Soluble VEGF receptor 1 (sFLT1) induces non-apoptotic death in ovarian and colorectal cancer cells

Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1/sFLT1) is an angiogenesis inhibitor that competes with angiogenic factors such as VEGF and Placental Growth Factor (PlGF). Imbalances of VEGF and sFLT1 levels can cause pathological conditions such as tumour growth or preeclampsia. We observed direct damage caused by sFLT1 in tumour cells. We exposed several kinds of cells derived from ovarian and colorectal cancers as well as HEK293T cells to sFLT1 in two ways, transfection and exogenous application. The cell morphology and an LDH assay revealed cytotoxicity. Additional experiments were performed to clarify how sFLT1 injured cells. In this study, non-apoptotic cell damage was found to be induced by sFLT1. Moreover, sFLT1 showed an anti-tumour effect in a mouse model of ovarian cancer. Our results suggest that sFLT1 has potential as a cancer therapeutic candidate.

3.0% H2O2 for 30 min. After blocking nonspecific reactivity with goat serum for 10 min at room temperature, the sections were incubated overnight at 4°C with a primary antibody against UK). The primary antibody was visualized using subsequent application of a secondary biotinylated antibody (Histofine SAB PO kit) and streptavidin-peroxidase (Histofine SAB PO kit). Immunostaining was developed using Envision DAB (Dako Ltd.) and the sections were counterstained with Mayer's haematoxylin. As a negative control, some sections were subjected to normal serum blocking with omission of the primary antibody. CD31: Tissues were frozen without fixation and sectioned at 100 μm thickness with a cryostat. Endogenous peroxidase was blocked by incubating the sections in 3.0% H2O2 for 30 min. After blocking nonspecific reactivity with rabbit serum for 10 min at room temperature, the sections were incubated overnight at room temperature in a rat anti-mouse CD31 monoclonal antibody (BD PharMingen, USA) diluted 1:200, followed by 30 min in biotin-labelled rabbit secondary anti-rat IgG antibody (Dako, USA) diluted 1:600. Immunostaining was developed using Envision DAB (Dako, USA), and the sections were counterstained with Mayer's haematoxylin.
As a negative control, some sections were subjected to normal serum blocking with omission of the primary antibody.

SI Figure Legend
Supplementary Figure S1 | Only excessive VEGF did not accelerate cell growth. To evaluate the effect of additional VEGF on cell proliferation, pLV-EGFP was transduced into HEK293T, SKOV3, HeyA8, and HT-29 cells, and at the same time as the transfection we added rVEGF into the culture media. The number of cells with or without additional rVEGF was not significantly different in HEK293T, SKOV3, HT-29, and HeyA8 cells. HeyA8 and SKOV3 cell lines, cell numbers were significantly lower in rVEGFR1 groups compared with control groups. *P<0.05 versus EGFP group. (c) Comparison of LDH leakage assay after transfection with pLV-EGFP or pLV-sFLT1. In MCF7 cells, the level of LDH release in pLV-sFLT1 transfected group was significantly higher than that of the pLV-EGFP transfected group. And in A549 cells, we also found a similar tendency. With addition of rVEGF to the sFLT1 groups, the levels of LDH release were restored to values near to those of the EGFP groups. Data are presented as percentage of control. *P<0.05 versus EGFP group. (d) Comparison of LDH leakage assay after treatment with rVEGFR1 or bevacizumab. In A549 cells, the level of LDH release was significantly higher. Furthermore, rVEGF restored the level of LDH release. *P<0.05 versus EGFP group. Quantification was determined by measuring the CD31-stained area with Image J software. CD31-positive, ring-like structures were considered as lumen. Scale bar, 100 µm. Data are shown as the means ± S.E.

Supplementary
Student's t test was performed. Statistically significant differences are indicated by asterisks: *, P < 0.05 significantly different from PBS-treated mice; **, P < 0.05 significantly different from SKOV3-EGFP mice.
Quantification was determined by counting five different fields per tumour, followed by averaging. Scale bar, 100 µm. Data are shown as the means ± S.E. Student's t test was performed. Statistically significant differences are indicated by asterisks: *, P < 0.05 significantly different from PBS treated mice; **, P < 0.05 significantly different from SKOV3-EGFP mice. (*5:P=9.1×10 -4 , *6:P=3.3×10 -6 , **7:P=2.2×10 -3 ) Supplementary Table S1 | The relationship between the concentration of sFLT1 in culture supernatants and the amounts of plasmid DNA. ELISAs. The relationship between the concentration of sFLT1 in culture supernatants and the amounts of plasmid DNA. Cells were seeded in 6-well plates at densities of 2×10 4 (HEK293T, HT-29 and HeyA8) and 4×10 4 (SKOV3) cells in 2mL of culture medium per well, respectively, and allowed to attach for 24 hours. Then, the cells were transfected with various concentrations of pLV-sFLT1. In HEK293T and HeyA8 cells, culture supernatants were collected 72 hours after transfection, and in SKOV3 and HT-29 cells, collected 96 hours after transfection. We determined the concentrations using commercial ELISA kits (R&D Systems) according to the manufacturer's specifications.

Supplementary Table S2 | The concentrations of sFLT1, VEGF and PlGF in supernatants of culture
dishes. ELISAs. The concentrations of sFLT1, VEGF and PlGF of supernatants of culture dishes revealed a correlation between lower concentration of VEGF and fewer cell numbers. We determined the concentrations using commercial ELISA kits (R&D Systems) according to the manufacturer's specifications. .

Supplementary Table S3 | Evaluation of side effects caused by sFLT1 in ovarian cancer model mice. (a)
Systolic and diastolic blood pressure were measured at five weeks after initial treatment (n = 4). In intraperitoneal treatment experiments, the mean blood pressure of neither the rVEGFR1-2000ng nor the rVEGR1-200ng groups was elevated compared to the control group. On the other hand, with injection of transfected cells, the mean blood pressure of the sFLT1 group tended to be higher than that of the EGFP group. (P=0.16) (b) Urine albumin and creatinine concentrations were analyzed at five weeks after initial treatment. Student's t test was performed against the PBS group or the SKOV3-pLV-EGFP group as control.
The ratios of urinary albumin/urinary creatinine in the rVEFGR1 group or SKOV3-pLV-sFLT1 group was similar to those in the control group. (pg/ml)