Combined treatment of human colorectal tumor cell lines with chemotherapeutic agents and ionizing irradiation can in vitro induce tumor cell death forms with immunogenic potential.
ABSTRACT Chemotherapeutic agents (CT) and ionizing radiation (X-ray) induce DNA damage and primarily aim to stop the proliferation of tumor cells. However, multimodal anti-cancer therapies should finally result in tumor cell death and, best, in the induction of systemic anti-tumor immunity. Since distinct therapy-induced tumor cell death forms may create an immune activating tumor microenvironment, this study examined whether sole treatment with CT that are used in the therapy for colorectal cancer or in combination with X-ray result in colorectal tumor cell death with immunogenic potential. 5-Fluorouracil (5-FU), Oxaliplatin (Oxp), or Irinotecan (Irino) in combination with X-ray were all potent inhibitors of colorectal tumor cell colony formation. This study then examined the forms of cell death with AnnexinA5-FITC/Propidium Iodide staining. Necrosis was the prominent form of cell death induced by CT and/or X-ray. While only a combination of Irino with X-ray leads to death induction already 1 day after treatment, also the combinations of Oxp or 5-FU with X-ray and X-ray alone resulted in high necrosis rates at later time points after treatment. Inhibition of apoptosis increased the amount of necrotic tumor cells, suggesting that a programmed form of necrosis can be induced by CT + X-ray. 5-FU and Oxp alone or in combination with X-ray and Irino plus X-ray were most effective in increasing the expression of RIP, IRF-5, and p53, proteins involved in necrotic and apoptotic cell death pathways. All treatments further resulted in the release of the immune activating danger signals high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70). The supernatants of the treated tumor cells induced maturation of dendritic cells. It is, therefore, concluded that combination of CT with X-ray is capable of inducing in vitro cell death forms of colorectal tumors with immunogenic potential.
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ABSTRACT: To investigate the fate of apoptotic cells in the germinal centers (GCs) of patients with systemic lupus erythematosus (SLE). Lymph node biopsy specimens obtained from 7 SLE patients with benign follicular hyperplasia, 5 non-SLE patients with benign follicular hyperplasia (non-SLE), 5 patients with malignant follicular lymphoma, and 3 patients with dermatopathic lymphadenitis were stained with monoclonal antibodies against macrophages (CD68) and follicular dendritic cells (CR2/CD21). TUNEL staining and transmission electron microscopy were performed to detect apoptotic cells. Confocal microscopy was used to evaluate the in vivo capacity of tingible body macrophages to remove apoptotic cell material. In a subgroup of patients with SLE, apoptotic cells accumulated in the GCs of the lymph nodes. The number of tingible body macrophages, which usually contained engulfed apoptotic nuclei, was significantly reduced in these patients. In contrast to what was observed in all controls, TUNEL-positive apoptotic material from SLE patients was observed to be directly associated with the surfaces of follicular dendritic cells (FDCs). Our findings suggest that in a sub-group of SLE patients, apoptotic cells are not properly cleared by tingible body macrophages of the GCs. Consequently, nuclear autoantigens bind to FDCs and may thus provide survival signals for autoreactive B cells. This action may override an important control mechanism for B cell development, resulting in the loss of tolerance for nuclear antigens.Arthritis & Rheumatism 02/2002; 46(1):191-201. · 7.48 Impact Factor
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ABSTRACT: A novel cell type has been identified in adherent cell populations prepared from mouse peripheral lymphoid organs (spleen, lymph node, Peyer's patch). Though present in small numbers (0.1-1.6% of the total nucleated cells) the cells have distinct morphological features. The nucleus is large, retractile, contorted in shape, and contains small nucleoli (usually two). The abundant cytoplasm is arranged in processes of varying length and width and contains many large spherical mitochondria. In the living state, the cells undergo characteristic movements, and unlike macrophages, do not appear to engage in active endocytosis. The term, dendritic cell, is proposed for this novel cell type.Journal of Experimental Medicine 06/1973; 137(5):1142-62. · 13.21 Impact Factor
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ABSTRACT: Although cancer progression is primarily driven by the expansion of tumor cells, the tumor microenvironment and anti-tumor immunity also play important roles. Herein, we consider how tumors can become established by escaping immune surveillance and also how cancer cells can be rendered visible to the immune system by standard therapies such as radiotherapy or chemotherapy, either alone or in combination with additional immune stimulators. Although local radiotherapy results in DNA damage (targeted effects), it is also capable of inducing immunogenic forms of tumor cell death which are associated with a release of immune activating danger signals (non-targeted effects), such as necrosis. Necrotic tumor cells may result from continued exposure to death stimuli and/or an impaired phosphatidylserine (PS) dependent clearance of the dying tumor cells. In such circumstances, mature dendritic cells take up tumor antigen and mediate the induction of adaptive and innate anti-tumor immunity. Locally-triggered, systemic immune activation can also lead to a spontaneous regression of tumors or metastases that are outside the radiation field - an effect which is termed abscopal. Preclinical studies have demonstrated that combining radiotherapy with immune stimulation can induce anti-tumor immunity. Given that it takes time for immunity to develop following exposure to immunogenic tumor cells, we propose practical combination therapies that should be considered as a basis for future research and clinical practice. It is essential that radiation oncologists become more aware of the importance of the immune system to the success of cancer therapy.Current Medicinal Chemistry 03/2012; 19(12):1751-64. · 4.07 Impact Factor
Standard tumor therapies as radiotherapy (RT) and
chemotherapy (CT) primarily aim to stop the tumor
cell’s proliferation. Since RT attacks the tumor locally
and CT acts systemically, those two treatments are
often combined. However, during the last years it has
become more and more evident that targeting the tumor
cells by either RT or CT is capable to induce anti-tumor
immunity (summarized in Hannani et al., 2011). The
standard treatments modify the tumor cells and may
thereby render them visible for the immune system (Frey
et al., 2012). Especially low and ultra-low doses of CT
Combined treatment of human colorectal tumor cell lines
with chemotherapeutic agents and ionizing irradiation
can in vitro induce tumor cell death forms with
Benjamin Frey1, Christina Stache1,2, Yvonne Rubner1, Nina Werthmöller1, Kathrin Schulz1,
Renate Sieber1, Sabine Semrau1, Franz Rödel3, Rainer Fietkau1, and Udo S. Gaipl1
1Department of Radiation Oncology, 2Department of Neuropathology, University Hospital Erlangen,
Friedrich-Alexander University of Erlangen-Nürnberg, Germany, and 3Department of Radiotherapy and
Oncology, University of Frankfurt, Germany
Chemotherapeutic agents (CT) and ionizing radiation (X-ray) induce DNA damage and primarily aim to stop the
proliferation of tumor cells. However, multimodal anti-cancer therapies should finally result in tumor cell death and,
best, in the induction of systemic anti-tumor immunity. Since distinct therapy-induced tumor cell death forms may
create an immune activating tumor microenvironment, this study examined whether sole treatment with CT that
are used in the therapy for colorectal cancer or in combination with X-ray result in colorectal tumor cell death with
immunogenic potential. 5-Fluorouracil (5-FU), Oxaliplatin (Oxp), or Irinotecan (Irino) in combination with X-ray were
all potent inhibitors of colorectal tumor cell colony formation. This study then examined the forms of cell death
with AnnexinA5-FITC/Propidium Iodide staining. Necrosis was the prominent form of cell death induced by CT and/
or X-ray. While only a combination of Irino with X-ray leads to death induction already 1 day after treatment, also
the combinations of Oxp or 5-FU with X-ray and X-ray alone resulted in high necrosis rates at later time points after
treatment. Inhibition of apoptosis increased the amount of necrotic tumor cells, suggesting that a programmed form
of necrosis can be induced by CT + X-ray. 5-FU and Oxp alone or in combination with X-ray and Irino plus X-ray were
most effective in increasing the expression of RIP, IRF-5, and p53, proteins involved in necrotic and apoptotic cell death
pathways. All treatments further resulted in the release of the immune activating danger signals high-mobility group
box 1 (HMGB1) and heat shock protein 70 (HSP70). The supernatants of the treated tumor cells induced maturation
of dendritic cells. It is, therefore, concluded that combination of CT with X-ray is capable of inducing in vitro cell death
forms of colorectal tumors with immunogenic potential.
Keywords: X-radiation, chemotherapeutic agents, colorectal cancer cells, necrosis, apoptosis, necroptosis, danger
signals, dendritic cell activation, immunogenicity
B. Frey and C. Stache contributed equally to this work.
Address for Correspondence: Priv.-Doz. Dr. Udo S. Gaipl, PhD, Department of Radiation Oncology, Radiation Immunobiology, University
Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Universitätsstr. 27, 91054 Erlangen, Germany.
Tel: 4991318532311. Fax: 4991318539335. Email: firstname.lastname@example.org
(Received 21 March 2012; revised 24 April 2012; accepted 08 May 2012)
Journal of Immunotoxicology, 2012; 9(3): 301–313
© 2012 Informa Healthcare USA, Inc.
ISSN 1547-691X print/ISSN 1547-6901 online
302 B. Frey et al.
Journal of Immunotoxicology
have been demonstrated to increase the immunogenicity
of human colon cancer cells (Kaneno et al., 2011).
More than 95% of malignant cancers of the gut
are colorectal carcinoma. Over time, the optimal
choice of adjuvant chemotherapy has changed from a
5-Fluorouracil (5-FU)-based chemotherapy alone to the
so-called FOLFOX chemotherapy regimen that contains
5-FU + Leucovorin (folinic acid) and Oxaliplatin (Oxp)
(Andre et al., 2004). Another combination partner besides
Oxp is Irinotecan (Irino) (Goodwin and Asmis, 2009).
Since the rates of local recurrence and of distant metasta-
ses of locally advanced rectal cancer are still high, inten-
sified neo-adjuvant radiochemotherapy (RCT) should
be applied. Cytostatic drugs such as Oxp and Irino are
good candidates for the development of those protocols
(Klautke and Fietkau, 2007). Whether CT acts as radio-
sensitizer has not been proven yet; this is even though, in
advanced colorectal cancer, 5-FU-based RCT improved
local control and had some influence on systemic relapses
and on overall survival compared to RT only (Glimelius et
al., 2008). The latter facts give strong hints that systemic
anti-tumor immune responses were induced by RCT.
Therefore, to gain a better understanding about the mode
of action of novel treatment schemes for colorectal cancer,
we examined the effects of 5-FU, Oxp, and Irino—alone
and in combination with ionizing radiation (X-ray)—
on clonogenic potential, mode of cell death, cell death
pathways, and in vitro immunogenic potential of human
colorectal tumor cell lines.
While many data exist concerning how chemotherapeu-
tic agents and X-ray induce a stop in tumor cell proliferation
after setting the DNA damage, inhibit cell cycle progres-
sion, and how CT sensitizes tumor cells to RT (Pauwels
et al., 2010), little is known about the forms of tumor cell
death inclusive of their immunogenic potential after expo-
sure to X-ray and/or CT. The two most well-known forms of
cell death are apoptosis and primary necrosis. When inter-
acting with professional phagocytes like macrophages,
apoptotic cells exert non- or anti-inflammatory effects,
while necrotic cells induce inflammation (Voll et al., 1997).
However, by exposing normally intracellular located pro-
teins on their surface like the endoplasmic reticulum (ER)-
derived chaperone calreticulin (CRT), apoptotic cells are
recognized by dendritic cells (DC) and thereby become
immunogenic (Obeid et al., 2007).
Under healthy conditions, apoptotic cells are swiftly
cleared by macrophages and do not proceed to second-
ary necrosis. However, in chronic autoimmune diseases
the clearance machinery of the body is overcharged and
apoptotic cells accumulate. The latter lose their mem-
brane integrity during time and are then called secondary
necrotic cells (Gaipl et al., 2006). A disturbed clearance of
the body’s own cells therefore might lead to chronic auto-
immune disease, as has been described by our group and
by others with respect to systemic lupus erythematosus
(Botto, 2001; Baumann et al., 2002). Under conditions
with massive cell death induction by CT in combina-
tion with RT, the resulting ‘immunotoxicity’ could be
beneficial when considering immune reactions that are
initiated not against healthy but against the cancer cells.
In the present study, we therefore first focused on
the cell death forms induced by CT and RT and on pos-
sible mechanisms leading to cell death induction. The
molecular characterization of pathways triggering the
sensitivity towards chemotherapeutic agents is essential
to improve therapies against cancer (Rohwer et al., 2010).
Many types of DNA damage are known to finally trigger
apoptosis (Roos and Kaina, 2006). However, besides
apoptosis, necrosis is also observed in cells derived from
human solid tumors after CT (Rogalska et al., 2010).
Since inhibition of CT-induced apoptosis in tumor cells
by pan-caspase inhibitors as zVAD-fmk (zVAD) some-
times does not result in the expected reduction of cell
death, alternative ways of programmed dying have been
suggested (Aresvik et al., 2010; Wu et al., 2011). Hitomi
et al. (2008) described a molecular pathway of pro-
grammed necrotic cell death, the so-called necroptosis.
Besides the occurrence of necrosis when apoptosis
is blocked, necroptosis is characterized by a Receptor-
Interacting Protein (RIP)-1 Kinase (RIPK1) dependent
formation of the complex IIb consisting of RIP-1 and RIP-3.
The initiation of programmed necrosis requires the kinase
activity of RIP-1 (Vandenabeele et al., 2010). We focused in
the current study on whether a blockade of RCT-induced
apoptosis may increase necrosis in colorectal tumor cells
and whether the total amount of RIP protein (RIPK1) is
modulated by the treatments. Since the mechanisms that
determine whether p53-dependent apoptosis occurs in
response to specific DNA damage are poorly understood,
we investigated besides the expression of RIPK1 that of the
tumor suppressor protein p53 and of (IFN)-regulatory fac-
tor-5 (IRF-5). We recently showed that in the radiosensitive
HCT15 colorectal tumor cell line, X-ray or hyperthermia
alone leads to a transient up-regulation of IRF-5, while in
less radiosensitive colorectal tumor cells (e.g. SW480 cells)
the expression of IRF-5 was only increased when both
death stimuli were combined (Mantel et al., 2010). IRF-5 is,
like p53, a tumor suppressor and assists cell arrest, apop-
tosis, and immune activation. It may further sensitize p53
mutated tumor cells to DNA-damage induced cell death
(Hu et al., 2005).
Of special note is that, besides the tumor cells
themselves, the tumor microenvironment strongly
contributes to tumor surveillance, growth, and spreading
(Schreiber et al., 2011). Cancer treatments should therefore
aim, in addition to tumor cell death induction, to break
down the immune suppressive tumor microenvironment
and to stimulate anti-tumor immune responses. Adaptive
anti-tumor immune responses are induced by DC that
present and cross-present tumor antigen with appropriate
co-stimulation to CD4+ and CD8+ T-cells, respectively
(Steinman and Cohn, 1973; Palucka et al., 2011). Danger
signals that are mainly released from necrotic tumor
cells are capable to activate DC via Toll-like receptors
(TLR) and to foster processing and cross-presentation
of tumor peptides by DC (Apetoh et al., 2007). Another
Radiochemotherapy modulates tumor cell immunogenicity 303
© 2012 Informa Healthcare USA, Inc.
mode of action is based on stimulating the differentiation
of myeloid-derived suppressor cells (MDSC) towards
DC. This was just recently discovered to be independent
of TLR4 and to be induced by exposing MDSC to ultra-
low concentrations of paclitaxel (Michels et al., 2012).
After CT with anthracyclines, the high-mobility group
box 1 (HMGB1) protein is preferentially released from
the stressed tumor cells (Apetoh et al., 2008). Of note is
that Oxp also induces similar immunogenic cell death in
colon cancer cells (Tesniere et al., 2010).
High mobility group (HMG) chromatin proteins are
normally located inside the cell and responsible for
chromatin stability. However, when present outside
the cell, those nuclear proteins trigger inflammation
and immune activation (Scaffidi et al., 2002). Besides
HMGB1, the heat shock protein 70 (HSP70) may act as
an immune activating signal, when present in the extra-
cellular space (summarized in Sherman and Multhoff,
2007). While intracellular located HSP have a protec-
tive function and participate in oncogenesis and in
resistance to CT, extracellular HSP link the innate and
adaptive immune system and carry immunogenic pep-
tides (Binder et al., 2001; Pockley and Multhoff, 2008;
Joly et al., 2010). HSP70 is capable of directly activating
natural killer (NK) cells and delivering tumor antigens
to DC; either leading to immune activation against the
tumor (Sherman and Multhoff, 2007). These are reasons
why here we examined how certain chemo-therapeutic
agents—alone or in combination with X-ray—induce the
release of HMGB1 and HSP70 in colorectal tumor cell
lines and how monocyte-derived DC get activated after
contact with supernatants (SN) of the treated tumor cells.
Materials and methods
Culture of colorectal tumor cells
The human colorectal adenocarcinoma tumor cell lines
SW480 (CCL-228) and SW48 (CCL-231) (both obtained
by LGC Standards GmbH, Wesel, Germany), as well as
HCT15 (ACC 357; DSMZ, Braunschweig, Germany) were
used for the experiments. The tumor cells were grown
in Dulbecco’s Modified Eagle’s Medium (DMEM; PAN-
Biotech GmbH, Aidenbach, Germany) supplemented
with 10% fetal bovine serum (FBS; Biochrom AG, Berlin,
Germany), 1% sodium pyruvate, 2 mM glutamine, 100 U
penicillin/ml, and 100 µg streptomycin/ml (termed D10
medium; Invitrogen, Darmstadt, Germany). The tumor
cells were cultured in cell culture flasks (Cellstar, Greiner
BioOne, Nürtingen, Germany) at 37°C in 5% CO2 and 90%
Treatment with chemotherapeutic agents and X-ray
The chemotherapeutics 5-FU, Oxp, and Irino were
obtained from the dispensary of the University Hospital
Erlangen and dissolved in 5% glucose (Oxp) or 0.9%
NaCl carrier solution (5-FU, Irino). The concentrations
used for the treatments were established in preliminary
clonogenic assays with the colorectal tumor cell lines.
In the studies reported here, the respective concentra-
tion utilized was the one wherein 60% of the clonogenic
potential of the colorectal tumor cells was still pres-
ent after a treatment for 6 h with the chemotherapeutic
agent (data not shown). This resulted in concentrations
being used of 60 µg 5-FU/ml, 10 µg Oxp/ml, and 10 µg
Irino/ml, values in the range of those previously used
for in vitro experiments (Schimanski et al., 2009). The
chemotherapeutics (5-FU at 1.2 µl/ml, Oxp at 2.0 µl/ml,
and Irino at 0.5 µl/ml) were added to the tumor cells in
D10 medium and the samples were then cultured for 6 h
at 37°C. Afterwards, the cell suspensions were washed
twice (once with phosphate-buffered saline [PBS, pH 7.4]
and once with D0 [D10 without FBS]) and re-suspended
in D10 medium for a further culture period.
An X-ray generator (GE Inspection Technologies,
Hürth, Germany) was used to irradiate the tumor cells.
The latter were exposed to a single dose of 5 Gy (120 kV,
21.5 mA, 0.7 min) of X-ray, representing an average
half-weekly dose in tumor therapy. For the combined
treatment of the cells with CT and X-ray, cells were first
treated with CT for 6 h and afterwards irradiated.
Determination of the clonogenic potential
The clonogenic assay was performed as previously
described (Mantel et al., 2010). Shortly, a single-cell sus-
pension of exponentially growing colorectal tumor cells
was used. Cells were counted, plated in growth-medium
into Petri dishes, and irradiated 12 h after plating. After
≈2 weeks, the tumor cells were stained with methylene
blue for 30 min. Colonies containing >50 cells were
counted using an automatic colony analyzer (University
Cell death detection and inhibition of apoptosis
After treatment and culture of the tumor cells, cell death
was analyzed by staining the cell suspension (2 × 105 cells)
with AnnexinA5 (AnxA5)-FITC/propidium iodide (PI) (1
µg/ml FITC-labeled AnxA5 [Genethor, Berlin, Germany]
and 20 µg/ml PI [Sigma Aldrich, St. Louis, MO]) in
Ringer’s solution). After 0.5 h at 4°C, the samples were
analyzed with a multi-color flow cytometer (Gallios®,
Beckman-Coulter, Brea, CA) and it’s associated Kaluza
1.1 Software®. A minimum of 10,000 events/sample were
Apoptotic cells expose phosphatidylserine (PS) on
their outer cell membrane. AnxA5 binds with high-
affinity to PS and allows one to distinguish between
viable and apoptotic cells. However, PS on the inner
leaflet of necrotic cells is also accessible to AnxA5
binding, since necrotic cells have lost their membrane
integrity. Therefore, necrosis needs to be differentiated
from apoptosis by co-staining with PI. The latter is able
to penetrate into cells that have lost their membrane
integrity, and intercalates into the cells’ DNA. In general,
viable cells are negative for AnxA5 and PI binding,
apoptotic cells are positive for AnxA5 and negative for PI
binding, and necrotic cells are positive for AnxA5-FITC
304 B. Frey et al.
Journal of Immunotoxicology
and PI binding. A representative scatter dot plot of AnxA5-
FITC and PI binding properties of SW480 cells 1 day
after treatment with 5 Gy is depicted in Supplementary
Figure 1. During programmed cell death, the cells further
shrink by releasing blebs (Bovellan et al., 2010), resulting
in cells with erratic surfaces and displaying higher side
scatter (SSc) and lower forward scatter (FSc) properties.
A representative scatter dot plot of SW480 cells 1 day
after treatment with 5 Gy is displayed in Supplementary
For inhibition of apoptosis, the tumor cells in D10
medium were treated for 2 h with 100 µM pan-caspase
inhibitor zVAD (carbobenzoxy-valyl-alanyl-aspartyl-[O-
methyl]- fluoromethyl-ketone, Bachem, Weil am Rhein,
Germany). Upon completion of the incubation and
washing of the cell suspension, the respective treatment
with CT and/or X-ray was performed.
Analyses of protein expression—Western blot assay
The amount of intracellular RIP (RIPK1), p53, t-Bid, Bax-
alpha, IRF-5, HMGB1, and HSP70 was semi-quantita-
tively determined by Western blot technique. The treated
tumor cells were washed with PBS and re-suspended in
lysis-buffer (1% NP-40, 0.5% sodium deoxycholate, 1%
SDS, protease inhibitor cocktail (Pierce Biotechnology,
Rockford, IL), 1 mM sodium orthovanadate, 1 mM
β-glycerophosphate, 1 mM NaF, and 0.1 M PMSF) to
generate total cell lysates. Protein concentrations were
determined using a BCA assay kit (Pierce). The amount
of HMGB1 and HSP70 in the supernatant (SN) of the cell
culture was also detected by Western blot technique.
All probes were separated over 10% SDS-PAGE gels and
transferred to PVDF membranes (Immobilon, Millipore,
Billerica, MA). The membranes were then blocked with
5% non-fat dry milk (Roth, Karlsruhe, Germany) in Tris-
buffered saline (TBS) solution containing 0.05% Tween
20 (Merck, Hohenbrunn, Germany; buffer hereafter
referred to as TBS-T).
For the detection of the various proteins, the mem-
branes were incubated with a specified primary antibody
at 4°C overnight. The antibodies were: anti-RIP (1:200),
anti-Bax (1:200, both from Santa Cruz Biotechnology,
Santa Cruz, CA), anti-p53 (1:2000), anti-Bid (1:1000),
anti-IRF-5 (1:1000 all from Cell Signaling Technology,
Danvers, MA), anti-HMGB1 (Millipore), and anti-HSP70
(1:2000, Pharmingen, Heidelberg, Germany). After incu-
bation, the membranes were washed with TBS-T and
probed with horseradish peroxidase-conjugated second-
ary goat anti-mouse IgG and goat anti-rabbit (both from
Millipore) for 1 h at 20°C. Thereafter, following another
washing step, the membranes were developed using the
ECL method wherein luminescence was visualized using
Amersham Hyperfilm ECL (GE Healthcare Limited,
Munich, Germany) and a Curix 60 film processor (Agfa,
Generation of immature human DC
Human peripheral blood mononuclear cells (PBMC)
were isolated from heparinized whole blood samples
from healthy human donors. All donors provided written
consent prior to the blood collection. Density gradient
separation of the whole blood samples using Lymphoflot
(Biotest AG, Dreieich, Germany) was conducted at 850 x
g for 20 min. The isolated PBMC were collected, washed
with PBS, and re-suspended in 10 ml D0 (D10 without
FBS) for further use.
PBMC (≈7 × 106) were seeded into individual
wells of 6-well plates (Greiner BioOne). After a 1.5-h
incubation at 37°C, non-adherent lymphocytes were
removed by thorough washing with pre-warmed D0. To
generate immature dendritic cells (iDC), the remaining
adherent monocytes were cultured for 6 days in RPMI
complete medium supplemented with 10% FBS,
2 mM L-glutamine, 100 U penicillin/ml, and 100 µg
streptomycin/ml (hereafter referred to as R10), as well as
the following cytokines: 250 U/ml IL-4 (Immuno Tools,
Friesoythe, Germany) and 800 U/ml GM-CSF (Leukine
sargramostim; Bayer Schering AG, Berlin). Fresh
medium containing 25 U IL-4/ml and 800 U GM-CSF/ml
was added on days 2 and 5 of culture.
Figure 1. Colony formation of colorectal tumor cells after treatment with X-ray and/or chemotherapeutic agents. The time interval between
application of 5-FU, Oxp, or Irino and irradiation with 5 Gy was 6 h. The data here were obtained from two independent experiments, each
performed in triplicate. The colony formation of the untreated cells was set to 100% and the reduction due to treatment is displayed in terms
of %. 5-FU, 5-fluorouracil; Gy, Gray; Irino, irinotecan; Oxp, oxaliplatin; w/o, untreated control. *p < 0.05, **p < 0.01 (against 5 Gy treatment
value). Median values (± SE) are displayed.
Radiochemotherapy modulates tumor cell immunogenicity 305
© 2012 Informa Healthcare USA, Inc.
Analyses of DC activation
At day 6 after isolation of the monocytes, the medium
was removed and replaced by SN from treated colorec-
tal tumor cells. As negative control, iDC were cultured
in medium only or as second background control with
SN from mock-treated tumor cells. After 16 h of incu-
bation, the DC were detached, centrifuged once, and
re-suspended in R10 medium. Staining was then done
with the fluorochrome-conjugated antibodies HLA-DR-
Pacific Blue (Beckman Coulter), CD103-Alexa 647 (eBio-
science, Frankfurt, Germany), CCR7-PC7 (Pharmingen),
CD80-PC7, and CD86-PerCP/Cy5.5
Biolegend, Fell, Germany) for 30 min at 4°C. Afterwards,
the cells were analyzed by multicolor flow cytometry in
the Gallios® system.
Representative Western blot data of at least two indepen-
dent experiments are displayed. The cell death data are
obtained from two independent experiments each per-
formed in triplicate. The colony formation experiments
were performed at least twice. The unpaired Student’s
t-test was used for statistical analyses and a p-value of
<0.05 was considered as significant (*) and one of <0.01
as highly significant (**).
Oxaliplatin in combination with X-ray is most effective
in the reduction of colony formation of colorectal
In the entire set of colorectal tumor cell lines (HCT15,
SW48, SW480) used for our in vitro testing, chemo-
therapeutic agents did significantly enhance the radio-
sensitivity of the tumor cells (Figure 1). In comparison
to mock-treated cells, only ~5% of the colorectal tumor
cells were capable to form colonies after irradiation.
The treatment with 5-FU or Irino alone reduced the
capability to ≈50% in comparison to the non-irradiated
cells. A combination with X-ray resulted in a significant
enhanced inhibition of colony formation in all three cell
lines compared to the X-ray treatment alone. Oxp had the
strongest effects: the sole application reduced the colony
formation to ≈15% and combination with radiation
resulted in reduction of colony formation to ≈0.2%. While
Irino and Oxp did not influence the size of the colonies,
treatment with 5-FU did not only reduce the number of
colonies, but also their size (data not shown).
After treatment with CT and/or X-ray colorectal tumor
cells expose phosphatidylserine (PS) and therefore
bind AnxA5 before losing their FSc/SSc properties
Figure 2a displays the amount of dead tumor cells
according to their FSc/SSc properties and Figure 2b the
amount of all AnxA5-FITC+ (apoptotic plus necrotic)
cells, detected by AnxA5-FITC/PI staining, both 24 h
after treatment with RT, CT, or RCT. The results indi-
cate that the treated tumor cells expose PS before los-
ing their FSc/SSc characteristics. Tumor cells treated
with 5-FU, 5-FU/5 Gy, Oxp, Oxp/5 Gy display compa-
rable FSc/SSc properties 24 h after treatment in com-
parison to non-treated cells, while they already show
significantly enhanced AnxA5 binding (Figure 2b). PS
exposure is therefore a very early event in cell death of
colorectal tumor cells, even preceding blebbing of the
Necrotic forms of cell death dominate over apoptotic
ones in colorectal tumor cells after treatment with CT
The treatment of colorectal tumor cells either with X-ray
and Irino alone or in combination resulted in fast necro-
sis induction, while, 24 h after all other treatments, only a
Figure 2. Cell death of colorectal tumor cells after treatment with X-ray and/or chemotherapeutic agents. The percentage of dead tumor
cells characterized by FSc/SSc properties 24 h after the indicated treatments is shown in (a). The amount of AnxA5 binding apoptotic plus
secondary plus primary necrotic tumor cells 24 h after the respective treatments is displayed in (b). Results of one of three independent
experiments each performed in triplicate are shown. AnxA5, annexinA5; FSc, forward scatter; 5-FU, 5-fluorouracil; Gy, Gray; Irino, irinotecan;
Oxp, oxaliplatin; prim, primary; SSc, sideward scatter; w/o, untreated control. Median values (± SE) are displayed.
306 B. Frey et al.
Journal of Immunotoxicology
slight enhanced amount of apoptotic cells was observed
(Figure 3a). Seventy-two hours after the respective treat-
ments, a clear induction of apoptosis and necrosis by
chemotherapeutic agents and X-ray was observed (Figure
3b). X-ray induced cell death (apoptosis plus necrosis) in
≈25% of the tumor cells (Figure 3b). As a single application
of a chemotherapeutic agent, 5-FU and Oxp were most
effective in tumor death induction 3 days after the treat-
ment. While Irino alone led to a fast induction of primary
necrotic cells (Figure 3a), the effects after 72 h were the
weakest compared to irradiation, Oxp, or 5-FU treatment
(Figure 3b), indicating that necrosis did not result from
accumulating apoptotic cells that undergo necrosis dur-
ing in vitro culture. The percentage of necrotic cells even
decreased during culture time after single treatment with
Irino, since viable tumor cells continue to proliferate (not
shown) and thereby reduce the percentage of apoptotic
and necrotic cells. However, combination of Irino with
irradiation resulted in the highest cell death rates like the
treatment with 5-FU/5 Gy. Regarding the forms of tumor
cell death, all treatments induced higher amounts of
necrotic (primary plus secondary necrotic ones) cells com-
pared to apoptotic ones (Figure 3b). In the case of 5-FU and
5-FU plus X-ray, primary necrosis dominated over second-
ary necrosis (Figure 3). Combinatory treatments always
induced increased amounts of apoptotic plus necrotic cells
in comparison to single applications (Figure 3).
Five days after treatment of colorectal tumor cells with
chemotherapeutic agents/X-ray, significantly higher
amounts of apoptotic plus necrotic cells were observed
after combinatory treatment with irradiation and 5-FU or
Irino in comparison to X-ray only exposure. A combination
of Oxp with 5 Gy resulted in similar amounts of apoptotic
plus necrotic cells compared to X-ray only (Figure 3c).
The highest amount of necrotic cells was observed when
applying 5-FU or Irinotecan with irradiation. In contrast,
Figure 3. Forms of colorectal tumor cell death after treatment with X-ray and/or chemotherapeutic agents. After treatment of colorectal
tumor cells with X-ray, 5-FU, Oxp, or Irino alone or in combination, the cells were stained with AnxA5-FITC/PI and cell death was analyzed
by flow cytometry. The percentage of apoptotic, primary, or secondary necrotic SW480 colorectal tumor cells (a) 24, (b) 72, and (c) 120 h
after treatment is displayed, respectively. Results of one of three independent experiments each performed in triplicate are shown. 5-FU,
5-fluorouracil; Gy, Gray; Irino, irinotecan; Oxp, oxaliplatin; w/o, untreated control; 1°, primary; 2°, secondary. Median values (± SE) are
Radiochemotherapy modulates tumor cell immunogenicity 307
© 2012 Informa Healthcare USA, Inc.
Oxp induced similar amounts of apoptotic, as well as
primary and secondary necrotic cells (Figure 3c).
Programmed necrosis may play a role in colorectal
tumor cell death induced by CT alone or in
combination with X-ray
The influence of the pan-caspase inhibitor zVAD on cell
death induction by CT and X-ray (Figure 4) was also
analyzed. Caspase inhibition usually retards apoptosis
in the tumor cells. As expected, 1 day after treatment of
colorectal tumor cells with CT plus X-ray, the amount
of apoptotic cells was slightly decreased in the presence
of zVAD after all treatments (data not shown). Of note
is that significantly increased amounts of necrotic cells
were observed after irradiation, treatment with 5-FU,
5-FU/5 Gy, or with Oxp, 48 h after adding the pan-cas-
pase inhibitor (Figure 4a). In the case of Oxp/5Gy, Irino,
or Irino/5Gy, significantly decreased amounts of apop-
totic tumor cells were still present (Figure 4b). However,
after a further 24 h, also increased amounts of necrotic
cells were observed when zVAD was present in the cul-
tures (Figure 4c), while the amount of apoptotic cells was
similar (Figure 4d).
The expression of proteins that are involved in
apoptosis and necrosis execution is modulated in
colorectal tumor cells after treatment with CT
To obtain further hints whether programmed necrosis
plays a role in cell death induction of colorectal tumor
cells by chemotherapeutic agents in combination with
X-ray, the expression of the RIP protein (RIPK1) was
analyzed (Figure 5a). At 24 h after application of CT
and/or X-ray, the expression of RIP was increased in
comparison to the untreated control after all treatments
apart from only irradiation. Two days after the respective
treatments, the expression of RIP was highest in tumor
cells that have been treated with 5-FU, 5-FU/5 Gy, Oxp,
or Oxp/5 Gy. A slightly increased expression was also
observed after treatment with 5 Gy or Irino/5 Gy. At 72 h
after the respective treatments, enhanced expression of
RIP remained when the tumor cells had received 5-FU,
5-FU/5 Gy, Oxp, or Oxp/5 Gy; a slight increase was still
observed after Irino/5 Gy (Figure 5a).
Furthermore, all treatments resulted in an increased
expression of p53, most pronounced after Oxp or Oxp/5 Gy
(Figure 5b). After 72 h, the levels of p53 were still elevated
when the tumor cells had been treated with 5-FU, 5-FU/5
Gy, Oxp, or Oxp/5 Gy (Figure 5b). Similar results were
obtained for expression of tBid and Bax-alpha (data not
shown), while the expression of the anti-apoptotic protein
Bcl-2 was not influenced (data not shown). Regarding IRF-
5, the highest expression was observed 24 h after treatment
with 5-FU, 5-FU/5 Gy, Oxp, Oxp/5 Gy, or Irino/5 Gy. After
72 h, the strongest expression of IRF-5 was seen after Oxp,
Oxp/5 Gy, and also after 5-FU, 5-FU/5 Gy (Figure 5c). Taken
together, the most prominent up-regulation of expression
of proteins involved in programmed cell death pathways
Figure 4. The influence of the pan-caspase inhibitor zVAD on
apoptosis and necrosis of colorectal tumor cells after treatment
with X-ray and/or chemotherapeutic agents. After treatment of
colorectal tumor cells with X-ray, 5-FU, Oxp, Irino alone, or in
combination, in each case in the presence or absence of 100 µM
zVAD, the cells were stained with AnxA5-FITC/PI and cell death
was analyzed by flow cytometry. The percentage of necrotic
and apoptotic cells (a, b) 2 and (c, d) 3 days after the treatments
is displayed, respectively. Results of one of three independent
experiments each performed in triplicate are shown. 5-FU,
5-fluorouracil; Gy, Gray; Irino, irinotecan; Oxp, oxaliplatin; w/o,
untreated control; zVAD, pan caspase inhibitor. *p < 0.05 (against
without zVAD treatment). Median values (± SE) are displayed.
308 B. Frey et al.
Journal of Immunotoxicology
was observed after treatment with 5-FU, 5-FU/5Gy, Oxp,
Oxp/5Gy, or Irino/5Gy (Figure 5).
X-ray and CT induce the release of danger signals by
colorectal tumor cells
The immunogenic potential of tumor cells is in part
determined by the release of danger signals. We
therefore analyzed the amount of extracellular HMGB1
and HSP70 after treatment of colorectal tumor cells
with irradiation and/or chemotherapeutic agents.
The cytoplasmic and nuclear expression of HSP70
and HMGB1 remained unchanged after all treatments
(not shown). However, all treatments resulted in an
increased release of HMGB1 into the SN of the tumor
cell cultures (Figure 6a). In the case of HSP70, the
highest amount of extracellular HSP70 was observed
after treatment with 5-FU, 5-FU/5 Gy, Oxp, Oxp/5 Gy
Supernatants of tumor cells treated with Oxp or Irino
alone or in combination with X-ray induce activation
of dendritic cells
We also incubated immature human dendritic cells (iDC)
with cell-free SN of the treated colorectal tumor cells
to test their potential to activate the antigen presenting
cells (APC). Surface markers of DC were analyzed by flow
cytometry. In Figure 7, the median surface expressions
of HLA-DR, CD103, CCR7, and CD80 are displayed. The
expression of HLA-DR was significantly increased after
incubation of DCs with SN of Oxp/5Gy, Irino, or Irino/5Gy
treated cells in comparison to only irradiated tumor cells.
Treatment with only Oxp enhanced also slightly, but not
significantly, HLA-DR expression on DC (Figure 7a). The
Figure 5. Expression of RIP, p53, and IRF-5 in colorectal tumor
cells after treatment with X-ray and/or chemotherapeutic agents.
The levels of the proteins (a) RIP, (b) p53, and (c) IRF-5 in SW480
colorectal tumor cells 24 or 72 h after treatment with X-ray, 5-FU,
Oxp, or Irino alone or in combination was analyzed by Western
Blot. Expression of Calnexin served as a loading control. The figure
displays representative data from three independent experiments.
5-FU, 5-fluorouracil; Gy, Gray; Irino, irinotecan; IRF-5, interferon
regulatory factor-5; Oxp, oxaliplatin; p53, tumor suppressor protein
(53 kDa); RIP (RIPK1), Receptor-Interacting Protein 1 Kinase; w/o,
Figure 6. Release of HMGB1 and HSP70 by colorectal tumor cells
after treatment with X-ray and/or chemotherapeutic agents. The
amount of the danger signals (a) HMGB1 and (b) HSP70 in the
cell culture supernatants of SW480 colorectal tumor cells 24 h after
treatment with X-ray, 5-FU, Oxp, or Irino alone, or in combination
was analyzed semi-quantitatively by Western blot. The figure
displays representative data from two independent experiments.
5-FU, 5-fluorouracil; Gy, Gray; HMGB1, high-mobility group
protein B1; HSP70, heat shock protein 70; Irino, irinotecan;
Oxp,oxaliplatin; w/o, untreated control.
Radiochemotherapy modulates tumor cell immunogenicity 309
© 2012 Informa Healthcare USA, Inc.
expression of CD103 was significantly increased after
contact of DC with SN of tumor cells treated with Irino,
or Irino/5 Gy compared to 5 Gy irradiated or untreated
tumor cells (Figure 7b). The expression of CCR7 was also
significantly enhanced after Irino, or Irino/5 Gy (Figure
7c). The treatment with Oxp or Oxp/5 Gy also slightly, but
not significantly, enhanced CCR7 and CD103 expression
on DC (Figures 7b and c). The DC activation marker
CD80 was significantly up-regulated on the surface of
DC after all treatments except 5 Gy and 5FU compared
to untreated tumor cells (Figure 7d). The most prominent
up-regulation was observed after contact with SN from
Oxp/5 Gy-, Irino-, or Irino/5 Gy-treated SW480 colorectal
tumor cells (Figure 7d); additionally the up-regulation
also differed significantly from 5 Gy-irradiated tumor
cells. Combinatory treatments resulted always in a
slight, but not significant, increased expression of
CD80 and CCR7 in comparison to single applications
(Figures 7c and d).
Before or after surgical resection of colorectal cancer, by
default, radiotherapy is applied to, respectively, reduce
the tumor volume or to attack/minimize tumor small
masses and metastases. To date, the radiosensitivity
of tumors has been mostly determined by analyzing,
after irradiation, the clonogenic potential of tumor cells
in vitro (Williams et al., 2008). As current treatment
of colorectal cancer includes surgery, chemotherapy,
and radiotherapy (reviewed in Asmis and Saltz, 2008),
we analyzed the effects on the clonogenic potential of
colorectal tumor cell lines from X-ray alone and in com-
bination with certain chemotherapeutic agents that are
clinically combined with X-rays.
In general, the tested chemotherapeutic agents
enhanced the radiosensitivity of the colorectal tumor
cells. The combination of X-ray with Oxp was most effec-
tive. However, besides the targeted effects of X-ray, non-
targeted and systemic effects also can occur (Turesson
et al., 2003; Rzeszowska-Wolny et al., 2009; Hildebrandt,
2010). Extra-nuclear and extra-cellular effects contribute
to (immune) biological consequences of X-ray (Hei et al.,
2010; Salomaa et al., 2010). Besides cytokines, immune
activating danger signals that are mainly released from
necrotic tumor cells after RCT are able to mediate sys-
temic responses. We analyzed the forms of colorectal
tumor cell death after X-ray or CT alone or after com-
binatory treatments. We observed that the prominent
in vitro form of colorectal tumor cell death was necrosis
(Figure 3). The membrane integrity of primary necrotic
tumor cells is lost rapidly, resulting in leakage of intracel-
lular contents. Furthermore, cells can become second-
arily necrotic when apoptotic cells lose their membrane
integrity at any time while executing cell death. Under
healthy conditions, secondary necrosis rarely occurs,
since apoptotic cells are cleared rapidly and in a non-
inflammatory manner (Gregory and Pound, 2011).
Figure 7. Surface expression of activation markers by dendritic cells after treatment with supernatants of colorectal tumor cells. The expression
of the DC activation markers (a) HLA-DR, (b) CD103, (c) CCR7, and (d) CD80 was analyzed by flow cytometry after incubation of immature
DC with cell culture supernatants from SW480 colorectal tumor cells treated with X-ray, 5-FU, Oxp, or Irino alone or in combination. Results
of one of two independent experiments each performed in duplicate are shown. DC, dendritic cells; 5-FU, 5-fluorouracil; Gy, Gray; Irino,
irinotecan; MFI, mean fluorescence intensity after binding of the respective antibody; Oxp, oxaliplatin; w/o, untreated control. *p < 0.05,
**p < 0.01 (against 5-Gy treatment); #p < 0.05, ##p < 0.01 (against w/o).
310 B. Frey et al.
Journal of Immunotoxicology
However, in a disease state like cancer, sporadic necro-
sis may contribute to the therapy-induced immune
response towards the tumor (Guerriero et al., 2011). In
the current studies, it was seen that total necrosis domi-
nated over apoptosis after the treatment of the colorectal
tumor cells with X-ray and/or CT.
The resistance of tumor cells to apoptotic cell death
is one important factor for cancer therapy resistance.
However, certain therapeutic settings may kill tumor
cells by inducing necrotic cell death forms instead of
apoptosis (Jiang et al., 2011). Moreover, alkylating DNA
damaging agents have been shown to foster regulated
forms of necrotic cell death (Zong et al., 2004). The
kinase activity of RIP1 is specifically required as an
upstream regulator of regulated or programmed necro-
sis, also called necroptosis (Yuan and Kroemer, 2010). In
the current studies, we observed an increased intracel-
lular expression of RIPK1 after treatment with 5-FU, Oxp
alone, or combined with X-ray, and after Irino plus X-ray
(Figure 5). We suggest that, in colorectal tumor cells,
necroptosis may result as an alternative form of cell
death (Christofferson and Yuan, 2010) after treatment
with X-ray and/or CT. We further demonstrated a sig-
nificantly increased rate of necrosis when apoptosis was
blocked in the treated colorectal tumor cells with zVAD
in comparison to no inhibition of caspases (Figures 4a
and c). Caspase-dependent apoptosis can be temporar-
ily efficiently-blocked by pan-caspase inhibitors like
zVAD. An increased amount of dead cells after treat-
ment with zVAD suggests that other forms of cell death
as apoptosis can be induced, most likely programmed
forms of necrosis (Yuan and Kroemer, 2010).
In the studies here, the forms of cell death after inhibi-
tion with zVAD 24, 48, and 72 h after the treatments were
analyzed. Regarding apoptosis inhibition, 24 h after treat-
ment, apoptosis was slightly inhibited after all treatments
(not shown). However, after 48 h, the amount of necrotic
cells already increased significantly after treatment with
X-ray, 5-FU, 5-FU/X-ray, and Oxp. After 72 h, the block-
ade of apoptosis in the Oxp/X-ray, Irino, or Irion/X-ray
treated cells also finally resulted in more necrotic cells.
We conclude that colorectal tumor cells can enter into
necrosis at variable time points after inhibition of apop-
tosis. Future work should focus on whether blockade
of RIPK1 with necrostatin-1 (Nec-1) and knock down
of RIP-3, being a central component of the complex IIb
leading to necroptosis (Yuan and Kroemer, 2010), can
reduce the RCT-induced necrosis in colorectal tumor
cells to further strengthen the hypothesis that colorectal
tumor cells have the potential to undergo necroptotic cell
Today it is well accepted that the immune system
contributes to tumor regression (Shurin et al., 2012).
Guerriero et al. (2011) demonstrated that HMGB1-
deficient tumors have an impaired ability to recruit
innate immune cells into CT-treated tumor tissue, indi-
cating that HMGB1 plays an important role in anti-tumor
immunity. In the current study, an increased amount of
extracellular HMGB1 after all treatments was observed
(Figure 6). Like HMGB1, extracellular HSP70 acts as an
immune activating danger signal and activates the innate
as well as the adaptive immune system (Multhoff, 2006;
Torigoe et al., 2009). We found there was a significantly
increased amount of extracellular HSP70 protein after
treatment of colorectal tumor cells with 5-FU, Oxp alone,
or in combination with X-ray. Danger signals are part of
immune modulating agents that trigger the activation
status of DC in the tumor microenvironment. Our labora-
tory recently showed that treatment of colorectal tumor
cells with X-ray plus hyperthermia leads to an HSP70
dependent activation of DC (Schildkopf et al., 2011).
However, the activation of DC by SN of colorectal tumor
cells treated with 5 Gy of X-ray plus Irino or Oxp did not
correlate with the amount of released HSP70.
We conclude that multiple mechanisms (mode of
cell death, danger signals, cytokines, chemokines) are
involved in the activation process of DC, and that the
combinations of RT with hyperthermia or CT result in
different activation mechanisms. In our preclinical in
vitro cell culture model system, SN of the combinatory
treatments with X-ray plus CT resulted in a slight, but
not significant increase of the activation markers CD103
and CD80, and the homing receptor CCR7 on human
monocyte-derived DC compared to single treatment
with the chemotherapeutic agent. The CCR7 receptor is
important for homing of DC in the lymph nodes where
specific activation of CD8+ cytotoxic T-cells (CTL) takes
place (Ohl et al., 2004). As has been previously discussed
with respect to histone deacetylase inhibitors (Dickinson
et al., 2010), X-ray plus CT may exert their clinical efficacy
by inducing cell death in the cancer cells or by priming
the tumor cells to other stimuli, such as those from the
immune system. As a primary additional effect upon
tumor cell death induction or as a result of it, the tumor
microenvironment might be beneficially altered by stan-
dard cancer treatments to foster an attack on the tumor
by immune cells. For this reason, it is also worth pro-
tecting DC and progenitor cells during cancer therapy
(Briegert et al., 2007). Table 1 reflects the complex mode
of action of X-ray treatments with chemotherapeutic
agents. Synergistic modes of action of various treatment
strategies may finally result in an efficient killing of tumor
cells, and may allow the immune system to execute anti-
tumor responses (Zitvogel et al., 2011). Immunogenic
tumor cell death induced by standard treatments may
render the tumor cells visible for the immune system,
and may result in a systemic anti-tumor immune defense
(Weiss et al., 2010).
Besides the general, but timely restricted, immune
suppressive features of CT and RT, specific anti-tumor
immune responses can be generated by modifying
the tumor, as well as its microenvironment, with
those standard therapies; immunogenic cancer cell
Radiochemotherapy modulates tumor cell immunogenicity 311
© 2012 Informa Healthcare USA, Inc.
death results. The latter has just recently been shown
in pre-clinical and clinical models to be induced by
Oxp in colorectal tumor cells (Tesniere et al., 2010).
Additionally, chemotherapeutic agents administered
in ultra-low doses may directly lead to maturation of
DC and consecutive effective tumor antigen cross-
presentation (Shurin et al., 2012). The tumor has to be
diminished by RT, CT, or surgery, and further its micro-
environment has to be modified by optimized com-
binations of standard tumor therapies alone or with
additional immune stimulation (Frey et al., 2009) to
make the shrunken tumor and its metastases attack-
able by immune cells (Gaipl, 2012).
Our data indicate that combinations of chemo-
therapeutic agents used for the treatment of colorectal
cancer with X-ray foster necrotic forms of tumor cell
death, the release of immune activating danger signals,
the maturation, and activation of DC. Future research
should also focus on the monitoring of danger signals
and activation status of DC in blood of tumor patients
The German Research Foundation (GA 1507/1-1 and
DFG -Graduiertenkolleg 1660: Key signals of the adap-
tive immune response), the German Federal Ministry of
Education and Research (BMBF; m4 Cluster, 01EX1021R
and GREWIS, 02NUK017G), and the European Com-
missions (DoReMi, European Network of Excellence,
contract number 249689) supported this work. This is
an article based upon original research presented at the
2nd International Conference on Cancer Immunotherapy
and Immunomonitoring (CITIM), Budapest, Hungary,
Declaration of interest
The authors report no conflicts of interests. The authors
alone are responsible for the content and writing of
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Oxp + X-ray+++o +++ ++
All compared to w/o: + [high(er) expression], − [no increased expression], o [slightly-increased expression]. a24 h after treatment; b120 h after
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