Extended Data Fig. 2: Cytotoxic effect of CARMA on tumor cell growth.
a, Protein expressions of CD3 and CD28 in THP-1 human monocyte cell line and PBMC-derived primary human monocytes (Mono). b, The procedure of CARMA generation. The details of the procedure were provided in the Methods section. Image was created with BioRender.com. c, The expression of CD74+ Inflam-TAMs and CD206+ Reg-TAMs before or after CD14 microbeads isolation. (n = 5 / group). d, mRNA and protein expression of INF-γ, TNF-α and IL-1β on monocyte before or after CD14 microbeads isolation. (n = 5 / group). e, Efficiency of virus infection of Flag-labeled anti-MSLN-CAR constructs to the human primary (Pri) macrophage (MΦ) and N-CARMA and anti-MSLN-CARMA were examined by FACS. Significance was calculated using One-way ANOVA with Tukey’s multiple comparisons test: (e) ***, p = 0.0001 (n = 4 / group). f, The Flag protein expressions on the primary T, primary macrophage, Anti-MSLN-CAR-T, and anti-MSLN-CARMA (MyD-CARMA) cells were examined by western blot. g, MSLN binding ability in control N-CARMA and anti-MSLN-MyD-CARMA. h, Morphological changes of monocyte/macrophage that were treated with PBS (Veh), 1 nM CSF1, GM-CSF, or PMA. i, Cell viability of primary macrophage (Mϕ) and different CARMA cells after the treatment with PBS, CSF1 (1 nM), GM-CSF (1 nM) or PMA (1 nM) for 24 h. i, We calculated cell viability by dividing the absorbance of the wells containing cells treated with various cytokines by the average absorbance of the control wells. (n = 5 / group). j, Expression of Inflam-TAMs/Reg-TAMs phenotype on CARMA with PBS, CSF1, GM-CSF, or PMA treatment for 24 h. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 4 / group). k, The expression of MSLN in the tumor and CARMA. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). l, The MyD-CAR construct in lentivirus was introduced into the PBMC-derived primary human monocyte by viral infection, and respective CAR-monocyte cells were generated. MyD-monocytes were treated with PBS, CSF1, PMA, or GM-CSF. After 24 h, the cells were washed with PBS and they were co-cultured with luciferase-labeled H2030BrM cells (E/T ratio: 30:1) followed by assaying cell viability by IVIS. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 3 / group). m, Real-Time Cytotoxicity Assay (RTCA) was used to evaluate cytotoxic function of CARMA. Target cells (H2030BrM) were first added to the culture wells, and cell index was recorded. After 25 h, effector cells (N-, CD3-, and MyD-CARMA) were added at a 30:1 (Effector: Target cells) ratio. The percentage of specific cell lysis was shown in the right panel. One-way ANOVA with Tukey’s multiple comparisons test: **, p = 0.001. (n = 4 / group). n, Primary N-, CD3-, or MyD-CARMA were cultured with luciferase-labeled H2030BrM cells (E/T ratio: 10:1) followed by assaying long-tern effect of cell viability by IVIS. Two-way ANOVA with Tukey’s multiple comparisons test: **, p = 0.0063 and ***, p = 0.0001. (n = 4 / group). The data are presented as the mean ± SD.
