Lenalidomide inhibits lymphangiogenesis in preclinical models of mantle cell lymphoma

Abstract

Lymphomas originate in and spread primarily along the lymphatic system. However, whether lymphatic vessels contribute to the growth and spreading of lymphomas is largely unclear. Mantle cell lymphoma (MCL) represents an aggressive non-Hodgkin's lymphoma. We found that MCL exhibited abundant intratumor lymphatic vessels. Our results demonstrated that the immunomodulatory drug lenalidomide potently inhibited the growth and dissemination of MCL in a xenograft MCL mouse model, at least in part, by inhibiting functional tumor lymphangiogenesis. Significant numbers of tumor-associated macrophages expressing vascular endothelial growth factor-C were found in both human MCL and mouse MCL xenograft samples. Lenalidomide treatment resulted in a significant reduction in the number of MCL-associated macrophages. In addition, in vivo depletion of monocytes/macrophages impaired functional tumor lymphangiogenesis and inhibited MCL growth and dissemination. Taken together, our results indicate that tumor lymphangiogenesis contributes to the progression of MCL and that lenalidomide is effective in decreasing MCL growth and metastasis most likely by inhibiting recruitment of MCL-associated macrophages.

Figure 1

MCL tumors exhibit abundant intratumor lymphatic vessels, and LEN decreases lymphangiogenesis in MCL. A. Immunohistochemical analysis of two lymphatic markers, VEGFR-3 and podoplanin (PDPN), and a blood vessel marker, CD34, in human MCL patient samples. The black arrow indicates an intratumor lymphatic vessel. The white arrow indicates a blood vessel. B. Schematic diagram showing different tumor regions. C. Immunofluorescence staining for PDPN, LYVE-1, and CD31 in sections of sham- and LEN-treated murine MCL tumors. Images were taken from peripheral regions of the tumor. White arrow indicates a LYVE-1⁺, PDPN⁺, and CD31^low lymphatic vessel with an open lumen. D. Quantification of LYVE-1⁺, CD31⁺ lymphatic vessels. Data represent the mean ± SEM (n = 20). *P < 0.05. E. Quantification of the depth of infiltrated lymphatic vessels. The depths were calculated as the distance from the tumor-host interface to the innermost lymphatic vessels. Data represent the mean ± SD (n = 20). ***P < 0.001. F. Immunoblots of Prox-1, PDPN, VEGFR-2, and VEGFR-3 in sham- and LEN-treated mouse MCL tumors. GAPDH is an internal control. Data represent two experiments. Scale bars, 100 μm.

Figure 2

LEN treatment inhibits MCL growth and dissemination. A. The tumor volume of Mino cell–derived MCL mouse xenografts in NSG mice was measured at different times and is presented as the mean ± SEM (n = 10). Scale bar, 10 mm. B. Tumors were weighed at harvest, and data are presented as the mean ± SD (n = 10). C. Schematic diagram showing inguinal lymph node, collecting lymphatic vessel, and axillary lymph node in mouse skin flaps. The yellow circle indicates the site of MCL xenograft. Green arrow shows the direction of lymphatic flow. D. Images and diameters of axillary lymph nodes. Data represent the mean ± SD (n = 10). Scale bar, 5 mm. E. H&E and immunostaining of the human B-cell marker, CD20, in sections of axillary lymph nodes from sham- and LEN-treated mice. Scale bars, 100 μm. F. Quantification of disseminated CD20⁺ Mino cells in the axillary lymph nodes using ImageJ. The relative staining density was calculated by comparing CD20⁺ areas per 10× field of LEN-treated samples with those of sham-treated samples. Data represent three independent experiments (Mean ± SEM, n=10, *P < 0.05).

Figure 3

LEN treatment results in non-functional tumor lymphatic vessels. A. MCL tumors immunostained for LYVE-1 and podoplanin (PDPN). The tumor tissues were collected 45 minutes after intratumor injection of FITC-dextran. Arrow indicates a FITC-dextran–filled lymphatic vessel. B. Percent of lymphatic vessels filled with FITC-dextran per 20× field in MCL tumors. Data represent the mean ± SEM (n = 6). C. Representative three-dimensional confocal images from sham- and LEN-treated tumor sections (1 mm in thickness) immunostained for lymphatic markers LYVE-1 and PDPN and MCL tumor marker CD20. Arrow indicates a tumor cell–containing lymphatic vessel. D. Percent of tumor cell–containing lymphatic vessels in MCL tumors. Data represent the mean ± SEM (n = 6). Scale bars, 50 μm. *P < 0.05. E. Two hours after intratumor injection of FITC-dextran, fluorescence analysis of a whole-mount skin flap showed that the FITC-dextran was present in the collecting lymphatic vessel (arrowheads) of sham-treated, but not LEN-treated mice. Arrow marks the direction of lymph flow. Scale bar, 2 mm. F. Reconstructed 3-dimenstional confocal images of PDPN⁺ lymphatic vessels surrounding MCL xenografts. Scale bar, 50 μm.

Figure 4

MCL tumors contain VEGF-C–expressing macrophages and LEN reduces the number of tumor-associated macrophages. A. Immunofluorescent staining of CD68, a macrophage marker, and VEGF-C in human MCL tumor sections. Arrows indicate VEGF-C⁺, CD68⁺ macrophages. Scale bar, 100 μm. B. Immunofluorescent staining of VEGF-C and macrophage markers F4/80 and CD11b in the peripheral regions of sham- and LEN-treated mouse MCL tumors. Arrow indicates a VEGF-C⁺ macrophage. Scale bar, 100 μm. C. F4/80⁺, VEGF-C⁺, and CD11b⁺ macrophages were quantified in randomly selected areas from the tumor peripheral regions. Data represent the mean ± SD (n = 6, *P < 0.05). D. Expression of human CCL5 in MCL tumor cells was analyzed by real-time PCR. Results represent relative units after normalized to the expression of 18S RNA. Data represent the mean ± SEM (n = 3, ***P < 0.001) from three independent experiments. E. Immunoblot analysis of human CCL5 levels in MCL tumor cells. GAPDH is an internal control. Data are representative of three experiments. F. Macrophage migration measured in response to Mino cell conditioned media (CM) with or without LEN treatment using a transwell assay. Results represent the mean ± SD (n = 9) from three independent experiments. **P < 0.01. G. Macrophage migration in response to sham-treated Mino cell conditioned media with a neutralizing antibody against CCL5 or isotype IgG (control). Results represent the mean ± SD (n = 9) from three independent experiments. *P < 0.05.

Figure 5

Clodrolip reduces the number of tumor-associated macrophages, inhibits tumor lymphangiogenesis, and impairs MCL growth and dissemination. A. Flow cytometry of CD115⁺ monocytes/macrophages in the peripheral blood from control liposome– (control) or clodrolip-treated mice bearing MCL tumors. Data represent the mean ± SD (n = 3). B. Quantification of F4/80⁺ cells from the peripheral regions of tumors. Data represent the mean ± SD (n = 6). C. Volume of MCL tumors. Data represent the mean ± SD (n = 6). D. Lymphangiography of whole-mount skin flaps showed that the FITC-dextran was present in the collecting lymphatic vessel (arrows) of control-treated but not clodrolip-treated mice. Scale bar, 2 mm. E-F. Representative immunostaining images and quantification of LYVE-1⁺ lymphatic vessels and infiltration depths in MCL xenografts treated with control liposomes or clodrolip. Scale bar, 100 μm. Lymphatic vessel density was determined from at least six representative images from each tumor. Data represent the mean ± SD (n = 6). G. Gross analysis of axillary lymph nodes in control liposome– and clodrolip-treated mice bearing MCL tumors. Red circles highlight the axillary lymph node region. Scale bar, 5 mm. Immunofluorescence staining for human CD20 and murine LYVE-1 of axillary lymph node sections. Scale bar, 100 μm. H. Quantification of disseminated CD20⁺ Mino cells in the axillary lymph nodes (n=6). Relative CD20⁺ positive staining density was presented as Mean ± SD values (n=6). *P < 0.05.