A pilot in vivo study: potential ovarian cancer therapeutic by placental extracellular vesicles

Abstract

The biological links between cancer and pregnancy are of interest due to parallel proliferative, immunosuppressive, and invasive mechanisms between tumour and placental cells. However, the proliferation and invasion of placental cells are strictly regulated. The understanding of this regulation is largely unknown. Placental extracellular vesicles (EVs) may play an important role in this regulation, as placental EVs are known to contribute to maternal adaptation, including adaptation of the vascular and immune systems. We have previously reported that placental EVs significantly inhibited ovarian cancer cell proliferation by delaying the progression of the cell cycle. We, therefore, performed this pilot in vivo study to investigate whether placental EVs can also inhibit ovarian tumour growth in a SKOV-3 human tumour xenograft model. A single intraperitoneal injection of placental EVs at 15 days post tumour implantation, significantly inhibited the growth of the tumours in our in vivo model. Signs of cellular necrosis were observed in the ovarian tumour tissues, but not in other organs collected from mice that had been treated with placental EVs. Expression of receptor-interacting kinase 1 (RIPK1) and mixed linkage kinase domain-like (MLKL), which are mediators of necroptosis were not observed in our xenografted tumours. However, extensive infiltration of CD169+ macrophages and NK cells in ovarian tumour tissues collected from placental micro-EVs treated mice were observed. We demonstrate here that inhibition of ovarian tumour growth in our xenograft model by placental EVs involves cellular necrosis and infiltration of CD169+ macrophages and NK cells into the tumour tissues.

Keywords: CD169+; necrosis; ovarian cancer; placental EVs; tumour regression; tumour xenograft model.

Figure 1. Growth chart of tumour

The size of tumours in CD-1 nude mice that had been treated with placental micro-EVs (red line) and nano-EVs (green line) was significantly reduced, compared to control (blue line).

Figure 2. Histology examination

Signs of necrosis were present in tumours (A–C), but not in liver (D–F) and spleen (G–I), collected from mice that had been treated with placental micro-EVs (A, D, G) and nano-EVs (B, E, I), compared with controls (C, F, I). Bar = 100 µm. The consistent finding was seen in tumour tissues collected from five untreated mice, and three micro-EVs treated, and five nano-EVs treated mice (Supplementary Figure S6).

Figure 3. Analysis of apoptosis associated proteins

No expression of RIPK1 (A–C) or MLKL (D–F) was present in tumours collected from mice that had been treated with placental micro-EVs (A,D) or nano-EVs (B,E), compared with untreated (C,F). Positive staining controls of spleen for the RIPK1 (G) and MLKL (H) antibodies. (I): antibody control. Bar = 100 µm. The consistent finding was seen in tumour tissues collected from five untreated mice, and three micro-EVs treated, and five nano-EVs treated mice

Figure 4. Immune response in tumour tissues

Significantly higher intensity of CD169 was present in tumours collected from mice that had been treated with placental micro-EVs (A), compared with mice that had been treated with placental nano-EVs (B) or controls (C), measured by semi-quantitative analysis (E). (D): antibody control. The consistent finding was seen in tumour tissues collected from four untreated mice, and three micro-EVs treated, and four nano-EVs treated mice (Supplementary Figure S7).

Figure 5. Immune response in tumour tissues

Significantly higher intensity of NKp46 was present in tumours collected from mice that had been treated with placental micro-EVs (A), compared with mice that had been treated with placental nano-EVs (B) or controls (C), measured by semi-quantitative analysis (E). (D): antibody control. The consistent finding was seen in tumour tissues collected from four untreated mice, and three micro-EVs treated, and four nano-EVs treated mice (Supplementary Figure S8).