Fig 1: Nerve profiles are less associated with vasculature in cancer-infiltrated bone. (A) For each nerve profile it was determined if it was associated within a distance of 25 µm (green hatched line) or unassociated (yellow hatched line) with vasculature. (B,C) Representative illustrations of nerve profiles found within 25 µm of a vascular structure in the bone marrow of human healthy tissue (B1,B2) and the bone marrow of patients with metastatic bone disease (MBD) (C1,C2). Sections were stained for protein gene product 9.5 (PGP9.5, red arrow heads) and CD34-positive endothelial cells (black arrowheads), while the adjacent section was stained for cytokeratin 7 and 19 [CK7/19, gray arrow heads, (C2)] to identify cancer cells. (D) The percentage of nerve profiles found proximate to a vascular structure was significantly higher in the healthy bone marrow compared to the cancer-infiltrated bone marrow. (E) Analysis of the three subgroups of cancer-infiltrated bone demonstrated a decreased association of nerve profiles and vascular structures in the MBD treated group and the MBD surgery group compared to controls. The comparison of healthy bone tissue with the MBD untreated subgroup did not reach significance (p = 0.0504). Comparisons were performed with Mann-Whitney U test or Kruskal–Wallis one-way analysis of variance followed by Dunn's multiple comparisons test as appropriate. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001. Scale bars: 25 µm.
Fig 2: Nerve profile density in bone sub-compartments and their association to the vasculature. (a) The number of nerve profiles per tissue area was significantly higher in the cortical pores compared to the bone marrow and periosteum. Each dot represents the value in a given biopsy (n = 10), and the horizontal line represents the mean. The data were analyzed by repeated measure ANOVA with Geisser-Greenhouse’s correction: **p < 0.01. (b) The percentage of TH+ nerve profiles relative to TH-/PGP9.5+ nerve profiles was higher in the cortical pores compared to the bone marrow and periosteum (not significant). The data was analyzed by a clustered logistic regression, addressing whether the odds between TH+ and TH-/PGP9.5+ nerve profiles were different between the compartments. (c) Most of the nerve profiles were closely associated with the vasculature. Still, the percentage of nerve profiles associated with vasculature relative to those without a vascular association was higher in the cortical pores compared to the bone marrow and periosteum. The data was analyzed by a clustered logistic regression, addressing whether the odds between nerve profiles with or without vascular association is different between the compartments. TH: tyrosine hydroxylase, PGP9.5: protein gene product 9.5, OR: odds ratio.
Fig 3: Association of nerve profiles to the vasculature. (a–h) Images showing double immunostaining of CD34+ vasculature in combination with PGP9.5+ or TH+ nerve profiles (red arrows) in the bone marrow. Large arteries with PGP9.5+ (a) or TH+ (e) nerve profiles, small arteries with PGP9.5+ (b) or TH+ (f) nerve profiles, capillaries with PGP9.5+ (c) or TH+ (g) nerve profiles, and sinusoids with PGP9.5+ (d) or TH+ (h) nerve profiles. (i) The percentage distribution of PGP9.5+ and TH+ nerve profiles associated with the respective vascular structures in the bone marrow, and (j) the number of nerve profiles/cluster profile for the respective vascular structures. TH: tyrosine hydroxylase, PGP9.5: protein gene product 9.5; lAr: large arteriole; sAr: small arteriole; Ca: capillary; Nv: Nerve profiles with no association to the vasculature. n = 11.
Fig 4: Nerve profiles are more abundant close to the bone surface. In the bone marrow, nerve and cluster profiles were quantified within and above 100 µm of the bone surfaces. (a,d) The density of PGP9.5+ (a) and TH+ (d) nerve profiles was significantly higher close to the bone surface. (b,e) The density of cluster profiles innervated by PGP9.5+ (b) and TH+ (e) nerve profiles was significantly higher close to the bone surface, indicating that innervated vascular structures are more abundant close to the bone surface compared to the distant bone marrow. (c,f) No difference was observed in the nerve profile/cluster profile ratio of the PGP9.5+ (c) and TH+ (f) innervation, indicating that the increase in innervation was not due to an increase in numbers of nerve profiles per vascular structure. The data was analyzed by clustered regression analysis. ***p < 0.001, n = 11. TH: tyrosine hydroxylase, PGP9.5: protein gene product 9.5.
Fig 5: Nerve profile density and tumor burden are not correlated. (A1) Illustration of the method. For each nerve profile it was determined if it was located within a distance of 25 µm to cancer cells. (A2,A3) Representative illustration of nerve profiles found within 25 µm of cancer cells in the bone marrow of a patient with metastatic bone disease (MBD). Sections were stained for protein gene product 9.5 (PGP9.5, red arrow heads) and CD34 [black arrow heads, (A2)], while an adjacent section was stained for cytokeratin 7 and 19 [CK7/19, gray arrow heads, (A3)] to identify cancer cells. (B) No difference was found in the percentage of nerve profiles found within 25 µm of cancer cells in between the three groups of cancer-infiltrated bone tissue. (C) No correlation was found between the nerve profile density and the tumor burden, neither overall nor in any of the subgroups. Analyses were performed with Kruskal–Wallis one-way analysis of variance followed by Dunn's multiple comparisons test and Spearman correlation. Data are presented as mean ± SEM. Scale bars: 25 µm.
Supplier Page from MilliporeSigma for Anti-PGP9.5 antibody produced in rabbit