Colorectal cancer (CRC) is currently one of the most prevalent malignant diseases (Siegel
CRC cell invasion of the liver starts at the hepatic sinusoids—narrow, sinuose, and highly specialized capillaries composed of hepatic-specific non-parenchymal cell types, such as liver sinusoidal endothelial cells (LSECs), resident macrophages (also known as Kupffer cells (KC)), and pericyte-like hepatic stellate cells (HSCs). The interaction of CRC cells with LSECs via adhesion molecules is one of the earliest events that initiate the prometastatic cascade. As a result, a proinflammatory and proangiogenic microenvironment is generated (Khatib
Intercellular adhesion molecule-1 (ICAM-1) is one of the adhesion molecules proposed as a key factor triggering this initial response. ICAM-1 is constitutively expressed in LSECs and is upregulated under inflammatory conditions (Volpes
In the early phases of experimental liver colonization of CRC cells, ICAM-1 prompts the secretion of different inflammatory mediators, such as IL-1β, IL-6, PGE2, and TNF-α (Benedicto
Despite this plethora of information, little is known about the role of COX-2 as a major force in the creation of the proinflammatory and proangiogenic microenvironment that boosts tumor progression during liver metastasis by means of resident cell recruitment. In this study, we aimed to shed some light on the ICAM-1-dependent COX-2-driven mechanisms during cancer cell/LSEC crosstalk in the early stage of liver colonization. Furthermore, we analyzed, for the first time, the COX-2 dependence of the prometastatic response of HSCs in the hepatic microenvironment generated by colonizing CRC cells.
BALB/c mice, 6-8 weeks old, were obtained from Charles River (Barcelona, Spain). Maintenance, care, and experimental conditions were developed under institutional guidelines and national laws for experimental animal care. All of the proceedings were approved by the Basque Country University Ethical Committee (CEID) in accordance with institutional, national, and international guidelines regarding the protection and care of animals used for scientific purposes.
Tumor cell secretomes were obtained by culturing 5.3×104 C26 tumor cells/cm2 on 24-wells plates in complete RPMI-1640 medium overnight. Then, the media was replaced with fresh serum-free RPMI-1640 medium. After 24 h of incubation, the media was collected and centrifuged at 4000 rpm for 5 min for the collection of C26 secretome. For COX-2 inhibition, cells were treated for 1 h with 20 µM celecoxib (CLX; Sigma-Aldrich, St. Louis, MO, USA) in 1% FBS containing RPMI-1640. Afterwards, the cells were activated as indicated with sICAM-1 (200 ng/mL; Life Technologies, Carlsbad, CA, USA) in 1% FBS containing RPMI-1640 for 18 h. Finally, the medium was replaced with serum-free RPMI-1640 medium and collected after 24 h, followed by 4000 rpm centrifugation for 5 min, and the supernatant was stored at −20ºC.
The isolation and culture of mouse LSECs and HSCs have been described elsewhere (Smedsrod and Pertoft, 1985). Briefly, livers were perfused using collagenase type IV, disaggregated, and were subjected to isopycnic centrifugation using a Percoll gradient (GE Healthcare, Chicago, IL, USA). KCs were removed by differential adhesion onto plastic. The obtained LSECs were cultured onto 24-well plates coated with 0.05 mg/mL collagen type I (Thermo Fisher Scientific) at 1.6×106 cell/mL in RPMI-1640 supplemented with 5% FBS. LSECs were incubated for at least 2 h at 37ºC prior to the start of experiments. HSCs were cultured in RPMI-1640 in uncoated plates for 18 h before experimentation. For the COX-2 inhibition assays, HSCs were treated with 20 µM CLX for 1 h before adding any treatment routine. C26 were pretreated with 20 µM CLX before sICAM-1 activation before their addition to LSEC cultures.
After isolation, LSECs were incubated for 3 h with RPMI-1640 supplemented with 5% FBS before experimentation. Next, the C26 cells were added to a 1:5 LSEC/tumor cell ratio with RPMI-1640 supplemented with 1% FBS for 6 h. In some experiments, the tumor cells were subjected to either ICAM-1 stimulation, COX-2 inhibition, or both before tumor cell addition to LSEC cultures.
The presence of PGE2 and VEGF were analyzed through an enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants following the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).
For the quantification of COX-2 activity, either cell monocultures or cocultures treated under the different experimental conditions were incubated for 30 min in the presence of arachidonic acid (10 µM), and the culture supernatant was collected for the analysis of PGE2 through ELISA.
For measurement of tumor cell proliferation, 1.56×104 tumor cells/cm2 were cultured for 24 h. Cells were treated with 20 µM CLX for 1 h. The media was changed to fresh media containing 1% FBS and supplemented with increasing concentrations of sICAM-1, ranging from 50 up to 200 ng/mL. Cell proliferation was measured after 24 and 48 h using an MTT assay (Sigma-Aldrich).
For the transwell migration assay, 2×104 cells/transwell of either LSECs or HSCs were cultured for 3 h in type I collagen-coated modified Boyden chambers (Greiner BioOne, Kremsmünster, Austria). Afterward, the cells were stimulated with different secretome preparations for 18 h before analysis of the cell migration. The inserts were cut off, fixed in 4% PFA, and stained with crystal violet (Sigma-Aldrich). For the quantification, 5-10 images were obtained at 200× magnification from each insert and were counted using ImageJ software (NIH, Bethesda, MD, USA).
Fibroblasts and macrophages were cultured overnight at 2×105 cells/mL on 24-well plates in DMEM-F12 and DMEM medium, respectively, supplemented with 10% FBS. The next day, cells were treated with mitomycin C (Fisher Scientific, Thermo Fisher Scientific; 5 µg/mL for fibroblasts and 1 µg/mL for macrophages) for 2 h. Afterwards, a scratch or wound was created using a 200 µL tip, and the medium was replaced with C26 secretome obtained after different treatments, diluted at a ratio of 1:2 in fresh RPMI, and supplemented with 1% FBS. Pictures were taken at 0 and 24 h. Quantification was carried out using ImageJ software (NIH) through the measurement of total wound area after 24 h compared with that at 0 h.
A total of 2×105 tumor cells diluted in PBS were injected intrasplenically (i.s.) in BALB/c mice anesthetized with Nembutal (50 mg/kg). The mice were randomly separated into four groups as follows: control tumor-bearing mice group, control tumor-bearing CLX-treated mice group, sICAM-activated tumor-bearing mice group, and sICAM-activated tumor-bearing CLX-treated mice group. CLX was administered daily through oral cleavage for 14 d (100 µg/kg). Then, the livers were collected and fixed in a zinc solution and embedded in paraffin for histological analysis. The metastatic liver area occupied by the tumor was quantified through H&E staining in 5 µm thick liver sections. Furthermore, the collagen deposition was analyzed through Picrosirius Red staining. All
Recruitment of the host cells was analyzed in the liver tissue by immunohistochemical analysis through alpha-smooth muscle actin (ASMA) and F4/80 labeling for HSCs and macrophages, respectively. For ASMA staining, 5 µm liver tissue sections were incubated at 95ºC for 30 min in a citrate buffer (pH 6.0) for antigen retrieval. Next, endogenous peroxidase was blocked through 40 min of incubation in 3% H2O2 containing 1× PBS, followed by 1 h of incubation in 5% FBS containing 1× PBS. Then, the samples were incubated with F(ab) antibody to inhibit unspecific binding, followed by overnight incubation with the mouse anti-human ASMA antibody (1:500; DakoCytomation, Agilent Technologies, Santa Clara, CA, USA). Next, the samples were washed with 1× PBS, incubated with rat anti-mouse secondary antibody (1:500; Invitrogen, Thermo Fisher Scientific), and visualized through HRP (1:500) and DAB Quanto substrate (Thermo Fisher Scientific). For the F4/80 staining, antigen retrieval was carried out by incubating the samples with 100 µL of proteinase K (final concentration 1.2 U/mL; Sigma-Aldrich) diluted in 1 mL of TE buffer (0.5% Triton X-100 diluted in 50 mM Tris with 1 mM EDTA). After peroxidase and unspecific binding blocking, as described above, the liver tissue sections were incubated overnight with rat anti-mouse F4/80 primary antibody (1:200; Bio-Rad, Hercules, CA, USA). After incubation with a biotin-conjugated secondary goat-anti rat antibody (1:500; Invitrogen, Thermo Fisher Scientific), the process proceeded as described above.
Data are expressed as mean ± standard deviation (SD) of three independent experiments. The statistical analysis was performed using SPSS version 13.0 (Professional statistic, Chicago, IL, USA). Individual comparisons were performed using a two-tailed, unpaired Student’s
The enhancement of uncontrolled tumor growth provided by the host microenvironment is a hallmark of metastasis. We have previously shown that C26 cell colonization of the liver correlates with ICAM-1 upregulation on LSECs (Arteta
Early steps of liver colonization occur in the liver sinusoids, and the interaction of tumor cells with LSECs represent a limiting step for the development of liver metastasis. Our group has previously shown that this interaction is highly dependent on ICAM-1 (Arteta
The recruitment of LSECs and HSCs is a crucial event for the establishment and growth of experimental CRC tumors (Vidal-Vanaclocha, 2011). We have previously shown that C26 secretomes promote LSEC and HSC migration, and that sICAM-1 pretreatment of tumor cells enhanced this effect (Benedicto
To further confirm the involvement of COX-2 on the promigratory effect exerted by sICAM-1-activated C26 cells, we carried out
Although the role of COX-2 in tumor cells has been extensively studied, the involvement of hepatic COX-2 activity during liver metastasis remains mostly unexplored. As HSCs are recruited by tumor cell secretomes to liver foci shortly after their establishment (Olaso
Next, we investigated the role of sinusoidal endothelium-derived ICAM-1 and the subsequent increase in COX-2 activity in tumor cells during experimental liver metastasis of CRC. To mimic the activation of tumor cells when interacting with LSEC ICAM-1, tumor cells were treated with sICAM-1 prior to their injection into mice.
We have previously demonstrated that
Interestingly, tumor microenvironment COX-2 inhibition reduced both the PGE2 and VEGF concentrations in the portal blood serum of tumor-bearing mice 14 days after tumor cell injection (Supplementary Fig. 2). It is worth noting that, at this late stage of tumor progression, COX-2 inhibition led to the restoration of portal PGE2 levels to those observed in healthy animals. Even though the effects on the portal VEGF concentration were less notorious, they were shown to be significant 14 days after tumor cell injection into mice treated with CLX.
Next, we analyzed whether CLX inhibition modified the stromal compartment of metastatic foci. In line with our previous work (Benedicto
COX-2 has been linked with CRC and with the metastatic spread of cancer cells to the liver (Yu
We have previously shown that cancer cell interaction with LSEC ICAM-1 in the sinusoids promotes liver metastasis. Here, we show that ICAM-1 seems to stimulate COX-2 in tumor cells, promoting cancer cell proliferation. Interestingly, COX-2 is the main mediator of PGE2, which is elevated in the blood plasma of cancer patients suffering from CRC liver metastasis (Narisawa
It is well known that during metastatic growth, cancer cells recruit stromal cells to create a favorable microenvironment for tumor progression (Brodt, 2016). In the liver, this is represented by the recruitment of HSCs and LSECs into the nascent foci. Here, we show that COX-2 activation in cancer cells promotes the secretion of attractant soluble factors that induce the migration of LSECs and HSCs. Interestingly, it has been reported that endothelial cell migration and tube formation is stimulated by PGE2. This finding may explain the reported reduction in LSEC migration when these cells are stimulated with CLX-treated C26 secretomes (Jana
Interestingly, COX-2 inhibition decreased the ability of C26-secretome-activated HSCs to promote LSEC and C26 cell migration. This effect could be linked with the reduced VEGF secretion by CLX-treated HSCs, as VEGF mediates the migration of LSECs and CRC cells (Valcárcel
In concordance with our
Regarding stromal cell interaction, HSCs and macrophages are key players that drive several steps in the development of liver metastasis (Olaso
The obtained data support the role of tumor COX-2 as a mediator in the early steps of liver metastasis, which both increases the response of tumor cells to ICAM-1 and drives the prometastatic phenotype of HSCs. COX-2 amplifies the creation of a favorable niche in the liver, leading to metastatic growth. These results uncover novel roles for COX-2 while suggesting a combination approach with chemotherapy drugs to reduce metastatic lesions of CRC.
This work was supported by the Department of Industry and Research of the Basque Government SAIOTEK S-PE12UN075 and S-PE11UN043 to B.A., IT-487-09 to E.O., and by the Spanish Science and Technology Ministry MINECOR18/P32.
The authors declare no conflicts of interest.