pd-1/pd-l1immunecheckpointinhibitionwithradiation in ... · pd-1/pd-l1 therapy often have tumors...

MOLECULAR CANCER THERAPEUTICS | CANCER BIOLOGYAND TRANSLATIONAL STUDIES

PD-1/PD-L1 ImmuneCheckpoint InhibitionwithRadiationin Bladder Cancer: In Situ and Abscopal EffectsAlexis Rompr�e-Brodeur1,2, Surashri Shinde-Jadhav1, Mina Ayoub1,3, Ciriaco A. Piccirillo3,4,7,Jan Seuntjens6, Fadi Brimo5, Jose Joao Mansure1,3, and Wassim Kassouf1,2,3

ABSTRACT◥

The combination of radiation with immune checkpoint inhi-bitors was reported in some cancers to have synergic effects bothlocally and distally. Our aim was to assess this combined therapyon both radiated and nonradiated bladder tumors and to char-acterize the immune landscape within the tumor microenviron-ment. Murine bladder cancer cells (MB49) were injected subcu-taneously in both flanks of C57BL/6 mice. Mice were randomlyassigned to the following treatments: placebo, anti-PD-L1 (fourintraperitoneal injections over 2 weeks), radiation to right flank(10 Gy in two fractions), or radiationþanti-PD-L1. Tumordigestion, flow cytometry, and qPCR were performed. Log-rank analysis was used for statistical significance. Radia-tionþanti-PD-L1 group demonstrated statistically significantslower tumor growth rate both in the radiated and nonirradiatedtumors (P < 0.001). Survival curves demonstrated superior

survival in the combination group compared with each treatmentalone (P¼ 0.02). Flow cytometry showed increased infiltration ofimmunosuppressive cells as well as CTL in the radiation andcombination groups (P ¼ 0.04). Ratio of immunosuppressivecells to CTL shifted in favor of cytotoxic activity in the combi-nation arm (P < 0.001). The qPCR analysis revealed down-regulation of immunosuppressive genes (CCL22, IL22, and IL13),as well as upregulation of markers of CTL activation (CXCL9,GZMA, and GZMB) within both the radiated and distant tumorswithin the combination group. Combining radiation withimmune checkpoint inhibitor provided better response in theradiated tumors and also the distant tumors along with a shiftwithin the tumor microenvironment favoring cytotoxic activity.These findings demonstrate a possible abscopal effect in urothe-lial carcinoma with combination therapy.

IntroductionBladder cancer is the fifth most common cancer in annual

incidence and represents a significant burden on healthcare sys-tems worldwide (1, 2). Around 30% of patients will present with amuscle invasive bladder cancer (MIBC). MIBC, locally advancedisease, and metastatic bladder cancer are associated with a 5-yearoverall survival of approximately 65%, 35%, and 5%, respective-ly (3, 4). Although, the standard treatment for localized MIBC wasradical cystectomy with lymph node dissection and formation of aurinary diversion, it offers a 5-year overall survival of only60% (3, 5). Furthermore, there are significant treatment-associated morbidity and mortality, as well as long-term urinary

and sexual impacts related to radical cystectomy. On the otherhand, radiation-based therapy is an attractive alternative as itspares the bladder allowing for maintenance of urinary and sexualfunctions. It is also well tolerated in older patients with morecomorbidities. However, it is associated with suboptimal diseasecontrol where 30% of patients will require a salvage cystectomy andhalf will develop metastasis (6).

Immune checkpoint inhibitor targeting the programmed celldeath protein 1 (PD-1) and programmed cell death ligand 1(PD-L1) have showed activity in unresectable and metastatic blad-der cancer (7–13). Drugs such as pembrolizumab and atezolizumabhave both been approved recently in the treatment of advanced andmetastatic bladder cancer with overall response rate of 15%–26% (8–10, 12). Patients presenting the highest response rate toPD-1/PD-L1 therapy often have tumors with elevated PD-1 andPD-L1 expression and are infiltrated with CD8þ cytotoxic tumor-infiltrating lymphocytes (9, 12–14). On the other hand, patientswith low expression of PD-1 and PD-L1 are often poor respondersto this therapy (13, 15).

Ionization radiation could allow for better response to immunecheckpoint inhibitors as it was demonstrated both in vitro and in vivothat bladder tumors expressed greater levels of PD-1 at their cellsurface in response to radiation (16–18). This could allow for a morepermissive immune microenvironment for immune checkpoint inhi-bitors. The combination of both modalities is currently being studiedthrough clinical trials (19–22).

Radiotherapy is also known to have systemic and immunologiceffects (23). Perhaps one of the most interesting of which is theabscopal effect, where metastatic tumor regression is observed at adistant site to the irradiated tumor. This phenomenon was firstreported in 1966 (24) and has now been reported in different cancertypes as melanoma, clear cell renal cell carcinoma, adenocarcinoma ofthe lung and esophagus, hepatocellular carcinoma, and cervical

1Urologic Oncology Research Program, Research Institute of the McGill Univer-sity Health Center, Montreal, Quebec, Canada. 2Department of Urology, McGillUniversity Health Center, Montreal, Quebec, Canada. 3Centre of Excellence inTranslational Immunology (CETI), Research Institute of the McGill UniversityHealth Centre, Montreal, Quebec, Canada. 4Department of Microbiology andImmunology, McGill University Health Center, Montreal, Quebec, Canada.5Department of Pathology, McGill University Health Center, Montreal, Quebec,Canada. 6Department of Medical Physics, McGill University Health Center,Montreal, Quebec, Canada. 7Program in Infectious Diseases and ImmunologyinGlobal Health, Centre for Translational Biology, Research Instituteof theMcGillUniversity Health Centre, Montreal, Quebec, Canada.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Wassim Kassouf, McGill University Health Centre, 1001Decarie Blvd, D02.7210, Montreal, Quebec H3G 1A4, Canada. Phone: 514-934-8246; Fax: 514-934-8297; E-mail: [email protected]

Mol Cancer Ther 2020;19:211–20

doi: 10.1158/1535-7163.MCT-18-0986

�2019 American Association for Cancer Research.

AACRJournals.org | 211

on June 6, 2021. © 2020 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst September 18, 2019; DOI: 10.1158/1535-7163.MCT-18-0986

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-18-0986&domain=pdf&date_stamp=2019-12-20http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-18-0986&domain=pdf&date_stamp=2019-12-20http://mct.aacrjournals.org/

carcinoma (25–29). The two main mechanisms proposed for theabscopal effect are mediated through cytokine stimulation by aug-menting tumor surveillance, inhibit tumor growth, or direct tumor-icidal properties, as well as through secondary immune system acti-vation by promoting presentation of dying tumor cells to dendriticcells, cross-presentation of tumor-derived antigens to T cells, andconsequent activation of antitumor T cell (30). Abscopal effect result-ing from the combination of immune checkpoint inhibitor withionizing radiation has been demonstrated in metastatic melanomaand in preclinical models with breast and colon cancer models(21, 31, 32).

In this context, our study aimed at identifying a possible abscopaleffect in bladder cancer and to explore this unique tumor microen-vironment when immune checkpoint inhibition is integrated withradiation.

Materials and MethodsMice

Seven-week-old male C57BL/6 mice were obtained from CharlesRiver Laboratory and maintained in a pathogen-free environment inthe animal facility of the McGill University Health Center ResearchInstitute (Quebec, Canada). All animal experiments were done accord-ing to the Animal Ethical Care Protocol no. 7886, approved by ouranimal facility.

Cell line and reagentMB49 is a murine bladder cancer cell line derived from C57BL/6

mice that we obtained as a gift from Dr. Peter Black (University ofBritish Colombia, Vancouver, Canada) and stably express luciferasevector for bioluminescence in vivo imaging and monitoring. TheMB49 cells were cultured in DMEM (Wisent) supplementedwith 10% FBS (Wisent) at 37�C. The thawed cells were typicallypassed 4–5 times prior to subcutaneous injection. Cell count wasperformedwithVi-cell-XRCellViabilityAnalyzer (BeckmanCoulter).The Anti-PD-L1 mAb 10F:9G2 from BioXCell was diluted with PBSto obtain 250 mg of mAb in 200 mL injectable aliquots.

In vitro and in vivo expression of PD-L1 in MB49 tumor cellline and treatments

MB49 cells were cultured in DMEM (Wisent) supplemented with10% FBS (Wisent) at 37�C. The thawed cells were passed twice beforetreatment. Cells were seeded in 100-mm petri dish and were irradiated5 or 10Gy (two doses of 5Gy) using the Faxitron X-Raymachine. Cellswere collected at varying timepoints (24 or 48 hours) post radiationdelivery and were stained for viability (eBioscience, 65-0865) and PD-L1 (BioLegend, 124321). Flow cytometry analyses were performedusing the BD LSRFORETSSA X-20 (BD Biosciences) and results wereanalyzed with FlowJo v10.

For the in vivo quantification of the PD-L1 protein expressionlevel, C57BL/6 mice were subcutaneously implanted with 0.5 � 106MB49 cells in the flank. Tumor growth was monitored with elec-tronic caliper measurements using an ellipsoidal approximationformula of the tumor volume calculated as length � width2, wherethe width represents the smallest measure. When tumors reached avolume of 0.3 cm3, mice subjects were treated with 10 Gy ofradiation in two doses of 5 Gy 24 hours apart with some micereceiving no radiation as a control. Mice were sacrificed 24 or48 hours post radiation delivery and tumors were flash frozen inliquid nitrogen with optimal cutting temperature for immunofluo-rescence analyses.

In vivo tumor growth experimental design and treatmentsC57BL/6 mice were injected subcutaneously in the right and left

flank with 0.5 � 106 MB49 cells diluted in PBS. The right flanktumor was referred to as the primary tumor and the left flank tumoras the distant tumor. Tumor growth was monitored with electroniccaliper measurements using an ellipsoidal approximation formulaof the tumor volume calculated as length � width2, where the widthrepresents the smallest measure. Mice were included in the studywhen they reached a tumor volume of 0.3 cm3. The mice wererandomly assigned to the following treatment arm: control, anti-PD-L1, radiation, and radiationþanti-PD-L1. The randomizationwas performed using the Research Randomizer online software athttps://www.randomizer.org/, the experimenters where not blindedto group assignment. Each group was composed of 8 mice to allowappropriate number for statistical analysis and encompass for anyunexpected loss of animal during the experiment period. Ionizingradiation was delivered to the radiation and anti-PD-L1þradiationgroups using the X-RAD SmART Small Animal Image GuidedIrradiation System from Precision X-ray Inc. The radiation treat-ments were done using fluoroscopic guidance to deliver a total of10 Gy specifically to the primary tumor (right flank) only in twoseparate fractions of 5 Gy given 24 hours apart. The control arm andthe radiation arm received 200 mL intraperitoneal injections ofPBS on the first and third day of the week for 2 weeks, with fourinjections in total, starting from the inclusion date and 1 hour afterreceiving the first radiation dose for the radiation group. The anti-PD-L1 and the anti-PD-L1þradiation groups received intraperito-neal injections of 250 mg of anti-PD-L1 in 200 mL of PBS on thefirst and third day of the week for 2 weeks, with four injections intotal starting from the inclusion date and 1 hour after receiving thefirst radiation dose for the anti-PD-L1þradiation group. The tumorvolumes were calculated every 48 hours using the same electroniccaliper device and formulas as described above until they reachedthe predetermined cut-off volume of 1.5 cm3 or if tumor ulcerationoccurred, at which point the mice were sacrificed and the tumorsextracted.

Tumor digestion and flow cytometry analysisOnce tumors reached their cut-off size, the mice were sacrificed,

and the subcutaneous tumors were extracted for tumor digestions.In each group, both the left and the right flank tumors of 5 micewhere sampled for the experiment. The tumors were minced, andthe immune cells were extracted using the GentleMACS Dissociator(Miltenyi Biotec). Negative cell selection of immune cells wasperformed using the Microbeads CD4þ T-Cell Isolation Kit(Miltenyi Biotec). All immunologic stainings were performedusing mAbs. The CD4þ cells were stained for regulatory T cells(Tregs) panel using CD4-APC antibodies (eBioscience, 17-0041-81), and then fixed and permeabilized using Foxp3 Staining BufferSet (Thermo Fisher Scientific, 00-5523-00) and stained with intra-cellular stain for Foxp3-FITC (eBioscience, 11-5773-80) at a ratioof 1:100 in PBS. The CD4� cells were stained for myeloid-derivedsuppressor cells (MDSCs) using Gr-1-APC.cy7 (BD Biosciences,557661) and CD11b-APC (BioLegend, 101211) extracellular anti-bodies at a ratio of 1:100 in PBS. Tumor-associated macrophages(TAM) were identified as CD11bþ Gr1�. The T effector cells weredetermined by first inducing the expression of IFNg using acytokine stimulation cocktail of PMA (1:10,000) þ ionomycin(1:2,000) þ golgi stop (1:1,000) at 37�C for 4 hours then stainingthe extracellular matrix with CD8-BV650 (BD Biosciences,563822) antibody at a ratio of 1:100 in PBS prior to fixation and

Rompr�e-Brodeur et al.

Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER THERAPEUTICS212



https://www.randomizer.org/https://www.randomizer.org/http://mct.aacrjournals.org/

permeabilization with PMA (1:10,000) þ ionomycin (1:2,000) þgolgi stop (1:1,000) then Foxp3 Staining Buffer Set (ThermoFisher Scientific, 00-5523-00) and stained with intracellular stainfor IFN-g-PE (BD Biosciences, 562020) antibody at a ratio of1:100 in PBS. Flow cytometry analyses were performed with theBD LSRFORETSSA X-20 (BD Biosciences) and analyzed withFlowJo v10.

qPCR analysisIn each group, both the left and the right flank tumors of 5 mice

where sampled for the qPCR experiment. At time of tumor sampling, asmall peripheral section of the tumor was isolated and preserved at�80�C. Mature RNA was isolated from the tumor using a miRNAExtraction Kit (Qiagen catalog no. 217004) according to the manu-facturer's instructions. The RNA quantification was then determinedby NanoDrop and a cDNA conversion kit using RT2 First Strand Kit(catalog no. 330401, Qiagen). The cDNA was platted of the RT2

Profiler PCR Array Mouse Cancer Inflammation & Immunity Cross-talk (Qiagen, catalog no. PAMM-181Z) in combination with RT2

SYBRGreen qPCRMastermix (catalog no. 330529). Data analysis wasdone via Gene Globe web portal at http://www.qiagen.com/geneglobe.Fold changes were calculated using DDCt method with a Ct � 2 ascutoff.

Tumor tissue immunofluorescenceFrozen tissues were cryo-cut (5 mm) andwere fixed with 4%PFA for

5 minutes. Slides were washed three times for 5 minutes with TBS þ0.1% Tween (TBST) and blocked with normal goat serum in PBS(Millipore, 20773) for 1 hour. Tissues were then stained using primaryantibodies for PD-L1 (eBioscience, 14-5982-81) and CTLA4 (Abcam,ab134090). Slides were washed as above and stained for secondaryantibodies anti-rat-IgG (Cell Signaling Technology, 4416S) and anti-rabbit IgG (Molecular probes, A11036). Slidesweremountedwith Pro-Long Diamond with DAPI (Molecular Probes, P36966) and imagedusing Olympus IX81 Microscope.

Statistical analysisStatistical analyses were performed using the GraphPad Prism

software. A sample size of 8 mice/arm was estimated to have >80%power to detect a minimum difference of 25% reduction in tumorvolume at a P value of 0.05 as published previously (33). Repetitivemeasures ANOVA model with Bonferroni correction was used tocompare tumor volume means across treatments. Kaplan–Meiercurves were used to illustrate differences in survival. Log-rank (Man-tel–Cox) test, mean difference, and Student t test were performed forthe evaluation of continuous variables. Pearson x2 independence testwas conducted for categorical variables. P < 0.05 was considered to bestatistically significant.

ResultsTimely relation of PD-L1 expression levels pre- and post-radiation in MB49 tumor cells

MB49 cells were exposed to varying doses of radiation (5 and 10Gy)and PD-L1 expression was examined using flow cytometry at time-points 24 and 48 hours post radiation treatment. PDL1 expressionlevels were compared with nonirradiated control MB49 cells (Fig. 1AandB). Through this experiment, we were able to demonstrate that thePD-L1 protein expression level rises after radiation and that thisreaction is greater at a dose of 10Gy and at 48 hours after the radiation.Once the rise in the expression level after radiation was quantified

in vitro, we confirmed in vivo that the subcutaneous tumors alsoincreased their PD-L1 transmembrane protein expression level afterbeing submitted to a total of 10 Gy of radiation (Fig. 1C). Once again, agreater effect was seen at the 48-hourmark after initiation of treatment.However, when measured in tumors at the endpoint of our radiatedmice cohort, median survival of 11 days, the transmembrane expres-sion level of PD-L1, CTLA-4, and DAPI was not different from thecontrol (Supplementary Fig. S1).

Tumor growth kinetics and survival analysisMonitoring the growth rate of the syngeneic tumors demonstrated

that the combination group receiving both the radiotherapy and theanti-PD-L1 treatment provided the best outcome (Fig. 2B andC). Theirradiated right flank tumors in the combination arm had a statisticallysignificant slower growth rate when compared with radiation alone(P< 0.001), which reproduces what was described previously in similarexperiments (18), were the addition of the immune checkpointinhibitor potentiated the effects of the ionizing radiation. Moreinterestingly, the nonirradiated left flank tumors in the combinationarm also demonstrated a statistically significant slower growth thanthe other treatments arms, notably when compared with radiationalone (P < 0.001).

Accordingly, the benefit of the radiation and anti-PD-L1 combi-nation treatment translated in an improved survival (Fig. 1D) com-pared with the anti-PD-L1 arm (P ¼ 0.02), the radiation alone group(P ¼ 0.005), and control group (P ¼ 0.002).

Tumor microenvironmentThe impact of each treatment on the immune landscape within the

tumor microenvironment was also investigated. Mainly, the recruit-ment of different immunosuppressive populations with known pro-tumor activity, such as MDSCs, TAMs, and CD4þ Tregs were eval-uated in addition to assessing recruitment of the antitumor effector Tcells (IFNg-producing CD8þ T cells). The gating strategies can befound in the Supplementary Data (Supplementary Fig. S2).

The data in Fig. 3 demonstrate that the group who receivedradiation alone attracted significantly more immunosuppressive celltypes, such asMDSCs, TAMs, andTregs, in both flanks (irradiated andnonirradiated flanks), as compared with control. However, this effectseems to be reversed by the concomitant anti-PD-L1 treatmentobserved in the combination group. On the other side of the spectrum,the antitumor-infiltrating cytolytic CD8þ T cells were also recruited inlarger number, both locally and in the distant tumor, in both theradiation group and the combination arm. Although not statisticallysignificant, we can observe a trend toward a greater attraction ofcytolytic CD8 T cells in the nonirradiated flank in the group receivingthe combination treatment (Fig. 3A). However, combination ofradiation with PD-L1 blockade reversed the immunosuppressiveenvironment created by the radiation toward an antitumor environ-ment, reflecting individual changes in the ratio of immunosuppressive-infiltrating cells versus cytolytic (IFNþCD8þT) cells at the primary anddistant (nonirradiated) tumor (Fig. 3B, last graphic).

Inflammation and immune profiling associated withcombination of radiation and PD-L1 blockade

The effects of combining ionizing radiation with immune check-point inhibitor were also studied at the molecular level across treat-ments, with further distinction on the differences between nonirra-diated left flank and radiated right flank across the treatment arms.First of all, we can note that the selected genes present in the arrayallowed the nonsupervised hierarchical clustering conjoint grouping of

Abscopal Effect in Urothelial Carcinoma

AACRJournals.org Mol Cancer Ther; 19(1) January 2020 213



http://www.qiagen.com/geneglobehttp://mct.aacrjournals.org/

Figure 1.

In vitro and in vivo levels of PD-L1 expression in MB49 tumor cells post radiation.A, In vitro dose/response study of MB49 tumors cells treated with 5 Gy of 10 Gy (twodoses of 5 Gy) radiation. PDL1 expression was measured by flow cytometry at timepoints 24 or 48 hours post radiation treatment. Data represented as meanfluorescence intensity (MFI). B, Histogram plot comparing PDL1 expression in MB49 cells at baseline, nonradiated tumor cells (blue), or 48 hours post 10 Gy ofradiation treatment (red). C, Immunofluorescence PD-L1 expression in MB49 tumor tissues of mice sacrificed at 24 and 48 hours post receiving 10 Gy of radiation.Magnification, 63�.D,Quantification of PDL1 expression in tissues represented asmean intensity (P

Figure 2.

Tumor growth rate across four group of treatment [control, anti-PD-L1 alone, radiation (Rad) alone, and combination]. A, A total dose of radiation of 10 Gy wasdelivered in two fractions of 5Gyonday 1 and 2, only to the rightflank. Anti-PD-L1 (250mgpermouse)was given intraperitoneally two times perweek for 2weeks. Forthe combination group, anti-PD-L1 was given 1 hour after radiation. B, Tumor growth curve showing tumor volume of irradiated flank (right). C, Tumor growth curveshowing tumor volume of nonirradiated flank (left) for indication of systemic response (abscopal effect). Error bars are SE, log-rank analysis for statisticalsignificance. D, Kaplan–Meier survival curve analysis and median survival of the four different groups of treatment.





http://mct.aacrjournals.org/

all the treatment arms who received radiation, both for the irradiatedand nonirradiated flanks (Fig. 4A).

In the single-gene analysis details, a pattern of downregulation ofimmunosuppressive (protumor) genes emerges more pronounced inthe combination (radiationþanti-PD-L1) group (Table 1). The com-plete list of genes and their varying expression in the differentconditions tested can be found in the Supplementary Data (Supple-mentary Table S1). Among the protumor genes, IL13, IL22, and theG-CSF3 were all downregulated significantly in the combined armwhen compared with the other treatment arms. These genes are,respectively, associated in urothelial carcinoma with potent immuno-suppression, high-risk features, and cancer progression (34–37). Thechemokine C-X-Cmotif ligand 12 (CXCL12), another protumor gene,was upregulated in all treatment arm (anti-PD-L1: 4.15-fold, radiation:right flank 2.94-fold, left flank 2.53-fold). However, its expressionremains at baseline in the combination arm. IL13, a potent immuno-suppressor in bladder cancer (34), is seen to undergo maximal down-regulation in the combination arm when compared with the othertreatment groups (irradiated right flank: �7.47-fold, nonirradiatedleft flank: �17.21-fold). IL22, whose high expression levels wereassociated with high risk bladder cancer in human (35), is alsomaximally downregulated in the radiationþanti-PD-L1 group (irra-diated right flank: �4.74-fold, nonirradiated left flank: �10.92-fold)when compared with the other treatment groups. G-CSF3, associatedwith bladder cancer progression (36), was found to be downregulatedonly in the radiationþanti-PD-L1 group (irradiated rightflank:�2.56-fold, nonirradiated left flank: �2.21-fold).

Conversely, few genes associated with cytolytic immune responsewere found to be upregulated in the mice who received radiationbut more so in the radiationþanti-PD-L1 group. The chemokineC-X-C motif ligand 9 (CXCL9), responsible for the attraction ofCD8þ T cell, Th1, and natural killer (NK) cells (38), was found to beupregulated at a higher degree in the radiationþanti-PD-L1 group

than in any other group with increases of 5.36-fold in the irradiatedright flank and 3.08-fold in the nonirradiated left flank. Moreover,the cytolytic enzymes GZMA and GZMB were also overexpressedin the radiationþanti-PD-L1 group (GZMA right flank: 5.56-fold,GZMB right flank: 3.01-fold).

DiscussionWhile the effects and limitations of ionizing radiation in urothelial

carcinoma are well known, the development of immunologic agentsfor advanced and metastatic cases provides us with new opportunitiesfor treatment development. In this study we aimed at studying theeffects of combining ionizing radiation with immune checkpointinhibitors both in a radiated tumor and on a distant tumor site,mimicking a distant tumor outside of the field of radiation, to studya potential novel abscopal effect in urothelial carcinoma. If it were to bethe case, this treatment combination could represent a superiorstrategy to the current bladder-sparing treatments whose local recur-rence rate stand between 25% and 30% and that cannot treat micro-metastases outside of the scope of radiation.

Observing the tumor growth curves in all groups we can notice aseparation of the curves of the combination arm as soon as day 6 afterinclusion, separation which was maintained throughout the study.This effect was reflected on the statistically significant difference inmedian survival conferred to the subjects receiving both the radiationand anti-PD-L1 therapy. These findings are comparable with thoseobtained by Wu and colleagues (18) who also observed that thecombination of radiation with immune checkpoint inhibitors on anectopic murine bladder tumor produces a significant improvement inthe tumor growth rates. However, their study did not address theimpact of the treatment on distant lesions and was designed with onlyone dose of 12Gyof radiation, whereaswe delivered two separate dosesof 5 Gy given 24 hours apart. This fractionation of the radiation is

Figure 3.

Tumor-infiltrating immune cells. A, Quantitative flow cytometry analysis showing the absolute number of recruited/infiltrating MDSCs (CD11bþGr1þ), TAM(CD11bþGr1�), Treg (CD4

þFOXP3þ), and CD8þT cells in live cells in the tumors. B, Corresponding ratio of MDSCs, TAM, and Treg to activated IFNþCD8þTcells. The ratio of immunosuppressive cells to IFNþCD8þTcells is the mean of all immunosuppressive cells (MDSCs, TAMs, and Treg) to activatedCD8þTcells (IFNþCD8þTcells). Sample size of n ¼ 5 tumors extracted from each flank in all treatment groups, error bars are SE, log-rank analysis forstatistical significance.






Figure 4.

qPCR analysis of extracted tumors. Gene expression fold changes (Ct � 2 folds) of protumor and antitumor immune-related genes within the tumormicroenvironments across treatments as compared with control. A, Heatmap of the qPCR analysis with the nonsupervised hierarchical clustering conjointgrouping. B, Graphic representation of expression fold changes in the different treatment arm with only the genes reaching Ct � 2 folds in at least onetreatment arm represented. Sample size of n ¼ 5 tumors extracted from each flank in all treatment groups, log-rank analysis for statistical significance. Lt, leftside; Rt, right side; XRT, radiation.






intended to help promote an abscopal effect as was previously dem-onstrated in preclinical models, notably of breast and coloncarcinoma (32, 39).

Themain interest of this growth kinetics relies in the improvementsprovided by the combination therapy of radiation with immunecheckpoint inhibitors being mirrored in the nonirradiated tumor.This significant improvement in the contralateral tumor, statisticallysignificant from the radiation or anti-PD-L1 arms, could represent anabscopal effect. It is also interesting to note that the contralateralnonirradiated tumor of the radiation arm also benefited from slowingof the tumor growth in the first few days immediately after radiation.Although this difference induced by radiation alone did not translate ina statistically significant separation of its growth curve from the othergroup, this trend echoes the first case studies published in the 1960swhere an abscopal effect was demonstrated with the use of radiother-apy alone (24).

Different studies have looked at the effect of PD-L1 and of radiation,as well as their combination, on the systemic immunity, whereas herewe present one of the few experiments detailing their combined effectsin the tumor microenvironment. Regarding the impact on systemiclymphoid organs, Oweida and colleagues evaluated the combination ofthe same two modalities in a head and neck squamous cell cancermodel, where the CD8þ population was found to be comparable in thespleen between their different study groups while the CD4þ popula-tion was increased in the group that received radiation (40). Inter-estingly, their study also highlighted a discrepancy between the locallymphoid tissues and the systemic lymphoid tissues where the anti-tumor response in the presence or abscence of anti-PD-L1 treatmentappears to be unchanged in the systemic lymphoid tissues. Similarresults were also found by another group investigating the combina-tion treatment with anti-PD-L1 and radiation in a melanoma modelwhere they observed only a limited CD8þ response to tumor antigenstimulation in nondraining lymph nodes, in clear discrepancy to thehigh response found in local lymph nodes (41). Vandeveer andcolleagues studied the impact of selective depletion of CD4 or CD8cells onMB49 tumor growth and determined that their role was criticalto the antitumoral effect of PD-L1 antibodies namely by the ability ofspleen-isolated T cell to recognize MB49 tumor antigens (42). Thesestudies together provide an understanding of the systemic impact ofanti-PD-L1 and radiation treatment which lays ground for the experi-ments performed in our study.

Studying the tumor microenvironment provided further informa-tion about the immunologic changes within both the radiated andnonirradiated tumor of the combination arm. As described in theliterature, the effect of ionizing radiation alone could induce bothimmune tolerance and activate cytotoxic immune response (43, 44). A

more immune tolerant environment when ionizing radiation is givenalone is visible in Fig. 2 with the ratio of the total immunosuppressivecells over IFNþ/CD8þ cells largely in favor of immunosuppression.However, the addition of the anti-PD-L1 treatment to radiation, asstudied with the combination arm, reversed this ratio and the immu-nosuppressive effect observed in the radiation alone group. In humansubjects, radiation was also shown to increase T-cell activation, leadingto CD8þ T-cell–dependent cytotoxicity toward cancer cells (45). Theaddition of immune checkpoint inhibitors appears to dampen theimpact on the immunosuppressive cell line populations observed inour radiation cohorts for two reasons. First, one of the proposedmechanisms in the literature for the abscopal effect relies on thesystemic immune activation through the presentation of tumor anti-gen to various effector cells (45, 46). Second, it was studied thatradiotherapy treatments will promote the cell surface expression ofPD-L1 receptor (18), which we were also able to confirm in our owncell line (Fig. 1). Those two observations together appear to explain therelatively immune-tolerant populations observed in the radiationgroup and, more importantly, the drastic change in immune cell linepopulations observed with the addition of anti-PD-L1 antibody in thecombination arm. The overall result is of a remarkably changed ratio ofimmune cell line populations toward antitumor activity in the tumormicroenvironment.

The effect of the combination of radiation with anti-PD-L1 on theimmune cell lines present in the tumormicroenvironment, promotinga less immunosuppressive phenotype and more antitumoral activity,were then explored using differential gene expression across thetreatment arms at the experimental endpoint. We observed a down-regulation of CXCL12, which plays a critical role in regulation oftumor growth, metastasis, recruitment ofMDSCs, and development ofchemoresistance (37). Similarly, IL13, a potent immunosuppressorcytokine in bladder cancer (34) and IL22 associated with high riskbladder cancer in human (35) showed lower expression in the com-bination group as compared with control. On the other hand, theupregulation of antitumor genes such asCXCL9who plays a key role inthe attraction of CD8þ, as well as Th1 andNK cells, favoring antitumorimmune activation was observed. It is to be noted that some genes ofinterests showed no significant difference in their expression levels atthe experimental endpoint between the different treatment arms,notably PD-L1 and CTLA-4, another important immune checkpointinhibitor target (22), nor FOXP3 whose role in radiation resistance isnow well described (47). While we could demonstrate with immuno-fluorescence in our subjects that the level of membrane expression ofPD-L1 increases with the effect of radiation, its expression level did goback to background levels at the endpoint of our study period(Supplementary Fig. S1). This finding is consistent with our qPCR

Table 1. Gene expression variation in the tumor microenvironment.

Genes Main immune effect Anti-PD-L1 XRT right XRT left XRTþAnti-PD-L1 right XRTþAnti-PD-L1 leftCXCL12 Immunosuppressive 4.15 2.94 2.53 n.s n.s.IL13 Immunosuppressive �5.25 �2.28 n.s. �7.47 �17.21IL22 Immunosuppressive �3.33 �2.83 �3.77 �4.74 �10.92CSF3 Immunosuppressive n.s n.s. n.s. �2.56 �2.21CXCL9 Cytolytic response n.s. 2.64 2.20 5.36 3.08GZMA Cytolytic response �2.16 3.54 n.s. 5.56 n.s.GZMB Cytolytic response n.s n.s. n.s. 3.01 n.s.

Note: Gene expression fold changes classified on theirmain immune effect in key selected genes fromFig. 4. Nonsignificant fold change (n.s.) represent a fold changeCt < 2 folds.Abbreviation: XRT, radiation.






results were no variation in the RNA expression level can be seen intumors at the endpoint.

Comparing our findings to the current body of literature onimmune checkpoint inhibitors and the immunomodulator effects ofradiation, we hypothesize that combining radiation with immunecheckpoint inhibitors can help reverse the tumor immune escapemechanisms in urothelial carcinoma. In our model, we showed thationizing radiation increases the urothelial carcinoma cell surfaceexpression of PD-L1, which could then promote immune tolerancetoward the cancer cells inside the tumor microenvironment and helpsuppress the activation of cytolytic lymphocytes. More importantly,increasing the cell surface expression of PD-L1 may generate whatappeared to be a more permissive environment for the PD-1/PD-L1axis antibody-mediated selective inhibition which then allowed forincreased recruitment of cytotoxic lymphocytes. At the experimentalendpoint, we observed a pattern of downregulation of the expressionlevel of immunosuppressive genes and upregulation of the cytolyticactivity genes; effectively reversing the tumor microenvironment fromimmune tolerance to antitumor activity. The work of Vandeveer andcolleagues provides insight into the role played by CD8þ and CD4þ Tcells in the antitumorocidal effect of anti-PD-L1 treatment on MB49cells (42). Pursuing their work and conducting depletion experimentswith these two cell lines would have provided further insight into thecausal implications of the immunologic processes at play within thetumor microenvironment, particularly in the contralateral nonirradi-ated tumor, and conceivably represents a limitation of our study.

In conclusion, these results represent a first demonstration of apotential abscopal effect in urothelial carcinomaby targeting the PD-1/PD-L1 axis. Currently, immune checkpoint inhibitors monotherapysuch as pembrolizumab and atezolizumab were approved as a second-line for the treatment of advanced andmetastatic urothelial carcinoma.Our results are encouraging, as the integration of immune checkpointinhibition may hold promise in patients treated with radiotherapy for

muscle-invasive bladder cancer. Further studies will be relevant inexploring this unique tumor microenvironment in depth to improvetumor outcomes. Given recent trials and translational research pro-jects, it will also be relevant to study the effect of different radiationfractionation schedules and their impact on the radiated tumors andabscopal tumors when combined with immunotherapy.

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

Authors’ ContributionsConception and design: A. Rompr�e-Brodeur, J.J. Mansure, W. KassoufDevelopment of methodology: A. Rompr�e-Brodeur, W. KassoufAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): A. Rompr�e-Brodeur, S. Shinde-Jadhav, M. Ayoub, J. Seuntjens,F. BrimoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Rompr�e-Brodeur, S. Shinde-Jadhav, M. Ayoub,C.A. Piccirillo, J.J. Mansure, W. KassoufWriting, review, and/or revision of themanuscript:A. Rompr�e-Brodeur, S. Shinde-Jadhav, C.A. Piccirillo, J. Seuntjens, F. Brimo, J.J. Mansure, W. KassoufAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): A. Rompr�e-Brodeur, W. KassoufStudy supervision: J.J. Mansure, W. KassoufOther (expertise with regards to animal irradiation and dosage): J. Seuntjens

AcknowledgmentsThe current work was supported by the operating grant no. 23025 of the Cancer

Research Society (CRS) awarded to W. Kassouf.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 9, 2018; revised April 2, 2019; accepted September 12, 2019;published first September 18, 2019.

References1. Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F. Bladder cancer

incidence and mortality: a global overview and recent trends. Eur Urol 2017;71:96–108.

2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016;66:7–30.

3. Stein JP, Lieskovsky G, Cote R, Groshen S, Feng AC, Boyd S, et al. Radicalcystectomy in the treatment of invasive bladder cancer: long-term results in1,054 patients. J Clin Oncol 2001;19:666–75.

4. Cronin KA, Ries LA, Edwards BK. The Surveillance, Epidemiology, and EndResults (SEER) program of the National Cancer Institute. Cancer 2014;120:3755–7.

5. Advanced Bladder Cancer (ABC) Meta-Analysis Collaboration. Neoadjuvantchemotherapy in invasive bladder cancer: update of a systematic review andmeta-analysis of individual patient data advanced bladder cancer (ABC) meta-analysis collaboration. Eur Urol 2005;48:202–5.

6. Ploussard G, Daneshmand S, Efstathiou JA, Herr HW, James ND, R€odel CM,et al. Critical analysis of bladder sparing with trimodal therapy in muscle-invasive bladder cancer: a systematic review. Eur Urol 2014;66:120–37.

7. Apolo AB, Ellerton JA, Infante JR, Agrawal M, Gordon MS, Aljumaily R, et al.Updated efficacy and safety of avelumab in metastatic urothelial carcinoma(MUC): pooled analysis from 2 cohorts of the phase 1b javelin solid tumor study.J Clin Oncol 2017;35:4528.

8. Balar AV, Castellano DE, O'Donnell PH, Grivas P, Vuky J, Powles T, et al.Pembrolizumab as first-line therapy in cisplatin-ineligible advanced urothe-lial cancer: results from the total keynote-052 study population. J Clin Oncol2017;35:284.

9. Balar AV, Galsky MD, Rosenberg JE, Powles T, Petrylak DP, Bellmunt J, et al.Atezolizumab as first-line therapy in cisplatin-ineligible patients with locally

advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase2 trial. Lancet 2017;389:67–76.

10. Bellmunt J, deWit R, VaughnDJ, Fradet Y, Lee J-L, Fong L, et al. Pembrolizumabas second-line therapy for advanced urothelial carcinoma. N Engl J Med 2017;376:1015–26.

11. Hahn NM, Powles T, Massard C, Arkenau H-T, Friedlander TW, Hoimes CJ,et al. Updated efficacy and tolerability of durvalumab in locally advanced ormetastatic urothelial carcinoma (UC). J Clin Oncol 2017;35:4525.

12. Rosenberg JE, Hoffman-Censits J, Powles T, van der Heijden MS, Balar AV,Necchi A, et al. Atezolizumab in patients with locally advanced and metastaticurothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single arm, phase 2 trial. Lancet 2016;387:1909–20.

13. Sharma P, Retz M, Siefker-Radtke A, Baron A, Necchi A, Bedke J, et al.Nivolumab in metastatic urothelial carcinoma after platinum therapy (check-mate 275): a multicentre, single-arm, phase 2 trial. Lancet Oncol 2017;18:312–22.

14. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJM, Robert L, et al.PD-1 blockade induces responses by inhibiting adaptive immune resistance.Nature 2014;515:568–71.

15. Powderly JD, Koeppen H, Hodi FS, Sosman JA, Gettinger SN, Desai R, et al.Biomarkers and associations with the clinical activity of PD-L1 blockade in aMPDL3280A study. J Clin Oncol 2013;31:3001.

16. Deng L, Liang H, Burnette B, Beckett M, Darga T, Weichselbaum RR, et al.Irradiation and anti–PD-L1 treatment synergistically promote antitumor immu-nity in mice. J Clin Invest 2014;124:687–95.

17. Teng F, Mu D, Meng X, Kong L, Zhu H, Liu S, et al. Tumor infiltratinglymphocytes (TILs) before and after neoadjuvant chemoradiotherapy and itsclinical utility for rectal cancer. Am J Cancer Res 2015;5:2064–74.






18. Wu C-T, ChenW-C, Chang Y-H, LinW-Y, ChenM-F. The role of PD-L1 in theradiation response and clinical outcome for bladder cancer. Sci Rep 2016;6:19740.

19. Sundahl N, DeWolf K, Rottey S, Decaestecker K, De Maeseneer D, Meireson A,et al. A phase I/II trial of fixed-dose stereotactic body radiotherapy withsequential or concurrent pembrolizumab in metastatic urothelial carcinoma:evaluation of safety and clinical and immunologic response. J TranslatMed 2017;15:150.

20. Hiniker SM, Reddy SA, Maecker HT, Subrahmanyam PB, Rosenberg-Hasson Y,Swetter SM, et al. A prospective clinical trial combining radiation therapy withsystemic immunotherapy metastatic melanoma. Int J Radiat Oncol Biol Phys2016;96:578–88.

21. Hu ZI, Ho AY, McArthur HL. Combined radiation therapy and immunecheckpoint blockade therapy for breast cancer. Int J Radiat Oncol Biol Phys2017;99:153–64.

22. Massari F, Di Nunno V, Cubelli M, Santoni M, Fiorentino M, Montironi R, et al.Immune checkpoint inhibitors for metastatic bladder cancer. Cancer Treat Rev2018;64:11–20.

23. Hall EJ, Giaccia AJ.Radiobiology for the radiologist. 8th ed.Philadelphia, PA:Lippincott Williams & Wilkins;2011.

24. Boyd W.The spontaneous regression of cancer. Springfield, Illinois:Charles CThomas;1966.

25. MacManus MP, Harte RJ, Stranex S. Spontaneous regression of metastatic renalcell carcinoma following palliative irradiation of the primary tumour. Ir JMed Sci1994;163:461–3.

26. Ehlers G, FridmanM. Abscopal effect of radiation in papillary adenocarcinoma.Br J Radiol 1973;46:220–2.

27. Ohba K, Omagari K, Nakamura T, Ikuno N, Saeki S, Matsuo I, et al. Abscopalregression of hepatocellular carcinoma after radiotherapy for bone metastasis.Gut 1998;43:575.

28. TakayaM, Niibe Y, Tsunoda S, Jobo T, ImaiM, Kotani S, et al. Abscopal effect ofradiation on toruliform para-aortic lymph node metastases of advanced uterinecervical carcinoma–a case report. Anticancer Res 2007;27:499–503.

29. Rees GJG, Ross CMD. Abscopal regression following radiotherapy for adeno-carcinoma. Br J Radiol 1983;56:63–6.

30. Siva S, MacManus MP, Martin RF, Martin OA. Abscopal effects of radiationtherapy: a clinical review for the radiobiologist. Cancer Lett 2015;356:82–90.

31. Postow MA, Callahan MK, Barker CA, Yamada Y, Yuan J, Kitano S, et al.Immunologic correlates of the abscopal effect in a patient with melanoma.N Engl J Med 2012;366:925–31.

32. DewanMZ, Galloway AE, Kawashima N, Dewyngaert JK, Babb JS, Formenti SC,et al. Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti–CTLA-4 antibody.Clin Cancer Res 2009;15:5379.

33. Shrivastava S, Mansure JJ, AlmajedW, Cury F, Ferbeyre G, PopovicM, et al. Therole of HMGB1 in radioresistance of bladder cancer. Mol Cancer Ther 2016;15:471–9.

34. MalekZadeh K, Nikbakht M, Sadeghi IA, Singh SK, Sobti RC. Overexpression ofIL-13 in patients with bladder cancer. Cancer Invest 2010;28:201–7.

35. Zhao T,WuX, Liu J. Association between interleukin-22 genetic polymorphismsand bladder cancer risk. Clinics 2015;70:686–90.

36. Tachibana M, Miyakawa A, Tazaki H, Nakamura K, Kubo A, Hata J, et al.Autocrine growth of transitional cell carcinoma of the bladder induced bygranulocyte-colony stimulating factor. Cancer Res 1995;55:3438–43.

37. Nagarsheth N, Wicha MS, Zou W. Chemokines in the cancer microenvi-ronment and their relevance in cancer immunotherapy. Nat Rev Immunol2017;17:559–72.

38. Muthuswamy R, Wang L, Pitteroff J, Gingrich JR, Kalinski P. Combination ofIFNa and poly-I:C reprograms bladder cancer microenvironment for enhancedCTL attraction. J Immunother Cancer 2015;3:6.

39. Formenti SC. Optimizing dose per fraction: a new chapter in the story of theabscopal effect? Int J Rad Oncol 2017;99:677–9.

40. Oweida A, Lennon S, Calame D, Korpela S, Bhatia S, Sharma J, et al. Ionizingradiation sensitizes tumors to PD-L1 immune checkpoint blockade in orthotopicmurine head and neck squamous cell carcinoma. Oncoimmunology 2017;6:e1356153.

41. Lugade AA, Moran JP, Gerber SA, Rose RC, Frelinger JG, Lord EM. Localradiation therapy of B16 melanoma tumors increases the generation of tumorantigen-specific effector cells that traffic to the tumor. J Immunol 2005;174:7516–23.

42. Vandeveer AJ, Fallon JK, Tighe R, Sabzevari H, Schlom J, Greiner JW.Systemic immunotherapy of non-muscle invasive mouse bladder cancer withavelumab, an anti-PD-L1 immune checkpoint inhibitor. Cancer ImmunolRes 2016;4:452–62.

43. McBrideWH, Chiang CS, Olson JL,Wang CC, Hong JH, Pajonk F, et al. A senseof danger from radiation. Radiat Res 2004;162:1–19.

44. Haikerwal SJ, Hagekyriakou J, MacManus M,Martin OA, Haynes NM. Buildingimmunity to cancer with radiation therapy. Cancer Lett 2015;368:198–208.

45. Lugade AA, Sorensen EW, Gerber SA, Moran JP, Frelinger JG, Lord EM.Radiation-induced IFN-g production within the tumor microenvironmentinfluences antitumor immunity. J Immunol 2008;180:3132.

46. Chakraborty M, Abrams SI, Coleman CN, Camphausen K, Schlom J, HodgeJW. External beam radiation of tumors alters phenotype of tumor cells torender them susceptible to vaccine-mediated T-cell killing. Cancer Res 2004;64:4328.

47. Liu S, Sun X, Luo J, Zhu H, Yang X, Guo Q, et al. Effects of radiation on Tregulatory cells in normal states and cancer: mechanisms and clinical implica-tions. Am J Cancer Res 2015;5:3276–85.






2020;19:211-220. Published OnlineFirst September 18, 2019.Mol Cancer Ther Alexis Rompré-Brodeur, Surashri Shinde-Jadhav, Mina Ayoub, et al.

and Abscopal EffectsIn SituBladder Cancer: PD-1/PD-L1 Immune Checkpoint Inhibition with Radiation in

Updated version

10.1158/1535-7163.MCT-18-0986doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2019/09/18/1535-7163.MCT-18-0986.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/19/1/211.full#ref-list-1

This article cites 45 articles, 13 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/19/1/211To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-18-0986http://mct.aacrjournals.org/content/suppl/2019/09/18/1535-7163.MCT-18-0986.DC1http://mct.aacrjournals.org/content/19/1/211.full#ref-list-1http://mct.aacrjournals.org/cgi/alertsmailto:[email protected]://mct.aacrjournals.org/content/19/1/211http://mct.aacrjournals.org/

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 200 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 900 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MarksOffset 18 /MarksWeight 0.250000 /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PageMarksFile /RomanDefault /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> > ]>> setdistillerparams> setpagedevice

pd-1/pd-l1immunecheckpointinhibitionwithradiation in ... · pd-1/pd-l1 therapy often have tumors...

Documents