Journal: Cancer Research Communications

Article Title: Induction of Triple-Negative Breast Cancer Cell Death and Chemosensitivity Using mTORC2-Directed RNAi Nanomedicine

doi: 10.1158/2767-9764.CRC-24-0261

Figure Lengend Snippet: Rictor knockdown blocks mTORC2 signaling without reactivation of PI3K. A, Western blot analysis of cells transfected with siRaptor, siRictor, or siControl. Antibodies used are shown to the left of each panel. B and C, CellTiter-Glo was used to measure cell number ( B ) and Caspase-3/7-Glo was used to measure caspase 3/7 (cell death signaling; C ) at 96 hours following siRNA transfection. Each point represents the average value of 3 replicates and midlines are the average values ± SD, normalized to the average value of siControl-transfected samples. One-way ANOVA was used. D, Western blot analysis of cells collected 72 hours after transfection. Antibodies used are shown at the left of each panel. All siRNA sequences designed against RAPTOR and RICTOR were validated for their ability to knockdown target genes. E, Phospho-kinase protein array was used on HCC70 cells treated with PP242 (500 nmol/L for 48 hours) or after siRNA transfection (50 nmol/L for 48 hours and 7 days after transfection). F, PI(3,4,5)P3 ELISA was used to measure PI3K activity from cells treated with PP242 or transfected with siRNA. PI(3,5)P2 was used as a substrate. Values shown are average ± SD, normalized to the average values in DMSO-treated samples. N = 3. One-way ANOVA was used.

Article Snippet: Human TNBC cell lines HCC70 (cat #CRL-2315, RRID: CVCL_1270), HCC1937 (cat #CRL-2336, RRID: CVCL_0290), BT20 (cat #HTB-19, RRID: CVCL_0178), MDA-MB-157 (cat #HTB-24, RRID: CVCL_0618), BT549 (cat #HTB-122, RRID: CVCL_1092), MDA-MB-231 (cat #HTB-26, RRID: CVCL_0062), MDA-MB-436 (HTB-130, RRID: CVCL_0623), HCC1599 (cat #CRL-2331, RRID: CVCL_1256), and HCC1187 (cat #CRL-2322, RRID: CVCL_1247) were purchased from the ATCC.

Techniques: Knockdown, Western Blot, Transfection, Protein Array, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Cancer Research Communications

Article Title: Induction of Triple-Negative Breast Cancer Cell Death and Chemosensitivity Using mTORC2-Directed RNAi Nanomedicine

doi: 10.1158/2767-9764.CRC-24-0261

Figure Lengend Snippet: si-NP carrier technology enables siRNA delivery to TNBC tumors. A, Schematic representation of core-forming 50B polymer and surface-forming 20kPEG-50B polymer, and their assembly with siRNA to generate the si-NP formulation. B, Cy5 fluorescence was measured ex vivo within organs and tumors collected from HCC70 tumor–bearing mice 24 hours after delivery of Cy5-si-NPs (1 mg/kg siRNA, i.v.). Values represent the average ± SD, N = 6. C, Flow cytometric quantitation of Cy5+ cells, with representative histogram shown on left and % Cy5+ on right. D, Cy5 geometric mean fluorescence intensity (gMFI). Data were measured from cells dissociated from HCC70 tumors 24 hours after delivery of Cy5-si-NP (1 mg/kg, i.v.) or vehicle. Values represent the average ± SD, N = 5–6. An unpaired t test was used. E, Confocal microscopy to visualize Cy5-siRNA in HCC70 tumor cryosections collected 24 hours after delivery of Cy5-siNP. Nuclei stained with DAPI.

Article Snippet: Human TNBC cell lines HCC70 (cat #CRL-2315, RRID: CVCL_1270), HCC1937 (cat #CRL-2336, RRID: CVCL_0290), BT20 (cat #HTB-19, RRID: CVCL_0178), MDA-MB-157 (cat #HTB-24, RRID: CVCL_0618), BT549 (cat #HTB-122, RRID: CVCL_1092), MDA-MB-231 (cat #HTB-26, RRID: CVCL_0062), MDA-MB-436 (HTB-130, RRID: CVCL_0623), HCC1599 (cat #CRL-2331, RRID: CVCL_1256), and HCC1187 (cat #CRL-2322, RRID: CVCL_1247) were purchased from the ATCC.

Techniques: Polymer, Formulation, Fluorescence, Ex Vivo, Quantitation Assay, Confocal Microscopy, Staining

Journal: Cancer Research Communications

Article Title: Induction of Triple-Negative Breast Cancer Cell Death and Chemosensitivity Using mTORC2-Directed RNAi Nanomedicine

doi: 10.1158/2767-9764.CRC-24-0261

Figure Lengend Snippet: In vivo delivery of siRictor-NPs blocks mTORC2 in the tumors and increases tumor cell death. A, HCC70 ( N = 4–5), CAL-85-1 ( N = 3), and HCC1937 ( N = 3) tumor–bearing mice were treated with si-NPs intravenously, and tumors were harvested for molecular analysis on day 7. Schematic timeline of treatment schedule is shown. B, Tumor volumes (T vol ) were tracked to the study endpoint. C, Western blot analysis of whole-tumor lysates. Antibodies used are shown to the left of each panel. D and E, Quantitation of the percentage of tumor cells that are Ki67+, cleaved (Cl.) caspase-3+, and P-Histone H3+ (P-HH3) based on IHC of HCC70 tumor sections. For quantitation, each point represents the average of 4–5 randomly chosen fields from a single tumor. Midline is the average ± SD of N = 3 tumors. An unpaired t test was used. F and G, CAL-85-1 tumor–bearing mice ( N = 4) were treated with si-NPs intravenously, and tumor volume was measured. Schematic timeline ( F ) of treatment schedule and tumor growth ( G ) is shown. Significance calculated using an unpaired t test of AUC analysis.

Article Snippet: Human TNBC cell lines HCC70 (cat #CRL-2315, RRID: CVCL_1270), HCC1937 (cat #CRL-2336, RRID: CVCL_0290), BT20 (cat #HTB-19, RRID: CVCL_0178), MDA-MB-157 (cat #HTB-24, RRID: CVCL_0618), BT549 (cat #HTB-122, RRID: CVCL_1092), MDA-MB-231 (cat #HTB-26, RRID: CVCL_0062), MDA-MB-436 (HTB-130, RRID: CVCL_0623), HCC1599 (cat #CRL-2331, RRID: CVCL_1256), and HCC1187 (cat #CRL-2322, RRID: CVCL_1247) were purchased from the ATCC.

Techniques: In Vivo, Western Blot, Quantitation Assay

Journal: Cancer Research Communications

Article Title: Induction of Triple-Negative Breast Cancer Cell Death and Chemosensitivity Using mTORC2-Directed RNAi Nanomedicine

doi: 10.1158/2767-9764.CRC-24-0261

Figure Lengend Snippet: siRictor-NPs treatment synergizes with chemotherapy to diminish tumor cell survival and tumor growth in TNBC. A and B, HCC70 cells were treated with 100 nmol/L si-NPs ± paclitaxel in increasing doses. Cell viability was measured by CellTiter-Glo 2 days after chemotherapy addition ( A ), and caspase 3/7 activity was measured by Caspase-Glo 1 day after chemotherapy addition ( B ). Statistical significance was calculated based on AUC, in which midline is the average ± SD and N = 3. An unpaired t test was used. C–I, HCC70 tumor–bearing mice ( N = 9–11) were treated with si-NPs intravenously and paclitaxel intraperitoneally, and the tumors were monitored until harvest for molecular analysis on day 52. C, Schematic timeline of treatment schedule. D, Tumor volume was measured throughout the 52-day treatment period. Values shown on tumor growth curve (left) are average ± SE. Statistical significance was calculated based on AUC (right), in which midline is the average ± SD and N = 9–11 tumors. Endpoint tumor volume was calculated at day 52. Midline is the average ± SD. One-way ANOVA was performed with the Tukey multiple comparison post hoc test. E, Kaplan–Meier analysis of tumor-bearing mice, defining survival as tumor volume under 500 mm 3 (paclitaxel-treated mice). Average time to exceed the defined tumor volume (T 50 ) for each group is shown within panel. Significance calculated using the log-rank (Mantel–Cox) test. F, Quantitation of the percentage of tumor cells that are Ki67+ ( N = 7–10 tumors) based on IHC of tumor sections. For quantitation, each point represents an average of 4–5 randomly chosen fields from a single tumor. Midline is the average ± SD. One-way ANOVA was performed with the Tukey multiple comparisons post hoc test. G and H , Quantitation of the percentage of tumor cells that are P-Histone H3 (P-HH3) Ser10+ ( N = 3–4) ( G ) based on IHC of tumor sections ( H ). For quantitation, each point represents an average of 4–5 randomly chosen fields from a single tumor. Midline is the average ± SD. One-way ANOVA was performed with the Tukey multiple comparisons post hoc test. I and J , Quantitation of the percentage of tumor cells that are cleaved (Cl.) caspase-3+ ( I ) based on IHC of tumor sections ( J ). For quantitation, each point represents an average of 4–5 randomly chosen fields from a single tumor. Midline is the average ± SD of N = 5–6 tumors. One-way ANOVA was performed with the Tukey multiple comparison post hoc test.

Article Snippet: Human TNBC cell lines HCC70 (cat #CRL-2315, RRID: CVCL_1270), HCC1937 (cat #CRL-2336, RRID: CVCL_0290), BT20 (cat #HTB-19, RRID: CVCL_0178), MDA-MB-157 (cat #HTB-24, RRID: CVCL_0618), BT549 (cat #HTB-122, RRID: CVCL_1092), MDA-MB-231 (cat #HTB-26, RRID: CVCL_0062), MDA-MB-436 (HTB-130, RRID: CVCL_0623), HCC1599 (cat #CRL-2331, RRID: CVCL_1256), and HCC1187 (cat #CRL-2322, RRID: CVCL_1247) were purchased from the ATCC.

Techniques: Activity Assay, Comparison, Quantitation Assay