Additional file 3: Table S1. of Tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC): the crucial contribution of immune cells (effector, regulatory) and cytokines (Th1, Th2) to immune-mediated tumour cell death induced by NAC

Comparison of Tumour-infiltrating Lymphocytes (TILs) in Primary Breast Tumours and Pre-NAC(1) ALN(2) Metastatic Tumours in Women with LLABCs(3). There was no significant difference between the levels of TILs in primary breast tumours and axillary metastatic tumour deposits. Table S2. Correlations of Tumour-infiltrating Lymphocyte Subsets in Primary Breast Tumours and ALN (1) Metastatic Tumours in Women with LLABCs(2) [Spearman’s Correlation Coefficient (rho)] (n = 20). There was a positive correlation between CD8+ T and CD56+ NK cells infiltrating primary breast cancers and the tumour deposits in metastatic ALNs (rho=0.514, p =0.020; rho=0.721, p < 0.001, respectively). There was no correlation, however, between CD4+, FOXP3+ and CTLA-4+ T cells infiltrating the primary and metastatic tumours. (DOCX 26 kb

Additional file 1: Table S3. of Tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC): the crucial contribution of immune cells (effector, regulatory) and cytokines (Th1, Th2) to immune-mediated tumour cell death induced by NAC

Patient and Tumour Characteristics, Responses to Neoadjuvant Chemotherapy (n = 33). (DOCX 56 kb

Effect of CD133 on cell motility.

<p>Transwell migration was measured after knockdown of CD133 in HT29 and sorting of SW480 into CD133+/− populations. Significantly fewer HT29^CD133− cells migrated across the membrane than HT29^ssc cells (Figure 6a, p = 0.04). Conversely, larger numbers of sorted CD133+ cells migrated than CD133− cells (Figure 6b, p = 0.03). A wounding assay was also undertaken and gene knockdown was associated with marked delay in closure of the wound which was visiually perceptible after only 24 hours (Figure 6c) and statistically significant (p<0.001).</p

Plasticity of CD133 expression.

<p>SW480 was sorted into CD133 positive and negative populations which were then cultured separately. (a) Gating was set so that only the extreme populations were collected which, on immediate retesting, were shown to be very pure populations. (b) After prolonged culture, both of the sorted populations became biphasic. The ratios of the CD133+ and CD133− cells became stable after three weeks and did not change after that (although they were not the same as the original parental cell line).</p

Evaluation of CD133+ populations by flow cytometry.

<p>Eight CRC cell lines were tested and showed varying size of the population of CD133+ cells. (a) Sample flow cytometry images are shown for Caco2 (left panels, >95% positive cells) and SW837 (right panels). Analysis was performed using AC133/1 antibody (top panel) and gating was performed for each cell line using an isotype control (bottom panel), PMTLin 1 is photmultiplier tube linear 1 which indicates side scatter) (b) shows that in two cell lines there were no CD133+ cells identified whilst in two cell lines nearly all cells were CD133+. The remaining cell lines had varying size population of CD133+ cells.</p

mRNA level in sorted populations.

<p>Q-RT-PCR analysis of the sorted populations showed that there was transcriptional repression of <i>CD133</i> in the CD133− population (although low levels of mRNA were still detectable).</p

Knockdown of CD133.

<p>HT29 cells were transfected with either CD133 specific siRNA (HT29^CD133−) or scrambled control ((HT29^ssc). Western blotting confirmed the loss of CD133 expression by gene knockdown. β-Actin shows equal protein loading.</p

Effect of CD133 on proliferation.

<p>Cell proliferation was evaluated after knockdown of CD133 in HT29 (Figure 4a) and sorting of SW480 into pure CD133+ and CD133− populations (Figure 4b). A time course assay was performed over several days with no association seen between CD133 and cell numbers.</p

Association of CD133 expression with features of stemness.

<p>CD133 gave resistance to staurosporine induced apoptosis. Figure 5a shows that after exposure to staurosporine there were fewer viable cells after transfection with CD133 specific siRNA (HT29^CD133−) than with scrambled control (HT29^ssc) (p = 0.01). There were no differences between the cells after exposure to DMSO. Figure 5b shows that the CD133+ population of SW480 showed cells showed significantly greater resistance than the CD133− population (p = 0.008). Figure 5c and 5d show CD133+ cells were significantly more clonogenic than CD133− cells (p<0.0001 and p<0.003) in 2D and 3D culture respectively. Figures 5e (colonies formed by CD133+ cells) and 5F (colonies formed by CD133− cells) show that both CD133+ and CD133− populations of SW480 were able to form colonies from single cells (typical colonies are shown).</p