Introduction
Human breast cancer cell lines have been used extensively in vitro and in mice to dissect the cellular mechanisms associated with tumour aggressiveness and metastasis. However, cell line xenografts typically fail to recapitulate tumour cell heterogeneity. By contrast, human breast tumours engrafted into mice as patient-derived xenografts (PDXs) generally show excellent reproducibility of morphological and genetic characteristics of the original tumour with minimal genetic drift and as such are clinically relevant platforms for preclinical studies [
1‐
5].
Aggressive cancers are known to display epithelial-mesenchymal plasticity (EMP), through which they can fluctuate between epithelial or mesenchymal states (or degrees of these) to assist survival in changing microenvironmental conditions [
6‐
8]. Epithelial to mesenchymal transition (EMT) describes a phenotypic change toward a more mesenchymal state resulting in more motile and invasive cancer cells. EMT has been shown to promote progression in several cancer types including breast [
9,
10], contributes to chemoresistance [
11,
12], and is prominent in circulating tumour cells (CTCs) [
7,
13‐
15]. Evidence for EMP has been demonstrated in numerous human breast cancer cell line studies and increasingly in breast cancer in vivo models [
16‐
19] and clinical material (reviewed in [
20]).
Invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) (also known as invasive carcinoma of no special type (IC NST)) have distinctive morphological features [
21]. ILC is typified by single-file epithelial tumour cells, a finer stromal infiltration and often a minimal sclerotic tissue reaction—the combination of which makes self-detection and screen-detection (mammography) more difficult than for the typically more palpable IDC tumours, which grow as masses of epithelial cells within a desmoplastic stroma. These patterns are intimately linked to E-cadherin: ILCs do not express
CDH1 due to the presence of inactivating mutations [
22,
23] or silencing via methylation [
24], or copy loss [
25], and hence grow as individual and linear arrays of tumour cells. By contrast, IDCs typically express
CDH1 and hence grow as cohesive tumour nests [
21].
A major initiating event in the transcriptional programming of EMT is E-cadherin repression [
26‐
28]. IDC expression of
CDH1 is downregulated when they undergo an EMT, which is associated with increased invasiveness [
29,
30]; thus for the purposes of this study, IDC could be considered as “EMT-positive”. Interestingly, EMT does not occur spontaneously in ILC cells [
31] and they tend to show less vimentin expression than IDC [
32]; thus, ILC could be considered
“EMT-negative”
. Similarly, not all IDC-derived cell lines undergo EMT upon
CDH1 silencing, while some do [
18].
Integrin switching is prominent in breast cancer EMT and has been linked to tumour aggressiveness [
33], and integrins have been shown to play important roles in tumour cell transmigration via EMT (reviewed in [
34]). Integrins are a large family of heterodimeric cell surface receptors that play a prominent role in the adhesive interactions between cells and their surrounding extra-cellular matrix (ECM), providing adhesion for stationary cells, as well as traction during cell movement [
35‐
38]. TGFβ-induced EMT in NMuMG mouse mammary cancer cells results in the downregulation of epithelial
ITGA6/
B4 expression (which mediates contact with the basement membrane) through epigenetically silencing of the gene encoding integrin β4 [
39]. ITGA3/B1, which binds laminin and also associates with E-cadherin, is required for progression through EMT in lung alveolar epithelial cells, where it integrates beta-catenin and transforming growth factor-β (TGFβ)–SMAD signalling to promote myofibroblast formation and lung fibrosis [
40]. Interactions between ITGA5/B1 integrin and fibronectin have been associated with EMT in EpH4 mouse normal mammary epithelial cells and human lung cancer cell lines [
41,
42]. In pancreatic carcinoma cells, increased expression of ITGA1/B1 or ITGA2/B1 integrins and their interactions with type I collagen facilitate the disruption of E-cadherin complexes and the nuclear translocation of beta-catenin and promote proliferation and motility [
43].
Indeed, ITGA2/B1 is widely expressed on epithelial cells and its levels are increased in several carcinoma cells from the epithelial origin [
44]. Growing evidence indicates that ITGA2/B1 can be a key pathway in cancer pathogenesis [
43,
45,
46]. Furthermore, Chen et al. showed that increased expression of ITGA2/B1 is positively correlated with increased metastatic ability in human squamous cell lung cancer cells when
i.v. inoculated in severe combined immunodeficiency (SCID) mice [
47].
It has been demonstrated that ILK can induce a complete EMT in various epithelial cell lines and thus be involved in the initiation of EMT in vivo, and the maintenance of the mesenchymal phenotype and disease progression [
48‐
51]. ILK can modulate the expression of not only E-cadherin, but also other epithelial markers such as CK18 [
52] and MUC1 [
53], as well as mesenchymal markers such as LEF1 [
54] and vimentin [
53,
54]. Therefore, ILK is able to initiate an EMT. Gain and loss of function strategies have shown that over-expression, and/or constitutive activation of ILK results in oncogenic transformation and progression to invasive and metastatic phenotypes [
55].
In the current study, the divergent cancer subtypes of ILC and IDC, which differ in regard to their EMT status, were studied as PDXs in order to examine the relationship between EMT, ITGA2/B1 and ILK in cancer progression, through serial passages in mice. The human tumoural material for both PDXs were obtained from bone metastases and thus may have already undergone cycles of EMT, and its reversal MET, to enable colonisation [
26].
We coupled this with an investigation into the pattern of ILK and integrin expression changes in PMC42-ET human breast cancer cells induced by epidermal growth factor (EGF) treatment to undergo an EMT in vitro, and assessed whether these integrin changes were necessary for EMT to occur.
Materials and methods
Patient material and creation of xenografts
Establishment of the PDXs used in this study was described previously [
56].
The ED03 xenograft was derived from a lobular breast cancer bone metastasis in a 40-year-old woman, 3.5 yrs. after initial diagnosis of her primary tumour. The EDW01 PDX was derived from a bone metastasis of an invasive ductal carcinoma of the breast presenting as clinically overt macrometastatic deposits in a 44-year-old woman.
Briefly, the tumour tissue derived initially from the bone metastasis deposits was diced into ~ 1 mm pieces, mixed with Matrigel® (BD Biosciences, Australia) and implanted bilaterally subcutaneously in SCID mice (ARC, Perth, Australia). For each passage, once tumour volumes reached 2000 mm3, mice were euthanised, and the tumours were removed. The tumour tissue was again chopped into chunks, mixed with Matrigel®, and implanted into fresh mice. This was repeated 6 times for EDW01 (total of 7 passages) and 10 times for ED03 (total of 11 passages). Portions were snap frozen for RNA extraction and formalin fixed and paraffin embedded for immunohistochemical analyses at each passage.
Immunohistochemistry (IHC)
A tissue microarray was created of randomised duplicate 2-mm diameter cores of tumour blocks corresponding to various passage numbers through mice of the ED03 and EDW01 PDX model systems. IHC was performed using the Ventana Discovery Ultra Automated Slide Preparation system. Details of antibodies used in this study can be found in Table
1. The membrane-associated proteins (E-cadherin, beta-catenin, P120-RasGAP, CD24, CD44, carbonic anhydrase IX (CAIX)) were scored as cytoplasmic or membranous, and whether they were heterogeneous or homogeneous in these areas. Vimentin staining was scored as positive if present in the cytoplasm of cells, whereas for Twist1 and Ki67, the proportion of positive nuclei was recorded.
Table 1
Antibodies used in this study
Antigen |
Vimentin^,# | Mouse Monoclonal IgG (V9) | 1:750 | Dako, Australia |
E-cadherin^,# | Mouse Monoclonal IgG (36) | 1:12,500 | BD Transduction Laboratories, USA |
N-cadherin (A-CAM) | Mouse Monoclonal IgG (GC-4) | 1:2000 | Sigma-Aldrich, Australia |
TWIST^ | Mouse/ 2C1a | 1:100 | Abcam, England |
Beta-catenin^ | Mouse monoclonal, clone 14 | 1:500 | BD Biosciences, Australia |
P120^ | Mouse monoclonal 98/pp120 | 1:200 | BD Biosciences, Australia |
CD24^ | Mouse monoclonal SN3b | 1:50 | Thermo Fisher Scientific, Australia |
CD44^ | Mouse monoclonal 156-3C11 | 1:75 | Abcam, England |
Ki-67^ | Mouse monoclonal MIB-1 | 1:100 | Dako, Australia |
CA-IX^ | Rabbit monoclonal EP161 | 1:100 | Cell Marque (Sigma Aldrich), USA |
ITGB1# | Mouse Monoclonal IgG | 1:5000 | Chemicon International (Fisher Scientific), USA |
ITGA2# | Rabbit Polyclonal | 1:1000 | Chemicon International (Fisher Scientific), USA |
Integrin-Linked Kinase (ILK)# | Rabbit monoclonal IgG | 1:4500 | Cell Signaling Technologies (Danvers, MA,USA) |
Pan-Actin, Ab-5# | Mouse Monoclonal IgG | 1:10,000 | Neomarkers (Invitrogen), USA |
Secondary antigen |
Biotinylated Immunoglobulin^,# | Polyclonal Rabbit Anti-Mouse | 1:200 | Dako, Australia |
IgG-HRP^,# | Goat Anti-Mouse | 1:20,000 | Dako, Australia |
IgG-HRP^,# | Goat Anti-Rabbit | 1:20,000 | Dako, Australia |
Quantification of IHC data shown in Supplementary Figures
2 and
4 was determined using ImageJ, where DAB brown-positive nuclei were separated from blue (total) nuclei, and using the colour threshold tool, a threshold applied to only select positive nuclei that was visible by eye. Using this threshold, the area taken up by positive nuclei was quantified. The ratio of “relative intensity per cell” was obtained by dividing the overall area of positivity for the IHC target (either pink or brown) by overall nuclear area.
Reverse transcriptase-quantitative PCR
RNA was extracted using the Qiagen RNeasy Mini prep Kit (Qiagen, Doncaster, VIC, Australia). cDNA synthesis and reverse transcriptase-quantitative PCR (RT-qPCR) were performed as previously described, using a specific reverse transcriptase (RT) primer in the cDNA synthesis step [
57,
58]. Expression levels indicated by raw cycle thresholds (CTs) of the genes of interest were subtracted from the raw CT of the ribosomal protein L32 (
RPL32) mRNA and plotted as dCT.
RPL32 CT values were observed to be unchanged by passage number in mice or PDX. Human-specific primers for various genes examined in this study are detailed in Table
2.
Table 2
QPCR primers for various genes examined in this study
5′ Hs L32 | Human | CAGGGTTCGTAGAAGATTCAAGGG |
3′Hs L32 | Human | CTTGGAGGAAACATTGTGAGCGATC |
Hs L32 RT | Human | CAGAAAACGTGCACATGAGCTGC |
5′ Hs CD24 | Human | GACTCAGGCC AAGAAACGTC TTCTAAA |
3′ Hs CD24 | Human | GTTGCCTCTCCTTCATCTTG TACATGAAA |
Hs CD24 RT | Human | GGGCGACAAAGTGAGACTGTCTAAAA |
5′ Hs CD44 | Human | CACAATGGCCCAGATGGAGAAA |
3′ Hs CD44 | Human | CTTCGACTGTTGACTGCAATGCAAA |
Hs RT CD44 | Human | GGCAATGTTGCAAGGGTTTGTGAAGACTT |
5′ Hs VIM | Human | CAGGCGATATATTACCCAGGCAAGAA |
3′ Hs VIM | Human | CTTGTAGGAGTGTCGGTTGTTAAGAA |
Hs VIM RT | Human | CTAAATCTTGTAGGAGTGTCGGTTGTT |
5′ Hs CDH1 | Human | GGCACAGATGGTGTGATTACAGTCAAAA |
3′ Hs CDH1 | Human | GTCCCAGGCGTAGACCAAGAAA |
Hs CDH1 RT | Human | CTCTGTCTTTGGCTGCAGCACTTTA |
5′ Hs ILK | Human | GATGCAGGACAAGTAGGACTGGAA |
3′ Hs ILK | Human | CAACCAGAGGCCTGCTGCTTT |
Hs ILK RT | Human | GCTGGGGTAGTACCATGACTG |
5′ Hs TWIST1 | Human | CTAGAGACTCTGGAGCTGGATAACTAAAAA |
3′ Hs TWIST1 | Human | CGACCTCTTGAGAATGCATGCATGAAAAA |
Hs TWIST1 RT | Human | GAGAAAGTCCATAGTGATGCCTTTCCTTT |
5′ Hs SNAI1 | Human | CCAGACCCACTCAGATGTCAAGAA |
3′ Hs SNAI1 | Human | GGCAGAGGACACAGAACCAGAAAA |
Hs SNAI1 RT | Human | CGCAGACAGGCCAGCTCAGGAAT |
5′ Hs SNAI2 | Human | CCCAATGGCCTCTCTCCTCTTT |
3′ Hs SNAI2 | Human | CATCGCAGTGCAGCTGCTTATGTTT |
Hs SNAI2 RT | Human | CATCGCAGTGCAGCTGCTTATGTTT |
5′ Hs ZEB1 | Human | GTTACCAGGGAGGAGCAGTGAAA |
3′ Hs ZEB1 | Human | GACAGCAGTGTCTTGTTGTTGTAGAAA |
Hs ZEB1 RT | Human | GACAGCAGTGTCTTGTTGTTGTAGAAA |
5′ Hs ZEB2 | Human | CCACCTGGAACTCCAGATGCTTTT |
3′ Hs ZEB2 | Human | GCCTTGCCACACTCTGTGCATTT |
Hs ZEB2 RT | Human | GCCTTGCCACACTCTGTGCATTT |
5′ Hs ITGA2 | Human | GACCTATCCACTGCCACATGTGAAAAA |
3′ Hs ITGA2 | Human | CCACAGAGGACCACATGTGAGAAAA |
Hs ITGA2 RT | Human | GTCAGAACACACACCCGTTGTGTAATA |
5′ Hs ITGB1 | Human | GACTGATCAGTTCAGTTTGCTGTGTGTTT |
3′ Hs ITGB1 | Human | CCCTGCTTGTATACATTCTCCACATGATTT |
Hs ITGB1 RT | | CCCTGCTTGTATACATTCTCCACATGATTT |
5′ Ms. ITGB1 | Mouse | GCGTGTGCAGGTGTCGTGTTT |
3′ Ms. ITGB1 | Mouse | GAAGGCTCTGCACTGAACACATTCTTT |
Ms ITGB1 RT | Mouse | GAAGGCTCTGCACTGAACACATTCTTT |
Small interfering RNA (siRNA)-mediated knockdown of
ITGB1,
ITGA2 and
ILK (Horizon, [formerly Dharmacon], Melbourne, Australia) was performed in PMC42-ET cells. These cells display a molecular phenotype of Basal B (E Tomaskovic-Crook and T Blick, unpublished observation), based on clustering of a limited number of the Basal B discriminator genes [
19] showing reliable data in an Affymetrix U133A analysis kindly performed by the laboratory of Joe Gray, Lawrence Berkeley National Laboratory, Berkeley, California [
59]. These IDC-derived cells express E-cadherin mRNA and protein but do not assemble it at the cell membrane [
27]. PMC42-ET were grown in RPMI with 10% foetal bovine serum (FBS, Thermo Fisher Scientific, Australia) at 37 °C with 5% CO
2.
The siRNA target sequences for
ITGB1,
ITGA2 and
ILK are presented in Table
3. A commercial non-targeting control sequence (control siRNA) was also used (siSTABLE Non-targeting siRNA #1, Horizon, [formerly Dharmacon], Melbourne, Australia). Briefly, PMC42-ET cells were transfected using DharmaFECT4 (Horizon, [formerly Dharmacon], Melbourne, Australia) and 100 nM siRNA targeting ITGB1, ITGA2, ILK or control siRNA. The transfection efficiency of the siRNA was inferred by the level of protein knockdown that was achieved, determined by Western blot. Specificity of the siRNA knockdown of ITGB1, ITGA2 or ILK was ascertained by the lack of effects seen with a control siRNA, or against the other targets studied. After 8 h, cells were then left either unstimulated or stimulated with 10 ng/ml EGF for 72 h. Controls included cells alone (no transfection), transfection reagent alone and the control siRNA. Protein and RNA were extracted 72 h post EGF-stimulation and analysed by Western immunoblotting and RT-qPCR, respectively. Recombinant EGF was purchased from BD Biosciences, (Bedford, MA, USA).
Table 3
Sequences of siRNA used in the current study
ITGB1 siRNA | AAGCTTTTAATGATAATTCAT |
ITGA2 siRNA | TCGCTAGTATTCCAACAGAAA |
ILK siRNA | CCTGACGAAGCTCAACGAGAA |
Western blotting
Western blotting for ITGB1, ITGA2, ILK, pan-actin, Vimentin, N-cadherin and E-cadherin in siRNA transfected PMC42-ET cells +/− EGF was performed as previously described [
57], with protein extracted using RIPA (radioimmunoprecipitation assay) buffer containing protease inhibitors. The RIPA-soluble fraction (supernatant after centrifugation) was analysed in all Western blots, with the insoluble pellet discarded. In regard to the contents of this RIPA buffer, 1.58 g Tris base and 1.8 g sodium chloride was dissolved in 150 ml of dH
20 and the pH adjusted to 7.4 with HCl. Twenty millilitres of 10% NP40 (Igepal) and 5 ml of 10% Na-deoxycholate (deoxycholic acid) was added and stirred until the mixture was clear. To this, 2 ml of 100 mM EDTA was added and the total volume adjusted to 200 mL with dH
20. One protease inhibitor cocktail tablet (Roche) was added to 10 mL RIPA immediately prior to use. Antibodies and their dilutions used for Western blotting are detailed in Table
1.
Cell matrix adhesion assay
Wells of 24-well plates (polystyrene, non-tissue culture treated; Nunc Inc., Naperville, IL) were coated with 100 μg/ml collagen-I, 100 μg/ml collagen-IV, 20 μg/ml fibronectin or 50 μg/ml laminin. Collagen 1, collagen IV, laminin (from Engelbreth-Holm Swarm murine sarcoma) and fibronectin (from bovine plasma) used in these assays were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Proteins were allowed to bind to the cells overnight at room temperature under the laminar flow hood, before the wells were rinsed with phosphate-buffered saline (PBS) and non-specific interactions were blocked for 1 h at 37 °C with 3% bovine serum albumin (BSA) in PBS, pH 7.4. PMC42-ET cells were transfected with siRNA. Eight hours later, cells were left either unstimulated or stimulated with EGF for 72 h, after which the cells were detached using 0.25% trypsin and allowed to attach to the various (ECM substrate-coated plates for 1 h. Cells attached to ECM was estimated by the average of cell counts from five random high-power fields under light microscopy (counted in situ on the substrate after washing).
Monolayer wound healing assay
This was performed as previously described [
57]. Briefly, PMC42-ET cells were plated in a 6-well plate set up in triplicate and incubated at 37 °C for 24 h to allow the formation of a confluent monolayer. The cells were then wounded by using a P200 pipette tip. The wounded monolayers were washed with complete media to remove detached cells. Images of the wounds were taken at 0, 24 and 48 h. Wound areas at each time point were analysed and quantitated using ImageJ software.
Boyden chamber migration assay
Boyden chamber migration assays were performed as previously described [
60]. Briefly, transmigration culture assays were performed using 8-μm pore Transwell chambers (Corning, USA). Polycarbonate membranes (8-μm pore size) of the upper compartment of 24-well chambers were coated with 100 μg/ml collagen I in serum-free media (SFM; Roswell Park Memorial Institute [RPMI]-1640 medium). siRNA-transduced PMC42-ET cells (+/− EGF) harvested by trypsinisation were re-suspended in SFM supplemented with 0.2% BSA, and the cell suspension (2.5 × 10
5 cells suspended in 250 μl SFM) was applied to the upper compartment in triplicate wells. The lower compartment was filled with 650 μl of chemoattractant (RPMI-1640 containing 10% FBS [Sigma-Aldrich]). After 24 h of incubation, the chambers were rinsed in PBS to eliminate non-adherent cells and the remaining non-migrated cells on the upper surface of the filter were removed carefully with a cotton swab. Migrated cells on the lower side of the filter were stained with 0.5% crystal violet (Sigma Aldrich, Australia) for 15 min. The crystal violet dye retained on the filters after washing was extracted with 10% acetic acid and cell migration was measured by reading the absorbance at 560 nm on a micro-titre plate reader (PolarStar Optima, BMG Labtech, Ortenberg, Germany).
For use of the ILK inhibitor QLT0267 in Boyden Chambers, sub-confluent monolayers of the PMC42-ET cells were left either untreated or treated for 24 h with this inhibitor (QLT, Inc., Vancouver, Canada) at a final concentration of 6.25 μM prior to assay. Invasion of PMC42-ET cells in vitro was assessed by the invasion of the cells through Collagen-I-coated Transwell inserts. The inhibitor was dissolved in DMSO (0.1%), which was used as the vehicle control.
Statistical analyses
Gene expression data across PDX passages were analysed using the two-tailed Mann-Whitney test (non-parametric) and Pearson’s correlation co-efficient, and the IHC intensity changes across PDX passages were analysed using Ordinary One-Way ANOVA. Cell matrix adhesion and Boyden chamber migration assay results were analysed using two-way ANOVA with Dunnett’s multiple comparison test. All statistical analyses were performed using GraphPad Prism v7 (GraphPad Software, San Diego, USA).
Discussion
We have shown that the EDW01 PDX model displayed evidence of EMT with progressive passages through mice, which was not seen in ED03. This is consistent with the known EMT status of IDC versus ILC, of which EDW01 and ED03 are examples, respectively. However, a comparison of the progression of such divergent cancer types with regard to EMT status has enabled the discovery of some unique findings. The partial EMT observed in EDW01 was associated with a rising hypoxia leading to Twist1 expression in early-mid passages, repressing E-cadherin expression and orchestrating vimentin upregulation, and accompanied by upregulation of ITGB1 and ITGA2 expression. The mesenchymal shift appeared to then return to the epithelial direction in later passages of EDW01; however, the increased integrin expression persisted. We present cell line data to support the association of ITGA2/B1 and the ILK signalling pathway with the observed EMT, but the EMT was not mediated by these. Assessment of ITGA2/B1 and ILK expression in a published Luminal breast cancer dataset was not consistent with the pro-aggressive implications of our association with EMT in the EDW-01 PDX, since ILK predicted improved regression-free survival post treatment. Thus, further analysis of this association in other models is required to refine the interpretation.
Although the EMT-associated ITGB1, ITGA2 and ILK are not essential for EGF-induced EMT in PMC42-ET cells (Fig.
6), they are necessary for breast cancer cell adhesion to ECM-substrates (Fig.
7) and cellular movement (Fig.
8). These molecules were more significantly upregulated in increasing EDW01 passages through mice than ED03 (Figs.
3 and
4); thus, they may have enabled ECM adhesion in this PDX. Indeed, previous studies demonstrate that ITGA2/B1 is primarily a receptor for collagen and laminin [
66] and expression is also associated with motility, invasiveness, and cellular differentiation of a variety of tumours [
67,
68]. This is in contrast to studies in which ITGA2 and ITGB1 have been found to suppress metastasis in models of mouse and human cancer [
69]. However, Dedhar and Saulnier [
70] showed that the expression of
ITGA2/B1 increased in the chemically transformed human osteosarcoma cells, and this integrin was implicated in tumour progression and metastasis. Similarly,
ITGA2/B1 expression accelerated either experimental metastasis or tumour dissemination of melanoma [
71] and rhabdomyosarcoma [
72,
73], gastric cancer [
74,
75] and colon cancer cells [
76]. Taken together, our data suggest that ITGA2/B1 contribute to the EMT phenotype observed increasingly in EDW01 over serial passages in mice.
The PDX models of ED03 and EDW01 were characterised as luminal A (ER positive, Her2 negative, PR low or absent) whereas the PMC42-ET breast cancer cell line used in this study was of the Basal B molecular phenotype [
63]. However, luminal A cell lines lack the degree of plasticity that is clearly evident in the EDW-01 PDX, so we felt it was justified to compare the PMC42-ET cell line that also exhibits plasticity. The PMC42-ET cells also largely exhibit an epithelioid appearance, despite constitutive vimentin-expression, and we have shown that they respond to EMT-inducing regimens such as EGF. We acknowledge that this is a limitation of our study which directly influences the conclusions that we can logically make in regard to the importance of ITGA2/B1 in cancer progression.
Actively growing tumours acquire areas of hypoxia, a product of imperfect angiogenesis coupled with rapid growth, which can facilitate cellular invasion via the induction of EMT [
77]. Vimentin and Twist1 positivity was observed in close proximity to necrotic areas in early passages and less commonly found at the centre of tumour ‘islands’ (Fig.
3). Of the E-cadherin repressor genes examined,
TWIST1, a target of HIF1A [
78‐
80] displayed the strongest correlative pattern of induction with
CAIX (Fig.
6b,
R2 = 0.81,
p = 0.04). Induction of
TWIST1 coincided with the repression of
CDH1 and induction of
VIM mRNA and therefore may be the instigator of the observed EMT in the EDW01 xenograft model. Indeed, hypoxia has been implicated in inducing EMT-related genes in another PDX model of serial transplantation. Wegner and colleagues [
81] demonstrate in their cervical cancer PDX model serially transplanted in mice that the EMT orchestrating gene
SNAI1 and stem cell markers were found to be increased in late compared to early passages along with hypoxic
CAIX gene expression, accompanied with an increase in tumour aggressiveness and proliferative rate. Their finding, in a different cancer type (cervical), adds further support to our suggestion that hypoxia may have been a major driving force in the observed progressive EMT in the EDW01 PDX model.
However, why did the mesenchymal shift return to epithelial in later passages of EDW01? Tumours in vivo have been found to adapt to low oxygen environments, such as reprogramming Akt signalling in the mitochondria [
82]. This coupled with the well-known ability of tumours to increase angiogenesis [
83] contributes to tumour cell survival and progression. Although beyond the scope of this investigation, the EDW01 PDX model provides a means to investigate these phenomena further, with relevance to understanding the progression of breast cancer in women.
Although many studies have associated EMT with therapy resistance [
11,
84], it is important to note that the EDW01 xenograft was not challenged by therapy; the EMT progression was spontaneous. Interestingly, considerable emphasis is being placed recently on the hybrid state of EMP recently [
6,
85‐
88], and this phenotype appears to manifest in the EDW01 xenografts (elevated vimentin and apparently only partially lost E-cadherin as shown in Figs.
3 and
4). A separate analysis of circulating tumour cells (CTCs) in the ED03 model indicate that despite the lack of any evidence of EMT in the primary xenograft tumours, the CTCs are enriched in mesenchymal gene expression, but also in epithelial genes (
CDH1 and
CD24), compared to the primary tumour, indicating a dysregulation of this axis and/or possibility of hybrid cells [
58].
Tumour-stroma crosstalk plays an integral role in EMT in vivo [
89]; similarly, we observed key changes in murine stroma in the PDX models examined in this study. We have previously demonstrated that EDW01 evoked greater expression of
MMPs (
-2,
-9,
-11 and
MT1-MMP) in the murine stroma than ED03 [
56]. Furthermore, EDW01 displayed MT1-MMP and MMP-13 at the tumour-stromal boundary, but did not express these factors or MMP-2 and MMP-9 within the tumour mass itself. As shown in the current study, the EDW01 tumours grew as islands traversed by thick collagenous stromal bands whereas the ED03 had delicate pericellular stroma dispersed throughout (Fig.
1). This pattern of growth may be directly attributable to the pattern of MMP expression of each of these PDX models—EDW01 lacked the capacity to invade as individual cells, possibly due to the lack of induction of intratumoural MMP-2 and MMP-9. Furthermore, the murine microenvironment (non-orthotopic) in which the EMT occurred in the EDW01 PDX over successive passages may have been conducive to this change. We found that murine (stromal)
Itgb1 expression aligned with human (tumoural) expression of the same integrin; in fact for EDW01, stromal
Itgb1 expression was approximately 22-fold higher than tumoural
ITGB1 at passage 7 (Fig.
4). This leads to speculation as to whether the murine microenvironment was the instigator of the EMT or a responder in this process. However, given that an EMT was not observed in the ED03 line, which was passaged through mice of the same genotype (SCID), it could be postulated that drivers of EMT came from within the tumour itself, lending further support to the notion that hypoxia was an initiating event.
Decreased CD44/CD24 expression ratio in later passages in both PDX lines was an unexpected finding, at least in EDW01, as CD24
+/high/CD44
low− phenotype is associated with the epithelial phenotype despite EMT being observed in this xenograft model. However, CD24 expression can also confer adhesive properties enabling invasion. In a meta-analysis of 16 studies of 5697 breast cancers, CD24 was found to be significantly associated with poorer survival [
90], presumably due to non EMP functions. In studies on breast cancer cell lines in vivo, CD24 was found to act as a ligand for P-selectin on the lung vascular endothelium [
64]. We found that
CD24, but not
CD44, correlated with
ITGA2 and
ITGB1 in both PDX models (Fig.
5b, ii versus iii), providing further suggestion, in addition to our PMC42-ET integrin knockdown/EGF studies (Fig.
6), that activation of these integrins is not necessarily intimately related to the EMT process. We recognise the limitation that the PMC42-ET cellular behaviour was examined in two-dimensional culture, and as such, our observations are hypothesis generating and require further validation.
Posttranslational cleavage of CD44 may explain the discrepancy between CD44 protein expression between the two PDXs—EDW01
CD44 gene expression is comparable with ED03 (Fig.
4) whereas CD44 membranous protein expression is strong and uniform in ED03 but somewhat weaker and more heterogeneous in EDW01 (Fig.
3, Supplementary Fig.
4–
5). CD44 may be shed from the cell by the action of MMPs, namely MMP-9 and MT1-MMP [
91,
92], resulting in the loss of cell membrane CD44. The CD44 antibody used in this study (clone 156-3C11, Abcam) recognises cell-membrane localised CD44. As previously mentioned in our earlier studies, EDW01 evoked greater expression of MMPs (-2, -9, -11 and MT1-MMP) in the murine stroma than ED03 [
56]. As shown in Supplementary Fig.
4, the homogeneous versus heterogeneous CD44 expression in ED03 compared with EDW01 associates with the interruption of growing tumour by murine stroma, as illustrated by Masson’s Trichrome staining, where connective tissue stains blue. A greater level of CD44 cleavage and shedding may have occurred in EDW01 PDX tumours than ED03, facilitated by stromal MMPs, resulting in the observed heterogeneous pattern. MMP-directed CD44 cleavage results in nuclear translocation of the intracellular CD44 domain [
93], which can result in the transcriptional activation of EMT-associated genes [
94,
95] and induction of stemness [
96]. Nuclear CD44 has been shown to occupy the
TWIST1 promoter [
96]; therefore, CD44 cleavage in EDW01 could have contributed to
TWIST1 transcriptional upregulation (Fig.
5a).
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.