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  • br F L induces apoptosis in


    (F) L80 induces apoptosis in vivo, as determined by TUNEL assay. TUNEL buffer without terminal deoxynucleotidyl transferase was used as a negative CTL. The extent of apoptosis is expressed as the percentage of total LY294002 that were TUNEL-positive. More than 1000 cells were analyzed per tumor from each mouse (bottom panel, ***p < 0.001). (G–H) Impact of L80 on expression of BCSC markers in vivo. Quantitation of fluorescence intensity of CD49f (G, **p < 0.01) and ALDH1A1 (H, **p < 0.01) signal is shown in the bottom panel, respectively. (I) Inhibition of tumor angiogenesis by L80 administration was determined by microvessel density assay. Tumor tissues were immunostained with CD31 (red) with DAPI, quantitative graphs represent the number of CD31-positive microvessels in intratumoral areas (***p < 0.001). (J) L80 administration resulted in a significant downregulation of vimentin expression. A quantitative graph of the vimentin signal intensity is shown in the right panel (***p < 0.001). The fluorescence intensities were analyzed using a histogram tool in the Carl Zeiss software package. Normal rabbit IgG or normal mouse IgG was used as negative controls. Data was analyzed by unpaired Student's t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    3.5. L80 attenuates tumor growth via the suppression of BCSC-like properties
    4T1 mammospheres were orthotopically injected into the duct of the fourth mammary gland of BALB/c female mice (Fig. 5A). L80 (20 mg/kg, every other day) or control solvent was administrated when the tumor volume reached approximately 50 mm3. Over the course of 27 days, 4T1 mammosphere-derived tumors subjected to L80 admin-istration exhibited a significant reduction in tumor growth (p < 0.001, Fig. 5B) and tumor burden (p < 0.01, Fig. 5C), when compared to their control groups. There was no significant change in mean body weight following L80 administration (NS; not significant, Fig. 5D). To confirm the inhibitory effect of L80 on tumor growth, Ki-67 proliferative index was undertaken using allograft tumor tissues. Immunofluorescence staining revealed that the animals receiving L80 exhibited a significant reduction in the number of Ki-67-positive cells (p < 0.001, Fig. 5E). Furthermore, the apoptotic index as determined by TUNEL assay re-vealed that a significant increase in the number of TUNEL-positive cells was observed in the L80-treated animal groups (p < 0.001, Fig. 5F).
    Our earlier in vitro observations demonstrated that L80 effectively targets BCSC-like traits (as shown in Fig. 4). To further investigate this effect, immunostaining analysis for mammary stem cell marker integrin alpha6 (CD49f) and ALDH1A1 was assessed in allograft tumors. Ani-mals receiving L80 exhibited a considerably lower level of CD49f (p < 0.01, Fig. 5G) and ALDH1A1 (p < 0.01, Fig. 5H) compared to their control counterparts. Since tumor progression is considered to arise in part by stimulation of tumor angiogenesis [36], whether the inhibitory effect of L80 on tumor growth was associated with sup-pression of angiogenesis was assessed a microvessel density (MVD) assay using the endothelial specific marker cluster of differentiation 31 (CD31). The number of CD31-positive microvessels in the intratumoral area was significantly reduced in the L80-treated groups (p < 0.001, Fig. 5I). The major epithelial-mesenchymal transition (EMT) factor vi-mentin is a well-known substrate of HSP90 and is responsible for cancer cell migration and invasion [37,38]. L80 administration caused a con-siderable downregulation of vimentin expression (p < 0.001, Fig. 5J), which may give rise to the inhibition of cell dissemination.
    3.6. L80 suppresses metastasis from primary tumors via dysregulation of STAT3 signaling
    We previously reported that BCSC-like characteristics are correlated with higher STAT3 activation and ALDH1 activity as well as enhance-ment of metastatic potential in TNBC in vivo [28]. After L80 adminis-tration, BCSC-enriched tumors exhibited a significant reduction in phospho-JAK2 (p < 0.001, Fig. 6A) and phospho-STAT3 (Tyr705) ex-pression (p < 0.01, Fig. 6B). We next evaluated the impact of L80 on metastasis in allografts derived from 4T1 mammospheres in vivo. BLI analysis revealed that mice receiving L80 exhibited a significant re-duction in bioluminescence intensity (p < 0.001, Fig. 6C). Re-presentative H&E staining further supported the evidence that L80 
    administration reduced lung and liver metastases (p < 0.01, Fig. 6D). The metastatic markers MMP-2 and MMP-9 are STAT3 downstream target genes and their presence promotes metastasis and angiogenesis via the degradation of extracellular matrix components [39–41]. Me-tastatic control mice exhibited significantly increased MMP-2 and MMP-9 serum levels when compared to normal BALB/c mice of the same age (p < 0.001) and these responses were markedly reduced by L80 administration (p < 0.01, Fig. 6E and F). Taken together, these findings suggest that L80 results in disruptions of STAT3 activity co-inciding with reduced MMP-2 and MMP-9 levels in serum, which may mitigate tumor angiogenesis and metastasis.