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Keywords:

  • Aberrant crypt foci;
  • azoxymethane;
  • colon cancer;
  • dysplasia;
  • rat

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Dysplasia represents a preneoplastic status in multistep colon carcinogenesis. Whereas laborious preparation of thin sections is required for its diagnosis, we here show that newly defined aberrant crypt foci (ACF) simply mark the majority of the dysplasia on the whole colon. Specifically, decoloring of the azoxymethane-treated rat colon after scoring classical ACF (cACF) resulted in visualization of a subset of aberrant crypts that remained densely stained. They were morphologically classified into three subtypes, of which two with compressed luminal openings proved highly correlated with dysplasia. Accordingly, we designated those foci harboring either of the two crypt subtypes as dysplasia-associated ACF (dACF). By serially applying different detection methods for known preneoplastic lesions to the same colon, we showed that most dACF had already been identified as cACF, and a few newly identified dACF contained an entire population of more advanced lesions, such as flat ACF and mucin-depleted foci. Consequently, integrative scoring of cACF and dACF enabled capture of all early lesions of the colon. Furthermore, 94% of the dACF showed dysplasia and 90% of the dysplastic lesions proved to be dACF. Thus, dACF is a promising marker for dysplasia, likely facilitating precise identification of the early stages of colon carcinogenesis.

Step-wise accumulation of genetic events underlies histological transition in colon carcinogenesis.[1-4] Aberrant crypt foci (ACF), clusters of enlarged thick crypts visualized by methylene blue (MB) staining, can be easily detected under a microscope at low magnification, and are the earliest identifiable lesions.[5, 6] As their incidence at an early time point is highly correlated with eventual tumor incidence,[7] they have been widely used in evaluating carcinogenicity of chemicals.[8] However, their specificity as tumor precursors has been questioned, partly due to their predominant location in the proximal colon, where tumors do not develop, and gradual disappearance over time.[9, 10] Moreover, ACF frequently harbor mutations in Kras, but rarely in adenomatous polyposis coli (Apc).[11-13] These findings strongly suggested that the majority of ACF might not be true preneoplastic lesions. Frequent Apc inactivation and β-catenin accumulation strongly suggest that dysplastic ACF[4, 14-16] or crypts with β-catenin accumulation[17-19] might be specific tumor precursors, but they need histological analysis to be identified.

There are other non-ACF lesions on the whole colon. Flat dysplastic ACF (fACF) are less densely stained foci by brief exposure to MB.[10] Flat dysplastic ACF are likely associated with Apc inactivation, as Apcmin/+ mice spontaneously develop equivalent lesions, but not classical ACF (cACF).[20] Mucin-depleted foci (MDF) are the stain-defect lesions by periodic acid–Schiff (PAS), which readily detects mucin.[9, 21] Unlike cACF, these two lesions develop predominantly in the distal colon and exhibit high-grade dysplasia. Moreover, chronological analysis has indicated that foci are likely tumor precursors.[9, 10] However, they manifest only a low sensitivity for dysplasia.

Feeding rats with a dietary mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), allows recapitulation of colon carcinogenesis, providing many insights into the molecular mechanisms.[14, 22-28] While characterizing PhIP-induced ACF, we noted that addition of a decoloring step could result in selective visualization of some stained foci. These foci tended to be more dysplastic than classical ACF, and the higher staining intensity of the foci could considerably, if not always, predict a higher likelihood of being dysplastic.[29] These findings raised the possibility that dysplasia could be identified as a new type of ACF. In this study, we verified this notion, and showed that the new ACF might be reasonably useful to evaluate the early stages of colon carcinogenesis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Animal studies

Five-week-old F344 male rats were purchased from Clea Japan (Tokyo, Japan). The rats were s.c. treated with azoxymethane (AOM; Nard Institute, Osaka, Japan) at a dose of 15 mg/kg twice at the age of 6 and 7 weeks, and killed 9 weeks after the last treatment. The institutional committee for ethics in animal experimentation approved the protocol for animal experiments. The experiments were carried out in accordance with the guidelines for animal experiments of the National Cancer Center (Tokyo, Japan).

Identification of cACF and dACF

The formalin-fixed colons were stained with 0.2% MB in PBS for 20 min, and the number of cACF[5] was counted. The colons were further stained with 0.2% MB for 20 min, and soaked in 70% methanol for 6 min for destaining.[29] Stained crypts were classified into three subtypes: crypts larger than surrounding normal ones with proportionally large and narrow openings were designated as c-type and cd-type, respectively, and crypts smaller than surrounding normal ones with narrower openings were designated as d-type. These three subtypes were readily distinguishable under a microscope, but objective evaluation was also carried out. The external diameter of normal (RN) and aberrant crypts (R), and the internal diameter of aberrant crypts (r) were measured on captured images to calculate relative luminal opening size and relative crypt size. Foci containing at least a single crypt of either cd-type or d-type crypts were diagnosed as dysplasia-associated ACF (dACF). The signal intensity ratio (SIR) of the foci was determined as previously described.[29]

Serial scoring of preneoplastic lesions in a whole mounted colon

The formalin-fixed colons were divided into four segments, which were subject to serial different staining. The mucosal surface was examined under an inverse light microscope, and pictures across the whole colon were taken at each step for later comparison of the images of the same foci. Fragments were first soaked in 1% Alcian blue (AB), pH 2.5, for 30 min, and subsequently in 0.2% MB for 5 s to detect MDF and fACF, respectively. We adopted the staining solely with AB for the detection of MDF,[21] instead of the original high iron diamine–AB staining,[9] to avoid interference of high iron diamine with MB in evaluating ACF. Subsequently, the number of cACF and dACF were counted.

Histological analysis

For vertical sections, the colons were cut into pieces 3 mm long and 6 mm wide, so that each dACF would be located in the center of the fragment. For horizontal sections, the colons were first cut longitudinally into two strips, and subsequently segmented laterally into 8-mm-long pieces. These pieces were embedded in paraffin and cross-sections were prepared in 4 μm. Serial sections were subject to H&E staining without prior knowledge of information on surface examination. Dysplastic lesions were further graded into three classes as previously described.[30] The AB-PAS staining was carried out for the detection of foci that had completely lost mucin, which we designated as mucin-depleted crypts (MDC). Crypts with an accumulation of β-catenin in the cytoplasm or nucleus were diagnosed as β-catenin accumulating crypts (BCAC).[19, 29]

Mutation analysis

Genomic DNA was selectively collected from BCAC by microdissection from the unstained serial sections of 20 independent samples. Extracted DNA was subject to amplification of exon 3 of the Ctnnb1 gene by PCR, as previously described.[16] Polymerase chain reaction products were purified and directly sequenced by the standard Sanger method.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Decoloring of MB-stained colon segregated three subtypes of aberrant crypts

We previously showed that more accurate detection of dysplasia might be achieved by destaining of cACF, although with laborious procedures, and found that the narrow opening might suggest the dysplastic nature of the foci.[29] We then hypothesized that stained foci harboring crypts with narrow opening, or dACF as tentatively designated, might be equivalent to dysplasia. To address this issue, we examined a rat colon treated with AOM. Starting with examining 161 induced cACF, we noted that decoloring gave rise to three morphologically distinct subtypes of stained crypts (Fig. 1a). We designated enlarged crypts with large openings, and small crypts with narrow openings as c-type and d-type, respectively, to emphasize specific features of cACF and dACF (Fig. 1B). Accordingly, enlarged crypts with narrow openings were designated as cd-type. Normal crypts and a subset of crypts within cACF turned invisible after decoloring. We designated the latter as h-type crypts (Fig. 1B), as such crypts constituted hyperplasia.[29] Thus, various types of crypts were differentially visualized through decoloring. We now redefined dACF as foci harboring at least single d- or cd-type crypt.

image

Figure 1. Crypt-based characterization of azoxymethane-induced dysplasia-associated aberrant crypt foci (dACF). (a) Typical images of the three crypt subtypes (closed arrowheads) constituting dACF: c-type, enlarged crypt and broad opening; cd-type, enlarged crypt and narrow opening; and d-type, small crypt and narrow opening. Each arrowhead points to a single crypt. Note that normal crypts in surrounding mucosa (open arrowheads) could serve as a reference with a diameter of approximately 30 μm. (b) Schematic view of the crypt subtypes. A ring-shaped image represents the morphology of epithelial lining. Dashed lines indicate invisible status. (c) Classification of aberrant crypts by objective and subjective methods. Note that visual judgment and strict quantification gave completely concordant results for randomly selected crypts (n = 66). r, internal diameter of aberrant crypts; R, external diameter of aberrant crypts; RN, external diameter of normal crypts. (d) Venn diagram for classical ACF (cACF) and dACF. Dysplasia-associated ACF (shaded box) was divided into two subgroups, cACF+ve and cACF−ve, depending on the presence of overlap with cACF (open box). Composition of crypt subtype in stained foci is indicated in parenthesis. (e) Breakdown of crypt subtypes within stained foci. Each axis of the bar chart depicts the number of aberrant foci, c-type crypt and d- or cd-type crypt.

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Dysplasia-associated ACF consisted of diverse combinations of crypt subtypes

The three crypt subtypes were morphologically so distinct that the right diagnosis could be made even by visual judgment. In fact, rigorous quantification of the size of both the crypt and its luminal openings for 66 randomly selected crypts gave completely concordant results (Fig. 1c). Direct comparison of the images of 71 dACF before and after decolorization revealed that 62 and 9 had been diagnosed as cACF+ve and cACF−ve, respectively (Fig. 1d). Nine newly identified cACF−ve consisted solely of d-type crypts, whereas 62 cACF+ve had various compositions of crypt subtypes. Most foci contained both cd- and c-type crypts (51/62), but some foci consisted of cd-type crypts alone (2/62), or with both c- and d-type crypts (9/62) (Figs 1d and 2a). Interestingly, cd- and d-type crypts never coexisted in the same foci, and the foci with more than two d-type crypts tended not to coexist with c-type crypts, which was not the case with c-type crypts (Fig. 1e). These observations strongly suggested that cd- and d-type crypts might have evolved independently. The SIR of the foci[29] was in fact more than 1.0 for all the stained lesions and tended to be higher in dACF than in cACF, but failed to completely distinguish between the two (Fig. S1), consistent with our previous study.[29] These observations confirmed the relevance of the size of the luminal opening, but not SIR of the foci, for the right diagnosis of dACF.

image

Figure 2. Histological features of dysplasia-associated aberrant crypt foci (dACF). (a) Representative images of dACF (left panel) and corresponding histological features with H&E (middle panel) and Alcian blue–periodic acid–Schiff (AB-PAS) (right panel) staining. A single crypt of c-type (enlarged crypt and broad opening; open arrowheads), cd-type (enlarged crypt and narrow opening; arrow), and d-type (small crypt and narrow opening; closed arrowheads) is individually indicated for classical ACF (cACF)+ve (#1–3). Classical ACF−ve consisted solely of d-type crypts (#4–6). Mucin expression was decreased in cd-type crypts (arrow) and completely depleted in d-type crypts (closed arrowheads). Accumulation of red granules indicative of Paneth cell differentiation (#4 and #5), consistent with severe dysplasia. (b–e) Comparison of cACF+ve and cACF–ve. The number of lesions with hyperplasia and dysplasia (b), and proportion of dysplastic lesions with each histological grade (c), with mucin depletion (d), and with β-catenin accumulation (e) are shown in the bar charts.

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Dysplasia-associated ACF histologically manifested dysplasia of all grades

Among 71 dACF, vertical sections were successfully prepared for 54 lesions, 45 cACF+ve and 9 cACF−ve (Fig. 2b). Histological analysis revealed that 51 dACF were dysplastic (94%) and 3 hyperplastic (6%). The cACF+ve lesions predominantly comprised moderate dysplasia and cACF−ve severe dysplasia (Fig. 2c). The SIR did not correlate with histological grades (Fig. S2), further declining its relevance. Notably, dysplastic crypts predominantly below the mucosal surface could be detected as dACF (#3–6 of Fig. 2a). Staining with AB-PAS clarified that mucin depletion was exclusively in cACF−ve lesions (Fig. 2d), and accumulation of red granules, indicative of Paneth cell differentiation due to Wnt activation,[31] only in cACF−ve (Fig. 2a and Fig. S3). Accumulation of β-catenin was observed in only a few cACF+ve, but in the majority of cACF−ve (Fig. 2e). These findings implied that dACF could manifest dysplasia of all grades, with cACF−ve representing Wnt-activated more advanced lesions than cACF+ve. Sequencing analysis of microdissected BCACs from 10 cACF+ve and 10 cACF−ve for Ctnnb1 mutation identified only one activating mutation from A to G substituting Asp at codon 32 with Asn in a lesion from cACF+ve, but not in any lesion from cACF−ve, implying non-genetic mechanisms for AOM-induced β-catenin accumulation, at least in early stages.

Dysplasia-associated ACF overlapped both cACF and advanced preneoplastic lesions

To relate dACF to other known preneoplastic lesions, the colons from three AOM-treated rats were serially stained to detect these lesions, and images of the same foci were directly compared (Fig. 3a). Staining with AB visualized four to five MDF per rat exclusively in the distal colon (Fig. 3b), as previously reported.[9, 21] Subsequent brief exposure to MB visualized fACF, which completely coincided with MDF in a total of 14 lesions from three rats (Fig. 3c). By longer exposure to MB and subsequent destaining, 200–300/rat cACF and ~120/rat dACF were visualized (Fig. 3c). Dysplasia-associated ACF tended to be in the more distal colon than cACF, in line with the dysplastic nature of dACF. Approximately 100 and 20 dACF were diagnosed as cACF+ve and cACF−ve, respectively (Fig. 3d), consistent with our pilot experiment. Taken together, dACF had a significant overlap with cACF and contained an entire population of MDF and fACF. Representative cases are shown in Figure 4; dACF-1 is cACF+ve (Fig. 4a–e) and dACF-2,3,4 are cACF−ve (Fig. 4f–q). Unlike dACF, all the other lesions were not constantly positive for these four foci. Of note, dACF-2 could be detected only as dACF, strongly suggesting higher sensitivity of dACF for dysplasia.

image

Figure 3. Serial scoring of multiple preneoplastic lesions on the same colon. (a) A schematic diagram of the serial detection of multiple preneoplastic lesions. The images of mucosal surfaces were captured at the end of each step (arrows) for comparison at a later time point. (b) Colons were cut open longitudinally and separated into four segments (I–IV). (c) Bar chart showing the number of preneoplastic lesions per segment. #1–3 corresponds to the colons of three individual F344 male rats. Note that dysplasia-associated aberrant crypt foci (dACF), flat ACF (fACF), and mucin-depleted foci (MDF) are more enriched in segments III and IV compared to classical ACF (cACF). (d) Venn diagram for the number of cACF, dACF, and fACF/MDF for each rat.

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image

Figure 4. Direct comparison between images of the same foci visualized by different methods: lesion dysplasia-associated aberrant crypt foci-1 (dACF-1) with mild dysplasia (a–e); lesions dACF-2 (f–j) and dACF-3 (k–o) with moderate dysplasia; and lesion dACF-4 with severe dysplasia (p–t). The left four columns show the images obtained by scoring preneoplastic lesions on the mucosal surface, and the diagnosis is indicated below the images as − (negative) or + (positive). The far right column depicts H&E staining of the corresponding lesions in horizontal sections, where the grade of dysplasia is shown below the images. Open arrowheads point to the area where the dysplastic lesions are supposed to be located in each image.

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Scoring dACF enables robust identification of dysplasia

We next wondered, conversely, if the dysplasia could appear as dACF. To address this issue, we thoroughly examined a defined part of the colon, both by histological analysis on horizontal sections, and by serially scoring preneoplastic lesions on the whole mounted colon. We scrutinized segment III of rat #1 (Fig. 5a) because it harbored the highest number of dACF across the samples (Fig. 3c). Of 64 dysplasia, 62 were successfully related between the images of H&E staining and those of the mucosal surface. It was revealed that 56 (90%) and 43 (69%) dysplasia were previously detected as dACF and cACF, respectively, whereas only 4 (6.5%) were diagnosed as fACF/MDF (Fig. 5b, Table S1). Dysplasia-associated ACF and cACF achieved the highest and second highest detection rate, respectively, but 42 of 43 cACF lesions were also detected as dACF, underscoring the superiority of dACF. The detection rates of fACF/MDF, MDC, and BCAC, were comparable to that of cACF only for severe dysplasia (Fig. 5b, Table S1), but still no higher than that of dACF. Representative cases of dysplasia are shown in Figure 5(c). Whereas all the four lesions were diagnosed as dACF, three and two were also diagnosed as BCAC and MDC, respectively. Notably, #58 and #62, identical to dACF-2 and dACF-3 (Fig. 4), respectively, were both diagnosed as MDC, but only #62/dACF-3 was diagnosed as MDF (Fig. 4), clearly indicating that MDF might be less sensitive for dysplasia below the mucosal surface. There was one lesion with severe dysplasia exceptionally undetectable as either dACF or cACF (Table S1). It turned out to be a single aberrant crypt undetectable as MDF despite obvious mucin depletion and β-catenin accumulation (Fig. S4), illustrating the limitation in detecting too small dysplasia as dACF. Taken together, dACF marked with high sensitivity pan-dysplasia of any depth in the mucosa, whereas the other advanced lesions marked dysplasia with modest sensitivity only when on the surface.

image

Figure 5. Robust identification of dysplasia by scoring dysplasia-associated aberrant crypt foci (dACF). (a) Schematic diagram of the comprehensive histological analysis. A fixed colon from rat #1 was longitudinally cut open and segment III (shaded area) was divided into 24 fragments of 8 mm width (open rectangle) to cover the whole area by horizontal sections. (b) Successful detection rate of dysplasia for each preneoplastic lesion. Note that dACF provides the highest sensitivity in all grades of dysplasia. (c) Histological features of four representative dACF. Images of serial sections from the same lesion are shown. Dashed line circles β-catenin accumulating crypts (BCAC) or mucin-depleted crypts (MDC). Note that Paneth differentiation is evident in #58 and #62 (dACF-2 and -3 in Fig. 4, respectively), consistent with severe dysplasia. AB-PAS, Alcian blue–periodic acid–Schiff; cACF, classical ACF; fACF, flat ACF; MDF, mucin-depleted foci.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

An ideal marker should be simply detected and easily diagnosed, let alone manifest high specificity and sensitivity. In terms of a marker for dysplasia, however, none of the known preneoplastic lesions of the colon meet all these criteria. As an extension of our previous study, we developed a more straightforward method that enables more specific and sensitive detection of dysplasia in this study. Indeed, 94% of the dACF showed dysplasia, while 90% of the dysplastic lesions proved to be dACF, obviously overwhelming cACF as a marker. Inter-relationships between dACF and various other lesions are summarized in Figure 6.

image

Figure 6. Dysplasia-associated aberrant crypt foci (dACF) among other preneoplastic lesions of the colon. A summary of the interrelationship between dACF and other preneoplastic lesions, both on unsectioned colon and in thin sections. The spectra of various lesions along multistep colon carcinogenesis clearly indicate that dACF successfully filled in the gap between mucin-depleted foci (MDF)/flat ACF (fACF) and classical ACF (cACF) in detection of dysplasia. c-type, enlarged crypt and broad opening; cd-type, enlarged crypt and narrow opening; d-type, small crypt and narrow opening.

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We developed the new marker by modifying diagnostic steps of the foci. Specifically, we discontinued strict measurement of foci's signal intensity because it proved dispensable and insufficient for detecting and separating dysplastic foci, respectively. Instead, we introduced crypt-based evaluation of the foci for diagnosis of dACF. The crypt subtypes were so distinct that even visual judgment gave concordant results with those obtained by objective quantification. In contrast, we felt that the right diagnosis might be difficult for fACF and MDF, although the visualizing procedure is simple. We were able to spot only relatively large lesions, which resulted in 100% coincident diagnosis of fACF and MDF in our hands, however, examination of the same samples by two experts resulted in a concordant rate as low as 42%,[32] implying difficulties in diagnosing small lesions for non-experts.

The potential of dACF might extend to a marker for tumorigenicity. Currently, incidence of cACF is widely used as a surrogate marker for eventual tumorigenesis based on their high correlation.[7] We showed that the number of dACF is approximately half that of cACF, with a large overlap with the cACF, which is still large enough to yield a proper dynamic range for quantitative evaluation. Given the close association of dACF with dysplasia, it is conceivable that the number of dACF could have more accurately predicted the incidence of eventual tumor development, had it been applied to a case where retinoids decreased the number of cACF, but not that of tumors.[33] It is also tempting to speculate that the relationship between cACF and dACF might account for the long-appreciated correlation between the incidence of cACF and eventual tumorigenesis, presumably through a correlation between the incidence of dACF and dysplasia, justifying the past studies using incidence of cACF as a marker for tumorigenicity.

Crypt-based analysis of dACF also clarified novel aspects of the early stages of colon carcinogenesis. The foci with more than two d-type crypts tended not to coexist with c-type crypts within the same foci, in line with the previous observation that adenomatous ACF developed from hyperplastic ACF,[34] while it was not the case with cd-type crypts. Together with a mutually exclusive relationship within the same foci, d- and cd-type crypts might constitute a distinct population that follows different pathways toward tumor development. To gain insights into the molecular differences between the two types, we compared cACF−ve and cACF+ve subsets of dACF, which contained exclusively d-type and predominantly cd-type, respectively. β-catenin accumulation and mucin depletion was significantly more frequent in cACF−ve, clearly indicating that cACF−ve are more advanced lesions. However, activating mutation of Ctnnb1 was rarely found in both subsets, strongly suggesting alternative mechanisms for Wnt activation in early stages, such as APC knockdown by SND1 overexpression.[35] Although d- and cd-type crypts were stained after decolorization of mucosa, underlying molecular mechanisms are poorly understood. Considering MB's high affinity to an acidic substrate, including phosphate,[36] higher staining intensity might reflect higher amounts of nucleic acids and phosphorylated protein in dysplastic foci. A puzzling feature of d-type crypts is that they are invisible before decolorization, although they are supposed to be stained by MB. This observation might be partly due to the fact that d-type crypts tend to reside predominantly below the stained normal crypts[22] (Fig. 2a, #3–6; Fig. S3, #7–10; Fig. 4h), consistent with the “bottom-up model” for colon carcinogenesis,[37] thereby requiring complete decolorization of surface mucosa. Taken together, further studies on crypt-based analysis would be warranted to elucidate molecular mechanisms underlying colon carcinogenesis.

In conclusion, we showed that dysplasia could be simply identified on an unsectioned colon as dACF. Based on their unique features, dACF might be established as novel standard preneoplastic lesions of the colon, by complementing or substituting cACF, the current gold standard in this field.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

We thank Ryoichi Masui and Miho Sasaki for their technical assistance, and Toshio Imai for helpful discussions. We are also grateful to the Central Animal Division in the National Cancer Center Research Institute for their support in animal studies. This work was supported in part by a Grant-in-Aid for Research on Global Health Issues from the Ministry of Health, Labor and Welfare of Japan (201204006) to H.N., a Grant-in-Aid for Cancer Research for the Third Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan (201220001) to Y.H., and a Grant-in-Aid for Scientific Research C from the Japan Society for the Promotion of Science (18590710) to M.O.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information
  • 1
    Aoki K, Taketo MM. Adenomatous polyposis coli (APC): a multi-functional tumor suppressor gene. J Cell Sci 2007; 120: 332735.
  • 2
    Levy DB, Smith KJ, Beazer-Barclay Y, Hamilton SR, Vogelstein B, Kinzler KW. Inactivation of both APC alleles in human and mouse tumors. Cancer Res 1994; 54: 59538.
  • 3
    Sparks AB, Morin PJ, Vogelstein B, Kinzler KW. Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer. Cancer Res 1998; 58: 11304.
  • 4
    Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996; 87: 15970.
  • 5
    Bird RP. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett 1987; 37: 14751.
  • 6
    McLellan EA, Bird RP. Aberrant crypts: potential preneoplastic lesions in the murine colon. Cancer Res 1988; 48: 618792.
  • 7
    Corpet DE, Tache S. Most effective colon cancer chemopreventive agents in rats: a systematic review of aberrant crypt foci and tumor data, ranked by potency. Nutr Cancer 2002; 43: 121.
  • 8
    Khare S, Chaudhary K, Bissonnette M, Carroll R. Aberrant crypt foci in colon cancer epidemiology. Methods Mol Biol 2009; 472: 37386.
  • 9
    Caderni G, Femia AP, Giannini A et al. Identification of mucin-depleted foci in the unsectioned colon of azoxymethane-treated rats: correlation with carcinogenesis. Cancer Res 2003; 63: 238892.
  • 10
    Paulsen JE, Loberg EM, Olstorn HB, Knutsen H, Steffensen IL, Alexander J. Flat dysplastic aberrant crypt foci are related to tumorigenesis in the colon of azoxymethane-treated rat. Cancer Res 2005; 65: 1219.
  • 11
    Yamashita N, Minamoto T, Ochiai A, Onda M, Esumi H. Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon. Gastroenterology 1995; 108: 43440.
  • 12
    Takahashi M, Wakabayashi K. Gene mutations and altered gene expression in azoxymethane-induced colon carcinogenesis in rodents. Cancer Sci 2004; 95: 47580.
  • 13
    Nucci MR, Robinson CR, Longo P, Campbell P, Hamilton SR. Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa. Hum Pathol 1997; 28: 1396407.
  • 14
    Nakagama H, Ochiai M, Ubagai T et al. A rat colon cancer model induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP. Mutat Res 2002; 506–507: 13744.
  • 15
    Jen J, Powell SM, Papadopoulos N et al. Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994; 54: 55236.
  • 16
    Ochiai M, Ushigome M, Fujiwara K et al. Characterization of dysplastic aberrant crypt foci in the rat colon induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Am J Pathol 2003; 163: 160714.
  • 17
    Yamada Y, Mori H. Pre-cancerous lesions for colorectal cancers in rodents: a new concept. Carcinogenesis 2003; 24: 10159.
  • 18
    Yamada Y, Yoshimi N, Hirose Y et al. Sequential analysis of morphological and biological properties of beta-catenin-accumulated crypts, provable premalignant lesions independent of aberrant crypt foci in rat colon carcinogenesis. Cancer Res 2001; 61: 18748.
  • 19
    Yamada Y, Yoshimi N, Hirose Y et al. Frequent beta-catenin gene mutations and accumulations of the protein in the putative preneoplastic lesions lacking macroscopic aberrant crypt foci appearance, in rat colon carcinogenesis. Cancer Res 2000; 60: 33237.
  • 20
    Paulsen JE, Steffensen IL, Loberg EM, Husoy T, Namork E, Alexander J. Qualitative and quantitative relationship between dysplastic aberrant crypt foci and tumorigenesis in the Min/+ mouse colon. Cancer Res 2001; 61: 50105.
  • 21
    Yoshimi N, Morioka T, Kinjo T et al. Histological and immunohistochemical observations of mucin-depleted foci (MDF) stained with Alcian blue, in rat colon carcinogenesis induced with 1,2-dimethylhydrazine dihydrochloride. Cancer Sci 2004; 95: 7927.
  • 22
    Nakagama H, Nakanishi M, Ochiai M. Modeling human colon cancer in rodents using a food-borne carcinogen, PhIP. Cancer Sci 2005; 96: 62736.
  • 23
    Ochiai M, Nakagama H, Watanabe M, Ishiguro Y, Sugimura T, Nagao M. Efficient method for rapid induction of aberrant crypt foci in rats with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Jpn J Cancer Res 1996; 87: 102933.
  • 24
    Creton SK, Zhu H, Gooderham NJ. The cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine activates the extracellular signal regulated kinase mitogen-activated protein kinase pathway. Cancer Res 2007; 67: 1145562.
  • 25
    Ubagai T, Ochiai M, Kawamori T et al. Efficient induction of rat large intestinal tumors with a new spectrum of mutations by intermittent administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in combination with a high fat diet. Carcinogenesis 2002; 23: 197200.
  • 26
    Ishiguro Y, Ochiai M, Sugimura T, Nagao M, Nakagama H. Strain differences of rats in the susceptibility to aberrant crypt foci formation by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: no implication of Apc and Pla2g2a genetic polymorphisms in differential susceptibility. Carcinogenesis 1999; 20: 10638.
  • 27
    Fujiwara K, Ochiai M, Ubagai T et al. Differential gene expression profiles in colon epithelium of two rat strains with distinct susceptibility to colon carcinogenesis after exposure to PhIP in combination with dietary high fat. Cancer Sci 2003; 94: 6728.
  • 28
    Igarashi M, Hippo Y, Ochiai M, Fukuda H, Nakagama H. AKT is critically involved in cooperation between obesity and the dietary carcinogen amino-1-methyl-6-phenylimidazo [4,5-b] (PhIP) toward colon carcinogenesis in rats. Biochem Biophys Res Commun 2014; 443: 8527.
  • 29
    Ochiai M, Watanabe M, Nakanishi M, Taguchi A, Sugimura T, Nakagama H. Differential staining of dysplastic aberrant crypt foci in the colon facilitates prediction of carcinogenic potentials of chemicals in rats. Cancer Lett 2005; 220: 6774.
  • 30
    Siu IM, Pretlow TG, Amini SB, Pretlow TP. Identification of dysplasia in human colonic aberrant crypt foci. Am J Pathol 1997; 150: 180513.
  • 31
    Joo M, Shahsafaei A, Odze RD. Paneth cell differentiation in colonic epithelial neoplasms: evidence for the role of the Apc/beta-catenin/Tcf pathway. Hum Pathol 2009; 40: 87280.
  • 32
    Femia AP, Paulsen JE, Dolara P, Alexander J, Caderni G. Correspondence between flat aberrant crypt foci and mucin-depleted foci in rodent colon carcinogenesis. Anticancer Res 2008; 28: 37715.
  • 33
    Zheng Y, Kramer PM, Lubet RA, Steele VE, Kelloff GJ, Pereira MA. Effect of retinoids on AOM-induced colon cancer in rats: modulation of cell proliferation, apoptosis and aberrant crypt foci. Carcinogenesis 1999; 20: 25560.
  • 34
    Otori K, Sugiyama K, Hasebe T, Fukushima S, Esumi H. Emergence of adenomatous aberrant crypt foci (ACF) from hyperplastic ACF with concomitant increase in cell proliferation. Cancer Res 1995; 55: 47436.
  • 35
    Tsuchiya N, Ochiai M, Nakashima K, Ubagai T, Sugimura T, Nakagama H. SND1, a component of RNA-induced silencing complex, is up-regulated in human colon cancers and implicated in early stage colon carcinogenesis. Cancer Res 2007; 67: 956876.
  • 36
    Tong C, Hu Z, Wu J. Interaction between methylene blue and calf thymus deoxyribonucleic acid by spectroscopic technologies. J Fluoresc 2010; 20: 2617.
  • 37
    Boman BM, Fields JZ, Cavanaugh KL, Guetter A, Runquist OA. How dysregulated colonic crypt dynamics cause stem cell overpopulation and initiate colon cancer. Cancer Res 2008; 68: 330413.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cas12446-sup-0001-FigS1.tifimage/tif3386K

Fig. S1. Signal intensity ratio (SIR) of the foci is insufficient for right diagnosis of dysplasia-associated aberrant crypt foci (dACF). A dot represents each lesion.

cas12446-sup-0002-FigS2.tifimage/tif5548K

Fig. S2. Lack of correlation between histological grade and signal intensity of stained foci.

cas12446-sup-0003-FigS3.tifimage/tif2790K

Fig. S3. Classical aberrant crypt foci (cACF)−ve subset of dysplasia-associated ACF (dACF) manifest severe dysplasia.

cas12446-sup-0004-FigS4.tifimage/tif1847K

Fig. S4. Dysplastic crypt exceptionally undetectable by scoring dysplasia-associated aberrant crypt foci (dACF).

cas12446-sup-0005-TableS1.docxWord document107K

Table S1. Characterization of comprehensively determined dysplastic lesions.

cas12446-sup-0006-FigureLegends.docxWord document88K 

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