Adiponectin and Breast Cancer Figure 1. Serum adiponectin distributions in wildtype and PyVT mice. The serum adiponectin concentrations were measured by an in- house sandwich ELISA assay using blood samples collected from the tail vein of FVB/N and C57BL/6J mice. The median and mean values were calculated and displayed in the table. doi:10.1371/journal.pone.0004968.g001 evidence suggested that adiponectin deficiency might result in the development of a basal-like subtype tumor, which could be aroused from a different origin or subgroups of stem cells that developed tumor more aggressively. Accelerated growth of primary tumor cells derived from adiponectin haplodeficient mice We next isolated the primary tumor cells from the PyVT mice, and examined their tumor development in athymic nude mice following the protocol described previously [39]. Since tumors originated from different torso regions of PyVT mice might show various degrees of aggressiveness and onset latencies, we collected only tumors from the axillary mammary glands and re-implanted the isolated tumor cells into the posterior glands of athymic nude mouse by intraductal inoculation. The transplantation was reproducible and tumor growth rate correlated with the number of transplanted cells. Consistent with those observed in PyVT mice, the tumor development of cells derived from adiponectin haplodeficient mice was more aggressive than those from mice with normal adiponectin expressions (Figure 5, A and B). The accelerated tumor growth was reproducibly observed even when the cells were re-implanted for multiple times in new batches of nude mice (data not shown), suggesting that serial transplantation preserved the molecular characteristics of the tumor origin. At the time of sacrifice, the total weights of the collected tumors from PyVT(+/2)/ADN(+/2) mice were heavier than those of PyVT(+/ 2)/ADN(+/+) mice (Table 2). Note that when the tumor cells were exposed to physiological adiponectin secreted from adipo- cytes in the mammary tissue of nude mice, the change in the tumor volume was smaller, but still significantly different between ADN(+/2) with ADN(+/+) groups. At ,3 weeks after tumor occurrence, the measurable tumor volumes were 5.5 and 2.8 fold higher in FVB/N male and female PyVT(+/2)/ADN(+/2) animals respectively than those of PyVT(+/2)/ADN(+/+) mice (Figure 3), whereas the differences for the implanted nude mice were approximately 3.2 and 2.1 fold for male and female tumor cells respectively (Figure 5). The results further suggest that the magnitude of tumor growth could be suppressed in the presence of endogenous expression of adiponectin by adipocytes. The lung tissues of mice implanted with male tumor cells showed elevated wet weights than those implanted with female tumor cells. Moreover, there was a significant difference between the two nude mice groups implanted with male PyVT(+/2)/ADN(+/+) and PyVT(+/2)/ADN(+/2) tumor cells, with much higher lung weights in the later group (Table 2). Massive lumps of metastatic tumor mass could be seen on the surface of the lungs from nude mice implanted with male PyVT(+/2)/ADN(+/2) tumor cells. Hematoxylin and eosin staining confirmed that the metastatic capacities of these tumor cells were much higher than those from other groups (Figure 6). We next compared the proliferation of the isolated primary tumor cells in culture by using [3H]-thymidine incorporation assay (Figure 5, C and D). Cells derived from PyVT(+/2)/ADN(+/2) mice showed dramatically enhanced DNA synthesis under both 0.5% FBS and 10% FBS DMEM culture conditions. Moreover, the fold changes of [3H]-thymidine incorporation between the two time points (24 hr and 48 hr) in ADN(+/2) group were greater than those of ADN(+/+) group. Similar results were also obtained by crystal violet staining and cell number counting (data not shown). These data demonstrated that tumor cells derived from adiponectin haplodeficient mice were more aggressive, and their intrinsic properties were well preserved even under conditions without any hormonal interference. Elevated PI3K/Akt/beta-catenin signalling in tumor cells derived from adiponectin haplodeficient mice We previously reported that chronic treatment of adiponectin could modulate GSK3beta/beta-catenin pathway in MDA-MB- PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4968