Endothelial cell–specific LAT1 ablation normalizes tumor vasculature
Jun-ichi Suehiro, Toru Kimura, Toshiyuki Fukutomi, Hisamichi Naito, Yasuharu Kanki, Youichiro Wada, Yoshiaki Kubota, Nobuyuki Takakura, Hiroyuki Sakurai
Jun-ichi Suehiro, Toru Kimura, Toshiyuki Fukutomi, Hisamichi Naito, Yasuharu Kanki, Youichiro Wada, Yoshiaki Kubota, Nobuyuki Takakura, Hiroyuki Sakurai
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Research Article Angiogenesis Vascular biology

Endothelial cell–specific LAT1 ablation normalizes tumor vasculature

Abstract

Some endothelial cells in the tumor vasculature express a system L amino acid transporter, LAT1. To elucidate the role of LAT1 in tumor-related endothelial cells, tumor cells were injected into endothelial cell–specific LAT1 conditional knockout mice (Slc7a5flox/flox; Cdh5-Cre-ERT2), and we found that the shape of the tumor vasculature was normalized and the size and numbers of lung metastasis was reduced. TNF-α–induced expression of VCAM1 and E-selectin at the surface of HUVEC, both of which are responsible for enhanced monocyte attachment and premetastatic niche formation, was reduced in the presence of LAT1 inhibitor, nanvuranlat. Deprivation of tryptophan, a LAT1 substrate, mimicked LAT1 inhibition, which led to activation of MEK1/2-ERK1/2 pathway and subsequent cystathionine γ lyase (CTH) induction. Increased production of hydrogen sulfide (H2S) by CTH was at least partially responsible for tumor vascular normalization, leading to decreased leakiness and enhanced delivery of chemotherapeutic agents to the tumor.

Authors

Jun-ichi Suehiro, Toru Kimura, Toshiyuki Fukutomi, Hisamichi Naito, Yasuharu Kanki, Youichiro Wada, Yoshiaki Kubota, Nobuyuki Takakura, Hiroyuki Sakurai

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Figure 4

Endothelial cell–specific LAT1 ablation suppressed CD11b-positive myeloid cell recruitment and endothelial VCAM1 expression in the premetastatic lung, inhibiting Ex-3LLC or B16F10 lung metastasis.

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Endothelial cell–specific LAT1 ablation suppressed CD11b-positive myeloi...
(A) Immunostaining of VCAM1 in lungs from control or Slc7a5iEC–KO mice intravenously injected with fluorescent-labeled Ex-3LLC cells. Nuclei were counterstained with DAPI (blue). Bar graph represented the numbers of VCAM1-positive cells (fluorescence intensity more than background)/hpf counted in 5 randomly selected slices and normalized to that of control. (B) Immunostaining of CD11b in lungs from control or Slc7a5iEC–KO mice intravenously injected with fluorescent-labeled Ex-3LLC cells. The numbers of CD11b cells/hpf were counted in 4 randomly selected slices and normalized to that of control. (C) Immunostaining of VCAM1 in lungs from control or Slc7a5iEC–KO mice intravenously injected with fluorescent-labeled B16F10 cells (control; n = 4, Slc7a5iEC–KO mice; n = 3). (D) Immunostaining of CD11b in lungs removed from control or Slc7a5iEC–KO mice intravenously injected with fluorescent-labeled B16F10 cells (control; n = 4, Slc7a5iEC–KO mice; n = 4). (E) Lung metastasis model by intravenous injection of Ex-3LLC cells in control or Slc7a5iEC–KO mice. Time course of tamoxifen administration and cancer cell injection are indicated at top. Middle images indicate representative images of whole lung and histological sections of the lung with hematoxylin and eosin staining. Scale bars (E–H): 1 cm (left); 2 mm (right). The ratio of ki-67 positive area in the bar graph was normalized to the control mice (= 100%) (mean ± SEM, control; n = 11, Slc7a5iEC–KO mice; n = 12). (F) Lung metastasis model by intravenous injection of B16F10 cells in control or Slc7a5iEC–KO mice (control; n = 7, Slc7a5iEC–KO mice; n = 8). (G) Lung metastasis model by intravenous injection of Ex-3LLC cells in control or GYY4137 loaded mice (control; n = 4, GYY4137 treated mice; n = 4). The ratio of ki-67 positive area was normalized to the control mice (= 100%). (H) Lung metastasis model by intravenous injection of B16F10 cells in GYY4137 loaded mice (control; n = 3, GYY4137 treated mice; n = 3). Bar graph showed mean ± SEM. P values were determined by 2-tailed, unpaired t test. *P < 0.05. Scale bars (A–D): 100 μm.