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Frank Fischer, Wolfgang Maier-Borst, Walter J. Lorenz


Im Neuenheimer Feld 280
D - 69120 Heidelberg, Germany

   Keywords:  blood, cancer, colon adenocarcinoma cells (CX1), hematogenous spread, laser induced fluorescence diagnosis.  








The hematogenous spread of cancer leads to new growth of cancer in distant sites of the body. Although only very few released cancer cells survive the passive transport in the blood stream, one surviving cancer cell can be sufficient to establish metastatic growth at a distant site. In order to allow precise diagnosis of the metastatic potential of the primary tumor in each individual case, the quantification of cancer cells released into the blood stream is a helpful piece of information. In photodynamic therapy, chemical compounds with fluorescent and photosensitizing properties are used for the diagnosis and therapy of primary tumor sites. Staining a primary tumor with a photosensitizer - as in photodynamic therapy - the tumor cells released from the primary tumor into the blood stream may contain sufficient amounts of photosensitizer in order to quantify the tumor cells using suitable fluorescence detection methods. We carried out initial  in vitro investigations in order to find suitable fluorescence detection methods for counting the tumor cells in blood. Laser induced fluorescence detection in form of standard flow cytometry showed promising results and is described in this contribution. We incubated colon carcinoma cells (CX1) with the photosensitizer Meso-Tetra(Hydroxyphenyl)chlorin (mTHPC). The stained tumor cells were then added to fresh whole human blood and counted with a standard flow cytometer. A procedure that involves a blood puncture and standard flow cytometry for quantifying hematogenous spread of cancer at the location of the blood puncture was shown to hold promise for such quantitave measurements.  



Every year millions of people die of cancer, the most common types in men being lung (33%), prostate (14%), colorectal (9%) and pancreas (5%) cancers; while in women the most common types are lung (24%), breast (18%), colorectal (11%) and ovarian (6%) and pancreas (5%) cancers (
Four consecutive phases of metastatic growth have been described: invasion; spread; establishment at a distant site; and growth of the new tumor (2). Three main routes of tumor cell dissemination are direct local invasion, lymphatic borne spread and hematogenous (vascular-borne) spread (3). In the latter, penetration by cells can occur into the neovasculature of the tumor’s own blood vessels, followed by passive dissemination into the circulation.  Otherwise, tumor cells located close to existing blood vessels of the host can penetrate the vessel’s wall and enter directly into the circulation (4).   
Most data concerning the rate of hematogenous metastasis were obtained from autopsy data (
In general, quantifying hematogenous released tumor cells could allow precise estimations of the metastatic potential of the primary tumor. So, in basic cancer research as well as in daily oncological practise, this quantification of the metastatic potential would be a helpful piece of information. We did initial in vitro investigations presented in this paper in order to develop a method to quantify hematogenous released tumour cells using laser induced fluorescence in the form of flow cytometry. In these initial in vitro investigations, we incubated colon carcinoma cells (CX1) with the photosensitizer meso-tetra(hydroxyphenyl)chlorin, mTHPC. The mTHPC stained tumor cells were added to fresh whole human blood and counted with a standard flow cytometer.  




1.  Tumor cells (CX1) and growing conditions Tumor cells (CX1) and growing conditions  
The adherent colon adenocarcinoma cell line CX1, established originally from a well-differentiated, solid colon adenocarcinoma of a 44 years old woman, was chosen because a high percentage of colon carcinomas are known to metastasize to the liver via the vena porta (3).  These cells were obtained from the Tumor Bank of the German Cancer Research Center, Heidelberg, Germany.  Unless otherwise described, the CX1 cells were grown as previously described (6). Briefly, 10% fetal calf serum in Ham's F12 cell medium was used with 20-25 mM (pH 7.4) Hepes antibiotics (penicillin/streptomycin) as cell culture medium. Viability of the CX1 cells was checked before every experiment by the trypan blue exclusion test (7). 
For the flow cytometric experiments, CX1 cells were incubated with 1 ug/ml meso-tetra(hydroxyphenyl)chlorin, mTHPC, for 24 h prior to adding them to blood or fresh medium. Incubation with the photosensitizer mTHPC was performed in nearly dark room conditions. After incubation, cells were harvested using 2 ml 0.05% trypsin-0.02% EDTA in phosphate buffered saline (Gibco BRL, Eggenstein, Germany) for 10 min, and were subsequently resuspended in medium in order to inactivate the trypsin. The cell concentration in the suspension was determined using a hemocytometer (Migge, Heidelberg, Germany) or an electronic cell counter (CASY 1, Model TTC, Schaerfe Systems, Reutlingen, Germany). Both methods gave essentially similar data.  However, standard deviations with the hemocytometer were ~20%, while the counter was only ~2%. Eventually the cell suspension was either diluted in medium or in blood in various concentrations. 
2.   Blood  
Blood was obtained from a 33 year-old healthy male volunteer by venipuncture using sodium heparin as anticoagulant (Vacutainer, Becton Dickinson Vacutainer Systems Europe, Meylan Cedex, France). The fresh whole blood was stored on a shaker (Contraves, Zurich, Switzerland) at room temperature (21oC) until required for use. Blood older than 6 h was discarded. 
3.   Flow cytometry  
An Epics Profile Analyzer 2.02 flow cytometer (Coulter, Krefeld, Germany) was used. Details of the flow cytometer can be found in (8). This standard flow cytometer detects two fluorescence emissions, using the same excitation wavelength. Furthermore, scattering of laser light in two directions, laser forward scattering (LFS) and laser sideward scattering (LSS) can be detected. For fluorescence excitation an all lines air-cooled argon ion laser (488 – 514 nm) was included in the instrument. For fluorescence emission detection a standard propidium iodide (PI) filter set was used. The PI-set allowed fluorescence emission detection in two different wavelength channels. Laser fluorescence 1 (LFL1) detected fluorescence light emitted between 540 nm and 560 nm and laser fluorescence 2 allowed emission detection at (630 20) nm. The sample (150 ul) flowed through the detection chamber at 20 ul/min. 
Various dilutions of the CX1 cell suspension were tested in order to allow precise detection of the CX1 cells without clumping the instrument. Finally, a 1:20000 dilution in medium was used for blood, CX1 suspensions in medium, and CX1 suspensions in blood. Flow cytometric analysis was performed right after dilution. All results described here were measured using the same adjustment of the flow cytometer sensitivity (photomultiplier voltages: SS = 450 V, FL1 = 1001 V, 
FL2 = 1200 V). 



Figure 1 shows an example of the flow cytometric plots of blood. In blood, nearly all counts are related to red blood cells. Therefore, the contribution of other blood cell components to the plots can be neglected. The red cells show low fluorescence in both emission channels (LF1 and LF2). However, higher emission intensities can be found in LFL2 than in LFL1. Forward as well as sideward scattering was detected for the red blood cells in a wide range of scattering intensities. This is thought to be due to the biconcave shape of an erythrocyte. 
Figure 2 shows scattering and fluorescence properties of CX1 cells incubated with mTHPC. 
Scattering is mainly located at small scattering intensities. A few cells give medium and high scattering intensities. A different cell cycle status changing the shape of the cells in solution might be a reason for this observation. Our observations showed that CX1 cells are able to grow not only if adherent to a cell flask bottom, but also ‘swimming’ in the medium. Alternatively, CX1 cells may stick together in the medium in order to build bigger aggregates, giving higher scattering intensities. In both fluorescence emission channels, LFL1 and LFL2, two fluorescence intensity peaks can be observed. Again, cell shape increase due to the cell cycle or aggregation of CX1 cells may be responsible for the higher fluorescence intensity counts. 
The flow cytometric analysis of a 1:1 mixture of blood and mTHPC containing CX1 cells is shown in Figure 3. While the scattering plot shows overlapping intensities for blood and CX1 cells, both fluorescence emission intensity plots show a clear difference between the fluorescence of blood and the fluorescence of the CX1 cells. 
While the blood fluorescence intensity signal remains unchanged, the mTHPC containing CX1 cells show a different fluorescence intensity pattern (Fig. 3). Out of the original two peaks only one fluorescence intensity peak remains. This single peak (for both fluorescence channels, LFL1 and LFL2) is located at the same position the lower fluorescence intensities could be observed for the mTHPC-CX1 cells in medium (Fig. 2). In blood either there seems to be no CX1 cell aggregation or the cells do not stick together anymore. 
Further optimizing of the flow cytometric procedure is possible. For example, the excitation wavelength could be changed to correspond to the main excitation peak of the photosensitizer used (i.e., ca. 420 nm for mTHPC (9)), the photomultiplier voltages could be changed for better distinction between blood and tumor cells, etc. However, it was shown that the difference in fluorescence intensity between blood cells and CX1 cells can be well distinguished. The difference can be used to quantify the CX1 cells in blood. 
In our opinion the critical point of the flow cytometric method described here, is that the number of tumor cells released from a primary tumor can be much lower than the number of red cells in blood. Keeping in mind that only 150 ul of a 1:20000 diluted blood-tumor cell suspension is injected into the flow cytometer, the in vivo detection limit for the tumor cells would be 1.3  x 105 tumor cells per milliliter of blood. 
Since, in vivo data on the number of tumor cells released into the blood stream for different kinds of tumors are very limited, in vivo experiments have to demonstrate whether a reduction of the tumor cell detection limit is necessary or not. A reduction of the detection limit could be achieved by changing the size of the injection chamber in order to allow higher volumes to flow through the flow cell. 



This work was supported by grants from the Tumorzentrum Mannheim/Heidelberg and from the German Cancer Research Center, Heidelberg. We appreciate the help in cell culture by Ulrike Bauder-Wuest. Furthermore, we appreciate Karl Hutter’s introduction into flow cytometry and his helpful hints and assistance during flow cytometric experiments. Furthermore, we thank Hans-Joerg Sinn for providing mTHPC and Roy Pottier for help in manuscript preparation. 




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