Description KEGG term p value FDR

Asthma rno05310 7.109735e-11 1.832813e-08

Hematopoietic cell lineage rno04640 1.234217e-10 1.832813e-08

Th1 and Th2 cell differentiation rno04658 4.763163e-09 4.634023e-07

Th17 cell differentiation rno04659 6.241108e-09 4.634023e-07

Staphylococcus aureus infection rno05150 1.040718e-08 6.181862e-07

Graft-versus-host disease rno05332 2.573079e-07 1.199658e-05

Autoimmune thyroid disease rno05320 3.231403e-07 1.199658e-05

Allograft rejection rno05330 3.231403e-07 1.199658e-05

Antigen processing and rno04612 1.702904e-06 5.619584e-05 presentation

Supplementary table 1. The top 10 up-regulated pathways following viral ILP compared with untreated controls. Significantly enriched pathways were identified using the WebGestaltR package. FDR = false discovery rate.

A B

C D

5000 100

) Isotype Isotype 3 4000 200 ug 80 200 ug 500 ug 500 ug 3000 60 1000 ug 1000 ug 2000 40

1000 Percent survival 20 Tumour volume (mm volume Tumour 0 0 0 5 10 15 20

Day 1 Day 5 Days from implantation Day 10 Days from implantation

Supplementary figure 1. (A) PD-L1 expression in BN175 cells in vitro (red histogram = isotype control; blue histogram = untreated; orange = IFNγ) (B) The J43 anti-PD-1 clone is cross reactive with the rat PD-1 protein. Beads conjugated with the rat PD-1 protein were analysed by flow cytometry (red histogram = isotype control; blue histogram = J43 anti-PD-1 antibody). Dose escalation of the J43 anti-PD-1 antibody in BN175 sarcoma. Indicated doses were given by intraperitoneal injection 3 times at 48-hour intervals. (C) Tumour growth curves. (D) Survival. No significant improvement in therapy was noted with dose escalation.

Supplementary figure 2. Individual growth curves of animals treated with VV-ILP and an isotype control. Iso = isotype.

A B

5000 ns 5000 ) ) 3 3 4000 4000

3000 3000

2000 2000

1000 1000 Tumour volume (mm volume Tumour Tumour volume (mm volume Tumour 0 0 ILP ILP + a-PD-1 0 20 40 60 80 Days from implantation

C D 5000 100 )

3 M M 4000 80

3000 60

2000 40

1000 Percent survival 20 Tumour volume (mm volume Tumour 0 0 0 20 40 60 80 0 20 40 60 80 Days from implantation Days from implantation

Supplementary figure 3. GLV-1h68 and anti-PD-1 antibodies are required for durable cure. Animals underwent treatment with the protocol shown in Figure 3 without the addition of GLV-1h68. (A) Tumour volumes at the time of surgery demonstrating no increased therapy with the addition of PD-1 inhibitors in the absence of GLV-1h68 (n = 9 animals per group). (B,C) Individual growth curves following ILP alone (B) or in combination with PD-1 inhibitors (C) prior to surgery (M denotes early termination due to symptomatic metastases) (n = 6 animals per group). The addition of PD-1 blockade to standard ILP did not prevent local and distant relapse and survival was not improved when compared with ILP alone (D) (p = 0.682).

A BN175 B SW872 C HT1080 20 20 20 24 hrs 48 hrs 15 15 15

10 10 10

5 5 **** 5 * Relative ATP release Relative ATP release Relative ATP release 0 0 0 0 1 0 1 0 1 0 1 0 1 0 1 10 10 0.1 10 0.1 10 0.1 10 0.1 10 0.1 100 0.1 100 100 100 100 100 Melphalan (uM) Melphalan (uM) Melphalan (uM) D E F BN175 SW872 HT1080 24 hrs 20 20 20 48 hrs * 15 15 15 **** ** 10 10 10 **** **** 5 5 5 Relative ATP release Relative ATP release Relative ATP release 0 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0.1 0.1 0.1 0.1 0.1 0.1 GLV-1h68 MOI GLV-1h68 MOI GLV-1h68 MOI

G H I BN175 SW872 HT1080 5 5 5

4 4 4 *** 3 3 3 ** 2 ** 2 *** 2 * ** **

CRT expression 1 CRT expression 1 CRT expression 1

0 0 0 0 1 0 1 0 1 10 10 10 100 100 100 Melphalan (uM) Melphalan (uM) Melphalan (uM)

J K L BN175 SW872 HT1080 5 5 5 ** ** 4 4 4 *** ** *** 3 3 3 ** *** 2 2 2 CRT expression 1 CRT expression 1 CRT expression 1

0 0 0 0 1 0 1 0 1 0.1 0.1 0.1 GLV-1h68 MOI GLV-1h68 MOI GLV-1h68 MOI

M N O BN175 SW872 HT1080 120 120 120 ** Control 100 ** ** * 100 100 10 uM *** *** ** 80 80 80 100 uM *** ** ** 60 * 60 ** 60 40 40 40 HMGB1 (pg/ml) HMGB1 (pg/ml) HMGB1 (pg/ml) 20 20 20

0 0 0 0 0 0 0.1 0.1 0.1 GLV-1h68 MOI GLV-1h68 MOI GLV-1h68 MOI Supplementary figure 4. Melphalan and GLV-1h68 induce markers of immunogenic cell death in vitro. (A-C) Secretion of ATP at 24 and 48 hours following treatment with of rat (A) and human sarcoma cell lines (B,C) with melphalan. Y axis = relative luminescence (n= 3 per group). (D-E) Secretion of ATP at the same time points following treatment with GLV-1h68. Y axis = relative median fluorescence (n= 3 per group). (G-L) Cell surface expression of CRT at 24 hours following treatment with melphalan (G-I) or GLV-1h68 (J-L) (n= 3 per group). (M-O) Secretion of HMGB1 at 72 hours following treatment with melphalan and GLV-1h68 (n= 2 per group). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Supplementary figure 5. Representative flow cytometry gating strategies used in the immunophenotyping of BN175 sarcomas following therapy. (A) NK cell panel – viable cells were stratified based on their expression of CD161 and CD3. NK cells were defined as CD161hi and CD3 negative (CD161hi population). NK cells were then stratified based on their expression of granzyme B and CD62L. (B) T cell panel – viable cells were stratified based on their expression of CD3. CD3 positive cells were then subdivided into CD4 positive and CD8 positive cells. CD4 positive cells were then stratified based on their expression of Foxp3. CD8 positive cells were then stratified based on their expression of granzyme B and CD62L.

A B CD3+ve CD4+ve Foxp3+ve CD3-ve CD161+ve 100000 250000

80000 200000

60000 150000

40000 100000 Cells/gram Cells/gram 20000 50000

0 0

ILP ILP Control a-PD-1 VV-ILP Control a-PD-1 VV-ILP

ILP + a-PD-1 ILP + a-PD-1 VV-ILP + a-PD-1 VV-ILP + a-PD-1

C Tumour immune profile D TDLN immune profile 800000 NK 8×107 700000 CD8 CD8 600000 CD4reg 6×107 CD4reg 500000 CD4eff CD4eff 400000 4×107 300000 Cells/gram Cells/gram 200000 2×107 100000 0 0

ILP ILP a-PD-1 a-PD-1 Control VV-ILP Control VV-ILP

ILP + a-PD-1 ILP + a-PD-1 VV-ILP + a-PD-1 VV-ILP + a-PD-1

Supplementary figure 6. Flow cytometry analysis of tumours collected on Day 12 following treatment (n = 3 animals per group). No significant increase in regulatory T cells (A) or CD3-CD161+ natural killer cells (B) was noted with any treatment. (C,D) Summaries of the alterations in the immune landscape in both the tumour (C) and tumour-draining lymph nodes (D) following treatment.

A B C

D E F

G H I

J K L

Supplementary figure 7. Immunohistochemistry analysis of tumours collected on Day 12 following treatment (n = 3 animals per group). (A-F) Representative images of CD8+ cell infiltration at the invasive margin (red dotted line) on immunohistochemistry from animals treated with an isotype control (A), anti-PD-1 antibody (B), ILP (C), ILP + anti- PD-1 antibody (D), viral ILP (E) and viral ILP + anti-PD-1 antibody (F) (n = 6 animals per group). (G-L) Representative images of CD8+ cell infiltration within the tumour parenchyma on immunohistochemistry from animals treated with an isotype control (G), anti-PD-1 antibody (H), ILP (I), ILP + anti-PD-1 antibody (J), viral ILP (K) and viral ILP + anti-PD-1 antibody (L) (n = 6 animals per group).

3.0 A Neutrophils B Eosinophils 2.5 6 Mast cells Il6 Gadd45b

Dendritic cells Log2 fold change vs. control Il1b 2.0 M2-like MΦ Nfkbiz 4 M1-like MΦ Rab20 Traf1 1.5 Monocytes Stat5a Nfkbia NK cells 2 Icam1 1.0 CD4 Treg cells Tnfrsf1b Relative abundance Sqstm1 CD4 Th cells Ifit1 0 0.5 CD8 T cells Skil Gnai3 B cells Cd86 Cdkn1a 0.0 -2

ILP ILP a-PD-1 Control a-PD-1 VV-ILP VV-ILP

ILP + a-PD-1 ILP + a-PD-1 VV-ILP + a-PD-1 VV-ILP + a-PD-1

6 10 6 CCL3 CXCL2 Il1a C CCL4 D E Log2 fold change vs. control Log2 fold change vs. control CXCL3 Log2 fold change vs. control CCL5 Il1b CXCL6 CCL6 4 Il6 4 CCL9 CXCL12 CCL11 CXCL13 Il15 CCL17 5 CXCL14 Il16 2 CCL20 2 CXCL16 CCL24 Il17b CCL27 Il17d CXCR2 0 0 Il24 CCR1 CXCR3 CCR3 0 CXCR4 Il34 CCR4 CCR6 CXCR6 Il36b -2 -2

ILP ILP ILP a-PD-1 a-PD-1 a-PD-1 VV-ILP VV-ILP VV-ILP

ILP + a-PD-1 ILP + a-PD-1 ILP + a-PD-1 VV-ILP + a-PD-1 VV-ILP + a-PD-1 VV-ILP + a-PD-1

6 6 6 Il1r1 Foxm1 F Il1r2 G H Klrb1 Log2 fold change vs. control Log2 fold change vs. control Il2rb Birc5 Log2 fold change vs. control Il2rg Itm2b Il3ra 4 Top2a 4 4 Il4r Il6r Tpx2 Cbx7 Il10ra Il10rb 2 Nme1 2 2 Cd2 Il11ra1 Ccnb1 Il13ra2 Il15ra Cep55 Satb1 Il17ra 0 0 0 Il17rd Tyms Il18r1 Saraf Il20rb Cenpf Il21r -2 -2 Fuca1 -2 Il36r Cdkn3

ILP ILP ILP a-PD-1 a-PD-1 a-PD-1 VV-ILP VV-ILP VV-ILP

ILP + a-PD-1 ILP + a-PD-1 ILP + a-PD-1 VV-ILP + a-PD-1 VV-ILP + a-PD-1 VV-ILP + a-PD-1

Supplementary figure 8. (A) Estimation of the relative abundance of immune subsets using CIBERSORT. (B) Fold change in expression of associated with an activated state in dendritic cells (C,D) Fold change in expression of CCL (C), CXCL (D) chemokines and their receptors compared with untreated controls. (E,F) Fold change in expression of interleukins (E) and their receptors (F) compared with untreated controls. (G) Fold change in expression of validated adversely prognostic pan-cancer genes compared with controls. (H) Fold change in expression of validated favourably prognostic pan-cancer genes compared with controls. Grey boxes denote no significant change (adjusted p value < 0.05) (n = 3 animals per group). Transcripts with no significant changes in any cohorts are not shown.