1.
Introduction
Current treatment of solid tumors is mainly based on their
surgical excision.Radiotherapy and chemotherapy are adjuvant treatment
modalities commonly used,depending on tumor type.PhotoDynamic Therapy (PDT) is
a method to treat cancer based on theinteraction between a photosensitizing
agent, light and molecular oxygen. There arenumerous types of photosensitizers,
which can be administered by intravenous, oralor topical routes. The
illumination of the tumor with visible light in order to activatethe
photosensitizer is taken after certain time of agent administration, leading
toproduction of Reactive Oxygen Species (ROS), determining cellular death by
necrosisand/or apoptosis [1]. Methylene Blue (MB) is a molecule that
has been consideredas a drug for photodynamic therapy (PDT). According to [2],MB has the potential to treat a variety
of cancerous and non-cancerous diseases,with low toxicity and no side effects.
An interesting feature of PDT is that it may activate the immune
systemagainst tumor cells. The phototoxic effects on cell membrane release a number
ofinflammatory mediators leading to innate immune system activation.
Localinflammation and neoplastic cell death provoke tumor antigen presentation[3]. Antigenpresentation
promotes activation of adaptive immune system and there issensitization of T lymphocytes
(CD8 +) leading to a specific response to the treatedtumor. This adaptive
immune response would be involved in the killing of tumorcells. In addition, B
lymphocytes are sensitized to tumor antigens, promoting a long-termtumor
control. The production of antitumor antibodies may be suggested,although this
has not yet been shown through experimental modelsin this study[4], we
aimed to verify if treating a primary Ehrlich tumor withMethylene Blue (MB) at
1% based PDT (MB-PDT) could present any systemicinfluence on the growth and
behavior of a second Ehrlich tumor implant in mice.
2.
Materials and Methods
2.1.
Animals
Male Swiss mice, weighing 25-35g, were obtained from the Animal
Facility ofthe Department of Pathology, School of Veterinary Medicine and
Animal Science,University of São Paulo, Brazil. During the experiment, animals
were kept inpolycarbonate boxes covered with wood shavings, kept in a room with
ventilation,exhaust and controlled climate with temperatures between 22 and 24
° C, relativehumidity 55% and light night/day cycle of 12 hours. Animals were
fed with abalanced diet and water ad libitum.
Animal use was in accordance with the ethicalprinciples of animal
experimentation of the Ethics Committee on Animal Use of theSchool of
Veterinary Medicine and Animal Science of the University of Sao Paulo,protocol
number 2235/2011.
2.2.
Experimental Design
Thirty Swiss male mice were inoculated with Ehrlich tumor cells on
thesubcutaneous region of the dorsum. Nine days after, Group 1 mice (n=10) receivedone session of MB-PDT (described
ahead), and Group 2 mice (n=10)
weresubmitted to surgical excision of primary tumor. A third group of mice, Group 3(n=10) received no treatment
and primary tumor kept growing. Twenty-four hoursafter MB-PDT, or surgery of
primary tumor, Group 1, Group 2 and Group 3 mice5received a second Ehrlich tumor implant on the left
footpad; these tumors weremeasured with a caliper for 17 days, when mice were euthanized[5]. At
necropsy,spleen and lymph nodes were weighed and processed for histological
analysis.Tumor samples were collected, routinely processed for embedding in
paraffin, andthe 5μm H&E sections were analyzed trough quantitative morphometry,
according to[6]. In addition, blood samples were takenfor leukocyte count before
euthanasia.
2.3.
Ehrlich Tumor Inoculation
Animals were physically restrained and with sterile syringes and
needleswere given 0.05 ml of suspension containing 2.5 x 106 tumor cells on
thesubcutaneous dorsal region (primary Ehrlich tumor) or left footpad (second
Ehrlichtumor).
2.4.
Ehrlich Tumor Treatment
Group 1-Methylene Blue Photodynamic
Therapy
At ninth day of primary tumor growth, the Group 1 animals received onesession of MB-PDT. Animals were
anesthetized with intraperitoneal administrationofketamine hydrochloride (100
mg/kg) associated with xylasin (20 mg/kg). Thetumor site was shaved and local
antisepsis was performed with iodized 70° alcohols.With sterile needles and
syringes, 0.05 ml of 1.0% methylene blue solution (Synth®) in
sterile 0.9% NaCl was administered by intratumoral injection. After 10
minutes,tumor was irradiated for 9 minutes at a dose of 54 J, using an InGaAIP
mediumdiode laser emitting visible red light at a wavelength of 660 nm and
power output of100 mW. During irradiation, all researchers
wore safety goggles.
Group 2-Surgical Excision
At ninth day of primary tumor growth, the dorsal Ehrlich tumors
from Group 2animals were surgically excised. Animals were anesthetized with
intraperitonealadministration of ketamine hydrochloride (100 mg/kg) associated
with xylasin (20mg/kg). The tumor site was shaved and local antisepsis
performed with iodized 70°alcohols. The tumors were surgically excised, with
margins of approximately 2 mm.Skin sutures were performed with nylon (4-0) in
simple uninterrupted sutures.Quantitative
morphometric analysis of the second Ehrlich tumorVolumetric Fraction (VF) of footpad cellular
componentsImages of H&E sections of the left footpad were taken
with a Nikon DXM1200c® camera coupled to a light microscope Nikon Eclipse E800®. With 20xmagnification
lenses, 8 fields were photographed and images were digitized byIMAGE PRO PLUS® software. Using open
source ImageJ software, a grid with 35points was overlapped on each field. Each
point was determined as tumor cells,inflammatory infiltrate, necrosis and other
(Vessels, Muscle, Subcutaneous, etc.).
Footpad Necrosis Area
Images of H&E sections of the left footpad were taken with a
Nikon DXM 1200c ®camera coupled to a light microscope Olympus model SZX2-ILLK®.
With 0,25xmagnification lenses, the entire footpad area was photographed using
opensource ImageJ software, total area and necrosis area were determined.
Statistical Analysis
For data analysis, the statistical software Minitab 16 ®
(Minitab Inc., PA, USA) and GraphPad Prism5.00 ®
(GraphPad Software, Inc., San Diego, USA) wereused. Normality was verified
using the Anderson-Darling test. Homoscedasticity wasverified by Bartlett test.
For parametric data, we used analysis of variance ANOVAfollowed by the Tukey's
test for comparison between experimental groups. Kruskal-Wallis test followed
by Dunn's test for comparison between groups was used fornon-parametric data.
The growth curve of the Ehrlich tumor was analyzed by two-wayANOVA. Differences
of p <0.05 were considered statistically significant. Datawere expressed
as mean ± standard deviation.
3.
Results
Analysis of Group 1 Ehrlich tumor growththe
primary Ehrlich tumor inoculated at the dorsum of Group 1 animals,which received MB-PDT, presented ulceration and
necrosis; the epithelium healedin a few days. Therefore, the tumors were not
completely cured, and continued togrow.The Group 2 animals, which received surgical excision of primary
tumor, didnot show any signs of local recurrence, with no further primary tumor
growth.The untreated animals of Group 3
showed continuous primary tumor growthto the end of the experiment.
Analysis of Second Ehrlich Tumor Growthas
previously stated, 24 hours after the initial treatment, animals from Group1,
Group 2 and Group 3 were inoculated with second Ehrlich tumor cells on the
leftfootpad, and the tumor growth was evaluated with a caliper for 17 days.
(Figure 1)shows the left
footpad growth curve (diameter in mm) by time (days) of mice bearingEhrlich
solid tumor treated either with PDT, surgery or no treatment (control). Dataare
presented as mean ± standard deviation. There was no significant differencebetween
groups PDT and Surgery and Control (p>0.05).
3.1.
Quantitative Morphometric Analysis
Volumetric Fraction of Footpad Cellular Components:
Tumor Cells and Inflammatory Infiltrate
The second tumor transplant was evaluated through morphometric
analysisusing images of H&E sections of the left footpad. (Figure
2)
3.1.2.
Footpad Necrosis Area
The necrotic areas in the left footpad were measured and the
results areshown in (Figure 3). Using the Kruskal-Wallis test the results are
significantdifferences in the necrotic area in different groups (p = 0.0007).
The Dunn testshowed a significantly smaller area of necrosis (p <0.05)
in the surgery groupcompared with PDT and control groups.
3.2.
Lymphoid Organ Analysis
Relative Spleen Weight
(Figure 4)illustrates data on relative spleen weight. The ANOVA
revealedsignificant difference between groups (p<0.0001).
The Tukey post test showed thatrelative spleen weight of the surgery group (Group 2) was significantly lower (p<0.05)
when compared to PDT (Group 1)
and control (Group 3) groups.
3.2.2.
Spleen Histological Analysis
(Figure 5) illustrates histological pattern found in mice bearing Ehrlich
solidtumor spleen. Surgery group spleens (Group 2) did not show any significantmorphological changes (Figure
5B). Group 1 (MB-PDT) (Figure
5A) and Group 3(control group)
(Figure 5C) mice revealed organ architecture preservation; however,lymphoid
follicles and its germinal center and marginal zone had apparently becomemore
prominent, with coalescent areas between follicles. Findings suggest
lymphoidhyperplasia in the spleen of Group 1 (MB-PDT) animals.
3.2.3.
Relative Lymph Node Weight
The ANOVA regarding the relative weight of the left popliteal
lymph noderevealed no significant differences between groups (p = 0.0622).
Left Popliteal Lymph Node Histological Analysis
Histological sections of popliteal lymph nodes showed similar
patternsbetween groups. There was marked hyperplasia of lymphoid follicles and
germinalcenter increase. There were many mononuclear cells phagocyting cellular
debris(tingible body macrophages) in lymphoid follicles. The pattern is
characteristic ofreactive lymph node hyperplasia. Para cortical and
interfollicular region hyperplasiawere observed and caused by increase of
lymphoid cells with eccentric nuclei,similar to plasma cells. There was also
sinusoidal histiocytosis characterized bymedullary cord hyperplasia and hyper cellularity
of medullary sinuses, filled withreticular cells. All groups had cells with the
phenotypic characteristics of the Ehrlichtumor, including atypical mitoses.
Those cells were observed in the sub capsular andinterfollicular regions,
although no large tumor proliferation foci that wouldcharacterize metastasis
were found.
Blood Leukocyte Count
The ANOVA for leukocyte count revealed no significant differences
betweengroups (data not shown).
4.
Discussion
In this study, we aimed to verify if treating primary tumor with
MB-PDT couldpresent any influence on the growth and behavior of a second tumor.
For thispurpose, a model of subsequent implants of Ehrlich tumor was used.While
the treatment of the solid tumor with MB-PDT caused necrosis in Group1 mice, the
footpad tumor growth curve analysis showed no significant differenceamong
experimental groups 1, 2 or 3, during the 17-day measurement. It is possibleto
see a trend on Group 1 approaching the Group 3 group from day 13. The finalmean
diameter of the surgery group is below the others, but comparing only thisfinal
diameter, there was no statistically significant difference.
The diameter increases of the footpad inoculated with Ehrlich
solid tumor ismainly due to the proliferation of tumor cells, but the inflammatory
reaction is alsoinvolved in increasing tumor size. According to literature, PDT
induces adaptiveimmunity against treated tumor and neoplastic cells that did
not die duringtreatment. Tumor immunity is systemic, long-term and does not
happen only onprimary tumor. The response is observed against metastasis and
recurrence ofspontaneous tumors. This was also seen on experimental models
withtransplantable cells inoculated at distant site from the primary tumor.Treated
fibro sarcomas MS-2 in mice with CASPcphotosensitizer and diode laser with an
irradiation scheme similar to the presentstudy[7]. There was no difference in the survival rate when compared to
surgicaltreatment, but when animals received a second tumor challenge,
surgically treatedanimals soon died while those treated with PDT survived for
up to 100 days[8]used BALB/c mice and
transplantable tumors(Colon 26 and EMT6). PDT was performed with HpD and Argon
laser. These tumorsproduce lung metastases but those diminished after 10 days
on PDT treatedanimals when compared to surgically treated mice.In the present
experimental model, a difference among the growth curve ofanimals previously
treated with MB-PDT, surgical excision or without treatment ofthe primary tumor
was expected. The lack of difference in tumor size can beexplained by
differences of inflammatory response; thus, tumor proliferation andnecrosis.
Although tumor growth curve did not differ between groups, themorphometric
analysis showed significant differences on the VF of footpad cellularcomponents
between surgically treated animals and animals treated with MB-PDTor without
treatment of the primary tumor, as described in literature [7-9].
Surgically treated animals had higher tumor proliferation, less
inflammatoryinfiltrate and a minor production of tumor necrosis. These results
indicate thatanimals treated with surgical excision of the primary tumor did
not develop acuteinflammation and the activation of innate immune system like
that produced by PDT.On surgically treated animals there was no induction of
adaptive immune responseagainst the treated tumor; therefore, tumor
proliferation was larger and occurswithout inducing important inflammation,
unlike that observed on animals treatedwith PDT.
Relative spleen weight showed a significant difference between
groups.Values were higher in MB-PDT and control groups, which may indicate a
morestimulated immune system in those animals. Histopathology showed
hyperplasia ofwhite pulp, confirming the suspicion of a greater immune
stimulation in thesegroups. These results corroborate with Koberlik et al [4]who demonstratedthe role of spleen cells
in the induction of tumor immunity produced by PDT throughtransferring spleen
cells from animals previously treated with PDT toimmunosuppressed animals. In
these animals, the PDT did not promote healing;however, when receiving spleen
cells, the cure rates were back to the levels of theimmune competent
animals.The analysis of the left popliteal lymph node, the lymphoid organ,
revealed nosignificant statistical difference between relative weights on
experimental groups.However, the significance level of p = 0.0622 found is
quite close to the valueconsidered to show significant difference among groups.
Kousis et al. [10]found similar results when assessing by flow
cytometry the cellular components oflymph node from animals treated with PDT.
After PDT, the population of CD8+ Tlymphocytes in the regional lymph node was
higher than in animals treated withsurgical excision of the tumor.
Histopathological analysis showed no differences on lymph node
architecturewithin different groups. The presence of cells with phenotypic
characteristics oftheEhrlich tumor were observed occupying the sub capsular
space in all animalsinoculated on the footpad, as noted earlier by Dagli et al.[11].
The resultsobtained clearly show that animals treated with surgical excision of
the primaryEhrlich tumor, when challenged with a new inoculation of tumor
cells, presentdifferent local and systemic immune responses than animals
treated with PDT or nottreated.The similarity of responses between animals
treated with MB-PDT and thosereceiving no treatment of the primary tumor can be
explained by the fact that theEhrlich tumor is immunogenic. As reported by Guerra (1983) [12]
when animalsbearing Ehrlich solid tumor after 12 days of inoculation receive a
second tumorchallenge, there is no progression of secondary tumor.Here, animals
were treated after 9 days of primary tumor progression. Inanimals treated with
surgical excision, the immune stimulation ceased before the 12days required to induceimmunity;
therefore, we observed growth of secondarytumor and a poor activation of the
immune system (seen in the analysis of lymphoidorgans and morphometry).
Animals treated with MB-PDT had a similar response to
untreatedanimals. When compared to surgically treated animals, they showed less
tumorproliferation and more inflammation andnecrosis of secondary tumor, in
addition togreater systemic response seen in the analysis oflymphoid organs.In
conclusion, the present study demonstrates that applying MB-PDT to aprimary
Ehrlich tumor leads to a different inflammatory and cell death behavior of
asecond Ehrlich tumor implant, possibly due to activation of adaptive immune
system.
These results led us to think that MB-PDT can possibly impact the
development ofmicro metastatic or metastatic growth in a variety of tumors.
Therefore,biotechnological applications of MB-PDT, mainly for the production of
cancervaccines, are currently under investigation.
research article 
