Targeted radionuclide therapy against GARP expressing T regulatory cells after tumour priming with external beam radiotherapy in a murine syngeneic model.

Purpose: Radiation therapy (RT) exerts its anti-tumour efficacy by inducing direct damage to cancer cells but also through modification of the tumour microenvironment (TME) by inducing immunogenic antitumor response. Conversely, RT also promotes an immunosuppressive TME notably through the recruitment of regulatory T cells (Tregs). Glycoprotein A repetitions predominant (GARP), a transmembrane protein highly expressed by activated Tregs, plays a key role in the activation of TGF-ß and thus promotes the immunosuppressive action of Tregs. The development of a theranostic approach targeting GARP combining imaging and targeted radionuclide therapy (TRT) was carried out. Methods: A preclinical model of 4T1 triple negative breast tumour-bearing BALB/c mice was used to show that GARP expression is increased after external beam radiation in the TME of our cancer model. We generated a theranostic probe through the bioconjugation of the chelating agent DOTAGA onto an anti-GARP monoclonal antibody. The bioconjugation with DOTAGA allows the radiolabelling of the DOTAGA-GARP conjugate with both Indium-111 for SPECT imaging and Lutetium-177 for TRT purposes. Results: We demonstrate that GARP expression is increased following RT in vivo and can be specifically detected and quantified using in vivo SPECT imaging with [111In]In-DOTAGA-GARP. In addition, 177Lu-DOTAGA-GARP limits tumour growth in our cancer model. Conclusion: This theranostic strategy may allow for the personalization of cancer treatments by early detection of activated Tregs infiltration following RT and identification of patients likely to respond to Tregs-targeted therapy via TRT.

CD206+ macrophages are relevant non-invasive imaging biomarkers and therapeutic targets in experimental lung fibrosis.

BACKGROUND: Interstitial lung diseases (ILDs) include a large number of diseases associated with progressive pulmonary fibrosis (PPF), including idiopathic pulmonary fibrosis (IPF). Despite the rarity of each of the fibrotic ILDs individually, they cumulatively affect a considerable number of patients. PPF is characterised by an excessive collagen deposition leading to functional decline. OBJECTIVES: Therapeutic options are limited to nintedanib and pirfenidone which are only able to reduce fibrosis progression. CD206-expressing M2 macrophages are involved in fibrosis progression, and whether they may be relevant therapeutic targets or biomarkers remains an open question. RESULTS: In our study, CD206+ lung macrophages were monitored in bleomycin-induced lung fibrosis in mice by combining flow cytometry, scRNAseq and in vivo molecular imaging using a single photon emission computed tomography (SPECT) radiopharmaceutical, 99mTc-tilmanocept. The antifibrotic effect of the inhibition of M2 macrophage polarisation with a JAK inhibitor, tofacitinib, was assessed in vivo. We demonstrate that CD206-targeted in vivo SPECT imaging with 99mTc-tilmanocept was able to accurately detect and quantify the increase in CD206+ macrophages from early to advanced stages of experimental fibrosis and ex vivo in lung biopsies from patients with IPF. CD206-targeted imaging also specifically detected a decrease in CD206+ lung macrophages on nintedanib and tofacitinib treatment. Importantly, early in vivo imaging of CD206+ macrophages allowed the prediction of experimental lung fibrosis progression as well as nintedanib and tofacitinib efficacy. CONCLUSIONS: These findings indicate that M2 macrophages may be relevant theranostic targets for personalised medicine for patients with PPF.

The GRP94 Inhibitor PU-WS13 Decreases M2-like Macrophages in Murine TNBC Tumors: A Pharmaco-Imaging Study with 99mTc-Tilmanocept SPECT.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancers and is not eligible for hormone and anti-HER2 therapies. Identifying therapeutic targets and associated biomarkers in TNBC is a clinical challenge to improve patients' outcome and management. High infiltration of CD206+ M2-like macrophages in the tumor microenvironment (TME) indicates poor prognosis and survival in TNBC patients. As we previously showed that membrane expression of GRP94, an endoplasmic reticulum chaperone, was associated with the anti-inflammatory profile of human PBMC-derived M2 macrophages, we hypothesized that intra-tumoral CD206+ M2 macrophages expressing GRP94 may represent innovative targets in TNBC for theranostic purposes. We demonstrate in a preclinical model of 4T1 breast tumor-bearing BALB/c mice that (i) CD206-expressing M2-like macrophages in the TME of TNBC can be specifically detected and quantified using in vivo SPECT imaging with 99mTc-Tilmanocept, and (ii) the inhibition of GRP94 with the chemical inhibitor PU-WS13 induces a decrease in CD206-expressing M2-like macrophages in TME. This result correlated with reduced tumor growth and collagen content, as well as an increase in CD8+ cells in the TME. 99mTc-Tilmanocept SPECT imaging might represent an innovative non-invasive strategy to quantify CD206+ tumor-associated macrophages as a biomarker of anti-GRP94 therapy efficacy and TNBC tumor aggressiveness.

GARP: A Key Target to Evaluate Tumor Immunosuppressive Microenvironment.

Glycoprotein-A repetitions predominant (GARP) is the docking receptor for latent transforming growth factor (LTGF-ß) and promotes its activation. In cancer, increased GARP expression has been found in many types of cancer. GARP is expressed by regulatory T cells and platelets in the tumor microenvironment (TME) and can be also expressed by tumor cells themselves. Thus, GARP can be widely present in tumors in which it plays a major role in the production of active TGF-ß, contributing to immune evasion and cancer progression via the GARP-TGF-ß pathway. The objective of this review is to highlight GARP expression and function in cancer and to evaluate the potential of membrane GARP as a predictive and therapeutic follow-up biomarker that could be assessed, in real time, by molecular imaging. Moreover, as GARP can be secreted, a focus will also be made on soluble GARP as a circulating biomarker.

[18F]FMISO PET/CT imaging of hypoxia as a non-invasive biomarker of disease progression and therapy efficacy in a preclinical model of pulmonary fibrosis: comparison with the [18F]FDG PET/CT approach.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor outcome and limited therapeutic options. Imaging of IPF is limited to high-resolution computed tomography (HRCT) which is often not sufficient for a definite diagnosis and has a limited impact on therapeutic decision and patient management. Hypoxia of the lung is a significant feature of IPF but its role on disease progression remains elusive. Thus, the aim of our study was to evaluate hypoxia imaging with [18F]FMISO as a predictive biomarker of disease progression and therapy efficacy in preclinical models of lung fibrosis in comparison with [18F]FDG. METHODS: Eight-week-old C57/BL6 mice received an intratracheal administration of bleomycin (BLM) at day (D) 0 to initiate lung fibrosis. Mice received pirfenidone (300 mg/kg) or nintedanib (60 mg/kg) by daily gavage from D9 to D23. Mice underwent successive PET/CT imaging at several stages of the disease (baseline, D8/D9, D15/D16, D22/D23) with [18F]FDG and [18F]FMISO. Histological determination of the lung expression of HIF-1α and GLUT-1 was performed at D23. RESULTS: We demonstrate that mean lung density on CT as well as [18F]FDG and [18F]FMISO uptakes are upregulated in established lung fibrosis (1.4-, 2.6- and 3.2-fold increase respectively). At early stages, lung areas with [18F]FMISO uptake are still appearing normal on CT scans and correspond to areas which will deteriorate towards fibrotic lesions at later timepoints. Nintedanib and pirfenidone dramatically and rapidly decreased mean lung density on CT as well as [18F]FDG and [18F]FMISO lung uptakes (pirfenidone: 1.2-, 2.9- and 2.6-fold decrease; nintedanib: 1.2-, 2.3- and 2.5-fold decrease respectively). Early [18F]FMISO lung uptake was correlated with aggressive disease progression and better nintedanib efficacy. CONCLUSION: [18F]FMISO PET imaging is a promising tool to early detect and monitor lung fibrosis progression and therapy efficacy.

The HSP GRP94 interacts with macrophage intracellular complement C3 and impacts M2 profile during ER stress.

The role of GRP94, an endoplasmic reticulum (ER) stress protein with both pro- and anti-inflammatory functions, has not been investigated in macrophages during ER stress, whereas ER stress has been reported in many diseases involving macrophages. In this work, we studied GRP94 in M1/LPS + IFNγ and M2/IL-4 primary macrophages derived from human monocytes (isolated from buffy coats), in basal and ER stress conditions induced by thapsigargin (Tg), an inducer of ER calcium depletion and tunicamycin (Tm), an inhibitor of N-glycosylation. We found that GRP94 was expressed on the membrane of M2 but not M1 macrophages. In M2, Tg, but not Tm, while decreased GRP94 content in the membrane, it induced its secretion. This correlated with the induction of a pro-inflammatory profile, which was dependent on the UPR IRE1α arm activation and on a functional GRP94. As we previously reported that GRP94 associated with complement C3 at the extracellular level, we analyzed C3 and confirmed GRP94-C3 interaction in our experimental model. Further, Tg increased this interaction and, in these conditions, C3b and cathepsin L were detected in the extracellular medium where GRP94 co-immunoprecipitated with C3 and C3b. Finally, we showed that the C3b inactivated fragment, iC3b, only present on non-stressed M2, depended on functional GRP94, making both GRP94 and iC3b potential markers of M2 cells. In conclusion, our results show that GRP94 is co-secreted with C3 under ER stress conditions which may facilitate its cleavage by cathepsin L, thus contributing to the pro-inflammatory profile observed in stressed M2 macrophages.

Chemoselective Synthesis of Amines from Ammonium Hydroxide and Hydroxylamine in Continuous Flow.

The chemoselective amination of alkyl bromides and chlorides with aqueous ammonia and hydroxylamine was achieved in continuous flow to produce primary ammonium salts and hydroxylamines in high yields. An in-line workup was designed to isolate the corresponding primary amine, which was also telescoped in further reactions, such as acylation and Paal-Knorr pyrrole synthesis. Monosubstituted epoxides are also compatible with the reaction conditions.