Single-Cell-Derived Primary Rectal Carcinoma Cell Lines Reflect Intratumor Heterogeneity Associated with Treatment Response

Abstract

Purpose: The standard treatment of patients with locally advanced rectal cancer consists of preoperative chemoradiotherapy (CRT) followed by surgery. However, the response of individual tumors to CRT is extremely diverse, presenting a clinical dilemma. This broad variability in treatment response is likely attributable to intratumor heterogeneity (ITH).

Experimental design: We addressed the impact of ITH on response to CRT by establishing single-cell-derived cell lines (SCDCL) from a treatment-naïve rectal cancer biopsy after xenografting.

Results: Individual SCDCLs derived from the same tumor responded profoundly different to CRT in vitro. Clonal reconstruction of the tumor and derived cell lines based on whole-exome sequencing revealed nine separate clusters with distinct proportions in the SCDCLs. Missense mutations in SV2A and ZWINT were clonal in the resistant SCDCL, but not detected in the sensitive SCDCL. Single-cell genetic analysis by multiplex FISH revealed the expansion of a clone with a loss of PIK3CA in the resistant SCDCL. Gene expression profiling by tRNA-sequencing identified the activation of the Wnt, Akt, and Hedgehog signaling pathways in the resistant SCDCLs. Wnt pathway activation in the resistant SCDCLs was confirmed using a reporter assay.

Conclusions: Our model system of patient-derived SCDCLs provides evidence for the critical role of ITH for treatment response in patients with rectal cancer and shows that distinct genetic aberration profiles are associated with treatment response. We identified specific pathways as the molecular basis of treatment response of individual clones, which could be targeted in resistant subclones of a heterogenous tumor.

Figure 1.

Tumor biopsy, clinical history, and cell line establishment. A, Clinical history of the rectal cancer study patient. B, Experimental workflow for establishment of SCDCLs. C, H&E staining of representative sections of the primary tumor biopsy (left panel) and the derived xenograft (right panel). D, CK20 and vimentin IHC staining of representative sections of the primary tumor biopsy (left panel) and the derived xenograft (right panel). E, PAS staining of representative sections of the primary tumor biopsy (left panel) and the derived xenograft (right panel). F, Morphology of the derived parental cell population. G, CK20 immunofluorescence of the established parental cell population. H, SKY analysis that is representative of the cytogenetic aberration profile of the parental population. Note the relative copy number loss of chromosome 18 and chromosome arm 17p and copy number increases of chromosome 20.

Figure 2.

Treatment response of SCDCLs. A, Survival fraction after irradiation with 4 Gy (SF4) of the parental cell population (passages 16 and 19) and 16 individual SCDCLs. B, Dose response of the parental cell population, the most resistant (1G10) SCDCL, and the most sensitive (2F4) SCDCL to irradiation alone (****, P< 0.0001, two-way ANOVA). C, Dose response of the parental cell population, the most resistant (1G10) SCDCL, and the most sensitive (2F4) SCDCL to irradiation in combination with 3 μmol/L 5-FU (***, P = 0.0003, two-way ANOVA) D, Dose response of the parental cell population, the most resistant (1G10) SCDCL, and the most sensitive (2F4) SCDCL to 5-FU alone. E, Growth curves of the parental cell line, SCDCL 1G10, and SCDCL 2F4. Each experiment was performed in triplicates and repeated three times.

Figure 3.

PyClone analysis of the tumor biopsy and derived cell lines. A, Violin plots showing the cellular prevalence of the identified PyClone clusters and selected associated mutations for the tumor biopsy, parental cell population, and most sensitive (2F4) and resistant (1G10) SCDCL. Cluster 0 is present at high frequency in all samples, while cluster 7 is absent from the sensitive SCDCL 2F4 and highly enriched in the resistant SCDCL 1G10 compared with both parental cell population and tumor biopsy. B, Cellular fractions of missense and nonsense mutations in the tumor biopsy, the parental cell population, and SCDCLs 1G10 and 2F4 that are most prevalent in the resistant SCDCL 1G10 or sensitive SCDCL 2F4, respectively.

Figure 4.

miFISH analysis of the tumor biopsy and the derived cell lines. Left, color charts of clonal imbalances based on relative gains and losses of the primary tumor biopsy (A), parental cell population (B), resistant SCDCL 1G10 (C), and sensitive SCDCL 2F4 (D). The color scheme is as follows: green, gains; red, losses; blue, unchanged relative to ploidy. The “Locus” column depicts the target gene for each probe. Each vertical line discerns specific signal patterns of the clones and indicates their prevalence in the population. Right, patterns of clonal evolution of the primary tumor biopsy (A), parental cell population (B), resistant SCDCL 1G10 (C), and sensitive SCDCL 2F4 (D). We show the percentage of dominant imbalance clones and their derivations with the most diploid clones left and progressive aneuploidy to the right. Clones derived by a single gain or loss change are connected by arrows.

Figure 5.

tRNA-seq of the parental cell population and SCDCLs. A, PCA analysis of tRNA-seq data showing a distinct clustering of the parental cell population (black); the three most resistant SCDCLs 1G10, 2D2, and 2G4 (each in triplicates; red); and the three most sensitive SCDCLs (2F4, 1D2, and 2F1; green). B, Heatmap of signaling pathway enrichment in the three most resistant SCDCLs (1G10, 2D2, and 2G4) compared with the three most sensitive SCDCLs (2F4, 1D2, and 2F1). C, RNA expression of TCF7L2 in the three most resistant SCDCLs (1G10, 2D2, and 2G4) and the three most sensitive SCDCLs (2F4, 1D2, and 2F1). D, Wnt reporter assay in the resistant (1G10) and sensitive (2F4) SCDCL and the parental cell population. E, GSEA normalized enrichment scores (NES) that correlate with resistance. Seven pathways exhibited NES that were significant in at least one GSEA comparison, and correlated with the resistance phenotype.