Metabolic imaging across scales reveals distinct prostate cancer phenotypes.

Hyperpolarised magnetic resonance imaging (HP-13C-MRI) has shown promise as a clinical tool for detecting and characterising prostate cancer. Here we use a range of spatially resolved histological techniques to identify the biological mechanisms underpinning differential [1-13C]lactate labelling between benign and malignant prostate, as well as in tumours containing cribriform and non-cribriform Gleason pattern 4 disease. Here we show that elevated hyperpolarised [1-13C]lactate signal in prostate cancer compared to the benign prostate is primarily driven by increased tumour epithelial cell density and vascularity, rather than differences in epithelial lactate concentration between tumour and normal. We also demonstrate that some tumours of the cribriform subtype may lack [1-13C]lactate labelling, which is explained by lower epithelial lactate dehydrogenase expression, higher mitochondrial pyruvate carrier density, and increased lipid abundance compared to lactate-rich non-cribriform lesions. These findings highlight the potential of combining spatial metabolic imaging tools across scales to identify clinically significant metabolic phenotypes in prostate cancer.

Multi-omics and imaging mass cytometry characterization of human kidneys to identify pathways and phenotypes associated with impaired kidney function.

Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.

Multi-modal molecular imaging maps the correlation between tumor microenvironments and nanomedicine distribution.

Gaining insight into the heterogeneity of nanoparticle drug distribution within tumors would improve both design and clinical translation of nanomedicines. There is little data showing the spatio-temporal behavior of nanomedicines in tissues as current methods are not able to provide a comprehensive view of the nanomedicine distribution, released drug or its effects in the context of a complex tissue microenvironment. Methods: A new experimental approach which integrates the molecular imaging and bioanalytical technologies MSI and IMC was developed to determine the biodistribution of total drug and drug metabolite delivered via PLA-PEG nanoparticles and to overlay this with imaging of the nanomedicine in the context of detailed tumor microenvironment markers. This was used to assess the nanomedicine AZD2811 in animals bearing three different pre-clinical PDX tumors. Results: This new approach delivered new insights into the nanoparticle/drug biodistribution. Mass spectrometry imaging was able to differentiate the tumor distribution of co-dosed deuterated non-nanoparticle-formulated free drug alongside the nanoparticle-formulated drug by directly visualizing both delivery approaches within the same animal or tissue. While the IV delivered free drug was uniformly distributed, the nanomedicine delivered drug was heterogeneous. By staining for multiple biomarkers of the tumor microenvironment on the same tumor sections using imaging mass cytometry, co-registering and integrating data from both imaging modalities it was possible to determine the features in regions with highest nanomedicine distribution. Nanomedicine delivered drug was associated with regions higher in macrophages, as well as more stromal regions of the tumor. Such a comparison of complementary molecular data allows delineation of drug abundance in individual cell types and in stroma. Conclusions: This multi-modal imaging solution offers researchers a better understanding of drug and nanocarrier distribution in complex tissues and enables data-driven drug carrier design.

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

Comparison of Osteosarcoma Aggregated Tumour Models with Human Tissue by Multimodal Mass Spectrometry Imaging.

Osteosarcoma (OS) is the most common primary bone malignancy and largely effects adolescents and young adults, with 60% of patients under the age of 25. There are multiple cell models of OS described in vitro that express the specific genetic alterations of the sarcoma. In the work reported here, multiple mass spectrometry imaging (MSI) modalities were employed to characterise two aggregated cellular models of OS models formed using the MG63 and SAOS-2 cell lines. Phenotyping of the metabolite activity within the two OS aggregoid models was achieved and a comparison of the metabolite data with OS human tissue samples revealed relevant fatty acid and phospholipid markers. Although, annotations of these species require MS/MS analysis for confident identification of the metabolites. From the putative assignments however, it was suggested that the MG63 aggregoids are an aggressive tumour model that exhibited metastatic-like potential. Alternatively, the SAOS-2 aggregoids are more mature osteoblast-like phenotype that expressed characteristics of cellular differentiation and bone development. It was determined the two OS aggregoid models shared similarities of metabolic behaviour with different regions of OS human tissues, specifically of the higher metastatic grade.

Intra- and intercellular signaling pathways associated with drug-induced cardiac pathophysiology.

Cardiac physiology and homeostasis are maintained by the interaction of multiple cell types, via both intra- and intercellular signaling pathways. Perturbations in these signaling pathways induced by oncology therapies can reduce cardiac function, ultimately leading to heart failure. As cancer survival increases, related cardiovascular complications are becoming increasingly prevalent, thus identifying the perturbations and cell signaling drivers of cardiotoxicity is increasingly important. Here, we discuss the homotypic and heterotypic cellular interactions that form the basis of intra- and intercellular cardiac signaling pathways, and how oncological agents disrupt these pathways, leading to heart failure. We also highlight the emerging systems biology techniques that can be applied, enabling a deeper understanding of the intra- and intercellular signaling pathways across multiple cell types associated with cardiovascular toxicity.

Evaluation of UV-C Decontamination of Clinical Tissue Sections for Spatially Resolved Analysis by Mass Spectrometry Imaging (MSI).

Clinical tissue specimens are often unscreened, and preparation of tissue sections for analysis by mass spectrometry imaging (MSI) can cause aerosolization of particles potentially carrying an infectious load. We here present a decontamination approach based on ultraviolet-C (UV-C) light to inactivate clinically relevant pathogens such as herpesviridae, papovaviridae human immunodeficiency virus, or SARS-CoV-2, which may be present in human tissue samples while preserving the biodistributions of analytes within the tissue. High doses of UV-C required for high-level disinfection were found to cause oxidation and photodegradation of endogenous species. Lower UV-C doses maintaining inactivation of clinically relevant pathogens to a level of increased operator safety were found to be less destructive to the tissue metabolome and xenobiotics. These doses caused less alterations of the tissue metabolome and allowed elucidation of the biodistribution of the endogenous metabolites. Additionally, we were able to determine the spatially integrated abundances of the ATR inhibitor ceralasertib from decontaminated human biopsies using desorption electrospray ionization-MSI (DESI-MSI).

Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 µm spatial resolution. Protein markers of proliferation (Ki-67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development.

Universal Sample Preparation Unlocking Multimodal Molecular Tissue Imaging.

A new tissue sample embedding and processing method is presented that provides downstream compatibility with numerous different histological, molecular biology, and analytical techniques. The methodology is based on the low temperature embedding of fresh frozen specimens into a hydrogel matrix composed of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) and sectioning using a cryomicrotome. The hydrogel was expected not to interfere with standard tissue characterization methods, histologically or analytically. We assessed the compatibility of this protocol with various mass spectrometric imaging methods including matrix-assisted laser desorption ionization (MALDI), desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS). We also demonstrated the suitability of the universal protocol for extraction based molecular biology techniques such as rt-PCR. The integration of multiple analytical modalities through this universal sample preparation protocol offers the ability to study tissues at a systems biology level and directly linking results to tissue morphology and cellular phenotype.

Pharmacology of the ATM Inhibitor AZD0156: Potentiation of Irradiation and Olaparib Responses Preclinically.

AZD0156 is a potent and selective, bioavailable inhibitor of ataxia-telangiectasia mutated (ATM) protein, a signaling kinase involved in the DNA damage response. We present preclinical data demonstrating abrogation of irradiation-induced ATM signaling by low doses of AZD0156, as measured by phosphorylation of ATM substrates. AZD0156 is a strong radiosensitizer in vitro, and using a lung xenograft model, we show that systemic delivery of AZD0156 enhances the tumor growth inhibitory effects of radiation treatment in vivo Because ATM deficiency contributes to PARP inhibitor sensitivity, preclinically, we evaluated the effect of combining AZD0156 with the PARP inhibitor olaparib. Using ATM isogenic FaDu cells, we demonstrate that AZD0156 impedes the repair of olaparib-induced DNA damage, resulting in elevated DNA double-strand break signaling, cell-cycle arrest, and apoptosis. Preclinically, AZD0156 potentiated the effects of olaparib across a panel of lung, gastric, and breast cancer cell lines in vitro, and improved the efficacy of olaparib in two patient-derived triple-negative breast cancer xenograft models. AZD0156 is currently being evaluated in phase I studies (NCT02588105).

AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation, chemotherapy and olaparib activity.

DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.

The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves survival of preclinical brain tumor models.

Poor survival rates of patients with tumors arising from or disseminating into the brain are attributed to an inability to excise all tumor tissue (if operable), a lack of blood-brain barrier (BBB) penetration of chemotherapies/targeted agents, and an intrinsic tumor radio-/chemo-resistance. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR. We report the primary pharmacology of the clinical-grade, exquisitely potent (cell IC50, 0.78 nM), highly selective [>10,000-fold over kinases within the same phosphatidylinositol 3-kinase-related kinase (PIKK) family], orally bioavailable ATM inhibitor AZD1390 specifically optimized for BBB penetration confirmed in cynomolgus monkey brain positron emission tomography (PET) imaging of microdosed 11C-labeled AZD1390 (Kp,uu, 0.33). AZD1390 blocks ATM-dependent DDR pathway activity and combines with radiation to induce G2 cell cycle phase accumulation, micronuclei, and apoptosis. AZD1390 radiosensitizes glioma and lung cancer cell lines, with p53 mutant glioma cells generally being more radiosensitized than wild type. In in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models, AZD1390 dosed in combination with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induced tumor regressions and increased animal survival compared to IR treatment alone. We established a pharmacokinetic-pharmacodynamic-efficacy relationship by correlating free brain concentrations, tumor phospho-ATM/phospho-Rad50 inhibition, apoptotic biomarker (cleaved caspase-3) induction, tumor regression, and survival. On the basis of the data presented here, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies.

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.

Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT.