Fig. 1: ALDH1B1 is required for tumor cell survival in confining space and distant metastasis.

a Schematic of the in vivo metabolic enzyme screen, using sequencing of viable tumor cells from lung-confining capillaries to identify essential genes. The cell isolation procedure is detailed in Supplementary Fig. 1a. b, c Volcano plots show altered genes (P < 0.05 and log₂ sgRNA fold change (FC) ≥ 1 or ≤ −1) in colored blue (pro-survival) or pink (pro-death). Ten candidates satisfied criteria (log₂ FC ≤ −2, good sgRNA count ≥ 5, P < 0.05) are labeled (b) and highlighted in pink (c) (n = 3 independent experiments). d Venn diagram of ten candidate genes associated with lung cancer metastasis (left). Box plots show ALDH1B1 expression in lung cancer patients with (n = 49) and without (n = 47) metastatic recurrence (GEO: GSE37745). The center line represents the median, box limits the upper and lower quartiles, and whiskers the minimum and maximum values (right). e, f Luciferase/mCherry-A549 cells expressing shNT or shALDH1B1 were tail-vein injected into mice. Representative bioluminescence images from n = 5 mice per group (e, left) and quantification (e, right). Representative images of dead tumor cells within capillaries from n = 5 mice per group (white) (f, left) and quantified percentages of dead tumor cells (f, right). DAPI, 4′,6-diamidino-2-phenylindole. g mCherry-expressing A549 cells expressing shNT or shALDH1B1 were tail-vein injected into mice. Representative images show tumor cells (red) localized outside capillaries from n = 5 mice per group (white) (left). The number of intravascular and extravascular tumor cells was quantified (right). h, i A549-luciferase cells expressing shNT or shALDH1B1 were intracardially injected into mice. Representative bioluminescence images from n = 7 mice per group (h, left) and quantification (h, right). Representative H&E-stained brain sections from n = 7 mice per group (i, left) and quantification of tumor areas and numbers (i, right). j Kaplan–Meier survival analysis of mice intracardially injected with A549 cells expressing shNT or shALDH1B1 (n = 6 per group). Data are presented as mean ± SD per mouse (e–i). P-value for each sgRNA was calculated using two-tailed Student’s t test, whereas P-value for each gene was calculated using one-tailed Student’s t test (negative selection) (b, c). P-values were calculated using unpaired two-tailed Student’s t test (d–i) and two-tailed log-rank test (j). NS not significant, Rel. relative. Source data are provided as a Source Data file.