Introduction
Breast tumors frequently comprise heterogeneous cancer cells with distinct morphologic and phenotypic features [
1,
2]. Intra-tumor heterogeneity can arise from stochastic genetic or epigenetic changes, or can be attributed to signals from the stroma within the tumor [
3,
4]. More recently, the cancer stem-cell hypothesis was proposed to explain these cancer cells heterogeneity and hierarchical organization [
5,
6]. From a clinical perspective, targeting specific cell lineage with metastatic proclivity remains a life-saving therapeutic challenge, as most breast tumors are invasive and result in a poor prognosis with decreased disease-free survival.
The variable expression of cell surface markers among cancer cells is being widely exploited to identify, isolate and characterize distinct cancer cell populations [
7,
8]. CD24, an anchored cell surface glycoprotein was recently identified as an ideal marker to isolate pure mammary epithelial cells that can be further isolated, along with staining for other cell surface markers, into stem/progenitor cells. In line with that finding, isolated Lin
−CD24
+CD49f murine mammary cells have been shown capable of generating functional mammary tissue in vivo [
9,
10]. As a ligand of p-selectin, CD24 serves as an adhesion molecule that facilitates the metastatic process by supporting the rolling of cancer cells on activated platelets and endothelial cells [
11,
12]. Recently it was suggested that although CD24 lacks an intracellular domain, it is involved in regulating cancer cell proliferation and gene expression. However the mechanisms mediating these effects remain elusive [
13].
Based on CD24 expression, we have recently identified two distinct subpopulations in the mammary carcinoma Mvt-1 cell line, which is derived from a primary mammary tumor in MMTV-VEGF/c-myc bi-transgenic female mice. Although several studies suggest that it is the lack of CD24 expression that characterizes breast cancer stem cells [
14,
15], it is known that cell-surface markers are not conserved among different tumors, due to differences in the driver mutations [
4]. Several questions remain to be on the role of CD24 in cancer and more specifically in tumor heterogeneity. First, does CD24 actively mediate tumorigenesis, or does it serve only as a surface marker for tumorigenic cells? Answering this would facilitate the design of better therapeutic strategies, i.e., inhibition/downregulation of CD24 or alternatively exploiting its expression for targeting specific cancer cells. Second, do CD24
+ cells act as stem/progenitor cells and are CD24
− cancer cells their progeny? Finally, are there specific genes that will discriminate between CD24
− and CD24
+ cells, and are there changes at the protein level in these subpopulations such as phosphorylation that result in activation of different signaling pathways?
To begin to elucidate the cellular differences between distinct cancer cell subpopulations, we isolated two cancer cell subpopulations based on CD24 expression and phenotypically characterized these cell subsets. Next, we turned to mouse models to determine the tumorigenic capacity of each subset. To investigate the role of CD24 in mediating tumorigenesis, we knocked down CD24 expression with an shRNA construct. In addition, we demonstrated a degree of hierarchy and plasticity in these cancer cells. We further analyzed the gene expression profile of each cell subset and tested the implication of these findings in vivo.
Our results suggest that CD24 cell surface expression on mammary epithelial cancer cells identify a subpopulation of cells that is enriched with stem/progenitor-cell properties. These CD24+ cells display highly tumorigenic properties with high metastatic capacity; moreover, these cells can differentiate in vivo, hence creating intra-tumor heterogeneity. CD24+ cells differ from their counterpart in their gene profile and are characterized by elevated extracellular matrix gene expression; this may enable them to modify the soil of the host tissue in order to invade and form metastasis.
Methods
Cell culture
The mouse mammary cancer cell line, Mvt-1, has been previously described [
16]. Cells were cultured in DMEM (Biological Industries, Beit Haemek, Israel) supplemented with 10 % fetal bovine serum (Biological Industries) and antibiotics (penicillin, streptomycin; Biological Industries) at 37 °C in a humidified atmosphere consisting of 5 % CO2 and 95 % air.
Animals
Female MKR mice and control mice on an FVB/N background were used in this study. The MKR mice are transgenic with a dominant-negative insulin-like growth factor-I receptor specifically targeted to the skeletal muscle with a resultant severe insulin resistance and hyperinsulinemia phenotype [
17]. Mice were kept on a 12-hour light/dark cycle with access to standard mouse chow and fresh water ad libitum. Mice studies were performed according to the protocol approved by the Technion Animal Inspection Committee. The Technion holds an National Institutes of Health (NIH) animal approval license number A5026-01.
Flow cytometry
The following antibodies were used for cell surface staining of the Mvt-1 cell line, Pacific-Blue-conjugated anti-CD24, PE-conjugated anti-CD29, AF647 (Alexa Fluor 647)-conjugated anti-CD61/β3 and AF647 (Alexa Fluor 647)-conjugated anti-CD49F (Biolegend, San Diego, CA, USA): 7-Amino actinomycin D (7-AAD, Biolegend) was used to gate live cells. Cells were stained at a concentration of 5 × 106 cells/ml of FACS buffer (PBS containing 0.1 % BSA) for 20 minutes on ice in the dark, after which the cells were washed twice and resuspended in FACS buffer containing 7-AAD. Stained cells were analyzed using the CyAn ADP Instrument (Dako-Cytomation, Glostrup, Denmark) and the FlowJo 7.25 analysis software. Intracellular staining was performed with the CytoWx/Cytoperm kit (BD PharMingen, San Diego, CA, USA), after CD24 cell surface staining. Cells were then washed with the Cytofix solution, and permeabilized with perm wash for 10 minutes on ice. To detect intracellular CD24, the antibody was added in combination with perm wash for 15 minutes on ice. Cells were washed twice and resuspended in FACS buffer until analysis. Flow cytometry-based cell sorting for CD24− and CD24+ cells was performed using FACSAria (BD Biosciences, San Jose, CA, USA).
Tumorspheres
CD24− and CD24+ single cell suspensions were prepared and plated in nonadherent conditions at 600 cells/cm2 in DMEM F12 HAM medium (Sigma, Rehovot, Israel) containing 20 ng/ml bFGF (Sigma), 20 ng/ml EGF (Sigma), 4 μg/ml of Heparin (Sigma) and B-27 supplement (1:50 dilution, GIBCO, Burlington, ON, USA), and cultured at 37 °C with 5 % CO2. Tumorsphere-forming efficiency (percentage) was calculated after 5 days as follows: (number of tumorspheres (>50 mm in diameter) per well/number of cells seeded per well)*100. To assess self-renewal, primary tumorspheres were centrifuged at 115 × g for 5 minutes, the pellet was resuspended in 300 μl of 0.5 % trypsin/0.2 % EDTA for 3 minutes at 37 °C. Tumorspheres were disaggregated into single cell suspension with the use of a 25-G needle and syringe, (trypsin was neutralized with medium containing serum). Cells were centrifuged at 580 × g for 5 minutes, the pellet was resuspended in ice-cold PBS, and single cell suspension was assured under a microscope. Single cells were plated at the same seeding density that was used in the primary generation. Tumorspheres (>50 mm in diameter) were measured after 5 days in culture. Self-renewal was calculated by dividing the number of secondary tumorspheres formed by the number of primary tumorspheres formed.
Quantitative PCR reaction for cDNA products
Quantitative PCR was performed using Absolute Blue SYBR-Green ROX mix (Thermo scientific, ABgene, Epsom, UK). RNA was extracted from treated Mvt-1 cells with the Total RNA Purification Kit (NORGEN Biotek Corp, Thorold, Canada) according to manufacturer’s instructions, followed by single-stranded cDNA synthesis using the Verso™ reverse transcriptase (Thermo Scientific, ABgene). The expression measurement of the designated genes was performed with the Rotor-GeneTM 6000 system (Corbett Research, Sydney, Australia) and its software, ver. 1.7. The relative gene copy number was normalized using B2M as the independent internal control gene, and calculated by the 2^-(Ct(n)-Ct(normalizer)) method, where Ct represents cycle threshold.
Proliferation
Cells were seeded in 96-well plates (1,000 cells/well) for 48 h. Proliferation of the cells was quantitated by the CyQuant (Invitrogen, Carlsbad, CA, USA) fluorimetric DNA assay according to the manufacturer’s recommendations.
Knockdown of CD24 by retroviral-based delivery of shRNA
The shRNA targeting sequence against mouse CD24, sense (5′-CCCAAATCCAAGTAACGCTACCATTCAAGAGATGGTAGCGTTACTTGGATTTGTTTTTA-3′) and antisense (5′-GGGTTTAGGTTCAT TGCGATG GTAAGT TCTCTACCATCGCAATGAACCTAAACAAAAAT-3′) or scrambled (control) oligonucleotides were annealed and then ligated into the BglII and HindIII sites of the psuper.retro.puro vector (OligoEngine, Seattle, WA, USA). Retroviral particles were generated and introduced as described above.
Syngeneic orthotopic tumor models
CD24− and CD24+ cells or knockdown cells were suspended in 100 μl PBS and then injected (5 × 104 cells/mouse; fewer cells were injected for the serial dilution experiments) into the left inguinal mammary fat pad (number 4) of 8-week-old female MKR mice. Tumor volume was monitored once a week with calipers; volume was calculated in mm3 by the formula: (width2 × length × 0.5). Following sacrifice, tumors were removed and weighed, then flash frozen in liquid nitrogen and kept at −80 °C for further analysis.
Generation of the green fluorescent protein-expressing (GFP) cell line
A construct containing GFP (NV-SV-40-puro-linkek-Ins-PGK-eGFP, a generous gift from Dr Neufeld, Technion, Haifa, Israel) sequence was transfected into the Lentiviral packaging cell line 293FT together with ViraPower packaging mix (Invitrogen) using the Lipofectamine 2000 reagent (Invitrogen). At 48 h post-transfection, the supernatant containing the viruses was collected and filtered through a 0.45-Am syringe filter. Viruses were used to infect Mvt1 cells in the presence of polybrene (Sigma, St Louis, MO, USA) at a final concentration of 8 μg/ml. Infected cells were selected and maintained with 2 μg/ml puromycin. CD24−/GFP+ and CD24+/GFP+ cells were sorted using FACSAria (BD Biosciences).
Tumor dissociation into single cells
Breast tumors were minced with scalpels and transferred to gentleMACS™ dissociator C-tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) containing 5 ml of DMEM (Biological Industries, Beit Haemek, Israel) supplemented with 10 % FBS (Biological Industries, Beit Haemek, Israel). C-tubes were then connected to the gentleMACS™ dissociator and tumor dissociation was performed according to the manufacturer’s instructions. Minced tumors were incubated in the C-tubes for 45 minutes with 300 units/ml collagenase I (Sigma-Aldrich, Rehovot, Israel) and 2 mg/ml dispase II (Roche Diagnostics, Mannheim, Germany) at 37 °C in a humidified atmosphere consisting of 5 % CO2 and 95 % air. Following incubation a second spin on the gentleMACS™ dissociator was performed, and the cells were then filtered through a 40-μm falcon strainer (Becton Dickinson, Franklin Lakes, NJ, USA).
mRNA-seq
Total RNA was isolated and purified from cells using the Total RNA Purification Kit (NORGEN Biotek Corp) according to the manufacturer’s instructions. cDNA libraries were prepared using 1 μg of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina). Briefly, polyadenylated RNA was purified using magnetic beads and fragmented according to the manufacturer’s instructions. After ligation of the paired-end adapter the approximately 200-bp fraction was amplified with 15 cycles of PCR. cDNA libraries were subjected to the Illumina HiSeq 2500 platform to 50-bp single reads sequencing.
Analysis of mRNA-Seq data
Custom-built software was used to map the reads to the mouse genome (mm9) and estimate the coverage of each gene. Briefly, the reads were clipped at both ends (2 at the 5′ end and 1 at the 3′ end) to remove potentially error-prone sites. The reads were then mapped to the genome using a suffix-array-based approach. The median of coverage across the transcripts was used as an estimate of gene expression. The expression values were quantile normalized, and ratios were calculated by comparing the mean of samples from CD24− cells against the mean of samples from CD24+ cells. The noise (or limits) of expression detection in the mRNA-seq data is the peak of the distribution of expression values (genes with high expression are in the long tail of this distribution). The expression values were regularized by adding the noise to the expression of each gene before the ratios were calculated. This ensures that genes with low expression do not contribute to the list of genes with large fold-changes. Genes expressed differentially between the groups were selected by using a p value <0.05 (calculated by the t test) and fold-change of at least 2 was required for the gene to be included in the list. The heat map was generated by custom software R.
Pathway analysis
We used Gene Ontology (GO) analysis [
18] to analyze and compare enriched pathways in CD24
− and CD24
+ cells. The Gorilla tool was used in order to identify enriched GO terms. As target list inputs, we used all the annotated genes as the background for the analysis. GO terms were selected with a conservative threshold of false detection rate (FDR) <0.2 and a
p value <0.05.
We injected10,000 cells from each subset through the tail vein of wild-type (WT) mice to assess lung metastatic activity. Mice were killed 28 days after injection, lungs were removed and fixed, and macrometastases were counted under a light-microscope.
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). The independent t test and the Mann-Whitney test was used for statistical analysis of unmatched groups; the Wilcoxon signed-rank test was used for matched group comparison, with p values <0.05 considered statistically significant.
Discussion
Breast tumors comprise heterogeneous cell populations that can be characterized and isolated with specific cell surface markers [
29,
30]. CD24, a known marker for mammary stem and progenitor cells, was proven useful to enrich mammary cancer stem cells [
31]. CD24
+ cells were shown to be highly tumorigenic, with capability to form mammary tumors even when implanted in low numbers [
9,
32]. From a clinical perspective, CD24 was recently suggested as a prognostic marker for invasive breast tumors and has been associated with shortened disease-free survival [
33,
34]. Here we studied the potential role of CD24 in the vascular epithelial growth factor (VEGF)/c-myc mutated Mvt-1 cell line. We report here that CD24 serves as a cell surface marker for highly metastatic tumorigenic cancer cells. Moreover, we demonstrated that the cancer process is orchestrated by stem/progenitor-cancer cells that account for tumor heterogeneity that is evoked by microenvironment stimuli. Using RNA-seq analysis we revealed a unique gene signature for the CD24
+ subset, with elevated expression of extracellular matrix genes that support the highly metastatic properties of these cells. These results suggest that the extracellular matrix genes are new therapeutic targets for invasive tumors.
CD24 expression is abundantly expressed in human solid tumors; its expression is associated with high tumorigenic capacity and worse prognosis [
35]. The role of CD24
+ cells in breast tumors is unclear, whereas studies have shown that CD24
− cells are widely described as tumor initiating cells with stem/progenitor-like properties [
15,
36]. Others have demonstrated that this CD24 phenotype is not universal for all cancer models but it is more diverse and is affected by the driver mutation [
4].
In the present study, we found heterogeneity within the Mvt1 cells by characterizing the cell-surface expression of CD24; we isolated two distinct subpopulations, CD24
− and CD24
+ cells. Along with other studies, CD24
+ cells have been found to have higher proliferation rates [
37], however, the underlying mechanism remain unclear. CD24 has already been suggested as a surface marker of mammary stem and progenitors cells [
9]. Using the tumorsphere assay, we demonstrated that the CD24
+ subpopulation is enriched with early stem/progenitors cells with self-renewal properties. Next, we tested the tumorigenic potential of these distinct populations in both WT and MKR mice. The MKR female mice serve as an ideal model to isolate insulin mitogenic effects; accelerated mammary gland development was found in these hyperinsulinemic female mice [
17,
19]. Furthermore, orthotopic mammary tumors displayed significantly accelerated growth in these mice compared to WT mice [
38,
39]. We report here that CD24
+ cells are highly tumorigenic, forming significantly larger tumors in both WT and MKR female mice. Although CD24
+ cells display high tumorigenic capacity, it remains to be determined whether CD24 serves as a marker for tumorigenicity or if it is functionally involved in tumorigenesis. CD24 is mostly considered as an adhesion molecule; by interacting with p-selectin. CD24 it promotes the initial steps of cell migration, and its expression in breast tumors is associated with metastasis [
34,
40].
Recent studies have suggested that CD24 is involved in intracellular signaling despite the lack of an intracellular domain [
41‐
43]. Using shRNA technology we were able to determine that in VEGF/c-myc mutated cancer cells and in the context of breast cancer, CD24 could serve as a key marker to identify a subpopulation of tumorigenic cancer cells, however it is not functionally involved in the induction of mitogenic pathways. CD24
+/CD24-KD cells remained highly proliferative, with ability to form tumorspheres with high efficiency. Importantly, CD24-KD had no effect on tumor growth, as implantation of both CD24
+/control and CD24
+/CD24-KD cells formed rapidly growing tumors. It was recently demonstrated that CD24
−/− cells are able to functionally reconstitute cleared mammary fat pads [
44]. These findings, along with our results, suggest CD24 is mainly a marker in the mammary epithelium. However, these data cannot absolutely exclude a role for CD24 in mediating the metastatic process. Whether it is the result of clonal evolution or hierarchical organization that follows the cancer stem cell model, mammary tumors along with other solid tumors constitute phenotypically and functionally heterogeneous cell populations [
6,
45,
46]. With the tumorsphere assay we identified stem/progenitor activity and self-renewal properties in the CD24
+ cells in vitro. We next tested these properties in vivo, having hypothesized that CD24
+ cells can differentiate and give rise to CD24
− cells. To validate this, we implanted CD24
+/GFP
+ cells into the mammary fat pad of both WT and MKR mice. FACS analysis of the labeled cancer cells revealed that CD24
+ cells fuels the cancer process by giving rise to the CD24
− cells that comprise the tumor bulk.
Next, we confirmed that CD24
− cells that were extracted from the CD24
+ tumors were morphologically and functionally distinct from their CD24
+ counterparts. Unlike the CD24
+ cells, these CD24
− cells (that originated in vivo from the CD24
+ cells) displayed a flatter, more rounded epithelial phenotype as oppose to the mesenchymal-like morphology of the CD24+ cells. These data are consistent with the concept of cancer stem cells having intra-tumor hierarchical organization as a result of cancer cell differentiation [
47]. These findings, suggest that mammary tumors develop in a multi-step process, which is dictated by CD24
+ cells that demonstrate directed plasticity towards the differentiated CD24
− cells. Moreover, the presence of about 30 % of CD24
+ cells in the CD24
+ inoculated tumors, suggests that the CD24
+ cells are capable of undergoing asymmetric divisions, thus, expanding both CD24
+ and CD24
− lineages. It is important to note that CD24
+ cells were not able to differentiate in vitro even when co-cultured with tumor-derived fibroblasts (data not shown). Other tumor microenvironment factors should be evaluated in order to determine which extrinsic mechanisms promote cancer cell differentiation.
In order to identify transcripts and pathways that may elucidate the enhanced tumorigenesis upon CD24
+ cells implantation and may serve in the future as therapeutic targets, we compared the CD24
− and CD24
+ cellular transcriptome. Our study identified 157 candidates with divergent expression between these two groups. Genes that are associated with the immature state (Tmem176b, Tmem176a, ATF5) [
22,
48] were significantly elevated in the CD24
+ cells. Our analysis also identified upregulation in genes that promote proliferation (AXL and DDR2) [
20,
49], migration and invasion (MRC2, FSCM and SERPINH1) [
24‐
26], and immune response-associated genes (SLPI and CHAC1) [
50,
51]. Most elevated pathways found in the CD24
+ are involved in matrix formation and remodeling. The GO: 0005578 ~ proteinaceous extracellular matrix term was recently identified as common to distinct circulating cancer cell populations [
28]. This finding suggests that CD24
+ cells possess highly metastatic and tumor-initiating properties. We confirmed these findings in vivo with the tail vein metastasis assay and by limited dilution transplantation we provided clear evidence for the stem-like properties of the CD24
+ cells.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DLR and MZ developed the ideas, and edited the manuscript. RR, SA, IG, SBS, RS, EJS, KBW, ZSO, and AC conducted the experiments. DLR wrote the manuscript. All authors contributed to the analysis of data. All authors have read and approved the final version of the manuscript.