STING and IRF3 in stromal fibroblasts enable sensing of genomic stress in cancer cells to undermine oncolytic viral therapy

Abstract

Cancer-associated fibroblasts (CAFs) perform diverse roles and can modulate therapy responses1. The inflammatory environment within tumours also influences responses to many therapies, including the efficacy of oncolytic viruses2; however, the role of CAFs in this context remains unclear. Furthermore, little is known about the cell signalling triggered by heterotypic cancer cell–fibroblast contacts and about what activates fibroblasts to express inflammatory mediators1,3. Here, we show that direct contact between cancer cells and CAFs triggers the expression of a wide range of inflammatory modulators by fibroblasts. This is initiated following transcytosis of cytoplasm from cancer cells into fibroblasts, leading to the activation of STING and IRF3-mediated expression of interferon-β1 and other cytokines. Interferon-β1 then drives interferon-stimulated transcriptional programs in both cancer cells and stromal fibroblasts and ultimately undermines the efficacy of oncolytic viruses, both in vitro and in vivo. Further, targeting IRF3 solely in stromal fibroblasts restores oncolytic herpes simplex virus function.

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Fig. 1: Cancer cell–CAF contact triggers the expression of ISGs and other inflammatory modulators.
Fig. 2: Production of IFNB1 in CAFs triggers the ISG response.
Fig. 3: Cytoplasmic DNA mechanisms converge on IRF3 to drive ISG expression following cancer cell–CAF contact.
Fig. 4: CAFs use transcytosis to detect genomic stress in cancer cells.
Fig. 5: Contact-mediated IRF3 activation undermines viral therapy.

Data availability

Microarray data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession numbers GSE121058. Previously published gene-expression data that were re-analysed here are available under accession codes GSE10332216 and GSE12153638 and GSE2215527. Source data for Figs. 15 and Extended Data Figs. 17 are available online. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Change history

  • 18 June 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank P. Chakravarty for assistance with Bioinformatics, S. Foo and R. Sadri for technical assistance, Joan Massagué for discussion, colleagues in our laboratory for support and advice and A. Blanchard for provision of compounds. E.N.A, E.L.M., A.R., S.D., S.H. and E.S. are supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001144), the UK Medical Research Council (FC001144) and the Wellcome Trust (FC001144). E.N.A. was additionally supported by the Wellcome Trust (096084/B/11/Z). A.R. was supported by the Spanish Society for Medical Oncology (Beca Fundación SEOM), E.L.M. was supported by a Biotechnology and Biological Sciences Research Council–GlaxoSmithKline CASE Fellowship. K.J.H., A.M. and D.M. are supported by The Royal Marsden and The Institute of Cancer Research National Institute for Health Research Biomedical Research Centre and a Cancer Research UK grant (A23275). T.K. was funded by Marie-Curie action (HeteroCancerInvaison # 708651) and the Japanese Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation.

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Contributions

Conceptualization and design: E.S., E.N.A., E.L.M., A.R. and K.J.H. Development of methodology: S.D., E.N.A., E.L.M. and A.R. Acquisition of data: E.N.A., S.D., E.L.M., S.H., T.K., D.M. and A.R. Analysis and interpretation of data: E.N.A., E.S., A.R., E.L.M., K.J.H. and A.M. Writing and editing: E.N.A, A.R., E.L.M., E.S., K.J.H. and A.M.

Corresponding author

Correspondence to Erik Sahai.

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Competing interests

E.S. and E.M. received research funds from GlaxoSmithKline through a Biotechnology and Biological Sciences Research Council–GlaxoSmithKline CASE Fellowship. The other authors declare no competing interests.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Direct cancer cell-CAF co-culture leads to production of Interferon stimulated genes, whereas co-culture with other cell types does not.

A) Scheme of experimental set-up of co-culture experiments highlighting ‘touch’ and ‘no touch’ assays. B-C) Gene expression heat map of cytokines (b) and IFN type 1 pathway genes (c) between direct and indirect co-cultures of A431 and VCAF2b. n=2 independent experiments for each cell type in different culture conditions (alone, direct co-culture or indirect co-culture). D) qRT-PCR of ISGs MX2 and OAS2 of the co-cultures of the non-transformed epithelial HaCat cell line with VCAF2b compared with A431/VCAF2b co-culture. Each dot is a biological replicate (n=3 independent experiments, except for OAS2 A431 control sample n=2). E) qRT-PCR of ISGs MX2 and OAS2 of co-cultures of A431 with HUVEC compared with A431/VCAF2b co-culture. Each dot is a biological replicate (n=3 independent experiments). F) qRT-PCR of ISGs MX2 and OAS2 of co-cultures of A431 with PBMC-derived macrophages from two different donors, compared with A431/VCAF2b co-culture. Each dot is a biological replicate (n=4 independent experiments for A431-Macrophages and n=3 for A431-VCAF2b). D-F Represented is mean and SD, mRNA levels were normalized against two housekeeping genes. See also Statistical Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 IFNB1 signals through IRF9 to produce ISGs.

A) qRT-PCR time course (96hrs) of OAS2 in direct, indirect and alone cultures of A431 and VCAF2b. Lapsed hours indicate number of hours after A431 cells were added to VCAF2b. mRNA levels were normalized against two housekeeping genes, each dot is a biological replicate (n=2). B) Additional micrographs of RNAScope combined with IF in cell co-culture or the cells on their own. IF: DAPI (grey), Keratin (magenta), RNA probes: MX2 (cyan), IFNB1 (yellow), scale bar is 50μm. Shown representative images from three biological replicates. C) Confocal micrographs of RNAScope combined with IF in A431/VCAF2b co-culture with defined borders (PDMS inserts used). IF: Fibronectin (magenta), DAPI (cyan), RNA probe: IFNB1 (white). Scale Bar 100µm. Red box highlights enlarged image of IFNB1 speckles in fibronectin positive CAF merge (left) and IFNB1 single channel (right). Blue box indicates zoomed in image of VCAF2b distant to the interface with A431, merge (left), IFNB1 single channel (right). Shown representative images from three biological replicates. D-E) qRT-PCR of OAS2 and MX2 in direct A431/VCAF2b (d) and VSCC10-VCAF10 (e) co-cultures after siRNA KD of IFNAR1/2 in both cells. mRNA levels shown were normalized to siRNA scramble control. Each dot is a biological replicate, (d) n = 5, (e) n = 2. One sample t test. F-G) qRT-PCR of IFNB1, OAS2 and MX2 in direct A431/VCAF2b (f) and VSCC10/VCAF10 (g) co-cultures after siRNA KD of two different IRF9 siRNA sequences in both cells. Each dot is a biological replicate (number of independent experiments shown in the graphs). mRNA levels shown were normalized to siRNA scramble control. For all graphs number of independent experiments is shown in the figure, represented is mean with SD. See also Statistical Source Data Extended Data Fig.2. Source data

Extended Data Fig. 3 IRF3 levels influence ISG production in vitro and in vivo, while not affecting invasion or matrix deposition.

A) qRT-PCR of MX2 and OAS2 after siRNA KD of IRF3 in A431-VCAF2b co-cultures using two different siRNA sequences. mRNA levels normalized to siRNA scramble control. One sample t test. B) Micrographs of IF staining of different culture conditions after cell-type specific siRNA KD of IRF3. Fibronectin (magenta), MX2 (yellow), DAPI (cyan). Representative images from three independent experiments, scale bar 50μm. C) qRT-PCR of IRF3 in A431-VCAF2b co-cultures showing siRNA efficacy. D) qRT-PCR of MX2 in co-cultures of A431 with VCAF2b par/OE after transfection with IRF3 siRNA. mRNA levels were normalized to siRNA scramble control. One sample t test. E) qRT-PCR of IRF3, TMEM173/STING and MB21D1/cGAS from subcutaneous tumours four days after injection of A431-VCA2b par or A431-VCAF2b OE in the flank of Balb/c nude mice. Each dot represents an individual tumour sample (n=4), from two independent experiments. mRNA levels were normalized against two housekeeping genes. F) IF images of tumours originating from injection of A431 cells alone or co-injected with VCAF2b in the flank of Balb/c nude mice. Images are representative from 5 tumours per group from two independent experiments. Scale bar 50 µm. G) Images show organotypic invasion assay with control siRNA or IRF3 KD in VCAF2b (green) A431 (magenta). Scale bar 100 µm. H) Quantification of images as seen in F each dot represents a replicate from two independent experiments. Unpaired t test. I) Confocal micrographs showing IF for Fibronectin in VCAF2b cultures with either control or IRF3 siRNA. White box highlights enlarged section shown below. Scale bar is 20µm. Representative images from two independent experiments. J) qRT-PCR of IFNB1 in A431/VCAF2b co-cultures after siRNA KD of IRF7 in both cells. mRNA levels were normalized to siRNA scramble control. For all graphs number of independent experiments is shown in the figure, represented is mean with SD. See also Statistical Source Data Extended Data Fig.3. Source data

Extended Data Fig. 4 CAFs present in human tumours express IRF3 and ISGs.

A) IF staining of IRF3 (red), DAPI (blue) in normal mucosa and oral SCC, showing IRF3 nuclear translocation. Yellow arrowheads indicate stromal cells. Images are representative of eight tumours. Scale bar 100 μm. B) Plot shows average ISG expression in IRF3/7 positive (expression of IRF3 or IRF7 >0) and IRF3/7 negative CAFs. ISG expression was calculated as the mean of MX1, MX2, OAS1, and OAS2. Quartiles, 90% value, and outliers are shown. Kolmogorov-Smirnov test. C) Plots show ACTA2, FAP, and CXCL12 expression in IRF3/7+ve and IRF3/7-ve CAFs. Each dot represents single cell value. Line at mean. D) qRT-PCR of MX2 in A431/VCAF2b co-cultures after cell-type specific siRNA KD of DNA sensors ZBP1/DDX41. mRNA levels normalized to siRNA control. One sample t test. E) qRT-PCR of MX2 of A431 or VCAF2b cells after treatment with cGAMP 20 µM or control. mRNA levels were normalized against two housekeeping genes. F) qRT-PCR of basal levels of STING/TMEM173 from A431 or VCAF2b cells. mRNA levels were normalized against two housekeeping genes. G) qRT-PCR of OAS2 and MX2 after treatment with DMSO, or Tonabersat 40 µM and Enoxolone 100 µM. mRNA levels were normalized to DMSO control. D-G) n=3 independent experiments. H) Images of calcein AM-parachute assays two hours after addition of labelled cells (in green in the title) to unlabelled cells; calcein (green) and DRAQ5(blue) are shown. Scale bar 25 μm. Magenta-dot indicates loaded cell, yellow-dot indicates transferred calcein from another cell. I) Sequential still images of time-lapse showing transcytosis of material from mCherryLifeAct expressing VCAF2b (blue) into A431-yPET cell (yellow). Representative images of two independent experiments. Scale bar 20 μm. J-L) Images showing A431 cells in co-culture with HUVEC cells (j), with macrophages derived from PBMCs (k) and HaCaT cells co-cultured with VCAF2b (l). The red line marks presence of keratin and location of Z projection. Phalloidin (cyan), keratin 7/17 (yellow). Scale bar 10 μm. Micrographs representative of two independent experiments. See also Statistical Source Data Extended Data Fig.4. Source data

Extended Data Fig. 5 Single cell cloning classifies A431 cells in inducers and non-inducers.

A) qRT-PCR of IFNB1 and MX2 in direct, indirect and alone A431/VCAF2b co-cultures after siRNA KD of E-cadherin. Each dot is a biological replicate (IFNB1 n=4, MX2 n=3). mRNA levels shown were normalized to siRNA scramble control. One sample t test. B) Images show DAPI staining of A431 cells treated with DMSO or 1μM AZD6733. Orange arrows show micronuclei. Images are representative of three independent experiments. Scale bar is 10 μm. C) Micrographs of IF staining of replicated cytoplasmic DNA labelled with BrdU (red), DAPI (blue) and Phalloidin (cyan) in A431 inducers and non-inducers. White arrows highlight presence of replicated DNA in the cytoplasm of Inducer cells. Images are representative of two independent experiments. Scale bar is 20μm. D) qRT-PCR comparing MX2 expression in direct co-cultures (Touch) of ‘inducer’ and ‘non-inducer’ A431 with VCAF2b. On the right, enlarged graph showing qRT-PCR comparing MX2 expression between direct (Touch) and indirect (No Touch) co-cultures of non-inducers A431 with VCAF2b. Each dot is a biological replicate (inducers n=5, non-inducers n=3). mRNA levels were normalized against two housekeeping genes. E) FACs plot showing equivalent numbers between inducers and non-inducers of VCAF2b cells with A431 cytoplasm fragments as seen in the GFP channel. Representative FACS plot from two independent experiments. For all graphs; each dot is a biological replicate, number of independent experiments is shown in the figure, represented is mean with SD. See also Statistical Source Data Extended Data Fig.5. Source data

Extended Data Fig. 6 Cancer cell-CAF direct co-culture confers resistance to oncolytic virus therapy.

A) Micrographs of 24h A431 inducers or non-inducers direct co-cultures with VCAF2b. Cells were infected using HSV-eGFP MOI=1 and fixed 48h later. HSV-eGFP (cyan), Vimentin (red), DAPI (blue), scale bar is 50 μm. Images from three independent experiments. B) Viral replication rates in PFU/hour from A431 Inducers-VCAF2b co-culture at 0->24h and 24->48h comparing direct and indirect co-cultures. Data from three independent experiments. Represented mean and SD. C) Micrographs of co-cultures with A431-yPet (red) and VCAF2b after 18h from Vaccinia-RFP (cyan) virus infection at MOI 0.5. Scale bar is 50μm. D) Micrographs of A431 and VCAF2b co-cultures 48h after Reovirus infection (MOI=1). IF for Reoδ3 (cyan), phalloidin (red), scale bar is 50μm. E) Micrographs of FaDu (stained with cytokeratin 7/17 antibody, red) and OCAF1 co-cultures after 48h infection with HSV-GFP (cyan) MOI=0.1. Scale bar is 50μm. F) Micrographs of A431 cells alone and in direct co-culture with VCAF2b and HUVEC cells after 48h of HSV-GFP infection (MOI=1). Scale bar is 50μm. G) Micrographs of A431 cells alone and in direct co-culture with VCAF2b and PBMC derived macrophages after 48h of HSV-GFP infection (MOI=1). Scale bar is 50μm. Panels c-g representative images from two independent experiments. See also Statistical Source Data Extended Data Fig.6. Source data

Extended Data Fig. 7 Overexpression of IRF3 in CAFs increases resistance to oncolytic virus therapy.

A) Western blot showing IRF3 levels before and after lentiviral transfection of siRNA resistant IRF3 to OCAF2 cells. IRF3 (50-55kDa) and loading control Anti-beta actin (43kDa). Representative plot from two independent experiments. B-D) Graphs representing tumour volume growth after co-injection of HSV treated FaDu-OCAF2 co-culture in Balbc nude mice. OCAF2 parental and IRF3 oe cells were previously incubated with Scramble or IRF3 siRNA for 24h before co-culture. MOI, A= 0.005, B = 0.00158 and C= 0.0005. n=36 mice in 12 different treatment groups of equal size, 3 mice per condition. Data from one independent experiment. E) Micrographs of IF staining for fibronectin (yellow), phospho-H2AX (magenta) with DAPI counterstain (cyan) of A431 inducers and non-inducers co-injected with VCAF2b in the flank of Balb/c nude mice. Representative images from four different tumours per group from two independent experiments. Scale bar is 50 µm. F) Quantification of p-H2AX relative to number of nuclei in percentage comparing tumours from Inducers and Non-Inducers cells. Each dot is a field of view (n=7 per group) from four different biological replicates from two independent experiments. Mann-Whitney test. G) Gene set enrichment analysis graphs obtained from comparing the gene expression data from VCAFb (Touch vs No Touch) and gene expression data obtained from patients resistant to radiation and CTLA-4. See also Statistical Source Data Extended Data Fig.7 and Unmodified Blot Extended Data Fig.7. Source data

Extended Data Fig. 8 cGAMP produced in cancer cells is transmitted to cancer associated fibroblasts leading to production of IFN stimulated genes.

Schematic overview of current understanding of signalling pathways.

Supplementary information

Supplementary Information

Supplementary Fig. 1

Reporting Summary

Supplementary Tables

Supplementary Tables 1–5: T1 gene expression of co-cultures, T2 ISGs and CAF markers, T3STR profiles, T4 siRNA sequences and T5 primers.

Supplementary Video 1

Movie of A431-yPET (yellow) and VCAF2b-Life Actin mCherry (blue) in direct co-culture, showing cytoplasmic transfer from the cancer cells to the CAFs. Scale bar is 10 µm.

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Arwert, E.N., Milford, E.L., Rullan, A. et al. STING and IRF3 in stromal fibroblasts enable sensing of genomic stress in cancer cells to undermine oncolytic viral therapy. Nat Cell Biol 22, 758–766 (2020). https://doi.org/10.1038/s41556-020-0527-7

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