Fig. 4

Fig. 4

Hypoxic tumor microenvironment in 3D lung adenocarcinoma spheroids regulates CCL2 mediated monocyte infiltration and promotes TAM polarization (A,B) Representative phase‐contrast (A), and Calcein‐AM fluorescence staining (B) images of 3D spheroids of BEAS‐2B, A549, and NCI‐H460 cells on day 10 showing THP‐1 monocytes infiltrations in normoxic and hypoxic conditions for 24 h. Scale bars: 100 μm. (C,D) Flow cytometric analysis of CD11b level (C) and its quantifications (D) indicated THP‐1 monocytes infiltration in 3D spheroids of BEAS‐2B or A549 or NCI‐H460 cells incubated in normoxic and hypoxic conditions for 24 h. (E,F) RT‐qPCR analysis showing the relative abundance of CCL2 (E), and mir‐210‐3p (F) gene expression in BEAS‐2B, A549, and NCI‐H460 cells spheroids under normoxic and hypoxic conditions. GAPDH and U6 snRNA were used as loading controls for CCL2, and mir‐210‐3p gene expression normalization, respectively. (G,H) RT‐qPCR analysis of CD206 (G), and CD163 (H) gene expression in BEAS‐2B, A549, and NCI‐H460 cell spheroids incubated with THP‐1 monocytes under normoxic and hypoxic conditions for 7 days. GAPDH was used as the loading control for normalization. (I) Representative phase‐contrast (upper panel) and Calcein‐AM fluorescence staining (lower panel) images of A549 cells spheroid transfected with control inhibitor or miR‐210‐3p inhibitor. Scale bars: 100 μm. (J) Flow cytometric analysis of CD11b level indicated THP‐1 monocytes infiltration in A549 cells spheroid transfected with control inhibitor or miR‐210‐3p inhibitor. (K,L) RT‐qPCR analysis of mir‐210‐3p (K) and CCL2 (L) gene expression in A549 cells spheroid transfected with control inhibitor or miR‐210‐3p inhibitor. GAPDH and U6 snRNA were used as loading controls for CCL2 and mir‐210‐3p gene expression normalization, respectively. Data represented as mean ± SD of three independent experiments. Statistical significance was analyzed by Student's t‐test, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns, nonsignificant.