Spatial epitope barcoding reveals subclonal tumor patch behaviors

Abstract

Intratumoral variability is a seminal feature of human tumors contributing to tumor progression and response to treatment. Current technologies are unsuitable to accurately track phenotypes and subclonal evolution within tumors, especially in response to genetic manipulations. Here, we developed epitope combinatorial tags (EpicTags), which we coupled to multiplexed ion beam imaging (EpicMIBI) for in situ tracking of barcodes within tissue microenvironments. Using this platform, we dissected the spatial component of cell lineages and phenotypes in a xenograft model of small-cell lung cancer. We observed emergent properties from mixed clones leading to the preferential expansion of subclonal patches for both neuroendocrine and non-neuroendocrine cancer cell states in this model. In tumors harboring a fraction of PTEN-deficient cancer cells, we uncovered a non-autonomous increase of subclonal patch size in PTEN wild-type cancer cells. EpicMIBI can facilitate in situ interrogation of cell-intrinsic and cell-extrinsic processes involved in intratumoral heterogeneity.

A multiplexed in vivo approach to identify driver genes in small cell lung cancer

“…We also investigated TSC2, the obligate partner of TSC1 within the tuberous sclerosis complex (Nellist et al, 1999). We generated populations of NCI-H82 SCLC cells independently expressing six unique combinations of short epitopes (epitope-combinatorial-tag, or Epi (Rovira-Clave et al, 2021)) and performed Cas9-RNA ribonucleoprotein nucleofection to generate TSC1 -KO (in Epi1 cells), TSC2 -KO (in Epi2 cells), and wild-type control cell lines (Epi3-6 cells, which received non-targeting Cas9-sgRNA ribonucleoprotein) ( Figures 4E and S10A ). We then pooled Epi1∼6-tagged NCI-H82 cells, cultured them for 21 days, and measured the relative change in epitope-tag representation using cytometry by time of flight (CyTOF) ( Figure 4E ).…”

“…Control and targeting sgRNAs were generated as previously described (Rovira-Clave et al, 2021). Briefly, for each region of interest, three sgRNAs were designed to hybridize approximately 150 bases apart, and 100 pmol of each sgRNA was resuspended in Tris-EDTA (Synthego) and mixed at a 1:1:1 ratio.…”

Single Cell lineage Tracing Identifies Cancer Testis Antigens as Mediators of Chemoresistance in Small Cell Lung Cancer

“…Single-cell RNA sequencing (scRNA-seq) has radically transformed our study and understanding of ITH. Indeed, scRNA-seq has generated important insights into SCLC tumors dynamics (14, 21, 28, 29), and combined with other genetic profiling has been used to generate pseudotime maps of ITH in response to growth and chemotherapy (13-15, 21, 23, 26, 30). While incredibly useful, pseudotime trajectories do not allow for the pinpointing of the populations critical for these tumor dynamics, rather phenotypic characteristics are inferred from the populations identified in the screening.…”

“…It has now been used to understand cellular lineages and heterogeneity in a number of cancer types including glioblastoma, breast, non-small cell lung cancer, leukemia, and melanoma (32, 35, 37–45). Currently, genetic barcoding has been used to understand spatial architecture in SCLC xenografts and the role of PTEN in regulating these clonal structures (30), an expanded use of barcoding techniques in both SCLC mouse models and xenografts will be required to shed light on broader SCLC growth dynamics and chemoresistance. While the use of genetic barcoding to barcode tumors in situ, by the use of genetically engineered animal models of cancer is a newer advance on the genetic barcoding technology (28, 35), and its utility in SCLC is still emerging.…”

Editorial: Defining the Spatial Organization of Immune Responses to Cancer and Viruses In Situ

“…Orthogonal interrogation with synergistic tools, such as RNA quantification methods including spatial transcriptomics, single-cell RNA sequencing, and other advanced techniques are needed to precisely define the genetic and protein topographies of human tissues. The development of multi-omic measurements in situ is a key fundamental advancement in this regard, including the quantification of metabolic states ( 21 ), nucleic acids and proteins ( 12 , 23 , 25 ), clonality ( 26 ), and epigenetic states ( 27 ). The continued advancement of computational methods is also vital for multi-scalar inferential analysis across different measurement modalities ( 28 ).…”