Formation of malignant, metastatic small cell lung cancers through overproduction of cMYC protein in TP53 and RB1 depleted pulmonary neuroendocrine cells derived from human embryonic stem cells

We recently described our initial efforts to develop a model for small cell lung cancer (SCLC) derived from human embryonic stem cells (hESCs) that were differentiated to form pulmonary neuroendocrine cells (PNECs), a putative cell of origin for neuroendocrine-positive SCLC. Although reduced expression of the tumor suppressor genes TP53 and RB1 allowed the induced PNECs to form subcutaneous growths in immune-deficient mice, the tumors did not display the aggressive characteristics of SCLC seen in human patients. Here we report that the additional, doxycycline-regulated expression of a transgene encoding wild-type or mutant cMYC protein promotes rapid growth, invasion, and metastasis of these hESC-derived cells after injection into the renal capsule. Similar to others, we find that the addition of cMYC encourages the formation of the SCLC-N subtype, marked by high levels of NEUROD1 RNA. Using paired primary and metastatic samples for RNA sequencing, we observe that the subtype of SCLC does not change upon metastatic spread and that production of NEUROD1 is maintained. We also describe histological features of these malignant, SCLC-like tumors derived from hESCs and discuss potential uses of this model in efforts to control and better understand this recalcitrant neoplasm. Overall design: Single-cell capture, reverse transcription, cell lysis, and library preparation were carried out by the WCM Epigenomics Core Facility using Chromium Single Cell 3' v3 kits according to the manufacturer's protocol (10x Genomics, USA). Single cell suspenions of RUES2-dervied RPM and RPM (T58A) day 50 cultures were prepared by incubating wells in 0.05% Trypsin/0.02% EDTA for 10-15 min. Digestions were then quenched with excess 0.1% BSA/PBS buffer and passed through 40µm mesh strainer. EGFP+ singlets were sorted at the WCM Flow Cytometry Core Facility on a BD Influx or Sony MA900 cell sorter. Each RUES2-dervied RPM and RPM(T58A) day 50 culture pools were isolated from 3 wells of a 12-well plate across two biological replicates, targeting ~50,000 viable, EGFP+ viable cells per replicate. Cell counts were then adjusted to 6000-9000 cells/50uL in PBS by Trypan Blue exclusion to achieve an estimated capture of 4000-5000 cells. Sequencing was performed by the WCM Genomics Resources Core Facility on a HiSeq 2500 (Illumina, paired-end protocol with 26 base pairs for read 1 and 98 bases for read 2). Single cell RNA sequencing from RP cultures were obtained from previous experiments (5). Transcript abundance was quantified by mapping sequencing reads to a custom human transcriptome reference (GENCODE v42, hg38) that included EGFP and was performed using simpleaf/alevin-fry (12).