immune system
Duijnhoven, Frederieke van
Citation
Duijnhoven, F. van. (2005, June 22). Local ablative therapies for colorectal liver metastases
and the immune system. Dept. of Surgery, Leiden University Medical Center, Leiden
University. Retrieved from https://hdl.handle.net/1887/2706
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Chapter 3
The effect of photodynam ic therapy on distant m etastases in
The effect of photodynam ic therapy on distant m etastases in
The effect of photodynam ic therapy on distant m etastases in
The effect of photodynam ic therapy on distant m etastases in
vivo
vivo
vivo
vivo
Introduction IntroductionIntroduction Introduction
Photodynamic therapy (PDT) of cancer involves the systemic administration of a tumour-localising photo-sensitising agent (photosensitiser). Upon tumour illumination by light of an appropriate wavelength the photosensitiser is activated and reacts with oxygen, producing reactive oxygen species. These reactive oxygen species lead to direct tumour cell damage and secondary effects like vascular damage and possibly activation of the immune system. The mobilisation of the immune system of the host to participate in the eradication of the treated tumour could be a very useful feature of PDT. A key element seems to be the induction of an inflammatory response at the treated site, associated with massive invasion of activated
myeloid cells1. Next to the non-specific activation of the immune system, tumour specific immune reactions
are being reported, suggesting the induction of tumour immunity by PDT. Several studies have been per-formed to show this. A study by Korbelik et al. with EMT6 mammary sarcoma in SCID and normal mice showed a significantly lower remission rate in SCID mice after PDT, suggesting that the difference in tumour
cures originated in the lack of activity of lymphoid cells2. After the transfer of T-lymphocytes of normal
(BALB/c) mice into the immune compromised mice, the recurrence rate was delayed drastically.
Hendrzak-Henion found similar results in a study with nude and T-cell depleted mice3. In two later studies, Korbelik
selectively depleted EMT6 sarcoma-bearing mice of specific myeloid (neutrophils, macrophages) and
lymphoid (T cells) populations4,5. Although immunodepletion of T cells did not affect the initial tumour
ablation by PDT, the tumour cure rate was significantly reduced, showing the importance of the immuno-logical contribution to the effect of PDT.
The role of NK cells in the effect of PDT is unclear, but as we have detected both the presence of NK
cells in CC531 tumours6 and anti-CC531 antibodies in serum from CC531 bearing rats (F.H. van Duijnhoven,
unpublished data), NK cells may contribute to an anti-tumour response via an antibody-dependent cell-mediated cytotoxicity.
Previously, our group showed that PDT is a very effective treatment modality for CC531 colorectal liver
metastases in a rat model7. In the current study we investigated the presence of a systemic immune
res-ponse after PDT by treating one of three tumours in a rat liver and then evaluating the effect on the two untreated tumours. W e also performed immunohistochemical staining on tumour and liver tissue to inves-tigate the presence of T lymphocytes, NK cells and macrophages in PDT treated and non-treated lesions.
Materials Materials Materials
Materials andandandand methods methods methods methods
Animals
Male Wag/Rij rats were used (Charles River, Zeist, The Netherlands). The animals had free access to food and water. The animals received care in accordance with established guidelines. The weight of the animals was followed throughout the experiment to monitor their general state. If weight loss was more than 20% the animal had to be taken out of the experiment for ethical reasons, but none reached this limit. The Animal Welfare Committee of the Leiden University Medical Center approved the study. Animals were randomly allo-cated to the experimental group (n = 15) or the control group (n = 15).
Tumour model
We used the colon adenocarcinoma cell line CC531, which is moderately differentiated, syngeneic and trans-plantable to Wag/Rij rats for tumour induction in the liver. CC531 is a 1,2-dimethylhydrazine-induced
adenocarcinoma of the rat colon6,8. Briefly, tumour cells were cultured on RPMI 1640 Dutch modification
culture medium (RPMI 1640 culture medium with 1 g/L sodium bicarbonate and 20 mM HEPES) supplemen-ted with 2 mM L-Glutamine, 10% heat inactivasupplemen-ted calf serum, 100 U/ml penicillin and 0.1 mg/ml strepto-mycin sulphate (all from Gibco, Grand Island, NY, USA). Cells were maintained by serial passage. Tumour cells were harvested with a solution of 0.25% (w/v) EDTA and 0.25% (w/v) trypsine in HBSS (Sigma, St. Louis, MO), washed three times in 0.9% (w/v) NaCl solution buffered with 1.4 mM phosphate (PBS) and adjusted to a
suspension containing 5 x 106 viable (trypan blue exclusion test) tumour cells per ml PBS. At day 0, three
liver tumours were induced in all rats by injecting 2.5x 105viable tumour cells per tumour (in 50 Fl
Figure 1. Location of the CC531 tumours that were induced in rat liver. For each tumour, 2.5 x 105 CC531 tumour cells were
injected subcapsulary. Tumour number 1 was in the upper right lobe; tumours number 2 and 3 were in the lower lobe ± 2 cm apart. Tumour number 3 was illuminated
Photodynamic therapy
At day 6 (day 0 = tumour inoculation) the photosensitiser metatetrahydroxyphenylchlorin (mTHPC) was ad-ministered to the animals of the experimental group via the tail vein; rats in the control group received the vehicle in which mTHPC was dissolved. The mTHPC solution was made by dissolving 2.2 mg mTHPC in 7.33 ml of a 30% polyethylene glycol 400, 20% ethanol and 50% water solution. The injected dose was 0.3 mg/kg. At day 7, during laparotomy, the surface area of all tumours was measured macroscopically. The tumour in the lower right lobe of each rat was illuminated (tumour number 3 in figure 1) using a diode laser (an AIGaInP laser diode by Diomed Ltd., Cambridge, UK) emitting light of 652 nm wavelength. The power output was set at a fluence rate of 200 mW. The output was measured at 80 mW (Gentec TPM 310, Gentec, Quebec, Canada). A bare tip fibre was used with a cross-section of 0.6 mm. After 200 seconds of irradiation, this
resulted in a light dose of 16 J/cm2. All surgical procedures were performed under clean but not sterile
conditions.
Immunohistochemical staining
2035, Leica, Nussloch, Germany) were cut from the paraffin-embedded tumours and transported onto APES-coated microscope slides. Sections were dried for one hour at approximately 70°C on a heated plateau (MEDAX Nagel GmbH, Kiel). Before rehydration, endogenous peroxidase activity was blocked by incubating
the slides for 20 minutes in methanol containing 0.3% H2O2.
Sections were also cut (Reichert Jung 2800 Frigocut, Leica) from the snap frozen tumours, transported onto Superfrost microscope slides and dried overnight at 60ºC. Sections were incubated for 60 minutes with different mouse-anti-rat monoclonal antibodies. To stain CC531 tumour cells we pre-treated the sections with 0.01 M citrate buffer and then used CC52 IgG1 for paraffin-fixed tissue, R73 IgG1 to stain T cells (directed against rat TCRαβ) and 3.2.3 IgG1 to stain natural killer (NK) cells (directed against CD161A) for snap frozen tissue. To detect the presence of activated macrophages, the antibodies ED1 and ED2 were
used9. The sections were then sequentially incubated for 30 minutes with horseradish
peroxidase(HRP)-conjugated rabbit-anti-mouse Ig anti-serum (Dako, Glostrup, Denmark), horseradish-peroxidase-conjugated swine-anti-rabbit Ig anti-serum (Dako) and finally for 10 minutes with 0.125 gram
3,3’-diamino-benzidine-tetrahydrochloride in a solution of 250 ml 0.05 M TrisHCl pH 7.6 with 15 Fl H2O2 30%.
A polyclonal rabbit anti-laminin antibody, which binds to rat laminin, followed staining with 3.2.3 and R73 antibodies for detection of laminin structures in the tumour. The sections were incubated with HRP-conjugated swine anti-rabbit Ig (DAKO) and streptavidine complex (DAKO). The immune complexes were visualized by incubation for 12 minutes in buffered Tris-HCl (pH 7.6) solution containing, per 100 ml, 40 mg 4-chloro-1-naphtol (Merck, Darmstadt, Germany) dissolved in 200 Fl dimethyl formamide (Baker, Deventer,
the Netherlands) and 300 Fl ethanol (Merck) and 100 Fl of a 30 % (v/v) H2O2 solution (Merck)10. In between all
steps, sections were washed three times with PBS for 5 minutes. Separately, the paraffin-embedded sections were also stained with haematoxylin-eosin (HE). The sections were counter stained with methyl green and then mounted using Pertex for CC52 and HE sections or Kaiser’s Glycerin (Merck) for R73 and 3.2.3 sections. For further evaluation of results, the following terms were used to define the various treatments: from rats with mTHPC administered tumour number 3 was illuminated (mTHPC, tumour 3) and tumours 1 and 2 were not illuminated (mTHPC, tumours 1 and 2); from control rats, tumours number 3 were illuminated (control, tumour 3) and tumours 1 and 2 were not illuminated (control, tumours 1 and 2).
Statistics
two instead of three groups, because of the difficulty of dividing the specimens in three discernible groups. The T cells and NK cells in surrounding liver tissue were quantified by counting cells in three randomly picked areas of the section at a magnification of 100×. This number was then divided by three, giving the
average number of cells in an area of 0,16 mm2 of the section. The cross section of each lesion was divided
in necrosis, stroma tissue and tumour epithelium. Percentages were assessed microscopically by two inde-pendent observers. All numbers are given as mean values ± SD. P values were determined with (paired) t-test for the numbers of T and NK cells in the liver and for percentages of lesion area. Macrophages, T and NK cells in different rats were compared with the Mann-Whitney U test and when samples were from one rat with the Wilcoxon signed rank test. A P value less than 0.05 was considered to be indicative of a significant difference between data.
Results ResultsResults Results Tumour growth
No complications occurred after laparotomy for tumour induction or PDT. The initial tumour induction was successful in all rats but one with three visible tumours 7 days after inoculation. In rat number 9, no tumour was measurable in the upper right lobe (i.e. tumour number 1 in figure 1) after 7 days, so this rat was ex-cluded from analysis for tumour 1.
Animals were killed at day 9, 14 and 21 after tumour inoculation. Tumour sizes and necrotic areas were measured. In all PDT treated tumours (mTHPC rats, tumour 3) there was a clearly defined necrotic area 2 days after illumination (day 9 after tumour inoculation), with no tumour in the central area. However, at the margins of 4 out of 5 treated lesions, small groups of tumour cells remained, indicating that the destruction range by PDT was not always complete (figure 2). At later time points as well, tumour cells were visible in the margin of most PDT treated lesions. The size and number of these groups of tumour cells however had not increased since day 2 after treatment, indicating that tumour cells had not proliferated further after PDT, possibly due to a lack of viability. In control rats the illuminated tumours (number 3) were of similar size as the non-illuminated tumours 1 and 2, showing that illumination without previous administration of photo-sensitiser did not affect tumour growth. Tumours 1 and 2 of both mTHPC and control rats all had grown at similar rates (figure 3a).
Figure 3. Effect of PDT with 16 J/cm2 fluence on tumour/lesion size. (a) Average tumour growth of tumours number 1 and 2
(both non-illuminated) in mTHPC and control group. Tumour/lesion area (mm2 ± SD) is measured directly before PDT and 2,
7 and 14 days after PDT. Administration of mTHPC at day 6 after tumour inoculation (*), illumination 24 hours later, at day 7 (**). (b) Distribution of lesion area taken up by tumour cells, stroma cells or necrosis in tumours 2 (non-illuminated) and 3 (illuminated) of mTHPC and control rats at 2, 7 and 14 days after PDT
0 2 0 4 0 6 0 8 0 1 00 1 2 0 1 4 0 1 6 0 0 5 1 0 1 5 2 0 2 5
days after inoculation
tu m o u r / le s io n s iz e ( m m 2 )
***
0 2 0 4 0 6 0 8 0 0 2 0 4 0 6 0 8 0 1 00 1 2 0 1 4 0 1 6 0 1 0 2 5 0 2 0 4 0 c o n tro l, tu m o u r 3 (illu m in a te d ) m TH P C , tu m o u r 2 m TH P C , tu m o u r 3 (P D T) c o n tro l, tu m o u r 1 c o n tro l, tu m o u r 2 m TH P C , tu m o u r 1 0 2 0 4 0 6 0 8 0 1 00 1 2 0 1 4 0 1 6 0 0 5 1 0 1 5 2 0 2 5days after inoculation
tu m o u r / le s io n s iz e ( m m 2 )
***
0 2 0 4 0 6 0 8 0 0 2 0 4 0 6 0 8 0 1 00 1 2 0 1 4 0 1 6 0 1 0 2 5 0 2 0 4 0 c o n tro l, tu m o u r 3 (illu m in a te d ) m TH P C , tu m o u r 2 m TH P C , tu m o u r 3 (P D T) c o n tro l, tu m o u r 1 c o n tro l, tu m o u r 2 m TH P C , tu m o u r 1 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 00 1 1 0 2 7 1 4 2 7 1 4 2 7 1 4 2 7 1 4 m TH P C , tu m o u r 3 m TH P C , tu m o u r 2 c o n tro l, tu m o u r 3 c o n tro l, tu m o u r 2treatm ent and tum our num b er at days 2 , 7 and 1 4
s iz e o f a re a ( m m 2 ) tu m o u r s tro m a n e c ro s is 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 00 1 1 0 2 7 1 4 2 7 1 4 2 7 1 4 2 7 1 4 m TH P C , tu m o u r 3 m TH P C , tu m o u r 2 c o n tro l, tu m o u r 3 c o n tro l, tu m o u r 2
treatm ent and tum our num b er at days 2 , 7 and 1 4
By comparing the surface areas of these tumours, no significant difference in tumour sizes could be detected between mTHPC and control rats: at day 9 (2 days after PDT) p = 0.26; at day 14 (7 days after PDT) p = 0.96;
at day 21 (14 days after PDT) p = 0.66 (figure 3a).These results indicate that although PDT was locally
effective, it did not affect the growth of distant tumours from the same cellular origin. Immunohistochemical staining
The effectiveness of PDT became clearly visible when assessing the lesion area in HE stained slides: after PDT, necrosis initially constituted over 90% of the lesion area, which corresponded to a lesion area size of 90
mm2, but in time necrosis decreased to 41% (p = 0.005; figure 3b). This was caused by an increase in
stroma, while the tumour epithelium area did not significantly increase in size. Since in untreated tumours, tumour epithelium did increase with time, the PDT treated tumour likely did not contain viable tumour cells. These percentages of necrosis in PDT treated tumours significantly differed from all other tumours (p < 0.05). Regarding area sizes of stroma, epithelium and necrosis, there were no significant differences between all non-treated tumours nor were there changes of these area sizes in time for these tumours.
Figure 4. Tumour sections immunohistochemically stained with 3.2.3. IgG1 for NK cells (brown) and polyclonal rabbit antibodies directed against laminin (blue), at 72x magnification. (a) PDT treated lesion at 2 days after treatment without NK cells in the treated necrotic lesion (N) and adjacent liver tissue (L). (b) Untreated tumour in the same liver with NK cells in tumour and advancing liver margin
N
L
N
Figure 5. Relative amount of NK cells in tumours 2 (non-illuminated) and 3 (illuminated) of mTHPC and control rats at 2, 7 and 14 days after PDT with a fluence of 16 J/cm2 . Sections from tumours were immunohistochemically stained for NK cells
with 3.2.3. IgG1. The number of NK cells in tumour was assessed by dividing sections in two groups, with ‘1’ indicating low number of NK cells and ‘2’ indicating high number of NK cells, as described in the materials and methods section. Error bars represent SD
At 2 days after PDT, there were no T cells present in the centre or margin of the PDT treated lesions, in significant contrast to all other tumours (p = 0.015) (figure 6). With time, stroma tissue increased at the margins of PDT treated tumours, containing a similar level of T cells as control tumours (figure 7). Apparently, after initial depletion of local tumour T cells upon PDT treatment, T cells quickly invaded the edges of the necrotic tumour area again. However, no increase of T cells in the nearby non-illuminated tumour 2 of mTHPC rats was observed. In the liver tissue, the number of T cells increased in time for all tumours, with no significant differences between the PDT treated tumours and the other tumours (figure 8), therefore this T cell increase was probably related to lesion/tumour area size.
0 0.5 1 1.5 2 2.5 3 3.5
2 day s 7 day s 14 day s
days after treatment
re la ti v e s c o re o f N K c e ll s
control, tumour 2 mTHPC, tumour 2 control, tumour 3 mTHPC, tumour 3
0 0.5 1 1.5 2 2.5 3 3.5
2 day s 7 day s 14 day s
days after treatment
re la ti v e s c o re o f N K c e ll s
Figure 6. Relative amount of T cells in tumours 2 (non-illuminated) and 3 (illuminated) of mTHPC and control rats at 2, 7 and 14 days after PDT with a fluence of 16 J/cm2. Sections from tumours were immunohistochemically stained for T cells
with R73 IgG1. The number of T cells in tumour was assessed by dividing sections in three groups, with ‘1’ indicating lowest number of T cells and ‘3’ indicating highest number of T cells, as described in the materials and methods section. Error bars represent SD 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
2 days 7 days 14 days
days after treatment
re la ti v e s c o re o f T -c e ll s control, tumour 2 control, tumour 3 mTHPC, tumour 2 mTHPC, tumour 3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
2 days 7 days 14 days
days after treatment
Figure 7. Tumour sections immunohistochemically stained with R73 IgG1 for T cells (brown) and polyclonal rabbit antibodies directed against laminin (blue), at 72x magnification. (a) PDT treated lesion 14 days after treatment with T cells in the reappearing stroma (S) at the margin of the necrotic lesion (N) and in the advancing liver margin (L). (b) T cells in untreated tumour at the same time point
Figure 8. Average number of T cells, immunohistochemically stained by R73 IgG1, in liver tissue surrounding tumours 2 (non-illuminated) and 3 (illuminated) of mTHPC and control rats at 2, 7 and 14 days after PDT with a fluence of 16 J/cm2.
Cells were counted in three randomly picked areas of the section, at a magnification of 100×. The average number of T cells per high power field (0.16 mm2) of the liver tissue is shown. Error bars represent SD
PDT resulted in a decreased amount of macrophages at two days after treatment in tumour and advancing liver margin. The necrotic centre of these tumours remained void of macrophages, although when stroma started reappearing at the lesion edges from day 7 after treatment, large numbers of macrophages entered the stroma again (figure 9a). For the nearby, non-illuminated tumours in these mTHPC rats, there was a lower amount of macrophages in the tumours as well at 2 days after treatment when compared to control rats, but at later time points (7 and 14 days) there was no difference anymore with these control groups. For both tumours 2 (non-illuminated) and tumours 3 (PDT) in mTHPC rats the amount of macrophages in the advancing liver margin did not increase in time. In all groups, there were hardly any macrophages in the tumour epithelium, even if massive amounts were present in the surrounding stroma. This phenomenon did not change over time or with treatment (figure 9b).
0 20 40 60 80
2 days 7 days 14 days
days after treatment
n u m b e r o f T c e ll s ( n /0 .1 6 m m 2 l iv e
r) control, tumour 2 mTHPC, tumour 2
control, tumour 3 mTHPC, tumour 3
0 20 40 60 80
2 days 7 days 14 days
days after treatment
n u m b e r o f T c e ll s ( n /0 .1 6 m m 2 l iv e
r) control, tumour 2 mTHPC, tumour 2
Figure 9. Tumour sections stained immunohistochemically with ED2 for macrophages (brown) and polyclonal rabbit antibodies directed against laminin (blue), at 72x (a) and 144x (b) magnification. (a) PDT treated tumour at 14 days after PDT with macrophages in the reappearing stroma at the margin of the lesion. (b) Untreated tumour at the same time point. Macrophages (thin arrow) were mainly found in the tumour stroma (S) that was separated from tumour epithelium (E) by laminin (thick arrow) and did not invade the epithelium compartment
S
E
Discussion DiscussionDiscussion Discussion
In this study, PDT treatment was successful in eradicating liver tumours, but had no effect on growth of non-illuminated tumours in the same liver nor resulted in increased numbers of T cells, NK cells or macrophages. At 21 days after PDT treatment, there was no significant regrowth of PDT treated tumours.
To achieve long-term anti-tumour efficacy with PDT an adequate immune response is indispensable, as
demonstrated by Korbelik et al.4. Selective depletion of T cells in EMT6 sarcoma-bearing mice did not affect
initial tumour ablation by PDT, but long-term tumour cure up to 90 days post-PDT was significantly reduced. Other studies showed that PDT of EMT6 mammary sarcoma in SCID mice resulted in lower remis-sion than the same treatment in immunocompetent mice. After transfer of T-lymphocytes into the SCID
mice, the recurrence rate was delayed drastically2,5. The reason that we did not find an indication for
immunological involvement in tumour response upon PDT treatment as compared to these studies may be the fact that in the mentioned studies tumours of completely different origin were investigated and the mice were observed for a longer time period after PDT treatment. Also, the degree or nature of tumour destruc-tion by PDT may influence the evolvement of an immune response.
An explanation for the apparent lack of a systemic immune response in our study as shown by the absence of infiltrating T cells in and the continuing growth of nearby tumours is the existence of a protective matrix surrounding the tumours. CC531 is known to form nodules of tumour cells, surrounded by stromal
cells with abundant extracellular matrix10. This division in two compartments, i.e. a tumour epithelium
compartment and a stromal compartment that are separated by a basal membrane-like structure, may prevent tumour antigens to contact with the immune system. Tumour cells that have not settled in tissue yet lack this protection and could therefore be sensitive to eradication by cells of the immune system. PDT may be able to evoke a tumour-specific immune response by disrupting tumour structure, thus enabling contact between cells of the immune system and tumour cells. However, this induced response may be unable to reach its target in untreated tumours while it may be able to prevent micro-metastases from growing into
tumours. This is in accordance with the previously mentioned findings of Korbelik et al.4,5 as well as with
studies by Chen et al.11,12 since in their studies, PDT treatment was effective in preventing local recurrence of
the treated tumours, i.e. the outgrowth of remaining tumour cells after severe disruption of the tumour structure.
single cell suspension of the tumour13. This indicated that these NK cells can enter the tumour more easily
after PDT, possibly due to the release of chemotactic factors upon PDT, but disruption of tumour structure may also contribute to these findings. In this same study, there was in fact a beneficial effect of these NK92MI cells on the recurrence rate of PDT treated tumours, decreasing the rate when compared to PDT treatment alone. As PDT disrupts the tumour structure, the administered NK cells may very well be able to destroy the remaining tumour cells and thus prevent outgrowth. However, since these NK cells were genetically altered so as to produce IL-2 and were injected peritumourally, it does not easily transfer to the physiological situation of an immune response. Previous studies in our group showed adoptively transferred
NK cells to be capable of infiltrating CC531 rat liver tumours after ex vivo IL-2 activation10.
The immunohistochemical staining of the tumours in our experiments did not show an increase in macrophages, T or NK cells shortly after PDT treatment. At 2 days after PDT, cells were hardly present in PDT treated tumours, since PDT induced a large area of necrosis and kills not only tumour cells but also other cells that are illuminated. Two weeks later, T cells in stroma at the margins of PDT treated areas were at a similar level as controls. More importantly, the number of T cells in non-illuminated tumours in animals with a PDT treated tumour did not differ at any time point from tumours of control rats. In a study by Qin et al. mice were given peritoneal haematoporphyrin derivative-based PDT, and lymphocytes were isolated from the peritoneal cavity at regular intervals. Three days after PDT, there was nearly complete depletion of peritoneal
T cells. At 10 days after PDT, the amount of T cells was normal again14. These results indicate a decrease in
quantity of T cells shortly after PDT, with levels returning to normal within two weeks after treatment. As we did not find an increased number of T cells in non-treated tumours that were nearby treated tumours, it is likely that the tumour infiltrating T cells were not tumour targeted T cells, but merely a component of the inflammatory response resulting from tumour growth in liver tissue.
In our study, NK-cells were initially present after inoculation of the tumour, but their number decreased in time. At 14 days, levels of NK cells in PDT treated and control tumours did not significantly differ. This is in accordance with the findings of Waterfield et al. that NK cell activity did not differ between treated and
control groups four days after whole body PDT15. Other studies show natural killer cells to have only a
contributory role in an effective PDT response, when given sub optimal doses of photosensitise3,5.
Upon PDT, neutrophils and other inflammation cells reportedly invade the treated area within several
hours after treatment1,16. In our experiments we did not find a large influx of macrophages at two days after
increase in macrophages in the advancing liver margin surrounding the lesion area. Also, the PDT treatment did not result in increased presence of inflammation cells at the nearby tumours.
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