Alarmin IL-33 orchestrates antitumoral T cell responses to enhance sensitivity to 5-fluorouracil in colorectal cancer

Rationale: Resistance to 5-fluorouracil (5-FU) chemotherapy remains the main barrier to effective clinical outcomes for patients with colorectal cancer (CRC). A better understanding of the detailed mechanisms underlying 5-FU resistance is needed to increase survival. Interleukin (IL)-33 is a newly discovered alarmin-like molecule that exerts pro- and anti-tumorigenic effects in various cancers. However, the precise role of IL-33 in CRC progression, as well as in the development of 5-FU resistance, remains unclear. Methods: High-quality RNA-sequencing analyses were performed on matched samples from patients with 5-FU-sensitive and 5-FU-resistant CRC. The clinical and biological significance of IL-33, including its effects on both T cells and tumor cells, as well as its relationship with 5-FU chemotherapeutic activity were examined in ex vivo, in vitro and in vivo models of CRC. The molecular mechanisms underlying these processes were explored. Results: IL-33 expressed by tumor cells was a dominant mediator of antitumoral immunity in 5-FU-sensitive patients with CRC. By binding to its ST2 receptor, IL-33 triggered CD4+ (Th1 and Th2) and CD8+ T cell responses by activating annexin A1 downstream signaling cascades. Mechanistically, IL-33 enhanced the sensitivity of CRC cells to 5-FU only in the presence of T cells, which led to the activation of both tumor cell-intrinsic apoptotic and immune killing-related signals, thereby synergizing with 5-FU to induce apoptosis of CRC cells. Moreover, injured CRC cells released more IL-33 and the T cell chemokines CXCL10 and CXCL13, forming a positive feedback loop to further augment T cell responses. Conclusions: Our results identified a previously unrecognized connection between IL-33 and enhanced sensitivity to 5-FU. IL-33 created an immune-active tumor microenvironment by orchestrating antitumoral T cell responses. Thus, IL-33 is a potential predictive biomarker for 5-FU chemosensitivity and favorable prognosis and has potential as a promising adjuvant immunotherapy to improve the clinical benefits of 5-FU-based therapies in the treatment of CRC.


Figures and Figure Legends
CRC patients were used to analyze the expression IL-33 and CD3 by IHC staining.
Representative micrographs and scores for IHC staining of IL-33 and CD3 in the peritumoral (P), edge, and tumoral (T) regions of CRC tissues (n = 10). Scale bars, 100 m. (C) The mRNA expression CXCL10 and CXCL13 in tumor tissues from 5-FU-sensitive (n = 10) and resistant (n = 10) CRC patients was detected by qPCR.
(D) Correlation between IL-33 and CD3 expression in the tumoral regions of 117 CRC tissues based on the IHC staining was analyzed by Pearson's correlation method.

CRC cell lines and cell culture
Human CRC cell lines, HCT116 and DLD-1, and mouse colon cancer cell line, CT26 were purchased from the American Type Culture Collection (Manassas, VA, USA).
All CRC cells were used within 15 passages and had been confirmed without mycoplasma contamination. Cells were cultured in DMEM (Gibco, Invitrogen Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin in 5% CO2 at 37°C.

Immunofluorescence staining
Tissue multiplex immunofluorescence staining was performed using a Pano-Panel Kit

Bioinformatics analysis
The Cancer Genome Atlas dataset for CRC was obtained from the UCSC Cancer Browser (https://genome-cancer.ucsc.edu). Gene expression levels based on the mRNA sequencing data of The Cancer Genome Atlas are shown as the mean ± standard error of the mean of triplicate determinations. Gene-set enrichment analysis was performed using GSEA 2.0.9 (http://www.broadinstitute.org/gsea/) according to The Cancer Genome Atlas mRNA sequencing data or RNA-sequencing data of CRC tissues and cells.

Co-culture of CD3 + T cells with CRC cells
For the co-culture experiments involving CD3 + T cells and CRC cell lines, CD3 + T cells isolated from the PMBCs of healthy donors and activated with 25 g/mL CD3/CD28 T cell Activator (STEMCELL) and 50 U/mL rhIL-2 for 48 h.
To examine the role of CRC cell-derived IL-33 in T cell responses, CD3 + T cells were pretreated with anti-IgG or 1 g/mL anti-ST2 neutralizing antibodies (R&D Systems) for 1 h, respectively, following by coculture with CRC cells with or without IL-33 overexpression for 72 h in 24-well Transwell system. Then CD3 + T cells were harvested for flow cytometry and CD4 + T sorting using MACS magnetic sorting system for western blotting.
To evaluate the effects of IL-33-induced T response on the sensitivity of CRC cells to 5-FU, wild type or 5-FU resistant CRC cells and CD3 + T cells were pretreated with anti-IgG or 1g/mL anti-ST2 neutralizing antibodies (R&D Systems) for 1 h, respectively. Then, CRC cells were alone cultured or cocultured with CD3 + T cells with or without rhIL-33 (10 ng/mL, Peprotech) treatment, followed by 5-FU (10 g/mL, Sigma-Aldrich) treatment in Transwell system. After 48 h, cell apoptosis was measured by flow cytometry analysis. Cell viability was evaluated at indicated time points by CCK-8 assay; vector or IL-33-overexpressing CRC cells and CD3 + T cells were pretreated with anti-IgG or 1 g/mL anti-ST2 neutralizing antibodies (R&D Systems) for 1 h, respectively. Then, CRC cells were alone cultured or cocultured with CD3 + T cells, followed by 5-FU (10 g/mL, Sigma-Aldrich) treatment in Transwell system. After 48 h, cell apoptosis was measured by flow cytometry analysis.
Cell viability was evaluated at indicated time points by CCK-8 assay.
To examine the influence of IL-33-mediated T cell responses and 5-FU treatment on the secretion of IL-33, CXCL10, and CXCL13 by CRC cells, as well as the influence on the feedback on T cell response, CRC cells were alone cultured or cocultured with CD3 + T cells pretreated with or without anti-ST2 neutralizing antibodies (1 g/mL) for 1 h following treatment with or without rhIL-33 (10 ng/mL, peprotech) for 72 h, which were then administrated with 5-FU (10 g/mL,

Sigma-Aldrich). After 48 h, CD3 + T cells were removed, and CRC cells in each group
were replaced with fresh medium and cultured for 72 h. Indicated supernatants were collected for the detection of the secretion of IL-33, CXCL10, and CXCL13 using ELISA and further treatment of CD3 + T cells for 72 h. Then CD3 + T cells were harvested for flow cytometry.

Total RNA extraction and quantitative RT-PCR (qPCR)
Total RNA was obtained from tissues or cells by using the TRIzol reagent (Invitrogen Corporation) according to the manufacturer's instructions. The concentration and purity of the RNA were assessed by using a NanoDrop 2000 instrument (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from total RNA by using a GoScript Reverse Transcription System (Promega, Madison, WI, USA).
RT-PCR was performed using GoTaq qPCR Master Mix (Promega). GAPDH was used as an endogenous control for normalization.

Western blot analysis
The western blot protocol was described elsewhere [2]. Nucleus proteins of CRC cells

ELISA
Secretion of the IL-33, CXCL10, and CXCL13 in the serum or culture supernatants was examined using commercially available ELISA kits (BioLegend, San Diego, CA and Abcam, Cambridge, MA, USA) according to the manufacturer's protocol.

GSH detection
The supernatants of CRC cells treated by indicated conditions were collected. The level of GSH in the supernatants was examined using a GSH detection kit (Jiangsu, KeyGEN Biotech, China) according to the manufacturer's protocol.

Transient transfection
Transfections were performed using the Lipofectamine 3000 Kit (Invitrogen Carlsbad, CA, USA) according to the manufacturer's instructions. Cells were grown to 50% to 60% confluence in 6-well plates and transfected with plasmid containing siRNAs.
Cells were harvested 48 h after transfection for subsequent confirming the transfection efficiency by western blot.

T cell migration assay
T cell migration was assessed by using a 24-well Transwell System with 3.0 m polycarbonate membranes (Corning, NY, USA). A total of 2.0 × 10 6 T cells in 100 l of serum-free RPMI 1640 medium and added to the upper chamber. 600 l medium alone and with rhCXCL10 (10 ng/mL, Peprotech), rhCXCL13 (10 ng/mL, Peprotech) or with 50% indicated culture supernatants from CRC cells was added to the lower chamber. After incubation at 37°C for 2 h, the cells that migrated into the lower chamber were harvested and counted using a hemocytometer. The negative control was T cells that migrated toward RPMI 1640 alone. The chemotactic index was calculated as the ratio of the number of T cells that migrated to conditioned medium divided by the number of those migrating to RPMI 1640 alone.

Statistical analysis
Statistical analyses were performed using SPSS version 19.0 or Prism 8 (Graph Pad Software Inc.). Data are expressed as the mean ± SD according to the distribution level. Differences between groups with normally distributed continuous variables were analyzed using independent-sample or paired t-test. The association between the expression levels of two markers was analyzed using Pearson's correlation coefficient.
The Kaplan-Meier method and log-rank test were used to plot survival curves and analyze differences in survival time between patient subgroups. Cox's proportional hazards regression model was used to evaluate the prognostic value of the risk factors.