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). Abundance of stromal area observed for EDW01 shown in here in Figure 1 did not visibly increase with subsequent passaging in mice (Supplementary Figure 1). Tumoural cores examined were generally representative of histology observed in the donor blocks from which the tissue microarray was assembled (Supplementary Figure 2).
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 until p7, where ERa was found to be low in some of the TMA cores. 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) 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 3). 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 VIM mRNA expression with each passage (Figure 4): passage 6 material displayed a significant increase (p=0.008), and passage 7 material also displayed significant 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 3). 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 VIM mRNA and protein in ED03 were almost negligible (Figure 3, 4 Supplementary Figure 3).
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 3). 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 4-5). By contrast, CD44 protein was heterogeneously expressed in EDW01. EDW01 exhibited higher mRNA abundance overall than ED03 (Figure 4), consistent with the appearance (Figure 3) and quantification of protein abundance by IHC where the increase in CD24 was found to be significant with increasing passage number (Supplementary Figure 5). Within EDW01, but not ED03, there were regions of tumour cells that appeared to lack both CD24 and CD44 (Figure 3), however these regions in EDW01 that are negative for CD44/CD24 do not increase over passage number (Supplementary Figure 4).
A parallel study of integrin expression in the PMC42-ET breast cancer cell line induced to undergo EMT with EGF indicated that ITGA2 and ITGB1, and their downstream regulator ILK, appeared to be upregulated (Supplementary Figure 6). 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 significantly higher expression (p=0.026) at passage 6 in comparison to passage 3 (Figure 4). Furthermore, ITGA2 mRNA expression in EDW01 xenografts (Figure 4) was significantly upregulated at passage 7 material when compared to passage 3 (p=0.024). ILK is activated by integrins including ITGA2/B1, 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 suggest that with serial passage EDW01 has accrued features consistent with EMT. The co-induction of the mRNA levels of ITGB1 and ITGA2 in EDW01 indicates 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) observed in this model system.
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 SNAI1, SNAI2, 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 5, of the E-cadherin repressor genes examined (SNAI1, SNAI2, TWIST1 and ZEB1/2), TWIST1 was more highly expressed in the EDW01 xenograft compared with ED03 (Figure 5A, i). ZEB1 and ZEB2 were not expressed at detectable levels in either PDX. Both TWIST1 and CAIX appeared to exhibit a similar expression pattern across the passages in the EDW01 PDX (Figure 5A, ii). Pearson correlation analyses of this data (shown in Figure 5B, i) indicated that this relationship was significant (R2=0.81, p=0.04). Furthermore, the pattern of membrane intensity of CAIX (Figure 5C), where we observed CAIX to be increase at p4 then drop at p6 in the EDW01 PDX, appeared to align with the CAIX (and TWIST1) gene expression data (shown in Figure 5A, ii). This suggests that Twist1 may be somewhat functionally involved in the hypoxia-induced EMT through consecutive passages of the EDW01 PDX, however further investigation is needed.
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 5B, iii).
Functional assessment of candidate genes ITGB1, ITGA2 and ILK in the PMC42-ET system
As expression of the ITGA2/B1 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-ET 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 4), . 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 6A, 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 6A); inhibition of ITGB1 also led to reduced ITGA2 protein expression (0.06 in Figure 6A)); and, ILK inhibition led to a reduction in ITGA2 protein expression by 60% (0.41 in Figure 6A). Inhibition of ITGB1 and ITGA2 by siRNA did not affect ILK protein levels. These data indicate a complex interplay between these three components.
We observed a a greater dynamic range in the induction of vimentin with EGF after 72 hours treatment in Supplementary Figure 6C compared to Figure 6B. This may be ascribed to differing behaviours of cells in culture over time and according to confluency when passaging but also differing gel/film exposure times between the two experiments. Despite the observable difference in regards to vimentin, one can deduce a clear and somewhat comparable downregulation of E-cadherin in both experiments.
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 6C) 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 6B). 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 6B). Similarly, the mesenchymal morphology caused by EGF treatment was not abrogated by any of the siRNAs (Figure 6C).
Together, seems to suggest 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 7A-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 8A) and the Monolayer Wound Healing assay (Figure 8B). Inhibition of ILK by a specific inhibitor, QLT0267, also significantly reduced cellular movement in the Boyden Chamber Assay (Figure 8C, 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 8A, B, p<0.05).
Evaluation of ITGB1, ITGA2 and ILK with respect to clinical parameters in breast cancers: analysis of a published dataset
Gene expression levels of ITGA2, ITGB1, ILK were assessed across a previously published breast cancer cohort  with respect to Regression Free Survival (RFS), Overall Survival (OS), Distant Metastasis Free Survival (DMFS) and Progression Free Survival (PFS), specifically examining Luminal A breast cancers. Of the genes examined, only ILK was found to be significant, and only for Regression Free Survival, with high expression being predictive of this clinical parameter. These results are shown in tabular form in Supplementary Figure 7. Kaplan-Meier curves for ILK in regards to RFS are shown in Figure 9. Its expression in Luminal A breast cancers is shown compared to RFS in other breast cancer molecular subtypes of Luminal B, Basal and Her-2. The results show that ILK specifically predicts improved RFS in Luminal A breast cancers.