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Microenvironment and Immunology
Antitumor Immunity Triggered by Melphalan IsPotentiated by
Melanoma Cell Surface–Associated
CalreticulinAleksandraM.Dudek-Peri�c1,GabrielaB. Ferreira2,
AngelikaMuchowicz3, JasperWouters4,Nicole Prada5, Shaun Martin1,
Santeri Kiviluoto6, Magdalena Winiarska3, Louis Boon7,Chantal
Mathieu2, Joost van den Oord4, Marguerite Stas8, Marie-Lise
Gougeon5,Jakub Golab3,9, Abhishek D. Garg1, and Patrizia
Agostinis1
Abstract
Systemic chemotherapy generally has been considered
immu-nosuppressive, but it has become evident that certain
chemother-apeutic drugs elicit immunogenic danger signals in dying
cancercells that can incite protective antitumor immunity. In this
study,we investigated whether locoregionally applied therapies,
such asmelphalan, used in limb perfusion for melanoma
(Mel-ILP)produce related immunogenic effects. In human
melanomabiopsies, Mel-ILP treatment upregulated IL1B, IL8, and IL6
asso-ciated with their release in patients' locoregional sera.
Althoughinduction of apoptosis in melanoma cells by melphalan in
vitrodid not elicit threshold levels of endoplasmic reticulum
and
reactive oxygen species stress associated with danger
signals,such as induction of cell-surface calreticulin,
prophylacticimmunization and T-cell depletion experiments showed
thatmelphalan administration in vivo could stimulate a CD8þ
Tcell–dependent protective antitumor response. Interestingly,the
vaccination effect was potentiated in combination withexogenous
calreticulin, but not tumor necrosis factor, a cyto-kine often
combined with Mel-ILP. Our results illustrate howmelphalan triggers
inflammatory cell death that can be lever-aged by immunomodulators
such as the danger signal calreti-culin. Cancer Res; 75(8);
1603–14. �2015 AACR.
IntroductionEvidence indicates that anticancer therapies capable
of harnes-
sing the host's immune system while inducing cancer cell
deathhold the highest therapeutic value (1, 2). Such therapies are
ofimmediate importance for antimelanoma therapy. Melanoma is
an aggressive cancer that typifies the paradox of being
highlyantigenic while simultaneously exerting potent
immunosuppres-sion (3).Moreover, melanoma has recently gainedwide
attentionfrom an immunotherapeutic standpoint owing to
promisingclinical effects of immune-checkpoint inhibitory drugs
(4). Allthis clearly advocates the need to further study the
antimelanomaimmune responses, and reveal additional strategies
capable ofaugmenting antimelanoma immunity.
In recent years,many anticancermodalities have been shown
topositively regulate immune-effector functions and induce
antitu-mor immunity (5). These include (i) strategies improving
thenatural killer (NK) cells'/dendritic cells' (DC)/T cells'
anticanceractivity, (ii) immunogenicity of the dying cancer cells,
and (iii)"resetting" microenvironment's immunocontexture (6).
Theabovementioned processes are strongly influenced by
certainimmune-effector cytokines exhibiting strong clinical
prognosticimpact (7). Moreover, immunogenicity as well as
vaccinationpotential has been recently linked, at least in part, to
"dangersignaling" operating on the cancer cell-level (8). Induction
ofdanger signaling mediates the spatiotemporally defined
"emis-sion" of specific "eat me" signals/damage-associated
molecularpatterns (DAMP) by the dying cancer cells, for example,
surfaceexposed (ecto-) calreticulin (CRT; ref. 9) and heat-shock
proteins(HSP)-70/90 (10), and secreted nucleotides, such as
adenosinetriphosphate (ATP; refs. 1, 11). Danger
signaling-potentiatingtherapies have been recently shown to
associate with favorableclinical outcome in cancer patients (5, 12,
13). Moreover, it hasbeen proposed that, combinatorial therapy with
exogenouslysupplied danger signals could hold great
immunogenicity-pro-moting potential (14).
1Cell Death Research and Therapy Laboratory, Department of
Cellularand Molecular Medicine, Faculty of Medicine, KU Leuven,
Leuven,Belgium. 2Laboratory of Clinical and Experimental
Endocrinology,Department of Clinical and Experimental Medicine, KU
Leuven, Leu-ven, Belgium. 3Department of Immunology, Center of
BiostructureResearch, Medical University of Warsaw, Warsaw, Poland.
4Transla-tional Cell and Tissue Research, Department of Imaging and
Pathol-ogy, Faculty of Medicine, KU Leuven, Leuven, Belgium.
5AntiviralImmunity, Biotherapy and Vaccine Unit, Infection and
EpidemiologyDepartment, Institute Pasteur, Paris, France.
6Laboratory of MolecularandCellular
Signaling,DepartmentofCellularandMolecularMedicine,Faculty of
Medicine, KU Leuven, Leuven, Belgium. 7Bioceros, CMUtrecht, the
Netherlands. 8Surgical Oncology, Department of Oncol-ogy, KU
Leuven, Leuven, Belgium. 9Institute of Physical Chemistry,Polish
Academy of Sciences,Warsaw, Poland.
Note: Supplementary data for this article are available at
Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Authors: Patrizia Agostinis and Abhishek D. Garg,
Laboratoryfor Cell Death Research and Therapy, Department of
Cellular and MolecularMedicine, KU Leuven, Campus Gasthuisberg,
O&N1, Herestraat 49, Box 802,3000 Leuven, Belgium. Phone:
32-16-33-06-50; Fax: 32-16-3-30735;
E-mail:[email protected]; and Abhishek D.
Garg,[email protected]
doi: 10.1158/0008-5472.CAN-14-2089
�2015 American Association for Cancer Research.
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Most of the chemotherapeutics tested so far as DAMPsinducers are
primarily used as systemic chemotherapeutics(15, 16) while
physicochemical modalities (such as radiother-apy/Hyp-PDT) are
primarily used as locoregional therapeutics(17–19). Considering
that immune responses following loco-regional therapy can differ
from those after systemic therapy(20), it is necessary that
anticancer immunity, danger signaling,and immune-effector
function–potentiating effects of loco-regionally applied
chemotherapeutics are also evaluated—aknowledge that is largely
missing and could have translationalsignificance (20, 21).
To this end, we studied the effects of melphalan (Mel),
theregionally applied (standard-of-care) chemotherapeutic
forextremities-associated melanoma (20, 22). Melphalan is an
alky-lating agent, used in the isolated limb perfusion
(ILP)/infusion(ILI) therapy (20, 22), for patients harboring
limb-localizedmalignancies (23). Melphalan-based ILP/ILI
(Mel-ILP/Mel-ILI)is considerably effective, with a significant
fraction of patients(25%–53%) displaying complete clinical
responses and variousothers showing partial responses (14%–39%;
ref. 22; clinicalmetadata analysis; Supplementary Table S1).
Hitherto, melano-ma cell-killing efficacy is postulated as the sole
contributor topatients' responsiveness towardmelphalan treatment
(24). How-ever, whether the promising antimelanoma efficacy
ofmelphalantherapy is associated with antitumor immunity remains
unex-plored. Thus, owing to these conjectures and a
gap-in-knowledgeabout regional chemotherapeutics, we studied
themechanisms ofmelphalan-induced melanoma cell death, the
inflammatory con-texture as well as the efficacy of
melphalan-induced inducedantitumor immunity/immune-effector
function against melano-ma. We also studied certain putative
immunomodulatory factorsthat are usable as combinatorial treatment
for augmenting anti-melanoma immunity.
Materials and MethodsMaterials and reagents
The following drugs were used: melphalan (Sigma;
M2011),thapsigargin (Enzo Life Sciences; BML-PE180-0001).
Hypericinwas prepared, purified, and stored as described previously
(25).Antibodies against the following proteins were used:
BiP/GRP78(Cell Signaling Technology; 3183), P-eIF2a (Cell Signaling
Tech-nology; 3597), eIF2a (Cell Signaling Technology; 21035),
MICA/B (Acris; AM26694AFN), actin (Sigma; A5441), CRT
(anti-CRT;Abcam;Ab92516),ULBP2 (Abcam;Ab88645),HSP90
(Stressgen;ADI-SPA-830), and HSP70 (Santa Cruz Biotechnology;
SC-24).The following secondary antibodies were used: goat
anti-mouse-DyLight680 (Thermo Scientific; 35519), goat
anti-rabbit-DyLight800 (Thermo Scientific; 35571), goat
anti-mouse-AlexaFluor 647 (Invitrogen; A21235), and goat
anti-rabbit-Alexa Fluor647 (Invitrogen; A21244). Western blot
detection was done onOdyssey.
Cell culture and treatmentsAll cellswere cultured inDMEM(D6546;
Sigma)with2mmol/L
glutamine, penicillin–streptomycin (P0781; Sigma) and 10%
fetalbovine serum (FBS) at 37�C under 5% CO2. A375 cells
wereobtained from the ATCC and authenticated through DNA
shorttandem repeat (STR) profiling. A375/K1735/MM031/B78 cellswere
incubated with melphalan (300 mmol/L or 600 mmol/Lfor B78) or
brefeldin A (50 ng/mL for B78 cells) for the indicated
times. For Hypericin-based photodynamic therapy
(Hyp-PDT)conditions, A375 cells were incubated for 16 hours with150
nmol/L Hypericin, whereas B78 were incubated for 2 hourswith 500
nmol/L Hypericin in media without FBS, followedby removal of
Hypericin, irradiation (2.70 J/cm2), and werecultured for indicated
times.
Measurement of ecto-CRT, ecto-HSP70, and ecto-HSP90After
treatment, cells were collected with TrypLE Express (Life
Technologies; 12604-021), washed with PBS and with FC
(FlowCytometry) buffer (2%FBS, 1%BSA in PBS), incubated for 1
hourat 4�Cwith primary antibodies, washed, and incubated for 1
hourat 4�C with secondary antibodies. After final washes, cells
wereincubated in FC buffer including 1 mmol/L Sytox Green
(LifeTechnologies; S7020) for 15 minutes and analyzed on AttuneFlow
Cytometer (Life Technologies). The permeabilized cellswere excluded
from the analysis due to intracellular staining, andthe fold
changes in the mean fluorescence intensity (MFI) foreach DAMP were
analyzed.
DC-maturation analysisHuman and murine immature DCs (iDC) were
prepared
according to previously described protocols (26, 27).
Theprotocol for coincubation of cancer cells with iDCs has
beenpreviously described (28, 29). Briefly, the DCs were
coculturedwith untreated or dying cancer cells (24-hour time point)
at a1:20 (DCs:cancer cells) ratio for 24 hours under
standardculture conditions. In some experiments, cancer cells
werepreincubated with blocking antibodies (1.25 mg/106 cells):
IgY(Promega, G116A), anti-HSP90 (Novus Bio; NB120-19104;antibodies
were present in the coculture media as well), coatedwith
recombinant CRT (rCRT; Abcam; ab15729; cells wereincubated with
rCRT at 4�C for 30 minutes followed by remov-al of unbound protein)
as described before (9) or in thepresence of 100 ng/mL soluble
recombinant TNF (rTNF;human: PeproTech, 300-01A; murine: PeproTech;
315-01A).For staining of human DCs, the following antibodies
wereused: anti-HLA-DR antibody (BD; MHLDR01) and anti-CD86(BD;
MHCD8605). For staining of murine DCs, the followingantibodies were
used: anti-MHC II antibody (eBioscience; 11-5321-81) and anti-CD86
(eBioscience; 17-0862-81).
T-cell proliferationThe protocol for triple culture of cancer
cells, DCs, and T cells
(1:1:50 ratio, respectively) has been previously described
(29).Briefly, the untreated or dying cancer cells (24-hour time
point)were cocultured with iDCs for 24 hours. Allogeneic T
cells(CD3þ), isolated from donors' blood according to the
manu-facturer's recommendations (Pan T Cell Isolation Kit II;
Milte-nyi Biotec; 130-095-130), labeled with eFluor 670
ProliferationDye (eBioscience; 65-0840-85), were added to the
coculturesfor an additional period of 5 days. Human IL2 was added
at day2 of the triple cocultures (25 U/mL). At the end of day 5,
cellswere stained for CD3, CD4, and CD8 [with antibodies
anti-CD3-eFluor450 (48-0038), anti-CD4-FITC (11-0049),
andanti-CD8-PE-Cy7 (25-0049); all from eBioscience]. Dead cellswere
excluded using the Fixable Live/Dead Yellow stain accord-ing to the
manufacturer's specifications (Invitrogen; L34959).Data acquisition
was performed on Gallios flow cytometer(Beckman Coulter) and the
Kaluza software (Beckman Coulter)was used for data analysis.
Dudek-Peri�c et al.
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Prophylactic mouse vaccinationMouse experiments were performed
in the animal facilities of
Warsaw Medical University (Warsaw, Poland) and KU Leuven(Leuven,
Belgium), according to the guidelines of the ethicalcommittees of
these universities. The prophylactic mice vaccina-tion was
performed according to the previously described proto-col (29).
Briefly, the mice were injected subcutaneously with 100mL
containing 500� 103 dying B78 cells (40% of apoptotic cells;in
indicated experiments, the cells were coated with
blockingantibodies or rCRT, as described above) or
coinjectedwithmurinerTNF, or with 100 mL of PBS into the left
flank. After 10 days, micewere rechallenged with untreated B78
cells into the right flank(50� 103 cells in 100 mL PBS) and tumor
growth was monitoredfor the next 40 days. Depletion of CD4þ or CD8þ
T cells wasperformed according to the previously described protocol
(30).To evaluate the elimination of T cells, blood was collected
viacheek pouch and the presence of CD4þ or CD8þ T cells wasdetected
through anti-CD4 (BD; 553047) and anti-CD8 (BD; cat.no., 553031)
staining, as described previously (30).
Statistical analysisData are presented as exact values,
percentages of cell population
or fold changes, specifically as indicated on each figure. Error
barsrepresent SEM. Depending on the type of experiments, as a
statis-tical analysisweperformed the Student t test,
one-wayANOVAwiththeDunnett post-test or two-wayANOVAwith
theBonferroni post-test, as indicated in thefigure legends. Fold
expressions of cytokinesin patients' samples were analyzed for
significance using either thetwo-tailed one sample t test (if
results hadGaussiandistribution) orthe two-tailed Wilcoxon rank-sum
test (if results did not haveGaussian distribution). Always �
represents P < 0.05; �� representsP < 0.01; and ���
represents P < 0.001.
ResultsMel-ILP evokes proinflammatory immune-effector
cytokinesproduction
A previous microarray/qRT-PCR analysis confirmed a signifi-cant
increase in IL6 levels, post-Mel-ILP in patients' biopsies
(31);this inspired us to further investigate whether clinical
melphalantreatment is associated with induction of certain other
majorcytokines. We first extended previous expression analysis
(31)to specific immune-effector cytokines in the tumor bed.
BeyondIL6 potentiation (31), we found significant increase in
levels ofIL1B and IL8 in the absence of significant changes in
IL10, TNF,and IFNG levels, in tumor samples taken 1 hour after
Mel-ILP(Fig. 1A).
Next, considering that Mel-ILP is a locoregionally
appliedtherapy, we wondered to what extent the Mel-ILP–induced
cyto-kine transcript pattern present in the tumor bed was mirrored
bythe locoregional plasma-associated cytokine pattern on the
pro-tein level. As early as 1 hour afterMel-ILP treatment, protein
levelsof IL6 and, to a lesser extent, IL1b increased significantly,
while wefailed to detect any significant increase in the levels of
IL12p70,IL8, TNF, IL10 (Fig. 1B and Supplementary Fig. S1A), and
IFNg(data not shown). Thus, the locoregional serum-associated
cyto-kine pattern largely mirrored the tumor bed–associated
transcriptpattern. Considering that samples were collected very
early (10–30 minutes/1 hour) after Mel-ILP, we suspected that
freshlytumor-infiltrating immune cells would not substantially
contrib-ute to the observed cytokine production. In line with this,
we
failed todetect increased immune cells' infiltration
followingMel-ILP (1 hour) after staining tumor sections for
CD68/CD3, specificmarkers of monocytes/macrophages, and T
lymphocytes, respec-tively (Supplementary Fig. S1B and S1C). This
suggests that Mel-ILP–triggered increase in
immune-effectors/proinflammatorycytokines is mostly the result of
the alteration in preexistingtumor microenvironment.
Melphalan-induced apoptosis in vitro is modulated by
thecombination of ER stress and ROS
A previous study indicated that post-Mel-ILP, signatures
ofendoplasmic reticulum (ER) stress (i.e., ATF3, GADD45A, andXBP1s)
were induced in patients' biopsies (31). Considering thatER stress
is a crucial stress response for eliciting cell death,
dangersignaling and cytokine production (32), we decided to
investigatethe ER stress-cell death cross-talk after melphalan
treatment.
We therefore studied the biochemical hallmarks of
melphalan-induced melanoma cell death in vitro using human (A375)
andmurine (B78) metastatic melanoma cell lines. Melphalan
time-dependently affected melanoma cell viability (Fig. 2A and
Sup-plementary Fig. S2A) and induced phosphatidylserine
exposure(Fig. 2B and Supplementary Fig. S2B), loss of
mitochondrialtransmembrane potential (Dcm; Fig. 2C and
Supplementary Fig.S2C), and significant activation of caspase-3
(Fig. 2D and Sup-plementary Fig. S2D). Furthermore, the pan-caspase
inhibitorzVAD-fmk abolished caspase-3 activation (Fig. 2E and
Supple-mentary Fig. S2E) and resulted in aprotection fromcell death
(Fig.2F and Supplementary Fig. S2F), thus indicating that
melphalaninduces apoptosis.
We next investigated whether melphalan induced ER stress
byevaluating markers of the unfolded protein response (UPR).
Mel-phalan-treated melanoma cells showed an increase in
BiP/GRP78content, a clear induction of eIF2a phosphorylation (Fig.
2G andSupplementary Fig. S2G) and of the spliced form of XBP1 (Fig.
2Hand Supplementary Fig. S2H), indicating the ability of
melphalanto activate the PERK and IRE1a arms of the UPR. Addition
of thechemical chaperone, TUDCA, which has been reported to
alleviateER stress (33), resulted in decreased levels of
phospho-eIF2a (Fig.2I) and a partial protection from
melphalan-induced cell death(Fig. 2J). This suggests that although
ER stress contributes to theinduction of apoptotic cell death after
melphalan treatment, othersignaling events are required to incite
apoptosis.
The presence of ER stress along with reactive oxygen
species(ROS) induction and caspase signalinghas been shown
toprovidethe biochemical prerequisite for efficient danger
signaling(9, 15, 28). Indeed, as reportedpreviously (34),melphalan
causeda significant increase in the intracellular levels of ROS in
mela-noma cells (Fig. 2K). Attenuation of ROS signaling by the
anti-oxidant N-acetylcysteine (NAC), neither significantly
protectedmelanoma cells frommelphalan-induced apoptosis (Fig. 2L),
norit affected the activation of ER stress (data not shown). In
contrast,the combination of TUDCA and NAC significantly blunted
mel-phalan-induced melanoma apoptosis (Fig. 2M).
These results underscore that ROS production and ER stress actin
concert to induce apoptosis in melanoma cells in response
tomelphalan.
Melphalan-induced apoptosis is associated with a defined
ERstress and ROS-dependent danger signaling
Melphalan treatment in vitro is able to induce ER stress
andROS—two most important apical prerequisites for danger
Melphalan, Antimelanoma Immunity, Inflammation
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signaling elicitation (28, 35) by apoptotically dying cells.
Toevaluate whether melphalan treatment induces danger
signalinginmelanoma cells and to reveal itsmolecular nature, we
analyzeda panel of well-established DAMPs and/or "eat me" signals
(9,10, 28, 36).
First, we measured CRT, HSP70, and HSP90 on the cell
surface(ecto-CRT, ecto-HSP70, and ecto-HSP90) of
nonpermeabilizeddying melanoma cells, and the secretion of ATP. The
effectsinduced by melphalan in A375 cells were compared with
Hyp-PDT, a previously characterized danger signaling-inducing
ther-apy (28, 29, 37), which caused fast preapoptotic ecto-CRT
andecto-HSP90, followed by HSP70 surface exposure (Fig. 3A).
Incontrast, melphalan-induced melanoma apoptosis was accom-panied
only by a significant ecto-HSP90 after 24 hours (Fig. 3A), aresult
that was confirmed in the murine B78 and K1735 cells plusin the
humanMM031 short-culturemelanoma cells (Supplemen-tary Fig.
S3A–S3C). Interestingly, melphalan treatment did notinduce ATP
secretion (Supplementary Fig. S3D and S3E). Of
note,melphalan-induced ecto-HSP90 was detected only when thewhole
population of dying cells entered late-apoptotic stage(according to
kinetics of caspase-3 activity; compare Fig. 3Aand Fig. 2D).
However, the population of ecto-HSP90þ cells waspartially
AnV�/7AAD� and AnVþ/7AAD� (preapoptotic or early/mid-apoptotic
cells; Fig. 3B), while the small population of ecto-CRTþ cells was
mostly AnVþ/7AAD� (early/mid-apoptotic cells;Supplementary Fig.
S3F). Thus, contrary to Hyp-PDT, melphalaninduced pre- or
early/mid-apoptotic ecto-HSP90 in a predomi-nantly
late/postapoptotic cell culture environment.
Because DAMPs emission has been shown to predominantlyrely on ER
stress-ROS signaling, and in some cases require caspasesignaling
(28), we decided to block these apoptotic mediators.Blocking
caspases by zVAD-fmk blunted melphalan-inducedecto-HSP90 (Fig. 3C
and Supplementary Fig. S3G), whereasattenuation of
melphalan-induced ER stress by TUDCA (Fig.3D), or ROS production by
NAC (Fig. 3E) exerted a dose-depen-dent decrease in ecto-HSP90.
Consistent with the effects of zVAD-fmk and the kinetics of DAMP
exposure, the combination ofTUDCA and NAC suppressed ecto-HSP90
(Fig. 3F), therebystrongly coupling cell death signaling reliant on
ER stress andROS with the mobilization of HSP90 at the plasma
membrane.
Despite inducing ROS and some features of ER stress, melpha-lan
did not increase ecto-CRT. Because in previous studies induc-tion
of robust ER stress, by thapsigargin and tunicamycin,
restoredecto-CRT after cisplatin treatment (38), we tested whether
aug-menting ER stress in melphalan-treated cells would elicit
ecto-CRT. To this end, we used various ER stress–inducing
agents:sarco/endoplasmic reticulum Ca2þ-ATPase (SERCA)
pumpinhibitor thapsigargin, the inhibitor of N-glycosylation
tunica-mycin, theproteasome inhibitor bortezomib, the glycolytic
inhib-itor 2-deoxy-D-glucose (2DG), and the reducing agent
dithiothrei-tol (DTT). Intriguingly, only the addition of high-dose
thapsi-gargin, but not other aforementioned ER stress
inducers,restored ecto-CRT after melphalan treatment (Fig. 3G
andSupplementary Fig. S3H). This effect could be dissociated froman
increased induction of cell death (Supplementary Fig. S2I)because
none of these agents enhanced melanoma killing when
Figure 1.Mel-ILP increases production ofproinflammatory
cytokines inmelanoma patients. A, relativeexpression of various
cytokines (IL1B,IL8, IL10, TNF, and IFNG) assessed onmRNA level
using qRT-PCR; RNA wasisolated from snap-frozen tumorsamples
collected pre- and post-Mel-ILP (the graph presents
relativeexpression of cytokines for eachpatient; statistical
analysis is describedin Materials and Methods). B, serasamples
isolated from locoregionallycirculating blood collected
beforeMel-ILP, after administration of melphalan(10–30 minutes),
and after Mel-ILP(1 hour) were tested for IL1b, IL6, IL8,IL10,
IL12p70, and TNF content (thegraph presents concentration of
eachcytokine for each patient; mean� SEMare added; respective
significantP values are mentioned forcorresponding conditions;
theWilcoxon matched-pairs signed-ranktest).
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added after the commitment phase of melphalan-inducedapoptosis
(i.e., after loss of mitochondria transmembranepotential and
caspase activation; Fig. 2B). Likewise, we won-
dered whether enhancing ROS levels could increase melphalanor
melphalan/thapsigargin-induced ecto-CRT. However, addi-tion of H2O2
failed to increase melphalan or melphalan/
Figure 2.Melphalan induces ER stress and ROS-dependent
apoptosis. Melphalan (Mel; 300 mmol/L)-treated A375 cells were
collected at indicated time points andinvestigated for percentage
of surviving cells (MUH assay; A), percentage of phosphatidylserine
exposing cells (stained with Annexin V, AnVþ) and
permeabilizedcells (PIþ; B), percentage of cells with decreased
mitochondrial transmembrane potential (Dcm, assessed by lower TMRM
staining; C), and increase in caspase-3activity in cell lysates
(RFU, relative fluorescent units; D). Treated A375 cells
coincubatedwith zVAD-fmk (25 mmol/L) collected at 24-hour time
point were tested forcaspase-3 activity in cell lysates (RFU,
relative fluorescent units; E) and percentage of permeabilized
(PIþ; F) cells. G and H, representativeWestern blot analysis
andcorresponding densitometric quantification showing kinetics of
BiP and eIF2a (total and phosphorylated) protein levels in
melphalan-treated A375 cells (G) andXBP1 splicing by RT-PCR (H). G
and H are representative results out of three independent
experiments. I, representativeWestern blot analysis of BiP and
eIF2a (totaland phosphorylated) protein levels of melphalan-treated
A375 cells (24 hours) coincubated with TUDCA [at 250 mg/mL (T250)
or 500 mg/mL (T500)]. J,corresponding percentage of
phosphatidylserine-exposing cells (AnVþ) and permeabilized cells
(PIþ). K, kinetics of ROS production by DCF-DA staining
ofmelphalan-treated A375 cells. L and M, effect of addition of NAC
(L) or NAC and TUDCA (added at the indicated concentrations; M)
tomelphalan-treated A375 cells(24-hour time point). Graphs show the
percentage of phosphatidylserine-exposing cells (AnVþ) and
permeabilized cells (PIþ). All graphs (A–F and J–M) show resultsof
three independent experiments (mean � SEM) and are statistically
analyzed with a two-way ANOVA; � , P < 0.05; �� , P < 0.01;
���, P < 0.001.
Melphalan, Antimelanoma Immunity, Inflammation
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Figure 3.Melphalan (Mel) induces ER stress andROS-dependent
danger signaling inmelanoma cells. A375 cells treatedwithmelphalan
(300 mmol/L) or Hyp-PDT (150 nmol/LHypericin; 2.70 J/cm2
irradiation) were evaluated at indicated time points for ecto-CRT,
ecto-HSP70, and ecto-HSP90 in nonpermeabilized cells (A;
threeindependent experiments, mean � SEM; and two-way ANOVA
analysis; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001).
(Continued on the following page)
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thapsigargin-induced ecto-CRT (Fig. 3I), and did not
exacerbatecell death (Supplementary Fig. S2J). Notably, addition of
eitherER stress inducers and/or H2O2 to melphalan-treated cellsdid
not affect ecto-HSP90 (Fig. 3H and J and SupplementaryFig.
S3I).
In aggregate, these observations confirm that while ROS and
ERstress are crucial for ecto-CRT and ecto-HSP90, the lack of a
robustER stress module compromises the
ecto-CRT–traffickingmechan-isms in melphalan-treated cells.
Melphalan-induced apoptosis is associated with the secretionof
proinflammatory chemokines
To determine whether melphalan treatment is additionallyable to
affect key cytokine or chemokine signaling in melanomacells, we
analyzed the supernatants of melphalan-treated A375cells for the
presence of key proinflammatory cytokines (IFNa,CXCL8/IL8, IL6, and
TNF), or chemokines (CCL2, CCL5,CXCL9, and CXCL10; refs. 39, 40).
A375 cells failed to releaseCCL5, CXCL9, CXCL10, IL6, and TNF under
basal conditions(data not shown). However, while neither melphalan-
nor Hyp-PDT treatments statistically influenced IFNa release, the
releaseof IL8 and CCL2 by A375 cells 24 hours after
melphalantreatment (Fig. 3H) was significantly increased. This
increasein IL8 and CCL2 was unique for melphalan because
Hyp-PDTinduced no CCL2 increase and induced even a
significantdecrease in IL8 (Fig. 3K).
Thus melphalan-induced apoptotic cell death of melanomacells in
vitro is associatedwith the induction of ecto-HSP90, aswellas the
secretion of proinflammatory chemokines, IL8 and CCL2.
Melphalan-treated cancer cells evoke moderate activation ofDCs
that is not reliant on ecto-HSP90 or ecto-CRT
Having established thatmelphalan treatment induces signatureof
danger signaling inmelanoma cells in vitro (Figs. 2 and 3) and
ashift toward a proinflammatory tumor microenvironment(Fig. 1),
wewondered about the direct interactions of such treatedmelanoma
cells with key immune cells.
To this end, we cocultured melphalan treated melanoma cellswith
iDCs and measured the phenotypic maturation (i.e.,increased surface
expression of HLA-DR and CD86) and func-tional stimulation of DCs.
In our experimental setting, LPStreatment of iDCs (Supplementary
Fig. S4A and S4B) was appliedas a positive control to test the
maturation potency of iDCs,whereas Hyp-PDT–treated cells served as
a control for the stim-ulation of fully mature DCs (28, 29).
Melphalan-treated mela-noma cells induced significant
DC-maturation, similarly to Hyp-PDT (fold changes: Fig. 4A;
percentage changes: SupplementaryFig. S4C). To establish the
relevance of ecto-HSP90 for themelphalan-treated cells-induced
DC-maturation, we blockedecto-HSP90 with a HSP90-specific antibody.
Despite the sugges-tive trend of decreased phenotypic maturation
with ecto-HSP90elimination (Fig. 4B), no statistical significance
was obtained. We
then wondered whether the immunostimulatory effects of
mel-phalan-treated humanmelanoma cells onDCs could be increasedby
coating of the dying melanoma cells with exogenous rCRT.However,
addition of rCRT to melphalan-treated human mela-noma cells didnot
alter phenotypicmaturation of coculturedDCs(Fig. 4C). We reasoned
that the proinflammatory cytokine TNFcould be a possible additional
candidate. This choice was moti-vated by our retrospective metadata
analysis of reported clinicaldata illustrating that the combination
of melphalan with TNF orTNF/IFNg (Supplementary Table S2) improves
patients' tendencyto achieve complete clinical responses within
ILP/ILI therapies(Supplementary Fig. S4H). Although high doses of
TNF and IFNggiven during ILP/ILI are known to be associated with
vasodisrup-tion and increased uptake of melphalan in tumors
(whichpotentiates melphalan's cytotoxicity; ref. 41), yet their
immuno-logic impact cannot be ruled out. However, addition of rTNF
tomelphalan-treated human melanoma cells did not increase
phe-notypic maturation of cocultured DCs (Fig. 4C), thus
suggestingthat to improve the interface betweenmelphalan-treated
cells andDCs, other factors are required.
We also quantified the levels of IL1b, IL12p70, IL6, TNF,
andIL10 in the cancer cell–DC coculture. Only Hyp-PDT–treated
A375cells stimulated a significant release of IL8, IL6, TNF, and
increasedIL1b secretion by human DCs (Fig. 4D). The
melphalan-treatedmelanoma cells stimulated a significant release of
IL8 by DCs andincreased secretion of IL1b and IL6 to not
significant levels; how-ever, it did not provoke the release of the
immunosuppressivecytokine IL10. These data point to the formation
of semi-matureDCs (42) (CD86highHLA-DRhighIL8highIL1blowIL6low)
after cocul-ture with melphalan-treated human melanoma cells.
We also wondered whether melphalan-treated cancer cellscould
affect the activation status of NK cells, as these immunecells
contribute to the direct elimination of cancer cells. In
vitrococulture ofmelphalan-treated A375 cells with peripheral
blood–isolated NKs neither increased the surface levels of NK
activating(NKp30, NKp46, and CD69) nor inhibitory (CD94)
receptorsas compared with untreated cancer cells (Supplementary
Fig.S5A–S5D). The absence of IFNg (and other important chemo-kines
and cytokines) further confirmed the lack of activation ofNK cells
(Supplementary Fig. S5E).Wenextmeasured the levels ofcancer
cell–associated surface molecules that are recognized byNKs, that
is,MICA/B andULBP2, before andafter the treatment. Incomparison
with the untreated A375 cells (Supplementary Fig.S5F),
themelphalan-treated cancer cells did not show any changein the
levels of MICA/B and ULBP2 (Supplementary Fig. S5G).This
observation could explain why dying cancer cells could notstimulate
NK cells in vitro.
Melphalan-treated melanoma cells increase
DC-mediatedproliferation of CD4þ and CD8þ T cells in the presence
of IFNg
To elucidate the functional impact of the semi-matureDCs induced
by melphalan-treated melanoma cells, we next
(Continued.) B, A375 cells treated with melphalan for 24 hours
were stained for ecto-HSP90, phosphatidylserine, and
permeabilization (the permeabilized cellswere excluded from the
analysis; three independent experiments, mean � SEM, and the
Student t test analysis; �� , P < 0.01; ��� , P < 0.001).
Effect of additionof zVAD-fmk (25 mmol/L; C), TUDCA (D), NAC (E),
and combination of TUDCA and NAC (F) was analyzed on
melphalan-induced ecto-HSP90 (24-hour timepoint) inA375 cells
(three independent experiments,mean�SEM, and2-wayANOVAanalysis; �
,P
-
investigated their T-cell activation capacity in vitro. For this
pur-pose, after 24-hour coculture of human iDCs with the
dyingcancer cells, T cells were added to the cell mixture and the
rateof T-cell proliferation and IFNg production were measured
asread-outs for T-cell activation (Fig. 4E–G).
Melphalan-treatedmelanoma cells, similar to Hyp-PDT–treated cells,
stimulatedproliferation of CD4þ and CD8þ T cells. This was
paralleled byan increased production of IFNg into the supernatant
of thecocultures (as compared with T cells alone), although the
mel-phalan-treated A375-mediated IFNg release by T cells was
lowerthan that induced by Hyp-PDT–treated cancer cells (Fig. 4G).
Wealso investigated whether antibody-based blockade of ecto-HSP90
or ectopic addition of rCRT or rTNF affects T-cell prolif-eration
in vitro. Consistentwith theDC-maturation results, neither
elimination of ecto-HSP90, nor addition of rCRT or rTNF,improved
T-cell activation mediated by the melphalan-treatedmelanoma cells
(Supplementary Fig. S4F and S4G).
Thus, DCs cocultured with melphalan-treated melanomacells
trigger increased (danger signals-independent) prolifera-tion of
CD4þ/CD8þ T cells in the presence of moderate IFNgproduction. These
results further substantiate the earlier con-clusion that
melphalan-treated melanoma cells induce semi-mature DCs.
Melphalan-triggered protective antitumor immunity ispotentiated
by rCRT but not by rTNF
To further explore whether melphalan-induced melanoma celldeath
has the ability to act as a "vaccine" and induce a protective
Figure 4.Melphalan (Mel)-treated A375melanoma cells elicit
semi-mature DCsand activate T cells. The phenotypicmaturation of
human iDCs (defined asincrease in CD86 and HLA-DRstaining) was
investigated after24-hour coincubation with untreatedor treated for
24 hours A375 cells (A;three independent experiments, mean� SEM,
and one-way ANOVA analysis;� , P < 0.05; �� , P < 0.01) or
untreated ormelphalan-treated A375 cells(24-hour timepoint) in the
presence ofcontrol antibody (IgY) or ecto-HSP90blocking antibody as
applicable(HSP90 IgY; B) or rCRT or rTNF(C; three independent
experiments,mean � SEM, and repeated measuresANOVA with the Tukey
post-testwithin control and melphalanconditions analysis). Graphs
A–Crepresent fold changes relative tocontrol-A375. D, supernatants
fromthe coculture of untreated or dyingA375 cells with iDCswere
investigatedfor the content of IL1b, IL6, IL8, IL10,IL12p70, and
TNF (three independentexperiments, mean � SEM, and one-way ANOVA
analysis). T cells culturedin the presence of iDCs and untreatedor
dying A375 cells were checkedfor proliferation of CD3þCD4þ (E)
andCD3þCD8þ (F) cells (representativeexperiment of
three-independentexperiments with one-way ANOVAanalysis for
conditions includingcancer cells; the Mann–Whitney t testfor
comparison between "T alone" and"LPS"). G, supernatants of this
triplecoculture were tested for IFNg content(representative
experiment of threeindependent experiments, mean ofduplicate; �
SEM, the Mann–Whitneyt test; � , P < 0.05; �� , P <
0.01;���/###, P < 0.001).
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anticancer response, we tested its immunization potential using
aprophylactic vaccination mice model.
We used themurine B78melanoma cells that uponmelphalantreatment
died apoptotically and displayed caspase-dependentecto-HSP90
(Supplementary Figs. S2 and S3A), induced semi-mature DCs (Fig. 5A
and B), which was unaffected by ecto-HSP90antibody-based blockage,
coating with rCRT or addition of rTNF(Fig. 5C and D). We thus
vaccinated C57BL/6 mice with mel-phalan-treated B78 cells or PBS
(placebo control), followed (10days later) by a rechallenge with
live B78 cells and tumor growthmonitoring. As a negative control,
we used a tolerogenic celldeath-inducer, brefeldin A (28, 43) and
compared the vaccinationefficacy ofmelphalan-treated cells with
that elicited by the immu-
nogenic cell death (ICD) inducer, Hyp-PDT (44).
Interestingly,melphalan-treated cancer cells exhibited the ability
to induce an"anticancer vaccination effect"—as many as 40% of the
micevaccinated with melphalan-treated cells rejected rechallenge
withlive tumor cells (Fig. 5D). This effect was considerably better
thanthe "vaccine" produced with brefeldin A (Fig. 5D), but not
asrobust as the Hyp-PDT–based vaccine, which protected 62% ofthe
mice from tumor formation following rechallenge (Fig. 5D).
To establishwhether the protective anticancer effect induced
bymelphalan-treated cancer cells is due to the stimulation of
anadaptive immune response, we depleted immunocompetentmice of CD4þ
or CD8þ T cells (antibody-based depletion; ascontrol, antibody
against b-galactosidase was used; depletion
Figure 5.Melphalan (Mel) induces inflammatorycell death
associated with anticancerimmunity. A–C, the phenotypicmaturation
of murine iDCs wasinvestigated after 24-hour incubationwith
untreated or treated for 24 hoursB78 cells or untreated or
melphalan-treated B78 cells (24-hour time point;A) in the presence
of control antibody(IgY) or ecto-HSP90 blocking antibodyas
applicable (HSP90 IgY; B) or rCRT orrTNF (C; three
independentexperiments, mean � SEM, graphs A–Crepresent fold
changes relative to crtl-B78 and one-way ANOVA analysis;�, P <
0.05; �� , P < 0.01; ���, P < 0.001).D, C57BL/6 mice were
vaccinated withB78 cells (collected at 24-hour timepoint after
respective treatments) orplacebo control (PBS); thereafter, 10days
later, these mice were injectedwith live B78 cells andmonitored for
thetumor growth (10mice/group; one-wayANOVA; � , P < 0.05; ���,
P < 0.001).Effect of elimination of CD4þ or CD8þ Tcells (E),
antibody-based blockage ofecto-HSP90 on surface of
melphalan-treated B78 cells (F), addition of rCRTor rTNF to
melphalan-treated B78 cellson the stimulation of anticancerimmunity
(G; number of mice per groupindicated on the graphs; one-wayANOVA;
� , P < 0.05; ���, P < 0.001).H, schematic representation
ofmelphalan-induced inflammation anddanger signaling associated
withimmunogenicity. In vivo (in melanomapatients) Mel-ILP increases
expressionof IL1B, IL6, and IL8 in the tumor bed andlocoregional
serum levels of IL1b andIL6 as early as 1 hour after Mel-ILP.In
vitro, melphalan inducesinflammatory cell death capable
ofstimulating semi-mature DCs as well asT-cell activation and
tangibleanticancer immunity in a prophylacticmice vaccination model
in vivo.
Melphalan, Antimelanoma Immunity, Inflammation
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results are presented on Supplementary Fig. S6B and
S6C).Remarkably, elimination of CD8þ T cells resulted in
abrogationof the melphalan-induced anticancer vaccination effect,
whereaselimination of CD4þ T cells was ineffective (Fig. 5E). This
obser-vation confirms that the vaccination potential of
melphalan-treated B78 cells is highly dependent on CD8þ T
cells.
To analyze the relevance of melphalan-induced ecto-HSP90
indefining in vivo immunogenicity, we carried out
prophylacticmicevaccination using melphalan-treatedmelanoma cells
coated withcontrol or with an HSP90-blocking antibody. This in vivo
exper-iment indicated that the vaccination effect of the
melphalan-treated melanoma cells does not rely on ecto-HSP90 (Fig.
5F).Furthermore, we wondered whether the immunogenic effect
ofmelphalan-treated murine melanoma cells could be potentiatedby
combinatorial addition of rCRT or rTNF. Remarkably,
coatingmelphalan-treated cells with rCRT significantly increased
theirimmunogenicity (Fig. 5G), while addition of rTNF did not
sig-nificantly increase the immunogenic properties of
melphalan-treated melanoma cells (Fig. 5G).
In conclusion, these in vivo studies show
thatmelphalan-treatedmurine melanoma cells are endowed with some
tumor-rejectingcapacity—which is possibly linked to the induction
of inflam-matory cell death inmelanoma associated with positive
immune-effector mechanisms; and which can be further potentiated in
vivoby combinatorial addition of rCRT.
DiscussionIn this study, we thoroughly describe melphalan as
inducer of
inflammatory cell death associated with immunogenicity in
mel-anoma. We show that melphalan-treated melanoma cells
favorinflammatory or immune-effector mechanisms in immune
cellsand/or tumormicroenvironment. This notion is supported by
thespectra of different cytokines detected inMel-ILP–treated
patients'samples and the observation that melphalan-treated
melanomacells induce semi-mature DCs, which, in turn, induce
moderateactivation of T cells. Importantly, melphalan-treated
melanomacells elicit noticeable, CD8þ T cells–dependent
"vaccine-like"antitumor immunity. These positive immune-mediated
antican-cer effects can be further elevated by a combinatorial
treatmentreconstituting ecto-CRT, an "eat me" signal, which is
otherwisepoorly trafficked to the plasma membrane after melphalan
treat-ment of melanoma cells.
We show that melphalan treatment was fairly efficient atinducing
ROS production and ER stress in melanoma cells, toan extent that
blocking these processes severely compromisedmelphalan-induced cell
death in vitro.Alongwith the induction ofan early ER stress
signature in patients' biopsies followingMel-ILPrevealed in a
previousmicroarray analysis (31) and the detectableupregulation of
IL6 and IL1b in the patients' sera as early as 1 hourafter Mel-ILP
found in this study, these findings highlight theability of
melphalan to rapidly tilt the balance toward a moreproinflammatory
tumor environment. Induction of ER stress andROS in a simultaneous
or concomitant fashion is a prerequisitefor efficient danger
signaling apically associated with the prea-poptotic surface
exposure of ecto-CRT (8, 16).However, our studyconclusively shows
that melphalan-induced ER stress was belowthe threshold required to
elicit ecto-CRT. Moreover, our dataunderscore that combining
melphalan treatment uniquely withthe SERCA inhibitor thapsigargin
restored ecto-CRT in melpha-lan-treated cells. This outlines the
importance of ER-Ca2þ release
over other ER stress–inducingmodalities in ecto-CRT
inductionorrestoration (of note,Hyp-PDT, apowerful enabler of
preapoptoticecto-CRT and ICD, also induces SERCA-photodamage–based
ER-Ca2þ release; ref. 45). Although, we did find that melphalan is
anefficient inducer of ecto-HSP90. Melphalan-induced
ecto-HSP90wasmediated by caspase signaling secondary to ER stress
andROSproduction—an interesting observation that deserves to
befurther explored, considering that the signaling
mechanismsunderlying ecto-HSP90 are elusive. However, our ex
vivo/in vivoobservations rule out a major role for ecto-HSP90 as a
dangersignal, thereby outlining that ecto-HSP90 is a more
context-dependent DAMP rather than a general one, as suggested
inprevious studies (46).
Prominently, on the immune-effectors front, the absence ofIL10
production followingMel-ILP in patients' samples and fromthe
DCs/NKs interacting with melphalan-treated cancer cells,further
indicates that melphalan does not actively promote
animmunosuppressivemicroenvironment.
Themelphalan-inducedinflammatory/immune-effector mechanisms
revealed here mighthave important prognostic implications for
melanoma, consid-ering that the immunomodulatory features induced
by melpha-lan, that is, high expression of HLA-DR, increased T-cell
activa-tion/IFNg production, and low presence of IL10, are also
positiveprognostic factors for malignant melanoma (7).
Moreover,increased IL6 production (another factor potentiated by
melpha-lan) was reported to associate with increased sensitivity
towardimmunotherapy against melanoma (47). Unfortunately, due tothe
low number of patients (with limited clinical follow-up)available
for this study (Supplementary Table S3), we could notobtain an
objective predictive or prognostic estimation for
mel-phalan-induced cytokines—a problem that should be addressedin
the future.
Nevertheless, our prophylactic immunization studies
convinc-ingly show that antitumor immunity may, at least partly,
con-tribute to the Mel-ILP/ILI's therapeutic effect against
melanoma.Immunogenicity of melphalan-based vaccines was
significantlybetter than the tolerogenic cell death inducer
brefeldin A but notas high as that of Hyp-PDT, a potent ICD inducer
(28). Thissuggests that certain immunogenicity-augmenting
strategiesmight be required to further increase the potential of
melpha-lan-based therapy. Indeed, melphalan treatment setting
lacked acrucial "eat me" signal, that is, ecto-CRT and a crucial
immune-effector cytokine on the level of cancer cells/immune cells,
knownto accentuate its therapeutic effect in the clinic, that is,
TNF.Additionof rCRTor rTNF in coculture assays
ofmelphalan-treatedcells/DCs/T cells did not affect DC-maturation/T
cells' prolifera-tion. These results are in linewith previous
studies showing that atleast ecto-CRT does not directly modulate
immune cell matura-tion (9). Remarkably, rCRT but not rTNF
significantly accentuatedthe immunogenic potential of
melphalan-treated melanomacells. This clearly shows that in the
melphalan treatment set-up,the combination of rCRT has a better
(immuno)therapeuticpotential than rTNF.
In conclusion, our study provides a comprehensive outlook(Fig.
5H) of the cell death and immunologic characteristics ofmelphalan,
a widely used locoregionally applied chemothera-peutic that, as
demonstrated by systemic chemotherapeutics, isnecessary to enable
the design of "smart" combinatorial immu-notherapies (especially in
case of melanoma). This advancementis direly needed because 40% to
50% of primary melanomaoccurs on the extremities and around 85.5%
of these patients
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develop recurrences (48). Our in vivo results indicate that
thestrategies aiming to potentiate the immunogenicity or
dangersignaling associated with melphalan should strive to
increaseecto-CRT. This could be obtained, either via combination
treat-ment with ER-Ca2þ release inducing ER stressors such as
thapsi-gargin or thapsigargin analogs such as G202 [prodrug
withinphase I clinical trial (49)] that could "intrinsically"
restore ecto-CRT; or via combination with exogenously supplied
rCRT. TheMel-ILP/ILI treatment schema represents an ideal
opportunity forthe latter combination treatment, as just such as
TNF, rCRT canalso be used in combination with melphalan for
short-termlocoregional treatment in extremities-associated
malignancies—a conjecture that should be investigated urgently in
the future.
Disclosure of Potential Conflicts of InterestA.D. Garg is a
consultant for Sotio. P. Agostinis is a consultant for Ono
Pharmaceutical and Sotio. No potential conflicts of interest
were disclosed bythe other authors.
Authors' ContributionsConception and design: A.M. Dudek-Peri�c,
J. Wouters, M. Stas, J. Golab, A.D.Garg, P. AgostinisDevelopment of
methodology: A.M. Dudek-Peri�c, G.B. Ferreira, J. Wouters,N. Prada,
M. Winiarska, C. Mathieu, M. Stas, M.-L. Gougeon, A.D.
GargAcquisition of data (provided animals, acquired and managed
patients,provided facilities, etc.): A.M. Dudek-Peri�c, G.B.
Ferreira, A. Muchowicz,J. Wouters, S. Martin, S. Kiviluoto, L.
Boon, C. Mathieu, J. van den Oord,M. Stas, M.-L. Gougeon, J. Golab,
A.D. GargAnalysis and interpretation of data (e.g., statistical
analysis, biostatistics,computational analysis): A.M. Dudek-Peri�c,
G.B. Ferreira, A. Muchowicz,J. Wouters, S. Kiviluoto, C. Mathieu,
M.-L. Gougeon, J. Golab, A.D. Garg,P. Agostinis
Writing, review, and/or revision of the manuscript: A.M.
Dudek-Peri�c,A. Muchowicz, J. Wouters, S. Kiviluoto, M. Winiarska,
L. Boon, C. Mathieu,M.-L. Gougeon, J. Golab, A.D. Garg, P.
AgostinisAdministrative, technical, or material support (i.e.,
reporting or organizingdata, constructing databases): A.M.
Dudek-Peri�c, J. Wouters, M. Stas,P. AgostinisStudy supervision:
A.M. Dudek-Peri�c, J. Golab, A.D. Garg, P. Agostinis
AcknowledgmentsThe authors thank Sofie Van Eygen and Frea Coun
for their excellent
technical assistance. The authors also thank all the blood
donors for theirsignificant contribution. Some of the figures were
prepared using ServierMedical Art (www.servier.com), for which the
authors would like toacknowledge Servier.
Grant SupportA.M. Dudek-Peri�c is supported by the Emmanuel van
der Schueren
scholarship awarded by the Kom op tagen Kanker Foundation,
Belgium.A.D. Garg and G.B. Ferreira are supported by a
FWO-Vlaanderen postdoc-toral fellowship. J. Wouters is funded by
the Melanoma Research Alliance(Team Science Research Award; USA).
J. Golab and M. Winiarska aresupported by European Commission 7th
Framework Programme FP7-REG-POT-2012-CT2012-316254-BASTION. This
work is supported by FWO-Vlaanderen (G0584.12N and K202313N) and
GOA/11/2009 grant of theKU Leuven to P. Agostinis. This article
represents research results of the IAP7/32 funded by the
Interuniversity Attraction Poles Programme, initiated bythe Belgian
State.
The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby markedadvertisement in accordance with 18 U.S.C. Section
1734 solely to indicatethis fact.
Received July 20, 2014; revised February 10, 2015; accepted
February 11,2015; published OnlineFirst March 11, 2015.
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Associated Calreticulin−Melanoma Cell Surface Antitumor Immunity
Triggered by Melphalan Is Potentiated by
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