TPP specifically binds to Hsp70, but not to other Heat shock proteins (HSPs).

<p> A peptide ELISA was performed to determine the <i>in vitro</i> binding capacity of peptides to different HSPs. Ninety six well plates that were coated either with Hsp70, Hsp60, Grp78, or Hsp27 were incubated with carboxyfluorescein (CF)-labeled TPP or a scrambled peptide at concentrations ranging from 100 to 25 ng/ml. Fluorescence was measured after 30 min at 27°C using a multiplate reader. Combinations of peptides and proteins are indicated as “+” in the lower panel. Differences in peptide binding (100 ng/ml) were evaluated using the Student’s <i>t</i>-test (** <i>p</i><0.01; *** <i>p</i><0.001) (n = 3).</p

TPP specifically binds to Hsp70 <i>in vitro</i>.

<p><b>A.</b> Western Blot analysis of whole cell lysates of 4T1 Hsp70 wild type (4T1 wt) and 4T1 knockout (4T1 Hsp70^−/−) cell lines showed a positive Hsp70 staining in 4T1 wt but not in 4T1 Hsp70^−/− cells using the cmHsp70.1 antibody; ß-actin was used as a loading control. <b>B.</b> Immunofluorescence staining and <b>C.</b> flow cytometric analysis of <i>in vitro</i> grown viable 4T1 wt and Hsp70^−/− cells proved the Hsp70 specificity of CF-labeled TPP. Binding of TPP to 4T1 wt cells was comparable to that of cmHsp70.1 antibody (left panel, green staining). Neither TPP nor cmHsp70.1 antibody did bind to 4T1 Hsp70^−/− cells (right panel). Cells were counter-stained with DAPI (blue staining). <b>C.</b> Representative flow cytometric histograms indicate a positive Hsp70 staining of 4T1 wt cells by cmHsp70.1 antibody and TPP. In contrast, 4T1 Hsp70^−/− cells were neither stained with cmHsp70.1 antibody nor with TPP; grey, isotype controls, open histograms, Hsp70 specific reagents. The numbers in the histograms show the proportion of Hsp70 membrane-positively stained cells.</p

Kinetics of TPP internalization.

<p>(Left panel) Flow cytometric analysis of memHsp70 positive human breast cancer lines MCF7, MDA-MB-231, T47D reveals an accumulation of the fluorescence intensity after incubation with CF-labeled TPP at 37°C between 1 and 60 min (solid lines). At 4°C (dashed lines) mean fluorescence intensity remained low within the same time frame. Cell lines with a high percentage of Hsp70 membrane positive cells (MCF7, MDA-MB-231) exhibit a higher and more rapid uptake of TPP, whereas the cell line with low Hsp70 membrane expression (T47D) exhibits only a low uptake of TPP. (Right panel) Confocal microscopy images were analyzed to provide a total count of fluorescent spots per cell after incubation with the TPP at 37°C for 30 min. Although fluorescent spots progressively accumulated in all three cell lines, this was most apparent in the MCF7 and MDA-MB-231 cell lines that express higher levels of memHsp70 than T47D cells.</p

TPP co-localizes with mitochondria after 90 min.

<p>MCF7, MDA-MB-231, and T47D cells were incubated with CF-labeled TPP (green) for 90 min, stained with the mitochondrial detection dye Mito-ID (purple) and then imaged using confocal microscopy. A proportion of internalized TPP co-localizes with mitochondria in all three tumor cell lines (Pearson’s coefficient: MCF7 r = 0.855, MDA-MB-231 r = 0.585, T47D r = 0.813). The Manders’ M1 coefficient was used to estimate the proportion of total TPP that co-localizes with mitochondria (40% in MCF7 cells, M1 = 0.407; 44% in MDA-MB-231 cells, M1 = 0.443). In contrast, 14% of TPP was co-localized to mitochondria in T47D cells (M1 = 0.141). These findings correlate with the differential Hsp70 membrane expression levels of the respective cancer cell lines. Images are representative single frames. Objective 63×; scale bar 10 µm.</p

Tumor cell lines expressing memHsp70 are able to bind TPP at 4°C.

<p>The memHsp70 status of a panel of human breast, colon, melanoma, lung, head & neck tumor cell lines was assessed using the cmHsp70.1 antibody (top row histograms). Incubation of tumor cells with carboxyfluorescein (CF)-labeled TPP at 4°C (bottom row histograms) results in a similar binding profile to that of cmHsp70.1 antibody in all tumor cell lines; grey, isotype controls, open histograms, Hsp70 specific reagents. The numbers in the histograms show the proportion of Hsp70 membrane-positively stained cells.</p

Localization of internalized TPP within cells.

<p>Confocal microscopy z-stacks were used to divide cells into quartiles using percentages of cell height from the adherent interface (0%) to the apex of the cells (100%). The number of fluorescent TPP spots in each quartile was counted in order to quantify the peptide distribution. For all time points and all cell lines investigated, TPP spots were found to be more prevalent in the quartile which included the adherent surface. This finding suggests that TPP is preferentially internalized and trafficked via this interface. The data shown are representative composite images of every z-stack frame counted in each quartile. Images were produced using a 63×/1.4 oil immersion lens on an inverted Zeiss 510 confocal microscope.</p

Uptake of TPP between 0 and 60 min follows an endosomal pathway.

<p>MCF7 cells take up CF-labeled TPP via an endosomal transport route in a time-dependent manner. Co-localization of TPP (green) with the endosomal marker proteins (Rab5, Rab7, LAMP1; each in red) is visible as a yellow signal. Co-localization of TPP with Rab5 can be seen within early endosomes at early time points (<30 min, arrows, upper left graph), but not at later time points (arrow heads, upper right graph). Between 30 and 60 min, TPP co-localizes with Rab7 vesicles (arrows, lower left graph). After 60 min, TPP co-localizes with LAMP1 positive lysosomes (arrows, lower right graph). Images are representative three-channel composites: brightfield, FITC, RFP. Objective 63×; scale bar 10 µm.</p

Specific uptake of TPP into tumor cells at 37°C.

<p>Human breast cancer cell lines expressing different levels of memHsp70 (MCF7, MDA-MB-231, T47D) were incubated with CF-labeled TPP or a scrambled peptide for 30 min at 37°C and the internalization of peptides imaged using confocal microscopy. <b>A.</b> TPP (green, right panel), but not the scrambled peptide (left panel) is internalized into the tumor cell lines. The appearance of the TPP (green, right panel) in well-defined, localized points suggests that the association of the peptide with intracellular vesicles is related to the memHsp70 expression status. The numbers shown as inserts indicate the percentage of memHsp70 positively stained cells. DAPI staining allows visualization of the nucleus (blue). Objective 2×; scale bar 50 µm. <b>B.</b> Individual cells were imaged as a z-stack in order to better illustrate the intracellular localization of TPP. Images are representative three-frame composites from approximately mid-way in the stack. Immunofluorescence staining, (left panel); brightfield, (right panel). Objective 63×; scale bar 10 µm.</p