Fatty Acid Oxidation Controls CD8þ Tissue-Resident Memory ...(1:1,000)orAlexaFluor555–conjugatedgoatanti-rabbitIgG(1:1,000; LifeTechnologies)at4 Cfor30minutes.Cellswerethenwashedwith

CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE

Fatty Acid Oxidation Controls CD8þ Tissue-ResidentMemory T-cell Survival in Gastric Adenocarcinoma A C

Run Lin1, Hui Zhang2,3, Yujie Yuan4, Qiong He5, Jianwen Zhou5, Shuhua Li5, Yu Sun5, Daniel Y. Li6,Hai-Bo Qiu7, Wei Wang8, Zhehong Zhuang9, Bin Chen1, Yonghui Huang1, Chuwei Liu4, Yingzhao Wang4,Shirong Cai4, Zunfu Ke3,5, and Weiling He4

ABSTRACT◥

The success of checkpoint inhibitors in cancer treatment isassociated with the infiltration of tissue-resident memory T(Trm) cells. In this study, we found that about 30% of tumor-infiltrating lymphocytes (TIL) in the tumor microenvironment ofgastric adenocarcinoma were CD69þCD103þ Trm cells. Trmcells were low in patients with metastasis, and the presence ofTrm cells was associated with better prognosis in patients withgastric adenocarcinoma. Trm cells expressed high PD-1, TIGIT,and CD39 and represented tumor-reactive TILs. Instead ofutilizing glucose, Trm cells relied on fatty acid oxidation for cellsurvival. Deprivation of fatty acid resulted in Trm cell death. In atumor cell–T-cell coculture system, gastric adenocarcinoma cellsoutcompeted Trm cells for lipid uptake and induced Trm cell

death. Targeting PD-L1 decreased fatty acid binding protein(Fabp) 4 and Fabp5 expression in tumor cells of gastric adeno-carcinoma. In contrast, the blockade of PD-L1 increased Fabp4/5expression in Trm cells, promoting lipid uptake by Trm cells andresulting in better survival of Trm cells in vitro and in vivo.PD-L1 blockade unleashed Trm cells specifically in the patient-derived xenograft (PDX) mice. PDX mice that did not respond toPD-L1 blockade had less Trm cells than responders. Together,these data demonstrated that Trm cells represent a subset ofTILs in the antitumor immune response and that metabolicreprogramming could be a promising way to prolong the lon-gevity of Trm cells and enhance antitumor immunity in gastricadenocarcinoma.

IntroductionGastric cancer is the second most common cancer worldwide, and

gastric adenocarcinoma accounts for 95% of the gastric cancer (1).Despite significant achievements in the management of gastric ade-nocarcinoma, the prognosis remains dismal with a 5-year survival rateof about 20% (2). Surgical resection remains thefirst choice for patients

with resectable tumors, but recurrence occurs in 20% to 50% of thepatients following gastric resection (2). More than 50% of patientspresent with locally advanced or metastatic gastric adenocarcinoma atdiagnosis, leaving chemotherapy as the main therapeutic option forthese patients (3). Thus, the development of novel therapeutic agentsand strategies for gastric adenocarcinoma is eagerly awaited due to itshigh morbidity and mortality.

The emergence of immunotherapy has changed the landscape ofcancer treatments (4). Immune checkpoint blockade of programmeddeath 1 (PD-1) is successful in the treatment of lung cancer,melanoma,kidney cancer, and various other cancers (5–7). Anti–PD-1 treatmentis effective for gastric cancer (8, 9). However, only 11.2% and 22% ofthe treated patients responded to such treatments in these two clinicaltrials, respectively. It is urgent to identify new therapeutic targets toapproach better outcomes for patients with gastric adenocarcinoma.

Tissue-resident memory T (Trm) cells are a T-cell subset thatresides in the tissue and does not circulate back to the blood orsecondary lymphoid organs (10, 11). Trm cells produce higheramounts of cytokines than their circulating counterparts and provideenhanced local immunity in response to infection (12, 13). CD8þ Trmcells are associated with antitumor immune responses (14, 15). Thepresence of Trm cells correlates with improved prognosis in patientswith cancer (16, 17). CD8þ Trm cells promote melanoma-immuneequilibrium in skin, whereas mice deficient in Trm cells' formation aremore susceptible to tumor development (18). These properties giveTrm cells great potential in the treatment of cancer. However, the rolesof Trm cells in GCA have not yet been reported, and the detailedmaintenance mechanism of CD8þ Trm cells in the tumor microen-vironment (TME) remains to be addressed.

In the current study, we found that Trm cells were present in theTME of gastric adenocarcinoma, which indicated better prognosis.Trm cells relied on lipid uptake and metabolism for cell survival.Cancer cells of gastric adenocarcinoma outcompeted Trm cells forlipid uptake and induced apoptosis of Trm cells, which could be

1Department of Radiology, The First Affiliated Hospital, Sun Yat-sen University,Guangzhou, Guangdong, China. 2Department of Rheumatology, The First Affil-iatedHospital, SunYat-senUniversity, Guangzhou, Guangdong, China. 3Instituteof Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University,Guangzhou, Guangdong, China. 4Department of Gastrointestinal Surgery, TheFirst Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.5Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University,Guangzhou, Guangdong, China. 6Department of Medicine, Columbia UniversityIrvingMedical Center, NewYork, NewYork. 7Department of Gastric Surgery, SunYat-sen University Cancer Center, Guangzhou, Guangdong, China. 8Departmentof Gastrointestinal Surgery, The Second Affiliated Hospital of GuangzhouUniversity of Chinese Medicine, Guangzhou, Guangdong, China. 9Departmentof Gastrointestinal Surgery, The Eighth Affiliated Hospital, Sun Yat-sen Univer-sity, Shenzhen, Guangdong, China.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

R. Lin, H. Zhang, and Y. Yuan contributed equally to this article.

Corresponding Authors: Weiling He, Department of Gastrointestinal Surgery,The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong510080, China. Phone: 86-20-87606865; E-mail: [email protected];Shirong Cai, Phone: 86-20-87755766; E-mail: [email protected]; andZunfu Ke, Institute of Precision Medicine, and Department of Pathology, TheFirst Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong510080, China. Phone: 86-20-87331780; E-mail: [email protected]

Cancer Immunol Res 2020;8:479–92

doi: 10.1158/2326-6066.CIR-19-0702

�2020 American Association for Cancer Research.

AACRJournals.org | 479

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

reversed by blocking PD-L1 on cancer cells. Anti–PD-L1 treatmentunleashed Trm cells specifically in the patient-derived xenograft(PDX) mice.

Materials and MethodsPatient samples

Primary tumor tissues were collected from patients with gastricadenocarcinoma with surgically resectable tumors in The First Affil-iated Hospital (cohort 1, n ¼ 180), Cancer Center, and The EighthAffiliated Hospital (cohort 2, n ¼ 152) of Sun Yat-sen University. Forcell isolation andmouse experiments, tumor tissues and blood sampleswere collected freshly from The First Affiliated Hospital, Sun Yat-senUniversity (cohort 3, n ¼ 43). Patients' clinical manifestations andlaboratory results were evaluated carefully, and patients with infectionor autoimmune diseases were excluded from this study. Demographicsof included patients (cohort 1, cohort 2, and cohort 3) are shown inSupplementary Tables S1–S3. This study was approved by the EthicsCommittee of The First Affiliated Hospital, Sun Yat-sen University.Consent was informed, and consent forms were obtained from allpatients. The studies were conducted in accordance with recognizedethical guidelines of the Declaration of Helsinki.

Cell isolationBlood samples were collected from patients with gastric adenocar-

cinoma. Peripheral blood mononuclear cells (PBMC) were isolated byFicoll–Hypaque through density gradient centrifugation. Freshly col-lected blood sampleswere dilutedwith PBS in a ratio of 1:1. The dilutedblood was then placed on top of Ficoll–Hypaque separation mediumcarefully, centrifuging at 1,500 rpm for 30minutes. Buffy coat layerwascollected and washed with PBS twice. To prepare single-cell suspen-sions, fresh tumor tissues were minced and digested with type Icollagenase (2 mg/mL, Sigma) and DNAse (50 U/mL) in RPMI1640 medium in 37�C. Digestion buffer was replaced with fresh bufferevery 30 minutes. Cells were then filtered through a cell strainer(70 mm) and washed with PBS. CD8þCD103Hi, CD8þCD103Med, orCD8þCD103Neg cells were sorted from tumor-infiltrating lympho-cytes (TIL) using a BD FACS Influx. CD8þ T cells were purified fromPBMCs by negative selection using the EasySep human total CD8þ

T Cell enrichment kit according to the manufacturer's instructions(STEMCELL Technologies Inc.; Catalog#19053). Cell purity waschecked by fluorescence-activated cell sorting meter (FACS; >96%,BD FACS Influx).

Autologous tumor cells were prepared as described previously (19).Resected gastric adenocarcinoma tumor samples were immediatelyprocessed into single-cell suspensions by mechanical dissociation andenzymatic digestion. Tumor tissues were minced and incubated withcollagenase I (2 mg/mL) and DNAse type I (50 U/mL). Tumor cellsdigested from tissue were filtered through a cell strainer. CD45�

population (nonhematopoietic) was further sorted by FACS from thetumor digest and used as autologous tumor cells. The staining detailsfor cell sorting were described below in the Flow cytometry section.

Circulating tumor cell enrichment and analysisCirculating tumor cells (CTC) were separated by the NanoVelcro

system (Cytolumina) as previously described (20). A total amount of5 ml blood specimens was collected from each of the patients withgastric adenocarcinoma, and the samples were processed within24 hours. Red blood cells were removed by incubating cells withred blood cell lysing buffer (BioLegend) at room temperature for5 minutes. Cells were then washed with PBS twice. IHC was applied to

visualize the captured cells on SiNW substrate. The captured cells werestained with 40, 6-diamidino-2-phenylindole (DAPI, nuclear marker),TRITC-conjugated anti-CD45 antibody (WBC marker; Abcam;1:400), and FITC-conjugated anti-CK antibody (cancer cell marker;Abcam; 1:100). Characteristic phenotypes and morphology of CTCswere scrutinized by an experienced pathologist. CTCs were identifiedby combination of the following criteria: positive staining of anti-CKand DAPI, and negative staining of anti-CD45.

Cell cultureThe human gastric cancer cell line SGC7901 and normal gastric

epithelial cell line GES-1 were obtained from the Type CultureCollection of Chinese Academy of Sciences in 2015 (Shanghai, China;ref. 21). These cells were authenticated and certified by cell viabilityanalysis, short tandem repeat profiling, and isoenzyme analysis, andwere also screened for mycoplasma contamination by Type CultureCollection of Chinese Academy of Sciences. Cells were not reauthen-ticated. Cells were grown in complete culture medium RPMI 1640supplemented with 10% FBS, 50 U/mL penicillin, and 50 mg/mLstreptomycin in a humidified atmosphere at 37�Cwith 5%CO2. Thesecells were cultured for a maximum of 15 passages after thawing fromour stocks that were frozen at passage 3.

Flow cytometryCells were isolated from tumor tissue of patients with gastric

adenocarcinoma as described above and were stained with thefollowing antibodies: FITC-conjugated anti-CD45, phycoerythrin-conjugated anti-CD3, APC-conjugated anti-CD8, BV421-conjugatedCD69, PE-CY7–conjugated CD103, BV605-conjugated PD-1, PE-conjugated TIGIT, FITC-conjugated CD39, PerCP5.5-conjugatedCD26 (all were 1:100 and purchased from BioLegend). Cells wereincubated with antibodies at 4�C for 30minutes and then washed withPBS twice. SGC7901 and GES-1 were stained with primary antibodiesagainst fatty acid binding protein (Fabp) 4 (1:200) and Fabp5 (1:200;both from Abcam) at 4�C overnight. Cells were then washed with PBStwice and incubated with Alexa Fluor 488–conjugated anti-mouse IgG(1:1,000) or Alexa Fluor 555–conjugated goat anti-rabbit IgG (1:1,000;Life Technologies) at 4�C for 30 minutes. Cells were then washed withPBS twice and analyzed by flow cytometry.

To measure cytokine production in T cells, cells were stimulatedwith 500 ng/mL phorbol myristate acetate, 1 mg/mL ionomycin, and5mg/mLBrefeldinA (Sigma-Aldrich) for 5 hours at 37�Cwith 5%CO2.Cells were collected, permeabilized, and fixed using the Fixation/Permeabilization Solution (BD Bioscience) on ice for 30 minutes.Cells were then washed with Perm/Wash Buffer and stained withV450-conjugated anti-IFNg (BD Biosciences) and FITC-conjugatedanti-TNFa antibodies (BioLegend) in Perm/Wash Buffer at 4�C for30 minutes. Cells were washed twice with Perm/Wash Buffer andanalyzed by flow cytometry.

To measure T-cell apoptosis, cells were stained with Pacific Blue–conjugated Annexin V and 7-AAD (BioLegend). Samples were ana-lyzed using a BD FACS ARIA (BD Bioscience). FlowJo (Tree Star)software was used for data analysis.

TransfectionOne day before the transfection, SGC7901 cells were seeded on

24-well culture plates. Fabp4, Fabp5, PD-L1 siRNA, and scramblesiRNA were purchased from Santa Cruz Biotechnology (Catalog#,sc-43592, sc-41237, and sc-39699). siRNA oligomers were diluted inOpti-MEM I Reduced Serum Medium without serum to a finalconcentration of 50 nmol/L. Lipofectamine 2000 (1 mL) was diluted

Lin et al.

Cancer Immunol Res; 8(4) April 2020 CANCER IMMUNOLOGY RESEARCH480

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

with Opti-MEM I Reduced SerumMedium (50 mL) and incubated for5 minutes at room temperature. The diluted oligomer was then mixedwith the diluted Lipofectamine 2000 and added to each well. Cells wereincubated at 37�C with 5% CO2. The siRNA–Lipofectamine complexwas removed after a 6-hour incubation, and the cells were cultured at37�C with 5% CO2 overnight in RPMI 1640 medium supplementedwith 10% FBS.

CocultureSorted CD8þCD103Hi, CD8þCD103Med, or CD8þCD103Neg T cells

were cocultured with autologous tumor cells or SGC7901 cells in48-well plates at a ratio of 5:1 in RPMI 1640 medium supplementedwith 10% FBS at 37�C with 5% CO2. T cells were stimulated with anti-CD3/CD28 antibodies (BioLegend) according to the manufacturer'sinstructions. PD-L1–blocking antibody (Bio X Cell, 1 mg/mL) wasincluded in some experiments. T cells were collected to determineapoptosis and lipid uptake by flow cytometry.

Cytotoxicity assayIn vitro cytotoxicity assays were performed by coculturing T cells

and cancer cells as described previously (22, 23). Sorted CD8þ T cellswere cocultured with autologous cancer cells for 16 hours at a ratio of4:1. After coculture, adherent and nonadherent cells were collected,stained with 7-AAD, and analyzed by flow cytometry to determine thenumber of dead tumor cells. The killing percentage was calculated bythe percentage of 7-AADþ cancer cells.

Western blotCells were lysed with RIPA Buffer including 1% v/v Halt Protease/

Phosphatase Inhibitor (Thermo Fisher Scientific). The BCA proteinassay (Thermo Fisher Scientific) was used for quantitation of totalprotein. A total of 30 mg protein was loaded on SDS–polyacrylamidegels (4% and 15%) and electrotransferred onto polyvinylidene difluor-ide membranes after separation. Membranes were blocked with 10%BSA in Tris-Buffered Saline Tween-20 buffer and incubated with anti-CD36 (1:1,000; Abcam) and anti–b-actin (1:2,000; Cell SignalingTechnology) primary antibodies at 4�C overnight. The membraneswere then washed with Tris-Buffered Saline Tween-20 and incubatedwith horseradish peroxidase–conjugated anti-rabbit IgG (1:2,500; CellSignaling Technology) at room temperature for 60 minutes. Signalswere detected by enhanced chemiluminescence (Thermo Fisher Sci-entific) with the Odyssey Fc imaging system (LI-COR Bioscience).

ImmunometabolismTo measure lipid uptake or glucose uptake, cells were incubated at

2 � 105 cells in complete medium containing either 1mmol/L Bodipy500 (Thermo Fisher) or 20mmol/L 2-NBDG (Thermo Fisher), for30minutes at 37�C in a cell incubator. To measure lipid content in thecells, Bodipy 493 (1mg/mL, Thermo Fisher) was added and incubatedfor 1 hour at 4�C. Cells were analyzed by flow cytometry. To furtherdetail the metabolic alterations in Trm cells, a Seahorse assay wasperformed to measure glycolysis and mitochondria oxygen consump-tion. T cells were incubated in a CO2-free incubator in RPMI 1640medium supplemented with glucose (20 mmol/L) and sodium pyru-vate (1 mmol/L) for 30 minutes. Measurements were performedusing an XF96 extracellular analyzer (Seahorse Bioscience). The Sea-horse XF Cell Mito Stress Test Kit was used for the measurement ofmitochondrial oxygen consumption rate. During the measurements,cells were treated with oligomycin (1 mmol/L), carbonyl cyanidep-trifluoromethoxyphenylhydrazone (1.5 mmol/L), and rotenone/antimycin A (1 mmol/L), respectively, as indicated.

ELISATo measure cytokines released by T cells, culture supernatant was

collected for the measurement of IFNg and TNFa using commercialELISA kits (Catalog# DIF50 and DTA00D) according to the manu-facturer's instructions (R&D Systems).

Immunofluorescence and immunohistochemistryParaffin-embedded tissues were cut into 4 mm sections. The slides

were deparaffinized with Xylene and rehydrated in 100% ethanol, 95%ethanol, 70% ethanol, and 50% ethanol sequentially. Antigen retrievalwas performed in a pressure cooker using Antigen Retrieval Buffer(Citrate Buffer pH 6.0). Slides were blocked with 5% BSA in PBS andthen incubated with primary antibody against CD8 (1:1,000) andCD103 (1:200; Abcam) at 4�C overnight. Slides were washed with PBStwice and incubated with Alexa Fluor 555–conjugated goat anti-rabbitIgG (1:500) and Alexa Fluor 488–conjugated anti-mouse IgG (1:500;Life Technologies) at room temperature for 30 minutes. Sections werecounterstained with DAPI in mounting medium. For IHC staining,slides were stained with primary antibody against CD103 (1:200) at4�C overnight. The sections were then incubated with an horseradishperoxidase–conjugated secondary antibody (1:500) for 1 hour at roomtemperature. Peroxidase was visualized with 3,30 diaminobenzidine,and the slides were counterstained with hematoxylin. Slides wereexamined using fluorescence microscopy (Zeisse), and the numberof CD8þCD103þ T cells was counted from 5 different high-powerareas.

Humanized PDX tumor modelSix- to 8-week-old NOD.Cg-PrkdcscidIl2rgtm1Sug/JicCrl (NOG)

mice (Vital River) were used to establish the PDX mouse modelas described previously (24). Tumor specimens of gastric adenocar-cinomawere collected and snap frozen in optimal cutting temperature.Tissue blocks were sectioned (4 mm) and fixed in precold acetone at4�C for 10 minutes. Tissue slides were then washed with PBS twiceand stained with Mayer hematoxylin solution for 5 minutes. Slideswere washed in warm running tap water for 10 minutes andcounterstained in eosin for 30 seconds. Slides were mounted withxylene-based mounting medium. Immune infiltrates in the TMEwere evaluated under light microscopy (Supplementary Fig. S1;Zeiss). Upon arrival, necrotic and supporting tissues were carefullyremoved using a surgical blade. Approximately 20 to 30 mg tissuefragments with immune infiltrates were implanted subcutaneouslyinto the flank region of NOG mice. Successful established mousemodels were monitored, and engrafted tumors were collected foranalysis. Established PDX mice were treated with an anti–PD-L1antibody (5 mg/kg) or isotype control (Bio X Cell). The mice weremonitored 3 times per week for evidence of morbidity and mortalityassociated with tumor growth and metastasis. All animal proce-dures were conducted in accordance with, and with the approval of,the Institute Animal Care and Use Committee of Sun Yat-senUniversity.

Statistical analysisData were presented as mean � SEM. Statistical analysis was

performed using SPSS (version 13.0 for windows; SPSS). Comparisonswere assessed using either the Student t test, paired Student t test, orone-way ANOVA with or without repeated measurements followedby Bonferroni multiple comparison posttest, as appropriate. The Coxunivariate and multivariate analyses were used to explore the influ-ences of different prognostic factors on overall survival. P values < 0.05were considered as statistically significant.

Tissue-Resident Memory T Cells in Gastric Adenocarcinoma

AACRJournals.org Cancer Immunol Res; 8(4) April 2020 481

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

ResultsTrm predicted better survival in gastric adenocarcinoma

Trm cells are associated with better survival in lung cancer andovarian cancer (17, 25). In this study, we investigated the role and thepredictive potential of Trm cells in gastric adenocarcinoma. Trm cellswere identified firstly in the TME of gastric adenocarcinoma by dualstaining of CD8 and CD103. It revealed that CD8þCD103þ Trm cellswere detectable in the TME of gastric adenocarcinoma (Fig. 1A), andCD103þ Trm cells were more abundant in primary tumor comparedwithmetastatic tumor as quantified by IHC (Fig. 1B). To further defineand assess Trmcells in gastric adenocarcinoma, single-cell suspensionsfrom tumor tissues were analyzed by flow cytometry. Trm cells wereidentified as CD69-positive or CD103-positive, or both. Because thevast majority of CD103þ T cells were simultaneously CD69þ (Fig. 1C;Supplementary Fig. S2), Trm cells were defined as CD103þ orCD69þCD103þ in this study. Enhanced frequency of Trm cells wasconfirmed in tumor withoutmetastasis asmeasured by flow cytometry(Fig. 1C and D).

To further investigate the tissue residency of Trm in the TME, aPDX tumor-bearingmousemodel was established by engrafting tumortissues from patients with gastric adenocarcinoma into the immuno-deficient mice. By gating on human CD45þCD8þ population, wefound that the CD69þCD103þ Trm cells resided in the TME and didnot recirculate back to the blood. The population of CD8þ T cells thatrecirculated back to the blood is CD69�CD103� (Fig. 1E and F;Supplementary Fig. S2). We next assessed the relationship betweenTrm cells and patient survival. CD103þ Trm cells were quantified (>5CD103þ TILs per 0.6 mm core) to stratify the cohort into high-densityversus low-density subsets as previously described (25). Using thisstrategy, we found that the presence of high-density CD103þTrm cellspositively correlated with survival in two cohorts of patients withgastric adenocarcinoma (Fig. 1G). The number of Trm cells wasnegatively correlated with the number of CTCs (SupplementaryFig. S3), indicating the potential function of Trm cells in suppressingtumor metastasis. Both univariate analysis and multivariate analysisperformed using the COX proportional hazard regression modelshowed that the number of CD103þ Trm cells was an independentprognosis-related marker for gastric adenocarcinoma (SupplementaryTables S4 and S5).

Tumor reactivity of Trm cells in gastric adenocarcinomaTILs highly express high immune inhibitory molecules (26). To

understand the expression patterns of the immune inhibitorymolecules in Trm cells, we evaluated the expression of PD-1, TIGIT,CD39, and CD26 in Trm cells from gastric adenocarcinoma speci-mens by flow cytometry (Fig. 2A and B). We found that theexpression of PD-1, TIGIT, and CD39 correlated with that ofCD103. CD103Hi subpopulation expressed the highest amountof inhibitory molecules, whereas CD26 expression did not correlateto that of CD103 (Fig. 2B–F). High PD-1/PD-L1 in the TMEpredicts better immune response and outcomes in patients withcancer to checkpoint blockade therapy (27). To investigate theantitumor response by Trm cells, we sorted the CD103Hi,CD103Med, and CD103Neg populations and cultured the cells withor without autologous tumor cells. We found that cytokine pro-duction of TILs from patients with gastric adenocarcinoma corre-lated with CD103 expression. CD103Hi cells produced loweramounts IFNg and TNFa when cultured alone as measured byELISA. On the contrary, CD103Hi cells produced higher amountsIFNg and TNFa in the supernatant when cultured with autologoustumor cells (Fig. 2G). These results were further confirmed by

intracellular cytokine production measured by flow cytometry(Fig. 2H and I). These data suggested that Trm cells are tumor-reactive TILs.

Increased lipid uptake and metabolism in Trm cellsMetabolic regulation is critical for T-cell activation and effector

functions (28). Specifically, lipid metabolism enhances CD8þ T-cellmemory (29). To understand themetabolic status of Trm cells, we firstmeasured the expression of CD36, which imports lipids into thecells (30), in CD103Hi, CD103med, and CD103Neg populations byWestern blot. CD103Hi Trm cells had the highest expression of CD36when compared with CD103Med and CD103Neg Trm cells (Fig. 3A).Along with increased CD36 expression, lipid content (measured byBodipy 493) was dramatically increased in CD103Hi Trm cells(Fig. 3B). In addition, by loading the cells with Bodipy 500 to measurelipid uptake, CD103Hi cells showed a clear increase in lipid uptake(Fig. 3B,D, and E). However, glucose uptake (measured by 2-NBDG)of CD103Hi Trm cells decreased when compared with CD103med orCD103Neg Trm cells (Fig. 3B and C). To further detail the metabolicalterations in Trm cells, a Seahorse assay was performed to measureglycolysis andmitochondria oxygen consumption. CD103Hi Trm cellsshowed decreased extracellular acidification rate (ECAR; Fig. 3F) andincreased oxygen consumption rate (OCR; Fig. 3G). CD103Hi Trmcells had significantly higher basal metabolic activity, ATP-coupledOCR, stronger spare mitochondrial capacity, and maximum respira-tory (Fig. 3H–K). These data implied that Trm cells underwent ametabolic reprogram and switched to mitochondria fatty acid oxida-tion to meet their energy requirements.

Lipid metabolism was required for the survival of Trm fromgastric adenocarcinoma

CD8þTrm cells tend to utilize mitochondrial fatty acid oxidation tosupport both their longevity and protective function (31). In this work,the role of lipid metabolism in the maintenance of Trm cells of gastricadenocarcinoma was explored. CD103Hi Trm cells showed increasedapoptotic rates in vitro (Supplementary Fig. S4), which can be reversedby supplementing free fatty acids (FFA). However, FFAs did notchange the apoptosis status of CD103Neg cells (Fig. 4A and B). FFAsalso enhanced cytokine production by CD103Hi Trm cells (Fig. 4CandD). Seahorse assay showed FFAs did not affect ECAR by CD103Hi

Trmcells. OCRbyCD103Hi Trm cells was significantly increasedwhenFFA was supplemented (Fig. 4E and F). These data suggested animportant role of lipid metabolism in Trm cell survival by improvingmitochondria activities in CD103Hi Trm cells. To further confirm thecontribution of lipidmetabolism to the survival of Trm cells in vivo, weestablished a PDX mouse model by engrafting tumor specimens frompatients with gastric adenocarcinoma. PDX mice were treated withetomoxir, which inhibits fatty acid oxidation (Fig. 4G). Consistentwith in vitro data, Etomoxir decreased the percentage of Trm cells inthe TME (Fig. 4H and I). The total number of Trm cells was reduceddramatically by etomoxir (Fig. 4J), whereas the number of non-Trmcells in the TME was not affected by etomoxir treatment (Fig. 4K).These data demonstrated that FFAs improved mitochondria functionof CD103Hi Trm cells, which was important for the survival of Trmcells.

Cancer cells deprive Trm cells of lipid uptake, inducing theirapoptosis

Metabolic shifting such as glucose deprivation in the TMEimpairs the antitumor effects of CD8þ T cells (32, 33). Thus, we nextexplored the metabolic alterations in the TME and the metabolic

Lin et al.

Cancer Immunol Res; 8(4) April 2020 CANCER IMMUNOLOGY RESEARCH482

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

Figure 1.

Trm cells in gastric adenocarcinoma. A, Immunofluorescence staining of CD8 (green) and CD103 (red) in tissue sections from gastric adenocarcinoma. Slides werecounterstained with DAPI. Representative images are shown. B, Tissue sections from primary or local metastasized gastric adenocarcinoma were stained with anti-CD103 antibody. Slideswere counterstainedwith hematoxylin. Representative images are shown (n¼ 180).C,Fresh tumor tissues fromprimary or localmetastasizedgastric adenocarcinomawere collected, and single-cell suspensionwas prepared. Cells were stainedwith antibodies against CD8, CD69, and CD103 and analyzed byflow cytometry. Representative counter plots gated on CD8þ cells are shown. FMO, fluorescenceminus one.D, Percentages of CD8þCD69þCD103þ T cells in primary(n¼ 24) or local metastasized (n¼ 8) gastric adenocarcinoma (mean� SEM, t test). E, Fresh collected tumor tissues from gastric adenocarcinoma were engraftedintoNOGmice to establish a PDXmodel. F,Mouse blood and tumorswere collected 3weeks after engraftment. Single cellswere prepared and analyzed for CD69 andCD103 expression on CD45þCD8þ T cells by flow cytometry. Representative counter plots of eight experiments are shown. G, Kaplan–Meier plots representing theprobability of overall survival (OS) in cohort 1 and cohort 2. ��� , P < 0.001.

Tissue-Resident Memory T Cells in Gastric Adenocarcinoma

AACRJournals.org Cancer Immunol Res; 8(4) April 2020 483

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

reprogramming in Trm cells fromTME of gastric adenocarcinoma. Bycoculturing CD103Hi and CD103Neg T cells with gastric adenocarci-noma cells, we found that gastric adenocarcinoma cells increased theapoptosis of CD103Hi Trm cells, whereas the CD103Neg T cells wereunaffected (Fig. 5A and B). Because lipid metabolism was importantfor Trm cells' survival (Fig. 4), we hypothesized that gastric adeno-carcinoma cells deprive lipid uptake of Trm cells and induce Trm cellapoptosis. We thus measured the expression of Fabp4 and Fabp5 ingastric adenocarcinoma specimens by Western blot. We found thatFabp4 and Fabp5 expression significantly increased in gastric adeno-carcinoma tumors when compared with para-cancer normal gastrictissue (Fig. 5C). Fabp4 and Fabp5 expression was also higher in gastricadenocarcinoma cells (SGC-7901) compared with healthy gastricepithelial cells (GES-1) as measured by flow cytometry (Fig. 5Dand E). In contrast to GES-1, lipid uptake increased in SGC-7901(Fig. 5F and G). Our previous study also shows gastric tumor cellsoutcompete CD8þ T cells for glucose consumption (34). Next, weinvestigated whether lipidmetabolism in cancer cells would affect lipiduptake and apoptosis of Trm cells. The results revealed that lipid

uptake by CD103Hi Trm cells was reduced when cocultured withcancer cells (Fig. 5H and I). In the tumor cell–T-cell coculture system,lipid uptake in Trm cells obviously increased with knockdown of bothFabp4 and Fabp5 in cancer cells when compared with knockdown ofeither Fabp4 or Fabp5 (Fig. 5J andK). Knockdown of Fabp4 or Fabp5in cancer cells decreased the apoptosis of Trm cells. And knockdown ofFabp4 and Fabp5 together further decreased the apoptosis of Trm cells(Fig. 5L and M). These data demonstrated that gastric adenocarci-noma cells could induce Trm cells' apoptosis by depriving lipid uptakeby Trm cells, which further confirmed that FFAs alone did not affectthe Trm cells in the TME (Supplementary Fig. S5).

PD-L1 regulated lipid competition in the TMEPD-1 altered T-cell metabolic reprogramming by inhibiting glucose

glycolysis and increasing mitochondria fatty acid oxidation (35). Weinvestigated if PD-L1, the ligand of PD-1, affects lipid uptake andmetabolism in cancer cells and Trm cells. In the tumor cell–T-cellcoculture system, we found that PD-L1 blockade decreased Fabp4/5expression in tumor cells, whereas Fabp4/5 expression in T cells was

Figure 2.

Tumor reactivity of Trm cells in gastricadenocarcinoma. A, Single-cell suspen-sions from gastric adenocarcinomawereanalyzed by flow cytometry. Gatingstrategy of CD8þCD103þ cells accordingto CD103 expression for subpopulationanalysis. Representative histogramplotsof gastric adenocarcinoma specimensare shown. B, Coexpression of PD-1,TIGIT, CD39, and CD26 on CD103 sub-populations was analyzed by flow cyto-metry. Representative histograms areshown. C–F, PD-1, TIGIT, CD39, andCD26 expression on CD103 subpopula-tions was summarized from 12 samples.CD103 subpopulations were FACSsorted. Cells were cultured with orwithout autologous tumor cells. Tumorreactivity of CD103 subpopulations wasevaluated by measuring IFNg and TNFain supernatant by ELISA (G) or intracel-lular cytokine measuring by flow cyto-metry (H and I). Data are mean � SEM.� , P < 0.05; �� , P <0.01; and ��� , P <0.001by one-way ANOVA. MFI, mean fluores-cence intensity; ns, not significant.

Lin et al.

Cancer Immunol Res; 8(4) April 2020 CANCER IMMUNOLOGY RESEARCH484

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

Figure 3.

Glucose and lipid metabolism in Trm cells from gastric adenocarcinoma. A, Single-cell suspensions were prepared from gastric adenocarcinoma–derivedtumor specimens. CD103 subpopulations were FACS sorted. CD36 expression on the CD103þ subpopulations was measured by Western blot. Representativebands are shown. B, Sorted CD103þ subpopulations were incubated with 2-NBDG (glucose uptake), Bodipy 493 (lipid content), and Bodipy 500 (lipid uptake).Cellswere analyzed by flow cytometry, and representative histograms are shown.C–E,Data are summarized from 12 samples. F andG, Sorted CD103 subpopulationswere analyzed with a Seahorse Bioscience XF96 analyzer. Glycolytic activity was measured by ECAR (F), and mitochondrial activities were measured by OCR (G).Summarized baseline respiration (H), respiration coupled to ATP production (I), respiratory spare capacity (J), andmaximal (Max.) respiration (K). Data aremean�SEM. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001; and ���� , P < 0.0001 by one-way ANOVA. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; MFI, meanfluorescence intensity; min, minute(s); ns, not significant.

Tissue-Resident Memory T Cells in Gastric Adenocarcinoma

AACRJournals.org Cancer Immunol Res; 8(4) April 2020 485

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

Figure 4.

Lipid metabolism regulated survival of Trm cells from gastric adenocarcinoma. A and B, CD103Hi and CD103Neg cells were cultured in the presence or absence ofpalmitate (Palm, fatty acid) or vehicle for 24 hours. The apoptotic rate was measured by flow cytometry. Data are from six experiments. C and D, CD103Hi cells werecultured in the presence or absence of Palm. IFNg and TNFa productionwasmeasured by flow cytometry. Representative count plots are shown. E and F, ECAR andmitochondrial activities were measured by OCR by Seahorse Bioscience XF96 analyzer. G, PDX was established as in Fig. 1. Mice were treated with etomoxir (CPT1inhibitor) for 1 week. CD8þCD69þCD103þ cells in TILs were measured by flow cytometry. H, Representative counter plots of CD8þCD69þCD103þ cells are shown.Percentages (I) and number (J) of CD8þCD69þCD103þ cells in the tumor tissue. K, Number of CD103� CD8þ T cells in the tumor tissue. Data are mean� SEM. � , P <0.05; ��, P < 0.01; and ��� , P < 0.001 by t test. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; min, minute(s); ns, not significant.

Lin et al.

Cancer Immunol Res; 8(4) April 2020 CANCER IMMUNOLOGY RESEARCH486

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

Figure 5.

Tumor cells deprived Trm cells of lipid uptake and induced their apoptosis.A, CD103Hi and CD103Neg subpopulations were sorted as in Fig. 2. CD103Hi and CD103Neg

were cocultured with gastric cancer cells (SGC7901) at a ratio of 5:1 for 24 hours. Cells were stained with Annexin V and 7-AAD. Apoptosis was measured by flowcytometry. B, Percentages of apoptosis summarized from 6 samples. C, Expressions of Fabp4 and Fabp5 in gastric adenocarcinoma and normal gastric tissue weremeasured by Western blot. Representative bands are shown. D and E, Fabp4 and Fabp5 expression in SGC7901 and normal gastric epithelial cells (GES-1) wasmeasured by flow cytometry. Representative histograms and data are from three experiments. F and G, SGC7901 and GES-1 cells were incubated with Bodipy 500,and the lipid uptake was measured by flow cytometry. Representative histograms and data are from three experiments. H and I, CD103Hi cells were cultured with orwithout SGC7901 and with Bodipy 500. Lipid uptake was measured by flow cytometry. Representative histograms and data are from three experiments. J and K,CD103Hi cells were cocultured with SGC7901 that were Fabp4, Fabp5, or dual knockdown. Lipid uptake was measured by incubating cells with Bodipy 500 andanalyzed by flow cytometry. Representative histograms and data were from three experiments. L and M, CD103Hi cells were cocultured with SGC7901 that wereFabp4, Fabp5, or dual knockdown for 24 hours. Cells were stained with Annexin V and 7-AAD for apoptosis analysis by flow cytometry. Representative histogramsand data are from three experiments. Data aremean� SEM. � ,P <0.05; �� , P <0.01; ��� , P <0.001; and ����, P <0.0001 by t test inB,E,G, and I and one-wayANOVA inK and M. KD, knockdown; MFI, mean fluorescence intensity; ns, not significant.

Tissue-Resident Memory T Cells in Gastric Adenocarcinoma

AACRJournals.org Cancer Immunol Res; 8(4) April 2020 487

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

increased when PD-L1 was blocked (Fig. 6A and B). Lipid uptake bycancer cells was reduced by blocking PD-L1 (Fig. 6C and D). Impor-tantly, lipid uptake in CD103Hi Trm cells was increased when PD-L1was blocked (Fig. 6E and F). We confirmed these data by knockingdown PD-L1 expression in tumor cells. Knockdown of PD-L1 expres-sion in tumor cells decreased lipid uptake by tumor cells and increasedlipid uptake in CD103Hi Trm cells (Supplementary Fig. S6). Lipiduptake by CD103Hi Trm cells was decreased when PD-1 was knockeddown (Supplementary Fig. S7). Meanwhile, the apoptotic rate ofCD103Hi Trm cells was decreased when PD-L1 was blocked(Fig. 6G and H). These data indicated that the inhibition of PD-L1in the TME could improve the survival of Trm cells and enhance

antitumor immune response. To further confirm this result, the PDXmodel was established as described in Fig. 1, and the tumor-bearingmice were treated with PD-L1–blocking antibody (Fig. 6I). After thetreatment, Trm cells in the TMEwere analyzed by flow cytometry. Wefound that the percentage of Trm cells was increased after PD-L1blockade. The absolute number of Trm cells was also increased byblocking PD-L1 in the PDX mice (Fig. 6J and K).

PD-L1 blockade unleashed Trm cells and their antitumor effectsThe success of anti–PD-1/PD-L1 in treatment of cancers has led to a

paradigm shift in the oncology field (36). We investigated how Trmcells were involved in antitumor immune response. We first tested

Figure 6.

PD-L1 blockade promoted Trm cell sur-vival. Gastric cancer cells (SGC7901)were treated with IFNg (10 ng/mL) for24 hours. CD103Hi CD8þ T cells werethen sorted by flow cytometry andcocultured with SGC7901 in the pres-ence of anti–PD-L1 antibody or isotypecontrol for 24 hours. A and B, Fabp4and Fabp5 expression in tumor cellsand T cells was measured by flowcytometry. Representative histogramsare shown, and data are from 5 patientsamples. C–F, Cells were incubatedwith Bodipy 500, and lipid uptake bytumor cells and T cells was assessed byflow cytometry. Representative histo-grams and data are from five experi-ments. G and H, Annexin V expressionin T cells was measured by flow cyto-metry. Representative histogramsgated on CD8þ cells and data arefrom 5 patient samples. I, PDX modelwas established as in Fig. 1. PDX-bearing mice were then treated withanti–PD-L1 or isotype for 2 weeks. Jand K, Single-cell suspension wasprepared from tumor xenografts,and CD8þCD69þCD103þ T cells in TILswere analyzed by flow cytometry. Per-centages (J) and cell numbers (K) areshown (n¼ 12). Data are mean� SEM.� , P < 0.05 and �� , P < 0.01 by t test inB, D, F, and H and paired t test in J andK. MFI, mean fluorescence intensity.

Lin et al.

Cancer Immunol Res; 8(4) April 2020 CANCER IMMUNOLOGY RESEARCH488

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

cytotoxicity of Trm cells to tumor cells. We found that CD103Hi Trmcells showed more effectively induced tumor cell death when com-pared with CD103Neg cells (Fig. 7A and B). In addition, PD-L1blockade enhanced cytotoxic function of CD103Hi Trm cells mostly(Fig. 7C and D). To further study CD103Hi Trm cells in antitumorimmune response in vivo, a PDX model was established as in Fig. 1.The tumor-bearing mice were treated with anti–PD-L1 antibody orisotype control (Fig. 7E). IFNg production in TILs was measured byflow cytometry after the treatment. We found that PD-L1 blockademainly affected the CD103Hi Trm cells through stimulating IFNgproduction, whereas there seemed to be no evident effects onCD103Neg (Fig. 7F andG).Wemonitored tumor growth bymeasuringthe tumor volume in mice treated with anti–PD-L1 antibody. SevenPDX mice responded to the anti–PD-L1 treatment, whereas tumorgrowth in 11 of the treated mice was not affected by anti–PD-L1treatment (Fig. 7H). The percentage of Trm cells was significantly

higher in the treatment-responsive mice compared with the nonre-sponsive mice (Fig. 7I and J). These data indicated that CD8þ

CD103Hi Trm cells contributed significantly to the antitumor immuneresponse to gastric adenocarcinoma.

DiscussionGastric cancer is one of the leading causes of cancer-related death.

There has been a paradigm shift in cancer therapy in the past 5 to10 years due to the successes of immunotherapy (37). Anti–CTLA-4and anti–PD-1/PD-L1 treatments represent the most successful ther-apeutics targeting immune system to cure cancer (38). Data fromclinical trials show the promising results using anti–PD-1/PD-L1antibody to treat gastric cancer (8, 9).However, due to the low responserate, there is an urgent need to develop novel strategies to improve thetherapeutic effect. In this study, we investigated the roles and the

Figure 7.

Cytotoxicity of Trm cells correlatedwithantitumor response to PD-L1 blockade.A and B, Autologous tumor cells werecocultured with or without CD103Hi andCD103Neg CD8þ T cells for 16 hours. Cellswere collected, and cell death was mea-sured by staining cells with 7-AAD. Rep-resentative FACS plots were gated onCD45� tumor cells. Data are from 5samples. C and D, Autologous tumorcells were cocultured with CD103Hi andCD103Neg CD8þ T cells in the presenceof anti–PD-L1 antibody or isotype con-trol for 16 hours. Cell death was mea-sured by staining cells with 7-AAD.Representative FACS plots were gatedon CD45� tumor cells. Data are from5 samples. E, A PDX model wasestablished and treated as in Fig. 6. Fand G, TILs from anti–PD-L1– orisotype-treated mice were measuredfor IFNg expression by flow cytometry.Percentages of IFNg-producing T cellsand representative counter plotsgated on CD103Hi and CD103Neg CD8þ

T cells (n ¼ 8). H, Tumor volume ofanti–PD-L1-treated mice. I and J,CD8þCD69þCD103þ cells in TILs fromanti–PD-L1 treatment-responsive (n ¼7) and nonresponsive (n ¼ 11) micewere analyzed by flow cytometry.Representative counter plots. Data aremean � SEM. � , P < 0.05; ��� , P < 0.001;and ���� , P < 0.0001 by one-wayANOVA in B, paired t test in D, andt test in G and J. ns, not significant.

Tissue-Resident Memory T Cells in Gastric Adenocarcinoma

AACRJournals.org Cancer Immunol Res; 8(4) April 2020 489

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

metabolic regulation of CD8þ Trm cells in gastric adenocarcinoma,focusing on the roles of fatty acid oxidation Trm cell death in gastricadenocarcinoma.We found that Trm cells were present in the TME ofgastric adenocarcinoma and predicted better survival. Fatty acidoxidation was required for the survival of Trm cells. Gastric adeno-carcinoma cells outcompeted Trm cells for lipid uptake, and PD-L1blockade reversed this competition, unleashing Trm cells, leading totumor regression.

Immune checkpoint blockade unleashes CD8þT cells and enhancestheir antitumor response in the TME (39). Trm cells are T cells thatreside in the tissue and provide stronger and faster immune responseduring antigen rechallenge (40). Trm cells can be harnessed to enhancethe efficacy of cancer vaccines (41). CD103þ Trm cells are present intheTMEandpredict better outcomes in breast cancer, lung cancer, andovarian cancer (16, 17, 25). In the current study, we first confirmed thepresence of CD103þ Trm cells in the TME of gastric adenocarcinomaby detecting the coexpression pattern of CD8 and CD103. Meanwhile,these cells were also CD69þ, which was in consistent with previousreports (17). Results from the PDX model proved that theCD69þCD103þ Trm cells were TILs that do not participate inlymphocyte recirculation. The percentage of CD8þ Trm cells waslower in patients with metastasis. Clinical data analysis showed thathigher density of Trm cell, as defined as >5 CD103þ TILs per 0.6 mmcore, was associated with better overall survival. We observed anegative correlation between the qualities of Trm cells and CTC,confirming the suppressive effect of Trm cells against metastasis.Together with the strong expression of the granzymes, perforin, andIFNg in Trm cells (17), these results demonstrate the critical role ofTrm cells in suppressing tumor growth and metastasis in gastricadenocarcinoma.

TILs highly express immune inhibitory molecules (42). To explorethe special status of Trm cells, we measured the expression of PD-1,TIGIT, and CD39 on TILs from patients with gastric adenocarcinoma.CD103Hi cells more highly expressed PD-1, TIGIT, and CD39. PD-1and TIGIT are highly expressed in TILs from gastric cancer (34).Coexpression of CD39 and CD103 identifies tumor-reactive T cells inhuman malignancies (43). CD39þ CD8þ T cells in human tumorinfiltrates are antigen specific, and CD8þ TILs without CD39 expres-sion could be defined as bystander (not responsive to tumors; ref. 44).The high CD39 expression on Trm cells indicates that Trm cells areantigen-specific T cells involved in the antitumor immune response. Inthis work, our results showed that CD103Hi Trm cells responded toautologous tumor cells by producing IFNg and TNFa, whereasCD103Neg T cells barely responded to autologous tumor cells. Fromthe PDX mouse experiments, we learned that the T cells that recircu-lated back to the blood were CD69�CD103�. Together, CD103Neg Tcells could be identified as the so-called bystander in the TME. Thesedata support the notion that Trm cells are the major effector T cells inresponse to cancer (43).

Energy demand is dramatically increased during T-cell activation,proliferation, and differentiation (28, 45). TILs display profoundmetabolic reprograming (19), whereas glucose uptake by cancer cellsoutcompetes TILs, which leads to impaired effector functions of CD8þ

T cells (33). Glucose deprivation suppresses effector functions of TILs,and metabolic reprogramming of TILs enhances their antitumorresponse (46). In the current study, we found that CD103Hi Trm cellsexpress the fatty acid translocase CD36. We also observed increasedlipid uptake and mitochondrial activities synchronous with decreasedglucose uptake and glycolytic activity in Trm cells. CD103Hi Trm cellsfrom TME of gastric adenocarcinoma displayed higher amounts ofapoptosis in in vitro culture, and FFAs rescued the Trm cells from

apoptosis. By inhibiting fatty acid oxidation, the proportions andquantities of Trm cells decreased in the TME of a PDX model forgastric adenocarcinoma. These findings are consistent with a previousreport showing that Trm cells rely on lipid metabolism for long-termsurvival (31). In the harsh TME, the supply of FFAs may be requiredfor Trm cell maintenance and long-term survival.

Due to the high proliferation rate, cancer cells rely on glucose togenerate energy in a fast but inefficient way through glycolysis (47).There is also an increased demand of lipids for the fast proliferatingcancer cells for energy production and newmembrane formation (48).Our study demonstrated that Fabp4/5 expression was increased ingastric adenocarcinoma cells and displayed increased lipid uptakecompared with healthy gastric epithelial cells. In a T-cell–tumor cellcoculture system, cancer cells suppressed the lipid uptake of Trm cellsand induced Trm apoptosis. These effects could be reversed byinhibiting cancer cells from lipid uptake. These results unearthed themetabolic competition between cancer cells and Trm cells for lipidconsumption, leading to the suppression of Trm cells.

PD-L1 is involved in the glucosemetabolismof cancer cells (33), andPD-L1 is expressed in the TME of all stages of gastric adenocarcino-ma (49), suggesting an important role of PD-L1 in the control ofmetabolism of gastric adenocarcinoma cells. However, the effects ofPD-L1 in the regulation of lipid metabolism remain unknown. Wefound that PD-L1 inhibition decreased Fabp4/5 expression in gastricadenocarcinoma cells, whereas Fabp4/5 expression was increased inthe T cells in the coculture system. The blockade of PD-L1 not onlysuppressed lipid uptake of cancer cells, but also rescued Trm cells fromapoptosis in the TME. These findings were verified in the PDXmodelstreated with anti–PD-L1 where an enrichment of Trm cells was seen.Taken together, cancer cells outcompete Trm cells for lipid uptakethrough PD-L1, which leads to the apoptosis of Trm cells in the TEM,dampening the antitumor immune response.

PD-1/PD-L1 blockade unleashes TILs and enhances antitumorresponses to suppress tumor from growing (50). We found thatCD103Hi Trm cells exhibited strong cytotoxic function in responseto autologous tumor cells and anti–PD-L1 enhanced the cytotoxicfunction of CD103Hi Trm cells. In the PDXmice treatedwith anti–PD-L1 antibody, the mice responding to the treatment showed dramat-ically higher percentage of Trm cells in the TME. In contrast, the micewith progressing tumors presented low frequencies of Trm. Check-point blockade targets tumor-specific T cells (51), and CD103þ TILsrepresent the tumor-reactive T cells (43). Trm cells contribute toantitumor immune response by targeting PD-1/PD-L1.

Taken together, our data suggested a distinct role for tumor-specificTrm cells in mediating antitumor immunity and predicting treatmentresponse to checkpoint blockade. Reprogramming lipidmetabolism ofTrm cells could be a promising therapeutic approach for gastricadenocarcinoma. Further investigation to explore the relevantmechanisms and potential clinical application targeting lipid metab-olism reprogramming of Trm cells is warranted.

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

Authors’ ContributionsConception and design: S. Cai, Z. Ke, W. HeDevelopment of methodology: W. HeAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): R. Lin, H. Zhang, Y. Yuan, Q. He, J. Zhou, S. Li, Y. Sun, H.-B. Qiu,W. Wang, Z. Zhuang, B. Chen, Y. Huang, C. Liu, Y. Wang, Z. KeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Lin, H. Zhang, Y. Yuan, D.Y. Li, W. He

Lin et al.

Cancer Immunol Res; 8(4) April 2020 CANCER IMMUNOLOGY RESEARCH490

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

Writing, review, and/or revision of the manuscript: R. Lin, H. Zhang, Y. Yuan,D.Y. Li, Z. Ke, W. HeAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): W. HeStudy supervision: S. Cai, Z. Ke, W. He

AcknowledgmentsThis work was supported by grants from the National Natural Science Foundation

of China (30900650, 81372501, 81572260, 81871994, and 81701834), GuangdongNatural Science Foundation (2011B031800025, S2012010008378, 2015A030313036,2017A010101030, and 2018A030310285), and the Guangzhou Science and Tech-

nology Planning Program (2014J4100132, 2015A020214010, 2012B031800115,2013B02180021, 2016A020215055, 201904010398, and 201902020018).

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

Received September 12, 2019; revised December 22, 2019; accepted February 14,2020; published first February 19, 2020.

References1. Ajani JA, Lee J, Sano T, Janjigian YY, Fan D, Song S. Gastric adenocarcinoma.

Nat Rev Dis Primers 2017;3:17036.2. Kim Y, Spolverato G, Ejaz A, Squires MH, Poultsides G, Fields RC, et al.

A nomogram to predict overall survival and disease-free survival after curativeresection of gastric adenocarcinoma. Ann Surg Oncol 2015;22:1828–35.

3. Sitarz R, Skierucha M, Mielko J, Offerhaus GJA, Maciejewski R, Polkowski WP.Gastric cancer: epidemiology, prevention, classification, and treatment.Cancer Manag Res 2018;10:239–48.

4. Drake CG, Lipson EJ, Brahmer JR. Breathing new life into immunotherapy:reviewofmelanoma, lung and kidney cancer.Nat RevClinOncol 2014;11:24–37.

5. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF,et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.N Engl J Med 2012;366:2443–54.

6. Barlesi F, Vansteenkiste J, Spigel D, Ishii H, Garassino M, de Marinis F, et al.Avelumab versus docetaxel in patients with platinum-treated advanced non-small-cell lung cancer (JAVELIN Lung 200): an open-label, randomised, phase 3study. Lancet Oncol 2018;19:1468–79.

7. Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, et al. Safety andtumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med2013;369:134–44.

8. KangYK, BokuN, SatohT, RyuMH,ChaoY,KatoK, et al. Nivolumab in patientswith advanced gastric or gastro-oesophageal junction cancer refractory to, orintolerant of, at least two previous chemotherapy regimens (ONO-4538–12,ATTRACTION-2): a randomised, double-blind, placebo-controlled, phase 3trial. Lancet 2017;390:2461–71.

9. Muro K, Chung HC, Shankaran V, Geva R, Catenacci D, Gupta S, et al.Pembrolizumab for patients with PD-L1-positive advanced gastric cancer(KEYNOTE-012): a multicentre, open-label, phase 1b trial. Lancet Oncol2016;17:717–26.

10. Mueller SN, Mackay LK. Tissue-resident memory T cells: local specialists inimmune defence. Nat Rev Immunol 2016;16:79–89.

11. Schenkel JM, Masopust D. Tissue-resident memory T cells. Immunity 2014;41:886–97.

12. Watanabe R, Gehad A, Yang C, Scott LL, Teague JE, Schlapbach C, et al.Human skin is protected by four functionally and phenotypically discretepopulations of resident and recirculating memory T cells. Sci Transl Med2015;7:279ra39.

13. Gebhardt T, Wakim LM, Eidsmo L, Reading PC, Heath WR, Carbone FR.Memory T cells in nonlymphoid tissue that provide enhanced local immunityduring infection with herpes simplex virus. Nat Immunol 2009;10:524–30.

14. Molodtsov A, Turk MJ. Tissue resident CD8 memory T cell responses in cancerand autoimmunity. Front Immunol 2018;9:2810.

15. Corgnac S, Boutet M, Kfoury M, Naltet C, Mami-Chouaib F. The emerging roleof CD8(þ) tissue resident memory T (TRM) cells in antitumor immunity: aunique functional contribution of the CD103 integrin. Front Immunol 2018;9:1904.

16. Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, et al. Single-cellprofiling of breast cancer T cells reveals a tissue-resident memory subsetassociated with improved prognosis. Nat Med 2018;24:986–93.

17. Ganesan AP, Clarke J, Wood O, Garrido-Martin EM, Chee SJ, Mellows T, et al.Tissue-resident memory features are linked to the magnitude of cytotoxic T cellresponses in human lung cancer. Nat Immunol 2017;18:940–50.

18. Park SL, Buzzai A, Rautela J, Hor JL, Hochheiser K, Effern M, et al. Tissue-resident memory CD8(þ) T cells promote melanoma-immune equilibrium inskin. Nature 2019;565:366–71.

19. Thommen DS, Koelzer VH, Herzig P, Roller A, Trefny M, Dimeloe S, et al.A transcriptionally and functionally distinct PD-1(þ) CD8(þ) T cell pool withpredictive potential in non-small-cell lung cancer treated with PD-1 blockade.Nat Med 2018;24:994–1004.

20. He W, Hou M, Zhang H, Zeng C, He S, Chen X, et al. Clinical significance ofcirculating tumor cells in predicting disease progression and chemotherapyresistance in patients with gestational choriocarcinoma. Int J Cancer 2019;144:1421–31.

21. Li G, Wang Z, Ye J, Zhang X, Wu H, Peng J, et al. Uncontrolled inflammationinduced by AEG-1 promotes gastric cancer and poor prognosis. Cancer Res2014;74:5541–52.

22. LaFleurMW,Nguyen TH, CoxeMA,Miller BC, Yates KB, Gillis JE, et al. PTPN2regulates the generation of exhausted CD8(þ) T cell subpopulations andrestrains tumor immunity. Nat Immunol 2019;20:1335–47.

23. WangW, GreenM, Choi JE, GijonM, Kennedy PD, Johnson JK, et al. CD8(þ) Tcells regulate tumour ferroptosis during cancer immunotherapy. Nature 2019;569:270–4.

24. Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, et al. High-throughput screening using patient-derived tumor xenografts to predict clinicaltrial drug response. Nat Med 2015;21:1318–25.

25. Webb JR, Milne K, Watson P, Deleeuw RJ, Nelson BH. Tumor-infiltratinglymphocytes expressing the tissue resident memory marker CD103 are associ-atedwith increased survival in high-grade serous ovarian cancer. ClinCancer Res2014;20:434–44.

26. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer 2012;12:252–64.

27. Nishino M, Ramaiya NH, Hatabu H, Hodi FS. Monitoring immune-checkpointblockade: response evaluation and biomarker development. Nat Rev Clin Oncol2017;14:655–68.

28. FoxCJ, Hammerman PS, ThompsonCB. Fuel feeds function: energymetabolismand the T-cell response. Nat Rev Immunol 2005;5:844–52.

29. Pearce EL, Walsh MC, Cejas PJ, Harms GM, Shen H, Wang LS, et al. EnhancingCD8 T-cell memory by modulating fatty acid metabolism. Nature 2009;460:103–7.

30. Silverstein RL, Febbraio M. CD36, a scavenger receptor involved in immunity,metabolism, angiogenesis, and behavior. Sci Signal 2009;2:re3.

31. Pan Y, Tian T, Park CO, Lofftus SY,Mei S, Liu X, et al. Survival of tissue-residentmemory T cells requires exogenous lipid uptake and metabolism. Nature 2017;543:252–6.

32. Scharping NE, Menk AV, Moreci RS, Whetstone RD, Dadey RE, Watkins SC,et al. The tumor microenvironment represses T cell mitochondrial biogenesis todrive intratumoral T cell metabolic insufficiency and dysfunction. Immunity2016;45:374–88.

33. Chang CH, Qiu J, O'Sullivan D, BuckMD, Noguchi T, Curtis JD, et al. Metaboliccompetition in the tumor microenvironment is a driver of cancer progression.Cell 2015;162:1229–41.

34. Chevrier S, Levine JH, Zanotelli VRT, Silina K, Schulz D, Bacac M, et al. Animmune atlas of clear cell renal cell carcinoma. Cell 2017;169:736–49.e18.

35. Patsoukis N, Bardhan K, Chatterjee P, Sari D, Liu B, Bell LN, et al. PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysisand fatty acid oxidation. Nat Commun 2015;6:6692.

36. Chen L, Han X. Anti-PD-1/PD-L1 therapy of human cancer: past, present, andfuture. J Clin Invest 2015;125:3384–91.

37. Ribas A. Releasing the brakes on cancer immunotherapy. NEngl JMed 2015;373:1490–2.

Tissue-Resident Memory T Cells in Gastric Adenocarcinoma

AACRJournals.org Cancer Immunol Res; 8(4) April 2020 491

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

38. Boutros C, Tarhini A, Routier E, Lambotte O, Ladurie FL, Carbonnel F, et al.Safety profiles of anti-CTLA-4 and anti-PD-1 antibodies alone and in combi-nation. Nat Rev Clin Oncol 2016;13:473–86.

39. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1blockade induces responses by inhibiting adaptive immune resistance. Nature2014;515:568–71.

40. Park CO, Kupper TS.The emerging role of resident memory T cells in protectiveimmunity and inflammatory disease. Nat Med 2015;21:688–97.

41. Nizard M, Roussel H, Diniz MO, Karaki S, Tran T, Voron T, et al. Induction ofresident memory T cells enhances the efficacy of cancer vaccine. Nat Commun2017;8:15221.

42. Baumeister SH, Freeman GJ, Dranoff G, Sharpe AH. Coinhibitory pathways inimmunotherapy for cancer. Annu Rev Immunol 2016;34:539–73.

43. Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in humansolid tumors. Nat Commun 2018;9:2724.

44. Simoni Y, Becht E, Fehlings M, Loh CY, Koo SL, Teng KWW, et al. BystanderCD8(þ) T cells are abundant and phenotypically distinct in human tumourinfiltrates. Nature 2018;557:575–9.

45. MacIver NJ, Michalek RD, Rathmell JC. Metabolic regulation of T lymphocytes.Annu Rev Immunol 2013;31:259–83.

46. Ho PC, Bihuniak JD, Macintyre AN, Staron M, Liu X, Amezquita R, et al.Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses.Cell 2015;162:1217–28.

47. Hsu PP, Sabatini DM. Cancer cell metabolism: Warburg and beyond. Cell 2008;134:703–7.

48. Rohrig F, Schulze A. The multifaceted roles of fatty acid synthesis in cancer.Nat Rev Cancer 2016;16:732–49.

49. Thompson ED, Zahurak M, Murphy A, Cornish T, Cuka N, AbdelfatahE, et al. Patterns of PD-L1 expression and CD8 T cell infiltration ingastric adenocarcinomas and associated immune stroma. Gut 2017;66:794–801.

50. Taube JM.Unleashing the immune system: PD-1 andPD-Ls in the pre-treatmenttumor microenvironment and correlation with response to PD-1/PD-L1 block-ade. Oncoimmunology 2014;3:e963413.

51. Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, et al.Checkpoint blockade cancer immunotherapy targets tumour-specific mutantantigens. Nature 2014;515:577–81.

Cancer Immunol Res; 8(4) April 2020 CANCER IMMUNOLOGY RESEARCH492

Lin et al.

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702

2020;8:479-492. Published OnlineFirst February 19, 2020.Cancer Immunol Res   Run Lin, Hui Zhang, Yujie Yuan, et al.   Survival in Gastric Adenocarcinoma

Tissue-Resident Memory T-cell+Fatty Acid Oxidation Controls CD8

  Updated version

  10.1158/2326-6066.CIR-19-0702doi:

Access the most recent version of this article at:

  Material

Supplementary

  http://cancerimmunolres.aacrjournals.org/content/suppl/2020/02/19/2326-6066.CIR-19-0702.DC1

Access the most recent supplemental material at:

  Cited articles

  http://cancerimmunolres.aacrjournals.org/content/8/4/479.full#ref-list-1

This article cites 51 articles, 5 of which you can access for free at:

  Citing articles

  http://cancerimmunolres.aacrjournals.org/content/8/4/479.full#related-urls

This article has been cited by 3 HighWire-hosted articles. Access the articles 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

  Permissions

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

.http://cancerimmunolres.aacrjournals.org/content/8/4/479To request permission to re-use all or part of this article, use this link

on March 17, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 19, 2020; DOI: 10.1158/2326-6066.CIR-19-0702