Limited Tumor Tissue Drug Penetration Contributes to Primary Resistance against Angiogenesis Inhibitors

Abstract

Resistance mechanisms against antiangiogenic drugs are unclear. Here, we correlated the antitumor and antivascular properties of five different antiangiogenic receptor tyrosine kinase inhibitors (RTKIs) (motesanib, pazopanib, sorafenib, sunitinib, vatalanib) with their intratumoral distribution data obtained by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). In the first mouse model, only sunitinib exhibited broad-spectrum antivascular and antitumor activities by simultaneously suppressing vascular endothelial growth factor receptor-2 (VEGFR2) and desmin expression, and by increasing intratumoral hypoxia and inhibiting both tumor growth and vascularisation significantly. Importantly, the highest and most homogeneous intratumoral drug concentrations have been found in sunitinib-treated animals. In another animal model, where - in contrast to the first model - vatalanib was detectable at homogeneously high intratumoral concentrations, the drug significantly reduced tumor growth and angiogenesis. In conclusion, the tumor tissue penetration and thus the antiangiogenic and antitumor potential of antiangiogenic RTKIs vary among the tumor models and our study demonstrates the potential of MALDI-MSI to predict the efficacy of unlabelled small molecule antiangiogenic drugs in malignant tissue. Our approach is thus a major technical and preclinical advance demonstrating that primary resistance to angiogenesis inhibitors involves limited tumor tissue drug penetration. We also conclude that MALDI-MSI may significantly contribute to the improvement of antivascular cancer therapies.

Keywords: angiogenesis; cancer; imaging mass spectrometry; matrix assisted laser desorption ionization; receptor tyrosine kinase inhibitor; resistance.

Figure 1

Graphical abstract of the study procedure. Ten serial frozen sections were cut from C26 and C38 mouse tumors treated with five different antiangiogenic RTKIs. Sections were used then to analyze drug dispersal by MALDI-MSI and, moreover, for HE staining and immunolabeling with antibodies against CD31, laminin, desmin, αSMA and the target receptors of RTKIs (VEGFR2, PDGFRα, PDGFRβ and FGFR1). An additional slide was also used for hypoxia detection. After scanning the tissue sections, the antitumor and antivascular properties of RTKIs were correlated with their tumor tissue distribution data obtained by MALDI-MSI.

Figure 2

Tumor tissue concentrations of antiangiogenic RTKIs. Signal intensities (normalized to TIC) of the appropriate RTKIs in treated tumors and the same non-specific normalized m/z values measured in control tumors were used to calculate intratumoral drug concentrations. Data are shown as box (first and third quartiles) and whisker (maximum to minimum) plots with the mean (horizontal bar) from 6 animals per group.

Figure 3

Representative images of drug distribution in C26 and C38 tumors after two weeks of treatment with different antiangiogenic RTKIs. Precursor ion signals of RTKIs were normalized to TIC.

Figure 4

In vivo growth inhibition of C26 and C38 tumors. Balb/C (C26) and C57Bl/6 (C38) mice were randomized to receive either vehicle (n=6) or 100 mg/kg RTKI (n=6/subgroup) treatment p.o. 5 times a week for two weeks. Out of the five different antiangiogenic RTKIs (motesanib, pazopanib, sorafenib, sunitinib and vatalanib), only sunitinib reduced significantly the in vivo growth of C26 mouse colon adenocarcinoma cells in Balb/C mice (a). In the C38 model, besides sunitinib, vatalanib also demonstrated a significant growth-inhibitory effect (b). Growth curves are means for six mice per group; bars, SEM. Mean tumor volumes at the beginning of treatments were 155.3±25.4 mm³ and 376.9±51.2 mm³ in the C26 and C38 models, respectively. *P=0.0018, **P=0.0048

Figure 5

Graph (a) and representative images (b) of hypoxic area ratios in the C26 model. Hypoxic area ratios are shown in the percentage of the total tumor section. Green, anti-pimonidazole staining for hypoxia; blue, nuclear staining with Hoechst. *P=0.0152

Figure 6

Microvessel areas of mouse C26 (a, b) and C38 (c, d) tumors. Antiangiogenic RTKI-treated C26 (a) and C38 (c) tumors were labeled with the endothelial cell marker CD31 (red) and with Hoechst33342 (as nuclear counterstain; blue). Microvessel areas of C26 (b) and C38 (d) tumors were calculated by counting the number of CD31-positive pixels in the total area of ten intratumoral regions. In (b and d), data are shown as box (first and third quartiles) and whisker (maximum to minimum) plots with the mean (horizontal bar) from 6 animals per group. *P<0.05; **P<0.03