Sybren Meyer

Milky spots in the greater omentum are predominant sites of local tumour cell proliferation and accumulation in the peritoneal cavity

Abstract The role that milky spots in the greater omentum play in tumour cell spread in the peritoneal cavity is presently not fully understood. To study whether intraperitoneally injected tumour cells appear preferentially in milky spots of the greater omentum and to study the changes in the greater omentum, and especially in the cell population of milky spots after tumour cell infiltration, the following study was performed. A detailed temporal sequences of changes in morphology and cellular composition in milky spots of the greater omentum of Wag/Rij rats 5, 15, 30, 60 min, 2, 4, 8, 16, 24 h, 2, 4, 8 days and 2 and 4 weeks after intraperitoneal administration of 2.0 × 106 CC 531 tumour cells was investigated by light microscopy and electron microscopy (pre-embedding labelling). Our data showed that the milky spots in the greater omentum were the sites to which tumour cells migrated preferentially from the peritoneal cavity. The tumour cells infiltrated the milky spots and formed clusters within. The cellular population in milky spots reacted by a very rapid influx of young macrophages during the first hour and an increase of the total number of cells (P < 0.01). After 4 h tumour cells were also located on the greater omentum outside the area of the milky spots. Around these tumour cell deposits, new milky spots are formed, which increased the total number of milky spots. The cells present in milky spots are not capable of reversing the growth of tumours and finally a solid omental cake of tumour cells is formed.

Enhanced killing capacity of human Kupffer cells after activation with human granulocyte/macrophage-colony-stimulating factor and interferon γ

In this study we investigated the effect of the cytokines human granulocyte/macrophage-colony-stimulating Factor (hGM-CSF) and interferon γ (IFNγ) on human Kupffer-cell-mediated cytotoxicity against the SW948 coloncarcinoma cell line. Kupffer cells were isolated from small liver wedge biopsies, taken from 14 patient who had had abdominal surgery for colon carcinoma or partial hepatectomy. The cells were incubated with hGM-CSF (100 ng/ml), or with IFNγ (100 U/ml) or with their combination and the perecentage cytotoxicity was determined using a recently described modified assay. Additional experiments were performed with tumour-necrosis-factor-α(TNFα)-sensitive U937 cells as target. The TNFα secretion of Kupffer cells was measured and we evaluated the effect of TNFα on colon tumour targets. We performed human-Kupffer cell-mediated cytotoxicity blocking experiments with anti-TNFα and used paraformaldehydefixed Kupffer cells to demonstrate lysis of TNFα-sensitive WEHI-164 cells and of SW948 cells. The overall cytotoxicity against SW948 caused by unactivated Kupffer cells (n=14), and by Kupffer cells activated with hGM-CSF (n=14), IFNγ (n=6) or their combination (n=6) was respectively: 19.5±2.6%, 25.3±2.9% 41±9.4% and 45.6±8% at E/T=1 and 28.2±2.9%, 35.6±3.2%, 55.6±9.7% and 62.8% at E/T=5. All differences were statistically significant (P<0.05). No growth-promoting activity by hGM-CSF on the SW948 tumour cells was observed. U937 cells were highly susceptible to Kupffer-cell-mediated cytotoxicity. The TNFα secretion by human Kupffer cells increased in parallel to their cytotoxicity after incubation with these cytokines. Soluble TNFα had only a slight anti-proliferative effect on SW948 cells, while specific anti-TNFα blocked Kupffer cell cytotoxicity by up to 80%. Finally, paraformaldehyde-fixed Kupffer cells were able to lyse WEHI-164 and SW948 cells. This indicates that expression of cell-associated TNFα is the main cytolytic mechanism of human-Kupffer-cell-mediated cytotoxicity. The implications for the use of hGM-CSF and IFNγ in vivo are discussed.

Ontogeny of milky spots in the human greater omentum: An immunochemical study

Milky spots in the human greater omentum are preformed specific accumulations of primarily macrophages within the stroma of the greater omentum. To obtain a better understanding of milky spots in the human greater omentum, the development and the earliest forms of milky spots in the human greater omentum were studied, with special attention to the macrophage population.

Novel isolation and purification method permitting functional cytotoxicity studies of macrophages from milky spots in the greater omentum

Milky spots in the greater omentum are well organized perivascular infiltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity. In milky spots, macrophages are the predominant cell type forming a distinct population of cells. To investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cytotoxicity of macrophages from milky spots in the greater omentum against tumor cells in vitro. In order to obtain a cell suspension, greater omenta of unstimulated healthy male WAG/RIJ rats were incubated in collagenase/DNase suspension and filtered. Subsequently, macrophages were isolated and purified using flow cytometry by sorting unstained cells on the basis of size and internal complexity. Macrophages and other cells were identified by routine May-Grünwald-Giemsa staining and by immunophenotyping with the specific macrophage monoclonal antibody ED 1. Furthermore, macrophage subtypes were characterized by ultrastructural analysis. Functional cytotoxicity of the isolated macrophages was assayed against the syngeneic CC 531 tumor cell line in a colorimetric MTT assay. From three greater omenta of healthy rats 1.16 +/- 0.16 x 10(6) macrophages were isolated with a purity of 83 +/- 2% and a viability of > or = 96%. The macrophages were of the exudate (monocytic), exudate-resident and resident cell type and were in equal proportions. The contaminating cells were mainly mesothelial. A maximum cytotoxicity of approximately 30% was reached with the macrophage fraction at an effector-to-target ratio of 10. Furthermore, it was established that the mesothelial cells did not exhibit cytotoxicity.

Isolation of rat and human Kupffer cells by a modified enzymatic assay.

A new rapid method is described for the isolation and purification of rat and human Kupffer cells without the need of liver perfusion techniques. Rat livers or small human liver wedge biopsies obtained peroperatively were incubated with pronase under continuous pH registration. Kupffer cells were subsequently separated from other nonparenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. Identification of Kupffer cells was achieved on the basis of ultrastructural analyses and immunophenotyping.

Cell-mediated immunity is enhanced by cytostatic drugs continuously released at the site of antigenic stimulation

SummaryImmunopotentiation by cytostatic drugs continuously released from osmotic minipumps, was investigated in a guinea-pig contact-sensitivity model. These pumps are designed to release their content within a period of 7 days. Vepesid (VP-16) and 5-fluorouracil were released into oxazolone-stimulated lymph nodes by subcutaneous implantation of pumps containing either of these drugs. The pumps were implanted at the intended sensitization site, 2 days before sensitization. Strong potentiation of T-cell-mediated immunity, evaluated by delayed-type hypersensitivity measurements, was observed with both drugs tested. Daily injections with VP-16 also caused an enhancement of the immune response. However, daily injections with 5-fluorouracil, a drug assumed to be cell-cycle-specific in its action, failed to potentiate delayed hypersensitivity to oxazolone. Intralesional administration of cytostatic drugs has been put forward as an effective treatment modality in various types of cancer. Therapeutic effects may depend on both local tumorcytotoxic and immunopotentiating activities. Our present results suggest that osmotic minipumps can be applied to broaden the applicability and effectiveness of local chemotherapy.

Granulocyte macrophage colony stimulating factor pretreatment induces an increase of rat Kupffer cells with enhanced cytotoxicity in vitro and prevention of tumor outgrowth in vivo.

Male Wag rats were pretreated for 7 days with 1000 U/ml recombinant murine granulocyte macrophage colony stimulating factor (rmGM-CSF). Rat Kupffer cells (KC) were Isolated by a enzymatic method. We injected decreasing numbers of CC531 tumor cells In the portal system. Mean KC yield increased from 1.5 ± 0.2 to 2.2 ± 0.2 (p < 0.05). Mean percentage of KC-mediated cytotoxicity against CC531 increased from 20.0 + 0.5 to 42 + 1.0 after rmGMCSF (p < 0.05). At 1 x 105 CC531 tumor cells we demonstrated prevention of formation of small foci of CC52+ tumor cells. We demonstrate increased isolated KC with enhanced cytotoxic capacity after rmGM-CSF. rmGM-CSF induced prevention of minimal residual disease in the rat liver.