Lactate dehydrogenases promote glioblastoma growth and invasion via metabolic symbiosis (2022)
Figure 3

Figure 3: Double knockout of LDHA and LDHB impairs GB lactate metabolism, cell viability and invasion.

A. Western blot analysis of LDHA and LDHB expression in P3 cells transduced with CRISPR-Cas9 lentiviral vectors with scramble sequence (sgControl) or against LDHA (sgLDHA), LDHB (sgLDHB) or both (sgLDHA/B). Knockout (KO) cells were exposed to 21% or 1% O2 for 48 h.

B. Lactate secretion of P3 cells KO for LDHA, LDHB or both, exposed to 21% or 1% (n = 4 independent experiments). Data are represented as mean ± s.d. and analyzed using Two-way ANOVA following by Tukey's multiple comparisons test: sgCont 21% vs sgCont 1%, P=0.002; sgLDHA 21% vs sgLDHA 1%, P<0.0001; sgLDHB 21% vs sgLDHB 1%, P=0.004; sgLDHA/B 21% vs sgLDHA/B 1%, P>0.99; sgCont/sgLDHA/sgLDHB vs sgLDHA/B, P<0.0001.

C. Intracellular lactate level analyzed with a fluorescent biosensor in P3 cells (control, KO for LDHA, LDHB, or both). Left, lactate level monitored in basal condition, followed by sequential exposure to OXPHOS block (5 mM sodium azide), 6 mM oxamate, and MCTs blockers (1 µM AR-C1558585 + 1 mM diclofenac). The response to oxamate and MCTs blockers was used to determine, Middle, the basal lactate level (expressed as biosensor occupancy) and, Right, the lactate production rate (n = 4, 33-44 cells analyzed per condition). Data are represented as mean ± s.e.m (Left) or as mean (Middle and Right) and analyzed using One-way ANOVA following Tukey's multiple comparisons test. Occupancy: sgCont vs sgLDHA, P=0.001; sgCont vs sgLDHB, P=0.005; sgCont vs sgLDHA/B, P<0.0001. Lactate production: sgCont vs sgLDHA, P=0.32; sgCont vs sgLDHB, P=0.01; sgCont vs sgLDHA/B, P<0.0001. See material and methods for analysis details and see also FigEV3D.

D. Left: P3 spheroid growth recorded over one week at 1% O2 (n = 3 independent experiments, one experiment including 16 spheroids per condition). Data are represented as mean and analyzed using Kruskal-Wallis test followed by Dunn's multiple comparisons test. Spheroid growth: sgCont vs sgLDHA, P=0.0003; sgCont vs sgLDHB, P=0.78; sgCont vs sgLDHA/B, P<0.0001. Dead area: sgCont vs sgLDHA, P=0.19; sgCont vs sgLDHB, P<0.0001; sgCont vs sgLDHA/B, P<0.0001. Right: Viability of spheroids at one week, incubated with calcein (green) or ethidium homodimer-1 (red). Scale bar: 200 µm.

E. P3 spheroid invasion in collagen I gel, incubated 24 h at 21% or 0.1% O2. Invasion rate is expressed as fold change from controls (n = 4 independent experiments, one experiment including 6-8 spheroids per condition). Data are represented as mean and analyzed using Two-way ANOVA following by Tukey's multiple comparisons test: sgCont 21% vs sgLDHA 21%, P=0.003; sgCont 0.1% vs sgLDHA 0.1%, P<0.0001; sgCont 21% vs sgLDHB 21%, P<0.0001; sgCont 0.1% vs sgLDHB 0.1%, P<0.0001; sgCont 21% vs sgLDHA/B 21%, P<0.0001; sgCont 0.1% vs sgLDHA/B 0.1%, P<0.0001. Images of representative of control or LDHA/B KO spheroids. Scale bar: 100 µm.

F-G. A first cohort of mice was sacrificed when one mouse reached a limit point, brains was extract, snap frozen in liquid nitrogen, sliced and stained. For the second cohort, each mouse was sacrificed when it reached a limit point allowing the draw of survival curves. Left: Immunofluorescence staining of Nestin (top and middle) and CD31 (bottom) of control and LDHA/B KO P3 tumor section. Scale bars: 2 mm (top), 250 µm (middle) and 100 µm (bottom). Right: Graphs represent tumor core and invasion area of control and LDHA/B KO P3 tumors in mm2 (n = 5 mice per group, average of 5-6 sections per tumor). Data are represented as mean. For F, data are analyzed using One-way ANOVA following by Dunnett's multiple comparisons test. Core: sgCont vs sgLDHA, P=0.48; sgCont vs sgLDHB, P=0.99. Invasion: sgCont vs sgLDHA, P=0.40; sgCont vs sgLDHB, P=0.81. For G, data are analyzed using unpaired t test. Core: sgCont vs sgLDHA/B, P=0.005. Invasion: sgCont vs sgLDHA/B, P=0.01.

H. Kaplan-Meier survival curves of xenotransplanted mice with P3 cells KO for LDHA (yellow), LDHB (green), LDHA/B (red) or control (blue) (n = 8 mice per group). Data are analyzed using Log-rank (Mantel-Cox) test: sgCont vs sgLDHA, P=0.06; sgCont vs sgLDHB, P=0.001; sgCont vs sgLDHA/B, P=0.0007.