P07.16.B A NOVEL METHOD ENABLING THE DETECTION OF INTRACELLULAR DRUGS BY SEQUENCING ON SINGLE-CELL LEVEL IN GLIOBLASTOMA

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Abstract BACKGROUND Glioblastoma is an aggressive and incurable primary brain tumor, primarily due to its resistance to standard therapies and imminent recurrence. The increasing importance of Single-Cell RNA Sequencing (scRNA-Seq) in deciphering tumor heterogeneity of glioblastoma (GB) unravels a further dimension to study its resistance to therapy: Whether an applied drug was taken up by a target cell or not, if so at what concentration, and what are the biological implications thereof for the development of drug resistance. In this proof-of-concept study, we used temozolomide (TMZ) as a model drug to detect drug uptake at cellular level resolution. MATERIAL AND METHODS TMZ conjugated with a barcoded DNA oligonucleotide (via a VC-PAB linker = TMZ-Oligo) was applied to LN-229, NCH421k, NCH705 and 671/12 GB cells at their IC-20 (inhibition concertation that produces a 20% viability loss). We applied an NT-Oligonucleotide (GFP) along with the IC-20 TMZ concentration to LN229 cells (GFP-TMZ). For a second control, GB cells were incubated with culture medium (BLANK). scRNA-Seq was performed using the 10x Genomics Chromium Single Cell Gene Expression assay kit, primers capturing the oligonucleotide sequence were added to the workflow. RESULTS 27152 BLANK, 7044 GFP-TMZ, and 5627 TMZ-Oligo cells were analyzed. In TMZ-Oligo cells, a variable number of TMZ-Oligo reads (maximum 2500 reads, median 800 reads) was detected, indicating technical success of the assay and corresponding to variable intracellular TMZ concentrations. On Uniform Manifold Approximation and Projection (UMAP) plots samples clustered into two main cell populations: One with BLANK single cells, along with GFP-TMZ and TMZ-Oligo sub-populations, corresponding to untreated cells, and treated resistant cells = unchanged. Another cluster with congruent abundance of GFP-TMZ and TMZ-Oligo cells was observed, suggesting TMZ-Oligo did not affect the efficacy of temozolomide in glioblastoma cells on a transcriptomic level. Differential analyses comparing changed and unchanged GB cells revealed significantly higher expression of mitochondrial proteins in changed cells. Comparing BLANK to GFP-TMZ and TMZ-Oligo cells in unchanged cells revealed a significant upregulation of MALAT1 in treated but unchanged cells, a long non-coding RNA known to convey TMZ resistance in GB. CONCLUSION We present a novel small molecule DNA barcoding technology that enables the quantification of drugs within single-cells, with the potential to elucidate mechanisms of drug resistance in glioblastoma at unprecedented spatial resolution. In addition, it can be re-purposed to study further drugs in more complex models and even other tumor entities, conveying a promising technology to decipher drug efficacy and resistance to therapy at single-cell level.

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