TGFβ blocks IFNα/β release and tumor rejection in spontaneous mammary tumors

Abstract

Type I interferons (IFN) are being rediscovered as potent anti-tumoral agents. Activation of the STimulator of INterferon Genes (STING) by DMXAA (5,6-dimethylxanthenone-4-acetic acid) can induce strong production of IFNα/β and rejection of transplanted primary tumors. In the present study, we address whether targeting STING with DMXAA also leads to the regression of spontaneous MMTV-PyMT mammary tumors. We show that these tumors are refractory to DMXAA-induced regression. This is due to a blockade in the phosphorylation of IRF3 and the ensuing IFNα/β production. Mechanistically, we identify TGFβ, which is abundant in spontaneous tumors, as a key molecule limiting this IFN-induced tumor regression by DMXAA. Finally, blocking TGFβ restores the production of IFNα by activated MHCII⁺ tumor-associated macrophages, and enables tumor regression induced by STING activation. On the basis of these findings, we propose that type I IFN-dependent cancer therapies could be greatly improved by combinations including the blockade of TGFβ.

Fig. 1

DMXAA does not induce a systematic regression of Spont-PyMT tumors. a Spont-PyMT mice were treated at day 0 with one i.p. injection of DMXAA or DMSO as a control. For each treated mouse, the changes in tumor size from baseline (d0) was calculated at each time point and expressed relative to the size at d0. The graphs in (a) provide an example of measurements obtained in one mouse treated with DMXAA i.p., with one curve per tumor. The average curve of all tumors of this mouse is shown in blue. The mouse clipart was created by N.B. b Each black curve corresponds to one mouse, and is calculated as the average relative tumor change for this mouse (as shown in a). The average curves for all the animals of a cohort are shown in red (DMXAA) or orange (CTRL). Overall progression (gray area) or regression (pink area) of tumor burden are indicated. Spont-PyMT mice: n = 30 (DMXAA), n = 25 (CTRL), from nine independent experiments. c The proportion of mice with, on average, regressing, stabilized or progressing tumors, are represented for these Spont-PyMT mice, in comparison with Trans-PyMT mice. Spont-PyMT mice: n = 30; Trans-PyMT mice: n = 26, from 9 to 10 independent experiments, respectively. d Intratumoral injection of DMXAA or ML RR-S2 CDA-induced tumor regression in Trans-PyMT mice, but not in Spont-PyMT mice. Trans- and Spont-PyMT mice were injected i.t. with DMXAA (1 × 500 µg, day 0) or ML RR-S2 (3 × 50 µg, at days 0, 3, and 6) or with HBSS i.t. as controls. Blue arrows: days of injections. The average fold change in tumor volume compared with baseline (day 0) is shown for each treated group. ML RR-S2 group (Spont-PyMT mice n = 4; Trans-PyMT mice n = 10), DMXAA (Spont-PyMT mice n = 3; Trans-PyMT mice n = 10), and HBSS/DMSO (Spont-PyMT mice n = 4; Trans-PyMT mice n = 6). The data are from three independent experiments for both tumor models. Source data are provided as a Source Data file

Fig. 2

DMXAA induces a weak immune infiltrate in Spont-PyMT tumors. a The proportion of CD45⁺ cells in Spont-PyMT or Trans-PyMT tumors before treatment (gray circles) and at days 1 to 8 after DMXAA i.p. injection (black circles) was determined by flow cytometry after dissociation of the tumors. b The proportion of neutrophils, TAM, monocytes, and CD8⁺ T cells among those CD45⁺ cells are shown. For day 0, Spont-PyMT n = 12 and Trans-PyMT n = 30 mice, for day 1 Spont-PyMT n = 6 and Trans-PyMT n = 12, for day 4 Spont-PyMT n = 9 and Trans-PyMT n = 24 and for day 8 Spont-PyMT n = 7 and Trans-PyMT n = 10. The data are from four to seven independent experiments for each group of mice. Results are expressed as mean ± SEM. Tukey’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001. Source data are provided as a Source Data file

Fig. 3

Lack of type I IFN triggering in Spont-PyMT tumors. a Spont-PyMT mice were injected i.p. with DMXAA and then killed after 3 h or 24 h to measure mRNA levels of cytokines and chemokines in tumors. The relative expressions in DMXAA-treated (black diamonds), compared with DMSO-treated (gray diamonds), mice are shown. Each dot corresponds to one tumor. Cumulative data from CTRL: n = 6 mice and DMXAA: n = 6 mice, from three independent experiments, are shown. Tukey’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001. b, c The relative expression of these cytokines/chemokines, compared with PBS (CTRL) injected mice, were measured in Spont-PyMT (gray circles) and Trans-PyMT (open circles) target lesions 3 h after (b) cGAMP (25 μg i.t.) injection (Spont-PyMT mice: n = 3 CTRL and n = 3 cGAMP; Trans-PyMT mice: n = 5 CTRL and n = 13 cGAMP) or (c) LPS (50 μg i.p.) injection (Spont-PyMT mice: n = 3 CTRL and n = 3 LPS; Trans-PyMT mice: n = 5 CTRL and n = 10 LPS) from three independent experiments. In b and c, the results are expressed as mean ± SEM. Source data are provided as a Source Data file

Fig. 4

Absence of IRF3 phosphorylation in Spont-PyMT tumors after DMXAA injection. a Spont-PyMT mice and Trans-PyMT mice received one i.p. injection of DMXAA. Three hours later, tumors were collected and tumors slices were stained with anti-EpCAM (blue) and anti-pIRF3 (red) mAbs. Representative images from Trans-PyMT tumors (left) and Spont-PyMT tumors (right) are shown. b In Trans-PyMT tumors, some pIRF3⁺ cells (red) are myeloid (F4/80⁺, green), or endothelial cells (CD31⁺, yellow). Scale bars = 50 µm. The data are representative of three Spont-PyMT and five Trans-PyMT mice, from five independent experiments

Fig. 5

TGFβ expression is associated with pSmad2/3 in Spont-PyMT tumors. a mRNA levels of Tgfβ were measured in untreated Spont-PyMT (gray diamonds) and Trans-PyMT (open circles) tumors, right after tumor dissociation. The relative expression, normalized to Trans-PyMT tumors, is shown. Each dot corresponds to one tumor. Cumulative data from Trans-PyMT mice: n = 10 and Spont-PyMT mice: n = 3. Student's t test. *p < 0.05; **p < 0.01; ***p < 0.001. b Tumors slices from Spont- and Trans-PyMT-untreated mice were stained with anti-EpCAM (blue) and anti-pSmad2/3 (red) Abs. c In Spont-PyMT tumors, pSmad2/3 ⁺ cells (red) may be myeloid cells (F4/80⁺, green), fibroblasts (gp38⁺, yellow), or tumor cells (EpCAM^+, blue). Note that the 2nd and 3rd images, not superimposed for clarity, are from the same image field. Images are representative of Spont-PyMT: n = 3 mice; Trans-PyMT: n = 3 mice, from three independent experiments for each group of mice. In b and c, scale bars = 50 µm. Source data are provided as a Source Data file

Fig. 6

Blocking TGFβ allows DMXAA-induced type I IFN production and tumor regression. a Spont-PyMT mice, treated with an anti-TGFβ (day − 4 and day − 1), received one i.p. injection of DMXAA. Three hours later, tumors were collected and tumor slices were stained with anti-EpCAM (blue) and anti-pIRF3 (red) mAbs. The image is representative of n = 4 anti-TGFβ-treated Spont-PyMT mice from three independent experiments. Scale bar = 50 µm. b Tumors from Spont-PyMT mice, treated with anti-TGFβ (black circles) or notβ (gray circles), and from Trans-PyMT mice (open circles), were analyzed by flow cytometry for the expression of MHCII on TAM and monocytes. The data are from anti-TGFβ-treated Spont-PyMT mice: n = 7; Spont-PyMT mice: n = 6 or 8; Trans-PyMT mice: n = 12 or 21, for TAM or monocytes respectively, collected in seven independent experiments. The results are expressed as mean ± SEM. Tukey’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001. c Tumors from anti-TGFβ-treated Spont-PyMT mice were pooled for each mouse (8–10 tumors), dissociated and F4/80⁺ cells were sorted with magnetic beads. F4/80⁺ and F4/80^neg fractions were restimulated in vitro with DMXAA (250 µg/ml, black circles) or not (gray circles) and IFNα was measured by ELISA in the supernatant after overnight culture. The results are expressed as mean ± SEM. Student t test. *p < 0.05; **p < 0.01; ***p < 0.001. d Spont-PyMT mice were treated with DMXAA (i.p. at day 0) or DMSO as a control, with anti-TGFβ (i.p. 200 µg), anti-IFNαR (i.p. 200 µg) and/or with anti-CD8 (i.p. 200 µg) Abs on day − 4, day − 1, day 1, day 4. The proportion of mice with an average regressing, stabilized or progressing tumors, is shown as in Fig. 1. DMSO-treated mice: n = 28; DMXAA-treated mice: n = 30; DMSO + anti-TGFβ: n = 6; DMXAA + anti-TGFβ: n = 9; DMXAA + anti-TGFβ + anti-IFNαR: n = 6; DMXAA + anti-TGFβ + anti-CD8: n = 5. Statistics are shown in Supplementary Table 2. Source data are provided as a Source Data file