Tracing Intratumoral Cellular Heterogeneity using genetic barcoding in small cell lung cancer - PROJECT SUMMARY
Tumors are often highly heterogeneous, with a number of functionally different cells existing within a single
tumor, giving some cells the ability to initiate new tumors, metastasize, and/or become resistant to
chemotherapy. Small Cell Lung Cancer (SCLC) is often a heterogeneous tumor, and is characterized by a low
5- year survival rate, high metastatic rates, and rapid acquisition of chemoresistance. The heterogeneous nature
of this tumor makes it difficult to study and to treat, as current attempts to understand tumor dynamics use a bulk
approach that averages the contribution of all cells within a tumor. Commonly upregulated in cancer, the core
pluripotency networks of Sox2, Oct4, and Nanog have a profound effect as master regulators of pluripotency,
and their expression in cancers leads to poor clinical outcomes. Sox2 in particular is often highly expressed in
SCLC, and is a marker of lung progenitor cells. Expression of these factors can reprogram cells to an induced
pluripotent state, and may contribute to enhanced plasticity in cancer. Therefore, I hypothesize that the
dysregulation of pluripotency factors imparts a high degree of cellular plasticity SCLC, driving its growth,
metastasis, and chemoresistance. I will test this hypothesis in two aims using a cellular barcoding lineage tracing
approach combined with single-cell RNA sequencing (scRNAseq). Aim 1 seeks to trace the contribution of
individual cells towards tumor growth and chemoresistance in xenograft models of SCLC. I will use a lentivirus
that will insert a unique genetic barcode, as well as a GFP tag in to SCLC cell lines that represent the two most
common molecular subtypes of SCLC. The cells will be sampled prior to xenografting, and will then be
xenografted in to nude mice. At the end of this aim, the endpoint xenograft, as well as the early tumor sample
will be evaluated by scRNAseq, which will provide data to evaluate gene networks responsible for tumor initiation,
progression, and chemoresistance. I will validate identified candidates by knockdown experiments in cell culture
to elucidate the role of the gene candidates, with a particular focus on progenitor and stem pathways. Aim 2 will
describe the genes responsible for growth, metastasis, and chemoresistance in a mouse model of SCLC. The
well-characterized SCLC Rblox/lox, p53lox/lox, p130lox/lox mouse model, bread to an H11lox-stop-lox-Cas9 mouse will be
used in combination with an adeno-associated virus containing a gRNA to Rosa26, and Rosa26 homology arms
flanking a GFP and barcode sequence. Shortly after tumor initiation by intratracheal Cre expression, the
barcoding-AAV will be injected into the trachea in order to barcode the newly-formed tumors. After tumors grow,
metastasize, and acquire chemoresistance, they will be dissected and primary tumors, metastases, and early
tumor samples collected by intratracheal brushing will undergo scRNA-seq to identify the genes responsible for
growth, metastasis, and chemoresistance. The successful completion of this work will identify the genes
responsible for tumor dynamics, develop the in vivo cellular barcoding approach and lead to the identification of
potential therapeutic targets.