WO2016123446A1

WO2016123446A1 - Label-free mr imaging of tumor malignancy

Info

Publication number: WO2016123446A1
Application number: PCT/US2016/015577
Authority: WO
Inventors: Jeff Bulte; Xiaolei Song; Raag Airan
Original assignee: Johns Hopkins University
Current assignee: Johns Hopkins University
Priority date: 2015-01-30
Filing date: 2016-01-29
Publication date: 2016-08-04
Anticipated expiration: 2017-07-30

Abstract

The present invention is directed to an MRI technique to assess tumor malignancy non-invasively. It is based on the principle that most malignant epithelial tumor cells express mucins that are heavily underglyocsylated. This phenomenon of aberrant expression has been well-documented in the literature. When benign tumor cells having normal mucin glycosylation with CEST MRI are imaged a strong CEST MRI signal effect is seen. When tumor cells lose their glycans and become malignant, the difference in MRI CEST signal is detectable. Thus, the intrinsic properties of tumor cell surface mucin glycosylation can be used as a surrogate marker for predicting tumor malignancy. This can be accomplished without injecting exogenous probes or labels, which have their own problems in terms of clinical approval, cost, and pharmacokinetics of binding to all tumor cells in question. Normal tissue can be used as a reference in determining changes in contrast associated with malignancy.

Description

LABEL-FREE MR IMAGING OF TUMOR MALIGNANCY

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

62/109,721 filed on January 30, 2015, which is incorporated by reference, herein, in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to medical imaging. More particularly the present invention relates to CEST magnetic resonance imaging.

BACKGROUND OF THE INVENTION

[0003] Mucins, a family of large molecular weight and heavily glycosylated proteins, constitute the mucous barrier at the epithelial surface, and play an important role in cell signal transduction. Alterations in mucin expression or glycosylation have long been associated with the development of cancer, as they are thought to influence cellular growth, invasion, metastasis, and immune surveillance. Mucin- 1, one of the cell-surface-associated mucins encoded by the MUC1 gene, is expressed aberrantly in -900,000 of the 1.4 million tumors diagnosed each year in the United States.

[0004] Studies have shown that MUCl-overexpressing breast, colon, and thyroid cancer cells are unresponsive to chemotherapeutic agents. Tumor-associated glycosylation changes have been observed for decades and are associated with tumor proliferation, metastasis, and angiogenesis. Most malignant epithelial tumor cells express mucins that are heavily underglycosylated, and alterations in mucin expression or glycosylation have long been associated with the development and prognosis of cancer. Cell-surface glycoproteins including mucins, in particular, Mucin-1 (MUC-1), have been used as a novel diagnostic and therapeutic target. MUC-1 is a marker of epithelial cell lines that is expressed in an underglycosylated form uMUC-1 in neoplastic cells derived from both epithelial and non-epithelial cell types. uMUC-1 is overexpressed in most malignant adenocarcinomas of epithelial origin (e.g. colon, breast, and ovarian cancer). The specificity of this marker for early tumorgenesis makes it a target of great interest for molecular imaging.

[0005] Under glycolycosylation of mucin implies that its hydroxyl content would be much reduced compared to normally glycosylated tissue. MUC-1, with a core protein mass of 120-225 kDa, increasing to 250-500 kDa with glycosylation, extends no less that 500 nm beyond the surface of the cell. However, in tumor cells that develop from normal cells, MUCl is often underglycosylated with fewer and truncated oligosaccharide side chains, identified as the tumor- associated underglycosylated MUCl (uMUCl) antigen (FIG. 1). The reduced glycosylation of tumor cells allows exposure of a highly immunogenic core peptide epitope of the uMUCl antigen, which has been exploited for the development of immunotherapeutic vaccines and targeted radiotheraputic drugs, and is also widely used as a serum diagnostic assay to detect ovarian, breast, and colon adenocarcinomas.

[0006] Targeted imaging agents against the uMUCl antigen recognizing the exposed peptide sequence on the tandem repeat have been developed, including radiolabeled agents and a dual- modality probe with the near-infrared fluorescence (NIRF) dye Cy5.5 conjugated to MRI- detectable superparamagnetic iron oxide nanoparticles. However, these approaches may not readily be adapted for clinical tumor staging as drug development and approval is a lengthy and costly process. In addition, the pharmacokinetics of the probes may be such that only a small fraction of the tumor can be targeted. An imaging technique that is "label-free" (i.e., that does not rely on administering an exogenous agent) and can sample the entire tumor would be extremely valuable.

[0007] Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is a non-invasive imaging technique that can detect biological agents via frequency-selective saturation of their exchangeable protons. It is highly sensitive, and can amplify signals from low- concentration agents with a factor between 102 and 106 compared to conventional proton spectroscopy. It has been used to detect both small molecules, such as glucose and glutamate and larger polymers, including glycogen and glycosaminoglycans.

[0008] It would therefore be advantageous to provide an efficient and effective form of CEST magnetic resonance imaging that enables detection of uMUC-1 expression.

BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 illustrates a schematic diagram depicting the different levels of glycosylation between normal mucin (left) and tumor-associated mucin (right). The oligosaccharide side- chains consist of a variety of glycans, e.g. GalNAc (triangles, which are O-linked to the core protein and contain sialic acid terminal residues (circles).

[0010] FIGS. 2A-2G illustrates graphical and image views for normally glycosylated mucin, which exhibits a strong CEST signal. FIG. 2 A illustrates a graphical view of a Z-spectra of 5 mg/ml mucin at different pH values. FIG. 2B illustrates a graphical view of calculated MTR_aSym values. FIG. 2C illustrates a graphical view of dependence of MTR_aSym on saturation power (B i) for pH=7.2. FIG. 2D illustrates a graphical view of MTRasym values for different mucin concentrations at pH=7.2 . FIG. 2D illustrates a graphical view of MTRasym values for different mucin concentrations at pH=6.6. FIG. 2F illustrates a graphical view of concentration- dependence of MTRasym at different offset frequencies. FIG. 2G illustrates a corresponding CEST image at 1.8 ppm.

[0011] FIGS. 3A-3E illustrate graphical and image views of a decrease of CEST signal following deglycosylation. Experiments were performed for native (normally glycosylated) mucin (M) and deglycosylated mucin (DM). Shown are the Z-spectra in FIG. 3 A, MTRasym values in FIG. 3B, and MTRasym image at 1.8 ppm in FIG. 3C. FIG. 3D illustrates an image of PAS glycoprotein staining. FIG. 3E illustrates an image view of SDS-PAGE.

[0012] FIGS. 4A-4H illustrate in vitro imaging of encapsulated cell lines. FIG. 4A illustrates a lOx bright-field image that shows individual microcapsules containing MCFIOA cells. FIG. 4B illustrates a graphical view of an averaged CEST spectra of the five cell lines at a saturation field strength (Β ι)=2.4 μΤ. FIG. 4C illustrates a graphical view of an averaged CEST spectra of the five cell lines at Β ι=3.6 μΤ. FIG. 4D illustrates an MT -weighted image showing the phantom layout. FIG. 4E illustrates a CEST contrast map at 1.8 ppm with Β ι=2.4 μΤ. FIG. 4F illustrates a CEST contrast map at 1.8 ppm with Β ι=3.6 μΤ. FIG. 4G illustrates a graphical view of a statistical comparison of MTRasym (1.8 ppm, Β ι=3.6 μΤ) for microcapsules with the 5 cell lines and without cells. Data represent means ± SE for 3 independently performed experiments, analyzed using one-way ANOVA (F5, 14=28.22, P<0.0001) followed by a Tukey-Kramer test for multiple comparisons. Capital letters on top (A-C) indicate groups that were significantly different from all other groups tested at P <0.05. FIG. 4H illustrates images of validation of MUCl glycosylation levels using immunostaining with an antibody detecting full-length MUCl (anti-MUCl antibody). Red=MUCl, blue=nuclei (DAPI).

[0013] FIGS. 5A-5D illustrate in vivo imaging of benign and malignant tumor xenografts. FIG. 5 A illustrates a T2w image, marked with regions of U87, LS174T, and control white matter (dashed square). FIG. 5B illustrates a CEST contrast map created by averaging 1.2 ppm and 0.9 ppm superimposed onto FIG. 5A for Β ι=3.6 μΤ. FIG. 5C illustrates a graphical view of MTRasym curves of the 3 ROIs marked in FIG. 5 A. FIG. 5D illustrates a graphical view of MTRasym values of the two cell lines showing a significant difference (p<0.05, t-test, n=3).

[0014] FIGS. 6A- 6C illustrate in vitro CEST images and spectrum for encapsulated LS174T uMUC-l⁺ and U87 uMUC-1^".

[0015] FIG. 7A illustrates in vivo mouse brain MTw images. FIG. 7B illustrates a CEST image at lppm and FIG. 7C illustrates a CEST MTRasym spectrum.

[0016] FIGS. 8A-8D illustrate graphical and image views of a Zspectra and MTRasym for deglycosylated and untreated mucin.

[0017] FIGS. 9A-9F illustrate microscopy, MTw image, CEST spectra and images, and imunostaining of encapsulated cell lines with different MUC-1 glycosylation levels. SUMMARY

[0018] The foregoing needs are met, to a great extent, by the present invention, wherein in one aspect a method for magnetic resonance imaging of a subject includes using a magnetic resonance imaging machine to generate CEST contrast image data for tissue. The method includes processing the CEST contrast image data to determine presence of a mucin in the tissue. The method also includes processing the CEST contrast image data to differentiate glycosylated and unglycosylated mucins and generate data related to the mucins present in the tissue.

Additionally, the method includes assessing cancer using the generated data related to the mucins present in the tissue.

[0019] In accordance with an aspect of the present invention, the method includes processing the CEST contrast image data with a non-transitory computer readable medium. The method also includes processing the CEST contrast image data with a computing device specifically designed for assessment of mucins in tissue. Additionally, the method includes using the data related to the mucins present in the tissue to non-invasively phenotype a tumor in the tissue, to detect early tumorgenesis, and to monitor tumor growth.

[0020] In accordance with yet another aspect of the present invention, a system for magnetic resonance imaging of a subject includes a magnetic resonance imaging machine configured to generate CEST contrast image data for tissue. The system includes a non-transitory computer readable medium programmed for processing the CEST contrast image data to determine presence of a mucin in the tissue. The non-transitory computer readable medium is also programmed for processing the CEST contrast image data to differentiate glycosylated and unglycosylated mucins and generate data related to the mucins present in the tissue and assessing cancer using the generated data related to the mucins present in the tissue.

[0021] In accordance with still another aspect of the present invention, the system includes a computing device. The computing device is specifically designed for the assessment of mucins in tissue. The non-transitory computer readable medium is programmed for processing the CEST contrast image data and loading the non-transitory computer readable medium on the computing device specifically designed for assessment of mucins in tissue. The non-transitory computer readable medium is programmed for using the data related to the mucins present in the tissue to non-invasively phenotype a tumor in the tissue. The non-transitory computer readable medium is also programmed for using the data related to the mucins present in the tissue to detect early tumorgenesis. The non-transitory computer readable medium is programmed for using the data related to the mucins present in the tissue to monitor tumor growth. The non-transitory computer readable medium is programmed for using normal tissue as a reference. Additionally, the non- transitory computer readable medium is programmed for using normal tissue as a reference during longitudinal follow-up studies and for calibrating and taking a ratio using normal tissue to gauge tumor contrast changes and malignancv. The magnetic resonance imaging machine and the non-transitory computer readable medium are networked together. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Drawings, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0023] The present invention is directed to a new MRI technique that can assess tumor malignancy non-invasively. It is based on the principle that most malignant epithelial tumor cells express mucins that are heavily underglyocsylated. This phenomenon of aberrant expression has been well-documented in the literature. When benign tumor cells having normal mucin glycosylation with CEST MRI are imaged a strong CEST MRI signal effect is seen. When tumor cells lose their glycans and become malignant the difference in MRI CEST signal is detectable. Thus, the intrinsic properties of tumor cell surface mucin glycosylation can be used as a surrogate marker for predicting tumor malignancy. This can be accomplished without the need for injecting exogenous probes or labels, which have their own problems in terms of clinical approval, cost, and pharmacokinetics of binding to all tumor cells in question.

[0024] As mucins are natural polymers rich in glycans, it was investigated whether

"mucCEST" imaging would be able to differentiate benign from malignant tumor cells based on their glycosylation level. In MUCl, a single core protein contains up to 120 tandem repeats, each of which has five potential sites of O-glycosylation; a single molecule can contain up to 600 oligosaccharide side chains. Glycosylation is initiated by the addition of an N- acetylgalactosamine (GalNAc) residue to a serine or threonine, followed by the sequential addition of carbohydrate residues, such as N-acetylglucosamine (GlcNAc), and then terminated by sialic acid, fucose, or galactose (FIG. 1). FIG. 1 illustrates a schematic diagram depicting the different levels of glycosylation between normal mucin (left) and tumor-associated mucin (right). The oligosaccharide side-chains consist of a variety of glycans, e.g. GalNAc (triangles, which are O-linked to the core protein and contain sialic acid terminal residues (circles).

[0025] As each chain contains 2-10 simple sugars with 4-5 -OH protons (8-50 total -OH protons per chain), it can be calculated that 1 nM of MUCl contains up to 30,000 nM

exchangeable protons that can participate in providing contrast on CEST imaging.

[0026] uMUC-l 's counterpart MUCl is a large polymer rich in glycans containing multiple exchangeable OH protons, which is readily detectable by Chemical Exchange Saturation

Transfer (CEST) MRI. Chemical deglycosylation of MUCl resulted in >75% reduction in CEST signal at 0.5-2 ppm. Three uMUCl+ human malignant cancer cell lines overexpressing uMUCl (BT20, HT29, and LS174T) showed a significantly lower CEST signal compared to the benign human epithelial cell line MCFIOA and the uMUCl - tumor cell line U87. When LS174T and U87 were bilaterally implanted in mouse brain, CEST MRI was able to make a clear distinction between the two types of tumors. These results suggest that mucCEST imaging can be used as a label-free surrogate marker to non-invasively assess mucin glycosylation and tumor malignancy. It should also be noted that normal tissue is used as a reference for longitudinal follow up studies. If the tumor contrast changes upon malignancy a ratio can be taken and calibrated using normal tissue (for instance breast glandular tissue).

[0027] EXEMPLARY EMBODIMENTS

[0028] The following exemplary embodiments are included by way of example. These examples are not intended to be limiting and are included merely to further illustrate the invention.

[0029] In an exemplary embodiment, a commercial mucin extract from porcine stomach (Sigma-Aldrich, M2378) was used to characterize the CEST properties. This crude product contains -1% bound sialic acid, which was obtained by digestion of hog stomach with pepsin. Mucin was dissolved in 0.01 M phosphate-buffered saline (PBS) at concentrations from 1.25 mg/ml to 10 mg/ml, and titrated using high concentration HCl/NaOH, to various pH values ranging from 6 to 8. The solutions were placed into 1 mm glass capillaries and assembled in a holder for CEST MR imaging. The samples were kept at 37 OC during imaging. [0030] The mucins were first prepared with reduced glycan chains, to mimic tumor mucins with lower glycosylation levels (FIG. l). Due to the complex O-linked glycosylation and polymerization of mucins, chemical deglycosylation is preferred over enzymatic methods. The oligosaccharide chains on mucins (Sigma- Andrich, M2378) were removed using anhydrous trifluoromethanesulfonic acid (TFMS) treatment, based on the protocol of the GLYCOFREE™ chemical deglycosylation kit (Glyko, GKK500). After treatment, both deglycosylated and untreated mucin were dialyzed against water (10K molecular weight cutoffs) overnight, lyophilized, and dissolved at -2.0 mg/ml in PBS (pH=7.2) for imaging. To verify the glycoprotein content, the deglycosylated mucin and the untreated mucin were further analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) on 4-15% polyacrylamide minigels (Bio- Rad, Gel #456-1083 S) stained with coomassie blue, with the glycosylation level was confirmed by periodic acid-Schiff (PAS) staining (Thermo Scientific, Pierce glycoprotein staining Kit, 24562).

[0031] Five human carcinoma cell lines with different MUC1 glycosylation levels were used. MCF10A, a benign human breast carcinoma and U87, a human glioblastoma cell line, were selected as uMUCl -negative cell lines. The three uMUCl -positive cell lines included BT20, a human breast carcinoma, and LS174T and HT29, both human colon carcinomas. U87, BT20 and LS174T cells were grown in Eagle's minimum essential medium (MEM) with non-essential amino acids in Earle's balanced salts solution, containing 10% fetal bovine serum (FBS), and 2% penicillin and streptomycin (all from Gibco, Grand Island, NY). HT29 cells were cultured using ATCC-formulated McCoy's 5a Medium Modified (Catalog No. 30-2007), containing 10% FBS. The control mammary epithelial cells, MCF10A, were grown in a Mammary Epithelial Cell Growth Medium kit (Lonza, CC-3150), which contains mammary epithelial cell basal medium and growth factors, with the addition of 100 ng/ml cholera toxin. Cultures were maintained at 37 °C in a humidified atmosphere of 5% C02 and 95% air. The cell media were changed every two to three days, and when cells were confluent, they were 1 :4 distributed to new flasks by removing cells from the surface of the culture flask gently with 0.05% trypsin EDTA and a sterile scraper.

[0032] To minimize cell sedimentation, variations in cell density, and pH changes due to cell death, the four cell lines were encapsulated in alginate-PLL-alginate microcapsules at the same density of 1000 cells/capsule. After the encapsulation, the cell-containing microcapsules were suspended in PBS, and immediately transferred to 5-mm MR tubes for CEST imaging. The empty microcapsules without cells were also imaged as controls. To validate the MUCl expression, immunohistological staining was performed with cells growing for two days in the Glass/Permanox chamber slides (Lab-Tek®), using an antibody that could detect full-length MUCl (anti-MUCl antibody, RabMAb, from Epitomics, Burlingame CA), with red=MUCl, and blue=nuclei (DAPI).

[0033] Balb/C NOD SCID male mice (n=3, 6-8 weeks old, -25 g weight) were initially anesthetized with intraperitoneal injection of a mixture of ketamine and xylazine (0.15 ml; 62.5 and 6.25 mg/kg, respectively). Mice were positioned in a stereotactic device (Stoelting Lab Standard). A small midline skin incision was made to expose the skull, and two 1 mm² holes were drilled, 2 mm to left and right of bregma. 1.5x105 MCF10A, LS174T or U87 cells were bilaterally injected to the striatum of each hemisphere at a depth of 2 mm, slowly over a period of 3-4 min with the syringe removed 30 s after completion to minimize back flow. These mice were subjected to MR imaging 2-3 weeks after implantation of tumor cells. During MR imaging, mice were anesthetized using 0.5-2% isoflurane.

[0034] Imaging experiments were performed on a Bruker 11.7T vertical bore scanner for the in vitro experiments and on a Bruker 9.4T horizontal bore scanner for the in vivo mice experiments, both using a transmit/receive volume coil. CEST images were acquired using a continuous wave (CW) saturation pulse of 3 sec. as preparation, followed by a Rapid Acquisition with Relaxation Enhancement (RARE) readout sequence. The saturation field strength (B i) was varied from 1.2 μΤ to 6.0 μΤ for investigating the CEST properties of normal mucin phantoms, with 2.4 μΤ and 3.6 μΤ chosen for the cell imaging and for in vivo imaging. The CEST z-spectra were acquired by incrementing the saturation frequency every 0.2 ppm from -6 to 6 ppm for phantoms, and every 0.25 ppm from -5 to 5 ppm for cells and in vivo. Another set of saturation weighted images with frequency incrementing every 0.1 ppm from -1 to 1 ppm, termed as Water Saturation Shift Reference (WASSR) were also collected for Bo mapping, using a 0.5 sec saturation pulse with B i of 0.5 μΤ. The other parameters are: TR/ effective TE=6000 ms/18 m for in vitro and 5000 ms/11.7 ms for in vivo experiments, matrix size =96x64, with slice thickness 1 mm. Following a pixel-by-pixel Bo correction, CEST contrast was quantified by MTRasym= (S-Δω- S+Am)/So with S+Δω, S-Δω, So representing the water signal with a saturation frequency offset at +Δω, -Δω or without saturation, respectively. For the encapsulated cells, MTRasym = ( -AwS+Aa)/ S-Δω was used to increase the dynamic range. [0035] In this exemplary embodiment, as the CEST signal is pH-dependent, the CEST z- spectra (FIG. 2A) and MTRasym spectra (FIG. 2B) were examined for 5 mg/ml normal

(glycosylated) mucin at pH= 5.8 to 7.8. FIGS. 2A-2G illustrate graphical and image views for normally glycosylated mucin, which exhibits a strong CEST signal. FIG. 2A illustrates a graphical view of a Z-spectra of 5 mg/ml mucin at different pH values. FIG. 2B illustrates a graphical view of calculated MTRasym values. FIG. 2C illustrates a graphical view of dependence of MTRasym on saturation power (B i) for pH=7.2. FIG. 2D illustrates a graphical view of

MTRasym values for different mucin concentrations at pH=7.2 . FIG. 2D illustrates a graphical view of MTRasym values for different mucin concentrations at pH=6.6. FIG. 2F illustrates a graphical view of concentration-dependence of MTRasym at different offset frequencies. FIG. 2G illustrates a corresponding CEST image at 1.8 ppm.

[0036] Mucin showed a broad CEST spectrum from 0.5 ppm-4 ppm, with the peak at ~ 1 ppm, which can be assigned to the numerous exchangeable -OH protons on the glycan side chains as reported previously for glucose, glycogen, glycosoaminoglycans, and poly-sialic acid. Similar to these reports, the MTRasym signal between 1 ppm and 2.5 ppm increases with decreasing pH. For mucin, the peak around 2.8 ppm increases below pH = 6 due to the amine protons entering the slow-to-intermediate exchange regime. FIG. 2C shows the MTRasym spectra for saturation field strengths (Bi) from 1.2-6.0 μΤ for solutions at pH=7.2. A dramatic increase of signal occurs between 0.5-2 ppm indicating fast exchange of hydroxyl groups. The CEST peak shifts slightly further from water as Bi increases, which is because of the increased direct water saturation moving the maximum to the left in the MTRasym calculation. Β ι=3.6 μΤ was chosen for all following experiments, since it shows a comparable CEST signal at -1.2 ppm with that at the higher B i value, but with a less broad spectrum. FIGS. 2D and 2F show the concentration dependence of CEST spectra for neutral (pH=7.2) and mildly acidic (pH=6.6) conditions (pH=7.2 and 6.6), as the extracellular pH is acidic for many malignant tumors. The 3.6 ppm peak from the backbone amides can be clearly observed in FIG. 2D. Even at the lowest concentration of 1.25 mg/ml, the MTR_aSym peaks reach >5%, which should be easily detectable. The MTRasym changes as a function of concentration for three saturation frequencies (FIG. 2F). The corresponding CEST image at 1.8 ppm (FIG. 2G) demonstrates strong CEST signal changes as a function of the concentration of normal glycosylated mucin.

[0037] FIGS. 3 A-3E illustrate graphical and image views of a decrease of CEST signal following deglycosylation. Experiments were performed for native (normally glycosylated) mucin (M) and deglycosylated mucin (DM). Shown are the Z-spectra in FIG. 3 A, MTRasym values in FIG. 3B, and MTRasym image at 1.8 ppm in FIG. 3C. FIG. 3D illustrates an image of PAS glycoprotein staining. FIG. 3E illustrates an image view of SDS-PAGE.

[0038] To further prove that the CEST contrast originated from the glycosyl groups, experiments were performed on chemically deglycosylated mucin, in order to mimic the underglycosylated MUC1 present on malignant tumor cells. Deglycosylated mucin could be easily differentiated from normal mucin in both the z-spectra and MTRasym spectra (FIGS. 3 A and 3B). In the z-spectra, there was no observable difference at the negative frequencies for the two samples, indicating that conventional magnetization transfer (MT) imaging may not able to specifically differentiate deglycosylated mucin from glycosylated normal mucin (FIG. 3 A). However, in the MTRasym spectra (FIG. 3B), there is a dramatic reduction in CEST contrast at the characterized frequency range for mucin, i.e. with values of >75% reduction at 0.5-2 ppm and of >50% reduction from 2-4 ppm. These large differences enabled the production of CEST images with a clear distinction of the two mucins (FIG. 3C). The residual CEST signal for deglycosylated mucin could either arise from the core proteins, or from sialic acid and GalNAc residues that were not completely cleaved off by chemical trifluoromethane sulfonic acid (TFMS) treatment. Near-complete deglycosylation was confirmed by SDS-PAGE with and without periodic acid-Schiff (PAS) glycoprotein staining, as normal (native) mucin exhibited a much higher intensity of glycosyl staining for proteins >260 kD (FIG. 3D). Furthermore, in the SDS-PAGE gel without PAS glycoprotein staining, only the deglycosylated mucin showed a protein band at a MW of -70 kD, whereas in the untreated mucin this was absent (FIG. 3E). Note that in FIG. 3E the region above 260 kD also did not stain, as the coomassie blue dye does not bind well to carbohydrate moieties. The absence of the band between 70-100 kD in the PAS- stained gel (FIG. 3D) indicates that the main protein content (70-100 kD) in the deglycosylated mucin is almost free of glycans, in agreement with its reduced CEST signal (FIG. 3B).

[0039] FIGS. 4A-4H illustrate in vitro imaging of encapsulated cell lines. FIG. 4A illustrates a lOx bright-field image that shows individual microcapsules containing MCF10A cells. FIG. 4B illustrates a graphical view of an averaged CEST spectra of the five cell lines at a saturation field strength (Β ι)=2.4 μΤ. FIG. 4C illustrates a graphical view of an averaged CEST spectra of the five cell lines at Β ι=3.6 μΤ. FIG. 4D illustrates an MT-weighted image showing the phantom layout. FIG. 4E illustrates a CEST contrast map at 1.8 ppm with Β ι=2.4 μΤ. FIG. 4F illustrates a CEST contrast map at 1.8 ppm with Β ι=3.6 μΤ. FIG. 4G illustrates a graphical view of a statistical comparison of MTRasym (1.8 ppm, Β ι=3.6 μΤ) for microcapsules with the 5 cell lines and without cells. Data represent means ± SE for 3 independently performed experiments, analyzed using one-way ANOVA (F5, 14=28.22, P<0.0001) followed by a Tukey-Kramer test for multiple comparisons. Capital letters on top (A-C) indicate groups that were significantly different from all other groups tested at P <0.05. FIG. 4H illustrates images of validation of MUCl glycosylation levels using immunostaining with an antibody detecting full-length MUCl (anti-MUCl antibody). Red=MUCl, blue=nuclei (DAPI).

[0040] Next, it was tested whether mucCEST imaging could differentiate between human cell lines expressing different levels of uMUCl . To prevent sedimentation of cells and achieve a homogeneous suspension, 5 cell lines were encapsulated in alginate-PLL-alginate hydrogels at the same density (-1000 cells/capsule, FIG. 4A). FIGS. 4B and 4C represent the average

MTRasym curves for the 5 cell lines, and for empty (no cells) control capsules at two different saturation field strengths. The 3 tumor cell lines expressing uMUCl (BT20, HT29, and LS174T) exhibited a significantly lower CEST contrast as compared to MCFIOA cells expressing MUCl (normally glycosylated) and U87, a uMUCl -negative cell line, at both saturation conditions, i.e., from 0.7 ppm to 3.8 ppm for Bi = 2.4 μΤ and from 0.7 ppm to 4.8 ppm for B i = 3.6 μΤ. Note that the MTRasym values above 2.5-3 ppm become negative (Fig. 4b), as it is normalized using the signal at the 'negative' frequency with respect to water, bringing more nuclear overhauser effect (NOE) contributions, especially at lower B i. [0041] CEST contrast maps showed a clearly differential MTRasym contrast at 1.8 ppm for both B i conditions (FIGS. 4E and 4F). Conventional MT-weighted (FIG. 4D) and T2-weighted images (not shown) only showed a speckled morphology of the capsules, and could not differentiate MCF10A from the other three uMUCl -positive cell lines. Multiple (n>3) independent encapsulation experiments were repeated for each cell line, and the CEST contrast at 1.8 ppm and 3.6 μΤ was analyzed by one-way ANOVA. The uMUCl -positive group showed a significant contrast difference from the uMUCl- negative group, with Fs, 14=28.22, P<0.0001 (FIG. 4G). Although there are subtle differences in the mucCEST spectra of the three uMUCl⁺ adenocarcinoma cell lines (BT20, HT29 and LS174T), a pair-wised comparison did not show any significant differences between them To validate the findings, immunohistological staining was performed using an antibody detecting full-length MUC1 (FIG. 4H). Only the MCF10A cell line demonstrated normal MUC1 expression.

[0042] FIGS. 5A-5D illustrate in vivo imaging of benign and malignant tumor xenografts. FIG. 5 A illustrates a T2w image, marked with regions of U87, LS174T, and control white matter (dashed square). FIG. 5B illustrates a CEST contrast map created by averaging 1.2 ppm and 0.9 ppm superimposed onto FIG. 5 A. FIG. 5C illustrates a graphical view of MTRasym curves of the 3 ROIs marked in FIG. 5 A. FIG. 5D illustrates a graphical view of MTRasym values of the two cell lines showing a significant difference (p<0.05, t-test, n=3).

[0043] Finally, the applicability of mucCEST imaging was tested for differentiating tumor cells in vivo. MCF10A, a benign human epithelial cell line, did not form tumors when implanted in the striatum of immunodeficient mice. U87, a tumor cell line without uMUCl expression (uMUCl-), was compared to malignant LS174T cells expressing uMUCl (uMUCl+). The observed size of the U87 tumors was smaller than for the LS174T in all the mice imaged (FIG. 5A), as a result from the different growth rates of the tumors. Similar to previous glycan studies, the average of 0.9 ppm and 1.2 ppm were used as the characteristic frequency for mucin in order to avoid overlap with the frequency ranges of amine and amide protons. The CEST contrast map (FIG. 5B) and CEST spectra (FIG. 5C) demonstrated that uMUCl+ LS174T cells displayed a significantly lower CEST signal compared to U87 (uMUCl-) cells in all three mice tested (p<0.05, FIG. 5D).

[0044] In this study, mucCEST MRI has been demonstrated as able to differentiate between tumor cells that are expressing normal vs. underglycosylated MUC1. Using extracted, purified mucins, these mucopolysaccharides display a broad peak from 0.5 ppm to 4 ppm, with a signal peak around ~1 ppm, owing to the abundance of glycan side chains. As a model for tumor cells expressing underglycosylated MUC1 (uMUCl), mucin was deglycosylated with as result a striking difference between the treated and untreated mucin, with the former showing a >75% reduction of CEST signal from 0.5 to 2 ppm. The hypothesis that underglycosylated human malignant tumor cell lines (BT20, HT29, and LS174T) showed a significantly lower CEST signal compared to a benign normally glycosylated human epithelial cell line (MCF10A) and to another uMUCl -negative cell line (U87), was tested and found in agreement. To validate in vivo detection, an uMUCl -positive cell line (LS174T) and an uMUCl -negative cell line (U87) were inoculated into the mouse brain, with as result a significantly lower contrast for LS174T compared to U87 tumors. The homogeneous environment of brain tissue was used instead of an orthotopical tumor model, as there are still challenges associated with high-field small animal CEST imaging, including motion artifacts, field inhomogeneity corrections, and susceptibility artifacts arising from air-tissue interfaces. Improved CEST imaging methods, better-equipped clinical scanners, and larger tumor volumes may allow future orthotopic imaging in patients, where longitudinal monitoring may allow for proper quantification. For instance, amide proton transfer (APT) CEST imaging has already been applied to monitor the response to neoadjuvant chemotherapy in breast cancer patients. For clinical mucCEST imaging of breast

adenocarcinoma , the large glycosylation contrast of normal breast glandular tissue may provide internal reference for quantification during longitudinal monitoring. As chemotherapy induces a consistent reduction in uMUC-1 levels, mucCEST MRI may be further explored as a noninvasive biomarker for an assessment of therapeutic efficacy. mucCEST imaging represents the first approach to differentiate label-free between tumor cells expressing and not expressing a single specific molecule, which has been widely studied and shown to play a significant role in tumor malignancy. CEST imaging has become an active new field, and new imaging schemes and pulse sequences are continuously being developed to improve the quantification and robustness of CEST imaging.

[0045] In another exemplary embodiment, LS174T (uMUC-1 positive, i.e., underglycosylated) and U87 (uMUC-1 negative, i.e., heavily glycosylated) tumor cells were encapsulated in

Alginate-PLLAlginate microcapsules5 at -1000 cells/capsule. Microcapsules suspended in 0.9% saline were loaded into 1 mm capillaries. Reference standards contained mucin from porcine stomach (Sigma, M2378) dissolved in 0.9% saline. [0046] CB 17-PRKDSCID/NCR female mice (6-8 weeks) were inoculated with 1.5x105 of LS174T and U87 cells in each cerebral hemisphere at 1 mm anterior, 2 mm lateral, and 2.5 mm ventral to bregma. CEST MRI was performed at 15-21 days post injection.

[0047] Encapsulated cells were imaged using a Bruker 500MHz 11.7T vertical scanner and in vivo experiments were performed on a Bruker 9.4T scanner with a modified RARE sequence: slice thickness=l mm, matrix size=96x48, FOV=1.15 cm x 0.55 cm for the in vitro and 1.7 cm x 1.6 cm for the in vivo studies, CW saturation pulse=3 sec, RARE factor=8, frequency range = -5 pm to 5 ppm (0.2 ppm increment) for encapsulated cells and -4.5 ppm to 4.5 ppm (0.3 ppm increment) in vivo, TR/TE= 6000 ms/14.05 ms for encapsulated cells and 5000 ms/11.72 ms in vivo, and NA=2. Bo inhomogeneity was corrected using WASSR6 with saturation pulse = 0.5 uT/50ms, and frequency range = -1 ppm to 1 ppm (0.1 ppm increment). Z-spectra were calculated from sample ROIs after Bo correction for each voxel using WAS SR.

100%*(S-Ao>-S+Aa>)/So was computed for each offset Δω.

[0048] Encapsulated cells showed differential CEST contrast depending on uMUC-1 expression, with the largest differences in CEST MTR_aSym spectrum between 2 and 4 ppm offset from water, in agreement with the measured contrast of isolated Mucin-1 (FIGS. 6A and 6B). FIGS. 6A- 6C illustrate in vitro CEST images and spectrum for encapsulated LS174T uMUC-l⁺ and U87 uMUC-r.

[0049] Similarly, differential CEST contrast was observed in vivo based on uMUC-1 expression, with uMUC-1 expressing cells showing a lower CEST contrast between 0.5 and 2 ppm (FIGS. 7A-7C). FIG. 7A illustrates in vivo mouse brain MTw images. FIG. 7B illustrates a CEST image at lppm and FIG. 7C illustrates a CEST MTR_aSym spectrum. The specific reduction of CEST contrast for LS174T cells is likely due to the different uMUC-1 glycosylation levels. The different chemical shifts of the maximal effects between the in vitro and in vivo preparations may be due to different pH buffering and also back-exchange effects to other protons such as amide.

[0050] Cell lines with differential expression of uMUC-1 show differential CEST contrast both in vitro and in vivo. The reduction of CEST contrast with uMUC-1 expression is likely due to the reduced number of hydroxyl groups as compared to heavy glycosylation. This negative contrast can potentially be used to non-invasively phenotype tumors and detect early

tumorigenesis based on uMUC-1 expression.

[0051] In another exemplary embodiment, due to the complicated O-linked glycosylation of mucin, chemical deglycosylation is preferred over enzymatic methods. The oligosaccharide chains on porcine stomach mucin (Sigma, M-2378) were removed using anhydrous

trifluoromethanesulfonic acid (TFMS) treatment. Both deglycosylated and untreated mucin were dialyzed against water, lyophilized and dissolved at a cone, of 4.0 mg/ml in PBS (pH=7.1) for imaging.

[0052] Three cell lines (MCF10A, non-tumorigenic human breast carcinoma; and LS174T and HT29, both human colon carcinomas) with different MUC-1 glycosylation levels were encapsulated in alginate-PLLalginate microcapsules at 1000 cells/capsule in order to minimize cell sedimentation and variations in cell density.

[0053] Images were taken on a Bruker 11.7T scanner, using a RARE sequence with CW saturation pulse of Β ι=3.6μΤ, T_sat=3 s and frequency incremented every 0.2 ppm from -6 to 6 ppm for phantoms and every 0.25 ppm from -5 to 5 ppm for cells; TR=6 s, effective TE=17-19 ms, matrix size=96x64. CEST contrast was quantified by MTRasym = (S-Ao>-S+Aa>)/So after a voxel -by-voxel Bo correction, with characterized mean Z-spectra and MTRasym spectra for sample ROIs plotted.

[0054] The untreated and deglycosylated mucin could be easily differentiated in both Zspectra and MTRasym spectra (FIGS. 8 A and 8B), with a significant reduction of CEST contrast over a broad chemical shift range, i.e. -80% reduction from 0.5 to 2 ppm and -50% loss from 2 to 4 ppm respectively. FIGS. 8A and 8B illustrate graphical views of a Zspectra and MTRasym for deglycosylated and untreated mucin. FIG.8C is a MTRasym contrast map at 1.8ppm peak. The deglycosylation was confirmed by SDSPAGE electrophoresis (FIG.8D), where deglycosylated mucin showed a MW of 70-100kD, whereas untreated mucin did not show any bands due to the MW being >260kD8. The CEST contrast produced by 3 cell lines with different MUC-1 expression was tested: LS174T and HT29, both expressing underglycosylated MUC-1 (i.e., "uMUC-1 positive"), and MCFlOA, expressing normally glycosylated MUC-1 (i.e. "uMUC-1 negative)". [0055] FIGS. 9A-9F illustrate microscopy, MTw image, CEST spectra and images, and imunostaining of encapsulated cell lines with different MUC-1 glycosylation levels. Both the MTRasym spectra (FIG. 9C) and contrast maps (FIG. 9D and 9E) clearly show that the underglycosylated MUC-1 tumor cell lines (LS174T and HT29) have a lower CEST contrast from 2 to 4 ppm. The MUC-1 glycosylation level was validated by immunostaining with an antibody detecting normally glycosylated MUC-1 (anti-MUCl antibody, Epitomics) with red=MUC-l, blue=nuclei (DAPI) (Fig 9F).

[0056] Deglycosylated and untreated mucin proteins could be easily differentiated by CEST MRI in vitro, with the deglycosylated sample showing >80% reduction in -OH peak. The MUC- 1 cancer marker also exhibits differential CEST contrast between 0.5 and 4 ppm depending on the glycosylation levels, which is lower for the two cell lines having underglycosylated MUC-1. The results suggest that CEST imaging of MUC-1 may potentially be used as a surrogate marker to non-invasively assess tumor malignancy and tumor progression.

[0057] In another exemplary embodiment, Oligosaccharide chains on mucin (Sigma, M-2378) were removed using trifluoromethanesulfonic acid(TFMS) treatment. Both deglycosylated and untreated mucin were dialyzed against water, lyophilized and dissolved at 4.0 mg/ml in PBS (pH=7.1). Three cell lines (MCFIOA, non-malignant human breast carcinoma; and LS174T and HT29, both human colon carcinomas) with different MUC-1 glycosylation levels were encapsulated at 1000 cells/capsule in order to minimize cell sedimentation and variations in cell density. Images were acquired on a Bruker 11.7T scanner, using a RARE sequence with a CW saturation pulse of Β ι=3.6 μΤ, T_sat=3 s, and with a frequency incremented every 0.2 ppm from -6 to 6 ppm for phantoms and every 0.25 ppm from -5 to 5 ppm for cells; TR/TE=6000 ms/17.5 ms, matrix size=96x64.

[0058] CEST contrast was quantified by

Untreated and

deglycosylated mucin could be easily differentiated in both the Z-spectra and MTR_aSym spectra with a significant reduction of CEST contrast over a broad chemical shift range, i.e. a -80% reduction from 0.5 to 2 ppm and a -50% loss from 2 to 4 ppm, respectively. Deglycosylation was confirmed by SDS-PAGE, where deglycosylated mucin showed a MW of 70-100kD, in contrast to untreated mucin which has an Mw >260kD6. The CEST contrast produced by LS174T and HT29 were compared, both expressing underglycosylated MUC-1 and MCF10A, expressing normally glycosylated MUC-1.

[0059] Both the MTRasym spectra and contrast maps clearly show that the underglycosylated MUC-1 tumor cell lines (LS174T and HT29) have a lower CEST contrast from 2 to 4 ppm, which corresponded to the expression levels seen on immunohistology. Both mucin extracts and encapsulated cells expressing MUC-1 exhibit differential CEST contrast depending on glycosylation levels, suggesting that CEST imaging may be used to assess mucin glycosylation as a surrogate marker for tumor malignancy.

[0060] In another exemplary embodiment, the sugar chains on a crude purification of porcine stomach mucin protein (Sigma-Aldrich, M-2378, St Louis, MO) by treatment with

trifluoromethanesulfonic acid (TFMS) and measured the CEST Z- and MTR_aSym-spectra of untreated mucin and deglycosylated mucin (FIGS. 8A and 8B). The CEST contrast produced by three cell lines with different MUC-1 expression were compared: two expressing

underglycosylated MUC-1 (i.e. uMUC-1 -positive; LS174T and HT29 human colon cancer cell- lines), and one expressing normally glycosylated MUC-1 (i.e. uMUC-1 -negative; MCF10A non- tumorigenic human breast cells). Measurements were performed in vitro by encapsulating live cells in alginate-PLL-alginate microcapsules (-1000 cells per capsule; FIG. 9A) in order to minimize variations in tumor cell density. Microcapsules were suspended in PBS and loaded into 5 mm MR tubes with their layout shown on the MT image in FIG. 9(b). MR images were acquired on a Bruker 11.7 T scanner using a 15 mm birdcage coil, and a rapid acquisition with relaxation enhancement sequence was used for CEST imaging with a CW saturation pulse of B i = 3.6 T, Tsat = 3 s and frequency sweeping every 0.25 ppm from 5 to +5 ppm, TR/TE = 6000 ms/ 19.09 ms, matrix size = 96 64 and, slice thickness = 1 mm.

[0061] The corrected CEST spectra (FIG. 9C) and MTR_asym maps (FIGS. 9D and 9E) indicated that the underglycosylated MUC-1 tumor cell lines (LS174T and HT29) showed a lower CEST contrast from 2 to 4 ppm. The MUC-1 glycosylation level of the three cell lines was further validated by immunostaining with an antibody detecting normally glycosylated MUC-1 (anti-MUCl antibody, RabMAb, Epitomics, Burlingame, CA, USA), as shown in FIG. 9F, with red = MUC-1 and blue = nuclei (DAPI).

[0062] The MUC-1 cancer marker exhibits differential CEST contrast between 0.5 and 4 ppm depending on the glycosylation level, which is lower for the two cell lines having

underglycosylated MUC-1. The present results suggest that CEST imaging of MUC-1 could possibly be used as a surrogate marker to noninvasively assess tumor malignancy. [0063] It should be noted that the pulse sequences, imaging protocols, described herein can be executed with a program(s) fixed on one or more non-transitory computer readable medium. The non-transitory computer readable medium can be loaded onto a computing device, server, imaging device processor, smartphone, tablet, phablet, or any other suitable device known to or conceivable by one of skill in the art. The computing device can be configured especially for use with the magnetic resonance imaging machine. The computing device can also be integrated into the magnetic resonance imaging machine either directly or via network. It is noted that the computing device to carry out the present invention can be a unique computing device designed especially for use with the present invention and to address specific needs of the execution of the present invention.

[0064] It should also be noted that herein the steps of the method described can be carried out using a computer, non-transitory computer readable medium, or alternately a computing device, microprocessor, or other computer type device independent of or incorporated with an imaging or signal collection device. An independent computing device can be networked together with the imaging device either with wires or wirelessly. Indeed, any suitable method of analysis known to or conceivable by one of skill in the art could be used. It should also be noted that while specific equations are detailed herein, variations on these equations can also be derived, and this application includes any such equation known to or conceivable by one of skill in the art. The computing device is unique to this application.

[0065] A non-transitory computer readable medium is understood to mean any article of manufacture that can be read by a computer. Such non-transitory computer readable media includes, but is not limited to, magnetic media, such as a floppy disk, flexible disk, hard disk, reel-to-reel tape, cartridge tape, cassette tape or cards, optical media such as CD-ROM, writable compact disc, magneto-optical media in disc, tape or card form, and paper media, such as punched cards and paper tape.

[0066] Although the present invention has been described in connection with preferred embodiments thereof, it will be appreciated by those skilled in the art that additions, deletions, modifications, and substitutions not specifically described may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for magnetic resonance imaging of a subject comprising:

using a magnetic resonance imaging machine to generate CEST contrast image data for tissue; processing the CEST contrast image data to determine presence of a mucin in the tissue; processing the CEST contrast image data to differentiate glycosylated and unglycosylated mucins and generate data related to the mucins present in the tissue; and assessing cancer using the generated data related to the mucins present in the tissue.

2. The method of claim 1 further comprising processing the CEST contrast image data with a non-transitory computer readable medium.

3. The method of claim 1 further comprising processing the CEST contrast image data with a computing device specifically designed for assessment of mucins in tissue.

4. The method of claim 1 further comprising using the data related to the mucins present in the tissue to non-invasively phenotype a tumor in the tissue.

5. The method of claim 1 further comprising using the data related to the mucins present in the tissue to detect early tumorgenesis.

6. The method of claim 1 further comprising using the data related to the mucins present in the tissue to monitor tumor growth.

7. The method of claim 1 further comprising using normal tissue as a reference.

8. The method of claim 7 further comprising using normal tissue as a reference during longitudinal follow-up studies.

9. The method of claim 7 further comprising calibrating and taking a ratio using normal tissue to gauge tumor contrast changes and malignancv.

10. A system for magnetic resonance imaging of a subject comprising: a magnetic resonance imaging machine configured to generate CEST contrast image data for tissue; a non-transitory computer readable medium programmed for, processing the CEST contrast image data to determine presence of a mucin in the tissue; processing the CEST contrast image data to differentiate glycosylated and unglycosylated mucins and generate data related to the mucins present in the tissue; and assessing cancer using the generated data related to the mucins present in the tissue.

11. The system of claim 10 further comprising a computing device.

12. The system of claim 11 further comprising the computing device being specifically designed for the assessment of mucins in tissue.

13. The system of claim 12 further comprising programming the non-transitory computer readable medium for processing the CEST contrast image data and loading the non-transitory computer readable medium on the computing device specifically designed for assessment of mucins in tissue.

14. The system of claim 10 further comprising the non-transitory computer readable medium being programmed for using the data related to the mucins present in the tissue to non-invasively phenotype a tumor in the tissue.

15. The system of claim 10 further comprising the non-transitory computer readable medium being programmed for using the data related to the mucins present in the tissue to detect early tumorgenesis.

16. The system of claim 10 further comprising the non-transitory computer readable medium being programmed for using the data related to the mucins present in the tissue to monitor tumor growth.

17. The system of claim 10 further comprising the non-transitory computer readable medium being programmed for using normal tissue as a reference.

18. The system of claim 17 further comprising the non-transitory computer readable medium being programmed for using normal tissue as a reference during longitudinal follow-up studies.

19. The system of claim 10 further comprising the non-transitory computer readable medium being programmed for calibrating and taking a ratio using normal tissue to gauge tumor contrast changes and malignancv.

20. The system of claim 10 wherein the magnetic resonance imaging machine and the non- transitory computer readable medium are networked together.