[PubMed] [Google Scholar] 18. (N-cadherin), an adhesion molecule that promotes invasion, was assessed by PCR. Expression of N-cadherin and its precursor form (pro-N-cadherin) was assessed by immunoblotting and flow cytometry. Pro-N-cadherin immunohistochemistry was performed on tumors obtained from patients pre- and post- neoadjuvant chemotherapy treatment. Results TNBC cells surviving short-term chemotherapy treatment exhibited increased invasive C1qtnf5 behavior and capacity to colonize metastatic sites compared to untreated tumor cells. The invasive behavior of chemo-resistant cells was associated with their increased cell surface expression of precursor N-cadherin (pro-N-cadherin). An antibody specific for the precursor domain of N-cadherin inhibited invasion of chemo-resistant TNBC cells. To begin to validate our findings in humans, we showed that the percent cell surface pro-N-cadherin (+) tumor cells increased in patients post- chemotherapy SKF-96365 hydrochloride treatment. Conclusions TNBC cells surviving short-term chemotherapy treatment are more invasive than bulk tumor cells. Cell surface pro-N-cadherin expression is associated with the invasive and chemo-resistant behaviors of this tumor cell subset. Our findings indicate the importance of future studies determining the value of cell surface pro-N-cadherin as: 1) a biomarker for TNBC recurrence and 2) a therapeutic target for eliminating chemo-residual disease. = 5.5 x 10-11; **, BT549, = 0.0001. C. Invasive potential of parental and chemo-residual SUM159 (left panel)and BT549 (right panel) tumor cells was measured in a Matrigel transwell assay. Top panel shows a representative field of crystal-violet stained invasive cells (100X magnification). Bottom panel shows quantitation of invasion, determined by counting the mean # invasive cells from triplicate wells [+/- standard error of the mean (SEM)] for each of the cell populations. Similar results were obtained in at least 3 independent trials for A.-C. **, SUM159; = 0.01; **, BT549- = 0.005, = 10), was assessed by luciferase signal, is indicated. *, = 0.03. B. After 34 d, animals were sacrificed, and lungs were removed and photographed (left panel). Macro-metastases were counted, and are reported as median number macroscopic metastases/mouse. Previous studies indicate that long-term chemotherapy selection models drive the growth of cancer stem-like cells [4-8]. We therefore sought to determine if chemo-resistant TN tumor cells emanating from our short-term chemotherapy treatment model exhibit cancer stem-like properties. As shown in Supplementary Figure 2, chemo-resistant tumor cells from our model did not exhibit an increased ability to grow as non-adherent spheres, a defining property of cancer stem-like cells. In fact, they had decreased ability compared to their non-treated parental counterparts. To measure their self-renewing activity, primary spheres were dissociated into single cells, and the efficiency of secondary sphere formation was determined. As shown in Suppl. Figure 2B, chemo-resistant tumor cells from our model did not exhibit increased self-renewing activity compared to parental tumor cells. Because cancer stem-like cells exhibit increased tumor-initiating activity, we next assessed the relative tumor-initiating ability of chemo-resistant and parental triple-negative tumor cells in an orthotopic mouse model. SUM159 cells obtained pre- and post-chemotherapy were injected in a limiting dilution study into the mammary fat pad of NSG mice (10 mice/group). Tumor volumes were assessed using calipers on a weekly basis until tumors reached a size of 100 mm3, at which point they were measured every 2-3 days until volumes reached 2000 mm3. As shown in Suppl. Figure 2C, tumor cells obtained post-chemotherapy treatment did not exhibit increased tumor-initiating activity compared to untreated TN tumor cells at any injection number. Furthermore, there were no differences in tumor growth rate between chemo-residual and parental grafts (Supplementary Figure 3). Long-term chemotherapy selection models drive an epithelial-mesenchymal transition in estrogen receptor-positive breast tumors, characterized by reduced epithelial adhesion marker (E-cadherin) and acquired mesenchymal adhesion marker (N-cadherin) expression. By SKF-96365 hydrochloride contrast, SKF-96365 hydrochloride triple-negative breast cancers are typically mesenchymal in nature, expressing significant N-cadherin prior to chemotherapy treatment. We performed real-time PCR to determine relative levels of N-cadherin in parental (untreated) and chemo-resistant SUM159 cells from our short term chemotherapy treatment model. As shown in Figure ?Figure3A,3A, SUM159 cells obtained post-chemotherapy treatment exhibited a seven-fold increase in N-cadherin mRNA levels compared to that observed in untreated SUM159 cells. Surprisingly, levels of N-cadherin protein (120 kDa) were equal in in SUM159 cells obtained pre- and post-chemotherapy treatment (Figure ?(Figure3B).3B). We did however observe that the N-cadherin antibody reacted with a higher molecular weight species, the expression of which was significantly increased in SUM159 SKF-96365 hydrochloride tumor cells obtained post-chemotherapy treatment compared to parental SUM159 tumor cells (Figure ?(Figure3B).3B). Based on the knowledge that N-cadherin is synthesized as a precursor protein (pro-N-cadherin) that is cleaved SKF-96365 hydrochloride by proteases to generate the mature form [11], we next investigated levels.
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