Tumor-initiating cells establish an IL-33-TGF-β niche signaling loop to promote cancer progression

Abstract

Targeting the cross-talk between tumor-initiating cells (TICs) and the niche microenvironment is an attractive avenue for cancer therapy. We show here, using a mouse model of squamous cell carcinoma, that TICs play a crucial role in creating a niche microenvironment that is required for tumor progression and drug resistance. Antioxidant activity in TICs, mediated by the transcription factor NRF2, facilitates the release of a nuclear cytokine, interleukin-33 (IL-33). This cytokine promotes differentiation of macrophages that express the high-affinity immunoglobulin E receptor FcεRIα and are in close proximity to TICs. In turn, these IL-33-responding FcεRIα⁺ macrophages send paracrine transforming growth factor β (TGF-β) signals to TICs, inducing invasive and drug-resistant properties and further upregulating IL-33 expression. This TIC-driven, IL-33-TGF-β feedforward loop could potentially be exploited for cancer treatment.

Fig. 1.. IL-33 is up-regulated in tumor cells adjacent to TGF-β–rich microenvironments in a mouse model of SCC.

(A) Fluorescent reporter illuminates higher TGF-β signaling activity in the invasive region of SCC. Note that increased TGF-β immunostaining (green) in the stroma correlates with the presence of TGF-β reporter⁺ cells (pink) at the invasive tumor front. (B) The proportion of TGF-β reporter⁺ cells and the intensity of TGF-β immunolabeling were measured by fluorescence microscopy of tumor sections with active and inactive TGF-β signaling (n = 7). (C) MA plot presentation of RNA-seq data comparison between TGF-β–responding and -nonresponding tumor basal cells. Biological replicates, n = 5. Red and blue dots indicate significantly up- and down-regulated genes, respectively (adjusted P < 0.01). Triangles indicate up-regulated NRF2-regulated genes involved in antioxidant responses (fig. S1H). Diamonds indicate genes encoding cytokines or chemokines (fig. S1I). (D) qPCR analysis of Il33 mRNA in FACS-isolated TGF-β reporter⁺ and reporter^neg tumor basal cells in vivo (the sorting strategy is shown in fig. S1G; n = 3). Data are expressed as relative mean expression with SEM and were analyzed with unpaired t test, ***P < 0.001. (E) Immunodetection of IL-33 protein in mosaic tissue with Tgfbr2 cKO (YFP⁺) and WT (YFP^neg) tumor cells. Mosaic tissues were induced by a partial activation of K14-CreER by limited dose of tamoxifen. (Right) Dotted lines denote the boundaries of WT and cKO tumor cell regions. (Graph) Quantification of IL-33 signal intensity (n = 3). Data are expressed as relative mean intensity with SD and were analyzed with unpaired t test, ***P < 0.001. Scale bars, 50 μm.

Fig. 2.. The NRF2-mediated antioxidant response induces extracellular release of IL-33.

(A) Immunolabeling of tumor tissues showing a range of IL-33 expression patterns. In well-differentiated SCC, IL-33 nuclear staining can be observed in both K5⁺ basal (arrows) and K5^dim suprabasal cells. In invasive SCC, cytoplasmic IL-33 staining can be observed in K5⁺ basal cells (arrowheads). (B) TGF-β reporter⁺ tumor cells at the invasive front exhibited cytoplasmic IL-33 localization (arrowheads), whereas suprabasal cells had nuclear IL-33 (arrows). (Graph) Quantification of IL-33 nuclear versus cytoplasmic localization in TGF-β reporter⁺ and reporter^neg tumor basal cells. (C) Immunostaining of invasive SCC tissue showing the correlation between nuclear NRF2 expression and cytoplasmic IL-33 localization (arrowheads). Note that nuclear IL-33–expressing K5^neg stromal cells are negative for NRF2. (D) Immunolabeling of human SCC section showing the correlation between cytoplasmic IL-33 localization and nuclear NRF2 expression (arrowheads). (E) Western blots showing that nonlethal oxidative stress induced by 500 μM H₂O₂ reduces IL-33 in total cell lysates over 3 hours. Arrowhead indicates an NRF2-specific band. Asterisk indicates a nonspecific band. (Graph) Quantification of IL-33 protein (n = 3). The relative intensity of IL-33 bands normalized based on the α-tubulin (α-tub) loading control is shown. Data are shown as mean with SD and were analyzed with unpaired t test, *P = 0.0195, **P < 0.01. (F) Western blot analysis of IL-33 protein in the concentrated conditioned medium (CM) of cells with and without H₂O₂ treatment. (G) Nrf2 (Nfe2l2) KD prevents H₂O₂-induced IL-33 protein reduction in total cell lysates. (Graph) Quantification of IL-33 protein (n = 4). Data are shown as mean with SD. (H) Western blots show that Keap1 KD cells increase NRF2 and decrease intracellular IL-33 protein levels regardless of oxidative stress. (I) Immunolabeling of IL-33 and NRF2 in scramble control, Nrf2 KD, or Keap1 KD cells with or without TGF-β treatment (50 pM for 36 hours). Note that Nrf2 KD results in an exclusive nuclear localization of IL-33, whereas TGF-β–treated and Keap1 KD cells show granular nuclear and cytoplasmic IL-33 localization. Dotted lines denote the tumor–stroma boundaries. Scale bars, 50 μm.

Fig. 3.. Depletion of tumor-derived IL-33 impairs tumor progression and paracrine TGF-β signaling.

(A) Immunolabeling of tumor sections showing efficient IL-33 protein depletion in Il33 shRNA-transduced (YFP⁺) cells. Note that some YFP^neg stromal cells maintain nuclear IL-33 (arrows). (B) HRAS^G12V-driven tumors in control and Il33 KD mice were sized at the time of euthanasia. Scramble control, n = 46; Il33 KD, n = 31. Approximation curves were drawn by applying the Michaelis-Menten kinetics curve. (C) Hematoxylin and eosin (H&E) staining of tumor sections showing smoother edges in Il33 KD tumors compared with control tumors, suggestive of reduced invasive capacity. Scale bars, 100 μm. (D) Immunolabeling of K10 showing a loss of differentiation property in control tumors, but not in Il33 KD tumors. (E) Detection of EdU incorporated 4 hours before euthanasia. (Graph) Quantification of EdU⁺ cells in integrin α6⁺ tumor basal cells of control and Il33 KD tumors (n = 3) (n = 806 to 1747 cells). Data are shown as mean with SEM and were analyzed with unpaired t test, **P = 0.004. (F) Flow cytometry analysis of TGF-β reporter (mScarlet) expression in YFP⁺ tumor epithelial cells. YFP+ cells in Il33 KD tumors show fewer and reduced TGF-β reporter activity compared with those in control tumors. (Graph) Proportion of TGF-β reporter⁺ tumor cells. Each dot indicates values from an individual tumor. Scramble control, n = 30; Il33 KD, n = 19. Data are shown as mean with individual values and were analyzed with unpaired t test, **P = 0.007. (G) Tumor-bearing mice were treated with cisplatin (10 mg/kg), and cells undergoing apoptosis (cleaved caspase-3⁺) in K5⁺ tumor basal cells were quantified 2 days after treatment (n = 3). Data are shown in box-and-whisker plots (midline, median; box, 25th and 75th percentiles; whiskers, min and max) and were analyzed with unpaired t test, **P = 0.0061, *P = 0.0334. (H) Volume of tumors after cisplatin administration. (Left) Spider plots showing the changes for each tumor. (Right) Normalized tumor volume of each condition. Scramble control, n = 3 (15 tumors), Il33 KD, n = 5 (24 tumors). Tu, tumor. St, stroma. Scale bars, 50 μm [except for (C)].

Fig. 4.. FcεRIα⁺ macrophages accumulate in the proximity of TGF-β–responding invasive tumor cells.

(A) Immunolabeling of tissue sections from different SCC stages showing clusters of FcεRIα⁺ cells accumulated within a 50-μm radius of TGF-β reporter⁺ invasive tumor cells (dotted circles and box). (B) Quantification of FcεRIα⁺ cells in the stroma adjacent to the tumor leading edge with TGF-β reporter high (≥30%) versus low (<30%) showing higher density of FcεRIα⁺ cells near TGF-β–responding cells. n = 4 mice, total 12 images that included both TGF-β reporter high and low areas and were analyzed with unpaired t test, **P = 0.0015. (C) Flow cytometry analysis of FcεRIα expression in different immune cell types in SCC. Gating strategy is shown in fig. S4D. (D) Immunolabeling of invasive SCC section showing that most FcεRIα staining overlaps with F4/80, an established murine macrophage marker, and present within a 50-μm radius of tumor leading edges. Note that F4/80⁺ cells farther than the 50-μm radius from tumor edges were negative for FcεRIα. Asterisks indicate rare FcεRIα⁺F4/80^neg cells. Dotted lines denote the tumor–stroma boundaries. (E) FACS plots of F4/80⁺FcεRIα⁺ (green dotted box) and F4/80⁺FcεRIα^neg (gray dotted box) macrophages in SCC. (F) MA plot presentation of RNA-seq data comparison between FcεRIα⁺ and FcεRIα^neg macrophages in SCC. Biological replicates, n = 3. Red and blue dots indicate significantly up- and down-regulated genes, respectively (adjusted P < 0.05). Dark red and dark blue dots highlight some of the “M2” and “M1” marker genes, respectively. (G) qPCR analysis of FACS-isolated macrophage populations in SCC (n = 3). Data are shown as mean with SD. Fcer1a, **P = 0.0019. Arg1, **P = 0.0032. Tgfb1, *P = 0.0266. (H) Immunolabeling of invasive SCC section showing that most FcεRIα⁺ cells overlap with TGF-β protein localization in the stroma. Magnified images are shown in fig. S4E. (I) Immunolabeling of human SCC section showing that FcεRIα⁺ cells cluster around invasive phospho-SMAD2 (pSMAD2)⁺ tumor cells (dotted circles). (J) Immunolabeling of human SCC section showing that most FcεRIα⁺ cells overlap with CD206, an established marker of alternatively activated macrophages, at the tumor-stroma interfaces, whereas CD206⁺FcεRIα^neg cells present in the stroma farther than a 50-μm radius from tumor edges. (Graph) Quantification of the density of FcεRIα⁺ cells and CD206⁺ cells 0 to 50 or 50 to 100 μm away from tumor edges. Data were analyzed with paired t test, **P = 0.0035. Tu, tumor. St, stroma. Scale bars, 50 μm.

Fig. 5.. IL-33-induced FcεRIα⁺ macrophages activate paracrine TGF-β signaling and epithelial cell invasion in vitro.

(A) Histogram presentation of ST2 expression in FcεRIα⁺ and FcεRIα^neg macrophages in SCC. (B) Flow cytometry analysis of FcεRIα and ST2 expression in CSF1- and IL-33–induced F4/80⁺ macrophages from bone marrow (BM)–derived cells (see fig. S5B). (C) qPCR analysis of CSF1-induced, IL-33–induced, and CSF1-induced→IL-4–treated macrophages in vitro (n = 3). Data are shown as relative mean expression with SD. (D) qPCR analysis of IL-33–induced macrophages treated with a MEK inhibitor (U0126, 0.5 –M), a JNK inhibitor (SP600125, 0.5 –M), a p38 inhibitor (SB202190, 0.5 –M), or an NF-κB inhibitor (BAY11-7082, 1 μM) during macrophage differentiation (n = 3). Data are shown as relative mean expression with SD and were analyzed with unpaired t test, **P = 0.0022. (E) qPCR analysis of IL-33–induced and IL-4–activated macrophages treated with the NF-κB inhibitor (n = 3). Data are shown as relative mean expression with SD and were analyzed with unpaired t test, ***P < 0.001. (F and G) qPCR analysis of IL-33–induced macrophages from ex vivo expanded hematopoietic progenitor cells that were LV transduced with scramble control, (F) Rela (NF-κB p65) shRNA, or (G) Fcer1a shRNA (see fig. S5E). (H) LV-transduced YFP⁺ MKs were cocultured with CSF1- or IL-33–induced YFP^neg macrophages (MΦs) for 24 hours. (Graph) Quantification of TGF-β fluorescent reporter intensity in YFP⁺ MKs. Data are shown in box-and-whisker plots (midline, median; box, 25th and 75th percentiles; whiskers, 5th and 95th percentiles with outliers); ***P < 0.001. ns, not significant. Scale bars, 50 μm. (I) Quantification of the circularity of YFP⁺ MKs cocultured with CSF1- or IL-33–induced MFs. Data are shown in box-and-whisker plots, ***P < 0.001. (J and K) Quantification of YFP⁺ MKs invaded through Matrigel-coated membranes. (J) MKs were cultured with CSF1- or IL-33–induced MΦs or with (K) IL-33–induced MΦs transduced scramble control or Tgfb1 shRNA (n = 3). Data are shown as mean with SD, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 μm.

Fig. 6.. FcεRIα⁺ macrophages mediate IL-33–induced paracrine TGF-β signaling and invasive tumor progression.

(A) No difference is observed in the frequency of F4/80⁺ macrophages in live, CD45⁺ cells in control and Il33 KD tumors. Scramble control, n = 30; Il33 KD, n = 20. Data are shown as mean with SEM. ns, not significant. (B) F4/80⁺ macrophages in Il33 KD tumors have a smaller FcεRIα⁺ST2⁺ double-positive population than control tumors. (Graph) Quantification of FcεRIα⁺ST2⁺ macrophages. Scramble control, n = 27; Il33 KD, n = 20. Data are shown as mean with SEM and were analyzed with unpaired t test, ***P < 0.001. (C) Immunolabeling of tumor sections showing fewer FcεRIα⁺TGF-β⁺ cells in the stroma of Il33 KD tumors compared with control. (Graph) Quantification of FcεRIα⁺ cells in the stromal area within a 50-μm radius of tumor edges analyzed with unpaired t test, *P = 0.0394. (D) Immunolabeling of tumor sections showing that sST2-overexpressing (OE) tumors have fewer FcεRIα⁺ cells in the adjacent stroma. (Graph) Quantification of FcεRIα⁺ cells in the stromal area within a 50-μm radius of tumor edges analyzed with unpaired t test, ***P < 0.001. (E) HRAS^G12V-driven tumors overexpressing sST2 or control were sized. Control, n = 6; sST2 OE, n = 6. Approximation curves were drawn by applying the Michaelis-Menten kinetics curve. (F) Immunolabeling of K10 showing a sustained differentiation property in sST2-overexpressing tumors. (G) Immunolabeling of IL-33 and FcεRIα in Nrf2 (Nfe2l2) or Keap1 KD tumors. Whereas Nrf2 KD tumors show mostly nuclear IL-33, Keap1 KD tumors show cytoplasmic IL-33 localization. (Graph) Quantification of FcεRIα⁺ cells in the stromal area within a 50-μm radius of the tumor leading edges (n = 3-4). Data are shown in box-and-whisker plots, *P = 0.0394 (control versus Il33 KD) or 0.0372 (Nrf2 KD versus Keap1 KD). (H) Immunolabeling of tumor sections showing tdTomato⁺ immune cells infiltrated from the circulation at 7 d after injection. Note that control tdTomato⁺ cells expressed FcεRIα (arrowheads) but ST2 KD cells were largely negative (arrows). Magnified images are shown in fig. S6I. (Graph) Quantification of tdTomato⁺ cells in the stroma and FcεRIα-expressing tdTomato⁺ cells (n = 4) analyzed with unpaired t test, **P = 0.0019. (I) FcεRIα⁺ cell depletion by anti-FcεRIα antibody injection in vivo. Quantification of cells expressing ST2 (a surrogate marker for FcεRIα) within the stromal area in a 50-μm radius of tumor basal cells (n = 3), ***P < 0.001. (J) Quantification of TGF-β fluorescent reporter intensity in K5 basal cells. Intensity values in each image were normalized by the median values (set as 1) (n = 3) and analyzed with unpaired t test, ***P < 0.001. (K) Tumors were sized before and 1 week after anti-FcεRIα antibody injection. Isotype control, n = 3, 10 tumors; anti-FcεRIα, n = 3, 10 tumors. **P = 0.0016. (L) Model of TIC-driven feedforward mechanism of invasive SCC progression. TGF-β–responding TICs release IL-33 through the NRF2-mediated antioxidant response, which induces differentiation of immature myeloid cells into FcεRIα⁺ macrophages in their close proximity. In turn, FcεRIα⁺ macrophages send reciprocal paracrine TGF-β signaling to TICs to promote invasive progression and drug resistance of SCC, and further induce the release of IL-33, establishing a self-reinforcing niche signaling loop between TICs and FcεRIα⁺ macrophages. Dotted lines denote the tumor–stroma boundaries. Scale bar, 50 μm.