DETECTION OF HEMATOGENOUS SPREAD
USING
LASER INDUCED FLUORESCENCE (LIF).
INITIAL IN VITRO RESULTS.
Frank Fischer,
Wolfgang Maier-Borst, Walter J. Lorenz
Keywords:
blood, cancer, colon adenocarcinoma cells (CX1), hematogenous spread, laser induced
fluorescence diagnosis.
| ABSTRACT |
| 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. |
| INTRODUCTION |
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. |
| MATERIAL AND
METHODS |
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). |
| RESULTS AND
DISCUSSION |
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. |
| ACKNOWLEDGEMENT |
| 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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