Interactions among Lung Cancer Cells, Fibroblasts, and Macrophages in 3D Co-Cultures and the Impact on MMP-1 and VEGF Expression

In vitro cell-based models of lung cancer are frequently employed to study invasion and the mechanisms behind metastasis. However, these models often study only one cell type with two-dimensional (2D) monolayer cell cultures, which do not accurately reflect the complexity of inflammation in vivo. Here, a three-dimensional (3D) cell co-culture collagen gel model was employed, containing human lung adenocarcinoma cells (HCC), human lung fibroblast cells (MRC-5), and macrophages. Cell culture media and cell images were collected, and matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor (VEGF) production was monitored under different cell culture conditions. We found that simulating hypoxia and/or serum starvation conditions induced elevated secretion of VEGF in the 3D co-culture model in vitro, but not MMP-1;the morphology of HCC in the 2D versus the 3D co-culture system was extremely different. MMP-1 and VEGF were secreted at higher levels in mixed cell groups rather than mono-culture groups. Therefore, incorporating lung cancer cells, fibroblasts, and macrophages may better reflect physiological metastasis mechanisms compared to mono-culture systems. Tumour stromal cells, macrophages, and fibroblast cells may promote invasion and metastasis, which also provides a new direction for the design of therapies targeted at destroying the stroma of tumor tissues

Systemic inflammation and pro-inflammatory cytokine profile predict response to checkpoint inhibitor treatment in NSCLC: a prospective study

Treatment with single agent immune checkpoint inhibitors (ICIs) has tremendously changed second line therapy in NSCLC. However, there are still no reliable biomarkers predicting response and survival in this group of patients. PD-L1 revealed to be a correlating, but no perfect marker. Therefore, we sought to investigate in this prospective study, whether inflammation status and cytokine profile could serve as additional biomarkers guiding treatment decision for single agent ICIs in NSCLC. 29 stage IV NSCLC patients receiving single agent PD-1 checkpoint-inhibitor in second line were prospectively enrolled. Inflammatory scores and cytokine profiles (IL-6, IL-8, IL-10, IFN-γ and TNFα) have been obtained before treatment and at the time of the first staging. Cytokine profiles were correlated with response and survival. Patients with signs of pre-therapeutic inflammation (elevated, NLR, SII, IL-6, IL-8) showed significantly lower response to ICI treatment and reduced PFS. Contrary, elevated levels of IFN-γ revealed to characterize a subgroup of patients, who significantly benefits from ICI treatment. Furthermore, low systemic inflammation and high levels of IFN-γ characterized patients with long term-response to ICI treatment. Pre-therapeutic assessment of inflammation and cytokine profiles has the ability to predict response and survival in NSCLC patients treated with single agent ICIs

Interactions among Lung Cancer Cells, Fibroblasts, and Macrophages in 3D Co-Cultures and the Impact on MMP-1 and VEGF Expression.

In vitro cell-based models of lung cancer are frequently employed to study invasion and the mechanisms behind metastasis. However, these models often study only one cell type with two-dimensional (2D) monolayer cell cultures, which do not accurately reflect the complexity of inflammation in vivo. Here, a three-dimensional (3D) cell co-culture collagen gel model was employed, containing human lung adenocarcinoma cells (HCC), human lung fibroblast cells (MRC-5), and macrophages. Cell culture media and cell images were collected, and matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor (VEGF) production was monitored under different cell culture conditions. We found that simulating hypoxia and/or serum starvation conditions induced elevated secretion of VEGF in the 3D co-culture model in vitro, but not MMP-1; the morphology of HCC in the 2D versus the 3D co-culture system was extremely different. MMP-1 and VEGF were secreted at higher levels in mixed cell groups rather than mono-culture groups. Therefore, incorporating lung cancer cells, fibroblasts, and macrophages may better reflect physiological metastasis mechanisms compared to mono-culture systems. Tumour stromal cells, macrophages, and fibroblast cells may promote invasion and metastasis, which also provides a new direction for the design of therapies targeted at destroying the stroma of tumor tissues

The expression of MMP1.

<p><b>(A) Expression of MMP-1 in 3D mono- and co-culture lung cancer models at 48 h detected by ELISA.</b> The expression of MMP1 in HCC & MRC-5 & macrophage co-culture group was higher than that in HCC & MRC-5 co-culture group, or MRC-5/HCC/macrophage mono-culture groups. There was almost no expression of MMP1 in the HCC/macrophage mono-culture group. <b>(B) Expression of MMP-1 in 3D mono- and co-culture lung cancer model at 48 h detected by Western blotting.</b> In Fig 1B, a, the molecular weight of MMP-1 is 52 kD. From the left to right, the lanes are: HCC mono-culture group (2 x 10⁵ cells); MRC-5 mono-culture group (2 x 10⁵ cells); MRC-5 and HCC co-culture group (2 x10⁵ cells); HCC mono-culture group (1 x 10⁶ cells); MRC-5 and HCC co-culture group (1 x 10⁶ cells); MRC-5 mono-culture group (1 x 10⁶ cells). Expression of MMP-1 in co-culture groups was higher than in mono-culture groups (both 2 x 10⁵ cells and 1 x 10⁶ cells). Expression of MMP-1 in the 1 x 10⁶ cell group was higher than the 2 x 10⁵ cell group, regardless of mono-culture or co-culture group designations. In Fig 1B, b, the mean IOD values of the Western blot are shown. <b>(C) Expression of MMP-1 under different co-culture conditions.</b> Expression of MMP1 under 10% FBS and O₂ (10% FBS cell culture medium with O₂) was higher than that under w/o FBS and w/o O₂ (without FBS and without O₂) at 7 different time points. Furthermore, the expression trend of MMP1 under the condition of w/o FBS and w/o O₂ continued to decline from 120 h.</p

Statistical analysis of the expression of VEGF in HCC, MRC-5, and macrophage mono-culture groups (pg/ml).

<p>Statistical analysis of the expression of VEGF in HCC, MRC-5, and macrophage mono-culture groups (pg/ml).</p

Statistical analysis of the expression of MMP-1 by ELISA assay (pg/ml).

<p>Statistical analysis of the expression of MMP-1 by ELISA assay (pg/ml).</p

The expression of VEGF.

<p><b>(A) Expression of VEGF in HCC, MRC-5, and macrophage mono-cultures groups.</b> Expression of VEGF in the HCC mono-culture group was significantly higher than expression in the MRC-5/macrophage mono-culture group under 10% FBS and O₂ culture conditions. <b>(B) Expression of VEGF in HCC, MRC-5, and macrophage co-culture groups compared with the HCC mono-culture group.</b> Expression of VEGF in the HCC & MRC-5 & Macrophage co-culture group was higher than in the HCC & MRC-5 co-culture group and the HCC mono-culture group cultured with 10% FBS and O₂ for 48 h. <b>(C) Expression of VEGF in HCC, MRC-5, and macrophage co-culture groups under different co-culture conditions.</b> The expression of VEGF in cells cultured w/o FBS (starved of FBS but with O₂), w/o FBS and w/o O₂ (without FBS and without O₂) was higher than that in 10% FBS or O₂ (10% FBS cell culture medium with O₂), while the expression of VEGF in the three different conditions first increased and then decreased.</p

Statistical analysis of the expression of MMP-1 by Western Blot (IOD value).

<p>Statistical analysis of the expression of MMP-1 by Western Blot (IOD value).</p