Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 5;36(1):1-12.
doi: 10.1038/onc.2016.182. Epub 2016 Jun 6.

VEGF-ablation therapy reduces drug delivery and therapeutic response in ECM-dense tumors

F Röhrig  1   2   3 , S Vorlová  2 , H Hoffmann  1   2   3 , M Wartenberg  4 , F E Escorcia  5 , S Keller  1 , M Tenspolde  1 , I Weigand  1 , S Gätzner  2 , K Manova  6 , O Penack  7 , D A Scheinberg  5 , A Rosenwald  4 , S Ergün  1 , Z Granot  8 , E Henke  1   2   3
Affiliations

VEGF-ablation therapy reduces drug delivery and therapeutic response in ECM-dense tumors

F Röhrig et al. Oncogene. .

Abstract

The inadequate transport of drugs into the tumor tissue caused by its abnormal vasculature is a major obstacle to the treatment of cancer. Anti-vascular endothelial growth factor (anti-VEGF) drugs can cause phenotypic alteration and maturation of the tumor's vasculature. However, whether this consistently improves delivery and subsequent response to therapy is still controversial. Clinical results indicate that not all patients benefit from antiangiogenic treatment, necessitating the development of criteria to predict the effect of these agents in individual tumors. We demonstrate that, in anti-VEGF-refractory murine tumors, vascular changes after VEGF ablation result in reduced delivery leading to therapeutic failure. In these tumors, the impaired response after anti-VEGF treatment is directly linked to strong deposition of fibrillar extracellular matrix (ECM) components and high expression of lysyl oxidases. The resulting condensed, highly crosslinked ECM impeded drug permeation, protecting tumor cells from exposure to small-molecule drugs. The reduced vascular density after anti-VEGF treatment further decreased delivery in these tumors, an effect not compensated by the improved vessel quality. Pharmacological inhibition of lysyl oxidases improved drug delivery in various tumor models and reversed the negative effect of VEGF ablation on drug delivery and therapeutic response in anti-VEGF-resistant tumors. In conclusion, the vascular changes after anti-VEGF therapy can have a context-dependent negative impact on overall therapeutic efficacy. A determining factor is the tumor ECM, which strongly influences the effect of anti-VEGF therapy. Our results reveal the prospect to revert a possible negative effect and to potentiate responsiveness to antiangiogenic therapy by concomitantly targeting ECM-modifying enzymes.

PubMed Disclaimer

Figures

Figure 1
Figure 1
MT6 sarcomas are resistant to VEGF ablation but react with vascular changes. (a, b) Treatment of established MT6 tumors with mG6-31 (5 mg/kg BW i.p., on days 12 and 18 indicated by black arrowheads) did not affect tumor growth, measured using a caliper or by weighting excised tumors. (c) Sections of tumors were stained for pan endothelial cell antigen (PanEC). Treated tumors display drastically reduced vessel density. Amount of PanEC-positive vessels was counted in whole tumor sections (n=4). Scale bar: 1.00 mm. (d) After mG6-31 treatment, remaining vessels showed increased coverage with NG2-positive pericytes, which was again quantified after imaging whole double immunofluorescent (PanEC and NG2) stained tumor sections (n=5). (e) Treated tumors showed stronger staining for CA IX, indicating increased hypoxia (scale bar: 50 μm, n=5).
Figure 2
Figure 2
VEGF ablation leads to therapeutic failure in MT6 fibrosarcoma. (a–c) Established MT6 sarcomas were treated with mG6-31 (5 mg/kg BW, i.p.) or a control antibody (IgG) and consecutively with two rounds of either doxorubicin or doxil (note: data from a six-armed study was divided into two graphs for clarity). Tumors did not react significantly to mG6-31 treatment alone, and animals pretreated with mG6-31 did respond less to cytotoxic treatment. Treatment days are indicated with black (mG6-31) and red (doxorubicin/doxil) arrowheads. (d–f) MT6 tumors treated with mG6-31 and injected with doxorubicin, doxil or 3H-paclitaxel. mG6-31 pretreatment reduced accumulation of all three drugs in the tumor. (g) 3D angiography using Alexa647-labeled Isolectin (IL-GS-IB4) shows reduction in perfused vessel density in mG6-31-treated tumors. (h) Distribution of H33342 in whole tumor sections was also reduced after mG6-31 treatment (n=4). * indicates statistical significance of doxorubicin/doxil treatment groups versus double treatment groups, *P<0.05, **P<0.01. All error bars: ±s.e.m.
Figure 3
Figure 3
Effect of VEGF ablation on drug distribution and drug tissue penetration is context dependent. (a) 4T1 tumors were treated with mG6-31 or a control IgG and consecutively treated with two rounds of doxorubicin. Treatment days are indicated with black (mG6-31) and red (doxorubicin/doxil) arrawheads. (b) Weight of 4T1 tumors excised 26 days after implantation and 14 days of treatment. (c, d) Pretreatment with mG6-31 did not change amounts of doxorubicin or 3H-paclitaxel delivered to 4T1 tumors and normal organs. Drug accumulation assays were performed on day 14 after tumor implantation, 2 days after the initial mG6-31 treatment. (e) Distribution of H33342 in whole tumor sections was improved after mG6-31 treatment. (f) Immunostaining for CD31 in 4T1 and MT6 tumors treated with mG6-31or an IgG control antibody (scale bar: 100 μm). (g, h) Quantification of relative area stained positive for CD31 and quantification of microvessel density in MT6 and 4T1 tumors treated with mG6-31 or control IgG. (i, j) Isolectin GS-4B staining of perfused vessels (Z-projections: 45 slides each with 0.98-μm spacing, scale bar: 100 μm) showed a more tortuous and convoluted vessel network in the MT6 tumors. mG6-31 reduced the degree of ramification in MT6 tumors. (k–m) Quantification of H33342 penetration depth from vessel surface and tissue volume supplied with detectable amounts of H33342 in correlation to vessel surface area in MT6 and 4T1 tumors treated with mG6-31 or control IgG. * indicates statistical significance versus control, '#' of double treatment groups versus both single treatment groups. *P<0.05; **,##P<0.01; ***,###P<0.001. All error bars: ±s.e.m., n=4 if not otherwise indicated. NS, not significant.
Figure 4
Figure 4
ECM quantity and characteristics differ significantly between responsive and non-responsive tumors. (a) Difference in the deposition of collagenous matrix proteins in 4T1 and MT6 tumors visualized by Masson-Goldner trichrome (MGTR) and picrosirius red staining (PSR) (scale bar: 50 μm). (b) Schematic view of ECM isolation process. (c) Protein quantification of matrix extracts from 4T1 and MT6 tumors, using either resolubilization in urea buffer (usECM) or resuspension of complete ECM extracts (cECM). (d) Schematic view of matrix permeability assay. (e, f). Permeability of cECM from 4T1 and MT6 tumors for doxorubicin in the transwell assay was recorded by continuous fluorescence measurement. (g) Relative permeability of usECM extracts calculated from the slope of the signal. (h) Relative mRNA expression of ECM proteins and lysyl oxidases (LOX, LOXL1–LOXL4) in 4T1 versus MT6 cells. (i) Overall lysyl oxidase activity measured in the supernatant of cultured MT6 and 4T1 cells. (j) Relative mRNA expression of lysyl oxidases in MT6 tumors versus the expression levels in 4T1 tumors. (k) Measurement of doxorubicin permeability of matrigel (MG) and collagen I-coated transwell membranes pretreated with rhmLOX or rhLOXL2. All error bars: ±s.e.m. NS, not significant.
Figure 5
Figure 5
Lysyl oxidase inhibition improves drug accumulation and distribution within tumors. (a) Treatment of established MT6 and 4T1 tumors with βAPN (100 mg/kg BW i.p., treatment days are indicated by arrowheads). (b) Doxorubicin accumulation in βAPN-treated MT6 and 4T1 tumors versus control tumors and in normal organs of βAPN-treated MT6-bearing C57Bl/6 J mice versus organs from control animals. (c) 3D confocal micrographs of MT6 and 4T1 tumors injected with H33342 and IL-IB4-A647 after βAPN treatment (Z-projections: 30 slides each with 0.90-μm spacing, SB: 100 μm). (d) Quantification of the H33342-positive area in MT6 tumors after βAPN treatment (n=4). (e) mRNA levels for the hypoxia marker CA IX and for VEGF-A in MT6 and 4T1 tumors after βAPN-treatment (n=5). (f) Whole mount sections of MT6 and 4T1 tumors display a strong reduction of central necrosis after APN treatment (NA=necrotic area, SB=1.00 mm). (g) Quantification of necrotic areas in MT6 and 4T1 tumor sections (n=8). (h) Relative permeability of usECM extracts obtained from control and APN treated MT6 and 4T1 tumors. Transwell inserts were coated with 3 μg/mm2 of respective usECM extracts and tested for doxorubicin permeability (n=3). * indicates statistical significance versus control. All error bars: ±s.e.m.
Figure 6
Figure 6
Lysyl oxidase inhibition reverses the negative effect of VEGF ablation on drug transport in MT6 fibrosarcoma. (a) Treatment of established MT6 tumors with doxorubicin (5 mg/kg BW i.p.), βAPN (100 mg/kg BW i.p.) and mG6-31 (5 mg/kg BW i.p) at the indicated days (black, blue and red arrowheads, respectively). Combining doxorubicin with βAPN improved efficacy; addition of mG6-31 reduced growth of the tumors further. (b) Schedule for short-term βAPN treatment of MT6 tumors. (c) H33342 penetration (n=9) was measured by 3D confocal imaging in MT6 tumors after short-term treatment. (d) Doxorubicin delivery into MT6 tumors of the different treatment groups was quantified by extraction (n=6–8). (e) Schematic model of the combined effect of vessel maturation and ECM permeability on drug delivery: The effect of antiangiogenic treatment is characterized by improvement of vessel maturation resulting in improved perfusion and transport through the individual blood vessel. In tumor tissue with high lysyl oxidase activity, drug diffusivity is strongly impaired and tissue drug penetration is widely independent from drug levels supplied by the individual vessels. Inhibition of lysyl oxidases can improve drug diffusion and restore increased drug delivery after antiangiogenic treatment. All error bars: ±s.e.m. # indicates statistical significance of double treatment group versus control and single treatment group, § of triple treatment group versus double treatment group. #,§P<0.05; ##P<0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kratz F. Drug delivery in oncology − challenges and perspectives. In: Kratz F, Steinhagen H, Senter P (eds). Drug Delivery in Oncology − Challenges and Perspectives in Drug Delivery in Oncology – from Research Concepts to Cancer Therapy, vol. 1. VCM: Weinheim, Germany; 2011, pp LIX-LXXXV.
    1. Gangloff A, Hsueh WA, Kesner AL, Kiesewetter DO, Pio BS, Pegram MD et al. Estimation of paclitaxel biodistribution and uptake in human-derived xenografts in vivo with (18)F-fluoropaclitaxel. J Nucl Med 2005; 46: 1866–1871. - PubMed
    1. Kesner AL, Hsueh WA, Htet NL, Pio BS, Czernin J, Pegram MD et al. Biodistribution and predictive value of 18 F-fluorocyclophosphamide in mice bearing human breast cancer xenografts. J Nucl Med 2007; 48: 2021–2027. - PubMed
    1. Staffhorst RW, van der Born K, Erkelens CA, Hamelers IH, Peters GJ, Boven E et al. Antitumor activity and biodistribution of cisplatin nanocapsules in nude mice bearing human ovarian carcinoma xenografts. Anticancer Drugs 2008; 19: 721–727. - PubMed
    1. Memon AA, Jakobsen S, Dagnaes-Hansen F, Sorensen BS, Keiding S, Nexo E. Positron emission tomography (PET) imaging with [11C]-labeled erlotinib: a micro-PET study on mice with lung tumor xenografts. Cancer Res 2009; 69: 873–878. - PubMed

Publication types

MeSH terms

Substances