Hidden RNA profiles revealed by malignant cell fraction-informed deconvolution

To understand cancer biology, it is crucial to unravel malignant- and microenvironmental cell states. Deconvolution of the bulk RNA profile from tumors can provide this insight, but is severely impaired by interpatient heterogeneity of the malignant cells. Bayesian statistics enables to inform RNA-deconvolution with malignant cell fraction estimates from e.g. DNA sequencing. We developed cell fraction-informed deconvolution, and evaluated this experimentally using 46 bulk RNA profiles from blood with 19 CyTOF determined cell fractions from this repository. Blood was drawn from patients undergoing surgery for glioblastoma, meningioma or astrocytoma, as well as healthy donors. PBMCs were isolated using gradient density centrifugation with lymphoprep (Axis-Shield). Multiple vials of PBMCs were cryopreserved per donor, which were partly used in a previous CyTOF study (Dusowa et al. Front Immunol. 2024). 48 Donors for which CyTOF data was available were included in the present study. In CyTOF only live cells are phenotyped, therefore only live cells were used for RNA isolation to obtain a matching dataset. First PBMCs were thawed through dropwise addition of IMDM (Gibco) with 10% fetal calf serum (FCS, Biowest), 100 IU Penicillin and 100 IU Streptomycin (Lonza) at 37 °C. Thawed cells were centrifuged at 500 g for 8 min at 4 °C. Cell pellets were taken up in cold sterile-filtered 0.1% BSA (Roche) in PBS (Fresenius Kabi) and again centrifuged. Washed cells were stained with ice cold 7-AAD in 0.1% BSA in PBS. Live cells were sorted at 5 °C using a FACS Aria Fusion (BD Biosciences) with IMDM with 20% FCS, 100 IU Penicillin and 100 IU Streptomycin as collection medium. After sorting 0.7-1 million live cells collection tubes were immediately centrifuged for 8 min at 500 g and 4 °C. The supernatant was poured off, the remaining liquid was used to resuspend the cell pellet before addition of TRIzol™ Reagent (Invitrogen; Thermo Fisher Scientific). RNA isolation was performed according to manufacturer’s protocol. Remaining TRIzol™ contaminations were removed using the buthanol extraction protocol described by Krebs et al (2009). RNA dissolved in water was mixed with water-saturated 1-buthanol (Sigma-Aldrich), briefly centrifuged, followed by removal of the upper organic layer. After repeating the previous step once with 1-buthanol, these steps were performed twice with water-saturated diethyl ether (Riedel-de Haën). The final diethyl ether layer was left to evaporate in a fume hood for 15 minutes. RNA quality was assessed using the Agilent RNA 6000 Pico Kit and the 2100 Bioanalyzer (Agilent Technologies). Samples included for library preparation had an RNA integrity number (RIN) value of at least 6.9 and a yield of at least 0.72 µg.