Methods and related aspects for grading glioblastoma aggressiveness

Abstract

Provided herein are methods of detecting an aggression level of a glioblastoma in a subject. In some embodiments, the methods include imaging the subject having the glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject. In some embodiments, the methods also include identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1.8-fold difference. Related aspects are also provided.

Description

METHODS AND RELATED ASPECTS FOR GRADING GLIOBLASTOMA AGGRESSIVENESSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 562,425, filed March 7, 2024, the disclosure of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made using Government support under grant no. EB030376, awarded by the National Institutes of Health. The Government has certain rights in this invention.BACKGROUND

[0003] Glioblastoma (GBM) is the most malignant primary brain cancer in adults. Despite recent therapeutic advances, GBM patients have a median survival rate of only 14-16 months. A major challenge has been radiological differentiation between tumors of varying aggressiveness, a distinction that plays a pivotal role in guiding treatment decisions. The abundant presence of fast proliferating cancer stem cells (CSCs) is a hallmark of GBM. Some advancements in developing targeted imaging agents against CSCs using radionuclides or magnetic resonance imaging (MRI) contrast agents have been made, but these approaches require a lengthy and expensive drug development process for clinical approval. An imaging technique that is ‘label-free’ (that does not rely on administering an exogenous agent) and can probe the aggressiveness of brain cancer during the initial diagnostic MRI session would be extremely valuable. Amide proton transfer (APT) chemical exchange saturation transfer (CEST) MRI is such a method that can report on the histopathological grade of adult diffuse gliomas, but it cannot differentiate between various degrees of GBM aggressiveness. One of the reasons why GBM is so invasive is an intricate biological reprogramming termed proneural-to-mesenchymal transition. This shifts the tumor toward mesenchymal traits, including extracellular matrix remodeling, cytoskeletal repatterning, and stem-like trait acquisition.

[0004] Accordingly, there is a need for additional methods, and related aspects, for grading GBM aggressiveness in subjects.SUMMARY

[0005] In one aspect, the present disclosure provides a method of detecting an aggression level of a glioblastoma in a subject. The method includes imaging the subject having the glioblastoma using a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test magnetic resonance (MR) image of the subject. The method also includes identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1.8-fold difference, thereby detecting the aggression level of the glioblastoma in the subject.

[0006] In some embodiments, the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique. In some embodiments, the brain cells in the control MR image comprise non-diseased brain cells. In some embodiments, the cancer stem cells comprise mesenchymal stem cells. In some embodiments, the cancer stem cells comprise mesenchymal stromal cells. In some embodiments, the brain cells in the control MR images are from the subject. In some embodiments, the brain cells in the control MR images are from a reference subject.

[0007] In some embodiments, the method further comprises generating a therapy recommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject. In some embodiments, the method further comprises administering at least one therapy that is effective against glioblastomas with the detected aggression level of the glioblastoma in the subject. In some embodiments, the method further comprises repeating the imaging and identifying steps at a subsequent time point to monitor the aggression level of the glioblastoma in the subject over time.

[0008] In another aspect, the present disclosure provides a method of detecting a glioblastoma with a high aggression level in a subject. The method includes imaging the subject having the glioblastoma using a CEST MRI technique inthe absence of introducing an exogenous MRI contrast agent into the subject to produce a test MRI of the subject. The method also includes identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, thereby detecting the glioblastoma with the high aggression level in the subject. In some embodiments, the overexpression level of mannose in and / or on the surfaces of the unlabeled mesenchymal stem cells of the glioblastoma of the subject comprises at least about a 1.8-fold difference relative to the identified expression level of mannose in and / or on the surfaces of the non-diseased brain cells in the control MR image.

[0009] In another aspect, the present disclosure provides a system that includes an MRI scanner configured to generate CEST MR images. The system also includes a controller operably connected to the MR scanner, which controller comprises, or is capable of accessing, computer readable media comprising non- transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: imaging a subject having a glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject using the MR scanner; and identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1 .8-fold difference.

[0010] In some embodiments, the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique. In some embodiments, the brain cells in the control MR image comprise non-diseased brain cells. In some embodiments, the cancer stem cells comprise mesenchymal stem cells. In some embodiments, the cancer stem cells comprise mesenchymal stromal cells. In some embodiments, the brain cells in the control MR images are from the subject. In some embodiments, the brain cells in the control MR images are from a reference subject.In some embodiments, the instructions further perform at least: generating a therapy recommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject.

[0011] In another aspect, the present disclosure provides computer-readable media comprising non-transitory computer-executable instructions which, when executed by at least one electronic processor, perform at least: imaging a subject having a glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject using an MR scanner; and identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1 .8-fold difference.

[0012] In some embodiments, the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique. In some embodiments, the brain cells in the control MR image comprise non-diseased brain cells. In some embodiments, the brain cells in the control MR image comprise non-diseased brain cells. In some embodiments, the cancer stem cells comprise mesenchymal stem cells. In some embodiments, the cancer stem cells comprise mesenchymal stromal cells. In some embodiments, the brain cells in the control MR images are from the subject. In some embodiments, the brain cells in the control MR images are from a reference subject. In some embodiments, the instructions further perform at least: generating a therapy recommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods, systems, and related computer readable media disclosed herein. The description provided hereinis better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation. It will be understood that like reference numerals identify like components throughout the drawings, unless the context indicates otherwise. It will also be understood that some or all of the figures may be schematic representations for purposes of illustration and do not necessarily depict the actual relative sizes or locations of the elements shown.

[0014] FIG. 1 is a flow chart that schematically depicts steps in a method of detecting an aggression level of a glioblastoma in a subject according to an exemplary embodiment.

[0015] FIG. 2 is a flow chart that schematically depicts steps in a method of detecting a glioblastoma with a high aggression level in a subject according to an exemplary embodiment.

[0016] FIG. 3 schematically depicts a system according to an exemplary embodiment.

[0017] FIGS. 4A-4E: LMAN1 and LMAN2 are enriched in mesenchymal GBM cells. (A) Expression of genes coding for regulators of mannose metabolism was compared between IDH-wt GBM and normal brain tissue. (B) Violin plots showing expression of LMAN1 , LMAN2, LMAN2L, and SLC35D2 in different GBM molecular subtypes. Correlation matrix showing Pearson’s coefficients between the mesenchymal marker CD44 or prominin and LMAN1 , LMAN2, LMAN2L, and SLC35D2 in GBM clinical specimens (C) and primary GBM neurospheres (D). (E) qRT-PCR to measure expression of LMAN1 , LMAN2, LMAN2L, and SLC35D2 in GBM neurospheres sorted into CD44 positive and negative populations. Statistical significance was calculated using unpaired, non-parametric, student’s t-test with Mann-Whitney post hoc test in panels A, B; statistical significance was calculated using Student’s t-test in panel E. Expression data was retrieved from the clinical databases using the GlioVis portal (http: / / gliovis.bioinfo.cnio.es). *p<0.05, **p<0.01 ; ***p<0.001 ; X=not determined.

[0018] FIGS. 5A-5F: Tissue array of grade IV GBM reveals the copresence of mannose and CD44, which correlate to tumor aggressiveness. Clinical tissue array containing 35 different grade IV GBM (patients aged 8-80 years)and 5 normal human cerebral tissue samples (for a total of 80 in duplicate) were stained for mannose (A), the mesenchymal marker CD44 (B) and H&E (C). Scatter plots with linear regression lines show the relationship between CD44 expression and aggressiveness (D), mannose levels and aggressiveness (E), and CD44 expression vs. mannose levels (F) in GBM grade IV tissue samples. Shaded gray areas represent 95% confidence intervals.

[0019] FIGS. 6A-6G: In vitro MANw CEST MRI signal corresponds to mannose and CD44 co-expression in mesenchymal GBM which is absent in proneural GBM. (A) Schematic illustration of the proneural-to-mesenchymal transition in GBM, resulting in elevated mannose N-linked glycan levels in CD44+mesenchymal cells that is absent in CD133+proneural cells. (B) Western blot showing expression of CD133 (proneural) and CD144 (mesenchymal) markers in GBM1a and M1123 cells. (C) qRT-PCR analysis of canonical proneural and mesenchymal markers in GBM1a and M1123 cells.. (D) Extreme limiting dilution assay (ELDA) to measure stem cell frequencies in GBM1a and M1123. (E) Mannose and CD44 staining in single representative GBM1a and M1123 3D neurospheres. Scale bar=200 pm. (F) In vitro MANw CEST MRI and (G) signal quantification of GBM1a and M1123 3D neurospheres. Scale bar in (F)=2 mm. *p<0.05, ** p<0.01 , and ***p<0.001 .

[0020] FIGS. 7A-7C: In vivo MANw CEST MRI signal corresponds to mannose and CD44 co-expression in mesenchymal GBM which is absent in proneural GBM. (A) Serial in vivo T2-weighted and MANw CEST MRI of a bilateral orthotopic GBM1a and M1123 mouse xenograft model at different time points post tumor implantation. (B) Quantified MANw CEST MRI signal at 1.2 ppm for the regions of interest drawn in A as an example for n=5 animals. (C) FITC-GNL and anti-CD44 staining in tissue sections representative for the brain slices shown in A.

[0021] FIGS. 8A-8G: Knockdown of LMAN1 and LMAN2 in M1123 cells decreases mannosylation and CEST contrast and prevents tumor growth in vivo. (A) Schematic outline of siRNA-mediated silencing of LMAN1 / 2. (B) qRT PCR analysis shows specific knockdown of LMAN1 and LMAN2 compared to control siRNA and LMAN cross-control siRNA. (C) Mannose staining and (D) quantification. (E) In vitro MANw CEST MRI signal at 1.2 ppm for wild type (WT) cells and LMAN1 / LMAN2 knockdown cells. (F) In vivo T2-weighted and MANw CEST MRI 7days post-implantation of LMAN1 / LMAN2-knockdown cells in the right hemisphere and WT M1123 cells in the left hemisphere. (G) Quantified MANw CEST MRI signal at 1.2 ppm for the regions of interest drawn in (F) as an example for n=3 animals. *p<0.05, ** p<0.01 , ***p<0.001 and ****p<0.0001 .DEFINITIONS

[0022] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0023] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0024] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In describing and claiming the methods, computer readable media, systems, and component parts, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.

[0025] About: As used herein, “about” or “approximately” or “substantially” as applied to one or more values or elements of interest, refers to a value or element that is similar to a stated reference value or element. In certain embodiments, the term “about” or “approximately” or “substantially” refers to a range of values or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value or element unless otherwisestated or otherwise evident from the context (except where such number would exceed 100% of a possible value or element).

[0026] Administering. As used herein, “administering” is defined herein as a means of providing an agent or a composition containing the agent to a subject in a manner that results in the agent being inside the subject's body. Such an administration can be by any route including, without limitation, oral, transdermal (e.g., vagina, rectum, oral mucosa), by injection (e.g., subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, intraocular), by inhalation (e.g., oral or nasal), or topical (e.g., eye drops, cream, etc.). Pharmaceutical preparations are, of course, given by forms suitable for each administration route.

[0027] Cancer. As used herein, “cancer” in a subject refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. In some circumstances, cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells.

[0028] Cancer Stem Cells: As used herein, “cancer stem cells” refer to cancer cells that have the ability to give rise to all cell types found in a particular cancer sample, including, for example, host cells, such as blood vessels, stromal cells, macrophages, and the like.

[0029] Control: As used herein, “control” refers to a standard or reference condition.

[0030] Detect: As used herein, “detect” refers to identifying the presence, absence or amount of the analyte to be detected.

[0031] Effective Amount: As used herein, “effective amount” of an agent refers to the amount of the agent sufficient to elicit a desired biological response or a measurable difference when compared to a control. As will be appreciated by one of ordinary skill in the art, the absolute amount of a particular agent that is effective for treating a disease, disorder, condition, or injury can vary depending on such factors as the agent to be delivered, the manner of administration, the age, body weight, and general health of the subject, the desired biological endpoint, the desired therapeuticeffect, and the like. Ultimately, an attending clinician will decide the appropriate amount and dosage regimen. For example, an “effective amount” of an agent can be an amount sufficient to produce a measurable image when the compound is used for imaging, or an amount sufficient to ameliorate the symptoms of a disease when the compound is used for therapy. One of ordinary skill in the art will further understand that an effective amount of an agent can be administered in a single dose, or can be achieved by administration of multiple doses.

[0032] Imaging: As used herein, “imaging” refers to using a method that is magnetic resonance (MR)-based (magnets that polarize, excite and saturate hydrogen nuclei in water molecules in tissue to produce a detectable signal) to form an image of a cell, tissue, tumor, part of body, and the like.

[0033] Magnetic Resonance Imaging As used herein, “magnetic resonance imaging” or “MRI” is meant to refer to a noninvasive diagnostic process that uses an MR scanner to obtain images of objects, tissues, or bodies. An MR scanner uses nuclear magnetic resonance to obtain images. The MR scanner may include, for example, (1 ) a body-encircling magnet that generates a strong, uniform magnetic field which interacts with radio waves to excite the nuclei of specific atoms, such as hydrogen, and (2) a detector that detects relaxation of the nuclei and transforms the detected signals into a visual image.

[0034] Mesenchymal Stem Cell: As used herein, “mesenchymal stem cell” refers to a stromal cell that has the ability to self-renew and also exhibits multilineage differentiation.

[0035] Mesenchymal Stromal Cell: As used herein, “mesenchymal stromal cell” refers to a spindle shaped plastic-adherent cell isolated from bone marrow, adipose, and other tissue sources, with multipotent differentiation capacity in vitro.

[0036] Reference: As used herein, “reference” refers to a standard or control condition.

[0037] Subject: As used herein, “subject” or “test subject” refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companionanimals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or pathology or a predisposition to the disease or pathology, or an individual that is in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject.” A “reference subject” refers to a subject known to have or lack specific properties (e.g., known ocular or other pathology and / or the like).

[0038] Therapeutic Agent: As used herein, “therapeutic agent” refers to a compound that has the potential of affecting the function of an organism. A therapeutic agent may decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of disease, disorder, or condition in a host organism or subject.

[0039] Treat: As used herein, the terms “treat,” treating,” “treatment,” and the like, are used interchangeably and are meant to decrease, suppress, attenuate, diminish, arrest, the underlying cause of a disease, disorder, or condition, or to stabilize the development or progression of a disease, disorder, condition, and / or symptoms associated therewith. The terms “treat,” “treating,” “treatment,” and the like, as used herein can refer to curative therapy, prophylactic therapy, and preventative therapy. The treatment, administration, or therapy can be consecutive or intermittent. Consecutive treatment, administration, or therapy refers to treatment on at least a daily basis without interruption in treatment by one or more days. Intermittent treatment or administration, or treatment or administration in an intermittent fashion, refers to treatment that is not consecutive, but rather cyclic in nature. Treatment according to the presently disclosed methods can result in complete relief or cure from a disease, disorder, or condition, or partial amelioration of one or more symptoms of the disease, disease, or condition, and can be temporary or permanent. The term “treatment” also is intended to encompass prophylaxis, therapy, and cure.DETAILED DESCRIPTION

[0040] Glioblastoma (GBM) is the most aggressive cancer known to men. Non-invasive assessment of aggressiveness is important for treatment planning, but current MRI protocols lack specificity. Amide proton transfer CEST MRI can gradediffuse gliomas, but not GBM aggression levels. GBM invasiveness arises from a shift from a pro-neural to mesenchymal phenotype. Accordingly, in certain aspects, the present disclosure provides methods of detecting aggression levels of glioblastoma in subjects that, for example, can be used to develop therapy recommendations as well as to monitor the progression of the disease over time.

[0041] To illustrate, FIG. 1 is a flow chart that schematically depicts steps in a method of detecting an aggression level of a glioblastoma in a subject according to an exemplary embodiment. As shown, method 100 includes imaging the subject having the glioblastoma using a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test magnetic resonance (MR) image of the subject (step 102). Method 100 also includes identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1.8-fold difference, thereby detecting the aggression level of the glioblastoma in the subject (step 104).

[0042] To further illustrate, FIG. 2 is a flow chart that schematically depicts steps in a method of detecting a glioblastoma with a high aggression level in a subject according to an exemplary embodiment. As shown, method 200 includes imaging the subject having the glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject (step 202). In addition, method 200 also includes identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, thereby detecting the glioblastoma with the high aggression level in the subject (step 204). In some embodiments, the overexpression level of mannose in and / or on the surfaces of the unlabeled mesenchymal stem cells of the glioblastoma of the subject comprises at least about a 1.8-fold difference relative tothe identified expression level of mannose in and / or on the surfaces of the nondiseased brain cells in the control MR image.

[0043] In some embodiments, the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique. In some embodiments, the brain cells in the control MR image comprise non-diseased brain cells. In some embodiments, the cancer stem cells comprise mesenchymal stem cells. In some embodiments, the cancer stem cells comprise mesenchymal stromal cells. In some embodiments, the brain cells in the control MR images are from the subject. In some embodiments, the brain cells in the control MR images are from a reference subject.

[0044] In some embodiments, the method further comprises generating a therapy recommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject. In some embodiments, the method further comprises administering at least one therapy that is effective against glioblastomas with the detected aggression level of the glioblastoma in the subject. In some embodiments, the method further comprises repeating the imaging and identifying steps at a subsequent time point to monitor the aggression level of the glioblastoma in the subject over time.

[0045] The present disclosure also provides various systems and computer program products or machine readable media. In some embodiments, for example, the presently disclosed subject matter also features a magnetic resonance imaging system. Magnetic resonance imaging systems are known in the art and commercially available. In certain aspects, the magnetic resonance imaging system comprises an imaging apparatus configured to perform a CEST MRI technique.

[0046] In some aspects, for example, the methods described herein are optionally performed or facilitated at least in part using systems, distributed computing hardware and applications (e.g., cloud computing services), electronic communication networks, communication interfaces, computer program products, machine readable media, electronic storage media, software (e.g., machineexecutable code or logic instructions) and / or the like. To illustrate, FIG. 3 provides a schematic diagram of an exemplary system suitable for use with implementing at least aspects of the methods disclosed in this application. As shown, system 300 includes at least one controller or computer, e.g., server 302 (e.g., a search engineserver), which includes processor 304 and memory, storage device, or memory component 306, and one or more other communication devices 314 and 316 (e.g., client-side computer terminals, telephones, tablets, laptops, other mobile devices, etc.) positioned remote from and in communication with the remote server 302, through electronic communication network 312, such as the Internet or other internetwork. Communication device 314 typically includes an electronic display (e.g., an internet enabled computer or the like) in communication with, e.g., server 302 computer over network 312 in which the electronic display comprises a user interface (e.g., a graphical user interface (GUI), a web-based user interface, and / or the like) for displaying results upon implementing the methods described herein. In certain aspects, communication networks also encompass the physical transfer of data from one location to another, for example, using a hard drive, thumb drive, or other data storage mechanism. System 300 also includes program product 308 stored on a computer or machine readable medium, such as, for example, one or more of various types of memory, such as memory 306 of server 302, that is readable by the server 302, to facilitate, for example, a guided search application or other executable by one or more other communication devices, such as 314 (schematically shown as a desktop or personal computer). In some aspects, system 300 optionally also includes at least one database server, such as, for example, server 310 associated with an online website having data stored thereon (e.g., acquired MR imaging data, etc.) searchable either directly or through search engine server 302. System 300 optionally also includes one or more other servers positioned remotely from server 302, each of which are optionally associated with one or more database servers 310 located remotely or located local to each of the other servers. The other servers can beneficially provide service to geographically remote users and enhance geographically distributed operations.

[0047] As understood by those of ordinary skill in the art, memory 306 of the server 302 optionally includes volatile and / or nonvolatile memory including, for example, RAM, ROM, and magnetic or optical disks, among others. It is also understood by those of ordinary skill in the art that although illustrated as a single server, the illustrated configuration of server 302 is given only by way of example and that other types of servers or computers configured according to various other methodologies or architectures can also be used. Server 302 shown schematically inFIG. 3, represents a server or server cluster or server farm and is not limited to any individual physical server. The server site may be deployed as a server farm or server cluster managed by a server hosting provider. The number of servers and their architecture and configuration may be increased based on usage, demand and capacity requirements for the system 300. As also understood by those of ordinary skill in the art, other user communication devices 314 and 316 in these aspects, for example, can be a laptop, desktop, tablet, personal digital assistant (PDA), cell phone, server, or other types of computers. As known and understood by those of ordinary skill in the art, network 312 can include an internet, intranet, a telecommunication network, an extranet, or world wide web of a plurality of computers / servers in communication with one or more other computers through a communication network, and / or portions of a local or other area network.

[0048] As further understood by those of ordinary skill in the art, exemplary program product or machine readable medium 308 is optionally in the form of microcode, programs, cloud computing format, routines, and / or symbolic languages that provide one or more sets of ordered operations that control the functioning of the hardware and direct its operation. Program product 308, according to an exemplary aspect, also need not reside in its entirety in volatile memory, but can be selectively loaded, as necessary, according to various methodologies as known and understood by those of ordinary skill in the art.

[0049] As further understood by those of ordinary skill in the art, the term "computer-readable medium" or “machine-readable medium” refers to any medium that participates in providing instructions to a processor for execution. To illustrate, the term "computer-readable medium" or “machine-readable medium” encompasses distribution media, cloud computing formats, intermediate storage media, execution memory of a computer, and any other medium or device capable of storing program product 308 implementing the functionality or processes of various aspects of the present disclosure, for example, for reading by a computer. A "computer-readable medium" or “machine-readable medium” may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks. Volatile media includes dynamic memory, such as the main memory of a given system. Transmission media includes coaxial cables, copper wire and fiber optics, including the wires thatcomprise a bus. Transmission media can also take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications, among others. Exemplary forms of computer-readable media include a floppy disk, a flexible disk, hard disk, magnetic tape, a flash drive, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave, or any other medium from which a computer can read.

[0050] Program product 308 is optionally copied from the computer-readable medium to a hard disk or a similar intermediate storage medium. When program product 308, or portions thereof, are to be run, it is optionally loaded from their distribution medium, their intermediate storage medium, or the like into the execution memory of one or more computers, configuring the computer(s) to act in accordance with the functionality or method of various aspects. All such operations are well known to those of ordinary skill in the art of, for example, computer systems.

[0051] To further illustrate, in certain aspects, this disclosure provides systems that include one or more processors, and one or more memory components in communication with the processor. The memory component typically includes one or more instructions that, when executed, cause the processor to provide information that causes at least one MR image and / or the like to be displayed (e.g., via communication device 214 or the like) and / or receive information from other system components and / or from a system user (e.g., via communication device 214 or the like).

[0052] In some aspects, program product 308 includes non-transitory computer-executable instructions which, when executed by electronic processor 304 perform at least: imaging a subject having a glioblastoma using a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject using an MRI scanner, and identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression levelcomprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1.8-fold difference.

[0053] System 300 also typically includes additional system components (e.g., MRI scanner 318) that are configured to perform various aspects of the methods described herein. In some of these aspects, one or more of these additional system components are positioned remote from and in communication with the remote server 302 through electronic communication network 312, whereas in other aspects, one or more of these additional system components are positioned local, and in communication with server 302 (i.e., in the absence of electronic communication network 312) or directly with, for example, desktop computer 314.EXAMPLE: HIGH MANNOSE CONTENT AS IMAGING BIOMARKER FORGLIOBLASTOMA CELLS WITH AN AGGRESSIVE MESENCHYMALPHENOTYPE

[0054] INTRODUCTION

[0055] Glioblastoma (GBM) is a very aggressive primary brain tumor with poor prognosis and high incidence of recurrence. Recent single cell analyses show that clinical GBM consists of cells representing four phenotypic states with the capacity to dynamically transition between states. Three of these states recapitulate neural cell types (i.e. astrocyte-like, oligodendrocyte-like and neural progenitor-like) and a fourth resembles mesenchymal cells (i.e. mesenchymal-like). These GBM phenotypes correlate with aggressivity, with the mesenchymal state associating with most aggressive disease, worse prognosis, resistance to current therapies and recurrence. Identifying key features of this mesenchymal transition can allow for the development of novel biomarkers to enable early detection of aggressive cell subset and improve patient outcome.

[0056] Protein glycosylation plays a critical role in multiple cellular processes including cell signaling, cell-extracellular matrix interactions, cell migration, immune modulation, and the maintenance of tissue structure and homeostasis. Among these, mannose-containing glycoproteins stand out due to their involvement in cellular recognition, trafficking, and immune response regulation. Aberrant glycosylation,including alterations in mannose residues, occurs during the epithelial-to- mesenchymal transition (EMT) and can enhance cell motility, invasiveness, and survival of tumor cells. Understanding glycoprotein biology, particularly the role of mannose-enriched glycans in tumor cells and their microenvironment, can allow for identification of novel biomarkers and therapeutic strategies to positively impact cancer patient outcome.

[0057] Currently, precision imaging in oncology has limitations, as dictated by the heterogeneity and spatiotemporal composition of tumor cells and their microenvironment. In this regard, recent advancements in molecular imaging may allow us to leverage aberrant glycan biology for diagnostic purposes. Chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) is an emerging molecular imaging technique that can detect metabolites and molecules that contain exchangeable amide, amine, and hydroxyl protons. The hydroxyl (OH) protons present in sugar residues have been exploited as an endogenous CEST MRI biomarker for mucin underglycosylation in adenocarcinoma and as exogenous glucose and dextran CEST MRI contrast agents for imaging tumor perfusion and blood-brain barrier leakage. We have recently shown that human mesenchymal stem cells (hMSCs) can be detected by mannose-weighted (MANw) CEST MRI by virtue of their cell membrane expressing high levels of mannose N-linked glycans. These findings prompted us to investigate if MANw CEST MRI can be used to differentiate aggressive GBM with a mesenchymal phenotype from less aggressive non- mesenchymal GBM, with the ultimate goal to develop the MANw CEST MRI signal as a surrogate imaging biomarker for tumor aggressiveness and recurrence in patients.

[0058] METHODS

[0059] Patient databases

[0060] Clinical and transcriptom ic data from control and glioma patient samples was retrieved from GlioVis database.

[0061] Glioblastoma tissue array

[0062] A tissue microarray, containing 35 cases of grade IV glioblastoma and 5 cases of normal cerebral tissue (duplicate cores per case) were obtained from Tissuearray. Com (GL805-L51 ). Tissue aggressiveness was quantified using acomposite scoring method incorporating H&E staining features. H&E-based aggressiveness scores were assigned based on histopathological characteristics, with higher scores corresponding to increased tumor proliferation, necrosis, and vascularization. CD44 expression and mannose levels were calculated by measuring mean fluorescence intensity (MFI) from fluorescence images, with values normalized on a 0-100% scale for comparison across samples.

[0063] Cell culture

[0064] Human 0913 (GBM1a) cells were initially established by Vescovi and colleagues and characterized by our group. M1123 cells were generously provided by Dr. Nakano (Ohio State University). 3D neurospheres were maintained in serum- free medium supplemented with epidermal growth factor (EGF) and fibroblast growth factor (FGF) and cultured in ultra-low-attachment 6-well plates (Millipore-Sigma) at 37°C under 5% CO2 / 95% air. Serum-induced differentiation was performed by introducing serum into the culture medium and transitioning the cells to adherent conditions in T-25 flasks.

[0065] For the ELDA, tumor cells were cultured in neurosphere medium containing EGF / FGF at decreasing cell densities ranging from 100 to 12.5 cells, with over 24 technical replicates for each dilution. The number of wells containing spheres was counted after 14 days, and the online ELDA tool was used to calculate stem cell frequencies.

[0066] Immunofluorescent staining for CD44 and mannose

[0067] GBM1a and M1123 neurospheres were transferred in 1-well cell culture chamber slides. Cells were first incubated in cold PBS containing 20 pg / ml GNL-FITC (FL-1241 , Vector Laboratories) at 4 °C for 50 minutes for the presence of mannose, then washed three times with PBS. They were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and permeabilized using 0.1 % Triton X-100 in PBS for 10 min. After blocking non-specific binding sites with 5% normal goat serum (NGS), cells were incubated with primary Alexa Fluor® 647 anti-CD44 antibody (1 :250, Abeam, EPR18668) according to the manufacturer’s protocol (3 h at RT in the dark). Excess antibody was removed by washing 3x with 10 mM phosphate buffered saline, pH=7.4 (PBS). Nuclei were stained with 4', 6- diamidino-2-phenylindole (DAPI) for 5 minutes. To stain the tissue array slides, theywere deparaffinized by sequential washes in xylene (2x5 minutes), followed by rehydration through a graded ethanol series (100%, 95%, 70%, and 50%, 5 minutes each) and a final rinse in distilled water. For antigen retrieval, slides were incubated in sodium citrate buffer (10 mM, pH=6.0) at 95-100°C for 20 minutes, then allowed to cool to RT before washing three times with PBS. For immunostaining, slides were blocked in 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature to minimize non-specific binding. Tissue arrays were then incubated with Alexa Fluor® 647 anti-CD44 antibody (1 :250) and 20 pg / ml of GNL-FITC for 1 hour at RT in the dark. Unbound antibody / lectin was removed by washing three times with PBS. Nuclei were counterstained with DAPI for 5 minutes, followed by a final PBS wash. All fluorescence imaging was performed using a Zeiss Axiovert 200 M inverted epifluorescence microscope, utilizing separate filter sets for DAPI, FITC, and Alexa Fluor® 647.

[0068] Knockdown of LMAN1 and LMAN2

[0069] For M1123 cells, the mannose-binding lectins LMAN1 and LMAN2 were knocked down using liposomal transfection with LMAN 1 / 2 siRNA (Millipore- Sigma, Cat# SASI_Hs01_00189318 and SASI_Hs01_00209153), and LMAN1 / 2 expression was quantified with qRT-PCR and normalized to GAPDH. siRNA transfections were performed using RNAiMax (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Briefly, GBM neurosphere single-cell suspensions (2.5x105 cells) were seeded onto ultra-low-attachment 24-well plates (Millipore-Sigma) in 800 pl of neurosphere medium. Lipofectamine RNAiMax reagent (12 pl) was added to 100 pl of Opti-MEM medium. In a separate tube, siRNA oligos were diluted in 100 pl of Opti-MEM to achieve a final concentration of 240 nM. Tubes were combined, mixed and incubated at RT for 15 minutes to allow lipid-siRNA complex formation. The mixture was then added dropwise to GBM neurospheres single-cell suspensions. Cells were collected 5 days after transfections for gene expression analysis.

[0070] qRT-PCR

[0071] Total RNA was extracted from the cells using a RNeasy mini kit (Qiagen, Germantown, MD). cDNA was made by reverse-transcribing 1 pg of total RNA using MuLV Reverse Transcriptase and Oligo (dT) primers (AppliedBiosystems, Waltham, MA). qRT-PCR was performed with the Bio-Rad CFX detection System (BioRad, Hercules, CA), and the expression of target genes was measured using the Power SYBR green PCR kit (Applied Biosystems). The samples were amplified in triplicate, and relative gene expression was analyzed using BioRad CFX manager software v3.1 and normalized to 18S RNA. The primer sequences used in this study were obtained from PrimerBank.

[0072] In vitro M ANw CEST M Rl

[0073] For in vitro MANw CEST MRI studies, 1 X106GBM1a and M1123 neurospheres with an average size of 500 pm were collected, rinsed three times with PBS, and loaded into 5 mm NMR tubes (Wilmad®, USA). CEST MRI was then conducted on an 11 .7 T Broker Biospin vertical bore scanner equipped with a 20 mm birdcage transmit / receive coil. A modified RARE (rapid acquisition with refocused echoes) sequence was employed, with the following parameters: repetition time (TR) / echo time (TE)=6, 000 / 5 ms, RARE factor=32, number of averages (NA)=2, slice thickness=1 mm, field of view (FOV)=16X16 mm, matrix size = 64x64, in-plane resolution^.25x0.25 mm, Bi=2.4 T, and saturation time (TSat)=4 s. Saturation frequencies ranged from -5 to +5 ppm in 0.2 ppm intervals, with O ppm referencing the water resonance. The total acquisition time was 20 min and 48 s. LMAN1 - LMAN2-, and dual LMAN1 / 2-knockdown neurospheres were subsequently imaged under identical conditions and compared with wt M1123. All experiments were performed as three independent replicates.

[0074] Animal Model

[0075] All animal procedures were approved by the Johns Hopkins University Animal Care and Use Committee. Immunodeficient NSG mice (NOD.Cg- PrkdcscidIl2rgtm1Wi' / SzJ) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and maintained as an in-house breeding colony. Male and female mice, aged 8 weeks (22-25 g), were housed under a 12-hour light / dark cycle with unrestricted access to food and water. During all surgery and imaging procedures, mice were anesthetized with isoflurane (2-3% for induction and 1-2% for maintenance in O2), using a calibrated vaporizer and scavenging system. Breathing rate and body temperature were continuously monitored and controlled throughout the scanning procedure. For stereotaxic injections, a burr hole was drilled at coordinates 0 mmcaudal and 2 mm lateral to the bregma on each side. A Hamilton syringe was used to inject 2.5 pL of single cell suspensions from neurospheres. Neurospheres were dissociated into single cells using gentle mechanical trituration and enzymatic treatment with Accutase. Spheres were first pipetted gently using a wide-bore tip, followed by incubation in pre-warmed StemPro™ Accutase™ cell dissociation reagent (Gibco, USA) at 37°C for 20 minutes, with occasional swirling to obtain a single-cell suspension. Cells (1.5X105M1123 or GBM1A in PBS) were injected into the right and left striatum at a depth of 2 mm below the endocranium, administered at a rate of 0.2 pL min-1. To further verify that the CEST signal is generated by mannose in mesenchymal cells, LMAN1 / 2 KD neurospheres were injected into the left striatum and wild-type M1123 neurospheres into the right striatum using the same procedure in three mice. To compare the perfusion properties of GBM1a and M1123 tumors, mice received 0.2 mmol / kg of a Gd-based contrast agent (Omniscan, Nycomed, Oslo, Norway) via a pre-inserted intravenous catheter. Dynamic T1 -weighted imaging was conducted before injection (baseline) and continuously for 10 minutes post-injection to evaluate perfusion and vascular permeability.

[0076] In vivo CEST and Gd-enhanced T1w MRI

[0077] MRI was carried out on days 1 , 8, and 16 post-transplantation using an 11 .7 T Bruker Biospec preclinical scanner (Bruker, Ettlingen, Germany) with a 23- mm volume transmit / receive coil. T2-weighted (T2w) MRI parameters were TR / TE=3, 000 / 5.6 ms, RARE factor=16, slice thickness=1 mm, FOV=1.6x1.5 cm, matrix=256x128, and NA=3. CEST MRI parameters were TR / TE=5,500 / 3.7 ms, RARE factor=23, single slice, slice thickness= 1 mm, FOV=1.6x1.6 cm, matrix=64x64, NA=1 , Bi=2.4 pT, and Tsat=3 s, with saturation frequencies ranging from -5 to +5 ppm in 0.25 ppm increments (water signal set to 0 ppm). The total acquisition time was 7 minutes. Tumor and brain regions of interest (ROIs) were manually drawn from T2-w images. This experiment was performed in five independent replicates.

[0078] Post-mortem tissue analysis

[0079] Mice were transcardially perfused with 10 mM PBS and 4% paraformaldehyde (PFA). The brain was removed and re-immersed in 4% PFA at 4°C for 24 h, then transferred to 30% sucrose for another 72 h. After embedding in optimal cutting temperature compound, the brain was cryosectioned into 10 pm sections. For GNL and anti-CD44 staining, brain sections were first rehydrated with PBS for 10 min and then incubated with 20 pg / ml FITC-GNL and 1 :250 Alexa Fluor® 647 anti-CD44 antibody for 1 h at RT. After washing 3x with PBS, sections were coverslipped using mounting medium containing DAPI. Fluorescence microscopy was performed using a Zeiss Axiovert 200 M inverted epifluorescence microscope.

[0080] Statistics and reproducibility

[0081] For statistical analysis of patient data, pairwise comparisons between group levels with corrections for multiple testing (p values with Bonferroni correction) were used. Pearson’s correlation analysis was performed to assess relationships between CD44 expression, mannosylation score, and aggressiveness scores, and linear regression models were applied to visualize trends with 95% confidence intervals. Data are presented as mean±standard deviation (SD and were analyzed using a one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test for multiple group comparisons, with significance thresholds set at *p<0.05, ** p<0.01 , and ***p<0.001 and ****p<0.0001 . Comparisons between tumors were performed using a two-tailed Student’s t-test. Sample sizes were determined to achieve adequate statistical power (>90%, p=0.01 ) for detecting expected effect sizes, estimated based on our preliminary data and prior experience with similar experiments. The number of independent repetitions for each experiment is given in the corresponding figure captions. All statistical analyses were conducted using GraphPad Prism 6.0. Investigators remained blinded to group allocations during and after experiments including MRI data processing.

[0082] RESULTS

[0083] Mesenchymal GBM cells express high levels of mannose and genes regulating mannosylation

[0084] Since mesenchymal transitions play important roles in GBM pathogenesis, we aimed to identify molecular events related to these transitions that coordinate changes in mannosylation. We queried expression of known genes involved in mannose regulation in 3 distinct clinical GBM transcriptom ic data sets. This analysis identified 13 genes related to mannosylation (LMAN1 , LMAN2,LMAN2L, SLC35D2, TMEM5 DPY19L1 , DPM3, DPM2, DPMI , DPY19L4, PIGB, FKTN, P0MT2) as being consistently upregulated in GBM compared to normal brain (Fig. 4A). Further analysis showed that expression of 4 of these 13 genes (LMAN1 , LMAN2, LMAN2L and SLC35D2) are elevated in mesenchymal GBM cells compared to the proneural and classical subtypes (Fig. 4B). We also identified positive correlations between LMAN1 , LMAN2, SLC35D2 and the mesenchymal marker CD44, but not the proneural marker Prom1 (CD133) in transcriptom ic datasets derived from clinical specimens (Fig. 4C) and GBM cells (Fig. 4D). Consistent with these transcriptom ic associations, fluorescence-activated cell sorting (FACS) followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) determined that LMAN1 and LMAN2 are enriched in CD44+GBM cells compared to their CD44’ counterparts (Fig. 4E). Kaplan-Meier survival analysis in both the TCGA- HG-U133 and REMBRANDT datasets demonstrated that low LMAN2 expression significantly improved survival in GBM patients compared to those with high LMAN2 expression (p=0.0025), indicating its potential prognostic relevance in I DH -wildtype GBM.

[0085] To compare mannosylation and CD44 expression between GBM and normal brain in patient samples, a tissue array consisting of 35 glioblastoma and 5 normal brain specimens was analyzed. Staining was performed using FITC- conjugated Galanthus nivalis lectin (GNL), which binds to (a-1 ,3) mannose residues, and an Alexa Fluor® 647-conjugated anti-CD44 antibody to assess the presence of mesenchymal GBM markers. Fluorescence imaging revealed a correlation between mannose and CD44 expression in GBM samples (Fig. 5A, B), whereas normal brain tissue exhibited little to no expression of these markers. Analysis of mannose levels and tumor aggressiveness demonstrated a positive correlation (r=0.76, p=0.0001 )(Fig. 5D), suggesting that elevated mannose levels are associated with GBM malignancy. Likewise, analysis of CD44 expression vs. tumor aggressiveness also revealed a positive correlation (r=0.71 , p=0.0001 ) (Fig. 5E), further supporting the notion that mesenchymal cells drive GBM progression. Combining these results, CD44 expression and mannose levels correlated with each other (r=0.65, p=0.0003)(Fig. 5F), indicating a potential mechanistic link between mannosylation and mesenchymal phenotypic transitions in GBM.

[0086] In vitro MANw CEST MRI of proneural and mesenchymal GBMcorrelates to mannose and CD44 co-expression

[0087] Mesenchymal transitions and changes in mannose levels are critical determinants of the tumor and stem cell phenotype in multiple cancers, including GBM. Our findings so far support the hypothesis that mesenchymal GBM cells express high levels of mannose compared to their proneural counterparts and that these molecular differences can be leveraged for label-free MANw CEST MRI detection (Fig. 6A). We used two well-characterized patient-derived human isocitrate dehydrogenase (IDH)-wt GBM neurosphere cell lines that represent the mesenchymal (M1123) and proneural subtypes (GBM1a). As shown previously, GBM1a expresses high levels of the proneural markers CD133, Sox2, and Ascii while M1123 expresses high levels of the mesenchymal markers CD44, vimentin (Vim) and Snail (Fig. 6B,C). Additionally, extreme limiting dilution assays (ELDA) demonstrated that M1123 cells display higher stem cell frequencies compared to GBM1a cells consistent with their more aggressive and higher tumor propagating phenotype (Fig. 6D). Consistent with these distinct molecular features of proneural and mesenchymal GBM cells, M1123 cells also express higher amounts of mannose as visualized by FITC-GNL staining in 3D neurospheres, but not in 2D cell cultures (Fig. 6E) Accordingly, the higher levels of mannose correspond to a significantly (p<0.05) higher MANw CEST signal at 1.2 ppm, i.e., the CEST signature of OH protons abundantly present in mannose (Fig. 63F). Importantly, we observed a significant decrease in mannose content and MANw CEST signal (Fig. 6G) in M1123 neurospheres upon serum-induced differentiation (2D culture), conditions that reduce the stem cell phenotype of GBM cells. Studies have shown that serumcontaining conditions promote this transition by activating pathways associated with lineage commitment and decreased self-renewal capacity.

[0088] In vivo MANw CEST MRI of proneural and mesenchymal GBM correlates with mannose levels and CD44 expression and differs from APT CEST MRI

[0089] We assessed the feasibility of MANw CEST MRI in differentiating brain tumors derived from proneural vs. mesenchymal neurospheres in vivo. Eight week old NOD SCID gamma mice received tumor cells in the left (GBM1a) and right (M1123) striatum. MR imaging was then performed on post-implantation days 1 , 8, and 16. M1123 cells exhibited a significantly higher growth rate concurrent with aprominent MANw CEST MRI signal that was absent in the GBM1a tumors and normal brain. The MANw CEST signal in M1123 tumors remained consistently elevated (>1.8-fold) compared to GBM1a tumors and normal brain for all three time points, with a characteristic mannose peak around 1.2 ppm (Fig. 7A,B). We also analyzed the obtained results for amide proton transfer (APT) CEST MRI, a technique approved by the FDA in 2018 for its ability to identify active high-grade brain cancer by detecting endogenous proteins and peptides. This is achieved by measuring the signal at 3.6 ppm (amide protons) in addition to the 1.2 ppm signal from hydroxyl protons. APT CEST MRI did not follow a consistent pattern in detecting GBM1a and M1123 tumors throughout the study. On day 1 post-tumor implantation, in contrast to MANw CEST MRI, APT CEST MRI did not show a statistically significant difference between the two tumors. However, a significantly higher APT CEST MRI signal was observed at day 8 in M1123 cells, which could be related to elevated mobile protein content associated with tumor progression. To the contrary, the APT CEST MRI signal was significantly higher in GBM1a compared to M1123 on day 16. Immunofluorescent post-mortem staining showed an excellent agreement between the area of MANw CEST MRI contrast and the co-presence of mannose and CD44, proving that the cells with high mannose content are indeed representing mesenchymal GBM cells (Fig. 7C). Gadolinium (Gd)-enhanced perfusion MRI, performed eight days post-tumor implantation, revealed no significant differences in perfusion characteristics between GBM1a and M1123 tumors, suggesting similar vascularization and blood-brain barrier permeability. However, MANw CEST MRI can detect GBM derived from mesenchymal neurospheres in vivo and may be used in tandem with APT MRI to detect both fast- and slow-growing GBM cells.

[0090] Mannose-binding lectins LMAN1 and LMAN2 impact mannose levels in mesenchymal GBM cells and tumor propagating capacity

[0091] Our computational analyses of GBM clinical specimens identified LMAN1 and LMAN2 as potential regulators of mannose homeostasis in mesenchymal GBM cells. To test this hypothesis, we examined the effects of LMAN1 and / or LMAN2 expression inhibition on mannose levels in M1123 neurosphere cells (Fig. 8A). qRT-PCR analysis demonstrated that the siRNA oligos block their intended gene targets (Fig. 8B) concurrent with a significant reduction in mannosestaining (Fig. 8C,D). We did not observe an additive effect when we knocked-down LMAN1 and LMAN2 simultaneously, suggesting that these genes regulate the same pathway (Fig. 8D). The decrease in mannose content after specific LMAN1 and LMAN2 knock-down correlated with a decrease in MANw CEST MRI signal in vitro (Fig. 8E). To further assess if LMAN1 and LMAN2 knock-down affects the CEST MRI signal in vivo, eight-weeks old NOD-SCID gamma mice received bilateral implants of M1123 cells transfected with siRNA against LMAN1 and LMAN2 or cells transfected with a scrambled control siRNA. Both T2w MRI and MANw CEST MRI performed 8 days after cell implantation detected tumor formation by the control M1123 cells but failed to detect tumor formation by the LMAN1 / 2-KD cells (Fig. 8F,G). Taken together, these findings show that mannose is directly responsible for the MANw CEST MRI signal in mesenchymal GBM cells with LMAN1 and LMAN2 playing critical roles in regulating tumor growth.

[0092] DISCUSSION

[0093] GBM remains one of the most aggressive and treatment-resistant brain tumors, with over 90% of patients experiencing tumor recurrence within 6-9 months despite aggressive treatment. Substantial efforts have been dedicated to characterizing the molecular landscape of GBM cells to understand mechanisms driving therapy resistance. Gene expression profiling studies in GBM have highlighted epithelial-to-mesenchymal transition (EMT) as a key contributor to poor patient outcomes, as mesenchymal-like cancer cells exhibit enhanced motility, selfrenewal capacity, resistance to apoptosis and immune surveillance. Single-cell transcriptom ic studies have further reinforced the link between mesenchymal transition and therapy resistance. High-resolution single-cell transcriptom ic analyses of GBM tumors before and after standard-of-care therapy has demonstrated critical changes in the tumor microenvironment and cell state transitions associated with sternness-driving events and acquisition of mesenchymal signatures.

[0094] One such key biological transition involves glycosylation, a crucial post-translational modification regulating tumor behavior. Among various glycosylation patterns, high mannose-linked glycosylation is a distinct feature of mesenchymal cells and may play a fundamental role in therapy resistance. Differences in N-glycan modification profiles between GBM stem-like cells indicate an enrichment of high-mannose-type N-glycans in aggressive glioblastomasubpopulations. This enhanced glycosylation is linked to the metabolic reprogramming of mesenchymal GBM, where elevated glycolysis sustains tumor progression and sternness. Consequently, high-mannose glycosylation has been proposed as a biomarker for aggressive GBM subtypes, underscoring the need for non-invasive imaging approaches capable of detecting these molecular alterations in vivo. Consistent with these observations, we show here that mesenchymal GBM cells express higher levels of genes involved in mannose regulation (Fig. 4) and mesenchymal GBM neurospheres display significantly higher MANw CEST signal then their proneural counterparts (Figs. 6,7). Importantly, these correlations were maintained in tissues resected from GBM patients (Fig. 5).

[0095] The currently available MR imaging sequences provide an overall satisfactory means of grading brain tumors, however they face inherent limitations in detecting and characterizing aggressive GBM subtypes. While conventional T1- weighted, T2-weighted, and contrast-enhanced imaging enable tumor localization, they lack specificity in differentiating tumor subtypes and assessing microenvironmental changes. Advanced techniques such as diffusion-weighted imaging (DWI) and perfusion imaging contribute to glioma grading by measuring water diffusion and vascularity, respectively, while magnetic resonance spectroscopy (MRS) further aids in tumor characterization by detecting metabolic markers such as choline and lactate. Functional MRI (fMRI) may also be used for preoperative planning, mapping eloquent brain regions to minimize surgical impact. However, these modalities do not capture any of the molecular features linked to tumor aggressiveness. APT CEST MRI, an FDA-approved molecular imaging technique, has emerged as a promising tool for assessing protein content and cellular proliferation in gliomas. APT CEST MRI detects mobile proteins and peptides, offering a label-free method for tumor grading. Despite its utility in distinguishing glioma grades, APT CEST MRI primarily reflects protein content without providing insights into specific metabolic pathways or tumor subtypes.

[0096] Like most cancers, GBM undergoes metabolic reprogramming to support the oncogenic phenotypes of survival, proliferation, and invasion. Aberrant glycosylation is a notable consequence of this metabolic rewiring, however, our understanding of the role of glycosylation in GBM pathology remains inadequate. We have shown here that MANw CEST MRI can distinguish between tumor cell subsetsboth in vitro and in vivo based on their differential mannose content (Fig. 6, 7). We also show that blocking expression of mannose-binding lectins significantly reduces MANw CEST MRI signal in vitro and in vivo (Fig. 8), demonstrating that mannose is directly responsible for MANw CEST MRI signal in mesenchymal GBM cells, proving causation of CEST MRI signal and GBM phenotype instead of a mere correlation. Along with APT CEST MRI, which captures broad protein content using in grading gliomas, MANw CEST MRI offers a more specific assessment of tumor aggressiveness by detecting high-mannose glycosylation in mesenchymal subtypes of GBM. Integrating MANw CEST MRI with the currently available MRI techniques including APT could enable a more comprehensive evaluation of GBM, improving detection of aggressive mesenchymal tumors, refining treatment stratification, and enhancing the monitoring of therapeutic responses. Once translated, this advancement may decrease the time interval between diagnosis and treatment, hopefully increasing patient survival. Since brain tumor patients already undergo routine MRI our findings are expected to be easily implemented with current clinical imaging protocols to advance GBM diagnosis.

[0097] Some further aspects are also defined in the following clauses:

[0098] Clause 1 : A method of detecting an aggression level of a glioblastoma in a subject, the method comprising: imaging the subject having the glioblastoma using a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test magnetic resonance (MR) image of the subject; and, identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1.8-fold difference, thereby detecting the aggression level of the glioblastoma in the subject.

[0099] Clause 2: The method of Clause 1 , wherein the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique.

[0100] Clause 3: The method of Clause 1 or Clause 2, wherein the brain cells in the control MR image comprise non-diseased brain cells.

[0101] Clause 4: The method of any of Clauses 1-3, wherein the cancer stem cells comprise mesenchymal stem cells.

[0102] Clause 5: The method of any of Clauses 1-4, wherein the cancer stem cells comprise mesenchymal stromal cells.

[0103] Clause 6: The method of any of Clauses 1-5, wherein the brain cells in the control MR images are from the subject.

[0104] Clause 7: The method of any of Clauses 1-6, wherein the brain cells in the control MR images are from a reference subject.

[0105] Clause 8: The method of any of Clauses 1-7, further comprising generating a therapy recommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject.

[0106] Clause 9: The method of any of Clauses 1-8, further comprising administering at least one therapy that is effective against glioblastomas with the detected aggression level of the glioblastoma in the subject.

[0107] Clause 10: The method of any of Clauses 1-9, further comprising repeating the imaging and identifying steps at a subsequent time point to monitor the aggression level of the glioblastoma in the subject over time.

[0108] Clause 11 : A method of detecting a glioblastoma with a high aggression level in a subject, the method comprising: imaging the subject having the glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject; and, identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, thereby detecting the glioblastoma with the high aggression level in the subject.

[0109] Clause 12: The method of Clause 11 , wherein the overexpression level of mannose in and / or on the surfaces of the unlabeled mesenchymal stem cellsof the glioblastoma of the subject comprises at least about a 1.8-fold difference relative to the identified expression level of mannose in and / or on the surfaces of the non-diseased brain cells in the control MR image.

[0110] Clause 13: A system, comprising: an MRI scanner configured to generate CEST MR images; and, a controller operably connected to the MR scanner, which controller comprises, or is capable of accessing, computer readable media comprising non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: imaging a subject having a glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject using the MR scanner; and identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1 .8-fold difference.

[0111] Clause 14: The system of Clause 13, wherein the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique.

[0112] Clause 15: The system of Clause 13 or Clause 14, wherein the brain cells in the control MR image comprise non-diseased brain cells.

[0113] Clause 16: The system of any of Clauses 13-15, wherein the cancer stem cells comprise mesenchymal stem cells.

[0114] Clause 17: The system of any of Clauses 13-16, wherein the cancer stem cells comprise mesenchymal stromal cells.

[0115] Clause 18: The system of any of Clauses 13-17, wherein the brain cells in the control MR images are from the subject.

[0116] Clause 19: The system of any of Clauses 13-18, wherein the brain cells in the control MR images are from a reference subject.

[0117] Clause 20: The system of any of Clauses 13-19, wherein the instructions further perform at least: generating a therapy recommendation report forthe subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject.

[0118] Clause 21 : A computer readable media comprising non-transitory computer executable instructions which, when executed by at least electronic processor, perform at least: imaging a subject having a glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject using an MR scanner; and identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1.8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1 .8-fold difference.

[0119] Clause 22: The computer readable media of Clause 21 , wherein the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique.

[0120] Clause 23: The computer readable media of Clause 21 or Clause 22, wherein the brain cells in the control MR image comprise non-diseased brain cells.

[0121] Clause 24: The computer readable media of any of Clauses 21-23, wherein the brain cells in the control MR image comprise non-diseased brain cells.

[0122] Clause 25: The computer readable media of any of Clauses 21-24, wherein the cancer stem cells comprise mesenchymal stem cells.

[0123] Clause 26: The computer readable media of any of Clauses 21-25, wherein the cancer stem cells comprise mesenchymal stromal cells.

[0124] Clause 27: The computer readable media of any of Clauses 21-26, wherein the brain cells in the control MR images are from the subject.

[0125] Clause 28: The computer readable media of any of Clauses 21-27, wherein the brain cells in the control MR images are from a reference subject.

[0126] Clause 29: The computer readable media of any of Clauses 21-28, wherein the instructions further perform at least: generating a therapyrecommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject.

[0127] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the methods, devices, systems, computer readable media, and / or component parts or other aspects thereof can be used in various combinations. All patents, patent applications, websites, other publications or documents, and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.

Claims

WHAT IS CLAIMED IS:1 . A method of detecting an aggression level of a glioblastoma in a subject, the method comprising: imaging the subject having the glioblastoma using a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test magnetic resonance (MR) image of the subject; and, identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1 .8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1.8-fold difference, thereby detecting the aggression level of the glioblastoma in the subject.

2. The method of claim 1 , wherein the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique.

3. The method of claim 1 , wherein the brain cells in the control MR image comprise non-diseased brain cells.The method of claim 1 , wherein the cancer stem cells comprise mesenchymal stem cells.

5. The method of claim 1 , wherein the cancer stem cells comprise mesenchymal stromal cells.

6. The method of claim 1 , wherein the brain cells in the control MR images are from the subject.

7. The method of claim 1 , wherein the brain cells in the control MR images are from a reference subject.

8. The method of claim 1 , further comprising generating a therapy recommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject.

9. The method of claim 1 , further comprising administering at least one therapy that is effective against glioblastomas with the detected aggression level of the glioblastoma in the subject.

10. The method of claim 1 , further comprising repeating the imaging and identifying steps at a subsequent time point to monitor the aggression level of the glioblastoma in the subject over time.

11. A method of detecting a glioblastoma with a high aggression level in a subject, the method comprising: imaging the subject having the glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject; and, identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, thereby detecting the glioblastoma with the high aggression level in the subject.

12. The method of claim 11 , wherein the overexpression level of mannose in and / or on the surfaces of the unlabeled mesenchymal stem cells of the glioblastoma of the subject comprises at least about a 1.8-fold difference relative to the identified expression level of mannose in and / or on the surfaces of the nondiseased brain cells in the control MR image.

13. A system, comprising: an MRI scanner configured to generate CEST MR images; and, a controller operably connected to the MR scanner, which controller comprises, or is capable of accessing, computer readable media comprising non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: imaging a subject having a glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject using the MR scanner; and identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1 .8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1 .8-fold difference.

14. The system of claim 13, wherein the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique.

15. The system of claim 13, wherein the brain cells in the control MR image comprise non-diseased brain cells.

16. The system of claim 13, wherein the cancer stem cells comprise mesenchymal stem cells.

17. The system of claim 13, wherein the cancer stem cells comprise mesenchymal stromal cells.

18. The system of claim 13, wherein the brain cells in the control MR images are from the subject.

19. The system of claim 13, wherein the brain cells in the control MR images are from a reference subject.

20. The system of claim 13, wherein the instructions further perform at least:generating a therapy recommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject.21 . A computer readable media comprising non-transitory computer executable instructions which, when executed by at least electronic processor, perform at least: imaging a subject having a glioblastoma using a CEST MRI technique in the absence of introducing an exogenous MRI contrast agent into the subject to produce a test MR image of the subject using an MR scanner; and identifying an overexpression level of mannose in and / or on surfaces of unlabeled cancer stem cells of the glioblastoma of the subject in the test MR image relative to an identified expression level of mannose in and / or on surfaces of brain cells in a control MR image, wherein a high aggression level is indicated when the overexpression level comprises at least about a 1 .8-fold difference and wherein a low aggression level is indicated when the overexpression level comprises less than about a 1 .8-fold difference.

22. The computer readable media of claim 21 , wherein the CEST MRI technique comprises a mannose-weighted (MANw) CEST MRI technique.

23. The computer readable media of claim 21 , wherein the brain cells in the control MR image comprise non-diseased brain cells.

24. The computer readable media of claim 21 , wherein the brain cells in the control MR image comprise non-diseased brain cells.

25. The computer readable media of claim 21 , wherein the cancer stem cells comprise mesenchymal stem cells.

26. The computer readable media of claim 21 , wherein the cancer stem cells comprise mesenchymal stromal cells.

27. The computer readable media of claim 21 , wherein the brain cells in the control MR images are from the subject.

28. The computer readable media of claim 21 , wherein the brain cells in the control MR images are from a reference subject.

29. The computer readable media of claim 21 , wherein the instructions further perform at least: generating a therapy recommendation report for the subject and / or a healthcare provider based, at least in part, on the detected aggression level of the glioblastoma in the subject.

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