Serum biomarkers for personalization of nanotherapeutics-based therapy in different tumor and organ microenvironments - PubMed (original) (raw)
Serum biomarkers for personalization of nanotherapeutics-based therapy in different tumor and organ microenvironments
Kenji Yokoi et al. Cancer Lett. 2014.
Abstract
Enhanced permeation and retention (EPR) effect, the mechanism by which nanotherapeutics accumulate in tumors, varies in patients based on differences in the tumor and organ microenvironment. Surrogate biomarkers for the EPR effect will aid in selecting patients who will accumulate higher amounts of nanotherapeutics and show better therapeutic efficacy. Our data suggest that the differences in the vascular permeability and pegylated liposomal doxorubicin (PLD) accumulation are tumor type as well as organ-specific and significantly correlated with the relative ratio of MMP-9 to TIMP-1 in the circulation, supporting development of these molecules as biomarkers for the personalization of nanoparticle-based therapy.
Keywords: Biomarker; EPR effect; Nanotherapeutics; PLD; Transport oncophysics.
Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
Conflict of interest statement
Conflict of Interest
The authors have no conflict of interest in this study to disclose.
Figures
Fig. 1
PLD accumulation and therapeutic efficacy. (A) Hematoxylin and eosin (H&E) staining and fluorescence imaging of accumulated PLD in 4T1 and 3LL tumors grown in brain. Bar indicates 100 μm. (B) Quantification of PLD accumulation in tumors. * Indicates p < 0.05 versus 4T1. The therapeutic efficacy of PLD was assessed by analyzing the survival of mice bearing tumors in the brain (C), or by measuring subcutaneous tumor growth (D). The mice were treated three times with PLD or PBS (control) at the times indicated by arrows. N.S. indicates no significant difference in the survival or tumor size. * Indicates p < 0.05 versus control.
Fig. 2
Immunofluorescence analysis of the 4T1 and 3LL tumors. (A) The amount of endothelial cells in brain tumors was evaluated by immunofluorescent analysis using a CD31 antibody. Bar indicates 100 μm. (B) Image quantification yielded no significant (N.S.) differences in the relative amount of endothelial cells. (C) A higher quantity of PLD extravasation was observed from vessels in 4T1 tumors than 3LL tumors. Bar shows 50 μm. (D) Amount of accumulated PLD to 4T1 or 3LL tumors as a function of time (* indicates p < 0.05 versus 4T1). (E) Cell proliferation, as evaluated by immunostaining with a Ki67 antibody, was significantly inhibited by PLD in 4T1 tumors (* Indicates p < 0.05 versus control), but not in 3LL tumors (N.S., no significant).
Fig. 3
Coverage of endothelial cells by the basement membrane and differential expression of MMP-9 by 4T1 and 3LL tumors in vitro and in vivo. (A) Immunofluorescence analysis of 4T1 and 3LL tumors in brain using antibodies to type IV collagen and CD31. (B) The coverage of endothelial cells by type IV collagen was quantified. * Indicates p < 0.05 versus 3LL tumors and normal tissue. (C) PLD extravasation was observed from only vessel that was not tightly covered by basement membrane (indicated by white ovals). Bar indicates 30 μm in A and 50 μm in C. (D) Protein expression analysis of 4T1 and 3LL cells in vitro was performed using a cytokine antibody array. Secretion of MMP-9 produced by 4T1 cells was higher than that secreted by 3LL cells (indicated by red ovals). (E) MMP-9 concentrations (conc.) in culture supernatant measured by ELISA. (F) Immunohistochemical analysis of MMP-9 expression in 4T1 and 3LL brain tumors. Bar indicates 100 μm. (G) Circulating MMP-9 concentrations in the mice bearing 4T1, 3LL brain metastases, or in normal mice, measured by ELISA. * Indicates p < 0.05 versus 4T1.
Fig. 4
Reduction of vascular permeability to PLD by MMP inhibitor. Batimastat or PBS was injected i.p. once a day for 3 days into the mice bearing 4T1 tumor, prior to i.v. injection of PLD. (A) Accumulation of PLD to the tumors was imaged ex vivo using an IVIS apparatus. (B) Accumulation of PLD to the tumors was abrogated by pre-treatment of mice with Batimastat. * Indicates p < 0.05 versus the other treatments. Confocal microscopic imaging of the tumors and image quantification of PLD accumulation. (C) The amount of type IV collagen. (D) And the coverage of endothelial cells by basement membrane (E). (F) Measurement of serum protein levels by ELISA yielded no significant differences in MMP-9 concentration (conc.). (G) However, a significant increase in TIMP-1 concentration was observed. * Indicates p < 0.05 versus batimastat.
Fig. 5
Organ-specific differences in vascular permeability to PLD. The accumulation of i.v. injected PLD to the tumors was (A) imaged and (B) quantified. Bar indicates 100 μm. * Indicates p < 0.05 versus brain and mfp. 4T1 tumors growing in brain, mfp, or liver were identified by accumulation of CD204-positive tumor-associated macrophages into the tumors. Macrophages in the liver (Kupffer cells) were also positively stained with a CD204 antibody. (C) The percentage of endothelial cells tightly covered by basement membrane in the tumors growing in the different organs. * Indicates p < 0.05 versus liver. (D) Intravital microscopy of the 4T1 tumors growing in mfp or liver probed with fluorescent dextran tracers of two sizes injected i.v. (Molecular Weight (MW) = 3000 Da (red) and =40,000 Da (green)). (E) Quantification of the fluorescent intensities of the tracers indicated by the white lines in D. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Organ specific differences in MMP-9 and TIMP-1concentration. (A) Serum concentration (conc.) of MMP-9 in the mice bearing 4T1 tumors in brain, mfp, or liver was measured by ELISA (graph). * Indicates p < 0.05 versus the others. MMP-9 expression in the 4T1 tumors growing in these organs was examined by immunohistochemical analysis using antibody to MMP-9. (B) Serum concentration (conc.) of TIMP-1 in these mice was measured using ELISA (graph). * Indicates p < 0.05 versus the others. TIMP-1 expression in these 4T1 tumors was examined by immunohistochemical analysis using antibody to TIMP-1. Bar indicates 100 μm. (C) PLD accumulation in the tumors is shown. * p < 0.05, 3LL brain tumors versus CT26 brain tumors. There was no significant difference in PLD accumulation to liver, regardless of the tumor type. (D) The serum concentration (conc.) of MMP-9 in the tumor bearing mice. * p < 0.05, 3LL brain tumors versus CT26 brain tumors; ** p < 0.05, 3LL liver tumors versus CT26 liver tumors. (E) The serum concentration (conc.) of TIMP-1. * p < 0.05, 3LL brain tumors versus 3LL liver tumors; ** p < 0.05, CT26 brain tumors versus CT26 liver tumors. (F) There was a significant correlation between PLD accumulation to the tumors with circulating MMP-9/TIMP-1 ratio evaluated by the linear regression analysis (_R_2 = 0.695).
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