Image-Guided Radiotherapy Targets Macromolecules through Altering the Tumor Microenvironment

Abstract

Current strategies to target tumors with nanomedicines rely on passive delivery via the enhanced permeability and retention effect, leveraging the disorganized tumor microvasculature to promote macromolecule extravasation and the reduced lymphatic and venous drainage that favor retention. Nonetheless, FDA approvals and clinical use of nanomedicines have lagged, reflecting failure to display superiority over conventional formulations. Here, we have exploited image-guided X-irradiation to augment nanoparticle accumulation in tumors. A single 5 Gy dose of radiation, below that required to significantly delay tumor growth, can markedly enhance delivery of macromolecules and nanoparticles. The radiation effect was independent of endothelial cell integrity, suggesting a primary role for damage to microvascular pericytes and/or interstitial extracellular matrix. Significantly, radiation-guided delivery potentiated the therapeutic effects of PEGylated liposomal doxorubicin on experimental tumors. Applied to patients, these results suggest repurposing image-guided radiotherapy as a tool to guide cancer nanomedicine delivery, enhancing local control for primary tumors and metastatic disease while limiting systemic toxicity.

Keywords: enhanced permeability and retention; ionizing radiation; nanoparticle delivery; tumor microenvironment.

Figure 1

Characterizing the radiation-enhanced delivery of macromolecules and nanoparticles. (A) Following 15 Gy “ablative” irradiation of a fluorescent MCF7^GFP-IBD xenograft tumor, the microvasculature visualized through the skin (negative contrast) appears grossly patent through 7 days, before eventually collapsing. (B) At 3 days after 15 Gy irradiation, the macromolecular blood pool agent AngioSense 750 extravasates into MCF7^GFP-IBD xenograft tumor parenchyma (green autofluorescence). (C) IVIS imaging of AngioSense 750 accumulation (black to yellow gradient) in MCF7^GFP-IBD hindlimb tumors demonstrates preferential accumulation and retention after irradiation. Times listed indicate time that has passed following AngioSense administration on day 3 after irradiation. (D) Quantitation of AngioSense 750 IVIS imaging demonstrates increasing accumulation/retention with radiation dose to MCF7^GFP-IBD flank tumors when probe was injected 3 days after irradiation. *p ≤ 0.05 compared to control, n = 5. (E) IVIS imaging of differential distribution (blue to red gradient) of AngioSPARK 680 PEGylated iron oxide nanoparticles after i.v. injection 3 days after 15 Gy irradiation of a MCF7^GFP-IBD flank tumor (dorsal view, upper panel; ventral view, lower panel). Note enhanced accumulation in irradiated tumor (arrow) compared to lungs, spleen, and/or liver (arrowheads). (F) By 3 h after injection, SAIVI PEGylated 100 nm latex particles remained in circulation in unirradiated tumor tissue, as indicated by endothelial staining with tomato lectin (green, upper panel). By contrast, SAIVI particles spread into the parenchyma in the region where the tumor had been irradiated with 5 Gy, 3 days prior (lower panel).

Figure 2

Radiation alters the tumor microenvironment to enhance delivery and retention. (A) Dose response of radiation-enhanced delivery examined by confocal microscopy of MCF7^GFP-IBD tumors injected with the endothelial stain tomato lectin and blood pool agent AngioSense demonstrates a threshold between 2 and 5 Gy at 3 days after irradiation. Scale bar = 100 μm. (B) Hematoxylin-stained (purple) tissue sections of MCF7^GFP-IBD xenograft tumors excised at 17 days after irradiation display dose-dependent changes in immunohistochemical staining (brown) for CD31 (endothelium), α-SMA (pericytes), Collagen IV (basement membrane), and Collagen I (extracellular matrix). Scale bar = 200 μm. (C) Quantification of immunohistochemical staining by percent of microscopic field. *p ≤ 0.05 relative to 0 Gy control, n = 3. (D) Transmission electron microscopy of thin sections of MCF7^GFP-IBD xenograft tumor excised 3 days after 5 Gy demonstrates disorganization of interstitial collagen (*). EC = endothelial cell, BL = basal lamina, L = vessel lumen. Scale bar = 2 μm.

Figure 3

Independence of radiation-enhanced delivery and endothelial integrity. (A) Immunohistochemistry of MCF7^GFP-IBD xenograft tumors excised 17 days after irradiation suggests simvastatin protects endothelial staining (brown, CD31), preventing depletion after treatment with 5 Gy. No significant effects were seen on α-SMA, Collagen IV, or Collagen I (brown). Purple = hematoxylin, nuclei. Scale bar = 200 μm. (B) Simvastatin protects endothelium against destruction after 15 Gy. (C) Relative quantification of IHC staining in MCF7^GFP-IBD tumor sections. % area denotes the area of an image stained calculated using an ImageJ macro (details in methods). *p ≤ 0.05 relative to no treatment control, n = 3. (D) In vivo imaging revealed that, despite endothelial integrity (green, tomato lectin), AngioSense still permeated from the vasculature into the MCF7^GFP-IBD tumor interstitium. Scale bar = 100 μm. (E) MCF7^GFP-IBD tumor retention of AngioSense, measured using IVIS fluorescence quantification, does not appear limited by the endothelial protection afforded by simvastatin treatment. AngioSense was administered 3 days after IR. *p ≤ 0.05 relative to no treatment control; ^#p ≤ 0.05 relative to statin only, at the same time point, n = 5.

Figure 4

Augmented delivery and therapeutic efficacy of the liposomal chemotherapy Doxil. Confocal imaging demonstrated improved homogeneous distribution of Doxil (yellow = nuclear doxorubicin) throughout irradiated MCF7^GFP-IBD xenograft tumors, using both (A) frozen sections (blue = DAPI) and (B) intravital imaging (red = AngioSense), compared to the perivascular distribution seen in controls. Scale bar = 30 μm. (C) C57Bl/6 mice bearing syngeneic B16F10 hindlimb tumors were treated with 5 Gy at day 0 and/or 10 mg/kg Doxil at day 3. Tumor growth was measured with calipers. *p ≤ 0.05 compared to no treatment, radiation only, and Doxil only. n = 4 for each data set.