Supplementary Figure S1. Raw flow cytometry data. (A) Representative flow scheme utilized

for immune profiling analyses. (B) Bar graph representing mean +/- SEM from tumors for the

indicated treatment groups, along with individual samples for each, denoting mean fluorescence

intensity (MFI) for LAG3+ CD8 cells. (C) Bar graph representing mean +/- SEM from tumors

for the indicated treatment groups, along with the individual samples for each, denoting MFI for

TIM3+ CD8 cells. (D) Representative flow plots representing the frequency of CD4 cells positive

for CD25 and FoxP3. (E) Bar graph quantifying frequencies observed in individual mice across

the indicated treatment groups. Bars denote mean +/- SEM with individual mice per group being

shown. (F) Bar graph representing the ratio of CD8:CD4 cells in tumor of the indicated groups.

Bars denote mean +/- SEM with individual mice per group being shown.

Supplementary Figure S2. Characterization of CAIX expression in the B16F10 model. (A)

Immunofluorescent staining of B16F10 tumor stained for the indicated markers. Pimonidazole =

hypoxia, Hoecsht33342 = nuclei. Scale bar = 50 μm. (B) Western blot analysis of CAIX and

CAXII levels in the indicated cell lines. β-actin serves as a loading control. (C) Micrographs of

immunohistochemically stained B16F10 shCar9 tumors for CAIX and CAXII. Scale bar = 100

µm left, 50 µm right.

Supplementary Figure S3. SLC-0111 in combination with immune checkpoint blockade in

B16F10 tumors. (A) Spider plots depicting B16F10 tumor growth from individual mice across

the indicated treatments. V+I – vehicle + isotype, S-SLC-0111, P-anti-PD-1, C-anti-CTLA-4,

S+P - SLC-0111+anti-PD-1, S+C – SLC-0111+anti-CTLA-4, P+C – anti-PD-1+anti-CTLA-4,

S+P+C – SLC-0111 + anti-PD-1 + anti-CTLA-4. (B) B16F10 tumors treated as indicated and

stained for CAIX by IHC. Scale bar = 50 μm. (C) Spider plots overlaying P+C treated mice

over S+P+C treated mice bearing B16F10 tumors. (D) Spider plots depicting B16F10 tumor

growth from individual mice across the indicated treatment groups. BB - anti-4-1BB, S+BB -

SLC-0111 + anti-4-1BB, P+BB - anti-PD-1 + anti-4-1BB, S+P+BB - SLC-0111 + anti-PD-1 +

anti-4-1BB. (E) Stacked bar graphs depicting tumor size distribution across the indicated

treatment groups at day 40. (F) Frequency of tumor free mice across the indicated treament

groups. (G) Kaplan-Meier curves displaying survival proportions within each treatment group

for the duration of the study. (H) Immunofluorescent staining of B16F10 tumor stained for the

indicated markers. Nuclei = Hoecsht33342 ; arrows indicate CD3 positive cells negative for

CAIX. Scale bar = 20 μm.

Supplementary Figure S4. CAIX inhibition leads to increased tumor necrosis and reduced

metastasis in the 4T1 model. (A) Growth of 4T1 shCar9 tumors in NODSCID mice. Bars

represent mean +/- SEM (B) Mass spectrometry detection of SLC-0111 in tumors (red) and

plasma (blue) from mice bearing orthotopic breast tumors dosed over the indicated range. (C)

Relationship of SLC-0111 accumulation in orthotopic tumors to decrease in tumor size at the

corresponding dose; r2=0.9823. (D) Spider plots of 4T1 tumor growth across the indicated

treatment groups. (E) Average tumor volume at endpoint day 29 for the indicated treatment

groups. Red bar represents mean volume. Circles represent individual mouse contribution to

group mean tumor volume. (F) IHC staining for CAIX in 4T1 tumors treated as indicated. Scale

bar = 50 μm. (G) Representative micrographs of H&E stained tumors for the indicated treatment

groups. Scale bar = 2000 µm. (H) Representative micrographs of H&E stained lung sections for

the indicated treatment groups. Arrows indicate regions of metastatic tumor growth. Scale bar =

2000 µm.

Supplementary Figure S5. Raw expression values for glycolytic genes in TCGA SKCM

and BRCA datasets. (A) Raw read counts from the TCGA_SKCM dataset for the indicated

genes across the hierarchical clusters displayed in Figure 6A. Shown are box and whisker plots

of log10 transformed FPKM values. (B) Raw read counts for the indicated genes across the

hierarchical clusters displayed in Figure 6B. Shown are box and whisker plots of log10

transformed FPKM values. (C) Hierarchical clustering according to high (red) and low (black)

metabolic activity. (D) Kaplan-Meier analyses of survival proportions based on clustering in (C).

(E) Shown are box and whisker plots of log10 transformed FPKM values displaying the

distribution of CA9 expression across the PAM50 subtypes. (F) Raw read counts from the

TCGA_BRCA dataset for the indicated genes across the hierarchical clusters in Figure 6. Shown

are box and whisker plots of log10 transformed FPKM values. (G) Raw read counts for the

indicated genes across the hierarchical clusters displayed in Figure 6. Shown are box and

whisker plots of log10 transformed FPKM values.

Supplementary Figure S6. Associations of CA9 expression with Th1 response in TCGA-

BLCA, TCGA-PAAD, TCGA-LUAD, TCGA-LUSC, TCGA-SARC datasets. (A)

Hierarchical clustering according to high (red) and low (black) Th1-related gene expression in

the TCGA-BLCA dataset. (B) Raw read counts for CA9 expression across the indicated clusters.

(C) Hierarchical clustering according to high (red) and low (black) Th1-related gene expression

in the TCGA-LUAD dataset. (D) Raw read counts for CA9 expression across the indicated

clusters. (E) Hierarchical clustering according to high (red) and low (black) Th1-related gene

expression in the TCGA-LUSC dataset. (F) Raw read counts for CA9 expression across the

indicated clusters. (G) Hierarchical clustering according to high (red) and low (black) Th1-

related gene expression in the TCGA-PAAD dataset. (H) Raw read counts for CA9 expression

across the indicated clusters. (I) Hierarchical clustering according to high (red) and low (black)

Th1-related gene expression in the TCGA-SARC dataset. (J) Raw read counts for CA9

expression across the indicated clusters.

Table S1: List of antibodies used in study Antibody Source Catalog # Dilution Required Flow Cytometry CD45.2 AF700 ThermoFisher Scientific 56-0454-81 1:200 CD11b-V500 BD Biosciences 562127 1:250 Gr1-AF488 ThermoFisher Scientific 53-5931-82 1:500 F4/80-e450 ThermoFisher Scientific 48-4801-82 1:150 SiglecF-BV786 BD Bioscience 740956 1:150 Ly6C-APC BD Bioscience 560595 1:200 CD206-PE ThermoFisher Scientific 12-2061-80 1:250 MHCII-PE-Cy5 ThermoFisher Scientific 15-5321-81 1:1000 Ly6G-PerCP-Cy5.5 BD Bioscience 560602 1:500 CD11c-APC-e780 ThermoFisher Scientific 47-0114-82 1:150 PD-L1-BV650 BD Bioscience 740614 1:150 CD3ε-BV421 BD Bioscience 562600 1:250 CD8α-PerCP-Cy5.5 ThermoFisher Scientific 45-0081-82 1:1000 CD4-e506 ThermoFisher Scientific 69-0042-82 1:1000 CD25-e660 ThermoFisher Scientific 50-0252-82 1:150 FoxP3-PE-Cy7 ThermoFisher Scientific 25-5773-82 1:100 GzmB-PE ThermoFisher Scientific 12-8898-80 1:150 EOMES-PE-eFluor610 ThermoFisher Scientific 61-4875-80 1:100 NK1.1-FITC ThermoFisher Scientific 11-5941-81 1:200 PD-1-APC-eFluor780 ThermoFisher Scientific 47-9985-80 1:100 CD19-BV650 BD Bioscience 563235 1:250 T-bet-PE-Cy7 ThermoFisher Scientific 25-5825-80 1:100 GATA-3-AF488 ThermoFisher Scientific 53-9966-41 1:100 KLRG1-FITC ThermoFisher Scientific 11-5893-80 1:250 CD44-AF700 Biolegend 103025 1:500 CD62L-PE-CF594 BD Bioscience 562404 1:250 TNF-α-APC ThermoFisher Scientific 17-7321-81 1:500 IFN-γ-PE-Cy7 ThermoFisher Scientific 25-7311-82 1:100 LAG-3-APC Biolegend 125209 1:100 TIM-3-PE-Cy7 Biolegend 134009 1:100 RORγT-APC ThermoFisher Scientific 17-6988-80 1:100 CD28-BV650 BD Bioscience 740466 1:125 CXCR5-APC-Cy7 Biolegend 145525 1:125 Bcl-6-APC-Cy7 BD Bioscience 563581 1:125 ICOS-BV786 Biolegend 313534 1:125 Western Blotting CAIX R&D Systems AF2344 1:400 β-actin Sigma A5441 1:10000 CAXII Abcam Ab218983 1:500 Immunohistochemistry CAIX (mouse) R&D Systems AF2344 1:100 CAIX (human) (M-75) Biosciences AB1001 1:100 GLUT-1 Abcam AB40084 1:100 MCT-4 (H-90) Santa Cruz SC-50329 1:100 CD3e (SP7) ThermoFisher Scientific RM-9107-S0 1:100 CD3 Abcam Ab5690 1:100 CAXII Abcam Ab218983 1:100 In vivo studies PD-1 (RMP1-14) BioXcell BP0146 See M&M CTLA-4 (9H10) BioXcell BE0131 See M&M CD137 R&D Systems MAB9371 See M&M Syrian Hamster IgG BioXcell BE0087 See M&M

In vitro studies CD3e (145-2c11) BD Biosciences 553058 See M&M CD28 (37.51) BD Biosciences 553294 See M&M

Table S2: Patient demographics and associations of CAIX staining with clinicopathological parameters

CAIX Staining Characteristic Negative, n (%) Positive, n (%) P 363(91) 37(9) Age 59.83(17.07) 58.65 0.689 Gender 0.485 Male 208 (57.3) 24 (64.9) Female 155 (42.6) 13 (35.1) Tumor Grade <0.001 1 130 (35.8) 3 (8.1) 2 110 (30.3) 10 (27.0) 3 90 (24.8) 13 (35.1) 4 33 (9.1) 11 (29.7) Location 0.006 Head and neck 55 (15.2) 4 (10.8) Trunk 95 (26.2) 4 (10.8) Extremity 89 (24.5) 5 (13.5) Lymph node 54 (14.9) 8 (21.6) Distant 64 (17.6) 16 (43.2) Unspecified 6 (1.7) 0 (0.0) Mitotic Rate 0.638 Unspecified 225 (62.0) 26 (70.3) <1 51 (14.0) 4 (10.8) 1-10 64 (17.6) 4 (10.8) >10 23 (6.3) 3 (8.1)

Table S3: CAIX expression is associated with increased melanoma grade and metastasis Parameter CAIX Positive CAIX Negative P value Frequency 37 363 Tumor Stage 1 or 2 13 (35%) 240 0.0003 3 or 4 24 (65%) 123 Metastasis Negative 13 (35%) 208 0.0003 Positive 24 (65%) 122

Table S4 : Hazard ratios from multivariate Cox regression analysis of TMA