Diphtheria toxin-mediated ablation of lymphatic endothelial cells results in progressive lymphedema Jason C. Gardenier 1, Geoffrey E. Hespe 1, Raghu P. Kataru 1, Ira L. Savetsky 1, Jeremy S. Torrisi 1, Gabriela García Nores 1, Joseph J. Dayan 1, David Chang 2, Jamie Zampell 1, Inés Martínez-Corral 3, Sagrario Ortega 4, and Babak J. Mehrara 1 1 Department of Surgery, Division of Plastic and Reconstructive Surgery, Memorial Sloan Kettering Cancer Center, New York, NY 10065 2 Section of Plastic and Reconstructive Surgery, The University of Chicago Medicine & Biological Sciences, Chicago, IL 60637 3 Department of Immunology, Genetics, and Pathology, Uppsala University, Uppsala, Sweden 75185 4 Transgenic Mice Core Unit, Spanish National Cancer Research Center, Madrid, Spain 28029
Supplemental Figures Supplemental Figure 1: Systemic DT administration results in variable lymphatic depletion in different tissue types. Immunofluorescent staining with LYVE-1 revealed variable destruction of lymphatic vessels in different tissues 24 hours after IP administration of DT (scale bars: trachea=200μm, diaphragm=200μm, mesenteric LN=500μm, cervical LN=200μm, and jejunum=100μm). Representative figures are shown above and quantification using imaging of LYVE-1 + area is shown below (p<0.001).
Supplemental Figure 2: Ablation of lymphatic vessels in the hindlimb result in lymphatic vessel fragments filling with leukocytes 1 week after hindlimb DT administration and progressive dermal thickening and fibrosis (A) Immunohistochemical localization of CD45 + cells demonstrating leukocytes filling the lumens of dermal lymphatics after DT administration. (B) Dual immunofluorescent staining of LYVE-1 and CD45 confirming that filled vessels are lymphatic vessel remnants (Scale bar=50μm). (C) Representative high power H&E stained cross-sections of the distal hindlimb of control (normal) and DT treated mice harvested 52 weeks after lymphatic ablation (Scale bar=200μm). (D) Quantification of dermal thickness in control and DT treated mice over time (p<0.01). (E) Representative immunohistochemical localization of type I collagen in control (normal) and DT treated mice harvested 52 weeks after lymphatic ablation (scale bar=50μm). (F) Quantification of type I collagen staining area in control and DT treated mice at various time points following lymphatic ablation (p<0.003).
Supplemental Figure 3: Expression of LYVE-1 by macrophages increases over time after lymphatic ablation. (A) Representative flow plots for LYVE-1 positivity gated on F4/80 + macrophages from digested hindlimb skin 1 week and 3 weeks after lymphatic ablation (5 animals were analyzed per group). (B) Absolute numbers of F4/80 + macrophages present in the distal hindlimb skin 1week and 3 weeks after lymphatic ablation as quantified by flow cytometry (p<0.05). (C) Quantification of the percentage of LYVE-1 + / F4/80 + macrophages 1 week and 3 weeks after lymphatic ablation (p<0.05).
Supplemental Figure 4: Clodronate liposome administration effectively depletes macrophages systemically, increases CD4+ cell infiltration and accelerates collecting lymphatic vessel sclerosis (A) Representative flow plots from spleens of mice after DT administration and 4 weeks of treatment with either clodronate liposomes or PBS liposomes showing effective reduction in F4/80 + macrophages (B) Quantification of macrophages (F4/80 + ) cells from spleens for each group (p<0.01). (C) Representative immunofluorescent images localizing CD4 + cells in the distal hindlimb tissues of control and macrophage depleted animals 4 weeks after DT administration (Scale bar=100μm). (D) Quantification of CD4 + cell infiltration in control and macrophage depleted animals (p<0.001). (E) Representative images of collecting lymphatics from control or macrophage depleted mice staining for podoplanin (green) and α-sma (red) harvested 4 weeks after DT administration (Scale bar=20μm). (F) Quantification of collecting vessel luminal cross-sectional area in control and macrophage depleted mice 4 weeks after DT administration (p<0.001). (G) Quantification of α-sma thickness in control and macrophage depleted animals 4 weeks after DT administration (p<0.005).
Supplemental Figure 5: CD4 mab administration reduces T cell infiltration in hind limb skin. (A) Representative flow plots of distal hindlimb single cell digests showing TCR-β + cells gated on CD45 + cells in isotype or CD4 mab treated mice. (B) Quantification of T cells in the two groups is shown below (p<0.05).
Supplemental Figure 6: A schematic model of the proposed phases of lymphedema. The two phases of reaction to lymphatic injury are shown with relative changes in lymphangiogenesis, fibrosis, and lymphatic function represented over time.