kpc mt4 cells  (Cold Spring Harbor Laboratory Meetings)

Journal: Cancer research

Article Title: Apolipoprotein E promotes immune suppression in pancreatic cancer through NF-κB-mediated production of CXCL1

doi: 10.1158/0008-5472.CAN-20-3929

Figure Lengend Snippet: (A) Dot plot of Ldlr, Vldlr, Lrp1, and Lrp8 in orthotopic KPC samples. Color represents average expression, while size of the dot represents expression frequency. (B) Dot plot of LDLR, VLDLR, LRP1, and LRP8 in human PDA. Color represents average expression, while size of the dot represents expression frequency. (C) Violin plot of normalized LDLR expression in human PDA. (D) Heatmap of differentially expressed genes in in vitro 7940b KPC cells treated with vehicle (n=3) compared to 7940b KPC cells treated with 0.3 μg/mL murine recombinant APOE (n=3) for 48 hours. High expression is in red, while low expression is in blue. (E) qRT-PCR analysis of Cxcl1 and Cxcl5 mRNA levels relative to Cyclophilin A housekeeping in 4 KPC cell lines (7940b, mT3, mT4, mT5). Dotted line represents fold induction compared to vehicle-treated cells normalized to 1. Statistical significance was determined using one-way ANOVA with Tukey’s test for multiple correction. (F) Survival analysis of PDA patients stratified by plasma CXCL1 levels. CXCL1 Low (n=38) and CXCL1 High (n=38). Statistical significance was determined using Log-rank (Mantel-Cox) test. (G) qRT-PCR analysis for Cxcl1 mRNA levels relative to Cyclophilin A housekeeping in wild-type fibroblasts (BLK6318) and cancer-associated fibroblasts (FB1), treated with vehicle (n=2-3) or 0.3 μg/mL recombinant ApoE (n=2-3) for 48 hours. Statistical significance was determined by two-tailed t tests. (H) qRT-PCR analysis of Cxcl1 and Cxcl5 mRNA levels relative to Cyclophilin A housekeeping in WT (n=6) and ApoE−/− (n=5) tumors. Statistical significance was determined using two-tailed t test. n.s. = not significant. (I) Co-immunofluorescence staining of CXCL1 (green), CK19 (red), αSMA (white), and DAPI (blue) in WT and ApoE−/− orthotopic KPC tumors. (J) Experimental design schematic. (K) qRT-PCR analysis of Cxcl1 mRNA levels relative to Cyclophilin A housekeeping in 7940b tumor cells alone control (n=6), 7940b cells cultured with WT macrophage conditioned media (n=6), 7940b cells cultured with ApoE−/− macrophage conditioned media (n=6), and 7940b cells cultured with ApoE−/− macrophage conditioned media with 0.3 μg/mL recombinant ApoE (n=3). Statistical significance was determined by two-tailed t tests between groups.

Article Snippet: We thank Dr. David Tuveson, at Cold Spring Harbor Laboratory for the generous gift of the mT3, mT4, and mT5 KPC cell lines.

Techniques: Expressing, In Vitro, Recombinant, Quantitative RT-PCR, Clinical Proteomics, Two Tailed Test, Immunofluorescence, Staining, Control, Cell Culture

Journal: Nature Communications

Article Title: Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade

doi: 10.1038/s41467-021-23731-7

Figure Lengend Snippet: a , b Volcano plots of the kinase activity prediction in neutrophils stimulated with ( a ) control or KPC-conditioned medium (KPC-CM) or ( b ) KPC-CM for 1 h in the presence of vehicle or 10 µM lorlatinib (data show results from three independent experiments). c Immunoblot analysis of p-STAT5, STAT5, p-STAT3, and STAT3 in neutrophils stimulated with control or KPC-CM in the presence of vehicle or lorlatinib (immunoblots are representative of one experiment performed three times). Sample integrity controls are vinculin and tubulin, respectively. Unprocessed scans of immunoblots are shown in Supplementary Fig. . d Transwell migration of neutrophils towards KPC-CM in presence of vehicle (V), 1 μM or 10 μM lorlatinib ( n = 3 independent experiments). e Representative immunofluorescence images of neutrophil/KPC-zsGreen co-cultures in the presence of vehicle or lorlatinib after 2 days of culture ( n = 3 independent experiments). f Orthotopic implantation of KPC mT4 cells into the pancreas. Mice were treated daily with vehicle (V) or 5 mg/kg lorlatinib (L) for 2 weeks starting 14 days after implantation. g Tumor weight of orthotopic PDAC tumors after treatment with vehicle or 5 mg/kg lorlatinib ( n = 12 mice per condition; data are from two independent experiments). h Representative IHC images of Ki67 in tumors from mice treated with vehicle control or lorlatinib ( n = 11 mice, vehicle; n = 6 mice, lorlatinib). Data are from two independent experiments. i Flow cytometry analysis of neutrophils in PDAC tumors from mice treated with vehicle or lorlatinib ( n = 11 mice, vehicle; n = 9 mice, lorlatinib; data are from two experiments). j – l Representative images of ( j ) neutrophils (Ly6G + ; purple) ( n = 10 mice, vehicle; n = 9 mice, lorlatinib), ( k ) picrosirius red ( n = 11 mice, vehicle; n = 10 mice, lorlatinib), and ( l ) fibroblasts (αSMA + ; green) ( n = 12 mice, vehicle; n = 10 mice, lorlatinib) in tumors from mice treated with vehicle control or lorlatinib. Data are from two independent experiments. Scale bars represent 100 µm ( h , j – l ) or 200 µm ( e ). Box-and-whisker plot shows the median (line), mean (plus sign), 25th and 75th percentiles (box), and 5th and 95th percentiles (whiskers); bar graphs represent mean and standard deviation; hypothesis testing performed using two-way ANOVA with Tukey’s method for multiple comparisons ( d , e ) or unpaired two-sided Student’s t test ( g – l ). Source data are provided as a Source Data file.

Article Snippet: Murine pancreatic cancer cells KPC mT4, generated in the Tuveson Laboratory (Cold Spring Harbor Laboratory, NY, USA), were isolated from PDAC tumor tissues obtained from Kras LSL-G12D/+ ;Trp53 LSL-R172H/+ ;Pdx1–Cre mice of a pure C57BL/6 background .

Techniques: Activity Assay, Control, Western Blot, Migration, Immunofluorescence, Flow Cytometry, Whisker Assay, Standard Deviation

Journal: Nature Communications

Article Title: Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade

doi: 10.1038/s41467-021-23731-7

Figure Lengend Snippet: a Intrasplenic injection of KPC mT4. Mice were treated daily with vehicle (V) or 5 mg/kg lorlatinib (L) for 2 weeks starting 7 days after implantation. b , c Quantification of hepatic metastatic burden after vehicle or lorlatinib treatment by ( b ) liver weight (%) of total body weight and ( c ) hematoxylin & eosin staining and average size of metastatic lesions ( n = 9 mice, vehicle; n = 11 mice; data are from two independent experiments). d Representative IHC images of Ki67 ( n = 9 mice per group). Data are from two independent experiments. e Flow cytometry analysis of neutrophils (CD11b + F4/80 neg Ly6C neg Ly6G + ) in hepatic metastases from vehicle- or lorlatinib-treated mice ( n = 8 mice, vehicle; n = 10 mice, lorlatinib; data are from two independent experiments). f – h Representative images of ( f ) neutrophils (Ly6G + ; purple) ( n = 8 mice, vehicle; n = 10 mice, lorlatinib), ( g ) picrosirius red ( n = 8 mice, vehicle; n = 9 mice, lorlatinib), and ( h ) fibroblasts (αSMA + ; green) ( n = 8 mice, vehicle; n = 10 mice, lorlatinib) in hepatic metastases from vehicle- and lorlatinib-treated mice. Data are from two independent experiments. i Intrasplenic injection of CRC organoids from villin CreER Apc fl/fl Kras G12D/+ Trp53 fl/R172H TgfbrI fl/fl (AKPT). Mice were treated with vehicle or 5 mg/kg lorlatinib starting 9 days after injection. Hepatic metastatic burden was determined 20 days after the start of treatment. j , k Quantification of hepatic metastases in mice after intrasplenic injection of CRC organoids and vehicle or lorlatinib treatment by ( j ) liver weight (%) of total body weight and ( k ) representative hematoxylin & eosin staining with quantification of the average metastatic lesion size ( n = 5 per group; data are from one experiment). Black lines show the outline of individual metastatic lesions. l – o Representative IHC and immunofluorescence images of ( l ) Ki67 ( n = 4 mice, vehicle; n = 5 mice, lorlatinib), ( m ) neutrophils (Ly6G + ; green) ( n = 5 mice per group), ( n ) picrosirius red ( n = 5 mice per group), and ( o ) fibroblasts (αSMA + ; green) ( n = 5 mice per group) in hepatic metastases from vehicle- and lorlatinib-treated mice. Data are from one experiment. Scale bars represent 5 mm ( c , k ) or 100 µm ( d , f – h , l – o ). Box-and-whisker plot shows the median (line), mean (plus sign), 25th and 75th percentiles (box), and 5th and 95th percentiles (whiskers); hypothesis testing performed using unpaired two-sided Student’s t test ( b – o ). Source data are provided as a Source Data file.

Article Snippet: Murine pancreatic cancer cells KPC mT4, generated in the Tuveson Laboratory (Cold Spring Harbor Laboratory, NY, USA), were isolated from PDAC tumor tissues obtained from Kras LSL-G12D/+ ;Trp53 LSL-R172H/+ ;Pdx1–Cre mice of a pure C57BL/6 background .

Techniques: Injection, Staining, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Nature Communications

Article Title: Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade

doi: 10.1038/s41467-021-23731-7

Figure Lengend Snippet: a Orthotopic implantation of KPC mT4 cells into the pancreas. Mice were treated daily with combinations of vehicle (V), 5 mg/kg lorlatinib (L), 200 μg control IgG and anti-Ly6G. b Flow cytometry analysis of neutrophils in PDAC tumors from mice treated with a combination of vehicle, lorlatinib, control IgG and anti-Ly6G ( n = 9 mice, vehicle + control IgG, and anti-Ly6G + lorlatinib groups; n = 10 mice, remaining groups). c Tumor weight of orthotopic PDAC tumors from mice treated with a combination of vehicle, lorlatinib, control IgG, and anti-Ly6G ( n = 9 mice, vehicle + control IgG; n = 10 mice, remaining conditions). Data are from two experiments. d Flow cytometry analysis of immune cells at day 28 in blood samples from mice with PDAC tumors treated with vehicle or 5 mg/kg lorlatinib for 14 days (see outline of the experiment in Fig. f) ( n = 9 mice, vehicle; n = 10 mice, lorlatinib (monocytes/neutrophils); n = 10 mice per group (T cells); n = 6 mice per group (B cells); data are from two independent experiments). e Flow cytometry analysis of immature (immNeu) and mature (matNeu) neutrophils in PDAC tumors at day 28 after treatment with vehicle or lorlatinib for 14 days ( n = 9 mice, vehicle; n = 10 mice, lorlatinib; data are from two independent experiments). f Flow cytometry analysis at day 28 of GMPs, pre-neutrophils (preNeu), immature and mature neutrophils in the bone marrow (BM) from mice with PDAC tumors treated with vehicle or lorlatinib for 14 days ( n = 12 mice per group; data are from two independent experiments). g Colony-forming unit – granulocyte macrophage (CFU–GM) assay of BM cells from healthy mice with vehicle or lorlatinib. Colonies with >20 cells were quantified after 3 days ( n = 4 independent experiments). h – j The number of colonies from CFU–GM ( h ) and flow cytometry analysis ( g ) of neutrophils on day 3 after CFU–GM culture in the ( i ) absence or ( j ) presence of recombinant G-CSF and lorlatinib ( n = 4 independent experiments). k CFU–GM of BM cells isolated on day 28 from mice with PDAC tumors after daily treatment with vehicle or lorlatinib for 14 days ( n = 4 mice per group). Scale bars represent 200 µm. Box-and-whisker plot shows the median (line), mean (plus sign), 25th and 75th percentiles (box), and 5th and 95th percentiles (whiskers); hypothesis testing performed using two-way ANOVA with Tukey’s ( b , c , g , h ) or Sidak’s ( d – f , i , j ) method for multiple comparisons or unpaired two-sided Student’s t test ( k ). Source data are provided as a Source Data file.

Article Snippet: Murine pancreatic cancer cells KPC mT4, generated in the Tuveson Laboratory (Cold Spring Harbor Laboratory, NY, USA), were isolated from PDAC tumor tissues obtained from Kras LSL-G12D/+ ;Trp53 LSL-R172H/+ ;Pdx1–Cre mice of a pure C57BL/6 background .

Techniques: Control, Flow Cytometry, Recombinant, Isolation, Whisker Assay

Journal: Nature Communications

Article Title: Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade

doi: 10.1038/s41467-021-23731-7

Figure Lengend Snippet: a Orthotopic implantation of KPC mT4 cells into the pancreas. Mice were treated daily with vehicle (V) or 5 mg/kg lorlatinib (L) for 2 weeks and PBS or 50 mg/kg gemcitabine twice per week starting 14 days after implantation. b Weight of PDAC tumors from mice treated with vehicle or lorlatinib in combination with PBS or Gemcitabine ( n = 8 mice per condition; data are from two independent experiments). c Orthotopic implantation of KPC mT4 cells into the pancreas. Mice were treated daily with vehicle (V) or 5 mg/kg lorlatinib (L) for 2 weeks and 250 μg control IgG or anti-PD-1 antibodies three times per week starting 14 days after implantation. d Weight of PDAC tumors from mice treated with combinations of vehicle, lorlatinib, control IgG, or anti-PD-1 ( n = 12 mice per condition, except n = 11 mice for control IgG + lorlatinib condition; data are from three independent experiments). e , f Flow cytometry analysis of ( e ) neutrophils and ( f ) CD8 + T cells in PDAC tumors from mice treated with combinations of anti-PD-1 and lorlatinib ( n = 10 mice, control IgG + vehicle; n = 11 mice, control IgG + lorlatinib; n = 12 mice, remaining groups; data are from three independent experiments). g Immunofluorescence analysis and quantification of neutrophils (Ly6G + ; purple) and T cells (CD8 + ; green) cells in PDAC tumors treated for 14 days with combinations of anti-PD-1 and lorlatinib ( n = 10 mice, control IgG + vehicle and anti-PD-1 + vehicle groups, n = 9 mice for remaining groups; data are from two independent experiments). h Flow cytometry plots and analysis of CD44 + CD69 + CD8 + T cells in tumors from mice treated with combinations of anti-PD-1 and lorlatinib ( n = 10 mice, control IgG + vehicle; n = 11 mice, control IgG + lorlatinib; n = 12 mice, remaining groups; data are from three independent experiments). Scale bar represent 100 µm. Box-and-whisker plot shows the median (line), mean (plus sign), 25th and 75th percentiles (box), and 5th and 95th percentiles (whiskers); hypothesis testing performed using two-way ANOVA with Tukey’s method for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: Murine pancreatic cancer cells KPC mT4, generated in the Tuveson Laboratory (Cold Spring Harbor Laboratory, NY, USA), were isolated from PDAC tumor tissues obtained from Kras LSL-G12D/+ ;Trp53 LSL-R172H/+ ;Pdx1–Cre mice of a pure C57BL/6 background .

Techniques: Control, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Combining activatable nanodelivery with immunotherapy in a murine breast cancer model

doi: 10.1016/j.jconrel.2019.04.008

Figure Lengend Snippet: In vitro release of IFN-α (A), IFN-β (B) in NDL and 4T1 murine mammary carcinoma, mT4 murine pancreatic cancer, MC-38 murine colon cancer cells, B16 murine melanoma, and HMGB 1 in NDL cell culture 24 h post treatment, respectively. Cells were preincubated for 5 min (type I IFN) or 1 min (HMGB 1) at 42°C prior to addition of media only (42°C) or a solution of 5 µg/mL Dox in media (Free Dox+42°C) at 42°C for another 5 min. * p < 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.

Article Snippet: The mT4 syngeneic mouse KPC pancreatic cancer cell line was isolated from KPC tumors derived from KPC-B6 background mice (Kras +/LSL-G12D ; p53 +/LSL-R172H ; PDX-Cre) and was a generous gift from Dr. David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Techniques: In Vitro, Cell Culture