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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
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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
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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
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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
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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.
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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.
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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.
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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.
Rompr�e-Brodeur et al.
Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER
THERAPEUTICS218
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-
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.
Abscopal Effect in Urothelial Carcinoma
AACRJournals.org Mol Cancer Ther; 19(1) January 2020 219
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://mct.aacrjournals.org/
-
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.
Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER
THERAPEUTICS220
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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
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