SCS macrophages restrict melanoma progression by blocking tumor vesicle interactions with B cells
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This study found that subcapsular sinus (SCS) macrophages in tumor-draining lymph nodes suppress melanoma progression by restricting interactions between tumor-derived extracellular vesicles (tEVs) and B cells. The researchers showed that tEVs carrying tumor material disseminate from primary tumors to lymph nodes via lymph vessels. SCS macrophages were identified as a major host cell type interacting with tEVs in lymph nodes. Depletion of SCS macrophages resulted in increased tEV-B cell interactions and enhanced tumor growth, suggesting SCS macrophages normally act as "tumor suppressors" by limiting pro-tumor immune responses.
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SCS macrophages restrict melanoma progression by blocking tumor vesicle interactions with B cells
- 1. SCS macrophages suppress melanoma by restricting tumor-derived vesicle–B cell interactions Ferdinando Pucci, Christopher Garris, Charles P. Lai, Andita Newton, Christina Pfirschke, Camilla Engblom, David Alvarez, Melissa Sprachman, Charles Evavold, Angela Magnuson, Ulrich H. von Andrian, Katharina Glatz, Xandra O. Breakefield, Thorsten R. Mempel, Ralph Weissleder, and Mikael J. Pittet Center for Systems Biology, Massachusetts General Hospital Research Institute, Harvard Medical School, Boston, MA 02114, USA. 2Graduate Program in Immunology, Harvard Medical School, Boston, MA 02115, USA. 3Department of Neurology, Massachusetts General Hospital Research Institute, Harvard Medical School, Charlestown, MA 02129, USA. 4Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA. 5Institute of Pathology, University Hospital Basel, 4031 Basel, Switzerland. 6Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital Research Institute, Harvard Medical School, Charlestown, MA 02129, USA Coach Professor: Dr. Yane-Shih Wang Sit-in Professor: Dr. Charles P. Lai Course Coordinator: Dr. Chi-Kuang Yao Dated: 20171122 Presenter: Gul Muneer Science 17 Mar 2016 DOI: 10.1126/science.aaf1328 1
- 2. SCS macrophages suppress melanoma by restricting tumor-derived vesicle–B cell interactions Ferdinando Pucci, Christopher Garris, Charles P. Lai*, Andita Newton, Christina Pfirschke, Camilla Engblom, David Alvarez, Melissa Sprachman, Charles Evavold, Angela Magnuson, Ulrich H. von Andrian, Katharina Glatz, Xandra O. Breakefield, Thorsten R. Mempel, Ralph Weissleder, and Mikael J. Pittet *Present address: Institute of Atomic and Molecular Sciences, Academia Sinica 2
- 3. Are vesicles a kind of cellular waste/debris? - “Vehicles of Intercellular Communication" 3
- 4. Tumor cell Tumor-Derived Extracellular Vesicles (tEVs) Exosome Ectosome Apoptotic Bodies Large Oncosome Cancer cells Immune cell Fibroblast Endothelial Cells TumorMicroenvironment 4
- 5. Dissemination? Distant Targets? Melanoma Tumor cells Tumor-Derived Vesicles Target cells Vesicle-host cell communication? Questions of the Study? 5
- 6. What is Lymph? What is Lymph Node? Lymph Node: Filtration or Detoxification? 6
- 7. Dissemination? mGluc⁺ Activity? Distant Tissues Mouse Bearing mGluc⁺ Melanoma mGluc⁺ Tumor Cells Membrane-bound Gaussia lucefrase 7
- 8. Fig. 1A. mGluc Luminescence activity (per µg tissue) Tumor (Primary) Cells Tumor-draining Lymph Node Non-draining Lymph Node Fig. S2B. Relative Luminescence Units per µl of lymph or plasma *=P<0.05 8
- 9. Dissemination Via dLNGFR⁺ CD63-eGFP ⁺ Host Cell Interaction? Mouse Bearing dLNGFR⁺ CD63-eGFP ⁺ Melanoma Lymph tdLN dLNGFR⁺ CD63-eGFP ⁺ Tumor Cells or Materials? Δ Low-affinity Nerve Growth Factor (Flow Cytometry analysis) (For Imaging Experiments) 9
- 10. Fig. 1B. dLNGFR⁺ cells in tdLN & ndLN cells Fig. 1C. dLNGFR⁺ cells in Lymphoid/Myeloid cell fractions Fig. 1D. dLNGFR⁺ cells in Macrophage subsets ****=P<0.0001 **=P<0.01 Fig. 1E. Multiphoton micrographs of explanted tdLN Fig. 1F. Outline of Lymph Collection, mGluc+ signal in Cell free Lymph and cells from Lymph ****=P<0.0001 tEVs disseminate Via Lymph Macrophages in Subcapsular Sinus of tumor draining lymph node were major host cell type interacting with tEVs. 10
- 11. UV Laster Photoconverted Cells in tumor stroma Photoconverted Cells in tumor stroma Non-photoconverted tdLN cells Photoconverted tdLN cells Cell trafficking? SCS Macrophages might originate from tumor area? -Track migration of tumor-associated host cells from tumor stroma to tdLN Time = 0 hour Time = 24 hours 11 Mouse Expressing Ubiquitous Photoconvertible Kaede Protein
- 12. Fig. S10B. Fraction of cells photoconverted in the tumor & tdLN Fig. S10C. photoconverted SCS Mø in tdLNs and TAMs after 24 hours Fig. S10D. Photoconversion in cell types in tdLNs after 24 hours Flow cytometry-based tracking of cell migration from tumor stroma to tdLN SCS macrophages did not originate from tumor stroma. SCS macrophages which are interacting with tEVs are resident macrophages 12
- 13. Melanin Pigment Staining Melanoma-free tdLNs [Melanin pigment staining (melanin pigment, dark brown)]n=13 Melanoma Patients Tumor depth <1 mm Tumor depth 1-2 mm Fig. S12. Melanin pigment staining & hemosiderin pigment staining (iron) of human melanoma-free (i.e. stage N0) sentinel LNs. Positive Melanin staining (brown) in macrophage-like cells (Macrophage-like populations) How about Homo sapiens? Cancer-free sentinel lymph node (CF-SLN) 13
- 14. Cancer-free sentinel lymph node (CF-SLN) mAb clone HMB-45 staining CF-SLNs (Melanoma Marker) n=13 Melanoma Patients Fig. 2A. Immunohistochemistry for the melanoma marker HMB-45 (red) in a tdLN from a melanoma-free (i.e., stage N0) patient. 14
- 15. Cancer-free sentinel lymph node (CF-SLN) mAb clone HMB-45 staining CF-SLNs (Melanoma Marker) n=13 Melanoma Patients Fig. 2C. HMB-45 immunohistochemistry (brown or red) in tdLNs from melanoma-free patients 15
- 16. mAb clone HMB-45 staining & CD 68 (Macrophage Marker) CF-SLNs (Melanoma Marker) n=13 Melanoma Patients Fig. 2B & D. Immunohistochemistry for HMB-45 melanoma and CD68 macrophage markers in sequential sections from a melanoma-free (i.e., stage N0) tdLN. Pie chart illustrating the fraction of patients containing 16 Cancer-free sentinel lymph node (CF-SLN)
- 17. Dissemination Via Irreversible YFP Expression in Cre Reporter Host Cell after Cre Horizontal Transfer? Cre-Reporter Mouse Bearing Cre⁺ Melanoma Lymph tdLN Cre⁺ Tumor Cells or Materials? 17 RNAs in tEVs can shape the fate of Macrophages?
- 18. Fig. S15D. qPCR quantification of Cre mRNA from either Cre+ B16F10 tumor cells or Cre+ B16F10- derived tEVs. (n = 3). Fig. S15E. CRE and HSP90 Western blot of tEVs purified from either control B16F10 or Cre+ B16F10 cells. Fig. 3A. Number of eYFP+ TAMs, SCS Mø and other cells on week 2 after challenging Cre-reporter mice with Cre+ B16F10 tumors. Fig. 3B. Multiphoton micrographs of LNs draining Cre+ tumors 18 tEVs do not modulate SCS MØ
- 19. Fig. S16. Multiphoton micrographs of tdLN WT mice (left), Cd169Dtr/Wt knockin mice (left), 2 days post DT injection Fig. 3C.Ttumor volume in ***P<0.001 Fig. S18A. Multiphoton micrographs of tdLN treated with PBS-Lip (right) or clodronate-Lip (left) and stained with anti- CD169 mAb (red) Fig. 3D. B16F10 tumor volume in WT mice treated with PBS-Lip or Clo-Lip Fig. 3E. Tumor volume in wild-type or Cd169Dtr/Wt mice, all Fig. 3F. Tumor volume in wild-type or Cd169Dtr/Wt mice, all **P<0.01 19 SCS Macrophage-tEV interactions regulate tumor progression?WT, SCS Mø preserved Cd169Dtr, SCS Mø ablated SCS MØ act as “tumor suppressors”.
- 20. Fig. S20B. dLNGFR+ tEVs by Rab35WT and Rab35S22N B16F10 tumor cells in vitro Fig. 3G. B16F10 tumor volume in wild-type or Cd169Dtr/Wt mice 20 tEV-SCS macrophage interactions regulate tumor progression? **=P<0.01 ***=P<0.001 ****=P<0.0001 Mouse Tumor Cells Sufficient tV production supports a causual Link between tEV-SCS macrophage interaction and tumor growth
- 21. Fig. 3H. multiphoton micrographs (2D projections) of tdLNs on d 3, 6 and 15 after B16F10 tumor challenge. Fig. 3H. Quantification of SCS Mø barrier disruption measured as CD169+ SCS macrophage number per field of view Fig. 3I. Multiphoton micrographs (2D projections) of inguinal LNs one week after starting i.p. Paclitaxel/Carboplatin injections, 3 times per week Fig. 3I. Quantification of SCS Mø barrier disruption measured as CD169+ SCS macrophage number per field of view 21 Whether tumor destroys SCS MØ network SCS macrophage barrier can be disrupted both during the natural course of tumor progression and upon chemotherapy.
- 22. Fig.4A. Cd169Dtr/Wt mice locally treated with DT s.c. on one side and challenged with B16F10 tumors on both flanks i.d Fig. S23A. Multiphoton imaging of LNs from mice injected with low doses (250 ng) of DT locally (left calf) Day 0: B16F10 i.d. both Flanks 22 tEV-SCS MØ interaction in tdLN but how this modulate distant tumor growth Day 2: DT i.d.
- 23. Fig. 4B. Multiphoton micrographs of tdLNs from CD63-eGFP+ B16F10 bearing mice (treated with PBS-Lip or Clo-Lip s.c.) and imaged at the indicated depth below the LN capsule 23 SCS macrophage act as “tEV gatekeepers”. Prevent spread of lymph-borne pathogens.
- 24. Fig. 4C. Distance between the indicated entities (CD169+ cells, CD63-eGFP+ tEVs and B220+ cells) and the LN capsule Fig. 4D. Quantification of dLNGFR+ lymphocyte subsets in tdLNs from dLNGFR+ B16F10 melanoma-bearing mice treated with PBS-Lip or Clo- Lip s.c. Fig. 4E. quantification of different cell types in B16F10 tumors from mice treated with PBS-Lip or Clo-Lip 24
- 25. Fig. 4F. B16F10 tumor volumes (d 9) in WT and Cd169Dtr/Wt mice treated with DT i.p. and/or with anti-CD20 depleting mAb Fig. 4G. B16F10 tumor volumes in WT recipient mice that received IgGs (25 μg) isolated from plasma of Cd169Dtr/Wt donor mice treated with PBS or DT i.p 25 Role of B-cells-tEV interaction SCS MØ suppress cancer progression by limiting pro-tumor Ig G responses.
- 26. Tumor Growth tEVs Lymph tdLN SCS MØ Cancer Progression, Cancer Treatment B cells IgGs Conclusion 26 SCS resident macrophages act as “flypaper and gatekeepers” against tumor dissemination by preventing tEVs-B-cell interaction.
- 27. Thank you very much For Your Patience! 27
Editor's Notes
- EVs were initially considered cellular debris or a way to excrete unneeded or toxic products from cells, but their ancient evolutionary origins and their conserved mechanisms of generation indicate that EVs perform essential physiological roles in cell-cell communication
- SCS MØ act as “tumor suppressors”.