Radiation impairs perineural invasion by modulating the nerve microenvironment

Abstract

Purpose: Perineural invasion (PNI) by cancer cells is an ominous clinical event that is associated with increased local recurrence and poor prognosis. Although radiation therapy (RT) may be delivered along the course of an invaded nerve, the mechanisms through which radiation may potentially control PNI remain undefined.

Experimental design: An in vitro co-culture system of dorsal root ganglia (DRG) and pancreatic cancer cells was used as a model of PNI. An in vivo murine sciatic nerve model was used to study how RT to nerve or cancer affects nerve invasion by cancer.

Results: Cancer cell invasion of the DRG was partially dependent on DRG secretion of glial-derived neurotrophic factor (GDNF). A single 4 Gy dose of radiation to the DRG alone, cultured with non-radiated cancer cells, significantly inhibited PNI and was associated with decreased GDNF secretion but intact DRG viability. Radiation of cancer cells alone, co-cultured with non-radiated nerves, inhibited PNI through predominantly compromised cancer cell viability. In a murine model of PNI, a single 8 Gy dose of radiation to the sciatic nerve prior to implantation of non-radiated cancer cells resulted in decreased GDNF expression, decreased PNI by imaging and histology, and preservation of sciatic nerve motor function.

Conclusions: Radiation may impair PNI through not only direct effects on cancer cell viability, but also an independent interruption of paracrine mechanisms underlying PNI. RT modulation of the nerve microenvironment may decrease PNI, and hold significant therapeutic implications for RT dosing and field design for patients with cancers exhibiting PNI.

Figure 1. GDNF-mediated cancer cell migration and a DRG co-culture model of PNI.

A. MiaPaCa2 pancreatic cancer cells were (**) grown in a culture insert adjacent to a DRG (*) in Matrigel. Bar, 500 µm. DRG becomes invaded by MiaPaCa2 (arrow) which track along a neurite. Bar, 250 µm. Area of invasion was calculated using Metamorph computer software, 8 days after insert removal. Bar, 50 µm. B. MiaPaCa2 reliably invades DRG, in contrast to the squamous cell carcinoma cell line QLL2 († p<0.05, t-test). C. MiaPaCa2 migrate towards both GDNF and DRG in Boyden chamber assays. D. DRG treated with anti-GDNF antibodies demonstrated decreased area of invasion by MiaPaCa2 (p = 0.15, t-test). Photomicrographs show representative decreased area of invasion (arrow) at day 8 with anti-GDNF antibody treatment in comparison to control. Bar, 250 µm.

Figure 2. Radiation to DRG alone impairs PNI in vitro.

A. DRG (*) remained viable without morphological change after a single dose of 16 Gy; however, a single dose of 24 Gy results in a loss of neurites (arrows) consistent with DRG death. Bar, 500 µm. B. DRG co-culture model of PNI was performed. Non-radiated MiaPaCa2 cells were grown in an insert adjacent to a DRG exposed to a single dose of radiation (4–16 Gy) in Matrigel. The insert was removed, and 8 days later the area of invasion was calculated using Metamorph computer software. Low doses of radiation to the DRG significantly suppressed invasion by untreated cancer cells († p<0.05, t-test). A photomicrograph of a DRG (*) 8 days after exposure to 4 Gy shows no PNI, in contrast to control, non-radiated DRG (area of invasion outlined). Bar, 250 µm. C. GDNF ELISA was performed on conditioned media from DRG 8 days after a single radiation exposure. Single doses of radiation as low as 4 Gy suppressed GDNF to levels below the limit of detection (31 pg/mL). D. GDNF supplementation partially rescues invasion following RT. DRG were grown in Matrigel supplemented with GDNF (150 ng) and then radiated to 4 Gy. GDNF was added (60 ng) to the DRG every other day, which partially restored the mean area of invasion (p<0.05, t-test).

Figure 3. Radiation of MiaPaCa2 cells impairs migration and PNI predominantly through compromised cellular viability.

A. Colony-forming assays of MiaPaCa2 cells show susceptibility to single-fraction radiation doses, with logarithmically diminishing surviving fractions up through 8 Gy. B. MiaPaCa2 cells were radiated at varying single fraction doses, 24 hours later serum starved overnight, and then added (2×10⁵) to the upper chamber of migration assays. The lower chamber contained control media, DRG, or GDNF (100 ng/mL). Radiation reduced the number of migrating cells towards DRG or GDNF in a dose-dependent fashion. MiaPaCa2 were radiated and simultaneously plated with migration assays, under identical conditions, and counted after 24 hours. Dose-dependent decreases in cancer cell number, attributable to radiation induced cell death, correlated with results from migration assay. C. Single fraction doses of radiation (2–8 Gy) were applied to MiaPaCa2 cells prior to their addition to the DRG co-culture model of PNI. Radiation doses as low as 2 Gy significantly decreased the area of invasion († p<0.05, t-test). MiaPaCa2 were radiated and simultaneously plated with DRG co-culture assays, under identical conditions, and counted after 8 days. Dose-dependent decreases in cancer cell number, attributable to radiation induced compromised cell viability, correlated with decreases in area of invasion. D. MiaPaCa2 colonies adjacent to the DRG (*) at day 8 show diminished viability with increasing radiation dose, consistent with cell numbers quantified in Fig. 3C. White bar, 1000 µm, black bar, 250 µm.

Figure 4. PNI in vivo is impaired following radiation.

A. Untreated MiaPaCa2 cells were implanted into the distal sciatic nerve of mice under anesthesia. Nine weeks later, there is gross thickening along the course of the nerve, consistent with PNI in comparison to a normal sciatic nerve. B. MRI (T2 weighted image) and hematoxylin and eosin (H&E) staining of a mouse injected with PBS into the sciatic nerve respectively show a thin, barely visible, sciatic nerve (bar, 1.0 cm) with normal nerve histology (bar, 100 µm). MRI and H&E of a non-radiated mouse after injection with non-radiated MiaPaCa2 cells respectively demonstrate a thick and enhancing right sciatic nerve extending toward the spine cord and extensive invasion of the nerve. MRI and H&E of a mouse radiated to 4 Gy to the sciatic nerve only, respectively show decreased nerve thickness and PNI, as compared with control. MRI and H&E after injection of a non-radiated mouse with MiaPaCa2 cells radiated to 4 Gy respectively show decreased nerve thickness and PNI as compared with control.

Figure 5. Radiation to the sciatic nerve alone impairs PNI and preserves nerve function in vivo.

A. Representative images of a mouse injected with PBS into the right sciatic nerve demonstrate normal bilateral hind limb function and a normal sciatic nerve in situ. Representative images of a mouse 7 weeks after injection of MiaPaCa2 into the right sciatic nerve demonstrate paralysis of the right hind limb and gross tumor (*) with PNI and sciatic nerve thickening (arrow) in situ. Representative images of a mouse that received 8 Gy of radiation to the right sciatic nerve which was then injected with non-radiated MiaPaCa2 cells. Seven weeks later, right hind limb function remains intact and a smaller gross tumor (*) with no sciatic nerve thickening (arrow) in situ. B. Radiation to the sciatic nerve, prior to cancer injection, preserves hind limb function. Mean right sciatic nerve function score of mice receiving 0 (control) or 8 Gy to the right sciatic nerve prior to injection of non-radiated MiaPaCa2 cells († p<0.05, t-test). A nerve score of four indicates normal hind limb function, and a score of one indicates complete paralysis. C. The sciatic nerve index (hindpaw span) was used as a measure of sciatic nerve function 7 weeks after MiaPaCa2 injection in the right sciatic nerve of mice receiving 0 (control) or 8 Gy of radiation to the right sciatic nerve prior to injection of non-radiated MiaPaCa2 cells. Radiation to sciatic nerves preserves nerve function in comparison to non-radiated nerves († p<0.05, t-test). D. GDNF production in sciatic nerves decreases 3 weeks after a single radiation dose (8 Gy) as compared with controls (0 Gy) (p = 0.13, t-test).

Figure 6. Radiation to cancer cells alone diminishes PNI in vivo.

A. Representative images of a non-radiated mouse 7 weeks after sciatic nerve injection with radiated (2 Gy) MiaPaCa2 cells demonstrate intact right hind limb function and diminished gross tumor (*) with normal sciatic nerve caliber (arrow) in situ, in contrast to non-radiated MiaPaCa2 cells (Fig. 5A). B. Radiation to cancer cells alone prevents sciatic nerve paralysis. Mean right sciatic nerve function scores were measured 7 weeks after mice underwent sciatic nerve injections with radiated (2 Gy) or control (0 Gy) MiaPaCa2 cells († p<0.05, t-test). C. The sciatic nerve index (hindpaw span) was used as a measure of sciatic nerve function 7 weeks after injection of radiated (2 Gy) or control (0 Gy) MiaPaCa2 cells into the right sciatic nerve. Mice injected with radiated cancer cells exhibit preserved neurological function as compared to mice receiving non-radiated control cancer cells († p<0.05, t-test). D. Mean gross tumor volumes assessed 7 weeks after sciatic nerve injections demonstrate that cancer cells receiving radiation (2 Gy) exhibited significantly smaller tumor volumes as compared with control (0 Gy) cancer cells († p<0.05, t-test).

Figure 7. RET is expressed widely in human adenoid cystic carcinoma (ACC), which frequently exhibit PNI.

A. Hematoxylin-eosin staining of an ACC specimen (10x) demonstrates well-formed ducts and nests of cells growing in hyalinized stroma in a mixed tubular and cribriform pattern. RET immunohistochemistry demonstrates widespread staining of ACC at both 10x (B.) and 20x (C.). D. RET immunohistochemistry at 40x demonstrates 3 cases of PNI, with RET positive expression by ACC cells (arrows) exhibiting PNI of small nerves (*).