cluster occurrence and the presence of LM was assessed within each line because the latter was evaluated in a previous study [26]. These associations were assessed by Fisher’s Exact test, and data were represented in 2 × 2 contingency table. To derive genetic signatures associated with CTC clusters and LM, genes that were differentially expressed and had some statistical evidence of being repeatable were considered; this signature was determined by using a linear contrast t test q–value (also called false discovery rate (FDR)) <0.25 or at least 16-fold average difference in expression between LM-positive and LM-negative PDX lines, which corresponds to a log2 contrast between LM-positive and LM-negative scoring greater than 4 in absolute value. To determine the prognostic value of the four-gene profile, gene-expression data of human BC [28] were each scored, taking the sum of the two “upregulated” genes minus the sum of the two “downregulated” genes (by using z-normalized expression values). All P values reported were two-sided, unless otherwise specified.