Additional file 1 of Inflammasome activation in peritumoral astrocytes is a key player in breast cancer brain metastasis development
Creators: name: Mészáros, Ádám
name: Molnár, Kinga
name: Fazakas, Csilla
name: Nógrádi, Bernát
name: Lüvi, Adél
name: Dudás, Tamás
name: Tiszlavicz, László
name: Farkas, Attila Elek
name: Krizbai, István Adorján
name: Wilhelm, Imola
Publisher: name: figshare
Year: 2024
Subjects: subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Mental Health

description: Additional file 1: Table S1. Antibodies used in the experiments. Table S2. Primers used for PCR. Fig. S1. Absence of detectable NLRP3 and IL-1β expression in astrocytes of the healthy human brain. Representative immunofluorescence micrographs (maximum intensity projections of z-stacks) showing the absence of NLRP3 and IL-1β staining in GFAP-positive cells in healthy human brain sections. Fig. S2. Upregulation of IL-1β in association with TNBC metastases in the mouse brain. Representative immunofluorescence micrographs (maximum intensity projections of z-stacks) showing tumor size-dependent upregulation of IL-1β expression in mouse TNBC brain metastases 7 days after inoculation of the tumor cells. Fig. S3. Proliferation of breast cancer cells in response to IL-1β. a Proliferation of human breast cancer cells in response to 10 ng/ml IL-1β. Graphs represent the average ± SEM (N = 3 independent experiments, each performed in duplicate, 5 different fields of view photographed from each well). *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 compared to control (MDA-TGL or MDA-BrM2, respectively) (ANOVA and Fisher’s LSD post hoc test). b Proliferation of mouse breast cancer cells in response to 10 ng/ml IL-1β. Graphs represent the average ± SEM (N = 3 independent experiments, each performed in duplicate, 5 different fields of view photographed from each well). **P ≤ 0.01, ****P ≤ 0.0001 compared to control (4T1-tdT) (ANOVA and Fisher’s LSD post hoc test). Fig. S4. Proliferation of breast cancer cells in response to astrocyte-conditioned media. a Representative phase contrast images showing the proliferation of human breast cancer cells cultured in human astrocyte-conditioned media. b Representative phase contrast images showing the proliferation of mouse breast cancer cells cultured in mouse astrocyte-conditioned media. Quantitative analyses are shown in Additional file 1: Fig. S5. Fig. S5. Proliferation of breast cancer cells in response to astrocyte-secreted factors (quantitative data). a Proliferation of human breast cancer cells cultured in human astrocyte-conditioned media. Graphs represent the average ± SEM (N = 3 independent experiments, each performed in duplicate, 5 different fields of view photographed from each well). **P ≤ 0.01, ****P ≤ 0.0001 compared to the same day’s control (MDA-TGL or MDA-BrM2, respectively) (ANOVA and Fisher’s LSD post hoc test). b Proliferation of mouse breast cancer cells cultured in mouse astrocyte-conditioned media. Graphs represent the average ± SEM (N = 3 independent experiments, each performed in duplicate, 5 different fields of view photographed from each well). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 compared to the same day’s control (4T1-tdT), ####P ≤ 0.0001 compared to the same day’s “4T1-tdT + act. MA-CM” (ANOVA and Fisher’s LSD post hoc test). Fig. S6. NLRP3 inhibition-induced changes in the expression of NLRP3 inflammasome-associated genes in astrocytes. Expression of NLRP3 and IL1B genes in human astrocytes cultured for 24 h in human TNBC cell-conditioned medium in the presence or absence of 1 μM MCC950. Graphs represent the fold change (normalized to GAPDH), average ± SEM (N = 3 independent experiments, each performed in triplicate). **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 compared to control (HA), #P ≤ 0.05, ####P ≤ 0.0001 compared to HA + MDA-TGL CM or HA + BrM2 CM, respectively (ANOVA and Fisher’s LSD post hoc test). Fig. S7. NLRP3 inhibition-induced reduction in gliosis associated with brain metastasis. a Schematic representation of the in vivo model (details presented in the “Materials and methods” section). b Percentage of GFAP-positive pixels compared to all pixels in brain sections of mice inoculated with 4T1-tdT TNBC cells (tumor-injected) or Krebs–Ringer solution (control) and treated with vehicle (DMSO in PBS) or 10 mg/kg MCC950 7 days after the injection of the tumor cells. Graphs represent the average ± SEM (N = 3 independent experiments, n = 8 sections/animal). **P ≤ 0.01 compared to “control, vehicle” (ANOVA and Fisher’s LSD post hoc test). c Representative immunofluorescence micrographs (maximum intensity projections of z-stacks) showing a reduction in peritumoral Iba1 expression in response to systemic MCC950 treatment in mouse TNBC brain metastases 7 days after inoculation of the tumor cells. d Ratio of Iba1-positive pixels in MCC950-treated animals compared to all pixels. Graphs represent the average ± SEM (N = 3 independent experiments, n = 6 sections/animal, 8 ROIs/6 sections). **P ≤ 0.01 (unpaired Student’s t test).