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. 2021 May 20:9:667645.
doi: 10.3389/fcell.2021.667645. eCollection 2021.

TGFβ1 in Cancer-Associated Fibroblasts Is Associated With Progression and Radiosensitivity in Small-Cell Lung Cancer

Jiaqi Zhang  1   2   3   4 , Jing Qi  2   3   4   5 , Hui Wei  1   2   3   4   6 , Yuanyuan Lei  7 , Hao Yu  1   2   3   4 , Ningbo Liu  1   2   3   4 , Lujun Zhao  1   2   3   4 , Ping Wang  1   2   3   4
Affiliations

TGFβ1 in Cancer-Associated Fibroblasts Is Associated With Progression and Radiosensitivity in Small-Cell Lung Cancer

Jiaqi Zhang et al. Front Cell Dev Biol. .

Abstract

Objective: Small-cell lung cancer (SCLC) is aggressive, with early metastasis. Cytokines secreted by cancer-associated fibroblasts (CAFs) within various tumors influences these features, but the function in particular of TGFβ1 (transforming growth factor beta 1) is controversial and unknown in SCLC. This study explored the influence of TGFβ1 in CAFs on the development, immune microenvironment, and radiotherapy sensitivity of SCLC.

Methods: SCLC specimens were collected from 90 patients who had received no treatment before surgery. Tumor and tumor stroma were subjected to multiplex immunohistochemistry to quantitate TGFβ1 and other immune factors in CAFs. Cell proliferation and flow cytometry apoptosis assays were used to investigate associations between TGFβ1 and proliferation and radiotherapy sensitivity. The immune factors in tumors were detected by immunohistochemistry in vitro and in vivo (mice).

Results: TGFβ1 levels on CAFs lower or higher than the median were found, respectively, in 52.2 and 47.8% of patients; overall survival of patients with TGFβ1-high levels (53.9 mo) was significantly longer than that of the TGFβ1-low group (26.9 mo; P = 0.037). The univariate and multivariate analyses indicated that a TGFβ1-high level was an independent predictor of increased survival time. TGFβ1-high levels in CAFs were associated with inhibition of growth, proliferation, antitumor immunity, and enhanced radiotherapeutic sensitivity and tumor immunity of tumor. TGFβ1-low levels promoted tumor cell growth and radiotherapy sensitivity in vivo and in vitro.

Conclusion: High levels of TGFβ1 in CAFs were associated with longer overall survival in patients with SCLC and enhanced radiotherapy sensitivity.

Keywords: TGFβ1; cancer-associated fibroblasts; immune microenvironment; prognostic marker; radiotherapy sensitivity; small-cell lung cancer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Prognostic role of TGFβ1 impact on OS and PFS in overall SCLC patients (A); and in patients receiving radiation therapy (B). OS, overall survival; PFS, progression-free survival.
FIGURE 2
FIGURE 2
The impact of CAFs on lung cancer cell proliferation and radiosensitivity. (A,B) The impact of CAFs on cell viability and proliferation of lung cancer cells were examined by CCK-8 assay and clonogenic survival assay. (C) Tumor volume and weight of xenografts were measured with calipers and electronic balance. The mean tumor volume in CAF and Control groups were 2122.52 ± 365.95 mm3 and 1467.10 ± 436.41 mm3, respectively. The mean tumor weight in CAF and Control groups were 1.61 ± 0.84 g and 0.79 ± 0.44 g, respectively. (D,E) The impact of CAFs on cell viability and proliferation of lung cancer cells after radiotherapy for 0, 2, 4, 6, 8 Gy were examined by CCK-8 assay and clonogenic survival assay. Statistical significance is shown (*p < 0.05; **p < 0.01). MEF, mouse fetal fibroblast cell; CAF, cancer associated fibroblast; LLC, Lewis lung cancer cell; KLN205, mouse squamous cell lung cancer cell.
FIGURE 3
FIGURE 3
The impact of TGFβ1 level produced by CAFs on lung cancer cell proliferation. (A,B) TGFβ1-knocked down CAFs promoted lung cancer cells viability and proliferation. Cell viability and proliferation were examined by CCK-8 assay and clonogenic survival assay, TGFβ1- overexpression CAFs enhanced the viability and proliferation of lung cancer cells. Statistical significance is shown (∗p < 0.05; ∗∗p < 0.01). shNC and oxNC, control groups corresponding to knockdown and overexpression of TGFβ1 recombinant lentiviral infection; shTGFβ1 and TGFβ1ox, knockdown and overexpression of TGFβ1 recombinant lentiviral infection; LLC, Lewis lung cancer cell; KLN205, mouse squamous cell lung cancer cell; H446, human small cell lung cancer cell.
FIGURE 4
FIGURE 4
TGFβ1 level produced by CAFs affected the radiosensitivity of lung cancer cells. (A,B) CAFs with TGFβ1 knocked down inhibited the radiosensitivity of lung cancer cells after radiotherapy of 0, 2, 4, 6, or 8 Gy detected by CCK-8 assay and clonogenic survival assay, CAFs with TGFβ1 overexpression enhanced the radiosensitivity of lung cancer cells. (C) TGFβ1-knocked down CAF inhibited the apoptosis of tumor cells after radiotherapy and the apoptosis of tumor cells were induced after co-cultured with TGFβ1 overexpression CAFs. Cell apoptosis rate was examined by Annexin V/PI staining and flow cytometry assays. Statistical significance is shown (*p < 0.05; **p < 0.01). shNC and oxNC, control groups corresponding to knockdown and overexpression of TGFβ1 recombinant lentiviral infection; shTGFβ1 and TGFβ1ox, knockdown and overexpression of TGFβ1 recombinant lentiviral infection; LLC, Lewis lung cancer cell; KLN205, mouse squamous cell lung cancer cell; H446, human small cell lung cancer cell.
FIGURE 5
FIGURE 5
Tumor volume and weight of xenografts were measured with calipers and electronic balance. (A) TGFβ1 level in CAFs affected the proliferation of lung cancer cells xenografts. (B) The mean tumor volume in shTGFβ1, shNC, TGFβ1ox and oxNC were 2341.62 ± 305.32 mm3, 1760.65 ± 428.63 mm3, 1143.87 ± 166.57 mm3, and 1917.78 ± 415.77 mm3, respectively. (C) The mean tumor weight in shTGFβ1, shNC, TGFβ1ox and oxNC were 1.59 ± 0.57 g and 0.94 ± 0.41 g, 0.59 ± 0.23 g and 1.01 ± 0.22 g, respectively. Statistical significance is shown (*p < 0.05; **p < 0.01). shNC and oxNC: control groups corresponding to knockdown and overexpression of TGFβ1 recombinant lentiviral infection; shTGFβ1 and TGFβ1ox: knockdown and overexpression of TGFβ1 recombinant lentiviral infection.
FIGURE 6
FIGURE 6
The impact of different TGFβ1 level in CAFs on tumor immunology. Immumohistochemical staining of lung cancer tissues in mice for TGFβ1, CD8, FOXP3, PDL1, CD56, CD11b, CD86, and CD206. TGFβ1 overexpression in CAFs inhibited the antitumor immunity of lung cancer xenograft. Statistical significance is shown (∗p < 0.05; ∗∗p < 0.01). shNC and oxNC, control groups corresponding to knockdown and overexpression of TGFβ1 recombinant lentiviral infection; shTGFβ1 and TGFβ1ox, knockdown and overexpression of TGFβ1 recombinant lentiviral infection.
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