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Densitometry analysis of the six chemokine array signals identified in Figure 1A was performed and normalized to the average intensity of 3 controls.

A. Close up views of the photomicrographs from Figure 4 show an association between the PC-3 cells and the wild type DRG neurites (Figure 4A), but fewer associations between the PC-3 cells and CCL2 -/- DRG neurites (Figure 4B). B. DRG's were harvested from wild type and CCL2 -/- mice and explanted in 15µl of growth-factor reduced Matrigel. Photographs were taken every other day. Neurite outgrowth rate was measured by measuring the area covered by the neurites and subtracting the center of the DRG (MetaMorph, Molecular Devices, Sunnyvale, CA).

Supplementary Figure

A. A schematic of the DRG and cancer cell co-culture model depicts that cancer cells initially attach to DRG neurites. Over time, the cancer cells migrate along the neurites towards the DRG and also proliferate. B. Close up views of the photomicrographs from Figure 3 show an association between the shControl PC-3 cells and the DRG neurites (Figure 3A), but fewer associations between the shCCR-2 PC-3 cells and DRG neurites (Figure 3B).

PC-3 migration towards DRG as the attractant is reduced by the addition of anti-CCL2 neutralizing antibody at 10 μg/ml to the DRG. In contrast, the addition of non-specific rat IgG at 10 μg/ml shows no inhibitory effects (* p

Sciatic nerve invasion length based on MRI imaging depicted in Figure 5 was assessed for mice injected with shControl or shCCR2 PC-3 cells. Images showing a thickened sciatic nerve were selected and analyzed with ImageJ (* p

A.Immunofluorescence microscopy was performed on control or shCdc42 MiaPaCa2 murine sciatic nerve tumors. Slides were stained with DAPI (blue) nuclear staining and primary antibodies (green) targeting total Cdc42, �-Pix, RET, and phospho-RET. B. Hematoxylin and eosin staining and GTP-Cdc42 immunohistochemistry of a control MiaPaCa2 murine sciatic nerve tumor.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Perineural invasion (PNI) is the ability of cancer cells to invade in, around, and along nerves. PNI is widely recognized as an adverse pathologic feature of many malignancies. Glial-derived neurotrophic factor (GDNF) is secreted by nerve and glial cells, and is important in neural development. We previously showed that GDNF activation of the RET/GFR-alpha tyrosine kinase in cancer cells induces chemotaxis towards nerves and PNI. Cdc42 is a member of the Rho family of G proteins and a key regulator of cell polarity. We demonstrate here that GDNF-mediated activation of RET/GFR-alpha leads to activation of Cdc42 as a mediator of cancer cell chemotaxis and PNI. Exposure of MiaPaCa2 cells to GDNF or dorsal root ganglia (DRG) activates Cdc42. SiRNA inhibition of Cdc42 impairs MiaPaCa2 chemotaxis towards GDNF or DRG in Boyden chamber assays. SiRNA inhibition of RET decreases Cdc42 activity under GDNF stimulation and MiaPaCa2 chemotaxis. To study dynamic interactions between nerves and cancer cells in vitro, we used a DRG and cancer cell co-culture model of PNI in matrigel. Analysis of individual cell trajectories reveals that cell velocity remains unchanged, while directional migration is disrupted with shRNA inhibition of Cdc42. The disruption of directional response impairs the ability of shRNA Cdc42 MiaPaCa2 cells to migrate along neurites, as visualized by time lapse microscopy and measured by area of PNI. MRI imaging of live mice with sciatic nerves injected with shRNA Cdc42 MiaPaCa2 cells reveals significantly diminished length and volume of PNI as compared with injected shRNA control cancer cells when matched for equivalent overall tumor volume. Murine sciatic nerves injected with shRNA Cdc42 MiaPaCa2 cells exhibited preserved hindlimb and hind paw neurologic function, and diminished perineural invasion by histopathologic examination as compared with shRNA control MiaPaCa2. To identify potential activators of Cdc42 in this model, we performed a comprehensive siRNA screen of guanine nucleotide exchange factors (GEFs), identifying specific GEFs which are necessary for MiaPaCa2 migration towards DRG in Boyden chambers. We identified and validated six GEFs ([FLJ10521][1], DOCK9, ARHGEF7, RASGRF1, ARHGEF5, PLEKHG1) that impair chemotaxis without affecting proliferation. These findings provide novel insights into the molecular mechanisms underlying cancer cell perineural invasion. The RET-GEF-Cdc42 axis may be an attractive target for novel therapies that may potentially interrupt perineural invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 460. doi:1538-7445.AM2012-460 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=FLJ10521&atom=%2Fcanres%2F72%2F8_Supplement%2F460.atom