Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression

Abstract

Molecular-genetic imaging is advancing from a valuable preclinical tool to a guide for patient management. The strategy involves pairing an imaging reporter gene with a complementary imaging agent in a system that can be used to measure gene expression or protein interaction or track gene-tagged cells in vivo. Tissue-specific promoters can be used to delineate gene expression in certain tissues, particularly when coupled with an appropriate amplification mechanism. Here we show that the progression elevated gene-3 (PEG-3) promoter, derived from a rodent gene mediating tumor progression and metastatic phenotypes, can be used to drive imaging reporters selectively to enable detection of micrometastatic disease in mouse models of human melanoma and breast cancer using bioluminescence and radionuclide-based molecular imaging techniques. Because of its strong promoter activity, tumor specificity and capacity for clinical translation, PEG-3 promoter-driven gene expression may represent a practical, new system for facilitating cancer imaging and therapy.

Figure 1

Cancer-specific PEG-Prom activity shown by bioluminescence imaging (BLI) in experimental metastasis models of human melanoma (Mel) and breast cancer (BCa). (a) BLI of a representative healthy control animal (Ctrl-2). (b) BLI showing firefly luciferase (Luc) expression observed in a representative Mel model (Mel-3). Each animal was imaged from four directions (V, ventral; L, left side; R, right side; D, dorsal views) in order to cover the entire body. Pseudo-color images from the two groups were adjusted to the same threshold. (c) Quantification of BLI signal intensity in the control group (Ctrl) and Mel group at 24 and 48 h after injection of pPEG-Luc/PEI polyplex. Quantified values are shown in Total flux (photons per second, p s⁻¹). *** P < 0.0001. (d,e) CT scans and gross anatomical views of lung from one representative animal from the control group (d) and the Mel group (e). (f,g) BLI of one representative animal from the control group (f, Ctrl-3) and the experimental breast cancer metastasis group (g, BCa-1). The pseudo-color images were adjusted to the same threshold. (h) Quantification of bioluminescent signal intensity in the Ctrl and BCa groups at 24 and 48 h after injection of pPEG-Luc/PEI polyplex. ** P = 0.0066. (i) A CT image and a macroscopic view of lung from a representative BCa animal. Displayed bioluminescent images (a,b,f,g) were obtained at 48 h after the systemic delivery of pPEG-Luc/PEI polyplex. Black arrows (e,i) indicate metastatic nodules observed in the lung. Scale bars, 5 mm.

Figure 2

Correlation between PEG-Prom-driven Luc expression and microscopic metastatic sites shown by histopathological analysis in a mouse model of melanoma metastasis (Mel). (a) BLI of a representative animal, Mel-2 from Supplementary Fig. 2b. These images were acquired at 48 h after the delivery of pPEG-Luc/PEI polyplex. (From left to right: ventral, left-sided, right-sided and dorsal views) (b–e) Hematoxylin and eosin (H&E) and firefly luciferase (Luc) staining of the formalin-fixed paraffin-embedded (FFPE) tissues collected from Mel-2. According to the imaging result (a, white solid or dotted rectangles and circles), organs tentatively associated with Luc expression were harvested for histological analysis. H&E staining confirms metastatic foci of melanoma cells in the lung (b, white solid rectangle in a), in the adrenal glands (c, white dotted rectangle in a), inside the thoracic cage adjacent to the sternum (d, white solid circle in a) and in abdominal inguinal adipose tissues adjacent to the urinary bladder (e, white dotted circle in a). Immunostaining of the consecutive sections with rabbit Luc-specific antibody (anti-Luc) shows precise correlation between the localization of microscopic metastasis and Luc expression (brown staining). Melanoma-associated melanin pigmentation was also observed in these organs (black arrows in ‘Macroscopic View’, b,c,d,e). Scale bars, 100 μm (H&E) and 2 mm (macroscopic views).

Figure 3

Histological confirmation of the microscopic metastatic sites detected by the PEG-Prom-driven BLI system in a human breast cancer metastasis model (BCa). (a) BLI of a representative animal, BCa-3 from Supplementary Fig. 3b, 24 h after the systemic delivery of pPEG-Luc/PEI polyplex (from left to right: ventral and dorsal views). The organs associated with the expression of Luc from a (black or white circles and rectangles), were collected for histological correlation. (b) H&E and Luc staining on cryosections of the lung from BCa-3, which correlates with bioluminescent light output shown in the white rectangle in a. Stained lung cryosections of a control animal (Ctrl-3) from Supplementary Fig. 3a are shown for comparison. (c–f) H&E and Luc staining of the FFPE-tissue sections collected from BCa-3. In the peripancreatic area (c, black circle in a), inside the thoracic wall adjacent to the sternum (d, white rectangle in a) and in the lymph node located in the abdominal inguinal adipose tissues adjacent to the bladder (e, white circle in a), H&E staining confirmed the presence of metastatic lesions. Thin layers of breast cancer cells were found inside the rib cage (f, black rectangle in a): between 1st–3rd ribs (f, upper panel) and 4th–7th ribs (f, lower panel). Luc staining on the consecutive sections demonstrated the co-localization of the metastatic sites and Luc expression. Another consecutive section of peripancreatic tissues was stained for human pan-cytokeratin (PAN CK) to ensure the precise co-localization of Luc expression and MDA-MB-231 cells (c). Scale bars, 100 μm.

Figure 4

Cancer-specific expression of HSV1-TK driven by PEG-Prom shown by SPECT-CT imaging in an experimental model of human melanoma metastasis (Mel). (a,b) CT, SPECT and co-registered [¹²⁵I]FIAU SPECT-CT images of lungs of the healthy control group (a, n = 3; Ctrl-1–3) and of the metastasis model of melanoma (b, n = 5; Mel-1–5). Images were acquired at 48 h after IV injection of [¹²⁵I]FIAU, which was 94 h after IV administration of pPEG-HSV1tk/PEI polyplex. Color scales are expressed as %ID/g (percentage injected dose per gram of tissue). (c) Quantification of lung SPECT images in a and b. ROIs of the same size and shape were drawn in the right lobes of the lung of each animal. Quantified radioactivity was expressed as Mean percentage ID per g (mean percentage injected dose per gram of tissue). ** P = 0.0070.

Figure 5

Detection and localization of metastatic masses by SPECT-CT imaging after the systemic administration of pPEG-HSV1tk. (a,c,e,g) Transverse, coronal and sagittal views of co-registered SPECT-CT images of Mel-2 (a) and Mel-3 (c,e,g) from Fig. 4b. All images were obtained at 24 h after [¹²⁵I]FIAU injection. (b,d,f,h) Gross anatomical details of the metastatic masses that were located based on the SPECT-CT images (a,c,e,g). Multiple metastatic sites were detected by imaging in Mel-2 (a, dotted circle). Necropsy of the corresponding area revealed melanoma masses under the brown adipose tissue in the upper dorsal area (b, dotted circle). (c) Accumulated radioactivity was detected adjacent to the thoracic mid-spine (arrow), which corresponded to a tumor at this location (d, arrow). Additional metastatic sites demonstrated by SPECT-CT imaging (e,g, arrow and dotted circle) correlated with melanoma masses uncovered immediately above the diaphragm (f, dotted circle) and in the left inguinal lymph node (h, arrow), respectively. (i,j) Cross-comparison of the PEG-Prom-mediated imaging and FDG-PET in a breast cancer metastasis model, BCa-1. Two nodules (Tu-1 and Tu-2) were detected by [¹²⁵I]FIAU-SPECT near the heart (i) and were confirmed by necropsy (j). While Tu-1 was also detected by [¹⁸F]FDG-PET, Tu-2, a smaller nodule attached to the heart, was not obvious in the PET image. SPECT images were acquired 48 h post-injection of [¹²⁵I]FIAU. The PET and SPECT images were acquired on the same day (i). (Tu, tumor; Ht, heart; Bf, brown fat) Color scales are expressed as %ID/g (percentage injected dose per gram of tissue). Scale bars, 10 mm.