It has been suggested that chemoresistance of chondrosarcoma (CHS), the cartilage tumor, is caused by the phenotypic microenvironmental features of the tumor tissue, mainly the chondrogenic ...extracellular matrix (ECM), and hypoxia. We developed and characterized a multicellular tumor spheroid (MCTS) of human chondrosarcoma HEMC-SS cells to gain insight into tumor cell biology and drug response. At Day 7, HEMC-SS spheroids exhibited a homogeneous distribution of proliferative Ki-67 positive cells, whereas in larger spheroids (Day 14 and Day 20), proliferation was mainly localized in the periphery. In the core of larger spheroids, apoptotic cells were evidenced by TUNEL assay, and hypoxia by pimonidazole staining. Interestingly, VEGF excretion, evidenced by ELISA on culture media, was detectable from Day 14 spheroids, and increased as the spheroids grew in size. HEMC-SS spheroids synthesized a chondrogenic extracellular matrix rich in glycosaminoglycans and type-2 collagen. Finally, we investigated the sensitivity of Day 7 and Day 14 chondrosarcoma MCTS to hypoxia-activated prodrug TH-302 and doxorubicin compared with their 2D counterparts. As expected, TH-302 exhibited higher cytotoxic activity on larger hypoxic spheroids (Day 14) than on non-hypoxic spheroids (Day 7), with multicellular resistance index (MCRI) values of 7.7 and 9.1 respectively. For doxorubicin, the larger-sized spheroids exhibited higher drug resistance (MCRI of 5.0 for Day 7 and 18.3 for Day 14 spheroids), possibly due to impeded drug penetration into the deep layer of spheroids, evidenced by its auto-fluorescence property. We have developed a model of human chondrosarcoma MCTS that combines an ECM rich in glycosaminoglycans with a high hypoxic core associated with VEGF excretion. This model could offer a more predictive in vitro chondrosarcoma system for screening drugs targeting tumor cells and their microenvironment.
Benzamide-based radioligands targeting melanin were first developed for imaging melanoma and then for therapeutic purpose with targeted radionuclide therapy (TRT).
IICF01012 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
IICF01012 to administer for the treatment of patients with pigmented metastatic melanoma.
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
IICF01012 (185 MBq at D0) to select patients who might benefit from
IICF01012 TRT in therapeutic part, i.e. patient presenting at least one tumour lesion with
IICF01012 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/m
, or 1600 MBq/m
, or 2700 MBq/m
or 4000 MBq/m
of
IICF01012 will be administered at D11 (± 4 days). The primary endpoint is the recommended therapeutic dose of
IICF01012, 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
IICF01012, 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.
This study is a first-in-human trial evaluating the
IICF01012 TRT in metastatic malignant melanomas with a diagnostic dose of the
IICF01012 to select the patients who may benefit from a therapeutic dose of
IICF01012, with at least one tumor lesion with
IICF01012 uptake and an acceptable AD to healthy organ.
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.
One of the current challenges in oncology is to develop imaging tools to early detect the response to conventional chemotherapy and adjust treatment strategies when necessary. Several studies ...evaluating PET imaging with 2-deoxy-2-
Ffluoro-D-glucose (
FFDG) as a predictive tool of therapeutic response highlighted its insufficient specificity and sensitivity. The
FFDG uptake reflects only tumor metabolic activity and not treatment-induced cell death, which seems to be relevant for therapeutic evaluation. Therefore, to evaluate this parameter
, several cell death radiotracers have been developed in the last years. However, few of them have reached the clinical trials. This systematic review focuses on the use of
FML-10 (2-(5-
Ffluoropentyl)-2-methylmalonic acid) as radiotracer of apoptosis and especially as a measure of tumor response to treatment. A comprehensive literature review concerning the preclinical and clinical investigations conducted with
FML-10 was performed. The abilities and applications of this radiotracer as well as its clinical relevance and limitations were discussed. Most studies highlighted a good ability of the radiotracer to target apoptotic cells. However, the increase in apoptosis during treatment did not correlate with the radiotracer tumoral uptake, even using more advanced image analysis (voxel-based analysis).
FML-10 PET imaging does not meet current clinical expectations for early detection of the therapeutic response to conventional chemotherapy. This review has pointed out the challenges of applying various apoptosis imaging strategies in clinical trials, the current methodologies available for image analysis and the future of molecular imaging to assess this therapeutic response.
Purpose
Pathological complete response to the neoadjuvant therapy (NAT) for triple negative breast cancer (TNBC) is predictive of prolonged patient survival. Methods for early evaluation of NAT ...efficiency are still needed, in order to rapidly adjust the therapeutic strategy in case of initial non-response. One option for this is molecular imaging of apoptosis induced by chemotherapy. Therefore, we investigated the capacity of
18
FML-10 PET imaging, an apoptosis radiotracer, to detect tumor cell apoptosis and early predict the therapeutic response of human TNBC.
Results
Initially, the induction of apoptosis by different therapies was quantified. We confirmed, in vitro, that paclitaxel or epirubicin, the fundamental cytotoxic drugs for breast cancer, induce apoptosis in TNBC cell lines. Exposure of TNBC models MDA-MB-231 and MDA-MB-468 to these drugs induced a significant increase (
p
< 0.01) of the apoptotic hallmarks: DNA fragmentation, membrane phospholipid scrambling, and PARP activation. Secondarily, apoptotic fraction was compared to the intracellular accumulation of the radiotracer.
18
FML-10 accumulated in the apoptotic cells after 72 h of treatment by paclitaxel in vitro; this accumulation positively correlated with the apoptotic fraction. In vivo,
18
FML-10 was rapidly cleared from the nontarget organs and mainly eliminated by the kidneys. Comparison of the in vivo
18
FFDG,
18
FFMISO, and
18
FML-10 uptakes revealed that the tumor accumulation of
18
FML-10 was directly related to the tumor hypoxia level. Finally, after the in vivo treatment of TNBC murine xenografts by paclitaxel, apoptosis was well induced, as demonstrated by the cleaved caspase-3 levels; however, no significant increase of
18
FML-10 accumulation in the tumors was observed, either on day 3 or day 6 after the end of the treatment.
Conclusions
These results highlighted that PET imaging using
18
FML-10 allows the visualization of apoptotic cells in TNBC models. Nevertheless, the increase of the chemotherapy-induced apoptotic response when using paclitaxel could not be assessed using this radiotracer in our mouse model.
Background
99m
Tc-NTP 15-5 is a SPECT radiotracer targeting proteoglycans (PG), components of the cartilaginous extracellular matrix. Imaging of PGs would be useful for the early detection of ...cartilage disorders (osteoarthritis, arthritis and chondrosarcoma, Aromatase Inhibitor associated arthralgia (AIA) in breast cancer), and the follow-up of patients under treatment. According to preclinical study results,
99m
Tc-NTP 15-5, is a good candidate for a specific functional molecular imaging of joints. We intend to initiate a first in-human study to confirm and quantify
99m
Tc-NTP 15-5 uptake in healthy joints.
Methods
As the clinical development of this radiotracer would be oriented toward the functional imaging of joint pathologies, we have chosen to include patients with healthy joints (unilateral osteoarthritis of the knee or breast cancer with indication of AI treatment). This phase I study will be an open-label, multicenter, dose-escalation trial of a radiopharmaceutical orientation to determine the recommended level of activity of
99m
Tc-NTP 15-5 to obtain the best joint tracer contrasts on images, without dose limiting toxicity (DLT). The secondary objectives will include the study of the pharmacology, biodistribution (using planar whole body and SPECT-CT acquisitions), toxicity, and dosimetry of this radiotracer. The dose escalation with 3 activity levels (5, 10, and 15 MBq/kg), will be conditioned by the absence at the previous level of DLT and of a visualized tracer accumulation on more than 80% of healthy joints as observed on scintigraphy performed at ≤ 2 h post-injection.
Discussion
This first in-human phase I trial will be proof-of-concept of the relevance of
99m
Tc-NTP 15-5 as a cartilage tracer, with the determination of the optimal methodology (dose and acquisition time) to obtain the best contrast to provide a functional image of joints with SPECT-CT.
Trial registration number
Clinicaltrials.gov
: NCT04481230. Identifier in French National Agency for the Safety of Medicines and Health Products (ANSM): N°EudraCT 2020-000495-37.
Purpose
This study aims to perform dosimetry for 99mTcNTP15‐5 radiotracer used in imaging of articular cartilage in rabbits and humans. The radiotracer (covered by a world patent WO 01/00621 A1) has ...been proposed in the previous years for the study of cartilage in osteoarthritis diseases. A sensitive imaging approach is essential to quantify osteoarthritis progression and monitor response to new therapies. 99mTcNTP15‐5 binds to cartilage proteoglycans whose decreased content is associated to a loss of biomedical function of cartilage. We have implemented the whole dosimetry study concerning this new radiotracer for rabbits and humans using the GATE Monte Carlo platform.
Materials and methods
Absorbed doses to critical organs are determined using the MIRD formalism. Biodistribution data are obtained by organ sampling, measuring the activity in organs for three rabbits sacrificed at various times postadministration, and by SPECT/CT imaging at different times after injection. Most important sources are cartilages (in knees and intervertebral discs), due to localization together with the liver and kidneys due to excretion of the agent. S‐values are calculated from rabbit’s CT scan and human CT scan using the GATE v8.0 Monte Carlo platform. Cumulated activity in humans is extrapolated from animals using the %kg‐dose/g method. Particular attention is given to dose calculation in bones, bone marrow and organs at risk.
Results
The dosimetry performed in rabbits shows highest absorbed doses for liver and kidneys with respectively 22.5 and 43.8 µGy per MBq of injected activity. In humans, we found absorbed doses for a maximum injected activity of 15 MBq/kg, that is, 1050 MBq for an adult of 70 kgs of 9.03 mGy for kidneys and 4.16 mGy for knee cartilages. Effective dose is 2.69 µSv/MBq.
Conclusions
The dosimetry profile of 99mTcNTP15‐5 in the context of preclinical trials is of major importance in order to make sure that organs at risk are not overexposed. GATE provides all the capability needed to calculate dose profiles for internal dosimetry. The extrapolation of the dose for a human model is a first step towards clinical trials.
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
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
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.
To investigate the molecular or cellular mechanisms related to the infection of epithelial colonic mucosa by pks-positive Escherichia coli (E. coli) using optical imaging.
We choose to evaluate the ...tumor metabolic activity using a fluorodeoxyglucose analogue as 2-deoxyglucosone fluorescent probes and to correlate it with tumoral volume (mm(3)). Inflammation measuring myeloperoxidase (MPO) activity and reactive oxygen species production was monitored by a bioluminescent (BLI) inflammation probe and related to histological examination and MPO levels by enzyme-linked immunosorbent assay (ELISA) on tumor specimens. The detection and quantitation of these two signals were validated on a xenograft model of human colon adenocarcinoma epithelial cells (HCT116) in nude mice infected with a pks-positive E. coli. The inflammatory BLI signal was validated intra-digestively in the colitis-CEABAC10 DSS models, which mimicked Crohn's disease.
Using a 2-deoxyglucosone fluorescent probe, we observed a high and specific HCT116 tumor uptake in correlation with tumoral volume (P = 0.0036). Using the inflammation probe targeting MPO, we detected a rapid systemic elimination and a significant increase of the BLI signal in the pks-positive E. coli-infected HCT116 xenograft group (P < 0.005). ELISA confirmed that MPO levels were significantly higher (1556 ± 313.6 vs 234.6 ± 121.6 ng/mL P = 0.001) in xenografts infected with the pathogenic E. coli strain. Moreover, histological examination of tumor samples confirmed massive infiltration of pks-positive E. coli-infected HCT116 tumors by inflammatory cells compared to the uninfected group. These data showed that infection with the pathogenic E. coli strain enhanced inflammation and ROS production in tumors before tumor growth. Moreover, we demonstrated that the intra-digestive monitoring of inflammation is feasible in a reference colitis murine model (CEABAC10/DSS).
Using BLI and fluorescence optical imaging, we provided tools to better understand host-pathogen interactions at the early stage of disease, such as inflammatory bowel disease and colorectal cancer.
Purpose
Dosimetry for melanoma‐targeted radionuclide therapy (TRT) with 131IICF01012, a melanin ligand, has been previously evaluated in mice bearing melanomas. In this study, activity distribution ...and dosimetry are performed on healthy rabbits (Fauve de Bourgogne) using SPECT‐CT imaging and ex vivo measurements.
Material and Methods
Ex vivo biodistribution (i.v. injection: 370 kBq/kg, n = 2 per point) is performed on blood, eyes, brain, lung, liver, kidneys, heart, stomach, and spleen. Dosimetry calculations follow the MIRD formalism: S values are calculated from CT images using the GATE Monte Carlo platform and activity distributions are obtained from SPECT‐CT imaging (i.v. injection: 37 MBq/kg n = 3 per point). A specific study is presented to assess dose to human retina.
Results
Time‐integrated activities based on SPECT‐CT are in accordance with ex vivo measurements except for spleen. Doses to liver and eyes are the most significant, with respectively, 6.38 ± 0.50 Gy/GBq (evaluated through SPECT‐CT imaging) and 45.8 ± 7.9 Gy/GBq (evaluated through ex vivo measurements). Characterization of ocular 131IICF01012 biodistribution in rabbits and quantification of melanin allowed to assess a dose of 3.07 ± 0.70 Gy/GBq to human retina.
Conclusion
This study sustains 131IICF01012 as a good candidate for melanoma TRT and open perspectives for personalized dosimetry calculation during phase I clinical transfer.
The biodistribution of dendronized iron oxides, NPs10@D1_DOTAGA and melanin-targeting NPs10@D1_ICF_DOTAGA, was studied in vivo using magnetic resonance imaging (MRI) and planar scintigraphy through ...177LuLu-radiolabeling. MRI experiments showed high contrast power of both dendronized nanoparticles (DPs) and hepatobiliary and urinary excretions. Little tumor uptake could be highlighted after intravenous injection probably as a consequence of the negatively charged DOTAGA-derivatized shell, which reduces the diffusion across the cells’ membrane. Planar scintigraphy images demonstrated a moderate specific tumor uptake of melanoma-targeted 177LuLu-NPs10@D1_ICF_DOTAGA at 2 h post-intravenous injection (pi), and the highest tumor uptake of the control probe 177LuLu-NPs10@D1_DOTAGA at 30 min pi, probably due to the enhanced permeability and retention effect. In addition, ex vivo confocal microscopy studies showed a high specific targeting of human melanoma samples impregnated with NPs10@D1_ICF_Alexa647_ DOTAGA.
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