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. 2013 May 28;110(22):9042-7.
doi: 10.1073/pnas.1219603110. Epub 2013 May 13.

PI3Kα activates integrin α4β1 to establish a metastatic niche in lymph nodes

Barbara Garmy-Susini  1 , Christie J Avraamides, Jay S Desgrosellier, Michael C Schmid, Philippe Foubert, Lesley G Ellies, Andrew M Lowy, Sarah L Blair, Scott R Vandenberg, Brian Datnow, Huan-You Wang, David A Cheresh, Judith Varner
Affiliations

PI3Kα activates integrin α4β1 to establish a metastatic niche in lymph nodes

Barbara Garmy-Susini et al. Proc Natl Acad Sci U S A. .

Abstract

Lymph nodes are initial sites of tumor metastasis, yet whether the lymph node microenvironment actively promotes tumor metastasis remains unknown. We show here that VEGF-C/PI3Kα-driven remodeling of lymph nodes promotes tumor metastasis by activating integrin α4β1 on lymph node lymphatic endothelium. Activated integrin α4β1 promotes expansion of the lymphatic endothelium in lymph nodes and serves as an adhesive ligand that captures vascular cell adhesion molecule 1 (VCAM-1)(+) metastatic tumor cells, thereby promoting lymph node metastasis. Experimental induction of α4β1 expression in lymph nodes is sufficient to promote tumor cell adhesion to lymphatic endothelium and lymph node metastasis in vivo, whereas genetic or pharmacological blockade of integrin α4β1 or VCAM-1 inhibits it. As lymph node metastases accurately predict poor disease outcome, and integrin α4β1 is a biomarker of lymphatic endothelium in tumor-draining lymph nodes from animals and patients, these results indicate that targeting integrin α4β1 or VCAM to inhibit the interactions of tumor cells with the lymph node microenvironment may be an effective strategy to suppress tumor metastasis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Integrin α4β1 is a biomarker of lymphatic vessels in premetastatic lymph nodes. (A) Lymph nodes from 0, 7, 14, and 21 d after s.c. LLC inoculation immunostained to detect Lyve-1+/Prox-1+ lymphatic vessels or cytokeratin+ metastases (arrowheads), counterstained with DAPI (blue). Dotted lines show lymph node edge. (B) Mean ± SEM Lyve-1+ pixels per field. (C) Mean ± SEM Prox-1+ pixels per field (n = 10, *P < 0.01, **P < 0.001). (D) Percentage of mice with lymph node (blue) and lung (red) metastases (n = 10, *P < 0.01, **P < 0.001). (E) Integrin α4β1, Lyve-1, and DAPI staining of inguinal lymph nodes from normal or tumor-bearing mice. Arrowheads, α4β1+Lyve-1+ vessels. (F) Mean α4β1+Lyve-1+ pixels per field ± SEM in lymph nodes from mice with and without LLC tumors (n = 10, *P < 0.001). (Scale bar, 50 µm.)
Fig. 2.
Fig. 2.
Integrin α4β1, a biomarker of lymphatic vessels in lymph nodes of patients with ductal breast carcinoma. (A) Podoplanin immunostaining (arrowheads) in axillary lymph nodes from patients without cancer (normal) and patients with nonmetastatic or metastatic ductal breast carcinoma. (Scale bar, 50 µm.) Inset, 600× magnification. (B) Podoplanin+, Lyve-1+, and CD31+ lymphatic vessels (brown, arrowheads) with hematoxylin counterstaining (blue) in serial sections of a metastasis+ axillary lymph node. (C) Podoplanin (red)/CD31 (green), podoplanin (green)/Lyve-1 (red), or podoplanin (red)/integrin α4β1 (green) fluorescence immunostaining (arrowheads) in an axillary lymph node from a patient with invasive breast carcinoma. (D) Percentage of area ± SEM of podoplanin+, Lyve-1+, or CD31+ vessels in normal (n = 35), premetastatic (n = 24), or metastatic (n = 33) human axillary lymph nodes. (E) Percentage of α4β1+ podoplanin+ lymphatic vessels in these same lymph nodes. (Scale bar, 50 µm.)
Fig. 3.
Fig. 3.
VEGF-C stimulates PI3Kα-dependent integrin α4β1 activation during lymph node lymphangiogenesis. (A) pAkt/Akt immunoblotting in VEGF-C–stimulated LECs. (B) Immunoblots of PI3K isoforms in cultured LECs and vascular endothelial cells. (C) pAkt/Akt immunoblotting in VEGF-C–stimulated LEC ± 500 nM PIK2α or control. (D) Lymph nodes treated with saline, VEGF-C, the PI3Kγ inhibitor (TG100–115), or the PI3Kα inhibitor (PIK2α). (E) Lyve-1+ pixels per field in VEGF-C, TG100-115, LY294002, or PIK2α treated lymph nodes from WT or mutant animals lacking PI3Kγ (p110γ−/−) animals (n = 10, **P < 0.001). (F–H) Saline- or VEGF-C–stimulated LEC adhesion to VCAM-1. (G) Saline- or VEGF-C–stimulated LEC adhesion to VCAM-1 ± 1 µM PI3Kα inhibitor PIK2α. (H) LEC adhesion to VCAM-1 of control and p110α (Hs_PIK3CA_8) siRNA transfected cells ± VEGF-C. (Scale bars, 50 µm.)
Fig. 4.
Fig. 4.
Lymph node lymphangiogenesis promotes tumor cell adhesion in lymph nodes. (A) Lyve-1+ lymphatic vessels in saline- and VEGF-C–stimulated inguinal lymph nodes from wild-type (WT) and integrin α4Y991A mice (n = 10). (B) Mean ± SEM Lyve-1+ pixels per field from A. (C and D) Inguinal lymph nodes were stimulated for 5 d with VEGF-C or saline, and then mice were inoculated in the footpad with red fluorescent LLC cells. (C) Lyve1+ lymphatic vessels (green) and tumor cells (red) in stimulated inguinal lymph nodes. (D) Mean ± SEM red fluorescent pixels per field in lymph nodes (n = 10). (E) Red fluorescent tumor cells (arrowheads) in VEGF-C– or saline-stimulated inguinal lymph nodes from WT or α4Y991A animals (n = 8). (F) Mean ± SEM red fluorescent pixels per field. (Scale bar, 50 µm.)
Fig. 5.
Fig. 5.
LEC integrin α4β1 promotes adhesion of tumor cells to lymphatic endothelium. (A) FACs analysis of α4β1 expression in LEC and LLC cells (black); isotype control (gray). (B) Adhesion of 5-(and-6)-[([(4-chloromethyl)benzoyl]amino)tetramethylrhodamine] (CMTMR)-labeled LLC cells (red) to LEC (brightfield) with and without anti-α4β1 or isotype control antibodies. (C) Mean ± SEM adherent LLC cells per field. (D) Mean ± SEM LLC cells transfected with α4 or control siRNA adherent to LEC. (E and F) Inguinal lymph nodes of mice were stimulated 5 d with VEGF-C and treated with anti-α4β1 or control antibodies 2 h before footpad inoculation with red fluorescent LLC cells (n = 10). (E) Mean ± SEM tumor cells (pixels per field). (F) Red fluorescent tumor cells in lymph nodes.
Fig. 6.
Fig. 6.
VCAM-1 promotes tumor cell adhesion to lymphatic endothelium. (A) Metastasis+ human axillary lymph nodes immunostained to detect VCAM-1. (B) VCAM-1 expression in cultured LLC cells and LEC (black line, gray line, IgG control). (C) VCAM-1 expression in LLC and PyMT tumor cells (red). (D) Mean adhesion to human LEC ± SEM of CMTMR-labeled LLC cells in the presence or absence of saline, antimurine VCAM-1, or isotype-matched control (cIgG) antibodies. (E) Mean adhesion to LEC ± SEM of VCAM-1 or control shRNA transfected LLC cells. (F–H) Mice stimulated with VEGF-C proximal to inguinal lymph nodes were injected in the footpad with red fluorescent LLC cells preincubated with anti–VCAM-1 or cIgG antibodies (F and G) or stably transfected with VCAM or control shRNAs (H) (n = 10). (F) Images of and (G and H) mean ± SEM red fluorescent tumor cells in lymph nodes.
Fig. 7.
Fig. 7.
Lymphatic endothelial cell integrin α4β1 promotes spontaneous metastasis. (A) Lyve-1 and H&E staining of lymph nodes and lungs from WT or α4Y991A mice with 15-wk-old (PyMT) tumors (arrowheads, metastases). (B) Mean ± SEM Lyve-1+ pixels per field in brachial (PyMT) lymph nodes (n = 10). (C) Incidence of lymph node metastasis from A. (D) Mean ± SEM metastatic nodules per lung from A (n = 10).
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