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. 2017 Dec 8;7(1):17225.
doi: 10.1038/s41598-017-17613-6.

Stromal Versican Regulates Tumor Growth by Promoting Angiogenesis

Keiichi Asano  1   2 , Courtney M Nelson  3 , Sumeda Nandadasa  3 , Noriko Aramaki-Hattori  3 , Daniel J Lindner  4 , Tyler Alban  3 , Junko Inagaki  5 , Takashi Ohtsuki  2 , Toshitaka Oohashi  1 , Suneel S Apte  3 , Satoshi Hirohata  6
Affiliations

Stromal Versican Regulates Tumor Growth by Promoting Angiogenesis

Keiichi Asano et al. Sci Rep. .

Abstract

The proteoglycan versican is implicated in growth and metastases of several cancers. Here we investigated a potential contribution of stromal versican to tumor growth and angiogenesis. We initially determined versican expression by several cancer cell lines. Among these, MDA-MB231 and B16F10 had none to minimal expression in contrast to Lewis lung carcinoma (LLC). Notably, tumors arising from these cell lines had higher versican levels than the cell lines themselves suggesting a contribution from the host-derived tumor stroma. In LLC-derived tumors, both the tumor and stroma expressed versican at high levels. Thus, tumor stroma can make a significant contribution to tumor versican content. Versican localized preferentially to the vicinity of tumor vasculature and macrophages in the tumor. However, an ADAMTS protease-generated versican fragment uniquely localized to vascular endothelium. To specifically determine the impact of host/stroma-derived versican we therefore compared growth of tumors from B16F10 cells, which produced littleversican, in Vcan hdf/+ mice and wild-type littermates. Tumors in Vcan hdf/+ mice had reduced growth with a lower capillary density and accumulation of capillaries at the tumor periphery. These findings illustrate the variability of tumor cell line expression of versican, and demonstrate that versican is consistently contributed by the stromal tissue, where it contributes to tumor angiogenesis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Expression, distribution and origin of versican GAGβ in cancer cell lines and tumor tissues. (A) Protein production of versican GAGβ in conditioned medium (CM) obtained from MDA-MB231, B16F10 and Lewis lung carcinoma (LLC) cancer cell lines and cell lysates (CL) was determined by Western blot analysis with anti-GAGβ antibody. Protein extract from an E13.5 mouse embryo (indicated as Embryo) was used as a positive control. Anti-β-actin antibody was used to indicate protein loading for cell lysates. Arrows indicate the predicted size of versican. (B) Expression of mouse versican V0 and/or V1 (Vcan V0/V1) and human versican V1 (VCAN V1) mRNA in cancer cells (cell) and corresponding tumors (tumor) was compared by quantitative reverse transcription PCR (qPCR). Each expression was normalized to Gapdh (for mouse versican) and RPLP2 (for human versican) mRNA levels. Please note that VCAN V1 RT-PCR determined the expression of tumor-derived VCAN V1 (human) in the MDA-MB231 xenograft tumors since they contain human-derived tumor cells and mouse-derived host cells. n = 4 for B16F10; n = 3 for LLC; n = 3 for MDA-MB231. Statistical significance was evaluated by two-tailed unpaired t-test. (C) Versican GAGβ content in B16F10-, LLC- and MDA-MB231-derived tumors was determined by Western blotting using anti-versican GAGβ antibody. Protein extract from E13.5 mouse embryo was used as the positive control. Arrows indicate the predicted size of versican. Note extensive versican proteolysis in all blots. (D) Versican GAGβ immunostaining (green) in B16F10- (upper panel), LLC- (middle panel) and MDA-MB231- (lower panel) tumors. Nuclei were counterstained with Hoechst dye (blue). The white dashed lines indicate the edge of each tumor. C: center of the tumor; P: periphery of the tumor. Arrows indicate the positive signal (green) in the tumors. Scale bars = 100 μm (E) RNA in situ hybridization (red signal) of mouse Vcan exon 7 (mouse Vcan V0 and/or V2) and exon 8 (mouse Vcan V0 and/or V1) in B16F10- (upper panels) and LLC-tumors (lower panels). Please note that both large exons of versican (i.e., exon 7 (Vcan V0 and/or V2) and exon 8 (Vcan V0 and/or V1)) were expressed in the tumor periphery. C: center; P: periphery. Arrows indicate the positive signals in B16F10-tumor. (F) In situ hybridization using mouse Vcan exon 7 (mouse V0/V2) and exon 8 (mouse V0/V1) to identify mouse-derived versican mRNA and human VCAN exon 7 (human V0/V2) in the MDA-MB231 xenograft tumor. The panel at right shows hematoxylin and eosin stain. Arrows indicate the positive signals in MDA-MB231 tumor. Please note that the positive signals indicate the versican expressing cell-derived from host (mouse) cells. Scale bars = 50 μm for in situ hybridization and 100 μm for hematoxyline-eosin staining. H, host tissue; P: periphery; C: center.
Figure 2
Figure 2
Localization of versican GAGβ in the vicinity of MDA-MB231 tumor vasculature. Immunofluorescence staining using anti-versican GAGβ, anti-CD31, anti-CD105, anti-F4/80 antibodies and biotinylated-hyaluronan binding protein (HABP) to compare the distribution of versican GAGβ (versican V0 and/or V1, green) with that of endothelial cells (CD105, CD31), macrophages (F4/80) or hyaluronan (each red) in the MDA-MB231 tumor. The arrows indicate overlapping localization of versican GAGβ with the vasculature, macrophages or hyaluronan, respectively. Nuclei were stained with Hoechst dye (blue). The white dashed lines indicate the edge of each tumor. Asterisks indicate the vessel-like structures. Scale bars = 100 μm.
Figure 3
Figure 3
ADAMTS-cleaved versican in the MDA-MB231 tumor vasculature. (A) Western blot analysis with anti-DPEAAE antibody for ADAMTS-cleaved versican in the MDA-MB231 tumor extract. Protein extract from an E13.5 mouse embryo (Embryo) was used as the positive control. Arrows indicate the predicted size of cleaved versican (220 kDa presumably arising from V0, 70 kDa from V1). (B) Immunostaining using anti-DPEAAE, anti-CD31, anti-CD105 anti-F4/80 antibodies and biotinylated hyaluronan-binding protein (HABP) compared localization of cleaved versican with that of vasculature, macrophages or hyaluronan. Arrows indicate some overlap of cleaved versican in endothelium and macrophages and show dissociation of cleaved versican from hyaluronan distribution. (C) Z-stack confocal imaging comparing the versican GAGβ (intact versican) with cleaved versican in the vascular endothelium. Cleaved versican overlapped with endothelium stain while the intact versican did not (arrows). Asterisks indicate capillaries. Nuclei were stained with Hoechst dye (blue). Scale bars = 100 μm in A, B; 10 μm in C.
Figure 4
Figure 4
Versican and versikine localization in the B16F10 and LLC tumor vasculature. (A) Immunofluorescence staining using anti-versican GAGβ or anti-DPEAAE (green), anti-CD31 (red), or Hoechst nuclear dye (blue) as indicated to compare the distribution of versican GAGβ (versican V0 and/or V1) or versikine (anti-DPEAAE) with that of endothelial cells (CD31). Boxed areas in gray-scale images are shown at higher magnification and in color. The arrows indicate localization of versican GAGβ or versikine in the vasculature. The vascular lumen is indicated by the asterisk. Scale bars = 100 μm.
Figure 5
Figure 5
Impaired B16F10 tumor growth and angiogenesis in Vcan hdf/+ mice. (A) Western blotting of tumors obtained from wild-type (WT) and Vcan hdf/+ mice consistently showing reduced versican (using anti-GAGβ) in the latter. Originally, the Western was performed on a single membrane and the extra lane in the middle was excised. (B) Comparison of tumor growth rate in wild-type and Vcan hdf/+ mice. Asterisks indicate the times after injection when tumor volume was statistically significant. n = 15 for each experiment. (C) Dot plots reporting tumor volumes in wild-type and Vcan hdf/+ mice. (D) Immunostaining of the tumor interior with anti-endomucin antibody (red) to identify capillaries (arrows). Sections were counterstained with hematoxylin. (E) Vessel counts obtained from wild-type and Vcan hdf/+ mice. n = 10 for each experiment. (F) Immunostaining of tumor vasculature using anti-endomucin (red). Nuclei were stained blue using Hoechst dye. White dashed lines Vcan hdf/+ timors indicates an abrupt boundary restricting most vasculature to the periphery (P); C, center.
Figure 6
Figure 6
Proposed models for host-derived versican contribution for tumor development. (A) Cartoon showing impaired vascular invasion in tumors growing in Vcan hdf/+ mice and accumulation of vasculature at the tumor periphery in Vcan hdf/+ mice. (B) A schematic outlining the suggested role of versican in the tumor stroma and relocation of cleaved versican (versikine) to the endothelial cells.
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