Identifying biomarkers of breast cancer micrometastatic disease in bone marrow using a patient-derived xenograft mouse model

After patients gave informed consent, human breast adenocarcinomas were prospectively collected using a protocol approved by the Institutional Review Board at Washington University in St. Louis, and transplanted into mice. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis. Briefly, after 3-week old female NOD-SCID mice were anesthetized, an inverted Y-shaped incision was made to expose the mammary glands. Using a dissecting microscope, the lymph-node and the vessel in the fat bridge between the fourth and fifth mammary fat pads were cauterized. The breast epithelium in this area was then excised to create the “cleared fat pad” into which human breast tissues were implanted without interference from the host’s mammary epithelium. At 2 weeks post clearance, 500,000 immortalized green fluorescent protein (GFP)-labeled human breast fibroblasts were injected into each cleared fat pad. After an additional 2 weeks, the humanized fat pads received tumor implants. Breast biopsies were prepared for engraftment by placing tissue in ice-chilled high glucose DMEM, immediately transporting it to the laboratory, and mincing into 1–2-mm pieces for implantation in up to five mice. Further details of the development and maintenance of xenograft lines has been previously described [9‐11]. Individual animals are designated using the labeling convention: primary tumor line – passage – unique animal ID (e.g. 17-B-1141). Clinical features and pathological characteristics of the five patient tumor xenograft lines that were used for the present studies are listed in Table 1.

Mice were killed when the primary xenograft tumor reached approximately 1.5 cm in size (approximately 6–8 weeks after implanting the tumor tissues). The femur and tibia were dissected from surrounding tissue, avoiding potential contamination, and flushed with cold PBS to isolate BM cells. Normal mouse BM samples were collected from non-tumor-bearing NOD-SCID mice, both with and without transplanted human fibroblasts. BM from the four long bones of each animal was pooled and cells pelleted for RNA extraction. Total RNA was isolated from samples using Trizol reagent (Invitrogen) according to manufacturer’s protocol. The extracted RNA was quantified and qualitatively assessed using an Agilent Bioanalyzer.

One microgram of RNA was used for synthesis of first-strand complementary DNA (cDNA) using the Retroscript (Ambion) kit with random hexamers. Resulting cDNA was diluted to an equivalent of 10 ng/μL of input RNA. qRT-PCR of the indicated genes was performed as described previously [8]. Human specific primer/probe sets for the genes tested were purchased from Applied Biosystems and the assay ids of the probes used are given in Additional file 1: Table S1. Each reaction consisted of 2 μL of cDNA, TaqMan Master Mix (Applied Biosystems) and primer/probe set in a total volume of 20 μL. For each transcript/sample, triplicate reactions were run in an ABI 7500 FAST Sequence Detection System. If a transcript was not detected in at least two replicates by cycle 40, it was considered absent in that sample and excluded from further analysis. Reactions with a cycle threshold (C _T) value difference >1.5 for the same probe were also excluded from further analysis. The C _T values of each gene were normalized to mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) C _T values for the same sample. These delta C_T (dC_T) values were then normalized to corresponding dC_T values of the non-tumor-bearing mice for the same transcript and fold change calculated using the ddC _T method. Transcripts that did not reach C_T in non-tumor-bearing control mouse samples after 40 cycles were assigned a C_T value of 40 for calculation purposes.

Gene expression profiling was performed as previously described [8]. Total RNA was used for two-cycle biotinylated cRNA target synthesis (Affymetrix). Resulting biotinylated cRNA was quantified and samples that yielded >15 μg of cRNA were used for GeneChip microarray hybridization. Fragmented, biotinylated cRNAs were hybridized to Affymetrix Human Gene 1.0 ST microarrays following standard protocols. Arrays were hybridized, washed, and scanned following the manufacturer's protocol. GeneChip CEL files were processed with the RMA algorithm and normalized using Partek Genomics Suite software. Differential patterns of gene expression were identified from annotated, normalized microarray data as detailed in the “Results” section. All data filtering, visualization, and analysis of variance (ANOVA) was performed using Partek Genomics Suite software. A schematic of data sets utilized and analysis workflows are presented in Fig. 1. Gene expression data are available at Gene Expression Omnibus [GEA:GSE57947].

To investigate the clinical relevance of PDX models for studying BM DTCs in patients with breast cancer, we utilized a set of previously characterized PDX mouse lines [9, 11, 15]. BM was collected from a total of 18 animals, spanning five different passages and representing initial implants from five different patients with a variety of molecular phenotypes (Table 1). All but one patient (7192) developed distant, clinical metastatic disease.

To allow for multiple molecular analyses with limited amounts of BM, we employed a molecular screen to detect BM DTCs in each animal, based on detection of human-specific GAPDH (hGAPDH) transcript. As shown in Fig. 2a, 10 of 18 (55%) animals analyzed had detectable expression of hGAPDH in their BM. hGAPDH expression clearly emanated from BM DTCs as other humanized xenograft animals and non-grafted controls, but fat pad humanized animals, had no detectable expression of hGAPDH (data not shown). Furthermore, all BM samples were assayed for GFP gene expression and found to be negative (data not shown), suggesting that hGAPDH expression emanated from actual DTCs and not from the GFP-labelled human fibroblasts that were implanted and that may have migrated from fat pad implantation. Although the actual number of human DTCs present in the BM of each mouse could not be calculated based on qRT-PCR data, assuming that hGAPDH expression levels per input mass of total RNA are proportional to DTC cell numbers, it is clear that WHIM17 mice maintained a much higher tumor burden in their BM, as compared to those from the WHIM12 line (Fig. 2a).

Among the DTC-positive mice, seven originated from the WHIM17 line and three originated from the WHIM12 line. These ten animals all developed distant solid metastases, primarily to the lung and liver (Table 1). In contrast, eight other animals, originating from lines WHIM11, WHIM12, WHIM13, and WHIM23 had neither detectable expression of hGAPDH in their BM nor any evidence of distant solid metastases, even after primary tumor growth had progressed to 1.5 cm at their greatest diameter at the time of sacrifice. The presence of BM DTCs and the development of distant metastases were highly correlated (p < 0.0001, Fisher’s exact test), consistent with clinical observations of DTCs and metastatic disease development in patients [1, 8]. In fact, although only 18 animals were analyzed in this study, detection of hGAPDH expression in BM was 100% specific and 100% sensitive for predicting metastatic spread of the xenograft tumor.

Data suggest that only those cancer cells that undergo extensive molecular and phenotypic adaptations, such as EMT, will successfully survive and proliferate in a foreign micro-environment [16]. We therefore examined the BM of the PDX mice for the expression of genes associated with both epithelial cell lineage and EMT using directed, qRT-PCR analyses for human-specific gene expression. EMT-associated transcripts included Snail1 (SNAI1), Gooscoid (GSC), and FOXC2. As expected, in control and non-metastatic mice without hGAPDH expression in BM, none of the epithelial and EMT marker genes were detected. In eight of the ten hGAPDH-positive mice, epithelial marker genes often used for DTC detection in human studies, i.e keratin17 (KRT17), mammaglobin (SCGB2A2), and EpCAM (TACSTD1) were also not detected. Keratin19 (KRT19) expression was detected in only one animal derived from the WHIM17 line (i.e. 17-B-1141). In contrast, expression of three EMT marker transcripts, Snail1 (SNAI1), Gooscoid (GSC), and FOXC2 were detected in many, albeit not all, of the seven WHIM17-derived animals (Fig. 2b) but in none of the WHIM12 mice. Since hGAPDH expression in the WHIM12 animals was also lower, this may simply reflect lower tumor (DTC) burden in the BM of these animals.

To better understand the molecular evolution of tumor metastasis we utilized one PDX line (WHIM17) to compare patterns of gene expression between primary xenograft tumor, BM DTC populations, and distant solid organ metastasis. Of specific interest were patterns of gene expression that were unique to DTC populations, and groups of genes that were common to both DTCs and solid organ metastasis, but distinct from those of the primary tumor itself.

Gene expression microarray analysis was performed on the WHIM17 primary xenograft tumor (17-B-29), a splenic metastasis that developed in that animal (17-B-29), and one DTC-positive BM sample from the same animal and six from different passages from the same line. Although human-specific microarrays were used for this analysis, it was expected that some patterns of gene expression in the mouse BM samples could originate from cross-hybridization of transcripts from murine BM cells. Therefore, we also profiled two BM samples from both control mice and non-engrafted mice with humanized mammary fat pads. Expression data from these animals was used as a baseline to identify human DTC-specific expression in the BM of each of the WHIM17 animals. To validate this in silico approach, we selected six transcripts of genes previously implicated in tumorigenesis and metastasis [17‐26] the expression of which was elevated at least threefold in all seven WHIM BM samples, as compared to control BM, and confirmed expression using human-specific primers and qRT-PCR (Fig. 3, Additional file 2: Table S2).

GLN3, ITGB3BP, MALAT1, and ITGB1BP1 were detected in the BM specimens from all WHIM17-derived mice, but not in the BM of mice derived from any other line. CD44 and ALCAM expression was detected in both mice that developed (WHIM17 and WHIM12) and mice that did not develop (WHIM23) metastatic disease. None of the non-tumor-bearing control mice with humanized mammary fat pads demonstrated expression of any of these genes (Fig. 3).

As shown in Fig. 4, global gene expression analysis of seven WHIM17 BM samples and corresponding tumor and metastatic lesion from the WHIM 17B29 animal showed specific clusters of genes with upregulated expression in both the primary tumor and the metastatic lesion. Surprisingly, there was considerable variability in gene expression among BM samples from the WHIM17 animals that appeared independent of tumor passage, pattern of metastatic spread, RNA quality, or other technical parameters. Not surprisingly, the WHIM17B29 BM expressed the greatest resemblance to the primary and metastatic lesion from the same animal, while four other BM samples shared a unique profile with a large number of transcripts that were over represented compared to primary tumor, metastasis, or other hGAPDH-positive BM samples.

We focused on clusters of 1979 unique “BM-specific” and 394 unique “metastasis-specific” transcripts to identify those that may be most relevant as biomarkers for the presence of DTCs. Additional file 3: Table S3 provides a complete list of those transcripts with significantly different expression between DTCs and primary tumors, and metastasis and primary tumor, while Tables 2 and 3 provide further filtered lists of those transcripts most highly associated with “metastasis” in the published literature in a comparison between metastases versus primary tumor and BM versus primary tumor. Many of these genes could be therapeutically targeted and are at different stages of clinical development (Tables 2 and 3). SLPI is appears in both gene sets indicating its enhanced expression in DTCs as well as metastatic tumor.

Since gene expression patterns strongly suggested that cells in PDX mouse BM are derived from their primary xenograft tumor, and that there is a robust association between their presence and metastatic outcome, we next investigated whether gene expression in the BM of mouse PDX models could also be detected in the BM of patients with breast cancer, prior to the development of overt metastatic disease. Using previously generated BM gene expression microarray data from a cohort of treatment-naïve, clinical stage II/III patients with breast cancer and healthy female controls [8], we examined the expression of 420 unique genes from the PDX BM data set to determine whether they could detect differences between these populations. From the original set of 1979 “BM-specific” transcripts identified in the xenograft model, we derived a set of 420 transcripts that both could be mapped to human microarray expression data probe sets and that were annotated in PubMed citations with the key words “metastasis”, “invasion”, and “epithelial mesenchymal transition” (Fig. 1). Globally (Fig. 5) the WHIM17 BM gene set did not distinguish healthy female BM from BM of patients with breast cancer and had no ability to classify those patients who did or did not experience a distant metastatic event. However, we did identify a subset of 17 genes with expression that after correcting for false discovery, could distinguish between healthy BM and BM from patients with breast cancer (Table 4, Fig. 1). Given that the expression of these genes (1) is frequently associated with biological processes such as tumor cell proliferation, invasion, and metastasis; (2) are detectable only in BM from PDX mice that develop DTCs in their BM and distant organ metastases; and (3) are expressed in patients with breast cancer, as compared to healthy human BM, we propose that they are excellent biomarker candidates for future prospective studies to evaluate whether they can stratify patients with breast cancer for risk of recurrent disease based upon detection and classification of BM DTCs.