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Concurrence of extracellular vesicle enrichment and metabolic switch visualized label-free in the tumor microenvironment

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Science Advances  25 Jan 2017:
Vol. 3, no. 1, e1600675
DOI: 10.1126/sciadv.1600675
  • Fig. 1 Coregistered lower dimensional multicontrast images of an unperturbed mammary tumor from a carcinogen-injected rat.

    (A) Composite χ(2)SHG(3)THG image reflecting local optical structure. (B) Composite AF(2)-AF(3) image reflecting local autofluorescence activity. (C) χ(3)CARS image at R2850. Comparison with the image at R3050 (see fig. S2A) reflects lipid-protein composition. (D) In situ chemical analysis revealing local (9-pixel average, 1.5 × 1.5 μm2) lipid-protein/water transition. The χ(3)CARS spectra in three panels plot χ(3)CARS intensity (arbitrary unit) against vibration frequency across the R2550-R3250 range. Molecular vibrations corresponding to the observed peaks of R2850, R2930, and R3010 are explicitly shown. In (A) to (C), the lipolytic region marked by broken squares can be directly compared to its counterparts in Figs. 2 and 4. The flow in one developing blood vessel can be visualized (see movie S1).

  • Fig. 2 Coregistered multicontrast images of an unperturbed mammary specimen from a control rat.

    (A) Composite χ(2)SHG(3)THG image reflecting local optical structure. (B) Composite AF(2)-AF(3) image reflecting local autofluorescence activity. Regular adipocytes are recognized as AF(2)-visible cytoplasm located at the periphery of large AF(3)-visible lipid droplets. (C) χ(3)CARS image at R2850. The comparison with the image at R3050 reflects local lipid-protein/water transition. (D) In situ chemical analysis revealing local (9-pixel average, 1.5 × 1.5 μm2) lipid-protein/water transition. In (A) to (C), the lipolytic region marked by broken squares can be directly compared to its counterparts in Figs. 1 and 4. Inset 1: A stroma-dominant region with stromal lipid dispersion and deformed lipids from mammary specimen of a control rat. Inset 2: Another stroma-dominant region with a mammary duct from normal-appearing mammary specimen of a carcinogen-injected rat.

  • Fig. 3 Macroscopic events in the tumor microenvironment identified by multicontrast imaging.

    Each image contains 380 × 380 pixels with 0.5-μm pixel size. (A) Natural angiogenesis (An). (B) Lymphangiogenesis (LA) near a blood vessel (BV), which can be differentiated from the LA by the presence of χ(3)R2850 contrast. Some native cells (red arrows) and non-native cells (blue arrows) are also marked. (C) Degraded basement membrane (DBM) from lymphatic vessels or mammary ducts/lobules. Some non-native cells (blue arrows) are also marked. (D) Collagen production and fibroblast activation demonstrated by paralleled formation of fibroblasts, collagen fibers, and angiogenic vessels (area marked by broken magenta line). A fibrosis feature of dense collagen is also identified. (E) Non-native cell recruitment. Tumor cells (red arrows), nontumor native stromal cells (magenta arrows), and non-native cells (blue arrows) can be discriminated against each other according to χ(3)THG contrast and χ(3)THG-AF(2)–covisible tumor-associated extracellular vesicles.

  • Fig. 4 Coregistered multicontrast images of an unperturbed precancerous mammary specimen from a carcinogen-injected rat.

    (A) Composite χ(2)SHG(3)THG image reflecting local optical structure. Enriched χ(3)THG-AF(2)–covisible tumor-associated extracellular vesicles are found in contrast to the control sample of Fig. 2A. (B) Composite AF(2)-AF(3) image reflecting local autofluorescence activity. In contrast to the tumor or malignant cells in Fig. 1B, none of the AF(2)-visible cells seems to have more than one of these vesicles inside the cell body. They are likely deformed adipocytes that are accepting these vesicles (see movie S2). (C) χ(3)CARS image at R2850. The comparison with the image at R3050 reflects local lipid-protein/water transition. (D) In situ chemical analysis revealing local (9-pixel average, 1.5 × 1.5 μm2) lipid-protein/water transition. Inset 1: Lipolytic region with deformed lipids and lipid breakdown from precancerous mammary specimen of another carcinogen-injected rat. This broken square–marked region can be directly compared to its counterparts in Figs. 2 and 3, indicating that the differences between the tumor (or precancerous) and nontumor samples are the enrichment of the tumor-associated extracellular vesicles and the emergence of the R3050 peak, just like the comparison between the main parts of Figs. 2 and 4. Inset 2: Stroma-dominant region with a mammary duct near the region of inset 1, which can be directly compared to inset 2 in Fig. 2 to reveal the same differences.

  • Fig. 5 Coregistered single-contrast images of an unperturbed human mammary tumor with various extracellular vesicles.

    (A) Multicontrast images of different pseudocolor representations. Red, χ(3)R3050 (upper left) or χ(3)R2850 (lower left); yellow, AF(2); green, χ(2)SHG; cyan, AF(3); magenta, χ(3)THG. Area α represents interstitial fluid consisting of a dilute protein solution, whereas area β is a lipid with high AF(2) signal. (B) Distribution of three distinct classes of vesicles (276 vesicles) automatically identified by a software algorithm [that quantifies the prominence of a vesicle and its full-width half-maximum (FWHM) size] and classified by cross-contrast visibility. (C) χ(3)CARS spectra of pure water, the interstitial fluid in area α, one class of vesicles averaged over eight vesicles, and a dense collagen region indicative of a progressive increase of protein content in water. Data from water and the dense collagen region are from other independent experiments. a.u., arbitrary units. (D) χ(3)CARS spectra of the lipid in area β and the other two classes of vesicles indicative of a progressive increase of lipid/water ratio.

  • Table 1 Longitudinal animal test of extracellular vesicles and χ(3)R3050 as quantitative breast cancer indicators within (A) a constant field of view (0.19 × 0.19 mm2) and (B) a larger field of view (0.57 × 0.57 mm2).

    Nontumor (control or normal), tumor, spatially precancerous (resected 2 to 5 mm away from a palpable tumor), temporally precancerous (with no palpable tumor), and two samples from an abnormal control rat are highlighted in green, red, violet, orange, and blue, respectively, in the first column. The tumor (on site), spatially precancerous, and normal-appearing samples (resected 10 mm away from a palpable tumor) linked by one palpable tumor are shaded together in the second column. For the diagnosis by visual inspection, “−” represents white and thin mammary gland, “+” represents palpable tumor mass with orange color and rice-like granular formation, and “+/−” represents slightly darker mammary gland suspicious of tumor development. Tumor-associated extracellular vesicles are automatically recognized from χ(3)THG images by a software procedure that quantifies the prominence of a candidate vesicle and its FWHM size. For the diagnosis by vesicle count, “+” represents more than six counts, and “−” represents less than six counts. Samples with inconsistent diagnosis from visual inspection and two quantitative cancer indicators (vesicle count and the presence of R3050 peak) are highlighted in red in the third, fourth, and fifth columns. For the classification of a sampling (imaging) site, “A” represents adipocyte region, “S” represents stromal region, and “T” represents tumor region. All images collected in the longitudinal animal study are included for unbiased statistical analysis.

    A
    Week_rat/
    sampling site
    Site related
    to tumor
    VisualVesicles, countR3050ClassTumor microenvironment
    events* and other biological
    structures/events (figure reference)
    1_control/a−, 1A&SLipid breakdown
    1_control/b−, 0A&SLipid breakdown
    1_control/c−, 1SCollagen production, lipolysis
    1_experiment/a−, 0A&SLipogenesis, lipid breakdown,
    stromal native cells
    1_experiment/b+/−+, >20+SHair follicle
    1_experiment/c+/−+, 9+SMammary duct, lipolysis
    1_experiment/d−, 0A&SNerve, stromal native cells
    2_control/a−, 0AFluorescent protein mass
    2_control/b−, 0SNative cells, lipolysis
    2_control/c−, 0ARegular adipocytes
    2_experiment/a−, 1SNative cells, mammary duct
    2_experiment/b−, 2SMammary duct, lipolysis,
    native cells (Fig. 2, inset 2)
    2_experiment/c−, 5SStromal native cells
    2_experiment/d−, 4A&SNerve
    2_experiment/e−, 0A&SNerve
    3_control/a−, 2ALipolysis
    3_control/b−, 0ARegular adipocytes
    3_control/c−, 4SCollagen production,
    native cells, nerve
    3_experiment/a++, >20+A&SLymphangiogenesis, stromal native
    cells, fluorescent protein mass
    3_experiment/b++, >20+T&SCollagen cross-linking, tumor (cells)
    3_experiment/c−, 1ARegular adipocytes
    3_experiment/d−, 0A&SStromal native cells
    3_experiment/e−, 4A&SCollagen ring
    4_control/a−, 0ARegular adipocytes, native cells
    among adipocytes, crystallization
    4_control/b−, 0A&SLipid breakdown, native cells
    4_experiment/aOn site++, >20+A&SLipid breakdown
    4_experiment/bOn site++, 10+A&SFibrosis, stromal native cells
    4_experiment/cOn site++, >20+TCollagen cross-linking, tumor (cells)
    4_experiment/d2–5 mm+/−+, 13+A&SLipid breakdown, crystallization
    5_control/a−, 0SLipolysis
    5_control/b−, 3A&SLipolysis
    5_control/c−, 0SStromal lipid dispersion, lipolysis
    5_experiment/aOn site++, 8+T/SBasement membrane degradation,
    reorganized collagen, tumor (cells)
    5_experiment/bOn site++, 18+A&SNon-native cell recruitment, collagen
    production, stromal native cells (Fig. 3E)
    5_experiment/cOn site++, 17+TBasement membrane degradation,
    non-native cell recruitment, reorganized
    collagen, tumor (cells) (Fig. 3C)
    5_experiment/dOn site++, >20+S&TAngiogenesis, blood cells, tumor (cells)
    5_experiment/eOn site++, 9+A&SLipid breakdown
    5_experiment/f10 mm−, 0A&SLymphatic vessel, stromal native cells
    5_experiment/g10 mm−, 0ARegular adipocytes
    6_control/a−, 1A&SVessel, stromal native cells
    6_control/b−, 0SLipolysis, stromal lipid dispersion (Fig. 2, inset 1)
    6_control/c−, 0A&SNative cells among adipocytes, crystallization
    6_experiment/aOn site++, 8+A&SAngiogenesis, lipolysis, stromal native cells
    6_experiment/bOn site++, >20+A&SAngiogenesis, stromal native cells
    6_experiment/c2–5 mm+/−+, 7+SMammary ducts
    6_experiment/d10 mm−, 0A&SStromal native cells, collagen production
    7_control/a+/−+, 13+SLymphangiogenesis, non-native cell
    recruitment (Fig. 3B)
    7_control/b+/−−, 5A&SAngiogenesis, non-native
    cell recruitment, lipolysis, stromal
    native cells (Fig. 3A)
    7_control/c−, 0ARegular adipocytes
    7_experiment/aOn site++, >20+A&SNon-native cell recruitment,
    reorganized collagen, nerve
    7_experiment/bOn site++, >20+A&SAngiogenesis, lymphangiogenesis,
    reorganized collagen,
    fibroblast activation, nerve
    7_experiment/c2–5 mm+/−+, 12+A&SLipolysis, stromal lipid dispersion
    7_experiment/e10 mm−, 5SStromal lipid dispersion
    8_control/a−, 2ANative cells among adipocytes
    8_control/c−, 0ARegular adipocytes
    8_experiment/aOn site++, 18+T/SReorganized collagen, tumor
    (cells) (Fig. 3D)
    8_experiment/b2–5 mm+/−+, 16+A&SMammary duct, stromal native
    cells, lipolysis (Fig. 4, inset 2)
    8_experiment/e2–5 mm+/−+, 16+A&SLipolysis (Fig. 4, inset 1)
    9_control/a−, 1ANative cells among adipocytes
    9_experiment/aOn site+−, 1TTumor necrosis
    9_experiment/bOn site+−, 0TTumor necrosis
    9_experiment/d2–5 mm+/−+, 15+A&SLipolysis, native cells, deformed adipocytes
    9_experiment/f10 mm−, 2A&SStromal native cells, lipolysis
    B
    Week_rat/
    sampling site
    Site related
    to tumor
    VisualVesicles/R3050ClassTumor microenvironment
    events* and other biological
    structures/events (figure reference)
    7_control/d10 mmA&SStromal native cells, lipolysis
    7_experiment/dOn site++A&SLymphangiogenesis, angiogenesis,
    non-native cell recruitment,
    reorganized collagen,
    fibroblast activation,
    fibrosis, nerve, blood cells,
    tumor (cells) (Fig. 1)
    8_control/bA&SNative cells (Fig. 2, main)
    8_control/dARegular adipocytes
    8_experiment/cOn site++T&SReorganized collagen, basement
    membrane degradation,
    lymphangiogenesis,
    non-native cell
    recruitment, angiogenesis,
    tumor (cells), native cells
    8_experiment/dOn site++A&SFibroblast activation,
    fibrosis, angiogenesis,
    reorganized collagen,
    non-native cell recruitment, native cells, lipolysis, tumor cells
    9_control/bANative cells
    9_experiment/cOn site+TTumor necrosis
    9_experiment/e2–5 mm+/−+A&SLipolysis, native cells, deformed
    adipocytes (Fig. 4, main)

    *Tumor microenvironment events are in bold font.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/1/e1600675/DC1

      table S1. Multicontrast nonlinear imaging to observe various tumor microenvironment events.

      table S2. New knowledge acquisition by dimension-increased multicontrast image analysis of a rat mammary tumor.

      table S3. Observed micrometer-sized vesicles in mammary tissue classified by cross-contrast visibility and chemical content.

      table S4. Macroscopic events observed in tumor microenvironments and in nontumor mammary samples.

      fig. S1. Schematic for label-free multicontrast nonlinear imaging.

      fig. S2. Coregistered multicontrast images of an unperturbed mammary tumor from a carcinogen-injected rat.

      fig. S3. Coregistered single-contrast images of an unperturbed normal human mammary specimen.

      movie S1. Sweeping of hyperspectral χ(3)CARS images from the middle-right corner of Fig. 1C.

      movie S2. Comparison of coregistered multicontrast images from the adipocyte-dominant control mammary tissue sample (Fig. 2, main) and the precancerous mammary sample (Fig. 4, main).

    • Supplementary Materials

      This PDF file includes:

      • table S1. Multicontrast nonlinear imaging to observe various tumor microenvironment events.
      • table S2. New knowledge acquisition by dimension-increased multicontrast image analysis of a rat mammary tumor.
      • table S3. Observed micrometer-sized vesicles in mammary tissue classified by cross-contrast visibility and chemical content.
      • table S4. Macroscopic events observed in tumor microenvironments and in nontumor mammary samples.
      • fig. S1. Schematic for label-free multicontrast nonlinear imaging.
      • fig. S2. Coregistered multicontrast images of an unperturbed mammary tumor from a carcinogen-injected rat.
      • fig. S3. Coregistered single-contrast images of an unperturbed normal human mammary specimen.

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      Other Supplementary Material for this manuscript includes the following:

      • movie S1 (.mp4 format). Sweeping of hyperspectral χ(3)CRS images from the middle-right corner of Fig. 1C.
      • movie S2 (.mp4 format). Comparison of coregistered multicontrast images from the adipocyte-dominant control mammary tissue sample (Fig. 2, main) and the precancerous mammary sample (Fig. 4, main).

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