Supplementary Methods
Dead cell removal
Following the generation of a single cell suspension, tumor cells were resuspended in
100μl of 1x binding buffer (Miltenyi-Biotec) and incubated for 15 minutes at room
temperature with 100μl Dead Cell Removal Microbeads (Miltenyi-Biotec). Magnetic
separation was carried out with a mini-MACS Separator (Miltenyi-Biotec) using a
positive selection MS column (Miltenyi-Biotec). The process was repeated to ensure
increased purity. At the end of the separation, viable tumor cells were resuspended in
HamF12:DMEM media. Viability was confirmed by utilizing trypan blue dye exclusion.
Each tumor specimen yielded approximately 2-3 million cells.
Limiting dilution assay and serial transplantation
Unsorted human colon cancer cells were injected at doses ranging from 1x104 to
2x106 cells. Fourteen out of the seventeen patient samples were injected over a range of
at least four cell doses with one mouse per dose resulting in an aggregate of between 8-17
mice per group for each injection dose. For purification experiments, colon cancer cell
suspensions were magnetically separated on the basis of CD133 expression prior to
injection. Purified cells were injected at doses ranging from 100 to 2x104 and 2x103 to
2.5x105 per mouse, for CD133+ and CD133- cells, respectively. There were between 410 mice per group for each injection dose.
Serial transplantation experiments were completed by taking tumors from primary
mice injected with bulk primary cells and separating the cells based on CD133
expression. The CD133+ and CD133- cells were re-implanted into secondary mice.
Tumors generated by the injection of CD133+ cells were subsequently injected into
tertiary mice.
Purity following magnetic bead separation
After passage through the mini-MACS Separator, CD133+ and CD133- cells were once
again evaluated by flow cytometry, using anti-CD133/2 (293C3) PE to determine the
efficiency of the separation. Purities ranged from 78 to 95% for the CD133+ cells
(median 89%) (Supplemental Fig. 3a and b) and from 85.3 to 99% for the CD133- cells
(median 92%) (Supplemental Fig. 3a and c).
Xenograft histopathology
Tissue sections taken from xenografts were fixed in 4% paraformaldehyde for 48
hours and then transferred to 70% ethanol. Once fixed, the tissue samples were processed
using Tissue-Tek VIP and embedded in paraffin. The blocks were sectioned at 4μm
thickness on a Microm HM 200 cryotome, and stained with haematoxylin-and-eosin
(H&E) as per standard histopathological technique.
Immunohistochemistry
Paraffin-embedded, 4m formalin fixed sections were dewaxed in xylene and
rehydrated with distilled water. The anti-human CD133 antibody was incubated at room
temperature overnight (Miltenyi-Biotec). All remaining antibodies were incubated for 1
hour at room temperature; Muc-1, Muc-2, Muc-5AC, Muc-6, CEA, ESA, CEACAM-1
(Vector), and MIB-1, p53, CK7, CK20 (Dako). All sections, with the exception of CEA
and ESA, were treated with heat-induced epitope retrieval technique using a 10mM
citrate buffer at pH 6.0. Antibody staining was followed by 30 minute incubations with a
biotinylated linking reagent (ID labs) and horseradich peroxidase-conjugated
ultrastreptavidin labeling reagent (ID labs). Development and counterstaining were done
with NovaRed solution (Vector) and Mayer’s hematoxylin (ID labs), respectively.
Statistical analysis
For limiting dilution assays, the frequency of the cancer initiating cell was calculated
using the Porter and Berry method, as previously described14,15,27. The calculation utilized
doses at which only a fraction of the injected mice demonstrated tumor growth.