Antitumor Immunity Triggered by Melphalan Is Potentiated by … · Microenvironment and Immunology Antitumor Immunity Triggered by Melphalan Is Potentiated by Melanoma Cell Surface–

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.

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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).






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.






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)






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).






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.






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






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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2015;75:1603-1614. Published OnlineFirst March 11, 2015.Cancer Res Aleksandra M. Dudek-Peric, Gabriela B. Ferreira, Angelika Muchowicz, et al.

Associated Calreticulin−Melanoma Cell Surface Antitumor Immunity Triggered by Melphalan Is Potentiated by

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