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The Precise Diagnosis of Cancer Invasion/Metastasis via 2D Laser Ablation Mass Mapping of Metalloproteinase in Primary Cancer Tissue Xiangchun Zhang, Ru Liu, Qing Yuan, Fuping Gao, Jiaojiao Li, Ya Zhang, Yuliang Zhao, Zhifang Chai, Liang Gao, and Xueyun Gao ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05584 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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The Precise Diagnosis of Cancer Invasion/Metastasis via 2D Laser Ablation Mass Mapping of Metalloproteinase in Primary Cancer Tissue Xiangchun Zhang1,2, Ru Liu2, Qing Yuan1,2, Fuping Gao2, Jiaojiao Li1,2, Ya Zhang1,2, Yuliang Zhao2, Zhifang Chai2, Liang Gao1,2*, Xueyun Gao1,2*

1Department

of Chemistry and Chemical Engineering, Beijing University of Technology,

Beijing 100124, China 2CAS

Key Laboratory for the Biological Effects of Nanomaterials and Nanosafety,

Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China

*To whom correspondence should be addressed: Email: [email protected]; [email protected]

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ABSTRACT: Cancer invasion and metastasis remain the major causes of over 90% of patient deaths. Molecular imaging methods such as computed tomography (CT)/magnetic resonance imaging (MRI) can precisely assess primary regional lymph node invasion and distant organ metastasis via body scanning; however, such diagnostic methods are often utilized too late for cancer therapy. To date, pathologic methods mainly provide information on differentiation/proliferation and potential drug therapy biomarkers of primary tumours rather than precisely reveal tumour regional invasion and distant metastasis in the body. We hypothesized that quantification of membrane type-1 matrix metalloproteinase (MT1-MMP) levels in primary tumour tissue will provide a precise assessment of tumour regional lymph node invasion and remote organ metastasis. In this work, we developed peptide-coated Au clusters with intrinsic red fluorescence and a specific mass signal. When these clusters labelled MT1-MMP in tumour tissue sections derived from the xenograft lung carcinoma model, human lung carcinoma and human renal carcinoma, we could directly observe MT1-MMP via optical fluorescence microscopy and quantitatively detect the MT1-MMP expression level via laser ablation inductively coupled plasma mass spectrometry 2D mapping (2D-LA-Mass Mapping). By observing and quantifying the MT1-MMP expression level in primary human lung carcinoma and human renal carcinoma tissue sections, we precisely assessed the risk of primary tumour invasion/metastasis. Importantly, the accuracy of this pathologic method was verified by CT/MRI molecular imaging of cancer patients and traditional haematoxylin

and

eosin

(H&E)

staining/immunohistochemistry

(IHC)/immunofluorescence (IF) pathologic studies of primary tumour tissues.

KEYWORDS: primary tumour invasion/metastasis, Au clusters, MT1-MMP, 2D-LA-Mass Mapping, lung carcinoma, renal carcinoma


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Despite major advances in the elucidation/treatment of cancer, cancer invasion and metastasis remain the primary causes of over 90% of patient deaths.1-3 Primary localized cancer mortality has significantly declined over the past ten years; however, the 5-year survival rate of metastatic cancer did not improve significantly compared with that of primary localized cancer in patients.4 To date, the dominant clinical diagnostic strategies for cancer regional invasion and distant metastasis are molecular imaging methods such as computed tomography (CT)/magnetic resonance imaging (MRI), which can precisely assess regional lymph node invasion and distant organ metastasis via body imaging. 5-7 However, the diagnostic results are too late for cancer patients as cancer invasion/metastasis have already occurred. The more effective strategy is to directly observe the primary tumour tissue to determine the invasion/metastasis risk by pathologic methods. If this method worked, it will provide a big benefit for primary cancer therapy even before cancer invasion/metastasis. To date, however, pathologic methods mainly provide differentiation and proliferation information on primary tumours rather than precisely reveal tumour regional invasion and distant metastasis information in the body.8 Although pathologic methods can determine biomarker expression levels of primary tumours for cancer therapy and prognosis, quantification of primary tumour-specific biomarkers for precise diagnosis of regional lymph node invasion and distant organ metastasis has never been achieved by pathologic studies.9,10 It would be beneficial if we could analyse specific biomarkers of primary tumour tissue and precisely assess the risk of primary tumour invasion/metastasis according to biomarker expression levels. Previous studies have revealed that when a primary tumour develops, tumour cells invade the surrounding normal tissues and lymph nodes.11-13 During this process, matrix metalloproteinases (MMPs) of tumour cells play a pivotal role in cleaving the extracellular matrix (ECM).14-16 For the MMP family, membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) is particularly important for tumour cell invasion because it directly degrades ECM macromolecules, including


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laminins 1 and 5, vitronectin, fibronectin, collagen types I, II, and III, fibrin and aggrecan.17-20 In addition, MT1-MMP promotes primary tumour invasion by activating pro-MMP-2 to degrade the ECM and promote angiogenesis.21,22 MT1-MMP has been used as a potential drug target for tumour therapy of various cancers, including cervical cancer,23 glioblastomas,24 prostate cancer,25 melanoma,21 lung cancer,26 and renal cancer.27 We hypothesized that quantitatively detecting the MT1-MMP level in primary tumour tissue may help researchers to effectively assess the invasion/metastasis risk even before CT/MRI body scanning. This strategy should provide a major advantage for cancer invasion/metastasis diagnostics compared to molecular imaging methods, which only detect cancer invasion/metastasis after they have occurred and which may provide this information too late for cancer therapy. Antibody-related immunohistochemistry (IHC) and immunofluorescence (IF) are key methods to assess biomarkers of tumour tissue sections in clinical applications.28-30 However, these methods have difficulty in quantitatively detecting MT1-MMP as the blurred colour changes in IHC or IF. For quantitative determination of the MT1-MMP expression levels in tumour tissue sections, mass spectrometry 2D imaging (MSI) could be used by labelling the biomarker with metal isotope–carrying antibodies.31-34 However, with these MSI methods, pathologists only observe the 2D virtual imaging of biomarkers in tumour tissue sections derived from isotope mass signals, and they cannot directly assess the biomarkers in tumour tissue by optical microscopy. Recent years, noble metal clusters have attracted much research attention due to their essential characteristics.35-37 Particularly, artificial peptides have been utilized to biomineralize metal clusters for targeting the enzymes including thioredoxin reductase 1,38 glutathione peroxidase-1,39 protein kinase A and casein kinase II.40 Herein, we establish a precise visible quantification method to detect MT1-MMP in primary tumour tissue sections by using metal clusters. Briefly, we developed peptide-coated metal clusters with intrinsic red fluorescence and a specific mass signal. When these clusters specifically labelled the


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MT1-MMP of formalin-fixed, paraffin-embedded (FFPE) tumour tissue sections, we could directly observe MT1-MMP via optical fluorescence microscopy and quantitatively determine the expression level via MSI. By observing and quantifying the MT1-MMP expression levels in primary tumour tissue sections, we effectively assessed the risk of primary tumour invasion/metastasis. Importantly, the accuracy of this pathologic method was verified by CT/MRI molecular imaging of cancer patients and the traditional haematoxylin and eosin (H&E) staining/IHC/IF pathologic methods of primary tumour tissues.

RESULTS AND DISCUSSION Illustrations of Au-CL Cluster Probes Applied in Tumour Pathological Analysis. We designed a functional peptide to construct Au cluster probes, as described in the Methods and

Supporting

Information

(Figures

S1-S3).

The

peptide

sequence

is

H2N-CCYHWKHLHNTKTFL-COOH (CL). The CC captures Au clusters, and the HWKHLHNTKTFL can specifically target MT1-MMP.41,42 The CL-coated Au (Au-CL) clusters have the following functions: 1) specific targeting of MT1-MMP by the CL peptide; 2) in situ fluorescent labelling of MT1-MMP protein on cells/tissues as Au clusters with red light emission; and 3) precise quantification of MT1-MMP membrane protein on cells/tissues as Au clusters with specific mass signals. Furthermore, a laser ablation inductively coupled plasma mass spectrometry 2D mapping (2D-LA-Mass Mapping) instrument was used to quantify the MT1-MMP level in primary tumour tissue sections. An illustration of Au-CL clusters in tumour cell/tissue analysis is shown in Scheme 1. Specific

Labelling

and

Quantitative

Detection

of

MT1-MMP

in

Lung

Adenocarcinoma PC-14 and A549 Cells at the Single Cell Level. Confocal laser scanning microscopy (CLSM) was employed to observe the specific labelling of MT1-MMP by Au-CL clusters in lung adenocarcinoma PC-14 and A549 cell lines. After


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the Au-CL clusters were cultured with these cell lines, the fluorescence imaging showed bright red signals in the PC-14 cell membrane, while there was relatively weak fluorescence on the A549 cell membrane (Figure 1A). For MT1-MMP, the specific targeting of Au-CL clusters was confirmed by an immunofluorescence co-localization assay.43 As depicted in Figure 1B, green fluorescence of PC-14 and A549 cell membranes derives from FITC-labelled MT1-MMP antibodies, while red fluorescence originates from the red emission of Au-CL clusters. The yellow colour on these cell membranes in the merged images reveals that Au-CL clusters target MT1-MMP. Moreover, a free CL peptide blocking study was employed to confirm the Au-CL specific targeting of MT1-MMP. PC-14 and A549 cells were first incubated with 5 mM free CL peptide for 1 h, after which Au-CL clusters were introduced into cultured cells. As expected, there was no fluorescence on the PC-14 and A549 cell membranes (Figure 1C) because the binding sites of MT1-MMP were occupied by free CL peptide.43 To accurately quantify MT1-MMP of the cell membrane by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), we first optimized the conditions for Au-CL labelling of PC-14 and A549 cells (Experimental Section and Figures S4, S5 in Supporting Information). The optimal conditions for Au-CL clusters labelling of PC-14 cells were derived by CLSM and inductively coupled plasma mass spectrometry (ICP-MS) (Figure S4). Similar studies were also performed for A549 cells (Figure S5). All the studies verified that the optimal conditions for Au-CL clusters labelling of lung adenocarcinoma cells (PC-14 or A549) were 60 min incubation time and 9 μM Au-CL clusters in cell culture. Under these optimized conditions, the Au-CL-labelled cells were first imaged by CLSM. Subsequently, the cells were ablated by the LA-ICP-MS system to acquire Au signals at the single cell level. Figure 1E and 1F show the Au signal counts of PC-14 and A549 cells at the single cell level, respectively. From the calibration curve (Figure S7), we calculated that the mass of Au in single cells ranged from 41.23 to 105.49 fg for PC-14 cells (Figure 1E and 1G) and from 10.50 to 25.99 fg for A549 cells


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(Figure 1F and 1G). As a single Au-CL cluster contained 26 Au atoms, the MT1-MMP in a single cell ranged from 8.05 to 20.59 amol for PC-14 and 2.05 to 5.07 amol for A549. The average MT1-MMP concentration was 16.30±2.94 amol for PC-14 cells and 3.41±0.70 amol for A549 cells (Figure 1G, S6, Table S1 and S2). This result is consistent with the data acquired from ICP-MS of large population of PC-14 and A549 cells (Figure S4 and S5). As depicted in Figure 1G, the expression level of MT1-MMP in PC-14 cells was higher than that of A549 cells at either the single cell level or the average level. The higher level of MT1-MMP of PC-14 indicated that this cell line is more invasive/metastatic than A549 cells. The MT1-MMP Expression Levels of Lung Adenocarcinoma PC-14 and A549 Cells were Positively Correlated with Their Invasion/Metastasis in Vitro. To verify the accuracy of the aforementioned LA-ICP-MS results, we evaluated the expression level of MT1-MMP in human lung adenocarcinoma PC-14 and A549 cells by a traditional immunoblotting method. As depicted in Figure 2A, PC-14 cells expressed more MT1-MMP (65 kDa band) than A549 cells. The grey value of the MT1-MMP band was analysed using ImageJ software. Figure 2B indicates that the expression level of MT1-MMP in A549 cells was 24.14% of that in PC-14 cells (MT1-MMP of PC-14 cells was defined as 100%). This immunoblotting result matched the LA-ICP-MS data in Figure 1G, Table S1 and S2. However, immunoblotting requires the lysis of large numbers of cell populations, and the protein may be lost in sample processing. Thus, this method may not precisely quantify MT1-MMP at the single cell level. The invasion and metastasis of lung adenocarcinoma cells (PC-14 and A549) were assessed with wound healing and invasion assays, respectively.44 Compared to A549 cells, PC-14 cells showed strong migration after scratching (Figure 2C). The wound healing rate of PC-14 cells was defined as 100%, and the PC-14 cells had a higher invasive ability than the A549 cells (46.67%, Figure 2D). Transwell assays were performed to further confirm the metastatic ability of PC-14 and A549 cells in vitro. The


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Transwell chambers were coated with Matrigel to imitate a natural 3D ECM environment in vivo.44 Highly metastatic tumour cells will degrade the Matrigel barrier, migrate and grow in a new site.45 As shown in Figure 2E, more PC-14 cells than A549 cells grew in new sites, and the metastatic rate of A549 cells was 32.51% of that of PC-14 cells, which was defined as 100% (Figure 2F). This study indicated that the invasion and metastasis of PC-14 cells were higher than those of A549 cells. The invasion and metastasis of PC-14 and A549 cells correlated well with the expression levels of MT1-MMP (Figure 1G and 2A). Our data indicate that the MT1-MMP expression level may be applied as a lung adenocarcinoma invasion/metastasis biomarker in vivo. Au-CL Labels MT1-MMP in Xenograft PC-14 and A549 Tumour Tissues and Quantitatively Detects MT1-MMP Expression Levels. To validate our MT1-MMP quantitation method in primary tumour tissue sections, we first applied this approach to lung adenocarcinoma tumours in a xenograft model. First, the mice bearing human lung adenocarcinoma primary tumours derived from PC-14 and A549 cells were sacrificed, and the tumour tissues were embedded in paraffin. Under the optimum tumour tissue labelling conditions (see Experimental Section and Figure S8 in the Supporting Information), xenograft tumour sections exhibited strong red fluorescence when Au-CL labelled the MT1-MMP of tumour tissues (Figure 3B and S9). In contrast, there was almost no visible red fluorescence in normal mouse lung tissue sections, suggesting that tumour tissue expresses higher levels of MT1-MMP than normal tissue. For xenograft tumour tissue sections, IF assays using an MT1-MMP antibody were also performed to compare the Au-CL cluster specificity for MT1-MMP. The fluorescence intensity and pattern of Au-CL-labelled MT1-MMP were almost the same as those of IF imaging (green fluorescence of MT1-MMP antibody, Figure 3A), indicating that Au-CL could specifically label MT1-MMP in tumour tissue (PC-14 and A549 xenograft tumours). Moreover, the fluorescence intensity of PC-14 xenograft tumour tissue sections was higher than that of A549 cells (see the statistics in Figure 3A and 3B), indicating that


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Au-CL clusters can distinguish MT1-MMP expression levels of PC-14 and A549 cells. After fluorescence microscopy analysis, the tumour tissues were scanned on a microscope slide using a 2D LA-ICP-MS system. Tumour mimic gelatin sections containing serial doses of Au standards were measured by a 2D LA-ICP-MS system to establish the standard curve. This standard curve was further corrected by the ICP-MS method. According to this curve, we calculated the MT1-MMP level of tumour tissue sections (see Experimental Section and Figure S10 in Supporting Information). As depicted in Figure 3C, the expression levels of MT1-MMP in PC-14 and A549 xenograft tissues can be virtually visualized by the 2D-LA-Mass Mapping method. As shown by the 2D-LA-Mass Mapping analysis, the colour of PC-14 xenograft tumour tissue sections was brighter than that of A549 xenograft tumour tissue sections, indicating that PC-14 xenograft tumours express more MT1-MMP than A549 tumours. The average mass signals of Au (corresponding to the MT1-MMP level) in PC-14 and A549 xenograft tissue sections were 83.71±5.15 fg and 22.27±4.17 fg (Au content corresponding to a 20 µm laser spot), respectively, showing a large difference in the MT1-MMP expression level. A similar result derived from IF analysis (Figure 3A and 3B) also confirmed that PC-14 cells have a higher MT1-MMP level than A549 cells in tumour tissues; however, the MT1-MMP levels of PC-14 and A549 tumours could not be precisely determined by the IF method. These findings implied that the 2D-LA-Mass Mapping method not only quantified the MT1-MMP levels of tumour tissues but also profoundly distinguished the expression level therein. This 2D-LA-Mass Mapping pathologic method is a better choice than the IF method for precise biomarker detection in tumour tissues. Is this 2D-LA-Mass Mapping result consistent with traditional H&E and IHC observations in xenograft tumour pathologic analysis? Herein, we used H&E to provide tumour differentiation, IHC to show tumour proliferation, and IHC to detect MT1-MMP levels in tumour tissues. As depicted in Figure 3E and 3F, PC-14 xenograft tumour tissues expressed higher levels of MT1-MMP and Ki67 than A549 xenograft tumour


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tissues. Additionally, the H&E data in Figure 3D revealed that the PC-14 xenograft tumour had a lower differentiation grade than the A549 xenograft tumour. All of these data derived from the H&E and IHC methods implied that PC-14 is more invasive/metastatic than A549, although H&E and IHC could not provide a quantitative assessment of tumour proliferation/differentiation and invasion/metastasis. In Figure 3, the normal mouse lung tissues showed negative staining for Ki67, MT1-MMP, and H&E staining. Briefly, the MT1-MMP data derived from the 2D-LA-Mass Mapping method are consistent with normal pathologic H&E and IHC observations of tumour tissues, and this method provided quantitative MT1-MMP levels in PC-14 and A549 tumour tissues. Quantitative Detection of MT1-MMP Levels in Primary Cancer Tissues and Precise Assessment of Their Invasion/Metastasis. Can 2D-LA-Mass Mapping of MT1-MMP be used to assess clinical primary cancer tissue? If yes, can the invasion/metastasis risk of primary cancer be determined by the MT1-MMP level in primary cancer tissue? To address these issues, we studied serial primary lung adenocarcinomas. The lung adenocarcinoma tumours of patients were first diagnosed via traditional H&E and IHC methods. (Table S3 in Supporting Information). As depicted in Figure 4A, 4B and S9, for cancer tissue sections derived from three patients, the fluorescence intensity and pattern of Au-CL-labelled MT1-MMP were similar to those of the MT1-MMP antibody (green fluorescence) by the IF method, indicating that Au-CL clusters could specifically label the MT1-MMP of cancer tissues from different patients. In Figure 4A and 4B, the primary cancer tissue sections of Patient 1 had higher red fluorescence than those of Patient 2 and Patient 3. From the 2D-LA-Mass Mapping (Figure 4C), we observed that the distribution and expression level of MT1-MMP in lung adenocarcinoma tissue of Patient 1 were stronger than those of Patient 2 and Patient 3. In lung adenocarcinoma tissue of Patient 3, there were few cytopathic lesions (Figure 4D, black dotted circles); therefore, it was difficult to observe the MT1-MMP signal in IF (Figure 4A and 4B) and IHC staining (Figure 4E and 4F). However, the few cancerous cells in lung


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adenocarcinoma tissue of Patient 3 could be visually distinguished by the 2D-LA-Mass Mapping of MT1-MMP (Figure 4C, white dotted circles). We randomly counted 20 sections from lung adenocarcinoma patients, and the average mass signals were 63.72±7.24 fg for Patient 1, 24.52±3.30 fg for Patient 2 and 4.88±6.60 fg for Patient 3. We further investigated whether this 2D-LA-Mass Mapping method was positively related to the traditional IHC method in a clinicopathologic study. Herein, we used the IHC method to determine the MT1-MMP level in primary tumour tissue (Figure 4E). As depicted in Figure 4E, the tumour tissues of Patient 1 expressed higher levels of MT1-MMP than those of Patient 2 and Patient 3. The IHC MT1-MMP data correlated well with the 2D-LA-Mass Mapping method data (Figure 4C). Note that the statistics data of H&E imaging in Figure 4D indicate that the proportions of abnormal cells in the tumour sections of Patient 1 and Patient 2 were similar, and Figure 4F shows that the tumour sections of Patient 2 had a high level of Ki67 compared with those of Patient 1. Note that Ki67 is a proliferative biomarker, and proliferative ability is not equal to invasive/metastatic ability.46,47 The H&E and Ki67 data derived from Patient 1 and Patient 2 make it difficult to provide a precise pathologic description of primary cancer. However, 2D-LA-Mass Mapping of MT1-MMP levels easily distinguished the different differentiated/proliferating primary cancers (Figure 4C, Table S3). As depicted from statistics of Figure 4C, the data dispersion of 2D-LA-Mass Mapping of 20 tumour tissue sections was lower than that of IF and IHC in Figure 4A, 4B and 4E, which indicates that the 2D-LA-Mass Mapping method is more accurate than the IF and IHC methods. As depicted from statistics of Figure 4C, the P value between Patient 1 and Patient 2 was lower than those in Figure 4A, 4B and 4E, demonstrating that 2D-LA-Mass Mapping can more sensitively distinguish the MT1-MMP level than traditional IF and IHC methods. Compared with H&E, IF and IHC, 2D-LA-Mass Mapping precisely sorted the MT1-MMP levels of primary tumours with different differentiation/proliferation statuses (Figure 4, Table S3).


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As previously discussed, MT1-MMP plays an important role in primary cancer invasion/metastasis and is a potential drug target in cancer therapy in phase II clinical studies.48,49 However, there are still no reported methods for direct quantification of this potential biomarker in primary cancer tissue to assist pathologists in predicting the risk of cancer invasion and metastasis. As 2D-LA-Mass Mapping of MT1-MMP is more precise than the standard H&E/IHC/IF methods in pathologic tumour tissue studies, we propose that quantitative data of 2D-LA-Mass Mapping of MT1-MMP in primary cancer tissue may provide an accurate prediction of regional invasion and remote organ metastasis. The lymph node status of primary tumours is a paramount indicator for evaluating cancer invasion/metastasis.50 Mediastinal lymph node (MLN) metastasis is associated with the degree of malignancy and invasion/metastasis of lung adenocarcinoma.51,52 Chest CT is a commonly used imaging modality for MLN staging in lung adenocarcinoma patients.53,54 For the primary lung adenocarcinoma of Patient 1 (2D-LA-Mass Mapping signal 63.72±7.24 fg in Figure 4C), CT imaging of the lung and mediastinal windows (Figure 5A and 5B) showed that the tumour is located in the right lower lobe of the right lung marked by a red dotted circle. We further observed the enlargement, calcification, fusion, and invasion of the vessel wall in the MLN of the lung from chest CT imaging. The primary tumour had metastasized into the MLN marked by a red dotted circle (Figure 5C, S11A and S11B). These results indicate that this adenocarcinoma patient is at high risk

of

remote

organ

metastasis.

To

prove

this,

we

used

T1-weighted

imaging (T1WI) and T2-weighted imaging (T2WI) of MRI to scan the patient. We observed a circular lesion near the central sulcus of the right frontal lobe of the brain of this primary lung adenocarcinoma patient (Figure 5D and 5E). The MRI results showed that the lung adenocarcinoma metastasized to the brain. For the same patient, the CT results of regional lymph node invasion and the MRI results of brain metastasis were correlated with the increased MT1-MMP levels in the primary tumour tissue in Figure 4C. For primary lung adenocarcinoma Patient 2 with a 2D-LA-Mass Mapping signal of


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24.52±3.30 fg (Figure 4C), the boundaries of the primary tumour were clear (Figure 5F and 5G). The MLN was enlarged only in the pulmonary hilar near the tumour and was not observed at the distal end of the hilum (Figure 5H, S11C and S11D), suggesting that the tumour started to invade close to the lymph node. However, the tumour had not yet metastasized to the remote brain after the patient was scanned by MRI (Figure 5I and 5J). For primary lung adenocarcinoma Patient 3 with a 2D-LA-Mass Mapping signal of 4.88±6.60 fg, we only observed a lesion on the lung window and found almost no lesions in the mediastinal window of the chest CT scan (Figure 5K and 5L), and as expected, no MLN was observed in the CT scan (Figure 5M, S11E and S11F). MRI scanning showed no lesions in the remote brain in Patient 3 (Figure 5N and 5O). Thus, we demonstrated that clinical samples of lung adenocarcinoma that express high levels of MT1-MMP protein are at high risk for regional lymph node invasion and remote brain metastasis. Au-CL Clusters Quantitatively Detects MT1-MMP Levels in Primary Renal Cancer and Precisely Assesses Cancer Invasion/Metastasis Risk. To verify that our 2D-LA-Mass Mapping method is applicable to other cancer tissues, we assessed clinical samples of primary renal cancer (normal H&E and IHC pathologic studies are shown in Supporting Information Table S4). After labelling by Au-CL clusters, the left area of the renal region showed bright red fluorescence, which had a good correlation with green fluorescence from the MT1-MMP antibody (Figure 6A, 6B and S12). Clinical renal cancer tissue has both cancerous and normal regions. As expected, the normal region, which barely expresses MT1-MMP protein, had no obvious fluorescence. As shown by 2D-LA-Mass Mapping of MT1-MMP, the tumour region and the normal region were clearly identified due to the strong difference in the mass signal (Figure 6C). The MT1-MMP level derived from 2D-LA-Mass Mapping of renal cancer tissue was positively correlated with the H&E, IHC of MT1-MMP, and IHC of Ki67, as shown in Figure 6D, 6E, 6F. The average mass signals of 20 tumour tissue sections of the cancer and junction regions were 35.23±3.40 fg and 17.24±2.82 fg, respectively. Almost no Au


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signal was detected in the normal region of this renal cancer tissue. Compared with the mass signal of lung adenocarcinoma tissue (63.72±7.24 fg in Figure 4C) where the invasion/metastasis happened, the mass signal of this primary renal cancer tissue is 35.23±3.40 fg. These data imply that primary renal cancer may not have remote organ metastasis. From the CT image, there was a space-occupying lesion, e.g., primary tumour, in the left kidney (Figure 7). We did not find close lymph node invasion or remote organ metastasis via CT/MRI scanning.

CONCLUSIONS In this study, we developed a method to construct Au clusters using CL peptides. Au-CL clusters exhibits red emission and can specifically target MT1-MMP in human lung adenocarcinoma cells (PC-14 and A549), xenograft lung adenocarcinoma tissue (PC-14 and A549), clinical lung adenocarcinoma tissues and renal cancer tissues. The MT1-MMP level of tumour cells can be viewed by fluorescence imaging and precisely quantified at the single cell level. With the combined fluorescence microscopy and 2D-LA-Mass Mapping method, MT1-MMP levels could be observed by fluorescence imaging and precisely detected in xenograft and human primary tumour tissues. Our mass mapping method of MT1-MMP in primary tumours had excellent accuracy compared with traditional H&E/IHC/IF assessment. By our 2D-LA-Mass Mapping method, non-professional staff can clearly observe the distribution of MT1-MMP in tumour tissues and precisely detect the expression levels of this marker compared with the data derived from the normal blurred colour change of the H&E/IHC/IF method. Recent years have witnessed great progress in single cell and tissue sections mass spectrometry.41,55-57 The key features of the Au-CL-based 2D-LA-Mass Mapping method are precise quantification of the invading MT1-MMP level in both single cells and primary tumour tissues. This feature will help pathologists assess the risk of primary tumour regional lymph node invasion/remote organ metastasis. Traditional pathologic


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methods,

including

H&E,

IHC,

and

IF,

can

identify

tumour

tissue

differentiation/proliferation for diagnosis and prognosis, and IHC/IF methods can also determine the biomarker expression levels for drug target therapy. Because there is no reported method to directly quantify tumour invasion/metastasis proteins in clinicopathologic observations, these traditional pathologic methods cannot provide exact information to effectively assess the risk of primary tumour invasion/metastasis without CT/MRI molecular imaging for patient scanning. Previous studies have found that MT1-MMP is a potential tumour biomarker for drug targets, but it has not been demonstrated to be an invasion/metastasis biomarker in clinical tissue samples.58-59 Here, utilizing H&E staining, IHC-Ki67, IHC-MT1-MMP and clinical CT/MRI, we demonstrated that the expression level of MT1-MMP in primary tumour tissue is well correlated with the tumour regional invasion/remote metastasis both for lung adenocarcinoma and renal cancer. Therefore, early diagnosis of primary tumour invasion/metastasis can be achieved by 2D-LA-Mass Mapping of MT1-MMP in tumour tissues. This method will have major benefits for clinical therapy and prognosis of primary tumour patients.

MATERIALS AND METHODS Materials. The peptide (H2N-CCYHWKHLHNTKTFL-COOH) was synthesized by a solid-phase method using Fmoc chemistry (Zhejiang Ontores Biotechnologies Co., Ltd, Purity: 98%). Human adenocarcinoma PC-14 and A549 cell lines were purchased from the Cancer Institute and Hospital, Chinese Academy of Medical Sciences. The cell culture media RPMI 1640 and Dulbecco’s modiﬁed Eagle’s medium (DMEM)/High Glucose and phosphate buffer solution (PBS) were purchased from HyClone, USA. Foetal bovine serum and trypsin-EDTA were acquired from Gibco, USA. Human MT1-MMP antibody, goat anti-rabbit IgG-FITC, and anti-rabbit IgG-HRP were obtained from Abcam Biotechnology. Sodium hydroxide (NaOH), nitric acid (HNO3, MOS grade),


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hydrogen peroxide (H2O2, MOS grade) and hydrochloric acid (HCl, MOS grade) were obtained from Beijing Chemical Reagent Co., China. Hydrogen tetrachloroaurate (III) (HAuCl4·3H2O) was purchased from Sinopharm Chemical Reagent Co., Ltd. Cell lysis buffer for immunoblotting, DAPI staining solution, anti-rabbit IgG-HRP, a Membrane Protein Extraction Kit, and a BCA Protein Assay Kit were obtained from the Beyotime Institute of Biotechnology, China. Rat tail type I collagen (COL-I) was obtained from BD Biosciences. Ultrapure water (18 MΩ) was used throughout the experiments. Synthesis and Determination of the Structure of Au-CL Clusters. Briefly, a volume of 117 μL of 25 mM HAuCl4 aqueous solution was slowly dripped into 1450 μL CL peptide aqueous solution in a 5 mL vial under vigorous stirring at room temperature (25 ºC). Subsequently, 240 μL of 0.5 M NaOH aqueous solution was added dropwise to the reaction vessel. Then, the reactant was sealed and protected from light at room temperature for 2 days to produce Au-CL clusters. All glassware was soaked in aqua regia (HCl: HNO3, volume ratio=3:1) before being used and further rinsed with ultrapure water. The as-synthesized Au-CL clusters were separated and purified to cut off aggregated clusters, free peptide and ions. First, 2 ml Au-CL clusters were added into the centrifugal filter (Millipore, Ultracel-30K) and centrifuged for 30 minutes at a centrifugal force of 8000 g. Then we collected the aqueous solution of the Au-CL clusters in the outer tube and added it to the Ultracel-10K centrifugal filter. Next, the sample was purified by adding H2O to cut off free peptide and ions until the pH of solution was at pH 9.0. Finally, we collected the supernatant and further purified it by HPLC (GE AKTA PURE). The chromatographic column is GE Healthcare Columu Superdex 200 10/300 GL (Figure S3). The components of the mobile phase are 0.5 M arginine and 1 M NaCl solution. After Au-CL clusters were purified and collected by UV absorption from HPLC system, the sample was analysed by MALDI-TOF MS. ICP-MS Analysis of the Au-CL Clusters Concentration. The concentration of the Au-CL clusters was determined by an ICP-MS analysis system (Thermo Elemental X7,


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USA). After collecting Au-CL clusters by HPLC, 1 mL nitric acid and 3 mL hydrochloric acid were introduced into the 5 μL Au-CL clusters in the glass vials for digestion. The next day, the mixed acid solution containing Au was evaporated to 0.1–0.2 mL and diluted to the appropriate concentration by 2% HNO3 and 1% HCl. A concentration of 20 ppb bismuth in a 2% HNO3 and 1% HCl solution was used as an internal standard. The Au calibration curve was determined by measuring different concentrations of Au standard solution (0.1, 0.5, 1, 5, 10 and 50 ng/mL containing 2% HNO3 and 1% HCl). Then, all samples were measured by an ICP-MS system. The calibration curve is presented in Figure S7C. The measurement was repeated three times for each sample. The Au-CL Clusters Targeting MT1-MMP on Lung Adenocarcinoma Cells. First, the PC-14 and A549 cells in culture dishes were fixed with 3.7% paraformaldehyde for 30 min. Next, the cells were washed with PBS three times, and 3% bovine serum albumin (BSA) in PBS solution was introduced to the cells to block the nonspecific recognition sites. Then, the cells were incubated with 9 µM Au-CL clusters for 60 min at 25 ºC. The cells in the blank control group were only treated with medium. Next, the nuclei were stained with 5 µg/ml DAPI for 5 min. Finally, the cells were imaged using an UltraVIEW Vox (PerkinElmer) confocal system attachment and a Nikon Ti-e microscope with a 60×1.4NA plan apochromat oil immersion lens. Au-CL Clusters and Antibody Co-localization Study on Cells. A fluorescent antibody was used to further confirm the Au-CL clusters located on the MT1-MMP protein epitope. PC-14 and A549 cells were fixed and blocked with 3% BSA solution. Then, the cells were incubated with anti-MT1-MMP antibody (a rabbit monoclonal antibody raised against peripheral blood mononuclear cells of human origin, dilution 1:180) in PBS for 2 h at 37 ºC, and goat anti-rabbit IgG-FITC (dilution 1:500) in 3% BSA solution was added to the cells for another 30 min at 37 ºC in the dark. Subsequently, 9 µM Au-CL clusters were incubated with the cells for 1 h. Finally, the cells were stained with DAPI and washed with PBS before observation by the CLSM system.


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Protein Binding Site Blocking Study. A binding site blocking study was performed to confirm the specificity of the Au-CL clusters for MT1-MMP. First, PC-14 and A549 cells were fixed with 3.7% paraformaldehyde. Then, 3% BSA was added to the cells for 30 min to block the nonspecific recognition sites. Next, the cells were incubated with 5 mM of free CL peptide solution for 1 h, followed by the addition of 9 µM Au-CL cluster solution for 60 min. Then, DAPI (5 µg/ml) was introduced into the cells to stain the nuclei. Finally, the cells were imaged by the CLSM system. Quantitative Analysis of MT1-MMP on Single PC-14 and A549 Cell via LA-ICP-MS. An NWR 213 laser ablation system (ESI, Fremont, USA) coupled with a NexION 300D ICP-MS instrument (PerkinElmer, Norwalk, USA) was employed in LA-ICP-MS measurements. The ablation gas was helium. The flow rate of helium was 0.6 L/min. After the cell was ablated, we injected argon through a Y-piece. During the ablation of NIST 612 glass, we tuned the

115In

signal intensity to the maximum, and the

UO/U ratio was stable at a low level. The operational parameters are depicted in Table S5. The STD mode was applied, and then, the signal intensities were recorded as a function of time (counts/s). PC-14 (1×105 cells/mL) and A549 cells (1×105 cells/mL) were seeded on a cover glass. Then, the cells were incubated with Au-CL clusters at 9 μM for 60 min. Subsequently, the cells were ablated. To ablate a single cell completely, we ablated 40 µm diameter areas at positions to ensure that the cell was completely covered while there was no chance of overlap with the adjacent cells. The Au standards (0, 28.93, 42.78, 93.61, 133.03 fg) were analysed under the same conditions in the sample analysis (Figure S7A and S7B). The Expression of MT1-MMP on PC-14 and A549 Cells was Detected by Immunoblotting. PC-14 and A549 cells were cultured in DMEM supplemented with L-glutamine (4 mM), penicillin (100 units/mL), streptomycin (100 μg/mL) and 10% foetal bovine serum. First, PC-14 and A549 cells (5 × 106) were washed with PBS and lysed with lysis buffer [Tris-HCl (pH 7.5), 1 mM Na2EDTA, 1 mM β-glycerophosphate,


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150 mM NaCl, 1 mM orthovanadate, 1% Triton, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 μg/ml leupeptin and 1 mM PMSF] for 10 min at 4 °C. Next, the cell membrane proteins were extracted by a Membrane Protein Extraction Kit. The protein concentration was confirmed by a BCA assay kit to ensure the same amount of protein was loaded in gel electrophoresis. Then, the protein extract was mixed with 1x loading buffer, heated at 100 °C for 5 min, and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, the resulting gels were transferred to PVDF membranes (Millipore), a rabbit monoclonal antibody was used as the primary antibody, and goat anti-rabbit HRP-labelled antibody was used as the secondary antibody. Finally, the protein bands were detected using the Amersham ECLTM Prime Western Blotting Detection Reagent (GE Healthcare, U.K.). Wound Healing Assay. PC-14 and A549 cells were grown in 6-well plates. After 24 h, the confluent cell monolayers were scratched in a straight line with a P200 pipet tip to create an empty gap. Then, the cells were washed with PBS buffer, and 2 mL of fresh serum-free medium was introduced into the cells for another 24 h. Then, the cells were fixed with 3.7% paraformaldehyde in PBS solution for 30 min. Next, the fixed cells were washed with PBS buffer and stained with 0.1% crystal violet for 8 min before imaging under a microscope. The wound width was measured to evaluate the wound healing ability. Transwell Invasion Assay. Transwell plates (Corning Costar, USA) with 24 wells and an 8.0 μm pore size Transparent PET Membrane were used for cell invasion assays. The insert membrane was coated with 100 µL COL-I (300 mg/mL in serum-free medium) and then dried in a cell incubator at 37 ºC for 12 h. The collagen coating was rehydrated for 1 h in serum-free medium. PC-14 and A549 cells were suspended at a density of 2×105 cells/mL in serum-free medium. A volume of 100 μL of PC-14 and A549 cells was introduced into the upper chamber. The medium containing 10% FBS (as a chemoattractant) was introduced into the lower chamber of the Transwell. Then, the


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Transwell plates were placed in the incubator for 24 h. After 24 h of incubation, the cells in the upper chamber were fixed with paraformaldehyde and stained (0.1% crystal violet). The cells attached to the Matrigel were removed from the upper chamber, while cells in the lower surface of the membrane were imaged under a microscope. The cells in at least five random microscopic fields were counted. Animal Xenograft Model. The animal care and experiments were conducted in compliance with the Chinese Academy of Medical Sciences guidelines and approved by the Institutional Animal Care and Ethics Committee (Approval No. SCXK2014-0004). Four-week-old male athymic nude BALB/c mice (body weight, approximately 18 g) were implanted with PC-14 and A549 cells (106 cells per mouse) into the rear flank of each mouse. After 21 days, the mice were sacrificed. Then, the tumours were fixed with formalin and embedded with paraffin. Pathological Study of Tumour Tissues. The FFPE tumour tissues were sectioned into 3-micron-thick sections. Then, the tissue sections were dewaxed in xylene and rehydrated using a graded series of ethanol. Next, sections were stained with H&E. The abnormal cells were counted using NDP.view 2 software and are reported as the proportion of abnormal cells in a field of view (400x). For IHC, FFPE tumour sections and normal sections were labelled by using a MT1-MMP antibody and Ki67 antibody. After dewaxing and rehydration, the sections were washed with PBS, and 3% H2O2 was used to deplete endogenous peroxide. Next, sections were heated in a microwave oven in sodium citrate buffer at 140 ºC for 3 min and then cooled for 15 min at room temperature. After that, goat serum was added to the tissues to block nonspecific adsorption sites at 37 ºC for 20 min. Subsequently, these tissue sections were incubated overnight with primary antibodies (1:80) at 4 ºC. The next day, the sections were washed extensively with PBS, and HRP-conjugated secondary antibody was added for 20 min at 37 ºC. After the sections were rinsed, DAB solution was applied and incubated for 5 min at room temperature, and sections were counterstained with haematoxylin. Digital images were


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acquired on an Olympus IX73 light microscope. For MT1-MMP and Ki67 staining quantification, positive cells were counted using NDP.view 2 software and are reported as the proportion of positive cells in a field of view (400x). For IF, after overnight incubation with an MT1-MMP antibody (1:180), the sections were washed with PBS. Then, a FITC-conjugated secondary antibody (goat anti-rabbit IgG-FITC) was introduced for 30 min at 37 ºC. Subsequently, the sections were imaged using an Olympus IX73 light microscope. The fluorescence intensity was calculated using fluorescence imaging software in a field of view (400x). Quantitative Analysis of MT1-MMP in Tumour Tissue Sections via 2D-LA-Mass Mapping. LA-ICP-MS was employed to quantify MT1-MMP expression in tissue sections. The paraffin-embedded tissues were cut into 3-micron-thick sections. Next, the sections were dewaxed, rehydrated and incubated with Au-CL clusters at 12 μM for 80 min. Then, the Au-CL-labelled tissue sections were thoroughly washed with PBS. After imaging by fluorescence microscopy, the tissues were ablated on a microscope slide by a 20 μm diameter spot using the LA-ICP-MS system. The tissue was ablated spot-by-spot along a scan line while the slide was moved under the fixed laser beam. The Au signals associated with each spot were simultaneously measured and recorded according to the position of each spot (Supporting video 1). Finally, a series of sequential scan lines yielded an Au count mapping of MT1-MMP on the selected tissue region. Statistical Data Analysis. All data are represented as the mean ± standard deviation. Statistical analyses were performed using SPSS software (SPSS version 22.0, USA). Statistical significance was calculated by the Kruskal-Wallis H test.

ASSOCIATED CONTENT Supporting Information Supporting information is available free of charge from the ACS Nano home page including characterization of clusters; optimization of the Au-CL-based labelling of lung


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adenocarcinoma cells and tissue sections; determination of the structure of Au-CL clusters ; the average amount of Au determined by ICP-MS; calibration curve from Au standards with LA-ICP-MS and ICP-MS; determine the binding ratio of Au-CL clusters to MT1-MMP; fluorescence imaging of the tumour tissue of lung adenocarcinoma xenografts and clinical samples; CT imaging of mediastinal lymph node invasion in clinical lung adenocarcinomas patients; pathological reports of clinical patients; operating conditions of LA-ICP-MS for cell and tissue ablation.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] ORCID Yuliang Zhao: 0000-0002-9586-9360 Xueyun Gao: 0000-0002-2267-9945 Author contributions X. Z. performed the experiments and wrote the draft; L. G., R. L. and Q. Y. synthesized and characterized the Au-CL clusters; F. G, J. L. and Y. Z. performed cell studies. Y. Z. and Z. C. participated in data analysis, and X. G. proposed the research idea, designed the experiments, and revised the manuscript. Conflicts of interest The authors declare no competing interests. ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (21425522, 21727817, 21390414, 51571185) and the Beijing Science and Technology Commission Special Project for Frontier Technology in Life Sciences (No. Z171100000417008). We thank Qingming Shu for the tumour tissue analysis and


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Yongyue Xu for the CT/MR imaging analysis (Chinese People’s Armed Police Force General Hospital).

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TOC Figure:


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Scheme 1. Illustration of the precise assessment of primary tumour invasion/metastasis risk via 2D-LA-Mass Mapping of the MT1-MMP expression level in single cells, xenograft tumours and human cancer tissues. (1) A single Au-CL cluster has 26 Au atoms and 9 CL peptides, emitting red fluorescence. (2) Au-CL clusters specifically target MT1-MMP on tumour cells and quantify MT1-MMP by mass spectrometry and fluorescence microscopy at the single cell level. (3) Pathological analysis and 2D-LA-Mass Mapping of MT1-MMP in a xenograft lung adenocarcinoma model. (4) Pathological analysis, 2D-LA-Mass Mapping of MT1-MMP and CT/MRI molecular imaging of cancer patients. FLI, Fluorescence imaging; IHC, antibody-related immunohistochemistry; CT, Computed tomography; MRI, Magnetic resonance imaging.


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Figure 1. Au-CL clusters specifically target MT1-MMP in lung adenocarcinoma PC-14 cells and A549 cells and single cell mass analysis of MT1-MMP in PC-14 and A549 cells. (A) CLSM images of cells marked by Au-CL clusters. (B) Cells marked by Au-CL clusters (red fluorescence) after MT1-MMP antibody labelling (green fluorescence). (C) Cells first marked by free CL peptides and then marked by Au-CL clusters. (D) Control cells. The blue is the DAPI for the cell nuclei. (E, F) Transient Au mass signals of single PC-14 cells and A549 cells labelled with Au-CL clusters. (G) Box plots of the Au mass of single PC-14 cells and A549 cells; each point represents an individual cell. P-values were calculated by the Kruskal-Wallis H test, ***P

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