DETAILED MATERIALS AND METHODS
Hypoxia measurements
Six-micrometer-thick
cryosections
of
tumor
xenografts
were
fixed
in
4%
paraformaldehyde, blocked in DAKO blocking solution (DakoCytomation), then incubated with
anti–HypoxyprobeTM-1 antibody (1:50; Chemicon International) followed by biotin conjugated
goat anti-mouse IgG (1:200) for AEC histochemistry (Sigma-Aldrich), and counterstained with
hematoxylin. The slides were washed and then mounted using Aquapolymount (Polyscience,
Warrington, PA). To quantify the hypoxic area, slides were coded and scored in a semi-blinded
fashion. Digital images of sections were captured by light microscopy at 2.5X magnification, and
the areas of hypoxia were scored and assessed by Optimas 6.2 image analysis software
(MediaCybernetics, San Diego, CA). The total hypoxic area for each section was divided by the
cross sectional area to obtain the proportion of hypoxia in each tumor.
Blood vessel density
To evaluate microvessel density, the vessels in the tumor tissues were stained using an
antibody to the endothelial marker CD31 (DeLisser et al., 1997). Six-micrometer-thick
cryosections were air dried, fixed with 4% paraformaldehyde and blocked with 10% donkey
serum for 30 minutes, followed by 10% normal rabbit serum for 30 minutes; rat anti-mouse
CD31 primary antibody (PECAM-1; HyCult Biotechnology) was used at 1:20 overnight at 4°C,
and donkey anti-rat Alexa Fluor 546-conjugated secondary antibody was used at dilution of
1:200 for 30 minutes. Nuclei were counterstained with DAPI. The slides were washed and
mounted using Fluorescent Mounting Medium (Dako Cytomation). Control sections received
PBS in place of primary antibody. To quantify the microvessel density, slides were coded and
scored in a semi-blinded fashion. Sections were examined at 10X magnification and images were
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captured using a Leica Opti-Tech epifluorescence microscope equipped with appropriate
excitation and emission filters. The total number of blood vessels for each section was divided by
the cross-sectional area to obtain the microvessel density (MVD; # blood vessels per mm2).
Cell sorting
To demonstrate the direct involvement of hypoxia on K-ras mutation of these tumor
cells, we used our previously reported approach to sort cells from Dks-8 xenografts based on
their proximity to perfused blood vessels (Herman et al., 1989; Yu et al., 2002). Briefly, the cells
most proximal to perfused vessels were labeled by cardiac injection of 0.25 mg/mouse Hoechst
33342 dye, in 200 µl volume, five minutes before euthanasia; mice had previously been injected
with Hypoxyprobe as described in Materials and Methods of main manuscript. This procedure
yields highly fluorescent cells immediately surrounding the vasculature and low fluorescence
intensity in more distal, unperfused areas (Herman et al., 1989; Yu et al., 2002). After enzymatic
disaggregation of each Dks-8 xenograft into a single-cell suspension, samples were fixed in 10%
formalin for 5 min, washed with PBS and sorted by fluorescence-activated cell sorting (using a
350-70nm UV channel with a FACSVantageSE™ instrument), and the cells displaying the 5%
highest (“Bright”) and 5% lowest (“Dim”) Hoechst fluorescence intensities were collected. Five
Dks-8 xenografts were used for this part of the study.
Cell suspensions containing 500 cells from “Bright” and “Dim” sorted populations were
used to generate cytospin slides, which were air-dried at room temperature (RT) for 15 minutes,
washed and blocked with 4% normal goat serum plus 2% bovine serum albumin (BSA) in PBS at
RT for 30 min. This was followed by incubation in Dako Protein block (Dako) for 15 min. Slides
were then incubated for one hour with anti-HypoxyprobeTM-1 antibody (Chemicon International
Corp.) and washed in PBS, followed by 30 min incubation with Secondary antibody (anti-mouse
2
Cy3). The slides were washed and mounted using Fluorescent Mounting Medium
(DakoCytomation). Images were captured using objective 20X of a Leica Opti-Tech
epifluorescence microscope equipped with appropriate excitation and emission filters. Images
were merged using Adobe Photoshop 5.0 (Adobe). Control sections received PBS in place of
primary antibody. Remaining sorted cells were pelleted and their DNA was extracted for K-ras
mutation analysis as described below.
K-ras mutation analysis
DNA from DLD-1 cells (which are heterozygously mutated in codon 13 (Shirasawa et
al., 1993)) was used as a positive control for codon 13 K-ras mutation. DNA was extracted from
cultured cells and xenografted tissues using a DNeasy Tissue Kit from QIAGEN Inc. (ON,
Canada). K–rasG13D mutations were determined using a PCR-RFLP analysis with the restriction
enzyme XcmI as described previously (Shahrzad et al., 2005). Briefly, ten µL of the PCR
reaction was digested with XcmI (10 U; New England Biolabs) for 20 hours at 37 °C, in a total
volume of 40 µL. When codon 13 is wild-type, the PCR product (165 bp) contains a restriction
site for XcmI, and digestion yields bands of 137 and 28 bp. If there is a mutation in either of the
first two bases of codon 13, the mutant PCR fragment will not be cut by XcmI, and will remain at
its original size of 165 bp. Digested products were visualized by electrophoresis on a 3% agarose
gel (containing 0.3 g/ml ethidium bromide). To enhance the 165 bp products, which represent
the mutated DNA, a second PCR-RFLP using the products of the first digestion as templates was
performed. The same primers from the first PCR could thus more selectively amplify the
undigested mutated DNA from the first digestion. Thus, a two-step PCR-RFLP led to selectively
amplification of the mutated K-ras, which was confirmed and identified by sequencing. The
bands corresponding to DNA fragments of length 165 bp, indicating undigested mutated DNA,
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were excised from 3% agarose gels and purified using a Qiagen QIAquick™ Gel Extraction Kit
(ON, Canada) before sequencing using an ABI Prism™ Big Dye® Terminator Cycle Sequencing
Ready Reaction Kit. Automated sequencing using ABI DNA sequencers was conducted at the
Guelph Molecular Supercentre at the University of Guelph, Guelph, ON, Canada.
Similar PCR-RFLP approaches (Shahrzad et al. 2005; modified from Toyooka et al.,
2003) were used to examine K-ras mutations at codon 61 in the xenografts (Supplementary Fig.
1). K-ras codon 61 letter 1 and 2 mutations were determined by a two-step PCR-RFLP analysis.
The PCR amplification produces a 271 bp fragment, which was digested with Bcl I. The
digestion products of the wild-type DNA are 25 bp and 246 bp fragments. The mutant DNA will
remain 271 bp. A second PCR was performed to amplify the undigested mutant DNA and a
second digestion was then completed on the new PCR DNA. K-ras codon 61 letter 3 mutations
were determined by a single-step PCR-RFLP (Shahrzad et al. 2005; modified from Toyooka et
al., 2003). The PCR product of 229 bp was digested with Bsp H1 or DraIII. Bsp H1 will cut if
the third letter in the codon is mutated from an A to a T, leading to fragments of 27 and 202 bp,
and Dra III will cut if the third letter is mutated from A to C leading to fragments of 26 bp and
203 bp. A second PCR cannot be performed to enhance any mutated PCR product due to the
cleavage of the mutant DNA.
ADDITIONAL EXPERIMENTS
In vitro oxygen and glucose deprivation
To test whether ischemic conditions of tumor microenvironment can affect nongenetically engineered cells, the parental cell line of Dks-8 cells (DLD-1) and CRC HCT116 cell
line, which both have one K-ras codon 13 allele mutated (Shirasawa et al., 1993) were exposed
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to ischemic conditions in vitro. Cells were maintained in standard culture conditions: DMEM
(Sigma-Aldrich, Oakville, ON, Canada) supplemented with 10% heat-inactivated fetal bovine
serum, 50 µg/mL gentamicin, 1 mmol/L sodium pyruvate, and 5 µL/mL of Fungizone
antimycotic, at 37°C in a humidified atmosphere containing 5% CO2. Glucose deprivation was
performed by substituting normal DMEM (1000 mg/L) with glucose-free DMEM (SigmaAldrich). Hypoxic conditions were achieved using a Modular Incubator Chamber (BillupsRothenberg, Inc., Del Mar, CA) modified to permit continuous flushing of the chamber with a
humidified mixture of 95% N2 and 5% CO2; the oxygen content in the chamber was kept at
<0.1% in all hypoxia experiments. 2 x 106 cells were seeded into 100 mm plates, which were
incubated under normal cell culture conditions overnight. Thereafter, the plates were assigned to
one of two groups—control and hypoglycemia plus hypoxia—and exposed to these conditions
for 24 or 48 hours. The cells were then harvested and tested for K-ras mutation. Each experiment
was repeated three times in duplicates. There was an enrichment in the amount of K-ras mutation
at codon 13 in the ischemia exposed HCT116 and DLD-1 cells (Supplementary Fig. 2). When
the same cells are grown as xenografts, the enrichment of additional K-ras mutations is even
greater (Shahrzad et al., 2005). These results thus demonstrate that two independent human CRC
cell lines, with different underlying mutation spectra (e.g. p53 status) can develop additional Kras mutations under appropriate conditions, and hence the results we report in this manuscript
are applicable to non-genetically engineered cells.
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SUPPLEMENTARY FIGURE LEGENDS
SF1: A) Two-step PCR-RFLP analysis of DNA from Dks-8 xenografts of LDM
cyclophosphamide treated mice (T) versus control mice (C) was used to detect K-ras mutations
for letter 1 and/or 2 at codon 61. Wild-type and mutant DNA produce a 246 bp and a 271 bp
product after digestion by Bcl I, respectively. All 10 treated and control tumors were wild-type
for letter 1 and/or 2 at codon 61 (5 examples are shown here). An undigested PCR product was
used to show the position of a mutant band on the gel. B) One-step PCR-RFLP analysis of DNA
from Dks-8 xenografts of LDM cyclophosphamide treated mice (T) versus control mice (C) was
used to detect K-ras mutations for letter 3 at codon 61. For the third letter of K-ras codon 61,
wild-type sequence remains uncut with a size of 229 bp and mutant DNA is digested by Bsp H1
and Dra III to produce fragments of 202 and 203 bp for point mutations of A to T and A to C,
respectively. All 10 treated and control tumors were wild-type for letter 3 at codon 61 (2
examples are shown here); DLD-1 and Dks-8 cell lines are used as known K-ras codon 61 wildtype controls for these experiments.
SF2: K-ras status of DLD-1 and HCT116 cells exposed to low oxygen and low glucose (LOLG)
in vitro for 24 and 48h. DNA from Dks-8 cells was used as negative control. In all gels, larger
fragments (165 bp) represent mutated K-ras codon, and smaller fragments (137 bp) represent
wild-type. Below each gel are values from densitometry quantification of the proportion of
mutant K-ras codon 13. There was enrichment in the amount of K-ras mutation in LOLG
exposed HCT116 and DLD-1 cells.
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