We have shown that the EDW01 PDX model displayed evidence of EMT with progressive passages through mice, which was not seen in ED03. This partial EMT 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 integrin α2β1 and the ILK signalling pathway with the observed EMT, but that the EMT was not mediated by these.
Although the EMT-associated ITGB1, ITGA2, and ILK are not essential for EGF-induced EMT in PMC42ET cells (Fig. 6) they are necessary for breast cancer cell adhesion to ECM-substrates (Fig. 7A-D) and cellular movement (Fig. 7E). These molecules were more significantly upregulated in increasing EDW01 passages through mice than ED03 (Fig. 3), thus they may have enabled ECM adhesion in this PDX. Indeed, previous studies demonstrate that the α2β1 integrin is primarily a receptor for collagen and laminin  and expression is also associated with motility, invasiveness, and cellular differentiation of a variety of tumours [66, 67]. This is in contrast to studies in which ITGA2 and ITGB1 have been found to suppress metastasis in models of mouse and human cancer . However, Dedhar and Saulnier  showed that the expression of α2β1 integrin increased in the chemically transformed human osteosarcoma cells, and this integrin was implicated in tumour progression and metastasis. Similarly, α2β1 integrin expression accelerated either experimental metastasis or tumour dissemination of melanoma  and rhabdomyosarcoma [71, 72], gastric cancer [73, 74] and colon cancer cells . Taken together, our data suggest that α2β1 integrins contribute to the EMT phenotype observed increasingly in EDW01 over serial passages in mice.
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 . 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. 2). Of the E-cadherin repressor genes examined, Twist1, a target of HIF-1α [77–79] displayed the strongest correlative pattern of induction with CAIX (Fig. 5B, R2 = 0.81, p = 0.04). Induction of Twist1 coincided with the repression of E-cadherin and induction of vimentin 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  demonstrate in their cervical cancer PDX model serially transplanted in mice, that the EMT orchestrating gene Snail1 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 supposition that hypoxia was 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 to have been found to adapt to low oxygen environments, such as reprogramming Akt signalling in the mitochondria . This coupled with the well-known ability of tumours to increase angiogenesis  contributes to tumour cell survival and progression. Although beyond the scope of this investigation, the EDW01 PDX model provides a means to investigate these phenomenon further, with relevance to understanding the progression of breast cancer in women.
We have previously shown that MDA-MB-468 xenografts, which express E-cadherin, exhibit hypoxia-related necrosis and subsequent EMT, features of which were lost upon E-cadherin knockdown . In the current study we also observed necrosis at p4 in EDW01 but not ED03, along with the hypoxic indicator gene CAIX induction (Fig. 5A, C). IDC, the classification of EDW01 PDX, generally proliferate at a higher rate than ILC , of which ED03 is classified. In our previous study, E-cadherin knockdown MDA-MD-468 xenografts grew slower than their control counterparts . In contrast, EDW01 (IDC) and ED03 (ILC) displayed similar Ki67 expression (Fig. 4C) and we did not find significant differences in implantation to harvest time durations between ED03 and EDW01 PDXs (data not shown). However, changes in daily growth rate cannot be ruled out between EDW01 and ED03, as apoptosis was not monitored. Regardless, the results from the current study reinforce a direct connection between E-cadherin tumoural expression and the appearance of hypoxia.
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 - Fig. 3, 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 (E-cadherin and CD24), compared to the primary tumour, indicating a dysregulation of this axis and/or possibility of hybrid cells .
Although the PDX models examined had varied ERα positivity in early passages, this increased in both models over serial transplantation in mice (Fig. 1). Mice used in this study for serial xenograft propagation were not administered estradiol, which is often used to foster the growth of ERα-positive xenografts, which generally have a lower engraftment rate than ERα negative tumours . ERα positivity is likely to have been maintained/increased because estradiol was not administered, as ERα undergoes ligand-dependent downregulation . Indeed, removal of estrogen for several weeks in PDXs has been found to increase ERα levels .
Tumour-stroma crosstalk plays an integral role in EMT in vivo , 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 . Furthermore, EDW01 displayed MT1-MMP and MMP-13 at the tumour-stromal boundary, but did not express these factors or MMP-2 and − 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. 1B). 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 intratumoral MMP-2 and − 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. 3). This leads to speculation as to whether the murine microenvironment was the instigator of the EMT or a reponder 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/CD44low− 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 5,697 breast cancers, CD24 was found to be significantly associated with poorer survival , 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 . We found that CD24, but not CD44, correlated with ITGA2 and ITGB1 in both PDX models (Fig. 5C versus 5D), providing further evidence in addition to our PMC42-ET integrin knockdown/EGF studies (Fig. 7B, C) that activation of these integrins is not necessarily intimately related to the EMT process.