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Cancer Therapy: Preclinical

In Situ Vaccination after AcceleratedHypofractionated Radiation and Surgery in aMesothelioma Mouse ModelLuis De La Maza1, Matthew Wu1, Licun Wu1, Hana Yun1, Yidan Zhao1, Mark Cattral2,Andrea McCart2, BC John Cho3, and Marc de Perrot1,4

Abstract

Purpose:How best to sequence and integrate immunotherapyinto standard of care is currently unknown. Clinical protocolswith accelerated nonablative hypofractionated radiation fol-lowed by surgery could provide an opportunity to implementimmune checkpoint blockade.

Experimental Design: We therefore assessed the impact ofnonablative hypofractionated radiation on the immune systemin combination with surgery in a mouse mesothelioma model.Blunt surgery (R1 resection) was used to analyze the short-termeffect, and radical surgery (R0 resection) was used to analyze thelong-term effect of this radiation protocol before surgery.

Results: Nonablative hypofractionated radiation led to a spe-cific immune activation against the tumor associated with signif-icant upregulation of CD8þ T cells, limiting the negative effect ofan incomplete resection. The same radiation protocol performed

7 days before radical surgery led to a long-term antitumorimmune protection that was primarily driven by CD4þ T cells.Radical surgery alone or with a short course of nonablativeradiation completed 24 hours before radical surgery did notprovide this vaccination effect. Combining this radiation protocolwith CTLA-4 blockade provided better results than radiationalone. The effect of PD-1 or PD-L1 blockade with this radiationprotocol was less effective than the combination with CTLA-4blockade.

Conclusions: A specific activation of the immune systemagainst the tumor contributes to the benefit of accelerated, hypo-fractionated radiation before surgery. Nonablative hypofrac-tionated radiation combined with surgery provides an opportu-nity to introduce immune checkpoint blockades in the clinicalsetting. Clin Cancer Res; 23(18); 5502–13. �2017 AACR.

IntroductionRecent advances in immunotherapy for solid tumors have

opened the door to a new field of therapy in oncology. One openquestion is how best to integrate immunotherapy into the currentstandard of care (including chemotherapy, radiotherapy, andsurgery). Several publications have shown the potential synergis-tic effect of combining immune checkpoint blockade withablative radiation in mice models, but clinical trials using thiscombination have so far been limited (1, 2). Part of the limitationis the lack of knowledge about the impact of standard therapy on

the immune system and the risk of toxicity when immunotherapyis added to these treatments.

The development of new highly conformal radiation techni-ques over the past 20 years allows precise targeting of the tumorand enables safe delivery of higher radiation doses per fraction(3). Traditionally, radiation is believed to work by direct andindirect damage to the DNA from ionizing radiation and relatedreactive oxygen species, causing cell death and tumor destruction.However, activation of the immune responses through cell death,tumor antigen release, and modification of the tumor microen-vironment may contribute to the benefit of radiation (4, 5).Ablative radiation (hypofractionated radiation doses of 8 Gy orhigher) delivered in a short course of 5 or fewer fractions appearsto augment antitumor immune responses, but this immunebenefit is complex and a nonlinear function of dose (6, 7).

In the clinical setting, an ablative dose of radiation is not alwaysachievable due to the risk of toxicity to the surrounding tissue (8).Therefore, surgery could be an important adjunct to the combi-nation of radiation and immunotherapy to achieve local tumorcontrol with an acceptable risk of toxicity from the radiation.Protocols using accelerated nonablative hypofractionated radia-tion followedby radical surgery havebeenused clinically for rectalcarcinoma and malignant pleural mesothelioma (9–12).

Malignant pleural mesothelioma is an aggressive malignancywith a median survival of less than 18 months despite aggressivetreatment with chemotherapy, surgery, and radiation (13, 14).Immunotherapy has shown some encouraging results, but ran-domized trials have yet to confirm its potential benefit (15, 16).

1Latner Thoracic Surgery Research Laboratories, Toronto General ResearchInstitute, University of Toronto, Toronto, Ontario, Canada. 2Department ofGeneral Surgery, Toronto General Hospital, University Health Network, Toronto,Ontario, Canada. 3Department of RadiationOncology, Princess Margaret CancerCentre, University Health Network, Toronto, Ontario, Canada. 4Division ofThoracic Surgery, Princess Margaret Cancer Centre, University Health Network,Toronto, Ontario, Canada.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Marc de Perrot, Toronto Mesothelioma Research Pro-gram, Division of Thoracic Surgery, Toronto General Hospital, 9N-961, 200Elizabeth Street, Toronto, Ontario M5G 2C4, Canada. Phone: 416-340-5549;Fax: 416-340-3478; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0438

�2017 American Association for Cancer Research.

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The protocol of accelerated hypofractionated radiation fol-lowed by surgery could be an ideal setting to implementimmunotherapy (17). We have thus developed a mouse modelto better understand the impact of this radiation protocol onthe immune system. We observed that a short course of hypo-fractionated radiation in combination with CTLA-4 blockadewas able to improve local control and induce an abscopal effectthrough an activation of the immune system with upregulationof activated CD8þ T cells and downregulation of regulatoryCD4þCD25þFoxp3þ T cells (Treg) in the tumor (18). In theseries of experiments reported herein, we therefore aimed todetermine the short- and long-term effects of nonablativehypofractionated radiation on the immune system when com-bined with surgery.

Materials and MethodsTumor cell lines and mice

AB12 and AE17 malignant pleural mesothelioma cell lineswere both derived from an asbestos-induced tumor in a BALB/c and C57BL/6 mouse, respectively. AB12 was kindly donated byDr. Jay Kolls, University of Pittsburgh (Pittsburgh, PA). AE17 wasobtained from the European Collection of Cell Cultures. AE17-OVA was developed by stably transfecting the parental cell line(AE17)with secretory ovalbumin (sOVA). The cell linewas kindlyprovided by Dr. Steven Albelda, University of Pennsylvania (Phi-ladelphia, PA), and Dr. Delia Nelson (University of WesternAustralia, Crawley, Australia).

AB12 and AE17 were grown in RPMI1640 culture media (LifeTechnologies Inc.) supplemented with 10% heat-inactivated FBS(Life Technologies Inc.), 2 mmol/L L-glutamine, 100 U/mL pen-icillin, 100 mg/mL streptomycin and nonessential amino acids.The transfected cell line AE17-OVA was maintained in the samemedium supplemented with 400 mg/L neomycin analogue G418(geneticin; Invitrogen). Cells were plated in tissue-culture coatedflasks (BD Biosciences Canada), grown in a 37�C and 5% CO2

environment, and passaged when 70% confluent.Eight- to 12-week-old BALB/c and C57BL/6 syngeneic mice

were purchased from Jackson Laboratories, and acclimatizedin the animal colony for 1 week before experimentation. Theanimals were housed in microisolator cages, 5 per cage, in a

12-hourlight/dark cycle. Sterile water and rodent food were givenad libitum. Animal care and experiments were performed inaccordance with institutional and Canadian Institute of Healthguidelines. All animal experiments were approved by the AnimalResearch Ethics Board at the Toronto General Research Institute(University of Toronto, Toronto, CA).

In vivo tumor growth experimentsMice were injected subcutaneously in the right flank with 1 �

106 AB12 cells, AE17 or AE17-OVA cells in 100 mL of PBS at day 0.For rechallenge experiments, cellswere injected subcutaneously inthe left flank with the same conditions. After removing furand cleaning the skin, injections were made with a syringe and25–27G needles. Tumor growth was monitored every 3 days.Tumor dimensions were measured using microcalipers. Tumorsize is expressed as tumor area in squared millimeters using thelongest length and the perpendicular width (length � width).Mice were sacrificed when tumor dimension reached 150mm2 orshowed signs of ulceration as per institutional ethics protocols.

Local radiotherapyRadiation was given using the X-Rad 225Cx small-animal

image-guided irradiator (Precision X-Ray). The irradiator has a225 kVp X-ray tube (Varian Associates) and a flat-panel silicondetector mounted on a 360� rotation C-arm gantry. The auto-mated stage is movable on the x, y, and z axis. It is all housed in aself-shielded cabinet and is remotely controlled by a computer(Dell Precision 690, Intel Xeon CPU running Windows XP). Themean targeting displacement error is�0.1mm in the x-y-z planes.Radiation was given to mice under isoflurane anesthesia. Toinitially visualize the animal, the tumor fluoroscopic mode wasused. To target precisely the tumor, a scout cone-beam CT wascreated at a 40 kVp tube potential and 0.5 mA current. Thetomography was then reconstructed at a 0.4-mm voxel size. Thebeam source was collimated to either a 1.5-cm or 2-cm diametercircular field. To confirm the area to be irradiated, the tumor wasthen visualized under fluoroscopic imagingwith the collimator inplace, immediately prior to delivery of treatment. Radiation wasdelivered at a tube potential of 225-kVP and a 13-mA current for adose rate of 3.02 Gy/minute. The daily dose was given from 2angles, half from above (180 degrees) and half from below (0degrees). Total dose was given in divided fractions over 3 daysaccording to treatment protocols. After radiation, mice wereplaced back in their cages and housing facilities.

Surgical resection of subcutaneous tumorsUnder general anesthesia with isoflurane, mice with flank

tumors were shaved and cleaned with isopropanol. Tear gel wasapplied on both eyes and a heating lamp was used to preventhypothermia. Skin around the tumor was infiltrated with Mar-caine (bupivacaine 0.25%) prior to incision. Two differentapproaches were used depending on the experiment. Blunt sur-gery was performed by blunt dissection removing all macroscopictumor, but no skin or surrounding healthy-looking tissue(R1 resection). Radical surgery was performed by removing theskin on top of the tumor and 0.5-cm margin of healthy-lookingsubcutaneous tissue around the tumor (R0 resection). Sterileprolene sutures (5-0 or 6-0) were used to close the wound.Marcaine was administered immediately after closing the woundfor postoperative analgesia and the mice were observed until

Translational Relevance

Protocols using an accelerated course of hypofractionatednonablative radiation followed by surgery might be an idealapproach to implement immunotherapy in clinical practice. Inthis series of experiments, we therefore analyzed the effect ofsurgery in combination with accelerated hypofractionatednonablative radiation in a murine mesothelioma model anddemonstrated that surgery did not preclude the potentialbeneficial effect of high-dose radiation on the immune systemas long as adequate time was provided between the end of theradiation and surgery to activate the immune system. To thebest of our knowledge, this is the first study providing exper-imental evidence supporting the use of induction radiationbefore surgery and demonstrating that surgery would not bedetrimental on the immune system if the timing between thedifferent therapeutic modalities was adequate.

Radiation and Surgery in Mesothelioma

www.aacrjournals.org Clin Cancer Res; 23(18) September 15, 2017 5503




complete recovery. Mice were then monitored at 6, 24, and 48hours after surgery and meloxicam 1 mg/kg was given subcuta-neously for postoperative analgesia.

In vivo depletion of CD4þ and CD8þ specific T cellsAnti-CD4 MAb from rat GK 1.5 hybridoma or anti-CD8 Mab

from 2.43 rat hybridoma (Bio X Cell) were diluted to a finalconcentration of 1 mg/mL with PBS or 2 mg/mL for doubledepletion. Intraperitoneal injections for 3 consecutive days with0.2 mL (0.2 mg) of purified Mab were performed. For doubledepletion the total volume injected was 0.2 mL, consisting of 0.1mL of each mAb at a concentration of 2 mg/mL. Examination ofperipheral lymphoid organs at day 6 revealed that >95% of thecells were depleted. The depleted condition was maintained with0.2 mg injections of mAb every 3 days.

Immune checkpoint blockadeMouse mAb (9D9) to CTLA-4, PD-1 or PD-L1 (Bio X Cell) was

diluted to a final concentration of 1 mg/mL with PBS and kept at4�C until further use. Intraperitoneal injections were made with0.2 mL (0.2 mg) of the purified antibody every 3 days for thelength of the specified treatment.

Tumor digestionTumors were removed and placed in 15-mL conical tubes filled

RPMI1640 culture media and stored on ice until further use.Tissue was chopped into 2-mm pieces and transferred to 15-mLconical tubes containing digestion media consisting inRPMI1640, DNAse (Roche 10104159001) and Liberase TM(Roche Diagnostics). Tubes were placed in a shaking water bathfor 30 minutes and when the pieces were soft and malleable thesolution was filtered and mashed through a 70-mm cell strainer.Cells were then washed with PBS and remaining cells werecounted and viability was assessed.

Flow cytometryCells were resuspended in FACS buffer, and stained for 30

minutes at 4�C with a-CD16/CD32 Fc block (BD, Pharmingen),and a combination of the following mouse-specific antibodies:CD3,CD4,CD8,CD44,CD45, CD69,CD137 (4-1BB), TIM3, PD-1, ICOS (BD, Pharmingen). Cells stained with tetramer wereincubated for 30 minutes with the Class I H-2Kb SIINFEKLtetramer prior to surface staining. All samples were then washedtwice with FACS buffer and analyzed immediately using a BD LSRII flow cytometer (BD Biosciences) and FlowJo V10 software(FlowJo LLC). Tumor samples were pooled and analyzed as asingle sample to have sufficient cells.

ImmunofluorescenceFrozen tissue samples on slides were fixedwith cold acetone for

10minutes. Paraffin embedded samples were deparaffinizedwithXylene, 100% ethanol, 95% ethanol, and 70% ethanol respec-tively and antigen retrieval was performed by immersing samplesin 100�C citrate buffer for 20minutes. Samples were blockedwith5%BSA in Tris-buffered saline for one hour before the addition ofprimary antibody. After incubating overnight at 4�C, sectionswere washed in TBSþ0.2% Tween 20. Slides were subsequentlyincubated for 1 hour at room temperature with the appropriatefluorescently labeled secondary antibody. Slides were furtherwashedwith TBSþ0.2%Tween20before addingmountingmedia

withDAPI nuclear stain. Coverslips were placed on top and sealedwith nail polish.

Fluorescently labeled cells or tissues were visualized with theWaveFX (Quorum Technologies Inc) confocal microscope sys-tem. Pictures were analyzed using ImageJ V1.47 (NIH, Bethesda,MD). Corrected total cell fluorescence (CTCF) was calculated bythe formula CTCF ¼ integrated density – (area of cell � meanfluorescence of background reading).

Ovalbumin ELISAAE17-OVA and AE17 cell culture supernatants were collected 3

days after seeding cells. Cells were then trypsinized and washedtwice with PBS. For cell lysates, cells were collected by centrifu-gation, 5 minutes at 1,000 � g. Cells were then subjected toultrasonication for 4 cycles on ice. Cell lysates were collected bycentrifugation at 1,500 � g for 10 minutes at 4�C to removecellular debris. Cell lysate or media was placed in wells coatedwith a biotin-conjugated antibody specific to OVA from an ELISAkit (Biomatik corporation). Samples were left for 2 hours to bindanti-Ova antibodies. Avidin conjugated tohorseradish peroxidase(HRP) was then added to each well and incubated for 1 hour.Finally TMB substrate solution was added and those wells con-taining OVA, biotin-conjugated antibody and enzyme-conjugat-ed avidin exhibited a change in color. Reaction was terminated bythe addition of sulphuric acid. Concentrations were determinedby four-parameter logistic test using a standard curve. Sampleswere measured in duplicate.

Statistical analysisStatistical analysis was performed with GraphPad Prism 5

(GraphPad Inc). More than two groups were compared usingone-way ANOVA analysis. Unpaired two-tailed Student t test wasused to analyze two groups. A P value of less than 0.05 wasconsidered statistically significant. Results have been presented asmean� SEM. �, P < 0.05; ��, P < 0.01; ��, P < 0.001 in all figures.

ResultsDevelopment of a model of nonablative hypofractionatedradiation

Our initial goal was to develop a mesothelioma model thatmimicked the clinical setting in which mice received acceleratednonablative hypofractionated radiation. To optimize the mod-el, we compared 4 different doses of local radiotherapy (LRT)targeting the tumor. These experiments were done in BALB/cmice with AB12 cell line following previous in vitro and in vivoexperiments performed by our group (18). A total of 1 � 106

AB12 tumor cells were inoculated subcutaneously in the rightflank and LRT was started 7 days after tumor inoculation. Onthe first day of LRT treatment, mice were randomized intothe following groups: (i) no treatment, (ii) 15 Gy over 3 days(5 Gy/fraction �3), (iii) 22.5 Gy over 3 days (7.5 Gy/fractions�3), (iv) 30 Gy over 3 days (10 Gy/fraction �3), and (v)22.5 Gy in a single dose.

Untreated mice showed rapid tumor growth up to 22 dayswhen animals were sacrificed. All four groups treated with LRTachieved tumor growth stabilization for at least 7 days beforetumor growth resumed. No mice were cured by LRT alone. Therewas no significant difference in tumor size among the four LRTtreatment groups, but mice irradiated with 30 Gy in 3 fractionsand 22.5 Gy in one fraction showed signs of distress and lost 10%

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to 15% of total body weight during the first 2 weeks aftertreatment. These results confirmed that the tumor model wassensitive to LRT and that these doses of radiation were notablative. We chose a radiation dose of 15 Gy in 3 fractions (5Gy � 3) for the following experiments.

Combination therapy with LRT and surgeryWe next analyzed the role of radiotherapy in combination with

blunt surgery (R1 resection). Mice were randomized the first dayof LRT into the following groups: (i) no treatment; (ii) LRT (5 Gy� 3); (iii) surgery; and (iv) LRT (5 Gy� 3) and surgery. In the LRTand surgery group, blunt surgery was performed 5 days aftercompletion of LRT.

Mice treated with blunt surgery alone had rapid tumor recur-rence and tumor growth rate was faster after resection comparedwith untreated tumors. In the groups treated with LRT alone andwith the combination LRT surgery, tumor growthwas significantlyslower than in untreated mice or mice treated with surgery alone.There was no difference between the treatment group LRT aloneand LRT surgery (Fig. 1).

Upregulation of tumor-infiltrating CD8þ T cells after localradiation

To evaluate the involvement of the immune system on tumorgrowth after LRT, tumor samples from 15 Gy (5 Gy� 3) radiatedmice were compared with untreatedmice on day 2, 7, and 12 after

Figure 1.

The effect of combination therapy with radiation (LRT) and surgery (Sx). On day 12 after tumor cell injection (AB12 cell line), mice were randomized to notreatment group (no Tx;A); blunt surgery alone on day 12 (Sx;B); LRT alone on days 12–14 (LRT;C); or combination group, LRT on days 12–14 and blunt surgery on day19 (LRTþSx; D ). Each mouse is presented individually from 1 to 5 in A–D, whereas E represents the mean (n ¼ 5) for each group. In E, the day of surgical removalof the tumor is day 0 for the groups treated with blunt surgery; otherwise, day 0 is the day of the inoculation of the tumor. Values shown in E are themean� SEM of 5mice per timepoint. �, P < 0.05 compared with untreated (n ¼ 5 per group).






LRT. Immunofluorescent staining revealed that the number ofCD3þCD8þ double positive T cells 2 days after LRT was notsignificantly different between radiated and untreatedmice.How-ever, on day 7 and 12 after LRT, the number of CD3þCD8þ T cellswas significantly higher in the LRT group compared with untreat-ed tumor (Fig. 2). Flow cytometry showed that the frequency of

tumor infiltrating CD45þ CD3þCD8þ T cells were 5� higher inirradiated than in untreated tumors on day 7 (Fig. 2).

Tumor-infiltrating CD8þ T cells are OVA-specificTo determine whether tumor-infiltrating CD8þ T cells induced

by radiationwere tumor antigen–specific, we inoculated C57BL/6

Figure 2.

Tumor-infiltrating CD3þCD8þ cells afterradiation (LRT) compared with untreatedtumors (no Tx). LRT was administered ondays 7–9 (15 Gy in 3 fractions) after injection ofAB12 cell lines. Immunofluorescent staining oftumor 2, 7, and 12 days after LRT compared withno Tx (n ¼ 5 per group). A, Images show DAPI(blue), CD3 (green) and CD8 (red) mergedstaining.B,Average cell count of 5 random�200magnified fields. � , P < 0.05; �� , P <0.005.C, FACS analysis of the treated and untreatedtumor 7 days after the first day of radiation.Doublets and dead cells were excluded beforegating on CD45/CD3. FACS confirms theincreased number of tumor-infiltratingCD3þCD8þ cells after LRT compared withuntreated control (no Tx).

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mice with the AE17-OVA cell line. LRT-treated tumors wereexcised and analyzed 10 days after the end of radiation.Tumor-infiltrating cells were stained with H-2Kb tetramers con-taining the OVA protein–derived peptide SIINFEKL.

After gating for live cells, the proportion of CD44þ and SIIN-FEKL tetramer cellswere identified among theCD3þCD8þdoublepositive population. Radiated tumors showed a trend towardgreater proportion of tetramer-specific CD8þ T cells comparedwith untreated tumors (Fig. 3). The result of this experiment givesfurther evidence that LRT promotes recruitment of lymphocytes

into the tumor using a different tumor cell line and mice strain.About 30% of the recruited lymphocytes are specific for the OVA-derived peptide SIINFEKL in the treated tumor as compared withonly 15% in the untreated group.

Expression of 4-1BB and PD-1 by tumor-infiltratinglymphocytes

To assess the change in phenotype of tumor-infiltrating lym-phocytes after a short course of nonablative radiation, we ana-lyzed the expression of the inhibitory receptor PD-1 and the

Figure 3.

CD8þ lymphocytes infiltrating AE17-OVA tumor are OVA specific, and upregulate 4-1BB and downregulate PD-1 expression after radiation (LRT). LRT wasadministered on days 9–11 (15 Gy in 3 fractions) after injection of AE17-OVA cell lines.A, Representative flow cytometry graph gated on CD3þ, CD8þ. There is a greaterproportion (50.8%) of CD44þ Tetramerþ double-positive cells in the radiated group than in the untreated tumor (21.4%). B, Graph comparing proportion oftumor-specific CD8þ T cells in radiated anduntreated tumor.C,4-1BBandPD-1 expression onCD8þ/Tetramerþ T cells 3 and9days after LRT. 4-1BBupregulation 3 daysafter LRT and downregulation of PD-1, 3 and 9 days after LRT. ��, P < 0.05; n.s, not significant comparing treated groups to untreated (n ¼ 4 per group).






activation marker 4-1BB in radiated tumor 3 and 9 days after LRTin C57BL/6 mice. The early activation marker 4-1BB was signif-icantly upregulated in the tumors 3 days after radiation comparedwith untreated controls, but this difference decreased over timeand was not statistically significant on day 9 after radiation (Fig.3). In parallel, radiation led to a significant reduction of CD8þ Tcells expressing PD-1 on day 3 and 9 after radiation comparedwith untreated controls (Fig. 3). These findings suggest thataccelerated hypofractionated radiationmay transform the immu-nosuppressive microenvironment of the tumor even though theradiation was not ablative.

Depletion of CD4þ T cells and CD8þ T cells partially abrogatesthe effect of LRT on tumor growth

To examine whether CD4þ T cells and CD8þ T cells played arole in the tumor response to radiotherapy, animalswere depletedof CD4þ T cells, CD8þ T cells, or both starting 1 day before LRTand throughout the length of the experiment. Mice were random-ized to the following groups: (i) no treatment, (ii) LRT only, (iii)LRT and CD4þ T-cells depletion, (iv) LRT and CD8þ T-celldepletion, and (v) LRT and double depletion.

Double depleted mice treated with LRT showed significantlygreater tumor size than animals treated with LRT only (Fig. 4).However, tumor size in double depleted mice remained signif-icantly smaller than untreated mice demonstrating that LRT stillhad an impact on tumor growth independently of CD4þ andCD8þ cells. Mice depleted in CD8þ T cells alone showed signif-icantly greater tumor size than mice depleted in CD4þ T cellsalone, suggesting that the benefit of LRT on the immune systemwas predominantly mediated by CD8þ T cells.

Long-term immunologic protective memory after LRT andsurgery

We next investigated the role of LRT before radical surgicalresection of the tumors (R0 resection) to assess the ability ofaccelerated nonablative hypofractionated radiation to generate aneffective immunologic memory response. C57BL/6 mice wereinoculated with AE17-OVA cells and after 9 days were random-ized into the following treatment groups (i) surgery only, (ii) LRTand surgery 24 hours later (LRT-Surg 24 hrs), (iii) LRT and surgery7 days later (LRT-Surg 7d). A total of 7 days was chosen based onthe time course of tumor infiltrating CD8þ T-cell upregulation(Fig. 2). Cured mice were rechallenged at 90 days with the sametumor delivered to the opposite flank.

All 10 mice in the surgery alone group were tumor free 90 daysafter treatment, and 9 mice in both groups treated with LRT andsurgery were tumor free. One mouse in each group treated withLRT and surgery was lost during surgery due to tumor infiltrationof the chest wall. After tumor rechallenge, tumor growth wassignificantly smaller in the LRT-Surg 7d group compared with theother 2 groups (Fig. 5). In the LRT-Surg 7d group, 3 of 9 micecompletely rejected the tumor, whereas no tumor rejectionoccurred in the other two groups. This finding suggests thataccelerated nonablative hypofractionated radiation 7 days beforesurgical removal of the tumors promotes a protective immuno-logic memory response.

Role of T cells in the long-term protection of radiated miceTo determine the role of CD4þ and CD8þ T cells in the long-

term protection of radiated mice, we first confirmed that curedmice were protected by rechallenging them a second time withAE17-OVA; 20of 21mice rejected their tumor. These 20micewerethen rechallenged again after randomization into the followinggroups: (i) CD4depletion (n¼6), (ii) CD8depletion (n¼7), (iii)Double depletion (n ¼ 7). Depletion of lymphocytes started oneweek before AE17-OVA cells inoculation.

Double depleted mice for CD4þ and CD8þ cells lost theirantitumor memory response and displayed rapid tumor growthrate, similar to untreated mice challenged for the first time. Incontrast, all CD8þ-depleted mice completely rejected the tumor(Fig. 5). CD4þ-depleted mice rejected 1 of 6 tumors and growthrate of the remaining 5 tumors was significantly slower thandouble depleted mice. These results suggest that memory CD4þ

T cells are critical to provide long-term benefit from radiation,whilememoryCD8þT cells contribute to rejection of the tumor inthe long-term but are not sufficient to provide long-term immuneprotection against the tumor. Hence, CD4þ T cells are sufficient tomount an effective immune response against the tumor in thelong-term despite the absence of CD8þ T cells.

CTLA-4 blockade improves the beneficial effect of LRTIn this experiment, the goal was to determine the role of

immunotherapy combined with a short course of nonablativeradiation in improving the therapeutic effect of radiation ontumor growth. We therefore compared the effect of immunecheck point blockade in combination with hypofractionatedradiation. Anti-CTLA-4, anti-PD-1, and anti-PD-L1 have beensuccessfully used in combination with radiation previously, but

Figure 4.

Radiation (LRT) and CD4þ CD8þ T-cell depletion. Tumorgrowth in mice treated with LRT and depletion of CD4þ,CD8þ, or double depletion. Mice were randomized to thefollowing groups 9 days after AE17-OVA tumor cell injection:(1) No treatment (no Tx), (2) radiation only (LRT), (3) LRT andCD4þ T-cell depletion (CD4), (4) LRT and CD8þ T-celldepletion (CD8), and (5) LRT and double depletion (Double).CD4þ, CD8þ, and double depletion was started 1 daybefore LRT and continued throughout the length of theexperiment. Values shown are themean tumor area in squaremillimeters of 5 mice per time point and are expressed asmean � SEM. � < 0.05 compared with untreated;x < 0.05 compared to LRT; ¶ compared with LRTþ depletionof CD4þ. N ¼ 5 per group.

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have not been compared to each other (19–22). Mesotheliomatumor–bearing mice were therefore randomized to: (i) no treat-ment, (ii) anti-PD1, (iii) anti-PDL1, (iv) anti-CTLA-4, (v) LRT, (vi)LRT and anti-PD1, (vii) LRT and anti-PDL1, and (viii) LRT andanti-CTLA-4. Treatment started on day 9 in all groups, with LRTand/or mAb injection. The mAb injection was repeated every 3days for a total of 3 doses. The AE17 and AB12 cell lines do notexpress PD-L1 (Supplementary Fig. S1).However, as shown in Fig.3, a large proportion of tumor-infiltrating lymphocytes expressPD-1, suggesting that targeting the PD1/PD-L1 pathwaymaybe ofvalue.

Treatment with mAbs alone had no significant impact ontumor growth. Mice treated with LRT and anti-CTLA-4 antibodyhad significantly smaller tumors comparedwithmice treatedwithLRT only. There was limited difference in tumor size between LRTalone and the combination group LRT and anti-PD1 or LRT andanti-PDL1 (Fig. 6). In an additional study, we observed that micetreated with LRT and anti-CTLA-4 had three distinct responsepatterns, no response, partial response, and good response (Fig.6). IFNg-producing CD8þ T cells infiltrating the tumor weresignificantly higher in the combination group LRT and anti-CTLA-4 compared with the untreated group (Fig. 6).

DiscussionPrevious work had shown that radiation could enhance the

abscopal effect in a tumor bearing host, improve the capacity of

adoptively transferred T cell to infiltrate the tumor, and syner-gize with immune checkpoint blockade to provide better localand distant control of the tumor (23–25). These previousexperiments, however, focused on treating the primary tumorwith radiation alone. Although surgery could be an importantadjunct to radiation to provide optimal control of the primarytumor, the potential impact of surgery in the context of hypo-fractionated radiation on the immune system has, to the best ofour knowledge, never been experimentally analyzed. Surgerycould potentially limit the beneficial effect of radiation on theimmune system by removing the source of tumor neoantigenrelease or by creating a nonspecific anti-inflammatory state inthe vicinity of the tumor.

In this study, we combined a nonablative dose of radiationwith surgery to treat the primary tumor and analyzed theimpact of both treatments on the immune system in the shortand long term. We observed that surgery alone led to a fastertumor growth compared with untreated mice suggesting thatthe nonspecific inflammatory reaction created by the surgicaldissection was detrimental in this mesothelioma model. Accel-erated, hypofractionated radiation prior to surgery, however,prevented the negative impact of surgery. We also found that ashort course of nonablative radiation before complete resectionof the tumor could provide an in situ vaccination with long-term protection if there was sufficient time for the immunesystem to generate a specific immune response against thetumor before surgery.

Figure 5.

AE17 OVA rechallenge 90 days after treatment. A, Curedmice were rechallenged with AE17-OVA delivered to theopposite flank 90 days after their initial treatment thatconsisted of (1) radical surgery (Sx alone), (2) LRT andsurgery after 24 hours (LRTþSx 24 hrs), or (3) LRT andsurgery after 7days (LRTþSx 7d).Mice treatedwith LRTandradical surgery after 7 days grew significantly smallertumors compared with those treated with surgery aloneand those treated with LRT and surgery after 24 hours.Values shownare themean tumor area in squaremillimetersof 10 mice per time point in the surgery group and 9 micein the other groups and are expressed as mean � SEM.� , P < 0.05 compared with LRT and surgery after 7 days.B, Mice initially treated with radiation and radical surgeryafter 7 days that had rejected a second tumor afterrechallenge were then depleted of CD4þ T cells (CD4),CD8þ T cells (CD8), or both (Double) and rechallengedone more time. Tumor size was significantly larger indouble-depleted mice compared with CD4 or CD8single-depleted mice. Tumor size was also significantlylarger in CD4-depleted mice compared with CD8-depletedmice but smaller than in double-depleted mice. Valuesshown are the mean tumor area in square millimeters of 6mice per time point in the CD4 group and 7mice in the othergroups and are expressed as mean � SEM. � , P < 0.05compared with CD8; x < 0.005 compared with CD4.






Radiation is traditionally delivered with normofractionateddoses of 2 Gy per fraction, generally for 5 to 6 weeks to obtain aradical dose. However, over the past decade hypofractionatedtreatment with higher dose delivered in a shorter time framehas been increasingly used in the adjuvant setting after surgeryto limit the inconvenience and cost related to this long treat-ment (26, 27). The data presented in these series of experimentssuggest that hypofractionated radiation treatment switched

from the adjuvant to the neoadjuvant setting may enhance theimmunoprotective effect, thus keeping the total dose to aminimum.

T cells are extremely sensitive to radiation and thereforetumor-infiltrating lymphocytes are damaged after each dose ofradiation (5, 28, 29). This toxic effect of radiation on T cellstherefore masks the potential benefit of radiation on theimmune system in conventional radiation due to the daily

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Figure 6.

Combination therapywith LRTandCTLA-4 shows a synergisticeffect on tumor growth. On day 9 after AE17-OVA tumor cellinjection, mice were randomized to (1) no treatment (no Tx),(2) anti-PD1 therapy (PD1), (3) anti-PDL1 therapy (PD-L1), (4)anti-CTLA-4 therapy (CTLA4), (5) radiation alone (LRT), (6)radiation and anti-PD1 therapy (LRTþPD1), (7) radiation andanti-PD-L1 therapy (LRTþPD-L1), and (8) radiation and anti-CTLA-4 therapy (LRTþCTLA-4). Treatment started on day 9 inall groups. LRT was administered on days 9–11. mAb injectionwas started on day 9 and repeated every 3 days for a total of 3doses. A, Treatment with anti-CTLA-4 alone, anti-PD-1 aloneand anti-PD-L1 alone had minimal effect on tumor growthcompared with no treatment. Tumor growth significantlyslowed in the combination group LRTþCTLA-4 comparedwiththe anti-CTLA-4 therapy group and the radiation-alone group(n ¼ 5 per group). The effect of anti-PD-1 and anti-PD-L1 incombination with radiation was not as significant. B, Individualcurves showing 3 response patterns in the combination groupLRTþCTLA4 (n¼ 10). C, CD8þ TILs producing IFNg (n¼ 4 pergroup). The proportion of CD8þ TILs producing IFNg wassignificantly higher in combination treatment with LRTþCTLA-4 compared with untreated mice. Although there was a trendtoward higher proportion of CD8þ TILs producing IFNg inLRTþCTLA-4 compared with LRT alone, this did not reachstatistical difference (P ¼ 0.7). No significant difference wasobserved between LRT alone and untreated mice (P ¼ 0.9).

De La Maza et al.





delivery of radiation over the course of several weeks. A shortcourse of radiation in contrast takes full advantage of theactivation of the immune system by allowing the lymphocytesfrom the systemic pool to reinfiltrate the tumor not long afterthe end of radiation and continue to rise once the radiation isstopped. In accord with previous studies, we observed that after7 days the number of infiltrating T cells in the tumor was at least5 times higher after radiation than in untreated tumors and thatthe rise in tumor-infiltrating lymphocytes was tumor antigenspecific (30, 31).

Lymphocytes infiltrating solid tumors after radiation have beenexamined in different murine models, including previous pub-lications from our group (4, 18). Radiation can reverse theimmunosuppressive state of the tumor by decreasing the numberof myeloid-derived suppressor cells, switching tumor-infiltratingmacrophage from an M2 to an M1 phenotype and creating aproinflammatory environment where danger signals and cyto-kines generate the migration of lymphocytes toward the tumor(30–32). Accordingly in our model, we observed that this shortradiation course rapidly upregulated the expression of 4-1BB anddecreased the expression of PD-1 on lymphocytes. Although theexpression of 4-1BB was only temporary, its impact on theresponse to radiation may be significant as expression of 4-1BBon lymphocytes can stimulate production of IL2 in a CD28-independent way, a critical step for activation of T cells and forprevention of an anergic state (33). In addition, 4-1BB alsoenhances CTL cytolytic activity (34). The decreased expression ofPD-1 on the other hand was prolonged suggesting the absence ofT cells' exhaustion over time (35, 36).

Double depletion of CD4þ and CD8þ T cells significantlyabolished some of the early therapeutic benefit of radiation ontumor growth in our model. This finding suggests that bothCD4þ and CD8þ T cells play an important role in the therapeuticresponse to radiation and contribute to the benefit of radiationin our model. These observations are in line with the literatureindicating that CD8þ T cells are required for the therapeuticeffects of ablative radiation (6, 37). The role of CD4þ T cells inthe early response to radiation has been more variable, possiblydue to the presence Treg such as CD4þCD25þFoxp3 within thetumor (18).

We and others have shown that Treg are upregulated in thetumor after a short course of radiation (18, 38). In our experience,the use of CTLA-4 blockade was able to block the upregulation ofTreg, thus enhancing the impact of radiation on the primarytumor and the abscopal effect at distant tumor sites (18). Thebenefit of CTLA-4 blockade in combination with radiation inthis series of experiments confirms our previous observationand supports the potential importance of Treg in generating theimmunosuppressive microenvironment in mesothelioma(39, 40).

In contrast to the early response to radiation, this series ofexperiments demonstrate the critical role of CD4þ T cells in thelong term. Mice previously protected against the tumor failed toreject the tumor when CD4þ and CD8þ T cells were depleted(double depletion). CD4þ depletion alone led to the develop-ment of slowly growing tumor suggesting that CD8þ T cells andother immune cells were still able to respond to the tumor butcould not prevent their development. On the other hand, micedepleted in CD8þ alone did not develop the tumor, demonstrat-ing that memory CD4þ cells are a key component in the preven-

tion of tumor recurrences in the long term. These findings rein-force the increasing evidence that CD4þ T cells are a key compo-nent to generate long-term protective antitumor immunity, whiletumor will typically escape isolated protection from CD8þ T cellsalone (41, 42).

A time frame of 7 days or potentially longer between radiationand surgery is important to reach the peak of the adaptive T-cellimmune response before surgery (6, 43).Mice treatedwith radicalsurgery alone or with radiation followed by radical surgery within24 hours displayed rapid tumor regrowth after tumor rechallengedespite their initial cure, demonstrating the absence of durableimmune protection without an adequate activation of theimmune system. This finding correlates with clinical observationwhere a time gap of at least 5 days between accelerated radiationand surgery in locally advanced rectal cancer was shown to be apowerful and independent prognostic factor for better survival(44).

This study has limitations inherent to the animal modelsand correlative studies in patients will be important. We arethus currently planning to modify our induction radiationprotocol to 15 Gy in 3 fractions. This approach would allowus to preserve the lung at the time of surgery and facilitatethe concurrent implementation of immune checkpointinhibitors.

In conclusion, wedemonstrated the importance of the immunesystem in the benefit of clinical protocols using accelerated,hypofractionated radiation followed by surgery. This benefit isassociated with CD8þ and CD4þ T cells and can lead to an in vivovaccinationwith long-termprotection. Thesefindings suggest thatthese protocols provide an opportunity to introduce immunecheckpoint blockade in clinical practice.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. De La Maza, M. Wu, L. Wu, M. de PerrotDevelopment of methodology: L. De La Maza, M. Wu, L. Wu, Y. Zhao,M. de PerrotAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. De La Maza, L. Wu, M. de PerrotAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.De LaMaza, L.Wu, Y. Zhao,M.Cattral,M. de PerrotWriting, review, and/or revision of the manuscript: L. De La Maza, M. Wu,L. Wu, Y. Zhao, M. Cattral, A. McCart, J. Cho, M. de PerrotAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. De La Maza, L. Wu, H. Yun, M. de PerrotStudy supervision: L. Wu, M. de PerrotOther (provided feedback over the course of the study re design, analysis, andconclusions): A. McCart

Grant SupportThis work was supported by the Mesothelioma Applied Research Founda-

tion, the Princess Margaret Hospital Foundation, and the Canadian Mesothe-lioma Foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 14, 2017; revised April 27, 2017; accepted June 5, 2017;published OnlineFirst June 12, 2017.






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2017;23:5502-5513. Published OnlineFirst June 12, 2017.Clin Cancer Res Luis De La Maza, Matthew Wu, Licun Wu, et al. and Surgery in a Mesothelioma Mouse Model

Vaccination after Accelerated Hypofractionated RadiationIn Situ

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