Melanoma-derived extracellular vesicles mediate lymphatic remodelling and impair tumour immunity in draining lymph nodes

Abstract

Tumour-draining lymph nodes (LNs) undergo massive remodelling including expansion of the lymphatic sinuses, a process that has been linked to lymphatic metastasis by creation of a pre-metastatic niche. However, the signals leading to these changes have not been completely understood. Here, we found that extracellular vesicles (EVs) derived from melanoma cells are rapidly transported by lymphatic vessels to draining LNs, where they selectively interact with lymphatic endothelial cells (LECs) as well as medullary sinus macrophages. Interestingly, uptake of melanoma EVs by LN-resident LECs was partly dependent on lymphatic VCAM-1 expression, and induced transcriptional changes as well as proliferation of those cells. Furthermore, melanoma EVs shuttled tumour antigens to LN LECs for cross-presentation on MHC-I, resulting in apoptosis induction in antigen-specific CD8⁺ T cells. In conclusion, our data identify EV-mediated melanoma-LN LEC communication as a new pathway involved in tumour progression and tumour immune inhibition, suggesting that EV uptake or effector mechanisms in LECs might represent a new target for melanoma therapy.

Keywords: exosome; immunotherapy; lymphangiogenesis; pre-metastatic niche; sentinel lymph node.

FIGURE 1

B16F10‐derived EVs are taken up by LN macrophages and LECs. (A) Microscopic detection of tdTomato in lymphatic sinuses of popliteal LNs 2 h after injection of B16F10‐tdTomato‐derived EVs into the hind paw. Dashed boxes in the upper panels are shown enlarged in the lower panels. (B) Representative FACS gating for popliteal LN macrophage subsets (SSM, MSM, MCM) (top panels, pre‐gated for living singlets) and histograms showing DiD intensity 16 h after injection of B16F10‐derived EVs labelled with DiD into the hind paw. (C) Representative FACS gating for popliteal LN stromal cell subsets (FRCs, LECs, BECs) (top panels, pre‐gated for living singlets) and histograms showing DiD intensity 16 h after injection of B16F10‐derived EVs labelled with DiD into the hind paw. (D, E) Quantification of DiD intensity in MSMs (D) and LECs (E) in popliteal, sacral and inguinal LNs. Each line represents an individual experiment using a pool of 2‐3 mice / condition (N = 3). (F) Quantification of DiD intensity in LN LEC subsets (ACKR4⁺ cLECs, CD44⁺ fLECs, Mrc1⁺ mLECs). Each line represents an individual experiment using a pool of 2–3 mice / condition (N = 3). (G) Representative histograms and quantification of GFP intensity in stromal cells (FRCs, LECs, BECs) of axillary and inguinal LNs draining B16F10 tumours expressing palmitoylated GFP (palmGFP) (N = 7 mice / condition). * p < 0.05, ** p < 0.01, **** p < 0.0001, two‐way ANOVA with Sidak's post‐test (including data matching by experiment in panels D‐F)

FIGURE 2

Uptake of B16F10‐derived EVs by LN LECs is partly dependent on VCAM‐1. (A, B) Representative histogram (left panels) and quantification (right panels) of α4 integrin (ITGA4, A, N = 5) and β1 integrin (ITGB1, B, N = 3)) on the surface of B16F10‐derived EVs detected by small particle flow cytometry. * p < 0.05, ** p < 0.01, Student's t‐test. (C) Representative immunofluorescence images of subcapsular (left) and medullary (right) regions of naïve and B16F10 tumour‐draining inguinal LNs stained for Lyve‐1 (red) and VCAM‐1 (green). (D) Example histograms and quantification of VCAM‐1 expression in LN LEC subsets by flow cytometry (N = 3). *** p < 0.001, **** p < 0.0001, one‐way ANOVA with Sidak's post‐test. (E) Re‐analysed single‐cell RNA sequencing data of human LN LECs (Takeda et al., 2019) demonstrating enrichment of VCAM‐1 transcripts in fLECs (LEC II) and mLECs (LEC IV). (F, G) B16F10‐derived EVs labelled with DiD were injected into tamoxifen‐treated Prox1‐CreER^T2 x Vcam1^fl/fl mice. Example histograms (left panels) and quantification (right panels) of DiD uptake in popliteal LN LECs (F) and MSMs (G) (Control = wildtype mice injected with control liposomes; N = 3–4 pools of 2–3 mice each / condition). * p < 0.05, unpaired Student's t‐test

FIGURE 3

B16F10‐derived EVs induce LN remodelling and LEC expansion. (A) Schematic of the experimental workflow to determine acute effects of B16F10‐derived EVs on draining LNs. Mice were treated for three consecutive days with 5 μg of B16F10‐derived EVs / hind paw, and the popliteal LNs were analysed 2 days after the last injection. (B, C) Weight (B) and total cellularity (C) of popliteal LNs (determined by FACS) (N = 4 mice / group). (D) Light sheet microscopy images (maximum intensity projections) of optically cleared LN wholemounts stained for Lyve‐1 (green) and Prox1 (red) after injection of control liposomes or B16F10‐derived EVs. (E) Total LEC number in popliteal LNs determined by FACS (N = 4 mice / group). (F) Representative images and quantification of Ki67⁺ LECs in popliteal LNs (N = 5–6 mice / group). Arrows point to Ki67⁺ Prox1⁺ nuclei. * p < 0.05, unpaired Student's t‐test. (G) Representative western blot of EVs and whole cell lysates (WCL) derived from B16F10 cells with or without ova. (H) Mice were treated with 10 μg EVs derived from B16F10 cells with or without ova / hind paw, and SIINFEKL cross‐presentation in popliteal LN LEC and BECs was determined by FACS 1 day later (N = 3–4 pools of three mice / condition). Control = mice injected with control liposomes. * p < 0.05, two‐way ANOVA with Dunnett's post‐test

FIGURE 4

B16F10‐derived EVs induce transcriptional changes in draining LN LECs. (A) Schematic representation of the experimental approach: Mice were treated every other day by intradermal EV injection for 2 weeks. Subsequently, draining inguinal LNs were collected, LECs isolated and subjected to scRNA‐seq. Naïve mice served as controls. (B) Unsupervised clustering of pooled naïve and EV‐injected LN LECs and mapping of the three major LN LEC subpopulations: LECs lining the ceiling and the floor of the subcapsular sinus and LECs lining medullary sinuses. (C) Number of differentially expressed genes within the three major subsets. Floor LECs (fLECs) showed by far the highest number of differentially expressed genes. (D) Dot plot representing the top 10 most strongly up‐ and downregulated genes in fLECs and their expression in all three LEC subsets. (E) Top 10 most significantly (by FDR) enriched gene ontology terms (cellular component and biological process) among the up‐ and downregulated genes in fLECs. (F) Example plots showing the expression of Msr1, Ltb and Bst2 in naïve (top) and EV‐injected (bottom) LN LECs

FIGURE 5

Primary tumour‐induced LN LEC expansion and tumour antigen cross‐presentation are mediated by EVs. (A) Representative western blot of Rab27a in whole cell lysates of parental B16F10‐ova cells and B16F10‐ova cells deficient of Rab27a. (B) EV release of parental B16F10‐ova cells and Rab27a^ko B16F10‐ova cells in vitro quantified by protein content of isolated EVs (N = 3). * p < 0.05, one‐sample t‐test. (C‐D) Mice were challenged with parental B16F10‐ova cells or Rab27a^ko B16F10‐ova cells and the draining axillary and inguinal LNs were analysed on day 14 after tumour cell injection by flow cytometry. Frequency of LECs among all stroma cells (C, N = 15–16 mice / condition, pooled from three independent experiments) and frequency of Ki67⁺ LECs (D, N = 9–10 mice / condition, pooled from two independent experiments). (E) Number of CD45⁻ CD31⁻ GFP⁺ tumour cells in LNs draining parental B16F10‐ova or Rab27a^ko B16F10‐ova tumours additionally expressing palmGFP (N = 5 mice / condition). (F) SIINFEKL cross‐presentation by LECs in LNs draining parental B16F10‐ova or Rab27a^ko B16F10‐ova tumours expressed as normalized geometric mean intensity values (N = 10–11 mice / condition, pooled from two individual experiments). (G) Frequency of T cells specific for ovalbumin, Pmel (gp100) and Dct (Tyrp2) among all CD8⁺ T cells in LNs draining parental B16F10‐ova (grey) and Rab27a‐deficient B16F10‐ova tumours (black) (N = 6–7 mice / condition). (H‐I) Representative FACS plots (H) and rate of apoptosis determined by AnnexinV (AnV) staining (I) among CD8⁺ T cells specific for ovalbumin, Pmel (gp100) and Dct (Tyrp2) in LNs draining parental B16F10‐ova (grey) and Rab27a‐deficient B16F10‐ova tumours (black) (N = 6–7 mice / condition). (J‐K) CD8⁺ T cells were isolated on day 14 from tumor‐draining LNs of mice bearing parental B16F10‐ova or Rab27a^ko B16F10‐ova tumours that were treated with an adoptive transfer of naïve OT‐1 T cells on day 10. Proliferation (assessed by CFSE dilution) was determined after 72 h in presence of anti‐CD3 / anti‐CD28 antibodies or of B16F10‐ova cells (J), and the capacity to kill B16F10‐ova target cells was determined after 16 h (K). Naïve and ex vivo stimulated effector OT‐1 T cells served as negative and positive controls (N = 5 / condition). * p < 0.05, ** p < 0.01, **** p < 0.0001, unpaired Student's t‐test