Journal: Cell reports

Article Title: Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling.

doi: 10.1016/j.celrep.2023.113369

Figure Lengend Snippet: Figure 6. Netrin-1 interference suppresses metastasis and increases survival in murine PDAC models (A) Mice were pretreated with mAbNtn1 for 2 days and intrasplenically injected with 50,000 Ink4a.1 cells. Antibody treatment (10 mg/kg) continued every 2 days until liver harvest at day 14. Percentage tumor area in the liver was quantified as before (n = 5). (B) Kaplan-Meier graph showing survival of mice in- jected intrasplenically with 20,000 Ink4a.1 cells using mAbNtn1 (n = 16) as adjuvant treatment or vehicle (n = 19). Note that three mAbNtn1-treated mice have not recurred beyond day 200. (C) Immunohistochemistry of CK17-stained livers from C57Bl/6J mice injected via portal vein injection with KPC3 cells (left) and treated with IgG control or mAbNtn1, n = 10/group. Percentage of tumor metastasis area was quantified as before, n = 5 sec- tions/mouse. Note the significant reduction in mice treated with anti-Netrin-1 antibody (right). Scale bars, 200 mm. (D) Normalized Netrin-1 expression in KPC3 cells, normal liver, hepatic KPC3 metastases from control IgG-treated mice, and hepatic KPC3 metastases from mAbNtn1-treated mice as quantified by western blot. (E) Kaplan-Meier plot showing survival of KPC mice treated with IgG vehicle and mAbNtn1 antibodies. The vehicle group (n = 17) had MS = 18 days, while the mAbNtn1 group (n = 11) had MS 42 days, a doubling of lifespan. All experiments were completed at least in duplicate. Statistical analysis was completed using two-tailed Student’s t test (***p < 0.001, ****p < 0.0001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-netrin 1 antibody [EPR5428] Abcam ab126729 Human/Mouse ELF3 Antibody R&D Systems AF5787 RXRa (D6H10) Rabbit mAb Cell Signaling 3085S ELF3 Polyclonal Antibody Invitrogen PA5-89261 Normal Rabbit IgG Cell Signaling 2729P RARa/Retinoic Acid Receptor a Antibody (C-1) Santa Cruz Biotechnology sc-515796 Mouse (G3A1) mAb IgG1 Isotype Control Cell Signaling 5415S Anti-Desmin antibody [Y66] Abcam ab32362 Smooth muscle actin Polyclonal antibody ProteinTech 14395-1-AP Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 532 Invitrogen A-11009 Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 555 Invitrogen A-21428 Anti-CD9 antibody [EPR2949] Abcam ab92726 UNC5B (D9M7Z) Rabbit mAb Cell Signaling 13851 Purified Mouse Anti-Flotillin-1 BD Biosciences 610820 Cytokeratin 17 Monoclonal Antibody (CK17) Thermo Fisher 606–540 RARg1 (D3A4) XP Rabbit mAb Cell Signaling 8965S Anti-Retinoid X Receptor beta/RXRB antibody Abcam ab221115 RXRg Antibody (G-6) Santa Cruz Biotechnology sc-514134 Actin Antibody (H-6) Santa Cruz Biotechnology sc-376421 Alix (3A9) Mouse mAb Cell Signaling 2171S Alexa Fluor 647 anti-mouse/human CD324 (E-Cadherin) Antibody Biolegend 147307 Vimentin (D21H3) XP Rabbit mAb Cell Signaling 5741S alpha-Smooth Muscle Actin Antibody (1A4/asm-1) - Azide and BSA Free Novus Biologicals NBP2-34522 mAbNtn1 Patrick Mehlan N/A Bacterial and virus strains Edit-R Mouse Unc5b hEF1a All-in-one Lentiviral sgRNA Horizon Discovery VSGM11942-247910466 Edit-R Mouse Elf3 mCMV All-in-one Lentiviral sgRNA Horizon Discovery VSGM11942-248070215 Edit-R Mouse Ntn1 Lentiviral sgRNA Horizon Discovery VSGM10144-246774776 Biological samples BxPC-3 ATCC CRL-1687 Capan-2 ATCC HTB-80

Techniques: Injection, Adjuvant, Immunohistochemistry, Staining, Control, Expressing, Western Blot, Two Tailed Test