Intraperitoneal alpha therapy with 224Ra-labeled microparticles combined with chemotherapy in an ovarian cancer mouse model.

A novel alpha-therapy consisting of 224Ra-labeled calcium carbonate microparticles (224Ra-CaCO3-MP) has been designed to treat micrometastatic peritoneal disease via intraperitoneal (IP) administration. This preclinical study aimed to evaluate its efficacy and tolerability when given as a single treatment or in combination with standard of care chemotherapy regimens, in a syngeneic model of ovarian cancer in immune competent mice. Female C57BL/6 mice bearing ID8-fLuc ovarian cancer were treated with 224Ra-CaCO3-MP 1 day after IP tumor cell inoculation. The activity dosages of 224Ra ranged from 14 to 39 kBq/mouse. Additionally, 224Ra-CaCO3-MP treatment was followed by either carboplatin (80 mg/kg)-pegylated liposomal doxorubicin (PLD, 1.6 mg/kg) or carboplatin (60 mg/kg)-paclitaxel (10 mg/kg) on day 14 post tumor cell inoculation. All treatments were administered via IP injections. Readouts included survival, clinical signs, and body weight development over time. There was a slight therapeutic benefit after single treatment with 224Ra-CaCO3-MP compared to the vehicle control, with median survival ratios (MSRs) ranging between 1.1 and 1.3. The sequential administration of 224Ra-CaCO3-MP with either carboplatin-paclitaxel or carboplatin-PLD indicated a synergistic effect on overall survival at certain 224Ra activities. Moreover, the combinations tested appeared well tolerated in terms of weight assessment in the first 4 weeks after treatment. Overall, this research supports the further evaluation of 224Ra-CaCO3-MP in patients with ovarian cancer. However, the most optimal chemotherapy regimen to combine with 224Ra-CaCO3-MP should be identified to fully exploit its therapeutic potential.

Effect of Particle Carriers for Intraperitoneal Drug Delivery on the Course of Ovarian Cancer and Its Immune Microenvironment in a Mouse Model.

Novel treatment strategies are needed to provide a better prognosis for ovarian cancer. For this purpose, the current study was designed to evaluate the effects of different types of particle drug carriers on tumor response and on the tumor immune microenvironment (TME) after intraperitoneal (IP) administration in a murine tumor model. Mice with ID8-fLuc ovarian cancer were injected IP with pegylated liposomes, hydroxyapatite, polystyrene, poly(lactic-co-glycolic acid) (PLGA) and calcium carbonate (CaCO3) microparticles to evaluate the effect of the candidate carriers without drugs. Our results show that several types of microparticle drug carriers caused hyperproliferation of the tumor when injected IP, as reflected in a reduced survival or an accelerated onset of ascites. Alterations of the product formulation of CaCO3 microparticles could result in less hyperproliferation. The hyperproliferation caused by CaCO3 and PLGA was largely driven by a strong innate immune suppression. A combination with chemotherapy was not able to sufficiently counteract the tumor progression caused by the drug carriers. This research points towards the importance of evaluating a drug carrier before using it in a therapeutic setting, since drug carriers themselves can detrimentally influence tumor progression and immune status of the TME. However, it remains to be determined whether the hyperproliferation in this model will be of relevance in other cancer models or in humans.

Radon-220 diffusion from 224Ra-labeled calcium carbonate microparticles: Some implications for radiotherapeutic use.

Alpha-particle emitting radionuclides continue to be the subject of medical research because of their high energy and short range of action that facilitate effective cancer therapies. Radium-224 (224Ra) is one such candidate that has been considered for use in combating micrometastatic disease. In our prior studies, a suspension of 224Ra-labeled calcium carbonate (CaCO3) microparticles was designed as a local therapy for disseminated cancers in the peritoneal cavity. The progenies of 224Ra, of which radon-220 (220Rn) is the first, together contribute three of the four alpha particles in the decay chain. The proximity of the progenies to the delivery site at the time of decay of the 224Ra-CaCO3 microparticles can impact its therapeutic efficacy. In this study, we show that the diffusion of 220Rn was reduced in labeled CaCO3 suspensions as compared with cationic 224Ra solutions, both in air and liquid volumes. Furthermore, free-floating lead-212 (212Pb), which is generated from released 220Rn, had the potential to be re-adsorbed onto CaCO3 microparticles. Under conditions mimicking an in vivo environment, more than 70% of the 212Pb was adsorbed onto the CaCO3 at microparticle concentrations above 1 mg/mL. Further, the diffusion of 220Rn seemed to occur whether the microparticles were labeled by the surface adsorption of 224Ra or if the 224Ra was incorporated into the bulk of the microparticles. The therapeutic benefit of differently labeled 224Ra-CaCO3 microparticles after intraperitoneal administration was similar when examined in mice bearing intraperitoneal ovarian cancer xenografts. In conclusion, both the release of 220Rn and re-adsorption of 212Pb are features that have implications for the radiotherapeutic use of 224Ra-labeled CaCO3 microparticles. The release of 220Rn through diffusion may extend the effective range of alpha-particle dose deposition, and the re-adsorption of the longer lived 212Pb onto the CaCO3 microparticles may enhance the retention of this nuclide in the peritoneal cavity.

Therapeutic Effect of α-Emitting 224Ra-Labeled Calcium Carbonate Microparticles in Mice with Intraperitoneal Ovarian Cancer.

BACKGROUND: Ovarian cancer patients with chemotherapy-resistant residual microscopic disease in the peritoneal cavity have a considerable need for new treatment options. Alpha-emitting radionuclides injected intraperitoneally may be an attractive therapeutic option in this situation as they are highly cytotoxic, while their short range in tissues can spare surrounding radiosensitive organs in the abdomen. Herein we evaluate the therapeutic efficacy of a novel α-emitting compound specifically designed for intracavitary radiation therapy. METHODS: The α-emitter 224Ra was absorbed on calcium carbonate microparticles. Immunodeficient, athymic nude mice with human ovarian cancer cells growing intraperitoneally were treated with different activity levels of 224Ra-microparticles. Tumor growth, survival, and tolerance of the treatment were assessed. Two tumor models based on the cell lines, ES-2 and SKOV3-luc, with different growth patterns were studied. RESULTS: In both models, intraperitoneal treatment with 224Ra-microparticles gave significant antitumor effect with either considerably reduced tumor volume or a survival benefit. An advantageous discovery was that only a few kilobecquerels per mouse were needed to yield therapeutic effects. The treatment was well tolerated up to a dose of 1000 kBq/kg with no signs of acute or subacute toxicity observed. CONCLUSIONS: Intraperitoneal α-therapy with 224Ra-microparticles demonstrated a significant potential for treatment of peritoneal micrometastases in ovarian carcinoma.

Ra-224 labeling of calcium carbonate microparticles for internal α-therapy: Preparation, stability, and biodistribution in mice.

Internal therapy with α-emitters should be well suited for micrometastatic disease. Radium-224 emits multiple α-particles through its decay and has a convenient 3.6 days of half-life. Despite its attractive properties, the use of 224 Ra has been limited to bone-seeking applications because it cannot be stably bound to a targeting molecule. Alternative delivery systems for 224 Ra are therefore of considerable interest. In this study, calcium carbonate microparticles are proposed as carriers for 224 Ra, designed for local therapy of disseminated cancers in cavitary regions, such as peritoneal carcinomatosis. Calcium carbonate microparticles were radiolabeled by precipitation of 224 Ra on the particle surface, resulting in high labeling efficiencies for both 224 Ra and daughter 212 Pb and retention of more than 95% of these nuclides for up to 1 week in vitro. The biodistribution after intraperitoneal administration of the 224 Ra-labeled CaCO3 microparticles in immunodeficient mice revealed that the radioactivity mainly remained in the peritoneal cavity. In addition, the systemic distribution of 224 Ra was found to be strongly dependent on the amount of administered microparticles, with a reduced skeletal uptake of 224 Ra with increasing dose. The results altogether suggest that the 224 Ra-labeled CaCO3 microparticles have promising properties for use as a localized internal α-therapy of cavitary cancers.

Preparation of 212Pb-labeled monoclonal antibody using a novel 224Ra-based generator solution.

INTRODUCTION: Alpha-emitting radionuclides have gained considerable attention as payloads for cancer targeting molecules due to their high cytotoxicity. One attractive radionuclide for this purpose is 212Pb, which by itself is a ß-emitter, but acts as an in vivo generator for its short-lived α-emitting daughters. The standard method of preparing 212Pb-labeled antibodies requires handling and evaporation of strong acids containing high radioactivity levels by the end user. An operationally easier and more rapid process could be useful since the 10.6h half-life of 212Pb puts time constraints on the preparation protocol. In this study, an in situ procedure for antibody labeling with 212Pb, using a solution of the generator nuclide 224Ra, is proposed as an alternative protocol for preparing 212Pb-radioimmunoconjugates. METHODS: Radium-224, the generator radionuclide of 212Pb, was extracted from its parent nuclide, 228Th. Lead-212-labeling of the TCMC-chelator conjugated monoclonal antibody trastuzumab was carried out in a solution containing 224Ra in equilibrium with progeny. Subsequently, the efficiency of separating the 212Pb-radioimmunoconjugate from 224Ra and other unconjugated daughter nuclides in the solution using either centrifugal separation or a PD-10 desalting size exclusion column was evaluated and compared. RESULTS: Radiolabeling with 212Pb in 224Ra-solutions was more than 90% efficient after only 30min reaction time at TCMC-trastuzumab concentrations from 0.15mg/mL and higher. Separation of 212Pb-labeled trastuzumab from 224Ra using a PD-10 column was clearly superior to centrifugal separation. This method allowed recovery of approximately 75% of the 212Pb-antibody-conjugate in the eluate, and the remaining amount of 224Ra was only 0.9±0.8% (n=7). CONCLUSIONS: The current work demonstrates a novel method of producing 212Pb-based radioimmunoconjugates from a 224Ra-solution, which may be simpler and less time-consuming for the end user compared with the method established for use in clinical trials of 212Pb-TCMC-trastuzumab.

Evaluation of CD146 as Target for Radioimmunotherapy against Osteosarcoma.

BACKGROUND: Osteosarcoma is a rare form of cancer but with a substantial need for new active drugs. There is a particular need for targeted therapies to combat metastatic disease. One possible approach is to use an antibody drug conjugate or an antibody radionuclide conjugate to target the osteosarcoma metastases and circulating tumor cells. Herein we have evaluated a radiolabeled monoclonal antibody targeting CD146 both in vitro and in vivo. METHODS AND RESULTS: A murine monoclonal anti-CD146 IgG1 isotype antibody, named OI-3, was developed along with recombinant chimeric versions with human IgG1 or human IgG3 Fc sequences. Using flow cytometry, selective binding of OI-3 to human osteosarcoma cell lines OHS, KPDX and Saos-2 was confirmed. The results confirm a higher expression level of CD146 on human osteosarcoma cells than HER2 and EGFR; antigens targeted by commercially available therapeutic antibodies. The biodistribution of 125I-labeled OI-3 antibody variants was compared with 125I-labeled chimeric anti-EGFR antibody cetuximab in nude mice with subcutaneous OHS osteosarcoma xenografts. OI-3 was able to target CD146 expressing tumors in vivo and showed improved tumor to tissue targeting ratios compared with cetuximab. Subsequently, the three OI-3 variants were conjugated with p-SCN-Bn-DOTA and labeled with a more therapeutically relevant radionuclide, 177Lu, and their biodistributions were studied in the nude mouse model. The 177Lu-labeled OI-3 variants were stable and had therapeutically relevant biodistribution profiles. Dosimetry estimates showed higher absorbed radiation dose to tumor than all other tissues after administration of the chimeric IgG1 OI-3 variant. CONCLUSION: Our results indicate that CD146 can be targeted in vivo by the radiolabeled OI-3 antibodies.

NCR1+ cells in dogs show phenotypic characteristics of natural killer cells.

No specific markers for natural killer (NK) cells in dogs have currently been described. NCR1 (NKp46, CD355) has been considered a pan species NK cell marker and is expressed on most or all NK cells in all species investigated except for the pig which has both a NCR1(+) and a NCR1(-) population. In this study peripheral blood mononuclear cells (PBMC) from 14 healthy dogs, 37 dogs with a clinical diagnosis, including a dog diagnosed with LGL leukemia, and tissue samples from 8 dogs were evaluated for NCR1(+) expression by a cross reacting anti bovine NCR1 antibody. CD3(-)NCR1(+) cells were found in the blood of 93 % of healthy dogs and comprised up to 2.5 % of lymphocytes in PBMC. In a selection of healthy dogs, sampling and immunophenotyping were repeated throughout a period of 1 year revealing a substantial variation in the percentage of CD3(-)NCR1(+) over time. Dogs allocated to 8 disease groups had comparable amounts of CD3(-)NCR1(+) cells in PBMC to the healthy individuals. All organs examined including liver, spleen and lymph nodes contained CD3(-)NCR1(+) cells. Circulating CD3(-)NCR1(+) cells were further characterized as CD56(-)GranzymeB(+)CD8(-). A CD3(+)NCR1(+) population was observed in PBMC in 79 % of the healthy dogs examined representing at the most 4.8 % of the lymphocyte population. In canine samples examined for CD56 expression, CD56(+) cells were all CD3(+) and NCR1(-). To our knowledge, this is the first examination of NCR1 expression in the dog. The study shows that this NK cell associated receptor is expressed both on populations of CD3(+) and CD3(-) blood lymphocytes in dogs and the receptor is found on a CD3(+) GranzymeB(+) CD8(+) leukemia. Our results support that CD56 is expressed only on CD3(+) cells in dogs and shows that NCR1 defines a different CD3(+) lymphocyte population than CD56(+)CD3(+) cells in this species. CD3(-)NCR1(+) cells may represent canine NK cells.

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors.

BACKGROUND: Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression. RESULTS: Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes. Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI. CONCLUSIONS: Our findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.

Differential gene expression in brain tissues of aggressive and non-aggressive dogs.

BACKGROUND: Canine behavioural problems, in particular aggression, are important reasons for euthanasia of otherwise healthy dogs. Aggressive behaviour in dogs also represents an animal welfare problem and a public threat. Elucidating the genetic background of adverse behaviour can provide valuable information to breeding programs and aid the development of drugs aimed at treating undesirable behaviour. With the intentions of identifying gene-specific expression in particular brain parts and comparing brains of aggressive and non-aggressive dogs, we studied amygdala, frontal cortex, hypothalamus and parietal cortex, as these tissues are reported to be involved in emotional reactions, including aggression. Based on quantitative real-time PCR (qRT-PCR) in 20 brains, obtained from 11 dogs euthanised because of aggressive behaviour and nine non-aggressive dogs, we studied expression of nine genes identified in an initial screening by subtraction hybridisation. RESULTS: This study describes differential expression of the UBE2V2 and ZNF227 genes in brains of aggressive and non-aggressive dogs. It also reports differential expression for eight of the studied genes across four different brain tissues (amygdala, frontal cortex, hypothalamus, and parietal cortex). Sex differences in transcription levels were detected for five of the nine studied genes. CONCLUSIONS: The study showed significant differences in gene expression between brain compartments for most of the investigated genes. Increased expression of two genes was associated with the aggression phenotype. Although the UBE2V2 and ZNF227 genes have no known function in regulation of aggressive behaviour, this study contributes to preliminary data of differential gene expression in the canine brain and provides new information to be further explored.

Loss of heterozygosity at the FLCN locus in early renal cystic lesions in dogs with renal cystadenocarcinoma and nodular dermatofibrosis.

Small, macroscopically visible cysts on the surface of the kidneys were observed in eight 6-8-week-old puppies diagnosed with renal cystadenocarcinoma and nodular dermatofibrosis (RCND). Histologic examination of the renal cortices in these puppies reveals numerous small cystic tubular changes. Hyperplastic change of the epithelial lining of cysts is frequently observed. By laser-capture microdissection we have sampled epithelial cells from such early renal cystic lesions in eight paternal half-sibs diagnosed with RCND. DNA was obtained from the laser-captured material, and all coding exons of the germline-mutated FLCN gene were sequenced to detect putative second hits. Samples from 31 independent hyperplastic epithelial cell sections of tubular microcysts of the RCND siblings were examined as well as normal control samples of the tissue sections. Loss of heterozygosity was detected in 35% of the transformed samples. The frequently observed loss of heterozygosity at the FLCN locus in atypical epithelial cells lining the cysts suggests that loss of heterozygosity/function of the FLCN gene may contribute to neoplastic transformation of renal epithelial cells at a very early age of RCND-affected dogs. The transformed renal epithelial cells seem to grow slowly in young puppies, which indicates that other mutational events are required for the development of tumors in adult dogs.

Second hits in the FLCN gene in a hereditary renal cancer syndrome in dogs.

In this study, samples from multifocal renal tumors from two dogs affected with renal cystadenocarcinoma and nodular dermatofibrosis (RCND) were collected for detection of putative second hits in the FLCN gene. Genomic DNA from the samples was typed at the previously identified disease-associated missense mutation and cDNA representing the entire coding region of the FLCN gene was sequenced for mutation detection. Second hits with predicted functional implications for the wild-type FLCN allele were observed in 12 of 17 (71%) of the kidney tumor samples. The type of mutation of the second hits varied between the tumors. Different alternative splice mutations were detected, as well as loss of heterozygosity at the germline mutation and loss of transcription product of the wild-type FLCN allele. In total, the frequency and wide spectrum of second hits identified in the tumor samples suggests a tumor suppressor function of FLCN in the kidneys of RCND-affected dogs. No mutations were detected in skin nodules sampled from the two dogs. This shows that the skin tumors of RCND-affected dogs may be caused by haploinsufficiency of the FLCN gene product.