Fig. 4: LH2b inactivation inhibits orthotopic lung tumor growth and metastasis.

a Quantification of glucosylgalactosyl-DHLNL (GG-DHLNL) in human LUAD and adjacent normal lung samples (n = 11 of each). Frozen tissue specimens obtained from Houston Methodist Hospital. b–d LH2a and LH2b mRNA levels in tissue specimens (dots) from The Cancer Genome Atlas cohort. LUAD (b, n = 516), lung squamous cell carcinoma (c, n = 501), and normal lung (d, n = 110). e Reverse transcriptase PCR (RT-PCR) analysis of LH2a and LH2b mRNA levels in murine K-ras/Tp53-mutant LUAD cell lines. L32 included as loading control. f Quantitative RT-PCR analysis of FOX2 mRNA levels in 344SQ cells transfected with control (siCTL) or FOX2 (#1 or #2) siRNAs. Values expressed relative to siCTL-transfected cells (n = 3). g RT-PCR analysis of LH2a and LH2b mRNA levels in cells described in (f). h Numbers of visible primary lung tumors (left plot, n = 9 or 10) and metastases to mediastinal nodes (middle plot, n = 9 or 10) or contralateral lung (right plot, n = 9 or 10) per mouse (dots). Mice were injected intra-thoracically with parental (WT) or CRISPR/Cas-9-edited 344SQ cells with LH2 exon 13 A deletion. i Subcutaneous tumor size in mice (dots, n = 10). Tumor area (length times width) determined at time of sacrifice, which was 3 weeks, 6 weeks and 4 weeks after injection of parental, Δexon 13 A, and L735D 344SQ cells, respectively. j Number of invasion projections per multicellular aggregate (dot) (n = 33 or 40 or 46 or 47). Multicellular aggregates were created in laser-ablated microwells, embedded in collagen, cultured for 3 days, stained, and imaged. Length of each invasive projection (dot) (k, n = 314 or 398 or 562 or 672). Mutants include exon 13 A deletion (Δ13 A) or loss of dimerization (L735D). Projection length in Δexon 13 A aggregates was less than that of parental cells and L735D mutants. l Numbers of visible primary lung tumors (left plot, n = 9 or 10) and metastases to mediastinal nodes (middle plot, n = 9 or 10) or contralateral lung (right plot, n = 9 or 10) per mouse (dots). Parental (WT) or CRISPR/Cas-9-edited H358 cells with LH2 exon 13A deletion. Results are mean values (±S.D.) from replicate biological samples. Error bars indicate ±S.D. p values, 2-tailed Student’s t test.