IL-33/NF-κB/ST2L/Rab37 positive-feedback loop promotes M2 macrophage to limit chemotherapeutic efficacy in lung cancer

IL-33 is a danger signal that binds to its receptor ST2L to promote tumor progression. This study identifies the IL-33/ST2L positive-feedback loop and the trafficking of ST2L membrane presentation in macrophages that contribute to lung tumor progression. Mechanistically, IL-33 induces ST2L upregulation by activating NF-κB, which binds to the promoter region of the ST2L gene. Moreover, Rab37, a small GTPase involved in membrane trafficking, mediates ST2L trafficking to the plasma membrane of M2 macrophages. This IL-33/NF-κB/ST2L/Rab37 axis promotes positive-feedback loops that enhance ST2L expression and membrane trafficking in M2 macrophages. Notably, neutralizing antibodies against IL-33 or ST2L block NF-κB activity, suppress M2 macrophage polarization, and synergistically inhibit tumor growth when combined with cisplatin treatment in vitro/vivo. Clinically, Rab37+/ST2L+/CD206+ tumor-infiltrating M2 macrophages correlate with advanced-stage lung cancer patients with poor response to chemotherapy. These findings unveil a positive-feedback mechanism and provide a basis for IL-33/ST2L-targeting therapy for cancer.


Quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay
Total RNA was extracted using Trizol reagent (Invitrogen).Purified RNA was converted into cDNA by reverse transcription.q-PCR was performed to analyze the mRNA expression of genes by using SYBR Green Master Mix (Invitrogen).

Protein extraction and Western blot analysis
The cells were harvested and lysed in RIPA buffer containing protease inhibitors cocktail (Sigma-Aldrich, St. Louis, MO, USA), and then cell lysates were centrifuged at 13,200 r.p.m. for 15 min at 4°C.Protein extracts were solubilized in loading buffer (60 mM Trisbase, 2% SDS, 10% glycerol, and 5% β-mercaptoethanol).A total of 50 μg of protein lysates was loaded onto 10% sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinyl difluoride (PVDF) membrane.The protein was identified by incubating the PVDF membrane with primary antibodies followed by horse radish peroxidase-conjugated secondary antibodies.

Real-time live confocal fluorescence microscopy
V5-His tagged Rab37 wild-type (homemade) and pLV-mIL1RL1-GFPSpark (ST2L expression vector, Sino Biological Inc.) were transfected and expressed for 24 h in RAW264.7 cells.The Rab37-RFP and ST2L-GFP signals in RAW264.7 cells were recorded in real-time live images and video before and after the IL-33 50 ng/mL treatment were captured through the 100x lens of the microscope.The time-lapse images were acquired using an Olympus FV3000 confocal microscope with FV31S-SW software (Olympus, Tokyo, Japan).
b Rab37-WT, Q89L, or T43N were PCR-amplified with designated mutation at the primer sequences and cloned into pcDNA3.1-V5/Hisexpression vector to generate V5/His-tagged Rab37 expression vector.c Mouse Rab37 cDNA was PCR-amplified and cloned into the pDsRed2-C1 expression vector to generate an RFP-tagged Rab37 expression vector.d IL1RL1 gene encodes ST2L.
The viruses were obtained from the RNAi core (Academia Sinica, Taipei, Taiwan).3. The antibodies and their reaction condition used in the current study.a Molecular weight is not applicable to this antibody in such an application.

Supplementary Table
b ChIP: Chromatin immunoprecipitation.
c DAPI is a commercial product for nuclear staining and Opal reagents are commercial products for immunofluorescence-IHC.