Journal: bioRxiv

Article Title: The Pdgfd-Pdgfrb axis orchestrates tumor-nerve crosstalk in pancreatic cancer

doi: 10.1101/2025.08.26.672505

Figure Lengend Snippet: ( A, B ) MFI of Pdgfd (A) and Pdgfrb (B) in mouse PDAC cell lines expressing dCas9-VPR for CRISPR activation (n = 7-8 cells). Scale bar, 30 µm. ( C ) Whole DRG invasion assay projection image with trajectories of tdTomato+ cancer spheroids moving towards centrally located DRG over six days. Scale bar, 800 µm. ( D ) Confocal image of DRG from (C) stained with beta-3 tubulin (gray) with invaded tdTomato+ (red) cancer cells. Scale bar, 100 µm. ( E ) Quantification of neuroinvasion index, average invasion speed, and distance invaded by cancer cells upregulating candidate genes (n = 8). ( F ) Neuroinvasion index, average invasion speed, and distance traveled by Pdgfd cancer cells in co-cultures treated with varying concentrations of SU16f (n = 7-8). ( G ) Neuroinvasion index, average invasion speed, and distance traveled by Pdgfrb cells treated with vehicle, 20 nM rPdgfd, or a combination of 20 nM rP and 20nM SU16f (n = 8). *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001 [Mann-Whitney test, mean + SD (A, B, E-G)]. Abbreviations: Scram = Scramble, rP/rPdgfd = recombinant Pdgfd.

Article Snippet: DRG-containing domes were placed into the incubator for 15 minutes at 37°C to solidify the Matrigel, after which 100 μL of prewarmed DMEM 10% FBS containing 12,000 cancer cells with or without recombinant Pdgfd protein (R&D Systems, 9738-SB-050) and SU16f (MCE, HY-108628) was added into the well.

Techniques: Expressing, CRISPR, Activation Assay, Invasion Assay, Staining, MANN-WHITNEY, Recombinant

Journal: bioRxiv

Article Title: The Pdgfd-Pdgfrb axis orchestrates tumor-nerve crosstalk in pancreatic cancer

doi: 10.1101/2025.08.26.672505

Figure Lengend Snippet: ( A ) Nerve subtype staining (left) and quantification (right) for Na v 1.8 (cyan) and TH (magenta) in human PDAC (n = 6 patients). **P < 0.01 (Mann-Whitney test, Mean SD). ( B ) Schematic of whole DRG invasion assay. (C) Still frame from live cell imaging of central DRG explant and whole DRG invasion assay at day 0. Cancer cells are visualized by mNeon reen expression. Scale bar, 1 mm. ( D ) End point (day 6) image of whole DRG invasion from (b, left) and DRG-negative control (right). ( E ) Compiled replicates for speed and distance analyses of Pdgfd, Pdgfrb, and control cancer cell invasion of DRGs over 6 days. ( F ) Compiled replicates for speed and distance analyses of Pdgfd cancer cell invasion of DRGs with or without SU16f treatment over 6 days. ( G ) Compiled replicates for speed and distance analyses of Pdgfrb cancer cell invasion of DRGs with or without exogenous recombinant Pdgfd over 6 days. Abbreviations: Scram = Scramble, rP/rPdgfd = recombinant Pdgfd.

Article Snippet: DRG-containing domes were placed into the incubator for 15 minutes at 37°C to solidify the Matrigel, after which 100 μL of prewarmed DMEM 10% FBS containing 12,000 cancer cells with or without recombinant Pdgfd protein (R&D Systems, 9738-SB-050) and SU16f (MCE, HY-108628) was added into the well.

Techniques: Staining, MANN-WHITNEY, Invasion Assay, Live Cell Imaging, Expressing, Negative Control, Control, Recombinant

Journal: bioRxiv

Article Title: The Pdgfd-Pdgfrb axis orchestrates tumor-nerve crosstalk in pancreatic cancer

doi: 10.1101/2025.08.26.672505

Figure Lengend Snippet: ( A ) Transwell invasion assay with scramble gRNA control and Pdgfd cells (n = 6). Scale bar, 200 µm. ( B ) Confocal imaging of 5-week orthotopically transplanted tumors (green) with scramble gRNA control and Pdgfd malignant cells (n = 5 mice). Scale bar, 800 µm. ( C ) Radial invasion assay across malignant (green) cell lines and conditions. Images show Pdgfrb (Ctrl) and Pdgfrb + 20 nM rPdgfd invasion assays at 4 days (left). Scale bar, 1 mm. Graph of invasion area over time (middle) for Pdgfrb, Pdgfrb + 20 nM rPdgfd, Pdgfrb + 20nM rPdgfd and 200nM SU16f, Pdgfd, and Scramble. Quantifi ation of AUC for invasion area (right) after 120 hours (n = 7-9). ( D ) Representative confocal image depicting invading Pdgfrb (Ctrl) and Pdgfrb + 20 nM rPdgfd cancer cells (green) stained with nuclei (blue) and Ki67 (magenta) in radial invasion assay at 5 days. Scale bar, 1 mm. Graph (right) shows Ki67+ invasion area for Pdgfrb line (Ctrl), Pdgfrb + 20 nM rPdgfd, and Pdgfrb + 20nM rPdgfd and 200nM SU16f (n = 6). *P < 0.05, **P < 0.01, ****P < 0.0001 [Mann-Whitney test, Mean SD (A-D)]. Abbreviations: Scram = Scramble, rP/rPdgfd = recombinant Pdgfd.

Article Snippet: DRG-containing domes were placed into the incubator for 15 minutes at 37°C to solidify the Matrigel, after which 100 μL of prewarmed DMEM 10% FBS containing 12,000 cancer cells with or without recombinant Pdgfd protein (R&D Systems, 9738-SB-050) and SU16f (MCE, HY-108628) was added into the well.

Techniques: Transwell Invasion Assay, Control, Imaging, Invasion Assay, Staining, MANN-WHITNEY, Recombinant

Journal: bioRxiv

Article Title: The Pdgfd-Pdgfrb axis orchestrates tumor-nerve crosstalk in pancreatic cancer

doi: 10.1101/2025.08.26.672505

Figure Lengend Snippet: ( A ) Representative images and growth curves of nerves from Na v 1.8-Cre tdTomato+ mice co-cultured with Pdgfd, Pdgfrb, or scramble gRNA control cancer cells (left). Scale bar, 200 µm. Quantification (right) of area under the curve (AUC) for neurite growth curves after 48 hours (n = 10-11). ( B) Images and growth curves of nerves grown in control media, treated with conditioned media (CM) from Pdgfd-overexpressing malignant cells, and treated with a combination of CM and 200 nM of Su16f (left). Scale bar, 200 µm. Quantification (right) of AUC for neurite growth curves after 48 hours (n = 4). ( C ) Images and growth curves of nerves treated with control vehicle, 2 nM, 20 nM, and 200 nM of rPdgfd (left). Scale bar, 200 µm. Quantification (right) of AUC for neurite growth curves after 48 hours. ( D ) Images and quantification of axonal area in DRGs treated with vehicle (Ctrl), 20 nM rPdgfd, 20 nM rPdgfd and 200 nM SU16f, and 200 nM SU16f (n = 10-12). Scale bar, 1 mm. *P < 0.05, **P < 0.01, ****P < 0.0001 [Mann-Whitney test, Mean SD (A-D)]. Abbreviations: Scram = Scramble, rP/rPdgfd = recombinant Pdgfd.

Article Snippet: DRG-containing domes were placed into the incubator for 15 minutes at 37°C to solidify the Matrigel, after which 100 μL of prewarmed DMEM 10% FBS containing 12,000 cancer cells with or without recombinant Pdgfd protein (R&D Systems, 9738-SB-050) and SU16f (MCE, HY-108628) was added into the well.

Techniques: Cell Culture, Control, MANN-WHITNEY, Recombinant

Journal: bioRxiv

Article Title: The Pdgfd-Pdgfrb axis orchestrates tumor-nerve crosstalk in pancreatic cancer

doi: 10.1101/2025.08.26.672505

Figure Lengend Snippet: ( A ) Flow plot and histogram showing Pdgfrb expression in EGFP+ glial cells cultured for 4 days. ( B ) Radial invasion assay with dissociated EGFP+ glial cells (left). Cultures were treated with vehicle (Ctrl), 20 nM rPdgfd, or 20 nM rPdgfd and 20 nM SU16f. Scale bar, 1 mm. Quantification (right) of invasion area at 96 hours (n=4). ( C ) Live confocal image of DRG invasion with Plp-EGFP+ DRG (cyan) and KPT cancer cells (yellow; left). Scale bar, 500 µm. Still frames with elapsed time in hours and minutes of an EGFP+ glial cell (cyan) pulling a cancer spheroid (yellow) towards the DRG (middle). Scale bar, 100 µm. Analysis of distance tdTomato+ cancer spheroids were pulled by EGFP+ glia over 24 hours (n = 37-51 spheroids per group). ( D ) Images of sciatic nerves injected with KPT cancer cells from mice treated daily with vehicle (Ctrl) or SU16f daily for 14 days (left). Scale bar, 2 mm. Quantification of sciatic invasion distance (right) at days 1 and 14 (n = 4-6 mice). ( E ) Sciatic nerve cross-section from Plp-EGFP mouse injected with KPT cancer cells. Pdgfrb (magenta) is absent in glial cells distal to invading cancer cells (left) and present in glial cells proximal to tdTomato+ cancer cells (right). Scale bar, 40 µm. ( F ) Schematic of proposed Pdgfd-Pdgfrb signaling model. *P < 0.05, **P < 0.01, ****P < 0.0001 [Unpaired t test, Mean SD (B); Mann-Whitney test, Mean SD (C, D)]. Abbreviations: rP/rPdgfd = recombinant Pdgfd.

Article Snippet: DRG-containing domes were placed into the incubator for 15 minutes at 37°C to solidify the Matrigel, after which 100 μL of prewarmed DMEM 10% FBS containing 12,000 cancer cells with or without recombinant Pdgfd protein (R&D Systems, 9738-SB-050) and SU16f (MCE, HY-108628) was added into the well.

Techniques: Expressing, Cell Culture, Invasion Assay, Injection, MANN-WHITNEY, Recombinant