Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer (2019)
Figure 8

Figure 8. Inhibition of the TGFβ-IL-6 paracrine signal on cancer cells and NK cells results in the therapeutic efficacy in TGFβR2-mutant PDA results. (A) Control shRNA or two different shRNAs against IL6RA were used to knock down IL6RA in the Tgfbr2-mutant cell line Tgfbr2mut1. Cell lysates were harvested and western blotting for IL6RA and tubulin. (B-C) 3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). Control IgG, 2G8 and IL-6 neutralizing antibody (each 100 ng/ml, n = 5/group). Scale bars = 50 μm. P values by t test are shown (C). (D-F) Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated. (G-N) Immunohistochemistry with tumor samples from (D) for Ki67 (G and K), P-STAT3 (H and L), ZAP70, and CD3 (I-J and M-N) was performed. NK cells are highlighted as red (ZAP70+CD3-) and T cells are highlighted as yellow (ZAP70+CD3+) by ImageJ (N). Scale bars outside the magnification boxes = 50 μm, scale bars inside the magnification boxes = 10 μm. n = 5/group, P values vs. control by t test are shown.

The paper explained

Problem

TGFβ is a druggable target, inhibition of which has shown efficacy in multiple tumor types especially in combination with immune checkpoint blockade. However, inhibition of the TGFβ signaling pathway in pancreatic cancer (PDA) has not been successful in preclinical or clinical studies. This is due to the fact that canonical TGFβ signaling directly suppresses epithelial cancer cell growth in a context-dependent manner. Previously we reported that TGFβ signaling in stromal cells in PDA is a significant contributor to desmoplasia, immune suppression, and metastasis. Importantly, in human PDA tumor cell-specific deficiency in canonical TGFβ signaling via the loss of TGFβR2 or the downstream protein SMAD4 is common (up to 60%). Thus, we hypothesized that TGFβ-mutant PDA is not subject to the tumor cell growth-suppressive activity of TGFβ yet TGFβ activation of stromal cells still drives tumor progression.

Results

We demonstrate that TGFβ induces cancer-associated fibroblasts to produce IL-6 that promotes tumorigenesis via STAT3 activation in PDA cells and also inhibits NK cell activity. Thus, TGFβR2 blockade in vivo inhibits STAT3 activation in PDA cells and alters the immune microenvironment. However, TGFβR2 blockade has therapeutic efficacy only in PDA that harbors tumor cell loss-of-function mutations in Tgfbr2. We further demonstrate that the efficacy of TGFβR2 blockade in TGFβR2-mutant tumors is due to the inhibition of TGFβ signaling in stromal cells and IL-6 signaling in cancer cells and NK cells.

Impact

Our study highlights the potential benefit of TGFβ blockade in PDA and the importance of stratifying PDA patients who might benefit from such therapy.