fatty acid oxidation controls cd8+ tissue-resident memory ... · 91 cells relied on fatty acid...

1

Fatty acid oxidation controls CD8+ tissue-resident memory T cell 1

survival in gastric adenocarcinoma 2

Run Lin1#, Hui Zhang2, 3#, Yujie Yuan4#, Qiong He5, Jianwen Zhou5, Shuhua 3

Li5, Yu Sun5, Daniel Y. Li6, Haibo Qiu7, Wei Wang8, Zhehong Zhuang9, Bin 4

Chen1, Yonghui Huang1, Chuwei Liu4, Yingzhao Wang4, Shirong Cai4*, Zunfu 5

Ke3, 5*, and Weiling He4* 6

7

1 Department of Radiology, The First Affiliated Hospital, Sun Yat-sen 8

University, Guangzhou, Guangdong 510080, China 9

2 Department of Rheumatology, The First Affiliated Hospital, Sun Yat-sen 10


3 Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen 12


4 Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun 14

Yat-sen University, Guangzhou, Guangdong 510080, China 15

5 Department of Pathology, The First Affiliated Hospital, Sun Yat-sen 16


6 Department of Medicine, Columbia University Irving Medical Center, New 18

York, NY 510080, USA 19

7 Department of Gastric Surgery, Sun Yat-sen University Cancer Center, 20

Guangzhou, Guangdong 510060, China 21

8 Department of Gastrointestinal Surgery, The Second Affiliated Hospital of 22

Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510120, 23

China 24

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9 Department of Gastrointestinal Surgery, The Eighth Affiliated Hospital，Sun 25

Yat-sen University, Shenzhen, Guangdong 518033, China 26

27

Running Title: Tissue-resident memory T cells in gastric adenocarcinoma 28

29

# R. Lin, H. Zhang, and Y. Yuan contributed equally to this work. 30

31

Conflict of interest statement: The authors declare no potential conflicts of 32

interest. 33

34

Funding 35

This work was supported by grants from the National Natural Science 36

Foundation of China (30900650, 81372501, 81572260, 81871994, 37

81701834), Guangdong Natural Science Foundation (2011B031800025, 38

S2012010008378, 2015A030313036, 2017A010101030, 2018A030310285), 39

and the Guangzhou Science and Technology Planning Program 40

(2014J4100132, 2015A020214010, 2012B031800115, 2013B02180021, 41

2016A020215055, 201904010398, 201902020018). 42

43

Valid unique e-mail address of all authors 44

Run Lin: [email protected] 45

Hui Zhang: [email protected] 46

Yujie Yuan: [email protected] 47

Qiong He: [email protected] 48

Jianwen Zhou: [email protected] 49




3

Shuhua Li: [email protected] 50

Yu Sun: [email protected] 51

Daniel Y. Li: [email protected] 52

Haibo Qiu: [email protected] 53

Wei Wang: [email protected] 54

Zhehong Zhuang: [email protected] 55

Bin Chen: [email protected] 56

Yonghui Huang: [email protected] 57

Chuwei Liu: [email protected] 58

Yingzhao Wang: [email protected] 59

Shirong Cai: [email protected] 60

Zunfu Ke: [email protected] 61

Weiling He: [email protected] 62

63

* Corresponding authors: 64

Shirong Cai 65

Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-66

sen University, Guangzhou, Guangdong 510080, China 67

Phone: (86) 13005199688 68

Email: [email protected] 69

70

Zunfu Ke 71

Institute of Precision Medicine, and Department of Pathology, The First 72

Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, 73

China 74




4

Phone: (86) 13570591359 75


77

Weiling He 78

Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-79

sen University, Guangzhou, Guangdong 510080, China 80

Phone: (86) 13560212999 81





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Abstract 83

The success of checkpoint inhibitors in cancer treatment is associated 84

with the infiltration of tissue-resident memory T cells (Trm). In this study, we 85

found that about 30% of tumor infiltrating lymphocytes (TILs) in TME of gastric 86

adenocarcinoma (GAC) were CD69+CD103+ Trm cells. Trm cells were low in 87

patients with metastasis and the presence of Trm cells was associated with 88

better prognosis in GAC patients. Trm cells expressed high PD-1, TIGIT, and 89

CD39 and represented tumor-reactive TILs. Instead of utilizing glucose, Trm 90

cells relied on fatty acid oxidation for cell survival. Deprivation of fatty acid 91

resulted in Trm cell death. In a tumor cell-T cell coculture system, GAC cancer 92

cells outcompeted Trm cells for lipid uptake and induced Trm cell death. 93

Targeting PD-L1 decreased fatty acid binding protein (Fabp) 4 and Fabp5 94

expression in tumor cells of GAC. In contrast, the blockade of PD-L1 95

increased Fabp4/5 expression in Trm cells, promoting lipid uptake by Trm 96

cells and resulting in better survival of Trm cells in vitro and in vivo. PD-L1 97

blockade unleashed Trm cells specifically in the patient-derived xenograft 98

(PDX) mice. PDX mice that did not response to PD-L1 blockade had less Trm 99

cells than responders. Together, these data demonstrated that Trm cells 100

represent a subset of TILs in the antitumor immune response and that 101

metabolic reprogramming could be a promising way to prolong the longevity 102

of Trm cells and enhance antitumor immunity in GAC. 103

104

Key Words: gastric adenocarcinoma; tissue-resident memory T cells; tumor 105

microenvironment; lipid metabolism; programmed death ligand 1. 106




6

Introduction 107

Gastric cancer is the second most common cancer worldwide and 108

gastric adenocarcinoma (GAC) accounts for 95% of the gastric cancer. (1) 109

Despite significant achievements in the management of GAC, the prognosis 110

remains dismal with a 5-year survival rate of about 20%. (2) Surgical resection 111

remains the first choice for patients with resectable tumors but recurrence 112

occurs in 20–50% of the patients following gastric resection. (2) More than 113

50% of patients present with locally advanced or metastatic GAC at diagnosis, 114

leaving chemotherapy as the main therapeutic option for these patients. (3) 115

Thus, the development of novel therapeutic agents and strategies for GAC is 116

eagerly awaited due to its high morbidity and mortality. 117

The emergence of immunotherapy has changed the landscape of 118

cancer treatments. (4) Immune checkpoint blockade of programmed death 1 119

(PD-1) is successful in the treatment of lung cancer, melanoma, kidney 120

cancer, and various other cancers (5-7). Anti-PD-1 treatment is effective for 121

gastric cancer.(8,9) However, only 11.2% and 22% of the treated patients 122

responded to such treatments in these two clinical trials respectively. It is 123

urgent to identify new therapeutic targets to approach better outcomes for 124

GAC patients. 125

Tissue-resident memory T (Trm) cells are a T-cell subset that resides 126

in the tissue and does not circulate back to the blood or secondary lymphoid 127

organs.(10,11) Trm cells produce higher amounts of cytokines than their 128

circulating counterparts and provide enhanced local immunity in response to 129

infection.(12,13) CD8+ Trm cells are associated with antitumor immune 130

responses.(14,15) The presence of Trm cells correlates with improved 131




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prognosis in cancer patients.(16,17) CD8+ Trm cells promote melanoma-132

immune equilibrium in skin while mice deficient in Trm cells formation are 133

more susceptible to tumor development.(18) These properties give Trm cells 134

great potential in the treatment of cancer. However, the roles of Trm cells in 135

GCA have not yet been reported and the detailed maintenance mechanisms 136

of CD8+ Trm cells in the tumor microenvironment (TME) remains to be 137

addressed. 138

In the current study, we found that Trm cells were present in the TME 139

of GAC, which indicated better prognosis. Trm cells relied on lipid uptake and 140

metabolism for cell survival. Cancer cells of GAC outcompeted Trm cells for 141

lipid uptake and induced apoptosis of Trm cells, which could be reversed by 142

blocking PD-L1 on cancer cells. Anti-PD-L1 treatment unleashed Trm cells 143

specifically in the patient-derived xenograft (PDX) mice. 144

145

Materials and Methods 146

Patient samples 147

Primary tumor tissues were collected from GAC patients with surgically 148

resectable tumors in The First Affiliated Hospital (cohort 1, n=180), Cancer 149

Center and The Eighth Affiliated Hospital (cohort 2, n=152) of Sun Yat-sen 150

University. For cell isolation and mouse experiments, tumor tissues and blood 151

samples were collected freshly from The First Affiliated Hospital, Sun Yat-sen 152

University (cohort 3, n=43). Patients’ clinical manifestations and laboratory 153

results were evaluated carefully and patients with infection or autoimmune 154

diseases were excluded from this study. Demographics of included patients 155

(cohort 1, cohort 2, cohort 3) are shown in Supplementary Table 1-3. This 156




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study was approved by the Ethics Committee of The First Affiliated Hospital, 157

Sun Yat-sen University. Consent was informed and consent forms were 158

obtained from all patients. The studies were conducted in accordance with 159

recognized ethical guidelines of Declaration of Helsinki. 160

161

Cell isolation 162

Blood samples were collected from GAC patients . Peripheral blood 163

mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque through density 164

gradient centrifugation. Freshly collected blood samples were diluted with 165

PBS in a ratio of 1:1. The diluted blood was then placed on top of Ficoll-166

Hypaque separation medium carefully, centrifuging at 1500 r.p.m for 30 167

minutes. Buffy coat layer was collected and washed with PBS twice. To 168

prepare single cell suspensions, fresh tumor tissues were minced and 169

digested with type I collagenase (2 mg/ml, Sigma, USA) and DNAse (50 U/ml) 170

in RPMI 1640 medium in 37 °C. Digestion buffer was replaced with fresh 171

buffer every 30 mins. Cells were then filtered through a cell strainer (70 μm) 172

and washed with phosphate-buffered saline (PBS). CD8+CD103Hi, 173

CD8+CD103Med or CD8+CD103Neg cells were sorted from tumor infiltrating 174

lymphocytes (TILs) using a BD FACS Influx. CD8+ T cells were purified from 175

PBMCs by negative selection using the EasySep™ human total CD8+ T Cell 176

enrichment kit according to the manufacturer’s instructions (STEMCELL 177

Technologies Inc., Vancouver, Canada, Catalog#19053). Cell purity was 178

checked by fluorescence-activated cell sorting meter (FACS) (>96%, BD 179

FACS Influx). 180




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Autologous tumor cells were prepared as described previously.(19) 181

Resected GAC tumor samples were immediately processed into single-cell 182

suspensions by mechanical dissociation and enzymatic digestion. Tumor 183

tissues were minced and incubated with collagenase I (2 mg/ml) and DNAse 184

type I (50U/ml). Tumor cells digested from tissue were filtered through a cell 185

strainer. CD45− population (non-hematopoietic) was further sorted by FACS 186

from the tumor digest and used as autologous tumor cells. The staining 187

details for cell sorting were described below in the Flow cytometry section. 188

189

CTC enrichment and analysis 190

Circulating tumor cells (CTC) were separated by the NanoVelcro 191

system (Cytolumina, USA) as previously described (20). A total amount of 5 192

ml blood specimens was collected from each of the GAC patients and the 193

samples were processed within 24 h. Red blood cells were removed by 194

incubating cells with red blood cell lysing buffer (BioLegend, USA) at room 195

temperature for 5 min. Cells were then washed with PBS twice. 196

Immunocytochemistry was applied to visualize the captured cells on SiNW 197

substrate. The captured cells were stained with 4', 6-diamidino-2-phenylindole 198

(DAPI, nuclear marker), TRITC-conjugated anti-CD45 antibody (WBC marker) 199

(Abcam, HongKong, 1:400) and FITC- conjugated anti-CK antibody (cancer 200

cell marker) (Abcam, HongKong, 1:100). Characteristic phenotypes and 201

morphology of CTCs were scrutinized by an experienced pathologist. CTCs 202

were identified by combination of the following criteria: positive staining of 203

anti-CK and DAPI, and negative staining of anti-CD45. 204

205




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Cell culture 206

The human gastric cancer cell line SGC7901 and normal gastric 207

epithelial cell line GES-1 were obtained from the Type Culture Collection of 208

Chinese Academy of Sciences in 2015 (Shanghai, China). (21) These cells 209

were authenticated and certified by cell viability analysis, short tandem repeat 210

(STR) profiling, and isoenzyme analysis, and were also screened for 211

mycoplasma contamination by Type Culture Collection of Chinese Academy 212

of Sciences. Cells were not reauthenticated. Cells were grown in complete 213

culture medium RPMI 1640 supplemented with 10% fetal bovine serum 214

(FBS), 50 U/mL penicillin, and 50 mg/mL streptomycin in a humidified 215

atmosphere at 37°C with 5% CO2. These cells were cultured for a maximum 216

of 15 passages after thawing from our stocks that were frozen at passage 3. 217

218

Flow cytometry 219

Cells were isolated from tumor tissue of GAC patients as described 220

above and were stained with the following antibodies: FITC-conjugated anti-221

CD45, PE-conjugated anti-CD3, APC-conjugated anti-CD8, BV421-222

conjugated CD69, PE-CY7-conjugated CD103, BV605-conjugated PD-1, PE-223

conjugated TIGIT, FITC-conjugated CD39, PerCP5.5-conjugated CD26 (all 224

were 1:100 and purchased from BioLegend, USA). Cells were incubated with 225

antibodies at 4 °C for 30 min and then washed with PBS twice. SGC7901 226

and GES-1 were stained with primary antibodies against fatty acid binding 227

protein (Fabp) 4 (1:200) and Fabp5 (1:200)(both from Abcam, Hong Kong) at 228

4 °C overnight. Cells were then washed with PBS twice and incubated with 229

Alexa Fluor 488-conjugated anti-mouse IgG (1:1000) or Alexa Fluor 555–230




11

conjugated goat anti-rabbit IgG (1:1000) (Life Technologies, USA) at 4 °C for 231

30 min. Cells were then washed with PBS twice and analyzed by flow 232

cytometry. 233

To measure cytokine production in T cells, cells were stimulated with 234

500 ng/ml phorbol myristate acetate (PMA), 1 μg/ml ionomycin and 5 μg/ml 235

Brefeldin A (Sigma–Aldrich) for 5 h at 37°C with 5% CO2. Cells were 236

collected, permeabilized and fixed using the Fixation/Permeabilization 237

Solution (BD Bioscience, USA) on ice for 30 min. Cells were then washed with 238

Perm/Wash™ Buffer and stained with V450-conjugated anti-IFNγ （ BD 239

Biosciences，USA） and FITC-conjugated anti-TNF-α antibodies (BioLegend, 240

USA) in Perm/Wash™ Buffer at 4 °C for 30 min. Cells were washed twice with 241

Perm/Wash™ Buffer and analyzed by flow cytometry. 242

To measure T cell apoptosis, cells were stained with Pacific blue-243

conjugated Annexin V and 7-AAD (BioLegend, USA). Samples were analyzed 244

using a BD FACS ARIA (BD Bioscience). FlowJo (Tree Star, USA) software 245

was used for data analysis. 246

247

Transfection 248

One day before the transfection, SGC7901 cells were seeded on 24-249

well culture plates. Fabp4, Fabp5, PD-L1 siRNA or scramble siRNA were 250

purchased from Santa Cruz, (Catalog#, sc-43592, sc-41237 and sc-39699). 251

siRNA oligomer were diluted in Opti-MEM I Reduced Serum Medium without 252

serum to a final concentration of 50 nM. Lipofectamine 2000 (1 μl) was diluted 253

with Opti- MEM I Reduced Serum Medium (50μl) and incubated for 5 min at 254

room temperature. The diluted oligomer was then mixed with the diluted 255




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Lipofectamine 2000 and added to each well. Cells were incubated at 37°C 256

with 5% CO2. The siRNA-Lipofectamine complex was removed after a 6 h 257

incubation and the cells were cultured at 37°C with 5% CO2 overnight in 258

RPMI 1640 medium supplemented with 10% FBS. 259

260

Coculture 261

Sorted CD8+CD103Hi, CD8+CD103Med or CD8+CD103Neg T cells were 262

cocultured with autologous tumor cells or SGC7901 cells in 48-well plates at a 263

ratio of 5:1. in RPMI 1640 medium supplemented with 10% FBS at 37°C with 264

5% CO2. T cells were stimulated with anti-CD3/CD28 antibodies (BioLegend, 265

USA) according to the manufacturer’s instructions. PD-L1 blocking antibody 266

(BioXcell, USA, 1μg/ml) was included in some experiments. T cells were 267

collected to determine apoptosis and lipid uptake by flow cytometry. 268

269

Cytotoxicity assay 270

In vitro cytotoxicity assays were performed by coculturing T cells and 271

cancer cells as described previously (22,23). Sorted CD8+ T cells were 272

cocultured with autologous cancer cells for 16 h at a ratio of 4:1. After 273

coculture, adherent and non-adherent cells were collected, stained with 7-274

AAD and analyzed by flow cytometry to determine the number of dead tumor 275

cells. The killing percentage was calculated by the percentage of 7-AAD+ 276

cancer cells. 277

278

Western blot 279




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Cells were lysed with RIPA Buffer including 1% v/v Halt 280

Protease/Phosphatase Inhibitor (Thermo Fisher Scientific, USA). TheBCA 281

protein assay (Thermo Fisher Scientific, USA) was used for quantitation of 282

total protein. A total of 30μg protein was loaded on SDS–polyacrylamide gels 283

(4% and 15%) and electrotransferred onto polyvinylidine difluoride 284

membranes after separation. Membranes were blocked with 10% bovine 285

serum albumin in TBST buffer and incubated with anti-CD36 (1:1000, Abcam, 286

Hong Kong), anti-β-actin (1:2000, Cell signaling technology, USA) primary 287

antibodies at 4°C overnight. The membranes were then washed with TBST 288

and incubated with horseradish peroxidase conjugated anti-rabbit IgG (1:2500, 289

Cell Signaling Technology, USA) at room temperature for 60mins. Signals 290

were detected by enhanced chemiluminescence (Thermo Fisher Scientific, 291

USA) with the Odyssey Fc imaging system (LI-COR Bioscience). 292

293

Immunometabolism 294

To measure lipid uptake or glucose uptake, cells were incubated at 295

2 × 105 cells in complete medium containing either 1 μM Bodipy 500 (Thermo 296

Fisher, USA) or 20 μM 2-NBDG (Thermo Fisher, USA), for 30 min at 37 °C in 297

a cell incubator. To measure lipid content in the cells, Bodipy 493 (1 μg/ml, 298

Thermo Fisher, USA) was added and incubated for 1 h at 4 °C. Cells were 299

analyzed by flow cytometry. To further detail the metabolic alterations in Trm 300

cells, a Seahorse assay was performed to measure glycolysis and 301

mitochondria oxygen consumption. T cells were incubated in a CO2 free 302

incubator in RPMI 1640 medium supplemented with glucose (20 mM) and 303

sodium pyruvate (1 mM) for 30 min. Measurements were performed using an 304




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XF96 extracellular analyzer (Seahorse Bioscience, USA). The Seahorse XF 305

Cell Mito Stress Test Kit was used for the measurement of mitochondrial 306

oxygen consumption rate. During the measurements, cells were treated with 307

oligomycin (1 μM), FCCP (1.5 μM), and Rotenone/Antimycin A (1 μM), 308

respectively as indicated. 309

310

ELISA 311

To measure cytokines released by T cells, culture supernatant was 312

collected for the measurement of IFNγ and TNFα using commercial ELISA 313

kits (Catalog# DIF50 and DTA00D) according to the manufacturer’s 314

instructions (R&D Systems, USA). 315

316

Immunofluorescence and immunohistochemistry 317

Paraffin-embedded tissues were cut into 4μm sections. The slides were 318

deparaffinized with Xylene and the rehydrated in 100% ethanol, 95% ethanol, 319

70% ethanol and 50% ethanol sequentially. Antigen retrieval was performed 320

in a pressure cooker using Antigen Retrieval Buffer (Citrate Buffer pH 321

6.0).Slides were blocked with 5% BSA in PBS and then incubated with 322

primary antibody against CD8 (1:1000) and CD103 (1:200) (Abcam, 323

HongKong) at 4℃ overnight. Slides were washed with PBS twice and 324

incubated with Alexa Fluor 555–conjugated goat anti-rabbit IgG (1:500) and 325

Alexa Fluor 488-conjugated anti-mouse IgG (1:500) (Life Technologies, USA) 326

at room temperature for 30 min. Sections were counter stained with DAPI in 327

mounting medium. For immunohistochemistry staining, slides were stained 328




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with primary antibody against CD103 (1:200) at 4°C overnight. The sections 329

were then incubated with an HRP-conjugated secondary antibody (1:500) for 330

1 h at room temperature. Peroxidase was visualized with 3,3’ 331

diaminobenzidine, and the slides were counterstained with hematoxylin. 332

Slides were examined using fluorescence microscopy (Zeisse) and the 333

number of CD8+CD103+ T cells was counted from 5 different high-power 334

areas. 335

336 Humanized PDX tumor model 337

Six- to eight-week-old NOD.Cg-PrkdcscidIl2rgtm1Sug/JicCrl (NOG) 338

mice (Vital River, China) were used to establish the PDX mouse model 339

used as described previously.(24) Tumor specimens of GAC were 340

collected and snap frozen in OCT. Tissue blocks were sectioned (4 μm) and 341

fixed in pre-cold acetone at 4 °C for 10 min. Tissue slides were then washed 342

with PBS twice and stained with Mayer hematoxylin solution for 5 min. Slides 343

were washed in warm running tap water for 10 min and counterstain in eosin 344

for 30 seconds. Slides were mounted with xylene based mounting medium. 345

Immune infiltrates in the TME was evaluated under light 346

microscopy(Supplementary Figure S1, Zeiss). Upon arrival, necrotic and 347

supporting tissues were carefully removed using a surgical blade. 348

Approximately 20–30 mg tissue fragments with immune infiltrates were 349

implanted subcutaneously into the flank region of NOG mice. Successful 350

established mouse models were monitored and engrafted tumors were 351

collected for analysis. Established PDX mice were treated with an anti-PD-L1 352

antibody (5mg/kg) or isotype control (BioXcell, USA). The mice were 353

monitored three times per week for evidence of morbidity and mortality 354




16

associated with tumor growth and metastasis. All animal procedures were 355

conducted in accordance with, and with the approval of the Institute Animal 356

Care and Use Committee of Sun Yat-sen University. 357

358

Statistical analysis 359

Data was presented as means ± standard error of mean (SEM). 360

Statistical analysis was performed using SPSS (version 13.0 for windows; 361

SPSS, Chicago, Illinois). Comparisons were assessed using either the 362

Student’s t-test, paired Student’s t-test, or one-way ANOVA with or without 363

repeated measurements followed by Bonferroni’s multiple comparison post-364

test, as appropriate. The Cox univariate and multivariate analyses were used 365

to explore the influences of different prognostic factors on OS. p values < 0.05 366

were considered as statistically significant. 367

368

Results 369

Trm predicted better survival in gastric adenocarcinoma. 370

Trm cells are associated with better survival in lung cancer and ovarian 371

cancer (17,25). In this study, we investigated the role and the predictive 372

potential of Trm cells in GAC. Trm cells were identified firstly in the TME of 373

GAC by dual-staining of CD8 and CD103. It revealed that CD8+CD103+ Trm 374

cells were detectable in the TME of GAC (Figure 1A), and CD103+ Trm cells 375

were more abundant in primary tumor compared to metastatic tumor as 376

quantified by IHC (Figure 1B). To further define and assess Trm cells in GAC, 377

single cell suspensions from tumor tissues were analyzed by flow cytometry. 378

Trm cells were identified as CD69-positive or CD103-positive, or both. Since 379




17

the vast majority of CD103+ T cells were simultaneously CD69+ (Figure 1C, 380

Supplementary Figure S2), Trm cells were defined as CD103+ or 381

CD69+CD103+ in this study. Enhanced frequency of Trm cells was confirmed 382

in tumor without metastasis as measured by flow cytometry (Figure 1C, 1D). 383

To further investigate the tissue residency of Trm in the TME, a PDX 384

tumor-bearing mouse model was established by engrafting tumor tissues from 385

GAC patients into the immunodeficient mice. By gating on human CD45+CD8+ 386

population, we found that the CD69+CD103+ Trm cells resided in the TME and 387

did not recirculate back to the blood. The population of CD8+ T cells that 388

recirculated back to the blood are CD69-CD103- (Figure 1E, 1F, 389

Supplementary Figure S2). We next assessed the relationship between Trm 390

cells and patient survival. CD103+ Trm cells was quantified (>5 CD103+ TILs 391

per 0.6 mm core) to stratify the cohort into high-density versus low-density 392

subsets as previously described (25). Using this strategy, we found that the 393

presence of high-density CD103+ Trm cells positively correlated with survival 394

survival in 2 cohorts of GAC patients (Figure 1G). The number of Trm cells 395

was negatively correlated with the number of circulating tumor cells (CTC) 396

(Supplementary Figure S3), indicating the potential function of Trm cells in 397

suppressing tumor metastasis. Both univariate analysis and multivariate 398

analysis performed using the COX proportional hazard regression model 399

showed that the number of CD103+ Trm cells was an independent prognosis-400

related marker for GAC (Supplementary table S4, Supplementary table S5). 401

402

Tumor reactivity of tissue-resident memory T cells in GAC 403




18

TILs highly express high immune inhibitory molecules (26). To 404

understand the expression patterns of the immune inhibitory molecules in Trm 405

cells, we evaluated the expression of PD-1, TIGIT, CD39, and CD26 in Trm 406

cells from GAC specimens by flow cytometry (Figure 2A, 2B). We found that 407

the expression of PD-1, TIGIT and CD39 correlated with that of CD103. 408

CD103Hi subpopulation expressed the highest amount of inhibitory molecules 409

while CD26 expression did not correlate to that of CD103 (Figure 2B-2F). 410

High PD-1/PD-L1 in the TME predicts better immune response and outcomes 411

in cancer patients to checkpoint blockade therapy (27). To investigate the 412

antitumor response by Trm cells, we sorted the CD103Hi, CD103Med and 413

CD103Neg populations and cultured the cells with or without autologous tumor 414

cells. We found that cytokine production of TILs from GAC patients correlated 415

with CD103 expression. CD103Hi cells produced lower amounts IFNγ and 416

TNFα when cultured alone as measured by ELISA. On the contrary, CD103Hi 417

cells produced higher amounts IFNγ and TNFα in the supernatant when 418

cultured with autologous tumor cells (Figure 2G). These results were further 419

confirmed by intracellular cytokine production measured by flow cytometry 420

(Figure 2H, 2I). These data suggested that Trm cells are tumor-reactive TILs. 421

422

Increased lipid uptake and metabolism in tissue-resident memory T cells. 423

Metabolic regulation is critical for T cell activation and effector 424

functions.(28) Specifically, lipid metabolism enhances CD8+ T cell memory 425

(29). To understand the metabolic status of Trm cells, we first measured the 426

expression of CD36, which imports lipids into the cells (30), in CD103Hi, 427

CD103med and CD103Neg populations by western blot. CD103Hi Trm cells had 428




19

the highest expressed of CD36 when compared to CD103Med and CD103Neg 429

Trm cells (Figure 3A). Along with increased CD36 expression, lipid content 430

(measured by Bodipy 493) was dramatically increased in CD103Hi Trm cells 431

(Figure 3B). In addition, by loading the cells with Bodipy 500 to measure lipid 432

uptake, CD103Hi cells showed a clear increase in lipid uptake (Figure 3B, 3D, 433

3E). However, glucose uptake (measured by 2-NBDG) of CD103Hi Trm cells 434

decreased when compared to CD103med or CD103Neg Trm cells (Figure 3B, 435

3C). To further detail the metabolic alterations in Trm cells, a Seahorse assay 436

was performed to measure glycolysis and mitochondria oxygen consumption. 437

CD103Hi Trm cells showed decreased extracellular acidification rate (ECAR, 438

Figure 3F) and increased oxygen consumption rate (OCR, Figure 3G). 439

CD103Hi Trm cells had significantly higher basal metabolic activity, ATP-440

coupled OCR, stronger spare mitochondrial capacity and maximum 441

respiratory (Figure 3H-3K). These data implied that Trm cells underwent a 442

metabolic reprogram and switched to mitochondria fatty acid oxidation to meet 443

their energy requirements. 444

445

Lipid metabolism was required for the survival of Trm from GAC. 446

CD8+ Trm cells tend to utilize mitochondrial fatty acid oxidation to 447

support both their longevity and protective function (31). In this work, the role 448

of lipid metabolism in the maintenance of Trm cells of GAC was explored. 449

CD103Hi Trm cells showed increased apoptotic rates in vitro (Supplementary 450

Figure 4), which can be reversed by supplementing free fatty acids (FFAs). 451

However, FFAs did not change the apoptosis status of CD103Neg cells (Figure 452

4A, 4B). FFAs also enhanced cytokine production by CD103Hi Trm cells 453




20

(Figure 4C, 4D). Seahorse assay showed FFAs did not affect ECAR by 454

CD103Hi Trm cells. OCR by CD103Hi Trm cells was significantly increased 455

when FFAs was supplemented (Figure 4E, 4F). These data suggested an 456

important role of lipid metabolism in Trm cell survival by improving 457

mitochondria activities in CD103Hi Trm cells. To further confirm the 458

contribution of lipid metabolism to the survival of Trm cells in vivo, we 459

established a PDX mouse model by engrafting tumor specimens from GAC 460

patients. PDX mice were treated with Etomoxir, which inhibits fatty acid 461

oxidation (Figure 4G). Consistent with in vitro data, Etomoxir decreased the 462

percentage of Trm cells in the TME (Figure 4H, 4I). The total number of Trm 463

cells was reduced dramatically by Etomoxir (Figure 4J), whereas the number 464

of non-Trm cells in the TME was not affected by Etomoxir treatment (Figure 465

4K). These data demonstrated that FFAs improved mitochondria function of 466

CD103Hi Trm cells, which was important for the survival of Trm cells. 467

468

Cancer cells deprive Trm of lipid uptake, inducing their apoptosis. 469

Metabolic shifting such as glucose deprivation in the TME impairs the 470

antitumor effects of CD8+ T cells (32,33). Thus, we next explored the 471

metabolic alterations in the TME and the metabolic reprogramming in Trm 472

cells from TME of GAC. By coculturing CD103Hi and CD103Neg T cells with 473

GAC cells, we found that GAC cells increased the apoptosis of CD103Hi Trm 474

cells, whereas the CD103Neg T cells were unaffected (Figure 5A, 5B). Since 475

lipid metabolism was important for Trm cells survival (Figure 4), we 476

hypothesized that GAC cells deprive lipid uptake of Trm cells and induce Trm 477

cell apoptosis. We thus measured the expression of Fabp4 and Fabp5 in GAC 478




21

specimens by western blot. We found that Fabp4 and Fabp5 expression 479

significantly increased in GAC tumors when compared to para-cancer normal 480

gastric tissue (Figure 5C). Fabp4 and Fabp5 expression was also higher in 481

GAC cells (SGC-7901) compared to healthy gastric epithelial cells (GES-1) as 482

measured by flow cytometry (Figure 5D, 5E). In contrast to GES-1, lipid 483

uptake increased in SGC-7901 (Figure 5F, 5G). Our previous study also 484

shows gastric tumor cells outcompete CD8+ T cells for glucose 485

consumption(34). Next, we investigated whether lipid metabolism in cancer 486

cells would affect lipid uptake and apoptosis of Trm cells. The results revealed 487

that lipid uptake by CD103Hi Trm cells was reduced when cocultured with 488

cancer cells (Figure 5H, 5I). In the tumor cell-T cell coculture system, lipid 489

uptake in Trm cells obviously increased with knockdown of both Fabp4 and 490

Fabp5 in cancer cells when compared to knockdown of either Fabp4 or Fabp5. 491

(Figure 5J, 5K). Knockdown of Fabp4 or Fabp5 in cancer cells decreased the 492

apoptosis of Trm cells. And knockdown of Fabp4 and Fabp5 together further 493

decreased the apoptosis of Trm cells. (Figure 5L, 5M). These data 494

demonstrated that GAC cells could induce Trm cells apoptosis by depriving 495

lipid uptake by Trm cells, which was further confirmed that FFAs alone did not 496

affect the Trm cells in the tumor microenvironment (Supplementary Figure 5). 497

498

PD-L1 regulated lipid competition in the tumor microenvironment. 499

PD-1 altered T-cell metabolic reprogramming by inhibiting glucose 500

glycolysis and increasing mitochondria fatty acid oxidation (35). We 501

investigated if PD-L1, the ligand of PD-1, affects lipid uptake and metabolism 502

in cancer cells and Trm cells. In the tumor cell-T cell coculture system, we 503




22

found that PD-L1 blockade decreased Fabp4/5 expression in tumor cells, 504

while Fabp4/5 expression in T cells was increased when PD-L1 was blocked 505

(Figure 6A, 6B). Lipid uptake by cancer cells was reduced by blocking PD-L1 506

(Figure 6C, 6D). Importantly, lipid uptake in CD103Hi Trm cells was increased 507

when PD-L1 was blocked (Figure 6E, 6F). We confirmed these data by 508

knocking down PD-L1 expression in tumor cells. Knockdown of PD-L1 509

expression in tumor cells decreased lipid uptake by tumor cells and increased 510

lipid uptake in CD103Hi Trm cells (Supplementary Figure 6). Lipid uptake by 511

CD103Hi Trm cells was decreased when PD-1 was knocked down 512

(Supplementary Figure 7). Meanwhile, the apoptotic rate of CD103Hi Trm cells 513

was decreased when PD-L1 was blocked (Figure 6G, 6H). These data 514

indicated that the inhibition of PD-L1 in the TME could improve the survival of 515

Trm cells and enhance antitumor immune response. To further confirm this 516

result, the PDX model was established as described in Figure 1 and the tumor 517

bearing mice were treated with PD-L1 blocking antibody (Figure 6I). After the 518

treatment, Trm cells in the TME was analyzed by flow cytometry. We found 519

that the percentage of Trm cells was increased after PD-L1 blockade. The 520

absolute number of Trm cells was also increased by blocking PD-L1 in the 521

PDX mice (Figure 6J, 6K). 522

523

PD-L1 blockade unleashed Trm and their antitumor effects 524

The success of anti-PD-1/PD-L1 in treatment of cancers has led to a 525

paradigm shift in the oncology field (36). We investigated how Trm cells were 526

involved in antitumor immune response. We first tested cytotoxicity of Trm 527

cells to tumor cells. We found that CD103Hi Trm cells showed more effectively 528




23

induced tumor cell death when compared to CD103Neg cells (Figure 7A, 7B). 529

In addition, PD-L1 blockade enhanced cytotoxic function of CD103Hi Trm cells 530

mostly (Figure 7C, 7D). To further study CD103Hi Trm cells in antitumor 531

immune response in vivo, a PDX model was established as in Figure 1. The 532

tumor bearing mice were treated with anti-PD-L1 antibody or isotype control 533

(Figure 7E). IFNγ production in TILs was measured by flow cytometry after 534

the treatment. We found that PD-L1 blockade mainly affected the CD103Hi 535

Trm cells through stimulating IFNγ production, while there seemed be no 536

evident effects on CD103Neg (Figure 7F, 7G). We monitored tumor growth by 537

measuring the tumor volume in mice treated with anti-PD-L1 antibody. Seven 538

PDX mice responded to the anti-PD-L1 treatment, while tumor growth in 11 of 539

the treated mice was not affected by anti-PD-L1 treatment (Figure 7H). The 540

percentage of Trm cells was significantly higher in the treatment-responsive 541

mice compared to the non-responsive mice (Figure 7I, 7J). These data 542

indicated that CD8+ CD103Hi Trm cells contributed significantly to the 543

antitumor immune response to GAC. 544

545

Discussion 546

Gastric cancer is one of the leading causes of cancer-related death. 547

There has been a paradigm shift in cancer therapy in the past 5-10 years due 548

to the successes of immunotherapy (37). Anti-CTLA4 and anti-PD-1/PD-L1 549

treatments represent the most successful therapeutics targeting immune 550

system to cure cancer (38). Data from clinical trials show the promising results 551

using anti-PD-1/PD-L1 antibody to treat gastric cancer (8,9). However, due to 552

the low response rate, there is a urgent need to develop novel strategies to 553




24

improve the therapeutic effect. In this study, we investigated the roles and the 554

metabolic regulation of CD8+ Trm cells in GAC, focusing on the roles of fatty 555

acid oxidation Trm cell death in GAC. We found that Trm were present in the 556

TME of GAC and predicted better survival. Fatty acid oxidation was required 557

for the survival of Trm cells. GAC cells outcompeted Trm cells for lipid uptake 558

and PD-L1 blockade reversed this competition, unleashing Trm cells, leading 559

to tumor regression. 560

Immune checkpoint blockade unleashes CD8+ T cells and enhances 561

their antitumor response in the TME (39). Trm cells are T cells that reside in 562

the tissue and provide stronger and faster immune response during antigen 563

rechallenge (40). Trm cells can be harnessed to enhance the efficacy of 564

cancer vaccines(41). CD103+ Trm cells are present in the TME and predict 565

better outcomes in breast cancer, lung cancer and ovarian cancer (16,17,25). 566

In the current study, we first confirmed the presence of CD103+ Trm cells in 567

the TME of GAC by detecting the coexpression pattern of CD8 and CD103. 568

Meanwhile, these cells were also CD69+, which was in consistent with 569

previous reports (17). Results from the PDX model proved that the 570

CD69+CD103+ Trm cells were TILs that do not participate in lymphocyte 571

recirculation. The percentage of CD8+ Trm cells was lower in patients with 572

metastasis. Clinical data analysis showed that higher density of Trm cell, as 573

defined as >5 CD103+ TILs per 0.6 mm core, was associated with better OS. 574

We observed a negative correlation between the qualities of Trm cells and 575

CTC, confirming the suppressive effect of Trm cells against metastasis. 576

Together with the strong expression of the granzymes, perforin and IFNγ in 577




25

Trm cells (17), these results demonstrate the critical role of Trm cells in 578

suppressing tumor growth and metastasis in GAC. 579

TILs highly express immune inhibitory molecules (42). To explore the 580

special status of Trm cells, we measured the expression of PD-1, TIGIT and 581

CD39 on TILs from GAC patients. CD103Hi cells more highly expressed PD-1, 582

TIGIT, and CD39. PD-1 and TIGIT is highly expressed in TILs from gastric 583

cancer(34). Coexpression of CD39 and CD103 identifies tumor-reactive T 584

cells in human malignancies.(43) CD39+ CD8+ T cells in human tumor 585

infiltrates are antigen specific and CD8+ TILs without CD39 expression could 586

be defined as bystander (not responsive to tumors) (44). The high CD39 587

expression on Trm cells indicates that Trm cells are antigen specific T cells 588

involved in the antitumor immune response. In this work, our results showed 589

that CD103Hi Trm cells responded to autologous tumor cells by producing 590

IFNγ and TNFα while CD103Neg T cells barely responded to autologous tumor 591

cells. From the PDX mouse experiments, we learned that the T cells that re-592

circulated back to the blood were CD69−CD103−. Together, CD103Neg T cells 593

could be identified as the so call bystander in the TME. These data support 594

the notion that Trm cells are the major effector T cells in response to cancer 595

(43). 596

Energy demand is dramatically increased during T cell activation, 597

proliferation and differentiation (28,45). TILs display profound metabolic 598

reprograming(19) while glucose uptake by cancer cells outcompetes TILs, 599

which leads to impaired effector functions of CD8+ T cells (33). Glucose 600

deprivation suppresses effector functions of TILs and metabolic 601

reprogramming of TILs enhances their antitumor response (46). In the current 602




26

study, we found that CD103Hi Trm cells express the fatty acid translocase 603

CD36. We also observed increased lipid uptake and mitochondrial activities 604

synchronous with decreased glucose uptake and glycolytic activity in Trm 605

cells. CD103Hi Trm cells from TME of GAC displayed higher amounts of 606

apoptosis in in vitro culture and FFAs rescued the Trm cells from apoptosis. 607

By inhibiting fatty acid oxidation, the proportions and quantities of Trm cells 608

decreased in the TME of a PDX model for GAC. These findings are consistent 609

with a previous report showing that Trm cells rely on lipid metabolism for long-610

term survival (31). In the harsh TME, the supply of FFAs may be required for 611

Trm cell maintenance and long-term survival. 612

Due to the high proliferation rate, cancer cells rely on glucose to 613

generate energy in a fast but inefficient way through glycolysis (47). There is 614

also an increased demand of lipids for the fast proliferating cancer cells for 615

energy production and new membrane formation (48). Our study 616

demonstrated that Fabp4/5 expression was increased in GAC cells and 617

displayed increased lipid uptake compared to healthy gastric epithelial cells. 618

In a T cell-tumor cell coculture system, cancer cells suppressed the lipid 619

uptake of Trm cells and induced Trm apoptosis. These effects could be 620

reversed by inhibiting cancer cells from lipid uptake. These results unearthed 621

the metabolic competition between cancer cells and Trm cells for lipid 622

consumption, leading to the suppression of Trm cells. 623

PD-L1 is involved in the glucose metabolism of cancer cells(33) and 624

PD-L1 is expressed in the TME of all stages of GAC (49), suggesting an 625

important role of PD-L1 in the control of metabolism of GAC cells. However, 626

the effects of PD-L1 in the regulation of lipid metabolism unknown. We found 627




27

that PD-L1 inhibition decreased Fabp4/5 expression in GAC cells, while 628

Fabp4/5 expression was increased in the T cells in the coculture system. The 629

blockade of PD-L1 not only suppressed lipid uptake of cancer cells, but also 630

rescued Trm cells from apoptosis in the TME. These findings were verified in 631

the PDX models treated with anti-PD-L1 where an enrichment of Trm cells 632

was seen. Taken together, cancer cells outcompete Trm cells for lipid uptake 633

through PD-L1, which leads to the apoptosis of Trm cells in the TEM, 634

dampening the antitumor immune response. 635

PD-1/PD-L1 blockade unleash TILs and enhance antitumor responses 636

to suppress tumor from growing.(50) We found that CD103Hi Trm cells 637

exhibited strong cytotoxic function in response to autologous tumor cells and 638

anti-PD-L1 enhanced the cytotoxic function of CD103Hi Trm cells. In the PDX 639

mice treated with anti-PD-L1 antibody, the mice responding to the treatment 640

showed dramatically higher percentage of Trm cells in the TME. In contrast, 641

the mice with progressing tumors presented low frequencies of Trm. 642

Checkpoint blockade targets tumor-specific specific T cells(51) and CD103+ 643

TILs represent the tumor reactive T cells(43). Trm cells contribute to antitumor 644

immune response by targeting PD-1/PD-L1. 645

Taken together, our data suggested a distinct role for tumor-specific 646

Trm cells in mediating antitumor immunity and predicting treatment response 647

to checkpoint blockade. Reprogramming lipid metabolism of Trm cells could 648

be a promising therapeutic approach for GAC. Further investigation to explore 649

the relevant mechanisms and potential clinical application targeting lipid 650

metabolism reprogramming of Trm cells is warranted. 651

652




28

Author contributions 653

Conception, design and study supervision: Shirong Cai, Zunfu Ke and Weiling 654

He. 655

Funding support: Run Lin, Zunfu Ke and Weiling He. 656

Acquisition of data: Run Lin, Hui Zhang, Yujie Yuan, Qiong He, Jianwen Zhou, 657

Shuhua Li, Yu Sun, Haibo Qiu, Wei Wang, Zhehong Zhuang, Bin Chen, 658

Yonghui Huang, Chuwei Liu, and Yingzhao Wang. 659

Data analysis and interpretation: Run Lin, Hui Zhang, Daniel Y. Li, Shirong 660

Cai, Zunfu Ke and Weiling He. 661

Writing, review, and/or revision of the manuscript: Run Lin, Hui Zhang, Yujie 662

Yuan, Daniel Y. Li, Zunfu Ke and Weiling He. 663

664

Final approval of manuscript: All authors. 665

666

Acknowledgements 667

Not applicable. 668

669




29

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849

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33

Figure legends 851

Figure 1. Tissue-resident memory T cells in gastric adenocarcinoma. (A) 852

Immunofluorescence staining of CD8 (red) and CD103 (green) in tissue 853

sections from gastric adenocarcinoma (GAC). Slides were counterstained with 854

DAPI. Representative images were shown. (B) Tissue sections from primary 855

or local metastasized GAC were stained with anti-CD103 antibody. Slides 856

were counterstained with hematoxylin. Representative images are shown 857

(n=180). (C) Fresh tumor tissues from primary or local metastasized GAC 858

were collected and single cell suspension was prepared. Cells were stained 859

with antibodies against CD8, CD69 and CD103 and analyzed by flow 860

cytometry. Representative counter plots gated on CD8+ cells are shown. (D) 861

Percentages of CD8+CD69+CD103+ T cells in primary (n=24) or local 862

metastasized (n=8) GAC (mean ± SEM, t test). (E) Fresh collected tumor 863

tissues from GAC were engrafted into NOG mice to establish a PDX model. 864

(F) Mouse blood and tumors were collected 3 weeks after engraftment. Single 865

cells were prepared and analyzed for CD69 and CD103 expression on 866

CD45+CD8+ T cells by flow cytometry. Representative counter plots of 8 867

experiments are shown. (G) Kaplan–Meier plots representing the probability 868

of overall survival (OS) in cohort 1 and cohort 2. ***p<0.001 869

870

Figure 2. Tumor reactivity of tissue-resident memory T cells in gastric 871

adenocarcinoma. (A) Single cell suspensions from gastric adenocarcinoma 872

(GAC) were analyzed by flow cytometry. Gating strategy of CD8+CD103+ cells 873

according to CD103 expression for subpopulation analysis. Representative 874

histogram plots of GAC specimens are shown. (B) Coexpression of PD-1, 875




34

TIGIT, CD39 and CD26 on CD103 subpopulations was analyzed by flow 876

cytometry. Representative histograms are shown. (C-F) PD-1, TIGIT, CD39 877

and CD26 expression on CD103 subpopulations was summarized from 12 878

samples. CD103 subpopulations were FACS-sorted. Cells were cultured with 879

or without autologous tumor cells. Tumor reactivity of CD103 subpopulations 880

was evaluated by measuring IFNγ and TNFα in supernatant by ELISA (G) or 881

intracellular cytokine measuring by flow cytometry (H, I). Data are mean ± 882

SEM. **p<0.01, ***p<0.001 by one-way ANOVA. MFI: mean fluorescence 883

intensity. ns: not significant. 884

885

Figure 3. Glucose and lipid metabolism in tissue resident memory T 886

cells from gastric adenocarcinoma. (A) Single cell suspensions were 887

prepared from gastric adenocarcinoma (GAC) derived tumor specimens. 888

CD103 subpopulations were FACS sorted. CD36 expression on the CD103+ 889

subpopulations was measured by western blot. Representative bans were 890

shown. (B) Sorted CD103+ subpopulations were incubated with 2-NBDG 891

(glucose uptake), Bodipy 493 (lipid content), and Bodipy 500 (lipid uptake). 892

Cells were analyzed by flow cytometry and representative histograms are 893

shown. (C-E) Data are summarized from 12 samples. (F, G) Sorted CD103 894

subpopulations were analyzed with a Seahorse Bioscience XF96 analyzer. 895

Glycolytic activity was measured by extracellular acidification rates (ECAR, F) 896

and mitochondrial activities were measured by oxygen consumption rate 897

(OCR, G). Summarized baseline respiration (H), respiration coupled to ATP 898

production (H), respiratory spare capacity (I) and maximal respiration (J). Data 899

are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way 900




35

ANOVA. ns: not significant. FCCP: carbonyl cyanide p-901

trifluoromethoxyphenylhydrazone. 902

903

Figure 4. Lipid metabolism regulated survival of tissue resident memory 904

T cells from gastric adenocarcinoma. (A, B) CD103Hi and CD103Neg cells 905

were cultured in the presence or absence of Palmitate (Palm, fatty acid) or 906

vehicle for 24 h. The apoptotic rate was measured by flow cytometry. Data are 907

from 6 experiments. (C, D) CD103Hi cells were cultured in the presence or 908

absence of Palm. IFNγ and TNFα production was measured by flow 909

cytometry. Representative count plots are shown. (E, F) Extracellular 910

acidification rates (ECAR) and mitochondrial activities were measured by 911

oxygen consumption rate (OCR) by Seahorse Bioscience XF96 analyzer. (G) 912

PDX was established as in Fig.1. Mice were treated with Etomoxir (CPT1 913

inhibitor) for 1 week. CD8+CD69+CD103+ cells in tumor infiltrated lymphocytes 914

were measured by flow cytometry. (H) Representative counter plots of 915

CD8+CD69+CD103+ cells are shown. Percentages (I) and number (J) of 916

CD8+CD69+CD103+ cells in the tumor tissue. (K) Number of CD103- CD8+ T 917

cells in the tumor tissue. Data are mean ± SEM. *p<0.05, **p<0.01, 918

***p<0.001 by t test. ns: not significant. FCCP: carbonyl cyanide p-919

trifluoromethoxyphenylhydrazone. 920

921

Figure 5. Tumor cells deprived tissue-resident memory T cell of lipid 922

uptake and induced their apoptosis. (A) CD103Hi and CD103Neg 923

subpopulations were sorted as in Fig. 2. CD103Hi and CD103Neg were 924

cocultured with gastric cancer cells (SGC7901) at a ratio of 5:1 for 24 h. Cells 925




36

were stained with Annexin V and 7-AAD. Apoptosis was measured by flow 926

cytometry. (B) Percentages of apoptosis summarized from 6 samples. (C) 927

Expressions of fatty acid binding proteins 4 and 5 (Fap4, Fabp5) in gastric 928

adenocarcinoma and normal gastric tissue were measured by western blot. 929

Representative bands are shown. (D, E) Fabp4 and Fabp5 expression in 930

SGC7901 and normal gastric epithelial cells (GES-1) was measured by flow 931

cytometry. Representative histograms and data were from 3 experiments. (F, 932

G) SGC7901 and GES-1 cells were incubated with Bodipy 500 and the lipid 933

uptake was measured by flow cytometry. Representative histograms and data 934

were from 3 experiments. (H, I) CD103Hi cells were cultured with or without 935

SGC7901 and with Bodipy 500. Lipid uptake was measured by flow cytometry. 936

Representative histograms and data of 3 experiments. (J, K) CD103Hi cells 937

were cocultured with SGC7901 that were Fabp4, Fabp5 or dual knockdown. 938

Lipid uptake was measured by incubating cells with Bodipy 500 and analyzed 939

by flow cytometry. Representative histograms and data were from 3 940

experiments. (L, M) CD103Hi cells were cocultured with SGC7901 that were 941

Fabp4, Fabp5 or dual knockdown for 24 h. Cells were stained with Annexin V 942

and 7-AAD for apoptosis analysis by flow cytometry. Representative 943

histograms and data were from 3 experiments. Data are mean ± SEM. 944

**p<0.01, ***p<0.001, ****p<0.0001 by t test in B, E G I and one-way ANOVA 945

in K, M. ns: not significant. 946

947

Figure 6. PD-L1 blockade promoted tissue-resident memory T cell 948

survival. Gastric cancer cells (SGC7901) were treated with IFNγ (10ng/ml) 949

for 24h. CD103Hi CD8+ T cells were then sorted by flow cytometry and 950




37

cocultured with SGC7901 in the presentence of anti-PD-L1 antibody or 951

isotype control for 24h. (A, B) Fabp4 and Fabp5 expression in tumor cells and 952

T cells were measured by flow cytometry. Representative histograms were 953

shown and data from 5 patient samples. (C-F) Cells were incubated with 954

Bodipy 500 and lipid uptake by tumor cells and T cells was assessed by flow 955

cytometry. Representative histograms and data were from 5 experiments. (G, 956

H) Annexin V expression in T cells was measured by flow cytometry. 957

Representative histograms gated on CD8+ cells and data were from 5 patient 958

samples. (I) PDX model was established as in Fig. 1. PDX bearing mice were 959

then treated with anti-PD-L1 or isotype for 2 weeks. (J, K) Single cell 960

suspension was prepared from tumor xenografts and CD8+CD69+CD103+ T 961

cells in tumor infiltrated lymphocytes were analyzed by flow cytometry. 962

Percentages and cell numbers are shown respectively (n=12). Data are mean 963

± SEM. *p<0.05, **p<0.01, by t test in B, D, F, H and paired t test in J, K. 964

965

Figure 7. Cytotoxicity of tissue-resident memory T cells correlated with 966

antitumor response to PD-L1 blockade. (A) Autologous tumor cells were 967

cocultured with or without CD103Hi, CD103Neg CD8+ T cells for 16h. Cells 968

were collected and cell death was measured by staining cells with 7-AAD. 969

Representative FACS plots were gated on CD45- tumor cells. Data were from 970

5 samples (B). (C, D) Autologous tumor cells were cocultured with CD103Hi, 971

CD103Neg CD8+ T cells in the presence anti-PD-L1 antibody or isotype control 972

for 16h. Cell death was measured by staining cells with 7-AAD. 973

Representative FACS plots were gated on CD45- tumor cells. Data were from 974

5 samples. (E) A PDX model was established and treated as in Fig. 6. (F, G) 975




38

Tumor infiltrated lymphocytes (TILs) from anti-PD-L1 or isotype treated mice 976

were measured for IFNγ expression by flow cytometry. Percentages of IFNγ 977

producing T cells and representative counter plots gated on CD103Hi and 978

CD103Neg CD8+ T cells (n=8). (H) Tumor volume of anti-PD-L1 treated mice. (I, 979

J) CD8+CD69+CD103+ cells in TILs from anti-PD-L1 treatment responsive 980

(n=7) and non-responsive (n=11) mice were analyzed by flow cytometry. 981

Representative counter plots. Data are mean ± SEM. *p<0.05, ***p<0.001, 982

****p<0.0001 by one-way ANOVA in B, paired t test in D and t test in G, J. ns: 983

not significant. 984




Published OnlineFirst February 19, 2020.Cancer Immunol Res Run Lin, Hui Zhang, Yujie Yuan, et al. cell survival in gastric adenocarcinomaFatty acid oxidation controls CD8+ tissue-resident memory T

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