Results: 5

1.
Figure 1

Figure 1. From: A non-peptidic cathepsin S activity-based probe for noninvasive optical imaging of tumor-associated macrophages.

Non-peptidic cysteine cathepsin activity-based probes. A) Schematic presentation of the mechanism of action of a quenched ABP. B) Structure of the cathepsin S selective aldehyde and nitrile inhibitors reported by the Ellman lab. C) Structures of the peptidic activity-based probes GB123 and the quenched GB137 and the non-peptidic probes BMV011 and the quenched BMV083. D) Labeling profile of GB123, BMV011 and BMV083 in living RAW cells. Cells were exposed to the indicated concentrations of probe for 3 hr, before being harvested, washed and lysed. 40 μg total protein was resolved on 15% SDS-PAGE and fluorescently labeled proteins were visualized by in-gel fluorescence scanning. E) Labeling profile of BMV083 in living human primary macrophages. Cells were exposed to the indicated concentrations of BMV083 for 3 hr, before being harvested, washed and lysed. 40 μg total protein was analyzed as described above. F) BMV083 labeling of RAW cell lysate (35 μg total protein) at pH 5.5 and 7.0 with indicated concentrations of probe for 1 hr. Labeled proteins were analyzed as described above. See also supplemental figures S1–S4.

Martijn Verdoes, et al. Chem Biol. ;19(5):619-628.
2.
Figure 2

Figure 2. From: A non-peptidic cathepsin S activity-based probe for noninvasive optical imaging of tumor-associated macrophages.

Live cell imaging of probe labeling. A–D) Live cell confocal microscopy time course of BMV083 cysteine cathepsin labeling. A) 4T1-luc-GFP cells were exposed to 1 μM BMV083 and imaged over time. Where indicated cells were preincubated with the cysteine cathepsin inhibitor GB111-NH2 (5 μM) for 1 hr before addition of probe (Scale bar 10μm). B) 4T1-luc-GFP cells were exposed to 1 μM BMV083 and harvested at indicated time points. 40 μg total protein was resolved on 15% SDS-PAGE and fluorescently labeled proteins were visualized by in-gel fluorescence scanning (MW = molecular weight). C) Mouse primary bone-marrow derived macrophages were exposed to 1 μM BMV083 and imaged over time (Scale bar 10μm). D) After final time point lysotracker was added (Scale bar 10μm). E) 4T1-luc-GFP cells and mouse primary bone-marrow derived macrophages (2 to 1 ratio) were co-cultured. After 90 hr the cells were exposed to 1 μM BMV083 for 2 hr. Cells were imaged (Red, Cy5 fluorescence; Green, GFP; Blue, DAPI staining. Scale bar 10μm) and harvested. 40 μg total protein was resolved on 15% SDS-PAGE and fluorescently labeled proteins were visualized by in-gel fluorescence scanning.

Martijn Verdoes, et al. Chem Biol. ;19(5):619-628.
3.
Figure 4

Figure 4. From: A non-peptidic cathepsin S activity-based probe for noninvasive optical imaging of tumor-associated macrophages.

Noninvasive optical imaging of cysteine cathepsin activity in a syngeneic orthotopic model for breast cancer. A) 4T1-luc-GFP cells were implanted into mammary fat pad number 2 and 7 of female BALB/c mice. On day 15, BMV083 (20 nmol) was injected (intravenous via tail vein). 10 hr post injection the mice were injected with luciferin (intraperitoneal) and fluorescence and luminescence images of the living mice were taken (Top panel, bright field image. Red arrowheads indicate the tumors; middle panel, luciferase bioluminescence (Luc); lower panel, BMV083 signal (Cy5). Ex vivo fluorescence image of excised tumors is shown. B) BMV083 labeling in tumors. The tumors were homogenized and the active cysteine cathepsins were labeled with BMV099 (1 μM for 1 hr, 40 μg total protein) before the proteins were resolved on 15% SDS-PAGE. Fluorescently labeled proteins were visualized by in-gel fluorescence scanning (top panel, invivo BMV083 labeling; bottom panel, ex vivo BMV099 labeling). C) In vivo BMV083 distribution. Organs were homogynized and the active cysteine cathepsins were labeled with BMV099 (1 μM for 1 hr, 40 μg total protein) before the proteins were resolved on 15% SDS-PAGE. Fluorescently labeled proteins were visualized by in-gel fluorescence scanning (top panel, invivo BMV083 labeling; bottom panel, ex vivo BMV099 labeling. *auto-fluorescent band. Tu = tumor, Lv = liver, Kd = kidney, Sp = spleen, Ht = hart, Lg = lungs, Pn = pancreas). See also supplemental figure S6.

Martijn Verdoes, et al. Chem Biol. ;19(5):619-628.
4.
Figure 5

Figure 5. From: A non-peptidic cathepsin S activity-based probe for noninvasive optical imaging of tumor-associated macrophages.

FACS analysis to determine cellular source of cysteine cathepsin activity in 4T1 syngeneic breast tumors. 4T1 cells were implanted into mammary fat pad number 2 and 7 of female BALB/c mice. On day 15, BMV083 (20 nmol) was injected (intravenous via tail vein). 10 hr post injection tumors were excised. A–C) Single cell suspensions of excised tumors were analyzed by FACS for F4/80 and GFP expression and probe fluorescence. 4 populations (F4/80GFP, F4/80GFP+, F4/80DIM and F4/80HI) were analyzed and sorted. A) F4/80 histogram (top panel) and GFP histogram of F4/80 population (lower panel). B) Equal cell numbers per population (depicted in A) of vehicle and BMV083 treated mice were lysed and the active cysteine cathepsins were labeled with BMV099 (1 μM for 1 hr) before the proteins were resolved on 15% SDS-PAGE. Fluorescently labeled proteins were visualized by in-gel fluorescence scanning (top panel, in vivo BMV083 labeling; lower panel, ex vivo BMV099 labeling, *auto-fluorescent band). C) Bar graph of quantification of mean Cy5 fluorescence per population (depicted in A) and in-gel fluorescence intensity of fluorescently labeled cysteine cathepsins (depicted in B) of vehicle and BMV083 treated mice. D, E) Single cell suspensions of excised tumors were analyzed by FACS for CD11b, F4/80, MMR and Gr-1 expression and probe fluorescence. D) FACS plot and bar graph of mean Cy5 fluorescence of four populations (CD11bF4/80, CD11b+F4/80, CD11b+F4/80DIM and CD11b+F4/80HI) of vehicle and BMV083 treated mice. E) FACS plot and bar graph of mean Cy5 fluorescence of four populations (MMRGr-1, MMRGr-1+, MMR+Gr-1 and MMR+Gr-1+) of CD11b+F4/80HI population depicted in D. See also supplemental figure S6.

Martijn Verdoes, et al. Chem Biol. ;19(5):619-628.
5.
Figure 3

Figure 3. From: A non-peptidic cathepsin S activity-based probe for noninvasive optical imaging of tumor-associated macrophages.

Noninvasive optical imaging of cysteine cathepsin activity with quenched activity-based probes in tumor bearing mice. A–C) 4T1-luc-GFP cells were injected subcutaneously on the back of athymic BALB/c nude mice, 8 days before imaging. GB137 or BMV083 (20 nmol) was injected (intravenous via tail vein) and fluorescent images of the living mice were taken over time. Red arrowheads indicate the tumor. A) Optimal fluorescence contrast for individual time points. Maximum and minimum of scale bar depicted below image. B) Comparison of GB137 and BMV083 fluorescence at fixed intensity. C) Time dependent tumor specific fluorescence (tumor - background) for BMV083 and GB137 treated mice (n = 5, data represent mean values ± standard errors). D) Ex vivo fluorescence imaging of cross-sections of excised tumors (upper panel). Tumor tissue was homogenized and 40 μg total protein was resolved on 15% SDS-PAGE. In vivo fluorescently labeled proteins were visualized by in-gel fluorescence scanning (middle panel). 4T1 tumors were homogenized and the active cysteine cathepsins were labeled with BMV099 (1 μM for 1 hr, 80 μg total protein) before the proteins were resolved on 15% SDS-PAGE. Fluorescently labeled proteins were visualized by in-gel fluorescence scanning (lower panel). E) Fluorescence intensity of end point of non-invasive optical imaging (shown in A), ex vivo tumor imaging and in-gel fluorescence labeling (shown in D). Intensity relative to BMV083 is depicted (n = 5, data represent mean values ± standard errors). See also supplemental figures S5 and S6.

Martijn Verdoes, et al. Chem Biol. ;19(5):619-628.

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