Protocol to co-culture SCLC cells with human CD8+ T cells to measure tumor cell killing and T cell activation

Abstract

Small-cell lung cancer (SCLC) is an aggressive malignancy with immunosuppressive tumor microenvironment limiting immunotherapy efficacy. Here, we present a protocol to assess T cell activation and the ability of lurbinectedin to enhance anti-tumor responses using in vitro co-cultures of SCLC cells and human CD8 T cells. We describe steps for cell seeding, treatment, co-culture setup, and assessing cell viability. This protocol provides a platform to study anti-tumor T cell responses and develop new SCLC therapies. For complete details on the use and execution of the protocol, please refer to Chakraborty et al.¹.

Keywords: Cancer; Cell culture; Immunology; Molecular Biology.

Graphical abstract

Figure 1

Plate map of the different co-culture conditions 96 well plate depicting the different conditions and plate set up for the co-culture experiment.

Figure 2

Determination of secreted level CXCL10 upon lurbinectedin treatment (A) Bar graphs showing variability and low secreted level of cytokine CXCL10 upon 16 and 24 h post-10 nM lurbinectedin treatment compared to vehicle treatment in DMS114 cells when the cell free media was not concentrated. The data represents the means ± SEM of 3 biological replicates (n = 3), one-way ANOVA was performed followed by paired two-tailed Student’s t-test to compare between two groups. ns, no significance; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. (B) Bar graphs showing low variability and significant increase in secreted level of cytokine CXCL10 upon 16 and 24 h post-10 nM lurbinectedin treatment compared to vehicle treatment in DMS114 cells upon concentration of cell free media with Pierce Protein Concentrators. The data represent the means ± SEM of 3 biological replicates (n = 3), one-way ANOVA was performed followed by paired two-tailed Student’s t-test to compare between two groups. ns, no significance; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.