Phase I study of [131I] ICF01012, a targeted radionuclide therapy, in metastatic melanoma: MELRIV-1 protocol.

BACKGROUND: Benzamide-based radioligands targeting melanin were first developed for imaging melanoma and then for therapeutic purpose with targeted radionuclide therapy (TRT). [131I]ICF01012 presents a highly favorable pharmacokinetics profile in vivo for therapy. Tumour growth reduction and increase survival have been established in preclinical models of melanoma. According the these preclinical results, we initiate a first-in-human study aimed to determine the recommended dose of [131I]ICF01012 to administer for the treatment of patients with pigmented metastatic melanoma. METHODS: The MELRIV-1 trial is an open-label, multicentric, dose-escalation phase I trial. The study is divided in 2 steps, a selection part with an IV injection of low activity of [131I]ICF01012 (185 MBq at D0) to select patients who might benefit from [131I]ICF01012 TRT in therapeutic part, i.e. patient presenting at least one tumour lesion with [131I]ICF01012 uptake and an acceptable personalized dosimetry to critical organs (liver, kidney, lung and retina). According to dose escalation scheme driven by a Continual Reassessment Method (CRM) design, a single therapeutic injection of 800 MBq/m2, or 1600 MBq/m2, or 2700 MBq/m2 or 4000 MBq/m2 of [131I]ICF01012 will be administered at D11 (± 4 days). The primary endpoint is the recommended therapeutic dose of [131I]ICF01012, with DLT defined as any grade 3-4 NCI-CT toxicity during the 6 weeks following therapeutic dose. Safety, pharmacokinetic, biodistribution (using planar whole body and SPECT-CT acquisitions), sensitivity / specificity of [131I]ICF01012, and therapeutic efficacy will be assessed as secondary objectives. Patients who received therapeutic injection will be followed until 3 months after TRT. Since 6 to 18 patients are needed for the therapeutic part, up to 36 patients will be enrolled in the selection part. DISCUSSION: This study is a first-in-human trial evaluating the [131I]ICF01012 TRT in metastatic malignant melanomas with a diagnostic dose of the [131I]ICF01012 to select the patients who may benefit from a therapeutic dose of [131I]ICF01012, with at least one tumor lesion with [131I]ICF01012 uptake and an acceptable AD to healthy organ. TRIAL REGISTRATION: Clinicaltrials.gov : NCT03784625 . Registered on December 24, 2018. Identifier in French National Agency for the Safety of Medicines and Health Products (ANSM): N°EudraCT 2016-002444-17.

Novel Radioiodinated and Radiofluorinated Analogues of FT-2102 for SPECT or PET Imaging of mIDH1 Mutant Tumours.

Isocitrate dehydrogenases (IDHs) are metabolic enzymes commonly mutated in human cancers (glioma, acute myeloid leukaemia, chondrosarcoma, and intrahepatic cholangiocarcinoma). These mutated variants of IDH (mIDH) acquire a neomorphic activity, namely, conversion of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate involved in tumourigenesis. Thus, mIDHs have emerged as highly promising therapeutic targets, and several mIDH specific inhibitors have been developed. However, the evaluation of mIDH status, currently performed by biopsy, is essential for patient stratification and thus treatment and follow-up. We report herein the development of new radioiodinated and radiofluorinated analogues of olutasidenib (FT-2102) as tools for noninvasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging of mIDH1 up- and dysregulation in tumours. Nonradiolabelled derivatives 2 and 3 halogenated at position 6 of the quinolinone scaffold were synthesised and tested in vitro for their inhibitory potencies and selectivities in comparison with the lead compound FT-2102. Using a common organotin precursor, (S)-[125I]2 and (S)-[18F]3 were efficiently synthesised by radio-iododemetallation and copper-mediated radiofluorination, respectively. Both radiotracers were stable at room temperature in saline or DPBS solution and at 37 °C in mouse serum, allowing future planning of their in vitro and in vivo evaluations in glioma and chondrosarcoma models.

Synthesis and In Vitro Comparison of DOTA, NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates.

The development of 64Cu-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [64Cu]Cu-15-5 complex prompted us to investigate its potential for the 64Cu-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with 64Cu, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [64Cu]Cu-15-5- and [64Cu]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88-94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition.

Simultaneous proteoglycans and hypoxia mapping of chondrosarcoma environment by frequency selective CEST MRI.

PURPOSE: To evaluate the relevance of CEST frequency selectivity in simultaneous in vivo imaging of both of chondrosarcoma's phenotypic features, that are, its high proteoglycan concentration and its hypoxic core. METHODS: Swarm rat chondrosarcomas were implanted subcutaneously in NMRI nude mice. When tumors were measurable (12-16 days postoperative), mice were submitted to GAG, guanidyl, and APT CEST imaging. Proteoglycans and hypoxia were assessed in parallel by nuclear imaging exploiting 99m Tc-NTP 15-5 and 18 F-FMISO, respectively. Data were completed by ex vivo analysis of proteoglycans (histology and biochemical assay) and hypoxia (immunofluorescence). RESULTS: Quantitative analysis of GAG CEST evidenced a significantly higher signal for tumor tissues than for muscles. These results were in agreement with nuclear imaging and ex vivo data. For imaging tumoral pH in vivo, the CEST ratio of APT/guanidyl was studied. This highlighted an important heterogeneity inside the tumor. The hypoxic status was confirmed by 18 F-FMISO PET imaging and ex vivo immunofluorescence. CONCLUSION: CEST MRI simultaneously imaged both chondrosarcoma properties during a single experimental run and without the injection of any contrast agent. Both MR and nuclear imaging as well as ex vivo data were in agreement and showed that this chondrosarcoma animal model was rich in proteoglycans. However, even if tumors were lightly hypoxic at the stage studied, acidic areas were highlighted and mapped inside the tumor.

Relevance of the combination of external beam radiotherapy with the hypoxia-activated prodrug ICF05016 in an experimental model of extraskeletal myxoid chondrosarcoma.

Currently, there is no gold standard treatment for Extraskeletal Myxoid Chondrosarcomas (EMC) making wide margin surgical resection the most effective alternative treatment. Nevertheless, in previous preclinical studies our lab demonstrated the potential of the hypoxia-activated prodrug (HAP) ICF05016 on EMC murine model inoculated with the H-EMC-SS human cell line. The aim of this study was to assess, in vivo, the relevance of the combination of this HAP with External Beam Radiotherapy (EBR). Firstly EMC-bearing mice were treated with 6 Gy or 12 Gy of EBR (single 6 MV photon). Then for combination of HAP and EBR, animals received 6 doses of ICF05016 (46.8 µmol/kg, intravenously) at 4-day intervals, with 6 Gy EBR performed 24 h after the 3rd dose of HAP. Animals were monitored throughout the study for clinical observations (tumour growth, side effects) and survival studies were performed. From tumour samples, PCNA, Ki-67 and p21 expressions were used as markers of proliferation and cell cycle arrest. Statistical significances were determined using Kruskall-Wallis and log rank tests. The radiosensitivity of the EMC model was demonstrated at 12 Gy with significant inhibition of tumour growth. Then, the HAP strategy potentiated EBR efficacy at a lower dose (6 Gy) by improving survival without generating side effects. Thus, results of this study showed the potential interest of ICF05016 for the combination with EBR in the management of EMC.

The Class I HDAC Inhibitor, MS-275, Prevents Oxaliplatin-Induced Chronic Neuropathy and Potentiates Its Antiproliferative Activity in Mice.

Oxaliplatin, the first-line chemotherapeutic agent against colorectal cancer (CRC), induces peripheral neuropathies, which can lead to dose limitation and treatment discontinuation. Downregulation of potassium channels, which involves histone deacetylase (HDAC) activity, has been identified as an important tuner of acute oxaliplatin-induced hypersensitivity. MS-275, a class I histone deacetylase inhibitor (HDACi), prevents acute oxaliplatin-induced peripheral neuropathy (OIPN). Moreover, MS-275 exerts anti-tumor activity in several types of cancers, including CRC. We thus hypothesized that MS-275 could exert both a preventive effect against OIPN and potentially a synergistic effect combined with oxaliplatin against CRC development. We first used RNAseq to assess transcriptional changes occurring in DRG neurons from mice treated by repeated injection of oxaliplatin. Moreover, we assessed the effects of MS-275 on chronic oxaliplatin-induced peripheral neuropathy development in vivo on APCMin/+ mice and on cancer progression when combined with oxaliplatin, both in vivo on APCMin/+ mice and in a mouse model of an orthotopic allograft of the CT26 cell line as well as in vitro in T84 and HT29 human CRC cell lines. We found 741 differentially expressed genes (DEGs) between oxaliplatin- and vehicle-treated animals. While acute OIPN is known as a channelopathy involving HDAC activity, chronic OIPN exerts weak ion channel transcriptional changes and no HDAC expression changes in peripheral neurons from OIPN mice. However, MS-275 prevents the development of sensory neuropathic symptoms induced by repeated oxaliplatin administration in APCMin/+ mice. Moreover, combined with oxaliplatin, MS-275 also exerts synergistic antiproliferative and increased survival effects in CT26-bearing mice. Consistently, combined drug associations exert synergic apoptotic and cell death effects in both T84 and HT29 human CRC cell lines. Our results strongly suggest combining oxaliplatin and MS-275 administration in CRC patients in order to potentiate the antiproliferative action of chemotherapy, while preventing its neurotoxic effect.

Design, synthesis and evaluation of targeted hypoxia-activated prodrugs applied to chondrosarcoma chemotherapy.

The tumor microenvironment in chondrosarcoma (CHS), a chemo- and radio-resistant cancer provides unique hallmarks for developing a chondrosarcoma targeted drug-delivery system. Tumor targeting could be achieved using a quaternary ammonium function (QA) as a ligand for aggrecan, the main high negative charged proteoglycan of the extracellular matrix of CHS, and a 2-nitroimidazole as trigger that enables hypoxia-responsive drug release. In a previous work, ICF05016 was identified as efficient proteoglycan-targeting hypoxia-activated prodrug in a human extraskeletal myxoid chondrosarcoma model in mice and a first study of the structure-activity relationship of the QA function and the alkyl linker length was conducted. Here, we report the second part of the study, namely the modification of the nitro-aromatic trigger and the position of the proteoglycan-targeting ligand at the aromatic ring as well as the nature of the alkylating mustard. Synthetic approaches have been established to functionalize the 2-nitroimidazole ring at the N-1 and C-4 positions with a terminal tertiary alkyl amine, and to perform the phosphorylation step namely through the use of an amine borane complex, leading to phosphoramide and isophosphoramide mustards and also to a phosphoramide mustard bearing four 2-chloroethyl chains. In a preliminary study using a reductive chemical activation, QA-conjugates, except the 4-nitrobenzyl one, were showed to undergo efficient cleavage with release of the corresponding mustard. However N,N,N-trimethylpropylaminium tethered to the N-1 or C-4 positions of the imidazole seemed to hamper the enzymatic reduction of the prodrugs and all tested compounds featured moderate selectivity toward hypoxic cells, likely not sufficient for application as hypoxia-activated prodrugs.

Linker structure-activity relationships in fluorodeoxyglucose chlorambucil conjugates for tumor-targeted chemotherapy.

Nitrogen mustards, such as chlorambucil (CLB), can cause adverse side-effects due to ubiquitous distribution in non-target organs. To minimize this toxicity, strategies of tumor-targeting drug delivery have been developed, where a cytotoxic warhead is linked to a tumor-cell-specific small ligand. Malignant cells exhibit marked glucose avidity and an accelerated metabolism by aerobic glycolysis, known as the Warburg effect, and recognized as a hallmark of cancer. A targeting approach exploiting the Warburg effect by conjugation of CLB to 2-fluoro-2-deoxyglucose (FDG) was previously reported and identified two peracetylated glucoconjugates 2 and 3 with promising antitumor activities in vivo. These results prompted us to investigate the importance of the spacer in this tumor-targeting glucose-based conjugates. Here we report the chemical synthesis and an in vitro cytotoxicity evaluation, using a 5-member panel of human tumor cell lines and human fibroblasts, of 16 new CLB glucoconjugates in which the alkylating drug is attached to the C-1 position of FDG via different linkages. We studied the structure-activity relationships in the linker, and evidenced the positive impact of an aromatic linker on in vitro cytotoxicity: compound 51 proved to be the most active FDG-CLB glucoside, characterized by a bis-aromatic spacer tethered to CLB through an amide function.

Preparation of core/shell NaYF4:Yb,Tm@dendrons nanoparticles with enhanced upconversion luminescence for in vivo imaging.

Upconverting nanoparticles (UCNPs) were successfully dendronized for fluorescence medical imaging applications. The structural and morphological characterizations of resulting core/shell NaYF4:Yb,Tm@dendrons nanoparticles were performed by means of X-ray diffraction, infrared spectroscopy and transmission electron microscopy. In vitro cytotoxicity assays have evidenced their low toxicity. In vivo fluorescence imaging study was performed in mice upon IR excitation, showing promising imaging capacities at low concentrations (0.5mg/mL) and low power (50mW/cm2).

The free fatty acid receptor G protein-coupled receptor 40 (GPR40) protects from bone loss through inhibition of osteoclast differentiation.

The mechanisms linking fat intake to bone loss remain unclear. By demonstrating the expression of the free fatty acid receptor G-coupled protein receptor 40 (GPR40) in bone cells, we hypothesized that this receptor may play a role in mediating the effects of fatty acids on bone remodeling. Using micro-CT analysis, we showed that GPR40(-/-) mice exhibit osteoporotic features suggesting a positive role of GPR40 on bone density. In primary cultures of bone marrow, we showed that GW9508, a GRP40 agonist, abolished bone-resorbing cell differentiation. This alteration of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation occurred via the inhibition of the nuclear factor κB (NF-κB) signaling pathway as demonstrated by decrease in gene reporter activity, inhibitor of κB kinase (IKKα/ß) activation, inhibitor of κB (IkBα) phosphorylation, and nuclear factor of activated T cells 1 (NFATc1) expression. The GPR40-dependent effect of GW9508 was confirmed using shRNA interference in osteoclast precursors and GPR40(-/-) primary cell cultures. In addition, in vivo administration of GW9508 counteracted ovariectomy-induced bone loss in wild-type but not GPR40(-/-) mice, enlightening the obligatory role of the GPR40 receptor. Then, in a context of growing prevalence of metabolic and age-related bone disorders, our results demonstrate for the first time in translational approaches that GPR40 is a relevant target for the design of new nutritional and therapeutic strategies to counter bone complications.

Evaluation of two (125)I-radiolabeled acridine derivatives for Auger-electron radionuclide therapy of melanoma.

We previously selected two melanin-targeting radioligands [(125)I]ICF01035 and [(125)I]ICF01040 for melanoma-targeted (125)I radionuclide therapy according to their pharmacological profile in mice bearing B16F0 tumors. Here we demonstrate in vitro that these compounds present different radiotoxicities in relation to melanin and acidic vesicle contents in B16F0, B16F0 PTU and A375 cell lines. ICF01035 is effectively observed in nuclei of achromic (A375) melanoma or in melanosomes of melanized melanoma (B16F0), while ICF01040 stays in cytoplasmic vesicles in both cells. [(125)I]ICF01035 induced a similar survival fraction (A50) in all cell lines and led to a significant decrease in S-phase cells in amelanotic cell lines. [(125)I]ICF01040 induced a higher A50 in B16 cell lines compared to [(125)I]ICF01035 ones. [(125)I]ICF01040 induced a G2/M blockade in both A375 and B16F0 PTU, associated with its presence in cytoplasmic acidic vesicles. These results suggest that the radiotoxicity of [(125)I]ICF01035 and [(125)I]ICF01040 are not exclusively reliant on DNA alterations compatible with γ rays but likely result from local dose deposition (Auger electrons) leading to toxic compound leaks from acidic vesicles. In vivo, [(125)I]ICF01035 significantly reduced the number of B16F0 lung colonies, enabling a significant increase in survival of the treated mice. Targeting melanosomes or acidic vesicles is thus an option for future melanoma therapy.

Pomegranate and its derivatives can improve bone health through decreased inflammation and oxidative stress in an animal model of postmenopausal osteoporosis.

PURPOSE: Recently, nutritional and pharmaceutical benefits of pomegranate (PG) have raised a growing scientific interest. Since PG is endowed with anti-inflammatory and antioxidant activities, we hypothesized that it may have beneficial effects on osteoporosis. METHODS: We used ovariectomized (OVX) mice as a well-described model of postmenopausal osteoporosis to study the influence of PG consumption on bone health. Mice were divided into five groups as following: two control groups sham-operated and ovariectomized (OVX CT) mice fed a standard diet, versus three treated groups OVX mice given a modified diet from the AIN-93G diet, containing 5.7% of PG lyophilized mashed totum (OVX PGt), or 9.6% of PG fresh juice (OVX PGj) or 2.9% of PG lyophilized mashed peel (OVX PGp). RESULTS: As expected, ovariectomy was associated with a decreased femoral bone mineral density (BMD) and impaired bone micro-architecture parameters. Consumption of PGj, PGp, or PGt induced bone-sparing effects in those OVX mice, both on femoral BMD and bone micro-architecture parameters. In addition, PG (whatever the part) up-regulated osteoblast activity and decreased the expression of osteoclast markers, when compared to what was observed in OVX CT animals. Consistent with the data related to bone parameters, PG consumption elicited a lower expression of pro-inflammatory makers and of enzymes involved in ROS generation, whereas the expression of anti-inflammatory markers and anti-oxidant actors was enhanced. CONCLUSION: These results indicate that all PG parts are effective in preventing the development of bone loss induced by ovariectomy in mice. Such an effect could be partially explained by an improved inflammatory and oxidative status.

Small rigid platforms functionalization with quaternary ammonium: targeting extracellular matrix of chondrosarcoma.

This work takes place in the "cartilage targeting strategy", consisting in using the quaternary ammonium (QA) function as a vector to proteoglycans (PGs) of extracellular matrix (ECM). The objective was to demonstrate that QA could address gadolinium based small rigid platforms (SRP) to PG-rich tumors. SRP were functionalized with QA, radiolabeled with (111)Indium and evaluated for biodistribution in vivo, respectively to non functionalized SRP, in two experimental models: (i) the HEMCSS human xenograft model; (ii) the Swarm rat chondrosarcoma (SRC) orthotopic model. The contribution of cellular uptake to tumoral accumulation of nano-objects was also determined from in vitro binding. In the SRC model expressing a highly and homogeneously distributed PG content, tumor accumulation and retention of SRP@QA were increased by 40% as compared to non-functionalized SRP. When considering the radiosensitizing potential of gadolinium based SRP, these results provide hopes for the radiobiological approach of highly resistant tumor such as chondrosarcoma. FROM THE CLINICAL EDITOR: In this study, gadolinium-based complexing DOTA-surfaced small polysiloxane nanoparticles were functionalized with quaternary ammonium derivatives that target the extracellular matrix of chondrosarcoma. The authors demonstrate in a rat model that the use of these constructs results in a 40% increase of tumor accumulation and retention compared to non-functionalized (and otherwise same) platforms. Similar approaches would be welcome additions to the clinical armamentarium addressing chondrosarcoma.

3D Coculture between Cancer Cells and Macrophages: From Conception to Experimentation.

A tumor is a complex cluster with many types of cells in the microenvironment that help it grow. Macrophages, immune cells whose main role is to maintain body homeostasis, represent in the majority of cancers the most important cell population. In this context, they are called tumor-associated macrophages (TAMs) because of their phenotype, which contributes to tumor growth. In order to limit the use of animals, there is a real demand for the creation of in vitro models able to represent more specifically the complexity of the tumor microenvironment (TME) in order to characterize it and evaluate new treatments. However, the two-dimensional (2D) culture, which has been used for a long time, has shown many limitations, especially in terms of tumor representation. The three-dimensional (3D) models, developed over the last 20 years, have made it possible to get closer to what happens in vivo in terms of phenotypic and functional characteristics. Due to their architectural similarity to in vivo tissues, they provide a more physiologically relevant in vitro system. Most recently, it is the development of 3D coculture models in which two or three cell lines are cultured together that has allowed a better representation of TME with cell-cell interactions. Unfortunately, there is no clear direction for the design of these models at this time. In this Review on the coculture between cancer cells and TAMs, an in-depth analysis is performed to answer multiple questions on the conception of these models: Which models to use, and with which material and cancer lineage? Which monocyte or macrophage lines should be added to the coculture? And how can these models be exploited?

In vivo efficacy of melanoma internal radionuclide therapy with a 131I-labelled melanin-targeting heteroarylcarboxamide molecule.

The development of alternative therapies for melanoma treatment is of great interest as long-term tumour regression is not achieved with new targeted chemotherapies on selected patients. We previously demonstrated that radioiodinated heteroarylcarboxamide ([131I]ICF01012) induced a strong anti-tumoural effect by inhibiting both primary tumour growth and dissemination process in a B16BL6 melanoma model. In our study, we show that a single injection of [131I]ICF01012 (ranging from 14.8 to 22.2 MBq) was effective and associated with low and transient haematological toxicity. Concerning pigmented organs, cutaneous melanocytes and skin were undamaged. In 30% of treated animals, no histological alteration of retina was observed, and in the remaining 70%, damages were restricted to the optic nerve area. Using the Medical Internal Radiation Dose methodology, we determined that the absorbed dose in major organs is very low (<4 Gy) and that a delivery of 30 Gy to the tumour is sufficient for an effective anti-tumoural response. Molecular analyses of treated tumours showed a strong radiobiological effect with a decrease in proliferation, survival and pro-angiogenic-related markers and an increase in tumour suppressor gene expression, melanogenesis and anti-angiogenic markers. All these features are in accordance with a tumour cell death mechanism that mainly occurs by mitotic catastrophe and provide a better understanding of in vivo anti-tumoural effects of [131I] radionuclide. Our findings raise [131I]ICF01012 a good candidate for disseminated melanoma treatment and strongly support transfer of [131I]ICF01012 to clinical trial.

Homologation of the Alkyl Side Chain of Antimitotic Phenyl 4-(2-Oxo-3-alkylimidazolidin-1-yl)benzenesulfonate Prodrugs Selectively Targeting CYP1A1-Expressing Breast Cancers Improves Their Stability in Rodent Liver Microsomes.

Phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) are a new family of antimitotic prodrugs bioactivated in breast cancer cells expressing CYP1A1. In this study, we report that the 14C-labeled prototypical PAIB-SO [14C]CEU-818 and its antimitotic counterpart [14C]CEU-602 are distributed in whole mouse body and they show a short half-life in mice. To circumvent this limitation, we evaluated the effect of the homologation of the alkyl side chain of the imidazolidin-2-one moiety of PAIB-SOs. Our studies evidence that PAIB-SOs bearing an n-pentyl side chain exhibit antiproliferative activity in the nanomolar-to-low-micromolar range and a high selectivity toward CYP1A1-positive breast cancer cells. Moreover, the most potent n-pentyl PAIB-SOs were significantly more stable toward rodent liver microsomes. In addition, PAIB-SOs 10 and 14 show significant antitumor activity and low toxicity in chorioallantoic membrane (CAM) assay. Our study confirms that homologation is a suitable approach to improve the rodent hepatic stability of PAIB-SOs.

Quaternary ammonium-melphalan conjugate for anticancer therapy of chondrosarcoma: in vitro and in vivo preclinical studies.

Cartilage tumours present ongoing therapeutic challenges due to their chondrogenic extracellular matrix that potentially hampers drug delivery, their low percentage of dividing cells, and their poor vascularity. In this context, and based on the affinity of the quaternary ammonium moiety for proteoglycans (PG), we developed a strategy that uses the quaternary ammonium function to selectively deliver DNA alkylating agents to the cartilage tumour tissue. We engineered the quaternary ammonium derivative of melphalan (Mel-AQ) and assessed its antitumoural activity in vitro and in vivo. In vitro, micromolar concentrations of Mel-AQ inhibited the proliferation of human HEMC-SS chondrosarcoma and Saos-2 osteosarcoma cell lines. Moreover, 24-h incubation with 20 µM Mel-AQ induced a 2.5-fold increase in S population and a 1.5-fold increase in subG0G1 population compared to controls. In vivo, Mel-AQ demonstrated antitumour activity in the orthotopic model of primary Swarm rat chondrosarcoma. When given to chondrosarcoma-bearing rats (three doses of 16 µmol/kg at days 8, 12 and 16 post-implant), Mel-AQ demonstrated an optimal antitumour effect at day 43, when tumour cell growth inhibition peaked at 69%. Interestingly, the treatment protocol was proved well tolerated, since the animals showed no weight loss over the course of the study. This antitumoural effect was assessed in vivo by scintigraphic imaging using (99m)Tc-NTP 15-5 developed in our lab as a PG-targeting radiotracer, and tumour tissue was analyzed at study-end by biochemical PG assay with Alcian blue staining. Mel-AQ treatment led to a significant decrease in the PG content of tumoural tissue. These experimental results highlighted the promising antitumour potential of Mel-AQ as a PG-targeting strategy for therapeutic management of chondrosarcoma.

Early detection and longitudinal monitoring of experimental primary and disseminated melanoma using [¹°F]ICF01006, a highly promising melanoma PET tracer.

PURPOSE: Here, we report a new and rapid radiosynthesis of (18)F-N-[2-(diethylamino)ethyl]-6-fluoro-pyridine-3-carboxamide ([(18)F]ICF01006), a molecule with a high specificity for melanotic tissue, and its evaluation in a murine model for early specific detection of pigmented primary and disseminated melanoma. METHODS: [(18)F]ICF01006 was synthesized using a new one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Melanoma models were induced by subcutaneous (primary tumour) or intravenous (lung colonies) injection of B16BL6 melanoma cells in C57BL/6J mice. The relevance and sensitivity of positron emission tomography (PET) imaging using [(18)F]ICF01006 were evaluated at different stages of tumoural growth and compared to (18)F-fluorodeoxyglucose ([(18)F]FDG). RESULTS: The fully automated radiosynthesis of [(18)F]ICF01006 led to a radiochemical yield of 61 % and a radiochemical purity >99 % (specific activity 70-80 GBq/µmol; total synthesis time 42 min). Tumours were visualized before they were palpable as early as 1 h post-injection with [(18)F]ICF01006 tumoural uptake of 1.64 ± 0.57, 3.40 ± 1.47 and 11.44 ± 2.67 percentage of injected dose per gram of tissue (%ID/g) at days 3, 5 and 14, respectively. [(18)F]ICF01006 PET imaging also allowed detection of melanoma pulmonary colonies from day 9 after tumour cell inoculation, with a lung radiotracer accumulation correlated with melanoma invasion. At day 21, radioactivity uptake in lungs reached a value of 5.23 ± 2.08 %ID/g (versus 0.41 ± 0.90 %ID/g in control mice). In the two models, comparison with [(18)F]FDG showed that both radiotracers were able to detect melanoma lesions, but [(18)F]ICF01006 was superior in terms of contrast and specificity. CONCLUSION: Our promising results provide further preclinical data, reinforcing the excellent potential of [(18)F]ICF01006 PET imaging for early specific diagnosis and follow-up of melanin-positive disseminated melanoma.

Multicellular tumor spheroids of LNCaP-Luc prostate cancer cells as in vitro screening models for cytotoxic drugs.

An increasing number of studies concerning solid cancers, including prostate cancer, are tending to demonstrate the predominant role of the interactions of tumor cells with their microenvironment, and underlining the relevance of therapeutic approaches co-targeting these two components. Artificial in vitro 3D culture models, such as spheroids, are therefore being designed to allow intercellular interactions between tumor cells and the matrix, under hypoxic conditions mimicking a microtumor. This project aims to develop and characterize a multicellular tumor spheroid (MCTS) model of human prostate cancer cells expressing PSMA, for in vitro drug screening. To this end, 1,000 cells/well were seeded in 100 µl of culture medium with 0.5% of methylcellulose in 96-well, non-adherent, V-shaped bottom plates. Bioluminescent imaging of the spheroids enabled the measurement of spheroid growth. From Day 7 of growth, immunofluorescence studies showed cellular proliferation (Ki-67), mainly located in the periphery of the spheroid section, associated with the formation of an apoptotic core (TUNEL). Scanning electron microscopy and fluorescent imaging (Lox-1 probe) showed the presence of an extracellular matrix and the installation of an oxygen gradient leading to the formation of a hypoxic area during growth. This hypoxia was correlated with increased VEGF excretion. Drug sensitivity was assessed on 2D and 3D cultures. The LNCaP-Luc spheroids are more resistant to docetaxel and TH-302, a hypoxia-activated prodrug, compared with cells grown in a monolayer. For docetaxel, this resistance increased with the spheroid growth stage, whereas the activity of TH-302 was potentiated by the hypoxic environment. In conclusion, the development of LNCaP-Luc cell MCTS provides a simple model mimicking a microtumor; it appears to be particularly well-suited to the validation of new therapeutic approaches targeting proliferation and the microenvironment.

99mTc-NTP 15-5 is a companion radiotracer for assessing joint functional response to sprifermin (rhFGF-18) in a murine osteoarthritis model.

With the emergence of disease modifying osteoarthritis drugs (DMOAD), imaging methods to quantitatively demonstrate their efficacy and to monitor osteoarthritis progression at the functional level are urgently needed. Our group showed that articular cartilage can be quantitatively assessed in nuclear medicine imaging by our radiotracer 99mTc-NTP 15-5 targeting cartilage proteoglycans. In this work, surgically induced DMM mice were treated with sprifermin or saline. We investigated cartilage remodelling in the mice knees by 99mTc-NTP 15-5 SPECT-CT imaging over 24 weeks after surgery, as wells as proteoglycan biochemical assays. OA alterations were scored by histology according to OARSI guidelines. A specific accumulation of 99mTc-NTP 15-5 in cartilage joints was evidenced in vivo by SPECT-CT imaging as early as 30 min post-iv injection. In DMM, 99mTc-NTP 15-5 accumulation in cartilage within the operated joints, relative to contralateral ones, was observed to initially increase then decrease as pathology progressed. Under sprifermin, 99mTc-NTP 15-5 uptake in pathological knees was significantly increased compared to controls, at 7-, 12- and 24-weeks, and consistent with proteoglycan increase measured 5 weeks post-surgery, as a sign of cartilage matrix remodelling. Our work highlights the potential of 99mTc-NTP 15-5 as an imaging-based companion to monitor cartilage remodelling in OA and DMOAD response.