Myeloid cells contribute to tumor lymphangiogenesis - PubMed (original) (raw)
Myeloid cells contribute to tumor lymphangiogenesis
Adrian Zumsteg et al. PLoS One. 2009.
Abstract
The formation of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) promotes tumor outgrowth and metastasis. Previously, it has been demonstrated that bone marrow-derived cells (BMDC) can contribute to tumor angiogenesis. However, the role of BMDC in lymphangiogenesis has largely remained elusive. Here, we demonstrate by bone marrow transplantation/reconstitution and genetic lineage-tracing experiments that BMDC integrate into tumor-associated lymphatic vessels in the Rip1Tag2 mouse model of insulinoma and in the TRAMP-C1 prostate cancer transplantation model, and that the integrated BMDC originate from the myelomonocytic lineage. Conversely, pharmacological depletion of tumor-associated macrophages reduces lymphangiogenesis. No cell fusion events are detected by genetic tracing experiments. Rather, the phenotypical conversion of myeloid cells into lymphatic endothelial cells and their integration into lymphatic structures is recapitulated in two in vitro tube formation assays and is dependent on fibroblast growth factor-mediated signaling. Together, the results reveal that myeloid cells can contribute to tumor-associated lymphatic vessels, thus extending the findings on the previously reported role of hematopoietic cells in lymphatic vessel formation.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Bone marrow transplantation strategies.
(A) For total bone marrow transplantations, 5×106 T cell-depleted total bone marrow cells from donor mice were injected i.v. into lethally irradiated (2×550 cGy) mice, as indicated. Semi-lethally irradiated (450 cGy) mice were injected with FACS-sorted 4×105 CD11b+ myeloid cells, 4×105 CD19b+ B cells or 4×104 common myeloid progenitors (CMP) cells. 4×105 CD11b+ myeloid cells were also transferred into non-irradiated mice. After 3 weeks mice were sacrificed, engraftment of transplanted bone marrow was evaluated by FACS and pancreata were analyzed by histology for the presence of bone marrow-derived cells at the tumor site. (B) Schematic illustration of syngeneic TRAMP-C1 tumor experiments. 5×105 TRAMP-C1 cells were injected into the flank of either C57BL/6 previously reconstituted with bone marrow of beta-actin-GFP transgenic mice or bone marrow of double-transgenic CD11b-Cre;Z/EG mice, and tumors were allowed to grow for 3 to 4 weeks. FACS analysis was used to assess bone marrow reconstitution or Cre recombinase-mediated GFP expression, respectively. Histological sections from TRAMP-C1 tumors were analyzed by immunofluorescence for the presence of GFP+ cells. (C–E) Flow cytometry-based strategy for cell sorting. (C) Within a scatter gate excluding lymphocytes, CD11bhigh/GFPhigh cells were isolated by FACS. (D) CD19+ was used as marker for the isolation of B lymphocytes. (E) CMP cells were sorted as lin−/Sca-1−/IL7Rα−/cKit+ as described in Methods.
Figure 2. BMDC integrate into tumor-associated lymphatic vessels.
(A) Lethally irradiated RT2;VC mice (5 mice) were reconstituted with GFP-labeled bone marrow. 20 µm histological pancreatic sections were stained for the lymphatic markers Podoplanin, Prox-1, LYVE-1 and for GFP as indicated and analyzed by confocal microscopy and subsequent 3D reconstitution. Representative tumor sections per lymphatic marker are shown. 3% of Podoplanin+ TLEC (7 Podoplanin+/GFP+ cells out of 227 Podoplanin+ cells) as well as 3.5% of Prox-1+ or LYVE-1+ TLEC (14 Prox-1+/GFP+ cells out of 400 Prox-1+ cells and 17 LYVE-1+/GFP+ cells out of 485 LYVE-1+ cells) are bone marrow-derived. TRAMP-C1 tumors were subcutaneously implanted in C57BL/6 mice (4 mice) previously reconstituted with GFP-labeled bone marrow. 7–20 µm histological tumor sections were stained as described above. 4.1% of Podoplanin+ TLEC (14 Podoplanin+/GFP+ cells out of 334 Podoplanin+ cells) as well as about 2.8% of LYVE-1+ TLEC (11 LYVE-1+/GFP+ cells out of 395 LYVE-1+ cells) are bone marrow-derived. Arrows indicate double-positive cells and arrowheads indicate double-positive cells shown in inset magnifications. Insets show merged and individual channels. DAPI stains nuclei (blue). Scale bars: 40 µm. (B) Tumors of GFP-labeled bone marrow-transplanted RT2;VC mice or TRAMP-C1 tumors grown in GFP-labeled bone marrow-transplanted C57BL/6 mice were enzymatically digested (3 mice each). Single cell suspensions were stained for the pan-endothelial marker CD31 and the lymphatic endothelial marker Podoplanin and analyzed by FACS (left panels). 9.4+/−4.1% (RT2;VC) and 10+/−4.6% (TRAMP-C1) of CD31+/Podoplanin+ TLEC were GFP+, indicating their bone marrow origin (middle left panels). As control, the anti-Podoplanin antibody was omitted resulting in no separation between TLEC and TBEC (middle right panels). Furthermore, similar analysis of tumors grown in non-transplanted mice showed no GFP+ cells within TLECs (right panels).
Figure 3. BMDC integrated into tumor lymphatics express vascular endothelial cadherin.
A representative tumor section from RT2;VC mice previously reconstituted with GFP-labeled bone marrow was stained for the lymphatic marker LYVE-1 (purple), for the cell junction molecule VE-cadherin (red), and for GFP (green) and analyzed by confocal microscopy and subsequent 3D reconstitution. As indicated by arrowheads and shown magnified in insets, VE-cadherin expression, indicative for homophilic cell-cell contact, is observed between bone marrow-derived (BMDTLEC) and host-derived TLEC (HTLEC). Note the continuous VE-cadherin staining between lymphatic endothelial cells in contrast to the cell-cell contact restricted staining of blood endothelial cells depicted by arrows. DAPI was used for nuclear counterstaining (blue). The DAPI-staining cells within the lymphatic vessel represent a cluster of disseminated tumor cells. BV: blood vessel. Scale bars: 20 µm.
Figure 4. Myeloid origin of bone marrow-derived TLEC.
(A) 20 µm histological pancreatic sections of GFP-labeled bone marrow-transplanted RT2;VC mice were stained for the lymphatic marker LYVE-1 (purple), for the macrophage marker F4/80 (red), and for GFP (green) as indicated and analyzed by confocal microscopy and subsequent 3D reconstitution. Two representative tumor sections are shown. Left panel: BMDC that have integrated into tumor lymphatics express the macrophage marker F4/80. Arrows indicate triple positive cells and the arrowhead indicate a triple-positive cell shown in inset magnifications. Right panel: not all integrated cells express F4/80. The arrowhead indicates an integrated cell (LYVE-1+/GFP+) that does not express F4/80. Inset magnifications of this double-positive cell are shown. Insets show merged and individual channels. (B) 20 µm histological pancreatic sections of RT2;VC mice transplanted with bone marrow isolated from either CX3CR1+/GFP (2 mice) or CD11bCre;Z/EG mice (3 mice), or adoptively transferred with FACS-sorted CD11b+ cells were stained for the lymphatic markers LYVE-1, Podoplanin or Prox-1 (red) as well as for GFP (green) and analyzed by confocal microscopy. Representative tumor sections are shown. In both transplantation settings, myeloid cells were found integrated into the lymphatic vasculature surrounding the tumors. Arrowheads indicate double-positive cells shown in inset magnifications (first inset: merged channels, second and third insets: red and green channel, respectively). DAPI stains nuclei (blue). Scale bars: 20 µm. (C) CD11b+ lineage tracing experiments demonstrate the myeloid origin of TLEC in a transplantation-independent setting. TRAMP-C1 tumors were grown subcutaneously in CD11b-Cre;Z/EG mice (3 mice). In these mice, cells that have passed through a CD11b+ myeloid lineage express GFP. Fluorescent triple staining of histological tumor sections reveal co-expression of LYVE-1 and Prox-1 by myeloid-derived GFP+ cells integrated into lymphatic vessels (arrowhead). The right panel represents a magnification of the relevant region with individual channels combined. Note that GFP+ cells not connected to vascular structures do express neither LYVE-1 nor Prox-1 (arrow). DAPI stains nuclei (blue). Scale bars: 20 µm.
Figure 5. Depletion of macrophages reduces peritumoral lymphatic vessel density.
(A) RT2;VC mice were treated with liposome-encapsulated Clodronate (ClodroLip). Pancreatic sections representing a total of 5 PBS vehicle control-treated mice (97 tumors) and 6 ClodroLip-treated (132 tumors) mice were analyzed. Successful depletion of intra- and extra-tumoral macrophages in ClodroLip-treated mice is illustrated by the reduction of F4/80 immunoreactivity (red). Co-staining with the lymphatic endothelial marker LYVE-1 (green) reveals a reduced coverage of tumors by lymphatic vessels in ClodroLip-treated mice vs. in PBS-treated mice. DAPI was used for nuclear counterstaining (blue). T: tumor. Scale bar: 50 µm. (B) Tumors of ClodroLip and control-treated mice were analyzed by immunofluorescence staining with antibodies against LYVE-1 for the extent of lymphatic vasculature surrounding the perimeter of the tumors. Tumors of control-treated mice were surrounded by 90% or more with lymphatic vessels (median 90%, mean 74.9%), whereas tumors of ClodroLip-treated mice had significantly lower coverage (median 70%, mean 61.1%; P<0.01, Mann-Whitney test). (C) Tumor-associated CD11b+ macrophages (TAM) and tumor cells were isolated from tumors of RT2;VC mice by flow cytometry, and mRNA levels for murine VEGF-C, VEGF-D, FGF-1 and FGF-2 were determined using quantitative RT-PCR and compared to levels in total tumors. Shown is the result of three independent cell isolations. ΔCT values have been calculated and normalized to internal control (RPL19) CT value. The results are displayed as mRNA copies per 1000 copies of control RPL19 mRNA to visualize the relative levels of mRNA of interest to internal RPL19 control mRNA.
Figure 6. Bone marrow-derived-macrophages form and contribute to lymphatic-like structures in vitro.
(A) In vitro generated macrophages showed a specific marker expression profile (CD11b+/F4/80+) (upper left panel). Tube formation on Matrigel was monitored by phase-contrast microscopy. At day 2, macrophages formed clusters. Between days 3 and 15, they developed into cord-like structures with numbers of branches increasing over time. Scale bar: 100 µm. (B) Immunofluorescence staining against Podoplanin (Pdpn) revealed that macrophages having formed cord-like structures express Podoplanin whereas single cells do not. Staining of the tubular structures in the absence of any primary antibody was used as a control (2°Ab). DAPI stains nuclei (blue). Scale bar: 100 µm. Quantitative RT-PCR analysis revealed that upon tube formation, macrophages up-regulate lymphatic markers (LYVE-1, Prox-1, VEGFR-3, Foxc2, Foxc1) and down-regulate hematopoietic/myeloid marker (CD45, CX3CR1). ΔΔCT corresponds to the difference between the normalized CT values of macrophages forming tubes (day 8) and macrophages not having yet formed tubes (day 1). (C) FGF-2 is required for the formation of cord-like structures by macrophages, as its specific exclusion from culture medium abrogated this process (left panel). Furthermore, analysis of mRNA levels revealed up-regulation of FGF receptors -1 and 2 during cord formation. (D) Immortalized Podoplanin+ murine lymphatic endothelial cells (SV-LEC) (i), GFP-labeled bone marrow-derived-macrophages (ii), and mixed cultures of macrophages and SV-LEC (iii-vi) were seeded in Matrigel. At day 5, cells were stained for Podoplanin (red) and analyzed by confocal microscopy. Mixed cultures demonstrate that bone marrow-derived macrophages contribute to SV-LEC-mediated cord formation: GFP+ cells (green) are found integrated into Podoplanin+ cord-like structures (iii–vi). Note the preferential integration of bone marrow-derived macrophages at the tips and branch points of sprouting cord-like structures formed by SV-LEC (magnified in panel vi). DAPI stains nuclei (blue). Scale bars: 100 µm (i–ii) and 50 µm (iii–vi).
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