Novel strategy of ovarian cancer implantation: Pre-invasive growth of fibrin-anchored cells with neovascularization

Abstract

Although direct adhesion of cancer cells to the mesothelial cell layer is considered to be a key step for peritoneal invasion of ovarian cancer cell masses (OCM), we recently identified a different strategy for the peritoneal invasion of OCM. In 6 out of 20 cases of ovarian carcinoma, extraperitoneal growth of the OCM was observed along with the neovascularization of feeding vessels, which connect the intraperitoneal host stroma and extraperitoneal lesions through the intact mesothelial cell layer. As an early step, the OCMs anchor in the extraperitoneal fibrin networks and then induce the migration of CD34-positive and vascular endothelial growth factor A (VEGF-A)-positive endothelial cells, constructing extraperitoneal vascular networks around the OCM. During the extraperitoneal growth of OCM, podoplanin-positive and α smooth muscle actin (αSMA)-positive cancer-associated fibroblasts (CAF) appears. In more advanced lesions, the boundary line of mesothelial cells disappears around the insertion areas of feeding vessels and then extraperitoneal and intraperitoneal stroma are integrated, enabling the OCM to invade the host stroma, being associated with CAF. In addition, tissue factors (TF) are strongly detected around these peritoneal implantation sites and their levels in ascites were higher than that in blood. These findings demonstrate the presence of neovascularization around fibrin net-anchored OCMs on the outer side of the intact peritoneal surface, suggesting a novel strategy for peritoneal invasion of ovarian cancer and TF-targeted intraperitoneal anti-cancer treatment. We observed and propose a novel strategy for peritoneal implantation of ovarian cancer. The strategy includes the preinvasive growth of fibrin-anchored cancer cells along with neovascularization on the outer side of the intact peritoneal surface.

Keywords: extraperitoneal neovascularization; mesothelial barrier; ovarian cancer cell mass; peritoneal invasion; tissue factor.

Figure 1

Ovarian cancer cell masses (OCMs) on the outer side of the intact peritoneal surface in patients with high‐grade serous carcinoma (cases #1 and 2). Staining of hematoxylin and eosin (A‐D), podoplanin (E, F), fibrinogen (G), transfer factor (TF) (H), CD34 (I, J), vascular endothelial growth factor A (VEGF‐A) (K, L), CD31 (M, N), and E‐cadherin (O). Case #2 (A, B), case #1 (C‐O). A, B, Extraperitoneal OCMs (black arrows) received straight or tortuous feeding vessels (white arrows) from the noninvaded host stroma that was lined by intact mesothelial cells (black arrowheads). White arrowheads in (A) and (B) show fibrous structures around OCMs. C‐O, numerous OCM (black arrows) were enveloped by the thin fibrous structures without destroying or attaching to intact peritoneal epithelium (black arrowheads). C‐F, migration of fibroblasts was observed along the fibrin‐net structures (white arrowheads). Podoplanin was highly expressed on intact mesothelial cells (black arrowheads). G, Adjacent sites of (C, E) immunoreactive fibrinogen were detected on the thin fibrous tissues (white arrowheads). H, TF was detected on these fibrous tissues. I‐L, CD34 and VEGF‐A expression was detected on the migrating cells (white arrowheads) alongside the fibrin‐net structures. M, N, Adjacent sites of (G), fine vascular networks (white arrows) containing red blood cells were constructed by CD31‐positive endothelial cells on the outside of the intact mesothelial cell layer (black arrowheads). O, Adjacent sites of (G) E‐cadherin, which designate epithelioid phenotype, were dominant in fibrin‐anchored OCMs on the outer side of the intact peritoneal surface. Bars show 100 μm (A, C, E, G), and 50 μm (B, D, F, H, I, K, M, O) and 25 μm (J, L, N)

Figure 2

Extraperitoneal neovascularization around ovarian cell mass (OCM) in case #1. Staining of podoplanin (A, D), vascular endothelial growth factor (VEGF‐A) (B), transfer factor (TF) (C), α smooth muscle actin (αSMA) (E), stromal cell‐derived factor‐1/C‐X‐C motif chemokine ligand 12 (SDF‐1/CXCL12) (F), and hematoxylin and eosin (G). A‐C, At a more advanced site than Figure 1. B, Expression of VEGF‐A was shown on stromal tissues around OCMs. C, TF was also highly expressed around OCM. D, E, Podoplanin‐positive and αSMA‐positive CAF (black arrows) were observed among OCM. F, OCMs and stromal cells expressed SDF‐1/CXCL12 (black arrows). G, Vascular dilatation was evident in the stromal tissues around the OCM and abundant extravasation of red blood cells was observed beneath mesothelial cells. A, D, G, Around the area where numerous vessels traverse beyond the boundary, the line of podoplanin‐positive mesothelial cells was disrupted (intact peritoneal epithelium: black arrowheads, disrupted mesothelial cell layer: white arrowheads). Bars show 100 μm (A, B, C), 50 μm (D, E, F, G)

Figure 3

Invasion of ovarian cell mass (OCM) toward peritoneal stroma in case #1. Staining of hematoxylin and eosin (A, D‐G), podoplanin (B, H), and transfer factor (TF) (C, I). A‐C, At a more advanced site than Figure 2. The boundary line of mesothelial cells widely disappeared (white arrowheads) and both peritoneal and OCM‐surrounding stromal tissues were completely integrated together (black arrows). D‐F, These integrated areas were fused together and invading OCM were predominantly observed around the feeding vessels (white arrows). G, H, OCMs invade the peritoneal stroma, being associated with CAF (black arrows). I, TF was highly expressed around OCM. Black arrowheads show intact mesothelial cell layer and white arrowheads show disrupted mesothelial cells. Bars show 200 μm (G, I), 100 μm (A, C, D, H), and 50 μm (B, E, F)

Figure 4

The ascitic and plasma levels of transfer factor (TF) in patients with ovarian cancer. The plasma and ascitic TF levels (n = 25 and n = 20, respectively) were measured in patients (total n = 33) with ovarian cancer. A, The median value of ascitic TF was significantly higher than that of plasma TF. B, In 12 cases, we could obtain both plasma and ascitic samples. It was also confirmed that the TF level in ascites was higher than that in blood. C, RT‐PCR was performed to confirm the presence of TF in the tissue of ovarian cancer (case #11). mRNA expression of TF was confirmed both in primary and peritoneal metastatic regions. Met, peritoneal metastasis; NC, negative control; PC, positive control (ovarian cancer cell line; SKOV3); Pri, primary lesion. *P < .001. **P = .001

Figure 5

A proposed new strategy for peritoneal implantation by ovarian cancer cells. A, Step I. Ovarian cell masses (OCMs) cause an inflammatory reaction on the peritoneal stroma, inducing the leakage of fibrinogen. B, Step II. OCM are anchored and surrounded by fine fibrin networks without adhering to the peritoneal epithelium, inducing the migration of fibroblasts and endothelial cells from the host's tissues toward the fibrin networks. C, Step III. Fibrin networks around OCM become mature stromal tissues, developing vascular networks, growing OCM, differentiating cancer‐associated fibroblasts (CAF), inducing inflammation around vessel‐insertion sites, and extending the opening area of the intact mesothelial cell layer. D, Step IV. The boundary layer of mesothelial cells disappears and both intraperitoneal host stromal tissues and extraperitoneal OCM‐surrounding stromal tissues are integrated together, enabling the OCM to invade the host stroma, being accompanied by CAF