Sequential loss of tumor vessel pericytes and endothelial cells after inhibition of platelet-derived growth factor B by selective aptamer AX102 - PubMed (original) (raw)

Sequential loss of tumor vessel pericytes and endothelial cells after inhibition of platelet-derived growth factor B by selective aptamer AX102

Barbara Sennino et al. Cancer Res. 2007.

Abstract

Inhibition of platelet derived growth factor (PDGF) can increase the efficacy of other cancer therapeutics, but the cellular mechanism is incompletely understood. We examined the cellular effects on tumor vasculature of a novel DNA oligonucleotide aptamer (AX102) that selectively binds PDGF-B. Treatment with AX102 led to progressive reduction of pericytes, identified by PDGF receptor beta, NG2, desmin, or alpha-smooth muscle actin immunoreactivity, in Lewis lung carcinomas. The decrease ranged from 35% at 2 days, 63% at 7 days, to 85% at 28 days. Most tumor vessels that lacked pericytes at 7 days subsequently regressed. Overall tumor vascularity decreased 79% over 28 days, without a corresponding decrease in tumor size. Regression of pericytes and endothelial cells led to empty basement membrane sleeves, which were visible at 7 days, but only 54% remained at 28 days. PDGF-B inhibition had a less pronounced effect on pancreatic islet tumors in RIP-Tag2 transgenic mice, where pericytes decreased 47%, vascularity decreased 38%, and basement membrane sleeves decreased 21% over 28 days. Taken together, these findings show that inhibition of PDGF-B signaling can lead to regression of tumor vessels, but the magnitude is tumor specific and does not necessarily retard tumor growth. Loss of pericytes in tumors is an expected direct consequence of PDGF-B blockade, but reduced tumor vascularity is likely to be secondary to pericyte regression.

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Figures

Figure 1

Figure 1

Specificity of AX102 and AX102-mediated reduction in tumor pericytes. A, in nitrocellulose filtration assays, AX102 bound with high affinities to PDGF-BB and PDGF-AB, but not to PDGF-AA, PDGF-CC, or VEGF165 (i). Cross-reactivity to rodent PDGF was shown in binding assays in which AX102 bound to mouse and rat PDGF-BB with equal affinity to human (ii). B, fluorescence microscopic images showing pericytes in LLC tumors, stained for α-SMA immunoreactivity, after 7 d of vehicle (i) or 50 mg/kg AX102 (ii). Measurements show a dose-dependent decrease after 7 d of AX102 but no change after scrambled aptamer AX104 (iii). *, P < 0.05, different from corresponding value for vehicle; †, P < 0.05, different from AX104. C, fluorescence microscopic images of LLC stained for α-SMA show pericytes in the vehicle group (i) and few pericytes remaining after AX102 (50 mg/kg) for 28 d (ii). Measurements show the large, rapid decrease in α-SMA immunoreactive pericytes after AX102 in LLC tumors (iii). D, fluorescence microscopic images of RIP-Tag2 tumors stained for α-SMA show abundant pericytes under baseline conditions (i) and a conspicuous decrease after AX102 for 28 d (ii). Quantification illustrates a smaller, more gradual decrease in RIP-Tag2 tumors (iii) than in LLC tumors (C-iii). *, P < 0.05, different from corresponding value for vehicle. †, P < 0.05, different from corresponding value for 1-d AX102. Bar in (D-ii) is 110 μm in (B), 125 μm in (C), and 150 μm in (D).

Figure 2

Figure 2

Reduction of four pericyte markers in LLC tumors after AX102. A, confocal microscopic images of LLC tumors comparing pericytes stained for α-SMA (i), NG2 (ii), PDGFR-β (iii), or desmin (iv). v, area densities of the four markers in untreated LLC tumors. B, comparison of α-SMA, NG2, desmin, and PDGFR-β immunoreactivities in LLC tumors after vehicle, 1 d of AX102, and 7 d of AX102. *, P < 0.05, different from corresponding value for vehicle. C, fluorescence microscopic images of LLC tumors showing a pericyte with both PDGFR-β and NG2 immunoreactivities in the vehicle group (i); a pericyte with PDGFR-β but not NG2 immunoreactivity after 1 d of AX102 (ii); and a tumor vessel that lacks pericytes with either marker after 7 d of AX102 (iii). D, transmission electron micrographs showing pericyte (red) envelopment of endothelial cells (green) in vehicle-treated LLC tumor (i) and absence of pericytes in LLC tumor vessels after AX102 for 7 d (ii). Bar in (D-ii) is 10 μm in (A), 15 μm in (C), and 1.9 μm in (D).

Figure 3

Figure 3

Blood vessel loss in LLC and RIP-Tag2 tumors after AX102, leaving tumor vessels with closely associated pericytes. A, fluorescence microscopic images of LLC tumors treated with vehicle (i) and AX102 (ii) for 28 d stained for CD31 (endothelial cells; green) and PDGFR-β (pericytes; red). Measurements of LLC tumors show a significant reduction in PDGFR-β at 2 d, but no reduction in CD31 until 4 d. CD31 decreased more slowly than PDGFR-β, but by 28 d both had decreased by the same amount (iii). B, fluorescence microscopic images of RIP-Tag2 tumors stained for CD31 and PDGFR-β compare the abundant blood vessels under baseline conditions (i) and marked reduction after 28 d of AX102 (ii). Measurements of RIP-Tag2 tumors compare reductions in PDGFR-β and CD31 after 7, 14, and 28 d of AX102 (iii). *, P < 0.05, different from corresponding value for vehicle. C, confocal microscopic images of endothelial cells (CD31; green) and pericytes (α-SMA; red) on tumor vessels. Most pericytes in LLC tumors were loosely associated with blood vessels under baseline conditions (i); after AX102, pericytes were tightly apposed to endothelial cells (ii). Similarly, most pericytes in RIP-Tag2 tumors were loosely associated with tumor vessels under baseline conditions (iii); after AX102 for 7 d, pericytes were absent on some tumor vessels and closely associated with others (iv). Bar in (C-iv) represents 110 μm in (A), 150 μm in (B), and 12 μm in (C).

Figure 4

Figure 4

Changes in pericyte-endothelial cell colocalization in tumor vessels after AX102. A, confocal microscopic images showing CD31 (green), PDGFR-β (red), and colocalization of CD31 and PDGFR-β (white) in LLC tumors. Under baseline conditions, about half of the CD31 immunoreactivity colocalized with PDGFR-β (i), but after AX102 for 7 d, the colocalization was reduced, and many vessels lacked PDGFR-β (ii). After 28 d, endothelial cell staining decreased to the low value for pericytes, and the colocalization of CD31 with PDGFR-β increased compared with the value at 7 d (iii). B, measurements show a significant reduction in colocalization after AX102 for 7 d followed by an increase toward baseline at 28 d. *, P < 0.05, different from corresponding value for vehicle. C, confocal microscopic images showing blood vessels in LLC tumors stained for PDGFR-β (red) and CD31 (green) with 10% colocalization (i), 40% colocalization (ii), and 90% colocalization (iii). D, frequency distribution of pericyte-endothelial cell colocalization, expressed as vessel profiles per square millimeter of section, based on measurements of 100 individual blood vessels in LLC tumors in each of the three treatment groups. Top, colocalization pattern in vehicle-treated tumors where most vessels had 40% to 90% coverage. Middle, a very different colocalization pattern after AX102 for 7 d, where most vessels had little or no coverage. Bottom, another pattern after AX102 for 28 d, where pericyte coverage was about the same across the entire range. Differences in bar height under the three treatment conditions reflect differences in tumor vascularity. Bar in (C-iii) is 70 μm in (A) and 11 μm in (C).

Figure 5

Figure 5

Reduction of vascular basement membrane after AX102. A, fluorescence microscopic images of tumors stained for basement membrane (type IV collagen; green) and pericytes (PDGFR-β; red). Regions of colocalization are in yellow. In vehicle-treated LLC tumors, most type IV collagen colocalized with pericytes (i). AX102 for 28 d led to a reduction in type IV collagen (ii). Time course analysis revealed that after AX102, the total amount of type IV collagen was essentially unchanged at 1 d, but was reduced by 12% at 7 d, and by 54% at 28 d (iii). B, in vehicle-treated RIP-Tag2 tumors, most type IV collagen was associated with pericytes (i), but after AX102, type IV collagen was reduced by 11% at 7 d and by 21% at 28 d (ii and iii). Measurements of LLC tumors (A-iii) and RIP-Tag2 tumors (B-iii) show that pericytes decreased more rapidly and to a greater extent than type IV collagen. C, staining of basement membrane in the same section of LLC tumors showed complete colocalization of type IV collagen (i) and nidogen (ii). Laminin had a similar distribution (iii). All three basement membrane proteins in LLC tumors decreased by about the same amount after AX102 for 7 or 28 d (iv). *, P < 0.05, different from corresponding vehicle group. Bar in (C-iii) represents 110 μm in (A), 150 μm in (B), and 120 μm in (C).

Figure 6

Figure 6

Increased growth of LLC tumors after AX102. Growth curves of LLC tumors after vehicle or AX102 (50 mg/kg) treatment for 26 d. The curves were obtained by combining data from two different experiments (N = 10 for each group). P < 0.05 between the two curves by repeated measures ANOVA.

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