- Research article
- Open Access
- Open Peer Review
The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model
© The Author(s). 2016
- Received: 4 January 2016
- Accepted: 29 June 2016
- Published: 7 July 2016
Colorectal cancer is one of the most common types of cancers worldwide. Recent studies have identified cancer-initiating cells (CICs) as a subgroup of replication-competent cells in the development of colorectal cancer. Although it is understood that an inflammation-rich tumor microenvironment presumably supports CIC functions, the contributory factors are not very well defined. The present study advances our understanding of the role of the eicosanoids leukotriene D4 (LTD4) and prostaglandin E2 (PGE2) in the tumorigenic ability of CICs and investigates the consequential changes occurring in the tumor environment that might support tumor growth.
In this study we used human HCT-116 colon cancer ALDH+ cells in a nude mouse xenograft model. Protein expression and immune cell was determined in tumor-dispersed cells by flow cytometry and in tumor sections by immunohistochemistry. mRNA expressions were quantified using RT-q-PCR and plasma cytokine levels by Multiplex ELISA.
We observed that LTD4 and PGE2 treatment augmented CIC-induced tumor growth. LTD4-and PGE2-treated xenograft tumors revealed a robust increase in ALDH and Dclk1 protein expression, coupled with activated β-catenin signaling and COX-2 up-regulation. Furthermore, LTD4 or PGE2 accentuated the accumulation of CD45 expressing cells within xenograft tumors. Further analysis revealed that these infiltrating immune cells consisted of neutrophils (LY6G) and M2 type macrophages (CD206+). In addition, LTD4 and PGE2 treatment significantly elevated the plasma levels of cysteinyl leukotrienes and PGE2, as well as levels of IL-1β, IL-2, IL-6, TNF-α and CXCL1/KC/GRO. In addition, increased mRNA expression of IL-1β, IL-6 and IL-10 were detected in tumors from mice that had been treated with LTD4 or PGE2.
Our data suggest that both LTD4 and PGE2 promote CICs in initiating tumor growth by allowing modifications in the tumor environment. Our data indicate that new therapeutic strategies targeting eicosanoids, specifically LTD4 and PGE2, could be tested for better therapeutic management of colon cancer.
- Colon cancer
- Cancer-initiating cells
Colorectal cancer (CRC) is a major healthcare burden and the fourth most common cause of cancer-related deaths in the Western world [1, 2]. The etiological factors and pathogenic mechanisms underlying the development of CRC are complex and heterogeneous . Many studies have demonstrated the pre-existing inflammatory milieu as the main cause for CRC progression . The best characterized example for the role of inflammation in cancer is manifested by inflammatory bowel disease cases where long-standing inflammation imposes a high risk of CRC development [5, 6]. Moreover, non-steroidal anti-inflammatory drugs (NSAIDs) reduce the long-term risk of cancer death, highlighting the importance of inflammation in cancer progression . It is believed that chronic inflammation facilitates tumor progression by establishing a milieu that promotes the growth of cancerous cells. Inflammatory cells recruited to inflammatory foci can release various pro-inflammatory mediators, including eicosanoids and cytokines, which can change the microenvironment to an abnormal milieu. An increase in immune cells can alter the microenvironment so that it becomes pro-inflammatory and acquires the capability to change the phenotype of epithelial cells to promote tumor growth and metastasis .
Several studies have demonstrated that eicosanoids, such as prostaglandins and leukotrienes, are important inflammatory mediators in the crosstalk between epithelial cells and the surrounding stromal cells in the tumor microenvironment [9, 10]. The importance of COX-2-derived PGE2 in tumor progression is well proven in mouse models of CRC [11–13]. PGE2 treatment has been shown to increase intestinal polyps in both ApcMin/+ mice and AOM-induced mice . Interestingly, the LTD4 receptor CysLT1R is highly expressed in human colon cancer and correlates negatively with patient survival [14, 15]. Moreover, LTD4 was found to induce proliferation and survival . By contrast, reduced expression of CysLT2R is associated with a poor prognosis in patients with CRC, and CysLT2R signaling promotes apoptosis and differentiation [14, 17]. Taken together, these studies show the key role of eicosanoids in CRC development.
Over the last decade, the emergence of new therapeutic targets has improved cancer therapy and has prolonged the lifespan of these patients. However, initial therapy and recovery are often complicated by the development of relapsed tumors. Cancer-initiating cells (CICs) are believed to be a small group of tumor cells that can form tumors . CICs have been identified in many different tumors such as those in the hematopoietic system, breast, brain, head, neck, and colon [19–22]. One important characteristic of CICs is their self-renewal capacity. Therefore, CICs are the most probable cause of tumor chemoresistance and recurrence, and may be accountable for the current failure of standard therapies [23, 24].
CICs can be identified by inducing stemness-selective conditions, or isolated based on the putative expression of stem cell markers. Multiple cell surface markers like CD133, CD44, CD24, CD29, CD166 and Lgr5 have been used by different investigators to isolate CIC subpopulations from colon carcinomas [20, 25–27]. Besides cell surface markers, the activity of certain pathways or enzymes is also used to identify stemness in cells. The activity of aldehyde dehydrogenase 1 (ALDH1), a detoxifying enzyme that oxidizes intracellular aldehydes, has also been used to identify normal colon stem cells . In a recent study, we showed that ALDH+ cells, compared with other cell surface markers, such as CD133 and CD44, could initiate more colonies than ALDH− cells, whereas this distinction was not apparent in positive and negative cells of CD133 and CD44 . This finding indicates that ALDH activity can be considered a reliable CIC marker.
Furthermore, in a recent study, we have shown that inflammatory lipid mediators can actually enhance the characteristic properties of CICs under certain conditions in vitro .
The aim of the current study was to elucidate whether enrichment of the tumor microenvironment by inflammatory lipid mediators such as LTD4 and PGE2 could promote the tumorigenic properties of CICs in vivo and to determine the changes occurring in the tumor environment that could modify CIC functions.
Reagents and antibodies
LTD4, PGE2, rabbit anti-human COX-2 and 5-LOX polyclonal antibodies were purchased from Cayman Chemical (Ann Arbor, MI, USA). The ALDEFLOUR (ALDH) kit was purchased from Stem Cell Technologies (Grenoble, France). Anti-human CD326 (EpCAM) MicroBeads was obtained from Miltenyi Biotec (Gladbach, Germany). Anti-mouse CD45-FITC-conjugated antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse LY6G-PE, CD4-PE, and F4/80-PE conjugated antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-mouse CD206-Alexaflour 647 conjugated antibody was obtained from AbD Serotech (Dusseldorf, Germany). Rabbit anti human β-catenin antibody, mouse anti-human ALDH and matrigel basement membrane matrix were obtained from BD Biosciences (Franklin Lakes, NJ, USA). The rabbit monoclonal anti-human Ki67 antibody was obtained from Thermo Fisher Scientific (Waltham, MA). Rabbit anti-human Dclk1 antibody and Rat anti-mouse F4/80 antibody were obtained from Millipore (Temecula, CA, USA) and AbD serotec (Raleigh, NC, USA), respectively. All other chemicals were of analytical grade and were obtained from Chemicon International (Temecula, CA) or Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.
HCT-116 cells (ATCC# CCL-247), derived from human colon carcinoma, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in monolayer culture in McCoy’s 5A modified medium (Gibco BRL, Grand Island, NY) supplemented with 10 % fetal bovine serum (FBS), 55 μg/ml streptomycin and 55 IU/ml penicillin. The cells were grown until 5 days to 70–80 % confluence at 37 °C in a humidified atmosphere of 5 % CO2. The cell line tested negative for mycoplasma (MycoTect® kit, Gibco BRL) at regular intervals.
FACS was used to sort the ALDH+ cells from the parental HCT-116 cell line for xenograft implantation and to quantify the presence of CD45+ and ALDH+ subpopulations in dissociated tumor cells. For FACS sorting of ALDH+ cells, HCT-116 cells were harvested using 0.25 % trypsin and 0.02 % EDTA. After resuspension of the cells in serum-free culture media, the cells were washed with 0.5 % BSA/PBS and stained with Aldefluor reagent (Stem Co Biomedical, Durham, NC, USA). The Aldefluor substrate was added to 1 × 106 cells/ml suspended in Aldefluor assay buffer and incubated at 37 °C for 40 min. At the same time, cells treated with the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) were stained to serve as the negative control. Cells with bright fluorescent ALDH signals were detected using a FACSCalibur or FACSAria flow cytometer (BD Biosciences). The ALDEFLUOR kit was used to sort ALDH+ cells with high ALDH enzymatic activity, as described previously [29, 30].
For analysis of CD45+ cells, single-cell suspensions from digested tumors were washed, resuspended in PBS, counted and divided into 1 × 106 cell aliquots for flow cytometry. Cells were washed again with 0.5 % BSA/PBS, resuspended in 100 μl of PBS with 1 % mouse serum, and incubated at 4 °C for 45 min for Fc-receptor blocking. Thereafter, each sample was exposed to 5 μl of anti-mouse CD45-FITC antibody for 45 min at 4 °C in the dark. Cells were washed with 0.5 % BSA/PBS, resuspended in 100 μl of PBS with 0.1 % μg/μl of 7-AAD (BD Pharmingen), and incubated at 4 °C for 10 min in the dark. Finally, 400 μl of PBS was added to each sample and read on a flow cytometer. The analysis was performed using the Summit v4.6.
The 5- to 6-week-old female nude mice (BalbC nu/nu) used in this study were purchased from Taconic Europe A/S (Ry, Denmark). The Regional Ethical Committee for Animal Research at Lund University, Sweden (M401-12) approved the animal experiments. To induce subcutaneous human colon cancer xenografts, FACS-sorted 1 × 104 ALDH+ HCT-116 cells were suspended in a 1:1 mixture of PBS:Matrigel (BD Biosciences), and 100 μl of the mixture was injected subcutaneously into each of both flanks of the mice. Tumor development was detected by palpation. The time taken for a palpable tumor to develop was recorded (10–14 days), and the tumor size was measured every three days using a digital vernier caliper. Once palpable tumors were established, the mice were randomly divided into three groups, and then were treated with vehicle, LTD4 or PGE2. The mice received daily subcutaneous injections of either ethanol (5 %) as vehicle, or 24.8 μg/kg/day of LTD4 or 17.6 μg/kg/day of PGE2. Tumor growth was monitored, and the tumor volume was estimated every third day. All mice were sacrificed after 48 days. The tumors were removed, measured, weighed, and photographed. Tumor tissues were fixed in 10 % buffered formalin, embedded in paraffin for immunohistochemistry analysis and/or processed further for tissue dissociation immediately for FACS analysis. Tumor volumes were estimated according to the formula (length × width2)/2.
Dissociation of xenograft tumors
After sacrificing the mice, the excised tumors were washed with PBS and minced using sterile scalpels. The minced tumor pieces were resuspended in RPMI medium supplemented with 10 % FBS and 2 mg/ml Collagenase P (Roche diagnostics, Basel, Switzerland). The tumor pieces were further dissociated using the gentleMACS™ Dissociator for 30 s and incubated at 37 °C for 2 h with rotation. Afterwards, the cell suspension was filtered through a 70-μm mesh and washed once with PBS and counted. The single cells were separated into mouse cells and epithelial tumor cells using CD326 (EpCAM) microbeads. The mouse cells and epithelial tumor cells were immediately stained for CD45, CD4, LY6G, F4/80, CD206 and ALDH, respectively, for FACS analysis.
Paraffin-embedded sections obtained from xenograft tumors were sectioned (4 μm) for immunohistochemical staining. All procedures were performed using a Dako automatic slide stainer according to the manufacturer’s instructions. Tumor sections were treated with 1–3 % hydrogen peroxide, blocked, and incubated with anti-ALDH, anti-COX-2 or anti-F4/80 (1:100 dilution each), anti-Dclk1 or anti-5-LOX (1:200 dilution each), or anti-β-catenin (1:300). Sections were incubated with biotinylated secondary antibody, followed by ABC reagent (Vector Laboratories Inc., Burlingame, CA). Signals were detected using DAB solution (Vector Laboratories). Tissues were counterstained with hematoxylin. The slides were scanned using the Aperio ScanScope CS system (Aperio Technologies Inc, Vista, CA, USA), and images were evaluated in a blinded fashion by two independent observers.
The immunoreactivity of β-catenin, COX-2, 5-LOX and F4/80 proteins in the tumor cells was determined based on the following procedure. Briefly, staining intensity was scored as 0 (negative), 1 (very weak), 2 (weak), 4 (medium) or 6 (strong). The extent of staining was scored as 0 (0 %), 0.5 (1–5 %), 1 (6–10 %), 2 (11–20 %), 3 (21–30 %), 4 (31–40 %), 5 (41–50 %), 6 (51–60 %), 7 (61–70 %), 8 (71–80 %), 9 (81–90 %) and 10 (91–100 %) according to the percentage of the positive staining area in relation to the whole carcinoma area. Next, the sum of the intensity score and extent score was regarded as the final staining scores for COX-2, 5-LOX, F4/80 and β-catenin proteins.
CysLTs, PGE2 and cytokine ELISA analysis
Blood was collected by cardiac puncture at the time of animal sacrifice, and a nonselective COX inhibitor, indomethacin, was immediately added to the blood samples together with the anticoagulant sodium citrate. The plasma was separated by spinning the samples at 5000 × g for 4 min. CysLTs and PGE2 plasma samples were measured using a competitive enzyme immunoassay obtained from Enzo Life Sciences (Solna, Sweden). All measurements were performed according to the manufacturer’s instructions.
Plasma cytokines were analyzed using a multiplex sandwich immunoassay format and the electro-chemiluminescence MSD ultrasensitive proinflammatory multiplex kit (Meso-Scale Discovery, Gaithersburg, MD). The MSD multispot array was run according to the manufacturer’s protocol. Briefly, 96-well plates pre-coated with capture antibodies for TNFα, IL-1β, IL-2, IL-4, IL-6, IL-10 and CXCL1/KC/GRO, INFγ were incubated with plasma samples for 2 h. Subsequently, detection antibodies were added, and the plate was incubated for another 2 h. After washing, the plate was read using an MS2400 imager (MSD).
Real-Time quantitative PCR
qPCR reactions employing TaqMan gene expression assays were used to measure tumor tissue expression of CysLT1R (Hs00272624_s1), PTGER2 (Hs00168754_m1), PTGRR4 (Hs00168761_m1), Arginase 1 (Mm00475988_m1), IL-1β (Mm00434228_m1; Hs00174097_m1), IL-6 (Mm00446190_m1; Hs00985639_m1) and IL-10 (Mm01288386_m1) genes (Applied Biosystems, Cambridge, United Kingdom) as described earlier .
Statistical analysis was performed using GraphPad Prism 5 software. Results are expressed as the mean ± SEM. All comparisons between the mean values were performed using either one-way analysis of variance (ANOVA) with Newman-Keuls post-hoc test, two-way ANOVA, or Student’s unpaired t test wherever applicable. P values less than 0.05 were considered to indicate statistical significance.
Both LTD4 and PGE2 affect the tumorigenic potential of ALDH+ cells
Both LTD4 and PGE2 stimulation increase the percentage of ALDH+ cells and Dclk1, β-catenin and COX-2 protein expression
Effect of LTD4 or PGE2 treatment on immune cells
Both LTD4 and PGE2 treatment increase IL-1 and IL-6 mRNA and cytokine secretion
Both LTD4 and PGE2 treatment increase plasma CysLTs and PGE2 secretion
CICs, which represent a small population of cancer cells with tumor initiating/stem-like properties, are widely recognized as a significant contributor of CRC development and progression . However, the mechanisms whereby CICs promote tumor growth are largely unknown. Nonetheless, it is presumed that CICs functions are predominantly impacted by their surrounding microenvironment enriched with several mediators, including inflammatory lipid molecules. The present study investigated this possibility to develop a better understanding of the role of the inflammatory lipid mediators LTD4 and PGE2 in CIC tumor growth .
In this study, we demonstrated that the inflammatory mediators LTD4 and PGE2 augmented tumor growth in mice injected with ALDH+ HCT-116 cells, CICs, a finding that is consistent with that in our previous study . Our results show that high ALDH-expressing colon cancer cells show all characteristics of CICs under both in vivo and in vitro conditions. Additionally, these cells were shown to have increased cancer stem cell characteristics in the presence of the inflammatory mediators LTD4 and PGE2 . Consistent with this finding, we also observed that the tumor-forming ability of ALDH-sorted CICs was increased markedly with LTD4 or PGE2 treatment in nude mice. In addition, we noted that ALDH+ HCT-116-sorted CICs have higher tumor-initiating ability than parental cells because the onset of tumors was quicker in mice injected with ALDH+ HCT-116 sorted cells than in mice injected with parental cells. LTD4 and PGE2 are known to mediate their responses on colon cancer cells through activation of their receptors, CysLTR1 and EP1-4 [33–35]. Interestingly, we have found that CICs (ALDH+) expressed high levels of CysLT1R, EP2 and EP4 receptors and the expression levels of CysLT1 and EP2 receptors were further increased in the presence of LTD4 and PGE2. These data are in good agreement with our previous findings that the abilities of LTD4 or PGE2 to promote CIC-induced colony growth is suppressed in the presence of their receptor antagonist .
Another in vivo property of CICs is their self-expansion in tumors, and we investigated whether LTD4 or PGE2 drives the self-expansion of ALDH-sorted cells in tumors. Interestingly, we observed that ALDH proteins were highly expressed in tumors from mice receiving either LTD4 or PGE2, further substantiating our previous notions that both of these inflammatory mediators play an important role in driving CIC expansion . It was also noteworthy here that the stimulatory effect of the LTD4 or PGE2 on CIC content was not restricted merely to ALDH-marked cells but to stimulation of the expansion of different CIC markers with similar potency. We crosschecked the data by detecting changes in Dclk1 protein, which is an exclusive marker for cancer stem cells of the intestine. The tumorigenic properties of Dclk1-marked intestinal cancer stem cells have been proven in ApcMin/+, suggesting their role in CRC .
To identify the potential factors that could be involved in tumor growth induced by LTD4 or PGE2, we examined some key proteins such as β-catenin, COX-2 and 5-LOX, which are strongly associated with colon cancer cell proliferation and CRC progression [16, 29]. LTD4 was demonstrated to trigger the nuclear accumulation of β-catenin in colon cancer cells in vitro, a finding that is linked to the simultaneous up-regulation of COX-2  and the effect of LTD4 on proliferation . With this in mind, we examined whether LTD4 or PGE2 could modify β-catenin signaling in CIC-induced tumors in vivo. Interestingly, we observed a high percentage of β-catenin accumulated in the cytoplasm and nuclear fractions of tumor cells of mice treated with LTD4 and PGE2. Increased levels of active β-catenin lead to its translocation from the cytosol to the nucleus where it can activate the TCF/LEF family of transcription factors . These transcription factors, in turn, regulate several genes associated with carcinogenesis, such as cyclin D1, c-myc, and COX-2 . Interestingly, we detected significantly high COX-2 protein expression within the tumors of mice treated with LTD4 or PGE2. Elevated COX-2 levels have been demonstrated in all constituent cells of neoplastic colon tissue compared with normal colon tissues . COX-2 overexpression confers resistance to apoptosis and facilitates cell proliferation, which can, together, aggravate a cell’s tumorigenic potential [38–40].
Taken together, our data suggest that inflammatory mediators encourage CICs to evoke tumor growth, possibly by stimulating β-catenin signaling, and concurrent up-regulation of COX-2 as well as proliferation.
Lipid inflammatory mediators, particularly leukotrienes, also play crucial role in leukocyte chemoattraction by the induction of rapid invasion and recruitment of inflammatory cells to the plasma membrane of endothelial cells . Thus, inflammatory cells might be partly involved in driving tumor growth in mice treated with LTD4 or PGE2. To explore this possibility, we examined the CD45+ cell count in tumors of mice receiving LTD4 or PGE2 treatment. It was noted that both LTD4 and PGE2 treatment intensified the percentage of CD45+ cells in the tumors, with a concurrent increase in ALDH+ cells. Furthermore, we found high levels of F4/80-positive staining, representing the total macrophage population within the tumor. In the tumor microenvironment, macrophages play an important role being highly enriched within the tumor as well as in the tumor stroma and secreting many factors known to induce neoplasia . Tumor-associated macrophages (TAMs) play an important role in cancer progression; accordingly, high levels of macrophage infiltration into the tumor tissues are associated with a poor prognosis in cancer patients [42, 43]. These macrophages play a crucial role in tumor immunity and possess potent immunosuppressive functions that contribute to tumor growth. Macrophages were also demonstrated to produce a wide array of cytokines, prostaglandins and leukotrienes [43, 44]. We also found that LTD4- and PGE2-treated mice had higher plasma levels of CysLTs and PGE2, possibly derived from tumor associated macrophages.
In addition, we found significantly higher plasma levels of proinflammatory cytokines IL-1β, IL-2, IL-6 in both LTD4- and PGE2-treated mice. A significant increase in TNF-α levels was also detected with PGE2 treatment. TNF-α, IL-1, and IL-6 have been demonstrated to promote colorectal and colitis-associated tumor development . Interestingly, TNF-α is known to induce β-catenin nuclear accumulation without APC mutations in gastric tumors . Thus, an increased cytokine level could be a possible explanation for driving the increased nuclear accumulation of β-catenin observed in tumors of mice treated with LTD4 or PGE2. Furthermore, increased levels of the chemokine CXCL1/KC/GRO were found in mice treated with LTD4. CXCL1/KC/GRO was shown to recruit and activate murine neutrophils and could modify tumor growth by numerous mechanisms . One interesting finding here is that mice receiving LTD4 or PGE2 displayed an increased plasma level of IL-10 and a decreased level of IFN-γ. This finding is interesting, particularly because IL-10 is known to inhibit IFN-γ, which has an anti-tumor effect . High production of IL-10 is also a characteristic feature of tumor-associated/M2 macrophages, adapted to suppress immune responses against tumors [43, 49]. These data fits well with our present finding that LTD4 as well as PGE2 greatly enhanced tumor infiltration by M2 macrophages (recognized by CD206 positive staining) and by increased tumor expression of arginase1. Interestingly, the tumor infiltration of M2 macrophages coincide with an increased tumor expression of the IL-10 in tumors from animals treated with either of the two lipid mediators. In fact, our data support a notion whereby the infiltrating macrophages within the tumor might have acquired a phenotype characterized by a high production of IL-10, which suppresses the anti-tumor actions of IFN-γ and encourages tumor growth. Overall, our data strongly support a tight correlation between host systemic immune responses and tumor growth.
We have shown that the lipid inflammatory mediators LTD4 and PGE2 not only stimulated CICs to self-expand but also induced several changes in the CIC tumor microenvironment, which cumulatively drives tumor growth from CICs. This finding deserves considerable attention, and future research should focus not only to target CICs but also associated inflammatory lipid mediators to design more effective therapies for colon cancer prevention.
ALDH: Aldehyde dehydrogenase; CICs: cancer initiating cells; COX-2: Cyclooxygenase 2; CRC: Colorectal cancer; IHC: Immunohistochemistry; LTD4: Leukotriene D4; PGE2: Prostaglandin E2
This work was supported by grants to AS from the Swedish Cancer Foundation, the Swedish Research Council, the Foundations at Skanes University Hospital, Gunnar Nilsson Foundation, and to KB from the Royal Physiographical Society in Lund.
KB: contributed to experimental design and manuscript drafting, xenograft mice model, FACS, IHC, qPCR and ELISA. NC and JO: contributed to experimental design, xenograft mice model and IHC. SS: contributed in the xenograft mouse model. BC: contributed to qPCR and IHC analysis. AS: involved in design, interpretation of the data, coordination of the project, revising of the manuscript and intellectual input. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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