Fig 1: Inhibition of stromal TGFβR2 reduces IL‐6 production and tumor cell STAT3 activation in PDA AMouse qPCR array analysis was performed with Colo357 and MiaPaca‐2 orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect changes in mouse stromal gene expression after treatment. A heat map was generated with ratios of gene expression (2G8 versus control).BELISA‐based Quansys mouse chemokine assay was performed with Capan‐1 (Cap), MiaPaCa‐2 (Mia), Colo357 (Colo), and C5LM2 (C5) orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect mouse chemokine changes after treatment. Change in mouse IL‐6 was shown.C, D KIC mice were treated for 4 weeks, and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Tumors from were collected for mouse IL‐6 ELISA (n = 6/group).E–K KIC mice were treated for 4 weeks, and KPC mice were treated for 55 days with Mac84 (control) or 2G8. The activation of SMAD2 (P‐Ser465/467) (E and H–I) and STAT3 (P‐Tyr705) (F and J–K) and expression of IL‐6R (G) were detected by immunohistochemistry (n = 4/group). Scale bars outside the magnification boxes = 50 μm, and scale bars inside the magnification boxes = 10 μm.Data information: All data are reported as mean ± SD. P values versus control by t‐test are indicated.
Fig 2: CAF‐secreted IL‐6 is regulated by TGFβ and activates STAT3 in pancreatic cancer cells A–CControl, M1 (LPS stimulated), and M2 (IL‐4 stimulated) RAW 264.7 cells, and NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 h. Cell lysates were harvested and blotted for P‐SMAD2 (P‐Ser465/467), SMAD2, and tubulin (A). Conditioned media (CM) was collected for mouse IL‐6 ELISA (B–C). P value by ANOVA is shown.D–FNIH 3T3 (D), pancreatic stellate cells (PSC) (E), and human CAF cell lines CAF‐PC1 and CAF‐PC2 (F) were treated with TGFβ (30 ng/ml) and/or IL‐1α (1 ng/ml) for 24 h. CM was collected for mouse or human IL‐6 ELISA. P values by t‐test are shown.G, HCAF‐PC2 cells were treated with TGFβ (30 ng/ml) (G) or IL‐1α (1 ng/ml) (H) for 24 h with siRNA‐mediated knockdown of JUNB, JUND, SMAD4, or RELA. CM was collected and subjected to human IL‐6 ELISA. P values by t‐test were shown.I, J KIC (mPLRB9), KPC (KPC‐M09), and KPfC (BMFA3) cell lines were treated with normal DMEM (CTRL), CM from NIH 3T3 (CM), CM from TGFβ‐treated NIH 3T3 (TGFβ‐CM), CM from TGFβ‐treated NIH 3T3 + 2G8 (TGFβ‐CM + 2G8) (I), normal DMEM + TGFβ (TGFβ), and CM from TGFβ‐treated NIH 3T3 + IL‐6 neutralizing antibody (TGFβ‐CM + IL‐6 Ab). Cell lysates were harvested and blotted for P‐STAT3 (P‐Tyr705), STAT3, P‐SMAD2 (P‐Ser465/467), SMAD2, and tubulin (J).K–N3D culture: cells were seeded on poly‐HEMA‐coated 96‐well plates and cultured for 4 days (5,000 cancer cells for monoculture, 3,000 cancer cells + 2,000 NIH 3T3 for co‐culture). IL‐6 neutralizing antibody (100 ng/ml). Scale bars = 50 μm. n = 5/group, P values by t‐test are shown.Data information: All data are reported as mean ± SD.Source data are available online for this figure.
Fig 3: Inhibition of the TGFβ‐IL‐6 paracrine signal on cancer cells and NK cells results in the therapeutic efficacy in TGFβR2‐mutant PDA results AControl shRNA or two different shRNAs against IL6RA were used to knock down IL6RA in the Tgfbr2‐mutant cell line Tgfbr2 mut1. Cell lysates were harvested and Western blotting for IL6RA and tubulin.B, C3D culture: Control or IL6RA knockdown Tgfbr2 mut1 cells were seeded on poly‐HEMA‐coated 96‐well plates and cultured for 4 days (5,000 cancer cells for monoculture, 3,000 cancer cells + 2,000 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–FSubcutaneous 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 2×/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti‐Asialo‐GM1 3 days in a row. For maintenance, 25 μg of control rabbit IgG or anti‐Asialo‐GM1 was given twice a 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–NImmunohistochemistry 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 versus control by t‐test are shown.Data information: All data are reported as mean ± SD.Source data are available online for this figure.
Fig 4: TGFβ‐induced IL‐6 from CAFs inhibits NK cell activity and promotes pancreatic cancer A–FSaline and 2G8‐treated MiaPaca‐2 and Colo375 xenografts were subjected to RNA‐seq analysis for mouse gene expression changes. A heat map was generated with gene clustering (A). IPA showing pathways most effected by 2G8 (B–C). Molecules most significantly effected by 2G8 treatment. Y‐axis values shown are log10(FPKM+1) of the transcript levels (D‐F).GAn in vitro NK cell cytotoxicity assay was performed. Human NKL cells were used as effector cells, and mouse pancreatic cancer BMFA3 cells were used as target cells in normal DMEM (control), conditioned medium collected from TGFβ (30 ng/ml)‐treated human pancreatic CAFs (CAF‐PC2; CAF TGFβ‐CM), CAF TGFβ‐CM + IL‐6 antibody (100 ng/ml), or normal DMEM + TGFβ (TGFβ). Living cells were labeled with CFSE, and dead cells were labeled with 7‐AAD. Samples were analyzed by flow cytometry. Cytotoxicity percentage was calculated using the formula (7‐AAD‐positive cells %)/(7‐AAD‐positive cells % + CFSE‐positive cells %) × 100%. n = 4/group, P values by t‐test are shown.H, IHuman pancreatic cancer cell line Colo357 was orthotopically implanted into NOD SCID mice. After tumor establishment, mice were randomized to receive rat IgG Mac84 (control), 2G8, or anti‐mouse IL‐6 antibody MP5‐20F3 (each 30 mg/kg 2×/week, n = 6/group) for 3 weeks. For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti‐Asialo‐GM1 3 days in a row. For maintenance, 25 μg of control rabbit IgG or anti‐Asialo‐GM1 was given twice a week throughout the whole study. Tumors were harvested for analysis, and metastatic burden was determined by histologic evaluation of H&E‐stained liver tissue. Ten sections of the anterior lobe of the liver (n = 6 per group) were scored for lesions. P values by t‐test are shown.Data information: Data in (G–I) are reported as mean ± SD.
Fig 5: CAFs are the major source of IL‐6 in PDA ASingle‐cell RNA sequencing was performed to profile cell populations in normal mouse pancreas (n = 2), early KIC (40‐day‐old, n = 2), and late KIC (60‐day‐old, n = 3) pancreata. Samples from the same stage were pooled. Violin plots of expression of Il6, Tgfbr1, and Tgfbr2 in distinct cell populations is shown.BThe expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC‐M01, KPC‐M09), and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 h) or M2 (20 ng/ml IL‐4 for 18 h) macrophages. Tubulin was used as a loading control.CPearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R. Source data are available online for this figure.
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