Expression and activity of the urokinase plasminogen activator system in canine primary brain tumors

Figures & data

Table 1 Clinicopathological characteristics of dogs with primary brain tumors and normal controls

Sample number	Sample ID	Breed, sex, age (years)	Tumor location	Diagnosis	Analyses performed
					IHC	WB	ZYM	PCR
1	CII.1	Brittany Spaniel, MN, 2	Lateral ventricle	Choroid plexus papilloma (Grade II)	Yes	No	Yes	Yes
2	CII.2	Golden Retriever, FS, 8	Cerebellopontomedullary angle		Yes	Yes	Yes	Yes
3	CIII.1	Mixed breed, FS, 2	Cerebellopontomedullary angle	Choroid plexus carcinoma (Grade III)	Yes	No	No	Yes
4	MenI.1	Min. Schnauzer, FS, 12	Cerebral convexity	Grade I meningioma	Yes	Yes	No	No
5	MenI.2	Mixed breed, FS, 12	Parasagittal		Yes	Yes	Yes	Yes
6	MenI.3	Mixed breed, FS, 15	Basilar		Yes	Yes	Yes	Yes
7	MenI.4	Rat Terrier, MN, 11	Olfactory		Yes	No	Yes	No
8	MenI.5	Llewellin Setter, MN, 11	Falcine		Yes	Yes	No	No
9	MenI.6	Boxer, MN, 11	Cerebellar convexity		Yes	Yes	Yes	Yes
10	MenI.7	Keeshond, MN, 8	Parasellar		Yes	Yes	Yes	Yes
11	MenI.8	Siberian Husky, MN, 7	Cerebral convexity		Yes	No	Yes	No
12	MenI.9	Am. Staff. Terrier, FS, 11	Parasagittal		Yes	Yes	Yes	Yes
13	MenI.10	Chow, MN, 5	Cerebellopontomedullary angle		Yes	Yes	Yes	Yes
14	MenI.11	Mixed breed, MN, 13	Unknown		Yes	Yes	Yes	Yes
15	MenI.12	German Shepherd, FS, 8	Cerebral convexity		Yes	No	Yes	Yes
16	MenI.13	Australian Shepherd, F, 7	Parasagittal		Yes	No	No	Yes
17	MenI.14	Labrador Retriever, MN, 9	Cerebellar convexity		Yes	No	No	No
18	MenII.1	Mixed breed, MN, 6	Cerebral convexity	Grade II meningioma	Yes	No	No	Yes
19	MenII.2	Golden Retriever, MN, 8	Parasellar		Yes	Yes	Yes	Yes
20	MenII.3	Whippet, MN, 10	Falcine		Yes	Yes	Yes	Yes
21	MenII.4	German Shepherd, MN, 12	Basilar		Yes	Yes	Yes	Yes
22	MenII.5	Mixed breed, FS, 11	Falcine		Yes	No	Yes	Yes
23	MenII.6	Boxer, FS, 10	Olfactory		Yes	No	No	Yes
24	MenII.7	Golden Retriever, FS, 12	Cerebral convexity		Yes	Yes	Yes	Yes
25	AstroII.1	Bullmastiff, FS, 6	Frontal lobe	Grade II astrocytoma	Yes	Yes	No	Yes
26	AstroII.2	Boxer, MN, 7	Frontoparietal lobe		Yes	Yes	Yes	Yes
27	AstroIII.1	Am. Staff. Terrier, MN, 6	Parietal lobe	Grade III astrocytoma	Yes	Yes	Yes	Yes
28	GBM.1	Beagle, FS, 6	Bilateral cerebral (butterfly)	Grade IV astrocytoma	Yes	Yes	Yes	Yes
29	GBM.2	Boxer, FS, 11	Parieto-temporal lobes		Yes	Yes	No	No
30	GBM.3	Boston Terrier, MN, 2	Temporal lobe		Yes	No	Yes	Yes
31	GBM.4	Boxer, MN, 8	Thalamus		Yes	Yes	Yes	Yes
32	OligoII.1	Boxer, MN, 9	Piriform lobe	Grade II oligodendroglioma	Yes	Yes	Yes	Yes
33	OligoII.2	Boxer, MN, 5	Fronto-olfactory lobes		Yes	Yes	No	No
34	OligoII.3	Mixed breed, FS, 8	Parietal lobe		Yes	No	Yes	Yes
35	OligoIII.1	Boston Terrier, MN	Piriform lobe	Grade III oligodendroglioma	Yes	No	Yes	Yes
36	OligoIII.2	English Bulldog, FS, 7	Temporal lobe		Yes	Yes	Yes	Yes
37	OligoIII.3	Am. Bulldog, FS, 6	Frontal lobe		Yes	Yes	Yes	Yes
38	Cont1	Beagle, FS, 5	NA	Normal brain	Yes	Yes	Yes	Yes
39	Cont2	Beagle, MN, 6	NA	Normal brain	Yes	Yes	Yes	Yes
40	Cont3	Hound Mix, MN, 7	NA	Normal brain	Yes	Yes	Yes	Yes
41	Cont4	Am. Staff. Terrier, MN, 5	NA	Normal brain	Yes	Yes	Yes	Yes
42	Cont5	Mixed breed, FS, 4	NA	Normal brain	Yes	Yes	Yes	Yes

Abbreviations: Am, American; F, female; FS, female spayed; IHC, immunohistochemistry; Min, miniature; MN, male, neutered; NA, not applicable; PCR, polymerase chain reaction; Staff, Staffordshire; WB, Western blotting; ZYM, casein–plasminogen zymography.

Figure 1 Immunohistochemical evaluation of uPAR expression in control canine brain tissues (A–F) and brain tumors (G–R).

Notes: No uPAR immunoreactivity is present in normal meninges (A, B; Cont1). Constitutive uPAR expression is seen in rare microglial (D—arrow) and endothelial cells in the cerebral cortex (C, D; Cont3), and within the choroid plexus endothelium (E, F—arrows; Cont4). In all choroid plexus tumors, the neoplastic epithelium was diffusely and intensely immunoreactive to uPAR (G, H; CII.1). Markedly intense uPAR immunoreactivity was observed in the tumor cells of GBM (I, J; GBM.3) and tumor cells and hyperplastic vasculature (L—arrows) of Grade III oligodendroglioma cases (K, L; OligoIII.2). Diffuse uPAR immunoreactivity present in tumor and stromal cells in a Grade II meningioma (M, N; MenII.5). In Grade I meningiomas (O–R), two patterns of uPAR immunoreactivity were observed: samples in which staining was limited to the tumor vascular endothelium (P—arrows; MenI.2), or cases that displayed variable immunoreactivity in tumor (Q—arrow), stromal, and vascular endothelial (Q—arrowhead) cells (Q, R; MenI.4 and MenI.8, respectively). Panels in top row stained with hematoxylin and eosin (H&E). Panels in bottom row stained with anti-uPAR antibody and fast red chromogen. The numeral after each tumor type and grade indicates the specific sample depicted. Bar =200 μm in all panels.
Abbreviations: GBM, glioblastoma; H&E, hematoxylin and eosin; NB, normal brain (cerebral cortex); NCP, normal choroid plexus; NM, normal meninges; uPAR, urokinase plasminogen activator receptor; CII, grade II choroid plexus tumor; OligoIII, grade III oligodendroglioma; MenII, grade II meningioma; MenI, grade I meningioma.

Figure 2 (A) Representative Western blots of uPAR protein in canine normal brain tissues and brain tumors. The numeral after each tumor type and grade indicates the specific sample depicted. (B) uPAR protein expression compared with β-actin concentrations in normal canine brain tissues and tumors.

Notes: uPAR expression is significantly greater in tumors compared with controls (*P<0.02) and significantly higher in GBM compared with all other tumor types and grades (**P<0.001).
Abbreviations: GBM, glioblastoma; NM, normal meninges; NB, normal brain (cerebral cortex); NCP, normal choroid plexus; uPAR, urokinase plasminogen activator receptor; MenI, grade I meningioma; MenII, grade II meningioma; CII, grade II choroid plexus tumor; OligoII, grade II oligodendroglioma; OligoIII, grade III oligodendroglioma; AstroII, grade II astrocytoma; AstroIV, grade IV astrocytoma.

Figure 3 Expression of urokinase plasminogen activator receptor (uPAR) mRNA in canine primary brain tumors as determined by real-time quantitative polymerase chain reaction.

Notes: Expression of uPAR is increased in brain tumors (*P<0.02) compared with normal brain tissues. Among tumors, uPAR expression was significantly higher in glioblastoma (***P<0.003) and Grade III oligodendrogliomas (**P<0.01). The numeral after each designation indicates the specific sample depicted.
Abbreviations: NB.Cont, normal brain control; MN.Cont, normal meninges control; NCP.Cont, normal choroid plexus control; CII, grade II choroid plexus tumor; CIII, grade III choroid plexus tumor; MenI, grade I meningioma; MenII, grade II meningioma; AstroII, grade II astrocytoma; AstroIII, grade III astrocytoma; GBM, glioblastoma; OligoII, grade II oligodendroglioma; OligoIII, grade III oligodendroglioma.

Figure 4 uPA activity in normal canine brain tissues and brain tumors as determined with casein–plasminogen zymography.

Notes: (A, B) Zones of lysis spanning the 45–55 kDa region correspond with uPA activity, which is present in all tumor types and grades. (C) Lysis inhibition of uPA occurs in the presence of the amiloride-treated gels (+P/+A), with tPA activity visible at ~70 kDa. The numeral after each tumor type and grade indicates the specific sample depicted.
Abbreviations: NB, normal brain; NCP, normal choroid plexus; NM, normal meninges; +P, plasminogen-treated gel; MenI, grade I meningioma; MenII, grade II meningioma; CII, grade II choroid plexus tumor; OligoII, grade II oligodendroglioma; OligoIII, grade III oligodendroglioma; AstroII, grade II astrocytoma; AstroIII, grade III astrocytoma; GBM, glioblastoma; +P/+A, plasminogen and amiloride-treated gel; tPA, purified human tissue plasminogen activator control; uPA, urokinase-type plasminogen activator control.