Figure 8.
Figure 8. Blocking CCR8 inhibits tumor cell entry into the lymph node from collecting lymphatics. (A–D) Metastases from control, pcDNA empty vector transfected MDAcl.13 cells (GFP, green) in the sentinel lymph nodes. (A) Stereomicroscope captured epifluorescent image of the intact lymph node showing metastases in the afferent lymphatic vessel and inside the lymph node parenchyma (boxed area). (B–D) Ex vivo multiphoton imaging of GFP-labeled metastases (green) combined with second harmonic generation (SHG, red), showing metastases scattered throughout the lymph node cortex. (B and C) View from the top of the lymph node (from the outside). (D) View from the lymph node parenchyma (from the inside). SHG visualizes collagen fibers in the lymph node capsule (red). (E–H) Metastases from MDAcl.13/MC148 tumors. (E) Stereomicroscope captured epifluorescent image showing metastasis in the afferent lymphatic vessel (in-transit metastasis) but not in the lymph node parenchyma. Junction of the lymphatic vessel and the lymph node is sharply demarcated, indicating tumor cell arrest at that location (boxed area). (F–H) ex vivo multiphoton images of metastases (GFP, green) in the afferent lymphatic vessel combined with SHG (red), which visualizes collagen fibers of the lymphatic vessel wall. (F and G) Lateral view of the vessel. (H) View into the vessel lumen. (I) Bar graph depicts the incidence of intranodal metastases and in-transit metastases by MDAcl.13 tumors upon inhibition of CCR8 with MC148 or upon down-regulation with shRNA. Incidence is calculated as percent of all samples positive for either intranodal or in-transit metastases (pcDNA control, n = 15; MC148, n = 15; sh control, n = 14; shCCR8(1), n = 12; shCCR8(2), n = 8). (J) Scanning electron microscopy micrograph of a lymph node cross section showing MDAcl.13/shCCR8(1) metastasis in the lymphatic vessel at the junction with the SCS (boxed area); similar site as depicted in E. (K and L) Lymphatic endothelium at the floor of the SCS bordering the tumor visualized by scanning electron microscopy at high magnification (K) or by immunofluorescent staining for LYVE-1 (L). Images are representative of at least n = 9 mice per group. Arrows, lymphatic endothelium; t, tumor. Bars: (A-H, J, and L) 100 µm; (K) 5 µm.

Blocking CCR8 inhibits tumor cell entry into the lymph node from collecting lymphatics. (A–D) Metastases from control, pcDNA empty vector transfected MDAcl.13 cells (GFP, green) in the sentinel lymph nodes. (A) Stereomicroscope captured epifluorescent image of the intact lymph node showing metastases in the afferent lymphatic vessel and inside the lymph node parenchyma (boxed area). (B–D) Ex vivo multiphoton imaging of GFP-labeled metastases (green) combined with second harmonic generation (SHG, red), showing metastases scattered throughout the lymph node cortex. (B and C) View from the top of the lymph node (from the outside). (D) View from the lymph node parenchyma (from the inside). SHG visualizes collagen fibers in the lymph node capsule (red). (E–H) Metastases from MDAcl.13/MC148 tumors. (E) Stereomicroscope captured epifluorescent image showing metastasis in the afferent lymphatic vessel (in-transit metastasis) but not in the lymph node parenchyma. Junction of the lymphatic vessel and the lymph node is sharply demarcated, indicating tumor cell arrest at that location (boxed area). (F–H) ex vivo multiphoton images of metastases (GFP, green) in the afferent lymphatic vessel combined with SHG (red), which visualizes collagen fibers of the lymphatic vessel wall. (F and G) Lateral view of the vessel. (H) View into the vessel lumen. (I) Bar graph depicts the incidence of intranodal metastases and in-transit metastases by MDAcl.13 tumors upon inhibition of CCR8 with MC148 or upon down-regulation with shRNA. Incidence is calculated as percent of all samples positive for either intranodal or in-transit metastases (pcDNA control, n = 15; MC148, n = 15; sh control, n = 14; shCCR8(1), n = 12; shCCR8(2), n = 8). (J) Scanning electron microscopy micrograph of a lymph node cross section showing MDAcl.13/shCCR8(1) metastasis in the lymphatic vessel at the junction with the SCS (boxed area); similar site as depicted in E. (K and L) Lymphatic endothelium at the floor of the SCS bordering the tumor visualized by scanning electron microscopy at high magnification (K) or by immunofluorescent staining for LYVE-1 (L). Images are representative of at least n = 9 mice per group. Arrows, lymphatic endothelium; t, tumor. Bars: (A-H, J, and L) 100 µm; (K) 5 µm.

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