Inhibition of COX-2 Impairs Colon Cancer Liver Metastasis through Reduced Stromal Cell Reaction

Abstract

Liver colonization is initiated through the interplay between tumor cells and adhesion molecules present in liver sinusoidal endothelial cells (LSECs). This crosstalk stimulates tumor COX-2 upregulation and PGE₂ secretion. To elucidate the role of the LSEC intercellular adhesion molecule-1 (ICAM-1) in the prometastatic response exerted by tumor and stromal COX-2, we utilized celecoxib (CLX) as a COX-2 inhibitory agent. We analyzed the in vitro proliferative and secretory responses of murine C26 colorectal cancer (CRC) cells to soluble ICAM-1 (sICAM-1), cultured alone or with LSECs, and their effect on LSEC and hepatic stellate cell (HSC) migration and in vivo liver metastasis. CLX reduced sICAM-1-stimulated COX-2 activation and PGE₂ secretion in C26 cells cultured alone or cocultured with LSECs. Moreover, CLX abrogated sICAM-1-induced C26 cell proliferation and C26 secretion of promigratory factors for LSECs and HSCs. Interestingly, CLX reduced the protumoral response of HSC, reducing their migratory potential when stimulated with C26 secretomes and impairing their secretion of chemotactic factors for LSECs and C26 cells and proliferative factors for C26 cells. In vivo, CLX abrogated the prometastatic ability of sICAM-1-activated C26 cells while reducing liver metastasis. COX-2 inhibition blocked the creation of a favorable tumor microenvironment (TME) by hindering the intratumoral recruitment of activated HSCs and macrophages in addition to the accumulation of fibrillar collagen. These results point to COX-2 being a key modulator of processes initiated by host ICAM-1 during tumor cell/LSEC/HSC crosstalk, leading to the creation of a prometastatic TME in the liver.

Keywords: CAF; Colorectal cancer; Cyclooxygenase-2; Hepatic stellate cells; Liver metastasis; Tumor microenvironment.

Fig. 1

Effect of COX-2 inhibition on tumor cell proliferation. The effect of COX-2 inhibition was analyzed in untreated colorectal cancer (CRC) cells and upon stimulation with recombinant sICAM-1 for 24 and 48 h, untreated or treated with 20 µM celecoxib (CLX). Tumor cells were treated with CLX for 1 h and stimulated with sICAM-1 for 24 and 48 h. Cell viability was quantified through MTT assay in three independent experiments. The differences between untreated and sICAM-1 stimulated cells were considered statistically significant at *p<0.05 using Student’s t-test. Differences between CLX-untreated versus CLX-treated cells were considered significant at ^#p<0.05 and ^##p<0.01 using Student’s t-test.

Fig. 2

Measurement of PGE₂ secretion and COX-2 activity in cell cultures. (A, B) C26 monocultures were stimulated with 200 ng/mL sICAM-1 and treated with 10 and 20 µM CLX for the analysis of PGE₂ production and COX-2 activity. (C, D) For the coculture experiments, C26 cells were treated with CLX before culturing them with freshly isolated primary LSECs for 6 h. Afterwards, the supernatants were collected and analyzed for PGE₂ concentration and COX-2 activity using ELISA and arachidonic acid metabolization, respectively. Differences between untreated and sICAM-1-stimulated cells were considered statistically significant at *p<0.05 using Student’s t-test. Differences between CLX-untreated versus CLX-treated cells were considered significant at ^#p<0.05 and ^##p<0.01 using Student’s t-test.

Fig. 3

The effect of COX-2 inhibition in tumor-mediated liver sinusoidal endothelial cell (LSEC) and hepatic stellate cell (HSC) migration. (A, B) Freshly isolated primary LSECs and HSCs were cultured in modified Boden chambers and were stimulated with either C26 secretomes or CLX-treated C26 secretomes for 24 h. Afterwards, the inserts were fixed and stained with crystal violet for the quantification of migrated LSECs and HSCs. (C, D) NIH 3T3 and J774.1 were plated in 24-well plates and stimulated with C26 or CLX-treated C26 secretomes for 24 h after creating a scratch. Closure of this area by each cell line was quantified through ImageJ software. Differences between the control cells versus C26-secretome-stimulated cells were considered statistically significant at *p<0.05 and **p<0.01 using Student’s t-test. Differences between C26-secretome-activated versus C26-activated and CLX-treated cells were considered significant at ^#p<0.05 by Student’s t-test.

Fig. 4

Analysis of HSCs’ protumoral effect upon COX-2 inhibition. COX-2 was inhibited in freshly isolated primary HSCs, and several processes linked with tumor progression were analyzed. CLX-treated HSCs were stimulated with C26 secretomes and their migration was analyzed (A). HSCs were treated with CLX and stimulated with C26 secretomes. Afterwards, soluble factors released by HSCs were collected. The migration of LSECs (B), C26 (C), and the proliferation of C26 (D) was measured in response to CLX-treated and C26-secretome-activated HSCs secretomes. Differences between untreated versus HSCs or C26-secretome-activated cells were considered statistically significant at *p<0.05 and **p<0.01 using Student’s t-test. Differences between HSC or C26-secretome-stimulated HSCs versus CLX-treated HSC or C26-secretome-stimulated HSC-treated cells were considered significant at ^#p<0.05 and ^##p<0.01 using Student’s t-test.

Fig. 5

Liver metastasis development in response to sICAM-1 stimulation and COX-2 blockade. C26 tumor cells were stimulated or not with sICAM-1, and injected in the distal pole of the spleen in mice. Mice were treated with oral cleavage of CLX for 14 days, and the development of liver metastasis was quantified in terms of metastatic area and foci number and size (A, B). The levels of VEGF and PGE₂ were analyzed in the plasma isolated from the portal blood seven and fourteen days after tumor cell injection into the control mice, C26-injected mice and C26-injected CLX-treated mice (C, D) (n=6). Differences in metastatic development between untreated C26 versus sICAM-1-treated C26 were considered statistically significant at *p<0.05 using Student’s t-test. Differences between CLX-untreated versus CLX-treated cells were considered significant at ^#p<0.05 and ^##p<0.01 using Student’s t-test.

Fig. 6

Differences in intratumor stromal compartment of liver metastasis in sICAM-1- and CLX-treated mice. The infiltration of ASMA- and F4/80-expressing cells was quantified in the metastatic foci of mice under different treatment routines (A, B). Furthermore, the accumulation of intratumoral fibrillar collagen was analyzed (C) (n=6). Differences between the untreated C26 versus sICAM-1-treated C26 cell intratumoral expression of ASMA, F4/80, and collagen were considered statistically significant for *p<0.05. Differences between CLX-untreated versus CLX-treated animals were considered significant at ^#p<0.05 and ^##p<0.01 using Student’s t-test.

Fig. 7

Tumor COX-2 is upregulated during early stage of liver colonization. Upon tumor cell/LSEC interplay, ICAM-1 stimulates COX-2 increase and PGE₂ secretion by CRC cells. COX-2 mediates the secretion of promigratory factors for LSECs, HSCs, and macrophages favoring the creation of a protumoral tumor microenvironment. Moreover, HSC COX-2 supports tumor growth, promoting LSEC migration, tumor cell proliferation, and migration. Therefore, the ICAM-1-mediated increase in COX-2 arises as a promoter of liver metastasis in CRC.