Ecto-calreticulin in immunogenicchemotherapy
Summary: The conventional treatment of cancer relies upon radiotherapyand chemotherapy. Such treatments supposedly mediate their effects viathe direct elimination of tumor cells. Nonetheless, there are circumstancesin which conventional anti-cancer therapy can induce a modality ofcellular demise that elicits innate and cognate immune responses, whichin turn mediate part of the anti-tumor effect. Although differentchemotherapeutic agents may kill tumor cells through an apparentlyhomogeneous apoptotic pathway, they differ in their capacity tostimulate immunogenic cell death. We discovered that the pre-apoptotictranslocation of intracellular calreticulin (endo-CRT) to the plasmamembrane surface (ecto-CRT) is critical for the recognition andengulfment of dying tumor cells by dendritic cells. Thus, anthracyclinesand g-irradiation that induce ecto-CRT cause immunogenic cell death,while other pro-apoptotic agents (such as mitomycin C and etoposide)induce neither ecto-CRT nor immunogenic cell death. Depletion of CRTabolishes the immunogenicity of cell death elicited by anthracyclines,while exogenous supply of CRT or enforcement of CRT exposure bypharmacological agents that favor CRT translocation can enhance theimmunogenicity of cell death. For optimal anti-tumor vaccination andimmunogenic chemotherapy, the same cells have to expose ecto-CRT andto succumb to apoptosis; if these events affect different cells, no anti-tumor immune response is elicited. These results may have far reachingimplications for tumor immunology because (i) ecto-CRT exposure bytumor cells allows for the prediction of therapeutic outcome and because(ii) the re-establishment of ecto-CRT may ameliorate the efficacy ofchemotherapy.
Keywords: apoptosis, endoplasmic reticulum, stress response, tumor immunology
Introduction
The armamentarium of clinical oncologists has been bolstered
over the past decades, and some childhood cancers and adult
hematological malignancies are now cured on a routine basis.
Nonetheless, advanced solid tumors, such as carcinoma,
sarcoma, melanoma, and glioblastoma, still pose major
difficulties in their treatment. After one or several distinct
lines of chemotherapy, in most cases only partial responses are
obtained, meaning that after an initial, pyrrhic success, tumors
will resume growth, select therapy-resistant variants, and seal
the patient’s fate. Even in those cases in which the tumor has
Michel ObeidAntoine TesniereTheocharis PanaretakisRoberta TufiNick JozaPeter van EndertFrancois GhiringhelliLionel ApetohNathalie ChaputCaroline FlamentEvelyn UllrichStephane de BottonLaurence ZitvogelGuido Kroemer
Immunological Reviews 2007
Vol. 220: 22–34
Printed in Singapore. All rights reserved
r 2007 The Authors
Journal compilation r 2007 Blackwell Munksgaard
Immunological Reviews0105-2896
Authors’ addresses
Michel Obeid1,2,3, Antoine Tesniere1,2,3, Theocharis Panaretakis1,2,3,
Roberta Tufi1,2,3, Nick Joza1,2,3, Peter van Endert4,5,
Francois Ghiringhelli2,3,6,7, Lionel Apetoh2,3,6, Nathalie Chaput2,3,6,7,
Caroline Flament2,3,6,7, Evelyn Ullrich2,3,6, Stephane de Botton2,3,8,
Laurence Zitvogel2,3,6,7�, Guido Kroemer1,2,3�1INSERM, U848, Villejuif, France.2Institut Gustave Roussy, Villejuif, France.3Faculte Paris Sud-Universite Paris 11, Villejuif, France.4INSERM, U580, Paris, France.5Faculte de Medecine Rene Descartes, Universite ParisDescartes, Paris, France.6INSERM, U805, Villejuif, France.7Centre d’investigation clinique Biotherapie, CBT507,Institut Gustave Roussy, Villejuif, France.8Service d’Hematologie Clinique, Institut Gustave Roussy,Villejuif, France.
�Guido Kroemer and Laurence Zitvogel share seniorauthorship.
Correspondence to:Dr Guido KroemerINSERM, U848Institut Gustave Roussy, PR138 rue Camille DesmoulinsF-94805 VillejuifFranceTel.: 133 1 42 11 60 46Fax: 133 1 42 11 60 47e-mail: [email protected]
Acknowledgements
G. K. is supported by a special grant from Ligue contre leCancer (equipe labellisee) as well as by grants fromEuropean Commission (Active p53, RIGHT, Trans-Death,Death-Train, ChemoRes) and by Institut National contre leCancer (INCa). L. Z. is supported by grants from INCa, andfrom European DC THERA. M. O. and A. T. receivefellowships from Fondation pour la Recherche Medicale;E. U. is supported by the Deutsche Forshungsgemeinshaft,L. A. received grants from Ligue contre le cancer, and F. A.from Poste d’Accueil INSERM.
22
apparently been removed completely (‘complete remission’),
micrometastases of dormant tumor cells (or cancer stem cells)
often lead to relapse and final therapeutic failure.
To overcome this difficulty, there are only two solutions.
First, we may design strategies to kill all cancer (stem) cells
efficiently, by the correct combination and schedule of
chemotherapeutic agents (1, 2). Second, we may attempt to
stimulate an immune response against the residual
(chemotherapy-resistant) cancer cells, using a panoply of
immunological vaccination and stimulation protocols
targeting tumor cells (3–7). Intuitively, these two strategies
appear incompatible, because chemotherapeutic regimens are
highly immunosuppressive, especially at escalating doses
meant to increase the anti-tumor efficacy. Moreover,
chemotherapy is considered to induce apoptosis as the
principal cell death mechanism, and apoptosis is believed to
be an immunologically silent (or even tolerogenic) cell death
modality. Indeed, if several million cells succumb to apoptosis
in each second of an adult healthy individual’s lifetime (i.e. in
an individual without autoimmune disease), then it appears
impossible that apoptotic cell death would elicit an immune
response (8–11). In response to endogenous stress, radiation,
chemotherapeutic agents, or attacks by the innate immune
system, tumor cells can die through a variety of biochemical
subroutines, including apoptosis and, less frequently, necrosis
and other cell death subtypes (terminal senescence, mitotic
catastrophe, autophagic cell death, etc.) (12–14). The
preponderant type of cell death induced by chemotherapy is
apoptosis, which is frequently but not unanimously viewed as
a non-immunogenic or tolerogenic modality of cell death (9).
This lack of immunogenicity would be explained by ‘silent’
macrophage-mediated [as opposed to dendritic cell (DC)-
mediated] clearance (9), active suppression of inflammatory
reactions (15, 16), or the lack of the ‘second signal,’ also called
‘danger signal’ (17). Thus, as an unwarranted side effect, anti-
cancer chemotherapy could specifically reduce the anti-tumor
immune response. As ‘apoptosis’ is itself non-uniform with
respect to the programmed signaling events responsible for cell
death (18, 19), we thought that it would be important to map
these pathways against the apparent immunologically non-
uniform outcomes of cell death (20–22). In doing so, it may be
possible to design methods for increasing the immunogenicity
of therapeutically induced cell death. Manipulations designed
to increase the immunogenicity of cell death may include
techniques to induce non-apoptotic tumor cell demise, to
prevent the clearance of apoptotic tumor cells, thus allowing
them to undergo secondary necrosis, the induction of local
stress leading to the expression of immunogenic heat shock
proteins (HSPs) as well as the exogenous supply of second
signals. In vitro, DCs pulsed with apoptotic bodies (23, 24) and
exosomes from tumor cells (25) appear to be more efficient in
cross-priming of CD81 T cells as compared with DCs exposed
to lysates or other derivatives obtained from non-stressed
tumor cells (26, 27). By paying attention to the mechanisms
of cell death, we hope to establish therapeutic interventions
that will feed the immune system with antigenic information
that may potentiate an anti-tumor response (28).
We have recently challenged the concept that chemotherapy-
induced cell death is always non-immunogenic. Indeed, some
strategies for tumor cell killing, for instance killing by
anthracyclines, do elicit immunogenic cell death (29). Thus,
syngenic tumor cells that have been treated with anthracyclines
in vitro and then are injected in vivo into immunocompetent
mice, can mediate anti-tumor vaccination, meaning that
they protect against a re-challenge with live cells from the
same (but not from an unrelated) tumor (29). Similarly,
treatment of established tumors with anthracyclines in vivo is
much more efficient in immunocompetent than in
immunodeficient mice (29, 30). This type of experiment
clearly demonstrates the possibility of an ‘immunogenic
chemotherapy,’ that is a chemotherapy designed such that
tumor cell death will elicit an anti-tumor immune res-
ponse (31).
The present review summarizes the current state-of-the art
in this field, laying special emphasis on the tumor-intrinsic
properties that are required to elicit an anti-tumor immune
response. One major tumor-intrinsic change that determines
the immune response against tumors is the exposure of
calreticulin (CRT) (30, 32), a protein that usually resides in
the lumen of the endoplasmic reticulum (ER) (endo-CRT)
(33–36) or on the cell surface (ecto-CRT) (37); CRT is the
principal topic of this review.
Anthracyclines elicit immunogenic cell death correlatingwith the exposure of CRT on the cell surface
To challenge the widely held view that apoptosis would be
a uniformly non-immunogenic cell death modality, we
elaborated the following experimental strategy. Immuno-
competent BALB/c (H2d) or C57Bl/6 (H2b) mice were
injected with dying histocompatible CT26 colon carcinoma
(H2d) or methylcholanthrene (MCA)205 sarcoma (H2b) cells,
respectively (Fig. 1). In the initial screening performed on CT26
cells, death was induced by prior culture of the cells with a
panel of chemotherapeutic agents, and the system was
calibrated in a way that the injected cells exhibited
Immunological Reviews 220/2007 23
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
approximately 70� 10% positivity for annexin V–fluorescein
isothiocyanate (FITC) (AnnV) staining (30). This reagent
detects the exposure of phosphatidylserine residues on the
surface of viable tumor cells [which exclude the vital dye 60-
diamino-2-phenylindole (DAPI)] and enters dead cells that
have permeabilized their plasma membrane (which
incorporate DAPI). Hence, the sum of dying (AnnV1DAPI� )
and dead (AnnV1DAPI1) cells was kept at approximately 70%,
for each of the chemotherapeutic regimes that caused cell death
in vitro, before subcutaneous injection of the cells (3� 106/
animal) into one flank. In spite of the fact that approximately
30% of the cells were still non-apoptotic (AnnV�DAPI� ), the
populations containing approximately 70% dying cells rarely
induced tumors, presumably because these non-apoptotic cells
were already programmed to die later, as a result of the
exposure to the damaging agents in vitro (30). Eight days later,
the same animals were re-challenged with live tumor cells
(1� 106/animal) into the opposite flank, and tumor growth
was then monitored (Fig. 1). The absence of tumor growth was
then scored as an indication of an anti-tumor immune response
elicited by dying tumor cells.
When used on CT26 cells, this system revealed that two
types of cell death induction could trigger a specific anti-tumor
immune response in 460% of the animals, meaning that
460% of the mice failed to develop any signs of tumor
growth when live, untreated tumor cells were inoculated 1
week after injection of the dying cells. This finding applies to
g-irradiation (32) as well as to the chemotherapy with distinct
anthracyclines such as doxorubicin (29), idarubicin, and
mitoxanthrone (30). In contrast, cell death induced by agents
that target the ER (thapsigargin, tunicamycin, brefeldin),
mitochondria (arsenite, betulinic acid, C2 ceramide), or
DNA (Hoechst 33342, camptothecin, etoposide, mitomycin
C) failed to induce immunogenic cell death and hence elicited
an immune response in o 40% of the mice. In those BALB/c
mice that were protected against a first challenge of live CT26
tumor cells, rechallenge with the same type of live tumor cells
(1� 106 cells 7–12 days after inoculation of dying cells)
failed to induce tumor growth, while injection of an unrelated
tumor did result in the formation of ever growing, lethal
tumors. This finding indicates that successful vaccination
against tumor development induced permanent, tumor-
specific immunity (29).
We have been able to recapitulate these results in MCA205
sarcoma cells (a tumor that was induced by exposing C57Bl/6
mice to the carcinogen MCA) (38) growing in C57Bl/6 mice
(Fig. 2A–D). Cell death induction of MCA205 cells by
mitomycin C or mitoxanthrone during 24 h (according to the
conditions described in Fig. 2) produced 50% of AnnV1 cells,
whereas g-irradiation led to o 20% of AnnV1 cells (Fig. 2B).
When injected into syngenic mice, such dying cells never
produced tumors. C57Bl/6 mice that were injected with
mitomycin C-treated MCA205 cells had a similar incidence of
tumors as mice that were injected with phosphate-buffered
saline (PBS)-treated MCA205 cells, meaning that mitomycin C
failed to induce immunogenic death of MCA205 cells. In
contrast, g-irradiation and treatment with mitoxanthrone
induced immunogenic cell death in this system (Fig. 2C,D).
In CT26 cells, we found that inducers of immunogenic cell
death (g-irradiation and anthracyclines) induced the early, pre-
apoptotic exposure of CRT on the plasma membrane surface
(30). This process must involve the translocation of pre-
formed CRT form inside of the cells (endo-CRT) to the
surface of the cell (ecto-CRT), because inhibitors of protein
synthesis failed to prevent CRT exposure (30). It is a rapid
process that occurs within minutes, well before apoptotic
Anti tumorImmunity No tumor
Live cells
Dying cells
Screening scheme for immunogenic cytotoxic agents
Day 0 Day 7
Fig. 1. Experimental protocol to identify immunogenic cytotoxic agents. Immunocompetent mice receive a subcutaneous injection of dying tumorcells in the absence of any adjuvant. Special care is used to avoid the carryover of cytotoxic agents. Six to 8 days later, the animals are challenged with livecells and are then monitored during several weeks for tumor growth. The absence of tumor development is interpreted as a sign of anti-tumorimmunity.
24 Immunological Reviews 220/2007
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
phosphatidylserine exposure, which manifests after 412 h
(30). Similarly, MCA205 exposed to g-irradiation (32) or
mitoxanthrone exhibited an increase in ecto-CRT, while
mitomycin C failed to induce ecto-CRT, as measured 4 h after
stimulation (Fig. 2A). Hence, in this model, CRT exposure also
correlates with immunogenicity.
These data underscore the notion that the immunogenicity
of cell death correlates with early CRTexposure. The kinetics of
CRT exposure are probably of the utmost importance. In
immunogenic cell death, CRT exposure occurs hours and days
before signs of apoptosis (such as phosphatidylserine
exposure) become manifest and the cells disintegrate. This
% DAPI AnnV% DAPI AnnV
Mitoxantrone
Mitomycin C
Tautomycin
Salubrinal
Control
0 20 40 60 80 100
+
Percent cells
Per
cent
tum
or-f
ree
mic
e
Days after rechallenge
C
n=10
n=12
n=12
n=12
n=12
n=12
D
γ-irradiation
Isotype
0 20 40 60 80 100Anti-CRTFITC Percent tumor-free mice
Cou
nts
BA
0 20 40 60
Co
0
20
40
60
80
100
0 20 40 60
CoSA
0 20 40 60
CoTA
0
20
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CoMCMTX
* *
+ +
n=10 n=10
n=10 n=10
n=12n=12
n=12 n=12
n=12γ-irradiation
−
Fig. 2. Immunogenic cell death of methylcholanthrene (MCA)205 sarcoma cells. Cells were treated in complete medium [RPMI1640supplemented with 10% fetal calf serum (FCS), 1 mM of sodium pyruvate, 10 mM of HEPES and 1% of penicillin–streptomycin] with either phosphate-buffered saline (PBS) (control), 20 mM salubrinal, 150 nM tautomycin, 30mM mitomycin C, 1 mM mitoxanthrone, or alternatively were g-irradiated(75 Gy). (A) Four hours later, the cells were stained with an anti-calreticulin (CRT) antibody (Abcam, Cambridge, UK), revealed with a secondary anti-rabbit immunoglobulin G (IgG) antibody conjugated to fluorescein isothiocyanate (FITC) (Molecular Probes, Invitrogen, Carlsbad, CA, USA) andsubjected to fluorescence-assisted cell sorting (FACS) analysis while gating on viable, 60-diamino-2-phenylindole (DAPI� ) cells with normal sidescatter and forward scatter characteristics. (B) Twenty-four hours later, cells were stained with DAPI plus an Annexin V–FITC conjugate (Miltenyi Biotec,Bergisch Gladbach, Germany) and again subjected to FACS analysis (means� SD of triplicates) while gating on all cells. (C) After 24 h of treatment, cellswere extensively washed in PBS and injected subcutaneously (25� 104 cells in 200 ml PBS) into the right flank of C57Bl/6 mice (n = number of mice),followed by the assessment of the anti-cancer immune response, as outlined in Fig. 1 (n represents the total number of mice used in all experiments),�Po 0.001 (Student’s t-test). (D) The percentage of animals that remains tumor-free after subcutaneous injection of live MCA205 cells into the leftflank (3� 104 cells in 200 ml PBS) was plotted against time.
Immunological Reviews 220/2007 25
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
outcome is different from the general exposure of all proteins
from the ER that can be seen in full-blown apoptosis,
presumably as a result of membrane blebbing (and perhaps
the mixture of intracellular and plasma membranes),
concomitant with phosphatidylserine exposure (39). Thus,
only the early wave of CRT exposure can discriminate between
immunogenic and non-immunogenic cell death.
Stimulation of anti-tumor vaccination by ecto-CRT
Ecto-CRT is a novel ‘eat-me’ signal (37, 40) allowing for the
engulfment of tumor cells by DCs, both in vitro and in vivo (30).
The treatment of CT26 cells with tautomycin, an inhibitor of
the protein phosphatase-1 (PP1), or with salubrinal, an
inhibitor of the complex formed by PP1 and its cofactor
GADD34 (41), causes CRT exposure without a major
induction of cell death and phosphatidylserine exposure (30).
Ecto-CRT can be detected by in situ immunofluorescence on live
cells. This method yields the most reproducible results,
presumably because it does not require prior trypsinization
and because it detects both CRT translocation to the surface and
its aggregation in discrete patches. In addition, CRT exposure
can be detected by fluorescence-assisted cell sorting (FACS)
analysis, after trypsinization and immunofluorsecent staining
of non-permeabilized cells, yielding a shift that affects a
portion of the population and that has to be compared with
isotype control staining, by superpositions of FACS histograms
from treated and untreated cells. CRT exposure could also
be measured on MCA205 cells treated with mitoxantrone,
g-irradiation, and PP1/GADD34 inhibitors (Fig. 2A). However,
tautomycin or salubrinal-treated MCA205 cells failed to
vaccinate against rechallenge with live tumor cells (Fig. 2C, D)
but rather formed tumors themselves, indicating that they had
not been programmed to die by the treatment. Hence, the
induction of CRT exposure is by itself not sufficient to mediate
tumor vaccination.
In a further series of experiments, we found that MCA205
cells that were subjected to the simultaneous treatment with
the lethal compound mitomycin C and the ecto-CRT-inducing
agent tautomycin became immunogenic, while neither of
the two agents alone induced immunogenicity of such cells
(Fig. 3A, B). A similar result was obtained when recombinant
B
Per
cent
of t
umor
-fre
e m
ice
Days after rechallenge
C
A
00 20 40 60
CO n=5
MC n=12
MC + r CRT n=12
r CRT n=12
*
0
20
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Cou
nts
50
40
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10
0100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103
100
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Fig. 3. Combination regimes for the induction of immunogenic cell death. (A) Methylcholanthrene (MCA)205 cells were cultured for 24 h with30mM mitomycin C and/or 150 nM tautomycin. Optionally, cells were treated with recombinant calreticulin (CRT) (3mg for 1� 106 cells) in 10ml offluorescence-assisted cell sorting (FACS) buffer [phosphate-buffered saline (PBS) with 2% fetal calf serum (FCS)] during the last 45 min of theexperiment. Then, the cells were washed three times with FACS buffer and subjected to the immunofluorescence detection of surface CRT (sameprotocol as in Fig. 2A) or (B, C) injected into C57Bl/6 mice to evaluate the efficacy of anti-cancer vaccination (same protocol as in Fig. 2D) (n representsthe total number of mice used in all experiments), �Po 0.001 (Student’s t-test).
26 Immunological Reviews 220/2007
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
CRT was adsorbed to the surface of live cells, a manipulation
that did not result in immunogenicity. However, adsorption of
CRT to the surface of mitomycin C-treated cells conferred
immunogenicity (Fig. 3A, C). Moreover, when chemical
inhibitors of the PP1/GADD34 complex were replaced by
small interfering RNAs (siRNAs) depleting PP1 and GADD34
(Fig. 4A), we found that this manipulation induced ecto-CRT
(Fig. 4B) yet failed to induce immunogenicity (Fig. 4C,D), unless
it was combined with mitomycin C (Fig. 4B,D). When CT26
colon cancer cells treated with mitomycin C were coated with
recombinant CRT, they elicited a potent anti-tumor immune
response (30). However, the admixture of cells treated with
mitomycin C alone and that of cells treated with recombinant
CRT alone did not result in an anti-cancer vaccine (Fig. 5). The
simplest interpretation of these results is that CRT must be on
the surface of those cells that are going to die (as opposed to
the surface of adjacent live cells) to render cell death
immunogenic. Altogether, these results can be interpreted in
Per
cent
tum
or-f
ree
mic
e
A
B
C
siRNA CO siRNA (PP1+GADD34) siRNA CO+MC siRNA (PP1+GADD34)+MC
DCO n=5siRNA (PP1+GADD34) n=6siRNA (PP1+GADD34)+CRT n=6siRNA CO n=5
Days after rechallenge
CO n=5siRNA (PP1+GADD34) + MC n=6siRNA (PP1+GADD34)+CRT n=6MC n=6MC+CRT n=6
Cou
nts
GADD34 75 kDa
GAPDH 36 kDa
siRNA
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CO GADD34
siRNA
CO CO PP1
*
00
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Anti-CRT-FITC
siRNA50
0100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103
10
20
30
40
Isotype
Fig. 4. Knockdown of PP1/GADD34 induces calreticulin (CRT) exposure and facilitates immunogenic cell death. CT26 cells were transfectedwith small interfering RNAs (siRNAs) designed to downregulate PPICa (50-rGrCrUrGrGrCrCrUrArUrArArGrArUrCrArGrATT-30) and GADD34[(50-rCrArGrGrArGrCrArGrArUrCrArGrArUrArGrATT-30) sequence] or with unrelated control (50rGrCrCrGrGrUrArUrGrCrCrGrGUrUrArArGrUTT-30).(A) Forty-eight hours later, the efficacy of the siRNAs were tested by immunobloting with specific antibodies. (B) Mitomycin C was optionally addedduring the last 24 h of the experiment, and the surface exposure of CRT was determined among viable [60-diamino-2-phenylindole (DAPI� )] cells byimmunofluorescence and cytofluorometry. (C) In addition, the capacity of such cells to vaccinate against tumor development was monitored, eitherwithout a cell death inducer or (D) after addition of mitomycin C, (n represents the absolute number of mice used in all experiments), �Po 0.001(Student’s t-test).
Immunological Reviews 220/2007 27
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
the sense that only the combination of ecto-CRT plus cell death
(but neither of these features alone) results in immunogenicity.
Apparently, CRT must be at the surface of the cells that are
going to die.
Immunogenic chemotherapy stimulated by ecto-CRT
When subcutaneous MCA205 sarcomas are established in
C57Bl/6 mice, they fail to respond to local injections of the
non-immunogenic cell death-inducer mitomycin C. Similarly,
such tumors continue to proliferate after intratumoral injection
of recombinant CRT protein or the ecto-CRT-inducer
tautomycin. However, the combinations of mitomycin C plus
recombinant CRT or mitomycin C plus tautomycin led to
permanent cure in all tested C57Bl/6 mice, provided that the
animals were immunocompetent (Fig. 6A). When the same
type of experiment was done in athymic nu/nu mice, the
combination therapy of mitomycin C plus recombinant CRT
or tautomycin lost its efficacy (Fig. 6B), supporting the notion
that this kind of immunochemotherapy truly relies on a T-cell-
dependent immune response. These data recapitulate similar
results obtained with CT26 colon cancers in BALB/c mice, in
which only the combination of cell death inducers (mitomycin
C or etoposide) plus ecto-CRT (provided as recombinant CRT
or induced by PP1/GADD34 inhibitors such as tautomycin,
salubrinal, or calyculin A) could cure established tumors (30).
In the CT26 model, we showed that animals cured of CT26
tumors by the injection of mitoxanthrone, etoposide, plus
recombinant CRT or etoposide plus calyculin were protected
against rechallenge with live CT26 cells but not with live cells
from the unrelated TSA mammary carcinoma. However, when
the established TSA tumor was successfully treated by similar
intratumoral injections, the mice became protected against
further challenge with live TSA cells (Fig. 6C–G). These results
confirm the specificity of this immunochemotherapy, which
leads to the establishment of a permanent anti-cancer immunity
against autologous (but not against unrelated) tumors.
As further evidence that immunochemotherapy triggered an
active immune response, we obtained the splenocytes from
BALB/c mice that had either been successfully vaccinated
against CT26 cancers (Fig. 7A) or had been cured from
established CT26 cancers (Fig. 7B). Upon adoptive transfer
into naive irradiated BALB/c hosts, such cells conferred
significant protection against subsequent challenge with live
CT26 (but not TSA) tumors. Altogether, these results support
the feasibility of an immunochemotherapy based on the
induction of immunogenic cell death.
A unique sequence of molecular events leading toimmunogenic tumor cell death
As shown here and in Obeid et al. (30), some cell death
inducers (e.g. anthracyclines) can elicit immunogenic cell
death, while most cell death inducers (e.g. etoposide,
mitomycin C) trigger non-immunogenic cell death. The only
biochemical difference between non-immunogenic and
immunogenic cell death that has been identified thus far
resides in the absence or presence of ecto-CRT (and CRT-
associated proteins such as ERp57) on the plasma membrane
surface. Importantly, CRTexposure was an early event (30), and
tumor cells were optimally immunogenic at relatively early
time point (Fig. 8), well before the cells have undergone
apoptosis. When cell death is immunogenic, ecto-CRT appears
on the surface, as the result of an active translocation of pre-
formed endo-CRT (30). This translocation is an early event that
becomes detectable within minutes, well before the cells show
phophatidylserine expression and manifest the morphological
changes that define apoptosis, namely chromatin condensation
(pyknosis) and nuclear fragmentation (karyorhexis).
Enucleated cells that obviously cannot manifest any nuclear
Perc
ent tu
mor-
free m
ice
0
Control n=10
MC n=11
MC + r CRT (together) n=10
r CRT n=7
MC + r CRT (admixed) n=10
4020 600
Days after rechallenge
100
80
60
40
20
*
Fig. 5. Calreticulin (CRT) exposure and death must occur on the sametumor cell to elicit tumor immunogenicity. CT26 cells were treatedwith mitomycin C (30 mM, 24 h), followed by an optional treatment withrecombinant CRT (3mg for 1� 106 cells) in 10ml of fluorescence-assistedcell sorting (FACS) buffer [phosphate-buffered saline (PBS) with 2% fetalcalf serum (FCS)] added during the last 45 min, washed, and theninjected into BALB/c mice to study their efficacy as anti-cancer vaccines(as in Fig. 1). In this experiment, either 3� 106 mitomycin C-treated,CRT-treated, or mitomcyin C plus CRT-treated cells were injected.Alternatively, 1.5� 106 mitomycin C-treated cells were mixed with1.5� 106 CRT-treated cells (admixed) and injected into BALB/c mice.One week later, the mice were rechallenged with live CT26 cells, and theappearance of tumors was monitored (n represents the total number ofmice used in all experiments), �Po 0.001 (Student’s t-test).
28 Immunological Reviews 220/2007
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
changes of apoptosis still acquire ecto-CRT in response to
anthracyclines (30), underlining that this effect is not linked
to the DNA damage response elicited by anthracyclines and that
it does not depend on the acquisition of apoptotic features.
Non-immunogenic cell death per se can be rendered
immunogenic by means of two manipulations resulting in
ecto-CRTexposure (Fig. 9B). First, CRT can be supplied from an
external source as a recombinant protein. This finding suggests
A PBS Mitomycin C PBS Mitomycin C
PB
S+
Tau
tom
ycin
Days Days
Tum
or v
olum
e (c
m3 )
Tum
or v
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e (c
m3 )
Tum
or v
olum
e (c
m3 )
Tum
or v
olum
e (c
m3 )
n=0Cured
n=0Cured
n=0Cured
n=4Cured
n=0Cured
n=4Cured
B
n=0Cured
n=0Cured
n=0Cured
n=0Cured
n=0Cured
n=0Cured
C
10
Days
0
2
4
6
8
TSACured Curedn=3n=5 CT26CT26n=2
TSAMitox
Intratumoral injection
Mitox
Intratumoral injection
D
E F
G
0
2
4
6
8
10
TSACured n=4 Cured n=3
Intratumoralinjection
CT26n=4
CT26
Intratumoralinjection
n=5 TSA
TSAEt+CaEt+Ca
n=5 Curedn=4 Cured
Intratumoral injection
CT26
n=4CT26
Intratumoral injection
n=4TSA
0
2
4
6
8
10
TSANaive
0
2
4
6
8
10
CT26Naive
Days
DaysDays
Days0 10 20 30 40 50 0 105 20 30 3515 25
0 4020 80 120 14060 100 2000 4020 80 120 14060 100 200
0 4020 80 120 14060 100 200
10
0
2
4
6
8
+r.
CR
T
PB
S+
Tau
tom
ycinT
umor
vol
ume
(cm
3 )+
r.C
RT
Et+r.CRT Et+r.CRT
n=1
Fig. 6. Immunogenic chemotherapy for the treatment of established tumors. (A, B) Immunogenic chemotherapy of methylcholanthrene(MCA)205 sarcomas. (A) Groups of five immunocompetent or (B) athymic nu/nu C57Bl/6 mice carrying MCA205 sarcomas received one singleintratumoral injection (arrows) of 50ml phosphate-buffered saline (PBS) alone or with 30mM mitomycin C, 15 mg/ml recombinant calreticulin (CRT)protein, and/or 150 nM tautomycin. Tumor growth was then monitored with a caliper. The numbers refer to the number of tumors that have beencured (up to day 40) after treatment. (C–G) Immunogenic chemotherapy of CT26 colon cancers and TSA breast cancer. Induction of an anti-tumorimmune response was evaluated by rechallenging BALB/c mice that had been cured from established CT26 cancers (E– G) using the indicatedtreatments (arrows), either with live CT26 cells or with unrelated TSA breast cancer cells (arrow heads). TSA tumors that developed were again treatedby the indicated agents, followed by rechallenge with the same tumor cells.
Immunological Reviews 220/2007 29
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
that CRT does not act as immunoadjuvant because of its
capacity to bind tumor-specific antigen peptides that would be
presented to the immune system (36, 42, 43). Rather,
(peptide-free) ecto-CRT absorbed to the surface of the tumor
cells is likely to act as an eat-me signal, hence allowing for the
phagocytosis of stressed or dying cells by DCs (37, 44, 45).
Second, CRT exposure can be enforced by pharmacological
manipulations. Inhibition of PP1 or more specifically of the
PP1/GADD34 complex causes the immediate translocation of
endo-CRT to the plasma membrane, and this confers
immunogenicity to non-immunogenic cell death. This finding
illuminates the tantalizing perspective to ameliorate existing
chemotherapies by rendering tumor cell death immunogenic
(46).
The principles of the ecto-CRT-based immunochemotherapy
appear clear with respect to its final outcome, i.e. establishment
of anti-tumor immunity. However, the details of this process
are poorly understood in its mechanistic details, as far as the
interaction between dying cells and the immune system is
concerned. Depletion of DCs abolishes the anti-tumor immune
response as efficiently as does the depletion of T cells (29, 30).
Based on the observation that the presence of ecto-CRT is
sufficient to induce phagocytosis of tumor cells by DCs (30),
the following scenario can be envisaged (Fig. 10). Upon rapid,
pre-apoptotic ecto-CRT exposure on their surface, tumor cells
(or their fragments) are taken up by DCs. However, this DC-
mediated uptake per se is not sufficient to cause optimal antigen
presentation. As a conditio-sine-qua non for DC-mediated antigen
presentation, tumor cells (or their fragments within DCs) have
to activate the apoptotic machinery. Yet-to-be-determined
factors emanating from disintegrating cells then stimulate DC
maturation and optimal antigen presentation to cytotoxic T
lymphocytes.
This scenario is compatible with the observation that only
the combination of ecto-CRT and chemotherapy-elicited cell
death (but neither of them alone) occurring in the same cells
(rather than adjacent cells) allows for anti-tumor vaccination in
vivo. It suggests that only a particular temporal sequence of
death-associated events (CRT exposure before late stage
apoptosis) can elicit an immune response at the DC level by
the concatenation of antigen uptake, DC maturation, and
effective antigen presentation.
Molecular mechanisms of CRT exposure: implicationsfor anti-cancer chemotherapy
CRT is a Ca21-binding protein with a high capacity for
buffering Ca21 yet low affinity for this divalent ion. As a
result, CRT is a modulator of Ca21 signaling and Ca21
homeostasis (47–49). CRT is a soluble protein that is mainly
located in the lumen of the ER, where it functions also as a
chaperone and lectin interacting with several proteins endowed
with disulfide isomerase activity, in particular Erp57 (50, 51).
A fraction of CRT resides outside of the ER. Cytosolic CRT may
be produced by retrotranslocation from the lumen of the ER
(52) or by inefficient CRT translocation to the ER (53).
MTXcured cured curedn=13
* * *
*p < 0.05 vs COA
Tumorbearing
0
20
40
60
80
100B
0
20
40
60
80
100
MTXn=10
Per
cent
tum
or-f
ree
mic
e
* * *
n=10 n=11n=11 n=13n=7
Naive MC+TA MC+r.CRT MC+TA MC+r.CRT
Fig. 7. Adoptive transfer of anti-cancer immunity. (A) Adoptive transfer of tumor immunity induced by vaccination with dying tumor cells.Splenocytes were obtained from BALB/c mice that had been successfully vaccinated against CT26 tumors by injecting dying tumor cells that had beenincubated in vitro either with mitoxanthrone, mitomycin C (MC) plus tautomycin (TA), or mitomycin C plus recombinant calreticulin (CRT).Splenocytes were retrieved 10 weeks after anti-cancer vaccination and 9 weeks after rechallenge with live tumor cells. Splenocytes (1� 107) wereadoptively transferred by tail vein injection into 6-week-old female BALB/c mice, which received 5� 105 CT26 cells subcutaneously 1 day later. Tumorgrowth was monitored. (B) Adoptive transfer of tumor immunity induced by immunochemotherapy of established cancers. Splenocytes from mice thathad been cured from established CT26 cancers by intratumoral injection of the indicated agents (10 weeks after treatment) were transferred as in (A)and evaluated for their capacity to prevent the growth of live CT26 cells in the recipients (n represents the absolute number of mice used in allexperiments), �Po 0.05 (Student’s t-test).
30 Immunological Reviews 220/2007
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
Cytosolic CRT is arginylated at the N-terminal aspargine
residue and can associate with stress granules (54). The
function of CRT outside of the ER is not clear. However, CRT
has been suggested to regulate nuclear protein transport (55),
signaling via nuclear steroid receptors, and integrin signaling.
In response to anthracyclines and PP1/GADD34 inhibitors, a
variety of rodent cancer cells translocate CRT to the cell surface,
including CT26 colon carcinoma, MCA205 sarcoma, TS/A
breast carcinoma (56), SV40-transformed mouse embryonic
fibroblasts in mouse, and PROb colon carcinomas in rat (57).
Similarly, a variety of human cancer cell lines (e.g. HeLa cervical
cancer, HCT116 colon cancer, A549 non-small cell lung
cancer) expose CRT in response to anthracyclines and PP1/
GADD34 inhibitors, suggesting that the signal transduction
pathways leading to CRT exposure are conserved in mammals
(data not shown). The molecular mechanisms through which
CRT translocates to the cell surface are being investigated in our
laboratory.
One point of convergence between the pathway elicited by
anthracyclines and the one elicited by PP1/GADD34 inhibitors
is the phosphorylation of eukaryotic initiation factor 2a
A
B
0
0
Per
cent
tum
or-f
ree
mic
eP
erce
nt tu
mor
-fre
e m
ice
C
n = 5
n = 5n = 5
n = 5
n = 18
n = 18n = 17
n = 16
Anti tumorImmunity No tumor
Livecells
Dyingcells
Day 0
Doxorubicin
24HTreatment time
4H2H0H
24HTreatment time
4H2H0H
Mitoxantrone
100
80
60
40
20
100
80
60
40
20
Fig. 8. Simultaneous injection of anthracycline-treated and livetumor cells for the assessment of immunogenicity of tumor cells.(A) Schematic representation of experimental protocol. 3� 106
anthracycline-treated CT26 cells and 5� 105 untreated CT26 cells wereinjected subcutaneously on the same day in opposite flanks. Tumordevelopment was then monitored. (B, C) Kinetics of anthracycline treatmentfor optimal immunogenicity. Cells were cultured with 25mM doxorubicin(B) or 1mM mitoxantrone (C) for the indicated period, immediately beforeextensive washing in sterile PBS and subcutaneous injection.
EtoposideMitomycin C
A
B Etoposide or Mitomycin C+ PPI/GADD34 inhibition
Etoposide
Recombinant Calreticulin Calreticulin
Anthracyclines
Non-immunogenic Immunogenic
Mitomycin C
ImmunogenicImmunogenic
Fig. 9. Strategies for the induction of immunogenic cell death.(A) Difference between immunogenic versus immunogenic cell death.While most cell death inducers (exemplified by etoposide and mitomycinC) induce non-immunogenic cell death, some triggers (exemplified byanthracyclines) induce immunogenic cell death. The discriminativebiochemical characteristic of immunogenic cell death is the pre-apoptotictranslocation of calreticulin (CRT) from inside the cell (endo-CRT) to thecell surface (ecto-CRT). (B) Conversion of non-immunogenic toimmunogenic cell death. To confer immunogenicity to non-immunogenic cell death, two strategies are possible. Either recombinantCRT is absorbed to the surface of the cells, or the translocation of CRT tothe cell surface is enforced by treatment with inhibitors of the PP1/GADD34 complex.
Immunological Reviews 220/2007 31
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
(eIF2a), a protein that is known to be phosphorylated as a
result of the unfolded protein response (UPR) of the ER (41,
58, 59). Phosphorylation of eIF2a on serine 51 results in an
arrest of protein synthesis, allowing cells to adapt to ER stress
(60). Mouse embryonic fibroblasts from eIF2aS51A/S51A
knockin mice (61) failed to expose CRT in response to both
anthracyclines and PP1/GADD34 inhibitors (data not shown).
These results strongly suggest that pleiotropic responses
conditioned by eIF2a phosphorylation at the level of the ER
control CRT exposure, which in turn determines the
immunogenicity of cell death. However, direct induction of
ER stress by thapsigargin, tunicamycin, or brefeldin A (which
all induce eIF2a phosphorylation) does not induce CRT
exposure, indicating that eIF2a phosphorylation is required
but not sufficient for CRT translocation to the plasma
membrane surface.
CRT exposure could be induced in a fraction of primary
cancer cells obtained by collagenase digestion of freshly
obtained human carcinomas. Similarly, circulating leukemia
cells from several patients treated by intravenous injection with
anthracyclines exhibited an increase in CRTexposure that could
be measured ex vivo (our unpublished observation). However,
CRT exposure could not be found in all human tumor cell lines
or in all primary carcinomas and acute myeloid leukemias,
pointing to a hitherto unsuspected heterogeneity in the
mechanisms of CRT exposure. The mechanisms accounting for
this deficient CRT exposure remain elusive. It is tempting to
speculate, however, that tumors that fail to expose CRT would
be refractory to the immunological component of chemo-
therapy and hence would demonstrate a poorer prognosis than
tumors that conserve an intact CRT translocation response. This
possibility is currently under intense investigation in our
laboratory.
Theoretically, tumors may avoid the CRT-dependent immune
response by an alternative strategy, namely by proteolytically
degrading CRT, which then does not appear on the surface and
is secreted into the tumor environment. Such CRT fragments,
which have been found in the serum of patients with hepatitis
C-induced hepatocellular carcinoma (62), might then
competitively block the interaction between ecto-CRT and its
diverse range of receptors on the surface of DCs, including
scavenger receptor A (SRA), scavenger receptor expressed by
endothelial cell-I (SREC-I) (63), and CD91 (37). However, this
possibility has to be investigated in further detail.
Abnormalities in CRT expression have been documented for
several kinds of neoplasia. In a subset of patients with acute
myeloid leukemia (AML), CRT undergoes a particular post-
translational modification, namely an acetylation of residue
K206. This acetylation is specifically found in those AML
patients that bear the t(8;21) translocation, which is
associated with favorable prognosis (64). Upregulation of CRT
has a positive prognostic impact in neuroblastoma (65), a type
of cancer that is likely to be influenced by the immune system
(66). Similarly, colon cancers with microsatellite instability
(which have a better prognosis than the dominant type of
colon cancer with chromosomal instability) show a relatively
high expression level of CRT (67). The expression of CRT (and
that of its associated protein ERp57) is downmodulated in a
fraction of primary laryngeal squamous cell cancer lesions, and
this state determines a trend to reduced infiltration by CD81 T
cells and poor patient survival (68). Unfortunately, no
information is available on the subcellular localization of CRT
in these cancers, before and after chemotherapy. Therefore, the
possible link between alterations in the expression level of CRT
and anti-tumor immune responses is still elusive.
Perspectives
As outlined in this article, it appears that the pre-apoptotic
acquisition of ecto-CRT is a major factor determining the
immune response elicited by dying tumor cells. This notion
has major implications for tumor immunology, at the
following levels:
Normal
CRT exposure
CRT exposure+apoptosis
Eat-me signal
for DC
Maturation signal
for DC
Fig. 10. Model of the crosstalk between dying cells and dendritic cells(DCs). In immunogenic cell death, pre-apoptotic calreticulin (CRT)exposure allows the tumor cell to be efficiently engulfed by DCs.Subsequent biochemical changes tied to late-stage apoptosis are requiredfor the induction of DC maturation as well as for optimal antigenpresentation.
32 Immunological Reviews 220/2007
Obeid et al � Ecto-calreticulin in immunogenic cancer cell death
� Screening for compounds that induce pre-apoptotic CRTexposure should identify those cytotoxic agents that areendowed with the capacity to induce immunogenic celldeath and hence can be used for immunogenicchemotherapy. For this reason, we are currently in theprocess of screening a variety of different chemot-herapeutic agents and cytotoxic components for theircapacity to induce ecto-CRT.
� Tumors that fail to expose CRT on the cell surfaceshould have a particularly bad prognosis in response tothose chemotherapies that usually elicit an ecto-CRT-dependent anti-tumor immune response. Therefore, weare attempting to correlate CRT exposure and therapeuticoutcome. Moreover, we are in the process of dissectingthe signal transduction pathway that leads to CRTexposure. Ideally, this knowledge should lead to thedesign of a diagnostic ‘chip’ that detects the propensity oftumors to expose CRT and hence predicts therapeuticoutcome.
� External supply of ecto-CRT or enforcement of CRTexposure can ameliorate the efficacy of thosechemotherapeutic regimes that usually fail to induce CRTexposure. Similarly, ecto-CRT may restore the anti-tumorimmune response in those cancers that are impaired intheir capacity to translocate CRT to the cell surface. Thechallenge is to continue the pre-clinical and pharmaco-logical development of CRT-exposing agents and tointroduce them into clinical trials.
� Immunogenic cell death can be induced bychemotherapeutic agents (such as anthracyclines) or localradiotherapy, and this treatment is sufficient to mediateanti-tumor immune responses. Thus, endogenous danger-associated molecular patterns including ecto-CRT arehighly efficient triggers of anti-tumor immunity.Nonetheless, it will be important to investigate thepossibility of further improving immunochemotherapy,for instance by providing additional costimulatory signalsor activation signals for the immune system.
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