Spontaneous Epithelial-Mesenchymal Transition and Resistance to HER-2-Targeted Therapies in HER-2-Positive Luminal Breast Cancer

Resistance to trastuzumab, a rationally designed HER-2-targeting antibody, remains a major hurdle in the management of HER-2-positive breast cancer. Preclinical studies suggest the mechanisms of trastuzumab resistance are numerous. Unfortunately, the majority of these studies are based around HER-2-positive (HER-2+) luminal cell lines. The role of epithelial to mesenchymal transition (EMT), a genetic program that confers a basal phenotype, may represent a novel mechanism of escape for HER-2+ luminal cells from trastuzumab treatment. Here we investigated this possibility using a model of clonal selection in HER-2+ luminal breast cancer cells. Following a random isolation and expansion of “colony clusters” from SKBR-3 cell lines, several colony clusters underwent a spontaneous EMT in-vitro. In addition to expression of conventional EMT markers, all mesenchymal colony clusters displayed a predominant CD44+/CD24- phenotype with decreased HER-2 expression and elevated levels of a β1-integrin isoform with a high degree of N-glycosylation. Treatment with a β1-integrin function-blocking antibody, AIIB2, preferentially decreased the N-glycosylated form of β1-integrin, impaired mammosphere formation and restored epithelial phenotype in mesenchymal colony clusters. Using this model we provide the first clear evidence that resistance to trastuzumab (and lapatinib) can occur spontaneously as HER-2+ cells shift from a luminal to a basal/mesenchymal phenotype following EMT. While the major determinant of trastuzumab resistance in mesenchymal colony clusters is likely the down regulation of the HER-2 protein, our evidence suggests that multiple factors may contribute, including expression of N-glycosylated β1-integrin.


Introduction
In recent years, research emphasizing the cellular hierarchy and architecture within the mammary gland has greatly enhanced our understanding of breast cancer (BC). Breast ducts have two main epithelial cell types: basal and luminal cells are bounded by a fibrous basement membrane and embedded in a growth mediating stroma rich for soluble factors [1]. Accumulating evidence points to a cellular hierarchy in the mammary gland, whereby stem cells (SC) residing in the basal layer give rise to basal and luminal cell types [2]. Together basal, luminal and SC provide the overall genetic background for four of five intrinsic subtypes, including: claudin-low (SC enriched), basal, and two luminal subtypes. The fifth intrinsic subtype is called HER-2-positive (HER-2+), and results from overexpression or amplification of the HER-2 oncogene [3].
Approximately, 15-25% of invasive breast tumors are HER-2+, a designation associated with aggressive clinical disease and poor overall prognosis [4]. As a subgroup, HER-2+ tumors can demonstrate luminal and/or basal BC features. Interestingly, a recent clinical study found that HER-2+ tumors with an underridding basal phenotype have a worse overall prognosis than basal tumors alone [5]. Nonetheless, the standard medical care for HER-2+ BC remains trastuzumab, a HER-2 directed antibody. In HER-2+ BC, trastuzumab in combination with conventional chemotherapy extends the disease and progression-free survival [6,7]. Unfortunately, rates of intrinsic resistance to trastuzumabbased treatment range from 30-50%. For those who initially respond, acquired resistance to trastuzumab typically occurs in less than one year [8,9].
Based on preclinical studies, potential mechanisms of trastuzumab resistance (TZR) include dysregulated mitogenic signaling from alternative signaling receptors or loss of tumor suppressor PTEN [9][10][11]. Interestingly, the HER-2+ cell lines used in most preclinical studies are of predominantly luminal phenotype [12]. This may significantly impact our current understanding of TZR, especially relating to recent ''cancer SC'' (CSC)-based models. For instance, HER-2+ luminal cell lines are typically enriched for a distinct CSC population defined by high aldehyde dehydrogenase (ALDH) activity. These CSC express the highest HER-2 levels and remain sensitive to trastuzumab treatment [13,14].
Interestingly, JIMT-1, the first commercial cell line established from a HER-2+ patient with intrinsic resistance to trastuzumab [15], is naturally enriched for a CD44+, CD24 negative (CD44+/ CD24-) CSC phenotype characteristic for basal/mesenchymal cells [16]. JIMT-1 also expresses high levels of b1-integrin, a basal adhesion molecule responsible for mediating most cell-matrix interactions with the breast basement membrane [17]. In breast development, b1-integrin is essential for proper SC function and tissue morphogenesis [18]. In BC, b1-integrin is implicated in nearly every aspect of tumor development and progression, including drug resistance and the induction of epithelial to mesenchymal transition (EMT) [19][20][21][22]. Interestingly, we recently identified b1-integrin as the strongest prognostic indicator of TZR in a cohort of HER-2+ metastatic BC. Additionally, when siRNA was used to knockdown b1-integrin in JIMT-1 cells, they failed to survive in culture [23].
One potential link between trastuzumab sensitive HER-2+ luminal cells and trastuzumab resistant basal/mesenchymal cells is EMT. EMT is an embryonic transcription network characterized by a CD44+/CD24-CSC phenotype and increased malignant behavior [24]. In a recent study, Oliveras-Ferraros et al, demonstrated the importance of TWIST and SLUG in maintaining the EMT phenotype of JIMT-1 cells. Accordingly, depletion of SLUG in JIMT-1 diminished the percentage of cells with a CD44+/CD24-CSC phenotype, and sensitized JIMT-1 to trastuzumab treatment [25]. A previous study by the same authors showed that spontaneous enrichment of the CD44+/CD24-CSC phenotype in JIMT-1 coincided with a global decrease in HER-2 expression [16]. Together, these results demonstrate a role for EMT in maintaining the intrinsic trastuzumab resistant phenotype of HER-2+ basal/mesenchymal cell line, JIMT-1. However, the role of EMT in trastuzumab sensitive, HER-2+/luminal cells remains to be investigated.
Here we investigated the role of EMT in TZR using luminal HER-2+ SKBR-3 cells transfected with an empty expression vector or a vector containing b1-integrin. When distinct colony clusters were isolated simultaneously from luminal SKBR-3 using multiple cloning rings, several underwent a spontaneous EMT in culture. Unlike conventional ''single cell'' cloning techniques, which yield very few expandable cell populations [26], all colony clusters of SKBR-3 survived the expansion phase in culture. Characterization of this ''de-novo'' EMT-phenotype in SKBR-3 revealed decreased HER-2 levels and expression of a heavily Nglycosylated variant of b1-integrin, associated malignant traits in BC cells [27]. Here we present original evidence that TZR can result from the spontaneous conversion of HER-2+ cells to a CD44+/CD24-/HER-2-low phenotype through EMT. Furthermore, our results suggest a function link between EMT, Nglycosylated b1-integrin and basal CSC in HER-2+ BC.

Materials and Methods
Cell culture and reagents SKBR-3 (American Type Culture Collection) and JIMT-1 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) were cultured in DMEM with 10% fetal bovine serum and 1% antibiotic/antimycotic (Invitrogen) in a 5% CO 2humidified incubator at 37uC (standard conditions). Trastuzumab (Genentech) was purchased from the pharmacy at the Cross Cancer Institute. Lapatinib Ditosylate (Biovision) was solubilized in DMSO. Rat-IgG1 and Human-IgG1 (Jackson Immuno Research Laboratories) served as control antibodies for AIIB2 and trastuzumab, respectively. N-Glycosidase-F (PGNase-F) was used as recommended by the manufacturer (New England Biolabs).

Clone isolation
SKBR-3 clones were selected from cells stably transfected with empty vector (SKBR-3/EV) or b1-integrin (SKBR-3/b1) as described previously [23]. Briefly, cells were seeded at low density onto 10cm plates (100,000 cells total). Using multiple 10mm cloning rings, ''colony clusters'' were isolated simultaneously from a single plate of cells after 10-12 days growth (standard conditions). Please refer to Methods S1 for cloning schematic and legend detailing the procedure.

Image capture and morphological assessment
A Seniscam camera attached to a Zeiss Axiovert 200M microscope with a Zeiss Plan-NEOFLUAR 5x/0.15 or 10x/0.3 lens was used unless otherwise stated. MetaMorph v7.0 was used to export images (TIFF). Adobe Photoshop CS4 was used to process images (levels and cropping). Error bars = 100 mm. Cell Profiler (www.cellprofiler.org) [28] was used for morphology analysis. Briefly, a pipeline to mask and outline cells was generated from randomly selected images. Cell masks were assessed for eccentricity (cell resemblance to a circle), generating a value between 0 (circle) to 1 (line) for each cell.

Gene expression profiling
Agilent Whole-Genome Arrays were analyzed with Genespring-GX-7.3 software. Sample preparation was performed as previously described [29]. Gene expression profiles from post-EMT (passage .5) cultures of SK-EV-C4, SK-b1-C1, and passage-matched cultures of SKBR-3/EV, SKBR-3/b1, were compared. Features selected had at least a 2X difference in at least one cell-cell comparison (one-way ANOVA, P-value .0.05). Graphs presented as fold change relative to gene expression in SKBR-3/EV.

Hierarchical clustering and pathway analysis
Expression profiles from SKBR-3/EV and SK-b1-C1 were compared. A differential gene list was generated to include genes differing by at least 1.5X, with a t-test P-value of .0.02 (unpaired, Bonferroni corrected). A total of 1940 entities met these criteria, including b1-integrin and HER-2. Canonical Pathways Analysis (Ingenuity Systems, www.ingenuity.com) was performed using a Fisher's exact test. Hierarchical clustering was performed using Euclidean distance metric and centroid linkage and displayed as a heat Map (Genespring).

AIIB2 production and purification
See Methods S1. Briefly, b1-integrin positive, AIIB2 hybridoma cells detected by indirect ELISA were grown in hyperflask culture vessels with 10 gas permeable layers (Corning). Rat-monoclonal antibody, AIIB2, was purified from the supernatant. AIIB2 treatment: Following overnight attachment, cells in standard conditions were treated for 72 h with AIIB2 or rat-IgG (monolayer culture). For mammosphere culture, growth media was supplemented with AIIB2 or IgG at indicated concentrations. Mammospheres were assessed as described above.

Trastuzumab and lapatinib sensitivity
Cells were seeded into 96-well microtiter plates (1,500/well in 100 mL) in standard conditions. Media was treated with trastuzumab, human IgG1, lapatinib or vehicle at indicated concentrations. After 72 h, cell proliferation was assessed as previously described [23]. Results presented relative to control condition.

Conditioned media
An equal number of SKBR-3/EV or SK-b1-C1 cells (500,000 cells/60 mm dish) were allowed to grow overnight in standard conditions. Media was collected the following day, spun at 500 g for 3 min to remove cellular debris and dead cells. Supernatant was mixed 1:1 with fresh standard media prior to use.

Statistics
Where applicable, results are presented as mean plus standard deviation (SD) from at least 3 independent experiments performed in triplicate or quadruplicate. Significance assessed using twotailed student t-tests with a P-value ,0.05 considered to be significant.

Results
De-novo SKBR-3 phenotype is mediated by spontaneous EMT, and results in conversion to a HER-2 negative basal/ mesenchymal phenotype When we cultured SKBR-3 cells as clones, phenotypic heterogeneity was apparent between colonies. Comparatively, no heterogeneity was observed between colonies in basal/mesenchymal cell line, JIMT-1 (data not shown). We used multiple cloning rings simultaneously on a single dish of cultured cells to isolate distinct clusters of SKBR-3 colonies (''colony clusters'') transfected with either an empty expression vector or one containing basal marker, b1-integrin. We also isolated colony clusters from trastuzumab resistant JIMT-1 cells known to have elevated levels of endogenous b1-integrin (Fig. S1).
To determine the molecular phenotype and identify heterogeneity among different SKBR-3 colony clusters, we used gene expression profiling. Gene expression data from luminal SKBR-3/ EV and SKBR-3/b1 cells, together with mesenchymal colony clusters, SK-EV-C4 and SK-b1-C1, was compared and then normalized to SKBR-3/EV for analysis. Data was analyzed for differences in HER receptors, integrins, and major SC markers (Pvalue cutoff = 0.05). In terms of HER receptors, EGFR levels remained similar between all cell lines. Alternatively, HER-2 and HER-3 levels were significantly decreased in mesenchymal colony clusters, SK-EV-C4 and SK-b1-C1 (Fig. S3). Interestingly SC related alpha6-integrin was upregulated in mesenchymal colony clusters but not in luminal SKBR-3/b1 cells (Fig. 2a).

N-glycosylation of b1-integrin plays a functional role in the SKBR-3 EMT phenotype
In this study, SK-b1-C1 cells gained expression of a high molecular weight variant of b1-integrin following EMT. To investigate the relationship between observed b1-integrin variants and EMT, we returned to an early passage of colony cluster SK-b1-C1. Using cell shape as a phenotypic marker for EMT, we monitored sequential passaged of SK-b1-C1 cells. Notably, the quantity of spindle-shaped cells increased exponentially between passage 4 and 5, marking the point of EMT in SK-b1-C1 (P-value ,0.001). Interestingly, by western blotting we confirmed that the molecular weight shift in b1-integrin coincided precisely with the expression of EMT markers and loss of HER-2 in SK-b1-C1 cells. EMT-inducer, TWIST-1, was expressed in passage 1 and 3 SK-b1-C1 cells, preceding the occurrence of EMT (Fig. 3a).
Alterations to the electrophoretic mobility of b1-integrin, typically corresponds to differences in N-glycosylation [27,32]. In this regard, it appears that the 105 KDa 'precursor' form of b1integrin becomes completely exchanged for the 130 KDa 'mature' form of the protein in passage 5 of SK-b1-C1 cells. To confirm if N-glycosylation accounts for the shift in electrophoretic mobility of b1-integrin, we used PGNase-F, an enzyme that hydrolyzes Nglycan chains from glycoproteins [33]. Lysates from SKBR-3/b1, SK-b1-C1, and SK-EV-C4 cells were treated with PGNase-F. Subsequent resolution of proteins by SDS-page confirmed increased N-glycan deposition on b1-integrin in mesenchymal colony clusters (Fig. 3b).
To evaluate a role for N-glycosylated b1-integrin in the basal/ mesenchymal phenotype of SK-b1-C1, we used AIIB2, a b1integrin antibody that may preferentially target the high molecular weight isoform of b1-integrin, and has been shown to revert the malignant phenotype in BC cells [37,38]. Here, we found that treatment of SK-b1-C1 cultures with 10 and 100 mg/ml of AIIB2 decreased the N-glycosylated variant of b1-integrin. Remarkably, this was accompanied by a restoration of round epithelial morphology in SK-b1-C1 cells (Fig. 3c). To characterize other changes in EMT marker proteins caused by AIIB2 treatment, we performed additional western blotting of lysate treated with AIIB2 or IgG control. After protein expression was quantified and analyzed, our results showed that AIIB2 treatment of SK-b1-C1 cells induced an extensive down-regulation of b1-integrin and EGFR proteins, as well as, a modest down-regulation of phosphorylated (p)-FAK (Tyr397), vimentin, and TWIST-1 (Fig. S7).
Next, we treated mammosphere cultures from colony cluster SK-b1-C1 with AIIB2 or IgG control, and assessed sphere formation following 10 days of treatment. Notably, treatment with 10 or 100 mg/ml of AIIB2 resulted in a dramatic 54%, and 59% reduction in the number of mammospheres formed, respectively (P-value ,0.001) (Fig. 3d). To determine if reduced mammosphere formation was the direct result of inhibited cell proliferation by AIIB2, we treated SK-b1-C1 cells with antibody. After 72 hours, both doses of AIIB2 treatment (10 and 100 mg/ml) resulted in a 19% reduction in cell proliferation in SK-b1-C1. These doses did not inhibit the proliferation of SKBR-3/EV cells (Fig. S7). This modest reduction in cell proliferation is unlikely to account for the greater than 50% reduction in mammosphere formation in SK-b1-C1 following AIIB2 treatment.
EMT results in spontaneous resistance to HER-2 targeted therapies in SKBR-3 colony clusters Finally, we used our model of spontaneous phenotype plasticity in luminal, HER-2+, trastuzumab sensitive SKBR-3 cells to investigate a role for EMT in resistance to trastuzumab. First, cell lines were treated with a common preclinical dose of trastuzumab, 10 mg/ml representing the ideal target serum level in patients [8]. Our results showed that, while luminal SKBR-3 cell lines remained sensitive to trastuzumab treatment, mesenchymal colony clusters were completely resistant (P-value ,0.001) (Fig. 4a). Notably, all colony clusters isolated from JIMT-1 cells retained a high level of resistance to trastuzumab treatment (Fig. S8). Interestingly, western blotting of SKBR-3/EV colony clusters revealed altered expression of several TZR-associated proteins simultaneously in mesenchymal colony clusters. For instance, p-FAK (Tyr397), p-Akt (Ser473), and Cyclin-D1 were elevated in SK-EV-C3 and SK-EV-C4, while the level of p27 (KIP1) was decreased (Fig. 4b).
To determine if trastuzumab sensitivity was correlated with HER-2 and/or b1-integrin expression we quantified HER-2 and b1-integrin receptor levels based on protein expression. We used western blotting of HER-2 and b1-integrin expression for several luminal (SKBR-3/EV, SK-EV-C1, SKBR-3/b1) and mesenchymal (SK-EV-C4, SK-b1-C1, JIMT-1) cell lines. Normalized protein expression values were correlated to trastuzumab sensitivity as determined by XTT assay. We found a statistically significant positive correlation (r = 0.75, P = 0.043) between trastuzumab resistance and b1-integrin expression across cell lines analyzed. Additionally, we found a statistically significant negative correlation (r = 20.92, P = 0.004) between trastuzumab resistance and expression of HER-2. Next, we analyzed whether, or not, the degree of b1-integrin N-glycosylation was correlated with trastuzumab resistance. Our results showed a trend towards higher levels of N-glycosylation and trastuzumab resistance, but this did not reach statistical significance (P = 0.081) (Fig. 4c).
Next, we looked to determine if the mixture of growth factors and cytokines secreted by SK-b1-C1 cells could mediate resistance to trastuzumab in luminal SKBR-3/EV cells. To accomplish this, SKBR-3/EV cells were cultured in the presence of conditioned media by SK-b1-C1 or SKBR-3/EV (as control). Following 72 h, we observed spindle (mesenchymal) morphology in SKBR-3/EV cells exposed to SK-b1-C1-conditioned media. Furthermore, western blotting showed downregulated HER-2 levels and increased expression of both precursor and mature b1-integrin isoforms following treatment with SK-b1-C1 conditioned media.
Lapatinib, a small molecule inhibitor of EGFR and HER-2 signaling, was recently approved for use in trastuzumab refractory BC [39]. Interestingly, b1-integrin mediated signaling has been reported to play a role in lapatinib resistance in HER-2+ BC [38].
To determine if mesenchymal SKBR-3 colony clusters displayed resistance to lapatinib therapy, cells were treated with established doses of lapatinib [40] and proliferation was assessed by XTT assay. Following 72h of treatment, JIMT-1 cells, along with all SKBR-3 mesenchymal colony clusters, were resistant to 2 and 5 mm of lapatinib treatment. Luminal colony clusters, on the other hand, remained sensitive to all concentrations of lapatinib tested (P-value ,0.001) (Fig. 4e). Together, these results suggest that mesenchymal colony clusters have shifted reliance away from the HER-signaling axis for growth and survival.

Discussion
Despite ranking as the second leading cause of cancer-related death in women worldwide, significant advances are being made in the diagnosis and treatment of BC [41]. For example, the discovery of molecular biomarkers has helped standardize the classification and management of the disease. Two routine clinical biomarkers assessed in BC, include: ER and HER-2. ER, a marker of differentiated luminal epithelium, is expressed in ,65% of all BCs. These tumors are generally well differentiated (low-grade) and less aggressive than ER-negative BCs. On the other hand, patients with basal or HER-2+ BC have poor survival outcomes. While HER-2 remains an important clinical biomarker, it fails to identify which HER-2+ patients are most likely to respond to treatment [42]. Consequently, high rates of drug resistance and cardiotoxictiy remain long standing problems associated with trastuzumab use [4].
Recent studies have found that BC cell lines, including those HER-2+, contain multiple CSC populations defined by distinct markers [43]. In this regard, CSC defined by a CD44+/CD24- phenotype are typically enriched in claudin-low cell lines that are HER-2 negative. However, emerging evidence suggests that these HER-2 negative CSC are not only present in HER-2+ cell lines, but may actually contribute to TZR [44]. In addition, the discordance rates for HER-2 expression between matching primary and metastatic tumors has been reported [45]. For example, in one study of 382 matched tumors, a discordance rate of 34% was observed. Notably, HER-2+ patients whose metastatic disease had converted to HER-2-negative on biopsy (23.6%) had a worse overall prognosis [46]. In another study, the HER-2 gene signature was diminished in residual tumor cells following treatment with conventional therapies, while the proportion of cells enriched for claudin-low, EMT/CSC gene expression, was increased [47]. Based on our evidence presented here, one potential explanation for these findings would be an EMTmediated loss of HER-2 expression during tumor progression. Furthermore, our results highlight a potential role for the heavily N-glycosylated, mature isoform of b1-integrin in EMT and the claudin-low phenotype in BC cells.
Given the down-regulation of HER-2 in mesenchymal SKBR-3 colony clusters, the findings of trastuzumab resistance may seem largely predictable. However, multiple studies have demonstrated that trastuzumab treatment can be effective in BC with low or negative HER-2 expression. A study by Paik et al, showed no correlation between HER-2 gene amplification and response to adjuvant trastuzumab treatment. Furthermore, the authors found patients with normal levels of the HER-2 (so called ''HER-2 negatives'') received benefit from adjuvant trastuzumab therapy [48]. A preclinical study by Diermeier et al, makes a similar conclusion that HER-2 expression status alone is not able to predict trastuzumab resistance in BC cell lines, including SKBR-3.
Here the authors showed that sensitivity to trastuzumab is not solely based on HER-2 receptor status, but rather influenced by co-expression of alternative HER receptors and ligands. For instance, SKBR-3 cells co-express elevated levels of EFGR (HER1) compared to luminal, HER-2+ BT-474 cells. As consequence, when treated with trastuzumab, BT-474 cells are more likely to enter a quiescent state compared to SKBR-3. Additionally, treatment of both cell lines with neuregulin (NRG), a HER ligand, was able to completely compensate for trastuzumab mediated inhibition of cell proliferation [49].
To investigate changes in HER receptors and ligands between different SKBR-3 colony clusters, we used gene expression profiling. Interestingly, we found decreased levels of HER-2 and HER-3 in mesenchymal colony clusters compared to luminal cell lines (Fig. S3). EGFR levels remained consistent throughout all SKBR-3 cell lines, indicating that EGFR is unlikely to account for the trastuzumab resistant phenotype in mesenchymal colony clusters. Notably, the levels of NRG were significantly upregulated in mesenchymal but not luminal colony clusters (Table 1). Together with modulation of p-AKT, p-FAK, Cyclin-D1, and p27 (KIP1) (Fig. 4b), up-regulated NRG levels represent an additional regulator of trastuzumab response that is modulated by EMT in SKBR-3 cells.
According to the ''intrinsic'' model for cell culture heterogeneity, phenotype divergence occurs inherently as cell-fate determinants are asymmetrically segregated during cell division. In comparison, the ''extrinsic'' model proclaims that changes in the local environment influence cell differentiation [50]. While our SKBR-3 model provides support for the latter, the conversion of SKBR-3 cells from a luminal to a claudin-low phenotype should be recognized in light of an important caveat. First, the fact that SKBR-3 cells are negative for two functionally important luminal markers, ER and E-Cadherin, may have facilitated their shift in phenotype. Second, a comprehensive survey of 51 BC cell lines showed the claudin-low phenotype to be enriched in 27% of cell lines. This is compared to less than 10% of breast tumors enriched for the same basal/mesenchymal traits (claudin-low) [3,12].
These figures suggest that cell culture conditions may provide a more favorable environment for propagation of the claudin-low phenotype. Future studies using additional cell lines, including: trastuzumab sensitive, HER-2+ BT-474 cells (ER and E-cadherin positive), are therefore warranted.
In conclusion, our data highlight the role of EMT in TZR in luminal HER-2+ BC. We demonstrated the functional significance of a heavily N-glycosylated variant of b1-integrin in the EMT phenotype. Furthermore, while variant N-glycosylation is an established hallmark of cancer, specific N-glycosylation targets remain a relatively untapped class of biomarkers and therapeutic targets [51]. Accordingly, most previous studies of b1-integrin have focused primary on intracellular signaling pathways and cellmatrix interactions. Our findings presented here represent a novel link between EMT, N-glycosylated b1-integrin and the therapeutic consequences of cell plasticity in luminal HER-2+ BC cells.
The authors of this publication declare that all experiments described herein comply with the law of the country in which they were performed. Figure S1 Isolation of colony clusters from HER-2+ SKBR-3 breast cancer cells.
[d] SKBR-3/EV cells were cultured for 72 h in the presence of conditioned media (CM) obtained from SKBR-3/EV or SK-b1-C1. Images and western blotting showed increased spindle morphology (d-i), decreased levels of HER-2, and increased levels of b1-integrin (d-ii) in SK-b1-C1-CM treated cells. Cell proliferation following CM-exposure was assessed as previously described, and showed resistance to trastuzumab in SKBR-3/EV cells exposed to SK-b1-C1-CM (d-iii).