Functional and anatomical relationships among primary afferent fibers, blood vessels, and cancers are poorly understood. However, recent evidence suggests that physical and biochemical interactions between these peripheral components are important to both tumor biology and cancer-associated pain. To determine the role of these peripheral components in a mouse model of cancer pain, we quantified the change in nerve and blood vessel density within a fibrosarcoma tumor mass using stereological analysis of serial confocal optical sections of immunostained hind paw. To this end we introduced the Discoma coral-derived red fluorescent protein (DsRed2) into the NCTC 2472 fibrosarcoma line using the Sleeping Beauty transposon methodology, thus providing a unique opportunity to visualize tumor-nerve-vessel associations in context with behavioral assessment of tumor-associated hyperalgesia. Tumors from hyperalgesic mice are more densely innervated with calcitonin gene related peptide (CGRP)-immunoreactive nerve fibers and less densely vascularized than tumors from non-hyperalgesic mice. As hyperalgesia increased from Day 5 to 12 post-implantation, the density of protein gene product 9.5 (PGP9.5)- immunoreactive nerves and CD31-immunoreactive blood vessels in tumors decreased, whereas CGRP-immunoreactive nerve density remained unchanged. Importantly, intra-tumor injection of a CGRP1 receptor antagonist (CGRP 8-37) partially blocked the tumor-associated mechanical hyperalgesia, indicating that local production of CGRP may contribute to tumor-induced nociception through a receptor-mediated process. The results describe for the first time the interaction among sensory nerves, blood vessels and tumor cells in otherwise healthy tissue, and our assessment supports the hypothesis that direct tumor cell-axon communication may underlie, at least in part, the occurrence of cancer pain.
Bibliographical noteFunding Information:
This work was supported by NIH R01-CA84233, RO1-CA72669, RO1-HL49997 and the University of Minnesota Cancer Center. NIH/5T 32-DEO 7288-02 and NIH R01-CA72669 also supported PWW. We thank John Oja and Jerry Sedgewick in the Biomedical Image Processing Laboratory in the Department of Neuroscience, Lisa Olson in Cutaneous Surgery in the Department of Dermatology, and Christine Lynch in the University of Minnesota Cancer Center for technical assistance, the University of Minnesota Cancer Center for support of the tissue culture facility, Clifford Steer (Department of Medicine) for technical assistance with DsRed2 transfection, and Jeremy Alley (Veterinary Pathobiology and Neuroscience) for further technical assistance.
- CGRP1 receptor antagonist
- Calcitonin gene-related peptide
- Cancer pain
- Endothelial cells
- Primary afferent
- Sleeping Beauty