Ecto-calreticulin in immunogenic chemotherapy

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


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

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Days after rechallenge

C

n=10

n=12

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γ-irradiation

Isotype

0 20 40 60 80 100Anti-CRTFITC Percent tumor-free mice

Cou

nts

BA

0 20 40 60

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CoSA

0 20 40 60

CoTA

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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.



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


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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Anti-CRT-FITC

Cou

nts

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40

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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).



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


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

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CO GADD34

siRNA

CO CO PP1

*

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siRNA50

0100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103

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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).



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


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).



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

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CT26n=4

CT26

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n=5 TSA

TSAEt+CaEt+Ca

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CT26

n=4CT26


n=4TSA

0

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TSANaive

0

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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

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4

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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.



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

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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).



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

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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.



(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.



� 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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Ecto-calreticulin in immunogenic chemotherapy

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