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We read with interest the study by Corry et al1 reporting that upregulation of IFNγ, IFNα and STAT-1 response pathways, downstream of double-stranded (ds) RNA and/or viral responses, is associated with favourable prognosis in stroma-rich colorectal cancers (CRCs).
Furthermore, in vitro stimulation of myeloid cells with poly(I:C), a synthetic dsRNA viral mimetic and toll-like receptor 3 (TLR3) agonist, effectively induces expression of STAT1 and its target genes. Importantly, administration of poly(I:C) enhances immune cell infiltration of liver metastases and reduces metastatic tumour burden in a CRC murine model.1
Thus, TLR3 targeting may represent a novel therapeutic option in patients with stroma-rich CRC, characterised by high stromal component, microsatellite stable (MSS) status and severe prognosis.2 3 However, TLRs are expressed by a variety of cell types, besides myeloid cells.4 We previously reported that TLR triggering on tumour cells enhances their chemokine production capacity ultimately favouring intratumoral immune cell recruitment.5 Whether tumour associated stromal cells (TASCs) may also be directly targeted by TLR3 or other TLR agonists remains to be addressed.
In this study, we investigated TLR expression profiles of CRC-derived TASCs in a publicly available single-cell RNA seq database, including 51 MSS CRCs (3and online supplemental table 1), and following in vitro expansion,6 (online supplemental table 2 and online supplemental figure 1) and assessed their capacity to respond to microbial stimuli.
Supplemental material
Substantial fractions of CRC-derived TASCs expressed heterogeneous levels of TLR3, TLR4 and TLR5. TLR1, TLR2 and TLR6 expression was also detected, although on fewer cells, while expression of other TLRs was negligible (figure 1A). Accordingly, in expanded TASCs TLR3, TLR4, TLR5 and TLR6 gene expression was detected in 8, 10, 5 and 11 out of 12 samples, respectively (figure 1B). Expression of TLR1 and TLR2 genes was also observed in three and two samples, respectively. No expression of TLR7, TLR8, TLR9 and TLR10 genes was detected (figure 1B).
CRC-associated TASCs do express functional TLRs. (A) Data relative to cells classified as ‘Fibroblasts’ (n=16 058) were retrieved from a publicly available scRNA seq database, including 51 human primary MSS CRCs across five different cohorts.3 This panel reports the normalised log-expression of TLR1-10 genes, as detected in 2763 cells. (B) TASCs were isolated from human primary CRC samples (n=12), as previously described.6 Following short in vitro expansion, expression of stromal cell markers was confirmed by phenotypical analysis by flow cytometry (see online supplemental figure 1). Gene expression of TLR1-10 was assessed on expanded TASC preparations (n=12) by quantitative qRT-PCR, using GAPDH as housekeeping gene. Each dot corresponds to one TASC preparation. Those further used for functional testing are identified by a specific colour. (C) To identify optimal stimulatory conditions, TASCs from one representative preparation were incubated with a panel of TLR agonists, including the TLR2-ligand peptidoglycan (PGN), the TLR3-ligand polyinosinic-polycytidylic acid, poly(I:C), the TLR4-ligand lipopolysaccharide (LPS), the TLR5-ligand flagellin (FLAG), the TLR2/TLR6 ligand synthetic diacylated lipoprotein (FSL-1),the TLR7/8 ligand Imiquimod (IMQ) and the TLR9-ligands CpG oligonucleotide type A (ODN 2216), and CpG oligonucleotide type B (ODN 2006), at the indicated titrated concentrations. After 4 hours cells were collected, and following RNA extraction, IL-6 gene expression was assessed by quantitative qRT-PCR, using as control GAPDH housekeeping gene. Cumulative data from three independent experiments are shown. (D) TASCs from five different preparations (each identified by a specific colour) were stimulated with poly(I:C), LPS, and FLAG at the indicated concentrations. After 4 hours, IL-6 expression was assessed as detailed above. (E, F) TASCs were stimulated with TLR agonists as described above. After overnight incubation culture supernatants were collected and IL-6 release was assessed by ELISA assays. (E) Cumulative data from three independent experiments performed with one single TASC preparation. (F) Data from five different TASC preparations, each identified by a specific colour. Statistical significance of observed differences was assessed by Mann-Whitney (C, E) or Friedmann (D, F) tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CRC, colorectal cancer; MSS, microsatellite stable; TASCs, tumour associated stromal cells.
TLR functionality was assessed on expanded TASCs, based on IL-6 production at baseline and upon stimulation by TLR agonists (see online supplemental methods). Consistent with TLR expression profiles, stimulation with poly(I:C) strongly upregulated IL-6 expression at both gene and protein levels at all concentrations tested (figure 1C–F). Similarly, exposure to lipopolysaccharide (LPS) and flagellin, TLR4 and TLR5 ligands, respectively, resulted in higher expression of IL-6 gene and protein, although to lower extents than poly (I:C). Instead, no significant response was observed upon stimulation with the TLR2/TLR6 agonist FSL-1, the TLR2 agonist PGN, the TLR7/8 agonist imiquimod and the TLR9 agonist ODNs.
Interestingly, TLR triggering in TASCs resulted in differential modulation of chemokine expression patterns (figure 2). Expression of myeloid cell-recruiting chemokines, including CCL2, CXCL1, CXCL2, CXCL5, CXCL6 and CXCL8, already detectable at baseline, was boosted at both gene and protein levels. However, while LPS-mediated and flagellin-mediated effects largely varied across different TASC preparations, possibly reflecting heterogeneous expression levels of TLR4 and TLR5, stimulation by poly (I:C) strongly enhanced chemokine production in all TASC samples (figure 2A,C). Remarkably, only poly (I:C) induced expression of T cell-recruiting chemokine genes, including CCL5, CXCL9, CXCL10 and CXCL11, whereas other TLR agonists showed poor or no capacity (figure 2B,D). Consistently, supernatants from poly(I:C)-stimulated TASCs significantly induced T cell migration in vitro, whereas those from LPS-stimulated or flagellin-stimulated TASCs displayed negligible effects (figure 2E).
TLR triggering induces chemokine production in TASCs. CRC-derived TASCs were stimulated with poly (I:C), LPS, and flagellin (FLAG) at the indicated concentrations, as described in figure 1. (A, B) After 4 hours, cells were collected, and following RNA extraction, chemokine gene expression was assessed by quantitative qRT-PCR, using GAPDH as housekeeping gene. Cumulative data (mean±SD) are reported. (C, D) After an overnight incubation, chemokine release in culture supernatants was assessed by Legendplex assay. Data from individual TASC preparations, each identified by a specific colour, are reported. Means are indicated by columns. (E) T cells were isolated from healthy donors and their capacity to migrate towards TASC supernatants was evaluated. Numbers of cells (total T cells, CD8+ or CD4+) migrated towards individual TASC preparations, each identified by a specific colour, are reported. Statistical significance of observed differences was assessed by Kruskal-Wallis (A, B) or Friedmann (C–E) tests. *p<0.05, **p<0.01, ***p<0.001. CRC, colorectal cancer; LPS, lipopolysaccharide; TASC, tumour associated stromal cells.
Our findings demonstrate that human CRC-associated TASCs express functional TLRs, enabling them to directly sense microbial stimuli and potentially therapeutic TLR ligands. Importantly, we provide further evidence in favour of TLR3 targeting as the most powerful approach to enhance chemokine production in various cell types of CRC microenvironment, including TASCs, in addition to myeloid1 and tumour cells,3 thereby effectively promoting recruitment of beneficial immune cells into tumour tissues.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by Ethikkommission Nordwest und Zentralschweiz, EKNZ, study protocol n. 2014-388, Comitato Etico Cantonale Ticino, 2020-00437 I CE 3598. Participants gave informed consent to participate in the study before taking part.
Acknowledgments
We thank the patients and their families for their consent to use their biological samples for this study and our colleagues at the Department of Surgery, Basel University Hospital for helping in the initial phase of the project. We are also thankful to Dr Valentina Cecchinato and Professor Mariagrazia Uguccioni, Institute of Research in Biomedicine (IRB), Bellinzona, for their help with migration assays, and to Dr Chiara Arrigoni and Professor Matteo Moretti for their support with TASC culture.
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Contributors Conceptualisation: JD, LC, GS, DC and GI. Methodology: JD, NSCR, VM, LC, ES, CB, MV, AC, RR and JG. Visualisation and original draft: JD, NSCR, GS and GI. Supervision: DC and GI. Review and editing: JD, GS, PEM-H, DC and GI. Guarantor and overall supervision: GI.
Funding This work was supported by Kurt und Senta Herrmann Stiftung, Krebsliga Beider Basel, Stiftung für Krebsbekämpfung, Gebert Rüf Stiftung and EOC AFRI Senior Research Grant to GI.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.