1. Supplemental Material

2. Supplemental Figure Legends
Table S1: Immune marker panels used in NanoString DSP. CAB# is custom antibody ID, which uniquely identifies this reagent.
Table S2: Clinicopathological characteristics of EGFR TKI treated NSCLC patients in Yale cohort A.
Table S3: Clinicopathological characteristics of NSCLC patients in Yale cohort B.
Table S4: Clinicopathological characteristics of ICIs treated melanoma patients in Yale cohort C.
Table S5: Multivariate analysis of the predictive markers for PFS in Yale melanoma ICIs treated cohort C.
Table S6: Multivariate analysis of the predictive markers for OS in Yale melanoma ICIs treated cohort C.
Figure S1: Representative images of NanoString DSP and AQUA compartment selection in NSCLC. The epithelial compartment was defined by cytokeratin positivity (green) which allowed the measurement of targets by both assays in the same compartment. Stroma was defined by cytokeratin negative DNA positive regions (purple).
Figure S2: NanoString DSP reproducibility across different cores in ITx melanoma cohort C. (A) CD8 reproducibility across 2 cores (B) CD68 reproducibility across 2 cores
Figure S3: NanoString DSP sequential compartment assignment. CD8 counts by NanoString DSP in tumor, CD68+ and CD45+ compartment. Compartment definition accuracy is affected by the order of barcode collection from each compartment. In overlapping compartments, measurement of a marker can be assigned to only one compartment.

3. Table S1 Table S2 Table S3 Yale NSCLC Cohort A Yale NSCLC Cohort B
n=43 %
Age
<70 31
72.1
≥70 12
27.9
Sex
Female 26
60.5
Male 17
39.5
Smoking history
Never smoker 13
30.2
Former/ Current smoker 30
69.8
Stage
I/II
III/IV
Histology
ADC 41
95.3
SCC 2
4.7
EGFR mutation
Yes 36
83.7
Exon 18 3
7.0
Exon 19 16
37.2
Exon 20
Exon 21 14
32.6
Unknown 1
2.3 No 6
EGFR TKI treatment
Afatinib
Erlotinib
Osimertinib
Regorafenib
Yale NSCLC Cohort B
n= 113 %
Age
<70 64
56.6
≥70 49
43.4
Sex
Female 59
52.2
Male 54
47.8
Smoking history
Never smoker 7
6.2
Former/ Current smoker
106
93.8
Stage
I/II 85
75.2
III/IV 28
24.8
Histology
ADC 66
58.4
SCC 22
19.5
Other 20
17.7
Unknown 5
4.4
Table S3
Yale Melanoma Cohort C
n= 58 %
Age
<70 38
65.5
≥70 20
34.5
Sex
Female 25
43.1
Male 33
56.9
Stage I 10
17.2 II 11
19.0
III 22
37.9 IV 12
20.7
Unknown 3
5.2
Itx treatment
Pembro
Nivo
Ipi+Nivo
Specimen
Primary
Met LN 15
25.9
Mutation
None 32
55.2
BRAF 17
29.3
NRAS 8
13.8
CKIT 1
1.7

4. Table S4 Cocktail A – Yale NSCLC cohort A
Cocktail B – Yale NSCLC cohort B
Cocktail C - Yale Melanoma Cohort C #
Target
CAB #
Working Conc. (ug/mL) 1
Beta-Catenin
963 2
0.5
2.5
CD8
1022
PMS2
934 3
B7-H3
959 4
CD4
941 5
FOXP3
268 6
Mouse IgG1
891
Phospho-p70S6K
336 7
CD68
1023 8
Phospho-RPS6
767
Rabbit IgG
836 9
CD14
397 10 S6
837 11
GZMB
1068
MLH1
937 12
Ki-67
314
CD34
1041 13
Beta-2-microglobulin
964
MSH2
919 14
Histone H3
387
1.3 15
Akt
962 16
CD3
1019 17
VISTA
940 18
PD1
1024
0.2 19
Phospho-STAT3
458 20
CD44
619 21
STAT3
957
MSH6
920 22
CD56
1020 23
PD-L1
1039 24
CD45
1018
P-AKT
624 25
CD19
642 26
CD20
706 27
Pan-Cytokeratin
886 28
CD45RO 77 29
Tim3
1038
Bcl-2
960 30
HLA-DR
1052 31 32 33 34 35
B7-H4
1053 36
Ik-Ba
907 37
BIM
894 38
IDO1
1033 39
CD11c
1036 40
Mouse IgG2a
1258 41
BCL6
880 42
c-Myc
878 43
Phospho-STAT5
882 44
CD163
1031

5. Table S5 Table S6 Predictive markers for PFS - Multivariate Analysis
Tumor ROI
Cutpoint
Hazard Ratio
95% CI
P value
CD68+ ROI
CD45+ ROI
IDO1
Low (1st and 2nd tertile) vs High (3rd tertile)
0.42
0.14 to 1.20
0.1
PDL1
0.51
0.19 to 1.27
0.15
B2M
Median
0.3869 to
0.0033
CD11c
0.7
0.31 to 1.48
0.36
CD8
0.62
0.25 to 1.43
0.27
HLADR
1.14
0.37 to 3.17
0.8
TIM3
0.79
0.33 to 1.79
0.58
0.91
0.42 to 1.91
0.82
0.96
0.36 to 2.35
0.93
0.86
0.19 to 3.07
0.83
CD3
0.87
0.20 to 3.48
0.85
Table S6
Predictive markers for OS - Multivariate Analysis
Tumor ROI
Cutpoint
Hazard Ratio
95% CI
P value
CD68+ ROI
CD45+ ROI
CD20
Median
0.39
0.10 to 1.22
0.11
PDL1
Low (1st and 2nd tertile) vs High (3rd tertile)
0.2
0.03 to 0.88
0.032
B2M
0.38
0.12 to 1.03
0.059
HLADR
0.29
0.05 to 1.64
0.17
CD4
0.46
0.14 to 1.36
0.16
MSH2
1.98
0.62 to 7.66
0.25
IDO1
0.5
0.09 to 2.09
0.35
MYC
2.15
0.70 to 7.08
PMS2
1.3
0.33 to 4.54
0.68
CD8
1.88
0.3 to 10.24
0.48
1.81
0.63 to 5.52
0.26
1.21
0.38 to 3.40
0.72
0.88
0.24 to 3.10
0.85
CD3
1.18
0.18 to 7.79
0.86
0.93
0.14 to 5.33
0.94

6. Figure S1
Figure S2 A B

7. Figure S3