In vivo growth of subclones derived from Lewis lung carcinoma is determined by the tumor microenvironment

Abstract

Heterogeneity is a fundamental feature of human tumors and plays a major role in drug resistance and disease progression. In the present study, we selected single-cell-derived cell lines (SCDCLs) derived from Lewis lung carcinoma (LLC1) cells to investigate tumorigenesis and heterogeneity. SCDCLs were generated using limiting dilution. Five SCDCLs were subcutaneously injected into wild-type C57BL/6N mice; however, they displayed significant differences in tumor growth. Subclone SCC1 grew the fastest in vivo, whereas it grew slower in vitro. The growth pattern of SCC2 was the opposite to that of SCC1. Genetic differences in these two subclones showed marked differences in cell adhesion and proliferation. Pathway enrichment results indicate that signal transduction and immune system responses were the most significantly altered functional categories in SCC2 cells compared to those in SCC1 cells in vitro. The number and activation of CD3⁺ and CD8⁺ T cells and NK cells in the tumor tissue of tumor-bearing mice inoculated with SCC2 were significantly higher, whereas those of myeloid cells were significantly lower, than those in the SCC1 and LLC1 groups. Our results suggest that the in vivo growth of two subclones derived from LLC1 was determined by the tumor microenvironment rather than their intrinsic proliferative cell characteristics.

Keywords: Cancer cell line; Lewis lung carcinoma; heterogeneity; immunological characterization; single cell-derived cell lines.

Figure 1

Clonal populations derived from the LLC1 cell line vary in population doubling time and dose response to cytotoxic drugs. A. Doubling time of LLC1 and clonal cell lines (SCDCLs) grown for 5 d. Doubling time was calculated using http://www.doublingtime.com/compute.php. Data shown as mean ± SEM from three independent experiments; p-values from one-way ANOVA, corrected for multiple comparisons using Dunnett’s method. B. In vivo tumor growth in C57BL/6N mice inoculated with the LLC1 cell line and five SCDCLs (n = 5). Data are mean ± SEM. p-values from two-way ANOVA. C. Expression of PD-L1 in SCDCLs after cytotoxic drug treatment. Each experiment was performed in triplicate and repeated three times. p-values from one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

WES analysis of LLC1, SCC1, and SCC2 cells. A. Venn diagram of unique and shared genetic variants. B. Number of somatic SNVs and the distribution of different mutation types. C. Mutation spectrum of LLC1 and the single-cell clonal lines SCC1 and SCC2. Color codes represent the fraction of different base substitutions. D. Number of somatic SNVs and the distribution of different mutation types. E. Circos plot of all detected mutations comparing LLC1 to SCC2 cells.

Figure 3

Transcriptomic heterogeneity in LLC1, SCC1, and SCC2 cells. A. Statistical histogram of differentially expressed genes. Up (red) and Down (blue) show the number of up- and downregulated genes, respectively, with significant differences. B. Common and unique differentially expressed genes (DEGs) among different comparison groups. C. Enriched top 30 GO terms when comparing SCC2 to SCC1 cells (total) by GO enrichment analysis; the y-axis shows the log10-transformed p-value. D. Signaling pathway enrichment of DEGs at KEGG Level 2 (KEGG Pathway Classification) in SCC2 compared to that in SCC1 cells.

Figure 4

Flow cytometry analysis of immune cell populations in the tumor microenvironment of LLC1, SCC1, and SCC2 tumor bearing mice. Representative plots of CD3⁺ T (A), CD3⁺CD4⁺ T (C), CD3⁺CD8⁺ T (E), and MDSC (CD11b⁺Gr-1⁺) (G) cell frequency among CD45-positive cells. Pooled data of CD3⁺ T (B), CD3⁺CD4⁺ T (D), CD3⁺CD8⁺ T (F), and MDSC (CD11b⁺Gr-1⁺) (H) cell frequency among CD45-positive cells (n = 5 per group, one-way ANOVA followed by Tukey’s multiple comparison test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 5

Representative plots of PD-1⁺ and Tim-3⁺PD-1⁺ (A) among CD8⁺ T cells. Pooled data of PD-1⁺ (B) and Tim-3⁺PD-1⁺ (C) among CD8⁺ T cells; and PD-1⁺ (D) and Tim-3⁺PD-1⁺ (E) among CD4⁺ T cells. Pooled data of NK cell frequency among CD45-positive cells (F). Pooled data of PD-1⁺ (G) and Tim-3⁺PD-1⁺ (H) among NK cells; PD-L1 frequency among CD45-positive cells (I) (one-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 6

SCDCLs show poor response to anti-PD-1 therapy. A. Selected transcription factors were validated in tumors by quantitative RT-PCR. B-D. SCDCLs have poor response to anti-PD-1 therapy.