3.2. Combinatorial treatment with TMZ and αCT11 does not increase GBM cell killing.
To test efficacy of Cx43 targeting in combination with TMZ, we first investigated the effect of αCT11 peptide on drug efficacy.Organoids were treated with combinations of TMZ (0, 10, 100, 1000 µM) and αCT11 (0, 4, 40, 400 µM) with the drug and media replenished after 4 days. After combinatorial treatment of GBM organoids for 7 days. Cell viability was assessed using the CellTiter Glo 3D (Promega) assay. The assay is used to quantify ATP levels in cells and has been previously verified for studying the drug efficacy of 3D cancer organoids (Dominijanni et al., 2021). Live/dead staining was used to qualitatively confirm results from the viability assay. These images can be found inSupplemental Figures 1-3 . The GBM organoids were grouped by three concentrations of TMZ (10, 100, or 1000 µM) and a control group (TMZ = 0), and the organoid viability is compared across three concentrations of αCT11 (4, 40, or 400 µM) and a control group ([αCT11] = 0) in each TMZ subgroup.
The viability of all 3 GBM cell lines decreased as TMZ concentration increased (Figure 2 ). In A172 and BT169 organoids, there is no statistically significant difference between cells treated with TMZ alone and αCT11 (Figure 2A and 2C ). The U87 organoids showed significantly decreased viability in the TMZ 10 µM + αCT11 4 µM condition, but in no other condition (Figure 2B ).