Dye Lipophilicity and Retention in Lipid Membranes: Implications for Single-Molecule Spectroscopy

Fluorescence studies of individual lipid vesicles rely on the proper positioning of probes in the lipid milieu. This is true for both positional tags and chemoselective fluorogenic probes that undergo chemical modification following reaction with an analyte of interest within the lipid environment. The present report describes lipophilicity and localization estimations for a series of BODIPY dyes bearing substituents of varying hydrophobicity. We also studied fluorogenic trap–reporter probes that undergo fluorescence emission enhancement upon trapping of reactive oxygen species (ROS), including lipid peroxyl radicals. We show that caution has to be taken to extrapolate ensemble partition measurements of dyes to the single-molecule regime as a result of the dramatically different lipid concentration prevailing in ensemble versus single-molecule experiments. We show that the mole fraction of dyes that remains embedded in liposomes during a typical single-molecule experiment may be accurately determined from a ratiometric single-particle imaging analysis. We further demonstrate that fluorescence correlation spectroscopy (FCS) provides a very rapid and reliable estimate of the lipophilic nature of a given dye under highly dilute single-molecule-like conditions. Our combined single-particle spectroscopy and FCS experiments suggest that the minimal mole fraction of membrane-associated dyes (<i>x</i>_m) as determined from FCS experiments is about 0.5 for adequate dye retention during single-molecule imaging in lipid membranes. Our work further highlights the dramatic effect that chemical modifications can have on chemoselective fluorogenic probe localization

<i>In silico</i> analyses using the Oncomine platform showed that expression of the ESR2 gene in the breast tumour microenvironment does not correlate with patient outcome (p = 0.9337) (A). Expression of TGFBR2 (B) and BMPR2 (C) showed a trend towards significance (p = 0.1046 and p = 0.2029) suggesting that high expression levels of these genes are associated with lower rates of patient survival.

<p><i>In silico</i> analyses using the Oncomine platform showed that expression of the ESR2 gene in the breast tumour microenvironment does not correlate with patient outcome (p = 0.9337) (A). Expression of TGFBR2 (B) and BMPR2 (C) showed a trend towards significance (p = 0.1046 and p = 0.2029) suggesting that high expression levels of these genes are associated with lower rates of patient survival.</p

Laser micro-dissection (LMD) was used to isolate areas of epithelium in normal (A), DCIS (B) and invasive (C) breast tissue from the same tissue section. Breast tissue images show examples of the areas captured for patient 4 as a representative example. The expression of miR–92 decreased during breast cancer progression with highest levels observed in normal breast epithelium, decreasing in DCIS and being lowest in invasive breast tissue (D). *denotes significance of p<0.01.

<p>Laser micro-dissection (LMD) was used to isolate areas of epithelium in normal (A), DCIS (B) and invasive (C) breast tissue from the same tissue section. Breast tissue images show examples of the areas captured for patient 4 as a representative example. The expression of miR–92 decreased during breast cancer progression with highest levels observed in normal breast epithelium, decreasing in DCIS and being lowest in invasive breast tissue (D). *denotes significance of p<0.01.</p

Down-regulation of miR92 expression in normal fibroblasts (NFs), but not cancer associated fibroblasts (CAFs), significantly enhances the invasive capacity of breast cancer epithelial cells. Matched NFs and CAFs were reverse transfected with either an inhibitor of miR92 or negative control (A) and a Matrigel™ invasion assay was used to assess effects on the behaviour of breast cancer epithelial cells 48 hours post-transfection. Down-regulation of miR92 significantly increased the invasion of MCF7 (B) and MDA-MB–231 (C) cells. Error bars are ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01; ***denotes significance of p<0.001.

<p>Down-regulation of miR92 expression in normal fibroblasts (NFs), but not cancer associated fibroblasts (CAFs), significantly enhances the invasive capacity of breast cancer epithelial cells. Matched NFs and CAFs were reverse transfected with either an inhibitor of miR92 or negative control (A) and a Matrigel™ invasion assay was used to assess effects on the behaviour of breast cancer epithelial cells 48 hours post-transfection. Down-regulation of miR92 significantly increased the invasion of MCF7 (B) and MDA-MB–231 (C) cells. Error bars are ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01; ***denotes significance of p<0.001.</p

Expression of ERβ1 and miR–92 are not inversely correlated in patients with primary breast cancer. Staining patterns of ERβ1 showed nuclear expression in the normal breast (A) epithelium (examples shown by red arrows) with no significant difference in nuclear staining during cancer progression (B). A shift in the localisation of ERβ1 staining was observed with a significant increase in cytoplasmic staining (green arrows) during progression to DCIS (C, E) and invasive breast cancer (D, E). Blue arrows show ERβ1-negative nuclei. Images of breast tissues show the staining patterns for patient 4 as a representative example. Horizontal lines represent the mean. Range ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01.

<p>Expression of ERβ1 and miR–92 are not inversely correlated in patients with primary breast cancer. Staining patterns of ERβ1 showed nuclear expression in the normal breast (A) epithelium (examples shown by red arrows) with no significant difference in nuclear staining during cancer progression (B). A shift in the localisation of ERβ1 staining was observed with a significant increase in cytoplasmic staining (green arrows) during progression to DCIS (C, E) and invasive breast cancer (D, E). Blue arrows show ERβ1-negative nuclei. Images of breast tissues show the staining patterns for patient 4 as a representative example. Horizontal lines represent the mean. Range ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01.</p

<i>In silico</i> analysis using BreastMark [26] shows luminal A breast cancer patients (n = 154) who express miR–92 have improved disease free survival (DFS) rate compared to patients with low expression levels with a hazard ratio of 0.49 (95% confidence intervals 0.28–0.84; p = 0.008).

<p><i>In silico</i> analysis using BreastMark [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139698#pone.0139698.ref026" target="_blank">26</a>] shows luminal A breast cancer patients (n = 154) who express miR–92 have improved disease free survival (DFS) rate compared to patients with low expression levels with a hazard ratio of 0.49 (95% confidence intervals 0.28–0.84; p = 0.008).</p

Expression of miR92 in normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs) in patient samples (A) and primary fibroblast cultures (B) by qRT-PCR. *denotes significance of p = 0.0014 between NFs (high and low expressing groups) and NFs and CAFs (ANOVA).

<p>Expression of miR92 in normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs) in patient samples (A) and primary fibroblast cultures (B) by qRT-PCR. *denotes significance of p = 0.0014 between NFs (high and low expressing groups) and NFs and CAFs (ANOVA).</p

Primers used for pyrosequencing.

<p>Primers used for pyrosequencing.</p

Selected characteristics of OCTANE trial 2 participants at baseline.

<p>Percentages use number of participants with a result as the denominator.</p>a<p>Race was collected as it could potentially be related to treatment response and was reported by the site investigators.</p>b<p>All women had screening CD4<200 cells/mm³, but at study entry, 58 women had CD4≥200 cells/mm³.</p>c<p>K103N.</p

Kaplan-Meier plot of time to early discontinuation of initial randomized treatment for any reason (toxicity, intolerance, VF, death), by randomized treatment arm.

<p>Kaplan-Meier plot of time to early discontinuation of initial randomized treatment for any reason (toxicity, intolerance, VF, death), by randomized treatment arm.</p