Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment.

ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.

Radiolabelling of Polyclonally Expanded Human Regulatory T Cells (Treg) with 89Zr-oxine for Medium-Term In Vivo Cell Tracking.

Regulatory T cells (Tregs) are a promising candidate cell therapy to treat autoimmune diseases and aid the longevity of transplanted solid organs. Despite increasing numbers of clinical trials using human Treg therapy, important questions pertaining to their in vivo fate, distribution, and function remain unanswered. Treg accumulation in relevant tissues was found to be crucial for Treg therapy efficacy, but existing blood-borne biomarkers are unlikely to accurately reflect the tissue state. Non-invasive Treg tracking by whole-body imaging is a promising alternative and can be achieved by direct radiolabelling of Tregs and following the radiolabelled cells with positron emission tomography (PET). Our goal was to evaluate the radiolabelling of polyclonal Tregs with 89Zr to permit their in vivo tracking by PET/CT for longer than one week with current preclinical PET instrumentation. We used [89Zr]Zr(oxinate)4 as the cell-labelling agent and achieved successful radiolabelling efficiency of human Tregs spanning 0.1-11.1 Bq 89Zr/Treg cell, which would be compatible with PET tracking beyond one week. We characterized the 89Zr-Tregs, assessing their phenotypes, and found that they were not tolerating these intracellular 89Zr amounts, as they failed to survive or expand in a 89Zr-dose-dependent manner. Even at 0.1 Bq 89Zr per Treg cell, while 89Zr-Tregs remained functional as determined by a five-day-long effector T cell suppression assay, they failed to expand beyond day 3 in vitro. Moreover, PET imaging revealed signs of 89Zr-Treg death after adoptive transfer in vivo. In summary, 89Zr labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as 89Zr-Tregs failed to expand and survive. Consequently, we conclude that indirect Treg labelling is likely to be the most effective alternative method to satisfy the requirements of this cell tracking scenario.

Chimeric antigen receptor-modified human regulatory T cells that constitutively express IL-10 maintain their phenotype and are potently suppressive.

Clinical trials of Treg therapy in transplantation are currently entering phases IIa and IIb, with the majority of these employing polyclonal Treg populations that harbor a broad specificity. Enhancing Treg specificity is possible with the use of chimeric antigen receptors (CARs), which can be customized to respond to a specific human leukocyte antigen (HLA). In this study, we build on our previous work in the development of HLA-A2 CAR-Tregs by further equipping cells with the constitutive expression of interleukin 10 (IL-10) and an imaging reporter as additional payloads. Cells were engineered to express combinations of these domains and assessed for phenotype and function. Cells expressing the full construct maintained a stable phenotype after transduction, were specifically activated by HLA-A2, and suppressed alloresponses potently. The addition of IL-10 provided an additional advantage to suppressive capacity. This study therefore provides an important proof-of-principle for this cell engineering approach for next-generation Treg therapy in transplantation.

PET Imaging of Small Extracellular Vesicles via [89Zr]Zr(oxinate)4 Direct Radiolabeling.

Exosomes or small extracellular vesicles (sEVs) are increasingly gaining attention for their potential as drug delivery systems and biomarkers of disease. Therefore, it is important to understand their in vivo biodistribution using imaging techniques that allow tracking over time and at the whole-body level. Positron emission tomography (PET) allows short- and long-term whole-body tracking of radiolabeled compounds in both animals and humans and with excellent quantification properties compared to other nuclear imaging techniques. In this report, we explored the use of [89Zr]Zr(oxinate)4 (a cell and liposome radiotracer) for direct and intraluminal radiolabeling of several types of sEVs, achieving high radiolabeling yields. The radiosynthesis and radiolabeling protocols were optimized for sEV labeling, avoiding sEV damage, as demonstrated using several characterizations (cryoEM, nanoparticle tracking analysis, dot blot, and flow cytometry) and in vitro techniques. Using pancreatic cancer sEVs (PANC1) in a healthy mouse model, we showed that it is possible to track 89Zr-labeled sEVs in vivo using PET imaging for at least up to 24 h. We also report differential biodistribution of intact sEVs compared to intentionally heat-damaged sEVs, with significantly reduced spleen uptake for the latter. Therefore, we conclude that 89Zr-labeled sEVs using this method can reliably be used for in vivo PET tracking and thus allow efficient exploration of their potential as drug delivery systems.

Radiolabelling Pt-based quadruplex DNA binders via click chemistry.

Guanine-rich sequences of DNA and RNA can fold into intramolecular tetra-helical assemblies known as G-quadruplexes (G4). Their formation in vivo has been associated to a range of biological functions and therefore they have been identified as potential drug targets. Consequently, a broad range of small molecules have been developed to target G4s. Amongst those are metal complexes with Schiff base ligands. Herein, we report the functionalisation of one of these well-established G4 DNA binders (based on a square planar platinum(II)-salphen complex) with two different radiolabelled complexes. An 111In-conjugate was successfully used to assess its in vivo distribution in a mouse tumour model using single-photon emission computed tomography (SPECT) imaging. These studies highlighted the accumulation of this Pt-salphen-111In conjugate in the tumour.

A preclinical pipeline to evaluate migrastatics as therapeutic agents in metastatic melanoma.

BACKGROUND: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancers' ability to metastasise. First anti-metastatic treatments have recently been approved. METHODS: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues. RESULTS: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology. CONCLUSIONS: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging.

Options for imaging cellular therapeutics in vivo: a multi-stakeholder perspective.

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics.

Non-invasive Reporter Gene Imaging of Cell Therapies, including T Cells and Stem Cells.

Cell therapies represent a rapidly emerging class of new therapeutics. They are intended and developed for the treatment of some of the most prevalent human diseases, including cancer, diabetes, and for regenerative medicine. Currently, they are largely developed without precise assessment of their in vivo distribution, efficacy, or survival either clinically or preclinically. However, it would be highly beneficial for both preclinical cell therapy development and subsequent clinical use to assess these parameters in situ to enable enhancements in efficacy, applicability, and safety. Molecular imaging can be exploited to track cells non-invasively on the whole-body level and can enable monitoring for prolonged periods in a manner compatible with rapidly expanding cell types. In this review, we explain how in vivo imaging can aid the development and clinical translation of cell-based therapeutics. We describe the underlying principles governing non-invasive in vivo long-term cell tracking in the preclinical and clinical settings, including available imaging technologies, reporter genes, and imaging agents as well as pitfalls related to experimental design. Our emphasis is on adoptively transferred T cell and stem cell therapies.

Spatiotemporal PET Imaging Reveals Differences in CAR-T Tumor Retention in Triple-Negative Breast Cancer Models.

Chimeric antigen receptor T cell therapy (CAR-T) has been rolled out as a new treatment for hematological malignancies. For solid tumor treatment, CAR-T has been disappointing so far. Challenges include the quantification of CAR-T trafficking, expansion and retention in tumors, activity at target sites, toxicities, and long-term CAR-T survival. Non-invasive serial in vivo imaging of CAR-T using reporter genes can address several of these challenges. For clinical use, a non-immunogenic reporter that is detectable with exquisite sensitivity by positron emission tomography (PET) using a clinically available non-toxic radiotracer would be beneficial. Here, we employed the human sodium iodide symporter to non-invasively quantify tumor retention of pan-ErbB family targeted CAR-T by PET. We generated and characterized traceable CAR T cells and examined potential negative effects of radionuclide reporter use. We applied our platform to two different triple-negative breast cancer (TNBC) models and unexpectedly observed pronounced differences in CAR-T tumor retention by PET/CT (computed tomography) and confirmed data ex vivo. CAR-T tumor retention inversely correlated with immune checkpoint expression in the TNBC models. Our platform enables highly sensitive non-invasive PET tracking of CAR-T thereby addressing a fundamental unmet need in CAR-T development and offering to provide missing information needed for future clinical CAR-T imaging.

Relationship of In Vitro Toxicity of Technetium-99m to Subcellular Localisation and Absorbed Dose.

Auger electron-emitters increasingly attract attention as potential radionuclides for molecular radionuclide therapy in oncology. The radionuclide technetium-99m is widely used for imaging; however, its potential as a therapeutic radionuclide has not yet been fully assessed. We used MDA-MB-231 breast cancer cells engineered to express the human sodium iodide symporter-green fluorescent protein fusion reporter (hNIS-GFP; MDA-MB-231.hNIS-GFP) as a model for controlled cellular radionuclide uptake. Uptake, efflux, and subcellular location of the NIS radiotracer [99mTc]TcO4- were characterised to calculate the nuclear-absorbed dose using Medical Internal Radiation Dose formalism. Radiotoxicity was determined using clonogenic and γ-H2AX assays. The daughter radionuclide technetium-99 or external beam irradiation therapy (EBRT) served as controls. [99mTc]TcO4- in vivo biodistribution in MDA-MB-231.hNIS-GFP tumour-bearing mice was determined by imaging and complemented by ex vivo tissue radioactivity analysis. [99mTc]TcO4- resulted in substantial DNA damage and reduction in the survival fraction (SF) following 24 h incubation in hNIS-expressing cells only. We found that 24,430 decays/cell (30 mBq/cell) were required to achieve SF0.37 (95%-confidence interval = [SF0.31; SF0.43]). Different approaches for determining the subcellular localisation of [99mTc]TcO4- led to SF0.37 nuclear-absorbed doses ranging from 0.33 to 11.7 Gy. In comparison, EBRT of MDA-MB-231.hNIS-GFP cells resulted in an SF0.37 of 2.59 Gy. In vivo retention of [99mTc]TcO4- after 24 h remained high at 28.0% ± 4.5% of the administered activity/gram tissue in MDA-MB-231.hNIS-GFP tumours. [99mTc]TcO4- caused DNA damage and reduced clonogenicity in this model, but only when the radioisotope was taken up into the cells. This data guides the safe use of technetium-99m during imaging and potential future therapeutic applications.

In Vivo PET Tracking of 89Zr-Labeled Vγ9Vδ2 T Cells to Mouse Xenograft Breast Tumors Activated with Liposomal Alendronate.

Gammadelta T (γδ-T) cells are strong candidates for adoptive immunotherapy in oncology due to their cytotoxicity, ease of expansion, and favorable safety profile. The development of γδ-T cell therapies would benefit from non-invasive cell-tracking methods and increased targeting to tumor sites. Here we report the use of [89Zr]Zr(oxinate)4 to track Vγ9Vδ2 T cells in vivo by positron emission tomography (PET). In vitro, we showed that 89Zr-labeled Vγ9Vδ2 T cells retained their viability, proliferative capacity, and anti-cancer cytotoxicity with minimal DNA damage for amounts of 89Zr ≤20 mBq/cell. Using a mouse xenograft model of human breast cancer, 89Zr-labeled γδ-T cells were tracked by PET imaging over 1 week. To increase tumor antigen expression, the mice were pre-treated with PEGylated liposomal alendronate. Liposomal alendronate, but not placebo liposomes or non-liposomal alendronate, significantly increased the 89Zr signal in the tumors, suggesting increased homing of γδ-T cells to the tumors. γδ-T cell trafficking to tumors occurred within 48 hr of administration. The presence of γδ-T cells in tumors, liver, and spleen was confirmed by histology. Our results demonstrate the suitability of [89Zr]Zr(oxinate)4 as a cell-labeling agent for therapeutic T cells and the potential benefits of liposomal bisphosphonate treatment before γδ-T cell administration.

Hydrodynamic Radii of Ranibizumab, Aflibercept and Bevacizumab Measured by Time-Resolved Phosphorescence Anisotropy.

PURPOSE: To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with µs time-resolved phosphorescence anisotropy. METHODS: Ruthenium-based dye Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2, whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity. RESULTS: The measured radii of 2.76±0.04 nm for ranibizumab, 3.70±0.03 nm for aflibercept and 4.58±0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements. CONCLUSIONS: Time-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.

Imaging tumour heterogeneity of the consequences of a PKCα-substrate interaction in breast cancer patients.

Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein-protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC-ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically <10 nm range to address the oncological challenge of tumour heterogeneity, is discussed.

3D super-resolution imaging with blinking quantum dots.

Quantum dots are promising candidates for single molecule imaging due to their exceptional photophysical properties, including their intense brightness and resistance to photobleaching. They are also notorious for their blinking. Here we report a novel way to take advantage of quantum dot blinking to develop an imaging technique in three-dimensions with nanometric resolution. We first applied this method to simulated images of quantum dots and then to quantum dots immobilized on microspheres. We achieved imaging resolutions (fwhm) of 8-17 nm in the x-y plane and 58 nm (on coverslip) or 81 nm (deep in solution) in the z-direction, approximately 3-7 times better than what has been achieved previously with quantum dots. This approach was applied to resolve the 3D distribution of epidermal growth factor receptor (EGFR) molecules at, and inside of, the plasma membrane of resting basal breast cancer cells.

Genetic Engineering of Therapeutic Cells with the Sodium Iodide Symporter (NIS) to Enable Noninvasive In Vivo Therapy Tracking.

Noninvasive long-term imaging of therapeutic cells in preclinical models can be achieved through introducing a reporter gene into the cells of interest. Despite important recent developments such as gene editing, cell engineering based on lentiviruses remains a mainstream tool for gene transfer applicable to a variety of different cell types.In this chapter, we describe how to use lentivirus-based genetic engineering to render different candidate cell therapies in vivo traceable by radionuclide imaging. We illustrate this reporter gene technology using the sodium iodide symporter (NIS), which is compatible with both positron emission tomography (PET) and single-photon emission computed tomography (SPECT). For preclinical experimentation, we fused NIS with a suitable fluorescent protein such as monomeric GFP or RFP to streamline cell line generation and downstream analyses of ex vivo tissue samples. We present protocols for reporter gene engineering of human cardiac progenitor cells, regulatory T cells, and effector T cells as well as for the characterization experiments required to validate NIS-fluorescent protein reporter function in these candidate therapeutic cells.

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer.

BACKGROUND: Small interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. METHODS: PEG12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. RESULTS: Our results showed that PEG12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. CONCLUSION: In conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

In Vitro and Preclinical Systematic Dose-Effect Studies of Auger Electron- and ß Particle-Emitting Radionuclides and External Beam Radiation for Cancer Treatment.

PURPOSE: Despite a rise in clinical use of radiopharmaceutical therapies, the biological effects of radionuclides and their relationship with absorbed radiation dose are poorly understood. Here, we set out to define this relationship for Auger electron emitters [99mTc]TcO4- and [123I]I- and ß--particle emitter [188Re]ReO4-. Studies were carried out using genetically modified cells that permitted direct radionuclide comparisons. METHODS AND MATERIALS: Triple-negative MDA-MB-231 breast cancer cells expressing the human sodium iodide symporter (hNIS) and green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used. In vitro radiotoxicity of [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- was determined using clonogenic assays. Radionuclide uptake, efflux, and subcellular location were used to calculate nuclear absorbed doses using the Medical Internal Radiation Dose (MIRD) formalism. In vivo studies were performed using female NSG mice bearing orthotopic MDA-MB-231.hNIS-GFP tumors and compared with X-ray-treated (12.6-15 Gy) and untreated cohorts. Absorbed dose per unit activity in tumors and sodium iodide symporter-expressing organs was extrapolated to reference human adult models using OLINDA/EXM. RESULTS: [99mTc]TcO4- and [123I]I- reduced the survival fraction only in hNIS-expressing cells, whereas [188Re]ReO4- reduced survival fraction in hNIS-expressing and parental cells. [123I]I- required 2.4- and 1.5-fold lower decays/cell to achieve 37% survival compared with [99mTc]TcO4- and [188Re]ReO4-, respectively, after 72 hours of incubation. Additionally, [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- had superior cell killing effectiveness in vitro compared with X-rays. In vivo, X-ray led to a greater median survival compared with [188Re]ReO4- and [123I]I- (54 days vs 45 and 43 days, respectively). Unlike the X-ray cohort, no metastases were visualized in the radionuclide-treated cohorts. Extrapolated human absorbed doses of [188Re]ReO4- to a 1 g tumor were 13.8- and 11.2-fold greater than for [123I]I- in female and male models, respectively. CONCLUSIONS: This work reports reference dose-effect data using cell and tumor models for [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- for the first time. We further demonstrate the tumor-controlling effects of [123I]I- and [188Re]ReO4- in comparison with external beam radiation therapy.

Multi-scale in vivo imaging of tumour development using a germline conditional triple-reporter system.

Imaging reporter genes are indispensable for visualising biological processes in living subjects, particularly in cancer research where they have been used to observe tumour development, cancer cell dissemination, and treatment response. Engineering reporter genes into the germline frequently involves single imaging modality reporters operating over limited spatial scales. To address these limitations, we developed an inducible triple-reporter mouse model (Rosa26LSL - NRL) that integrates reporters for complementary imaging modalities, flfluorescence, bioluminescence and positron emission tomography (PET), along with inducible Cre-lox functionality for precise spatiotemporal control of reporter expression. We demonstrated robust reporter inducibility across various tissues in the Rosa26LSL - NRL mouse, facilitating effective tracking and characterisation of tumours in liver and lung cancer mouse models. We precisely pinpointed tumour location using multimodal whole-body imaging which guided in situ lung microscopy to visualise cell-cell interactions within the tumour microenvironment. The triple-reporter system establishes a robust new platform technology for multi-scale investigation of biological processes within whole animals, enabling tissue-specific and sensitive cell tracking, spanning from the whole-body to cellular scales.

Combined Vorinostat and Chloroquine Inhibit Sodium-Iodide Symporter Endocytosis and Enhance Radionuclide Uptake In Vivo.

PURPOSE: Patients with aggressive thyroid cancer are frequently failed by the central therapy of ablative radioiodide (RAI) uptake, due to reduced plasma membrane (PM) localization of the sodium/iodide symporter (NIS). We aimed to understand how NIS is endocytosed away from the PM of human thyroid cancer cells, and whether this was druggable in vivo. EXPERIMENTAL DESIGN: Informed by analysis of endocytic gene expression in patients with aggressive thyroid cancer, we used mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake, and NanoBRET to understand the mechanisms of NIS endocytosis in transformed cell lines and patient-derived human primary thyroid cells. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and gene expression in BALB/c mice. RESULTS: We identified an acidic dipeptide within the NIS C-terminus that mediates binding to the σ2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine (CQ) modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor vorinostat/SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. CONCLUSIONS: NIS internalization is specifically druggable in vivo. Our data, therefore, provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. See related commentary by Lechner and Brent, p. 1220.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.