Effect of NGF neutralizing antibody on the ability of pancreatic cancer cells to migrate towards neurites extended from dorsal root ganglia (DRG).

<p>A) Representative images (top panel: bright field; bottom panel: green fluorescence) of the migration of Mia PaCa-2 cells towards nerves treated with different concentrations of NGF neutralizing antibody. Arrows indicate the cancer cells that migrated towards the neurites. B) Quantification of the effect on the migration of cancer cells using relative invasion index. *: p ≤ 0.01; ** ≤ 0.001; *** ≤ 0.0001 (compared to no antibody control).</p

Effect of TRKA inhibitor GW441756 on the ability of pancreatic cancer cells to migrate towards neurites extended from DRG.

<p>A) Representative images (top panel: bright field; bottom panel: green fluorescence) of the migration of Mia PaCa-2 cells towards nerves treated with different concentrations of GW441756. Arrows indicate the cancer cells that migrated towards the neurites. B) Quantification of the effect on the migration of cancer cells using relative invasion index. *≤ 0.01; ** ≤ 0.001; *** ≤ 0.0001 (compared to no drug control).</p

Reciprocal signaling between pancreatic cancer cells and neurites extended from the PC-12 cells is reduced when the NGF-TRKA signaling pathway is disrupted.

<p>A) Treatment with the NGF neutralizing antibody or the TRKA inhibitor (GW441756) reduces the neurite outgrowth induced by conditional medium from pancreatic cancer cells. B) Quantification of neurite outgrowth assay shown in A. Conditioned Media (CM) collected from MiaPaCa2 or BxPC3 cells were treated with a neutralizing NGF antibody and a TRKA inhibitor and neurite (yellow arrows) extension from the PC-12 cells was visualized under a bright field microscope. Recombinant NGF protein was used as a positive control. *≤ 0.01; ** ≤ 0.001; *** ≤ 0.0001 (compared to Conditioned Media).</p

Knocking down the expression of the NGF-TRKA signaling pathway genes using siRNA oligonucleotides.

<p>The cells were treated with the siRNA oligonucleotides for 72 hours and then subjected to Western blotting (A and B) or RT-PCR analysis (C and D) for NGF, p75^NTR and TRKA expression in Mia PaCa-2 (A and C) or BxPC-3 (B and D) cells. SF: siLentFect (transfection reagent), NT: Non-targeting siRNA control. *: p ≤ 0.05; ** ≤ 0.01.</p

Knocking down the expression of the NGF-TRKA signaling pathway proteins or inhibiting the activity of TRKA reduces the growth and migratory activity of pancreatic cancer cells.

<p>A) Cell growth inhibition by siRNA treatment in Mia PaCa-2 and BxPC-3 cells. Cell viability was measure 72 hours post siRNA treatment and compared to Non-targeting (NT) siRNA control. B) Cell growth inhibition by TRKA inhibitor GW441756. Cells were treated with the inhibitor for 72 hours. C) siRNA knockdown of the expression of NGF, p75^NTR or TRKA reduced migration of pancreatic cancer cells. D) GW441756 inhibited the migration of pancreatic cancer cells in a dose dependent manner. *: p ≤ 0.05; ** ≤ 0.01 (compared to Non-targeting control).</p

Effect of siRNA knockdown of the expression of NGF, TRKA, or p75^NTR on the ability of pancreatic cancer cells to migrate towards neurites extended from DRG.

<p>A) Representative images (top panel: bright field; bottom panel: green fluorescence) of the migration of Mia PaCa-2 cells towards nerves treated with siRNA oligonucleotides targeting the indicated genes. Arrows indicate the cancer cells that migrated towards the neurites. B) Quantification of the effect on the migration of cancer cells using relative invasion index. SF: siLentFect (transfection reagent), NT: Non-targeting siRNA control. ** ≤ 0.001; *** ≤ 0.0001 (compared to Non-targeting siRNA control).</p