Cryobiology 61 (2010) 268–274

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Cryobiology

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Immunoregulatory effects of freeze injured whole tumour cells on humandendritic cells using an in vitro cryotherapy model q

Mohamed Ismail a,⇑, Richard Morgan a, Kevin Harrington b, John Davies c, Hardev Pandha a

a Postgraduate Medical School, University of Surrey, Guildford, Surrey GU2 7WG, UKb Targeted Therapy Laboratory, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKc The Royal Surrey County Hospital, Guildford, Surrey GU2 7XX, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 June 2010Accepted 6 September 2010Available online 21 September 2010

Keywords:CryotherapyDendritic cellsCytokine

0011-2240/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.cryobiol.2010.09.004

q Statement of funding: This project was funded by⇑ Corresponding author. Address: 1 Gill Avenue, G

+44 1483688699.E-mail address: ms18273@gmail.com (M. Ismail).

Tumour cryotherapy has been described as both immunostimulatory and immunoinhibitory in previousstudies. However, previous studies have not accurately reproduced the precise conditions of current clin-ical cryotherapy. The objective of this study is to assess the immunological effects of cryotreated wholetumour cells on dendritic cells (DC) maturation and function using an in vitro model. Prostate cancer cellswere cooled using Endocare cryo-system to mimic temperatures achieved during clinical cryotherapy.Human DC were prepared from cluster of differentiation (CD) 14 monocytes and matured with lipopoly-saccharide (LPS). Cryotreated cancer cells were added to DC on day 3. On day 7, DC were harvested andphenotyped. Cytokine gene expression was assessed using real time quantitative polymerase chain reac-tion (RT-PCR). Functional activity of DC was assessed in allogenic mixed lymphocyte reaction (MLR) andthe molecular changes using gene microarray technology. There was statistically significant upregulationof costimulatory molecules and maturation markers (CD86, CD83, CD80 and CL II) in DC loaded withcryotreated whole tumour cells compared to both control DC and DC matured with LPS (P < 0.001). Therewas a significant increase in stimulatory cytokines gene expression (IL-2, IL-12, IL-15, IL-18 and IFN-c).However, IL-10 and TGF-b expression reduced significantly. The effect of different freezing temperaturewas equal. cDNA microarray analysis showed upregulation of interleukin 1 (IL-1) and cycline dependentkinase inhibitor 1A (CDKN1A (p21) and downregulation of Caspase 8 and BCL2. Overall, our findings sug-gest that the effect of cryotherapy is generally stimulatory to DC which may enhance anti-tumour effects.Therefore, the combination of cryotherapy and DC vaccine may represent a novel method to increase theefficacy of cryotherapy especially at the peripheral zones of the prostate where cells are exposed to sub-lethal temperature.

� 2010 Elsevier Inc. All rights reserved.

Introduction

Cryotherapy is a procedure which includes exposure to sub-zero freezing temperature resulting in tissue necrosis. Systemicanti-tumour immune responses have been postulated followingfew clinical reports observing regression of metastatic diseaseand symptomatic relief following prostate cryotherapy [24]. Localtumour destruction by cryotherapy results in the release of cryone-crotic tissue and tumour antigens and enhances the uptake ofthese antigens by local DC resulting in tumour specific immuneresponse and tumour eradication [5]. Cryoimmune response hasbeen studied in several animal models and both immunostimula-tory and immunoinhibitory effect was noticed [19,10,6,20,13].

ll rights reserved.

Prostate project foundation.uildford GU2 7WW, UK. Fax:

The precise mechanism of the immunostimulatory effect was notclear. Early cytokine mediated response was reported [6,20].Involvement of T cell immunity and enhanced natural killer (NK)cell cytotoxicity was also described [2]. Other reports suggestedthe development of anti-tumour antibodies following cryotherapy[29]. On the contrary, other reports suggest suppressed immunityand enhanced tumour growth and metastases following cryotherapy[10,6].

Dendritic cells (DC) are bone marrow derived cells that are spe-cialized in antigen processing and presentation. Immature DC re-side in the peripheral tissues sensing their microenvironment forantigens. They are highly endocytic with poor antigen presentingcapacity. Upon maturation, DC undergo phenotypic and functionalchanges. They show increased expression of major histocompati-bility complex (MHC), adhesion and costimulatory molecules(CD80 and CD86). Mature DC have increased antigen presentingcapacity and are more capable of migration to local lymph nodeswhere they act as efficient antigen presenting cells (APC) to prime

M. Ismail et al. / Cryobiology 61 (2010) 268–274 269

naïve antigen specific T cells [1]. The ability of DC to process tu-mour antigens and present them to T cells to induce tumour spe-cific immune response renders them the ideal immune cells fortumour immunotherapy. Dendritic cell immunotherapy has beenshown to have significant anti-tumour effects in numerous phaseI and phase II trials [3]. Recently a randomized phase III study ofDC-type autologous vaccine was shown to improve overall survivalin patients with metastatic prostate cancer, resulting in Food andDrug Administration (FDA) approval of this approach [9]. Ex vivogeneration and loading of DC remains the preferable method inDC based vaccine therapy. Such vaccines are usually deliveredintradermally, relying on efficient DC migration from injectionsites to the regional lymph node [25]. Cryonecrotic tissue repre-sents an excellent milieu for APC. den Brok et al. [5] demonstratedthat in situ tumour destruction by cryotherapy was more efficientthan radiofrequency in loading DC with tumour antigen. Intratu-moral administration of ex vivo generated DC combined with cryo-therapy of the primary tumour was shown to induce tumourspecific Th1 type immune response [28,15]. The objectives of thisstudy are to investigate the immunological effects of cryotreatedwhole tumour cells on DC maturation and function and to identifythe mechanisms of immune modulation by cryotherapy. We alsoassessed the molecular changes in DC following exposure to frozenprostate cancer cells using gene microarray technology.

Materials and methods

Cell line and preparation of samples for cryotreatment

DU145 and PC-3 prostate cancer cell line were obtained fromAmerican Type culture collection (ATCC). Cells were grown in cul-ture medium (RPMI 1640 with 10% fetal calf serum, 1% L-glutamineand 1% penicillin/streptomycin all from Sigma–Aldrich, Poole, UK)and incubated in 37 �C with 5% CO2. Cells were washed and sus-pended in fresh culture medium prior to treatment.

Freezing protocol

Cells were treated using the Cryocare system™ (Endocare, Inc.,Irvine, CA) as described before [12]. Cells were cooled to 0, �5, �10and �20 �C for 10 min and then thawed to room temperature. Allcells groups were exposed to the same experimental conditions.

Generation of monocyte derived DC

A buffy coat was obtained from the National Blood Service in40 ml transfusion bag. Peripheral blood mononuclear cells (PBMCs)were resuspended in culture medium (RPMI-1640 containing 10%FCS, 100 U/ml penicillin 100 lg/ml streptomycin and 50 lm 2-mercaptoethanol, all from Sigma–Aldrich, Poole, UK) at a final con-centration of 3 � 106 cells/ml. Four millilitres of cell suspensionwere incubated in 6 well plate for 2 h at 37 �C. Non-adherent cellswere discarded and 4 ml of fresh culture medium, 100 ng/ml gran-ulocyte macrophage colony stimulating factor (Berlex, New Jersey,USA) and 50 ng/ml interleukin-4 (Peprotech, EC Ltd., London, UK)was added to each well. The cells were characteristically dendriticin appearance. GM-CSF and IL-4 were added every 3 days up to7 days. On day 4, LPS 1 mg/ml (Sigma–Aldrich, Poole, UK) wasadded to half of the wells to induce DC activation and maturation.

Tumour lysate

DU145 and PC-3 cells were re suspended in serum free RPMI-1640 medium at a density of 1 � 106 cells/ml. Cell suspensionwas frozen at �80 �C, interrupted by four freeze–thaw cycles.

The lysate was centrifuged at 690g for 10 min and supernatantwas passed through 0.2 lm filter. Protein contents of the lysatewere determined by Bio-Rad’s protein assay (Bio-Rad, Herts, UK).

Treatment protocol

Cryotreated prostate cancer cells were added to both immatureDC and DC matured by LPS at day 3, at least 2 h after the addition ofLPS. DC:cancer cells ratio was 5:1. Two hundred microlitres of tu-mour lysate (= 200 lg/ml protein) was added to the remainingwells at the same time.

DC staining and phenotyping

Phenotypic analysis of DC was performed by flow cytometryusing anti-CD14, CD11c, CD86, CD80, CCR7, CD1a, CL II, CD209and CD83 antibodies. Briefly, cells were resuspended in 300 llFACS buffer (450 ml sterile distilled water, 50 ml of 10� calcium/magnesium free PBS, 3 g bovine serum albumin, 3 g sodium azide,all from Sigma–Aldrich, Poole, UK) and stained with FITC, PE, orPECY5 conjugated mAb (all from BD Biosciences, Oxford, UK). Cellsthen washed with FACS buffer and fixed with 150 ll FACS FIX(450 ml sterile distilled water, 60 ml of 10� calcium/magnesiumfree PBS, 15 ml 38% formaldehyde solution, all from Sigma–Aldrich,Poole, UK). Stained DC were analyzed on Epics XL (Beckman-Coul-ter, High Wycombe, UK).

Real time quantitative RT-PCR

RT-qPCR analysis was performed using Stratagene Mx3005PqPCR system. GAPDH was used as a house keeping gene and allreactions were performed in duplicated in 96-well plate. Two fourmicrolitres of PCR mixture contained 1 ll cDNA, 1 ll of each cyto-kine primer, 10.5 ll nuclease free water and 12.5 ll SYBR� GreenJumpStart™ Taq ReadyMix™ (Sigma–Aldrich, Poole, UK). Primerswere designed by Primer3 software and obtained from Eurogentecgroup (Eurogentec Ltd., UK). RT-qPCR efficiency was calculatedusing standard curve. Thermal cycling conditions were as follow:1 cycle (10 min at 95 �C) followed by 40 cycles (30 s at 95 �C,1 min at 60 �C and 30 s at 72 �C) followed by 1 cycle (1 min95 �C, 30 s at 55 �C and 30 s at 95 �C). The 2�DDCT method was usedto calculate the difference in CT value between the treated sampleand untreated control and is expressed as a fold changes in geneexpression relative to the untreated cells. The results then normal-ized to an endogenous reference gene (GAPDH) whose expressionis constant in all groups.

�DDCT ¼ ðCT ; target� CT reference geneÞtreated � ðCT ; target

� CT reference geneÞuntreated

Cytokine assay

Supernatant was collected at different time points (2, 6, 24 and72 h) and stored in aliquots at �80 �C. Cytokine levels secreted inthe supernatant from the treated DC were quantified using Bio-Plex cytokine assay (Invitrogen, Paisley, UK) following the manu-facturers’ protocol. The multiplex bead-based assay is designedto quantitate IL-2, IL-6, IL-12(P70), IL-15 and IFN-c.

Mixed lymphocyte reaction (MLR)

Functional activity of DC was assessed in allogenic MLR. Allo-genic human T cells (responder) was isolated from a buffy coat,and added in triplicate in graded concentration (104–106 cells perml) in a rounded bottomed 96-well plate. DC (1 � 104/ml) were

270 M. Ismail et al. / Cryobiology 61 (2010) 268–274

then added into each well. Plates were incubated at 37 �C in 5% CO2

for 5 days. Proliferation of T cells was measured after adding 3H-thymidin for 18 h. Cells were harvested using Filtermate harvester(PerkinElmer, Massachusetts, USA) and the amount of 3H-thymidinincorporated into the DNA is measured using MicroBeta TriLuxcounter (PerkinElmer, Massachusetts, USA). The counts were ex-pressed as a count per minute (cpm).

cDNA microarray analysis for gene expression profile

Total RNA was isolated using RNeasy_Plus mini kit (Qiagen,west Sussex, UK) according to the manufacturer’s protocol. cRNAfor each sample was synthesized by using One-Color Low RNA In-put Linear Amplification Kit PLUS (Invitrogen, Paisley, UK) and waslabeled with Cy3 by direct labeling method. Following purificationby RNeasy_Plus mini kit, cRNA was fragmented by incubation inthe fragmentation buffer at 60 �C for 30 min. The fragmented andlabeled cRNA was hybridized to the human whole genom 1884 � 44 K cDNA chip (Invitrogen, Paisley, UK). After washing, theslides was scanned using Agilent’s microarray scanner (Agilent,Cheshire, UK) and gene expression profiles were analysed by Genespring GX 7.3.1 software.

Results

Cryotreated prostate cancer cells induce upregulation of costimulatorymolecules on cultured DC

On day 7, mean fluorescence intensity (MFI) of surface markersexpression was measured and compared between six groups,immature DC (control), DC matured with LPS (LPS), DC loaded withuntreated whole tumour cells (UT), DC loaded with tumour lysate(Lys), immature DC loaded with cryotreated whole tumour cells (F)and DC loaded with LPS and cryotreated whole tumour cells(F(LPS)).

LPS induced upregulation of costimulatory molecules and mat-uration markers (CD80, CD83, CD86, Class-II and CCR7) (P < 0.001)and downregulation of CD14 and CD209 molecules (Fig. 1). Expo-sure of DC to cryotreated tumour cells at �10 �C resulted in 8-foldincrease in the expression of CD86 compared to the control(P < 0.001). Upregulation of CD86 induced by cryotreated tumourcells was equivalent to LPS response. Further increase in CD86expression was noted when cryotreated cells were added to DC

0 100 200 300 400 500 600 700

CD14

CD11c

CD86

CL II

CD209

CD83

CD80

CD1a

CCR7

DCsDCs+LPS

MFI(% of control)

CD

mol

ecul

e

Fig. 1. The effect of LPS on different CD molecules expression by dendritic cells.

matured with LPS (Fig. 2). There was a statistically significant in-crease in CD86 expression in DC loaded with cryotreated cells com-pared to cells loaded with untreated tumour cells or tumour lysate(Fig. 2). Furthermore, DC loaded with cryotreated tumour cells ex-pressed significant levels of CD80 and CD83 compared to othertreatments in a similar trend to CD86. Cryotreated tumour cellsand tumour lysate induced more than 2-fold upregulation of sur-face expression of Class-II molecule compared to control DC(P < 0.01). On the contrary Class-II expression was minimallyupregulated by untreated whole tumour cells (P > 0.05). DC loadedwith cryotreated tumour cells showed a significant upregulation ofCCR7 molecule compared to the control and it was equivalent toLPS stimulated DC response (data not shown). CD209 expressionwas significantly downregulated following exposure to tumour ly-sate and cryotreated tumour cells (data not shown).

Cryotherapy results in increased stimulatory cytokine expression inprostate cancer cells

We next looked at the cytokine response to tumour cells whichhad been exposed to freeze injury. mRNA level of immunostimula-tory (IL-1b, IL-2, IL-6, IL-12p40, IL-15, IL-18 and TNF-a) andimmunoinhibitory (IL-10 and TGF-b) cytokines in response to cryo-therapy were assessed using RT-qPCR.

DU145 and PC-3 cells were evaluated and showed similar re-sults overall; representative data for DU145 is shown in Fig. 3. Bothcell lines expressed IL-18, IL-1b and IL-15 at relatively high levels,while they expressed lower levels of IL-12, IL-6 and TGF-b andminimal levels of IL-2 and IL-10 (data not shown). mRNA expres-sion for IL-2, IL-15 IL-18 and TNF-a was significantly upregulatedin response to freezing injury with the highest expression beingat �10 �C (P < 0.001). IL-6, and IL-12 mRNA expression were clearlyupregulated at all treatment temperatures and predominantly at�5 �C (P < 0.001). In the contrary, immunoinhibitory cytokines(IL-10 and TGF-b) mRNA expression was downregulated uponfreezing. The change in expression was insignificant (P > 0.05) ex-cept for IL-10 at �10 �C. IL-1b mRNA expression was the only pro-inflammatory cytokine tested which is reduced with freezing inDU145 cells whereas in PC-3 cells the mRNA levels increased sig-nificantly at �10 �C (data not shown).

DC exposed to cryotreated whole tumour cells express high levels ofimmunostimulatory cytokines

We investigated the effects of cryotreated tumour cells on DC.Pro- (IL-1b, IL-2, IL-6, IL-12, IL-15, IL-18, IFNc, TNF-a, and CCR-7)and anti- (IL-10, TGFb) inflammatory cytokine gene expressionwas quantitatively assessed in the six experimental groups usingRT-PCR. DC loaded with cryotreated tumour cells expressed higherlevels of IL-12p40, TNF-a, IL-1b IFN-c and CCR7 (P < 0.001) com-pared to control group. IL-15 and IL-18 mRNA expression levelswere statistically insignificant (Fig. 4). Upregulation of proinflam-matory cytokine induced by cryotreated tumour cells was superiorto LPS, tumour lysate and untreated tumour cell response(P < 0.001). Stimulation of DC with LPS followed by cryotreated tu-mour cells resulted in a strong upregulation of most of immuno-stimulatory cytokines (IL-1b, TNFa, IFN-c, IL-12p40, CCR7, IL-6and IL-15 P < 0.001). IL-2 and IL-6 mRNA expressions were stronglyupregulated in DC loaded with tumour lysate (P < 0.001). Un-treated tumour cells did not result in any significant change inthe level of cytokines tested. mRNA levels of the Immunoinhibitorycytokines (IL-10 and TGF-b) showed downregulation trends never-theless, changes were statistically insignificant (P > 0.05) (data notshown).

Fig. 2. The effects of LPS, untreated tumour cells, tumour lysate and cryotreated tumour cells at �10 �C on cell surface marker expression on DC. Cells were loaded at day 3with various agents and harvested at day 7. Cells were then washed, stained and analysed using FACS machine. Mean fluorescence intensity as a percent of control is plottedagainst treatment. (Control = immature DC, LPS = DC matured with LPS, UT = DC loaded with untreated tumour cells, Lys = DC loaded with tumour lysate, F = DC loaded withcryotreated tumour cells at �10 �C and F(LPS) = DC matured with LPS and then loaded with cryotreated cells.)

Fig. 3. Cytokine gene expressions by RT-PCR in DU145 cells in response to freezing injury. Cells were treated for 10 min at three different target temperatures of (0, �5 and�10 �C) and actively thawed to room temperature. Cells then incubated over night at 37 �C, 5% CO2. mRNA expression of the immunostimulatory cytokines was significantlyupregulated upon freezing (P < 0.001) whereas the immunoinhibitory cytokines were downregulated but the changes were insignificant except for IL-10 in cells cooled to�10 �C (P < 0.05).

M. Ismail et al. / Cryobiology 61 (2010) 268–274 271

Cytokine secretion in supernatant from DC exposed to various stimuli

Cytokine secretion was evaluated in the supernatant collectedfrom different groups of DC at different time intervals (Fig. 5). fivegroups of DC were compared, immature DC (control), LPS stimu-lated DC (LPS), DC loaded with untreated tumour cells (UT), DCloaded with tumour lysate (Lys) and DC loaded with cryotreatedtumour cells (F). Supernatants were collected at 2, 6, 24 and 72 hpost treatment. IL-6, IL-15, IL-12p70, IL-2 and IFN-c cytokine levelsin the supernatant were assessed using Bio-Plex cytokine assay.Significant levels of IL-6 were detected in the supernatant of allgroups. There was a significant reduction in IL-6 production uponexposure to cryotreated tumour cells compared to the control DC(P = 0.0028). There was a significant increase in IL-15 secretionby DC pulsed with cryotreated tumour cells compared to the con-

trol DC and DC matured by LPS (P < 0.001). IFN-c levels were signif-icantly increased in DC loaded with cryotreated tumour cellscompared to control (P = 0.0212). There were no significant differ-ence in IFN-c levels in the supernatant from DC loaded with cryo-treated tumour cells and LPS stimulated DC. IL-12p70 levels weresignificantly reduced in the F group compared to control(P = 0.0175). There was no significant change in IL-2 levels in thesupernatant collected from different groups (P = 0.0517).

DC exposed to cryotreated cells acquire potent T cell stimulatorycapacity

We assessed the capacity of DC exposed to cryotreated tumourcells to prime naïve T cells in allogenic MLR. Human T cells wereisolated from peripheral blood and stimulated in vitro by immature

IL1ββ IL-2 IL-6 IL-15 IL-18 IFN-g TNF- α IL-12 CCR-705

1015202530354045505560

ControlLPSUTLys.FF(LPS)

Cytokine

Fold

chn

ge in

gene

exp

ress

ion

Fig. 4. Cytokine gene expressions in DC by RT-PCR. RNA was extracted from different groups of DC and reversed transcribed using cDNA synthesis kit. Fold change in geneexpression was measure in comparison to control. DC loaded with cryotreated tumour cells showed statistically significant upregulation of IL-1b, IL-12p40, IFN-c, TNFa, andCCR7 (P < 0.001). Further upregulation was noticed after the addition of LPS. There were no significant changes in IL-10 and TGF-b levels (data not shown).

Fig. 5. IL-6, IL-15, IL-2, IFN-c and IL-12P70 cytokines levels in supernatant 72 h following DC stimulation detected by Bio-Plex cytokine assay. There was a significant increasein IL-15 secretion by DC loaded with cryotreated tumour cells (P < 0.001). IL-6 levels showed a significant reduction (P = 0.0028). IFN-c and IL-2 levels showed an increasingtrend following exposure to cryotreated tumour cells (P = 0.0212 and P = 0.0517 respectively). IL-12p70 reduced significantly (P = 0.0175).

272 M. Ismail et al. / Cryobiology 61 (2010) 268–274

DC (control), DC mature with LPS (LPS), DC pulsed with tumourcells cooled at �10 (F), tumour lysate (Lys) or untreated tumourcells (UT) (Fig. 6). Proliferative response was then measured. Astrong stimulation of allogenic T cell proliferation was observedwith DC that has been pulsed with cryotreated tumour cells com-pared to DC pulsed with tumour lysate or untreated tumour cells(P < 0.001 and P < 0.05 respectively). The stimulatory activity washigher in DC matured with LPS (P < 0.05) (data not shown).

Fig. 6. The effect of DC exposed to cryotreated tumour cells on T cell proliferation inMLR. Stimulator/effector ratio was 1:10. Following exposure to cryotreated cells, DCgained high stimulatory capacity on T cells proliferation compared to the controlgroup (P < 0.001).

Gene expression profile by gene microarray in DC loaded withcryotreated tumour cells

We studied the molecular changes in DC loaded with cryotreat-ed cells (F), Lysate (Lys), LPS (LPS) and untreated tumour cells (UT).Immature DC served as a control. Total numbers of differentiallyexpressed genes at the mRNA level were 50, 59, 48 and 67 inLPS, UT, Lys and F groups respectively with more than twofoldchange in gene expression. All reported genes were statisticallysignificant (P < 0.05). Altered genes were sorted on their biological

function which showed that cryotherapy regulates importantgenes involved in DC maturation and activation (Table 1). Interleu-

M. Ismail et al. / Cryobiology 61 (2010) 268–274 273

kin 1 (IL-1) was upregulated 57-fold in DC loaded with cryotreatedtumour cells. It represents a key cytokine for DC activation duringearly stages of inflammation. It also stimulates CD40 ligand leadingto increased cytokines secretion by activated DC.

Fas was upregulated approximately 35-fold in DC loaded withcryotreated tumour cells compared to the control DC. It was previ-ously reported that DC express Fas receptor that can induce T cellapoptosis via Fas–FasL dependent mechanism [18]. DC loaded withcryotreated cells showed 6-fold increase in cycline dependent ki-nase inhibitor 1A (CDKN1A (p21)) expression compared to control.CDKN1A is required for DC maturation and function [14]. Tumournecrosis factor receptor superfamily, member 11b (osteoproteger-in) was 4-fold upregulated in DC loaded with cryotreated cells. Itwas demonstrated that osteoprotegerin expression was controlledby NF-jB and it may play a role in regulating immune response[23]. Other genes which was upregulated in the (F) group is vascu-lar endothelial growth factor (VEGF 3.83-fold) and CXCR4 (3.46-fold). CXCR4 is a chemokine receptor that is expressed in matureDC and is responsible for DC migration. It promotes DC survivalvia altering the balance between key anti- and pro-apoptotic pro-teins and enhances its function [8]. A number of important genesinvolved in the induction of apoptosis were downregulated in DCexposed to cryotreated cells such as Caspase 8 (0.426-fold) andBCL2 (0.455-fold).

Discussion

The aim of prostate cryotherapy is to destroy neoplastic tissue andpreserve vital structures around the prostate. It is technically chal-lenging to achieve lethal critical temperature in all the prostatic tis-sue as this will result in a high percentage of complications.Therefore complete ablation of prostate cancer tissue sometimes failsand results in local disease recurrence. In an attempt to overcome thisissue, several reports described an adjunctive therapy to cryotherapyin order to increase the sensitivity of cells to cryogenic injury [11].One approach was intratumoral administration of DC following cryo-ablation of the primary tumour or cryoimmunotherapy [28].

In this study the effect of cryotreated prostate cancer cells onDC maturation and function was addressed and the mechanismresponsible for the DC stimulation by cryotherapy identified.

DC can be activated by endogenous signals such as stressedcells, virally infected, necrotic or apoptotic cells [7,22,5]. DCprimed with tumour freeze/thaw lysate resulted in the develop-ment of active immune response and the regression of the primarytumour in cancer patients [17]. Whole tumour cell provides manytumour specific and tumour associated antigens and is ideal for DCpriming [26]. den Brok et al. [4] demonstrated that tumour debriscreated by radiofrequency and cryotherapy comprises an effectiveantigen source for local DC which was able to induce DCmaturation.

Table 1Differentially expressed genes in DC loaded with LPS (LPS), untreated tumour cells(UT), cryotreated tumour cells (�10) and tumour lysate (Lys). Numbers represent foldchange in gene expression compared to control DC. Empty cells means genes were notdifferentially expressed.

Gene name GenBank LPS UT Lys F

IL-1b NM_000576 19.16 14.29 9.96 56.94FAS NM_000043 2.07 11.09 2.14 34.55CDKN1A NM_000389 4.38 5.20 2.45 5.82Osteoprotegerin NM_002546 3.43 – 3.99 4.37CXCR4 NM_001008540 – – – 3.46VEGF NM_001025366 2.15 2.52 – 3.83Caspase 8 NM_033356 – – – 0.42BCL2 NM_000633 – 0.46 0.47 0.45

We demonstrated that prostate cancer cells cooled to �10 �Cwere more effective in inducing DC maturation than tumour lysateand untreated tumour cells. Flowcytometric data revealed 8-foldincrease in CD86 marker expression and more than double ofclass-II expression. Maturation was further increased by treatingDC with LPS. The nature of maturation stimuli provided by cryo-treated cells is not well identified. Two cytokines are importantfor DC maturation, TNF-a and IL-1b, both cytokines were not de-tected in the supernatant of necrotic tumour cells which inducedDC maturation [22]. Salio et al. [21] demonstrated that the stimu-lation was mainly as a consequence of mycoplasma contamination.Cell lines used in this experiment tested negative for mycoplasmainfection. Matzinger [16] proposed that stressed cells release alarmsignals responsible for DC activation. Todryk et al. [27] have shownthat heat shock protein (HSP) 70 expression by tumour cells wasassociated with increased infiltration of DC into the tumour tissueand resulted in increased antigen uptake by immature DC.

In order to explore the mechanism by which cryotreated tu-mour cells stimulate maturation signal in DC, we evaluated cyto-kine gene expression by prostate cancer cells in response tocryotherapy. Immunostimulatory cytokines (IL-2, IL-15, IL-12p40,IL-18 and TNF-a) were significantly upregulated upon freezing at�10 �C. Freezing injury resulted in upregulation of IL-1B in PC-3cell line (data not shown). Immunoinhibitory cytokines was signif-icantly downregulated. These data suggest that cryotherapy cre-ates immunostimulatory environment through increasedproduction of proinflammatory cytokines for the immune cells tofunction. To explore further the nature of these maturation signalson DC function, we examined cytokine gene expression by DC ex-posed to different maturation stimuli. DC loaded with cryotreatedtumour cells showed increased expression of stimulatory cytokines(IL-1b, IL-12p40, IL-15, TNFa, IFN-c) and chemotactic marker CCR7compared to DC exposed to tumour lysate or untreated tumourcells. The expression was higher than LPS stimulated DC.

DC loaded with cryotreated tumour cells are functionally moreactive than DC stimulated by tumour lysate or untreated tumourcells in MLR. The effect was similar to LPS stimulated DC. Freezingtemperature has no effect on the level of DC maturation, and tem-perature of �5, �10 and �20 �C produced the same effect (data notshown).

Gene microarray data revealed that DC exposed to cryotreatedtumour cells showed upregulation of important genes that are in-volved in DC maturation and function such as IL1 b, Fas, CDKN1A(p21), osteoprotegrin and CXCR4. Genes responsible for apoptoticdeath pathway such as BCL2 and Caspase 8 were downregulated.These finding provide a strong evidence that cryogenic lesion isimmunostimulatory for DC in the tumour microenvironment.

Overall, our data suggest that the effect of cryotherapy is gener-ally stimulatory to dendritic cells which may favour anti-tumourimmune response. Freezing injury stimulate DC in two differentways. First it provides a pool of antigen from the cryo destructedtumour tissue and second, by creating an immunostimulatorycytokine environment which enhance DC maturation. Therefore,the combination of cryotherapy and DC vaccine may represent anovel method to increase the efficacy of cryotherapy especially atthe peripheral zones of the prostate where cells are exposed tosub-lethal temperature. Further in vivo study of this potentialimmunotherapy in a murine model of cryotherapy is warranted.

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