American Association for Cancer Research
00085472can142464-sup-136677_1_supp_2870761_nk2t8l.pdf (497.43 kB)

Supplementary figures 1-7 from Full-Length Semaphorin-3C Is an Inhibitor of Tumor Lymphangiogenesis and Metastasis

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journal contribution
posted on 2023-03-30, 23:04 authored by Yelena Mumblat, Ofra Kessler, Neta Ilan, Gera Neufeld

Supplementary figures 1-7. Contains the 7 supplementary figures (1S to 7S) in PDF format. Fig. 1S: Characterization of the expression of class-3 semaphorin receptors in LEC Fig. 2S: Semaphorins that fail to contract LEC nevertheless induce contraction of other cell types. Fig. 3S: Measurement of cell contraction using the xCELLigence dual plate (RTCA) machine and FR-sema3C induced changes in in thentent of M2 macrophages in the tumors. Fig. 4S: Inhibition of BrdU incorporation in lEC by FR-sema3C. Fig. 5S: The effect of FR-sema3C on the Distribution of intrtumoral and peritumoral lymphatics Fig. 6S: The effect of tumor size on the development of lymph node metastases. Fig. 7: P65-Sema3C promotes the survival of A549 lung cancer cells.



Semaphorins play important regulatory roles in diverse processes such as axon guidance, angiogenesis, and immune responses. We find that semaphorin-3C (sema3C) induces the collapse of the cytoskeleton of lymphatic endothelial cells (LEC) in a neuropilin-2-, plexin-D1-, and plexin-A1–dependent manner, while most other semaphorins, including antiangiogenic semaphorins such as sema3A do not. Sema3C is cleaved, like other class-3 semaphorins, by furin-like pro-protein convertases (FPPC). Cleaved sema3C (p65-Sema3C) was unable to induce the collapse of the cytoskeleton of LEC. FPPC are strongly upregulated in tumor cells. In order to examine the effects of full-length sema3C on tumor progression, we therefore generated an active point mutated furin cleavage-resistant sema3C (FR-sema3C). FR-sema3C inhibited potently proliferation of LEC and to a lesser extent proliferation of human umbilical vein–derived endothelial cells. FR-sema3C also inhibited VEGF-C–induced phosphorylation of VEGFR-3, ERK1/2, and AKT. Expression of recombinant FR-sema3C in metastatic, triple-negative LM2-4 breast cancer cells did not affect their migration or proliferation in vitro. However, tumors derived from FR-sema3C–expressing LM2-4 cells implanted in mammary fat pads developed at a slower rate, contained a lower concentration of blood vessels and lymph vessels, and metastasized much less effectively to lymph nodes. Interestingly, p65-Sema3C, but not FR-sema3C, rendered A549 lung cancer cells resistant to serum deprivation, suggesting that previously reported protumorigenic activities of sema3C may be due to p65-Sema3C produced by tumor cells. Our observations suggest that FR-sema3C may be further developed into a novel antitumorigenic drug. Cancer Res; 75(11); 2177–86. ©2015 AACR.

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