Fig 1.
Omental fat CM enhances pancreatic cancer cell growth.
(A) XTT assay demonstrating a significant increase (P< .01) in proliferation of pancreatic cancer cells after incubation with omental fat CM, n = 7; (B) Omental fat CM markedly increased pancreatic cancer cell colony formation capacity (P< .01). The upper panel graphs represent the average of four repeated independent experiments ±SD, and the lower panel depicts representative images of cell colonies in soft agar (magnification, X100); (C) Omental fat CM-induced S-phase population in pancreatic cancer cells; a more pronounced effect was seen in PANC-1 cells than in MIA-PaCa-2 cells (P< .05). Bar plots display the data of 7 independent experiments.
Fig 2.
Omental fat CM enhances pancreatic cancer cell migration and invasion.
(A) Wound healing scratch assay demonstrating the effect of omental fat CM on PANC-1 and MIA-PaCa-2 cell migration; Scale bar = 200 μm. (B); Modified Boyden chamber assays depicting the effects of omental fat CM on pancreatic cancer cell migration (P< .001); (C) Matrigel invasion chamber demonstrating a significant increase in invasion of PANC-1 and MIA-PaCa-2 cells pre-treated with omental fat CM (P< .05); (D) Increased invasion of PANC-1 and MIA-PaCa-2 cells by using omental fat CM as a chemoattractant (P < .01). The upper panel graphs represent the average of 5 repeated experiments ± SD, and the lower panel depicts representative images (magnification, X100).
Fig 3.
Omental fat CM augments pancreatic cancer cell chemoresistance.
(A) XTT assay demonstrating a significant increase in survival of gemcitabine-treated pancreatic cancer cells following incubation with omental CM (P< .05), n = 4; (B and C) Annexin-V/PI FACS analysis demonstrating a marked reduction in gemcitabine-induced apoptosis of PANC-1 (B; P< .05) and MIA-PaCa-2 cells (C; P< .05) pretreated with omental fat CM, n = 5.
Fig 4.
Tumor growth is promoted by omental fat in vivo.
(A) Facilitation of tumor growth and weight of PANC-1 tumors in mice following pre-treatment with omental fat CM (n = 15). Graphs represent the average of three repeated experiments ±SD (P< .05); (B) Representative tumor and mice images. (C) Marked increase in proliferation (Ki-67) and microvessel density (CD31) by human omental fat CM. Representative immunohistochemistry (IHC) images are shown on the left (H&E, x200; Ki-67, x200; CD31, x200). IHC quantification is shown on the right. n = 10 in tissues of each site; Scale bar = 100 μm.
Table 1.
Cancer-related proteins identified by LC-MS/MS analysis of human omental fat (OF) versus subcutaneous fat (SC).
Fig 5.
Molecular characterization of omental fat CM-treated PANC-1 cells.
(A) PCA of gene expression microarray data. The PCA graphs of global gene expression data were computed using Partek GS, version 6.6; RM control samples are shown as red spheres, n = 3; CM samples are shown as yellow spheres, n = 9. (B) Affymetrix microarray hierarchical clustering performed on mRNA of PANC-1 cells treated with omental fat CM compared to RM. A colored bar indicating the standardized log2 intensities accompanies the expression profile. (C) qRT-PCR validation of the expression levels of OPN in PANC-1 cells pretreated with omental fat CM compared to RM, n = 6. Cells were analyzed by Western blot for the expression of OPN protein levels. α-actinin was used as a protein loading control, n = 6.
Table 2.
Cancer-related genes identified by mRNA array analysis of PANC-1 cells pre-treated with omental fat conditioned medium (CM) versus control regular medium (RM).