new title: anti-tumor immunity triggered by melphalan is potentiated … · 2015. 3. 11. · 1...

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Title: Anti-tumor immunity triggered by melphalan is potentiated by melanoma

cell surface associated calreticulin

Running title: Melphalan, anti-melanoma immunity and inflammation

Aleksandra M. Dudek-Perić1, Gabriela B. Ferreira

2, Angelika Muchowicz

3, Jasper Wouters

4,

Nicole Prada5, Shaun Martin

1, Santeri Kiviluoto

6, Magdalena Winiarska

3, Louis Boon

7,

Chantal Mathieu2, Joost van den Oord

4, Marguerite Stas

8, Marie-Lise Gougeon

5, Jakub

Golab3,9

, Abhishek D. Garg1,*

, Patrizia Agostinis1,*

1Cell Death Research and Therapy Laboratory, Department of Cellular and Molecular

Medicine, Faculty of Medicine, KU Leuven, Leuven, Belgium; 2Laboratory of Clinical and

Experimental Endocrinology, Department of Clinical and Experimental Medicine, KU

Leuven, Leuven, Belgium; 3Department of Immunology, Center of Biostructure Research,

Medical University of Warsaw, Poland; 4

Translational Cell and Tissue Research, Department

of Imaging and Pathology, Faculty of Medicine, KU Leuven, Leuven, Belgium; 5Institute

Pasteur, Antiviral Immunity, Biotherapy and Vaccine Unit; Infection and Epidemiology

Department, Paris, France; 6Laboratory of Molecular and Cellular Signaling, Department of

Cellular and Molecular Medicine, Faculty of Medicine, KU Leuven, Leuven, Belgium;

7Bioceros, CM Utrecht, The Netherlands;

8Surgical Oncology, Department of Oncology, KU

Leuven, Leuven, Belgium; 9Institute of Physical Chemistry, Polish Academy of Sciences,

Warsaw, Poland.

Conflicts of interests: Patrizia Agostinis and Abhishek D. Garg collaborate with and/or

provide consultancy to Sotio. Patrizia Agostinis has received consultancy fees from Ono

Pharmaceuticals.

Research. on March 30, 2021. © 2015 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 11, 2015; DOI: 10.1158/0008-5472.CAN-14-2089

http://cancerres.aacrjournals.org/

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Financial Support: A.M.D.P. is supported by the Emmanuel van der Schueren scholarship

awarded by the Kom op tagen Kanker foundation, Belgium. A.D.G. and G.B.F. are supported

by a FWO-Vlaanderen post-doctoral fellowship. J.W. is funded by the Melanoma Research

Alliance (Team Science Research Award; USA). J.G. and M.W. are supported by European

Commission 7th

Framework Programme FP7-REGPOT-2012-CT2012-316254-BASTION.

This work is supported by FWO-Vlaanderen (G0584.12N and K202313N) and GOA/11/2009

grant of the KU Leuven to P.A. This paper represents research results of the IAP7/32 Funded

by the Interuniversity Attraction Poles Programme, initiated by the Belgian State.

Corresponding authors:

Prof. Patrizia Agostinis;

Laboratory for Cell Death Research and Therapy, Department of Cellular and Molecular

Medicine

University of Leuven (KU Leuven), Campus Gasthuisberg, O&N1, Herestraat 49, Box 802,

3000 Leuven, Belgium ; fax: +32 16 3 30735 ; e-mail: [email protected]

Dr. Abhishek D. Garg;

Laboratory for Cell Death Research and Therapy, Department of Cellular and Molecular

Medicine

University of Leuven (KU Leuven), Campus Gasthuisberg, O&N1, Herestraat 49, Box 802,

3000 Leuven, Belgium ; fax: +32 16 3 30735 ; e-mail: [email protected]

Word Count (Introduction-Discussion): 4990

Number of Figures: 5



mailto:[email protected]:[email protected]://cancerres.aacrjournals.org/

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Abstract

Systemic chemotherapy generally has been considered immunosuppressive, but it has become

evident that certain chemotherapeutic drugs elicit immunogenic danger signals in dying

cancer cells that can incite protective antitumor immunity. In this study, we investigated

whether loco-regionally applied therapies such as melphalan used in limb perfusion for

melanoma (Mel-ILP) produces related immunogenic effects. In human melanoma biopsies,

Mel-ILP treatment upregulated IL-1B, IL-8 and IL-6 associated with their release in patients'

loco-regional sera. While induction of apoptosis in melanoma cells by melphalan in vitro did

not elicit threshold levels of endoplasmic reticulum (ER) and ROS stress associated with

danger signals such as induction of cell-surface calreticulin, prophylactic immunization and T

cell depletion experiments showed that melphalan administration in vivo could stimulate a

CD8+ T cell-dependent protective anti-tumor response.

Interestingly, the vaccination effect was potentiated in combination with exogenous

calreticulin, but not tumor necrosis factor, a cytokine often combined with Mel-ILP. Our

results illustrate how melphalan triggers inflammatory cell death that can be leveraged by

immunomodulators such as the danger signal calreticulin.




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Abbreviations:

7-AAD – 7 aminoactinomycin D; AnV – Annexin V; APC(s) – antigen presenting cell(s);

ATP - adenosine triphosphate; BrefA – brefeldin A; CD – cluster of differentiation; CR –

complete response; CRT – calreticulin; DAMP(s) – damage-associated molecular pattern(s);

(i)DC(s) – (immature) dendritic cell(s); (ecto-) – exposed; ER – endoplasmic reticulum; HLA-

DR – human MHC class II cell surface molecule; HSP70 – heat-shock protein, 70kDa; HSP90

– heat-shock protein, 90kDa; Hyp – Hypericin; Hyp-PDT – Hypericin-based photodynamic

therapy; ICD – immunogenic cell death; IFNγ – interferon gamma; IL – interleukin; ILI –

isolated limb infusion; ILP – isolated limb perfusion; Mel – Melphalan; Mel-ILI – Melphalan-

based ILI; Mel-ILP – Melphalan-based ILP; MHC-II - major histocompatibility complex class

II; NAC – N-acetylcysteine; NK(s) – natural killer cell(s); PBMC(s) – peripheral blood

mononuclear cell(s); PI – propidium iodide; PR – partial response; PS – phosphatidylserine;

ROS – reactive oxygen species; Tg – thapsigargin; TNF – tumor necrosis factor; TUDCA –

tauroursodeoxycholate; UPR – unfolded protein response.




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Introduction

Evidence indicates that anti-cancer therapies capable of harnessing the host’s immune system

while inducing cancer cell death hold the highest therapeutic value (1,2). Such therapies are of

immediate importance for anti-melanoma therapy. Melanoma is an aggressive cancer that

typifies the paradox of being highly antigenic while simultaneously exerting potent

immunosuppression (3). Moreover, melanoma has recently gained wide attention from an

immunotherapeutic standpoint owing to promising clinical effects of immune-checkpoint

inhibitory-drugs (4). All this clearly advocates the need to further study the anti-melanoma

immune responses, and reveal additional strategies capable of augmenting anti-melanoma

immunity.

In recent years, many anti-cancer modalities have been shown to positively regulate immune-

effector functions and induce anti-tumor immunity (5). These include (i) strategies improving

the natural killer (NK) cells’/dendritic cells’ (DCs)/T cells’ anti-cancer activity, (ii)

immunogenicity of the dying cancer cells, and (iii) “resetting” microenvironment’s

immunocontexture (6). The above mentioned processes are strongly influenced by certain

immune-effector cytokines exhibiting strong clinical prognostic impact (7). Moreover,

immunogenicity as well as vaccination potential has been recently linked, at least in part, to

“danger signaling” operating on the cancer cell-level (8). Induction of danger signaling

mediates the spatiotemporally defined ‘emission’ of specific ‘eat me’ signals/damage-

associated molecular patterns (DAMPs) by the dying cancer cells, e.g. surface exposed (ecto-)

calreticulin (CRT) (9) and heat-shock proteins (HSP)-70/90 (10), and secreted nucleotides,

like adenosine triphosphate (ATP) (1,11). Danger signaling-potentiating therapies have been

recently shown to associate with favorable clinical outcome in cancer patients (5,12,13).

Moreover, it has been proposed that, combinatorial therapy with exogenously supplied danger

signals could hold great immunogenicity-promoting potential (14).




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Most of the chemotherapeutics tested so far as DAMPs-inducers are primarily used as

systemic chemotherapeutics (15,16) while physicochemical modalities (like

radiotherapy/Hyp-PDT) are primarily used as (loco-)regional therapeutics (17-19).

Considering that immune responses following (loco-)regional therapy can differ from those

after systemic therapy (20); it is necessary that anti-cancer immunity, danger signaling and

immune-effector function potentiating effects of (loco-)regionally-applied chemotherapeutics

are also evaluated – a knowledge that is largely missing and could have translational

significance (20,21).

To this end, we studied the effects of Melphalan (Mel), the regionally-applied (standard-of-

care) chemotherapeutic for extremities-associated melanoma (20,22). Mel is an alkylating

agent, employed in the isolated limb perfusion (ILP)/infusion (ILI) therapy (20,22), for

patients harboring limb-localized malignancies (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 various others showing partial responses (14-39%) (22)

(clinical metadata analysis, Suppl. Table 1). Hitherto, melanoma cell-killing efficacy is

postulated as the sole contributor to patients’ responsiveness towards Mel-treatment (24).

However, whether the promising anti-melanoma efficacy of Mel-therapy is associated with

anti-tumor immunity remains unexplored. Thus, owing to these conjectures and a gap-in-

knowledge about regional chemotherapeutics, we studied the mechanisms of Mel-induced

melanoma cell death, the inflammatory contexture as well as the efficacy of Mel-induced

induced anti-tumor immunity/immune-effector function against melanoma. We also studied

certain putative immunomodulatory factors that are employable as combinatorial treatment

for augmenting anti-melanoma immunity




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Materials and methods

Materials and reagents

The following drugs were used: Melphalan (Sigma, M2011), Thapsigargin (Tg; Enzo Life

Sciences, BML-PE180-0001). Hypericin was prepared, purified, and stored as described

previously (25). Antibodies against the following proteins were used: BiP/GRP78 (Cell

Signaling Technology, 3183), P-eIF2α (Cell Signaling Technology, 3597), eIF2α (Cell

Signaling Technology, 21035), MICA/B (Acris, AM26694AFN), actin (Sigma, A5441),

calreticulin (anti-CRT; Abcam, Ab92516), ULBP2 (Abcam, Ab88645), HSP90 (Stressgen,

ADI-SPA-830), HSP70 (Santa Cruz Antibodies, 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-Alexa Fluor®647 (Invitrogen,

A21235) and goat anti-rabbit-Alexa Fluor®647 (Invitrogen, A21244). Western Blot detection

was done on Odyssey.

Cell culture and treatments

All cells were cultured in DMEM (D6546, Sigma) with 2 mM glutamine, Penicillin-

Streptomycin (P0781, Sigma) and 10% fetal bovine serum at 37°C under 5% CO2. A375 cells

were obtained from ATCC and authenticated through DNA STR-profiling.

A375/K1735/MM031/B78 cells were incubated with Mel (300 µM/600 µM for B78) or

brefeldin A (BrefA; 50 ng/mL for B78 cells) for the indicated times. For Hyp-PDT

conditions, A375 cells were incubated for 16 hr with 150 nM Hypericin while B78 were

incubated for 2 hr with 500 nM Hypericin in media without FBS, followed by removal of

Hypericin, irradiation (2.70 J/cm2) and were cultured for indicated times.




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Measurement of ecto-CRT, ecto-HSP70 and ecto-HSP90

After treatment, cells were collected with TrypLE Express (Life Technologies, 12604-021),

washed with PBS and with FC (Flow Cytometry) buffer (2% FBS, 1% BSA in PBS),

incubated for 1 hr at 4°C with primary antibodies, washed and incubated for 1 hr at 4°C with

secondary antibodies. After final washes cells were incubated in FC buffer including 1 µM

Sytox Green (Life Technologies, S7020) for 15 min and analyzed on Attune Flow Cytometer

(Life Technologies). The permeabilised cells were excluded from the analysis due to

intracellular staining, and the fold changes in the mean fluorescence intensity (MFIs) for each

DAMP were analyzed.

DC-maturation analysis

Human and murine iDCs were prepared according to previously described protocols (26,27).

The protocol for co-incubation of cancer cells with iDCs has been previously described

(28,29). Briefly, the DCs were co-cultured with untreated or dying cancer cells (24 hr time

point) at a 1:20 (DCs:cancer cells) ratio for 24 hours under standard culture conditions. In

some experiments cancer cells were pre-incubated with blocking antibodies [1,25 µg/106

cells]: IgY (Promega, G116A), anti-HSP90 (Novus Bio, NB120-19104; antibodies were

present in the co-culture media as well), coated with recombinant CRT (rCRT; Abcam,

ab15729; cells were incubated with rCRT at 4°C for 30 minutes followed by removal of

unbound protein) as described before (9) or in the presence of 100 ng/mL soluble recombinant

TNF (rTNF; human: PeproTech, 300-01A; murine: PeproTech, 315-01A). For staining of

human DCs the following antibodies were used: anti-HLA-DR antibody (BD, MHLDR01)

and anti-CD86 (BD, MHCD8605). For staining of murine DCs the following antibodies were

used: anti-MHC II antibody (e-Biosciences, 11-5321-81), anti-CD86 (e-Biosciences, 17-0862-

81).




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T cell proliferation

The 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 hr time point)

were co-cultured with iDCs for 24 hr. Allogeneic T cells (CD3+), isolated from donors’ blood

according to the manufacturer’s recommendations (Pan T Cell Isolation Kit II; Miltenyi

Biotec, 130-095-130), labeled with eFluor® 670 Proliferation Dye (eBioscience, 65-0840-85)

were added to the co-cultures for an additional period of 5 days. Human IL2 was added at day

2 of the triple co-cultures (25 U/mL). At the end of day 5, cells were stained for CD3, CD4

and CD8 (with antibodies anti-CD3-eFluor®450 (48-0038), anti-CD4-FITC (11-0049) and

anti-CD8-PE-Cy7 (25-0049) all from eBioscience). Dead cells were excluded using the

Fixable Live/Dead Yellow stain according to the manufacturer’s specifications (Invitrogen,

L34959). Data acquisition was performed on GalliosTM

flow cytometer (Beckman Coulter)

and the KaluzaTM

software (Beckman Coulter) was used for data analysis.

Prophylactic mouse vaccination

Mouse experiments were performed in the animal facilities of Warsaw Medical University

and KU Leuven, according to the guidelines of the ethical committees of these universities.

The prophylactic mice vaccination was performed according to the previously described

protocol (29). Briefly, the mice were injected subcutaneously with 100 µL containing 500x103

dying B78 cells (40% of apoptotic cells; in indicated experiments the cells were coated with

blocking antibodies or rCRT, as described above, or co-injected with murine rTNF, or with

100 µL of PBS into the left flank. After 10 days mice were re-challenged with untreated B78

cells into the right flank (50x103 cells in 100 µL PBS) and tumor growth was monitored for

the next 40 days. Depletion of CD4+ or CD8

+ T cells was performed according to the

previously described protocol (30). To evaluate the elimination of T cells, blood was collected




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via cheek pouch and presence of CD4+ or CD8

+ T cells was detected through anti-CD4 (BD,

553031CD8) and anti-CD8 (BD cat. 553047) staining, as described previously (30).

Statistical analysis

Data are presented as exact values, percentages of cell population or fold changes, specifically

as indicated on each figure. Error bars represent SEM. Depending on the type of experiments,

as a statistical analysis we performed Student t-test, 1-way ANOVA with Dunnett’s post-test

or 2-way ANOVA with Bonferroni post-test, as indicated in the figure legends. Fold

expressions of cytokines in patients’ samples were analyzed for significance using either the

two-tailed one sample t-test (if results had Gaussian distribution) or the two-tailed Wilcoxon

rank sum test (if results did not have Gaussian distribution). Always * represents p-value <

0.05; ** p-value < 0.01; *** p-value < 0.001.




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Results

Mel-ILP evokes pro-inflammatory immune effector cytokines production

A previous microarray/qRT-PCR analysis confirmed a significant increase in IL6 levels, post-

Mel-ILP in patients’ biopsies (31); this inspired us to further investigate whether clinical Mel-

treatment is associated with induction of certain other major cytokines. We first extended

previous expression analysis (31) to specific immune-effector cytokines in the tumor bed.

Beyond IL6 potentiation (31), we found significant increase in levels of IL1B and IL8 in the

absence of significant changes in IL10, TNF and IFNG levels, in tumor samples taken 1 hr

post-Mel-ILP (Fig. 1A).

Next, considering that Mel-ILP is a (loco-)regionally-applied therapy, we wondered to what

extent the Mel-ILP-induced cytokine transcript-pattern present in the tumor bed was mirrored

by the (loco-)regional plasma-associated cytokine pattern on the protein level. As early as 1 hr

after Mel-ILP treatment, protein levels of IL6 and, to a lesser extent, IL1β increased

significantly, while we failed to detect any significant increase in the levels of IL12p70, IL8,

TNF, IL10 (Fig. 1B and Suppl. Fig. 1A) and IFNγ (data not shown). Thus, the loco-regional

serum-associated cytokine pattern largely mirrored the tumor bed-associated transcript

pattern. Considering that samples were collected very early (10-30 min/ 1 hr) post-Mel-ILP,

we suspected that freshly tumor-infiltrating immune cells would not substantially contribute

to the observed cytokine production. In line with this, we failed to detect increased immune

cells’ infiltration following Mel-ILP (1 hr) after staining tumor sections for CD68/CD3,

specific markers of monocytes/macrophages and T lymphocytes, respectively (Suppl. Fig. 1B-

C). This suggests that Mel-ILP triggered increase in immune-effectors/pro-inflammatory

cytokines is mostly the result of the alteration in pre-existing tumor microenvironment.




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Mel-induced apoptosis in vitro is modulated by the combination of ER stress and ROS

A previous study indicated that post-Mel-ILP, signatures of endoplasmic reticulum (ER)

stress (i.e. ATF3, GADD45A and XBP1s) were induced in patients’ biopsies (31). Considering

that ER stress is a crucial stress response for eliciting cell death, danger signaling and

cytokine production (32), we decided to investigate the ER stress-cell death crosstalk post-

Mel-treatment.

We therefore studied the biochemical hallmarks of Mel-induced melanoma cell death in vitro

using human (A375) and murine (B78) metastatic melanoma cell lines. Mel time-dependently

affected melanoma cell viability (Fig. 2A, Suppl. Fig. 2A) and induced phosphatidylserine

(PS) exposure (Fig. 2B, Suppl. Fig. 2B), loss of mitochondrial transmembrane potential

(ΔΨm) (Fig. 2C, Suppl. Fig. 2C) and significant activation of caspase-3 (Fig. 2D, Suppl. Fig.

2D). Furthermore, the pan-caspase inhibitor zVAD-fmk abolished caspase-3 activation (Fig.

2E, Suppl. Fig. 2E) and resulted in a protection from cell death (Fig. 2F, Suppl. Fig. 2F), thus

indicating that Mel induces apoptosis.

We next investigated whether Mel induced ER stress by evaluating markers of the unfolded

protein response (UPR). Mel-treated melanoma cells showed an increase in BiP/GRP78

content, a clear induction of eIF2α phosphorylation (Fig. 2G, Suppl. Fig. 2G) and of the

spliced form of XBP1 (Fig. 2H, Suppl. Fig. 2H), indicating the ability of Mel to activate the

PERK and IRE1α arms of the UPR. Addition of the chemical chaperone, TUDCA, which has

been reported to alleviate ER stress (33), resulted in decreased levels of phospho-eIF2α (Fig.

2I) and a partial protection from Mel-induced cell death (Fig. 2J). This suggests that although

ER stress contributes to the induction of apoptotic cell death after Mel-treatment, other

signaling events are required to incite apoptosis.




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The presence of ER stress along with ROS induction and caspase signaling has been shown to

provide the biochemical pre-requisite for efficient danger signaling (9,15,28). Indeed, as

reported previously (34), Mel caused a significant increase in the intracellular levels of ROS

in melanoma cells (Fig. 2K). Attenuation of ROS signaling by the antioxidant N-

acetylcysteine (NAC), neither significantly protected melanoma cells from Mel-induced

apoptosis (Fig. 2L), nor it affected the activation of ER stress (data not shown). In contrast,

the combination of TUDCA and NAC significantly blunted Mel-induced melanoma apoptosis

(Fig. 2M).

These results underscore that ROS production and ER stress act in concert to induce apoptosis

in melanoma cells in response to Mel.

Mel-induced apoptosis is associated with a defined ER stress and ROS-dependent

danger signaling

Mel-treatment in vitro is able to induce ER stress and ROS - two most important apical pre-

requisites for danger signaling elicitation (28,35) by apoptotically dying cells. To evaluate if

Mel-treatment induces danger signaling in melanoma cells and to reveal its molecular nature,

we analyzed a panel of well-established DAMPs and/or ‘eat me’ signals (9,10,28,36).

Firstly, we measured CRT, HSP70 and HSP90 on the cell surface (ecto-CRT, ecto-HSP70,

ecto-HSP90) of non-permeabilised dying melanoma cells, and the secretion of ATP. The

effects induced by Mel in A375 cells were compared to Hypericin-based photodynamic

therapy (Hyp-PDT) a previously characterized danger signaling-inducing therapy (28,29,37),

which caused fast pre-apoptotic ecto-CRT and ecto-HSP90, followed by HSP70 surface

exposure (Fig. 3A). In contrast, Mel-induced melanoma apoptosis was accompanied only by a

significant ecto-HSP90 after 24 hr (Fig. 3A), a result that was confirmed in the murine B78

and K1735 cells plus in the human MM031 short-culture melanoma cells (Suppl. Fig. 3A-C).




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Interestingly, Mel-treatment did not induce ATP secretion (Suppl. Fig. 3D-E). Of note, Mel-

induced ecto-HSP90 was detected only when the whole population of dying cells entered late-

apoptotic stage (according to kinetics of caspase-3 activity) (compare Fig. 3A and Fig. 2D).

However, the population of ecto-HSP90+ cells was partially AnV

-/7AAD

- and AnV

+/7AAD

-

(pre-apoptotic or early/mid-apoptotic cells) (Fig. 3B), while the small population of ecto-

CRT+ cells was mostly AnV

+/7AAD

- (early/mid apoptotic cells) (Suppl. Fig. 3F). Thus

contrary to Hyp-PDT, Mel induced pre- or early/mid-apoptotic ecto-HSP90 in a pre-

dominantly late/post-apoptotic cell culture environment.

Since DAMPs emission has been shown to predominantly rely on ER stress-ROS signaling

and in some cases require caspase signaling (28), we decided to block these apoptotic

mediators. Blocking caspases by zVAD-fmk blunted Mel-induced ecto-HSP90 (Fig. 3C,

Suppl. Fig. 3G), whereas attenuation of Mel-induced ER stress by TUDCA (Fig. 3D), or ROS

production by, NAC, (Fig. 3E) exerted a dose dependent decrease in ecto-HSP90. Consistent

with the effects of zVAD-fmk and the kinetics of DAMP exposure, the combination of

TUDCA and NAC suppressed ecto-HSP90 (Fig. 3F), thereby strongly coupling cell death

signaling reliant on ER stress and ROS with the mobilization of HSP90 at the plasma

membrane.

Despite inducing ROS and some features of ER stress, Mel did not increase ecto-CRT. Since

in previous studies induction of robust ER stress, by thapsigargin and tunicamycin, restored

ecto-CRT, post-cisplatin treatment (38), we tested whether augmenting ER stress in Mel-

treated cells would elicit ecto-CRT. To this end, we used various ER stress inducing agents:

SERCA pump inhibitor thapsigargin (Tg), the inhibitor of N-glycosylation tunicamycin

(Tunica), the proteasome inhibitor bortezomib (Borte), the glycolytic inhibitor 2-deoxy-D-

glucose (2DG), and the reducing agent dithiothreitol (DTT). Intriguingly, only the addition of

high dose Tg, but not other aforementioned ER stress inducers, restored ecto-CRT post-Mel-




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treatment (Fig. 3G, Suppl. Fig. 3H). This effect could be dissociated from an increased

induction of cell death (Suppl. Fig. 2I) since none of these agents, enhanced melanoma killing

when added after the commitment phase of Mel-induced apoptosis (i.e. after loss of

mitochondria transmembrane potential and caspase activation, Fig. 2B). Likewise, we

wondered whether enhancing ROS levels could increase Mel or Mel/Tg-induced ecto-CRT.

However, addition of H2O2 failed to increase Mel or Mel/Tg-induced ecto-CRT (Fig. 3I); and

did not exacerbate cell death (Suppl. Fig. 2J). Notably, addition of either ER stress inducers

and/or H2O2 to Mel-treated cells did not affect ecto-HSP90 (Fig. 3H, 3J, Suppl. Fig. 3I).

In aggregate these observations confirm that while ROS and ER stress are crucial for ecto-

CRT and ecto-HSP90, the lack of a robust ER stress module compromises the ecto-CRT

trafficking mechanisms in Mel-treated cells.

Mel-induced apoptosis is associated with the secretion of pro-inflammatory chemokines

To determine whether Mel-treatment is additionally able to affect key cytokine or chemokine

signaling in melanoma cells, we analyzed the supernatants of Mel-treated A375 cells for the

presence of key pro-inflammatory cytokines (IFNα, CXCL8/IL8, IL6 and TNF), or

chemokines (CCL2, CCL5, CXCL9, CXCL10) (39,40). A375 cells failed to release CCL5,

CXCL9, CXCL10, IL6 and TNF under basal conditions (data not shown). However, while

neither Mel- nor Hyp-PDT-treatments statistically influenced IFNα release, the release of IL8

and CCL2 by A375 cells 24 hr after Mel-treatment (Fig. 3H) was significantly increased. This

increase in IL8 and CCL2 was unique for Mel since Hyp-PDT induced no CCL2 increase and

even a significant decrease in IL8 (Fig. 3K).

Thus Mel-induced apoptotic cell death of melanoma cells in vitro is associated with the

induction of ecto-HSP90, as well as the secretion of pro-inflammatory chemokines, IL8 and

CCL2.




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Mel-treated cancer cells evoke moderate activation of dendritic cells which is not reliant

on ecto-HSP90 or ecto-CRT

Having established that Mel-treatment induces signature of danger signaling in melanoma

cells in vitro (Fig. 2-3) and a shift towards a pro-inflammatory tumor microenvironment (Fig.

1), we wondered about the direct interactions of such treated melanoma cells with key

immune cells.

To this end, we co-cultured Mel-treated melanoma cells with immature dendritic cells (iDCs)

and measured the phenotypic maturation (i.e. increased surface expression of HLA-DR and

CD86) and functional stimulation of DCs. In our experimental setting, LPS treatment of iDCs

(Suppl. Fig. 4A-B) was applied as a positive control to test the maturation potency of iDCs,

whereas Hyp-PDT-treated cells served as control for the stimulation of fully mature DCs

(28,29). Mel-treated melanoma cells induced significant DC-maturation, similarly to Hyp-

PDT (Fold changes: Fig. 4A; Percent changes: Suppl. Fig. 4C). To establish the relevance of

ecto-HSP90 for the Mel-treated cells-induced DC-maturation, we blocked ecto-HSP90 with a

HSP90-specific antibody. Despite the suggestive trend of decreased phenotypic maturation

with ecto-HSP90-elimination (Fig. 4B), no statistical significance was obtained. We then

wondered whether the immunostimulatory effects of Mel-treated human melanoma cells on

DCs could be increased by coating of the dying melanoma cells with exogenous recombinant

CRT (rCRT). However, addition of rCRT to Mel-treated human melanoma cells did not alter

phenotypic maturation of co-cultured DCs (Fig. 4C). We reasoned that the pro-inflammatory

cytokine TNF could be a possible additional candidate. This choice was motivated by our

retrospective metadata analysis of reported clinical data illustrating that the combination of

Mel with TNF or TNF/IFNγ (Suppl. Table 2) improves patients’ tendency to achieve

complete clinical responses within ILP/ILI therapies (Suppl. Fig. 4H). Although high doses of

TNF and IFNγ given during ILP/ILI are known to be associated with vasodisruption and




17

increased uptake of Mel in tumors (which potentiates Mel’s cytotoxicity) (41), yet their

immunological impact cannot be ruled out. However, addition of rTNF to Mel-treated human

melanoma cells did not increase phenotypic maturation of co-cultured DCs (Fig. 4C), thus

suggesting that to improve the interface between Mel-treated cells and DCs, other factors are

required.

We also quantified the levels of IL1β, IL12p70, IL6, TNF and IL10 in the cancer cell-DC co-

culture. Only Hyp-PDT-treated A375 cells stimulated a significant release of IL8, IL6, TNF

and increased IL1β secretion by human DCs (Fig. 4D). The Mel-treated melanoma cells

stimulated a significant release of IL8 by DCs and increased secretion of IL1β and IL6 to not

significant levels; however it did not provoke the release of the immunosuppressive cytokine

IL10. These data point to the formation of semi-mature DCs (42)

(CD86high

HLA-DRhigh

IL8high

IL1βlow

IL6low

) after co-culture with Mel-treated human

melanoma cells.

We also wondered whether Mel-treated cancer cells could affect the activation status of NK

cells, as these immune cells contribute to the direct elimination of cancer cells. In vitro co-

culture of Mel-treated A375 cells with peripheral blood-isolated NKs neither increased the

surface levels of NK activating (NKp30, NKp46, CD69) nor inhibitory (CD94) receptors as

compared to untreated cancer cells (Suppl. Fig. 5A-D). Absence of IFNγ (and other important

chemokines and cytokines) further confirmed the lack of activation of NK cells (Suppl. Fig.

5E). We next measured the levels of cancer cell-associated surface molecules that are

recognized by NKs i.e. MICA/B and ULBP2, before and after the treatment. In comparison to

the untreated A375 cells (Suppl. Fig. 5F) the Mel-treated cancer cells did not show any

change in the levels of MICA/B and ULBP2 (Suppl. Fig. 5G). This observation could explain

why dying cancer cells could not stimulate NK cells in vitro.




18

Mel-treated melanoma cells increase DC-mediated proliferation of CD4+ and CD8

+ T

cells in the presence of IFNγ

To elucidate the functional impact of the semi-mature DCs induced by Mel-treated melanoma

cells, we next investigated their T cell activation capacity in vitro. For this purpose, after 24 hr

co-culture of human iDCs with the dying cancer cells, T cells were added to the cell mixture

and the rate of T cell proliferation and IFNγ production were measured as read-outs for T cell

activation (Fig. 4E-G). Mel-treated melanoma cells, similar to Hyp-PDT-treated cells,

stimulated proliferation of CD4+ and CD8

+ T cells. This was paralleled by an increased

production of IFNγ into the supernatant of the co-cultures (as compared to T cells alone),

although the Mel-treated A375-mediated IFNγ release by T cells was lower than that induced

by Hyp-PDT-treated cancer cells (Fig. 4G). We also investigated whether antibody-based

blockade of ecto-HSP90 or ectopic addition of rCRT or rTNF affects T cell proliferation in

vitro. Consistent with the DC-maturation results, neither elimination of ecto-HSP90, nor

addition of rCRT or rTNF, improved T cell activation mediated by the Mel-treated melanoma

cells (Suppl. Fig. 4F-G).

Thus, DCs co-cultured with Mel-treated melanoma cells trigger increased (danger signals-

independent) proliferation of CD4+/CD8

+ T cells in presence of moderate IFNγ production.

These results further substantiate the earlier conclusion that Mel-treated melanoma cells

induce semi-mature DCs.

Mel-triggered protective anti-tumor immunity is potentiated by rCRT but not by rTNF

To further explore whether Mel-induced melanoma cell death has the ability to act as a

“vaccine” and induce a protective anti-cancer response, we tested its immunization potential

using a prophylactic vaccination mice model.




19

We used the murine B78 melanoma cells that upon Mel-treatment died apoptotically and

displayed caspase-dependent ecto-HSP90 (Suppl. Fig. 2 and 3A), induced semi-mature DCs

(Fig. 5A and 5B), which was unaffected by ecto-HSP90 antibody-based blockage, coating

with rCRT or addition of rTNF (Fig. 5C-D). We thus vaccinated C57BL/6 mice with Mel-

treated B78 cells or PBS (placebo control), followed (10 days later) by a re-challenge with

live B78 cells and tumor growth monitoring. As a negative control, we used a tolerogenic cell

death-inducer, Brefeldin A (BrefA) (28,43) and compared the vaccination efficacy of Mel-

treated cells to that elicited by the ICD-inducer, Hyp-PDT (44). Interestingly, Mel-treated

cancer cells exhibited the ability to induce an “anti-cancer vaccination effect” - as many as

40% of the mice vaccinated with Mel-treated cells rejected rechallenge with live tumor cells

(Fig. 5D). This effect was considerably better than the “vaccine” produced with BrefA (Fig.

5D), but not as robust as the Hyp-PDT-based vaccine, which protected 62% of the mice from

tumor formation following rechallenge (Fig. 5D).

To establish whether the protective anti-cancer effect induced by Mel-treated cancer cells is

due to the stimulation of an adaptive immune response, we depleted immunocompetent mice

of CD4+ or CD8

+ T cells (antibody-based depletion; as control, antibody against β-

galactosidase was used; depletion results are presented on Suppl. Fig. 6B-C). Remarkably,

elimination of CD8+ T cells resulted in abrogation of the Mel-induced anti-cancer vaccination

effect, whereas elimination of CD4+ T cells was ineffective (Fig. 5E). This observation

confirms that the vaccination potential of Mel-treated B78 cells is highly dependent on CD8+

T cells.

To analyze the relevance of Mel-induced ecto-HSP90 in defining in vivo immunogenicity, we

carried out prophylactic mice vaccination using Mel-treated melanoma cells coated with

control or with an HSP90-blocking antibody. This in vivo experiment indicated that the

vaccination effect of the Mel-treated melanoma cells does not rely on ecto-HSP90 (Fig. 5F).




20

Furthermore, we wondered whether the immunogenic effect of Mel-treated murine melanoma

cells could be potentiated by combinatorial addition of rCRT or rTNF. Remarkably, coating

Mel-treated cells with rCRT significantly increased their immunogenicity (Fig. 5G), while

addition of rTNF did not significantly increase the immunogenic properties of Mel-treated

melanoma cells (Fig. 5G).

In conclusion, these in vivo studies show that Mel-treated murine melanoma cells are

endowed with some tumor-rejecting capacity – which is possibly linked to the induction of

inflammatory cell death in melanoma associated with positive immune effector mechanisms;

and which can be further potentiated in vivo by combinatorial addition of rCRT.




21

Discussion

In the present study, we thoroughly describe Melphalan (Mel) as inducer of inflammatory cell

death associated with immunogenicity in melanoma. We show that Mel-treated melanoma

cells favor inflammatory or immune effector mechanisms in immune cells and/or tumor

microenvironment. This notion is supported by the spectra of different cytokines detected in

Mel-ILP-treated patients’ samples and the observation that Mel-treated melanoma cells

induce semi-mature DCs, which in turn induce moderate activation of T cells. Importantly,

Mel-treated melanoma cells elicit noticeable, CD8+ T cells-dependent “vaccine-like” anti-

tumor immunity. These positive immune-mediated anti-cancer effects can be further elevated

by a combinatorial treatment reconstituting ecto-CRT, an ‘eat me’ signal, which is otherwise

poorly trafficked to the plasma membrane after Mel-treatment of melanoma cells.

We show that Mel-treatment was fairly efficient at inducing ROS production and ER stress in

melanoma cells, to an extent that blocking these processes severely compromised Mel-

induced cell death in vitro. Along with the induction of an early ER stress signature in

patients’ biopsies following Mel-ILP revealed in a previous microarray analysis (31) and the

detectable upregulation of IL6 and IL1β in the patients’ sera as early as 1 hr post-Mel-ILP

found in this study, these findings highlight the ability of Mel to rapidly tilt the balance

towards a more pro-inflammatory tumor environment. Induction of ER stress and ROS in a

simultaneous or concomitant fashion is a prerequisite for efficient danger signaling apically

associated with the pre-apoptotic surface exposure of ecto-CRT (8,16). However, our study

conclusively shows that Mel-induced ER stress was below the threshold required to elicit

ecto-CRT. Moreover, our data underscore that combining Mel-treatment uniquely with the

SERCA inhibitor Tg restored ecto-CRT in Mel-treated cells. This outlines the importance of

ER-Ca2+

release over other ER stress-inducing modalities in ecto-CRT induction or

restoration (of note, Hyp-PDT, a powerful enabler of pre-apoptotic ecto-CRT and ICD, also




22

induces SERCA-photodamage-based ER-Ca2+

release) (45). Though, we did find that Mel is

an efficient inducer of ecto-HSP90. Mel-induced ecto-HSP90 was mediated by caspase

signaling secondary to ER stress and ROS production– an interesting observation that

deserves to be further explored, considering that the signaling mechanisms underlying ecto-

HSP90 are elusive. However, our ex vivo/in vivo observations rule out a major role for ecto-

HSP90 as a danger signal, thereby outlining that ecto-HSP90 is a more context-dependent

DAMP rather than a general one, as suggested in previous studies (46).

Prominently, on the immune effectors front, the absence of IL10 production following Mel-

ILP in patient samples and from the DCs/NKs interacting with Mel-treated cancer cells,

further indicates that Mel does not actively promote an immunosuppressive

microenvironment. The Mel-induced inflammatory/immune effector mechanisms revealed

here, might have important prognostic implications for melanoma, considering that the

immunomodulatory features induced by Mel, i.e. high expression of HLA-DR, increased T

cell activation/IFNγ production and low presence of IL10, are also positive prognostic factors

for malignant melanoma (7). Moreover, increased IL6 production (another factor potentiated

by Mel) was reported to associate with increased sensitivity towards immunotherapy against

melanoma (47). Unfortunately due to the low number of patients (with limited clinical follow-

up) available for this study (Suppl. Table 3), we could not obtain an objective predictive or

prognostic estimation for Mel-induced cytokines – a problem that should be addressed in the

future.

Nevertheless our prophylactic immunization studies convincingly show that anti-tumor

immunity may at least partly contribute to the Mel-ILP/ILI’s therapeutic effect against

melanoma. Immunogenicity of Mel-based vaccines was significantly better than the

tolerogenic cell death inducer BrefA but not as high as that of Hyp-PDT, a potent ICD inducer

(28). This suggests that certain immunogenicity-augmenting strategies might be required to




23

further increase the potential of Mel-based therapy. Indeed, Mel-treatment setting lacked a

crucial “eat me” signal i.e. ecto-CRT and a crucial immune effector cytokine on the level of

cancer cells/immune cells, known to accentuate its therapeutic effect in the clinic i.e. TNF.

Addition of rCRT or rTNF in co-culture assays of Mel-treated cells/DCs/T cells did not affect

DC-maturation/T cells’ proliferation. These results are in line with previous studies showing

that at least ecto-CRT does not directly modulate immune cell maturation (9). Remarkably,

rCRT but not rTNF significantly accentuated the immunogenic potential of Mel-treated

melanoma cells. This clearly shows that in the Mel-treatment set-up, the combination of rCRT

has a better (immuno)therapeutic potential than rTNF.

In conclusion, our study provides a comprehensive outlook (Fig. 5H) of the cell death and

immunological characteristics of Melphalan, a widely used (loco-) regionally applied

chemotherapeutic which, as demonstrated by systemic chemotherapeutics, is necessary to

enable the design of “smart” combinatorial immunotherapies (especially in case of

melanoma). This advancement is direly needed since 40-50% of primary melanoma occurs on

the extremities and around 85.5% of these patients develop recurrences (48). Our in vivo

results indicate that the strategies aiming to potentiate the immunogenicity or danger signaling

associated with Mel should strive to increase ecto-CRT. This could be obtained, either via

combination treatment with ER-Ca2+

release inducing ER stressors like Tg or Tg-analogs like

G202 (pro-drug within phase I clinical trial (49)) that could “intrinsically” restore ecto-CRT;

or via combination with exogenously supplied rCRT. The Mel-ILP/ILI treatment schema

represents an ideal opportunity for the latter combination treatment, as just like TNF, rCRT

can also be employed in combination with Mel for short-term (loco-)regional treatment in

extremities-associated malignancies – a conjecture that should be investigated urgently in the

future.




24

Acknowledgements

We thank Sofie Van Eygen and Frea Coun for their excellent technical assistance. Here, we

would like as well to thank all the blood donors for their significant contribution. A.M.D.P. is

supported by the Emmanuel van der Schueren scholarship awarded by the Kom op tagen

Kanker foundation, Belgium. A.D.G. and G.B.F. are supported by a FWO post-doctoral

fellowship. J.W. is funded by the Melanoma Research Alliance (Team Science Research

Award; USA). J.G. and M.W. are supported by European Commission 7th

Framework

Programme FP7-REGPOT-2012-CT2012-316254-BASTION. This work is supported by

FWO (G0584.12N and K202313N) and GOA/11/2009 grant of the KU Leuven to P.A. This

paper represents research results of the IAP7/32 Funded by the Interuniversity Attraction

Poles Programme, initiated by the Belgian State. Some of the figures were prepared using

Servier Medical Art (www.servier.com), for which the authors would like to acknowledge

Servier.




25

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28

Figure legends

Figure 1. Melphalan-based isolated limb perfusion (Mel-ILP) increases production of

pro-inflammatory cytokines in melanoma patients. (A) Relative expression of various

cytokines (IL1B, IL8, IL10, TNF, IFNG) assessed on mRNA level using qRT-PCR; RNA was

isolated from snap-frozen tumor samples collected pre- and post-Mel-ILP (the graph presents

relative expression of cytokines for each patient; statistical analysis is described in materials

and methods). (B) Sera samples isolated from loco-regionally circulating blood collected

before Mel-ILP, after administration of Mel (10-30 min) and after Mel-ILP (1 hr) were tested

for IL1β, IL6, IL8, IL10, IL12p70 and TNF content (the graph presents concentration of each

cytokine for each patient, mean ± SEM are added; respective significant p-values are

mentioned for corresponding conditions; Wilcoxon matched-pairs signed rank test).

Figure 2. Melphalan induces ER stress and ROS-dependent apoptosis. Mel (300µM)-

treated A375 cells were collected at indicated time points and investigated for: (A) percentage

of surviving cells (MUH assay), (B) percentage of phosphatidylserine exposing cells (stained

with Annexin V, AnV+) and permeabilized cells (PI

+), (C) percentage of cells with decreased

mitochondrial transmembrane potential (ΔΨm, assessed by lower TMRM staining) and (D)

increase in caspase-3 activity in cell lysates (RFU-relative fluorescent units). Treated A375

cells co-incubated with zVAD-fmk (25 µM) collected at 24 hr time point were tested for (E)

caspase-3 activity in cell lysates (RFU-relative fluorescent units) and (F) percentage of

permeabilized (PI+) cells. (G) Representative Western blot and corresponding densitometric

quantification showing kinetics of BiP and eIF2α (total and phosphorylated) protein levels in

Mel-treated A375 cells (H) XBP1 splicing by RT-PCR (G and H are representative results,

out of three independent experiments). (I) Representative Western blot of BiP and eIF2α (total

and phosphorylated) protein levels of Mel-treated A375 cells (24 hr) co-incubated with




29

TUDCA [at 250 µg/mL (T250) or 500 µg/mL (T500)], (J) corresponding percentage of

phosphatidylserine exposing cells (AnV+) and permeabilized cells (PI

+). (K) Kinetics of ROS

production by DCF-DA staining of Mel-treated A375 cells, (L) Effect of addition of NAC or

(M) NAC and TUDCA (added at the indicated concentrations) to Mel-treated A375 cells (24

hr time point). Graphs show the percentage of phosphatidylserine exposing cells (AnV+) and

permeabilized cells (PI+). All graphs (A-F and J-M) show results of three independent

experiments (mean ± SEM) and are statistically analyzed with 2 way-ANOVA; * represents

p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.

Figure 3. Melphalan induces ER stress and ROS-dependent danger signaling in

melanoma cells. A375 cells treated with melphalan (Mel; 300 µM) or Hypericin-PDT (Hyp-

PDT; 150 nM Hypericin; 2.70 J/cm2 irradiation) were evaluated at indicated time points for:

(A) ecto-CRT, ecto-HSP70 and ecto-HSP90 in non-permeabilized cells (three independent

experiments, mean ± SEM, and 2-way ANOVA analysis, * represents p-value < 0.05; ** p-

value < 0.01; *** p-value < 0.001.). (B) A375 cells treated with Mel for 24 hr were stained

for ecto-HSP90, phosphatidylserine and permeabilisation (the permeabilised cells were

excluded from the analysis; three independent experiments, mean ± SEM, and Student’s t-test

analysis, ** p-value < 0.01; *** p-value < 0.001). Effect of addition of (C) zVAD-fmk (25

µM), (D) TUDCA, (E) NAC and (F) combination of TUDCA and NAC was analyzed on

Mel-induced ecto-HSP90 (24 hr time point) in A375 cells (three independent experiments,

mean ± SEM, and 2-way ANOVA analysis, * represents p-value < 0.05; ** p-value < 0.01;

*** p-value < 0.001). (G-H) Effect of addition of various ER stressors (the full names and

concentrations are indicated in the materials and methods), or (I-J) Tg and H2O2 (added at the

indicated concentrations) on Mel-induced ecto-CRT (G and I) and ecto-HSP90 (H and J) at 24

hr time point in A375 cells (three independent experiments, mean ± SEM, and 2-way




30

ANOVA analysis, * represents p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001). (K)

Supernatants from A375 cells (collected at 24 hr time point) were tested for IL8, CCL2 and

IFNα (three independent experiments, mean ± SEM, and 1-way ANOVA analysis, ** p-value

< 0.01; *** p-value < 0.001).

Figure 4. Melphalan-treated A375 melanoma cells elicit semi-mature DCs and activate T

cells. The phenotypic maturation of human iDCs (defined as increase in CD86 and HLA-DR

staining) was investigated after 24 hr co-incubation with: (A) untreated or treated for 24 hr

A375 cells (three independent experiments, mean ± SEM, and 1-way ANOVA analysis, *

represents p-value < 0.05; ** p-value < 0.01) or untreated or Mel-treated A375 cells (24 hr

time point) in the presence of (B) control antibody (IgY) or ecto-HSP90 blocking antibody as

applicable (HSP90 IgY) or (C) rCRT or rTNF (three independent experiments, mean ± SEM,

and Repeated Measures ANOVA with Tukey’s post-test within ctrl and Mel conditions

analysis). Graphs A-C represent fold changes relative to crtl-A375. (D) Supernatants from the

co-culture of untreated or dying A375 cells with iDCs were investigated for the content of

IL1β, IL6, IL8, IL10, IL12p70 and TNF (three independent experiments, mean ± SEM, and 1-

way ANOVA analysis). T cells cultured in the presence of iDCs and untreated or dying A375

cells were checked for proliferation of (E) CD3+CD4

+ and (F) CD3

+CD8

+ cells

(representative experiment of three-independent experiments with 1-way ANOVA analysis

for conditions including cancer cells; Mann-Whitney t-test for comparison between “T alone”

and “LPS”); (G) Supernatants of this triple co-culture were tested for IFNγ content

(representative experiment of three-independent experiments, mean of duplicate; ± SEM,

Mann-Whitney t-test; * represents p-value < 0.05; ** p-value < 0.01; ***/### p-value <

0.001).




31

Figure 5. Melphalan induces inflammatory cell death associated with anti-cancer

immunity. (A-C) The phenotypic maturation of murine iDCs was investigated after 24 hr-

incubation with: (A) untreated or treated for 24 hr B78 cells, or untreated or Mel-treated B78

cells (24 hr time point) in the presence of (B) control antibody (IgY) or ecto-HSP90 blocking

antibody as applicable (HSP90 IgY) or (C) rCRT or rTNF (three independent experiments,

mean ± SEM, graphs A-C represent fold changes relative to crtl-B78 and 1-way ANOVA

analysis, * represents p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001). (D) C57BL/6

mice were vaccinated with B78 cells (collected at 24 hr time point after respective treatments)

or placebo control (PBS); thereafter, 10 days later, these mice were injected with live B78

cells and monitored for the tumor growth (10 mice per group; 1-way ANOVA, * represents p-

value < 0.05; *** p-value < 0.001). Effect of (E) elimination of CD4+ or CD8

+ T cells, (F)

antibody-based blockage of ecto-HSP90 on surface of Mel-treated B78 cells, (G) addition of

rCRT or rTNF to Mel-treated B78 cells on the stimulation of anti-cancer immunity (number

of mice per group indicated on the graphs; 1-way ANOVA, * represents p-value < 0.05; ***

p-value < 0.001). (H) Schematic representation of Mel-induced inflammation and danger

signaling associated with immunogenicity: In vivo (in melanoma patients) Mel-ILP increases

expression of IL1B, IL6 and IL8 in the tumor bed and loco-regional serum levels of IL1β and

IL6 as early as 1 hr post-Mel-ILP. In vitro, Mel induces inflammatory cell death capable of

stimulating semi-mature DCs as well as T cell activation and tangible anti-cancer immunity in

a prophylactic mice vaccination model, in vivo.




Published OnlineFirst March 11, 2015.Cancer Res Aleksandra M Dudek-Peric, Gabriela B Ferreira, Angelika Muchowicz, et al. melanoma cell surface associated calreticulinAnti-tumor immunity triggered by melphalan is potentiated by

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