Figure 2. The brain tumor microenvironment contributes to increased de novo lipid synthesis in breast tumors.

a) Schematic workflow used for organotypic slice culture experiments. Organotypic slice cultures were prepared from breast cancer tumors isolated from mouse orthotopic brain (permitted to reach a volume of ~60 mm³) or MFP (permitted to reach a volume of ~100–120 mm³). Slices were cultured for 3 days in vitro (DIV) prior to their use in an experiment.

b) Immunofluorescence (IF) staining of organotypic slice cultures prepared from breast cancer tumors isolated from orthotopic brain or MFP lesions established in NSG female mice from GFP-positive BT474 cells. Staining for α-smooth muscle actin (SMA) was performed on slices from MFP tumors, and staining for glial fibrillary acidic protein (GFAP) was performed on slices from brain tumors. DAPI staining and GFP fluorescence from tumor cells is also assessed as indicated. (scale bar = 100 μM).

c, d) The percent labeling of even isotopomers of saponified palmitate measured by gas chromatography–mass spectrometry (GCMS) in organotypic slice cultures exposed to ¹³C-glucose for 72 hours. Organotypic slice cultures were prepared from BT474 (c) or MDAMB361 (d) brain and MFP tumors established in Nude female mice. Slices cultures analyzed were obtained from 2–3 independent tumors from different mice. (BT474 brain tumor slices, n=5; BT474 MFP tumor slices, n =4; MDAMB361 brain tumor slices, n=9; MDAMB361 MFP tumor slices, n=8).

e) Western blot assessment of FASN expression in parental BT474 cells never exposed to the brain microenvironment (BT474 Parental), and in multiple independent cell line isolates from orthotopic brain BT474 tumors established in NSG mice and cultured for 10 days in vitro (BT474 Brain 10DIV). β-actin expression was also assessed as a loading control.

f) The percent labeling of even isotopomers of saponified palmitate measured by GCMS from parental BT474 cells, and cell line isolates from orthotopic BT474 brain tumors described in (e), that were cultured in vitro with ¹³C-glucose for 48 hours. Two independent isolates (BT474 Brain 1, BT474 Brain 2) were analyzed. (n=3 cell culture biological replicates).

Data in panels c, d and f represent means ± SEM.