Radiation-guided drug delivery to mouse models of lung cancer

Abstract

Purpose: The purpose of this study was to achieve improved cancer-specific delivery and bioavailability of radiation-sensitizing chemotherapy using radiation-guided drug delivery.

Experimental design: Phage display technology was used to isolate a recombinant peptide (HVGGSSV) that binds to a radiation-inducible receptor in irradiated tumors. This peptide was used to target nab-paclitaxel to irradiated tumors, achieving tumor-specificity and enhanced bioavailability of paclitaxel.

Results: Optical imaging studies showed that HVGGSSV-guided nab-paclitaxel selectively targeted irradiated tumors and showed 1.48 ± 1.66 photons/s/cm(2)/sr greater radiance compared with SGVSGHV-nab-paclitaxel, and 1.49 ± 1.36 photons/s/cm(2)/sr greater than nab-paclitaxel alone (P < 0.05). Biodistribution studies showed >5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel-treated groups as compared with either nab-paclitaxel or SGVSGHV-nab-paclitaxel at 72 hours. Both Lewis lung carcinoma and H460 lung carcinoma murine models showed significant tumor growth delay for HVGGSSV-nab-paclitaxel as compared with nab-paclitaxel, SGVSGHV-nab-paclitaxel,and saline controls. HVGGSSV-nab-paclitaxel treatment induced a significantly greater loss in vasculature in irradiated tumors compared with unirradiated tumors, nab-paclitaxel, SGVSGHV-nab-paclitaxel, and untreated controls.

Conclusions: HVGGSSV-nab-paclitaxel was found to bind specifically to the tax-interacting protein-1 (TIP-1) receptor expressed in irradiated tumors, enhance bioavailability of paclitaxel, and significantly increase tumor growth delay as compared with controls in mouse models of lung cancer. Here we show that targeting nab-paclitaxel to radiation-inducible TIP-1 results in increased tumor-specific drug delivery and enhanced biological efficacy in the treatment of cancer.

Figure 1. TIP-1 receptor targeting studies

a, Proteins were co-precipitated with the HVGGSSV peptide-biotin on streptavidin. Protein was separated by PAGE and transfers were incubated with polyclonal antibody to TIP-1. Shown is the autoradiograph of TIP-1 protein at 1, 4 and 24 hours after irradiation with 3 Gy compared to TIP-1 protein in untreated controls (0 Gy). b, Tumor sections were stained with anti-TIP-1 antibody. Shown are immunohistochemical sections of untreated control and irradiated tumor. c, LLC tumors grown in both hind limbs were treated with 3 Gy (left hind limb) or 0 Gy (right hind limb). Intravenous injection of Alexa fluor 750 labeled rabbit IgG antibody (control) and rabbit anti-TIP-1 polyclonal antibody; guinea pig IgG antibody (control) and guinea pig anti-TIP-1 polyclonal antibody; mouse IgG antibody (control) and mouse anti-TIP-1 monoclonal antibody. Shown are near-infrared (NIR) fluorescence images acquired approximately 72 hrs post-injection of mice. d. Bar graph of radiance for anti-TIP-1 antibodies vs. control IgG in irradiated tumors. Shown are the mean and SEM for 6-8 animals in each group. Unpaired Student’s t test (p≤0.08).

Figure 2. HVGGSSV-nab-paclitaxel targeted to irradiated tumors

a, LLC tumors grown in both hind limbs were treated with 3 Gy (left hind limb) or 0 Gy (right hind limb). Near-infrared (NIR) images acquired 72 hrs post-injection of mice after intravenous injection of Alexa fluor 750 labeled HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, and unconjugated nab-paclitaxel. All images normalized to the same scale. Radiance was measured for both irradiated (3 Gy) and untreated (0 Gy) tumors. b, Bar graph of radiance for all treatment groups. The color scale bar indicates radiance in units of photons/s/cm²/sr. Shown are the mean and SEM for five animals in each group. Unpaired Student’s t test (p<0.01). c, To determine whether HVGGSSV specifically binds to TIP-1 in tumor microvasculature, TIP-1 blocking studies were also done using the same tumor model in nude mice and NIR imaging. Shown are NIR images of TIP-1 blocked and unblocked (control) mice. Tumors on the left hind limb were irradiated with 3 Gy and mice were given 50 μg of rabbit anti-TIP-1 polyclonal antibody intravenously 4 hrs later. Alexa fluor 750 labeled HVGGSSV-nab-paclitaxel was injected 2 hrs after antibody administration. Near-infrared imaging showed pre-blocking the TIP-1 receptor by administration of rabbit anti-TIP-1 IgG polyclonal antibody, followed by injection of HVGGSSV-nab-paclitaxel. d, Radiance was measured for both TIP-1 blocked and unblocked tumors as compared to pre-blocking with control IgG. Bar graph of radiance for both treatment groups, with mean and SEM for three animals in each group. Unpaired Student’s t test (p<0.01).

Figure 3. Colocalization HVGGSSV-nab-paclitaxel with tumor vascular endothelium

Cryosections of LLC tumors from each treatment group were stained for vascular marker von Willebrand factor (vWF) (green) 3 hrs after treatment with a, 3 Gy and b, 0 Gy. HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, and nab-paclitaxel labled with Alexa fluor 594 (red) prior to injection.

Figure 4. Biodistribution of targeted HVGGSSV-nab-paclitaxel

a, Biodistribution of paclitaxel in tumor and tissues in each treatment group compared to nab-paclitaxel. Shown are the mean and SEM from three animals in each group. b, Tumor/plasma concentration ratios at 72 hrs post-injection. Shown are the mean and SEM from three animals in each group. c, Shown are paraffin sections immunohistochemically stained for paclitaxel presence (brown), and counterstained with hematoxylin (blue). Nuclei stained brown were scored as positive and nuclei that stained blue were scored as negative. Scale bar, 50 μm.

Figure 5. Therapeutic efficacy of targeted HVGGSSV-nab-paclitaxel in LLC and H460 xenografts

Tumor growth delay studies: a, LLC murine lung carcinoma bearing C57 mice or b, H460 bearing nude mice were treated with either 3 Gy or 0 Gy and injected i.v. 5 hrs later with either targeted HVGGSSV-nab-paclitaxel, nab-paclitaxel, SGVSGHV-nab-paclitaxel or saline. Arrows indicate daily irradiation with 3 Gy every other day. Shown are graphs of fold volume increase with mean and SEM from five animals in each group. HVGGSSV-nab-paclitaxel combined with irradiation resulted in significantly greater tumor growth delay compared to tumors treated with SGVSGHV-nab-paclitaxel and irradiation or nab-paclitaxel and irradiation (p<0.01, Kruskal-Wallis). Shown are immunohistochemical stains of LLC tumor sections from each treatment group taken at 72 hrs after treatment initiation. c, Tissue slices were probed for active caspase-3 to identify cell death, and d, endothelial cell marker von Willebrand Factor (vWF) for vascular endothelium. All sections counterstained with hematoxylin. Brown stained nuclei were scored as positive and nuclei that stained blue were scored as negative. Paraffin sections, scale bar indicates 50 μm.

Figure 5. Therapeutic efficacy of targeted HVGGSSV-nab-paclitaxel in LLC and H460 xenografts

Tumor growth delay studies: a, LLC murine lung carcinoma bearing C57 mice or b, H460 bearing nude mice were treated with either 3 Gy or 0 Gy and injected i.v. 5 hrs later with either targeted HVGGSSV-nab-paclitaxel, nab-paclitaxel, SGVSGHV-nab-paclitaxel or saline. Arrows indicate daily irradiation with 3 Gy every other day. Shown are graphs of fold volume increase with mean and SEM from five animals in each group. HVGGSSV-nab-paclitaxel combined with irradiation resulted in significantly greater tumor growth delay compared to tumors treated with SGVSGHV-nab-paclitaxel and irradiation or nab-paclitaxel and irradiation (p<0.01, Kruskal-Wallis). Shown are immunohistochemical stains of LLC tumor sections from each treatment group taken at 72 hrs after treatment initiation. c, Tissue slices were probed for active caspase-3 to identify cell death, and d, endothelial cell marker von Willebrand Factor (vWF) for vascular endothelium. All sections counterstained with hematoxylin. Brown stained nuclei were scored as positive and nuclei that stained blue were scored as negative. Paraffin sections, scale bar indicates 50 μm.