Wild-type C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). APJ-KO mice were gifts from Prof. Fukamizu [16]. All mice were used between 8 and 11 weeks of age. Animals were housed in environmentally-controlled rooms in an animal experimentation facility at Osaka University. All mouse experiments were performed under the guidelines of the Osaka University Committee for Animal and Recombinant DNA Experiments (Approval number 4784).

B16/BL6, a murine melanoma cell line, was purchased from the Riken Cell Bank (Tsukuba, Japan) and maintained in DMEM (Sigma–Aldrich, St. Louis, MO, United States) supplemented with 10% fetal bovine serum (FBS, Sigma–Aldrich) and 1% penicillin/streptomycin (Life Technologies, Tokyo, Japan). B16/BL6 cells were starved and then stimulated with LY364947 (10 μM; Selleck Biotech, Huston, TX, United States) or dimethyl sulfoxide (Nacalai Tesque, Inc., Kyoto, Japan) for 24 h. Methods for the generation of apelin-overexpressing cancer cells were previously described [12].

To establish APJ-KO cells using a CRISPR/Cas9 genome editing system, single-guide RNAs were designed using a CRISPRdirect Web application (https://crispr.dbcls.jp) for the mAPLNR first exon using pSpCas9(BB)-2A-Puro (PX459) V2.0 (plasmid number 62988; Addgene, Watertown, MA, United States). The two-pair gene-specific single-guide RNA sequences were 5′-CAC CGA GGC CAG TCA AAC TCC CGG T - 3′(sense), 5′- AAA CAC CGG GAG TTT GAC TGG CCT C -3′ (antisense), 5′-CAC CGA GGT GCA GGC TTG GCG AAA T′ (sense), and 5′- AAA CAT TTC GCC AAG CCT GCA CCT C-3′ (antisense). The oligos were ligated into a BbsI site of PX459. The constructed targeting vector was transfected into a B16/BL6 cell line. Clones were selected, and elimination of APJ expression at mRNA and protein levels was detected through real-time quantitative PCR (RT–qPCR) and western blot analysis, respectively (as described below).

B16/BL6 cells (1 × 10⁷ cells/mL per mouse in 150 µL PBS) were inoculated subcutaneously into WT C57BL/6 or APJ-KO mice (8–11 weeks of age). Tumors were dissected 14 days after implantation. Tumor volumes were measured with calipers every 5–7 days and calculated as follows: width × width × length × 0.5.

In APJ western blot analysis, cell extracts were prepared with GPCR extraction and stabilization reagent (Thermo Fisher Scientific, Waltham, MA, United States) for APJ or with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) according to the manufacturer’s protocol. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and blocked with 5% skimmed milk in 0.1% Tris-buffered saline and Tween 20. Membranes were subsequently incubated with antibodies against APJ (5H5L9; Thermo Fisher Scientific), p-Smad2, Smad2, p-Smad3, Smad3 (Cell Signaling Technology, Danvers, MA, United States), or glyceraldehyde-3-phosphate dehydrogenase (Millipore, Temecula, CA, United States), and then developed with horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, United States). Antibody binding was visualized using electrochemiluminescence reagents (GE Healthcare, Milwaukee, WI, United States).

Cell migration was evaluated using a transwell assay. For the analysis of cell migration, cells were suspended in DMEM at a density of 1 × 10⁶ cells/mL and added to the upper chamber (200 μL/chamber); 750 μL of medium (DMEM containing 10% FBS) was added to the lower chamber. After 14 h, cells on the top membrane surface of the upper chamber were removed using a cotton swab, and the infiltrating cells on the bottom surface were fixed in 4% paraformaldehyde for 1 min, stained with hematoxylin and eosin, and visualized and counted using a microscope.

Each well of a 96-well plate was coated with 60 μL of growth factor–reduced UltiMatrix (R&D Systems, Inc., Minneapolis, MN, United States) for 30–60 min at 37°C. Melanoma cells were seeded onto the gel surface at a density of 4.0 × 10⁴ cells per well in a volume of 200 μL medium. Network formation was analyzed after a 15-h incubation. Images were analyzed with ImageJ and Angiogenesis analyzer software [17].

Tumor sections were rehydrated in PBS and blocked with normal goat serum in PBS for 20 min at room temperature. Primary antibodies used were: rabbit anti-APJ (5H5L9; Thermo Fisher Scientific; 1:200), hamster anti-CD31 (Millipore; 1:200), and rat anti-GFP (BioLegend, San Diego, CA, United States; 1:200) with overnight incubation. Following three washes with PBS, sections were incubated with Alexa Fluor-conjugated secondary antibody (Thermo Fisher Scientific) and diluted 1:200 in blocking solution for 60 min. After three washing steps with PBS, sections were mounted using Vectashield (Vector Laboratories, Burlingame, CA, United States) under coverslips. Stained sections were examined using confocal microscopy (TCS/SP8 or TCS/SP5; Leica Microsystems, Wetzlar, Germany).

To quantify the length and density of blood vessels, CD31-positive immunofluorescence images were analyzed with the computational analysis tool, Angiotool (version 0.6; National Institutes of Health, National Cancer Institute, Bethesda, MD, United States) [18]. It performs the analysis of vascular morphometric parameters like vessel length and density. Tumor sections were scanned under low-power magnification (×200) to identify areas of highest CD31-positive vessel density. All images shown are representative of more than five independent experiments.

Briefly, total RNA was extracted from cells using RNeasy-plus mini kits (Qiagen, Hilden, Germany) and reverse-transcribed using a PrimeScript RT reagent kit (Takara, Shiga, Japan) according to the manufacturer’s protocol. Quantitative real-time PCR analysis was performed with TB Green Premix Ex Taq II (Takara) using a LightCycler 96 System (Roche Diagnostics GmbH, Penzberg, Germany). Primers are described in Supplementary Table S1. Results were normalized to GAPDH using a comparative threshold cycle method.

We examined melanoma cells for the expression of APJ protein. We found that B16/BL6 (B16) melanoma cells strongly expressed this compared with murine ECs (MS-1), which were previously reported to express APJ (Figure 1A). As observed in cultured cells, immunohistochemical staining in tumor tissues of C57/BL6 wild-type mice subcutaneously inoculated with B16 cells overexpressing green fluorescent protein (GFP) revealed that B16 melanoma cells (GFP positive) expressed APJ protein (Figure 1B); APJ expression was equivalently observed in tumor ECs.

To analyze the effect of apelin on APJ in cancer cells, we overexpressed apelin in B16 cells (B16/apelin; Figure 2A; p < 0.01). When cell proliferation was compared in mock-transfected B16 cells and those transfected with apelin, we found that apelin significantly promoted the proliferation of apelin-overexpressing B16 cancer cells (Figure 2B; p < 0.05). Next, to evaluate whether or not cell proliferation by apelin reflects in vivo tumor growth, we transplanted cancer cells into APJ-KO mice to eliminate the effect of apelin on cells other than cancer cells, especially ECs. We subcutaneously inoculated B16/apelin or mock-transfected control cells into APJ-KO mice and found that B16/apelin mice showed significantly higher tumor growth than B16/mock-transfected control mice (Figures 2C, D; p < 0.05). In this tumor model, the density of blood vessels in B16/apelin tumors did not significantly differ from that in B16/mock-transfected control tumors (Figure 2E). Taken together, these data suggested that APJ deficiency in the tumor host microenvironment does not affect tumor angiogenesis when B16 cells were used as tumor models. An excess amount of apelin induced the growth of B16 melanoma cells by affecting APJ in tumor cells.

We found that overexpression of apelin in B16 melanoma cells induced the growth of tumors. Therefore, we next observed whether or not a lack of APJ in B16 cells would affect tumor growth. To assess this, we knocked out the APJ gene in B16 cells by a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) method and established two sublines (B16/APJ-KO#1, 2). The mRNA (Figure 3A) and protein (Figure 3B) levels of APJ in cell lines were analyzed. When cells were cultured, we found no significant difference in cell growth between wild-type (WT) and APJ-KO B16 cell lines (Figure 3C).

Next, these cancer cell lines were subcutaneously inoculated into C57/BL6 WT mice and the growth of tumors was evaluated. All three tumors (B16/WT, B16/APJ-KO#1, 2) showed similar tumor growth. We calculated the tumor volume on day 14 after the inoculation of tumor cells. Unexpectedly, we found that the elimination of APJ from B16 cells did not affect the growth of tumors (Figure 3D). Tumor vascular development is a major factor in regulating tumor growth in the tumor microenvironment and therefore we next evaluated the vascular density of each tumor. Initially, we suspected that APJ deficiency induced tumor angiogenesis and compensated tumor proliferation that should be observed in the presence of an apelin/APJ system on B16 cells. However, unexpectedly, tumors generated by B16/WT cells showed a higher vascular density than tumors by B16/APJ-KO cells (Figures 3E, F; p < 0.01). This suggested that progression of tumor growth by B16/APJ-KO is induced by factors other than angiogenesis factors.

In order to maintain the same volume between B16/WT and B16/APJ-KO tumors, we postulated that blood supply other than through tumor vessels is required. Therefore, we speculated that vascular mimicry (VM) may be induced in B16/APJ-KO tumors and indeed we detected abundant periodic acid Schiff (PAS)-positive, but CD31-negative, vascular-like structures in tumors generated by B16/APJ-KO cells compared to those of B16/WT cells (Figures 4A, B).

It is widely accepted that a change in cell shape is involved in vascular mimicry by tumor cells [19]. We found that APJ-KO B16 cells showed a more spindle-like shape compared to WT cells (Figure 4C). Several studies suggested that VM by B16 cells requires cancer cell migration. We therefore compared the migration of B16/WT and B16/APJ-KO cell lines using transwell assays. We found that B16/APJ-KO (#1 and 2) cells showed more transmigration than B16/WT cells (Figures 4D, E). Moreover, an in vitro tube formation assay that mimics vascular formation indicated that the lack of APJ more effectively generated tube-like structures in B16 cells (Figures 4F, G).

Thus far, we found that a lack of APJ in B16 cells meant that these showed a spindle-like phenotype with high migrating potency. We hypothesized that this phenotypic change may be induced by mechanisms involved in epithelial–mesenchymal Transition (EMT). Signaling affected by transforming growth factor (TGF)-β is a key factor for EMT. Therefore, we examined the expression of TGF-β and transcription factors associated with EMT. We found that TGF-β mRNA was expressed in B16/APJ-KO cells at a higher level than in B16/WT cells (Figure 5A). Correlating with this expression, expression of the mesenchymal maker, Zeb1, was also higher in APJ-KO melanoma and B16/apelin cells than in B16/WT cells (Figure 5B). By contrast, when smooth muscle actin (SMA) mRNA expression was observed, a slightly higher level was detected in B16/apelin compared to B16/mock-transfected cells; however, an increase in SMA mRNA was not detected in B16/APJ-KO cells. Since the expression of another EMT-related molecule was not altered in B16/apelin or B16/APJ-KO cells, as far as we could determine, this phenotypic change by APJ or apelin may have been induced by partial EMT. Recently, it has been reported that partial EMT status prior to the acquisition of full mesenchymal phenotype provides maximal stemness, tumor initiation capacity, and the ability to adapt to environmental changes [20].

Next, we investigated the relationship of TGF-β/Smad signaling with Zeb1 expression. At first, phosphorylated Smad2 and Smad3 (pSmad2 and pSmad3) proteins were measured by western blotting in B16 cells. However, we found that the ratio of pSmad2/Smad2 and pSmad3/Smad3 proteins were not altered by the absence of APJ (Figures 5C, D). Next, we further observed the relationship between Zeb1 and TGF-β/Smad signaling by using an inhibitor of TGF-β/Smad signaling pathways. We showed that an inhibitor of the TGF-β1 receptor (LY364947) affected B16/WT and B16/APJ-KO cells since the mRNA level of plasminogen activator inhibitor-1, which is an end-product of the Smad signaling pathway, was inhibited. In this setting, however, Zeb1 expression was not suppressed by LY364947 in the B16/APJ-KO group (Figure 5E). Therefore, we concluded that APJ negatively regulated Zeb1 expression in B16 cells and therefore attenuation of APJ in B16 cells promoted Zeb1 expression to induce EMT; however, this APJ signaling is independent of TGF-β/Smad signaling pathways.