Research ArticleImaging

Intravital microscopy of osteolytic progression and therapy response of cancer lesions in the bone

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Science Translational Medicine  01 Aug 2018:
Vol. 10, Issue 452, eaao5726
DOI: 10.1126/scitranslmed.aao5726
  • Fig. 1 Generation and characterization of a tissue-engineered ossicle in vivo.

    (A) Macroscopic overview of melt electrospun mPCL-CaP scaffold by bright-field microscopy. Box: Magnification shown in (B). Scale bar, 1 mm. (B) Maximum intensity projection of mPCL-CaP scaffold by multiphoton-excited SHG detection (z depth of 100 μm). Box: Magnification. Scale bar, 100 μm. (C) Schematic representation of the model. Nude mice were implanted subcutaneously with an mPCL-CaP scaffold embedded in fibrin glue and BMP7 (10 μg per mouse) that generated a mature, vascularized ossicle over time. (D) Localization of the ossicle (mTEBC) by micro–computed tomography (μCT) at day 30. Ve, vertebra. Box: Representative 3D reconstruction and bright-field image shown on the right. Scale bars, 5 and 1 mm, respectively. (E to H) Histological overview, cortical detail, and tartrate-resistant acid phosphatase (TRAP) staining showing mTEBC maturation over time [d10 (E), d20 (F), d30 (G), and d40 (H); d, day after implantation]. Scale bars, 1 mm (overviews) and 100 μm (zooms). Insets show higher magnifications of boxed regions in (H). Scale bar, 25 μm. (I) Quantification by bone histomorphometry. Parameters analyzed: osteoblast surface (percentage of total bone surface; Ob. S/BS, %), osteoclast surface (percentage of total bone surface; Oc. S/BS, %), erosion surface (percentage of total bone surface; ES/BS, %), bone volume (percentage of tissue volume; BV/TV, %), and trabecular separation (Tb. Sp.). Mean ± SD is shown (n = 4 to 6 per group). Bone cortex measurements: five cortical areas per mTEBC, 350 × 150 μm each; medullary region measurements, four areas per mTEBC, 250 × 250 μm each. *P < 0.05; ***P < 0.001 by one-way ANOVA, followed by Tukey’s post hoc test.

  • Fig. 2 Analysis of the mTEBC morphology.

    (A) Maximum intensity projection of the SHG signal to detect the cortical bone of the ossicle ex vivo at days 10, 20, 30, and 40. Horizontal (xy; left) and orthogonal view (xz; right) are shown (z depth of 150 μm). Scale bar, 100 μm. (B and C) Quantification of cortical bone thickness by MPM or stereomicroscope [image in (C)]. Mean ± SD; six areas per mTEBC were averaged; three ossicles from independent mice per time point. (D) Measurement of the signal intensity in dependence of the laser power at the outer and inner cortical bone surface. Scanning wavelength, 1090 nm. One representative of two independent experiments is shown. (E) z-power adaptation (z-PA). Representative orthogonal view (xz) images of increased intensity and signal distribution are shown. Scanning wavelength, 1090 nm. (F) Quantification of surface porosity, represented as mean ± SD; individual data points are shown (six areas per mTEBC were averaged; three ossicles from independent mice per time point). (G) Histology (hematoxylin and eosin) of the ossicle compared to the mouse tibia, vertebra, rib, and calvaria. Scale bar, 50 μm. (H) 3D SHG reconstruction of the indicated bones (z depth of specimens: TEBC, 1 mm; tibia, 800 μm; vertebra, 1 mm; rib, 400 μm; calvaria, 800 μm). Yellow dashed line, cavity. Box: Magnification of cortical bone. Red dashed line, cortical bone. Data are means ± SD (n = 4 to 6 independent samples). (I) Left: Intravital detection of blood vessels in the mTEBC [70-kDa Alexa Fluor 750–conjugated dextran (Dx)]. Center and right: MPM detection of blood vessels (laminin) in cleared mTEBC, tibia, and calvaria ex vivo. Right: Quantification (mean ± SD, n = 3). Scale bar, 100 μm. *P < 0.05; ***P < 0.001 by one-way ANOVA, followed by Tukey’s post hoc test.

  • Fig. 3 PC3 tumor cell administration and monitoring by histology and iMPM.

    (A) Schematic representation of the model. PC3 cells were injected into the mature ossicle 30 days after implantation into nude mice. A dorsal skinfold chamber (DSFC) was applied 10 to 14 days after tumor cell implantation, adjacent to the ossicle to enable iMPM. (B) Histology of PC3 cells in mTEBC and patient biopsy. T, tumor. Scale bar, 100 μm. (C) iMPM detection of PC3 cells at different scanning depths (z-stack at z = 0, −50, and −96 μm). Overviews shown as xy and xz, yz orthogonal sections, and detail with subcellular resolution (dotted box). Dashed line in top left denoted the edge of the ossicle with fluorescence signal originating from the tumor. (D) XZ intensity profile of individual channels with increasing scanning depth into the ossicle. (E) Quantification of interphase (IF), mitotic, or apoptotic cells in 3D stacks captured by iMPM. Mean ± SD is shown (n = 5 independent lesions). Nucleus, H2B/eGFP (green); cytoplasm, DsRed2 (red); collagen fibers: bone, SHG (cyan). Scale bar, 100 μm.

  • Fig. 4 Monitoring of osteolysis and dynamics of Cat K+ cells.

    (A) TRAP staining of PC3 lesion in mTEBC. Box: Inset (magnification). Arrowheads indicate osteoclasts. Scale bar, 100 μm. (B) Evaluation of osteolysis by ex vivo μCT and SHG detection by MPM. Magnifications of numbered boxed regions are shown. Scale bar, 1 mm. (C) iMPM detection of an osteolytic lesion. Merged xy and xz, yz orthogonal views, single channels, and details are shown. Box: Magnification. Nucleus, H2B/eGFP (green); cytoplasm, DsRed2 (red); collagen fibers: bone, SHG (cyan); bone remodeling, OsteoSense (OS; white); osteoclast/phagocyte, cathepsin K (Cat K; yellow). Solid arrowhead, mitotic event; void arrowhead, apoptotic nucleus; asterisk, Cat K+ cells. Scale bar, 100 μm. (D) Imaging analysis, xy intensity profile for single channels and quantification of the distance between tumor border (first layer of cells, DsRed2) and osteoclast/phagocyte (Cat K) or bone (OS). Scatter diagrams represent measurements from different regions in three independent lesions. (E) Dynamics of Cat K+ events monitored by time-lapse iMPM and analyzed by tracking. “+” and diamonds indicate the start, red dots indicate the final position of each cell, and numbers indicate individual cells. Right: Heat map of the speed of 15 representative cells. Scale bar, 20 μm. (F and G) Cat K signal detected over time by time-lapse iMPM (F) and particle imaging velocimetry (PIV) obtained by whole-field analysis (G). Two sequential frames obtained at different time points (0 and 8 min) from movie S2 are shown. Scale bar, 10 μm. Magnitude, vector map, and speed frequency distribution (three to four areas per tumor, from three mice).

  • Fig. 5 Response to zoledronic acid therapy monitored by iMPM.

    (A) Schematic representation of the experimental schedule. Ten to 14 days after PC3 cells injection, mice were implanted with a DSFC and treated with ZA (20 μg per mouse in saline by a single intravenous injection) the subsequent day. (B and C) Longitudinal monitoring of osteolytic progression by SHG detection at iMPM in control (vehicle, saline) and ZA-treated mice and quantification of the resorbed area of lesions represented in (B). Scale bar, 100 μm. Note the different scale of the images representing control and ZA-treated tumors to accommodate the different size of the osteolytic region. (D) Imaging analysis of micro-osteolytic lesion progression in ZA-treated mice. (E to F) Quantification of the microspeed of bone resorption after administration of ZA in individual subregions (E) and corresponding average microspeed per subregion over time representing the changes from days 0 to 9 (F). Data are means ± SD (n = 10 microregions from three independent osteolytic lesions). μregion, microregion; bone μres, bone microresorption. (G) Density of Cat K signal over time. Mean ± SD (n = 3 independent lesions per time point). Scale bar, 100 μm. No significant difference was identified by one-way ANOVA, followed by Tukey’s post hoc test. (H) PIV analysis of Cat K signal in control and ZA-treated mice; two representative frames and magnitude map. Scale bar, 20 μm. Area, 65 × 65 μm. (I and J) Average speed in control and ZA-treated mice. Mean ± SD. For image analysis, the quantification of PIV was obtained from three to four areas per lesion, from four mice per group. Speed distribution obtained from representative cells highlighted by red outline in (I). ***P < 0.001, unpaired two-tailed Student’s t test. (K) PC3 cell replicative status. Representative images taken at the onset and 6 days after ZA application (left) and quantification of the ratio between mitotic and apoptotic cells at different time points (right diagram); mean ± SD (n = 4 independent lesions per time point). Scale bar, 10 μm. No significant difference was identified by one-way ANOVA, followed by Tukey’s post hoc test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/452/eaao5726/DC1

    Materials and methods

    Fig. S1. Optimization of tissue-engineered ossicle generation.

    Fig. S2. Longitudinal characterization of mTEBC maturation.

    Fig. S3. Functional analysis of bone marrow in TEBC and bone marrow transplantation experiments.

    Fig. S4. 3D organization and functionality of blood vessels in mTEBC.

    Fig. S5. Topology and position of solid PC3 tumors in mTEBC.

    Fig. S6. C4-2B and MDA-MB-231 tumor cell administration and monitoring by μCT, x-ray, histology, and iMPM.

    Fig. S7. Analysis of the bone morphologies ex vivo.

    Fig. S8. Imaging analysis of PC3-mediated osteolytic lesions by iMPM.

    Fig. S9. Distribution of osteoclasts and Cat K+ cells within mTEBC.

    Fig. S10. PIV analysis of response to zoledronate therapy monitored by iMPM.

    Movie S1. 3D reconstruction of PC3 lesion in mTEBC.

    Movie S2. 3D reconstruction of a PC3 osteolytic lesion in mTEBC.

    Movie S3. Kinetics of a Cat K+ cell.

    Movie S4. Kinetics of Cat K+ cell overview.

    Movie S5. Kinetics of a Cat K+ cell after ZA treatment.

    References (5559)

  • The PDF file includes:

    • Materials and methods
    • Fig. S1. Optimization of tissue-engineered ossicle generation.
    • Fig. S2. Longitudinal characterization of mTEBC maturation.
    • Fig. S3. Functional analysis of bone marrow in TEBC and bone marrow transplantation experiments.
    • Fig. S4. 3D organization and functionality of blood vessels in mTEBC.
    • Fig. S5. Topology and position of solid PC3 tumors in mTEBC.
    • Fig. S6. C4-2B and MDA-MB-231 tumor cell administration and monitoring by μCT, x-ray, histology, and iMPM.
    • Fig. S7. Analysis of the bone morphologies ex vivo.
    • Fig. S8. Imaging analysis of PC3-mediated osteolytic lesions by iMPM.
    • Fig. S9. Distribution of osteoclasts and Cat K+ cells within mTEBC.
    • Fig. S10. PIV analysis of response to zoledronate therapy monitored by iMPM.
    • Legends for movies S1 to S5
    • References (5559)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). 3D reconstruction of PC3 lesion in mTEBC.
    • Movie S2 (.mov format). 3D reconstruction of a PC3 osteolytic lesion in mTEBC.
    • Movie S3 (.avi format). Kinetics of a Cat K+ cell.
    • Movie S4 (.avi format). Kinetics of Cat K+ cell overview.
    • Movie S5 (.avi format). Kinetics of a Cat K+ cell after ZA treatment.

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