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Cancer Res (2022) 82 (12_Supplement): 1475.
Published: 15 June 2022
...Aidan O'Brien; Jason W. Hoskins; Daina R. Eiser; Katelyn E. Connelly; Jun Zhong; Thorkell Andresson; Irene Collins; Pancreatic Cancer Cohort Consortium; Pancreatic Cancer Case-Control Consortium; Stephen J. Chanock; Alison P. Klein; H. Efsun Arda; Laufey T. Amundadottir In western nations...
Journal Articles
Cancer Res (2021) 81 (18): 4723–4735.
Published: 16 September 2022
... complication of cancer with a dire prognosis. Although any malignancy can spread to the leptomeninges, breast cancer, particularly the HER2+ subtype, is its most common origin. HER2+ breast LC (HER2+ LC) remains incurable, with few treatment options, and the molecular...
Includes: Supplementary data
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The presence of OPCs reduces HER2<sup>+</sup> LC cell viability.  A,  IF im...
Published: 16 September 2022
Figure 1. The presence of OPCs reduces HER2+ LC cell viability. A, IF images of various CNS cell types immuno-panned from human iPSCs and stained with the indicated antibodies. Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. B, Annexin V FACS-based analysis of Lepto1 and Lepto2 cells (seeded at 0.5 × 105 density/well of a 24-well plate in the bottom chamber) cocultured with the indicated human CNS cell populations (seeded at 0.5 × 105 density/well of 24-well plate in the top inserts). Coculture with OPCs increased the proportion of apoptotic Lepto cells (n = 3; **, P < 0.001 relative to control Lepto cells exposed to no CNS cell types). C, Viability of Lepto1 and Lepto2 (seeded at 0.1 × 105 density/well of a 96-well plate in the bottom chamber) lines cocultured with various CNS cell types (derived in Fig. 2A ; all seeded at 0.1 × 105 density/well of a 96-well plate in the top inserts) for 48 hours, measured using CellTiter-Glo assays (n = 3; **, P < 0.001 relative to control Lepto cells exposed to no CNS cell types). D, IF images of mCherry: LUC-labeled (red) Lepto cells stained with Annexin V (green) and procaspase-3 (magenta) after 48 hours of treatment with or without OPC-conditioned medium. Scale bar, 50 μm. E, Schematic showing the protocol used for the in vivo characterization of the effects of OPCs on Lepto cell growth. mCherry: LUC-labeled Lepto cells (100K; red) were injected into the cisternae magna of adult NOD/SCID mice on day 0, and OPCs (100K; green) were injected on days 7 and/or 14. Tumor growth was monitored by BLI from days 14 to 50, with representative images acquired starting on day 28. F, Left, quantitative analyses showing that the mice that received OPCs exhibited reduced tumor growth (n = 6; ***, P < 0.001 relative to mice with OPCs implanted on days 7 and 14). Right, Kaplan–Meier curves showing the overall survival of mice implanted with Lepto cells on day 0 only (solid red line) or co-implanted with OPCs on day 7 (dashed green line) or on days 7 and 14 (solid green line; ***, P < 0.001). G, Histopathologic analyses of the H&E-stained axial spinal cord sections from control Lepto infused and Lepto+ OPC (OPC infusion on D7 and D14) co-infused mice (left) and ×20 magnified regions showing Lepto deposition. Figure 1. The presence of OPCs reduces HER2+ LC cell viability. A, IF images of various CNS cell types immuno-panned from human iPSCs and stained with the indicated antibodies. Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. B, Annexin V FACS-based analysis of Lepto1 and Lepto2 cells (seeded at 0.5 × 105 density/well of a 24-well plate in the bottom chamber) cocultured with the indicated human CNS cell populations (seeded at 0.5 × 105 density/well of 24-well plate in the top inserts). Coculture with OPCs increased the proportion of apoptotic Lepto cells (n = 3; **, P < 0.001 relative to control Lepto cells exposed to no CNS cell types). C, Viability of Lepto1 and Lepto2 (seeded at 0.1 × 105 density/well of a 96-well plate in the bottom chamber) lines cocultured with various CNS cell types (derived in Fig. 2A; all seeded at 0.1 × 105 density/well of a 96-well plate in the top inserts) for 48 hours, measured using CellTiter-Glo assays (n = 3; **, P < 0.001 relative to control Lepto cells exposed to no CNS cell types). D, IF images of mCherry: LUC-labeled (red) Lepto cells stained with Annexin V (green) and procaspase-3 (magenta) after 48 hours of treatment with or without OPC-conditioned medium. Scale bar, 50 μm. E, Schematic showing the protocol used for the in vivo characterization of the effects of OPCs on Lepto cell growth. mCherry: LUC-labeled Lepto cells (100K; red) were injected into the cisternae magna of adult NOD/SCID mice on day 0, and OPCs (100K; green) were injected on days 7 and/or 14. Tumor growth was monitored by BLI from days 14 to 50, with representative images acquired starting on day 28. F, Left, quantitative analyses showing that the mice that received OPCs exhibited reduced tumor growth (n = 6; ***, P < 0.001 relative to mice with OPCs implanted on days 7 and 14). Right, Kaplan–Meier curves showing the overall survival of mice implanted with Lepto cells on day 0 only (solid red line) or co-implanted with OPCs on day 7 (dashed green line) or on days 7 and 14 (solid green line; ***, P < 0.001). G, Histopathologic analyses of the H&E-stained axial spinal cord sections from control Lepto infused and Lepto+ OPC (OPC infusion on D7 and D14) co-infused mice (left) and ×20 magnified regions showing Lepto deposition. More
Images
Combination treatment with a pan-Aurora kinase inhibitor and anti-GMCSF neu...
Published: 16 September 2022
Figure 5. Combination treatment with a pan-Aurora kinase inhibitor and anti-GMCSF neutralizing antibodies reduces Lepto cell growth in vivo. A, RT-qPCR analysis of Aurora A transcript levels in nodular HER2+ LC, primary tumor (PT2), metastatic tumor (MT2), normal breast, and normal brain tissues. The HER2+ LC tissues exhibited the highest Aurora A transcript levels. B, Dose-dependent inhibition of Lepto cell viability by CCT137690, measured by CellTiter-Glo assays. The IC50 value is shown. C, IF images of mCherry: LUC-labeled (red) Lepto cells stained with Annexin V (green) after 24 hours of treatment with CCT137690 (100 nmol/L) or 0.1% DMSO (CTL). Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. D, Percentages of tumorsphere-initiating cells after 24-hour treatment (as shown in Supplementary Fig. S4A, top row), measured by CCK assays. The number of DMSO-treated cells was set to 100 (n = 3; **, P < 0.01, compared with DMSO-treated cells). E, Viability of primary tumorspheres after 2-day treatment (as shown in Supplementary FIg. S4A, middle row), measured by CCK assays. The number of DMSO-treated cells was set to 100 (n = 3; **, P < 0.01, compared with DMSO-treated tumorspheres). F, Viability of secondary tumorspheres 12 days after dissociation of treated primary tumorspheres (as shown in Supplementary Fig. S4A, bottom row), measured by CCK assays (**, P < 0.01, compared with secondary tumorspheres from DMSO-treated primary tumorspheres). G, Schematic showing the protocol used to monitor the effects of the intrathecal administration of anti-GMCSF neutralizing antibodies and CCT137690 ((8 μg/g and 100 mg/kg, respectively, on days 5, 10, and 15) in mice implanted with mCherry: LUC-labeled Lepto cells (100K). Tumor growth was monitored by BLI from days 15 to 50. H, Left, BLI-based quantification of mCherry: LUC-labeled Lepto tumor growth in mice treated with anti-GMCSF antibodies alone or with CCT137690 (n = 9). Control animals were treated with vehicle (PBS for antibodies and 0.1% DMSO for CCT137690). Right, survival analysis of the same mice. Combination treatment with anti-GMCSF antibodies and CCT137690 (anti-GMCSF+CCT137690) significantly reduced tumor growth and increased survival. Treatment with anti-GMCSF antibodies alone also significantly reduced tumor growth but to a lesser extent (**, P < 0.01). I, H&E-stained sagittal brain tissue sections from Lepto-bearing NOD/SCID mice treated with vehicle, Anti-GMCSF, CCT137690, and CCT137690+anti-GMCSF. Red arrows, presence of Lepto-derived tumor mass. Figure 5. Combination treatment with a pan-Aurora kinase inhibitor and anti-GMCSF neutralizing antibodies reduces Lepto cell growth in vivo. A, RT-qPCR analysis of Aurora A transcript levels in nodular HER2+ LC, primary tumor (PT2), metastatic tumor (MT2), normal breast, and normal brain tissues. The HER2+ LC tissues exhibited the highest Aurora A transcript levels. B, Dose-dependent inhibition of Lepto cell viability by CCT137690, measured by CellTiter-Glo assays. The IC50 value is shown. C, IF images of mCherry: LUC-labeled (red) Lepto cells stained with Annexin V (green) after 24 hours of treatment with CCT137690 (100 nmol/L) or 0.1% DMSO (CTL). Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. D, Percentages of tumorsphere-initiating cells after 24-hour treatment (as shown in Supplementary Fig. S4A, top row), measured by CCK assays. The number of DMSO-treated cells was set to 100 (n = 3; **, P < 0.01, compared with DMSO-treated cells). E, Viability of primary tumorspheres after 2-day treatment (as shown in Supplementary FIg. S4A, middle row), measured by CCK assays. The number of DMSO-treated cells was set to 100 (n = 3; **, P < 0.01, compared with DMSO-treated tumorspheres). F, Viability of secondary tumorspheres 12 days after dissociation of treated primary tumorspheres (as shown in Supplementary Fig. S4A, bottom row), measured by CCK assays (**, P < 0.01, compared with secondary tumorspheres from DMSO-treated primary tumorspheres). G, Schematic showing the protocol used to monitor the effects of the intrathecal administration of anti-GMCSF neutralizing antibodies and CCT137690 ((8 μg/g and 100 mg/kg, respectively, on days 5, 10, and 15) in mice implanted with mCherry: LUC-labeled Lepto cells (100K). Tumor growth was monitored by BLI from days 15 to 50. H, Left, BLI-based quantification of mCherry: LUC-labeled Lepto tumor growth in mice treated with anti-GMCSF antibodies alone or with CCT137690 (n = 9). Control animals were treated with vehicle (PBS for antibodies and 0.1% DMSO for CCT137690). Right, survival analysis of the same mice. Combination treatment with anti-GMCSF antibodies and CCT137690 (anti-GMCSF+CCT137690) significantly reduced tumor growth and increased survival. Treatment with anti-GMCSF antibodies alone also significantly reduced tumor growth but to a lesser extent (**, P < 0.01). I, H&E-stained sagittal brain tissue sections from Lepto-bearing NOD/SCID mice treated with vehicle, Anti-GMCSF, CCT137690, and CCT137690+anti-GMCSF. Red arrows, presence of Lepto-derived tumor mass. More
Images
Modulation of GMCSF expression alters Lepto cell proliferation <em>in v</em>...
Published: 16 September 2022
Figure 3. Modulation of GMCSF expression alters Lepto cell proliferation in vitro and in vivo. A, Diagram showing the lentiviral Tet-On 3G-inducible GMCSF ORF expression cassette used in this study. B, Left, Western blot analysis of GMCSF in Lepto cell lysates collected 48 hours after 5 μg/mL DOX administration to induce GMCSF overexpression. β-Actin served as a loading control. Right, FIJI-based quantification of the Western blots shows DOX mediated overexpression of GMCSF in Lepto cells. C, FACS-based analysis of ZsGreen1expression in Lepto cells cultured for 48 hours with (red) or without (blue) 5 μg/mL DOX. D, Fluorescence imaging of Lepto cells 48 hours after 5 μg/mL DOX or vehicle (PBS) treatment, showing robust green fluorescence of Lepto cells after GMCSF induction. Scale bar, 50 μm. E, Viability of Lepto cells cultured with or without 5 μg/mL DOX and/or OPCs for 48 hours, measured by CellTiter-Glo assays (n = 3; **, P < 0.001). F, Annexin V FACS-based analysis of apoptosis in Lepto cells grown under the conditions shown in E (n = 3; **, P < 0.001). G, Diagram showing the lentiviral Tet-On 3G-inducible GMCSF-shRNA expression cassette used in this study. H, Western blot analysis of GMCSF in Lepto cell (Lepto1, left; Lepto2, right) lysates collected 48 hours after 5 μg/mL DOX administration to induce shGMCSF expression. Tubulin served as a loading control. I, FIJI-based quantification of the Western blots in H shows DOX-mediated overexpression of shGMCSF in Lepto1 and 2 cells (*, P < 0.01). J, ELISA-based quantification of GMCSF concentrations in the media of Lepto1 and 2 cells conditionally expressing shGMCSF (***, P < 0.01). K, Proliferation rates of control Lepto cells and Lepto cells expressing shGMCSF over 6 days. shGMCSF-expressing Lepto cells showed prolonged doubling times relative to control Lepto cells. L, Heatmap of the tumor-seeding capacities (per eight xenografted animals) of control Lepto cells versus Lepto cells conditionally expressing shGMCSF. M, H&E-stained sagittal brain tissue sections from NOD/SCID mice implanted with Lepto1 cells (100K) constituitively overexpressing GMCSF alone, Lepto1 cells (100K) conditionally overexpressing shGMCSF (DOX; ON), and Lepto1 cells constituitively overexpressing GMCSF as well as conditionally overexpressing shGMCSF (DOX; ON). Red arrows, presence of Lepto-derived tumor mass. N, Heatmap of the tumor-seeding capacities (per four xenografted animals) of control Lepto cells versus constitutive GMCSF overexpressing Lepto cells versus GMCSF (constitutive overexpression) combined with conditionally expressing shGMCSF Lepto cells. Figure 3. Modulation of GMCSF expression alters Lepto cell proliferation in vitro and in vivo. A, Diagram showing the lentiviral Tet-On 3G-inducible GMCSF ORF expression cassette used in this study. B, Left, Western blot analysis of GMCSF in Lepto cell lysates collected 48 hours after 5 μg/mL DOX administration to induce GMCSF overexpression. β-Actin served as a loading control. Right, FIJI-based quantification of the Western blots shows DOX mediated overexpression of GMCSF in Lepto cells. C, FACS-based analysis of ZsGreen1expression in Lepto cells cultured for 48 hours with (red) or without (blue) 5 μg/mL DOX. D, Fluorescence imaging of Lepto cells 48 hours after 5 μg/mL DOX or vehicle (PBS) treatment, showing robust green fluorescence of Lepto cells after GMCSF induction. Scale bar, 50 μm. E, Viability of Lepto cells cultured with or without 5 μg/mL DOX and/or OPCs for 48 hours, measured by CellTiter-Glo assays (n = 3; **, P < 0.001). F, Annexin V FACS-based analysis of apoptosis in Lepto cells grown under the conditions shown in E (n = 3; **, P < 0.001). G, Diagram showing the lentiviral Tet-On 3G-inducible GMCSF-shRNA expression cassette used in this study. H, Western blot analysis of GMCSF in Lepto cell (Lepto1, left; Lepto2, right) lysates collected 48 hours after 5 μg/mL DOX administration to induce shGMCSF expression. Tubulin served as a loading control. I, FIJI-based quantification of the Western blots in H shows DOX-mediated overexpression of shGMCSF in Lepto1 and 2 cells (*, P < 0.01). J, ELISA-based quantification of GMCSF concentrations in the media of Lepto1 and 2 cells conditionally expressing shGMCSF (***, P < 0.01). K, Proliferation rates of control Lepto cells and Lepto cells expressing shGMCSF over 6 days. shGMCSF-expressing Lepto cells showed prolonged doubling times relative to control Lepto cells. L, Heatmap of the tumor-seeding capacities (per eight xenografted animals) of control Lepto cells versus Lepto cells conditionally expressing shGMCSF. M, H&E-stained sagittal brain tissue sections from NOD/SCID mice implanted with Lepto1 cells (100K) constituitively overexpressing GMCSF alone, Lepto1 cells (100K) conditionally overexpressing shGMCSF (DOX; ON), and Lepto1 cells constituitively overexpressing GMCSF as well as conditionally overexpressing shGMCSF (DOX; ON). Red arrows, presence of Lepto-derived tumor mass. N, Heatmap of the tumor-seeding capacities (per four xenografted animals) of control Lepto cells versus constitutive GMCSF overexpressing Lepto cells versus GMCSF (constitutive overexpression) combined with conditionally expressing shGMCSF Lepto cells. More
Images
GMCSF acts as an oncogenic autocrine driver contributing to HER2<sup>+</sup>...
Published: 16 September 2022
Figure 2. GMCSF acts as an oncogenic autocrine driver contributing to HER2+ LC cell growth. A, Cytokine XL array-based analyses of conditioned media from OPCs and/or Lepto cells cultured in media supplemented with hCSF. The secreted factors identified in the media of monocultured OPCs and OPCs cocultured with Lepto cells are listed in Supplementary Tables S4 and S5. B, Top, control and GMCSF-specific blots from the array shown in A . Bottom, density-based quantification of the GMCSF blots shown in the top panel. C, RT-qPCR analysis of GMCSF transcript levels in Lepto1 and Lepto2 cells, as well as in the indicated iPSC-derived CNS cell types. The Lepto lines exhibited the highest GMCSF mRNA levels among all cell types analyzed (n = 3; *, P < 0.001 relative to OPCs). D, RT-qPCR analysis of GMCSF transcript levels in HER2+ LC tumor, HER2+ breast metastatic tumor (MT2), primary tumor (PT2), normal breast, and normal brain tissues. The HER2+ LC tumor tissues exhibited the highest GMCSF transcript levels among all tissues analyzed (n = 3; **, P < 0.001). E, Left, IHC analysis of patient HER2+ LC specimens showing pGMCSFRα (orange) in tumor cells but not surrounding brain tissue. Top, low magnification image showing tumor and surrounding normal brain tissue. Bottom, high magnification image showing the selected tumor region. Scale bar, 100 μm. Right, FIJI-based quantification of the IHC image analyses from n = 3 patient HER2+ LC specimens showing higher levels of pGMCSFRα in tumor cells but not in the surrounding brain tissue. F, Left, Western blot analysis of the indicated signaling proteins in extracts from Lepto cells cultured alone or with OPCs for 48 hours. Tubulin was used as the loading control. Right, heat map showing FIJI-based quantification of the Western blots. Compared with monocultured Lepto cells, Lepto cells cocultured with OPCs exhibited lower pGMCSFRα levels and less growth factor phosphorylation/activation (pSTAT5, pAKT, and pERK1/2). Tubulin served as a loading control. G, Quantification of Annexin V-positive Lepto cells grown under the indicated conditions. Cells cultured with OPC-conditioned media or anti-GMCSF neutralizing antibodies were significantly more apoptotic than control cells grown in hCSF-supplemented media alone (n = 6). ***, P < 0.001. H, Schematic showing the protocol used to monitor the effects of the intrathecal administration of anti-GMCSF neutralizing versus control IgG antibodies (8 μg/g on days 5, 10, and 15) in mice implanted with mCherry:LUC-labeled Lepto cells (100K). Tumor growth was monitored by BLI from days 15 to 50. I, IHC analysis of brain sections from Lepto cell–implanted NOD/SCID mice treated with anti-GMCSF antibodies or control IgG. Anti-GMCSF antibody treatment suppressed Lepto tumor growth. J, Left, BLI-based quantification of mCherry:LUC-labeled Lepto tumor growth in mice treated with anti-GMCSF antibodies or control IgG (n = 6). Antibody treatment blocked tumor progression. Right, survival analysis of the same mice (***, P < 0.001). Figure 2. GMCSF acts as an oncogenic autocrine driver contributing to HER2+ LC cell growth. A, Cytokine XL array-based analyses of conditioned media from OPCs and/or Lepto cells cultured in media supplemented with hCSF. The secreted factors identified in the media of monocultured OPCs and OPCs cocultured with Lepto cells are listed in Supplementary Tables S4 and S5. B, Top, control and GMCSF-specific blots from the array shown in A. Bottom, density-based quantification of the GMCSF blots shown in the top panel. C, RT-qPCR analysis of GMCSF transcript levels in Lepto1 and Lepto2 cells, as well as in the indicated iPSC-derived CNS cell types. The Lepto lines exhibited the highest GMCSF mRNA levels among all cell types analyzed (n = 3; *, P < 0.001 relative to OPCs). D, RT-qPCR analysis of GMCSF transcript levels in HER2+ LC tumor, HER2+ breast metastatic tumor (MT2), primary tumor (PT2), normal breast, and normal brain tissues. The HER2+ LC tumor tissues exhibited the highest GMCSF transcript levels among all tissues analyzed (n = 3; **, P < 0.001). E, Left, IHC analysis of patient HER2+ LC specimens showing pGMCSFRα (orange) in tumor cells but not surrounding brain tissue. Top, low magnification image showing tumor and surrounding normal brain tissue. Bottom, high magnification image showing the selected tumor region. Scale bar, 100 μm. Right, FIJI-based quantification of the IHC image analyses from n = 3 patient HER2+ LC specimens showing higher levels of pGMCSFRα in tumor cells but not in the surrounding brain tissue. F, Left, Western blot analysis of the indicated signaling proteins in extracts from Lepto cells cultured alone or with OPCs for 48 hours. Tubulin was used as the loading control. Right, heat map showing FIJI-based quantification of the Western blots. Compared with monocultured Lepto cells, Lepto cells cocultured with OPCs exhibited lower pGMCSFRα levels and less growth factor phosphorylation/activation (pSTAT5, pAKT, and pERK1/2). Tubulin served as a loading control. G, Quantification of Annexin V-positive Lepto cells grown under the indicated conditions. Cells cultured with OPC-conditioned media or anti-GMCSF neutralizing antibodies were significantly more apoptotic than control cells grown in hCSF-supplemented media alone (n = 6). ***, P < 0.001. H, Schematic showing the protocol used to monitor the effects of the intrathecal administration of anti-GMCSF neutralizing versus control IgG antibodies (8 μg/g on days 5, 10, and 15) in mice implanted with mCherry:LUC-labeled Lepto cells (100K). Tumor growth was monitored by BLI from days 15 to 50. I, IHC analysis of brain sections from Lepto cell–implanted NOD/SCID mice treated with anti-GMCSF antibodies or control IgG. Anti-GMCSF antibody treatment suppressed Lepto tumor growth. J, Left, BLI-based quantification of mCherry:LUC-labeled Lepto tumor growth in mice treated with anti-GMCSF antibodies or control IgG (n = 6). Antibody treatment blocked tumor progression. Right, survival analysis of the same mice (***, P < 0.001). More
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OPC-derived TPP1 is a candidate regulator of GMCSF.  A,  Venn diagram of un...
Published: 16 September 2022
Figure 4. OPC-derived TPP1 is a candidate regulator of GMCSF. A, Venn diagram of unique and shared secreted proteins identified in the hCSF of mono- or cocultured astrocytes, OPCs, and Lepto cells. B, Relative TPP1 protein levels in control hCSF (no cells) and hCSF from the indicated cell cultures. C, Relative quantification (RQ) of GMCSF levels in media from Lepto cells cultured in OPC-conditioned hCSF or in hCSF with exogenous TPP1 (50 or 100 ng/mL), as measured by ELISA (**, P < 0.01). D, Lepto cell viability following treatment with OPC-conditioned hCSF or hCSF with exogenous TPP1, as shown in C , measured by CellTiter-Glo assays (**, P < 0.01). E, ELISA-based analysis of GMCSF concentrations in the media of Lepto cells conditionally overexpressing GMCSF (following treatment with 5 μg/mL DOX) and cultured with OPCs or TPP1 (50 ng/mL). Both conditions reduced GMCSF levels in culture media (**, P < 0.01). F, Representative BLI images of NOD/SCID mice on day 26 post-implantations of Lepto cells (100K) alone or with OPCs (100K or 200K). G, BLI-based quantification of tumor growth showing OPC density-dependent suppression of Lepto tumor growth (***, P < 0.001). H, Kaplan–Meier curves showing the density-dependent effects of OPC implantation on the survival of mice bearing Lepto tumors (***, P < 0.001). I, H&E-stained brain tissue sections from NOD/SCID mice implanted with Lepto cells (100K) alone or with OPCs (200K). J, ELISA-based analysis of TPP1 levels in serum extracted from mice on days 8, 16, 24, and 32 postimplantations of with Lepto cells (100K) alone (Control) or with OPCs (100K or 200K). K, ELISA-based analysis of GMCSF levels in serum extracted from mice on days 8, 16, 24, and 32 postimplantations with Lepto cells (100K) alone (Control) or with OPCs (100K or 200K). L, Viability of OPCs extracted from CSF samples collected between days 5 and 20 after co-implantation with Lepto cells into NOD/SCID mice, measured by CellTiter-Glo assays. M, Left, BLI-based quantification of tumor growth in Lepto1 bearing NOD/SCID mice co-implanted with OPC-shGFP (100K), OPC-shTPP1 (100K), and OPC-shTPP1 (200K) shows density-dependent elevation of Lepto1 tumor growth (***, P < 0.001). Right, Kaplan–Meier curves showing survival of mice bearing Lepto1-derived tumors co-implanted with OPC-shGFP (100K), OPC-shTPP1 (100K), and OPC-shTPP1 (200K; ***, P < 0.001). N, Left, ELISA-based analysis of TPP1 levels in serum extracted from mice on days 8, 16, 24, and 32 postimplantations with Lepto1 cells (100K), followed by co-implantation with 100K OPCs-shGFP, (100K and 200K) OPCs-shTPP1. Right, ELISA-based analysis of GMCSF levels in serum extracted from mice on days 8, 16, 24, and 32 postimplantations with Lepto1 cells (100K), followed by co-implantation with 100K OPCs-shGFP, (100K and 200K) OPCs-shTPP1. O, H&E-stained coronal brain tissue sections from NOD/SCID mice co-implanted with Lepto1 cells (100K) and with OPCs-shGFP or with OPCs-shTPP1 (100K). Red arrows, presence of Lepto-derived tumor mass. P, H&E-stained sagittal brain tissue sections from NOD/SCID mice co-implanted with Lepto1 cells (100K) and with OPCs-shGFP or with OPCs-shTPP1 (100K). Red arrows, presence of Lepto-derived tumor mass. Figure 4. OPC-derived TPP1 is a candidate regulator of GMCSF. A, Venn diagram of unique and shared secreted proteins identified in the hCSF of mono- or cocultured astrocytes, OPCs, and Lepto cells. B, Relative TPP1 protein levels in control hCSF (no cells) and hCSF from the indicated cell cultures. C, Relative quantification (RQ) of GMCSF levels in media from Lepto cells cultured in OPC-conditioned hCSF or in hCSF with exogenous TPP1 (50 or 100 ng/mL), as measured by ELISA (**, P < 0.01). D, Lepto cell viability following treatment with OPC-conditioned hCSF or hCSF with exogenous TPP1, as shown in C, measured by CellTiter-Glo assays (**, P < 0.01). E, ELISA-based analysis of GMCSF concentrations in the media of Lepto cells conditionally overexpressing GMCSF (following treatment with 5 μg/mL DOX) and cultured with OPCs or TPP1 (50 ng/mL). Both conditions reduced GMCSF levels in culture media (**, P < 0.01). F, Representative BLI images of NOD/SCID mice on day 26 post-implantations of Lepto cells (100K) alone or with OPCs (100K or 200K). G, BLI-based quantification of tumor growth showing OPC density-dependent suppression of Lepto tumor growth (***, P < 0.001). H, Kaplan–Meier curves showing the density-dependent effects of OPC implantation on the survival of mice bearing Lepto tumors (***, P < 0.001). I, H&E-stained brain tissue sections from NOD/SCID mice implanted with Lepto cells (100K) alone or with OPCs (200K). J, ELISA-based analysis of TPP1 levels in serum extracted from mice on days 8, 16, 24, and 32 postimplantations of with Lepto cells (100K) alone (Control) or with OPCs (100K or 200K). K, ELISA-based analysis of GMCSF levels in serum extracted from mice on days 8, 16, 24, and 32 postimplantations with Lepto cells (100K) alone (Control) or with OPCs (100K or 200K). L, Viability of OPCs extracted from CSF samples collected between days 5 and 20 after co-implantation with Lepto cells into NOD/SCID mice, measured by CellTiter-Glo assays. M, Left, BLI-based quantification of tumor growth in Lepto1 bearing NOD/SCID mice co-implanted with OPC-shGFP (100K), OPC-shTPP1 (100K), and OPC-shTPP1 (200K) shows density-dependent elevation of Lepto1 tumor growth (***, P < 0.001). Right, Kaplan–Meier curves showing survival of mice bearing Lepto1-derived tumors co-implanted with OPC-shGFP (100K), OPC-shTPP1 (100K), and OPC-shTPP1 (200K; ***, P < 0.001). N, Left, ELISA-based analysis of TPP1 levels in serum extracted from mice on days 8, 16, 24, and 32 postimplantations with Lepto1 cells (100K), followed by co-implantation with 100K OPCs-shGFP, (100K and 200K) OPCs-shTPP1. Right, ELISA-based analysis of GMCSF levels in serum extracted from mice on days 8, 16, 24, and 32 postimplantations with Lepto1 cells (100K), followed by co-implantation with 100K OPCs-shGFP, (100K and 200K) OPCs-shTPP1. O, H&E-stained coronal brain tissue sections from NOD/SCID mice co-implanted with Lepto1 cells (100K) and with OPCs-shGFP or with OPCs-shTPP1 (100K). Red arrows, presence of Lepto-derived tumor mass. P, H&E-stained sagittal brain tissue sections from NOD/SCID mice co-implanted with Lepto1 cells (100K) and with OPCs-shGFP or with OPCs-shTPP1 (100K). Red arrows, presence of Lepto-derived tumor mass. More
Journal Articles
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Genome-wide CRISPR/Cas9 screening identifies METTL3 as a driver of <span class="search-highlight">cancer</span> m...
Published: 05 July 2022
Figure 1. Genome-wide CRISPR/Cas9 screening identifies METTL3 as a driver of cancer metastasis. A, Schematic diagram showing the process for CRISPR/Cas9 screening of key regulators of cancer metastasis. B, Heatmap showing the dropout genes involved in dynamic m6A modification in GeCKO v2-transduced cells and input cells. C, Read counts of sgRNAs targeting METTL3 in GeCKO-transduced cells and input cells. D, Diagram showing the establishment of highly metastatic ESCC sublines (KYSE150-Luc-LM5 and EC9706-Luc-LM3) via intravenous injection in a mouse model. E, Western blot analysis of METTL3 protein expression in KYSE150-Luc-LM5 and KYSE150-Luc cells as well as EC9706-Luc-LM3 and EC9706-Luc cells. F and G, Representative IHC images of METTL3 staining in 114 ESCC tissues and 66 paired normal tissues. H and I, Expression pattern of METTL3 in 40 ESCC tumors and the corresponding metastatic tissues. J, Kaplan–Meier survival patients with ESCC stratified by METTL3 expression. K, The box plots depict the expression level of METTL3 in ESCA, HNSCC, and HCC, respectively, in UALCAN database. The box indicates the concentration area of the data, and the two whiskers indicate the data extension area. The whisker upper extreme is the highest value of the data set, and the whisker lower extreme is the lowest value of the data set. And the horizontal bar in the middle of the box indicates the middle value of the entire data set (median). L, Analysis of METTL3 in patients with early (I–II) and advanced (III–IV) ESCA, HNSCC, and HCC through the UALCAN website. Bars, SDs. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figure 1. Genome-wide CRISPR/Cas9 screening identifies METTL3 as a driver of cancer metastasis. A, Schematic diagram showing the process for CRISPR/Cas9 screening of key regulators of cancer metastasis. B, Heatmap showing the dropout genes involved in dynamic m6A modification in GeCKO v2-transduced cells and input cells. C, Read counts of sgRNAs targeting METTL3 in GeCKO-transduced cells and input cells. D, Diagram showing the establishment of highly metastatic ESCC sublines (KYSE150-Luc-LM5 and EC9706-Luc-LM3) via intravenous injection in a mouse model. E, Western blot analysis of METTL3 protein expression in KYSE150-Luc-LM5 and KYSE150-Luc cells as well as EC9706-Luc-LM3 and EC9706-Luc cells. F and G, Representative IHC images of METTL3 staining in 114 ESCC tissues and 66 paired normal tissues. H and I, Expression pattern of METTL3 in 40 ESCC tumors and the corresponding metastatic tissues. J, Kaplan–Meier survival patients with ESCC stratified by METTL3 expression. K, The box plots depict the expression level of METTL3 in ESCA, HNSCC, and HCC, respectively, in UALCAN database. The box indicates the concentration area of the data, and the two whiskers indicate the data extension area. The whisker upper extreme is the highest value of the data set, and the whisker lower extreme is the lowest value of the data set. And the horizontal bar in the middle of the box indicates the middle value of the entire data set (median). L, Analysis of METTL3 in patients with early (I–II) and advanced (III–IV) ESCA, HNSCC, and HCC through the UALCAN website. Bars, SDs; **, P < 0.01; ***, P < 0.001. More
Images
Schematic diagram depicting the role of FoxM1/Rb in breast <span class="search-highlight">cancer</span> progressi...
Published: 05 July 2022
Figure 8. Schematic diagram depicting the role of FoxM1/Rb in breast cancer progression and metastasis. In the breast tumor cells, high FoxM1 expression and intact Rb protein generate FoxM1/Rb repression complex that plays critical role in accumulation of the poorly differentiated cells, cancer st... More
Images
Ribosome heterogeneity plays a role in tumorigenesis and <span class="search-highlight">cancer</span> progression...
Published: 05 July 2022
Figure 1. Ribosome heterogeneity plays a role in tumorigenesis and cancer progression. Ribosome biogenesis begins in the nucleolus where repeats of rDNA reside. RNA Pol I transcription factors, such as UBTF and SL1 bind to active clusters of rDNAs to initiate RNA Pol I transcription and pre-rRNA b... More
Images
Comparing lung-specific rules versus pan-<span class="search-highlight">cancer</span> rules in predicting drug re...
Published: 05 July 2022
Figure 3. Comparing lung-specific rules versus pan-cancer rules in predicting drug response within lung samples in gCSI ( A ) and GDSC2 ( B ). Figure 3. Comparing lung-specific rules versus pan-cancer rules in predicting drug response within lung samples in: a. gCSI, and b. GDSC2. More
Images
Erlotinib's logic model validation on PDX samples (lung <span class="search-highlight">cancer</span>). <em>Y</em>...
Published: 05 July 2022
Figure 6. Erlotinib's logic model validation on PDX samples (lung cancer). Y-axis is erlotinib response based on angle between control and treated PDXs (higher angle represents higher response). ε is (1-minimum angle) and normalization is used to show differences at low angles. P value is based on Wilcoxon signed-rank test. Figure 6. Erlotinib's logic model validation on PDX samples (lung cancer). Y-axis is Erlotinib response based on angle between control and treated PDXs (higher angle represents higher response). ε is (1-minimum angle) and normalization is used to show differences at low angles. P value is based on Wilcoxon signed-rank test. More
Images
PKM1 and PKM2 expression in normal and <span class="search-highlight">cancerous</span> human prostate tissue.  A,...
Published: 05 July 2022
Figure 7. PKM1 and PKM2 expression in normal and cancerous human prostate tissue. A, Representative IHC staining of PKM2 and PKM1 expression in low- and high-grade human prostate tumors and normal human prostate tissue is shown. Staining scored as low, intermediate, and high expression for PKM2 ... More
Journal Articles
Cancer Res can.21.2908.
Published: 06 July 2022
...Lisa M. Kim; Paul Y. Kim; Yemarshet K. Gebreyohannes; Cheuk T. Leung Many advanced therapeutics possess cytostatic properties that suppress cancer cell growth without directly inducing death. Treatment-induced cytostatic cancer cells can persist and constitute a reservoir from which recurrent...
Includes: Supplementary data
Journal Articles
Cancer Res (2022) 82 (13): 2444–2457.
Published: 05 July 2022
...Figure 1. Genome-wide CRISPR/Cas9 screening identifies METTL3 as a driver of cancer metastasis. A, Schematic diagram showing the process for CRISPR/Cas9 screening of key regulators of cancer metastasis. B, Heatmap showing the dropout genes involved in dynamic m6A modification...
Includes: Supplementary data
Journal Articles
Cancer Res (2022) 82 (13): 2458–2471.
Published: 05 July 2022
...Figure 8. Schematic diagram depicting the role of FoxM1/Rb in breast cancer progression and metastasis. In the breast tumor cells, high FoxM1 expression and intact Rb protein generate FoxM1/Rb repression complex that plays critical role in accumulation of the poorly differentiated cells, cancer...
Includes: Supplementary data
Journal Articles
Cancer Res (2022) 82 (13): 2357–2360.
Published: 05 July 2022
... addressed if a peak time could be established in treating metastatic colorectal cancer by varying the treatment times for irinotecan ( 7 ). Irinotecan was chronomodulated with a peak delivery rate at 1 of 6 clock hours staggered by 4 hours on day 1. One hundred ninety-nine patients (130 males and 63 females...
Journal Articles
Cancer Res (2022) 82 (13): 2344–2353.
Published: 05 July 2022
...Figure 1. Ribosome heterogeneity plays a role in tumorigenesis and cancer progression. Ribosome biogenesis begins in the nucleolus where repeats of rDNA reside. RNA Pol I transcription factors, such as UBTF and SL1 bind to active clusters of rDNAs to initiate RNA Pol I transcription and pre-rRNA...