A co-culture model with brain tumor-specific bioluminescence demonstrates astrocyte-induced drug resistance in glioblastoma

Abstract

Background: Although several studies suggest that stromal fibroblasts mediate treatment resistance in several cancer types, little is known about how tumor-associated astrocytes modulate the treatment response in brain tumors. Since traditionally used metabolic assays do not distinguish metabolic activity between stromal and tumor cells, and since 2-dimensional co-culture system does not recreate the formidable complexity of the microenvironment within 3-dimensional structures such as solid tumor tissue, we instead established a glioblastoma (GBM) cell-specific bioluminescent assay for direct measurements of tumor cell viability in the treatment of clinical relevant drugs.

Methods: Using lentiviral transfection, we established a panel of human GBM cell lines constitutively expressing a fusion transgene encoding luciferase and the enhanced green fluorescence protein (eGFP). We then initiated co-cultures with immortalized astrocytes, TNC-1, and the eGFP/Luc GBM cell lines. Next, we treated all eGFP/Luc GBM cell lines with Temozolomide (TMZ) or Doxorubicin, comparing co-cultures of glioblastoma (GBM) cells and TNC-1 astrocytes with mono-cultures of eGFP/Luc GBM cells. Cell viability was quantitated by measuring the luciferase expression.

Results: Titration experiments demonstrated that luciferase expression was proportional to the number of eGFP/Luc GBM cells, whereas it was not influenced by the number of TNC-1 cells present. Notably, the presence of TNC-1 astrocytes mediated significantly higher cell survival after TMZ treatment in the U251, C6, A172 cell lines as well as the in vivo propagated primary GBM tumor cell line (P3). Moreover, TNC-1 astrocytes mediated significantly higher survival after Doxorubicin treatment in the U251, and LN18 glioma cell lines.

Conclusion: Glioma cell-specific bioluminescent assay is a reliable tool for assessment of cell viability in the brain tumor cell compartment following drug treatment. Moreover, we have applied this assay to demonstrate that astrocytes can modulate chemo sensitivity of GBM tumor cells. These effects varied both with the cell line and cytotoxic drug that were used, suggesting that several mechanisms may be involved.

Figure 1

Bioluminescence expression is proportional to the number of tumor cells in spheroids formation. A: eGFP expressing glioma spheroids initiated from an increasing number of 172 glioma cells (500, 1000, 2000, 4000, 8000, 16000, 32000, 64000, 96000, 128000) per well. B and C: Bioluminescence measurements for increasing cell numbers of U251 and A172 glioma cells demonstrated a linear relationship (R² = 0.9993 for U251 cells and R² = 0.9987 for A172 cells).

Figure 2

TNC-1 cells have no influence on bioluminescence expression of tumor cells. A: mixing a varying number of A172 eGFP/Luc tumor cells (500, 800, 1600, 3200, 6400, 8000, 16000, 32000, 48000 and 64000) with a varying number of stromal TNC-1 cells (100, 500, 1000, 2000, 4000, 56000, 810000, 210000, 200000, 30000 and 40000) respectively, initiate homogeneous spheroids. B: 8000 A172 eGFP/Luc tumor cells were co-cultured with TNC-1 with an increasing cell numbers (0, 500, 1000, 2000, 4000, 6000, 8000, 10000, 200000, 30000 and 40000) demonstrated TNC-1 has no influence on bioluminescence value changes of A172 tumor cells indicating bioluminescence intensity is proportional to tumor cells in the co-culture spheroids.

Figure 3

Cell viability measured by bioluminescence versus the MTS assay after treatment with TMZ. A and B: LN18 eGFP positive cells on mono-culture spheroids treated with TMZ at doses as indicated were counted by flow cytometry, and compared to measurements by MTS assay and bioluminescence assay, respectively. C and D: LN18 eGFP positive cells on co-culture spheroids with TNC-1 cells treated with TMZ at doses as indicated were counted by flow cytometry, and compared to measurements by MTS assay and bioluminescence assay, respectively. E, F, G and H: Comparison of MTS with bioluminescence intensity measurements in the LN18, A172, U251 and C6 glioma cell lines co-cultured with TNC-1 cells after treatment with TMZ at doses as indicated.

Figure 4

TNC-1 cells modulate sensitivity of TMZ to GBM tumor cells. A, B, C and D: TNC-1 attenuated the response of U251, C6, P3 and A172 tumor cells to TMZ treatment respectively. E and F: The cell survival of LN18 and HF66 to TMZ was not significantly different in the presence or absence of TNC-1.

Figure 5

TNC-1 cells modulate the sensitivity of Doxorubicin to GBM tumor cells. A, B and C: TNC-1 attenuated the response of U251 and LN18 to Doxorubicin at the concentration of 0.5, 1 and 2 μM. HF66 seemed to be protected by TNC-1 at different concentration of doxorubicin, but not significant (p > 0.05) except at the concentration of 1 μM (p < 0.05). D, E and F: TNC1 has no effect on C6, A172 and P3 tumor cells. However, with increasing concentrations of TMZ more A172 cells were eliminated in the presence of TNC-1 compared to spheroids of tumor cells only.