Longitudinal investigation of permeability and distribution of macromolecules in mouse malignant transformation using PET

Abstract

Purpose: We apply positron emission tomography (PET) to elucidate changes in nanocarrier extravasation during the transition from premalignant to malignant cancer, providing insight into the use of imaging to characterize early cancerous lesions and the utility of nanoparticles in early disease.

Experimental design: Albumin and liposomes were labeled with (64)Cu (half-life 12.7 hours), and longitudinal PET and CT imaging studies were conducted in a mouse model of ductal carcinoma in situ. A pharmacokinetic model was applied to estimate the tumor vascular volume and permeability.

Results: From early time points characterized by disseminated hyperproliferation, the enhanced vascular permeability facilitated lesion detection. During disease progression, the vascular volume fraction increased 1.6-fold and the apparent vascular permeability to albumin and liposomes increased ∼2.5-fold to 6.6 × 10(-8) and 1.3 × 10(-8) cm/s, respectively, with the accumulation of albumin increasing earlier in the disease process. In the malignant tumor, both tracers reached similar mean intratumoral concentrations of ∼6% ID/cc but the distribution of liposomes was more heterogeneous, ranging from 1% to 18% ID/cc compared with 1% to 9% ID/cc for albumin. The tumor-to-muscle ratio was 17.9 ± 8.1 and 7.1 ± 0.5 for liposomes and albumin, respectively, indicating a more specific delivery of liposomes than with albumin.

Conclusions: PET imaging of radiolabeled particles, validated by confocal imaging and histology, detected the transition from premalignant to malignant lesions and effectively quantified the associated changes in vascular permeability.

Fig. 1

PET and histological images at the time of transition. Ai) Ex vivo PET image acquired 48 hours after injection of Cu-labeled liposomes at week 5 (scalebar 5 mm). Aii) H&E showing increased mitotic cells (M), indicating an area of transformation to carcinoma. This area corresponds geometrically with the region in Ai) with high tracer uptake (20 ×, scalebar 200 μm). Aiii) Low-power H&E of the MIN-O precancer filling the precleared fat pad with early carcinoma of the same tumor (scalebar 1.5 mm) as in Ai). Regions with increased liposome uptake in Ai) correspond geometrically with regions in the H&E section with high cellular density and less residual fat. Aiv) High-power H&E of acinar and ductal structures with high intra-lesional heterogeneity (20 ×, scalebar 200 μm, A-acini). Bi) Ex vivo PET image at 48 hours after injection of Cu-labeled albumin is homogenous with lower uptake than observed with liposomes in Ai) (identical image settings) (scalebar 5 mm). Bii) Precancerous region with well differentiated cells and ducts (20 ×, scalebar 200 μm). Biii) Low-power H&E of the same lesion as in Bi) with the MIN-O precancer filling the precleared fat pad (scalebar 1.5 mm). Biv) The edge of the precancerous region at higher power (20×, scalebar 200 μm). Ci and ii) Maximum-intensity-projection (MIP) images at 48 hours of the same mice as in Ai) and Bi) respectively. D) PET measurements in tumor and striated muscle correspond well with biodistribution data. White arrows indicate tumors. PET image color scale from 0–100%.

Fig. 2

Longitudinal development and malignant transformation. MIP images of a single animal as the tumors progressed at Ai) 3, Aii) 5, Aiii) 7 and Aiv) 8 weeks after transplantation of premalignant tissue from a donor. Filled white arrows indicate tumors, red arrows bladder and yellow arrows fiducial markers. B). A rapid increase in tumor growth was observed after week 5, reflecting a transition from premalignant to malignant. C) Tumor doubling time (in days) in the pre-transition phase (3–5 weeks), transition (5–7 weeks), post-transition phase (7–8 weeks). D) Images and histology post-liposome (i–iii) and -albumin (iv–vi) injection at 8 weeks after implantation. i, iv) Co-registered PET and CT images of an animal 18 hours post-liposome (i) or -albumin (iv) injection. ii, v) H&E histomorphology of tumors in i, iv demonstrate densely packed tumor cells and large cystic regions (scalebar 1.5 mm), iii, vi) High-power CD31 section of the tumor center demonstrates large collapsed vessels (10×, scalebar 150 μm). PET image color scale from 0 to 100% as in A.

Fig. 3

Tracer kinetics and biodistribution. A) Circulation of PET-labeled liposomes and albumin averaged over all weeks and animals. B) Tumoral liposome accumulation (average over animals after blood radioactivity is subtracted) vs time after injection as a function of weeks after implantation. Transition to a malignant phenotype between 5 and 7 weeks increases accumulation in step-wise fashion. C) Tumoral albumin accumulation (average over animals after blood radioactivity is subtracted) vs time after injection as a function of weeks after implantation. Transition to a malignant phenotype between 5 and 7 weeks gradually increases accumulation. D) Biodistribution after injection of albumin and liposomes in week 8. *P-value < 0.05 (image data analyzed with ANOVA and biodistribution data with Mann-Whitney test)

Fig. 4

Tumor accumulation and heterogeneity. A) Ratio of whole tumor mean tracer accumulation (at peak over time) after vs before tumor transition for the same animals and both tracers. B) Spatial mean, minimum and maximum of tumor concentration after subtracting the radioactivity in the tumor vasculature (peak over time and averaged over animals) versus weeks after implantation for albumin and liposomal tracers. Mean uptake of both tracers increased with disease progression. Maximum tumor uptake of the liposomal tracer is higher after the transition from the premalignant to malignant phenotype than with the albumin tracer reflecting greater heterogeneity. Spatial minimum accumulation of both tracers is < 1% ID/cc. C) -D) Tumor PET accumulation images and surface plots of liposomes (C) and albumin (D) at 5 weeks (upper row (i–ii)) and 8 weeks (lower row (iii–iv)). In i) and iii) the color map is described in the methods section. In ii) and iv) color map is scaled to 0–100% for each tumor. Both before and after the transition the albumin tracer accumulates faster than the larger-sized liposomal tracer, visualized by a higher intensity at 6 and 18 hours. Liposomal accumulation is heterogeneous with minimum in tumor center at week 8. Orange/red lines delineate the tumor tissue, and z-axes are in %ID/cc. *P-value < 0.05 in A) paired t-test, in B) ANOVA).

Fig. 5

Vascular volume fraction and permeability. A) Estimated vascular volume fraction based on PET images versus weeks after implantation. B) Vascular volume fraction estimated by CD31-stained sections versus weeks after implantation. C) Mean tumor apparent permeability (AP) of both tracers increases with increasing tumor volume. AP of the albumin tracer is significantly higher than liposomes at all points (p<0.05). D) Change in AP over time for each tracer. *p<0.05 tracers at the same time-point (ANOVA).

Fig. 6

Tracer kinetics and accumulation on a microscopic level. A) Confocal images of tumor tissue captured at 0, 18 and 28 hours (3D) after injection of Alexa-555-liposomes and lectin and at 18 hours in striated muscle. B) Corresponding images after injection of Alexa-555-albumin and lectin in tumor tissue and in striated muscle (3D of 0 hr tumor). C) The kinetics of the labeled liposomes in different regions within the tumor, where the interstitium shows increasing fluorescence intensity over time compared to the 0-hour time-point, indicating retention of the tracer. D) The kinetics of the Alexa-555-labeled albumin, which accumulates and clears more rapidly than in C), indicating an enhanced permeability but lower retention of the albumin. Scalebar all images 50 μm.