Histological comparison of the ED03 and EDW01 xenografts with increasing passages through mice
The ED03 PDXs was serially passaged up to passage (p) 11, whereas the EDW01 PDX was passaged up to p7. Histologically, the ED03 xenografts displayed a diffuse growth pattern with minimal visible tumour stroma, often growing in long cords of cells, consistent of ILC. By contrast, EDW01 PDX revealed histology consistent with IDC, with clearly visible stromal septae separating growing tumour islands (Figure 1). In ED03 the stromal collagen was evident only under higher magnification as it was finer and more pericellular compared with EDW01, in which the thicker stromal cords separated lobules of tumour. This stroma was of murine origin, as it did not stain with human specific vimentin antibody (Figure 1).
Assessment of estrogen receptor (ERa) across serial passages.
We investigated the expression of ERa in the ED03 and EDW01 PDX models. As shown in Figure 2 (low power in part A, higher power shown in part B), immuno-reactivity to ERa in the ED03 PDX was approximately 99% tumour cells, and this level of staining was maintained across passages 2-7. The EDW01 PDX also displayed almost 100% positivity for ERa at p3, however this progressively declined to approximately 40% in passage 6 and 7. These relative changes are plotted in Figure 2C. Progesterone receptor (PR) expression was negative in ED03 in all passages and was weak (<15%) in EDW01 at p3, disappearing by p4. HER-2 in both PDXs was negative (data not shown). The clinical approximated subtypes of breast cancer (defined according to the 2011 St Gallen International Breast Cancer Conference) classifies both ED03 and EDW01 as Luminal A, since Ki67 is less than 14% in both PDXs (Figure 3) .
Immunohistochemistry (IHC) and RT-qPCR quantification of EMT markers
To assess any changes in EMT status over sequential passaging, key effector molecules implicated in the EMT process (vimentin/E-cadherin, Twist1, beta-catenin, P120-RasGTPase activating protein [P120-RasGAP], CD24/CD44) and the proliferative marker Ki67 were screened in the ED03 and EDW01 PDX models across the series of passages using IHC (Figure 3, Supplementary Figure 2) and human-specific RT-qPCR (Figure 4).
E-cadherin immunostaining was almost completely absent in ED03 original patient material, consistent with its lobular carcinoma derivation . We subsequently confirmed a putative somatic missense variant (p.His128Asn, data not shown). Consistent with this, less than 1% of cells expressed E-cadherin in any ED03 PDX passage in mice (Figure 3, top left panel). This was reflected in a relative E-cadherin staining intensity index per cell score of the range 0-0.2 compared with 0.2-0.9 for EDW01 (Supplementary Figure 1). Similarly, beta-catenin was not detectable within the ED03 PDXs, p120-RasGAP was aberrant, with staining observed to be mostly cytoplasmic. By contrast, EDW01 PDXs displayed strong E-cadherin immunostaining (Figure 3, top right panel) and readily detectable RNA levels (Figure 4). However, this was accompanied by a progressive increase in human-specific vimentin mRNA expression with each passage (Figure 4): passage 6 material displayed an approximate 8-fold increase (P=0.008), and passage 7 material displayed an approximate 12-fold increase (P=0.024) in comparison to passage 3 material. This was consistent with a significant (p>0.05) increase in vimentin protein intensity per cell index across the passages for EDW01, derived from IHC (Supplementary Figure 1). E-cadherin positive tumour cells transitioning to vimentin positivity (possibly remaining E-cadherin positive, see high magnification inset, Figure 3) were observed in EDW01 PDXs whereas vimentin mRNA and protein in ED03 were almost negligible (Figure 3, 4B, Supplementary Figure 1).
As shown in Figure 3, positive nuclear Twist1 expression was seen only in EDW01 xenograft tumours, adjacent to regions of necrosis (as indicated by black arrows), however nuclear Twist1 positivity did not increase in abundance across the passages (Supplementary Figure 1). Beta-catenin and P120-RasGAP was also mainly observed in EDW01 at the cell membrane, and corresponded with E-cadherin staining.
We went on to further examine the expression of breast cancer stem cell markers CD44 and CD24, as upregulation of CD44 and downregulation of CD24 is observed in breast cancer cell line EMT . ED03 displayed a homogeneous CD44 IHC pattern, which was relatively consistent throughout the passages at the protein and mRNA level (Figure 3, 4, Supplementary Figure 2-3). By contrast, CD44 protein was heterogeneously expressed in EDW01. Although CD24 was upregulated with increasing passages in both models, EDW01 exhibited higher mRNA abundance overall than ED03 (Figure 4), consistent with the appearance (Figure 3) and quantification of protein abundance by IHC (Supplementary Figure 3). Within EDW01, but not ED03, there were regions of tumour cells that appeared to lack both CD24 and CD44 (Figure 3).
A parallel study of integrin expression in the PMC42-ET breast cancer cell line induced to undergo EMT with EGF revealed that ITGA2 and ITGB1, and their downstream regulator ILK, were consistently upregulated (Supplementary Figure 4). Hence ILK and these integrins were examined further in the PDX models. Increases in ITGA2 (p7 significantly higher than p3) and ITGB1 (p6 significantly higher than p3) were observed in ED03 xenograft material, which were maintained (Figure 4). However, similar to the increased vimentin seen with each passage in EDW01, the levels of human ITGB1 mRNA in the xenografts increased with successive passage, demonstrating an approximately 26-fold increase (p=0.026) at passage 6 in comparison to passage 3 (Figure 4). Whilst passage 7 showed approximately a 38-fold increase in ITGB1 mRNA levels when compared to passage 3, this did not reach statistical significance. However, ITGA2 mRNA expression in EDW01 xenografts (Figure 4) was significantly upregulated by approximately 33-fold at passage 7 material when compared to passage 3 (p=0.024). ILK is activated by integrins including a2b1, and mediates a number of signalling responses in relation to survival and proliferation in addition to induction of EMT . A trend was observed towards upregulation of ILK mRNA expression in both ED03 and EDW01 xenografts (Figure 4).
Murine (stromal) ITGB1 displayed a similar pattern of upregulation as human (tumoural) ITGb1 (Figure 4).
These findings illustrate that with serial passage EDW01 has accrued features consistent with EMT. The co-induction of the mRNA levels of ITGB1 and ITGA2 in EDW01 implies that they may be important for the EMT process and/or phenotype, because they track with the indices of EMT (decreased E-cadherin and increased vimentin) clearly observed in this model system.
E-cadherin expressing xenograft tumours exhibit more necrosis
Cores of tumours from the EDW01 series at passage 3 exhibited noticeably more necrosis than any other passage from this line, whereas necrosis was minimal or absent in all PDXs across the various passages in the ED03 line (data not shown). We have previously demonstrated that E-cadherin expression is associated with high proliferative rate and observed an association with E-cadherin expression and the appearance of necrotic tissue in actively growing xenografts . We investigated whether an association existed between E-cadherin expression in the EDW01 line and the proportion of necrosis. As shown in Figure 5, a trend was observed such that in the cores that had detectable necrosis (measured as % total area of the core), E-cadherin was generally expressed at a high level, and more highly expressed than vimentin, measured as % total area of the core.
Further investigation of EMT drivers and markers in the EDW01 xenograft model.
Hypoxia is a common driver of EMT in breast cancer, and E-cadherin repressor genes have been implicated in this process . We sought to examine the pattern of Snail1, Snail2, Twist1 and Zeb1/2 expression through the serial passages in mice in the ED03 and EDW01 xenograft models, in comparison to the hypoxic indicator gene carbonic anhydrase 9 (CAIX).
As shown in Figure 6, of the E-cadherin repressor genes examined (Snail1, Snail2, Twist1, Zeb1 and Zeb2), Twist1 was more highly expressed in the EDW01 xenograft compared with ED03 (Figure 6A, i). Zeb1 and Zeb2 were not expressed at detectable levels in either PDX. Twist1 displayed the highest correlation with CAIX in the EDW01 xenograft model, with both exhibiting a progressive increase from p2 to p5 which was then reduced in p6 to p7 (Figure 6A, i). Membrane intensity of CAIX (Figure 6C) aligned with the expression data, suggesting Twist1 may drive hypoxia-induced EMT through consecutive passages of the EDW-01 PDX.
CD24 is an epithelial-associated marker with relevance to breast cancer stem cells, where its expression is reduced in comparison to luminal breast cancer cells ; it’s expression has been shown to indirectly stimulate cell adhesion to fibronectin, collagens I and IV, and laminin through the activation of integrin activity . Interestingly, the expression pattern of CD24 with ITGB1 was significantly positively correlated in the ED03 series (R2=0.9, p=0.0012) and in the EDW01 series (R2=0.96, p=0.023). CD24 was also positively correlated with ITGA2 in the ED03 series (R2=0.84, p=0.0035) and this reached near significance in the EDW01 series (R2=0.76, p=0.054) (Figure 6C). No significant or near significant correlations were observed for CD44 with integrins in either PDX systems (Figure 6B, iii).
Functional assessment of candidate genes ITGB1, ITGA2 and ILK in the PMC42 system
As expression of the α2β1 integrin components were associated with the EMT observed in EDW01 xenografts over serial passages through mice, we tested whether they could be a “driver” of the EMT, using the PMC42 EMT model system.
Although already somewhat mesenchymal , PMC42-ET cells treated with EGF in vitro undergo a further EMT in which ILK, ITGB1 and ITGA2 are upregulated (supplementary Figure 1), . We examined the effects of siRNA knockdown of ITGA2, ITGB1 and ILK on late stage mesenchymal gene expression, cell adhesion and cell migration.
As shown in Figure 7A, individual siRNA knockdown of ITGB1, ITGA2 or ILK resulted in the expected reduction of expression of the target genes. In addition, ILK inhibition also led to the reduction of ITGB1 protein levels by 80% (0.2 in Figure 7A); inhibition of ITGB1 also led to reduced ITGA2 expression (0.06 in Figure 7A)); and, ILK inhibition led to a reduction in ITGA2 expression by 60% (0.41 in Figure 7A). Inhibition of ITGB1 and ITGA2 by siRNA did not affect ILK protein levels. These data indicate a complex interplay between these three components
To determine whether the cells with suppressed ITGB1, ITGA2 or ILK were able to undergo EMT with EGF treatments, cellular morphology and protein expression was examined. After 72 hours of EGF treatment, cellular morphology (Figure 7B) revealed a clear acquisition of spindle-shapes and breaking apart of cellular islands, consistent with an EMT. Protein expression of the “classical” indicators of EMT, vimentin, E-cadherin, and N-cadherin were measured by Western immunoblotting (Figure 7C). E-cadherin was dramatically reduced in the “cells alone” treated with EGF compared to the untreated “cells alone” control, whereas slight increases in vimentin and N-cadherin were observed in these untransfected cells, consistent with EGF-induced EMT of these cells as previously reported [27, 63].
No observable differences in the responses of vimentin, N-cadherin or E-cadherin protein levels to 72hrs of EGF treatment were seen following treatment with ITGB1, ITGA2 or ILK siRNA, when compared to treatment with control siRNA (Figure 7C). Similarly, the mesenchymal morphology caused by EGF treatment was not abrogated by any of the siRNAs (Figure 7B).
Together, this indicates that these candidates do not directly mediate the EMT induced by EGF in this breast cancer cell line.
We then investigated what effect the stepwise increase in integrin expression, observed in the EDW01 xenografts (Figure 4) may have had on growth and invasion/motility of these tumours over serial passages in mice, inferred by parallel analyses in the PMC42-ET cells. We focused on cell adhesion to various substrates and migration, properties known to be mediated by integrins and ILK, and these were again examined in PMC42-ET cells.
PMC42-ET cells require ITGB1, ITGA2 and ILK for maximal adherence to collagen I, collagen IV, laminin and fibronectin substrates, as knockdown of these molecules significantly reduced adhesion in comparison to control siRNA (Figure 8A-D, p<0.05). When stimulated with EGF, the attachment of the PMC42-ET cells treated with ITGB1, ITGA2, and ILK siRNA was also significantly abrogated (p<0.001).
Coordinated regulation of cell adhesion and adhesion complex remodelling are crucial for cell movement. ITGB1, ITGA2, and ILK siRNA-mediated silencing in PMC42-ET cells caused them to be significantly less migratory, as shown by the assessment of migration using the Boyden Chamber Assay (Figure 9A) and the Monolayer Wound Healing assay (Figure 9B). Inhibition of ILK by a specific inhibitor, QLT0267, also significantly reduced cellular movement in the Boyden Chamber Assay (Figure 9C, p<0.001). EGF treatment caused these cells to increase their migration, whereas ITGB1, ITGA2, and ILK silencing each significantly reduced migration in both assays under EGF-stimulated conditions (Figure 9A, B, p<0.05).