Ascitic complement system in ovarian cancer

Abstract

Ovarian cancer spreads intraperitoneally and forms fluid, whereby the diagnosis and therapy often become delayed. As the complement (C) system may provide a cytotoxic effector arm for both immunological surveillance and mAb-therapy, we have characterised the C system in the intraperitoneal ascitic fluid (AF) from ovarian cancer patients. Most of the AF samples showed alternative and classical pathway haemolytic activity. The levels of C3 and C4 were similar to or in the lower normal range when compared to values in normal sera, respectively. However, elevated levels of C3a and soluble C5b-9 suggested C activation in vivo. Malignant cells isolated from the AF samples had surface deposits of C1q and C3 activation products, but not of C5b-9 (the membrane attack complex; MAC). Activation could have become initiated by anti-tumour cell antibodies that were detected in the AFs and/or by changes on tumour cell surfaces. The lack of MAC was probably due to the expression of C membrane regulators CD46, CD55 and CD59 on the tumour cells. Soluble forms of C1 inhibitor, CD59 and CD46, and the alternative pathway inhibitors factor H and FHL-1 were present in the AF at concentrations higher than in serum samples. Despite the presence of soluble C inhibitors it was possible to use AF as a C source in antibody-initiated killing of ovarian carcinoma cells. These results demonstrate that although the ovarian ascitic C system fails as an effective immunological surveillance mechanism, it could be utilised as an effector mechanism in therapy with intraperitoneally administrated mAbs, especially if the intrinsic C regulators are neutralised.

Figure 1

CD59 in ascites fluid (AF) has acyl-chains attached to it. The Triton X-114 (TX-114) partitioning was used to separate soluble and phospholipid-containing forms of CD59 in AF. TX-114 (0.5%) was added to cell-free AF at +4°C. After an incubation at +37°C, the detergent and water phases were separated by centrifugation. The native AF sample, detergent and water phases of AF were run in nonreduced SDS–PAGE (10%), transferred onto nitrocellulose membranes and probed with an anti-CD59 mAb (BRIC229). CD59 was detected principally in the detergent phase, indicating that the majority of CD59 molecules still contained membrane phospholipids.

Figure 2

Immunoblotting analysis of C regulators in the concentrated cell culture supernatants of Caov-3, PA-1, SK-OV-3 and SW626 ovarian cancer cells. Confluent cultures of ovarian cancer cells were incubated with serum-free cell culture medium for 48 h. The centrifuged and filtered (0.45 μm) cell culture supernatants were concentrated (100 ×). Aliquots (20 μl) were electrophoresed in 10% nonreducing SDS–PAGE slab gels and blotted onto nitrocellulose membranes. The membranes were incubated with antibodies to CD46 (J4-48), CD55 (BRIC 230 or 216), CD59 (BRIC 229) and SP40,40.

Figure 3

Complement-mediated lysis of PA-1 and Caov-3 cells with ascitic fluids from ovarian cancer patients. Caov-3 and PA-1 cells were labelled with ⁵¹Cr. After sensitising with the anti-CD59 C-activating mAb (YTH53.1; 350 μg ml⁻¹), the cells were exposed to ascites fluid (•) or normal human serum (○). The AF and serum dilutions are indicated on the x-axis. The released radioactivity was counted from the supernatants after a 30 min incubation at 37°C. Cell lysis was determined as a percentage of the maximum releasable radioactivity: ((released radioactivity−spontaneous release)/(maximum released radioactivity−spontaneous release)) × 100%.

Figure 4

Immunohistochemical analysis of the expression of membrane bound C regulators on ovarian tumour cells isolated from AF. Cytotospin specimens were fixed and stained with the mAbs BRIC 216 (A), J4-48 (B) and BRIC 229 (C) directed against DAF, MCP and CD59, respectively. An irrelevant mouse IgG was used as a negative control (D). The bound mAbs were detected using an immunoperoxidase staining kit. Original magnification, × 200. Membrane cofactor protein and CD59 were strongly expressed by all the isolated tumour cells (B and C), while DAF expression was weaker and more heterogeneous (A).

Figure 5

Immunohistochemical analysis of the C deposits on ovarian tumour cells isolated from AF. The cell specimens were stained with polyclonal antibodies against C1q (A) and C3d (B) and a mAb directed against a TCC neoantigen (C). Normal rabbit serum was used as a negative control (D). The bound Abs were detected using an immunoperoxidase staining kit, and the slides were counterstained with haematoxylin. Original magnifications, × 40 (A) and × 200 (B, C and D). Deposits of C1q (A) and C3d (B), but not of C5b-9 (C), could be detected. No reactivity was seen when the primary antibody was replaced with normal rabbit serum (D).

Figure 6

Immunoblotting analysis of IgG in AF reacting with ovarian cancer cell membrane antigens. Cell membranes from Caov-3, PA-1, SK-OV-3 and SW626 cells were isolated and electrophoresed in 5–15% SDS–PAGE gels and transferred onto nitrocellulose filters. The nitrocellulose strips were incubated with the AF samples (diluted 1 : 800). Bound IgG was detected by the enhanced chemiluminescence system.