Determination of spatial distributions of pharmaceuticals in tissue samples via mass spectrometry imaging of metal isotopes

Abstract

Systems and methods are disclosed for determining the spatial distributions of radionuclides in tissue samples via mass spectrometry imaging. After administration of a radiopharmaceutical that contains a radionuclide, a mass spectrometry imaging system, such as an imaging mass cytometer or a multiplexed ion beam imaging system, is employed to interrogate a tissue sample to obtain a dataset. The radiopharmaceutical includes, or generates via radioactive decay of the radionuclide, a metal isotope detectable by the mass spectrometry imaging system. The dataset is processed to determine a spatial distribution of the metal isotope within the tissue sample, thereby characterizing the spatial distribution of the radionuclide delivered within the tissue sample. The tissue sample may be stained with a biomarker reagent that includes an additional metal isotope attached to a biomarker ligand, enabling the spatially multiplexed and co-registered analysis. The present methods can be adapted to infer and assess localization of non-radioactive pharmaceuticals.

Description

DETERMINATION OF SPATIAL DISTRIBUTIONS OF PHARMACEUTICALS IN TISSUE SAMPLES VIA MASS SPECTROMETRY IMAGING OF METAL ISOTOPESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 635,544, titled “DETERMINATION OF SPATIAL DISTRIBUTIONS OF RADIONUCLIDES IN TISSUE SAMPLES VIA MASS SPECTROMETRY IMAGING OF METAL ISOTOPES” and filed on April 17, 2024, which is incorporated herein by reference in its entirety, and to Canadian Patent Application No. 3,235,517, titled “DETERMINATION OF SPATIAL DISTRIBUTIONS OF RADIONUCLIDES IN TISSUE SAMPLES VIA MASS SPECTROMETRY IMAGING OF METAL ISOTOPES” and filed on April 17, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] The present disclosure relates to the field of radiopharmaceutical-based treatment and diagnostic imaging. The present disclosure also relates to the field of mass spectrometry imaging, such as imaging mass cytometry and multiplexed ion beam imaging.

[0003] Targeted radionuclide therapy (TRT) combines cyto-lethal radioisotopes, including radiometals such as lutetium-177, yttrium-90, and actinium-225, with molecules that target tumor-selective biomarkers for cancer treatment^]. TRT represents an excellent treatment option for many forms of cancer[4] including highly aggressive cancers refractory to multiple other forms of treatment^]. Precision TRT aims to deliver radionuclides to specific tumor antigens to interrupt or eliminate crucial tumorigenic processes and subpopulations, yet techniques to demonstrate localization of TRT to intratumoral cell subpopulations and techniques to assess the microenvironmental response of target tissue are limited.

[0004] In the preclinical setting, efforts to visualize the distribution of TRT in tumors and its biological effects can be conducted using a combination of phosphor imaging (autoradiography) and immunohistochemistry (IHC) [6], However, the resolution provided by conventional phosphor-based autoradiography is extremely limited (mm-resolution) relative to the cellular-level resolution provided byconventional immunohistochemistry visualized using digital microscopes [7], Additionally, most tissue processing services are not equipped to handle radioactive tissues and most radiopharmaceutical development teams are unequipped to embed and stain tissues for IHC.

[0005] Radioluminescence microscopy (RLM) is an emerging technique that drastically improves on the resolution provided by phosphor autoradiography to image radionuclides on single cells in suspension, and has an estimated resolution of 20-25 pM, approaching single-cell level resolution for tissue imaging [8], RLM specializes in imaging p- particles and enables imaging live aqueous systems for pharmacokinetic studies, but its applications outside of p' particle imaging and ease of pairing with IHC are limited.

[0006] Microautoradiography implements thin photographic emulsions to achieve excellent resolution but can be limited by low sensitivity and requires separate tissue sections if IHC analysis is desired, which limits the ability to co-register the images provided by each technique at a single-cell level [9],

[0007] Despite many advances in imaging radioactivity distribution within tissues with high resolution [10,11], a single-cell level resolution technique to multiplex radionuclide distribution within tissues, antigens targeted by precision TRT, antigens for tumor phenotyping, and antigens for microenvironmental response has not yet been realized. As a consequence of the limited capabilities of conventional radiobiological techniques to visualize radiation and radiation response at cellular- level resolution, the potential utility of therapeutic effects induced by targeted radionuclide therapies directed to tumor subpopulations or the tumor microenvironment (e.g., cancer stem cells, bystander effect, abscopal effect, etc.) has not yet fully been realized.SUMMARY

[0008] Systems and methods are disclosed for determining the spatial distributions of radionuclides in tissue samples via mass spectrometry imaging. After administration of a radiopharmaceutical that contains a radionuclide, a mass spectrometry imaging system, such as an imaging mass cytometer or a multiplexed ion beam imaging system, is employed to interrogate a tissue sample to obtain a dataset. The radiopharmaceutical includes, or generates via radioactive decay of the radionuclide, a metal isotope detectable by the mass spectrometry imaging system.The dataset is processed to determine a spatial distribution of the metal isotope within the tissue sample, thereby characterizing the spatial distribution of the radionuclide delivered within the tissue sample. The tissue sample may be stained with a biomarker reagent that includes an additional metal isotope attached to a biomarker ligand, enabling the spatially multiplexed and co-registered analysis. The present methods can be adapted to infer and assess localization of non-radioactive pharmaceuticals.

[0009] Accordingly, in a first aspect, there is provided a method of characterizing a spatial distribution of a therapeutic radiopharmaceutical within a sample via mass spectrometry imaging, the method comprising: employing a mass spectrometry imaging system to interrogate the sample to obtain a mass spectrometry imaging dataset, the sample having been obtained from a subject having been administered the therapeutic radiopharmaceutical comprising a radionuclide, wherein the therapeutic radiopharmaceutical comprises, or generates via radioactive decay of the radionuclide, a metal isotope detectable by the mass spectrometry imaging system; and processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal isotope within the sample.

[0010] In some example implementations, the method further comprises associating the spatial distribution of the metal isotope within the sample with the spatial distribution of the radionuclide delivered within the sample.

[0011] In some example implementations, the method further comprises generating an image showing the spatial distribution of the metal isotope within the sample.

[0012] In some example implementations of the method, the therapeutic radiopharmaceutical comprises a radioligand, the radioligand comprising the radionuclide and a targeting ligand configured to specifically bind to a target.

[0013] In some example implementations of the method, the metal isotope is a carrier isotope labelling the targeting ligand, the carrier isotope having been employed during production of the radionuclide.

[0014] In some example implementations of the method, the metal isotope is a decay product of the radionuclide. The decay product may be a metastable metal isotope.

[0015] In some example implementations of the method, the metal isotope is the radionuclide. The metal isotope may be a stable metal isotope labelling the targeting ligand. The stable metal isotope may not be associated with production of the radionuclide.

[0016] In some example implementations of the method, the therapeutic radiopharmaceutical comprises both the metal isotope and the radionuclide, wherein the metal isotope and the radionuclide are bound to a non-specific carrier molecule.

[0017] In some example implementations of the method, the metal isotope is a first metal isotope, and wherein the therapeutic radiopharmaceutical further comprises a second metal isotope that is different from the first metal isotope, the second metal isotope being detectable by the mass spectrometry imaging system, and wherein the mass spectrometry imaging dataset is processed to determine a spatial distribution of the second metal isotope within the sample. The method may further include employing both the spatial distribution of the first metal isotope within the sample and the spatial distribution of the second metal isotope within the sample to infer a spatial distribution of the radionuclide delivered within the sample.

[0018] The therapeutic radiopharmaceutical may include a radioligand, the radioligand comprising a targeting ligand labeled with the radionuclide, wherein the targeting ligand is configured to specifically bind to a target. The first metal isotope may be a first carrier isotope labelling the targeting ligand, and the second metal isotope may be a second carrier isotope labelling the targeting ligand, the first carrier isotope and the second carrier isotope having been employed during production of the radionuclide.

[0019] In some example implementations of the method, the first metal isotope is a carrier isotope labelling the targeting ligand, and the second metal isotope is a decay product of the radionuclide, the carrier isotope having been employed during production of the radionuclide. In some example implementations of the method, the first metal isotope is a carrier isotope labelling the targeting ligand, and the second metal isotope is the radionuclide, the carrier isotope having been employed during production of the radionuclide. In some example implementations of the method, the first metal isotope is a carrier isotope labelling the targeting ligand, and the second metal isotope is a stable metal isotope labeling the radionuclide, the carrier isotope having been employed during production of the radionuclide, wherein the stable metal isotope is not associated with production of the radionuclide.

[0020] In some example implementations of the method, the sample has been stained with a metal-tagged reagent, the metal-tagged reagent comprising an additional metal isotope conjugated to a biomarker ligand, the additional metal isotope being detectable by the mass spectrometry imaging system, the method further comprising: processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal-tagged reagent within the sample. The method may further include generating a multiplexed image showing the spatial distribution of the metal isotope and the spatial distribution of the metal-tagged reagent within the sample. The method may further include generating a spatially resolved multidimensional measurement characterizing the spatial distribution of the metal isotope and the spatial distribution of the metal-tagged reagent within the sample.

[0021] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a type of cell specifically targeted by the therapeutic radiopharmaceutical. The biomarker ligand may be configured to specifically bind to cancer stem cells. The biomarker ligand may be configured to specifically bind to a type of immune cell.

[0022] In some example implementations, the method further comprises processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing co-localization of the radionuclide and the type of cell specifically targeted by the therapeutic radiopharmaceutical.

[0023] In some example implementations, the method further comprises processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing off-target delivery of the radionuclide.

[0024] In some example implementations of the method, the type of call targeted by the therapeutic radiopharmaceutical is known to reside in a first tissue region that is associated with pathology, and the method further comprises: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing on-target delivery of the radionuclide within a second tissue region. The second tissue region may be a healthy tissue region.

[0025] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a selected subcellular structure.

[0026] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a marker of microenvironmental cellular response to the therapeutic radiopharmaceutical. The method may further include processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing microenvironmental cellular response to therapeutic radiopharmaceutical.

[0027] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a marker of cellular apoptosis. The biomarker ligand may be configured to specifically bind to a marker of pathway activation. The biomarker ligand may be configured to specifically bind to a marker associated with a resistance mechanism.

[0028] In some example implementations of the method, the therapeutic radiopharmaceutical comprises a metal nanoparticle formed from the metal isotope, the radionuclide being bound to the nanoparticle.

[0029] In some example implementations of the method, the mass spectrometry imaging system is an imaging mass cytometry system.

[0030] In some example implementations of the method, the mass spectrometry imaging system is a multiplexed ion beam imaging system.

[0031] In another aspect, there is provided a method of characterizing a spatial distribution of a tracer radiopharmaceutical within a sample via mass spectrometry imaging, the method comprising: employing a mass spectrometry imaging system to interrogate the sample to obtain a mass spectrometry imaging dataset, the sample having been obtained from a subject having been administered the tracer radiopharmaceutical comprising a radionuclide, wherein the tracer radiopharmaceutical comprises, or generates via radioactive decay of the radionuclide, a metal isotope detectable by the mass spectrometry imaging system; and processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal isotope within the sample.

[0032] In another aspect, there is provided a method of characterizing a spatial distribution of a pharmaceutical within a sample via mass spectrometry imaging, the method comprising: employing a mass spectrometry imaging system to interrogate the sample to obtain a mass spectrometry imaging dataset, the sample having been obtainedfrom a subject having been administered the pharmaceutical, wherein the pharmaceutical is non-radioactive and further comprises a passive, non-therapeutic metal isotope detectable by the mass spectrometry imaging system; and processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal isotope within the sample.

[0033] The pharmaceutical may comprise a therapeutic payload that is absent of a metal. The pharmaceutical may comprise a therapeutic payload that includes a metal that is different from the non-therapeutic metal isotope. The pharmaceutical may be a diagnostic or theranostic reagent labeled with the passive, non- therapeutic metal isotope.

[0034] In some example implementations of the method, the biomarker ligand is configured to specifically bind to cancer stem cells.

[0035] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a type of immune cell.

[0036] In some example implementations, the method further comprises processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing co-localization of the pharmaceutical and the type of cell specifically targeted by the pharmaceutical.

[0037] In some example implementations, the method further comprises processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing off-target delivery of the pharmaceutical.

[0038] In some example implementations of the method, the type of call targeted by the pharmaceutical is known to reside in a first tissue region that is associated with pathology, the method further comprising processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing on-target delivery of the pharmaceutical within a second tissue region. The second tissue region may be a healthy tissue region.

[0039] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a selected subcellular structure.

[0040] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a marker of microenvironmental cellular response to the pharmaceutical.

[0041] In some example implementations, the method further comprises processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing microenvironmental cellular response to pharmaceutical.

[0042] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a marker of cellular apoptosis.

[0043] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a marker of pathway activation.

[0044] In some example implementations of the method, the biomarker ligand is configured to specifically bind to a marker associated with a resistance mechanism.

[0045] In some example implementations of the method, the mass spectrometry imaging system is an imaging mass cytometry system.

[0046] In some example implementations of the method, the mass spectrometry imaging system is a multiplexed ion beam imaging system.

[0047] A further understanding of the functional and advantageous aspects of the disclosure can be realized by reference to the following detailed description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Embodiments are described with reference to the accompanying drawings. In the drawings, like reference numbers can indicate identical or functionally similar elements.

[0049] FIG. 1 schematically illustrates an example imaging mass cytometry system for performing methods disclosed herein.

[0050] FIGS. 2A, 2B and 2C present a sensitivity comparison between phosphor autoradiography and IMC. FIG. 2A shows serial dilutions of [177Lu]LuCl3 spotted on an agarose-coated microscope slide and measured via phosphor imaging and imaging CyTOF (cytometry by time of flight). In FIG. 2B, data is presented as the mean pixel intensity (log ) in a 0.075 cm2circular ROIs drawn using imaged, while in FIG. 2C, Lu-176 is measured through IMC.

[0051] FIG. 3 demonstrates imaging mass cytometry (IMC)-based detection of potential isotopes introduced to tumors by Lu-177 targeted radionuclide therapy. IMC was performed on specimens from tumors treated with 3.7 MBq [177Lu]Lu-DOTA- RW03 (top row) and 14.8 MBq [177Lu]Lu-DOTA-RW03 (bottom row). Channels assessed include (panels a & d) Hf-177, (panels b & e) Lu-175, and (panels c & f) Lu-176. Signal intensity is in-line with the isotopic abundance of the carrier lutetium used to generate Lu-177 (<1 % Hf-177, 16% Lu-175, 83% Lu-176) and the injected dose. Hafnium-177 (stable daughter isotope of Lu-177) signal was not observed above background levels. FOV = 770 pm.

[0052] FIG. 4 demonstrates visualization of DNA, Lu-176, and actin by multiplexed IMC in (panel a) untreated HT-29 tumors and (panel b) tumors treated with 14.8 MBq [177Lu]Lu-DOTA-RW03. FOV = 770 pm.

[0053] FIG. 5 shows gross morphology of CD133 IMC (panel a) and CD133 IHC (panel b) and high magnification images of CD133 IMC (panel a-i) and CD133 IHC (panel b-i). FOV = (panel a & b) 700 pm, (panel a-i & b-i) 900 pm.

[0054] FIG. 6 shows colocalization of Lu 176 to CD133 expressing cells as visualized by IMC. In untreated HT-29 tumors (panels a-d) and HT-29 tumors treated with 14.8 MBq [177Lu]Lu-DOTA-RW03 (panels e-h), Lu176 signal (panels a,e), CD133 expression (panels b,f), and CC3 (cleaved caspase 3) expression (panels c,g) were detected by IMC. Panel g shows CD133 and Lu176 signal colocalized in regions denoted by arrows. FOV = 770 pm.

[0055] FIG. 7 shows IMC-based visualization of microenvironmental markers for biological response to treatment. Untreated HT-29 tumors (panels a-c) and HT-29 tumors excised 14 d into [177Lu]Lu-DOTA-RW03 therapy (panels d-f) were stained and acquired to visual Lu176 signal (a,d), p-yH2AX expression (panels b,e), and cleaved caspase 3 expression (panels c,f). FOV = 1.7 mm.

[0056] FIGS. 8A and 8B show IMC visualization of tissue architecture (low magnification, FIG. 8A) and single-cells (FIG. 8B) to include the antigenic target (CD133; panel i), carrier isotopes introduced by TRT (Lu176; panel ii), and antigens to differentiate cells of various tissue origins and phenotypes (DNA, panel iv; PanCK, panel v; and CD49b, panel vi). Arrows indicate Lu176 and CD133 overlap in panel Hi..

[0057] FIGS. 9A, 9B, 9C, 9D and 9E compare immunohistochemical staining and CyTOF to assess biological response to treatment. Samples prepared for IHC using different methods produce poorly co-registered adjacent slides leading to (FIG. 9A) catastrophic damage in some samples. The resolution provided by phosphor autoradiography (FIG. 9C) is unsuited to co-localizing with IHC stains and to assess radiation distribution in tumors. At higher magnifications (FIG. 9B) IHC coregistration worsens in identically prepared samples and conventional autoradiography (FIG. 9D) is unusable for co-registering radionuclides with cell-level biology. A single slide assessed using imaging CyTOF (FIG. 9E) reveals multiple biology markers and contaminant radioisotopes delivered by TRT at cellular resolutions. FOV = (FIG. 9E) 1000 pm.DETAILED DESCRIPTION

[0058] Various embodiments and aspects of the disclosure will be described with reference to details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well- known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure.

[0059] As used herein, the terms “comprises” and “comprising” are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms “comprises” and “comprising” and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0060] As used herein, the term “exemplary” means “serving as an example, instance, or illustration,” and should not be construed as preferred or advantageous over other configurations disclosed herein.

[0061] As used herein, the terms “about” and “approximately” are meant to cover variations that may exist in the upper and lower limits of the ranges of values, such as variations in properties, parameters, and dimensions. Unless otherwise specified, the terms “about” and “approximately” mean plus or minus 25 percent or less.

[0062] It is to be understood that unless otherwise specified, any specified range or group is as a shorthand way of referring to each and every member of a range or group individually, as well as each and every possible sub-range or sub-group encompassed therein and similarly with respect to any sub-ranges or sub-groups therein. Unless otherwise specified, the present disclosure relates to and explicitly incorporates each and every specific member and combination of sub-ranges or subgroups.

[0063] As used herein, the term "on the order of", when used in conjunction with a quantity or parameter, refers to a range spanning approximately one tenth to ten times the stated quantity or parameter.

[0064] Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood to one of ordinary skill in the art. Unless otherwise indicated, such as through context, as used herein, the following terms are intended to have the following meanings:

[0065] As used herein, the term “mass spectrometry imaging” refers to systems and methods that can employ mass spectrometry for the spatially-resolved detection of non-endogenous mass elements, such as metal isotopes, liberated from a sample in a spatially-resolved manner. Non-limiting examples of mass spectrometry imaging include imaging mass cytometry and multiplexed ion beam imaging.

[0066] As used herein, the term “sample” refers to a biological sample having a defined spatial extent suitable for analysis via metal-tag-based mass spectrometry imaging. In some example implementations, a biological sample includes cellular material, such as a tissue section, a cell monolayer, or a cell preparation (e.g. “a tissue sample”). A sample may be a thinly sectioned biological tissue, for example, from 10 nm to 1 mm in thickness, orfor example, from 1 micrometers to 100 micrometers in thickness, or, for example, from 1 millimeter to 10 mm in thickness. In other example implementations, a sample may be an un-sectioned tissue sample. A tissue sample may, in some example implementations, be embedded in a material, such as, for example, paraffin. In some cases, a sample is a collection of cells. In some example implementations, a sample is one or more selected cells from a group of cells.

[0067] As used herein, the term “radiopharmaceutical” refers to a therapeutic or non-therapeutic pharmaceutical composition that can be administered to a subject, and which includes a radionuclide. A radiopharmaceutical may be a targetingradiopharmaceutical, such as a radioligand that includes a targeting ligand configured to selectively bind to a target, or may be a non-targeting radiopharmaceutical that is absent of a targeting ligand.

[0068] As used herein, the term “metal isotope” refers to a metal isotope having a mass that is distinguishable via mass spectrometry imaging relative to elemental species present in a normal tissue sample. Non-limiting examples of metal isotopes include transition metals and isotopes thereof, such as lanthanides, and isotopes thereof.

[0069] As used herein, the term “targeting ligand”, when used in reference to a radioligand employed for targeted radionuclide therapy, is component, such as a molecule, that is configured to selectively bind to a target to facilitate the targeted delivery of a radionuclide to the target. Non-limiting examples of targeting ligands include antibodies, antibody fragments (such as, for example, a Fab fragment, scFV, a nanobody, minibody, diabobdy or other fragment), engineered proteins such as affibodies and DARPins, peptides, aptamers, miniproteins (natural or engineered), and small molecules.

[0070] As used herein, the term “biomarker ligand”, when used in reference to a metal-tagged biomarker reagent having a metal tag detectable by mass spectrometry imaging, is a component, such as a molecule, that is configured to selectively bind to a biomarker to facilitate imaging of the biomarker via the detection of the metal tag. Non-limiting examples of biomarker ligands include antibodies, antibody fragments (such as, for example, a Fab fragment, scFV, a nanobody, minibody, diabobdy or other fragment), engineered proteins such as affibodies and DARPins, miniproteins (natural or engineered), peptides, aptamers, and small molecules.

[0071] As used herein, the term “radioligand” refers to a radionuclide bound (e.g. attached, bonded, conjugated) to a targeting ligand.

[0072] As used herein, the term “carrier isotope” refers to a metal isotope that is associated with production of a radionuclide, and which is incorporated into a radiopharmaceutical with the radionuclide.

[0073] As used herein, the phrases “carrier free” and “no-carrier-added” refer to radiopharmaceuticals that are absent of carrier isotopes.

[0074] The present inventors, seeking to address the aforementioned problems involving the inability to achieve single-cell level resolution imaging of radionuclidedistributions within tissue samples, realized that this problem could be solved with the use of imaging mass cytometry (IMC) and other mass spectrometry imaging modalities, such as multiplexed ion beam imaging.

[0075] Mass spectrometry imaging methods that involve mass labels, such as imaging mass cytometry and multiplexed ion beam imaging, are performed by ablating a material from a biological sample, such as a tissue sample, and analyzing the ablated material by mass spectrometry. The typical workflow for mass spectrometry imaging involves, prior to ablation, staining of the sample with reagents that include specific ligands, such as antibodies, labeled with distinct metal isotopes (elemental labels), which are easily differentiated by mass spectrometry. Examples of metal isotope tags that are commonly employed in mass spectrometry imaging and attached (e.g. conjugated) to a given ligand (e.g. antibody) include Gd, Nd, Tb, Eu, Gd, Dy, Ho, Sm, Er, Yb, among many others.

[0076] By recording the location of where ablation was performed on the sample and the quantity of metal isotope detected by a mass spectrometer, an image of the spatial distribution of the target in the sample can be measured. The mass spectrometer can detect many different metal isotope tags simultaneously, enabling highly multiplexed imaging to be performed. While antibodies are typically employed as the biomarker ligand when performing mass spectrometry imaging, other types of ligands (selective molecules) can be employed to provide selective recognition, such as peptides, aptamers, and small molecules. Mass spectrometry imaging enables the simultaneous and multiplexed detection of multiple parameters or biomarkers with single cell resolution.

[0077] An imaging mass cytometer generally includes three main components or subsystems: a laser ablation subsystem, an ionization subsystem, and a mass spectrometer. The laser ablation subsystem, which includes a scanning subsystem for scanning the focused laser beam relative to the sample, is employed to generate an ablation (aerosol) plume from the sample. Examples of suitable lasers include solid state lasers, fiber lasers, and often with an excitation wavelength in the ultraviolet. The laser ablation subsystem, and associated scanning mechanism (e.g. a galvo scanning system including motorized scanning mirrors, or motorized linear translation stage-based scanning, and / or acousto-optic-based scanning systems) employ rapid acquisition of distinct ablation plumes as the laser beam is scanned across the sample. For example, an imaging mass spectrometer that employs alaser ablation ICP-MS system may ablate spots on a micron scale, thereby enabling the detection of the elemental or isotopic composition among millions of spots across a single sample.

[0078] The ionization system ionizes the atoms in the ablation plume to form elemental ions, thereby enabling their detection by the mass spectrometer based on their mass / charge ratio. An example of a suitable ionization subsystem is inductively coupled plasma (ICP). A transfer conduit is typically provided between the laser ablation subsystem and the ionization subsystem to capture the ablated plume and transfer the captured ablated plume to the ionization subsystem. The ions produced by the ionization system are then passed into the mass spectrometer, which may be, for example, a time of flight (TOF) or a magnetic sector mass spectrometer.

[0079] Another mass spectrometry imaging modality is multiplexed ion beam imaging (Ml Bl), which also employs a mass spectrometer for metal isotope mass analysis, but differs from an imaging mass cytometer in that ablation of the sample is performed using an ion beam that is scanned across the sample, as opposed to a laser beam, and where secondary-ion mass spectrometry (SIMS) is employed to detect the metal tags. The primary ion beam sputters the metal isotope tags and tissue-endogenous elements, releasing secondary ions that are quantified through mass spectrometry (e.g. a TOF mass spectrometer).

[0080] In some example implementations, electrospray ionization (DESI) may be employed for sample introduction and ionization instead of laser ablation. In other example implementations, a MALDI-TOF system may be adapted for metal isotope detection and employed to perform the methods described herein.Mass spectrometry imaging for Detection Spatial Distribution of Metal Isotopes Delivered through Radionuclide Therapy

[0081] The present inventors envisioned that the capability of imaging mass cytometry and other mass spectrometry imaging modalities to provide antigen- multiplexed images of tumors on a single slide using metal-isotope labeled antibodies could be adapted to facilitate the visualization of the spatial distribution of radionuclides administered to a subject. The present inventors reasoned that if a radiopharmaceutical that was administered to a subject included a metal isotope, or generated a metal isotope via radioactive decay, then mass spectrometry imaging (e.g. imaging mass cytometry or multiplexed ion beam imaging) could be employedto detect, in a tissue sample obtained from the subject, the spatial distribution of the metal isotope within the tissue sample, which would in turn facilitate, for example, an assessment, inference and / or visualization of the spatial distribution of the radiopharmaceutical with subcellular resolution. Moreover, the present inventors realized that in such cases, mass spectrometry imaging could also be employed to spatially map, on a subcellular level, biomarkers such as target antigen biomarkers, tumor phenotyping biomarkers, and / or biomarkers associated with the local microenvironmental response, to obtain multiplexed image data that is co-registered and show both the spatial distribution of the radiopharmaceutical and the spatial distribution of the biomarkers within the tissue sample.

[0082] Accordingly, in various example embodiments of the present disclosure, a mass spectrometry imaging system is employed to interrogate a tissue sample obtained from a subject having been administered a therapeutic radiopharmaceutical that includes a radionuclide. The therapeutic radiopharmaceutical includes, or generates via radioactive decay of the radionuclide, a metal isotope detectable by the mass spectrometry imaging system. The dataset obtained from the mass spectrometry imaging system is processed to determine a spatial distribution of the metal isotope within the tissue sample, thereby facilitating an assessment, inference and / or visualization of the spatial distribution of the radionuclide within the tissue sample. Furthermore, when the tissue sample is stained with metal-tagged reagents that are specific to biomarkers, the mass spectrometry imaging dataset can be processed to provide multiplexed and spatially co-registered visualizations of the spatial distribution of the radiopharmaceutical and biomarkers within the sample, at single-cell resolution.

[0083] It will be understood that there are many different types of radiopharmaceuticals that include, or generate via radioactive decay, a metal isotope that is detectable via mass spectrometry imaging, such that after administration of the radiopharmaceutical to a subject and collection of a tissue sample, the spatial distribution of the metal isotope is detectable via mass spectrometry imaging, thereby enabling the assessment, inference and / or visualization of the spatial distribution of the radiopharmaceutical within the tissue sample. The following sections of the present disclosure describe these different types of radiopharmaceuticals and the detection, via mass spectrometry imaging, of metalisotopes associated with each type of radiopharmaceutical, for the determination of the spatial distribution of the radiopharmaceutical in a tissue sample.

[0084] Targeted radionuclide therapy (TRT, also known as radioligand therapy, RLT) employs radiopharmaceuticals that contain a targeting ligand that specifically binds to a selected target for the targeted delivery of local radiotherapy. Such targeting radiopharmaceuticals, also known as radioligands, include a radionuclide bound (e.g. attached, bonded, conjugated) to the targeting ligand. TRT thus combines cyto-lethal radioisotopes, including radiometals such as lutetium-177, yttrium-90, and actinium-225, with targeting ligands that target cell-selective biomarkers, such as tumor-specific markers for cancer treatment. Precision TRT aims to deliver radionuclides to specific tumor antigens to interrupt or eliminate crucial tumorigenic processes and subpopulations. Techniques to demonstrate and quantify the localization of TRT to intratumoral cell subpopulations and techniques to assess the microenvironmental response of target tissue have been limited prior to methods of the present disclosure.

[0085] Examples of targeting ligands that are employed to form radioligands for targeted radionuclide therapy include, but are not limited to, antibodies, peptides, aptamers, and small molecules. For example, peptide therapeutics can be labelled with radioactive ligands, such as a somatostatin receptor binding peptide for neuroendocrine tumors. The radionuclide may be connected (attached, bonded) to the targeting ligand by several means, such as, but not limited to, a chelator (e.g. DOTA or DTPA) or a linking molecule (e.g. conjugation) that contains metals such as an oligo, peptide, nanoparticle, quantum dot, nanotube, or polymer or secondary probe or reagent (e.g. Horse Radish Peroxidase I DAB, oligo I labeled oligo), or can be included or doped within the probe or biomolecule. In some example embodiments, the radioligand can be provided in the form of a targeting ligand that is attached to a nanoconstruct comprising or encapsulating the radioligand, such as encapsulated in a nanoparticle, or a nanosphere.

[0086] In some example embodiments of the present disclosure, mass spectrometry imaging, such as imaging mass cytometry or multiplexed ion beam imaging, is employed to determine, in a tissue sample, the spatial distribution of a radioligand administered to a subject. The radioligand includes, or generates via radioactive decay of the radionuclide, a metal isotope detectable by the mass spectrometry imaging system. The mass spectrometry imaging dataset is processed to determinethe spatial distribution of the metal isotope, thereby facilitating the inference of the spatial distribution of the radioligand within the tissue sample. The metal isotope that is detected by the mass spectrometry imaging system to characterize the spatial distribution of the radioligand within the tissue sample may take on many different forms, for example, depending on the type of radioligand that is administered to the subject, as described further below.

[0087] In one example embodiment, the metal isotope detected by the mass spectrometry imaging system may be the cytotoxic radionuclide, provided that the tissue sample is obtained and interrogated by the mass spectrometry imaging system prior to substantial radioactive decay of the radionuclide.

[0088] In another example embodiment, the metal isotope detected by the mass spectrometry imaging system may be a decay product of the radionuclide. For example, one or more decay products of the radionuclide may be stable or metastable metal isotopes that are detectable by the mass spectrometry imaging system.

[0089] Some methods of radioligand fabrication involve the incorporation of isotopes of the cytotoxic radionuclide. Such radioligands are known as “carrier-added” radioligands and include, in addition to the radionuclide (the cytotoxic radioisotope) and the targeting ligand, a carrier isotope that is associated with production of the radionuclide. The carrier isotope may be incorporated into the radiopharmaceutical during the process of labeling of ligand with the radionuclide. For example, one method of production of the radionuclide Lutetium-177 is the irradiation, within a nuclear reactor, of the stable isotope Lutetium-176. This production method results in a reasonable specific activity due to the high cross-section for the neutron capture reaction. However, the final preparation consists of a mixture of both of the Lu-176 and Lu-177 isotopes. The residual stable isotope Lu-176 that is bound, with the radionuclide Lu-177, to the targeting ligand, is referred to as the carrier isotope.

[0090] The methods disclosed herein may be employed to measure and / or assess the spatial distribution, in a tissue sample, the spatial distribution of a wide variety of radioligands administered to a subject (or a tissue sample). Non-limiting examples of radioligands include Lu177-DOTATATE; Lu177-PSMA; In111- or 1123-low-density- lipoproteins for liver receptor binding; radioiodine labelled biomolecules; rhenium skin cancer therapy Rh188 brachytherapy, Y90 radioembolization or internalradiation therapy with or without microspheres; and samarium-153-lexidronam (EDTMP).

[0091] As demonstrated in the examples provided below, the present inventors have found that a carrier isotope of a carrier-added radioligand can be detected in a tissue sample after administration of the radioligand to a subject, with the carrier isotope playing the role of a surrogate, in the tissue sample, of the radionuclide that was delivered to the subject. Accordingly, in some example embodiments, a tissue sample may be obtained from a subject having been administered a carrier-added radioligand that includes, in addition to the cytotoxic radionuclide and the targeting ligand, a carrier isotope associated with production of the radionuclide. A mass spectrometry imaging system that is capable of detecting the carrier isotope is employed to interrogate the tissue sample to obtain a mass spectrometry imaging dataset, and the dataset may be processed to determine a spatial distribution of the carrier isotope. The spatial distribution of the carrier isotope within the tissue sample may be associated with the spatial distribution of the radionuclide delivered within the tissue sample.

[0092] The use of a carrier isotope as a surrogate for detection of the radionuclide via mass spectrometry imaging may be beneficial for a number of reasons. Firstly, the carrier isotope may be a stable or metastable isotope that persists in the tissue sample long after administration of the radioligand to the subject, unlike the radionuclide that may have a relatively short half-life, such as hours or days. Secondly, the identical chemical properties shared between carrier isotope and the radioactive isotope (e.g. as in the case of the carrier isotopes Lu-175 and Lu-176, and the primary cytotoxic radionuclide Lu-177, which are employed in the example below), may be beneficial in ensuring that the carrier isotope acts as a true surrogate for the radionuclide within the tissue sample.

[0093] The use of carrier isotopes for the assessment of the spatial distribution of a radioligand within a tissue sample is demonstrated in the examples below, in which a TRT ligand ([177Lu]Lu-DOTA-RW03) was administered to tumor xenograft mice and potential isotopes delivered to tumors by the TRT were visualized ex vivo through imaging mass cytometry. Although imaging mass cytometry was unable to visualize the stable daughter isotope of the TRT radionuclide (Hf-177), the carrier-isotopes (Lu-175 & Lu-176) incorporated into [177Lu]Lu-DOTA-RW03 during radiolabeling were readily visualized.

[0094] More recently, radiopharmaceuticals for targeted radionuclide therapy have been produced in the absence of carrier isotopes, yielding a “no-carrier-added” (n.c.a.) radioligand such as n.c.a. Lutetium-177. Such n.c.a. TRT radioligands are considered to provide significantly higher radionuclide quality due to the high specific activity (as a consequence of the near absence of “cold” atoms of the same element in the preparation) and the high purity of the radionuclide. For example, no-carrier- added Lutetium-177 can be produced via a modified manufacturing method involving the neutron irradiation of highly enriched Ytterbium-176. The neutron irradiation of Yb-176 results in short-lived Yb-177, which decays to the radionuclide Lu-177 without contamination from the metastable isotope Lu-177m that can persist in carrier-added Lu-177 preparations.

[0095] In the absence of a detectable carrier isotope in a no-carrier-added radioligand, the aforementioned method involving the use of mass spectrometry imaging to infer the spatial distribution of a radionuclide may be adapted to detect a metal isotope that is present in the no-carrier-added radioligand, or a metal isotope that is generated by radioactive decay of the radionuclide. For example, in the case of administration of a no-carrier-added radioligand, the metal isotope detected by the mass spectrometry imaging system may be the cytotoxic radionuclide, provided that the tissue sample is obtained and interrogated by the mass spectrometry imaging system prior to substantial radioactive decay of the radionuclide. Alternatively, the metal isotope detected by the mass spectrometry imaging system may be a decay product of the radionuclide. For example, one or more decay products of the radionuclide may be stable or metastable metal isotopes that are detectable by the mass spectrometry imaging system.

[0096] In other example implementations, the metal isotope that is detected by the mass spectrometry imaging system may be a metal isotope tag (label) that is incorporated into the radioligand. For example, any of the stable metal isotopes that are detectable by the mass spectrometry imaging system may be incorporated into the radioligand (e.g. bound to the targeting ligand) as passive metal isotope tags that are locally delivered, along with the radionuclide. The detection of the spatial distribution of such metal isotope tags therefore enables an assessment, inference or determination of the spatial distribution of the cytotoxic radionuclide delivered to the subject.

[0097] While the preceding example embodiments pertain to targeted radionuclide therapy, the present methods may also be adapted for use with non-targeting radiotherapy that involves the use of radiopharmaceuticals that do not include a targeting ligand. Such non-targeting radiopharmaceuticals employ physiological mechanisms to facilitate preferential uptake or accumulation of the radionuclide within a desired anatomical region (in some example implementations, such radiopharmaceuticals can be provided as nanoparticles), or are only released or activated in a specific environment or by localized activation such as with ultrasound guided microbubbles or local hyperthermia. The consumption or injection of radioactive rare-earth or high mass metal containing metabolites may label cells or tissues that uptake and incorporate these rare-earth containing molecules or metabolites with specific or increased metabolic requirements or uptake. Any of the preceding example methods that employ detection, via mass spectrometry imaging, of a metal isotope associated with the radionuclide may be employed to detect or assess the spatial distribution of the radionuclide. For example, the metal isotope detected by the mass spectrometry imaging system may be the cytotoxic radionuclide, a decay product of the radionuclide, or an additional metal isotope tag that is incorporated into the non-targeting radiopharmaceutical.

[0098] It will also be understood that the metal isotope may be present in the radiopharmaceutical according to many different forms. For example, the metal isotope, which is detected by the mass spectrometry imaging system as, for example, the cytotoxic radionuclide, a decay product of the cytotoxic radionuclide, a carrier isotope, and / or an additional metal isotope tag, may be present in the form of a nanoconstruct, such as encapsulated in a nanoparticle, or a nanosphere.

[0099] It will be understood that in some example implementations, two or more different metal isotopes may be employed to detect, assess, infer or visualize the spatial distribution, within a tissue sample, of a radionuclide delivered to a subject. For example, two or more different carrier isotopes may be detected by the mass spectrometry imaging system, and the spatial distributions of the different carrier isotopes may be employed to infer, assess and / or visualize the spatial distribution of the radionuclide within the tissue sample. In another example, a first metal isotope detected by the mass spectrometry imaging system may be a carrier isotope, and a second metal isotope detected by the mass spectrometry imaging system may be a decay product of the radionuclide, and the spatial distributions of the different metalisotopes may be employed to infer, assess or visualize the spatial distribution of the radionuclide within the tissue sample. In another example, a first metal isotope detected by the mass spectrometry imaging system may be a carrier isotope, and a second metal isotope detected by the mass spectrometry imaging system may be the radionuclide, and the spatial distributions of the different metal isotopes may be employed to infer, assess and / or visualize the spatial distribution of the radionuclide within the tissue sample. In another example, a first metal isotope detected by the mass spectrometry imaging system may be a carrier isotope, and a second metal isotope detected by the mass spectrometry imaging system may be a stable metal isotope tag incorporated into the radiopharmaceutical, and the spatial distributions of the different metal isotopes may be employed to infer, assess and / or visualize the spatial distribution of the radionuclide within the tissue sample. In another example, a first metal isotope detected by the mass spectrometry imaging system may be a first stable metal isotope tag incorporated into the radiopharmaceutical, and a second metal isotope detected by the mass spectrometry imaging system may be a second stable metal isotope tag incorporated into the radiopharmaceutical, and the spatial distributions of the different metal isotopes may be employed to infer, asses and / or visualize the spatial distribution of the radionuclide within the tissue sample. In another example, a combination of multiple metal isotopes detected by the metal- tag-based mass spectrometry based system may be used as a barcode tag where combinations of different metals or their ratios correspond to a tag corresponding to a specific reagent and / or ligand of interest.Modification of Mass spectrometry imaging System for Enhanced Sensitivity of Detection of Metal Isotopes Delivered through Radionuclide Therapy

[0100] It may be beneficial, in some example implementations, to modify the metal-tag-based mass spectrometry imaging system, and / or the method of ablation and aerosol capture of the mass spectrometry imaging system, to achieve a sufficiently high signal-to-noise ratio for detecting a metal isotope in a tissue sample that is associated with a radionuclide administered to a subject. For example, in the case of an imaging mass cytometry system, larger tissue regions could be laser ablated for increased sample introduction, or regions could be ionized through additional imaging passes in a MIBI system.Modification Tissue Sample Preparation Method for Enhanced Sensitivity of Detection of Metal Isotopes Delivered through Radionuclide Therapy

[0101] It may be beneficial, in some implementations, to modify the sample preparation steps that are employed during preparation of the tissue sample, to achieve a sufficiently high signal-to-noise ratio for detecting a metal isotope in a tissue sample that is associated with a radionuclide administered to a subject. For example, approaches can be taken to decrease metal loss through decreased washes, and for example, tissue processing during antigen retrieval including the use of reagents which do not utilize EDTA or other chelating reagents. Alternatively, tissue samples can be processed to further crosslink antibodies, targeting reagents, metal chelators, or metal containing reagents into the tissue or with the tissue in a polymer / gel.Use of Mass Spectrometry Imaging for Co-Registered Imaging of Spatial Distribution of Radionuclides and Biomarkers

[0102] In some example embodiments, mass spectrometry imaging is employed to spatially map, on a subcellular level, one or more biomarkers, such as target antigen biomarkers (e.g. disease-specific antigen biomarkers such as PSMA and CA19), tumor phenotyping biomarkers, and / or biomarkers associated with the local microenvironmental response, to obtain multiplexed images that are co-registered and show both the spatial distribution of the radiopharmaceutical and the spatial distribution of the biomarkers within the tissue sample. The tissue sample is stained, prior to performing mass spectrometry imaging, with one or more metal-tagged reagents that each include an additional metal isotope conjugated to a biomarker ligand that selectively binds with a respective biomarker, where the additional metal isotope is also detectable by the mass spectrometry imaging system. The mass spectrometry imaging dataset is processed to determine a spatial distribution of the metal-tagged reagents within the tissue sample, and a multiplexed dataset can be obtained characterizing the spatial distribution of the both the metal isotope indicative of the radionuclide and the spatial distribution of the metal-tagged reagents within the tissue sample, in an inherently co-registered manner, without the need for image registration.

[0103] In some example implementations, the mass spectrometry imaging dataset may be employed to generate a spatially multiplexed image showing the spatialdistribution of the metal isotope associated with the radionuclide and the spatial distribution of the one or more metal-tagged biomarker reagents. In other example implementations, the mass spectrometry imaging dataset may be processed to determine one or more measures characterizing the spatial distribution of the metal isotope associated with the radionuclide and the spatial distribution of the one or more metal-tagged biomarker reagents, with or without also generating an image. In some example implementations, a tissue sample may be randomly spatially sampled instead of rasterizing, thereby obtaining data that can be employed to compute one or more measures but which may not properly form an image.

[0104] It will be understood that the biomarker ligand associated with a given metal- tagged biomarker reagent may be an antibody, or another type of selective molecule capable of achieving selective binding / recognition, such as, for example, peptides, aptamers, and small molecules. For example, a biomarker ligand may be a peptide hormones, a synthetic ligand, or other non-antibody ligand, including a functional ligand.

[0105] The multiplexed image data thus facilitates the visualization of both the spatial distribution of the radionuclide delivered within the tissue sample and the spatial distribution of the biomarker, and / or the computation of one or more measures associated with the spatial distribution of the radionuclide delivered within the tissue sample and the spatial distribution of the biomarker, thereby enabling a wide variety of analyses. For example, as described further below, the spatial distributions of the metal isotope associated with the radionuclide and the metal isotope(s) associated with the one or more metal-tagged reagents can be employed to assess co-localization of the radionuclide with the intended radiotherapy target, off-target delivery of the radionuclide, and microenvironmental response to the radionuclide therapy. In some example embodiments, the multiplexed image data, and / or the computation of one or more measures associated with the spatial distribution of the radionuclide delivered within the tissue sample and the spatial distribution of one or more biomarkers, may be employed to determine and / or perform one or more additional treatment steps (additional therapeutic interventions).

[0106] Accordingly, the methods disclosed herein can be employed to facilitate the visualization of radiation and radiation response at cellular-level resolution, the potential utility of therapeutic effects induced by targeted radionuclide therapies directed to tumor subpopulations or the tumor microenvironment (e.g., cancer stemcells, bystander effect, abscopal effect, etc.). These and other potential applications and benefits are described in further detail below.

[0107] Examples of the use of co-registered biomarker and radionuclide imaging via imaging mass cytometry are provided in the examples below, in which an experimental assessment was made of the capability of imaging mass cytometry to collected single-cell level resolution, multiplexed images of tumors including the carrier isotopes delivered to the tumor through TRT and biomarkers for the tumor microenvironmental response to treatment, tissue differentiating markers, and CD133 (the antigenic target of the TRT).

[0108] Biomarkers of interest that may be simultaneously imaged could include, for example, therapeutic targets including functional markers and disease or tumour specific isoforms, variants, fusion proteins, markers, glyocproteins, or antigens. Nonlimiting examples of such biomarkers include Axl, EGFR, HER2 / ErbB2, CA125, CEA, CD44v6, CD146, MUC-1 , MAGE, MG7, TROP-2, Nectin-4, CLDN18, PDGFRa / p, PSMA, PSA, PSCA, CD105, CD133, CEACAM5, c-MET, EphA3, FAP, FGFR2, LRRC15, TAG-72, NCAM, vWF, many immune cell subtype markers and more. The identification and co-localization of these and other markers on specific cell types or in combination with cell activities of interest, such as pathway activation, metabolic state, or their location or surrounding environment may be performed. For example, markers of cell type expressing specific targets, such as somatostatin receptor subtype 2 (SSRT2) or other markers of neuroendocrine cells or tumours can be employed to determine the colocalization with radionuclide labelled somatostatin analogues.

[0109] In one example implementation, a colocalization measure can be computed by segmenting co-registered images into features of interest such as cells, cell types, tumour or stromal compartments, blood or lymph vessels, neurons, or other tissue features and the expression or presence of targets, labelled reagents, cellular context quantified per region of interest and the distances between these features computed to identify distance to or exposure to reagents. These could include absolute or proportion of marker positivity, or scaled expression of various markers, distances between single cell or tissue features such as touching, relative, or empiric distances, or spatial patterns and variance.Computation of Measures Based on Spatial Distribution of Metal Isotope Indicative of Radionuclide and Biomarkers

[0110] In some example embodiments, the mass spectrometry imaging data characterizing the spatial distribution of the metal isotope that is representative of the spatial distribution, in a tissue sample, of a radionuclide delivered to a subject, and also the spatial distribution of a biomarker determined by staining the tissue sample with one or more metal-tagged biomarker reagents, in an inherently co-registered manner, may be processed to determine one or more measures.

[0111] For example, the spatial distribution of the metal isotope that is representative of the spatial distribution of a radionuclide and the spatial distribution of a biomarker specific to the intended radiotherapy target may be processed to assess (e.g. determine a measure of) co-localization of the radionuclides with the intended target. This may be performed, for example, by the identification of measurements (locations or pixels) which contain both radionuclide and metal isotope representing markers of interest, or by identifying the spatial distance and Euclidean distance between measurements containing features of interest. In addition, biological features of interest, such as cells or tissue compartments such as tumor lesions or blood vessels may be identified in the lesions and their radionuclide content and distance metrics can be utilized as analysis features.

[0112] In one example, the spatial distribution of the metal isotope that is representative of the spatial distribution of a radionuclide, and the spatial distribution of a biomarker specific to the intended radiotherapy target, may be processed to determine one or more distance measures, for example, using various distance metrics to other tissue features, for example, in Euclidean and / or other space, dependent on physical parameters such as known diffusion metrics, and radiation exposure calculation.

[0113] In another example implementation, the spatial distribution of the metal isotope that is representative of the spatial distribution of a radionuclide, and the spatial distribution of a biomarker specific to the intended radiotherapy target, may be processed to assess (e.g. determine a measure) of off-target delivery of the radionuclides. This may be performed, for example, by identifying correlation / anti- correlation or distance with or to specific cell types or cell states that are either targets or not targets (e.g. markers of healthy cells), or are features known to be sensitive to off-target effects. In some example implementations, the spatialdistribution of the metal isotope that is representative of the spatial distribution of a radionuclide, and the spatial distribution of a biomarker specific to the intended radiotherapy target, could be processed to detect the presence of (or a measure associated with) radionuclides delivered to locations that are absent of biomarker targets.

[0114] In another example implementation, the spatial distribution of the metal isotope that is representative of the spatial distribution of a radionuclide, and the spatial distribution of a biomarker specific to the intended radiotherapy target, may be processed to assess (e.g. determine a measure of) on-target delivery within in a different or healthy tissue. This may be performed, for example, by measuring correlation with or distance to single cells or tissue features containing biomarkers measuring the therapeutic target of interest, but also the presence of the target within pathologic or healthy tissues or cells.

[0115] In another example implementation, the spatial distribution of the metal isotope that is representative of the spatial distribution of a radionuclide, and the spatial distribution of a biomarker configured to specifically bind to a marker of microenvironmental cellular response to the therapeutic radiopharmaceutical, may be processed to assess (e.g. determine a measure of characterizing) microenvironmental cellular response to therapeutic radiopharmaceutical. Nonlimiting examples involving the characterization of microenvironmental cellular response to therapeutic radiopharmaceutical include a detection or assessment of cellular proliferation, apoptosis, pathway activation, and the presence of a resistance mechanism. For example, such an assessment could include measurement of cell or tissue types or states in the vicinity of target or therapy such as blood vessels, angiogenesis, immune cells, fibrogenesis, matrix deposition, metabolic or cellular tissue state including pathway or morphology indicative of response or resistance to therapy such as DNA damage, cell cycle arrest or state enrichment, apoptosis, or proliferation, pathway activation, invasion, epithelial-mesenchymal transition.

[0116] In another example implementation, the spatial distribution of the metal isotope that is representative of the spatial distribution of a radionuclide, and the spatial distribution of a biomarker configured to specifically bind to a marker of microenvironmental cellular response to the therapeutic radiopharmaceutical, may be processed to assess the co- or anti-correlation of multiple therapeutic reagents for combination therapy in the same targets, or synergistic treatment of different areas,cell or tissue states within one patient. This may be performed, for example, by the spatial measurement of diverse biomarkers of tissue state or response in spatial relationships to radionuclides and targets of interest or alternatively, the environment or state of target expressing tissues that do not contain measurable radionuclide therapy. This could indicate the prognostic or predictive expression of radionuclide sensitive or resistant or refractory cells and tissues, or even the presence of tissue which should be targeted by an alternative treatment. Alternative treatments could include different targets of interest, sensitivity to untargeted therapies, or the need for a different therapeutic cargo delivered from the same targeted therapy, alternatively this could identify microenvironment targeted therapies needed to enable proper delivery such as the need for VEGFa targeting therapeutics or vessel normalization and stabilization treatment, immune-modulatory, or matrix / tension altering treatments.Embodiments Involving Radiopharmaceuticals for Diagnostic Imaging

[0117] While the preceding example embodiments have been disclosed within the context of radionuclide-based therapy involving the detection of radiopharmaceuticals administered for providing local radiation therapy, it will be understood that the methods disclosed herein may be employed to detect the spatial distribution of diagnostic (e.g. tracer) radiopharmaceuticals or other non-radioactive but metal-containing or tagged therapeutics in a tissue sample.

[0118] In one example workflow involving diagnostic radiopharmaceuticals, and a diagnostic radioligand having a metal isotope could be delivered and employed for imaging (e.g. MRI / PET, immunoPET), and a tissue sample could subsequently be measured and analyzed via the present example methods involving the use of mass spectrometry imaging. Examples of diagnostic radiopharmaceuticals that could be adapted to include a metal isotope label include those against targets listed above or therapeutics such as [111 ln-DTPA-D-Phe1]-octreotide, ln-111 pentetreotide; In111 - or 1123-low-density-lipoproteins for liver receptor binding; 111-lndium-Capromab Pendetide; radioactive copper, gallium, yttrium, zirconium reagents for PET or SPECT imaging; radioiodine for thyroid cell or cancer uptake,177Lu-PSMA-617 (Pluvicto),177Lu-DOTATATE, [89Zr]Zr-Df-HER2-Fab-PAS200 in breast cancer,223Ra-Dichloride (Xofigo), 68Ga-PSMA-11,161Tb-DOTATOC, or188Re- Hydroxyethylidene Diphosphonate (HEDP).Embodiments Involving Non-radioactive Therapeutic Pharmaceuticals Labeled with Metal Isotope

[0119] Many of the example embodiments disclosed above pertain to methods of characterizing the spatial distribution of a radiopharmaceutical, where the radiopharmaceutical includes, or generates via radioactive decay of the radionuclide, a metal isotope detectable by a mass spectrometry imaging system (and also pertain to the co-registered imaging of spatial distribution of radionuclides and biomarkers, and the computation of measures based on spatial distribution of the metal Isotope indicative of the radionuclide and biomarkers). However, in some example embodiments, the methods and applications disclosed above may be adapted to facilitate the characterization of a non-radioactive pharmaceutical (a pharmaceutical that is absent of a radionuclide) that is labeled with a stable metal isotope facilitating detection via mass spectrometry imaging.

[0120] Accordingly, in some example implementations, a non-radioactive pharmaceutical that includes a metal isotope label may be administered to a subject, and a tissue sample subsequently obtained from the subject may be processed by a mass spectrometry imaging system to infer a spatial distribution of the nonradioactive pharmaceutical within the sample.

[0121] While some non-radioactive pharmaceuticals include a metal component as a therapeutic payload, some example embodiments of the present disclosure involve the detection of non-radioactive pharmaceuticals that are labeled by a non- therapeutic, passive (inert) metal isotope label (tag). The payload may be absent of a metal, or may include a metal that is different from the metal isotope label.

[0122] In some example implementations, the passive metal isotope is employed to label a non-radioactive pharmaceutical that includes a therapeutic payload. The therapeutic payload of the non-radioactive pharmaceutical, which is labeled with a non-therapeutic passive metal isotope to facilitate detection by imaging mass spectrometry, may be absent of a metal, or may include a metal that is different from the metal isotope label. . Examples of non-radioactive pharmaceuticals that include a non-metallic therapeutic payload, and which could be labeled with a non-therapeutic passive metal isotope, include, but are not limited to, antibodies, antibody fragments (such as, for example, a Fab fragment, scFV, a nanobody, minibody, diabody or other fragment), engineered proteins such as affibodies and DARPins, peptides,aptamers, miniproteins (natural or engineered), and small molecules. Examples of non-radioactive pharmaceuticals that include a metallic therapeutic payload, and which could be labeled with a non-therapeutic passive metal isotope, include, but are not limited to, cisplatin, carboplatin, oxiplatin, nedaplatin, phenanthriplatin, picoplatin, straplatin, or other platinum containing therapies, ruthenium complexes such as BOLD-100, NAMI-A [lmH][trans-RuCI4(dmso-S)(lm)], KP1019 / KP1339, RAPTA, RAED; colloidal silver; metal containing nanoparticles for magnetic hyperthermia therapy such as lanthanum strontium manganite; cerium oxide nanoparticles. In other example implementations, the passive metal isotope is employed to label a non-radioactive pharmaceutical that is employed as a diagnostic or a theranostic reagent. Examples of such non-radioactive diagnostic or theranostic pharmaceuticals include, but are not limited to, imaging reagents for MRI, PET, SPECT, X-ray / CT or dual modality imaging which could include antibodies, antibody fragments (such as, for example, a Fab fragment, scFV, a nanobody, minibody, diabobdy or other fragment), engineered proteins such as affibodies and DARPins, peptides, aptamers, miniproteins (natural or engineered), metabolites, hormones, and small molecules similar to those currently in use.

[0123] Other non-limiting examples of diagnostic and / or theranostic reagents that can serve as payloads to be labeled by a passive metal isotope include: 16p- [18F]fluoro-5a-dihydrotestosterone (FDHT) to image prostate cancer antigen or androgen receptor expression in prostate cancer; 6-fluoro-[18F]DOPA (FDOPA), [123l]FP-p-CIT and [99mTc]TRODAT-1, 9-fluoropropyl-(+)-DTBZ (AV-133) used to map dopamine receptors; Fluorodeoxyglucose ([18F]FDG or FDG) to measure glucose uptake and glucose consuming cancers; 16a-[18F]fluoroestradiol (FES): 21- [18F]fluoro-furanyl-nor-progesterone (FFNP), or Copper-Diacetyl-bis(N4- methylthiosemicarbazone) (Cu-ATSM), Gadolinium containing MRI imaging reagents such as Gadopentetate Dimeglumine (Magnevist), Gadoteridol (ProHance), Gadodiamide (Omniscan), Gadobenate Dimeglumine (MultiHance), Gadoversetamide (OptiMARK), Gadobutrol (Gadavist), Gadofosveset Trisodium (Ablavar), Gadoxetate Disodium (Eovist); superparamagnetic iron oxide (SPIO) nanoparticles and agents such as Ferumoxytol (Feraheme), Ferucarbotran (Resovist), Ferristene (Endorem), Ferumoxsil (Lumirem, GastroMARK), Feruglose (Clariscan); Technetium-99m (99mTc) agents: "mTc-Sestamibi (Cardiolite, e.g. for cardiac perfusion imaging, tumor imaging), "mTc-Medronate (MDP, e.g. for boneimaging (skeletal scintigraphy)), "mTc-Tetrofosmin (e.g. for myocardial perfusion imaging), "mTc-Pertechnetate (e.g. for thyroid, salivary gland, and gastric mucosa imaging), "mTc-Macroaggregated Albumin (MAA, e.g. for lung perfusion imaging), "mTc-Red Blood Cells (RBC, e.g. for blood pool imaging for gastrointestinal bleeding and cardiac function), "mTc-DMSA (Dimercaptosuccinic acid, e.g. for renal cortical imaging, "mTc-HMPAO (Hexamethylpropyleneamine oxime, e.g. for brain perfusion imaging, "mTc-Labeled White Blood Cells (e.g. for infection or inflammation imaging); lndium-111 (111ln) agents:111ln-Pentetreotide (Octreoscan, e.g. for imaging of neuroendocrine tumors),111ln-Labeled White Blood Cells (e.g. for infection and inflammation imaging),111ln-DTPA - Cisternography (e.g. for cerebrospinal fluid imaging),111ln-PSMA (e.g. for Prostate cancer imaging (in development or specialized applications)),2O1TI-Chloride (e.g. for myocardial perfusion imaging, tumor imaging (used as a potassium analog)),67Ga-Citrate (e.g. for imaging of infection, inflammation, and tumors (especially lymphoma)), iodine or barium containing contrast reagents for X-ray and CT imaging, and gold nanoparticles as contrast reagents for photoacoustic or other imaging.

[0124] The present example embodiments involving the use of mass spectrometry imaging to characterize spatial distribution of a non-radioactive pharmaceutical within a tissue sample, via the detection and imaging of a passive, non-therapeutic metal label, may be beneficial in a wide range of research and clinical applications.

[0125] For example, in cases in which the passive metal isotope is employed to label a non-radioactive pharmaceutical that includes a therapeutic payload, the nonradioactive pharmaceutical can include a non-radioactive antibodies, antibody fragments (such as, for example, a Fab fragment, scFV, a nanobody, minibody, diabobdy or other fragment), engineered proteins such as affibodies and DARPins, peptides, aptamers, miniproteins (natural or engineered), and small molecules, all which can be labelled with a passive metal isotope.

[0126] Non-limiting examples of currently available reagents which can be labelled with a passive metal isotope and which are used for imaging for diagnostics include all theranostic reagents such as HER2 labelling Ga-ABY-025, [89Zr]Zr-trastuzumab, [18F]FBEM-trastuzumab, 68Ga]Ga-DOTA-F(ab')2-trastuzumab. In general a metal tag can be added to any antibody therapeutic or a metal can be incorporated into a small molecule inhibitor. Some examples include the conversion of therapeutic reagents into theranostic reagents such as anti-EGFR Cetuximab (Erbitux), anti-TR0P2 Sacituzumab Govitecan (Trodelvy), anti-Nectin-4 Sacituzumab Govitecan (Trodelvy), Amivantamab (Rybrevant), or even anti-SARS-CoV-2 Spike protein Sotrovimab or any other target. Alternatively, metals could be incorporated into peptide receptor antagonists or analogs such as Semaglutide (Ozempic, Wegovy, Rybelsus), Octreotide (Sandostatin), Octreotide (Sandostatin), Teriparatide (Forteo), or small molecule inhibitors such as lodine-125 / lodine-131 -labeled Gefitinib, Erlotinib, Sorafenib or Technetium-99m-labeled Lapatinib, or Lutetium-177-labeled Sunitinib. Gallium-68 or Lutetium-177 labeled Bombesin analogs, Technetium-99m- lablled EC20 folate for folate receptor targeting, PSMA -11 PSMA-1007 PSMA-617 ligands, labelled DOTATATE, labelled Fluciclovine (Laxumin), Fibroblast activation protein inhibitors (FAPI), SPECT, CXCR4 receptor inhibitors Gallium-68-labeled Pentixafor and Lutetium-177-labeled Pentixather, labelled RGD peptides or RP748 inhibitors for integrin imaging, PARP inhibitors and more.

[0127] In some example embodiments, multiplexed image data, and / or the computation of one or more measures associated with the spatial distribution of the non-radioactive pharmaceutical delivered within the tissue sample and the spatial distribution of one or more biomarkers, may be employed to determine and / or perform one or more additional treatment steps (additional therapeutic interventions), examples of which are described below.Example Applications

[0128] One example application of the methods disclosed herein is therapeutic monitoring. One non-limiting example of therapeutic monitoring is “window of opportunity” testing, by measuring, via mass spectrometry imaging, the presence, spatial distribution, or absence of a metal isotope associated with a radiopharmaceutical and its location within cells or tissues of specific phenotype, or within cells or tissues with specific activity associated with therapeutic response or clinical outcome. Measurements would be completed on a sample or biopsy taken from a patient shortly after treatment with metal containing molecule or therapeutic. The resulting measurements would inform localization and response to the treatment and determine if the patient should continue on the treatment or if another treatment should be employed for therapeutic benefit.

[0129] Another non-limiting example of therapeutic monitoring is surveying for resistance mechanisms being present or arising. For example, such a method couldinvolve the identification of phenotypes arising during treatment that are resistant to therapy and the exclusion of a metal isotope associated with a pharmaceutical (e.g. a radiopharmaceutical) from the tissue mass, or, for example, the absence of a metal isotope associated with a pharmaceutical (e.g. a radiopharmaceutical) from the cell compartment or interior such as membrane, cytoplasm or nucleus, or from cells of a particular phenotype or from location containing target or antigen. In this instance, the measurement and analysis output would identify patients who should stop treatment or receive alternative therapeutic options.

[0130] In one example application, the methods disclosed herein could be employed to facilitate a determination of the spatial distribution, in a tissue sample, of DOT AT ATE reagents, which are metal isotope labeled peptide hormones which bind to somatostatin receptors in neuroendocrine tumors, or antigen targeting radionuclide therapies such as Lu177-PSMA or Ac225-PSMA reagents which are radionuclide prostate tumour specific antigen therapies.

[0131] In yet another example, the methods disclosed herein could be employed to facilitate and confirm the removal of cell types for autoimmune disease, for example, by measuring the presence of a pharmaceutical such as Santi-IL-17 Secukinumab (Cosentyx), anti-TNF Adalimumab (Humira) or Adalimumab (Humira), CD20 with Rituximab (Rituxan) or other therapeutically administered antibodies against immune cell populations, inflammatory cytokines, or reactive antigens using therapeutic antibodies or alternative reagents imaging mass cytometry or other methods. In this case, the presence of metal labelled cells would indicate that therapeutic targets are still present within the patient or tissue and treatment should continue or has failed or is unresponsive.

[0132] In another example implementation, a metal tagged chemotherapy agent or a metal tagged targeted treatment can be employed with mass spectrometry imaging to assess (and optionally to determine subsequent therapeutic actions for improving or facilitating) removal of bone marrow before bone marrow transplant. Non-limiting examples of suitable metal tagged targeted treatments include radioligand antibodies (tagged with a metal isotope or generating a detectable metal isotope by radioactive decay), metal-isotope-tagged antibody drug conjugates, metal-isotope-labelled small molecules, or naturally bone-marrow targeting radioactive elements (such as strontium-89, samarium-153, rhenium-186, and radium-223). Mass spectrometry imaging could be employed to determine the spatial distribution of the metal isotopeassociated with the delivered drug, in combination with the measurement of immune cell or cell phenotype content which the therapy was attempting to remove from the bone marrow. In some example implementations, bone marrow biopsies, aspirates, or cell smears could be imaged by imaging mass cytometry or spectrometry to quantify the presence of therapeutic reagents in relation to their targets such as metastatic cancer cells (residual disease) and their antigens, or the presence of cell types impacting immune rejection or autoimmune disease, such as host stem or other cell populations that could impact transplantation success or result in graft versus host disease. Measurement of such cell populations would indicate the failure to remove residual cancer cells, or to thoroughly deplete or remove the stem cell populations which are being replaced in a transplant. Their co-localization with therapy would inform cellular resistance (if therapy and target co-detected) versus therapeutic delivery failure (if target present but spatially separate from therapy), or therapy success if targets are absent. This can impact therapeutic decisions to continue or increase cancer treatment to capture all or maximal residual disease, or stop in the case of resistance and failure, or to increase radiation to deplete bone marrow for successful transplantation thereby avoiding graft-versus-host disease.

[0133] In other example implementations, the methods disclosed herein could be employed to screen for therapeutic reagents with specificity, stability, penetration, or delivery features of interest, or those which elicit a specific cellular or tissue response. For example, therapeutic pharmaceuticals could be screened by labelling various therapeutic pharmaceuticals of interest with metal isotopes, and upon treatment of tissues (e.g. via any suitable mode of pharmaceutical delivery), measuring the presence of metal isotopes and therefore inferring the spatial distribution of the therapeutic pharmaceuticals, and optionally their association with target antigens or cells as well as the cellular activity or response of cells containing or localized near reagent in the tissues. This could be completed in patients and imaged in biopsy samples such as in a Window of Opportunity clinical trial, or in tumour explant samples grown ex vivo. Scoring of location, amount, and cellular activity can therefore determine features of the therapeutic pharmaceuticals associated with specificity, penetration, stability, tissue response, toxicity and more.

[0134] It will be understood that a pharmaceutical (including a radiopharmaceutical) that includes, or generates via radioactive decay of a radionuclide, a metal isotope detectable by a mass spectrometry imaging system,can be locally or systemically delivered according to a wide variety of methods, including, for example, intravenously, orally, through localized injection or implantation, or topically to the skin or other accessible surface. In other example methods, a pharmaceutical (including a radiopharmaceutical).

[0135] In other example implementations, the methods disclosed herein could be employed to test the response to a treatment by a variety of samples or conditions or individual tumours / lesions at the same time, such as in drug development applications, including high-throughput drug screening / discovery. For example, response to a treatment could be assessed by creating multiple lesions or conditions within a single tissue and measuring their response to individual treatment with each location indicating a different condition. Such model systems could include arrayed or pooled cell models such as cell lines, organoids, or intact tissue models that have been modified using libraries of genetic tools with or without associated barcodes.Example Mass spectrometry imaging System

[0136] Referring now to FIG. 1 , an example imaging mass cytometer system (which is an example of a mass spectrometry imaging system) is shown. A laser ablation and scanning subsystem 100 employed to scan a tissue sample 105 and generate an aerosol plume 101. The aerosol is delivered to an ionization subsystem 120 via a transfer conduit 110. The elemental ions generated by the ionization subsystem are delivered to the mass spectrometer 130, which performs mass analysis. As shown in the figure, the laser subsystem 100, ionization subsystem 120, and mass spectrometer 130 are operably connected to and controlled by the control and processing circuitry 200.

[0137] The example control and processing circuitry 200 may include a processor 210, a memory 215, a system bus 205, one or more input / output devices 220, and a plurality of optional additional devices such as communications interface 225, external storage 230, and a data acquisition interface 235. In one example implementation, a display (not shown) may be employed to provide a user interface for facilitating input to control the operation of the system 200. The display may be directly integrated into a control and processing device (for example, as an embedded display), or may be provided as an external device (for example, an external monitor). The control and processing system 200 may include or be connectable to a console 280 that provides an interface enabling an operator tocontrol the system. The console may include, for example, one or more input devices, such, but not limited to, a keypad, mouse, joystick, touchscreen, and may optionally include a display device.

[0138] The methods described herein, such as methods of generating multiplexed images characterizing the spatial distribution of a metal isotope associated with the delivery of a radionuclide and one or more additional biomarkers, or other example methods described herein, can be implemented via processor 210 and / or memory 215. As shown in FIG. 1 , executable instructions represented as control module 250 are processed by control and processing circuitry 200. Such executable instructions may be stored, for example, in the memory 215 and / or other internal storage.

[0139] The methods described herein can be partially implemented via hardware logic in processor 210 and partially using the instructions stored in memory 215. Some embodiments may be implemented using processor 210 without additional instructions stored in memory 215. Some embodiments are implemented using the instructions stored in memory 215 for execution by one or more microprocessors. Thus, the disclosure is not limited to a specific configuration of hardware and / or software.

[0140] It is to be understood that the example system shown in the figure is not intended to be limited to the components that may be employed in a given implementation. For example, the system may include one or more additional processors. Furthermore, one or more components of control and processing circuitry 200 may be provided as an external component that is interfaced to a processing device. Furthermore, although the bus 205 is depicted as a single connection between all of the components, it will be appreciated that the bus 205 may represent one or more circuits, devices or communication channels which link two or more of the components. For example, the bus 205 may include a motherboard. The control and processing circuitry 200 may include many more or less components than those shown.

[0141] Some aspects of the present disclosure can be embodied, at least in part, in software, which, when executed on a computing system, transforms an otherwise generic computing system into a specialty-purpose computing system that is capable of performing the methods disclosed herein, or variations thereof. That is, the techniques can be carried out in a computer system or other data processing system in response to its processor, such as a microprocessor, executing sequences ofinstructions contained in a memory, such as ROM, volatile RAM, non-volatile memory, cache, magnetic and optical disks, or a remote storage device. Further, the instructions can be downloaded into a computing device over a data network in a form of compiled and linked version. Alternatively, the logic to perform the processes as discussed above could be implemented in additional computer and / or machine- readable media, such as discrete hardware components as large-scale integrated circuits (LSI's), application-specific integrated circuits (ASIC's), or firmware such as electrically erasable programmable read-only memory (EEPROM's) and field- programmable gate arrays (FPGAs).

[0142] A computer readable storage medium can be used to store software and data which when executed by a data processing system causes the system to perform various methods. The executable software and data may be stored in various places including for example ROM, volatile RAM, nonvolatile memory and / or cache. Portions of this software and / or data may be stored in any one of these storage devices. As used herein, the phrases “computer readable material” and “computer readable storage medium” refers to all computer-readable media, except for a transitory propagating signal perse.

[0143] While many of the examples disclosed herein (and the system shown in FIG. 1 involve the use of an imaging mass cytometer to perform spatially resolved detection and imaging of metal isotopes, it will be understood that many other systems may be employed in the alternative. Non-limiting examples include multiplexed ion beam imaging (Ml Bl).EXAMPLES

[0144] The following examples are presented to enable those skilled in the art to understand and to practice embodiments of the present disclosure. They should not be considered as a limitation on the scope of the disclosure, but merely as being illustrative and representative thereof.Example 1 : Demonstration of Visualization of Carrier Isotopes Delivered during Targeted Radionuclide Therapy by Imaging Mass Cytometry (CyTOF)

[0145] In the present example, it is demonstrated that IMC can be adapted, according to the methods described above, to image the distribution of metalisotopes delivered by radioimmunotherapy to tumors during treatment, and to simultaneously image these metal isotopes with the antigen targeted by the treatment, and with epitopes for microenvironmental biomarkers of interest, all on a single tissue section.

[0146] The results shown below confirm that IMC has the sensitivity to visualize carrier isotopes delivered to tumors during treatment with targeted radionuclide therapy, and given identical chemical properties shared between carrier and radioactive isotopes (as for Lu-175, Lu-176, and Lu-177), these results indicate that carrier isotopes can act as surrogates to visualize radionuclide distribution in tumors at single-cell resolution.MethodsGeneral Materials

[0147] All reagents were ACS grade and used as received. Ultrapure 18.2 Q water was used for preparing all buffers. [177Lu]LuCls, received from McMaster Nuclear Reactor, was prepared via neutron irradiation of176Lu enriched lutetium (III) chloride followed by dissolution in high purity HCI (0.1 M), for > 51 .8 GBq / mg with an activity concentration greater than 11.1 GBq / mL. Lu-176 enriched LuCh contained a Lu-175 isotopic abundance of 17.1 %, Lu-176 abundance of 82.9%, <100 ppm of iron, <200 ppm of indium, and 310 ppm of manganese.Samples for Ex vivo Analysis

[0148] All tumor and tissue samples were collected in a previous study. Briefly, BALB / c nu / nu mice were inoculated with HT-29 tumors and 7 days post inoculation, mice were administered a single dose of either 3.7 or 18.5 MBq [177Lu]Lu-DOTA- RW03, a single dose of 18.5 MBq [177Lu]Lu-DOTA-RW03 followed by a second dose of 3.7 MBq [177Lu]Lu-DOTA-RW03 1 week later (cumulative dose of 22.5 MBq), or a vehicle control. Mice were euthanized and tissues collected 14 days post-injection (p.i) of the vehicle, 3.7 MBq [177Lu]Lu-DOTA-RW03, 18.5 MBq [177Lu]Lu-DOTA- RW03, and 22.5 MBq [177Lu]Lu-DOTA-RW03. Mice were also collected 28 days p.i. of 18.5 MBq [177Lu]Lu-DOTA-RW03. Additionally, a human skin sample was provided by STTARR (UHN, Toronto) as a CD133 negative control.Ex Vivo Tissue Preparation

[0149] Tumors were excised from mice and cut into 2 halves using a scalpel. One half of the tumor was used for immunohistochemistry and CyTOF; these sampleswere placed in 10% formalin for 24-48 h. Tissues were exchanged into 70% ethanol over a gradient (10% for 4 h, 20% for 4 h, then 70%) where they remained until >10 half-lives decay (approximately 70 days). Following decay, tissues were embedded into paraffin wax using routine procedures. For autoradiography, the other half of the tumor was embedded in Tissue-Tek optimum cutting temperature (OCT) compound (Tissue-Tek, Sakura, Torrance, CA) and frozen over liquid nitrogen vapor.CD133 Immunohistochemistry

[0150] Immunohistochemical analysis (IHC) of CD133 was performed by Wax-lt histology services Inc. Antigen retrieval was performed in citrate buffer (10 mM, pH 6.0, 0.05% Tween) for 25 minutes at 98°C. CD133 ab (CST #64326) was used at a concentration of 0.55 pg / ml with an overnight incubation followed by incubation with polymer anti-rabbit secondary for 30 mins. Tissue blocking was performed using the Wax-lt in-house protein block and peroxide was blocked with 3% H2O2. Slides were subsequently developed using DAB and counterstained with hematoxylin. Coverslips were mounted with Permount. Adjacent slides were also stained with an isotype control antibody to confirm specific anti-CD133 antibody binding.Autoradiography and Imaging CyTOF: Calibration Curve of Lu-177

[0151] To 100 mL of stirring ultrapure water was added 2 g agarose (Invitrogen Ultrapure Agarose, #16500100) and heated to 90°C for 30 minutes. The melted agarose was cooled to 70°C for 10 minutes, and pre-warmed glass microscope slides (70°C) (VWR, MENZJ1820AM NZ) were dipped into the agarose, and left to cool at room temperature overnight. To 100 pL of Trypan Blue (Thermofischer, 15250061) was added 0.4 MBq [177Lu]LuCl3. A calibration curve was generated through serial % dilutions into Trypan Blue. To test for accuracy, 20 pL of each calibration curve solution was tested for activity using a gamma counter. Onto the agarose coated microscope slides was spotted ~0.1 pL of each [177Lu]LuCl3 dilution (highest dilution contained 350 nM Lu-176). The [177Lu]LuCl3 spotted microscope slides were laid face down on a syran-wrap coated phosphor plate (Fujifilm BAS cassette, Screen BAS-IP MS 2040 E Multipurpose Standard) for 2 days. The plate was imaged at 25 pM pixel size using an Amersham Typhoon. Regions of interest (ROI) analysis was conducted using ImageLab (Bio-Rad).Antibody Panel

[0152] An antibody panel was designed to target epitopes on CD133, epitopes specific for cell cycle (ki67), cell death (cleaved caspase 3) and phosphorylation-based signaling (gH2AX), and to distinguish epithelial (pan-cytokeratin), endothelial (CD31 ), mesenchymal (SMA or FAP) and immune cell types (CD45)Preparation and Staining

[0153] Tissue samples were formalin-fixed and paraffin-embedded at McMaster University. The antibody panel was used to stain the tissue sections. Tissue sections were dewaxed in xylene overnight and rehydrated in a graded series of alcohol (ethanokdeionized water 100:0, 90:10, 80:20, 70:30, 50:50, 0:100; 5 min each). In a 95 °C water bath, heat-induced epitope retrieval was conducted in Tris-EDTA buffer at pH 9 for 20 min. The tissue sections were immediately cooled and then blocked with 3% BSA and 5% goat serum in tris-buffered saline (TBS) for 1 h. Samples were incubated overnight at 4 °C in primary antibody at 7.5 g I-1diluted in TBS, 0.1 % Triton X-100 and 1 % BSA. Tissue samples were washed twice with TBS and 0.1 % Triton X-100, and twice with TBS, and dried before IMC measurements. imaging Mass Cytometry

[0154] Images were acquired using a Hyperion Imaging System (Fluidigm). The tissue sections were laser-ablated in a rastered pattern at 200 Hz, and preprocessing of the raw data was completed using commercial acquisition software (Fluidigm). IMC acquisition stability was monitored by interspersed acquisition of isotope-containing polymer (Fludigm). All successful image acquisitions were processed, and where applicable, signal spillover between channels was corrected using functions from the CATALYST R package28 (v.1 .5.6). Data were filtered to exclude detector noise and censored at the 99th percentile to remove outliers. Channels were then individually normalized and merged.ResultsSensitivity Comparison to Phosphor imaging

[0155] The sensitivity of IMC was compared to the sensitivity of a conventional phosphor-based autoradiography through imaging serial dilutions of [177Lu]LuCl3 spotted on an agarose-coated microscope slide (FIG. 2A). Phosphor imaging ROI analysis (FIG. 2B) demonstrated good linearity through the entire concentration range of the calibration curve following exposure for 2 days.IMC for Visualizing Carrier Isotopes Delivered to Tumors During C77Lu]Lu- DOTA-RW03 Treatment

[0156] The capability of IMC to visualize potential isotopes (Hf-177, Lu-175, and Lu- 176) delivered to target tumor tissue by TRT was assessed in HT-29 tumorspecimens excised from mice 14- days into treatment with either 3.7 MBq or 14.8 MBq [177Lu]Lu-DOTA-RW03, a CD133-targeted radioimmunotherapy (FIG. N2). In both specimens, the signal intensity of hafnium-177 (stable isotope following Lu-177 decay) was not present above the background (FIG. 3, panels a and d). Channels for Lu-175 (panels b and e) and Lu-176 (panes c and f) detected signal significantly above the background in both tumor specimens. The detected signals in the Lu-175 and Lu-176 channels arose in the same pixels, and the signal intensity in the Lu-176 channel exceeded that of Lu-175 which is in-line with the isotopic abundances of Lu- 175 and Lu-176 (16% and 83%, respectively) in the carrier-added Lu-177 used for treatment. The total signal intensity was greater in specimens from mice that received 14.8 MBq relative to 3.7 MBq of treatment. Together these results confirmed that IMC has the sensitivity to visualize carrier isotopes delivered to tumors during treatment with targeted radionuclide therapy, and given identical chemical properties shared between carrier and radioactive isotopes (as for Lu-175, Lu-176, and Lu-177), these results indicate that carrier isotopes can act as surrogates to visualize radionuclide distribution in tumors at single-cell resolution.Multiplexed imaging of carrier isotopes introduced by TRT and ex vivo metal- labeled antibodies

[0157] The capability of IMC to simultaneously visualize carrier isotopes introduced to tumors during TRT and metal- labeled antibodies (that target biological markers of interest, here DNA and actin) introduced to tissue sections ex vivo was assessed in untreated HT-29 tumors and tumors treated with 14.8 MBq [177Lu]Lu-DOTA-RW03 (FIG. 4).

[0158] In untreated tumors Lu-176 intensity was not found to exceed background levels (FIG. 4, panel a) and in treated tumors Lu-176 signal intensity was detected. The carrier isotope introduced by TRT was successfully visualized in tandem with DNA and actin on a tumor specimen embedded on a single microscope slide. In panel b of FIG. 4, IMC images show cell-sized clusters of Lu-176 in areas of distinct cell nuclei and pixel-sized, non-background Lu-176 signal in areas absent of distinct cell nuclei, which potentially reflect areas of necrotic and non-necrotic tumor tissue. These results confirmed that IMC can simultaneously visualize metal isotopes introduced to tumors through TRT with biomarkers stained ex vivo on a single tissue slide.Comparing CD133 immunohistochemistry and imaging CyTOF

[0159] The distribution of CD133, the epitope targeted by [177Lu]Lu-DOTA-RW03 treatment, was compared in tumors imaged by IMC and CD133 immunohistochemistry (FIG. 5). Images produced using IMC and IHC both clearly visualize the distribution of CD133 between the cell surface and cytoplasm and both modalities visualized circular clusters with increased CD133 expression on vascular structures within the tumor.Ex vivo CD133 staining colocalizes with176Lu in tumors treated with TRT

[0160] The capacity for IMC to colocalize carrier isotopes delivered to tumors through TRT with epitope-positive target cells stained ex vivo was assessed in untreated tumors and tumors treated with 14.8 MBq [177Lu]Lu-DOTA-RW03 (FIG. 6). In untreated tumors Lu-176 signal was not observed and CD133 (Nd-146) was abundant in HT-29 tumors (panel a). In tumors treated with 14.8 MBq [177Lu]Lu- DOTA-RW03, IMC detected both Lu-176 introduced during TRT and the CD133- targeted Nd-146 labeled antibody introduced ex vivo, and the Lu-176 signal was highly restricted to Nd 146+ pixels in the image (panel b). These results demonstrate the IMC can be used to confirm localization of TRT to antigenic cells of interest within tumors.Multiplexed imaging CyTOF compared to overlayed autoradiography and IHC

[0161] The capability of IMC to simultaneously image the intratumoral distribution of TRT and microenvironmental markers of interest (here, yH2Ax and cleaved caspase- 3) was assessed in untreated tumors and tumors treated with 14.8 MBq [177Lu]Lu- DOTA-RW03 (FIG. 7). While the peak yH2AX concentration is known to much early into treatment with TRT than in tumors assessed here (excised 14 days into treatment), [177Lu]Lu-DOTA-RW03 treated tumors showed increased yH2AX foci and cleaved caspase-3 (a marker for cellular apoptosis) relative to untreated control tumors.

[0162] In FIGS 8A-8E, multiplex IMC was extended to include additional antigens that can be used to distinguish tissues of distinct phenotypes towards applications in tissue barcoding and evaluating treatment response.Multiplexed imaging CyTOF compared to overlayed autoradiography and IHC

[0163] The workflow, resolution, and reliability of multiplexed IMC to visualize TRT and microenvironmental markers of interest in tumors was compared to a conventional method for co- registration of IHC images for different antigens usingadjacent tissue sections and autoradiography (FIG. 9A). Previously, HT-29 tumors from mice treated with [177Lu]Lu-DOTA-RW03 were examined using autoradiography to visualize the distribution of radioactivity in tumors and an attempt was made to coregister these images at low magnifications with adjacent tissue sections stained using IHC for various microenvironmental markers of interest (FIG. 9A). Currently, tissue embedding and IHC services compatible with radioactive tissue are not widely available, and co-registration of autoradiography with IHC images begins with sectioning tumor specimens for analysis through each technique, which introduces considerable organization challenges when assessing multiple sections within a single tumor. Furthermore, for each method, different tissue processing methods may be required (i.e. , flash frozen and formalin) which can lead to differing grosstissue geometry (and co-registration challenges) or tissue data (representative images shown in FIG. 7A). While IHC stains on identically processed sections can be easily co-registered at low-resolutions (FIG. 9A), co-registration at a single-cell level of resolution cannot be readily achieved due to the thickness of adjacent tissue slices created for each IHC stain.

[0164] Due to the low resolutions afforded by conventional phosphor radiography, co-registration of autoradiography and IHC images in adjacent tissue slices is challenging and results provide limited insights into understanding the distribution of TRT within tumors with respect to antigens (target or microenvironmental) of interest (FIG. 9C). At a single-cell level of resolution, results from co-registration of conventional autoradiography with IHC on adjacent tissue sections are meaningless (FIG. 9D). In contrast, IMC enables visualization of multiple antigens on a single (formalin fixed) tissue slide (as shown in FIG. 9E) and through visualizing carrier isotopes delivered to tumors during TRT, IMC perfectly co-registers the intratumoral distribution of TRT and antigens of interest at single cell resolution.Discussion

[0165] The potential utility of therapeutic effects induced by targeted radionuclide therapies directed to tumor subpopulations or the tumor microenvironment (eg., cancer stem cells, bystander effect, abscopal effect, etc.) has not yet fully been realized, in part due to the limited capabilities of conventional radiobiological techniques to visualize radiation and radiation response at cellular-level resolution. In the present example, it is demonstrated that IMC has the capability to image the distribution of metal isotopes delivered by radioimmunotherapy to tumors duringtreatment and showed that these isotopes can be simultaneously imaged with the antigen targeted by the treatment and with epitopes for microenvironmental biomarkers of interest on a single tissue section. While IMC did not afford the sensitivity to detect the daughter nuclide hafnium-177 following in tumors with [177Lu]Lu-DOTA-RW03 (a CD133-targeted radionuclide therapy), carrier isotopes Lu-176 and Lu-175 that were incorporated into radiopharmaceutical during labelling were readily visualized.

[0166] The present example demonstrates that the cellular distribution Lu-175 and Lu-176 carrier atoms is virtually identical in tumors treated with [177Lu]Lu-DOTA- RW03, which strongly indicated that these isotopes represent the distribution of Lu-177 given their identical chemistries. The example approach employed in the present example should be capable of visualizing any radiopharmaceutical where carrier- added isotopes were used for radiolabeling.

[0167] For the work presented in this example, the distribution of [177Lu]Lu-DOTA- RW03 in HT-29 tumors was evaluated by IMC as this tumor model robustly expresses the target antigen (CD133) in the majority of cells. The target antigen CD133 is selective for cancer stem cells in a wide variety of tumors[13-15] and [177Lu]Lu-DOTA-RW03 represents a TRT strategy aimed to eliminate this critical cell subpopulation in refractory tumors.

[0168] To better understand the therapeutic effects of cancer stem cell (CSC) TRT in the preclinical setting, mouse models with orthotopic tumors can be employed to capture both the pharmacokinetic accessibility and microenvironmental factors in the tumor tissue of origin, and patient-derived xenografts (PDX) can be employed that recapitulate patient tumor CSC populations and tumor heterogeneity (i.e. often < 1 % of total cells). Given the powerful capability of IMC to differentiate heterogeneous tumor phenotypes through antigen multiplexing, the present example may be extended to utilize IMC to evaluate CD133 TRT for treatment of PDX mouse models.

[0169] The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the claims are not intended to be limited to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure.REFERENCES1. Giesen C, Wang HAO, Schapiro D, Zivanovic N, Jacobs A, Hattendorf B, et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods. 2014;11 :417-22.2. Elenbaas B, Spirio L, Koerner F, Fleming MD, Zimonjic DB, Donaher JL, et al. Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev. 2001 ;15:50-65.3. Goldsmith SJ. Targeted Radionuclide Therapy: A Historical and Personal Review. Semin Nucl Med. 2020;50:87-97.4. Bilinski P, Webb M. An exceptional response to 177LuPSMA undermined by neuroendocrine transformation. Urol Case Rep. 2020;34:101467.5. Tagawa ST, Sun M, Sartor AO, Thomas C, Singh S, Bissassar M, et al. Phase I study of 225 Ac-J591 for men with metastatic castration-resistant prostate cancer (mCRPC). JCO. 2021 ;39:5015-5015.6. Staudacher AH, Bezak E, Borysenko A, Brown MP. Targeted a-therapy using 227Th-APOMAB and cross-fire antitumour effects: preliminary in-vivo evaluation. Nucl Med Commun. 2014;35:1284-90.7. Solon EG. Autoradiography techniques and quantification of drug distribution. Cell Tissue Res. 2015;360:87-107.8. Natarajan A, Tiirkcan S, Gambhir SS, Pratx G. Multiscale Framework for Imaging Radiolabeled Therapeutics. Mol Pharmaceutics. 2015;12:4554-60.9. Radionuclide targeting and dosimetry at the microscopic level: the role of microautoradiography | SpringerLink [Internet], [cited 2021 Oct 27], Available from: https: / / link.springer.eom / article / 10.1007 / BF0242670110. Cabello J, Bailey A, Kitchen I, Prydderch M, Clark A, Turchetta R, et al. Digital autoradiography using room temperature CCD and CMOS imaging technology. Phys Med Biol. 2007;52:4993-5011.11. Esposito M, Mettivier G, Russo P. 14C autoradiography with an energy-sensitive silicon pixel detector. Phys Med Biol. 2011 ;56:1947-65.12. Tagawa ST, Vallabhajosula S, Christos PJ, Jhanwar YS, Batra JS, Lam L, et al. Phase 1 / 2 study of fractionated dose lutetium-177-labeled anti-prostate-specificmembrane antigen monoclonal antibody J591 ( 177 Lu-J591) for metastatic castration-resistant prostate cancer. Cancer. 2019;125:2561-9.13. Brugnoli F, Grassilli S, Al-Qassab Y, Capitani S, Bertagnolo V. CD133 in Breast Cancer Cells: More than a Stem Cell Marker. J Oncol [Internet], 2019 [cited 2020 Apr 17];2019. Available from: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6766124 / 14. Beier D, Schulz JB, Beier CP. Chemoresistance of glioblastoma cancer stem cells - much more complex than expected. Mol Cancer. 2011 ;10:128.15. Mizrak D, Brittan M, Alison MR. CD133: molecule of the moment. J Pathol. 2008;214:3-9.16. Jackson HW, Fischer JR, Zanotelli VRT, Ali HR, Mechera R, Soysal SD, et al.The single-cell pathology landscape of breast cancer. Nature. 2020;578:615-20.

Claims

CLAIMS1 . A method of characterizing a spatial distribution of a therapeutic radiopharmaceutical within a sample via mass spectrometry imaging, the method comprising: employing a mass spectrometry imaging system to interrogate the sample to obtain a mass spectrometry imaging dataset, the sample having been obtained from a subject having been administered the therapeutic radiopharmaceutical comprising a radionuclide, wherein the therapeutic radiopharmaceutical comprises, or generates via radioactive decay of the radionuclide, a metal isotope detectable by the mass spectrometry imaging system; and processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal isotope within the sample.

2. The method according to claim 1 further comprising associating the spatial distribution of the metal isotope within the sample with the spatial distribution of the radionuclide delivered within the sample.

3. The method according to claim 1 or 2 further comprising generating an image showing the spatial distribution of the metal isotope within the sample.

4. The method according to any one of claims 1 to 3 wherein the therapeutic radiopharmaceutical comprises a radioligand, the radioligand comprising the radionuclide and a targeting ligand configured to specifically bind to a target.

5. The method according to claim 4 wherein the metal isotope is a carrier isotope labelling the targeting ligand, the carrier isotope having been employed during production of the radionuclide.

6. The method according to claim 4 wherein the metal isotope is a decay product of the radionuclide.

7. The method according to claim 6 wherein the decay product is a metastable metal isotope.

8. The method according to claim 4 wherein the metal isotope is the radionuclide.

9. The method according to claim 4 wherein the metal isotope is a stable metal isotope labelling the targeting ligand.

10. The method according to claim 9 wherein the stable metal isotope is not associated with production of the radionuclide.11 . The method according to any one of claims 1 to 3 wherein the therapeutic radiopharmaceutical comprises both the metal isotope and the radionuclide, wherein the metal isotope and the radionuclide are bound to a non-specific carrier molecule.

12. The method according to any one of claims 1 to 3 wherein the metal isotope is a first metal isotope, and wherein the therapeutic radiopharmaceutical further comprises a second metal isotope that is different from the first metal isotope, the second metal isotope being detectable by the mass spectrometry imaging system, and wherein the mass spectrometry imaging dataset is processed to determine a spatial distribution of the second metal isotope within the sample.

13. The method according to claim 12 wherein further comprising employing both the spatial distribution of the first metal isotope within the sample and the spatial distribution of the second metal isotope within the sample to infer a spatial distribution of the radionuclide delivered within the sample.

14. The method according to claim 12 or 13 wherein the therapeutic radiopharmaceutical comprises a radioligand, the radioligand comprising a targeting ligand labeled with the radionuclide, wherein the targeting ligand is configured to specifically bind to a target.

15. The method according to claim 14 wherein the first metal isotope is a first carrier isotope labelling the targeting ligand, and the second metal isotope is a second carrier isotope labelling the targeting ligand, the first carrier isotope and the second carrier isotope having been employed during production of the radionuclide.

16. The method according to claim 14 wherein the first metal isotope is a carrier isotope labelling the targeting ligand, and the second metal isotope is a decay product of the radionuclide, the carrier isotope having been employed during production of the radionuclide.

17. The method according to claim 14 wherein the first metal isotope is a carrier isotope labelling the targeting ligand, and the second metal isotope is the radionuclide, the carrier isotope having been employed during production of the radionuclide.

18. The method according to claim 14 wherein the first metal isotope is a carrier isotope labelling the targeting ligand, and the second metal isotope is a stable metal isotope labeling the radionuclide, the carrier isotope having been employed during production of the radionuclide, wherein the stable metal isotope is not associated with production of the radionuclide.

19. The method according to any one of claims 1 to 18 wherein the sample has been stained with a metal-tagged reagent, the metal-tagged reagent comprising an additional metal isotope conjugated to a biomarker ligand, the additional metal isotope being detectable by the mass spectrometry imaging system, the method further comprising: processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal-tagged reagent within the sample.

20. The method according to claim 19 further comprising generating a multiplexed image showing the spatial distribution of the metal isotope and the spatial distribution of the metal-tagged reagent within the sample.

21. The method according to claim 19 further comprising generating a spatially resolved multi-dimensional measurement characterizing the spatial distribution of the metal isotope and the spatial distribution of the metal-tagged reagent within the sample.

22. The method according to any one of claims 19 to 21 wherein the biomarker ligand is configured to specifically bind to a type of cell specifically targeted by the therapeutic radiopharmaceutical.

23. The method according to claim 22 wherein the biomarker ligand is configured to specifically bind to cancer stem cells.

24. The method according to claim 22 wherein the biomarker ligand is configured to specifically bind to a type of immune cell.

25. The method according to any one of claims 22 to 24 further comprising: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing co-localization of the radionuclide and the type of cell specifically targeted by the therapeutic radiopharmaceutical.

26. The method according to any one of claims 22 to 24 further comprising: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing off-target delivery of the radionuclide.

27. The method according to any one of claims 22 to 24 wherein the type of call targeted by the therapeutic radiopharmaceutical is known to reside in a first tissue region that is associated with pathology, the method further comprising: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing on-target delivery of the radionuclide within a second tissue region.

28. The method according to claim 27 wherein the second tissue region is a healthy tissue region.

29. The method according to any one of claims 19 to 21 wherein the biomarker ligand is configured to specifically bind to a selected subcellular structure.

30. The method according to any one of claims 19 to 21 wherein the biomarker ligand is configured to specifically bind to a marker of microenvironmental cellular response to the therapeutic radiopharmaceutical.31 . The method according to claim 30 further comprising: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing microenvironmental cellular response to therapeutic radiopharmaceutical.

32. The method according to claim 30 or 31 wherein the biomarker ligand is configured to specifically bind to a marker of cellular apoptosis.

33. The method according to claim 30 or 31 wherein the biomarker ligand is configured to specifically bind to a marker of pathway activation.

34. The method according to claim 30 or 31 wherein the biomarker ligand is configured to specifically bind to a marker associated with a resistance mechanism.

35. The method according to any one of claims 1 to 34 wherein the therapeutic radiopharmaceutical comprises a metal nanoparticle formed from the metal isotope, the radionuclide being bound to the nanoparticle.

36. The method according to any one of claims 1 to 34 wherein the mass spectrometry imaging system is an imaging mass cytometry system.

37. The method according to any one of claims 1 to 34 wherein the mass spectrometry imaging system is a multiplexed ion beam imaging system.

38. A method of characterizing a spatial distribution of a diagnostic radiopharmaceutical within a sample via mass spectrometry imaging, the method comprising:employing a mass spectrometry imaging system to interrogate the sample to obtain a mass spectrometry imaging dataset, the sample having been obtained from a subject having been administered the diagnostic radiopharmaceutical comprising a radionuclide, wherein the diagnostic radiopharmaceutical comprises, or generates via radioactive decay of the radionuclide, a metal isotope detectable by the mass spectrometry imaging system; and processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal isotope within the sample.

39. A method of characterizing a spatial distribution of a pharmaceutical within a sample via mass spectrometry imaging, the method comprising: employing a mass spectrometry imaging system to interrogate the sample to obtain a mass spectrometry imaging dataset, the sample having been obtained from a subject having been administered the pharmaceutical, wherein the pharmaceutical is non-radioactive and further comprises a passive, non-therapeutic metal isotope detectable by the mass spectrometry imaging system; and processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal isotope within the sample.

40. The method according to claim 39 wherein the pharmaceutical comprises a therapeutic payload that is absent of a metal.

41. The method according to claim 39 wherein the pharmaceutical comprises a therapeutic payload that includes a metal that is different from the non-therapeutic metal isotope.

42. The method according to claim 39 wherein the pharmaceutical is a diagnostic or theranostic reagent labeled with the passive, non-therapeutic metal isotope.

43. The method according to any one of claims 39 to 42 further comprising associating the spatial distribution of the metal isotope within the sample with the spatial distribution of the pharmaceutical delivered within the sample.

44. The method according to any one of claims 39 to 43 further comprising generating an image showing the spatial distribution of the metal isotope within the sample.

45. The method according to any one of claims 39 to 44 wherein the pharmaceutical comprises a targeting ligand configured to specifically bind to a target.

46. The method according to any one of claims 39 to 45 wherein the sample has been stained with a metal-tagged reagent, the metal-tagged reagent comprising an additional metal isotope conjugated to a biomarker ligand, the additional metal isotope being detectable by the mass spectrometry imaging system, the method further comprising: processing the mass spectrometry imaging dataset to determine a spatial distribution of the metal-tagged reagent within the sample.

47. The method according to claim 46 further comprising generating a multiplexed image showing the spatial distribution of the metal isotope and the spatial distribution of the metal-tagged reagent within the sample.

48. The method according to claim 46 further comprising generating a spatially resolved multi-dimensional measurement characterizing the spatial distribution of the metal isotope and the spatial distribution of the metal-tagged reagent within the sample.

49. The method according to any one of claims 46 to 48 wherein the biomarker ligand is configured to specifically bind to a type of cell specifically targeted by the pharmaceutical.

50. The method according to claim 49 wherein the biomarker ligand is configured to specifically bind to cancer stem cells.51 . The method according to claim 49 wherein the biomarker ligand is configured to specifically bind to a type of immune cell.

52. The method according to any one of claims 49 to 51 further comprising: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing co-localization of the pharmaceutical and the type of cell specifically targeted by the pharmaceutical.

53. The method according to any one of claims 49 to 51 further comprising: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing off-target delivery of the pharmaceutical.

54. The method according to any one of claims 49 to 51 wherein the type of call targeted by the pharmaceutical is known to reside in a first tissue region that is associated with pathology, the method further comprising: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing on-target delivery of the pharmaceutical within a second tissue region.

55. The method according to claim 54 wherein the second tissue region is a healthy tissue region.

56. The method according to any one of claims 46 to 48 wherein the biomarker ligand is configured to specifically bind to a selected subcellular structure.

57. The method according to any one of claims 46 to 48 wherein the biomarker ligand is configured to specifically bind to a marker of microenvironmental cellular response to the pharmaceutical.

58. The method according to claim 57 further comprising: processing the spatial distribution of the metal isotope within the sample and the spatial distribution of the metal-tagged reagent within the sample to generate a measure characterizing microenvironmental cellular response to pharmaceutical.

59. The method according to claim 57 or 58 wherein the biomarker ligand is configured to specifically bind to a marker of cellular apoptosis.

60. The method according to claim 57 or 58 wherein the biomarker ligand is configured to specifically bind to a marker of pathway activation.61 . The method according to claim 57 or 58 wherein the biomarker ligand is configured to specifically bind to a marker associated with a resistance mechanism.

62. The method according to any one of claims 39 to 61 wherein the mass spectrometry imaging system is an imaging mass cytometry system.

63. The method according to any one of claims 39 to 61 wherein the mass spectrometry imaging system is a multiplexed ion beam imaging system.

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