Clinically-translated silica nanoparticles as dual-modality cancer-targeted probes for image-guided surgery and interventions

Abstract

Early diagnosis and treatment of melanoma are essential to minimizing morbidity and mortality. The presence of lymph node metastases is a vital prognostic predictor, and accurate identification by imaging has important implications for disease staging, prognosis, and clinical outcome. Sentinel lymph node (SLN) mapping procedures are limited by a lack of intraoperative visualization tools that can aid accurate determination of disease spread and delineate nodes from adjacent critical neural and vascular structures. Newer methods for circumventing these issues can exploit a variety of imaging tools, including biocompatible particle-based platforms coupled with portable device technologies for use with image-guided surgical and interventional procedures. We describe herein a clinically-translated, integrin-targeting platform for use with both PET and optical imaging that meets a number of key design criteria for improving SLN tissue localization and retention, target-to-background ratios, and clearance from the site of injection and the body. The use of such agents for selectively probing critical cancer targets may elucidate important insights into cellular and molecular processes that govern metastatic disease spread. Coupled with portable, real-time optical camera systems, we show that pre-operative PET imaging findings for mapping metastatic disease in clinically-relevant larger-animal models can be readily translated into the intraoperative setting for direct visualization of the draining tumor lymphatics and fluorescent SLN/s with histologic correlation. The specificity of this platform, relative to the standard-of-care radiotracer, (18)F-FDG, for potentially discriminating metastatic disease from inflammatory processes is also discussed in the setting of surgically-based or interventionally-driven therapies.

Fig. 1

Schematic of SLN mapping in the head and neck using ¹²⁴I-cRGDY-PEG-C dots. (a) Injection of ¹²⁴I-cRGDY-PEG-C dots about an oral cavity lesion with drainage to preauricular and submandibular nodes. (b) ¹²⁴I-cRGDY-PEG-ylated core-shell silica nanoparticle with surface-bearing radiolabels and peptides and core-containing reactive dye molecules (insets).

Fig. 2

Specific binding and internalization of cRGDY-PEG-C dots in a human melanoma cell line (M21). (a) Specific binding of cRGDY-PEG-C dots to M21 cells. Binding of cRGDY-PEG-dots to M21 cells and α_νβ₃-integrin receptor blocking by flow cytometry using an anti-α_νβ₃ antibody before particle probe incubation. Non-specific binding using media alone and a scrambled peptide-bound construct, cRADY-PEG-dots (controls) is shown. (Adapted from J. Clin. Invest., 2011, 121, 2768–2780) (b–d) cRGDY-PEG-C dots colocalize with endosomal and macropinocytosis markers using confocal microscopy. (b) Uptake of cRGDY-PEG-C dots into M21 cells (red puncta) with Hoechst counterstaining (blue). (c) LysoTracker Red labeling of acidic organelles (green puncta) with Hoechst counterstaining. (d) Colocalization of cRGDY-PEG-C dots with LysoTracker Red staining (yellow puncta). (e) Colocalization of cRGDY-PEG-C dots with FITC-dextran staining (yellow areas). Surface-bound cRGDY-PEG-C dots are shown around the cell periphery (arrowheads). 63× magnification images.

Fig. 3

Minimally invasive surgery utilizing a handheld fluorescence camera system. (a) ArteMIS^™ handheld camera fluorescence imaging system for open and laparoscopic procedures. (b) Minimally invasive surgery using laparoscopic tools. (c) System components (top to bottom): camera, laparoscope, and ring light. (d) Handheld gamma probe for radiodetection. (e) Optical imaging of a serial dilution of 10 nm Cy5.5-containing cRGDY-PEG-C dots (exposure = 60 ms; gain = 125; laser power = 85%; camera distance = 175 mm).

Fig. 4

Imaging of metastatic disease in a spontaneous melanoma miniswine model. (a) Whole-body ¹⁸F-FDG PET-CT sagittal and axial views demonstrating primary tumor (green arrow) and single SLN (white arrow) posteriorly within the right (Rt) neck after i.v. injection. ant, anterior. (b) High-resolution PET-CT scan reveals bilateral nodes 1 hour after subdermal, 4-quadrant, peritumoral injection of ¹²⁴I-cRGDY-PEG-C dots (SLN, arrow; left-sided node, arrowhead). (c, d) Gross images of the cut surfaces of the black-pigmented SLN (asterisk, c) and contralateral metastatic node (arrowhead, d) in the left posterior neck. (e) Low-power view of H&E-stained SLN demonstrating scattered melanomatous clusters (white arrowhead). (f) Corresponding high-power view of H&E-stained SLN, revealing melanoma cells (yellow arrowheads) and melanophages (white arrowhead). (g) Low-power image of a melanoma-specific marker, HMB-45 (white arrowhead), in representative SLN tissue. (h) High-power image of HMB-45-stained SLN tissue. (i) Low-power view of H&E-stained contralateral lymph node showing scattered melanomatous clusters (white arrowhead). (j) High-power image of contralateral node showing infiltration of melanomatous cells (yellow arrowheads). (k) Low-power image of representative normal porcine nodal tissue. (l) High-power image of representative normal porcine nodal tissue. Scale bars: 1 mm (e, g, i, k); 20 μm (f, h, j, l). (Adapted from J. Clin. Invest., 2011, 121, 2768–2780).

Fig. 5

Image-guided SLN Mapping in a spontaneous melanoma miniswine model: Pre-operative PET imaging. (a, b) Axial CT images reveal a left pelvic soft tissue mass (a, blue arrow) and left flank SLN (b, blue arrow). (c, d) Axial ¹⁸F-FDG PET images show localized activity within the tumor (c, black arrow) and left flank SLN (d, black arrow) following i.v. tracer injection. (e) Axial and (f) coronal ¹²⁴I-cRGDY-PEG-C dot co-registered PET-CT images show site of local injection about the pelvic lesion (e, white arrow). (g) Corresponding axial and (h) coronal co-registered PET-CT images localize activity to the SLN (g, white arrow). Large bladder uptake is also evident in both images. (i) Radioactivity levels of the primary tumor, SLN (in vivo, ex vivo), and a site remote from the primary tumor (i.e., background), using a handheld gamma probe.

Fig. 6

Image-guided SLN mapping in a spontaneous melanoma miniswine model: Real-time intraoperative optical imaging with correlative histology. Intraoperative SLN mapping was performed on the animal shown in Fig. 5. (a–i) Two-channel NIR optical imaging of the exposed nodal basin. Local injection of Cy5.5-incorporated particles displayed in dual-channel model (a) RGB color (green) and (b) NIR fluorescent channels (white). (c–f) Draining lymphatics distal to the site of injection. Fluorescence signal within the main draining proximal (c, d), mid (e), and distal (f) lymphatic channels (yellow arrows) extending toward the SLN (‘N’). Smaller caliber channels are also shown (arrowheads). Images of the SLN displayed in the (g) color and (h) NIR channels. (i) Color image of the exposed SLN. (j–m) Images of SLN in the color and NIR channels during (j, k) and following (l, m) excision, respectively. (n) Low power view of H&E stained SLN shows cluster of pigmented cells (black box) (bar = 1 mm). (o) Higher magnification of (n) reveals rounded pigmented melanoma cells and melanophages (bar = 50 μm). (p) Low power view of HMB-45-stained (red) SLN confirms presence of metastases (black box, bar = 500 μm). (q) Higher magnification in (p) reveals clusters of HMB-45+ expressing melanoma cells (bar = 100 μm).

Fig. 7

Discrimination of inflammation from metastatic disease: comparison of ¹⁸F-FDG and ¹²⁴I-cRGDY-PEG-C dot tracers. (a–d) Imaging of inflammatory changes using ¹⁸F-FDG-PET with tissue correlation. (a) Axial CT scan of the ¹⁸F-FDG PET study shows calcification within the left posterior neck (yellow arrows). (b) Fused axial ¹⁸F-FDG PET-CT reveals hypermetabolic activity at this same site (yellow arrows). Increased PET signal is also seen in metabolically active osseous structures (asterisks). (c) Lowand (d) high-power views of H&E-stained calcified tissue demonstrate extensive infiltration of inflammatory cells. (e–k) Metastatic disease detection following injection of ¹²⁴I-cRGDY-PEG C dots about the tumor site. (e) Pre-injection axial CT scan of ¹²⁴I-cRGDY-PEG-C dots shows calcified soft tissues within the posterior neck (yellow arrows). (f) Co-registered PET-CT shows no evident activity corresponding to calcified areas (arrow), but demonstrates a PET-avid node on the right (arrowhead). (g) Axial CT at a more superior level shows nodes (arrowheads) bilaterally and a calcified focus (yellow arrow). (h) Fused PET-CT demonstrates PET-avid nodes (N) and lymphatic drainage (curved arrow). Calcification shows no activity (arrow). (i) Low- and (j) high-power views confirm the presence of nodal metastases. (k) Single frame from a three-dimensional (3D) PET image reconstruction shows multiple bilateral metastatic nodes (arrowheads) and lymphatic channels (solid arrow) draining injection site (white asterisk). Bladder activity is seen (dashed arrow) with no significant tracer accumulation in the liver (black asterisk). Bladder activity is seen with no significant tracer accumulation in the liver. Scale bars: 500 μm (c, d); 100 μm (i, j).

Fig. 8

3D integrated ¹⁸F-FDG and ¹²⁴I-cRGDY-PEG-C dot PET-CT. (a–c) 3D volume rendered images were generated from CT and PET imaging data shown in Fig. 7. (a) PET-CT fusion image (coronal view) shows no evident nodal metastases (asterisks). Increased activity within bony structures is identified. (b, c) High-resolution PET-CT fusion images showing coronal (b) and superior views (c) of bilateral metastatic nodes (open arrows) and lymphatic channels (curved arrows) within the neck following local injection of ¹²⁴I-cRGDY-PEG-C dots.

Fig. 9

Assessment of treatment response after radiofrequency ablation (RFA) using ¹²⁴I-cRGDY-PEG-C dots (a–c) Single-dose ¹²⁴I-cRGDY-PEG-C dot localization of the SLN. (a) Baseline coronal CT (white arrowhead), (b) PET (black arrowhead), and (c) fused PET-CT images (white arrowhead) following a peritumoral injection. (b–d) Tumor ¹²⁴IcRGDY- PEG-C dot activity. (b) PET-avid exophytic left pelvic mass (black arrow). (c, d) Combined PET-CT images showing a PET-avid lesion (white arrow) and ¹²⁴I-cRGDY-PEG-C dot flow within a draining lymphatic channel (asterisk) towards the SLN (curved arrow). (e, f) Pre-ablation axial CT images locate the SLN (e, white arrowhead) prior to RFA electrode placement (f, arrow) into the node (below crosshairs). (g) Pre-ablation fused PET-CT reveals increased SLN activity (posterior to cross-hairs). (h) Postablation PET-CT scan shows mildly reduced activity at the SLN site, anterior to the needle tip. (i) Corresponding pre-ablation H&E staining of core biopsy tissue from the SLN confirms pigmented tumor infiltration (bar = 200 μm). (j) High magnification of boxed area in (i) reveals large, rounded pigmented clusters of melanoma cells (bar = 50 μm). (k) Post-ablation H&E staining shows necrotic changes within a partially tumor-infiltrated node (box) and multifocal hemorrhages (bar = 500 μm). (l) High magnification of (k) reveals significant tissue necrosis (arrowheads) within the metastatic node, in addition to lymphoid tissue (bar = 50 μm). (m) TUNEL staining of metastatic SLN before ablation (bar = 20 μm). (n) Post-ablation TUNEL staining demonstrating focal areas of necrosis (red) with adjacent scattered tumor foci and normal nodal tissue (NT) (bar = 500 μm). (o) High magnification of boxed area in (n) shows positive TUNEL staining (red area), consistent with necrosis (bar = 20 μm).