Fibronectin expression in metastatic tumours and normal tissues

EMT is a key step in the initiation of invasion and metastases of high-risk breast cancer. TGF-β is a major inducer of EMT and promotes ECM production35,36. The treatment of 4T1 breast cancer cells with TGF-β resulted in a fibroblast-like mesenchymal phenotype (Fig. 2a), accompanied by the appearance of the features of EMT, including upregulation of the mesenchymal marker fibronectin and downregulation of the epithelial marker E-cadherin, as determined by quantitative PCR (Fig. 2b). Fibronectin expression in metastatic tumours and normal tissues was determined by western blot in both primary and metastatic breast cancers induced by inoculating 4T1–GFP-Luc2 cancer cells in the mammary fat pad of the Balb/c mouse tumour model. As shown in Fig. 2c,d (Supplementary Fig. 1), both the primary and metastatic tumours isolated from different tissues exhibit increased levels of fibronectin. The fibronectin expression in the metastatic tumours was relatively higher than that in the primary tumours. In contrast, fibronectin expression in the normal lung and brain tissues was much lower than that in the tumours, except the liver, where plasma fibronectin is normally produced.

Figure 2: Expression of fibronectin in cells, tissues and tumours. (a) The morphology of 4T1 cells with and without TGF-β induction (15 ng ml−1, 5 days). Images were taken by phase-contrast microscopy; all scale bars, 50 μm. (b) RT–PCR analysis demonstrates that treatment with TGF-β induces upregulation of fibronectin and downregulation of E-cadherin, which are characteristic features of EMT, data represent the mean±s.d., n=3. (c) Representative western blots showing fibronectin expression in normal tissues, and in primary and metastatic 4T1 breast tumours in Balb/c mice. (d) Densitometric analysis of the expression of fibronectin normalized to that of β-actin. Values represent mean±s.d., n=3. Full size image

CREKA binds to fibrin–fibronectin complexes in metastases

The binding specificity of CREKA to the fibrin–fibronectin complexes in metastatic tumours was determined in the mouse tumour model using the fluorescence probe CREKA-Cy5.0 (Fig. 1b). Figure 3a and Supplementary Fig. 2 depict the bright-field and fluorescence images of the major organs and tissues with metastases in the mice, 4 h post injection of CREKA-Cy5.0. Tumour metastases labelled with green fluorescent protein (GFP) appear green in various organs and tissues, including brain, liver, lung, lymph node and spleen. Strong red fluorescence from CREKA-Cy5.0 was observed in the metastases, while the normal tissues showed little fluorescence (Fig. 3a,b). The overlay of GFP and Cy5.0 fluorescence images demonstrated specific binding of the targeting peptide to the metastases. In contrast, the non-targeted probe CERAK(Cys-Glu-Arg-Ala-Lys)-Cy5.0 was unable to differentiate between the metastases and normal tissues, as shown in Supplementary Fig. 3.

Figure 3: CREKA peptide specifically binds to fibronectin-related complexes in the metastatic tumours. (a) Mice bearing spontaneous metastatic 4T1–GFP-Luc2 breast tumours developed by orthotopic implantation were i.v. injected with CREKA-Cy5.0 (0.3 μmol kg−1). After 4 h, the mice were killed and tissues with metastases were imaged using the Maestro FLEX In Vivo Imaging System. (b) Fluorescence intensity ratios between metastatic tumours and normal tissues (T/N ratio), collected from different mice, represent the mean±s.d., n=5. (c) Frozen sections of metastatic tumours in the different organs shown in a were stained for fibronectin (scale bar, 100 μm). Full size image

Immunostaining of the metastatic tumour sections with an antibody against fibronectin showed abundant fibronectin expression in the tumour ECM, as shown in Fig. 3c. The fluorescence from CREKA-Cy5.0 co-localized with the fibronectin immunostaining in the metastatic tumours, while the non-specific peptide probe showed little binding in the tumour sections and no co-localization with the fibronectin staining (Supplementary Fig. 4).

MRI of metastases in mice with intracardiac cell inoculation

Next, we assessed the effectiveness of molecular MRI with CREKA-Tris(Gd-DOTA) 3 for detecting metastatic tumours in mice bearing 4T1–GFP-Luc2 or MDA-MB-231-Luc breast cancer metastases and further validated it by cryo-fluorescence imaging. The metastatic tumour model was developed by intracardiac inoculation of mice with breast cancer cells (Fig. 4a)37,38,39. Metastatic tumours, including micrometastases, were observed in the lung, liver, lymph node, adrenal gland, bone and brain, using bioluminescence imaging 2 weeks after inoculation. This was also confirmed by fluorescence cryo-imaging40 that has the ability to detect single fluorescence-labelled cancer cells (Fig. 4b-d). High-resolution 3D fluorescence images showing the GFP-labelled tumours and Cy5.0-labelled peptide binding in whole mice were acquired and constructed with fluorescence cryo-imaging after contrast-enhanced MRI (Fig. 4c). The tumour detection with cryo-imaging was found to correlate well with the bioluminescence imaging; the former is also more sensitive at detecting very small tumours. Figure 4d shows the number and size distribution of metastatic tumours detected by cryo-imaging. Figure 4e shows representative fat-suppressed T 1 -weighted FLASH MR images of the mice before and after injection of the contrast agent and correlation of tumour enhancement with fluorescence imaging. The targeted contrast agents provided robust contrast enhancement in metastatic tumours, which were clearly visible in MR images. As shown in Fig. 4e, the MRI-detected metastases are located in the bone marrow, adrenal gland and lymph nodes in the thigh and shoulder. The metastases detected by molecular MRI correlated well with those visualized by bioluminescence imaging and post-mortem high-resolution cryo-fluorescence imaging of GFP and CREKA-Cy5.0, which validates the effectiveness of molecular MRI in the detection of small metastatic tumours using the targeted contrast agent. Supplementary Fig. 5 demonstrates examples of MR images enhanced by a non-targeted contrast agent in the same tumour model, indicating that the non-targeted contrast agent is unable to provide significant contrast enhancement to detect small metastatic tumours. The targeted contrast agent CREKA-Tris(Gd-DOTA) 3 also provided robust contrast enhancement in small metastatic tumours derived from MDA-MB-231 human triple negative breast cancer cells (Supplementary Fig. 6).

Figure 4: MRI and fluorescence cryo-imaging of metastases in mice with intracardiac-injected 4T1–GFP-Luc2 breast cancer cells. (a) Illustration of MRI and fluorescence cryo-imaging of metastases in mice with intracardiac injection of cancer cells. (b) Representative bioluminescent images of mice bearing metastases. (c) Representative whole-body tumour distribution in mice bearing metastases revealed by cryo-imaging of GFP-labelled tumours and followed by segmentation using Amira software. (d) Size distribution of the metastatic tumours after the intracardiac cancer cell inoculation (n=4). (e) The selected MR images from the 3D data set before and after injection of CREKA-Tris(Gd-DOTA) 3 , and the subtraction images of the pre-injection from the post-injection images, and the enlarged subtraction MR images of metastatic sites and their corresponding cryo-images post-injection of CREKA-Cy5.0 (tumours are indicated by arrow; all scale bars, 1 mm). Full size image

Additional comprehensive analysis of the contrast-enhanced MR images in correlation with fluorescence cryo-imaging revealed robust tumour signal enhancement by CREKA-Tris(Gd-DOTA) 3 for distinct visualization of small metastatic tumours and micrometastases (0.53–19.50 mm3 in volume) in the lung, liver, muscle, bone marrow, lymph nodes and adrenal gland (Fig. 5a–c and Supplementary Fig. 7). The targeted contrast agent resulted in 76–122% signal increase in these metastases, while only 20–48% increase in the background noise was seen in the normal tissues (P<0.05) (Fig. 5d). The signal enhancement in the metastases was significantly higher than the increase in background noise, rendering clear tumour delineation, including micrometastases, in the MR images. In contrast, no significant contrast enhancement was observed in metastases in mice injected with the non-targeted contrast agent, CERAK-Tris(Gd-DOTA) 3 (Supplementary Fig. 5 and Supplementary Fig. 8). The signal increase in metastases in the mice injected with the non-targeted contrast agent was only 31–41%, which is not significantly different from the noise increase in the normal tissues (22–38%, P>0.05) (Supplementary Fig. 8).

Figure 5: Analysis of tumour metastases detected by MRI and cryo-imaging. The (a) size of the given metastatic tumours in different tissues from mice tumour model of intracardiac injection, (b) their representative cryo-images, and (c) pre-injection MRI, 25 min post-injection MRI and subtraction images of the pre-injection and post-injection MRI are shown (tumours are indicated by arrows; all scale bars, 1 mm). (d) The contrast enhancement ratios of metastases in different tissues, r=Signal post-injection /Signal pre-injection . The mean±s.d. were calculated from the tumours (n=3–6) in different mice imaged by CREKA-Tris(DOTA) 3 -enhanced MRI (*P<0.05, **P<0.01, Student’s t-test). Full size image

Molecular MRI in mice with spontaneous metastasis

We next evaluated the ability of molecular MRI with CREKA-Tris(Gd-DOTA) 3 in imaging micrometastases spontaneously developed in mice bearing orthotopic 4T1–GFP-Luc2 breast cancer xenografts in the mammary fat pad in co-registration with high-resolution fluorescence cryo-imaging (Fig. 6a). This metastatic tumour model closely mimics the physiological symptoms of naturally developed breast cancer metastases2,37,38. Metastatic tumours formed in the mice after the primary tumors were surgically excised39. High-resolution 3D MR images, which were contrast-enhanced by CREKA-Tris(Gd-DOTA) 3 , were acquired and co-registered with fluorescence cryo-images of both GFP and CREKA-Cy5.0. Representative co-registration of the contrast-enhanced MR images and cryo-images of bright-field and fluorescent channels are shown in Fig. 6b. A high quality of co-registration was achieved, because there was <100 μm difference between the MRI and cryo-images (Fig. 6c). The targeted contrast agent provided strong contrast enhancement in the metastases (Fig. 6b). Most of the micrometastases labelled with GFP were detected by MRI with CREKA-Tris(Gd-DOTA) 3 . Fluorescence cryo-imaging had the resolution and sensitivity to detect a single GFP-labelled metastatic cancer cell and was able to detect all micrometastases expressing GFP, while molecular MRI was limited by its resolution in detecting the smallest metastases. These metastases were also recognized by CREKA-Cy5.0, as shown in red in the cryo-images, further validating the effectiveness of molecular MRI. Figure 6d shows contrast-enhanced MR images of micrometastases validated by the whole-body image co-registration with both GFP and Cy5.0 fluorescence cryo-imaging. CREKA-Tris(Gd-DOTA) 3 was able to produce robust contrast enhancement in the MR images of micrometastases as small as 0.125 mm3, which enabled the detection of micrometastases in the lung, liver, bone marrow, lymph nodes and other distant organs (Fig. 6d).

Figure 6: MRI of spontaneous metastases in mice with orthotopic implant of 4T1–GFP-Luc2 breast cancer cells. (a) MRI and fluorescence cryo-imaging of cancer metastasis in mouse bearing metastasis, 40 days after cell inoculation in the mammary fat pad. (b) Representative sections showing whole-body co-registration of MRI and fluorescence cryo-images (arrows indicate metastases, auto-fluorescence from intestine and stomach is masked; Scale bars, 10 mm). (c) The co-registration quality of MRI and cryo-images. (d) Representative co-registered MRI (post-injected with the targeted contrast agent) and cryo-images of micrometastases in different organs or tissues (Scale bars, 1 mm). Full size image

Sensitivity of MRI in detecting cancer micrometastasis

In addition, we also assessed the sensitivity of molecular MRI with CREKA-Tris(Gd-DOTA) 3 in detecting metastatic tumours, in comparison with fluorescence cryo-imaging. To precisely segment the GFP-labelled tumours, healthy mice were used as controls and bright-field images were also examined to differentiate between tissues with auto-fluorescence and real metastases. Figure 7a shows the metastatic tumours detected with GFP (green) and Cy5.0 (red) fluorescence cryo-imaging and high-resolution contrast-enhanced MRI in a mouse. The tumour size distribution of spontaneous metastases based on GFP fluorescence imaging is shown in Fig. 7b. To quantify the sensitivity of the probes, contrast-to-noise ratio (CNR) was used to ascertain whether a tumour was detected or not. Here the CNR decision threshold was set to 4, which is the most commonly used value41,42. Figure 7c shows the size and number of metastases in one mouse, detected by different imaging modalities. MRI with CREKA-Tris(Gd-DOTA) 3 was able to detect 91% of the GFP-labelled metastases with size >0.5 mm3, in comparison to 94% in fluorescence imaging with CREKA-Cy5.0. When the minimally detectable tumour size was set to 0.125 mm3, molecular MRI had a sensitivity of 79% (Fig. 7c). Fluorescence imaging with CREKA-Cy5.0 was slightly more sensitive than molecular MRI in detecting micrometastases, because a considerably lower concentration of the fluorescence probe is needed to generate detectable signal than molecular MRI. Nevertheless, a sufficient amount of CREKA-Tris(Gd-DOTA) 3 was able to bind to the abundant fibrin–fibronectin complexes in the tumour ECM to generate significant signal enhancement in detecting micrometastasis with a volume >0.5 mm3 by MRI with high sensitivity comparable to fluorescence imaging. We also tested contrast enhancement of a non-targeted contrast agent CERAK-Tris(Gd-DOTA) 3 along with a non-specific fluorescence probe CERAK-Cy5.0 in the same spontaneous mouse model. MRI with this non-targeted agent was able to detect only large metastases.

Figure 7: Comparison between the sensitivity of molecular MRI and fluorescent imaging in detecting micrometastases. (a) Whole-body distribution of metastatic tumours (GFP), and Cy5.0 (CREKA-Cy5.0) binding or MRI tumour enhancement by CREKA-Tris(DOTA-Gd) 3 (positive) tumours after segmentation. (b) Size distribution of metastatic tumours in mice after orthotopic cancer cell inoculation. (c) Sensitivity of molecular MRI in detecting micrometastases, based on volumes of 82 metastatic tumours analysed. Full size image

To further evaluate tumor detection with molecular MRI, we performed free-response receiver operating characteristic (FROC) studies where the reader of the MR images was blinded to cryo-imaging results. FROC has been used in a variety of medical imaging studies to quantitatively evaluate image quality for tumour detection. Readers identify an arbitrary number of abnormalities per examination and provide the location and confidence level for each perceived abnormality43. Data are analysed by comparing with gold standard cryo-imaging. A preliminary FROC study was performed on a 4T1–GFP-Luc2 intracardiac-injected metastatic tumour model. As shown in Supplementary Fig. 9, the FROC curve indicated that the observer could blindly identify 83% metastatic tumours at a rate of 0.22 false positives per examination. In general, undetected tumours had a lower CNR and smaller size than the detected tumours.

Histological analysis in correlation with molecular MRI

Finally, we performed histological analysis of the cryo-imaged mouse sections to further verify specific binding of the molecular probes in metastatic tumours. Haematoxylin and eosin (H&E) staining of the whole-body sections from cryo-imaging also demonstrated the presence of metastatic tumours in correlation with GFP fluorescence imaging and molecular MRI. Figure 8 shows an example of multimodal examination of a mouse section with metastases. H&E staining revealed the high cell density in the metastatic tumours, which correlated with the strong fluorescence signal and MRI contrast enhancement, in comparison to normal tissues (Fig. 8b,c). Immunostaining further validated the abundant presence of fibronectin in the tumour ECM (Fig. 8d). Co-localization of fibronectin immunostaining with CREKA-Cy5.0 verified the binding of the peptide to fibrin–fibronectin complexes. By comparison, healthy tissues had low contrast enhancement in the post-injection MR images, low Cy5.0 fluorescence signal (Fig. 8a,c) and little fibronectin immunostaining (Fig. 8e).