Download 9 General discussion and future perspectives

9
General discussion and future
perspectives
CHAPTER 9
Introduction
Colorectal cancer (CRC) is one of the most prevalent solid cancers in both men
and women in developed countries. Annually, 1.2 million cases are recorded
worldwide, and more than 600.000 patients die of this disease.1 While surgical
resection of the primary colorectal carcinoma is the preferred treatment, it has been
associated with higher risk for metastases development.2-4 Approximately 20-25%
of all patients already have metastatic disease upon diagnosis of CRC.5 However,
another ~10-25% of patients who do not have visible evidence of metastases at
the time of diagnosis and in whom the tumor is removed surgically, will develop
metastases within 5 years.6 This supports the presence of minimal residual disease
that grows out into metastases after surgery. The liver is the major target organ for
development of colorectal metastases, and accounts for ~70% of colorectal-related
deaths.7
Previously, it was demonstrated that peritoneal surgery resulted in vascular
damage in the liver that caused the exposure of sub-endothelial extracellular
matrix (ECM) proteins.4 These are ligands for adhesion molecules like integrins
that are expressed on the cells surface.8 Importantly, adhesion of tumor cells to
the exposed ECM was observed. This supported that performing surgery in the
abdominal cavity initiated systemic inflammatory responses, which enabled tumor
cell adhesion in the liver. Therefore, I investigated the relation between surgeryinduced inflammation and liver metastases development in more detail to gain
new insights in this phenomenon (chapter 2-6). Furthermore, in chapters 7 and
8 it is shown that surgery-induced liver metastases outgrowth can be prevented
with peri-operative monoclonal antibody therapy. The implications for future
perspectives are furthermore discussed in this chapter.
Role of integrin molecules in metastases development
Development of metastases is usually a highly inefficient process. Firstly, tumor
cells need to detach from the primary tumor by down regulating the expression of
adhesion molecules, and enter lymphatic or blood vessels. Secondly, disseminated
tumor cells have a limited life span when they are unable to adhere, and can be
rapidly cleared by the immune system. However, previous studies supported
that surgical trauma increased the risk of metastases development.9, 10 It was
demonstrated that animals undergoing laparotomy (opening of the peritoneal
cavity) developed more liver metastases compared to non-operated control
animals.4 Therefore, it was previously proposed that during surgery tumor cells
may be spilled from the primary tumor without the need of reduced expression
of adhesion molecules on cells surface.4 Because of high expression of adhesion
molecules on tumor cells and surgery-induced exposure of ECM proteins, to
which these cells adhere preferentially, metastases formation is increased. It was
demonstrated in animal models that blockade of integrins a2 or b1 prevented
tumor cell adhesion in the liver or peritoneal cavity, respectively, whereas a5
was not involved.2, 4 Importantly, when integrin a2 on tumor cells was blocked,
development of liver metastases was successfully prevented, supporting that
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integrin a2 was essential for tumor cell adherence. In chapter 2 we investigated
the expression of integrins a2, a5 and b1 in primary colorectal tumors and
correlated this with the survival of patients. In this retrospective study we found
that high expression of integrin a2 positively correlated with lower overall survival.
Moreover, patients with high integrin a2 expression in the primary tumor had
higher risk of metastases development. Unfortunately, this did not reach statistical
significance, which may be due to limited numbers of participating patients. In a
previous study higher expression levels of integrin a2 in colorectal liver metastases
was observed, compared to lung metastases.11 Therefore I speculate, the liver may
contain a specific composition of ECM proteins, which favors the adhesion of
tumor cells with high levels of a2 integrins expression. Adhesion of these cells may
result in formation of liver metastases. Alternatively, it is also possible that because
of high expression of integrin a2 tumor cells from colorectal cancers are able to
adhere easily in the first organ they pass, which is the liver, and grow out into
metastases. We did not find any correlation between integrins a5 or b1 expression
and patients’ survival. Previously, it was shown that blockade of integrin b1 was
not sufficient to prevent tumor cells adherence in the liver.4 Strikingly, tumor cells
adhesion to the peritoneal cavity was inhibited when cells were incubated with
antibody against integrin b1.2 Furthermore, it was demonstrated that high levels
of integrin a5 gave rise to kidney metastases formation.12 All these data indicate
that expression of specific integrins on tumor cells facilitate metastases formation
in different target organs.
Because expression of integrins on tumor cells mediates metastases formation,
blockade of integrins on tumor cells may represent an attractive therapeutic
strategy. However, resection of the primary tumor unavoidably introduces a
wound in the peritoneal cavity and the intestines. Wound healing is a process in
which the integrins also play a central role.13, 14 Thus, blockade of integrins on tumor
cells with the intention to prevent tumor cells adhesion, may impair wound healing
as well. Hampered wound healing may cause post-surgical anastomic leakage.
Since patients with anastomic leakage were shown to have serious complications
and poorer survival,15 I strongly caution against these therapies before extensive
research has been performed to prove safety of this approach.
Role of surgery-induced inflammation in development of liver metastases
Previous studies demonstrated that surgery caused the release of inflammatory
mediators, including reactive oxygen species (ROS).16-18 These compounds were
suggested to be involved in tumor development by facilitating tumor cells
adhesion.19 Incubation of endothelial monolayers with ROS for 12 hours was shown
to result in up-regulation of adhesion molecule expression on endothelial cells such
as endothelial-selectin (E-selectin), inter-cellular adhesion molecule-1 (ICAM-1)
and vascular cell adhesion molecule-1 (VCAM-1).19 These adhesion molecules were
suggested to be involved in enhanced tumor cells adhesion to endothelial cells. In
chapter 3 we now demonstrate that incubation of endothelial monolayers with
comparable amounts of ROS resulted in damage of the monolayers. Subsequently,
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intercellular gaps were formed that led to exposure of sub-endothelial ECM
components to which tumor cells adhered preferably. Furthermore, we showed that
surgery led to impairment of liver vasculature both in experimental animal models
and in human liver samples. Treatment of animals with a ROS scavenger prevented
vascular damage. Importantly, when rats were treated with a ROS scavenger we
observed significantly less tumor cell adherence in the livers compared to rats that
were treated with the vehicle only. When liver resident macrophages, Kupffer cells
(KCs) were depleted, we also observed less tumor cells adhesion, suggesting that
KCs are involved in ROS production. Unfortunately, treatment with ROS scavenger
was not sufficient for prevention of metastases development. Previous studies
demonstrated that KCs recognize and kill malignant cells in a ROS dependent
manner.20, 21 Thus, scavenging of ROS during surgery likely acts as a double-edged
sword. First, surgery stimulates the production of ROS by KCs, which leads to
disruption of endothelial lining. This causes the exposure of sub-endothelial ECM
proteins to which circulating tumor cells bind preferably. Second, interruption of
ROS production and/or scavenging system may imbalance the tumor cell killing
by KCs, which also requires ROS. This may finally lead to outgrowth of metastases
from adhered tumor cells. Thus, overall scavenging of ROS may not have beneficial
clinical effects in prevention of liver metastases formation.
Role of bacterial products in metastases development
Resection of the primary colorectal tumor furthermore resulted in translocation
or spillage of bacterial products from the gut lumen to the peritoneal cavity.15,
22-26
Interestingly, patients with positive bacterial translocation or anastomic
leakage had poor clinical outcome.15, 24 Bacterial products are potent activators of
strong immune responses, which may be involved in mediating damage to liver
vasculature. This subsequently may lead to liver metastases formation. Because
in our experimental laparotomy model the intestines were not resected, the
additional effects of bacterial components on metastases formation were not
investigated. Therefore, we developed a second experimental colectomy model in
which a small part of the colon was removed surgically, that was followed by endto-end anastomosis (chapter 4). Smear samples were taken from the peritoneal
surface of the colon of rats undergoing colectomy at the begin and the end of the
surgical procedure. Bacterial outgrowth was significantly increased in the samples
that were taken from the anastomosis. Moreover, we observed decreased levels
of tight junction protein expression in the livers of rats that received colectomy,
compared with the livers of control rats or rats that underwent laparotomy.
Subsequently, enhanced numbers of adhered tumor cells were detected in the
livers of rats that underwent colectomy, compared to the livers of control rats or rats
from the laparotomy group. Importantly, rats from the colectomy group developed
significantly more liver metastases. Previous studies demonstrated that severity of
surgical trauma correlated with development of liver metastases development.27
Thus, one explanation might be that rats undergoing colectomy developed more
liver metastases because of higher extent of surgical trauma. However, in our
colectomy model we also found significantly increased bacterial contamination
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after surgery, which also is observed in patients. This may lead to systemic exposure
to bacterial products. Importantly, patients with bacterial translocation or anastomic
leakage after surgery had significantly shorter disease-free survival and lower overall
survival. Moreover, local and systemic recurrence rates were higher in patients
with anastomic leakage after surgery.15 This indicated that exposure to bacterial
components during surgery has a negative effect on long term clinical outcome.15, 24,
28
Therefore, we propose that enhanced tumor development in our colectomy model
is mainly caused by bacterial products that are released or spilled during resection
in the colon.
Bacterial products are potent initiators of inflammatory immune responses through
interacting with Toll-like receptors (TLRs). For instance, the bacterial outer membrane
component LPS is the main ligand for TLR4. This receptor is expressed by wide variety
type of immune cells such as polymorphonuclear cells (PMNs) and KCs.29 Previously,
it was demonstrated that abdominal surgery led to attraction of high numbers of
PMNs to the peritoneal cavity.30 Interestingly, depletion of PMNs prevented local
recurrence of tumors. Furthermore, it was demonstrated that incubation of PMNs
with mesothelial monolayers enhanced the expression of adhesion molecules on
mesothelial cells. These molecules mediated increased tumor cells adherence.31
Thus, these data suggested that PMNs might be involved in development of tumors.
Therefore, in chapter 5 we investigated the potential role of PMNs in initiation of
liver metastases development. In the livers of rats that had received laparotomy,
increased numbers of PMNs were observed, compared to the livers of control
rats. Moreover, the numbers of PMNs was further increased when rats underwent
colectomy. As we observed enhanced bacterial translocation in rats after colectomy
(see above), exposure to bacterial components likely led to sequestration of PMNs
in the liver. This is supported by previous studies that showed that LPS injection
resulted in accumulation of PMNs in the livers of mice.32, 33 Importantly, the numbers
of adhered tumor cells in the livers of rats either undergoing laparotomy of
colectomy were elevated as well, compared to control rats. This suggested a relation
between high numbers of PMNs and tumor cells adherence. As was demonstrated
previously,34 incubation of PMNs with LPS rapidly induced ROS release. In chapter 3
we observed that ROS had detrimental effects on endothelial monolayers. Therefore,
we investigated whether incubation of endothelial monolayers with PMNs and LPS
causes endothelial damage. When endothelial monolayers were incubated with
PMNs and LPS, formation of intercellular gaps were observed. This consequently
led to exposure of sub-endothelial ECM proteins, as we demonstrated in chapter 3.
Furthermore, injection of rats with LPS resulted in high numbers of both PMNs and
tumor cells in their livers. This suggested that LPS led to significant attraction of PMNs
to the liver that subsequently caused damage to the liver vasculature, resulting in
increased tumor cell adhesion. Importantly, both PMN and tumor cell accumulation
that was caused either by laparotomy or LPS administration (without surgery) was
prevented when rats were treated with a ROS scavenger. Thus, these data suggested
that activation of PMNs by LPS led to ROS production, which subsequently caused
damage to the liver vasculature and facilitated tumor cell adhesion.
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Additionally, it was shown that LPS activates KCs.35 Furthermore, previously it was
suggested that macrophages play a role in liver metastases development.21 Therefore,
we evaluated the involvement of KCs in initiation of metastases development (chapter
6). When rats were injected with LPS (in the absence of surgery), tumor cell adherence in
the livers was ameliorated significantly, compared to the livers of rats that were injected
with saline. A previous study suggested that LPS increased the expression of adhesion
molecules on tumor cells.36 Therefore, we tested whether LPS influenced tumor cells
adhesion to either different ECM coatings or endothelial layers. Incubation of tumor cells
with varying concentrations of LPS for different time points, did not affect the adherence
of tumor cells on various ECM coatings (data not shown). Moreover, incubation of tumor
cells or endothelial monolayers with LPS did not enhance tumor cell adherence to
endothelial layers. These data indicated that tumor cell adhesion that was increased by
LPS injection of rats was not caused by enhanced or altered adhesion to the endothelial
lining.
KCs are however in close proximity of sinusoidal endothelial cells.37 Interaction of LPS
with its receptor on KCs was furthermore shown to induce the release of inflammatory
mediators including ROS.29, 35, 38 Subsequently, release of ROS by KCs may be harmful for
the integrity of liver vasculature. Therefore, we tested the effect of LPS to co-cultures
of endothelial cells and macrophages. We found that addition of LPS had detrimental
effects on endothelial monolayers and caused intercellular gaps formation. Moreover,
endothelial damage was prevented when ROS scavengers were added. This indicated
that tumor cell adherence that was enhanced by LPS injection was due to damaged liver
vascular lining. Importantly, depletion of KCs or using a ROS scavenger in vivo significantly
reverted tumor cell adherence that was enhanced by LPS injection. Thus, LPS-mediated
tumor cells adherence is ROS dependent and KCs play an important role in this process.
In chapter 5 we observed that the highest numbers of tumor cells had adhered
after 1.5 hours following laparotomy, while the numbers of PMNs was maximal after
6 hours. This suggested that tumor cells adherence might be PMNs independent.
Because we found that KCs play a pivotal role in tumor cells adherence, we studied
whether KCs are involved in accumulation of PMNs and tumor cells after laparotomy
or LPS injection. Interestingly, the livers of rats in which KCs were depleted contained
significantly less PMNs and tumor cells after laparotomy or LPS injection (Figure 1).
Thus, KCs play a regulatory role in sequestration of PMNs and tumor cells in the liver.
Therefore I hypothesize that activation of KCs results in release of ROS, which damages
the liver vasculature. Subsequently, sub-endothelial ECM is exposed to which both
PMNs and tumor cells adhere in high numbers (Figure 2). Additionally, as soon as PMNs
are adhered and activated they may also release ROS and exacerbate liver vasculature
damage. Our data are supported by a previous study, which demonstrated that PMNs
sequestration in the liver during inflammation is diminished when KCs were depleted
or non-functional.39 Furthermore, it was demonstrated that increased binding of
PMNs in the liver vasculature after LPS treatment was due to altered interaction with
ECM.33 The authors speculated that LPS-induced ROS production led to modification
to ECM in the liver that facilitated the increased PMNs adherence.
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GENERAL DISCUSSION AND FUTURE PERSPECTIVES
Figure 1: KCs regulate the accumulation of PMNs and tumor cells in the liver. a: the numbers of PMNs
and tumor cells in the liver of control and KCs depleted rats after laparotomy. b: PMNs and tumor cells
in the livers of control or KCs depleted rats, that were treated with saline or LPS. green=His48+ cells;
blue=cell nudei. *p<0.05, **p<0.01, ***p<0.001
Antibody therapy
Thus, surgery leads to undirected activation of KCs resulting in undesired damage
of liver vasculature and increased tumor cell adhesion. However, directed activation
of KCs that specifically target tumor cells was suggested to be advantageous in
prevention of liver metastases development.40 Anti-tumor immune responses
can be stimulated with the use of monoclonal antibodies (mAb). Previously it was
demonstrated that mAbs may reduce tumor growth directly by inhibiting tumor cell
proliferation, induction of programmed cell death (apoptosis) or sensitizing tumor
cells for chemotherapy.41 Alternatively, mAbs may also control tumor progression
indirectly by involving the immune system. One of the indirect mechanisms is
complement dependent cytotoxicity (CDC).42 In this process, mAb binding to
antigens activates molecules of the classic complement pathway, resulting in
perforation of the cell membrane, leading to cell death. Additionally, mAb can form
a bridge between tumor cells and Fc receptor-expressing immune cells, which can
lead to lysis of tumor cells via a process referred to as antibody-dependent cellular
cytotoxicity (ADCC).43 Traditionally, natural killer (NK) cells have been considered
as main effector cells for ADCC.44 After formation of immune synapses between the
mAb-opsonized target cells and NK cells, target cells are eliminated by directed
release of cytotoxic compounds from NK cells.
Alternatively, a role for macrophages was proposed in tumor targeting
antibody immunotherapy. A previous study demonstrated that liver metastases
development was successfully prevented by mAb treatment, which was dependent
on the presence of either FcgRI or FcgRIV.45 Since monocytes/ macrophages are the
only cells that express both receptors in mice, a role for these cells was strongly
supported. Recognition of mAb-coated target cells by macrophages or monocytes
leads to phagocytosis and subsequent degradation in a process that is referred as
antibody-dependent phagocytosis (ADPh).46
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Before
KCs
Endothelial cells
Hepatocytes
After
PMNs
Tumor cells
ROS
ECM
LPS
Figure 2: Schematic overview of the liver microvasculature before and after surgery. Surgery-released
factor(s) such as LPS cause the initiation of systemic immune responses that result in activation of the
liver resident macrophages, KCs. Activated KCs release ROS that damages the sinusoidal endothelial
cells and leads to exposure of sub-endothelial extracellular matrix (ECM) proteins. Circulating PMNs
and tumor cells preferably adhere to exposed ECM. Additionally, adhered PMNs can produce ROS as
well, and may exacerbate the damage to the sinusoidal microvasculature, stimulating the adherence of
tumor cells that grow out in liver metastases.
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GENERAL DISCUSSION AND FUTURE PERSPECTIVES
In a previous study, co-localization of tumor cells and KCs in the liver was observed.46
Interestingly, treatment with mAb in vivo increased co-localization with 14%.
Nonetheless, even though mAb therapy only marginally increased co-localization
of tumor cells and KCs, massive impact on survival was observed, as mAb treated
animals did not develop liver metastases, in contrast to isotype-treated controls.45,
46
Since depletion of KCs was shown to abolish the anti-tumoral effects of mAbs,
an essential role for KCs in tumor prevention was proposed.45, 46 However, the
exact mechanism of how KCs are involved in mAb-mediated prevention of tumor
development remained unknown. To understand these results and investigate
the mechanisms of effective mAb therapy in more detail, we therefore performed
intravital microscopy (in chapter 7). Kupffer cells were able to sample small parts of
tumor cells when mice were treated with PBS or an isotype control, but phagocytosis
of whole tumor cells was limited. Still, these results explained why co-localization
between Kupffer cells and tumor cells was observed with immunohistochemistry
experiments. By contrast, Kupffer cells phagocytosed complete tumor cells rapidly
when mice were treated with tumor specific mAb. Thus, although no difference
was observed in the numbers of tumor cells that were in contact with KCs, a
significantly increased number of tumor cells were ingested by KCs of mice that had
been treated with specific anti-tumor mAb. Furthermore, the fate of tumor cells 24
hours after injection was investigated. Although red fluorescent dye was still visible
(indicative of tumor material), particles were significantly smaller in mAb-treated
mice, supporting breakdown inside macrophages. 3D reconstruction confirmed
that tumor cells in the livers of untreated mice poorly co-localized with Kupffer
cells. Furthermore, large clusters of tumor cells were observed, which supports
outgrowth. In contrast, most tumor material was encapsulated by Kupffer cells in
mAb-treated animals, indicating effective degradation of tumor cells.
Recently, mice were injected with tumor cells and mAb against tumor cells in the
peritoneal cavity.47 After peritoneal lavage, interactions between cells were studied
microscopically. Interestingly, formation of immune synapses between tumor
cells and immune cells after mAb treatment was demonstrated, suggesting the
elimination of tumor cells via ADCC.48 In contrast, we observed the liver of mice
constantly after injection of tumor cells with an intravital microscope. We found
that treatment with mAb led to effective uptake of tumor cells by KCs through
ADPh within minutes after injection. Moreover, depletion of KCs abrogated mAbmediated anti-tumoral response. This strongly indicated that mAbs therapy leads
to ADPh in the liver and KCs are the main effector cells in this process.
Importantly, previous studies demonstrated the presence of circulating tumor cells
in peripheral blood of colorectal cancer patients.49-56 Moreover, the numbers of
circulating tumor cells were increased during or after surgery, especially in portal
blood, suggesting the dissemination of tumor cells by surgical manipulation.
Increased numbers of circulating tumor cells furthermore correlated with
poor patient prognosis.57, 58 Thus peri-operative mAb treatment may eliminate
disseminated tumor cells. Successful mAb therapy however depends on the
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expression of an (specific) antigen by tumor cells. Epidermal growth factor receptor
(EGFR) is up regulated in 60-80% of colorectal cancer cases and was therefore
suggested as a potential target for mAb therapy.59 The anti-EGFR mAb Cetuximab
is an antibody that is already approved for clinical use. It was demonstrated that
binding of Cetuximab to EGFR prevented cell proliferation and therefore inhibited
tumor progression.60 However, in patients with established colorectal tumors
Cetuximab treatment resulted in disappointing response rates of 11% that were
increased to 23% when mAb therapy was combined with chemotherapy.59 Previous
studies suggested that therapeutic actions of Cetuximab was mainly dependent
on the RAS/RAF signaling cascade.60, 61 Interference with RAS/RAF signaling results
in disruption of several processes such as cell cycle progress and apoptosis, which
finally prevents tumor growth.60 Mutational changes in these proteins impair the
response to anti-EGFR mAb therapy, which is likely the cause of disappointing
clinical results. However, anti-EGFR mAb may be used successfully in patients with
colorectal tumors who undergo surgical resection of the tumor, as binding of mAbs
to EGFR on circulating tumor cells may result in phagocytosis by macrophages.
In chapter 8, we demonstrate that incubation of tumor cells with a mAb against
EGFR (Zatulumumab) effectively enhanced tumor cell phagocytosis and killing by
macrophages. This was only dependent on surface expression of EGFR on malignant
cells. Importantly, cell lines with mutated proteins of RAS or RAF were also efficiently
phagocytosed and killed. Subsequently, after phagocytosis, lysosomal fusion with
the phagosome led to cancer cell degradation. Thus, anti-EGFR mAbs may be
used successfully for prevention of surgery-induced liver metastases. Importantly,
mutations of cell signaling proteins do not affect this process. As we showed in
animal models, surgery-induced liver metastasis is successfully prevented by mAbs
that target tumor specific antigens. Therefore, I propose that clinical studies must
be implemented as soon as possible to investigate this in patients.
Conclusions
In this thesis I show that surgery-induced inflammation and/or bacterial products
that are spilled during surgery cause the activation of KCs. Activated KCs release
the inflammatory mediator ROS that damages the sinusoidal endothelial lining
and disrupts liver microvasculature. Subsequently, this leads to exposure of subendothelial ECM proteins. Circulating tumor cells and PMNs adhere to exposed
ECM via their adhesion molecules like integrin proteins (Figure 2). However,
experimental therapy using an anti-oxidant to prevent metastases formation
was not successful. This may be due to interference with ROS dependent tumor
cell killing by macrophages. Importantly, injection of tumor specific mAb led to
effective phagocytosis of tumor cells by KCs and therefore was sufficient for
prevention of liver metastases formation.
Future perspectives and recommendations
Detailed knowledge of inflammatory responses that occur in the period during and
immediately after surgery is pivotal for development of new therapeutic strategies
to prevent metastases development. We demonstrated that abdominal surgery
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GENERAL DISCUSSION AND FUTURE PERSPECTIVES
and the bacterial component LPS – is spilled during surgery - stimulated tumor cell
adhesion. Thus, improvement of surgical techniques to minimize surgical trauma,
bacterial translocation and spillage of tumor cells may improve patients’ outcome.
However, surgical resection of the primary colorectal tumor inevitably leads to
tissue damage. Therefore, additional therapies are required to prevent surgeryinduced inflammatory responses. For instance, prevention of the interaction
between LPS and its receptor TLR4 on immune cells may avoid undesired activation
of macrophages that facilitates tumor cell adherence. Effects of LPS scavengers or
TLR4 antagonists on tumor cells adherence should be investigated in more detail.
Furthermore, spillage of bacteria may also result in enhanced concentration of
other bacterial components such as lipoproteins, lipopeptides or flagellin. Each of
these bacterial components can trigger different TLRs such as TLR1, 2, 5 and 6 and
initiate similar potent immune responses, compared to TLR4.29 To find out which
bacterial product(s) or TLRs are involved in enhanced tumor cells adhesion, animals
with specific knock outs of these receptors or TLR signaling pathway proteins
may be used. Detailed knowledge about the role of bacterial products in tumor
development may serve to design novel targeted therapies. Moreover, clinical
studies can be performed investigating whether selective decontamination of the
digestive tract, eliminating the bacterial presence in the intestines, may prevent
recurrence of local or distant metastases.
Furthermore, we demonstrated that undirected activation of KCs during surgery
facilitated tumor cell adhesion. However, we also showed that KCs are the most
important effector immune cells in mAb therapy against circulating tumor cells.
Thus, directed activation of KCs may improve their anti-tumoral actions and
prevent tumor formation. Therefore, I hypothesize that pre- or peri-operatively
administration of mAbs against tumor antigens, which induce efficient ADPh, will
inhibit metastases development. Clinical studies should be executed to investigate
the effects of mAbs on surgery-induced liver metastases development.
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