Journal: bioRxiv

Article Title: Aging promotes lung cancer metastasis through epigenetic ATF4 induction

doi: 10.1101/2024.07.03.601209

Figure Lengend Snippet: a. Schematic depicting the fate of isotope-labeled [1,2- 13 C]-D-Glucose via glycolysis (atoms shown as blue circles) or via the pentose phosphate pathway (PPP) (atoms shown as grey circles). (b,c). Fractions of (b) pyruvate and (c) lactate isotopomers derived from [1,2- 13 C]-D-Glucose in KP-Y and KP-O cultures. ( n = 6). d . Schematic depicting the fate of 13 C atoms (dark pink circles) from [U 13 C]-L-Glutamine tracing. e. Mass isotopomer analysis of TCA cycle intermediates glutamate, fumarate, citrate and aspartate derived from 8 h [U 13 C]-L-Glutamine stable isotope tracing in KP-Y and KP-O cultures ( n = 6 and n =4 respectively). f. Mass isotopomer analysis of TCA cycle intermediates glutamate, fumarate, citrate and aspartate derived from 8 h [U 13 C]-L-Glutamine stable isotope tracing in KP-O ( n = 4) and KP-O sh Atf4 .2 ( n = 4). g . Seahorse analysis of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in KP-O and KP-Y primary cultures ( n = 10 and 11 respectively). Data are normalized to protein content. h . Relative viability assessed by CellTiter-Glo (relative luminescence units) of KP-Y and KP-O cultures after 72 h treatment with CB-839. i. Heatmap representing the relative viability values for each indicated primary culture treated with CB-839 and the indicated compounds. All data points are relative to vehicle (veh)-treated controls ( n = 3). j . Relative viability of KP-Y and KP-O cultures in 3D, pretreated for 72 h with 1 µM ISRIB followed by 0.1 µM CB-839 treatment. Cell viability is measured after 48 h of addition of CB-839 ( n = 3). k. Heatmap representing the relative viability values for each indicated primary culture grown in 3D conditions and treated with CB-839 and the indicated compounds. All data points are relative to vehicle-treated controls ( n = 3). (l-m) . Intravenous injections of KP-Y and KP-O primary cultures expressing a GFP-luciferase reporter in mice. Mice were administered 200 mg/kg CB-839 p.o twice/day every other day for the duration of the experiment (n = 3-5 / group). l. Left, quantification of lung metastasis burden as average radiance from in the lungs 12 days after injection. Right, representative lung ex vivo In Vivo Imaging System (IVIS) images (Scale bar, radiance, p s −1 cm −2 sr −1 ). m. Left, hematoxylin& eosin (H&E) quantification of lung metastasis burden (total tumor area/total area). Right, representative H&E-stained lung sections (Scale bar, 1000 µm). (n-o). Subcutaneous injection of KP-Y and KP-O primary cultures expressing a GFP-luciferase reporter in mice. Mice were administered 200 mg/kg CB-839 p.o twice/day every other day once the tumors reached 100 mm 3 in size and for the duration of the experiment ( n = 12, 11, 13, and 9 mice respectively). n. Tumor growth of subcutaneous tumors of mice (KP-Y n = 24, KP-Y + CB-839 n = 22, KP-O n = 26 and KP-O + CB-839 n = 18 tumors). o . H&E quantification of lung metastases foci ( n = 12, 11, 13, and 9 mice respectively). Data presented as mean values ± s.e.m. Statistical significance was assessed by multiple unpaired t-tests ( b,c,e,f,g), two-way ANOVA (h) and one-way ANOVA with Tukey’s multiple comparisons test (j,l,m,o) .

Article Snippet: For CB-839 studies, mice were randomized and subjected to treatment with either 200 mg/kg body weight CB-839 (#HY-12248, MedChemExpress) or vehicle.

Techniques: Labeling, Derivative Assay, Expressing, Luciferase, Injection, Ex Vivo, In Vivo Imaging, Staining

Journal: bioRxiv

Article Title: Aging promotes lung cancer metastasis through epigenetic ATF4 induction

doi: 10.1101/2024.07.03.601209

Figure Lengend Snippet: a. KP-Y and KP-O primary cultures were pre-treated for 72 h with 1 µM ISRIB followed by 0.1 µM CB-839 treatment for another 72 h. Cell viability was assessed by CellTiter-Glo (relative luminescence units). All values were normalized to their respective vehicle treated control ( n = 3). b. KP-Y and KP-O (sg Tom or sg Atf4 ) primary cultures were treated with 0.0625 µM CB-839 for 72 h. Cell viability was assessed by CellTiter-Glo (relative luminescence units). All values were normalized to their respective vehicle treated control ( n = 3). c. Correlation analysis between ATF4 expression and CB-839 sensitivity in human lung adenocarcinoma (LUAD) cell lines ( n = 45) from Dependency Map (DepMap) portal. Primary cell lines are depicted with grey squares, metastatic cell lines with pink circles. (d-f) KP-Y and KP-O primary cultures were subcutaneously injected in mice. Mice were administered 200 mg/kg CB-839 p.o twice/day every other day for the duration of the experiment once the tumors reached 100 mm 3 in size. Complementary results presented in n,o. d. Tumor weight of the tumors formed by KP-Y and KP-O primary cultures after subcutaneous injections e. Quantification of lung metastasis burden (total tumor area/total area). f . Left, hematoxylin and eosin (H&E)-stained lung sections showing metastatic foci in the lungs (black and red arrows). Right, close-up of the area identified by the red arrow in the left panel (Scale bar, 1000 µm and 50 µm). Data presented as mean values ± s.e.m. Statistical significance was assessed by simple linear regression (c) , ordinary one-way ANOVA with Tukey’s multiple comparisons test (a,b,d,e ).

Article Snippet: For CB-839 studies, mice were randomized and subjected to treatment with either 200 mg/kg body weight CB-839 (#HY-12248, MedChemExpress) or vehicle.

Techniques: Control, Expressing, Injection, Staining