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. 2021 Jan 27;16(1):e0245292. doi: 10.1371/journal.pone.0245292

Urinary 8-iso PGF2α and 2,3-dinor-8-iso PGF2α can be indexes of colitis-associated colorectal cancer in mice

Yusuke Miyazaki 1, Tatsuro Nakamura 1, Shinya Takenouchi 1, Akane Hayashi 1, Keisuke Omori 1, Takahisa Murata 1,*
Editor: Hiroyasu Nakano2
PMCID: PMC7840041  PMID: 33503019

Abstract

Early diagnosis of colorectal cancer is needed to reduce the mortal consequence by cancer. Lipid mediators play critical role in progression of colitis and colitis-associated colon cancer (CAC) and some of their metabolites are excreted in urine. Here, we attempted to find novel biomarkers in urinary lipid metabolite of a murine model of CAC. Mice were received single administration of azoxymethane (AOM) and repeated administration of dextran sulfate sodium (DSS). Lipid metabolites in their urine was measured by liquid chromatography mass spectrometry and their colon was collected to perform morphological study. AOM and DSS caused inflammation and tumor formation in mouse colon. Liquid chromatography mass spectrometry-based comprehensive analysis of lipid metabolites showed that cyclooxygenase-mediated arachidonic acid (AA) metabolites, prostaglandins, and reactive oxygen species (ROS)-mediated AA metabolites, isoprostanes, were predominantly increased in the urine of tumor-bearing mice. Among that, urinary prostaglandin (PG)E2 metabolite tetranor-PGEM and PGD2 metabolite tetranor-PGDM were significantly increased in both of urine collected at the acute phase of colitis and the carcinogenesis phase. On the other hand, two F2 isoprostanes (F2-IsoPs), 8-iso PGF2α and 2,3-dinor-8-iso PGF2α, were significantly increased only in the carcinogenesis phase. Morphological study showed that infiltrated monocytes into tumor mass strongly expressed ROS generator NADPH (p22phox). These observations suggest that urinary 8-iso PGF2α and 2,3-dinor-8-iso PGF2α can be indexes of CAC.

Introduction

Six hundred million people die of colorectal cancer per year all over the world. Inflammatory bowel disease (IBD), which is characterized by relapse and remission of intestinal mucosal inflammation, is a major risk of colitis-associated colorectal cancer (CAC) [1]. Statistical research showed that 5-year survival rate of colorectal cancer is depending greatly on disease stage at diagnosis; 90% at localized stage and 14% at distant stage [2]. Thus, early diagnosis of colorectal cancer is critical to prolong lifespan for patients.

Sigmoidoscopy and colonoscopy are often performed for diagnosis of colorectal cancer [3]. These diagnostic methods are burdensome for patients, and they need well-experienced specialists and equipments to perform [4, 5]. Recent studies reported that serum micro RNA-141 or osteopontin [6, 7], microsatellite instability or sialyl-Tn antigen in biopsy specimens [8, 9] were useful for biomarkers of colorectal cancer. However, these procedures are invasive, and they also require special equipment. More convenient biomarkers for CAC are still required.

Researchers have used murine colorectal carcinogenesis models to investigate the mechanisms of the onset/progression of CAC. Azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis and CAC model has been often used because of its convenience and stability of incidence [10]. Previous studies showed that sustained and abundant infiltrations of neutrophils and macrophages were observed in the colonic mucosal tissue of AOM/DSS-treated mouse [11–13]. Other studies reported that reactive oxygen species (ROS) produced by infiltrating immune cells into lamina propria [14] damaged DNA, which in turn promotes carcinogenesis in AOM/DSS-treated mouse [15].

Lipid mediators are bioactive substances produced from polyunsaturated fatty acid of the cell membrane and they regulate inflammation and carcinogenesis. Cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) mediate oxygenation of PUFA, such as arachidonic acid (AA), and synthesize lipid mediators. In human colon cancer, protein expressions of a COX isoform, COX-2, and a LOX isoform, 5-LOX, were increased [16]. Experimental studies showed that pharmacological inhibition of COX-2 ameliorated CAC induced by administration of AOM/DSS [17]. Gene deletion of microsomal prostaglandin (PG)E synthase inhibits the development of carcinogen-induced colon cancer [18]. On the other hand, the deficiency of PGD synthase aggravated AOM/DSS-induced colitis and CAC in mice [19]. These reports indicate that lipid mediators critically regulate the progression and onset of colitis/CAC.

Since lipid mediators and their related metabolites are excreted into urine, we generated AOM/DSS-induced colitis and CAC model mice for exploration of novel urinary index of CAC in this study and we discovered candidate substances for biomarker of CAC.

Materials and methods

Reagents

The following reagents were used: 6-keto PGF1α-d4, thromboxane (TX)B2-d4, PGF2α-d4, PGE2-d4, PGD2-d4, leukotriene (LT)C4-d5, LTB4-d4, 5(S) HETE-d8, 12(S) HETE-d8, 15(S) HETE-d8, PAF C16-d4, Oleoylethanolamide-d4, tetranor-PGEM, tetranor-PGEM-d6, tetaranor-PGDM, tetranor-PGDM-d6, LTE4, LTE4-d5, 11-dehydro-TXB2, 11-dehydro-TXB2-d4, 8-iso PGF2α, 8-iso PGF2α-d4, 2,3-dinor-8-iso PGF2α (Cayman Chemical, USA); azoxymethane, DEXTRAN SULFATE SODIUM SALT REAGENT GRADE, ethanol, methanol, acetonitrile, hexane, acetic acid, formic acid, LabAssayTM Creatinine, hydrogen peroxide, citric acid monohydrate, trisodium citrate dehydrate, mayer’s hematoxylin solution (FUJIFILM Wako, Japan); 3,3'-diaminobenzidine, tetrahydrochloride (DOJINDO, Japan); TritonX-100 (MP Biomedicals); normal donkey serum (Merck Chemicon, USA).

Induction of colitis and CAC

Seven- to nine-weeks old female wild type C57BL/6 mice were used. All experimental procedures in this study were approved by the Institutional Animal Care and Use Committee at the University of Tokyo (P11-576 and P08-258). Mice were intraperitoneally administrated the large intestine-specific carcinogen AOM (12 mg/kg). Five days later, mice were provided 2% DSS in drinking water ad libitum for 4 days, followed by a 17-day-off period (Fig 1A). This cycle was performed three times. Colon tissue was sampled at day 5, day 26, day 47 and day 60. No randomization was used to allocate mice to any groups and confounders were not controlled. The number of mice were 13 in vehicle group and 8–26 in AOM/DSS administration group depending on the phase of day 5, 26, 47 and 60. Disease activity index (DAI) and body weight were checked every 3 days. The DAI was scored according to stool condition as followed: 0, normal; 1, soft but formed; 2, very soft; 3, diarrhea; 4, bloody diarrhea. These parameters were also used in previously published paper [19] and appropriate to assess animals’ health and well-being.

Fig 1. AOM/DSS-induced colitis and CAC in mice.

Fig 1

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(A) Schematic figure of AOM/DSS administration. (B) DAI and (C) body weight (n = 8–26). Body weight is showed as a value relative to the ratio of the body weight at day 0. (D) Representative histological images (hematoxylin & eosin staining) of colon on day 5, 26, 47 and 60 compared with vehicle treatment group. Black arrow, normal or damaged mucosa; white arrow, infiltrating cell; black arrowhead, macrophage; white arrowhead, neutrophil; grey arrowhead, lymphocyte. Scale bar, 100 εm. (E) Histological scoring of colon inflammation (n = 6–10). **, p<0.01; ***, p<0.001. (F) Colon length on day 47 and 60 compared with vehicle treatment group (n = 8–13) and (G) macroscopic images of a whole colon. White arrowhead, tumor. Scale bar, 1 cm. ***, p<0.001.

Histopathological assessment of colitis and CAC

Mice were properly euthanized by cervical dislocation. Colon tissue was fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Tissue was sectioned at 4 μm and stained with hematoxylin and eosin in a basic protocol. For assessment of colitis severity, histological scoring was performed as shown in Table 1.

Table 1. Histological scoring.

Cell infiltration Score
Low frequency in the lamina propria 0
High frequency in the lamina propria 1
Extending into the submucosa 2
Transmural extension of the infiltration 3
Tissue damage Score
No mucosal damage 0
Surface mucosal erosion 1
Focal ulceration 2
Extensive mucosal damage and extention into deeper structures of the bowel wall 3
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The extent of cell infiltration and tissue damage was assessed. The sum of these scores was used as an index of colon inflammation.

Measurement of lipid metabolites

Urine was serially collected from the same mouse by metabolic cages and preserved at -80°C. Sample solutions were prepared by mixing 100 μl urine, 850 μl deionized water, 50 μl internal standards (the composition is shown in S1 and S2 Tables) and 10 μl formic acid. Solutions were loaded onto methanol-conditioned and water-equilibrated solid-phase extraction (SPE) cartridges (Oasis HLB, Waters, USA). Followed by wash of cartridges by 5% (v/v) acetonitrile or water and hexane, the lipid metabolites absorbed to the cartridges were eluted with methanol and reconstituted in 80% (v/v) methanol. Samples were injected to LCMS-8030 (Shimadzu, Japan).

For comprehensive analysis, the liquid chromatographic separation was performed using a Phenomenex Kinetex C8 column (Shimadzu) and using a mobile phase consisting of 0.05% (v/v of water) formic acid (solvent A) and 0.05% (v/v of acetonitrile) formic acid (solvent B) (The gradient program is shown in S3 Table). The production amount of each lipid metabolites was determined in area under the curve (AUC).

For absolute measurement, the liquid chromatographic separation was performed using an Inertsil ODS-3 column (GL Sciences, Japan) and using a mobile phase consisting of 0.02% (v/v) acetic acid (solvent A) and acetonitrile (solvent B) (The gradient program is shown in S3 Table and other detailed settings are shown in S4 Table). Absolute value of 2,3-dinor-8-iso PGF2α was adjusted for 8-iso PGF2α-d4 as previously described [20]. The concentration value was calculated by correcting with the amount of creatinine in urine.

In some experiments, the concentrations of 8-iso PGF2α and 2,3-dinor-8-iso PGF2α were measured in plasma and colon tissues of naive or AOM+DSS-treated mice. Briefly, plasma was deproteinized by mixing with the same equivalent of organic solvent (metnaol: acetonitrile = 1:1, v/v) contatining 5% 5N HCl. The colon tissues or polyp were homogenized in 300 μl methanol. After centrifusion, the supernatants were cleaned by SPE and the analytes were injected into LC-MS/MS, as descrived above. Because both 8-iso PGF2α and 2,3-dinor-8-iso PGF2α could not be detected in plasma by LCMS-8030, we utilized successor model LSMS-8060 to measure plasma and colonic concentrations of F2-IsoPs. From the standard curve, the lower limit of quantification of 8-iso PGF2α was 0.11 ng/ml and that of 2,3-dinor-8-iso PGF2α was 0.19 ng/ml, respectively. The tissues concenctrations were expressed as pg per mg tissue weight.

Immunostaining

Paraffin-embedded colon tissue sections were deparaffinized and treated with blocking reagent (5% normal donkey serum and 0.1% TritonX-100 in PBS) for 40 minutes at room temperature. The sections were incubated overnight at 4°C with anti-COX-2 goat polyclonal antibody (diluted 1:100, Santa Cruz, USA), and then incubated with biotinylated anti-goat IgG horse antibody (diluted 1:200, VECTOR, USA) for 2 hours. Antibodies were visualized by treatment with hydrogen peroxide and 3,3'-diaminobenzidine, tetrahydrochloride. Counterstain was performed by hematoxylin. For p22phox staining, anti-p22phox mouse monoclonal antibody (diluted 1:200, Santa Cruz, USA) was applied as primary antibody. For ROS detection, freshly prepared frozen tissue sections were incubated with 2 μM dihydroethidium for 30 minutes at 37°C.

Statistical analysis

No criteria for including and excluding were set and no animals and data points were excluded. Results were expressed as the mean ± SEM. DAI and body weight were assessed by two-way ANOVA, followed by Bonferroni post-tests for comparison between more than two groups. Comprehensive analysis was assessed by the unpaired Student t test for comparison between two groups. Other data evaluations were conducted using one-way ANOVA, followed by Tukey test for comparison between more than two groups. We analyzed the result by GraphPad Prism version 3.02 (GraphPad Software, San Diego, California, USA, www.graphpad.com).

Results

Induction of colitis and CAC by administration of AOM/DSS

We generated colitis and CAC model by single injection of AOM followed by repeated cycles of DSS administration as shown in Fig 1A. Almost all of the animals didnot die unexpectedly. Vehicle group of mice drinking normal water did not represent any symptoms of colitis indicated as disease activity index (DAI, Fig 1B). Their body weight was gradually increased (Fig 1C). Administration of AOM/DSS increased DAI and decreased body weight of mice (Fig 1B and 1C). Their symptoms were exacerbated just after the beginning of each DSS administration and they gradually relieved after that (Fig 1B and 1C). On day 60; 14 days after the end of third administration of DSS, the disease scores subsided to the same level as vehicle group.

Histopathological study showed that vehicle group showed no mucosal damage of colon (representative pictures are shown in Fig 1D and histological scores are summarized in Fig 1E) and little infiltration of immune cells. Repeated cycles of DSS administration damaged colonic mucosa (shown by a black arrow) and infiltrated mononuclear cells and neutrophils into the lamina propria. The severity of their manifestations was increased in DSS administration cycle-dependent manner (Fig 1D and 1E). The colon length was strongly shortened in the phase of third DSS administration (Fig 1F, day 47). In contrast, 14 days after the last DSS administration (day 60), mucosal damage and infiltration of immune cells were almost completely disappeared and the histological score and the colon length were recovered to the level of vehicle treatment (Fig 1D–1F, day 60).

On the third DSS administration cycle, some tumors were formed in the distal area of colon (Day 47, Fig 1D, black arrow and Fig 1F). We also confirmed the presence of tumors in colon by morphological study (Fig 1G). The tumors resided in the colon even after the remission of inflammation (day 60). Thus, the AOM/DSS administrations induced CAC after day 47.

Comprehensive analysis of lipid metabolites in urine

To explore the candidates of urinary indexes of CAC, we performed comprehensive analysis of lipid metabolites excreted in the urine of AOM/DSS-treated mice on day 60. We detected 32 lipid metabolites in 131 types of that we measured (Fig 2 and S5 Table, which shows detected lipid mediators). Among them, arachidonic acid (AA)-derived metabolites accounted for 88% (28 types) of the detected metabolites (Fig 2 and S5 Table). The amounts of 10 lipid metabolites were significantly increased in the urine of AOM/DSS administrated mice compared with that of vehicle treated mice (Fig 2).

Fig 2. Increased urinary lipid metabolites in CAC mice.

Fig 2

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Lipid metabolites were comprehensively measured in the urine of vehicle or DSS administrated mice (n = 7 each). dihy, dihydro; te, tetranor; PG, prostaglandin; TX, thromboxane; AA, arachidonic acid. *, p<0.05; **, p<0.01; ***, p<0.001.

Upon inflammation, PGs are synthesized from AA by COX, mainly COX-2, activity. Three types of PGE2 metabolites (Fig 2A–2C), two types of PGD2 metabolites (Fig 2D and 2E) and three types of PGF2αmetabolites (Fig 2F–2H) were significantly increased in the urine of CAC-bearing mice. A metabolite of PGI2 metabolite, 6,15-diketo-13,14-dihydro-PGF1α (Fig 2I) was also significantly increased in the CAC mice urine. The other enzymatic oxidative products, AA-LOX metabolite; 18-carboxy-dinor-LTB4 (S5 Table, p = 0.009) and EPA-CYP metabolite; 17,18-DiHETE (S5 Table, p = 0.04) were significantly increased in the urine of CAC-bearing mice. In addition to oxidative enzyme, ROS catalyzes non-enzymatic oxidation of AA and produces isoprostanes [21]. 8-iso PGF2α (Fig 2J) was significantly increased in the CAC mice urine. A metabolite of 8-iso PGF2α, 2,3-dinor-8-iso PGF2α tended to be increased in the CAC-bearing mice urine (p = 0.083, Fig 2K). From these results, we focused on AA-derived COX-2 and/or ROS metabolites as candidates of urinary CAC index.

Expression of COX-2 and p22phox in mucosal tissue and tumor tissue

We next assessed the expression of COX-2 and a main subunit of NADPH oxidase (NOX), p22phox in the AOM/DSS stimulated colon tissues. As previously reported [17, 22], COX-2 was expressed in the inflamed colonic epithelial cells on day 5, 26 and 47 (Fig 3A). It was highly expressed in the inflamed colonic epithelial cells on day 5 and colorectal tumor cells in carcinogenesis period (Fig 3A).

Fig 3. The colon expression of enzyme related lipid metabolite production.

Fig 3

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Representative images of immunohistochemistry of (A) COX-2 and (B) p22phox. Areas surrounded by squares indicate COX-2 or p22phox-positive cells. Scale bar, 50 μm.

A NOX isoform NOX1 was reported to be expressed in human colonic epithelial cells and colorectal cancer cells [23] and another NOX isoform NOX2 was expressed in innate immune cells, such as neutrophils and macrophages [24]. As shown in Fig 3B, the protein expression of p22phox was observed in the mononuclear cells infiltrated into lamina propria during DSS administration cycle and which was higher in the colorectal tumor tissue (day 60) than inflamed mucosal tissue (day 5–47, Fig 3B).

Absolute measurement of lipid metabolites in the development of CAC

Both tetranor-PGEM and tetranor-PGDM are abundant urinary metabolites of PGE2 or PGD2, respectively [25, 26]. In addition, it is reported that two types of F2-isoPs, 8-iso PGF2α and 2, 3-dinor-8-iso PGF2α, are detectable in several biological fluids [27]. We next measured the absolute concentration of tetranor-metabolites and F2-isoPs. Both tetranor-metabolites transiently increased on the acute phase of colitis (day 1–2) and carcinogenesis period (day 47, 60) (Fig 4A and 4B). On the other hand, the concentrations of F2-isoPs were significantly increased only in the carcinogenesis period (Fig 4C and 4D).

Fig 4. The absolute concentration of lipid metabolites.

Fig 4

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The urinary concentration of tetranor-PGEM (A), tetranor-PGDM (B), 8-iso PGF2α (C) and 2,3-dinor-8-iso PGF2α (D) in acute colitis and tumor formation period (n = 6–17). The concentration of creatinine in urine was measured to calculate the correction value. (E) Plasma levels of 8-iso PGF2α (n = 3: control, n = 6: CAC). (F) The levels of 2,3-dinor-8-iso PGF2α in colon tissues and polyp (n = 3: control, n = 5: inflaimed colon tissue without polyp, n = 3: polyp). The lower limit of quantification (LLOQ) of 8-iso PGF2α or 2,3-dinor-8-iso PGF2α were 0.19 or 0.11 ng/ml, respectiely. The values below LLOQ were excluded from obtained data (n = 4: control, n = 8: CAC). *, p<0.05; **, p<0.01; ***, p<0.001 compared with day 0.

To verify that 8-iso PGF2α and 2, 3-dinor-8-iso PGF2α were increased in the carcinogenesis period, we measured both F2-IsoPs concentrations in plasma and colonic tissues. The levels of 8-iso PGF2α in all plasma samples and that of 2, 3-dinor-8-iso PGF2α in some samples were below the lower limit of quantification (LLOQ). Mean concentrations of 2, 3-dinor-8-iso PGF2α in plasma samples (n = 3: control, n = 6: CAC) were comparable between control and CAC mice (Fig 4E). The levels of 8-iso PGF2α in some colon tissues and polyp were also below the LLOQ (n = 1/4: control, n = 3/8: inflamed colon, n = 4/7: polyp). Unexpectedly, mean concentrations of 8-iso PGF2α, except below the LLOQ, were comparable between control colon, inflamed colon tissues and polyp (Fig 4F). The levels of 2, 3-dinor-8-iso PGF2α in all colon samples were below the LLOQ.

Discussion

In this study, we performed qualitative and quantitative analysis of the urinary lipid metabolites in AOM/DSS-induced colitis and CAC model mice. We found that the urinary levels of 8-iso PGF2α and 2,3-dinor-8-iso PGF2α were significantly increased in the phase of CAC. Considering that 2,3-dinor-8-iso PGF2α is a metabolite of 8-iso PGF2αand is chemically stable, 2,3-dinor-8-iso PGF2α would be an optimal candidate of urinary biomarker of CAC.

The significant increases of F2-isoPs in urine were observed in the phase of tumor development but not acute/chronic inflammation. NADPH oxidase (NOX) is a group of membrane-associated enzyme, consisting of two membrane protein p22 and gp91 and catalyze one-electron reduction of oxygen [28]. As a result of this reaction, ROS is generated, and oxidative stress occurs due to disturbing redox balance by overproduced ROS. Overproduced ROS caused DNA damage and genetic mutation which leaded carcinogenesis in vivo [15]. At the same time, ROS mediates metabolism of AA to isoprostanes including 8-iso PGF2α via free radical-catalyzed peroxidation [21]. Previous study has demonstrated that myeloid derived suppressor cells (MDSCs) from tumor-bearing mice expressed the enhanced levels of NOX subunits, such as gp91 and p22, and produced more ROS than that from tumor-free mice [29]. In addition, as previous report showing that MDSCs massively accumulated in the CAC region [30], we also observed a NOX subunit p22phox-positive cells in the colonic lamina propria of mice with polyp. Thus, phase-dependent increase of F2-isoP extraction would be considerd by NOX-expressed MDSC accumulation in CAC region. However, we colud not find the increased levels of F2-IsoP in plasma and CAC region in this study. This may be because the concentration of F2-IsoP produced in local region by ROS-generating cells was too small to quantify in mice. Indeed, the levels of 8-iso PGF2α were below the LLOQ in all plasma samples and some colon samples. More sensitive methods are needed for measuring local F2-IsoPs levels.

The concentration of tetranor-PGEM and tetranor-PGDM were significantly increased in the urine of early phase of colitis inflammation and CAC. It is reported that PGE2 synthase m-PGES was expressed higher in tumor stroma cells of AOM-induced CAC model mice [18]. Urinary excretion of tatranor-PGEM was increased in patients suffering from ulcerative colitis [31] and colorectal cancer [32]. The expressions of two types of PGD synthase H-PGDS and L-PGDS are increased in the inflamed intestinal tumor tissue in mice and human [19, 33]. It is reasonable that urinary concentration of major PGE2 or D2 metabolites is high in mice with colitis and CAC in this study. On the other hand, there are numerous studies showing the production and contribution of PGE2 or PGD2 in various types of inflammatory diseases other than CAC, such as viral-induced fever, food allergy, cystic fibrosis and lung metastasis [34–37]. Thus, urinary tetranor-PGEM and/or tetranor-PGDM may lack a specificity as a biomarker of CAC although it can be utilized as a supportive index reflecting inflammatory condition.

In the present study, we found candidates of urinary biomarker of CAC using AOM/DSS-induced CAC mouse model which represents similar characteristic with human CAC; diseased area of colon, infiltration of granulocytes, accumulation of β-catenin and mutation of K-Ras gene [38, 39]. However, it is still required to investigate the clinical usefulness of the urinary CAC marker found in this study. Further investigations using human urine sample are necessary to confirm this issue.

In conclusion, we identified urinary 8-iso PGF2α and 2,3-dinor-8-iso PGF2α as candidates of CAC biomarkers using murine colitis and CAC model. In addition, we found that urine is more appropriate for detecting CAC biomarker than plasma. Our findings may contribute to the improvement of early diagnosis of CAC.

Supporting information

S1 Table. The composition of internal standards mixture for comprehensive analysis.

(DOCX)

Click here for additional data file. (16.9KB, docx)
S2 Table. The composition of internal standards mixture for absolute measurements.

(DOCX)

Click here for additional data file. (16.4KB, docx)
S3 Table. Gradient program for comprehensive analysis and absolute measurement.

(DOCX)

Click here for additional data file. (17.4KB, docx)
S4 Table. Ion mode, m/z value and elution time of each substance for absolute measurement.

Ion mode was selected fromnegative (-) or positive (+).

(DOCX)

Click here for additional data file. (17.2KB, docx)
S5 Table. Lipid metabolites detected in the urine of CAC mice model.

The values are shown as fold increase ± SE compared with the average of vehicle treated mice. DGLA, dihomo-γ-linoleic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; COX, cyclooxygenase; LOX, lipoxygenase; CYP, cytochrome P450; PG, prostaglandin; TX, thromboxane; LT, leukotriene; DiHETE, dihydroxyeicosatetraenoic acid; OxoEDE, oxoeicosadienoic acid; -, non-enzymatic oxidation.

(DOCX)

Click here for additional data file. (19.4KB, docx)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (19H03569, 17H01509, 18K08809 to TM, 18K14603 to TN); Shimadzu Science Foundation (to TM); Kobayashi Foundation (to TM); The Morinaga Foundation for Health & Nutrition (to TM); Takeda Science Foundation (to TM).

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Decision Letter 0

Hiroyasu Nakano

3 Sep 2020

PONE-D-20-25696

Urinary 8-iso-PGF2α and 2,3-dinor-8-iso-PGF2α can be indexes of colitis-associated colorectal cancer in mice Lipid mediators as biomarker of colitis-associated colorectal cancer

PLOS ONE

Dear Dr. Murata

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

While the reviewer appreciates the importance of the study, the reviewer has several concerns that need to be addressed before publication. Specifically, the cellular source of PGF2alpha and its metabolites is not directly investigated in the study. Hence, the authors need to investigate whether colon cancer cells or cancer tissues produce PGF2alpha and its metabolites. Moreover, it would be intrigued to determine the concentrations of PGF2alpha and its metabolites in the plasma of untreated and AOM/DSS-treated mice.

==============================

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Hiroyasu Nakano, M.D., Ph.D.

Academic Editor

PLOS ONE

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Reviewer #1: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

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Reviewer #1: Yes

**********

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Reviewer #1: Miyazaki et al established a murine model of inflammation-induced colon cancer (CAC), and searched for novel lipid biomarkers in the urine of this model mouse. They identified contents of several metabolites derived from arachidonic acid are increased in the CAC urine. Some of them (PGD and E metabolites) are also increased in the inflammation period, and not candidates for biomarkers. They finally found that 8-iso-PGF2αand 2,3-dinor-8-iso-PGF2α are increased only in the CAC period, and concluded that these two PGF2 metabolites are candidate biomarkers for colorectal cancer. They also stained the mouse cancer tissues and showed that the expression of COX-2 and NADPH oxidase is increased in the cancer cells, and this can explain the increase of the metabolites.

This is an interesting study that utilizes a mouse oncogenic model to find biomarkers. The biosynthesis and metabolic inactivation of arachidonic acid-derived lipid mediators are conserved among various animal species, this strategy is reasonable. This group has a long history of measuring PG and PG metabolites, and the manuscript is simply and clearly written. The reviewer, however, has to raise several important issues to be addressed in the revised manuscript.

Major points

The authors claim that 8-iso-PGF2alpha and 2,3-dinor-8-iso-PGF2 are possible biomarkers based on the specific increase of these metabolites in CAC phase. They did not succeed in convincing the reviewer that the colon cancer cells can generate and metabolize PGF2 alpha. Quantification of PGF2alpha and its metabolites in the cancer tissues, hopefully, lazar-dissected tissues, is required. If they think that cancer cells produce PGF2alpha, which enters into the blood and is metabolized in the other tissues, they should measure PGF2alpha and metabolites in the plasma.

Regarding the application to the human situation, they should measure these metabolites in the urine of human colon cancer patients and show some representative results.

Minor points

Statistic analyses are missing in Fig. 1E, F, and Fig. 2.

**********

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PLoS One. 2021 Jan 27;16(1):e0245292. doi: 10.1371/journal.pone.0245292.r002

Author response to Decision Letter 0

21 Dec 2020

Responses to the reviewers’ comments of the manuscript “Urinary 8-iso-PGF2α and 2,3-dinor-8-iso-PGF2 can be indexes of colitis-associated colorectal cancer in mice”.

Comment 1-1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

<Response>

We revised the manuscript and supplemental files according to the PLOS ONE style templates and they correctly meet PLOS ONE’s requirement.

Comment 1-2 As part of your revisions, please provide additional details pertaining to your animal research procedures. Please revise your Methods to address the following points: (1) sample size: how many animals per group and how did you determine the numbers (power analysis? pilot study? previously published data/findings?);

<Response>

We added the descriptions in manuscript in line 89-91 for (1); “The number of mice were 13 in vehicle group and 8 – 26 in AOM/DSS administration group depending on the phase of day 5, 26, 47 and 60.”

Comment 1-3 (2) provide complete information about all methods undertaken to minimize pain and distress of the animals in your work. (3) Please discuss your monitoring parameters (physical and behavioral signs to assess health and well-being), in addition to your humane endpoints (criteria used to determine when to euthanize animals in cases where animals become sick/moribund). (4) Please state the rate of mortality for animals who died unexpectedly (and state cause(s) of death). (6) Please also discuss supportive care that you provided to the animals.

<Response>

We used eight weeks old C57BL/6J male mice (20-22 g). Mice were housed under standard laboratory conditions (22±1°C, 12-hour light/dark cycles, food and water ad libitum). Animal care and handling procedures were in accordance with International Association for the Study of Pain (IASP) guidelines for the use of animals in pain research and the protocols for animal care and use were approved by the Institutional Animal Care and Use Committee at the University of Tokyo (P11-576 and P08-258). All effort was made to limit the number of animals used. We set humane endpoint to prevent or alleviate pain and/or distress when 20% body weight loss, disability in food intake and water drinking are observed. However, all the mice used in the current study did not exceed the criteria and did not die unexpectedly. We revised the manuscript as follows:

Line 108 for (2); “Mice were properly euthanized by cervical dislocation.”

Line 93 for (3); “These parameters were also used in previously published paper [19] and appropriate to assess animals’ health and well-being.”

Line 173 for (4); “Almost all of the animals have not died unexpectedly.”

Line xx for (6);

Comment 1-4 (7) Lastly, please complete and submit the ARRIVE Guidelines checklist (Essential 10) with your revision: https://arriveguidelines.org/resources/author-checklists

<Response>

We filled in the ARRIVE Guidelines checklist. And we added the following descriptions in manuscript; “Seven- to nine-weeks old female wild type C57BL/6 mice were used.” In line 82; “No randomization was used to allocate mice to any groups and confounders were not controlled.” In line 88; “No criteria for including and excluding were set and no animals and data points were excluded.” in line 161.

Reviewer #1:

Comment 2-1 Miyazaki et al established a murine model of inflammation-induced colon cancer (CAC), and searched for novel lipid biomarkers in the urine of this model mouse. They identified contents of several metabolites derived from arachidonic acid are increased in the CAC urine. Some of them (PGD and E metabolites) are also increased in the inflammation period, and not candidates for biomarkers. They finally found that 8-iso-PGF2αand 2,3-dinor-8-iso-PGF2α are increased only in the CAC period, and concluded that these two PGF2 metabolites are candidate biomarkers for colorectal cancer. They also stained the mouse cancer tissues and showed that the expression of COX-2 and NADPH oxidase is increased in the cancer cells, and this can explain the increase of the metabolites.

This is an interesting study that utilizes a mouse oncogenic model to find biomarkers. The biosynthesis and metabolic inactivation of arachidonic acid-derived lipid mediators are conserved among various animal species, this strategy is reasonable. This group has a long history of measuring PG and PG metabolites, and the manuscript is simply and clearly written. The reviewer, however, has to raise several important issues to be addressed in the revised manuscript.

Major points

The authors claim that 8-iso-PGF2alpha and 2,3-dinor-8-iso-PGF2 are possible biomarkers based on the specific increase of these metabolites in CAC phase. They did not succeed in convincing the reviewer that the colon cancer cells can generate and metabolize PGF2 alpha. Quantification of PGF2alpha and its metabolites in the cancer tissues, hopefully, lazar-dissected tissues, is required. If they think that cancer cells produce PGF2alpha, which enters into the blood and is metabolized in the other tissues, they should measure PGF2alpha and metabolites in the plasma.

<Response>

Thank you. 8-iso-PGF2� is an oxidized metabolite of arachidonic acid not PGF2��and 2,3-dinor-8-iso-PGF2� is a metabolite of 8-iso-PGF2�. As following your suggestion, we measured the concentrations of these two metabolites in plasma and polyps of AOM+DSS-treated mice (day 60). As shown in below Figure, the levels of 2, 3-dinor-8-iso PGF2α (left figure) in plasma and that of 8-iso-PGF2α (right figure) in colon tissues were unexpectedly comparable between control and CAC mice. The concentrations of 8-iso-PGF2α in all plasma samples and that of 2, 3-dinor-8-iso PGF2α in all colon samples were below the lower limit of quantification (LLOQ). From these results, we could not find the increase levels of F2-IsoPs in plasma and CAC region in this study. This may be because the local production of 8-iso-PGF2� by ROS-generating cells was too small to quantify in mice. Indeed, the levels of 8-iso-PGF2α in some colon tissues and polyp were below the LLOQ (n=1/4: control, n=3/8: inflamed colon, n=4/7: polyp). It needs more sensitive methods for measuring local F2-IsoPs levels. In other words, urine is more appropriate for detecting CAC biomarkers than plasma. We added these results as Figure 4E-F and added the descriptions in the material and method (line 137-146), results (line 255-264), and discussion section (line 298-303).

Comment 2-2 Regarding the application to the human situation, they should measure these metabolites in the urine of human colon cancer patients and show some representative results.

<Response>

We attempted to obtain urine sample from patients in cooperating with Prof. Mamoru Watanabe, predecessor of Advanced Clinical Center for IBD in Tokyo Medical and Dental University Hospital in Japan. However, we could not obtain the data for this manuscript because it will take time to approve clinical studies and sampling. Please understand the situation.

Comment 2-3

Statistic analyses are missing in Fig. 1E, F, and Fig. 2.

<Response>

We performed statistical analysis and added in Fig. 1E, F, and Fig.2.

Attachment

Submitted filename: 20201216 Response to reviewers.docx

Click here for additional data file. (312.5KB, docx)

Decision Letter 1

Hiroyasu Nakano

26 Dec 2020

Urinary 8-iso PGF2α and 2,3-dinor-8-iso PGF2α can be indexes of colitis-associated colorectal cancer in mice

PONE-D-20-25696R1

Dear Dr. Murata

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Hiroyasu Nakano, M.D., Ph.D.

Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

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Reviewer #1: No

Acceptance letter

Hiroyasu Nakano

18 Jan 2021

PONE-D-20-25696R1

Urinary 8-iso PGF2α and 2,3-dinor-8-iso PGF2α can be indexes of colitis-associated colorectal cancer in mice

Dear Dr. Murata:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. The composition of internal standards mixture for comprehensive analysis.

    (DOCX)

    Click here for additional data file. (16.9KB, docx)
    S2 Table. The composition of internal standards mixture for absolute measurements.

    (DOCX)

    Click here for additional data file. (16.4KB, docx)
    S3 Table. Gradient program for comprehensive analysis and absolute measurement.

    (DOCX)

    Click here for additional data file. (17.4KB, docx)
    S4 Table. Ion mode, m/z value and elution time of each substance for absolute measurement.

    Ion mode was selected fromnegative (-) or positive (+).

    (DOCX)

    Click here for additional data file. (17.2KB, docx)
    S5 Table. Lipid metabolites detected in the urine of CAC mice model.

    The values are shown as fold increase ± SE compared with the average of vehicle treated mice. DGLA, dihomo-γ-linoleic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; COX, cyclooxygenase; LOX, lipoxygenase; CYP, cytochrome P450; PG, prostaglandin; TX, thromboxane; LT, leukotriene; DiHETE, dihydroxyeicosatetraenoic acid; OxoEDE, oxoeicosadienoic acid; -, non-enzymatic oxidation.

    (DOCX)

    Click here for additional data file. (19.4KB, docx)
    Attachment

    Submitted filename: 20201216 Response to reviewers.docx

    Click here for additional data file. (312.5KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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