Detection of colorectal dysplasia using fluorescently labelled lectins

Abstract

Colorectal cancer screening using conventional colonoscopy lacks molecular information and can miss dysplastic lesions. We tested here the ability of fluorescently labelled lectins to distinguish dysplasia from normal tissue when sprayed on to the luminal surface epithelium of freshly resected colon tissue from the Apc^min mouse and when applied to fixed human colorectal tissue sections. Wheat germ agglutinin (WGA) showed significantly decreased binding to adenomas in the mouse tissue and in sections of human colon from 47 patients. Changes in WGA binding to the human surface epithelium allowed regions containing normal epithelium (NE) or hyperplastic polyps (HP) to be distinguished from regions containing low-grade dysplasia (LGD), high-grade dysplasia (HGD) or carcinoma (C), with 81% sensitivity, 87% specificity and 93% positive predictive value (PPV). Helix pomatia agglutinin (HGA) distinguished epithelial regions containing NE from regions containing HP, LGD, HGD or C, with 89% sensitivity, 87% specificity and 97% PPV. The decreased binding of WGA and HPA to the luminal surface epithelium in human dysplasia suggests that these lectins may enable more sensitive detection of disease in the clinic using fluorescence colonoscopy.

Full text

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Scientific RepoRts | 6:24231 | DOI: 10.1038/srep24231
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Detection of colorectal dysplasia
using uorescently labelled lectins
Joe Chin-Hun Kuo1,2, Ashraf E. K. Ibrahim3,4, Sarah Dawson5, Deepak Parashar6,
William J. Howat1, Kiran Guttula3, Richard Miller7, Nicola S. Fearnhead7, Douglas J. Winton1,
André A. Neves1 & Kevin M. Brindle1,2
Colorectal cancer screening using conventional colonoscopy lacks molecular information and can miss
dysplastic lesions. We tested here the ability of uorescently labelled lectins to distinguish dysplasia
from normal tissue when sprayed on to the luminal surface epithelium of freshly resected colon tissue
from the Apcmin mouse and when applied to xed human colorectal tissue sections. Wheat germ
agglutinin (WGA) showed signicantly decreased binding to adenomas in the mouse tissue and in
sections of human colon from 47 patients. Changes in WGA binding to the human surface epithelium
allowed regions containing normal epithelium (NE) or hyperplastic polyps (HP) to be distinguished from
regions containing low-grade dysplasia (LGD), high-grade dysplasia (HGD) or carcinoma (C), with 81%
sensitivity, 87% specicity and 93% positive predictive value (PPV). Helix pomatia agglutinin (HGA)
distinguished epithelial regions containing NE from regions containing HP, LGD, HGD or C, with 89%
sensitivity, 87% specicity and 97% PPV. The decreased binding of WGA and HPA to the luminal surface
epithelium in human dysplasia suggests that these lectins may enable more sensitive detection of
disease in the clinic using uorescence colonoscopy.
Progression of colorectal cancer (CRC) from low- to high-grade dysplasia1 provides an opportunity for prophy-
lactic removal2,3 of low-risk adenomas, which has been shown to reduce mortality4. However, colonoscopy misses
>20% of adenomatous polyps5–7, including high-grade lesions in the proximal colon8,9. Moreover, in inam-
matory bowel diseases (IBD) dysplasia may appear normal, requiring the entire colon to be randomly biopsied
for eective surveillance10,11. Furthermore, colonoscopy cannot distinguish between dysplasia and hyperplasia,
which is non-neoplastic and does not always require excision12, but frequently requires dierentiation from dys-
plasia using biopsy or polypectomy. Since polypectomy carries a low risk of fatal bleeding and colon perforation13
there is a need for the development of methods that can distinguish hyperplasia from dysplasia at colonoscopy.
Targeted molecular imaging agents can enhance contrast between non-neoplastic and neoplastic tissues,
improving the detection of dysplasia14. Fluorescently-labelled antibodies, injected intracardially in an animal
model, and small peptides applied topically in patients, have enhanced detection of colon neoplasia using con-
focal laser microendoscopy (CLM)15,16. However, CLM has a microscopic eld-of-view and can only examine
small regions of the colon. Wide-eld uorescence imaging, which can be integrated into conventional endo-
scopes, could allow rapid screening of the entire colon. is has been achieved using small peptides labelled
with near-infrared uorophores and applied topically for the detection of colon neoplasia in animal models17.
However, the molecular targets of these peptides are unknown, and therefore they may lack specicity.
Changes in glycosylation provide potential biomarkers of colon dysplasia18,19. Mucins cover the entire colonic
mucosa and changes in their expression and glycosylation are associated with progression to CRC20,21 and can be
associated with a poor prognosis22–25. Sialic acid content changes in colonic neoplasia26 and hyperplastic tissue
secretes mucus rich in sialomucins27. erefore imaging agents that bind specic glycan moieties may be useful
in distinguishing normal from dysplastic tissues as well as hyperplasia from dysplasia.
We have shown previously that topically applied uorescently labelled lectins can detect glycosylation changes
in freshly resected oesophagus, potentially allowing endoscopic identication of oesophageal dysplasia28. Lectins
1Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, UK.
2Department of Biochemistry, University of Cambridge, Cambridge, UK. 3Department of Pathology, Division of
Molecular Histopathology, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK. 4MRC, Laboratory of
Molecular Biology, Hills Road, Cambridge, UK. 5Cambridge Clinical Trials Unit, University of Cambridge, Cambridge,
UK. 6Statistics and Epidemiology Unit & Cancer Research Centre, Division of Health Sciences, Warwick Medical
School, The University of Warwick, Coventry, UK. 7Cambridge Colorectal Unit, Addenbrooke’s Hospital, Cambridge,
UK. Correspondence and requests for materials should be addressed to A.A.N. (email: andre.neves@cruk.cam.ac.uk)
Received: 11 December 2015
Accepted: 22 March 2016
Published: 13 April 2016
OPEN

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are a family of glycan-specic proteins29 that are relatively non-toxic and inexpensive to produce. We have inves-
tigated here the potential of uorescently labelled lectins to detect dysplasia elsewhere in the gastrointestinal tract,
in this case the colon, using lectins that have been reported previously to show changes in binding to colorectal
neoplasia, including Helix pomatia agglutinin (HPA)30, Artocarpus integrifolia or jackfruit lectin (JFL)31, Arachis
hypogaea or peanut agglutinin (PNA)32, Glycine max or soybean agglutinin (SBA)33, and Triticum vulgaris or
wheat germ agglutinin (WGA)31. Using freshly resected colon tissue from the Apcmin mouse and formalin xed
paran embedded (FFPE) human tissue sections, we show that some of these lectins, when uorescently labelled,
can distinguish between normal and dysplastic tissue from their dierential binding to colorectal luminal surface
epithelium. Since this surface is accessible to endoscopic examination, these lectins have the potential to be trans-
lated to the clinic for detecting colorectal dysplasia using uorescence colonoscopy.
Results
Lectin binding to freshly resected intestines from the Apcmin mouse. Fluorescently-labelled
WGA was sprayed onto the luminal surface epithelium. Adenomas, which occur less frequently in the colon
as compared to the small intestine in this animal model34, were easily identiable (Fig.1a(ii), black arrows).
Macroscopically, WGA showed binding to normal colon epithelium (Fig.1b; row 3, column 1) and decreased
binding to adenomas (Fig.1a(i), black arrows). Although lectin binding decreased with distance along the small
intestine (Fig.1a(i)) contrast between the adenomas and surrounding normal tissue was maintained. Microscopic
examination conrmed that WGA uorescence was limited to the luminal surface epithelium, as would be
expected from topical application (Fig.1b; columns 1 and 3). Unlike in the human disease, adenomas in the
intestines from Apcmin mice can display a covering layer of normal epithelial cells (Fig.1b; row 3)35. However,
similar to what we observed subsequently in human colorectal tissue sections, WGA binding was diminished in
adenomas in relation to normal tissue (Fig.1b; row 4). Binding of WGA was quantied and expressed as the mean
uorescence intensity (MFI) ratio of lectin-to-background (Fig.1c). is paired analysis showed a reduction in
WGA binding to adenomas in the colon (Fig.1c(i)), and similarly in the small intestine (Fig.1c(ii)).
Lectin binding to xed tissue sections from human colon. Next, binding of uorescently-labelled
WGA and other lectins to the luminal surface epithelium of xed human colon tissue sections was investigated
(Fig.2). Lectin binding was compared with histological assessment of paran-embedded colorectal samples
derived from adenoma lesions collected from 47 patients. ROIs representative of the pathology classes present
(Fig.3a), were analysed (Fig.3b(i),c(i) and Supplementary Fig. 1). Lectin uorescence signals were averaged to
give a score for each class. WGA and HPA binding showed signicant dierences across the dierent pathology
classes (P < 0.001). WGA showed highest binding to hyperplasia (Fig.3b(i)) and decreased binding in the pro-
gression from normal epithelium to dysplasia (LGD and HGD) and carcinoma (C). A similar trend was observed
for HPA binding (Fig.3c(i)), although HPA bound minimally to hyperplasia. Both lectins showed variable bind-
ing to normal epithelium (Supplementary Fig. 2c), which may reect partial loss of mucus due to FFPE tissue pro-
cessing. Normal epithelium, immediately adjacent to HGD or C, showed minimal HPA binding but this increased
dramatically with distance (> 5 mm) (white arrows, row 4, column 4 in Fig.3a; Supplementary Fig. 2a). WGA
binding showed no such dierences (row 4, column 3 in Fig.3a, Supplementary Fig. 2b). Soybean agglutinin
(SBA) binding showed a signicant decrease with disease progression (P = 0.05), however binding to all classes
was relatively weak (Supplementary Fig. 1). Jackfruit lectin (JFL) and peanut agglutinin (PNA) also showed rel-
atively low binding (Supplementary Fig. 1) and no signicant trends were observed (P = 0.064 and P = 0.259 for
JFL and PNA, respectively).
Lectin binding to samples from individual patients was averaged to give each pathology class a score for each
patient (Fig.3b(ii–iv)and, Supplementary Fig. 1). e same trend of decreased binding with disease progression
was observed for WGA and HPA. Outliers that showed high HPA binding (circled in Fig.3c(i,ii) were no longer
outliers as these patients also showed very high mean HPA binding to normal epithelium (Fig.3c(ii)).
Analysis of lectin sensitivity and specicity. WGA and HPA showed good sensitivity and specicity in
distinguishing non-dysplastic (normal or hyperplasia) from dysplastic epithelium (LGD or HGD, third row in
Table1). SBA, PNA and JFL showed low sensitivity and specicity (Table1). WGA showed a remarkable ability
to distinguish between hyperplasia and dysplasia or carcinoma (HP v LGD, HGD, C), with 100% sensitivity and
100% specicity, which was consistent with its increased binding to hyperplasia (Fig.3b). ese data suggest that
uorescently labelled WGA and HPA could potentially be used to distinguish normal from neoplastic luminal
surface epithelium using uorescence endoscopy, especially for severe lesions, and that WGA could be used to
distinguish between hyperplastic and dysplastic polyps, in particular those with severe high-grade dysplasia.
Mucin histochemistry. Alcian blue (AB)–periodic-acid Schi (PAS) combination stain (AB-PAS) was used
to determine whether WGA and HPA were binding to acidic mucins (sialomucins and sulfomucins) stained blue
by AB, or neutral mucins, stained deep-red/magenta by PAS (Fig.3). e AB staining pattern closely resembled
that observed for WGA, but not HPA (Figs3 and 4). Regions of hyperplasia showed the strongest staining of
luminal surface epithelium, with decreased staining in the progression from normal tissue to dysplasia and car-
cinoma (Fig.4a) (P < 0.001). ere was a strong correlation between WGA binding and AB staining (R = 0.79,
P < 0.0001, Pearson product moment correlation; Fig.4b), consistent with the specicity of WGA for acidic
glycans (sialic acids)33. Signicant correlations between WGA binding and AB staining were observed for normal
epithelium, LGD and HGD but not for HP and carcinoma (Supplementary Table 2a). AB staining, similarly to
WGA binding, could distinguish between non-neoplastic epithelium (normal or hyperplasia) and neoplasia with
high sensitivity and specicity (Supplementary Table 3). As with WGA binding, normal epithelium showed a wide
range of AB staining (Fig.4a), which again might be explained by loss of mucus during tissue processing. ere

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Figure 1. Images of freshly resected intestines from Apcmin mice that have been incubated with
uorescently-labelled WGA. (a) Macroscopic images; (i) far-red uorescence image (Exλ = 615–665 nm,
Emλ = 695–770 nm); (ii) bright-eld greyscale image. Black arrows indicate visible adenomas in the colon and
small intestine ((i) and (ii)); intestines displayed in the proximal to distal direction, le to right on the plate. Scale
bar in (ii) represents 1 cm. (b) Fluorescence microscopy of WGA binding to the luminal surface epithelium of
normal intestine (N) and adenomas (A). WGA is shown in yellow in columns 1 and 3. Nuclei were stained with
DAPI (in blue). e same sections were counter stained with haematoxylin and eosin (H&E) (columns 2 and 4).
e dashed lines on the H&E stained images represent the normal cell layer that covered some adenomas in
the colon and small intestine35. e white arrow indicates lack of WGA binding to a normal cell layer on the
surface of an adenoma. Row 4 shows adenoma tissue with no overlying normal cell layer that is devoid of
WGA uorescence. Scale bar, 250 m (row 1, column 2). (c) Regions of interest representing normal tissue and
adenomas were analysed for WGA binding, which is expressed as the ratio of lectin mean uorescence intensity
versus the background uorescence (MFI ratio), and averaged to give a score for each mouse (individual lines);
(i) colon and (ii) small intestine. e P value is from a two-tailed paired t-test.

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was no correlation between HPA binding and AB staining, consistent with HPA’s lack of specicity for acidic
glycans34 (Fig.4c and Supplementary Table 2). PAS staining was weak across all the classes and showed no corre-
lation with disease progression (P = 0.525, Supplementary Fig. 3a) or with WGA or HPA binding (Supplementary
Fig. 4b,c, Supplementary Table 2b). Deposits of PAS positive material were observed within the lumen of HGD
and C (row 4, column 2 in Fig.3a, Supplementary Fig. 4) and appeared to correlate with glandular regions that
showed very strong WGA binding (row 4, column3 in Fig.3a). ese regions occurred mostly deep beneath the
luminal surface epithelium and therefore were excluded from analysis. e luminal surface epithelium of HGD
and C were mostly devoid AB-PAS staining (column 2, row 3 and 4 in Figs3a and 4a, Supplementary Fig. 3a).
Discussion
Fluorescently labelled WGA sprayed onto the luminal surface of freshly resected intestines from the Apcmin mouse
showed decreased binding to adenomas. is is in agreement with the reported reduced staining by WGA of
glycosylated mucus proteins, particularly mucin 2 (Muc2), in the Apcmin mouse36. Muc2 expression is known to
be down regulated in both mouse37 and human38 colorectal tumours when compared to healthy colonic tissue.
is suggested to us that spraying of WGA onto the luminal surface of the human colon in situ, when used in
conjunction with uorescence colonoscopy, would have the potential to enhance detection of dysplasia, as we
have demonstrated previously for the oesophagus28. To investigate relevance to the human disease we analysed
the capability of uorescently labelled WGA and other lectins to distinguish dysplastic or neoplastic surface epi-
thelium from normal or hyperplastic surface epithelium in xed sections of human colon. WGA distinguished
epithelial regions containing NE or HP from regions containing LGD, HGD or carcinoma, with 81% sensitivity,
87% specicity and 93% positive predictive value (PPV). HPA distinguished epithelial regions containing NE
from regions containing HP, LGD, HGD or carcinoma, with 89% sensitivity, 87% specicity and 97% PPV.
Lectin binding to abnormal and diseased colorectal epithelium has been studied extensively in the past using
conventional lectin histochemistry methods. However, these studies typically focused on binding to cross sec-
tions of the colonic mucosa as a whole rather than specically to the luminal surface epithelium32,39,40. We have
shown here that the luminal surface epithelium of high-grade dysplasia (HGD) and carcinoma is largely devoid
of mucus and low in lectin binding. Conversely, deep beneath the mucosal surface, the glandular lumen of these
lesions appear to contain material that stains with periodic-acid Schi (PAS), which detects neutral mucins, and
which binds all the lectins studied here (Fig.3a and Supplementary Fig. 4). is may represent mucus secreted
by these advanced lesions.
Limitations of the study include the lower numbers of adenomas in the colon when compared with the small
intestine in the Apcmin mouse model and some loss of luminal surface mucus in the human FFPE material.
Figure 2. Binding of uorescently labelled lectins to colorectal luminal surface epithelium. e gure
shows a representative example of the binding of WGA conjugated to AF647 to colorectal tissue sections.
(a) Luminal surface epithelium was dened as the sole region of interest (ROI), which would be visible at
colonoscopy. (b) ROIs of dened length (ca. 500 m) and thickness (20 m) were dened at the luminal surface
epithelium, using an automated image analysis system (Ariol™), as illustrated by the white dashed-line box. e
insets (b,c) are 3.5 × magnications of the ROIs indicated by the white arrow and triangle in (a), respectively.
Scale bars = 250 m (a) and 70 m (b,c) m.

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e latter was evident when staining normal tissues with AB (Supplementary Fig. 2c). is surface mucus,
which was responsible for the observed binding of WGA to normal epithelium, is lost in advanced dysplasia
Figure 3. Quantitative analysis of uorescently labelled lectin binding to colorectal luminal surface
epithelium. (a) Colorectal tissue sections were stained with wheat germ agglutinin (WGA) or Helix pomatia
agglutinin (HPA). Lectin binding to the dierent pathology classes is shown in yellow in columns 3 and 4
from le. e same sections were stained with a nuclear stain (DAPI, in blue in columns 3 and 4) and with
haematoxylin and eosin (H&E; column 1). Regions of interest (in column 1) containing normal (N, green
arrows), hyperplasia (HP, dark blue arrows; row 1), low-grade dysplasia (LGD, yellow arrows; row 2), high-
grade dysplasia (HGD, grey arrows; row 3) and carcinoma (C, black arrows; row 4) are indicated. Alcian blue
(AB) – periodic acid Schi (PAS) combination stain (column 2) was applied to immediately adjacent tissue
sections to visualize the presence of acidic (blue) and neutral (magenta) mucins. White arrows (column 4)
indicate normal epithelium distant (> 5 mm) from carcinoma. Insets in the carcinoma tissue sections (row 4)
indicate WGA and HPA binding to PAS positive luminal necrosis as well as luminal malignant glands invading
the bowel wall, deep within the carcinoma tissue. Scale bars (column 1), 1 mm. WGA (b) and HPA (c) binding
to colorectal tissues was quantied as the ratio of mean lectin uorescence intensity versus the background
uorescence (MFI ratio), which generated a score (y-axis) for the dierent pathology classes (x-axis), for each
sample in patient-unmatched analyses (b(i),c(i)). e P value represents the Jonckheere-Terpstra test for
trend. ese data were averaged to give a single score for each pathological class in each patient for the patient-
matched analyses (ii-iv) for both WGA (b) and HPA (c). Circled data points in (c(i)) correspond to those in
(c(ii)). Statistical signicance (in b(ii),c(ii)) was determined by Wilcoxon matched-pairs signed rank test.

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and carcinoma41,42. e luminal surface mucus may be better preserved using alcohol xation methods or by
using frozen tissue sections41 or ideally freshly resected unxed colon samples, as were used for the studies with
intestines from Apcmin mice (Fig.1). Fresh colorectal tissue sections have a thicker mucus layer than their xed
counterparts41,43.
A further limitation was the limited sample size of the patient-paired data. Samples from each individual
patient oen did not contain more than two pathological classes, reecting the limited heterogeneity of sporadic
CRC. erefore, even though the patient-matched analyses conrmed the trends observed for unmatched data
(Fig.3b,c), the analysis of signicance, sensitivity and specicity could not be determined in patient-matched
data.
e presence of acidic mucins, stained by AB, showed a signicant decrease with disease progression, similar
to that shown by WGA binding (Fig.4), which can be explained by the specicity of WGA for sialic acid44, a
major terminal moiety of acidic mucins. ere was no signicant trend for PAS staining, indicating no dierences
in neutral mucin content on the luminal surface epithelium, and there was no correlation with lectin binding
(Supplementary Fig. 3).
Comparison
Sensitivity (n)Specicity
HPA JFL PNA SBA WGA HPA JFL PNA SBA WGA
N v (HP, LGD, HGD, C)0.89 (88) 0.22 (99) 0.62 (96) 0.50 (91) 0.65 (97) 0.87 0.67 0.73 0.76 0.77
N v (LGD, HGD, C)0.85 (65) 0.73 (64) 0.73 (62) 0.76 (74) 0.81 (81) 0.89 0.25 0.62 0.63 0.79
HP v (LGD, HGD + C)0.91 (61) 0.91 (51) 0.84 (61) 0.95 (51) 1.00 (54) 0.24 0.44 0.39 1.00 1.00
(N, HP) v (LGD, HGD)0.69 (63) 0.61 (74) 0.50 (60) 0.64 (72) 0.71 (72) 0.59 0.61 0.63 0.82 0.87
(N, HP) v (LGD, HGD, C)0.78 (88) 0.68 (99) 0.64 (96) 0.74 (91) 0.81 (93) 0.59 0.54 0.60 0.79 0.87
Positive predictive value Negative predictive value
N v (HP, LGD, HGD, C) 0.97 0.74 0.92 0.81 0.87 0.64 0.17 0.27 0.43 0.48
N v (LGD, HGD, C) 0.95 0.68 0.88 0.86 0.93 0.70 0.29 0.38 0.48 0.56
HP v (LGD, HGD, C) 0.50 0.70 0.50 0.78 1.00 0.75 0.78 0.77 1.00 1.00
(N, HP) v (LGD, HGD) 0.55 0.49 0.44 0.81 0.88 0.73 0.72 0.69 0.66 0.69
(N, HP) v (LGD, HGD, C) 0.73 0.57 0.69 0.86 0.93 0.67 0.65 0.55 0.64 0.69
Table 1. Statistical analysis of lectin performance in distinguishing dysplastic or neoplastic from non-
neoplastic tissues. Lectin binding to non-neoplastic colon epithelium (N, normal and/or HP, hyperplasia) was
compared with dysplasia (LGD, low-grade or HGD, high-grade dysplasia) or dysplasia grouped with neoplasia
(C, carcinoma). Sensitivity is the probability of a positive test result given the patient really has the disease.
Specicity is the probability of a negative test result in the true absence of disease. Positive predictive value is the
probability that a patient with a positive test result has the disease. Negative predictive value is the probability
that a patient with a negative test result does not have the disease. Abbreviations: HPA, Helix pomatia agglutinin;
JFL, jackfruit lectin; PNA, peanut agglutinin; SBA, soybean agglutinin; WGA, wheat germ agglutinin. Values in
parenthesis are sample numbers.
Figure 4. Quantitative analysis of acidic mucins on the luminal surface epithelium of the dierent
colorectal pathology classes and correlation with lectin binding. (a) Tissue sections were stained with
Alcian blue (AB)–periodic acid Schi (PAS) combination stain and the resulting colour analysed for the
presence of acidic mucins, by using a trained algorithm selective for the “blue” colour produced by AB staining
(Fig.3a, column 2). AB signals were averaged to generate scores (y-axis) for the dierent pathology classes
(x-axis) in each sample in unmatched patient analyses. e P value represents the Jonckheere-Terpstra test
for trend. Abbreviations: N, normal; HP, hyperplasia; LGD, low-grade dysplasia; HGD, high-grade dysplasia;
C, carcinoma. Linear regression analysis of acidic mucin staining, as a function of WGA (b) and HPA (c)
uorescence, for unmatched patient analyses. WGA, wheat germ agglutinin. HPA, Helix pomatia agglutinin.
Dashed lines (b,c) represent the 95% condence interval hyperbolas for the linear best ts (solid lines).

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e removal of LGD at colonoscopy is crucial for reducing mortality in sporadic CRC4. Moreover, in patients
with IBD, LGD can occur as at mucosal lesions, which are oen dicult to detect at colonoscopy45. Although
in the cohort analysis WGA and HPA showed low sensitivity for distinguishing normal and LGD (0.57 for WGA
and 0.33 for HPA; Supplementary Table 1), in the patient-matched data, decreased binding was observed between
normal and LGD in all patients for WGA (n = 16; P < 0.0001 by Wilcoxon test) and in all but one for HPA
(P < 0.0001 by Wilcoxon test). WGA binding could also distinguish between hyperplasia and neoplasia, which is
explained by its binding to sialic acids44.
HPA has specificity for alpha-N-acetylgalactosamine (α -GalNAc), the immune determinant sugar of
histo-blood group A46. For this reason, HPA may react dierently according to ABO blood group type. We have
found this not to be the case here, as normal colon sections from patients with dierent blood group types (Fig.3;
Supplementary Table 4) did not show dierential staining with HPA (Supplementary Fig. 5).
Hyperplasia is oen accompanied by an increase in acidic mucins that have high levels of sialic acids (sialomu-
cins)27, a trend also observed in this study (Fig.4). Hyperplastic and dysplastic polyps are dicult to distinguish
in routine endoscopy, due to their similar appearance, and as a consequence all suspicious polyps of a minimal
size (> 5 mm) are resected. Moreover, hyperplastic polyps (HP), which have reduced potential for malignant
transformation12, can appear similar at colonoscopy to sessile serrated adenomas (SSA), which can be precursors
to CRC47,48. Nevertheless, the removal of small polyps during endoscopy still carries a risk of colon bleeding and
perforation13. Fluorescently labelled WGA and HPA have the potential to identify dysplastic polyps and to distin-
guish them from hyperplasia or normal tissue.
We have shown previously that uorescently labelled lectins can be used for endoscopic identication of
dysplasia in Barrett’s oesophagus28. Similarly to what was observed here, WGA and HPA showed high binding to
oesophageal mucosa and Barrett’s and low binding to dysplastic tissue. Although the contrast observed here in
the colon and previously in the oesophagus is negative, this is not an issue in the context of endoscopic surveil-
lance since any regions with confounding factors that lead to loss of binding (false positives) would inevitably be
biopsied. In contrast, confounding factors that lead to loss of binding of an imaging agent that generates positive
contrast, i.e. that binds to diseased areas (false negatives), could result in failure to detect the presence of disease.
Lectins of plant or animal origin are potentially toxic. However, both WGA and HPA are components of
foodstus, wheat germ and edible snail, respectively, and in the case of WGA, part of a basic, gluten-containing
diet. Moreover, most studies that have investigated lectin toxicity have been conducted using much higher lectin
doses than those used here and over much longer periods of time49. erefore we do not anticipate any toxicity
with the use of these lectins. Furthermore, any potential toxicity could be reduced by washing o the lectin with
a large molar excess of a lectin-binding monosaccharide (GlcNAc for WGA and GalNAc for HPA) following the
imaging session28.
In conclusion, uorescently-labelled lectins, particularly, WGA or HPA, may be useful in the secondary sur-
veillance setting of sporadic CRC, to enhance detection of dysplasia using uorescence colonoscopy and, in par-
ticular with WGA, to allow hyperplasia to be distinguished from dysplasia.
Methods
Apcmin mice. All experiments were conducted in accordance with the Animals (Scientic Procedures) Act
of 1986 (United Kingdom) and were designed with reference to the UK Co-ordinating Committee on Cancer
Research Guidelines for the Welfare of Animals in Experimental Neoplasia. e work was approved by the
Cancer Research UK Cambridge Institute Ethical Review Committee. e small intestine, caecum and colon
were removed post mortem from Apcmin mice (n = 10) aged between 120–140 days, ushed with ice-cold blocking
buer (PBS containing 1% foetal bovine serum, FBS), incubated for 15 min with AlexaFluor™ − 647 (AF647) con-
jugated WGA (Life Technologies, Paisley, UK) at 5 g/ml, by clamping the two ends of the intestines at 20 °C. e
clamped intestines were immersed in PBS during incubation to avoid dehydration and subsequently ushed once
with ice-cold blocking buer. A rapid xation was then performed, by ushing the intestines with 10% neutral
buered formalin (NFB; 4% Formaldehyde in PBS, Sigma-Aldrich, Buchs SG, Switzerland). Aer a further wash
with ice-cold PBS, the intestines were sectioned, dissected and pinned luminal side uppermost on a wax plate
and imaged using an IVIS200™ camera (Perkin Elmer, Hopkinton, MA, USA), with a Cy55 lter set (Exλ = 615–
665 nm, Emλ = 695–770 nm). e intestines were then xed for 24 h with 10% NFB, replaced with 70% ethanol
for 24 h at 4 °C and subsequently processed and embedded in paran blocks. Tissue sections were mounted using
ProLong™ Gold Anti-fade reagent with DAPI (Life Technologies) for 24 h at room temperature, and examined
by uorescence microscopy, using a 20× lens, producing a mosaic of images that captured the entire tissue sec-
tion. Fluorescence micrographs were analysed using an Ariol™ imaging system (Leica Microsystems Ltd, Milton
Keynes, UK). AF647 uorescence was false-coloured in yellow.
Human samples. Colonoscopy biopsies or colonic resections were performed between 2008 and 2012
(Supplementary Table 4). Lectin binding was compared with histological assessment on 100 formalin-xed
paran-embedded (FFPE) colorectal samples derived from adenoma lesions collected from 47 patients (32
males, 15 females; mean age of 68.8 ± 8.6 yr., range 53–95 yr.). Informed written consent was obtained from
all subjects. Approval was obtained from a local ethics committee (Cambridgeshire Local Research Ethics
Committee, CLREC, ref. 06/Q0108/307). All the histological procedures were carried out in accordance with the
guidelines approved by the CLREC. All experimental protocols were approved by the CLREC. Normal epithelium
(NE) occupied 38.1% of the area of the tissue sections, hyperplastic polyps (HP) 16.1%, low-grade (LGD) dys-
plasia 24.4%, high-grade (HGD) dysplasia 13.1%, and carcinoma (C) 8.3%. Mean lesion size was 16.0 ± 14.2 mm
(range 2–60 mm), located in the caecum (3%), ascending (17%), transverse (14%), descending (11%) and sig-
moid (35%) colon and in the rectum (20%). H&E-stained, colorectal tissue sections (5-m), from 47 patients,

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8
Scientific RepoRts | 6:24231 | DOI: 10.1038/srep24231
were reviewed by a senior histopathologist (A.I.), and identied as normal colon (NE; n = 64), hyperplasia (HP;
n =27), low-grade dysplasia (LGD; n = 41), high-grade dysplasia (HGD; n = 22) or carcinoma (C; n = 14).
Lectin histochemistry. AF647 conjugated lectins (Life Technologies) were used on a Duolink™ (Olink
Bioscience, Uppsala, Sweden) system with Shandon Sequenza™ racks and cover plates (ermo Fisher Scientic,
Waltham, MA, USA). Deparanised slides, washed and blocked at 4 °C using lectin binding buer (LBB; 20 mM
HEPES, 150 mM NaCl, 1 mM CaCl2, MgCl2 and MnCl2, and 1% FBS, pH 7.4), were stained with lectin (5 g/ml)
for 15 min at 37 °C and then washed in cold LBB buer, then LBB buer with no serum, before mounting with
ProLong™ Gold with DAPI. Fluorescence was imaged using a 20× lens that captured the entire tissue section.
For ease of visualization, AF647 and DAPI uorescence were false-coloured in yellow and blue, respectively.
Alcian blue - periodic acid Schi combination staining. Deparanised slides were incubated in Alcian
Blue (AB) (pH 2.5) for 10 min, washed in water, incubated in 0.5% periodic acid Schi reagent (PAS) for 5 min,
washed in water, and further incubated in PAS (ermo Fisher Scientic) for 15 min and then washed in water.
e slides were counterstained in Mayers Haematoxylin for 45 s, rinsed with water, dehydrated through 2 changes
of 100% ethanol and cleared with 2 changes of xylene and mounted with DPX mountant (Sigma-Aldrich). Slides
were scanned into an Ariol™ imaging system and regions of interest (ROI) within 20 m of the luminal surface
epithelium were dened (Fig.2). Lectin binding to slide surface that was not covered by tissue was dened as
background. AB – PAS staining was analysed using a trained algorithm optimised for quantifying the AB and PAS
signals. Average ROI intensities were normalised against background signal.
Statistics. The significance of lectin binding and AB and PAS staining were assessed using the
Jonckheere-Terpstra test, using 5000 permutations to calculate the reference distribution. As some patients had
multiple samples but few had complete data, bootstrapping was used to repeatedly sample the data to ensure that
all the data were used whilst maintaining the assumption of independence. 1000 bootstraps were used for lectin,
AB and PAS staining and the median P-value over the 1000 bootstraps was taken. Recursive partitioning was
used to provide cut-os in lectin binding to the dierent stages of disease progression. Predicted disease stages
were compared with the true disease stages in terms of sensitivity and specicity, as well as positive and negative
predictive values.
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Acknowledgements
is work was supported by grants from Cancer Research UK (17242, 16465) to KMB.
Author Contributions
J.C.K. carried out lectin staining, image analysis, and wrote the manuscript. A.E.K.I. and K.G. carried
histopathology classication of samples. Statistical analysis of lectin histochemistry was performed by S.D. and
D.P. e algorithms for image analysis were developed by W.J.H. Patient selection and clinical translation advice
provided by N.S.F. and R.M. Apcmin mice, related protocols and technical advice were provided by D.J.W. e
project was designed and directed by A.A.N. and the data interpreted by A.A.N. and K.M.B. e manuscript was
revised by K.M.B. with contributions from all co-authors.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Kuo, J. C.-H. et al. Detection of colorectal dysplasia using uorescently labelled lectins.
Sci. Rep. 6, 24231; doi: 10.1038/srep24231 (2016).
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