Intravital Imaging Reveals Transient Changes in Pigment Production and Brn2 Expression during Metastatic Melanoma Dissemination

Pigment containing melanosomes can be identified by visible light emission following near IR illumination. A, B16F2 cells stimulated with α-MSH were imaged by transmitted light (i) and laser scanning microscopy following illumination at 800 nm (ii) merged image of emission at 410 to 450 nm (cyan), 500 to 550 nm (green), and 565 to 615 nm (red). Scale bar, 25 μm. Emission and excitation spectra of near IR–stimulated visible emission are shown in iii and iv, respectively. A.u., arbitrary unit. B, transmitted light and NIRVE images (dark red) of B16 cells are shown overlaid (left). A cell with a long process and high NIRVE signal is visible surrounded by cells with low NIRVE signal. Transmission electron microscopy images of the same area are shown. Middle left, a low magnification image of the NIRVE-positive cell and surrounding cells. Inset panels 1 to 3, high-magnification images of the areas indicated on the low-magnification image. White, orange, and red asterisks are placed adjacent to stage 2, 3, and 4 melanosomes, respectively. C, top, changes in pigment following α-MSH or H89 treatment. Bottom, NIRVE signal in green following α-MSH or H89 treatment; red, filamentous actin (F-actin). Scale bar, 50 μm. D, ear skin of mouse showing collagen (blue, collected between 420–490 nm following illumination at 800 nm) and NIRVE signal (red, collected between 575-630 nm following illumination at 800 nm). See also Supplementary Movie S2. Arrows, pigment-producing melanocytes. *, some NIRVE signal is also collected.

Monitoring pigment expression during melanoma dissemination. A, high resolution imaging of subcutaneous B16F2-GFP-CAAX tumor. GFP (green) and NIRVE signal (red; collected between 410–530 nm following illumination at 790 nm). Scale bar, 25 μm. B, left, motion analysis of intravital imaging of B16F2-GFP melanoma—:white areas are nonmotile, distinct areas of color indicate motile cells (arrows). Right, NIRVE signal from the same region with inset showing the positions of motile cells relative to the NIRVE signal (see also Supplementary Movies S3 and S4). Scale bar, 25 μm. C, histograms showing the quantification of pigment content in both motile and nonmotile tumor cells in the primary tumor, cells that had entered the vasculature, lymph node metastases (LN met), and lung metastases (lung met). Values are normalized to 1 for the primary tumor. *, P < 0.01 Kruskil-WallisANOVA test. D, i, macroscopic image showing the highly pigmented nature of the primary tumor, a lymph node metastasis, and lung metastases. ii, images of B16F2-GFP-CAAX tumor cells in primary tumor, blood, and lymph node. Merge shows GFP (green), NIRVE signal (red), and collagen (blue); scale bar, 100 μm.

Brn-2 promoter activity during melanoma dissemination. A, left, motion analysis of intravital imaging of B16F2 Brn-2:GFP/mRFP-CAAX melanoma; white areas are nonmotile; distinct areas of color indicate motile cells (arrows). Scale bar, 20 μm. Right, GFP signal from the Brn-2 promoter (green), mRFP-CAAX (red), and reflectance (blue) in same region (arrows, motile cells; see also Supplementary Movie S5). B, histograms showing the quantification of pigment content in both motile and nonmotile tumor cells in the primary tumor, cells that had entered the vasculature, lymph node metastases, and lung metastases. Values are normalized to 1 for the primary tumor. *, P < 0.01 Kruskil-WallisANOVA test. C, intravital image of B16F2 Brn2::GFP/mRFP-CAAX tumor. Green, GFP; blue, mRFP-CAAX; red, NIRVE signal. D, scatter plot showing the relationship between NIRVE signal and GFP expression from the Brn-2 promoter. Each cross represents the values in individual cells imaged in vivo.

Flow cytometry analysis of pigment content A, FACS profile of B16F2 cells with or without stable GFP expression (left). Effect of H89 or α-MSH on violet laser stimulated 450 nm emission is shown by contour plots. B, i, B16F2-GFP CAAX tumor cells isolated from xenograft tumors in nude mice. Contour plots show GFP (515–45 nm) against Violet (430–70 nm) emission. Blue line, gate used to sort GFP-expressing cells; magenta lines, the gates used to sort the top and bottom 10% pigmented cells. ii, pigment-high and pigment-low populations were reintroduced into the tail vein of mice and the resulting lung colonies were imaged. Representative images are shown with NIRVE signal (black) and GFP (green). iii, quantification of pigment levels assessed on a cell by cell basis in colonies generated from pigment-high and pigment-low populations. Relative frequency of pigment levels is shown (data from 12 mice in three independent experiments). C, i, B16F2 Brn2::GFP/mRFP-CAAX tumor cells isolated from xenograft tumors in nude mice. Contour plots show red (600–40 nm) against GFP (515–45 nm) emission. Blue line, gate used to sort mRFP-expressing cells; green lines, the gates used to sort the top and bottom 10% Brn2::GFP cells. ii, Brn2::GFP-high and Brn2::GFP-low populations were reintroduced into the tail vein of mice and the resulting lung colonies were imaged. Two representative images are shown with mRFP-CAAX signal (red) and Brn2::GFP (green). *, highlights three cells that have converted back to high levels of Brn2::GFP expression. iii, quantification of Brn2::GFP levels assessed on a cell by cell basis in colonies generated from Brn2::GFP-high and Brn2::GFP-low populations. Relative frequency of Brn2::GFP levels is shown (data from 10 mice in three independent experiments). *, P < 0.01 Mann-Whitney test.