Journal: eLife

Article Title: Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability

doi: 10.7554/eLife.44235

Figure Lengend Snippet: ( A ) Diagram of experimental models used to test the effect of tumor tissue of origin on TIF metabolite levels. Principal component analysis ( B ) and hierarchical clustering ( C ) of PDAC subcutaneous allograft TIF and LUAD subcutaneous allograft TIF samples based on LC/MS measurements of 104 metabolite concentrations. ( D ) Volcano plot depicting the log 2 fold change in metabolite concentration between PDAC and LUAD TIF for metabolites measured using stable isotope dilution. A fold change of 1.5 and raw p-value of 0.01 assuming unequal variance were used to select significantly altered metabolites indicated in pink. For all panels, n = 5 for PDAC subcutaneous allograft TIF samples and n = 10 for LUAD subcutaneous allograft TIF samples. 10.7554/eLife.44235.034 Figure 5—source data 1. Concentrations of 104 metabolites determined by both external standard calibration and stable isotope dilution in all subcutaneous PDAC and LUAD TIF samples in .

Article Snippet: For subcutaneous xenograft studies, 12 week old C57BL/6J animals purchased from Jackson Laboratories (IMSR Cat# JAX:000664, RRID: IMSR_JAX:000664 ) were injected with 100,000 murine PDAC or LUAD cancer cells (suspended in a volume of 100 μL of Matrigel (Corning, 354234) brought to 10 mg/ml with RPMI-1640 (Corning, 50–020-PC) into the subcutaneous space on the flank of the mice.

Techniques: Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Isotope Dilution

Journal: eLife

Article Title: Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability

doi: 10.7554/eLife.44235

Figure Lengend Snippet: ( A ) Diagram of experimental models used to test the effect of tumor tissue of origin on TIF metabolite levels. Principal component analysis ( B ) and hierarchical clustering ( C ) of PDAC subcutaneous allograft TIF and LUAD subcutaneous allograft TIF samples based on LC/MS measurements of 67 metabolites quantified using isotope-labeled internal standards. For all panels, n = 5 for PDAC subcutaneous allograft TIF samples and n = 10 for LUAD subcutaneous allograft TIF samples. 10.7554/eLife.44235.035 Figure 5—figure supplement 1—source data 1. Concentrations of 66 metabolites determined by only stable isotope dilution in all subcutaneous PDAC and LUAD TIF samples in .

Article Snippet: For subcutaneous xenograft studies, 12 week old C57BL/6J animals purchased from Jackson Laboratories (IMSR Cat# JAX:000664, RRID: IMSR_JAX:000664 ) were injected with 100,000 murine PDAC or LUAD cancer cells (suspended in a volume of 100 μL of Matrigel (Corning, 354234) brought to 10 mg/ml with RPMI-1640 (Corning, 50–020-PC) into the subcutaneous space on the flank of the mice.

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Isotope Dilution

Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: Low 15‐hydroxyprostaglandin dehydrogenase (15‐ PGDH ) expression is implicated in poor pancreatic ductal adenocarcinoma ( PDAC ) prognosis. A, Workflow diagram of patients who underwent pancreatic resection and contributed samples for immunohistochemical ( IHC ) analysis. B, Representative IHC staining of 15‐ PGDH expression in 107 PDAC tissues. Scale bar = 100 μm. C,D, Relationship between 15‐ PGDH expression and relapse‐free survival (C) or overall survival (D) using the Kaplan‐Meier method

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: 15‐Hydroxyprostaglandin dehydrogenase (15‐ PGDH ) downregulation by interleukin‐1β ( IL ‐1β) enhances pancreatic ductal adenocarcinoma cell growth. A,B, Expression of HPGD (the gene coding 15‐ PGDH protein, upper panel) or 15‐ PGDH (lower panel) in PK ‐8 cells (A) or S2‐013 cells (B) after treatment with si RNA targeting 15‐ PGDH or with control si RNA , evaluated by quantitative RT ‐ PCR (upper panel) or Western blot analysis (lower panel). Data are presented as the treated/control cell ratio. C,D, PK ‐8 cells (C) or S2‐013 cells (D) transfected with si RNA s targeting 15‐ PGDH or with control si RNA were incubated for up to 96 hours and assayed for cell number; data are presented as the treated/control (time = 0) cell ratio. E,F, Expression of 15‐ PGDH in PK ‐8 cells or S2‐013 cells after IL ‐1β (E) or tumor necrosis factor‐α ( TNF ‐α) (F) treatment for 24 and 48 hours and distilled water treatment for 48 hours as a control was evaluated by Western blotting. G, Column graph showing relative 15‐ PGDH levels in PK ‐8 cells or S2‐013 cells after IL ‐1β and TNF ‐α treatment for 24 and 48 hours, and distilled water treatment for 48 hours as a control, were evaluated using ImageJ software. H, Expression of HPGD and IL 1B in six PDAC patients determined by quantitative RT ‐ PCR . Data were normalized to the ACTB mRNA level and are shown as the mean ± SD of three independent experiments. **P < .01

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Transfection, Incubation, Software

Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: Tumor‐associated macrophages are inversely correlated with pancreatic ductal adenocarcinoma ( PDAC ) cells harboring high 15‐hydroxyprostaglandin dehydrogenase (15‐ PGDH ) expression. A,B, Representative immunohistochemical ( IHC ) staining of 15‐ PGDH (upper panel) and CD 163 (lower panel) expression in high 15‐ PGDH (A) and low 15‐ PGDH (B) serial PDAC specimens. Scale bar = 200 μm. C, Graph showing Pearson's correlation between the expression of 15‐ PGDH and the number of CD 163‐positive cells in 107 PDAC patients. D, Schematic representation of the findings of this study. IL ‐1βR, interleukin‐1β receptor

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: CTC-derived pancreatic cancer models serve as research tools and are suitable for precision medicine approaches

doi: 10.1016/j.xcrm.2024.101692

Figure Lengend Snippet: Highly efficient retrieval of CTCs from patients with pancreatic cancer (A) Comparison of CTC capture rates using EpCAM antibody-coated beads and beads with additional antibodies in PDX-derived primary PDAC cultures (SiC024). EMT induction in PDAC cultures using macrophage-conditioned media (MCM) served two purposes: mimicking clinical heterogeneity and simulating metastatic conditions. During metastasis, cancer cells often downregulate EpCAM expression, rendering EpCAM-only isolation procedures less effective. To model CTC analysis, 100 PDAC cells were spiked into 5 mL of blood obtained from healthy individuals. Beads coated with varying antibody combinations (EpCAM alone, EpCAM + CD47, EpCAM + FAP, and a triple combination of EpCAM, CD47, and FAP antibodies) were employed to isolate CTCs. The graph illustrates the count of isolated spiked-in PDAC cells as a model for CTCs ( n = 5). Boxplots show median values as horizontal lines within the boxes, with whiskers representing the 95% confidence intervals (CI). (B) Comparison of CTC retrieval efficiency of 100 cultured PDAC cells (SiC024) spiked into 5 mL of healthy blood. Three methods were used: triple antibody-coated beads, VAR2-coated beads, and immunodensity cell isolation by RosetteSep. The graph shows the number of retrieved CTCs ( n = 5). (C) Analysis of CTC retrieval from four patients with PDAC. The same volume of patient-derived blood (5 mL) was used for each isolation. Beads coated with EpCAM or triple antibody combination or VAR2 were compared for effectiveness of CTC retrieval. For one patient, enough blood was available to perform the VAR2 beads-based isolation in duplicate. The chart shows the count of isolated CTCs and the fold increase in VAR2 bead-based isolation compared to EpCAM bead-based isolation. (D) Comparison of triple-antibody cocktail-coated beads and VAR2-coated beads for isolating CTCs from patients with PDAC. The left graph illustrates CTC retrieval numbers, the middle graph presents the count of contaminating white blood cells (WBCs), and the right panel displays representative immunostainings ( n = 20). (E) VAR2-based retrieval of CD45 – CK + CTCs for individuals with various conditions, including healthy controls, pancreatitis, intraductal papillary mucinous neoplasms (IPMNs), different stages of PDAC (resectable local, locally advanced, and advanced), neuroendocrine tumors (NETs), and bile duct tumors (cholangiocarcinoma). Boxplots display median values as horizontal lines, with whiskers indicating the 95% CI. (F) Kaplan Meier analysis for overall survival in patients with PDAC patients ( n = 71), dichotomized by the “median CTC numbers” (65 CTCs). Statistical analysis by log rank test. (G) Assessment of the sensitivity of the VAR2-based retrieval method versus serum CA 19-9 levels for detecting local PDAC (left) and non-metastatic PDAC (right), respectively. The upper limit of normal (ULN) of 37 U/mL for CA 19-9 is highlighted in green. In one patient, no CTCs were detected (highlighted in orange). (H) Cox proportional hazards survival regression (Cox regression) analysis for overall 1-year survival. Hazard ratios and 95% confidence interval (CI) are shown. Definition of variables: CTC numbers ≥65/5 mL, disease stage I–IV, gender (M/F), age >65 years, CA 19.9 > 37 U/mL, C-reactive protein >3 mg/L. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001. Please also see Figure S1 .

Article Snippet: PDAC cells (1×10 5 in 50 μL Matrigel) were implanted subcutaneously into 6-8-week-old BALB/c nude mice (Charles River) as described previously., , Once PDAC tumors had reached ∼150 mm 3 , mice were randomly assigned to treatments as outlined in E. Mice were housed according to the institutional guidelines, and the procedures were approved by local animal ethics committees, i.e., SJTU approval n° A-2020-004 and Italian Ministry of Health approval n° 942/2021-PR.

Techniques: Comparison, Derivative Assay, Expressing, Isolation, Cell Culture, Cell Isolation

Journal: Cell Reports Medicine

Article Title: CTC-derived pancreatic cancer models serve as research tools and are suitable for precision medicine approaches

doi: 10.1016/j.xcrm.2024.101692

Figure Lengend Snippet: CTC-derived cultures are more aggressive compared to PDX-derived cultures (A) Invasion assay with fetal bovine serum as chemoattractant for comparing CTC-derived PDAC models versus PDX-derived cultures. Photographs on the left show invaded cells through a Matrigel-coated Boyden chamber, stained with crystal violet. Quantification is presented on the right. Boxplots show the median as a line within the box and use whiskers to represent the 95% CI. (B) In vivo metastasis assay involved injecting 100,000 luciferase-positive (Luc + ) CTC- versus PDX-derived cells into the spleen, followed by splenectomy on day 7 and subsequent tracking of Luc + liver metastasis until day 28. Quantification displayed in the upper-left panel. CTC numbers per mouse were assessed using the VAR2-coated beads method (upper-right panel). Ex vivo liver imaging, including quantification and representative ex vivo liver samples, is shown in the lower panel. (C) Immunohistochemical analysis for cytokeratin 19 (brown) to identify metastases in the liver and lungs of mice, injected with CTC-derived and PDX-derived PDAC cells. (D) Response of six representative CTC-derived models to gemcitabine (GEM), nab-paclitaxel (Abraxane; ABX), or their combination, as assessed by in vitro 3D sphere formation. (E) Corresponding patient’s clinical response for their individually allocated treatment regimen. ∗ p < 0.05 and ∗∗∗ p < 0.001. Please also see Figure S3 .

Article Snippet: PDAC cells (1×10 5 in 50 μL Matrigel) were implanted subcutaneously into 6-8-week-old BALB/c nude mice (Charles River) as described previously., , Once PDAC tumors had reached ∼150 mm 3 , mice were randomly assigned to treatments as outlined in E. Mice were housed according to the institutional guidelines, and the procedures were approved by local animal ethics committees, i.e., SJTU approval n° A-2020-004 and Italian Ministry of Health approval n° 942/2021-PR.

Techniques: Derivative Assay, Invasion Assay, Staining, In Vivo, Luciferase, Ex Vivo, Imaging, Immunohistochemical staining, Injection, In Vitro

Journal: Cell Reports Medicine

Article Title: CTC-derived pancreatic cancer models serve as research tools and are suitable for precision medicine approaches

doi: 10.1016/j.xcrm.2024.101692

Figure Lengend Snippet: CTC-derived cultures are functionally heterogeneous (A) Gating for FACSorting of SiC002 CTC-derived PDAC cells based on their mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine methyl ester (TMRM; left panel). The right panel shows the oxygen consumption rate (OCR) indicative of mitochondrial respiration in ΔΨm high cells versus low cells after sorting. (B) Mitochondrial membrane potential (ΔΨm) stained with TMRM in CSC-enriched sphere versus adherent cultures derived from a diverse set of CTC- and PDX-derived models, respectively ( n = 5). (C) ΔΨm stained with TMRM in CD133 + CSCs versus CD133 – differentiated cancer cells; n = 3. (D) qPCR analysis for mRNA levels of stemness-associated genes in ΔΨm high versus low cells sorted from SiC002 and SiC007 CTC-derived models; n = 4 (unpaired two-tailed t test). (E) Representative gating strategy of PDAC cells with low, medium, and high ΔΨm levels for FACSorting. (F) Representative images (left) and quantification (right) of sphere formation capacity on day 7 following ΔΨm sorting in two different CTC-derived PDAC models; n = 6 (unpaired two-tailed t test). (G) In vivo tumorigenicity of graded cell numbers for two different CTC-derived PDAC models after sorting for ΔΨm levels. Images of explanted tumors (left) and extreme limiting dilution analysis (right). (H) ΔΨm stained with TMRM for CXCR4 + versus CXCR4 – cells. Representative flow cytometry plots of CXCR4 gating and TMRM staining (left), quantification of mean fluorescence intensity (MFI) of TMRM staining (right); n = 3. (I) Immunofluorescence for CXCR4 (white) and TMRM (red) staining. DAPI serves as a nuclear stain. Representative images are on the left, with quantification on the right for two different CTC-derived PDAC models; n = 41 to 208 cells (horizontal lines indicate the mean values). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Please also see Figure S4 .

Article Snippet: PDAC cells (1×10 5 in 50 μL Matrigel) were implanted subcutaneously into 6-8-week-old BALB/c nude mice (Charles River) as described previously., , Once PDAC tumors had reached ∼150 mm 3 , mice were randomly assigned to treatments as outlined in E. Mice were housed according to the institutional guidelines, and the procedures were approved by local animal ethics committees, i.e., SJTU approval n° A-2020-004 and Italian Ministry of Health approval n° 942/2021-PR.

Techniques: Derivative Assay, Membrane, Staining, Two Tailed Test, In Vivo, Flow Cytometry, Fluorescence, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: CTC-derived pancreatic cancer models serve as research tools and are suitable for precision medicine approaches

doi: 10.1016/j.xcrm.2024.101692

Figure Lengend Snippet: Pancreatic CTCs comprise a subset of CXCR4 + CSCs (A) VAR2-based retrieval of CXCR4 + CD45 – CK + CTCs from individuals with various conditions, including healthy controls, pancreatitis, IPMNs, different stages of PDAC (resectable local, locally advanced, and advanced), NETs, and bile duct tumors (cholangiocarcinoma). The content of CD45 – CK + CXCR4 + CTCs among total CTCs at baseline is shown (left). Boxplots indicate median values with horizontal lines, and whiskers represent the 95% CI. Representative image of a retrieved CD45 – CK + CXCR4 + CTC (right). (B) Kaplan-Meier analysis for overall survival in patients with PDAC dichotomized for the “median of CXCR4 + CTC content” (8.9%). Statistical analysis by log rank test. (C) Cox proportional hazards survival regression (Cox regression) analysis for overall 1-year survival, including the variable “CXCR4 + CTC content.” Hazard ratios and 95% CI are shown on a logarithmic scale. (D) Single-cell droplet digital PCR (ddPCR) analysis. Upper panel: schematic illustration of the workflow. Lower panel: ddPCR results of freshly retrieved CXCR4 + versus CXCR4 – CTCs for the indicated genes plotted ( n = 9 each). (E) Flow cytometry illustrating the content of CXCR4 + cells for PDX- versus CTC-derived cultures. (F) Invasive capacity of CXCR4 + versus CXCR4 – cells for CTC- and PDX-derived cultures, respectively. Cells sorted for CXCR4 were placed in the upper chamber of a Boyden chamber, and invasion toward 20% fetal bovine serum (FBS) in the lower chamber was recorded ( n = 4–8). (G) In vivo tumorigenicity of CXCR4 + versus CXCR4 – cells for two representative CTC-derived models. Graded doses (1 to 10 3 cells/injection) were transplanted into immunocompromised nude mice. Table providing quantification (upper panel), estimate of stem cell frequency in vivo for CXCR4 – (black) versus CXCR4 + (red) CTC-derived PDAC cells by limiting dilution assay (lower panel). Depicted is a log-fraction plot of the limiting dilution model. The slope of the line is the log-active cell fraction. The dotted lines give the 95% CI. Analysis was performed at https://bioinf.wehi.edu.au/cgi-bin/limdil/limdil.pl . ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Please also see Figure S5 .

Article Snippet: PDAC cells (1×10 5 in 50 μL Matrigel) were implanted subcutaneously into 6-8-week-old BALB/c nude mice (Charles River) as described previously., , Once PDAC tumors had reached ∼150 mm 3 , mice were randomly assigned to treatments as outlined in E. Mice were housed according to the institutional guidelines, and the procedures were approved by local animal ethics committees, i.e., SJTU approval n° A-2020-004 and Italian Ministry of Health approval n° 942/2021-PR.

Techniques: Digital PCR, Flow Cytometry, Derivative Assay, In Vivo, Injection, Limiting Dilution Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone

doi: 10.1002/stem.572

Figure Lengend Snippet: PDACs injected intralesionally gradually disappear from bone within 2–4 weeks. Luciferase/GFP-expressing PDACs were analyzed by live animal imaging and immunohistochemistry. (A, B): Myelomatous severe combined immunodeficient (SCID)–rab mice were engrafted with myeloma cells from patient 1 and were intralesionally treated with luciferase/enhanced green fluorescent protein (EGFP)–expressing PDACs, which were examined by (A) live animal imaging and (B) bioluminescence analysis. (C) Myelomatous bone sections were immunohistochemically stained for EGFP 5 days and 5 weeks after injection of PDACs; note that clusters of PDAC were visible at day 5, whereas at 5 weeks, PDACs were occasionally detected with fibroblastoid-like and occasionally osteoblast-like, morphologies. (D): Nonmyelomatous SCID-rab mice were injected (via intrabone route) with PDACs and were examined by live animal imaging. (E): Hosts were subcutaneously injected with 5 × 106 PDACs (using HyStem-C hydrogel carrier) and examined by live animal imaging. See “Materials and Methods” section for the acquisition and processing of images. Abbreviations: OB, osteoblasts; PDAC, placenta-derived adherent cell.

Article Snippet: Placenta-Derived Adherent Cell PDACs were obtained from Celgene Cellular Therapeutics (Warren, NJ).

Techniques: Injection, Luciferase, Expressing, Imaging, Immunohistochemistry, Staining, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone

doi: 10.1002/stem.572

Figure Lengend Snippet: PDACs inhibit growth of H929 myeloma cells in bone but not in subcutaneous microenvironment. H929 myeloma cells were engrafted in the implanted bone in Severe combined immunodeficient (SCID)–rab mice (A–D) or injected subcutaneously in SCID mice (E, F). On establishment of myeloma, vehicle (CONT) or PDACs (1 × 106 cells per mouse) were injected into the implanted bone or subcutaneous tumor (eight mice per group). In SCID-rab mice engrafted with myeloma, (A) changes in BMD of the implanted bone; (B) representative x-ray radiographs of the myelomatous bones from three of eight mice from each group, taken before treatment and at the end of the experiment (final); (C) numbers of tartrate-resistant acid phosphatase-expressing osteoclasts and osteocalcin-expressing osteoblasts in the myelomatous bone sections; and (D) human Ig (hIg) levels before treatment and at the end of the experiment. (E, F): In SCID mice with subcutaneous myeloma tumors, (E) hIg levels before treatment and at the end of the experiment and (F) subcutaneous tumor volume. Abbreviations: BMD, bone mineral density; CONT, control vehicle; PDAC, placenta-derived adherent cell; pre-Rx, prior to treatment.

Article Snippet: Placenta-Derived Adherent Cell PDACs were obtained from Celgene Cellular Therapeutics (Warren, NJ).

Techniques: Injection, Expressing, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone

doi: 10.1002/stem.572

Figure Lengend Snippet: PDACs are capable of trafficking to myelomatous bone. Luciferase/enhanced green fluorescent protein (EGFP)–expressing PDACs were intravenously injected into SCID–rab mice engrafted with H929 myeloma cells; at indicated times, labeled PDACs were examined by live animal and ex vivo imaging and by immunohistochemical analysis for GFP. (A): Live animal imaging and ex vivo imaging of implanted bones in individual mice. Bioluminescence activity was detected in implanted bones of 10 of 18 mice (numbers 2, 5, 6, 7, 8, 9, 12, 13, 15, and 18). (B): Sections of implanted myelomatous bones were immunohistochemically stained for GFP, which detected PDACs in these bones 1 and 7 days after they were injected. See “Materials and Methods” section for the acquisition and processing of images. Abbreviations: MM, multiple myeloma; PDAC, placenta-derived adherent cell.

Article Snippet: Placenta-Derived Adherent Cell PDACs were obtained from Celgene Cellular Therapeutics (Warren, NJ).

Techniques: Luciferase, Expressing, Injection, Labeling, Ex Vivo, Imaging, Immunohistochemical staining, Activity Assay, Staining, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone

doi: 10.1002/stem.572

Figure Lengend Snippet: PDACs induce apoptosis in osteoclast precursors and inhibit osteoclast formation. Osteoclast precursors were cultured alone (CONT) or cocultured with PDACs in noncontact conditions in osteoclast medium. (A): Numbers of apoptotic (annexin V positive) or nonviable (PI positive) osteoclast precursors after 4 days of culture. (B): Representative photos demonstrating the effects of PDACs on osteoclast apoptosis (top panel) and viability (middle panel). Total numbers of TRAP-expressing osteoclast precursors are visualized on the bottom panel. (C): Numbers of multi-nucleated and TRAP-expressing osteoclasts after 8 days of culture. (D): Representative photos demonstrating the effects of PDACs on OC differentiation. See “Materials and Methods” section for the acquisition and processing of images. Abbreviations: CONT, control vehicle; OC, osteoclast; PDAC, placenta-derived adherent cell; PI, propidium iodide; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Placenta-Derived Adherent Cell PDACs were obtained from Celgene Cellular Therapeutics (Warren, NJ).

Techniques: Cell Culture, Expressing, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone

doi: 10.1002/stem.572

Figure Lengend Snippet: Survival of myeloma cells is poorer in coculture with PDACs than with MSCs. (A): The luciferase-expressing myeloma cell lines U266 and JB were cultured in a 96-well plate alone (CONT; 104 cells per well) or were cocultured for 7 days with PDACs or fetal MSCs (104 cells per well, for each cell type) and then subjected to bioluminescence quantification for measurement of growth rate [31]. Note that MSCs and PDACs supported growth of myeloma cells but the effect of MSCs was more profound. Indicated p values are compared with CONT group. (B): Similar to (A), a panel of seven luciferase-expressing myeloma cell lines (BN, JB, ARP1, U266, DNC, HLE, H929; 104 cells per well) were cocultured for 7 days with fetal MSCs, Pt. MSCs, or PDACs and then subjected to bioluminescence quantification. Results are expressed as fold of myeloma cell growth in coculture with fetal MSCs. Note reduced growth of myeloma cell lines in coculture with PDACs (p < .01, comparing all tested cell lines using analysis of variance). (C): CD138-selected myeloma cells (5 × 105 cells per well) from six patients were cocultured for 7 days in a 24-well plate in conditions allowing limited cell-cell contact with fetal MSCs or PDACs (105 cells per well, for each cell type) and then subjected to trypan blue analysis. Survival of myeloma cells was slightly, but not significantly, lower in coculture with PDACs than with fetal MSCs. (D): Annexin V/PI analysis of CD138-selected myeloma cells (from patient D) revealed a higher percentage of apoptotic myeloma cells in coculture with PDACs than with fetal MSCs. Abbreviations: CONT, control vehicle; MSC, mesenchymal stem cell; PDAC, placenta-derived adherent cell; Pt. MSC, patient mesenchymal stem cell.

Article Snippet: Placenta-Derived Adherent Cell PDACs were obtained from Celgene Cellular Therapeutics (Warren, NJ).

Techniques: Luciferase, Expressing, Cell Culture, Derivative Assay

Journal: Science (New York, N.Y.)

Article Title: Modulation of the Proteostasis Network Promotes Tumor Resistance to Oncogenic KRAS Inhibitors

doi: 10.1126/science.abn4180

Figure Lengend Snippet: (A) Schematic illustration of labeling and detection of misfolded and aggregated proteins with PROTEOSTAT dye. Upon intercalation into the cross-beta spine typically found in misfolded and aggregated proteins, PROTEOSTAT dye emits strong fluorescence. (B and C) Representative images (B) and quantification (C) of PROTEOSTAT (magenta) and DAPI (blue) staining in iKrasP cells at different time points after KrasG12D inactivation by Dox-withdrawal (Off Dox) until the cells acquired resistance to KrasG12D inactivation (iKrasR cell). (D and E) Representative images (D) and quantification (E) of PROTEOSTAT (magenta) staining in spontaneous tumors from the Dox-inducible, KrasG12D-driven PDAC mouse model (iKras GEMM) treated with doxycycline (Dox, 2g/L, n=5), Dox withdrawal for 3 days (n=4) or relapsed after 30 weeks of Dox-withdrawal (n=7). (F) Quantification of PROTEOSTAT intensity in parental MIA-PaCa-2 (MIAP) cells treated with DMSO or 30nM sotorasib for 2 days or in sotorasib-resistant MIA-PaCa-2 (MIAR) cells treated with 30nM sotorasib. MIAR cells were generated in vitro by continued sotorasib treatment until the cells acquired resistance. (G and H) Representative images (G) and quantification (H) of PROTEOSTAT (magenta) and DAPI (blue) staining in MIA-PaCa-2 xenograft tumors treated with vehicle (n=4), sotorasib (30mg/kg for 1 day, n=3), or relapsed after 9 weeks of sotorasib treatment (30mg/kg, n=4). Data represent average fluorescence intensity of PROTEOSTAT/cell from each image (C and F) or tumor (E and H) and are presented as mean ± SD from n≥10 images. Scale bar: 20μm. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (C, E, F and H) was used to calculate P values. n.s., not significant, * P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Oncogenic KRAS Inactivation Reprograms Proteostasis To understand the impacts of oncogenic KRAS on proteostasis, we used primary mouse PDAC cells derived from our previously generated, doxycycline (Dox)-inducible, Kras G12D -driven PDAC mouse model ( 55 ), which are hereafter designated as iKras cells.

Techniques: Labeling, Fluorescence, Staining, Generated, In Vitro

Journal: Neoplasia (New York, N.Y.)

Article Title: PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

doi: 10.1016/j.neo.2022.100793

Figure Lengend Snippet: Tumor growth and survival of BxPC3-HAS3 and BxPC3-wild tumors in response to radiation and PEGPH20 therapy. A Treatment plan. Treatment was initiated when the tumor size reached approximately 500 mm3 and consisted of a single dose of 2.5 Gy radiation and/or 1mg/kg PEGPH20 given every 3 days. B Growth kinetics of BxPC3-wild type tumors C Kaplan-Meier survival curve for BxPC3-wild type tumors (n = 10 per group). Survival refers to the time before reaching the maximally allowed tumor volume of 2,500 mm 3 . Asymmetric 95% CIs (shaded region) were determined by the profile method. D Growth kinetics of BxPC3-HAS3 tumors E Kaplan-Meier survival curve for BxPC3-wild type tumors (n = 10 per group). F Growth rates estimated from a Gompertz logistic growth model of the data in B and D. * indicates pair-wise comparisons that are statistically significant. G Hazard ratios of the treatment effect calculated (lower numbers favor the second treatment listed). * indicates Hazard Ratios that are statistically different from 1, taking into account the number of comparisons by the Bonferroni correction.

Article Snippet: PEGPH20 and BxPC3 PDAC (human pancreatic adenocarcinoma) cells lines transduced with hyaluronan synthase 3 (HAS3) were provided by Halozyme Inc. Out of the three hyaluronan synthase isoenzymes, HAS3 is the isoenzyme most responsible for extracellular HA accumulation and therefore the isoenzyme most likely to have a vascular impact on the tumor microenvironment.

Techniques:

Journal: Neoplasia (New York, N.Y.)

Article Title: PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

doi: 10.1016/j.neo.2022.100793

Figure Lengend Snippet: EPR imaging showed increased pO 2 after PEGPH20 treatment. EPR imaging was performed in BxPC3-HAS3 tumor bearing mice treated with control buffer or PEGPH20 (n = 7 each group) before (before the first injection on day 0) and after treatment (3 h after the 2nd PEGPH20 injection on day3). A, Representative oxygen map obtained by EPR imaging and T2-weighted anatomical image. B, Histograms of pO 2 distribution within the tumor of each group. C and E, The mean pO 2 of each tumor before and after treatment are shown by treatment group. Individual values are shown. D and F, pO 2 change (%) from the baseline (pre-treatment) of each group. Data are shown as mean ± SE. G and H, Histological assessment of pimonidazole staining. BxPC3-HAS3 tumors treated with control buffer or PEGPH20 were surgically resected 3h after the 2nd PEGPH20 injection on day3. Frozen sections of tumors were stained for exogenous hypoxia marker pimonidazole (pimo). G, Representative pimo staining of each group (scale bar = 1mm). H, Quantification of pimo positive area. I , Comparison of ΔHF10 between control and PEGPH20 groups evaluated by EPR imaging. Data are shown as mean ± SE (n = 5 per group). Statistical significance between groups was determined by paired t -test for C and by Student's t -test for D and F . * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Article Snippet: PEGPH20 and BxPC3 PDAC (human pancreatic adenocarcinoma) cells lines transduced with hyaluronan synthase 3 (HAS3) were provided by Halozyme Inc. Out of the three hyaluronan synthase isoenzymes, HAS3 is the isoenzyme most responsible for extracellular HA accumulation and therefore the isoenzyme most likely to have a vascular impact on the tumor microenvironment.

Techniques: Imaging, Control, Injection, Staining, Marker, Comparison

Journal: Neoplasia (New York, N.Y.)

Article Title: PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

doi: 10.1016/j.neo.2022.100793

Figure Lengend Snippet: DCE-MRI showed improved intratumor perfusion by PEGPH treatment. DCE-MRI was performed in BxPC3-HAS3 tumor bearing mice treated with control buffer or PEGPH20 (n = 7, n = 8 each group) before (before the first injection on day 0) and after treatment (3 h after the 2nd PEGPH20 injection on day3). A, Representative images of Gd-DTPA intensity with T2-weighted anatomical image B, Representative time-intensity kinetics of Gd-DTPA. C, The mean K trans value of each tumor before and after treatment are shown by treatment group. Individual values are shown. D, K trans change (%) from the baseline (pre-treatment) of each group. Data are shown as mean ± SE. Statistical significance between groups was determined by paired t -test for C and by Student's t -test for D . * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Article Snippet: PEGPH20 and BxPC3 PDAC (human pancreatic adenocarcinoma) cells lines transduced with hyaluronan synthase 3 (HAS3) were provided by Halozyme Inc. Out of the three hyaluronan synthase isoenzymes, HAS3 is the isoenzyme most responsible for extracellular HA accumulation and therefore the isoenzyme most likely to have a vascular impact on the tumor microenvironment.

Techniques: Control, Injection

Journal: Neoplasia (New York, N.Y.)

Article Title: PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

doi: 10.1016/j.neo.2022.100793

Figure Lengend Snippet: USPIO MRI shows increased blood volume after PEGPH20 treatment. MRI using USPIO was performed in BxPC3-HAS3 tumor bearing mice treated with control buffer or PEGPH20 (n = 3 each group) before (before the first injection on day 0) and after treatment (3 h after the 2nd PEGPH20 injection on day3). A, Representative imaging of USPIO intensity with T2-weighted anatomical image. Center four slices of each tumor are shown. B, Blood volume (%) of region of interest in each tumor (blue line in Fig. 3 A) before and after treatment are shown by treatment group. Individual values are shown. C, Blood volume change (%) from the baseline (pre-treatment) of each group. Data are shown as mean ± SE. Statistical significance between groups was determined by paired t -test for B and by Student's t -test for C .

Article Snippet: PEGPH20 and BxPC3 PDAC (human pancreatic adenocarcinoma) cells lines transduced with hyaluronan synthase 3 (HAS3) were provided by Halozyme Inc. Out of the three hyaluronan synthase isoenzymes, HAS3 is the isoenzyme most responsible for extracellular HA accumulation and therefore the isoenzyme most likely to have a vascular impact on the tumor microenvironment.

Techniques: Control, Injection, Imaging

Journal: Neoplasia (New York, N.Y.)

Article Title: PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

doi: 10.1016/j.neo.2022.100793

Figure Lengend Snippet: Photoacoustic imaging showed increased oxygen saturation after PEGPH20 injection. Photoacoustic imaging was performed in BxPC3-HAS3 tumor bearing mice treated with control buffer or PEGPH20 for 25 min after the bolus-dose injection (control; n = 1, PEGPH20 1 mg/kg: n=1, PEGPH20 10 mg/kg: n=1). A, Anatomical image (B mode) and parametric image of oxygen saturation of hemoglobin (sO 2 ) at 20 min after the injection of control buffer (top row), PEGPH20 1 mg/kg (middle row), PEGPH20 10 mg/kg (bottom row). B, sO 2 and total hemoglobin (Hb) of region of interest from the images in Fig. 4 A (purple line) are quantified and plotted. C, Bar plot of change in sO 2 (%) and % change of total Hb 20 min after injection.

Article Snippet: PEGPH20 and BxPC3 PDAC (human pancreatic adenocarcinoma) cells lines transduced with hyaluronan synthase 3 (HAS3) were provided by Halozyme Inc. Out of the three hyaluronan synthase isoenzymes, HAS3 is the isoenzyme most responsible for extracellular HA accumulation and therefore the isoenzyme most likely to have a vascular impact on the tumor microenvironment.

Techniques: Imaging, Injection, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

doi: 10.1016/j.neo.2022.100793

Figure Lengend Snippet: HP [1- 13 C] Pyruvate MRI shows decreased Lac/Pyr ratio after PEGPH20 treatment. HP [1- 13 C] Pyruvate MRI was performed in BxPC3-HAS3 tumor bearing mice treated with control buffer or PEGPH20 (n = 4, n = 5 each group) before (before the first injection on day 0) and after treatment (3 h after the 2nd PEGPH20 injection on day3). A, Representative kinetics of [1- 13 C] pyruvate and [1- 13 C] lactate and its anatomical 1 H image. B, Lac/Pyr ratio of each tumor before and after treatment are shown by treatment group. Individual values are shown. C, Lac/Pyr ratio change from the baseline (pre-treatment) of each group. Data are shown as mean ± SE. Statistical significance between groups was determined by paired t -test for B and by Student's t -test for C . * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Article Snippet: PEGPH20 and BxPC3 PDAC (human pancreatic adenocarcinoma) cells lines transduced with hyaluronan synthase 3 (HAS3) were provided by Halozyme Inc. Out of the three hyaluronan synthase isoenzymes, HAS3 is the isoenzyme most responsible for extracellular HA accumulation and therefore the isoenzyme most likely to have a vascular impact on the tumor microenvironment.

Techniques: Control, Injection