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. 2008 Aug 13;3(8):e2916.
doi: 10.1371/journal.pone.0002916.

Live Imaging of Cysteine-Cathepsin Activity Reveals Dynamics of Focal Inflammation, Angiogenesis, and Polyp Growth

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Free PMC article

Live Imaging of Cysteine-Cathepsin Activity Reveals Dynamics of Focal Inflammation, Angiogenesis, and Polyp Growth

Elias Gounaris et al. PLoS One. .
Free PMC article

Erratum in

  • PLoS ONE.2008;3(10).doi.org/10.1371/annotation/499b225f-e661-4124-aa2f-60bef89bd14a.. Plough, Hidde L [corrected to Ploegh, Hidde L]

Abstract

It has been estimated that up to 30% of detectable polyps in patients regress spontaneously. One major challenge in the evaluation of effective therapy of cancer is the readout for tumor regression and favorable biological response to therapy. Inducible near infra-red (NIR) fluorescent probes were utilized to visualize intestinal polyps of mice hemizygous for a novel truncation of the Adenomatous Polyposis coli (APC) gene. Laser Scanning Confocal Microscopy in live mice allowed visualization of cathepsin activity in richly vascularized benign dysplastic lesions. Using biotinylated suicide inhibitors we quantified increased activities of the Cathepsin B & Z in the polyps. More than (3/4) of the probe signal was localized in CD11b(+)Gr1(+) myeloid derived suppressor cells (MDSC) and CD11b(+)F4/80(+) macrophages infiltrating the lesions. Polyposis was attenuated through genetic ablation of cathepsin B, and suppressed by neutralization of TNFalpha in mice. In both cases, diminished probe signal was accounted for by loss of MDSC. Thus, in vivo NIR imaging of focal cathepsin activity reveals inflammatory reactions etiologically linked with cancer progression and is a suitable approach for monitoring response to therapy.

Conflict of interest statement

Competing Interests: Dr Weissleder is a shareholder of VisEn Medical in Woburn, Mass.

Figures

Figure 1
Figure 1. Infiltration of polyps by pro-inflammatory cells.
(Α) Histology of polyps from APCΔ468 mouse. (a–b) CEA stained paraffin sections counterstained with Gill's II Hematoxylin; (c&d) methylene blue staining; arrows point to (a) granulocytes, (b) mast cells, (c) plasma cells, (d) lymphocytes. (B) Immuno-fluorescence of polyps from APCΔ468 mice. Cryosections were stained with (a) DAPI, (b) CD11b-AlexaFluor 488, and (c) Gr1-AlexaFluor 594. Arrows point to polyp, and arrowheads to the adjacent healthy villus; note accumulation of CD11b+ cells and/or Gr1+ cells in the polyps. (C) FACS analysis of leukocytes prepared from micro-dissected polyps, and from adjacent tissue (n = 4), and of intestinal tissue from age-matched healthy control mice (n = 3). Mean values and SEM are shown for frequencies of CD11b+ and of CD11b+Gr1+ cells in 6 month-old mice.
Figure 2
Figure 2. Flow cytometric analysis of polyp infitrate.
(A) FACS analyses of pro-inflammatory cells. (a) Exemplar FACS analyses of mononuclear cells prepared from the intestine of (a&c) a 5 month-old APCΔ468 mouse and (b&d) an age matched wt C57BL/6J mouse; note increases in the frequencies of (a) CD11b+Gr1+ and (c) CD11b+F4/80+ myeloid type cells in the polyposis intestine, compared to wt tissue (b&d respectively). (B) Summary of FACS analysis of MNCs from wt (black bars) or APCΔ468 (open bars) intestine, showing mean frequencies and absolute numbers of cells with SEM values; n = 6 for APCΔ468, n = 3 for wt control mice, per age group.
Figure 3
Figure 3. In vivo molecular imaging of polyps in APCΔ468 mice.
A small loop of the intestine of each APCΔ468 and wt animals was surgically exposed and underwent laser scanning Intravital Fluorescent Microscopy (IVFM) 24 hour after injection with the ProSense-680. ProSense-680 image of cathepsin activity in (a) healthy intestine, (b) APCΔ468 polyp. AngioSense 750 image of vasculature in (c) healthy intestine, (d) APCΔ468 polyp. Spectrally resolved auto-fluorescence of (e) healthy intestine, (f) APCΔ468 polyp at 505–510 nm. Z-stack slice 42 for the healthy intestine and slice 51 for the APCΔ468 polyp were imaged using an UplanApo 10× objective with 2× electronic zoom (pixel size 1.05 µm with resolution 2.42 µm). (g) The mean volume of particles visualized at 694 nm (ProSense-680); green bar represents APCΔ468 polyps (128,900±104,800 µm3 (n = 92)), red bar healthy intestine (4,538±797.5 µm3 (n = 81)). (h) The mean volume of particles visualized at 790 nm (AngioSense-750); green bar represents vasculature in polyps (11270±3207 µm3, n = 101), red bar represents vasculature in healthy intestine (4538±798 µm3, n = 81). Calculated with the Image J “3D particle analysis” plug-in. (i) Linear regression, correlating the size of ProSense-680 particles in polyps (green dots, 1/slope = 0.004540, r2 = 1) with those in healthy intestine (red dots, 1/slope = 0.004539, r2 = 0.9999); note that the increase in intensity corresponds to the increase in the number of cathepsin active cells. (j) “Calculated centers of intensity” of particles in the z-axis of the APCΔ468 adenoma (green diamonds) and healthy intestine (red triangles); note distribution throughout the z- stack, and that the total intensity of the particles in the APCΔ468 adenoma is at least 2 orders of magnitude higher (mean total intensity 1.322×107±8.881×106 units) than the particles in the wt intestine (mean total intensity 8592±1257 units, P<0.0001 one sample t test).
Figure 4
Figure 4. The cellular source of cathepsin activity.
Cryosections of ProSense-680 in vivo stained intestine from APCΔ468mice were stained with antibodies to CD11b (AlexaFluor 488), Gr1 (AlexaFluor 594) and DAPI. The merged images of CD11b with DAPI (a, CD11b green, DAPI gray), Gr1 with DAPI (b, Gr1 red, DAPI gray), and ProSense-680 with DAPI (c, ProSense-680 blue, DAPI gray) were produced with the “RGB gray” plug-in of Image J. The “colocalization finder” plug-in produced images where the colocalized pixels appear white while the ProSense-680 was red (d&e), the CD11b was green (d, colocalization analysis of ProSense-680 and CD11b staining) and the Gr1 was green (e, colocalization analysis of ProSense-680 and Gr1 staining). ×400 magnification. Arrows mark a CD11b+Gr1 ProSense 680+ cell. Representative FACS dot-plots of MNCs prepared from polyposis intestine and ex vivo stained with ProSense-680 followed by CD11b and Gr1 staining. The live MNCs were gated for ProSence-680+ cells, which were analyzed for CD11b+Gr1+ (f, MDSCs) and CD11b+ F4/80+ (g, macrophages) cells. Cumulative results of 6 FACS experiments showing % of CD11b+Gr1+ ProSense-680+ and CD11b+F4/80+ProSense 680+ among total infiltrating MNCs. Note that among the ProSense-680+ cells (11±0.69% of total MNCs) over 75% were either CD11b+Gr1+ (3.4±0.6% of total MNCs) or CD11b+F4/80 (5.0±0.34% of total MNCs).
Figure 5
Figure 5. Quantification of active cysteine cathepsins using a specific active site directed probe.
Polyps from APCΔ468, Ctsb−/− APCΔ468, and anti-TNFα treated APCΔ468 mice were micro-dissected, pooled, and extracts were incubated with DCG-04 prior to electrophoresis on a 4–12% gradient SDS gel and western blotting; healthy adjacent regions were similarly analyzed. Active Cathepsins were visualized with the use of chemiluminescence reagents. (a) A representative blot. (b) Average Optical Densities (OD) from each band of three independent blots, measured with Image J software; values were normalized with the OD of the β-actin protein, detected using a specific antibody. Open bars: Cathepsin Z, black bars: Cathepsin B.
Figure 6
Figure 6. Cathepsin B deficiency or anti-TNFα treatment attenuate polyposis.
(a) Non-linear regression analysis of polyp number and diameter, assuming Gaussian distribution; APCΔ468 Ctsb−/− (continued line, open squares), and APCΔ468 (dotted line, closed triangles). Note that Cathepsin B−/− mice had fewer and smaller polyps. (b) Frequencies of ProSense-680 active leukocytes amongst total MNCs prepared from the intestine of APCΔ468 (open bar, 6.7%±086%) or APCΔ468Ctsb−/− (filled bar, 11%±0.69%, P = 0.0037; unpaired t test with Welsh correction). (c) Frequencies of ProSense-680 active CD11b+Gr1+ (mean 0.56%) or CD11b+F4/80+ (mean 4.46%) cells, from the intestines of APCΔ468 (open bar) and APCΔ468Ctsb−/− mice (filled bars); P<0.001, n = 6, 2way ANOVA. Note that Cathepsin B deficiency predominantly impacted the abundance of CD11b+Gr1+ cells. (d) Attenuation of polyposis in anti-TNFα treated mice (solid line, open squares, n = 6), as compared to the APCΔ468 (dotted line & closed triangles). (e) Frequencies of CD11b+Gr1+ amongst total intestine live MNCs; APCΔ468Ctsb−/− intestine (light gray bar, 0.56±0.15%, P<0.001), anti-TNFα treated (dark gray bar, 0.71±0.22%, P<0.001), untreated APCΔ468 (open bar, 4.2±0.093%), wt control intestine (black bar, 0.15±0.051%). (f) Frequencies of CD11b+F4/80+ in the APCΔ468 (5.03±0.78%), APCΔ468Ctsb−/− intestine (5.36±0.92%), and anti-TNFα treated intestine (dark gray bar, 3.0±0.67%).
Figure 7
Figure 7. Imaging polyps in mice deficient for Cathepsin B or responding to effective anti-TNFα therapy.
Spectral separation of images from, (a&b) APCΔ468 mice, (c&d) Ctsb−/−APCΔ468, (e&f) anti-TNFα treated APCΔ468 mice; ProSense-680 (green; CathepsinB), AngioSense-750 (red; blood vessels). The objective used was the UplanApo 4× (pixel size 5.4 µm, lateral resolution 12.42 µm). (g) Mean particle volumes in the Z stacks of fluorochrome 680 (open bar: APCΔ468, 120554±86906 µm; gray bar: APCΔ468Ctsb−/−, 26609±5268 µm3; anti-TNFα treated APCΔ468, 8639±1570 µm3; P<0.0001 one sample test). (h) Mean particle volumes in the Z stacks of fluorochrome 750 (open bar: APCΔ468, 15501±2144 µm3; gray bar: APCΔ468Ctsb−/−, 6963±1236 µm3, anti-TNFα treated, 3700±1444 µm3; P<0.0001 one sample test).

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References

    1. Amos-Landgraf JM, Kwong LN, Kendziorski CM, Reichelderfer M, Torrealba J, et al. A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer. Proc Natl Acad Sci U S A. 2007;104:4036–4041. - PMC - PubMed
    1. Boivin GP, Washington K, Yang K, Ward JM, Pretlow TP, et al. Pathology of mouse models of intestinal cancer: consensus report and recommendations. Gastroenterology. 2003;124:762–777. - PubMed
    1. Bertagnolli MM. APC and intestinal carcinogenesis. Insights from animal models. Ann N Y Acad Sci. 1999;889:32–44. - PubMed
    1. de Visser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer. 2006;6:24–37. - PubMed
    1. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science. 1990;247:322–324. - PubMed

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