Background
The insulin-like growth factor receptor (IGFIR) is a transmembrane receptor tyrosine kinase [
1] that is primarily activated by its cognate ligands, IGF1 and IGF2, and can be activated by insulin albeit with much lower affinity [
2]. Since the early 1980s, when interest in IGF1R’s role in breast cancer development began [
3,
4], preclinical studies demonstrated that IGF1R is involved in cell transformation in addition to mediating tumor-promoting functions such as proliferation, antiapoptosis and cancer cell dissemination [
5‐
7]. Case control and prospective studies further supported the role of the IGF1R axis in breast cancer patients [
8]. This repertoire placed IGF1R as a promising target for cancer therapy. However, recent results from clinical trials indicated that IGF1R antagonists failed to fulfill the high expectation [
9,
10]. It has now become clear that predictive biomarkers are warranted in order to identify patients that will benefit from anti-IGF1R therapy.
Recently, based on CD24 cell surface expression we were able to distinguish, sort and maintain two distinct subpopulations of the metastatic mammary carcinoma cell line, Mvt1 that overexpresses the c-Myc and VEGF oncogenes [
11]. CD24 is an anchored cell surface glycoprotein, mainly associated with the progression of invasive tumors through P-selectin binding, which is expressed by activated endothelial cells and platelets [
12,
13]. More recently, it was shown that intracellular CD24 promotes tumorigenesis by the distribution of the ARF-NPM interaction and p53 inactivation [
14]. Furthermore, it was shown that CD24 can serve as an important indicator for poor prognosis for many of the most common malignancies including breast cancer [
15,
16].
Our recently published results demonstrated that CD24+ cells displayed more spindle-like cells that resemble a mesenchymal phenotype compared to the rounded epithelial morphology of their CD24− counterparts. Moreover, CD24+ cells displayed highly tumorigenic and metastatic properties in vivo. Implantation of CD24+ cells into the mammary fat pad of FVB/N female mice and the hyperinsulinemic MKR female mice resulted in more rapidly growing tumors. Furthermore, this CD24+ subset formed metastatic lesions at a higher rate from both primary tumors and following tail vein injection.
CD24 is widely used to isolate pure mammary epithelial cells and along with other cell surface markers it can further serve to isolate stem/progenitor cells. It was shown that Lin
-CD24
+CD49f murine mammary cells were able to generate in vivo functional mammary tissue [
17,
18]. In accordance with these findings, we have demonstrated that CD24
+ cells possess cancer stem/progenitor-like properties both in vitro and in vivo. These cells are driven to differentiate in vivo and partly account for intratumor heterogeneity [
19]. Whereas CD24 can serve in certain cases as a marker for stemness, the IGF1R was found to play a crucial role in maintaining pluripotent properties of human embryonic stem cells [
20]; the self-renewal property of cancer stem cells [
21].
In this study, we determined whether the cell surface expression of CD24 may serve as a valuable biomarker for tumor sensitivity to anti-IGF1R therapy. To begin to elucidate this issue we compared the effects of the IGF1R-knockdown (KD) on CD24- and CD24+ cells morphology and phenotype in vitro. Moreover, we compared the tumorigenic and metastatic capacity in vivo of these distinct subsets following IGF1R-KD. We further screened for transcripts that may illuminate on the discrepancy in the effects obtained between these subsets. Finally, we tested the feasibility of the results on the human mammary cancer MCF7 cell line.
We anticipate that this study may constitute the starting point for future attempts to clinically circumvent the negative effects of IGF1R overstimulation in breast cancer patients.
Methods
Cell culture
Mouse and human mammary cancer cell lines, Mvt-1, and MCF7 cell line have been previously described [
22,
23]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries, Beit Haemek, Israel) supplemented with 10 % fetal bovine serum (FBS) (Biological Industries) and antibiotics (penicillin:streptomycin; Biological Industries) at 37 °C in a humidified atmosphere consisting of 5 % CO2 and 95 % air.
Knockdown of IGF1R and IR by lentiviral-based delivery of shRNA
Vectors (GIPZ) encoding the following microRNA-adapted short hairpin RNAs (shRNA) 5’-TGACTGTGAAATCTTCGGC-3’ (mouse/human IGF1R), 5’- TTAGTTCCATGATGACCAG-3’ (mouse IGF1R) packed in high-titer lentiviral particles were purchased from Open Biosystems (Huntsville, AL, USA). These vectors or a vector containing a scrambled shRNA sequence (control shRNA; Open Biosystems) were infected in the presence of 8 μg/ml polybrene (Sigma-Aldrich, Rehovot, Israel) into Mvt-1 or MCF7 cells, all three vectors contained a green fluorescent protein (GFP) marker and puromycin resistance gene. Stable knockdown of the IGF1R was achieved by selection with 2 μg/ml puromycin (Sigma-Aldrich).
Protein extraction and Western blot analysis
Western blot and densitometric analysis was carried out for protein detection in cells and tumor tissues as previously described [
24]. Antibodies toward IGF1R and β-actin were purchased from Cell Signaling Technology, Danvers, MA, USA, antibody toward SLPI was purchased from Thermo Fisher Scientific, Rockford, IL, USA, and the matched secondary antibody conjugated with horseradish peroxidase was purchased from Jackson Laboratories, Bar Harbor, ME, USA.
Animals
Female MKR mice and control mice on an FVB/N background were used in this study. The MKR mice are transgenic mice 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 [
25]. 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 a National Institutes of Health (NIH) animal approval license number A5026-01.
Syngeneic orthotopic tumor models
CD24- and CD24+ cells or knockdown cells were suspended in 100 μl phosphate-buffered saline (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 and the 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 for further analysis.
Flow cytometry
Pacific Blue-conjugated anti-mouse CD24 or PerCP/Cy5.5-conjugated anti-human CD24 (Biolegend, San Diego, CA, USA) were used for cell surface staining of the cells. 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 fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.1 % bovine serum albumin [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 (Tree Star, Ashland, OR, USA). Flow cytometry-based cell sorting for CD24- IGF1R-KD and CD24+ IGF1R-KD cells was performed using FACSAria (BD Biosciences, San Jose, CA, USA).
Tumorspheres
CD24- IGF1R-KD and CD24+ IGF1R-KD cell suspensions were prepared and plated in nonadherent conditions at 600 cells/cm2 in DMEM F12 HAM medium (Sigma-Aldrich, Rehovot, Israel) containing 20 ng/ml basic fibroblast growth factor (bFGF) (Sigma-Aldrich), 20 ng/ml epidermal growth factor (EGF) (Sigma-Aldrich), 4 μg/ml of heparin (Sigma-Aldrich) and B-27 supplement (1:50 dilution, GIBCO, Burlington, ON, Canada), and cultured at 37 degree with 5 % CO2. Tumorsphere-forming efficiency (TFE) (%) was calculated after 5 days as follows: (number of tumorspheres (>50 mm in diameter) per well/number of cells seeded per well)*100. In order 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 min 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 min, 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. Following 5 days in culture tumorspheres (>50 mm in diameter) were measured.
Quantitative PCR reaction for cDNA products
Quantitative PCR was performed using Absolute Blue SYBR-Green ROX mix (Thermo Fisher 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 Fisher 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 independent internal control gene, and calculated by the 2^-(Ct(n)-Ct(normalizer)) method.
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 medium (Biological Industries, Beit Haemek, Israel) supplemented with 10 % FBS (Biological Industries). 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 min with 300 unit/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).
A total of 10,000 cells from each subset were injected through the tail vein of WT mice to assess lung metastatic activity. Mice were sacrificed 28 days following injection, lungs were removed and fixed, macrometastases were counted under the light microscope.
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). 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
Over the past few decades, findings regarding the role of the IGF1R in cancer have continued to accumulate. The IGF1R promotes cancer cell proliferation and survival and, on the other hand, prevents apoptosis [
1,
29,
30]. Moreover, the importance of the IGF1R in establishing and maintaining a transformed phenotype was well described [
31,
32]. These evidences positioned the IGF1R as a promising target for treating cancer. Hence, investigators along with pharmaceutical companies invested great efforts in developing therapeutic strategies toward the IGF system and specifically toward the IGF1R. Despite the great success in preclinical studies, results from recent and ongoing clinical trials have been distinctly disappointing [
10]. It has become clear that predictive biomarkers are required in order to select patients who would benefit from anti-IGF1R therapy. In this study, we suggest that CD24 cell surface expression in cancer cells may predict sensitivity to anti-IGF1R therapy. Our results demonstrate that IGF1R-KD specifically in CD24
+ cancer cells alters their morphology and phenotype and, more importantly, markedly inhibited their tumorigenic capacity.
In the current study, we demonstrate that IGF1R-KD had minimal effect on mammary tumors that formed by inoculation of the heterogeneous Mvt1 cell line. Recently, we found that the Mvt1 cells are comprised of two subpopulations that differ by their CD24 cell surface expression, therefore termed CD24
- and CD24
+ cells [
19]. Furthermore, our recent results, demonstrated that the CD24
+ subset is highly tumorigenic; these cells formed rapidly growing mammary tumors and metastatic lesions compared to their CD24
- counterparts. Moreover, these cells displayed early stem/progenitor properties in vitro and in vivo [
19]. Thus, we tested whether CD24 cell surface expression could serve as a new marker to predict response to anti-IGF1R therapy. We found that IGF1R-KD induces a rounded epithelial morphology in the CD24
+ cells that are normally characterized by mesenchymal-like morphology, whereas CD24
- subset was not affected by the IGF1R-KD and maintained their epithelial morphology despite the IGF1R-KD. The ability of cells to generate tumorspheres when growing in suspension is a common method to identify cells with stem/progenitor properties [
33]. The greater capacity of CD24
+ cells to form tumorspheres and the inability of CD24
- cells to do so was the most profound phenotype discrepancy between these two populations in vitro [
19]. Here we show that IGF1R-KD completely abolished CD24
+ cells capacity to form tumorspheres.
In the pursuit of tailor-made medicine, the identification of biomarkers that can imply who should receive therapy and what therapy is greatly warranted [
34,
35] Following the disappointing results of anti-IGF1R phase 3 clinical trials in unselected patients, the search for predictive biomarkers that can identify suitable patients who can benefit from anti-IGF1R therapy is now under intensive investigation [
10]. In order to further investigate whether CD24 cell surface expression could implicate tumor sensitivity to anti-IGF1R therapy, we inoculated CD24
- and CD24
+ cells with and without IGF1R-KD into the mammary fat pad of WT and the hyperinsulinemic MKR female mice. In accordance with our in vitro results, IGF1R-KD cells had a dramatic effect specifically on the CD24
+ tumors. CD24
+ IGF1R-KD cells formed significantly smaller tumors compared with the control CD24
+, cells whereas IGF1R-KD had no effect on CD24
- tumor growth. Western blot analysis confirmed significant reduction of the IGF1R in both CD24
- and CD24
+ tumors compared to the control tumors. It is important to note that the PI3K/Akt pathway and MAPK pathway failed to show a reduction, this may be attributing to other endogenous factors which can activate both pathways, and alternatively, this can reflect the specific time point of sacrifice since this protein modification is dynamic.
SLPI is a protease inhibitor and mediates a broad array of activities that may not be related to its antiprotease functions [
36], including several malignancy-promoting functions [
37]. In the search for proteins that enhance tumor formation and metastasis, Wagenblast et al, identified SLPI as a driver of metastatic formation in a mouse model for breast cancer. Additionally, SLPI emerged as the most significantly gene in human breast cancer patients that had lung-metastatic relapses [
28]. Using RNA-seq analysis, we have identified SLPI among the top three upregulated genes in the highly tumorigenic CD24
+ Mvt1 cell subset [
19], and suggests that SLPI may play a role in the more aggressive cancers derived from CD24
+ cells. Taken together we hypothesized that IGF1R-KD in the CD24
+ significantly attenuates the tumorigenic capacity by regulating SLPI levels. Our results, confirmed a significant reduction in SLPI expression specifically in the CD24
+ IGF1R-KD tumors compared with CD24
+ control tumors. However, SLPI levels were comparable between CD24
- control and CD24
- IGF1R-KD tumors. We screened several other candidates that may elucidate the specific antitumorigenic effect of IGF1R-KD in the CD24
+ tumors. We identified a significant upregulation in CTGF, specifically in the CD24
+ IGF1R-KD tumors. CTGF is a member of the CCN family of proteins that are involved in adhesion, apoptosis, extracellular matrix production, and growth arrest of multiple cell types; this is mostly attributed to their ability to interact and activate integrins [
27,
38]. Though the role the CCN proteins play in cancer is controversial, analysis of 122 human breast tumors suggested CTGF as a tumor suppressor [
27]. We further demonstrated that this effect is not restricted to the tumor process and that SLPI and CTGF expression is directly regulated by IGF1R. QRT-PCR analysis of CD24
+ cells in vitro following IGF1R-KD demonstrated that IGF1R downregulation results with a profound reduction in SLPI levels and upregulation in CTGF levels. In accordance with our in vivo results IGF1R-KD had no effect on both SLPI and CTGF mRNA levels in the CD24
- subset. The altered expression observed for both SLPI and CTGF following IGF1R-KD were dramatically greater in vivo. SLPI levels were reduced by approximately 10-fold in the CD24
+ IGF1R-KD tumors (compared with the CD24
+ control tumors), whereas the in vitro results demonstrated only a 50 % reduction. These in vivo changes in SLPI levels matched the difference in SLPI levels between CD24
+ and CD24
- cells [
19]. Recently, we demonstrated that CD24
+ cells differentiate in vivo in response to intratumor stimuli into distinct CD24
- cell population [
19]. This, along with recent reports that suggest a role for the IGF1R in maintaining stemness [
20], encouraged us to test whether IGF1R-KD induced rapid differentiation in vivo toward the CD24
- phenotype. FACS analysis of cancer cells dissociated from CD24
+ control and IGF1R-KD tumors, revealed that IGF1R is essential for the maintenance of stem/progenitors-like cancer cells that fuel the cancer process as recently described [
19,
39].
There is some “heterogeneity” in the results of preclinical and clinical studies regarding anti-IGF1R therapies. Burtrum et al, demonstrated that IGF1R inhibition in vivo, resulted in a significant reduction in tumor growth following inoculation of the adenocarcinoma MCF7 cell line [
5]. In contrast, primary tumors formed by MDA-MB-231 cells with IGF1R-KD grew as rapidly as the control tumors [
40]. It is important to note that these cells known as CD24
- cells [
41]. Our results indicate that CD24 expression in breast tumors may indicate sensitivity to anti-IGF1R therapy. Hence, to further investigate this, we determined CD24 cell surface expression in the mammary carcinoma MCF7 cell line. We found in accordance with previous studies [
41,
42] that MCF7 are CD24
+ cells. To test the feasibility of our results in CD24
+ human breast cancer cells, we downregulated IGF1R in the MCF7 cells and identified a marked reduction in SLPI levels.
Dissemination of tumors cells from the primary tumors and colonialization in distant sites, accounts for the vast majority of cancer-related death [
43]. Using the tail vein metastasis assay we demonstrated that IGF1R-KD abolished the metastatic capacity of the Mvt1 CD24
+ cells.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DL developed ideas and drafted the manuscript. RR developed ideas, edited and drafted the manuscript, conducted the experiments, and contributed to the analysis of data. SBS conducted the experiments and contributed to the analysis of data and revised the manuscript. ZSO conducted the experiments and edited the manuscript. All authors have read and approved the final version of the manuscript.