Journal: Cancer Research

Article Title: Targeting the Dependence on PIK3C3-mTORC1 Signaling in Dormancy-Prone Breast Cancer Cells Blunts Metastasis Initiation

doi: 10.1158/0008-5472.CAN-23-2654

Figure Lengend Snippet: Genome-wide knockout CRISPR screens define the common and differential fitness genes in the 4T1 and 4T07 cells. A, Schematic outline for the performed screens’ pipeline. B, Enumeration of the revealed cell line–specific and common (core) fitness genes. C, Ranked differential gene fitness score between the 4T1 and 4T07 cells. A value >0 denotes a 4T1-specific fitness gene; a value <0 denotes a 4T07-specific fitness gene. D, Schematic representation integrating the 4T1-specific fitness genes (green) and proliferation suppressor genes (positively selected genes; gray) in the canonical PI3K-AKT pathway. E and F, Gene set enrichment analysis comparing the enrichment of genes previously found to be induced by AKT upregulation ( E ) or PTEN downregulation in the two cell lines ( F ). NES, normalized enrichment scale.

Article Snippet: For experiments in immunocompetent mice using the 4T07 model, 2 × 10 5 4T07 cells were resuspended in 50 μL of sterile PBS and injected into the fat pad of the fourth mammary gland of 8- to 10-week-old BALB/C mice (The Jackson Laboratory, #0651).

Techniques: Genome Wide, Knock-Out, CRISPR

Journal: Cancer Research

Article Title: Targeting the Dependence on PIK3C3-mTORC1 Signaling in Dormancy-Prone Breast Cancer Cells Blunts Metastasis Initiation

doi: 10.1158/0008-5472.CAN-23-2654

Figure Lengend Snippet: Pik3ca and Pik3c3 are differential fitness genes for the 4T1 and 4T07 cells. A, Schematic representation of the two-color competition assay. B, Representative images showing 4T1 cells expressing either Pik3ca -gRNA or Rosa26 -gRNA (both in green) cocultured independently with 4T1 cells expressing Rosa26 -gRNA (magenta) at passages zero (P0) and 10 (P10). Scale bar, 100 μm. C, Graph showing the representation of the green 4T1 cell populations over serial passages in reference to P0. P values indicate the difference between the control condition and experimental conditions at P10. D, Proliferation assay comparing the 4T1-4T07 cells’ response to BYL719. E, Proliferation assay comparing the 4T1-4T07 cells’ response to MK-2206. F, Representative images showing 4T07 cells expressing Pik3c3 -gRNA, Pik3r4 -gRNA, or Rosa26 -gRNA (all in green) cocultured independently with cells expressing Rosa26 -gRNA (magenta) at passages zero (P0) and 10 (P10). Scale bar, 100 μm. G, Graph showing the representation of the green 4T07 cell populations over serial passages in reference to P0. P values indicate the difference between the control condition and experimental conditions at P10. H, Schematic for the timeline of assessing the effect of VPS34IN1 on the 4T07 and 4T1 cells’ survival in I and J . The 4T07 and 4T1 cells were seeded and cultured on BME, in which they form spheroids, and were then treated with VPS34IN1 or DMSO. I, Representative images of 4T07 and 4T1 3D spheroids at the experimental endpoint after treatment with either the vehicle (DMSO) or VPS34IN1 at 5 and 10 μmol/L. J, Quantifications of cell death burden as indicated by positive staining for SYTOX.

Article Snippet: For experiments in immunocompetent mice using the 4T07 model, 2 × 10 5 4T07 cells were resuspended in 50 μL of sterile PBS and injected into the fat pad of the fourth mammary gland of 8- to 10-week-old BALB/C mice (The Jackson Laboratory, #0651).

Techniques: Competitive Binding Assay, Expressing, Control, Proliferation Assay, Cell Culture, Staining

Journal: Cancer Research

Article Title: Targeting the Dependence on PIK3C3-mTORC1 Signaling in Dormancy-Prone Breast Cancer Cells Blunts Metastasis Initiation

doi: 10.1158/0008-5472.CAN-23-2654

Figure Lengend Snippet: mTORC1 is more active in 4T07 cells compared with 4T1 cells. A and B, Western blot analysis for the PI3K–AKT–mTORC1 pathway readouts in the 4T1-4T07 cells upon treatment with BYL719 ( A ) or MK-2206 ( B ). C, Quantification of the relative PI3K and mTORC1 activity in the two cell lines from the performed Western blots in A and B . D, OncoPrints for somatic alterations in genes involved in the PI3K–AKT–mTOR pathway in 4T1 and 4T07 cells. E, Western blot analysis assessing mTORC1 activity in the 4T1-4T07 cells under different nutritional conditions. AA, amino acids.

Article Snippet: For experiments in immunocompetent mice using the 4T07 model, 2 × 10 5 4T07 cells were resuspended in 50 μL of sterile PBS and injected into the fat pad of the fourth mammary gland of 8- to 10-week-old BALB/C mice (The Jackson Laboratory, #0651).

Techniques: Western Blot, Activity Assay

Journal: Cancer Research

Article Title: Targeting the Dependence on PIK3C3-mTORC1 Signaling in Dormancy-Prone Breast Cancer Cells Blunts Metastasis Initiation

doi: 10.1158/0008-5472.CAN-23-2654

Figure Lengend Snippet: Differential lysosomal positioning between the 4T1 and 4T07 cells. A and B, Immunostaining for the lysosomal marker Lamp1 and mTORC1 markers Rptor and pMtor (S2448) in the 4T1-4T07 cells under basal culture conditions, amino acid starvation, or amino acid refeeding. C, Quantification of the mean fluorescence intensity of mTORC1 (pMtor signal) on the lysosomes in the two cell lines under the three different conditions. D, Graph representing the percentage of peripheral lysosomes in the two cell lines under the three different conditions. ∗∗∗∗ , P < 0.0001.

Article Snippet: For experiments in immunocompetent mice using the 4T07 model, 2 × 10 5 4T07 cells were resuspended in 50 μL of sterile PBS and injected into the fat pad of the fourth mammary gland of 8- to 10-week-old BALB/C mice (The Jackson Laboratory, #0651).

Techniques: Immunostaining, Marker, Fluorescence

Journal: Cancer Research

Article Title: Targeting the Dependence on PIK3C3-mTORC1 Signaling in Dormancy-Prone Breast Cancer Cells Blunts Metastasis Initiation

doi: 10.1158/0008-5472.CAN-23-2654

Figure Lengend Snippet: PIK3C3 mediates peripheral lysosomal positioning and mTORC1 activity in murine and human breast cancer cells. A, Western blot analysis for the VPS34IN1 (1 μmol/L) effect on 4T07 and 4T1 cells’ mTORC1 activity. B, Quantification of mTORC1 activity in A . C, Immunostaining for Lamp1 and pMtor in 4T07 and 4T1 cells under the effect of VPS34IN1 (1 μmol/L for 2 hours). D, Quantifications of the percentage of peripheral lysosomes in the VPS34IN1-treated cells in comparison with the control condition (DMSO-treated). E, Immunostaining for LAMP2 and pmTOR in the PDX-1915 cells under the effect of VPS34IN1 (2 μmol/L) or DMSO for 2 hours. ns, nonsignificant. F, Quantifications of the percentage of peripheral lysosomes in E . G, Quantifications of the percentage of tubulated lysosomes in E . H, Immunostaining for LAMP2 and pmTOR in the CAMA-1 cells under the effect of VPS34IN1 (2 μmol/L) or DMSO for 2 hours. I, Quantification of the percentage of peripheral lysosomes in H . J, Immunostaining for LAMP2 and pmTOR in the T47D cells under the effect of VPS34IN1 (2 μmol/L) or DMSO for 2 hours. K, Quantification of the percentage of peripheral lysosomes in J . L, Immunostaining for LAMP2 and pmTOR in the HCC70 cells under the effect of VPS34IN1 (2 μmol/L) or DMSO for 2 hours. M, Quantification of the percentage of peripheral lysosomes in L .

Article Snippet: For experiments in immunocompetent mice using the 4T07 model, 2 × 10 5 4T07 cells were resuspended in 50 μL of sterile PBS and injected into the fat pad of the fourth mammary gland of 8- to 10-week-old BALB/C mice (The Jackson Laboratory, #0651).

Techniques: Activity Assay, Western Blot, Immunostaining, Comparison, Control

Journal: Cancer Research

Article Title: Targeting the Dependence on PIK3C3-mTORC1 Signaling in Dormancy-Prone Breast Cancer Cells Blunts Metastasis Initiation

doi: 10.1158/0008-5472.CAN-23-2654

Figure Lengend Snippet: Inhibiting the Pik3c3-mTORC1 axis decreases the metastatic burden in vivo preferentially in the 4T07 model. A, Schematic of the experiment investigating the effect of Pik3c3 inhibition on the metastatic burden in the 4T07 model. B, Graph showing tumor weight of mice treated with either VPS34IN1 (50 mg/kg/day) or vehicle. C, Graph showing the number of colonies retrieved from the lungs of mice treated with either VPS34IN1 (50 mg/kg/day) or vehicle. D, Schematic of the experiment investigating the effect of Pik3c3 inhibition on the metastatic burden in 4T1 tumor–bearing BALB/c mice. E, Representative hematoxylin and eosin stainings of lungs obtained from mice in D . Black arrows, metastatic lesions. F–H, Quantifications of the metastatic burden: absolute number of lesions ( F ), number of lesions normalized to lung area ( G ), and percentage of different sizes of metastases ( H ) in the vehicle- and VPS34IN1-treated mice (50 mg/kg/day).

Article Snippet: For experiments in immunocompetent mice using the 4T07 model, 2 × 10 5 4T07 cells were resuspended in 50 μL of sterile PBS and injected into the fat pad of the fourth mammary gland of 8- to 10-week-old BALB/C mice (The Jackson Laboratory, #0651).

Techniques: In Vivo, Inhibition

Journal: Communications Biology

Article Title: Fibroblast growth receptor 1 is regulated by G-quadruplex in metastatic breast cancer

doi: 10.1038/s42003-024-06602-x

Figure Lengend Snippet: A Representative bioluminescence imaging (BLI) images of control and erdafitinib (Erd) treated (50 mg/kg/po/qd) mice bearing 4T07 pulmonary tumors monitored by bioluminescence 13 days following tail vein inoculation. B Bioluminescent values from pulmonary regions of interest (ROI) were normalized to the values at the initial day of injection. Data are the individual values for each mouse ( n = 5) per treatment group. C Kaplan-Meier analyses of control and erdafitinib treated mice, bearing 4T07 pulmonary tumors, resulting in the indicated p -value. D 4T07 spheroids expressing luciferase were formed in non-adherent round bottom plates and then plated onto a bed of matrix in the presence or absence of FGF2 (20 ng/ml) or the indicated FGFR inhibitors (100 nM). E D2.A1 cells expressing luciferase were similarly used in spheroid culture, treated, and analyzed as in panel D . F D2.OR cells expressing luciferase were similarly used in spheroid culture, treated, and analyzed as in panels D and E . Luminescence values at day 6 were normalized to the non-stimulated (NS) conditions. Scale bars in panels D –F are 1000 μm. Data are the mean ± s.e.m. ( n ≥ 3) where * p < 0.05. G Following 3D culture, D2.OR spheroids were trypsinized and single cells were plated on tissue culture plastic. Colony formation in 10 cm 2 tissue culture dishes was visualized by crystal violet staining 14 days later as a measure of dormant cell survival.

Article Snippet: D2.A1 (1 × 10 6 ) and 4T07 (5 × 10 5 ) were delivered into the lateral tail vein of 4 to 6-week-old BALB/c female mice purchased from Jackson Laboratories.

Techniques: Imaging, Control, Injection, Expressing, Luciferase, Staining