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Lung cancer–associated pulmonary hypertension: Role of microenvironmental inflammation based on tumor cell–immune cell cross-talk

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Science Translational Medicine  15 Nov 2017:
Vol. 9, Issue 416, eaai9048
DOI: 10.1126/scitranslmed.aai9048
  • Fig. 1. PA enlargement and pulmonary vascular remodeling detected in patients with lung cancer.

    (A) Distribution of PA size in 519 patients with non–small cell and small cell lung cancer. The blue bars indicate PA diameter ≥ 28 mm. (B) Distribution of PASP in 70 patients with non–small cell and small cell lung cancer. The orange bars indicate PASP ≥ 34 mmHg. (C) Representative photomicrographs of elastica van Gieson–stained SCC and AC lung cancer tissues, followed by (D) quantification of medial wall thickness of pulmonary vessels in nontumor (N) and tumor (T) regions of SCC and AC lung cancer tissues, shown as a percentage of vessels that fall into the range specified on the y axis. Scale bars, 20 μm; n = 14. (E) Representative photomicrographs of von Willebrand factor (vWF) (brown)–stained and α-smooth muscle actin (SMA) (violet)–stained SCC and AC lung cancer tissues, followed by (F) quantification of pulmonary vascular muscularization (given in percentage). N, nonmuscularized pulmonary vessels; P, partially muscularized pulmonary vessels; M, fully muscularized pulmonary vessels. Scale bars, 20 μm; n = 14. (G) Medial wall thickness of pulmonary vessels from biopsies obtained from lung cancer patients with COPD (n = 10) grouped by stage of COPD. *P < 0.05, **P < 0.01, ***P < 0.001 in (D), (F), and (G) compared with nontumor regions of SCC or AC.

  • Fig. 2. Features of PH in Sftpc-cRaf-BxB transgenic mice.

    (A) Schematic of experimental design. Measurements were taken at age 7, 8, and 9 months (mo) for WT and Sftpc-cRaf-BxB lung tumor–bearing mice. (B) Representative hematoxylin and eosin–stained sections of mouse lungs. Scale bar, 500 μm. (C) Echocardiographic measurements and (D) representative echocardiographic images. (E) Physiological measurements of RVSP, PVR, PO2, SAP, and (F) RV hypertrophy [RV/(LV + S)] and RV weight (RV/body weight) of the mice. For WT, n = 5; for Sftpc-cRaf-BxB, n = 5 to 8. (G) Representative photomicrographs of vWF (brown)–stained and α-SMA (violet)–stained tissues, followed by (H) quantification of pulmonary vascular muscularization (given as a percentage for each range of vessel sizes). Scale bars, 20 μm. (I) Representative photomicrographs of elastica van Gieson–stained tissues, followed by (J) quantification of medial wall thickness of pulmonary vessels. Scale bars, 20 μm. (K) Representative photomicrographs of Sirius red staining (right). Scale bars, 20 μm. Quantitative image analysis of Sirius red staining (collagen deposition) in the RV (left). For WT, n = 5; for Sftpc-cRaf-BxB, n = 9. *P < 0.05, **P < 0.01, ***P < 0.001 compared with WT; §P < 0.05, §§P < 0.01, §§§P < 0.001 compared with the nontumor part of Sftpc-cRaf-BxB.

  • Fig. 3. Features of PH in KRasLA2 transgenic mice.

    (A) Schematic of the experimental design. Measurements of 3-, 4-, and 5-month-old WT and KRasLA2 lung tumor–bearing mice. (B) Representative hematoxylin and eosin–stained sections of mouse lungs. Scale bar, 500 μm. (C) Echocardiographic measurements. (D) Physiological measurements of RVSP, SAP, PO2, and PVR of the mice. For WT, n = 7; for KRasLA2, n = 10. (E) Representative photomicrographs of vWF (brown)–stained and α-SMA (violet)–stained tissues, followed by (F) quantification of pulmonary vascular muscularization (given as a percentage for each range of vessel sizes). Scale bars, 20 μm. (G) Representative photomicrographs of elastica van Gieson–stained tissues, followed by (H) quantification of medial wall thickness of pulmonary vessels. Scale bars, 20 μm. (I) Representative photomicrographs of Sirius red staining (right). Scale bars, 20 μm. Quantitative image analysis of Sirius red staining (collagen deposition) in the RV (left). For WT, n = 7; for KRasLA2, n = 10. *P < 0.05, **P < 0.01, ***P < 0.001 compared with WT; §P < 0.05, §§P < 0.01, §§§P < 0.001 compared with nontumor regions of KRasLA2.

  • Fig. 4. Features of PH in LLC1 lung tumor mice.

    (A) Schematic of the experimental design. C57BL/6 mice were intravenously injected with saline (WT) or LLC1 cells. After 10, 14, and 18 days from the initial injection, WT and LLC1 lung tumor–bearing mice underwent echocardiographic and physiological measurements. (B) Representative hematoxylin and eosin–stained sections of mouse lungs. Scale bar, 500 μm. (C) Echocardiographic measurements. (D) Physiological measurements of RVSP, SAP, PO2, and PVR of the mice. For WT, n = 9; for LLC1, n = 10. (E) Representative photomicrographs of vWF (brown)–stained and α-SMA (violet)–stained tissues, followed by (F) quantification of pulmonary vascular muscularization (given as a percentage for each range of vessel sizes). Scale bars, 20 μm. (G) Representative photomicrographs of elastica van Gieson–stained tissues, followed by (H) quantification of medial wall thickness of pulmonary vessels. Scale bars, 20 μm. (I) Representative photomicrographs of Sirius red staining (right). Scale bars, 20 μm. Quantitative image analysis of Sirius red staining (collagen deposition) in RV (left). For WT, n = 9; for LLC1, n = 10. *P < 0.05, ***P < 0.001 compared with WT; §P < 0.05, §§P < 0.01, §§§P < 0.001 compared with nontumor tissue of LLC1.

  • Fig. 5. Role of immune and inflammatory cells in lung cancer–associated PH.

    Representative photomicrographs and quantification of (A) CD3+ (lymphocytes) and (B) CD68+ (macrophages) cells in pulmonary vessels from nontumor (N) and tumor (T) SCC and AC tissues. Scale bars, 20 μm; n = 7. *P < 0.05, **P < 0.01, compared with nontumor tissues of SCC or AC. NSG mice were injected with saline (NSG-control), LLC1 (NSG-LLC1) cells, or A549 (NSG-A549) cells. Twelve and 20 days after injection, the NSG-control (n = 7), NSG-LLC1 (n = 9), and NSG-A549 (n = 9) lung tumor–bearing mice underwent echocardiographic and physiological measurements. (C) Representative photographs of mouse lungs. Scale bars, 2 mm. (D) Representative echocardiographic images and echocardiographic measurements of (E) NSG-A549 and (F) NSG-LLC1 lung tumor–bearing mice. Physiological measurements (RVSP and PO2), RV hypertrophy [RV/(LV + S)], and PVR of (G) NSG-A549 and (H) NSG-LLC1 lung tumor–bearing mice.

  • Fig. 6. Tumor cell and immune cell cross-talk driving pulmonary vascular remodeling.

    (A) Proliferation and migration of human donor PASMCs and PAAFs subjected to CM and derived from AC tumor cells (A549), macrophages, or AC tumor cells cocultured with macrophages as indicated. (B) Cytokine array of different CM as indicated. Strongly regulated cytokines are highlighted. 1, IL-8; 2, CCL2; 3, GM-CSF; 4, IL-1RA; 5, CCL3; 6, CCL5. (C) Relative amounts of protein for each cytokine detected; n = 2. (D) Schematic diagram of the triple transgenic mice used to down-regulate IKK2 (Sftpc-cRaf-BxB/Ikk2DN) in epithelial cells. (E) Representative FACS images and (F) histological quantification of macrophage (F4/80 and CD68 antibodies for FACS and immunostaining, respectively) and T lymphocyte (CD3 antibody for both FACS and immunostaining) composition in mouse lung tissues. (G) Representative photomicrographs of elastica van Gieson–stained tissues (right), followed by quantification of medial wall thickness of pulmonary vessels (left). Scale bars, 20 μm; n = 3. **P < 0.01 compared with Sftpc-cRaf-BxB.

  • Fig. 7. Role of PDE5 up-regulation in lung cancer–associated PH.

    Expression of PDE5 mRNA in (A) PASMCs and (B) PAAFs that were stimulated with basal medium (BM) or indicated cytokines for 24 hours (n = 4). (C) Representative photomicrographs of PDE5 (green), α-SMA (red), and DAPI (nuclei, blue) in pulmonary vessels from human lung tissue without (Donor) or with lung cancer. Scale bars, 100 μm. (D) Migration and (E) apoptosis of human PASMCs and PAAFs that had been stimulated with BM or indicated cytokines in the absence or presence of the PDE5 inhibitor sildenafil; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with BM; §P < 0.05, §§P < 0.01 compared with cytokine stimulation in the absence of sildenafil. C57BL/6 mice were treated with sildenafil or vehicle and intratracheally instilled with LLC1 cells. (F) Echocardiographic measurements. (G) Physiological measurements. For WT, n = 5; for LLC1 treated with vehicle or sildenafil, n = 8 each. (H) Representative photomicrographs of elastica van Gieson–stained tissues (bottom), followed by quantification of medial wall thickness of pulmonary vessels (top). Scale bars, 20 μm; n = 4. §P < 0.05, §§P < 0.01 compared with vehicle-treated LLC1 mice.

  • Table 1. Baseline characteristics of all lung cancer patients with available CT data.

    SCLC, small cell lung cancer; NSCLC, non–small cell lung cancer.

    VariablePA ≥ 28 mmPA < 28 mmP
    Number250 (48%)269 (52%)
    Age (years)66.5 ± 9.664.4 ± 10.00.015
    Female23% (58)40% (107)<0.001
    Body mass index26.7 ± 11.924.2 ± 5.20.015
    Nasal oxygen dependency8 (110)1 (126)0.013
    Arterial PO2*65.6 ± 10.970.7 ± 9.0<0.001
    Histological classification151174
    NSCLC2930
    SCLC7065
    Not otherwise specified31% (33)42% (44)
    Non-COPD†9% (10)8% (8)
    COPD stage†32% (34)26% (28)
    GOLD stage I21% (22)19% (20)
    GOLD stage II7% (7)6% (6)
    GOLD stage III
    GOLD stage IV

    *Only PO2 measurements in the absence of nasal oxygen are included.

    †Lung function was not obtained in all patients.

    • Table 2. Baseline characteristics of lung cancer patients with available echocardiography data.
      VariablePASP ≥ 34 mmHgPASP < 34 mmHgP
      Number32 (46%)38 (56%)
      Age (years)68.1 ± 8.368.7 ± 10.10.780
      Female53% (17)34% (13)0.111
      Body mass index24.6 ± 4.925.0 ± 6.50.802
      Nasal oxygen dependency4 (25)0 (28)0.043
      Arterial PO2*67.6 ± 17.0 (22)69.9 ± 12.4 (21)0.608
      Histological classification1525
      NSCLC26
      SCLC157
      Not otherwise specified37% (7)29% (8)
      Non-COPD†11% (2)25% (7)
      COPD stage†16% (3)21% (6)
      GOLD stage I26% (5)18% (5)
      GOLD stage II11% (2)7% (2)
      GOLD stage III
      GOLD stage IV

      *Only PO2 measurements in the absence of nasal oxygen are included.

      †Lung function was not obtained in all patients.

      Supplementary Materials

      • www.sciencetranslationalmedicine.org/cgi/content/full/9/416/eaai9048/DC1

        Fig. S1. Dendritic cell recruitment in the pulmonary vasculature of human lung cancer tissues.

        Fig. S2. Role of immune/inflammatory cells in lung cancer–associated vascular remodeling.

        Fig. S3. Induction of pulmonary vascular cell proliferation by CM from cocultures of macrophages/T lymphocytes with lung cancer cells.

        Fig. S4. Secreted factors in the CM of macrophage/SCC cocultures.

        Fig. S5. Reduction of tumor growth by IKK2 down-regulation in epithelial cells.

        Fig. S6. Proposed mechanism of PH development in lung cancer.

        Table S1. Demographic data, tumor type, and tumor stage for samples undergoing histological examination.

      • Supplementary Material for:

        Lung cancer–associated pulmonary hypertension: Role of microenvironmental inflammation based on tumor cell–immune cell cross-talk

        Soni Savai Pullamsetti, Baktybek Kojonazarov, Samantha Storn, Henning Gall, Ylia Salazar, Janine Wolf, Andreas Weigert, Nefertiti El-Nikhely, Hossein Ardeschir Ghofrani, Gabriele A. Krombach, Ludger Fink, Stefan Gattenlöhner, Ulf R. Rapp, Ralph Theo Schermuly, Friedrich Grimminger, Werner Seeger,* Rajkumar Savai*

        *Corresponding author. Email: rajkumar.savai{at}mpi-bn.mpg.de (R.S.); werner.seeger{at}mpi-bn.mpg.de (W.S.)

        Published 15 November 2017, Sci. Transl. Med. 9, eaai9048 (2017)
        DOI: 10.1126/scitranslmed.aai9048

        This PDF file includes:

        • Fig. S1. Dendritic cell recruitment in the pulmonary vasculature of human lung cancer tissues.
        • Fig. S2. Role of immune/inflammatory cells in lung cancer–associated vascular remodeling.
        • Fig. S3. Induction of pulmonary vascular cell proliferation by CM from cocultures of macrophages/T lymphocytes with lung cancer cells.
        • Fig. S4. Secreted factors in the CM of macrophage/SCC cocultures.
        • Fig. S5. Reduction of tumor growth by IKK2 down-regulation in epithelial cells.
        • Fig. S6. Proposed mechanism of PH development in lung cancer.
        • Table S1. Demographic data, tumor type, and tumor stage for samples undergoing histological examination.

        [Download PDF]

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