Supplementary Table 1. Characteristics of 44 Ascites Samples with Gastric Adenocarcinoma for Whole Exome Sequencing and RNA Sequencing

Supplementary Table 1. Characteristics of 44 Ascites samples with gastric adenocarcinoma for whole exome sequencing and RNA sequencing

Treatm Tum Differentiati Lauren Signet ent or Exome RNA Location of on of Response for Matche Sample Sex Age classificat ring before Perce seque seque primary tumor primary treatment d DNA ion cell collecti ntage ncing ncing tumor on IP-004 M 82 Gastric Poorly Diffuse No Yes Non responder 80% Yes No Yes IP-007 M 33 Gastric Poorly Diffuse Yes Yes Non responder <5% No No Yes IP-008 M 62 Gastric Poorly Diffuse Yes No No treatment 95% No No Yes IP-009-2 M 40 Gastric Poorly Diffuse No No Non responder 85% Yes Yes Yes IP-010-1 F 58 Gastric Poorly Diffuse Yes Yes Responder 80% Yes Yes Yes IP-013 F 64 Gastric N/A N/A N/A No No treatment 40% Yes No Yes IP-020 M 77 Siewert type II Moderately Intestinal No Yes Responder 10% Yes No Yes IP-022 M 63 Gastric Poorly Diffuse No Yes Non responder <5% Yes Yes No IP-023 F 72 Gastric Poorly Diffuse Yes No No treatment <5% Yes Yes No IP-024-1 F 47 Gastric N/A N/A N/A Yes Responder 85% Yes Yes Yes IP-026-1 M 70 Gastric Poorly Diffuse No Yes Non responder 60% Yes Yes Yes IP-029 M 69 Siewert type II Moderately Intestinal No Yes Non responder 10% Yes Yes No IP-031 M 73 Gastric Poorly Diffuse No Yes Responder 80% Yes Yes Yes IP-032-1 F 62 Gastric Poorly Diffuse No Yes Responder <5% Yes Yes Yes IP-033-2 M 53 Siewert type III Poorly Diffuse Yes Yes No treatment 10% Yes Yes Yes IP-036 F 59 Siewert type II Poorly Diffuse No Yes No treatment 20% Yes No Yes IP-038 M 46 Siewert type II Moderately Intestinal No Yes Non responder 20% Yes Yes Yes IP-039-1 M 58 Siewert type II Moderately Intestinal No Yes Non responder <5% Yes Yes Yes IP-040 M 63 Siewert type II Moderately Intestinal No Yes Non responder 50% Yes No Yes IP-042 F 58 Siewert type II Poorly Diffuse No Yes Non responder <5% Yes Yes No IP-043 M 33 Gastric Poorly Diffuse No Yes Non responder 20% Yes No Yes IP-045 F 63 Gastric Poorly Diffuse Yes Yes Non responder 10% Yes No Yes IP-046 M 47 Gastric Poorly Diffuse Yes Yes Responder 95% No No Yes IP-051 F 43 Gastric Poorly Diffuse Yes No Non responder 10% Yes No Yes IP-052-1 M 70 Siewert type II Moderately Intestinal No Yes Responder <5% Yes Yes Yes IP-054 M 69 Siewert type II N/A N/A N/A Yes Non responder 85% Yes No Yes IP-056 F 44 Gastric Poorly Diffuse Yes Yes Responder 40% Yes No Yes IP-057 M 71 Siewert type II Poorly Diffuse Yes Yes Non responder 75% Yes Yes Yes IP-058-2 M 49 Siewert type III Moderately Intestinal No Yes Responder 60% Yes No Yes IP-062 F 61 Gastric Poorly Diffuse Yes Yes Responder 30% No No Yes IP-064-1 M 69 Siewert type III Poorly Diffuse Yes No Non responder 80% No No Yes IP-067-1 F 35 Gastric Poorly Diffuse Yes Yes Responder 80% No No Yes IP-067-2 F 35 Gastric Poorly Diffuse Yes Yes Responder 85% Yes No Yes IP-068 F 59 Siewert type II Poorly Diffuse No Yes No treatment 90% No No Yes IP-070 F 68 Gastric Poorly Diffuse No Yes Responder 95% No No Yes IP-073 M 66 Gastric Poorly Diffuse Yes Yes Responder 85% No No Yes IP-104 F 47 Gastric N/A N/A N/A Yes Responder 20% Yes No Yes IP-107-2 F 34 Gastric Poorly Diffuse Yes Yes Non responder N/A Yes No Yes IP-109 F 59 Gastric Poorly Diffuse Yes Yes Responder <5% Yes No Yes IP-116 M 61 Siewert type I Poorly Diffuse No Yes No treatment <5% Yes No Yes IP-118-1 F 54 Gastric N/A N/A N/A Yes Non responder 10% Yes No Yes IP-123-1 M 55 Gastric Moderately Intestinal No Yes Non responder N/A Yes No Yes IP-124-1 F 65 Gastric Poorly Diffuse Yes Yes Responder 70% Yes No Yes

Supplementary Figure S1.

Supplementary Figure S1. Tumor cell percentage in Representative PC samples determined by immunoflurocent staining of EpCAM (Green, tumor cells) and CD45 (Red, immune cells). Supplementary Figure S2

Matched gDNA Without 2.85 2.85 With 2.60 2.60 2.42 2.23 2.23 2.28 2.23

Mutation rate per Mb Mutation rate 2.12 3.04 0 50 100 150 0 5 10 15 20 25 30 35 01234567

All G2 G3 ESCA STAD SRC NOS AEG Diffuse Gastric Intestinal

B All Site Subtype Grade Histological type 60 60 60 60 ************ **** ****** **** ****** ****** 60 ****** ****** ****** **** **** ****** **** ****

40 40 40 40 40 G2

20 SRC 20 Gastric 20 20

20 Intestinal Observed spectrum (%) Observed spectrum (%) Observed spectrum (%) Observed spectrum (%) Observed spectrum (%)

0 0 0 0 0 C>A C>G C>T T>A T>C T>G C>A C>G C>T T>A T>C T>G C>A C>G C>T T>A T>C T>G C>A C>G C>T T>A T>C T>G C>A C>G C>T T>A T>C T>G 60 60 ****** **** ****** **** **** ****** 60 **** ****** ****** **** 60 **** ****** ****** ****

40 40 40 40 Untranscribed G3

Transcribed NOS AEG

20 Diffuse 20 20 20 Observed spectrum (%) Observed spectrum (%) Observed spectrum (%) Observed spectrum (%)

0 0 0 0 C>A C>G C>T T>A T>C T>G C>A C>G C>T T>A T>C T>G C>A C>G C>T T>A T>C T>G C>A C>G C>T T>A T>C T>G

Supplementary Figure S2. Mutation load and the transcription-induced mutational strand bias. (A) Mutation loads of the ascites samples in this study across histopathological subtypes and in the context of esophageal carcinoma (ESCA) and primary gastric cancer (STAD) from TCGA. The red solid line indicates the median value of mutation load, which is also labeled in the figure. Red and blue points denote ascites samples with or without matched germline DNA for mutation calling, respectively. (B) Mutational spectrum in transcribed and untranscribed strands in ascites samples across different histopathological subtypes.

1 Supplementary Figure S3

2 Supplementary Figure S3. WGD and aneuploidy analysis. (A) Density plot showing the distribution of tumor ploidy by histological subtypes for the TCGA-STAD dataset. (B) Profiles of DNA copy number alterations of the ascites samples in this study for all tumors together and across different histopathological subtypes. (C) Burden of DNA copy number losses (left part) or gains (right part) between different histological subtypes of this study. P value was calculated by non-parametric Mann-Whitney U test.

3 VHL TGFBR2 MLH1 PBRM1 4.50 p = 0.014 p = 0.0064 p = 0.0045 p = 0.0064 6 3.6 3.5 4.25

3.2 4.00 3.0 4

3.75 2.8 2.5

2 Gene expression 3.50 2.4 2.0

3.25 1.5 CN loss Other CN loss Other CN loss Other CN loss Other

BAP1 JAK2 CD274 PDCD1LG2 5 3 p = 0.0032 p = 0.0089 p = 0.01 p = 0.037 3 4 4 2 3 2

2 3 1 1 Gene expression 1

0 0 CN loss Other CN loss Other CN loss Other CN loss Other

SRC PTK6 p = 0.008 5 p = 0.017 5 4

4 3

2 3 Gene expression 1 2 CN gain Other CN gain Other

Supplementary Figure S4. Changes of gene expression levels in PC specimens had copy number alterations. CN, copy number; Other, specimens without detectable copy number alterations. Supplementary Figure S5

Histological.Type RNAseq Grade Subtype clustering Site cluster1 Tumor cell % cluster2 RNAseq clustering Histological.Type 1 N/A Intestinal 0.8 Diffuse Grade 0.6 N/A G2 0.4 G3

0.2 Subtype N/A 0 NOS SRC

Site AEG I AEG II AEGIII Gastric

Tumor cell % N/A <10 10~30 IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP IP 30~60 118 116 104 043 038 032 007 033 0107 073 067 056 054 010 123 109 064 062 051 045 040 039 036 026 020 013 58 124 067 070 068 057 052 046 031 024 009 004 008 60~90 >90 2 1 1 1 1 1 2 2 1 1 1 2 2

Supplementary Figure S5. Unsupervised Consensus clustering of ascites samples by top-1000 most variable genes from RNA-seq.

4 Supplementary Figure S6

Gra Histological type

4 4 index2 index2 index2 index2 index1 immune low index1 immune low Histological.Type immune high Histological.Type immune high Grade 2 Grade 2 Subtype index1 Subtype index1 SITE percentage low SITE percentage low batch 0 0 percentage high batch percentage high percentage percentage Histological.Type Histological.Type −2 −2 N/A N/A Diffuse Diffuse −4 Intestinal −4 Intestinal Grade Grade N/A N/A G2 G2 G3 G3

Subtype Subtype N/A N/A NOS NOS SRC SRC

SITE SITE AEG I AEG I AEG II AEG II AEGIII AEGIII Gastric Gastric

batch batch RNA333 RNA333 RNA435 RNA435

percentage percentage N/A N/A <10 <10 10~30 10~30 30~60 30~60 60~90 60~90 >90 >90 IP−032−1 IP−038 IP−039−1 IP−062 IP−036 IP−040 IP−123 IP−020 IP−045 IP−116 IP−58−2 IP−043 IP−109 IP−052−1 IP−064−1 IP−007 IP−051 IP−004 IP−026−1 IP−0107−2 IP−033−2 IP−068 IP−070 IP−067 IP−067−2 IP−010−1 IP−046 IP−056 IP−008 IP−031 IP−124 IP−073 IP−009−2 IP−057 IP−032−1 IP−038 IP−039−1 IP−062 IP−036 IP−040 IP−123 IP−020 IP−045 IP−116 IP−58−2 IP−043 IP−109 IP−052−1 IP−064−1 IP−007 IP−051 IP−004 IP−026−1 IP−0107−2 IP−033−2 IP−068 IP−070 IP−067 IP−067−2 IP−010−1 IP−046 IP−056 IP−008 IP−031 IP−124 IP−073 IP−009−2 IP−057

Subtype SITE

4 4 index2 index2 index2 index2 index1 immune low index1 immune low Histological.Type immune high Histological.Type immune high 2 Grade 2 Grade Subtype index1 Subtype index1 SITE SITE percentage low percentage low 0 batch 0 batch percentage high percentage high percentage percentage Histological.Type Histological.Type FABP1 −2 −2 N/A N/A Diffuse REG1A Diffuse Intestinal −4 Intestinal REG3A −4 Grade Grade UTS2 N/A N/A G2 MT1H G2 G3 G3 SAA1 Subtype Subtype CCDC91 N/A N/A NOS NOS CD34 SRC SRC ITLN1 SITE SITE AEG I CHRD AEG I AEG II AEG II MARK1 AEGIII AEGIII Gastric KLF15 Gastric

batch SOX8 batch RNA333 RNA333 B4GALNT4 RNA435 RNA435

percentage LYPD1 percentage N/A N/A PRRC2A <10 <10 10~30 MUC5B 10~30 30~60 30~60 AQP5 60~90 60~90 >90 CAPN6 >90

AC020914.1

MOGAT2

RAB26

SLC6A8

GPT IP−008 IP−046 IP−051 IP−010−1 IP−073 IP−024−1 IP−056 IP−068 IP−013 IP−033−2 IP−057 IP−020 IP−064−1 IP−032−1 IP−039−1 IP−040 IP−036 IP−038 IP−116 IP−123 IP−045 IP−062 IP−104 IP−58−2 IP−052−1 IP−118−1 IP−004 IP−026−1 IP−0107−2 IP−124 IP−009−2 IP−031 IP−043 IP−007 IP−109 IP−070 IP−054 IP−067 IP−067−2 IP−057 IP−067 IP−067−2 IP−010−1 IP−073 IP−008 IP−046 IP−056 IP−043 IP−124 IP−0107−2 IP−007 IP−109 IP−051 IP−064−1 IP−009−2 IP−052−1 IP−068 IP−031 IP−033−2 IP−004 IP−58−2 IP−026−1 IP−116 IP−070 IP−038 IP−032−1 IP−039−1 IP−020 IP−045 IP−062 IP−123 IP−036 IP−040

Supplementary Figure S6. Unsupervised hierarchical clustering of ascites samples by RNA-seq expression data.

5 EMT TGF-beta pathway Wnt pathway

EMT Wnt RNAseq clustering TGF_beta RNAseq clustering Histological type RNAseq clustering Histological type Grade Histological type Grade Subtype Grade Site Subtype Subtype Tumor cell % Site EMP3 * Site Tumor cell % FN1 10 Tumor cell % NOTCH1 VIM * NOG PPARD LRRC15 CDKN1C ADAM17 7 INHBA 8 LEFTY2 RBPJ MARVELD2 TJP1 MYC OCLN LTBP2 8 NCSTN 6 ITGA11 6 BCAR3 CTNNB1 ADAMTS12 WWTR1 NCOR2 5 COL10A1 BMPR1A 6 KAT2A MYH11 4 SMAD1 CUL1 COL6A3 CDH1 * GSK3A 4 NAP1L3 ID3 FZD8 ADAM12 2 SMURF1 4 GNAI1 3 SPOCK1 TGIF1 WNT5B DACT1 HIPK2 PTCH1 0 2 FAP SKIL 2 NKD1 GPC6 ARID4B WNT1 CDH2 EMT TRIM33 DKK4 1 MSRB3 HEY1 Mesenchymal XIAP BNC2 SMAD3 0 WNT6 0 PDGFRB Epithelial ACVR1 HEY2 AXL SMURF2 TGF_beta DLL1 MMP2 FNTA NOTCH4 Wnt SYT11 MAP3K7 1 DKK1 ADAMTS2 E.vs.M SMAD7 AXIN2 1 COL5A1 BMPR2 cluster2 HDAC11 COL5A2 M APC JAG2 cluster2 AEBP1 E PPM1A LEF1 FSTL1 IFNGR2 TCF7 THBS2 TGFBR1 CCND2 POSTN BMP2 FRAT1 CALD1 PMEPA1 FZD1 COL8A1 SMAD6 JAG1 NID2 JUNB AXIN1 CNRIP1 RHOA CSNK1E ANTXR1 TGFB1 * PSEN2 SULF1 ENG * APC ANGPTL2 SERPINE1 SKP2 FBN1 RAB31 * DVL2 LOXL2 THBS1 * HDAC2 PCOLCE HDAC1 TP53 COL1A1 SPTBN1 * HDAC5 COL3A1 KLF10 MAML1 SPARC FURIN NUMB COL1A2 CTNNB1 IP−0107−2 IP−58−2 IP−068 IP−040 IP−007 IP−104 IP−051 IP−109 IP−045 IP−062 IP−020 IP−123 IP−043 IP−038 IP−032−1 IP−039−1 IP−052−1 IP−064−1 IP−036 IP−118−1 IP−026−1 IP−033−2 IP−013 IP−116 IP−046 IP−009−2 IP−057 IP−010−1 IP−124 IP−067 IP−067−2 IP−008 IP−024−1 IP−004 IP−031 IP−056 IP−073 IP−054 IP−070 COL6A2 NCOR2 VCAN SLC20A1 OLFML2B * SKI PMP22 CDK9 * CMTM3 * UBE2D3 GYPC ID1 ZEB2 PPP1R15A CNOT1 ID2 CTNND1 FKBP1A CTNNA1 PPP1CA F11R Hippo pathway AP1G1 IP−013 IP−033−2 IP−116 IP−040 IP−026−1 IP−036 IP−038 IP−109 IP−007 IP−051 IP−020 IP−062 IP−039−1 IP−104 IP−032−1 IP−045 IP−123 IP−064−1 IP−043 IP−118−1 IP−008 IP−054 IP−068 IP−052−1 IP−073 IP−024−1 IP−031 IP−046 IP−004 IP−010−1 IP−070 IP−0107−2 IP−067 IP−067−2 IP−124 IP−009−2 IP−057 IP−056 IP−58−2 DYNC1LI2 COL6A1 HTRA1 Hippo RICTOR RNAseq clustering CLDN4 CDH1 * Histological type SPINT1 * Grade ERBB3 * PRSS8 Subtype CGN Site 6 ESRP1 * AMPK pathway Tumor cell % ESRP2 ATP8B1 FAT4 5 GALNT3 MST1 AMPK STK3 HOOK1 TEAD2 4 MARVELD3 RNAseq clustering GRHL2 TEAD4 IRF6 Histological type TEAD1 3 NF1 MYO5B Grade CDS1 TAZ MAP7 Subtype LATS2 2 IP−026−1 IP−116 IP−040 IP−062 IP−036 IP−123 IP−013 IP−007 IP−109 IP−020 IP−032−1 IP−045 IP−038 IP−039−1 IP−104 IP−064−1 IP−051 IP−043 IP−033−2 IP−118−1 IP−58−2 IP−052−1 IP−124 IP−0107−2 IP−009−2 IP−057 IP−004 IP−056 IP−073 IP−067 IP−067−2 IP−024−1 IP−046 IP−070 IP−008 IP−054 IP−010−1 IP−031 IP−068 E.vs.M LATS1 Site NF2 Tumor cell % FAT1 * 1 PCK1 YAP1 * TEAD3 * 0

APOC3 IP−007 IP−109 IP−020 IP−104 IP−038 IP−039−1 IP−051 IP−064−1 IP−043 IP−118−1 IP−036 IP−062 IP−123 IP−032−1 IP−045 IP−073 IP−009−2 IP−046 IP−58−2 IP−067 IP−067−2 IP−056 IP−068 IP−057 IP−0107−2 IP−070 IP−010−1 IP−031 IP−124 IP−052−1 IP−008 IP−054 IP−026−1 IP−040 IP−004 IP−024−1 IP−013 IP−033−2 IP−116 G6PC 10 Hippo ACADL CYP4A11 1 CYP7A1 8 FOXA2 cluster2 Proliferation signature HNF4A EHHADH 6 ONECUT2 CCND2 Proliferation signature MMP9 4 ANGPTL4 RNAseq clustering GADD45G Chromatin remodeling Histological type ACADM CPT2 2 Grade RUVBL1 Subtype ACSL1 * 0 pathway Site CYP27A1 * PDK4 AMPK Tumor cell % ACOX1 7 PCK2 1 NEIL3 G6PC3 RNAseq clustering PIF1 cluster2 6 ACSL5 Histological type POLE2 CAT ASPM CPT1A Grade CDKN3 5 BAX Subtype MCM2 GADD45A Site 4 TOB1 * UHRF1 Tumor cell % 5 CDK1 DNMT1 PRMT1 ARID1A CENPF 3 APOE * KAT8 TUBA1A * ATF4

IP−070 IP−008 IP−054 IP−004 IP−031 IP−010−1 IP−057 IP−024−1 IP−009−2 IP−046 IP−052−1 IP−58−2 IP−067 IP−067−2 IP−056 IP−073 IP−068 IP−0107−2 IP−124 IP−039−1 IP−026−1 IP−118−1 IP−007 IP−040 IP−033−2 IP−064−1 IP−036 IP−062 IP−104 IP−109 IP−038 IP−020 IP−032−1 IP−043 IP−051 IP−045 IP−123 IP−013 IP−116 DOT1L 4 TK1 * 2 KMT2C CDC20 * SETD2 TOP2A * WDR5 HPRT1 1 KMT2D 3 PCNA KMT2B ARID1B HMGB2 ARID2 RAN Proliferation 2

IP−58−2 IP−124 IP−056 IP−0107−2 IP−004 IP−068 IP−070 IP−009−2 IP−046 IP−010−1 IP−031 IP−067 IP−073 IP−067−2 IP−024−1 IP−057 IP−040 IP−051 IP−116 IP−032−1 IP−045 IP−109 IP−104 IP−118−1 IP−036 IP−062 IP−052−1 IP−007 IP−039−1 IP−123 IP−020 IP−038 IP−008 IP−054 IP−064−1 IP−026−1 IP−043 IP−013 IP−033−2 KMT2A signature EZH2 IP−031 IP−068 IP−073 IP−070 IP−067 IP−067−2 IP−0107−2 IP−007 IP−040 IP−124 IP−056 IP−013 IP−046 IP−58−2 IP−043 IP−033−2 IP−026−1 IP−064−1 IP−009−2 IP−010−1 IP−057 IP−036 IP−062 IP−039−1 IP−104 IP−051 IP−109 IP−116 IP−038 IP−123 IP−045 IP−020 IP−032−1 IP−054 IP−008 IP−024−1 IP−118−1 IP−004 IP−052−1 1 Hedgehog pathway 1 cluster2

Hedgehog HIF1 pathway RNAseq clustering Histological type Grade Subtype HIF1 Site Stemness RNAseq clustering Tumor cell % Histological type MYH9 ETS2 8 Grade LDB1 * RNAseq clustering ACHE Histological type Subtype RTN1 Site THY1 Grade SHH 6 Tumor cell % AMOT Subtype OPHN1 Site BHLHE40 CNTFR DDIT4 PTCH1 Tumor cell % ALDOA HEY1 4 PGK1 8 NRCAM CD44 TMEM45A SCG2 MYC STC2 HEY2 KLF4 8 NKX6−1 CTNNB1 TMEFF1 CRMP1 2 BNIP3 6 PLG HIF1A TGFB3 UNC5C NOTCH1 ALDH4A1 GLI1 EZH2 6 KDM3A SLIT1 BMI1 4 L1CAM 0 KDM5B MXD1 VLDLR PRM1 ETS1 * CDK5R1 NES 4 SERPINE1 * GLI2 Hedgehog IVNS1ABP 2 GLI3 ABCG2 VEGFA PU5F1 SSRP1 CDK6 1 ZSCAN4 EPRS 2 ADGRG1 cluster2 CD34 TFRC 0 TLE1 NANOG CCNB1 NRP1 * ZFP42 VEGFA * HIF1 DPYSL2 RASA1 * HLF 0 MECOM 1 TLE3 * TIST1 CA9 NRP2 SOX2 Category EGLN3 IP−008 IP−054 IP−068 IP−124 IP−057 IP−052−1 IP−056 IP−067 IP−067−2 IP−070 IP−009−2 IP−010−1 IP−073 IP−004 IP−024−1 IP−031 IP−046 IP−013 IP−116 IP−0107−2 IP−58−2 IP−104 IP−007 IP−109 IP−043 IP−064−1 IP−020 IP−032−1 IP−038 IP−039−1 IP−033−2 IP−118−1 IP−045 IP−123 IP−026−1 IP−036 IP−062 IP−040 IP−051 cluster2 PML LGR5 Catego CELSR1 IP−070 IP−004 IP−013 IP−0107−2 IP−068 IP−58−2 IP−052−1 IP−010−1 IP−057 IP−073 IP−024−1 IP−031 IP−009−2 IP−124 IP−064−1 IP−033−2 IP−056 IP−116 IP−118−1 IP−026−1 IP−036 IP−039−1 IP−045 IP−123 IP−020 IP−062 IP−043 IP−032−1 IP−038 IP−051 IP−104 IP−109 IP−007 IP−040 IP−046 IP−067 IP−067−2 IP−008 IP−054 Yamanaka Factor NF1 IP−043 IP−109 IP−038 IP−039−1 IP−036 IP−051 IP−020 IP−104 IP−032−1 IP−045 IP−062 IP−123 IP−118−1 IP−033−2 IP−064−1 IP−0107−2 IP−007 IP−056 IP−124 IP−013 IP−040 IP−026−1 IP−116 IP−052−1 IP−054 IP−070 IP−004 IP−008 IP−031 IP−057 IP−009−2 IP−046 IP−068 IP−073 IP−010−1 IP−024−1 IP−58−2 IP−067 IP−067−2 Normal Stem Cell Cancer Stem Cell r y

RNAseq clustering Histological type Grade Subtype Site Tumor cell % 1 N/A N/A N/A AEG I N/A cluster2 Intestinal G2 NOS AEG II <10 Diffuse G3 SRC AEGIII 10~30 Gastric 30~60 60~90 >90

Supplementary Figure S7. Expression profiles of genes in the key signaling pathways of gastric cancer.

6 T.cells.CD4. Monocytes memory.resting p = 0.0036 0.5 p = 0.0025 0.6

0.4

0.3 0.4

0.2 0.2 0.1 Relative fraction

0.0 E M E M

Myeloid dendritic Cytotoxic NK cells Fibroblasts cells lymphocytes 5 p = 3.3e−06 5 p = 4.3e-06 p = 2.5e-06 3 p = 1.2e-07 0.75 4 4

2 3 0.50 3 2 2 0.25 1 1 1

Absolute abundance 0.00 0 E M E M E M E M Supplementary Figure S8. The Cellular composition of PC microenvironment differed between two molecular subtypes. Upper panel, comparison of the CIBERSORT relative fractions of T cells CD4 memory resting cells and monocytes between two molecular subtypes; bottom panel, comparison of the MCP-Counter absolute abundance of cytotoxic lymphocytes, myeloid dendritic cells, NK cells, and fibroblasts between two subtypes. P values were calculated by the non-parametric Mann-Whitney U test.