1. No category

Immune biological rationale for hyperthermia
in cancer treatment
P. Schildkopf, B. Frey, O. J. Ott, F. Mantel, R. Sieber, E.-M. Weiss, R. Fietkau, and U. S. Gaipl
Priv.-Doz. Dr. Udo S. Gaipl (PhD)
Associate Professor
Head of Radiation Immunobiology
University Hospital Erlangen
Department of Radiation Oncology
Head: Prof. Dr. R. Fietkau
19.11.2010 14.09.2010
Barcelona,
Schildkopf et al., Curr Med Chem, 2010
What we hear about hyperthermia (HT)
• Modifies blood-circulation
• Modifies cell membranes (“smooth” membrane)
• Leads to acidosis (due to increased metabolism)
• Leads to loss of ATP (due to increased metabolism)
• Leads to retardation of DNA-replication (sensitizer for RT)
• Leads to pain relief
• Direct cytotoxic action:
¾T > 41°C lead to cell damages (preferentially tumor cells)
¾T < 41°C induce stress proteins
• Modulation of the innate and adaptive immune system (39-43°C)
Levine DB et al., HSS J, 2008
Heat to heal cancer – the Coley´s Toxin
(end of the 19th century)
Complete remission
of tumor in cancer
patients with high
fever.
Birth of immune
therapy as
treatment option
for cancer.
Coley´s Toxin:
mixture of killed
bacteria.
William Bradley Coley
(1862-1936)
surgeon and oncologist
However, strong
side effects.
Gaipl et al., Biological rationale of hyperthermia, in Hyperthermia in Oncology, Uni-Med Verlag AG, 2010
Biological modes of
Main aims of
action of hyperthermia anti-tumor therapy
• Direct cytotoxicity
• Radiosensitization
• Chemosensitization
• Systemic effects
• Immune modulation
• Stop proliferation
of tumor cells
• Kill tumor cells
• Keep residual tumor
cells in check
• Induce immunogenic
tumor cell death and
immune stimulation
Adapted from Torigoe et al., Int J Hyperthermia, 2009
Dendritic cells as main players in
immune activation
Adapted from Curtin et al., PLoS MEDICINE, 2009
Dendritic cells as main players in
immune activation
Distinct tumor
microenvironment
DC attraction and
migration
Uptake of antigen an
co-stimulation
Antigen presentation,
T cell activation
CTL response against
tumor
Modified from Milani et al. and Multhoff et al.
HT as immune therapy for cancer –
the role of heat-shock proteins
crosspresentation
(MHC class I)
Inside the cell: thermo tolerance
Outside the cell: immune activating danger signal
Adapted from Shi et al., J Immunol, 2006
HT fosters specific
cross-priming of dendritic cells
specific (Me275)
cross-presentation (CTL)
“hot” (HT treated tumor cells)
T cells co-cultured with tumor (Me275) loaded DC
Tumor was “cold” or “hot”
CTL lysis (MHC class I dependent) of melanoma cells (Me275 or K562)
Demaria S. et al., Int. J. Radiation Oncology Biol. Phys, 2004
Local applications may lead to
systemic effects
Latin: ab (position away from) and scopus (mark or target)
w/o
Flt3-L
RT +/Flt3-L
“secondary”
tumor
“primary”
tumor
Flt3-L: DC growth factor
RT: single dose of 2 Gy
RT
RT plus Flt3-L
Demaria S. et al., Int. J. Radiation Oncology Biol. Phys, 2004
Abscopal effect of RT – Reduction of tumor
growth outside the field of irradiation
w/o
Flt3-L
RT
RT plus Flt3-L
Nude mice:
since 2003:
BSD 2000-3D
Deep Regional Hyperthermia
(RHT)
since 2005:
BSD 500
Local Hyperthermia (LHT)
Interstitial Hyperthermia (IHT)
since 12/2007:
BSD 2000-3D MRI
Deep Regional Hyperthermia
c/w Magnetom Symphony (1.5 T)
11
Current definition of locally applied
hyperthermia (HT)
• Heating of tumor tissue
• No alternative but additive tumor therapy
• Temperature range: 40-44°C
• Therapeutic time: 60 minutes
Schildkopf et al., Strahlenther Onkol, 2010
Hyperthermia and colony formation
SW480 tumor cells
5Gy
HT in combination with
X-ray (5Gy) reduces colony
formation of colorectal
tumor cells
HCT15
1
colony formation fraction
colony formation fraction
SW480 tumor cells
w/o
**
0,1
0,01
0,001
0,0001
0,00001
w/o
HT
5 Gy
5 Gy
+ HT
10 Gy 10 Gy
+ HT
1
**
SW480
0,1
0,01
0,001
0,0001
0,00001
w/o
HT
5 Gy
5 Gy
+ HT
10 Gy 10 Gy
+ HT
Schildkopf et al., Biochem Biophys Res Commun, 2010
Hyperthermia and cell death
HT in combination with X-ray induces
cell death in colorectal tumor cells
HCT15 colorectal tumor cells; 72 hours after treatment;
HT: 41.5°C for 1h; time interval between treatments: 4 hours
Schildkopf et al., Biochem Biophys Res Commun, 2010
Hyperthermia and cell death
Pictures provided by
Marco Vitale et al.
Apoptotic cell
Necrotic cell
HT alone and most notably in combination with X-ray
induces mainly necrosis in colorectal tumor cells
HCT15 colorectal tumor cells; 72 hours after treatment;
HT: 41.5°C for 1h; time interval between treatments: 4 hours
Schildkopf et al., Biochem Biophys Res Commun, 2010
Necrosis – one prominent form of tumor cell
death after RT plus HT
HCT15
d)
50
apoptotic cells [%]
apoptotic cells [%]
a)
40
**
30
20
**
10
0
b)
w/o
HT
5 Gy 5 Gy 10 Gy 10 Gy
+ HT
+ HT
SW480
50
30
*
20
**
0
w/o
HT
40
**
30
**
20
*
10
0
w/o
HT
5 Gy 5 Gy 10 Gy 10 Gy
+ HT
+ HT
Colorectal tumor cells; 72 hours after treatment;
HT: 41.5°C for 1h; time interval between treatments: 4 hours
necrotic cells [%]
necrotic cells [%]
**
5 Gy 5 Gy 10 Gy 10 Gy
+ HT
+ HT
SW480
**
50
**
10
e)
HCT15
*
40
**
50
**
**
40
30
*
20
10
0
w/o
HT
5 Gy 5 Gy 10 Gy 10 Gy
+ HT
+ HT
Schildkopf et al., Strahlenther Onkol, 2010
Combinations of HT plus RT increase the
expression of PUMA
SW480
24h
PUMA
actin
1.5
1
0.5
48h
w/o HT 5Gy 5Gy 10Gy10Gy
+ HT
+ HT
SW480
1.5
1
0.5
0
4
3
2
1
0
48h
w/o HT 5Gy 5Gy 10Gy10Gy
+ HT
+ HT
HCT15
PUMA
actin
PUMA
actin
PUMA (18 kDa) content
(densitometric value)
PUMA (18 kDa) content
(densitometric value)
PUMA (18 kDa) content
(densitometric value)
PUMA
actin
0
HCT15
w/o HT 5Gy 5Gy 10Gy10Gy
+ HT
+ HT
PUMA (18 kDa) content
(densitometric value)
24h
4
3
2
1
0
w/o HT 5Gy 5Gy 10Gy10Gy
+ HT
+ HT
Schildkopf et al., Strahlenther Onkol, 2010
Combinations of HT plus RT increase the
expression of RIP-1
SW480
SW480
1.5
1
0.5
48h
w/o HT 5Gy 5Gy 10Gy10Gy
+ HT
+ HT
SW480
2
1.5
1
0.5
0
2
1.5
1
0.5
0
48h
w/o HT 5Gy 5Gy 10Gy10Gy
+ HT
+ HT
HCT15
RIP-1
actin
RIP-1
actin
RIP-1 (74 kDa) content
(densitometric value)
RIP-1
actin
2
0
HCT15
RIP-1 (74 kDa) content
(densitometric value)
RIP-1 (74 kDa) content
(densitometric value)
RIP-1
actin
24h
w/o HT 5Gy 5Gy 10Gy10Gy
+ HT
+ HT
RIP-1 (74 kDa) content
(densitometric value)
24h
2
1.5
1
0.5
0
w/o HT 5Gy 5Gy 10Gy10Gy
+ HT
+ HT
Schildkopf et al., Strahlenther Onkol, 2010
HT plus RT activate programmed apoptotic
and necrotic cell death pathways
X-ray
plus HT
via mitochondrial permeability transition
PUMA
bax
activation
RIP-1
IRF-5
activ
ation
p53#
caspase3/7
downregulation
bcl-2
blocking
tumor cell death
Schildkopf et al., Curr Med Chem, 2010
Synergistic effects of radiotherapy and HT
Tumor
Hyperthermia
Ionising
Radiation
pO2
- pH +
aggregation PROTEINS oxidation
S phase CELL CYCLE G2, M, G1
modulation CELL DEATH induction
Characteristics of the tumor
microenvironment:
Reduced blood flow and blood vessel density, chaotic
vasculature with areas of acidosis, hypoxia and
energy deprivation in form of ATP
Hyperthermia adds to
radiotherapy!
Schildkopf et al., Autoimmunity, 2009 and Biochem Biophys Res Commun, 2010
Hyperthermia (41.5°C) prolongs the G2 cell
cycle arrest induced by irradiation
48h after treatment
24h after treatment
70
50
30
10
0
20 Gy 20Gy+ HT HT+ 20 Gy
**
cells in G2 phase [%]
late Sphase
cells in G2 phase [%]
clonogenic potential
According: Fritz-Niggli et al., 1988
90
60
**
50
40
30
20
10
0
20 Gy
2.5
5
7.5
10
12.5
dose (Gy)
15
cells in G2 phase [%]
early SPhase
G1-phase
mitosis
G 2-phase
0
20Gy+HT HT+ 20 Gy
HCT15
90
80
70
60
50
40
30
20
10
0
**
**
*
w/o
HT
*
5 Gy 5 Gy 10 Gy 10 Gy
+ HT
+ HT
Adapted from Apetoh et al., Cancer Res, 2008
Immunogeneic tumor cell death –
Calreticulin and HMGB1
Calreticulin: phagocytosis of dying tumor cells by DC
HMGB1: mediates cross-presentation of tumor Ag by DC
Modified from: Obeid et al., Nature medicine, 2007
Various forms of cell death –
immunogenic cell death
chemotherapeutic
+/- HT
26.2.2007
43.0
T1
T2
T3
dead tumor cells
T4
42.5
irradiation
42.0
41.5
41.0
40.5
40.0
39.5
39.0
12:00
Tue 29 Jan 2008
15:00
Time
tumor free
animals
dead
tumor cells
anti-tumor
immunity
vital
tumor cells
Obeid et al., Nature medicine, 2007
and
Schildkopf et al., unpublished data
Immunogenic tumor cell death calreticulin on the tumor cell surface
Colorectal carc. cells
24h after X-ray and
X-ray + HT (41.5°C)
count
Correlation between CRT exposition
and immunogenicity:
log FITC
Calreticulin
Schildkopf et al., Biochem Biophys Res Commun, 2010
HT additionally fosters the release of
other danger signals like HMGB1
HT
24 hours
RT
tumor cell
HMGB1
(32 kDa)
w/o
5Gy
Examine the
supernatant
HMGB1: high mobility group box 1 protein
10Gy HT 5Gy HT+ 10Gy HT+
+HT 5Gy +HT 10Gy
HT in combination with RT induces the
release of HMGB1
SW480 colorectal tumor cells; 24 hours after treatment;
HT: 41.5°C for 1h; time interval between treatments: 4 hours
Modified from: Kono et al., Nat Rev Immunol, 2008
and
Gaipl et al., Curr Top Microbiol Immunol, 2006
Various forms of cell death –
immunogenic cell death induced by HT
chemotherapeutics
ionizing
irradiation
hyperthermia
apoptosis
hidden
DAMP
surface
modifications
e.g.
HMGB1, or
HSP70
viable
tumor cell
hidden
DAMP
necrosis
secondary
necrosis
eat
-m
es
i gn
annexinA5
als
of
e
s
ea MP
l
re DA
l i ke
phagocytosis
cal
ret
icu
li n
cells of the
innate
immune
system
non- or anti-inflammatory
response
immature
DC
DC
maturation
peptide-MHC
complex
mature
DC
inflammatory
response
antigens of
the dying cells
co-stimulatory
molecule
inflammatory response
activation of B and T cells
anti-tumor immunity
The tumor cell as target for the immune
system – Natural killer cells
• NK cells were originally identified on a functional basis:
capability of killing certain tumor cell lines in absence of a
deliberate previous stimulation
• NK cells do not express clonally distributed receptors for
antigen
• NK display cytolytic activity against tumor or virusinfected cells
• NK cells release cytokines and chemokines that mediate
inflammatory responses (connection to adaptive immunity)
The tumor cell as target for the immune
system – Natural killer cells
NK cell
tumor cell
lack of MHC I
inhibitory
receptor
activating
receptors
tumor escape!
Modified from Burd et al, J Cell Phys, 1998
NK cells are involved in heat-mediated
anti-tumor effects of HT
Nude mice, human breast cancer tumor
No HT
With HT
Green dots: TUNEL positive dead tumor cells
With HT,
but NK cell depleted
TAKE HOME MESSAGES
HT induces immunogenic cancer cell
death forms (release of danger signals)
Locally applied HT has systemic effects
and may lead to immune activation
HT activates DC
HT primes CTL
HT activates NK cells
Thanks to …
University Hospital Erlangen
Department of Radiation Oncology
Director: Prof. Dr. R. Fietkau
Group: Radiation Immunobiology
Dr. Benjamin Frey
Petra Schildkopf
Eva-Maria Weiss
Renate Sieber
Roland Wunderlich
Kathrin Schulz
Barbara Lödermann
Frederick Mantel
Sonja Stangl
Carolin Muth
OA Dr. Oliver Ott
Co-operation partners
Prof. Dr. Patrizia Rovere-Querini and Prof. Dr. Angelo Manfredi (H. S. Raffaele, Milano, Italy)
Prof. Dr. Ian Dransfield and Dr. Sandra Franz (MRC, Edinburgh, UK)
Dr. Bent Brachvogel (University of Cologne, Germany)
Dr. Ernst Pöschl (University of East Anglia Norwich, UK)
Prof. Dr. Reinhard Voll and Prof. Dr. Dr. Martin Herrmann (FAU of Erlangen-Nuremberg, Germany)
Prof. Dr. Evelyn Ullrich (FAU of Erlangen-Nuremberg, Germany)
Prof. Dr. Gabriele Multhoff (TU Munich, Germany)
Prof. Dr. Ludwig Keilholz (Hospital of Bayreuth, Germany)
PD Dr. Franz Rödel (University of Frankfurt, Germany)
PD Dr. Peter Kern (Capio Deutsche Klinik GmbH, Germany)
Dr. Martin Schiller and Petra Heyder (University of Heidelberg, Germany)
Thank you for your attention!
RT
CT
HT
IT
Cure
tumors:
turn the
heat on!