R di i M dRadiation ModuRadiation Modub l ffAbscopal Effect inAbscopal Effect in p

B il R R P 1 4 C i B éBertil R.R. Persson,1,4 Catrin BauréusBertil R.R. Persson, Catrin Bauréus1 4Crister Ceberg 1,4 Henrietta NCrister Ceberg, Henrietta N

1 21Medical Radiation Physics, 2Neurosurgy , gLundLund

Correspondence tCorrespondence t

I d iIntroductionIntroductionWe studied radiation modulated immune response in intracranial N29 tumorsWe studied radiation modulated immune response in intracranial N29 tumorsb bi i i l f ti f di ti th (5 ll 15 G ) dby combining a single fraction of radiation therapy (5 as well as 15 Gy) andimmunization with interferon‐gamma (IFN) transfected immunogenic tumorimmunization with interferon gamma (IFN) transfected immunogenic tumorcells In a parallel study we also studied the abscopal effect of radiation therapycells. In a parallel study we also studied the abscopal effect of radiation therapyin a model of collaterally implanted N29 tumors on both hind legs in rats, withy p g ,and without the combination of immunizationand without the combination of immunization.

l d h dMaterials and Methods Figure 1. Number of livingMaterials and Methods Figure 1. Number of livingtime for the rats withtime for the rats with 

Animal model In two parallel studies we used 8Animal model In two parallel studies, we used 8

inbred female and male Fischer 344 rats, inoculatedwith N29 rat glioma cells The N29 cells werewith N29 rat glioma cells. The N29 cells were

i l d i d f t d l d i CNS f6

previously derived from tumors developed in CNS ofpregnant Fischer rats exposed to ethyl‐N‐nitrosurea,pregnant Fischer rats exposed to ethyl N nitrosurea,and have since then been successively propagated

4and have since then been successively propagatedboth in vitro and in vivo.Intracranial tumors resembling human glio‐ 2

1Intracranial tumors, resembling human glio‐bl l if d l d i l f

1blastoma multiforme, were developed in a total of44 animals after inoculation by a stereotactic 044 animals after inoculation by a stereotacticinjection of 5000 N29 cells into the head of the right

0

injection of 5000 N29 cells into the head of the right 0                 50              100    

caudate nucleus. days aft

Extracranial tumors were induced in a total of 81Extracranial tumors were induced in a total of 81i l b b i l i f i 2 Sanimals by a subcutaneous inoculation of 200 000 Figure 2. Set‐up

N29 cells into the right hind leg and 50 000 cells in treatment of the eN29 cells into the right hind leg, and 50 000 cells inth l ft hi d lthe left hind leg.Treatment combinations The animals in the twoTreatment combinations The animals in the twogroups were further divided into 4 sub groups; (I)groups were further divided into 4 sub‐groups; (I)untreated controls, (II) radiotherapy alone, (III)( ) py ( )immunotherapy alone and (IV) radiotherapy andimmunotherapy alone, and (IV) radiotherapy andi th bi d D t th diff timmunotherapy combined. Due to the differentgrowth pattern and radiation response, thegrowth pattern and radiation response, thetreatment schedule and evaluation methodstreatment schedule and evaluation methodsdiffered for the intra‐ and extracranial tumors,,respectively Sub‐optimal non‐curative dose levelsrespectively. Sub‐optimal, non‐curative dose levels

h f hwere chosen for each group.Radiotherapy (RT) was given locally with collimatedRadiotherapy (RT) was given locally with collimatedfields of Co 60 gamma rays Animals with intra

Figure 3. Average tufields of Co‐60 gamma rays. Animals with intra‐ rats with extraccranial tumors were treated on day 7 after the

ats t e t acy

inoculation with a single fraction of 5 Gy or 15 Gyinoculation, with a single fraction of 5 Gy or 15 Gy.l h l dAnimals with extracranial tumors were treated

around day 30 with 4 daily fractions of 5 Gy eacharound day 30, with 4 daily fractions of 5 Gy each.I i ti (IFN ) i i t it lImmunization (IFN) was given as intraperitonealinjections of 3∙106 IFN‐gamma‐gene transfected N29injections of 3 10 IFN gamma gene transfected N29tumor cells The cells were given a sterilizing dose oftumor cells. The cells were given a sterilizing dose of70 Gy just before the injections. Animals with intra‐y j jcranial tumors were immunized 1 h after irradiationcranial tumors were immunized 1 h after irradiation

d 7 ft i l ti A i l ith ton day 7 after inoculation. Animals with extra‐cranial tumors were immunized 5 days before RT,cranial tumors were immunized 5 days before RT,and then two more times with 14 day intervalsand then two more times with 14‐day intervals.Treatment evaluation For the intracranial tumors,,the effect of the treatment was evaluated in termsthe effect of the treatment was evaluated in termsf i l i Th i l b d d il f Figure 4 Specific therapeuof survival time. The animals were observed daily for Figure 4. Specific therapeu

abscopal effect (SAE) for rasymptoms of the growing tumor and euthanized

abscopal effect (SAE) for rasymptoms of the growing tumor, and euthanizedh th t b k i t t dwhen the pre‐set breakpoint symptoms appeared

(keeping their heads turned to one side, rotating, or( p g , g,losing weight) For the extracranial tumors thelosing weight). For the extracranial tumors, the

ff l d ftreatment effect was evaluated in terms of tumorgrowth The subcutaneous tumors were estimatedgrowth. The subcutaneous tumors were estimatedb lli id ith l th d idth dby an ellipsoid, with length and width as measuredby using a caliper. The measured tumor volume, TV,by using a caliper. The measured tumor volume, TV,was fitted by an exponential growth modelwas fitted by an exponential growth model,

( )TV=TV0∙eTGR∙(t‐t

0), where t is the time after inoculation0 0 ,

in days and t is the time of the first irradiation TGRin days and t0 is the time of the first irradiation. TGRi th t th t t t i d 1 Whis the tumor growth‐rate constant in days‐1. Whenthe tumor reached a volume of about 9 cm3, thethe tumor reached a volume of about 9 cm , theanimals were euthanizedanimals were euthanized.

L d U i i / F l f S iLund University / Faculty of ScieLund University / Faculty of Scie

l d I R dulated Immune‐Response andulated Immune‐Response andph l lRats with Contra lateral tumoursRats with Contra‐lateral tumours

K h 1 4 G G f ö 1 4 P M k f R höld 1s‐Koch,1,4 Gustav Grafström,1,4 Per Munck af Rosenschöld,1s Koch, Gustav Grafström, Per Munck af Rosenschöld,2 4 3 4 f f 2 4Nittby 2,4 Bengt Widegren 3,4 and Leif G Salford 2,4Nittby, Bengt Widegren, and Leif G. Salford,

3 4gery, 3Tumour Immunology, 4Rausing Laboratory, Biomedical Centre, g y, gy, g y, ,d University 221 85 LUND Swedend University, 221 85 LUND, Swedento: Bertil R.R. Persson, bertil r.persson@med.lu.seto: Bertil R.R. Persson, bertil_r.persson@med.lu.se

C l iConclusionsConclusionsWe found a strong enhancement of the therapeutic effect in intracranial N29We found a strong enhancement of the therapeutic effect in intracranial N29t i t b bi i i l f ti f di ti th dtumors in rat by combining a single fraction of radiation therapy andimmunization with IFN transfected immunogenic tumor cells. In theimmunization with IFN transfected immunogenic tumor cells. In theextracranial model with collaterally implanted N29 tumors in rats weextracranial model with collaterally implanted N29 tumors in rats wedemonstrated the abscopal effect of radiation therapy. In this model enhancedp pytherapeutic and abscopal effect was found by radiation therapy only but notherapeutic and abscopal effect was found by radiation therapy only, but noi ifi f d i h RT bi d i h i hsignificant synergy was found with RT combined with immune‐therapy.

R l d Di ig rats and median survival

Results and Discussiong rats and median survivalintracranial tumours Results and Discussionintracranial tumours.

Intracranial tumors For the animals with intracranialIntracranial tumors For the animals with intracranialt th b f li i i l f ti ftumors, the number of living animals as a function oftime after inoculation is shown in Figure 1 for the153±31 (1SD) d N 8 time after inoculation is shown in Figure 1 for therats given combined treatment with RT and

153±31 (1SD) days, N=8rats given combined treatment with RT andimmunization. The median survival times are alsogiven in the figure The survival times of the treatedgiven in the figure. The survival times of the treated

d h d lgroups were compared to the untreated controls,119 35 (1SD) d N 8 and analyzed with the Mann‐Whitney test using a119±35 (1SD) days, N=8 and analyzed with the Mann Whitney test using a

i ifi l l f 0 05 It th f d th tsignificance level of =0.05. It was then found, thatRT alone (5 or 15 Gy) had no significant effect on the( y) gsurvival time IFN alone increased the survival time

          150              200              250survival time. IFN alone increased the survival time

h ( ) h b fer inoculation

with 60% (p=0.04). The combination of RT at 5 Gyp yand IFN increased the survival time with 87%f h di i and IFN increased the survival time with 87%( 0 003) i ldi 75% l t i i

p for the radiation(p=0.003), yielding 75% complete remissionsextracranial tumors.(p=0.03). Also the 15 Gy RT combined with IFN(p 0.03). Also the 15 Gy RT combined with IFNyielded an increased survival rate although not asyielded an increased survival rate, although not aseffectively as the 5 Gy RT and IFN treatment.y y Extracranial tumors For the animals with extra‐Extracranial tumors For the animals with extra‐

i l t Fi 2 th t th tcranial tumors, see Figure 2, the tumor growth rates(as measured by % per day) derived from the fit of(as measured by % per day) derived from the fit ofthe exponential growth model are displayed inthe exponential growth model are displayed inFigures 3 and 4. The tumor growth rates of theg gtreated groups were compared to the untreatedtreated groups were compared to the untreated

l d l d i h h icontrols, and analyzed with the t‐test using asignificance level of =0 05 It was then found thatsignificance level of 0.05. It was then found, thatRT (20 G ) l h d i ifi t ff t th tRT (20 Gy) alone had a significant effect on the tumorgrowth rate (p<0.0001), while IFN alone had noumor growth rate for g (p ), significant effect The combination of RT and IFNcranial tumors.  significant effect. The combination of RT and IFN

c a a tu o s.

also had a significant effect on the tumor growth rateg g(p=0 001) however when compared to a hypo‐(p=0.001), however, when compared to a hypoth ti ll dditi ff t f di th dthetically additive effect of radiotherapy andimmunization, there was no observable synergeticimmunization, there was no observable synergeticeffect due to the combined therapyeffect due to the combined therapy.Comparison By treatment of single intracranial N29p y gglioma tumors with RT in combination withglioma tumors with RT in combination withi i i i IFN f d llimmunization using IFN transfected tumor cells,75% complete tumor remissions has previously been75% complete tumor remissions has previously beendemonstrated (1) That effect of radiation wasdemonstrated (1). That effect of radiation wasrelated to diminishing of the tumor´s immune‐gsuppression and enhanced infiltration of activated Tsuppression and enhanced infiltration of activated T‐ll ff i h h i lcells affecting the tumor. In the present extracranial

model with two contra‐lateral tumors it wasutic effect (STE) and specific model with two contra lateral tumors it wash th i d th t ti t d T ll h ld l ff t

utic effect (STE) and specificts with extracranial tumors hypothesized that activated T‐cells should also affectts with extracranial tumors.

the left lateral un‐irradiated tumor. The results of RTthe left lateral un irradiated tumor. The results of RTalone appear as an abscopal effect with TGRalone appear as an abscopal effect with TGRdecrease also in the contra‐lateral un‐irradiatedtumor which account for the abscopal effect But intumor which account for the abscopal effect. But inthi d l i ifi t b l ff t f dthis model no significant abscopal effect was foundby radiation therapy combined with immunizationby radiation therapy combined with immunizationusing IFN transfected tumor cells This mightusing IFN transfected tumor cells. This mightindicate that factors other than immunological aregresponible for the radiation induced abscopal effectresponible for the radiation induced abscopal effect.

R fReferencesReferences11B Persson et al., Radiation Research 173:433‐440, 2010

/ M di l R di i Ph ience / Medical Radiation Physicsence / Medical Radiation Physics