(American Journal of Pathology. 1999;154:1831-1834.)
© 1999 American Society for Investigative Pathology
Colossal Crypts Bordering Colon Adenomas in ApcMin Mice Express Full-Length Apc
Matthew Bjerknes and
Hazel Cheng
From the Department of Anatomy and Cell Biology,
University of Toronto, Toronto, Ontario, Canada
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Abstract
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Enlarged but nondysplastic crypts are frequently
observed at the margins of colon tumors, forming what has been
called a transitional epithelium. It is now thought that this is a
reactive state and not a preneoplastic condition as previously
suggested. We have used the mouse familial adenomatous polyposis
model, ApcMin, to study
these abnormal adenoma-associated crypts. We report that these
nondysplastic crypts are enormous (as much as 10 times normal length)
and branch more frequently than normal crypts. They express wild-type
Apc protein and display the wild-type Apc allele. We
conclude that the colossal crypts at adenoma margins have normal
Apc gene function, consistent with the
suggestion that their phenotype is a reactive state. The cause remains
an open question, but the dramatic epithelial response hints at
the presence of potent epithelial trophic factors in the
vicinity of colon tumors.
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Introduction
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It has long been recognized that the colonic epithelium
at the margins of adenocarcinomas, although not neoplastic, is not
normal. The crypts in this transitional region are enlarged and often
branched.1-5
However, as a similar state is observed at
the margins of an array of lesions, it is now believed that it
represents a reactive state and not a preneoplastic
condition6,7
and has since been largely ignored. We have
noticed in studies of a mouse model of familial adenomatous
polyposis8
that although the dysplastic epithelium forming
the bulk of adenomas is Apc negative, there is often a layer of
Apc-positive nondysplastic crypts underlying the tumor.9
These Apc-positive crypts are often cut in cross section when the tumor
is sectioned orthogonally to the surface. This observation raised
questions about the relationship of the Apc-positive crypts to the
overlying tumor and to the surrounding normal epithelium. We show here
that they represent greatly elongated crypts, with normal Apc gene
function, that extend under the tumor from the margins.
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Materials and Methods
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Isolation of Intact Epithelium and Scanning Electron Microscopy
Fifteen week-old ApcMin mice (Jackson
Laboratories, Bar Harbor, ME) were used. Intact epithelium was
isolated, from small segments of colon each containing an adenoma, by
intraventricular perfusion of 30 mmol/L EDTA in PBS followed by
vibration into 4% paraformaldehyde in PBS.10
After
fixation for 30 minutes on ice, the isolated epithelium was washed in
PBS and then rinsed and stored in methanol at -20°C.
The colon adenomas and adjacent mucosa were dissected from the vibrated
segments and fixed for 30 minutes in 2.5% glutaraldehyde in PBS. The
adenomas were then processed for scanning electron microscopy (SEM) by
post-fixing in 1% osmium tetroxide in 0.1 mol/L cacodylate HCl
buffer, pH 7.4, and then dehydrated through a graded series of ethanol,
critical-point dried,11
and sputter coated with gold
palladium.
Immunofluorescence
Colossal crypts were microdissected from the isolated epithelium,
rinsed in 1% Nonidet P-40 (NP40) in PBS, freeze-thawed at -20°C to
increase permeability, rinsed in 1% NP40, and blocked at room
temperature for 1 hour in PBS containing 10% newborn calf serum and
1% NP40. The crypts were incubated overnight at 4°C in rabbit
polyclonal antibodies specific for the carboxy terminus of Apc protein
(Santa Cruz Biotechnology, Santa Cruz, CA), washed at room temperature
with 1% NP40 for 3 hours, incubated overnight at 4°C with
Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA), and then washed with 1% NP40. Controls,
which were negative, included omission of the primary anti-Apc
antibody, omission of both antibodies, and Apc peptide competition.
Cryosections from colon adenomas were incubated with the anti-Apc
antibodies and then stained with Cy3-conjugated goat anti-rabbit IgG as
described.9
Testing for Loss of Apc Heterozygosity by Polymerase Chain Reaction
Single colossal crypts were microdissected from the isolated
epithelium. Small sheets of dysplastic epithelium were microdissected
from the adenomas. The microdissected tissues were placed in individual
polymerase chain reaction (PCR) tubes and digested overnight at 50°C
in 10 µl of lysis buffer containing 1 mg/ml proteinase K (Boehringer
Mannheim, Indianapolis, IN), 10 mmol/L Tris/HCL (pH 8), 50 mmol/L KCl,
0.45% NP40, and 0.45% Tween 20. The next morning the proteinase K was
heat inactivated (10 minutes at 95°C), the tubes were centrifuged for
5 minutes, and the supernatant was transferred to new tubes. Loss of
heterozygosity (LOH) of the Apc gene was checked using
PCR.12
Briefly, after digestion with HindIII
the ApcMin allele PCR product generates a 144-bp
fragment whereas the wild-type allele PCR product generates a 123-bp
fragment. Thus, two DNA bands appear on gels (12% nondenaturing
polyacrylamide gel electrophoresis) from phenotypically normal tissue
from ApcMin mice. In contrast, as the vast
majority of adenomas in ApcMin mice result from
cells that have lost the wild-type allele,9,12
the
dysplastic epithelium from adenoma displays LOH, and only one band is
evident from the ApcMin allele.
Branching Crypt Frequency
Crypts from the epithelium isolated from four of the
ApcMin mice were randomly selected and
mounted on slides. The frequency of crypts that were branching was
determined using an oil immersion 100x objective. During the scoring,
colossal crypts were identified by their size (usually 5 to 10 times
longer than normal) and were recorded separately. The frequencies of
branching normal and colossal crypts were compared with a two-tailed
test for the equality of binomial
means.13
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Results and Discussion
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Sections through colon adenomas from ApcMin
mice often display a layer of nondysplastic, Apc-expressing crypts
underlying the dysplastic tissue, but the relationship of these
Apc-positive crypts to surrounding normal tissue was unclear. Some
sections suggest that the crypts bordering adenomas are enlarged and
extend under and sometimes into the margins of the tumor (Figure 1, A and B)
. When isolated, these crypts
were found to be enormous, ranging from 2 to 10 times longer than
normal crypts (Figure 1, C and D)
. The branching frequency of these
colossal crypts (11.8%; 12 of 102 crypts scored were branching) was
significantly higher (P = 10-5)
than that of normal crypts (2.4%; 99 of 4048 normal crypts scored were
branching).
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Figure 1. Demonstration of the presence of colossal crypts at the margin of colon
adenomas from ApcMin mice and their
Apc status. All scale bars represent 250 µm. A
and B: Laser scanning confocal micrographs of frozen sections
through colon adenomas from ApcMin mice. The
sections have been stained with an antibody specific for the carboxy
terminus of Apc. In A, note the absence of staining in the
dysplastic tissue and the layer of Apc-positive crypts underlying the
tumor (arrowhead). There are also
some enlarged crypts, sectioned in a more longitudinal plane, at the
left tumor margin. In B, note the large Apc-positive branching
nondysplastic crypt at the tumor margin
(arrowhead). C:
Photomicrograph of intestinal epithelium isolated from an
ApcMin mouse. A group of crypts from normal
colon is labeled 1. Compare them with the two sets of colossal crypts
from the margins of an adenoma, labeled 2. Note the branching colossal
crypt on the right. A normal crypt-villus unit isolated from proximal
jejunum, labeled 3, is shown for size comparison. D: Scanning
electron micrograph showing the base of a large adenoma from the colon
(note the honeycomb-like array of vacant pits in
the lamina propria, from which crypts have been extracted by the
isolation procedure). A few colossal crypts,
which have been extracted from the margins of the tumor, remain
attached to the tumor by surface epithelium. Many of the crypts are
branched. The crypts shown are ~500 µm long
(approximately double the normal crypt
height), although they appear smaller in this
view due to foreshortening and partial obstruction of the crypt tops.
Dysplastic epithelium is visible in the upper right corner. E
and F: Fluorescence micrographs of isolated colossal
(E) and normal crypts
(F) stained for Apc protein with an
antibody specific for the carboxy terminus. This demonstrates that the
cells in colossal crypts are making full-length Apc. The figures are at
the same magnification. G: Ethidium-bromide-stained gel showing
PCR product from single colossal crypts (lanes 1 and
2) and groups of normal crypts
(lanes 3 and 4). Note the
presence of both wild-type
(123-bp) and
ApcMin
(144-bp) alleles in both
colossal and normal crypts. This confirms the antibody results
presented in E and F. H: Demonstration of the
loss of heterozygosity that occurs in dysplastic epithelium from
ApcMin mice. Lane 1 shows PCR
product from normal epithelium (with both
Apc alleles) whereas
lanes 2 and 3 show product from isolated dysplastic
epithelium from adenomas (demonstrating only the
ApcMin allele).
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Crypt branching is the usual means of crypt replication with consequent
epithelial growth.14,15
The most appealing interpretation
of the observation of an increased frequency of branching among the
colossal crypts is that they are cycling faster than are normal crypts,
potentially adding to tumor bulk with time. However, simply observing
an increase in branching frequency is not sufficient to prove that the
crypts are cycling faster. For example, if colossal crypts require a
longer time to complete the branching process, due perhaps to their
large size, then an increased proportion of branching crypts would be
expected even if normal and colossal crypts have similar crypt cycle
times.
Abnormalities in the frequency of branching non-neoplastic crypts have
been reported in murine (ApcMin) and
human (familial adenomatous polyposis) APC± colon.16-18
It seems unlikely that this effect is directly related to the increased
branching frequency among the colossal crypts found at tumor margins as
the general effect is small and age dependent in mouse
colon.17,18
In humans, dysplastic crypts have a greatly
increased branching frequency in comparison with non-neoplastic
crypts.16-18
Others have reported a 19-fold increase in
branching rates of non-neoplastic crypts in these
patients,18
although we found no evidence of
this17
when obviously dysplastic crypts were excluded from
counts (dysplastic crypts are frequent even when care is taken to
sample only regions containing no macroscopic lesions).
Unusual tubular structures have also been observed near the margins of
a wide variety of ulcers.19
These highly contorted
structures grow out from the base of neighboring crypts and are
composed of a specialized nonproliferative cell lineage.19
In contrast, the colossal crypts are independent structures with a well
defined crypt base indicating that they are probably a distinct
phenomenon.
Dysplastic epithelium from colon adenomas displays LOH of the
Apc locus (Figure 1H)
, as is usually the case in tumors from
ApcMin mice.9,12
The dysplastic
epithelium also does not stain with the anti-Apc antibody specific for
the carboxy terminus of Apc protein (Figure 1, A and B)
. In contrast,
the colossal crypts do stain (Figure 1, A, B, and E)
and have wild-type
Apc alleles (Figure 1G)
indicating normal Apc
gene function. Furthermore, the Apc staining pattern, which normally
increases in intensity from crypt base to top (Figure 1F
20
), appears normal in colossal crypts (Figure 1E)
,
suggesting a normal differentiation program in the colossal crypt
cells.
As loss of function of the Apc gene, usually by
LOH,9,12
is the cause of dysplasia in this mouse model,
our findings are consistent with previous conclusions that the
nondysplastic colossal crypts found on the borders of tumors are indeed
reactive and are not directly involved in
tumorigenesis,6,7
although they probably add to tumor
bulk. This raises the question of the signal inducing the exceptional
size and branching frequency of these crypts. Is it a growth factor
originating from the tumor, or could it be a cytokine released as a
result of immune interactions in the tumor? The response of the
epithelium is so dramatic that the underlying cause should be
investigated, as it likely involves potent epithelial trophic factors
that may have clinical application, for example, in inducing epithelial
repair.
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Footnotes
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Address reprint requests to Dr. Matthew Bjerknes, Department of Anatomy and Cell Biology, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada. E-mail:
bjerknes{at}crypt.med.utoronto.ca
Supported by the Medical Research Council and the Natural Sciences and Engineering Research Council of Canada.
Accepted for publication February 25, 1999.
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Abstract
Introduction
