Comparison of TIC marker expression between an original tissue sample, matched spheroids and differentiated cells.

<p>(A) comparison of TIC marker expression between tissue, spheroids and differentiated cultures from the same patient (N = 1 for tissue (stats not shown), N = 4 for spheroids and N = 3 for differentiated cells, mean ± SEM, paired sample t-test, * = P≤0.05). (B) TIC marker expression comparing original spheroids with spheroids produced from differentiated cells when placed under low adherence conditions (N = 3, mean ± SEM, paired sample t-test, * = P≤0.05).</p

Flow cytometric profiling of spheroids under co-culture conditions.

<p>(A) Original sample profiles expressed as a percent of total cells, and as a percent of EpCAM⁺ cells. (B) Samples following one passage with 18Co cells expressed as a percent of total cells, and as a percent of EpCAM⁺ cells.</p

TIC marker expression in colorectal liver metastases.

<p>(A) Shows mean (±SEM) expression of TIC markers analysed by flow cytometry from patients undergoing resection for CRLM. N = 62 for ALDH combinations, N = 44 for CD26 combinations. (B) Mean (±SEM) TIC marker expression comparing males vs females. Male N = 39 and 26 for ALDH and CD26 combinations respectively, female N = 22 and 17 for ALDH and CD26 combinations respectively (independent t-tests, * = P≤0.05). (C) TIC marker expression in EpCAM⁺ cells stratified based on patient chemotherapy status. Chemo naïve N = 14, chemo-treated N = 36. (mean ± SEM, Mann Whitney-U test, * indicates P = 0.05). (D) Representative dot plot for CD26 (x-axis) and CD133 (y-axis) in a chemo-naïve (left) and chemo-treated (right) patient.</p

Serial passaging of CLRM tissue in NOD-SCID mice.

<p>(A) Tumor characterisation following engraftment of liver metastases into NOD-SCID mice. Haematoxylin/Eosin staining of the original hepatic metastatic lesion (upper panel) and tumor tissue following 2 passages in NOD-SCID mice (lower panel) (x20 objective). (B) Flow cytometric analysis of marker expression following each passage (Tissue N = 12, Passage 1 N = 11, Passage 2 N = 7, Passage 3 N = 6 and passage 4 N = 2, * = P≤0.05, t-test, mean ± SEM). (C) Tumor growth rates (passage 1) following the first NOD-SCID implantation (N = 11). (D) flow cytometric analysis of fast vs slow growing samples (directly from patient), (N = 5/group, mean ± SEM). (E) flow cytometric analysis of fast vs slow growing samples (following one passage), (N = 5/group, mean ± SEM, * = P≤0.05).</p

Flow cytometry data (EpCAM^<b>+</b> cells only) showing TIC populations by gender and chemotherapy status.

<p>Flow cytometry data (EpCAM^<b>+</b> cells only) showing TIC populations by gender and chemotherapy status.</p

Patient demographics.

<p>Patient demographics.</p

Analysis of the spheroid reforming ability of sorted spheroid TIC populations.

<p>(A) Gating strategy for cell sorting of samples based on TIC marker expression, and typical spheroid formation. (B) Mean percent of cells that form spheres from each population (N = 3 from a single patient sample, mean ± SEM, a paired sample t-test, * = P≤0.05). Spheroid forming ability was determined by the number of spheroids formed as a percentage of sorted cells. (C) Expression of TIC markers in spheroids grown from sorted spheroid TIC populations (N = 3. Mean ± SEM, paired sample t-test).</p