Combining SHP2 inhibition with CX3CR1 inhibition shrinks tumors and normalizes the tumor microenvironment
NF1 loss increases RAS-MAPK signaling in myeloid cell dominant benign nerve tumors that cause significant clinical burden, in which MEK inhibitor therapy is only partially effective. We studied SHP2 inhibition, which targets RAS-MAPK and has additional immunomodulatory effects, in a validated preclinical model system and found that both MEK and SHP inhibitors shrank tumor volume and tumor immune compartments. However, neither inhibitor was durable or efficacious in all mice. To identify druggable targets, paraspinal tumors from mice treated for 30 days with either vehicle, MEK inhibitor, or SHP2 inhibitor were collected, dissociated to achieve single-cell suspension and transcriptionally profiled. Overall design: Nf flox/flox;DhhCre mice were treated for 30 days with 1.5 mg/kg/day of the MEK inhibitor PD-032590, 10mg/kg/day of the SHP2 inhibitor RMC-4550, or vehicle control. Mice were anesthetized with isoflurane, perfused with ice cold phosphate-buffered saline (PBS) lacking calcium and magnesium. Paraspinal tumors were collected within 5 minutes of death and stored in L-15 medium until tissue was cut into 1-mm3 pieces and placed into dissociation medium in 50 mL tubes containing 20 mL L-15 medium, 10 mg collagenase type I, and 50 mg Dispase II in a 37°C incubator for 2 hours, with 170 rpm shaking. The dissociation process was stopped by adding 30 mL DMEM containing 10% FBS. Samples were centrifuged at 800g for 5 minutes at 4°C. Supernatants were removed and cell pellets were resuspended into 50 mL of 1× PBS containing 0.1% BSA. Suspensions were sequentially filtered with 70 µm, 40 µm, and 20 µm cell strainers and centrifuged at 600g for 5 minutes at 4°C. Pellets were resuspended into 100 µL of 1× PBS/0.1% BSA. Trypan blue–negative viable cells were counted to determine the cell concentration for droplet-based scRNA-seq.