M Timmers

Interactions between rat colon carcinoma cells and Kupffer cells during the onset of hepatic metastasis

Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK‐positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.

The F‐Actin Content of Multiple Myeloma Cells as a Measure of Their Migration

Abstract: One of the main characteristics of multiple myeloma (MM) cells is their specific homing and growth in the bone marrow (BM). For their homing, MM cells need chemotactic signals to be attracted towards the BM and to be activated. Profound knowledge of the different chemokines for MM cells and their signal transduction pathways is necessary to interfere in this process. We studied here an extra possible tool for the investigation of the different chemokines and their pathways. The 5T experimental mouse model was used to investigate the migration of MM cells towards BM stromal cells. We studied the changes of the F‐actin content in the 5TMM cells in the presence of BM stromal cell conditioned medium and we correlated this with their migratory capacity. F‐actin became polarized when the cells were migrating, in contrast to nonmigrating cells. This polarization could not only be seen by fluorescence and confocal laser scanning microscopy, but also could be quantified by fluorometry and flow cytometry. The correlation between the F‐actin content of the MM cells and their migration capacity thus makes its quantification a useful tool in studying their migratory behavior.

Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy.

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.

CC531s colon carcinoma cells induce apoptosis in rat hepatic endothelial cells by the Fas/FasL-mediated pathway

The mechanisms involved in colorectal carcinoma with liver metastasis are not well known. Metastasizing colon carcinoma cells express more FasL than primary colon carcinoma cells and cancer cells induce apoptosis in hepatocytes by the Fas/FasL pathway. Therefore, this study focused on Fas/FasL expression and functionality in rat liver sinusoidal endothelial cells (LSECs) and CC531s colon carcinoma cells in vitro and in vivo. RT‐PCR and immunochemistry revealed Fas and FasL in LSECs and CC531s, respectively. Functionality of Fas was assessed in vitro by incubation with human recombinant FasL (1–100 ng/ml) with or without enhancer. At concentrations of 10 and 100 ng/ml with enhancer, respectively 21% and 44% of endothelial cells showed signs of apoptosis using Hoechst 33342/propidium iodide staining and electron microscopy. In co‐cultures, apoptosis could be detected in endothelial cells neighboring the CC531s and could be inhibited by an antagonistic FasL antibody. Moreover, 18 h after mesenteric injection of CC531s, the sinusoidal endothelium revealed disruption. In conclusion, (i) CC531s cells induce apoptosis in LSECs in vitro by using Fas/FasL; (ii) CC531s cells damage the sinusoidal endothelial lining in vivo; and (iii) this might provide FasL‐positive tumor cells a gateway towards the hepatocytes.

Cytotoxic reactions of CC531s towards liver sinusoidal endothelial cells: a microscopical study

Colorectal cancer cells can induce apoptosis in cells of various tissues [1]. Apoptosis can be induced by a number of factors such as Fas inducing apoptosis through the Fas/FasL pathway; other factors involve the TRAIL pathway and TNF. It is known that metastasizing colon cancer cells express more FasL then primary carcinoma cells [2]. We investigated whether a rat colon carcinoma cell line CC531s could induce apoptosis in liver sinusoidal endothelial cells (LSECs). LSECs and CC531s were co-cultured for 18 hrs and cells were visualized by SEM and TEM. Apoptosis was visualized by markers such as Hoechst and Propidium iodide. Furthermore, cells were recorded by time lapse video microscopy with and without an antagonistic antibody for FasL.

Confocal laser scanning microscopic study of the killing of metastatic colon carcinoma cells by Kupffer cells in the early onset of hepatic metastasis

During hepatic metastasis, tumor cells are exposed to the sinusoidal environment, involving endothelial cells, Kupffer cells, pit cells (NK cells) and fat-storing cells [1]. It is known that Kupffer cells and pit cells play a direct role in killing metastasizing colon carcinoma cells [1,2]. Only few studies describe the interactions of Kupffer cells and tumor cells within the first 24 hrs of metastasis [3-6]. The importance of this early phase lies in the substantial but not complete killing of tumor cells. Also, at later stages, when different elements of host defense are involved, it is apparent that the local immune system is not capable of preventing metastasis. Investigating rare cellular events is facilitated by studying the full depth of thick sections with confocal laser scanning microscopy. Complicated preparation and histochemical procedures and a low sampling-volume [7] characterize classical microscopic methods. In contrast, confocal laser scanning microscopy (CLSM) has the ability to study thick sections (100 micrometers), gathering 3D information and allowing the use of different fluorescent probes for different variables [8]. Localization of cells can be done by immunohistochemistry, by labeling cells in vitro prior to injection or by labeling them in vivo. The anti-metastatic function of Kupffer cells was studied by labeling tumor cells with the lipophilic probe DiO in vitro and Kupffer cells with fluorescent latex particles in vivo.

Comments on augmentation of local antitumor immunity in liver by interleukin-2 gene transfer via portal vein: a possible explanation for contradictory in vivo and vitro results of interleukin-2 treatment in a rat model of colon carcinoma metastasis.

Tanji et al1 have demonstrated in a rat model that interleukin-2 (IL-2) gene transfer to the liver prevents liver metastasis of colon carcinoma cells. IL-2 gene transfer was performed by intraportal injection of an adenovirus containing the human IL-2 gene (AdCMVhIL-2), before the administration of RCN-9 syngeneic colon carcinoma cells. Controls included injection of either adenovirus containing the -galactosidase gene or PBS. The authors suggested that hepatic natural killer (NK) cells, which are located in the liver sinusoids,2,3 are responsible for this effect. Indeed, they demonstrated that hepatic NK cells isolated from AdCMVhIL-2 rats showed an increased cytotoxic activity against the NK-sensitive YAC-1 target cell line, confirming the results of Shiratori et al.4 However, they were unable to show an increased cytotoxic effect of these isolated hepatic NK cells against the RCN-9 colon carcinoma cells.1 In other words, the in vivo effects of preventing liver metastasis of RCN-9 colon carcinoma cells in AdCMVIL-2 treated rats did not coincide with an increased activity of hepatic NK cells of these rats against the same target cell line.