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.


Introduction
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][4][5][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.

Animals
Male Wag/Rij rats were purchased from Harlan (The Netherlands) and were 8-12 weeks of age, weighting 240-260 g. Animals had free access to food and water. The local university ethical committee for animal welfare approved all animal experiments.

Liver Metastasis Induction
Rats were anaesthetized using Nembutal at a dose of 60 mg/100 g body weight (Leo Pharmaceutical Products, Brussels, Belgium). Early stages of liver metastasis were generated by intramesenterical injection of 1 × 10 6 CC531s cells suspended in 0.5 ml PBS under sterile conditions. 1, 8 and 24 hrs after injection, the liver was perfusion-fixed (9 ml/min) using 2% paraformaldehyde prepared in PBS. 100 µm sections were made and

Confocal Microscopy
Liver sections were studied by a Leica TCS SP CLSM (Leica TCS NT software version 1.6.587) equipped with Ar/ HeNe lasers and installed on a Leica DM-IRBE inverted microscope [10].

Results and Discussion
During the first day of metastasis, tumor cells arriving subsequently in the liver were mainly localized in the portal area's [11]. At one hour, Kupffer cells were observed in contact with tumor cells [3]. Protrusions from Kupffer cells surrounding the tumor cells could be observed using CLSM (Fig. 1) as was verified with electron microscopy [data not shown; [3]].
Eight hours and 24 hrs after tumor cell administration, tumor cells phagocytosed by Kupffer cells could be frequently observed (Fig. 2). In contrast, 1 hr post injection, phagocytosed tumor cells were rarely observed, illustrating the importance of tumor cell binding prior to elimination [12]. The observation of phagocytosed tumor cells was facilitated by the presence of DiO in Kupffer cells marked by TRITC-labeled latex beads, as DiO does not transfer from labeled to unlabeled cells [10]. Large vacuoles (8 µm) could be observed surrounded by smaller vacuoles indicating the uptake of intact tumor cells [3,6,12] and their subsequent lysosomal degradation (Fig. 2) [5].
One day after the injection, non-phagocytosed tumor cells were seldom observed, showing the importance of the immune system present in the liver during this phase (first day) of metastasis [1,[4][5][6]. These results show that CLSM is a useful tool to study the interactions occurring between Kupffer cells and tumor cells in large volumes of tissue [10].