A Functional Role for VEGFR1 Expressed in Peripheral Sensory Neurons in Cancer Pain

Abstract

Cancer pain is a debilitating disorder and a primary determinant of the poor quality of life. Here, we report a non-vascular role for ligands of the Vascular Endothelial Growth Factor (VEGF) family in cancer pain. Tumor-derived VEGF-A, PLGF-2, and VEGF-B augment pain sensitivity through selective activation of VEGF receptor 1 (VEGFR1) expressed in sensory neurons in human cancer and mouse models. Sensory-neuron-specific genetic deletion/silencing or local or systemic blockade of VEGFR1 prevented tumor-induced nerve remodeling and attenuated cancer pain in diverse mouse models in vivo. These findings identify a therapeutic potential for VEGFR1-modifying drugs in cancer pain and suggest a palliative effect for VEGF/VEGFR1-targeting anti-angiogenic tumor therapies.

Graphical abstract

Figure 1

Expression of VEGFR1 and VEGFR2 in Sensory Neurons and Nociceptive Sensitization by VEGF-A (A and B) Typical examples (A) and quantitative representation (B) of large- and small-diameter sensory neurons (arrows and arrowheads, respectively) showing co-labeling of anti-VEGFR1 or anti-VEGFR2 with markers for subtypes of sensory neurons in sections of mouse DRG (n = 32 sections analyzed from five mice). (C) Western blot analysis on mouse DRG or lysates of neuron-enriched DRG cultures. (D) Co-immunostaining of anti-VEGFR1 or anti-VEGFR2 with a marker for peripheral nerves (PGP9.5) in mouse paw skin biopsies. (E and F) Time course (E) and dose dependence (F) of modulation of mechanical sensitivity at the plantar surface of the hind paw following intraplantar application of VEGF-A. Shown in (F) is the integral of entire stimulus-response frequency curves (area under the curve [AUC]) to application of graded mechanical force (0.07–0.6 g) at different time points following administration of VEGF-A or saline in the hind paw (n = at least 4 mice per dose); an increase in the AUC is indicative of exaggerated pain sensitivity. (G and H) Time course (G) and dose dependence (H) of effects of intraplantar injection of VEGF-A or vehicle on paw withdrawal latency to noxious heat (n = at least 4 mice per dose). (I) Analysis of Evan’s blue extravasation in mouse skin explants after cutaneous injection of PBS (vehicle), histamine (1 μg), or various doses of VEGF-A (1–10 ng). (J) Typical examples (upper traces) and summary (lower graph) of mean firing rates of mechanoreceptive nociceptors at 30 min after exposure to vehicle (PBS) or various doses of VEGF-A in the paw skin-saphenous nerve preparation (n = at least 10 fibers/dose). Negative control lacking primary antibodies and bright-field images of H&E staining showing tissue morphology in sections (not adjacent, but derived from the same animals as the immunostained sections) are shown in (A) and (D). ^∗p < 0.05 as compared with basal, †p < 0.05 as compared with corresponding data point for the vehicle group, ANOVA followed by post hoc Fisher’s test. Data are presented as mean ± SEM. Scale bars represent 50 μm in (A) and (D). See also Figure S1.

Figure 2

Delineating VEGF Family Ligands and Their Receptors in Nociceptive Sensitization (A) Schematic representation of known ligand specificity and tools employed. (B) Effects of intraplantar delivery of neutralizing antibodies against VEGFR1 or VEGFR2 on intraplantar VEGF-A-induced mechanical hypersensitivity (left) and thermal hyperalgesia (right) (n = 8 mice/group). (C) Effects of a VEGFR1-neutralizing antibody on mechanoreceptive nociceptors in skin-nerve preparation (n = 8 fibers/group). (D) Effects of intraplantar injection VEGF-A (100 pg) in VEGFR1-Tk^−/− mice and WT controls (n = 8 mice/group). (E) Effects of intraplantar neutralizing antibodies against NRP1 or NRP2 on VEGF-A-induced mechanical hypersensitivity (left) or thermal hyperalgesia (right) (n = 6 mice/group). (F–H) Effects of intraplantar application of VEGF-E (F), VEGF-B (G), or PLGF-2 (H) on mechanical sensitivity in hind paw of mice (n = at least 4 mice per dose). ^∗p < 0.05 as compared with vehicle in (D) and compared with basal in all other panels; ^†p < 0.05 as compared with corresponding control group; ANOVA followed by post hoc Fisher’s test. Data are presented as mean ± SEM. See also Figure S2.

Figure 3

Signaling Pathways Underlying Nociceptive Sensitization by VEGF-A (A) A schematic overview of intracellular signaling mediators activated by VEGFRs (in oval symbols) and their respective pharmacological inhibitors in square boxes. (B) Western blots showing VEGF-A-induced phosphorylation of ERK1/2 in neuron-enriched cultured DRG neurons (n = 3 independent experiments). (C) Effects of hind-paw injection of pharmacological inhibitors on mechanical hypersensitivity (left) and thermal hypersensitivity (right) evoked by intraplantar injection of VEGF-A (1 ng). Shown are effects following single-dose intraplantar injection: L-NAME (18.5 nmoles, NOS inhibitor), U71322 (20 pmoles, PLCγ inhibitor), LY294002 (1 nmole, PI3K inhibitor), PP2 (200 pmoles, Src Kinase inhibitor), PD98059 (18.7 nmoles, MEK inhibitor), SB203580 (30 nmoles, p38 MAPK inhibitor), vehicle (1% DMSO). (D) Experiments comparing the above data with effects of intraplantar combinations of half-maximal doses of PLCγ inhibitor and anti-VEGFR1 antibody on VEGF-A-induced mechanical hypersensitivity to 0.4 g von Frey force (upper) and thermal hyperalgesia (lower). (E and F) Effects of VEGF-A (1 ng, intraplantar) in mice lacking Prkg1 selectively in nociceptive neurons of the DRG (SNS-Prkg1^−/− mice; E) or in mice lacking Trpv1 (Trpv1^−/− mice; F) (n = 6–8 mice/group). (G) Western blots on Src phosphorylation in DRG cultures (three independent experiments). (H) TRPV1 expression in membranes of distal branches of sciatic nerve in mice receiving intraplantar injections of VEGF-A or vehicle (n = 3 mice/group). (I) Effects of VEGF-A (1 ng, intraplantar) in mice lacking Trpa1 (Trpa1^−/− mice) (n = 8 mice/group). ^∗p < 0.05 as compared with basal value, ^†p < 0.05 as compared with corresponding control, ANOVA followed by post hoc Fisher’s test. Data are presented as mean ± SEM. See also Figure S3.

Figure 4

Role of VEGFR1 in Cancer Pain following Osteolytic Sarcoma Cell Implantation in the Calcaneus Bone (A) Western blot analyses for VEGFR1 or VEGFR2 expression in ipsilateral L3–L4 DRG of tumor-bearing mice or sham-treated mice and their quantitative densitometric analysis (n = 3 independent experiments; ^∗p = 0.02; Student’s t test). (B) Typical examples and quantitative summary of VEGFR1 expression in PGP9.5-positive peripheral nerves overlying bone metastases in tumor-affected or sham hind paw (n = 6 mice/group). Negative controls for immunostaining and H&E-stained sections from same animals (not adjacent sections) are included to judge morphology. epi, epidermis; der, dermis. (C and D) Whole-mount images (C) or cryosection (D) of a DRG 3 weeks after injection of lentivirions expressing EGFP and shRNA. (E) Western blot analysis of VEGFR1 expression in L3–L4 DRGs injected ipsilaterally (ipsi) with lentivirions, using contralateral DRGs as control. (F–H) Tumor-induced mechanical hypersensitivity (F) and tumor-induced hypertrophy and sprouting of epidermal sensory nerves expressing the marker protein PGP9.5 (G and H) in mice injected with lenti-VEGFR1-shRNA as compared with lenti-non targeting shRNA in the DRG (n = 5 mice/group). ^∗p < 0.05 as compared with sham in (B), (H) and compared with basal in (F); ^†p < 0.05 as compared with lenti-non-targeting control; ANOVA followed by post hoc Fisher’s test. Scale bars represent 50 μm in (B), (D), and (G) and 250 μm in (C). Data are presented as mean ± SEM. See also Figure S4.

Figure 5

Role of VEGFR1 in Cancer Pain following Hind Paw Implantation of C57BL6-Isogenic Lewis Lung Carcinoma Cells (A) Western blot analysis on DRGs from SNS-Vegfr1^−/− and Vegfr1^fl/fl (control) mice. (B–G) Analysis of SNS-Vegfr1^−/− and Vegfr1^fl/fl mice (n = 6–8 mice/group) with respect to intraplantar VEGF-A-induced mechanical (B) and thermal hypersensitivity (C), tumor-associated hypersensitivity (D), tumor-induced hypertrophy and sprouting of PGP-9.5-immunoreactive sensory nerves (E and F), and tumor growth (G), including negative control for immunostaining and an H&E-stained section. (H and I) Tumor-induced mechanical hypersensitivity (H) and tumor growth (I) in VEGFR1-Tk^−/− and WT mice. ^∗p < 0.05 as compared with sham in (F) and compared with basal in other panels, ANOVA followed post hoc Fisher’s test; in (D) and (H), ANOVA for repeated-measures was performed; ^†p < 0.05 as compared with corresponding control group, ANOVA for random measures followed by post hoc Fisher’s test. Scale bar represents 50 μm in (E). Data are presented as mean ± SEM.

Figure 6

Role of VEGFR1 in Pain Associated with PDAC (A and B) Examples (A) and quantitative analyses (B) of nerves (arrows) showing anti-VEGFR1 immunoreactivity in PDAC biopsies from patients. Arrowheads indicate blood vessels. n = 21 sections analyzed from 13 biopsies. (C and D) Typical examples (C) and quantitative summary (D) of VEGFR1 expression in PGP9.5-positive nerves in human PDAC and pancreas of healthy donors (n = 95 sections from 30 patients and 7 donors). (E and F) Typical examples (E) and quantitative summary (F) of the relation between VEGFR1 immunoreactivity in human PDAC biopsies and subjective pain rating reported by the patients (n = 79 sections from 30 patients). (G) Western blot analysis of VEGFR1 expression in DRGs of mice with Advillin-Cre-mediated pan-DRG VEGFR1 deletion (Adv-Vegfr1^−/−) and controls (Vegfr1^fl/fl). (H) VEGF-A-induced mechanical hypersensitivity (left) and thermal hyperalgesia (right) in Adv-Vegfr1^−/− and Vegfr1^fl/fl mice (n = at least 4 mice/group). (I and J) Comparison of early post-operative pain (red arrow), tumor-associated hypersensitivity (blue arrow) to 0.008 g of abdominal von Frey application (I) and integral of responses to all von Frey forces tested (right) and tumor mass (J) between Adv-Vegfr1^−/− mice, SNS-Vegfr1^−/− mice, and Vegfr1^fl/fl mice (n = at least 8 mice/group). ^∗p < 0.05, t test in (D) and (F). ^∗p < 0.05 as compared with basal in (H) and (I); ANOVA for repeated-measures followed post hoc Fisher’s test; ^†p < 0.05 as compared with corresponding control group, ANOVA for random measures followed by post hoc Fisher’s test. Scale bar represents 100 μm in (A) and (C) and 50 μm in (E). Data are presented as mean ± SEM. See also Figure S5.

Figure 7

Role of Diverse VEGFR1 Ligands and Sequestering Agents in Cancer Pain in the Calcaneus Osteolytic Sarcoma Implantation Model (A) ELISA-based analysis of VEGF-A and PLGF-2 in hind paw (n = 4 mice each). (B) Effects of intraplantar application of VEGF-A-sequestering antibody (upper), PLGF-2-sequestering antibody (lower), or control IgGs on tumor-induced mechanical hypersensitivity and tumor growth (n = at least 6 mice/group). (C) Effects of intraplantar application of soluble-VEGFR1 (sFlt1) or control protein on tumor-induced mechanical hypersensitivity and tumor size (n = 5–6 mice/group). (D) Typical example (left) and densitometric quantification (right) of tyrosine phosphorylation of VEGFR1 in mouse hind paw in sham or tumor-bearing mice and its modulation by local application of anti-Flt1 or reverse peptide 8 days post-tumor implantation (n = 3 mice/group). (E) Effects of intraplantar application of anti-Flt1 or reverse peptide on tumor-induced mechanical hypersensitivity and tumor growth (n = 6–8 mice). ^∗p < 0.05, ANOVA followed post hoc Fisher’s test as compared with basal, ANOVA of repeated-measures performed in (B), (C), and (E); ^†p < 0.05 as compared with corresponding control group and ^#p < 0.01 as compared with reverse peptide in (D), ANOVA for random measures followed by post hoc Fisher’s test. Data are presented as mean ± SEM. See also Figure S6.

Figure 8

Effects of Local or Systemic Immunological Blockade of VEGFR1 or VEGFR2 on Cancer Pain (A and B) Effects of a regime of local, intraplantar injections of either IgG or anti-VEGFR1 or anti-VEGFR2 antibodies (5 μg/dose) on tumor-induced mechanical hypersensitivity (A) and tumor-induced hypertrophy and sprouting of PGP9.5-immunoreactive epidermal sensory nerves (B) in the calcaneus osteolytic sarcoma implantation model (n = at least 6 mice/group). (C–E) Analysis of tumor-induced mechanical hypersensitivity in the ipsilateral mouse paw (C), spontaneous pain (D), and sprouting of the femur periosteal peptidergic nociceptive fibers (E) induced by implantation of mammary cancer cells in the mouse femur bone in mice receiving a regime of systemic injections of either control IgG or the VEGFR1-neutralizing MF-1 clone or the VEGFR2-neutralizing DC101 clone (40 mg/kg body weight per injection, n = 8 mice/group). In all panels, ^∗p < 0.05 as compared with basal; ANOVA of repeated-measures, ^†p < 0.05 as compared with control IgG, ^#p < 0.01 as compared with sham; only in (D), p < 0.05 between MF-1 and DC101 groups is indicated by $ sign; ANOVA followed by post hoc Fisher’s test. Scale bars represent 50 μm and 25 μm in (B) and (E), respectively. Data are presented as mean ± SEM. See also Figure S7 and Movies S1, S2, S3, and S4.