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Page 10 of 14 Anand et al. J Cancer Metastasis Treat 2019;5:6 I http://dx.doi.org/10.20517/2394-4722.2018.98

Figure 5. Analyses of incidence of metastases, and total metastatic load, in nude mice with 4T1 breast tumors. Mice from four different
treatment groups [vehicle only, capecitabine (CPBN) only, vehicle + photodynamic therapy (PDT) and CPBN + PDT] were quantitatively
analyzed as follows. (A) Incidence of metastases by week 3. Note that the first three treatment groups showed 60%-70% of mice with
metastases, whereas the CPBN + PDT treatment group showed only 17% of mice with metastases; (B) metastatic tumor load per mouse,
calculated by comparing the cumulative bioluminescence units (BLUs) from each mouse with the average cumulative BLUs from no-PDT
controls. The metastatic load of 14% in CPBN + PDT group is much lower than the other three groups (96%-111%)

hematoxylin and eosin. Figure 6A clearly shows the presence of metastatic tumor nests (marked with dotted
lines) in the lung of a mouse that was positive by IVIS imaging. Keratin 14 (K14, a marker for epithelial
breast tumors at ectopic sites; Figure 6A), was expressed only within tumor nests in the lungs of tumor-
bearing mice and not in the lungs from control mice [Figure 6A; bottom]. Comparing this to the primary
tumor (which also stained strongly for K14; Figure 6B), the pattern of K14 expression was interesting, in that
the most intense expression was observed in the tumor periphery, with far fewer positive cells in the central/
stromal (hypoxic) tumor regions (Figure 6B; top vs. bottom), possibly due to hypoxia and necrosis in the
center and the proliferative nature of the tumor periphery, respectively. This observation further supports
the assertion that the primary breast tumor was the origin of the K14-positive, metastatic tumor nests in the
lungs. Histology of cutaneous metastatic lesions also clearly confirmed the presence of metastatic carcinoma
in skin (Figure 6C; righthand image marked with dotted lines). Further exploration of how CPBN-PDT
exerts its inhibitory effect on 4T1 metastatic spread is underway.

DISCUSSION
In this study we have shown, in a murine model of BCA, that pretreatment with CPBN prior to ALA-
mediated PDT causes selective enhancement of PpIX levels within 4T1 tumors, and improves the
treatment outcome of PDT by enhancing tumor cell death. We also showed, using histological and