Previously, our lab and others [4
] have shown that intraperitoneal injection of B16 melanoma tumor cells resulted in macroscopic foci of black-pigmented tumor cells on the omentum. Upon closer microscopic analysis using the whole mount technique and GFP expressing B16 tumor cells injected i.p., we found that the tumor foci were localized to distinct areas on the omentum, which coincided with immune cells. Five additional GFP+
] tumor cell lines including ID8, an ovarian line, bound preferentially to these areas (). Interestingly, a human ovarian tumor cell line (OVCAR-3), labeled with the fluorescent marker CFSE, also selectively bound to these same areas of the mouse omentum (). This result suggests that direct peritoneal metastatic seeding is similar in a xenograft system. Next, we monitored tumor burden over time within the same mouse by injecting B16 cells transfected with luciferase i.p. Following infusion with luciferin and using an in vivo imaging system (IVIS), the photon count, which represents tumor burden, was determined at both 24 h (data not shown) and 96 h (). At 96 h, the tumor-bearing omentum, along with a control omentum from a naïve mouse, was excised and digital images obtained (). Even through the pigmented skin of a C57BL/6 mouse, light from the tumor growing on the omental tissue is clearly visible in an intact mouse. These data demonstrate the possibility of monitoring the presence and subsequent growth of peritoneal metastases on the omentum, which can be utilized to examine potential tumor immunotherapy.
Fig. 1 Both mouse and human ovarian tumor cells bind selectively to the immune aggregates of the omentum and the IVIS system can be utilized to visualize growth of tumors on the omentum. 2 × 105 mouse ID8/Cherry (a, b) or CFSE-labeled human OVCAR-3 ( (more ...)
The omentum, like the rest of the peritoneum, is reported to be covered by a layer of mesothelial cells [15
]. We have been able to identify these cells using whole mount by staining for the adhesion molecule, VCAM-1 (CD106) [4
], and these VCAM-1+
cells are selectively found over the immune aggregates. Therefore, either the mesothelial cells do not cover the entire omentum, or alternatively, the mesothelial cells on the immune aggregates express much higher levels of VCAM-1. Either of these possibilities makes these mesothelial cells strong candidates for initiating the selective adhesion of tumor cells on the immune aggregates. Indeed, confocal microscopy studies have revealed that the tumor cells initially bind to these outer mesothelial cells (data not shown).
We have identified another unique feature of the immune aggregates, which is the presence of an intricate extracellular matrix that appears to be absent from the surrounding areas of the omentum. The whole mount technique provides an excellent visualization of this as shown in , which depicts an omental immune aggregate stained for collagen I. This extracellular matrix also expresses collagen III (data not shown) and is only seen in this dense configuration within the immune aggregates. Since we have observed openings in the overlying layer of mesothelial cells, this matrix is probably accessible to metastatic tumor cells, and thus could also be involved in the attachment process.
Fig. 2 GFP splenocytes home to immune aggregates, which possess a collagen matrix overlay. Omenta were removed and stained with anti-CD45 to label immune cells (red) and with anti-collagen type I (yellow). Overlaid images from the same field of view illustrate (more ...)
The ability of the cells within these aggregates to generate adaptive immunity to i.p. antigens has not received extensive study, and there is very little information available on whether or not these immune cells respond to the presence of metastatic tumor cells. However, there is evidence that some ovarian cancer patients have T cells present in the peritoneal cavity that are capable of reacting with tumor cells [16
]. This raises the possibility that the preferential homing of tumor-reactive cells to the immune aggregates on the omentum and other peritoneal tissues might provide an advantage for adoptive immunotherapy of metastatic disease. We have found that lymphocytes injected into the peritoneal cavity, like tumor cells, also preferentially localize to the immune aggregates (). In addition, the percentage of T cells found in the peritoneal lavage and on the omentum that expresses the integrin adhesion molecules VLA-4 (α
1) and LFA-1 (α
2), which bind to VCAM-1 and ICAM-1, respectively, is much larger than in lymphoid organs (). Therefore, VLA-4 and LFA-1 expressing immune cells may preferentially home to omental immune aggregates due to the presence of VCAM-1+
mesothelial cells covering the immune aggregates.