Adhesion of pancreatic cancer cells in a liver-microvasculature mimicking coculture correlates with their propensity to form liver-specific metastasis in vivo

Abstract

Organ-specific characteristic of endothelial cells (ECs) is crucial for specific adhesion of cancer cells to ECs, which is a key factor in the formation of organ-specific metastasis. In this study, we developed a coculture of TMNK-1 (immortalized liver sinusoidal ECs) with 10T1/2 (resembling hepatic stellate cells) to augment organ-specific characteristic of TMNK-1 and investigated adhesion of two pancreatic cancer cells (MIA-PaCa-2 and BxPC-3) in the culture. MIA-PaCa-2 and BxPC-3 adhesion in TMNK-1+10T1/ 2|coating culture (TMNK-1 monolayer over 10T1/2 layer on collagen coated surface) were similar. However, in TMNK-1+10T1/ 2|gel (coculture on collagen gel surface), MIA-PaCa-2 adhesion was significantly higher than BxPC-3, which was congruent with the reported higher propensity of MIA-PaCa-2 than BxPC-3 to form liver metastasis in vivo. Notably, as compared to BxPC-3, MIA-PaCa-2 adhesion was lower and similar in TMNK-1 only culture on the collagen coated and gel surfaces, respectively. Investigation of the adhesion in the representative human umbilical vein ECs (HUVECs) cultures and upon blocking of surface molecules of ECs revealed that MIA-PaCa-2 adhesion was strongly dependent on the organ-specific upregulated characteristics of TMNK-1 in TMNK-1+10T1/ 2|gel culture. Therefore, the developed coculture would be a potential assay for screening novel drugs to inhibit the liver-microvasculature specific adhesion of cancer cells.

Figure 1

Images of the cells in the collagen coated well cultures on day 4. (a) Monolayer morphology of 10T1/2 cells (red) without TMNK-1. (b) 10T1/2 cells (red) show a dispersed morphology containing cytoplasmic processes (indicated by arrows) with TMNK-1. (c) Monolayer of TMNK-1 (green) over 10T1/2 cells. Scale bar represents 200 μm.

Figure 2

BxPC-3 and MIA-PaCa-2 adhesion in the TMNK-1 only and TMNK-1 + 10T1/2 cultures in the collagen coated wells. Columns and error bars represent mean ± SEM of two independent experiments. Each independent experiment had six wells for each culture condition. Statistical significance is shown using symbols *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Figure 3

Images of the cells in the collagen gel cultures on day 4. (a) Monolayer morphology of 10T1/2 cells (red) without TMNK-1. (b) 10T1/2 cells (red) show a dispersed morphology containing cytoplasmic processes (indicated by arrows) with TMNK-1. (c) TMNK-1 cells (green) form monolayer over 10T1/2. (d) Haematoxylin and eosin (HE) staining of vertical cross section of TMNK-1 only culture. (e) HE staining of TMNK-1 + 10T1/2 culture. Some 10T1/2 cells in the culture can be distinguished by their elongated morphology beneath the TMNK-1 layer. Scale bars represent 200 μm (for (a), (b), and (c)) and 100 μm (for (d) and (e)).

Figure 4

BxPC-3 and MIA-PaCa-2 adhesion in the TMNK-1 only and TMNK-1 + 10T1/2 in the collagen gel cultures. Columns and error bars represent mean ± SEM of two independent experiments. Each independent experiment had six wells for each culture condition. Statistical significance is shown using symbols *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Figure 5

Expression of TMNK-1 phenotypic marker VAP-1 (a) and LYVE-1 (b) in various culture conditions. (a1), (a2), (b1), and (b2) and (a3), (a4), (b3), and (b4) show representative images showing the expression of the respective markers in the collagen coated and gel cultures, respectively. Similarly, (a5) and (b5) and (a6) and (b6) show the average pixel intensity of the immunostaining in the cultures. Intensity was averaged from at least five images taken from two samples for each condition. Columns and error bars represent mean ± SEM. Statistical significance is shown using symbols *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Figure 6

Expression of TMNK-1 phenotypic marker Stabilin-1 (a) and ICAM-1 (b) in various culture conditions. (a1), (a2), (b1), and (b2) and (a3), (a4), (b3), and (b4) show representative images showing the expression of the respective markers in the collagen coated and gel cultures, respectively. Similarly, (a5) and (b5) and (a6) and (b6) show the average pixel intensity of the immunostaining in the cultures. Intensity was averaged from at least five images taken from two samples for each condition. Columns and error bars represent mean ± SEM. Statistical significance is shown using symbols *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Figure 7

BxPC-3 and MIA-PaCa-2 adhesion in the HUVECs only and HUVECs + 10T1/2 in the collagen gel cultures. Columns and error bars represent mean ± SEM of two independent experiments. Each independent experiment had six wells for each culture condition. Statistical significance is shown using symbols *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Figure 8

Adhesion of BxPC-3 (a) and MIA-PaCa-2 (b) in the TMNK-1 only and TMNK-1 + 10T1/2 in the collagen gel cultures upon blocking of VAP-1 ((a1) and (b1)) and ICAM-1 ((a2) and (b2)). In the VAP-1 blocking experiment, two peptides were used: GGGGGGGGK (P1, as control) and GGGGKGGGG (P2, effective peptide), which bound poorly and efficiently with VAP-1, respectively [25]. ICAM-1 was blocked with anti-ICAM-1 antibody, while control was an isotype IgG antibody. Columns and error bars represent mean − SD of two to three independent experiments. Each independent experiment had four to six wells for each culture condition. Statistical significance is shown using symbols *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Figure 9

Adhesion of BxPC-3 (a) and MIA-PaCa-2 (b) in the HUVECs only and HUVECs + 10T1/2 in the collagen gel cultures upon blocking of VAP-1 ((a1) and (b1)) and ICAM-1 ((a2) and (b2)). In the VAP-1 blocking experiment, two peptides were used: GGGGGGGGK (P1, as control) and GGGGKGGGG (P2, effective peptide), which bound poorly and efficiently with VAP-1, respectively [25]. ICAM-1 was blocked with anti-ICAM-1 antibody, while control was an isotype IgG antibody. Columns and error bars represent mean − SD of two to three independent experiments. Each independent experiment had four to six wells for each culture condition. Statistical significance is shown using symbols *(P < 0.05), **(P < 0.01), and ***(P < 0.001).