Multivariable Cox regression proportional hazards analysis of 1421 CRCs.

<p>Multivariable Cox regression proportional hazards analysis of 1421 CRCs.</p

Panel A: Myc positive IHC staining; Panel B: Myc negative IHC staining; Panel C: Kaplan Meier analysis (Log Rank test p = 0.01); Panel D: Univariable Cox regression.

<p>Panel A: Myc positive IHC staining; Panel B: Myc negative IHC staining; Panel C: Kaplan Meier analysis (Log Rank test p = 0.01); Panel D: Univariable Cox regression.</p

Multivariable Cox regression proportional hazards analysis of 1421 CRCs including myc IHC status and MMR/BRAF IHC phenotype interaction terms.

<p>MMRd – DNA mismatch repair deficient; MMRp – DNA mismatch repair proficient; BRAFwt – BRAF wild type; BRAFV600E – BRAFV600E mutant.</p

Clinical and pathological characteristics of 1421 consecutive CRC patients (2004–2009).

<p>*Reports on the significance of differences between myc positive and negative groups for each variable, using either Pearson chi-square test (with continuity correction for 2×2 tables) for categorical variables or Mann-Whitney U test for age.</p

Myc IHC on whole section CRCs from 2004, confirming TMA findings.

<p>Panel A: Kaplan Meier curves showing superior overall survival of CRCs with MYC over-expression compared to myc negative CRCs (Log Rank test p<0.01); Panel B: Univariable Cox regression analysis showing MYC over-expression significantly correlated with improved overall survival [hazard ratio = 0.30 (95%CI = 0.15–0.60), p<0.01].</p

Multivariable binary logistic regression showing adjusted effect of MMR/BRAF IHC phenotype on myc over-expression in 1421 CRCs.

<p>Multivariable binary logistic regression showing adjusted effect of MMR/BRAF IHC phenotype on myc over-expression in 1421 CRCs.</p

ApoA-II plus lipid increased lipid uptake and cell growth may be via SR-B1 in pancreatic cancer.

<p>(A) HPDE6, PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cells were cultured and SR-B1 expression was measured by western blotting. The band intensity of the proteins was normalized with β-actin and HPDE6 was defined as 1. Values are the mean of two separate experiments. (B) Expression of SR-B1 by IHC on normal pancreas (NP) and human PDAC tissues. Inset, IgG represents the isotyped matched primary antibody to anti SR-B1. Graph shows the quantification of SR-B1 expression by the percentage of stained cells X intensity from 3 separate patients and NP was defined as 1. (C) Expression of SR-B1 and ApoA-II by IHC in corresponding xenografted PDAC tumours at 48 h after tail vein injection of lipid with or without ApoA-II. Inset, IgG represents the isotyped matched primary antibody to anti-ApoA-II. (D) Effects of anti SR-B1 alone in HPDE6, PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cell growth. (E) Cells were pretreated with or without the SR-B1 antibody (2.5 μg/mL) for 2 hours and then treated with ApoA-II, lipid and lipid plus ApoA-II for 48 hours. Proliferation was measured by crystal violet assay. Values are mean ± SD; <i>n</i> = 4. *^{, ‡, #} and ± p< 0.05 vs. control, ApoA-II, lipid and lipid+ApoA-II respectively, with the use of analysis of variance.</p

SMOFlipid ApoA-II targets pancreatic ductal adenocarcinoma (PDAC).

<p>(A) Spectral reflectance fluorescence images of 8 week old xenografts taken 48h after tail vein injection of PBS, DiD labeled SMOFlipid without or with ApoA-II using Carestream molecular imaging. Note the widespread distribution of DiD is better localized to the tumours when ApoA-II is included with lipid. Arrows indicate the site of tumours. (B) Spectral fluorescence images of explanted tumours and organs taken 48h after injection from mice after a tail vein injection of either PBS, lipid/DiD (n = 2) or lipid/DiD plus ApoA-II (n = 2) (right panel) along with the H&E photo micrograph of the tumour from two mice (left panel), retained the morphological characteristics with the original patient’s PC tumours in lipid and lipid plus ApoA-II treated mice. Note the uptake of fluorescence by the tumours, liver and spleen. (C) Graph represents the uptake by the tumour relative to the uptake of the liver as a fraction of area. These results were then normalised to the value for mice receiving lipid only (defined as 1) when the tumour uptake for the mice receiving lipid and ApoA-II had 3.4 fold increased uptake. Values are mean ± SD; <i>n</i> = 3.</p

ApoA-II reduces the size of lipid.

<p>Cryo-TEM micrograph picture of the SMOFlipid emulsion without ApoA-II (A) and after addition of ApoA-II (B). Note the bi-layer structure of the lipid surface of the nanoparticle like structures in presence of ApoA-II (black arrows). (C) Spectral flourescence color photograph of a 0.5% agarose gel run for 45 min at 90 V in 1 × TBE buffer. SMOFlipid labeled with DiD did not migrate through the well but reconstituted labeled SMOFlipid with ApoA-II migrated as a band.</p

Confocal micrographs of CFPAC-1 cells demonstrating the influence of ApoA-II on lipid uptake.

<p>SMOFlipid was labelled with DiD (red), ApoA-II was labelled green with a secondary antibody and DAPI stained the nuclei blue. (A1) cells treated with ApoA-II alone, (A2) cells treated with lipid alone, (A3) cells treated with reconstituted lipoproteins after adding ApoA-II to DiD labeled SMOFlipid (SMOF/A-II) demonstrating increased uptake and (A4) cells treated with (SMOF/A-II) after pretreating with anti-SR-B1antibodies which reduced the lipid uptake (scale bar, 20 μm). (B) Cells were pretreated with unlabeled lipid at 1:10 dilution and (B2) after 2 h SMOF/A-II was added, it appeared to be endocytosed in cytoplasmic vesicles as shown in (B3) and (B4). ApoA-II (green) is attached with the cell membrane (yellow arrow head) and as well in the cytoplasm in part associated with the lipid (red arrow head) in Differential Interference Contrast (DIC) overlay image (scale bar 25 μm). (C) Live cell imaging experiment demonstrated greater uptake of lipid in cells when reconstituted lipoproteins after adding ApoA-II to DiD labeled SMOFlipid was added to the media for lipid concentrations of 1:20 and 1:10 (scale bar 100 μm). (D) The intensity of the DiD staining was greater when 4 samples were examined with eight to twelve areas of interest for each study where ApoA2 increased the uptake of lipid 25.6±2.2, <i>P</i> = 0.02 and 54.8±2.2, <i>P</i> = 0.004 for dilutions of 1:20 and 1:10 respectively.</p