Figure 3: Autologous serum conserves the integrity of tumour explants.

(a) Activation levels of major RTKs by RPPA profiling of patient tumours (n=5). Quantification of RTK activation was performed by measuring the signal intensity of individual analytes normalized to negative control. The line within each notch box represents the median, and the lower and upper boundaries of the box indicate first and third quartiles, respectively. Error bars (whiskers) represent the interquartile range. (b) Serum growth factor (EGF, HGF, VEGF and MCSF) profiles of HNSCC patients by ELISA (n=8). Horizontal line represents median and error bars indicate the interquartile range. (c) The dose-dependent effect of AS in HNSCC was measured by Ki-67. *P<0.001 by one way analysis of variance (ANOVA; n=9) compared with no AS control. (d) Tumour slices cultured in the presence or absence of autologous ligands for 72 h and stained with hematoxylin and eosin stain (top) and Ki-67 (bottom). Scale bar, 50 μm. (e) Box plot shows ATP utilization (*P<0.05 by t-test, n=6) at 72 h in the presence of AS. (f) Box plot shows fold increase in Ki-67-positive cells cultured with AS and EGF (*P<0.05 and **P<0.01, t-test, n=8). (g) Impact of AS on the balanced activation of different signalling receptors close to T₀ baseline. Tumour explants were treated with 2% AS, 1 ng ml⁻¹ per h EGF or 8% FBS+2% BSA (BSA Control) for 72 h. Tumours were stained for pEGFR (top), pMet (middle) and pERK1/2 (bottom). Scale bar, 100 μm. (h) Graph shows quantification of effects of different treatments on the proliferation and phosphorylated EGFR status in the explants. HNSCC samples were cultured in the presence of 2% AS or EGF up to 6 h for pEGFR and 72 h for detecting proliferation. Appropriate controls (no serum, no antibody and antibody alone) were included. Anti-EGF was added 1 h before stimulation. The effect was assessed by pEGFR and Ki-67 staining. All data (n=8) are represented as mean±s.d. **P<0.01 by t-test. (i) Box plot shows percent pERK positivity (n=8) **P<0.01 (by analysis of variance). Horizontal line represents median and error bars indicate the interquartile range. Graphs shows comparison of the capacity of AS, HS and EGF in activating (j) EGFR (*P<0.02 by t-test, n=9) and (k) in maintaining phospho-Met expression (*P<0.0001 by t-test, n=7). (l) Global RTK profiles of cultured HNSCC tumour explants and corresponding T₀ baseline was compared following stimulation with 2% AS for 72 h. Total cell lysates were applied to array slides precoated with different antibodies against RTK pathways. Signal was detected by chemiluminiscence method.