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1.
ACS Pharmacol Transl Sci ; 4(2): 953-965, 2021 Apr 09.
Article in English | MEDLINE | ID: mdl-33860213

ABSTRACT

Lipophilicity is explored in the biodistribution (BD), pharmacokinetics (PK), radiation dosimetry (RD), and toxicity of an internally administered targeted alpha-particle therapy (TAT) under development for the treatment of metastatic melanoma. The TAT conjugate is comprised of the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate), conjugated to melanocortin receptor 1 specific peptidic ligand (MC1RL) using a linker moiety and chelation of the 225Ac radiometal. A set of conjugates were prepared with a range of lipophilicities (log D 7.4 values) by varying the chemical properties of the linker. Reported are the observations that higher log D 7.4 values are associated with decreased kidney uptake, decreased absorbed radiation dose, and decreased kidney toxicity of the TAT, and the inverse is observed for lower log D 7.4 values. Animals administered TATs with lower lipophilicities exhibited acute nephropathy and death, whereas animals administered the highest activity TATs with higher lipophilicities lived for the duration of the 7 month study and exhibited chronic progressive nephropathy. Changes in TAT lipophilicity were not associated with changes in liver uptake, dose, or toxicity. Significant observations include that lipophilicity correlates with kidney BD, the kidney-to-liver BD ratio, and weight loss and that blood urea nitrogen (BUN) levels correlated with kidney uptake. Furthermore, BUN was identified as having higher sensitivity and specificity of detection of kidney pathology, and the liver enzyme alkaline phosphatase (ALKP) had high sensitivity and specificity for detection of liver damage associated with the TAT. These findings suggest that tuning radiopharmaceutical lipophilicity can effectively modulate the level of kidney uptake to reduce morbidity and improve both safety and efficacy.

2.
Eur J Nucl Med Mol Imaging ; 48(11): 3408-3421, 2021 10.
Article in English | MEDLINE | ID: mdl-33772332

ABSTRACT

PURPOSE: There is significant interest in the development of targeted alpha-particle therapies (TATs) for treatment of solid tumors. The metal chelator-peptide conjugate, DOTA-TATE, loaded with the ß-particle emitting radionuclide 177Lu ([177Lu]Lu-DOTA-TATE) is now standard care for neuroendocrine tumors that express the somatostatin receptor 2 (SSTR2) target. A recent clinical study demonstrated efficacy of the corresponding [225Ac]Ac-DOTA-TATE in patients that were refractory to [177Lu]Lu-DOTA-TATE. Herein, we report the radiosynthesis, toxicity, biodistribution (BD), radiation dosimetry (RD), and efficacy of [225Ac]Ac-DOTA-TATE in small animal models of lung neuroendocrine neoplasms (NENs). METHODS: [225Ac]Ac-DOTA-TATE was synthesized and characterized for radiochemical yield, purity and stability. Non-tumor-bearing BALB/c mice were tested for toxicity and BD. Efficacy was determined by single intravenous injection of [225Ac]Ac-DOTA-TATE into SCID mice-bearing human SSTR2 positive H727 and H69 lung NENs. RD was calculated using the BD data. RESULTS: [225Ac]Ac-DOTA-TATE was synthesized with 98% yield, 99.8% purity, and displayed 97% stability after 2 days incubation in human serum at 37 °C. All animals in the toxicity study appeared healthy 5 months post injection with no indications of toxicity, except that animals that received ≥111 kBq of [225Ac]Ac-DOTA-TATE had chronic progressive nephropathy. BD studies revealed that the primary route of elimination is by the renal route. RD calculations determined pharmacokinetics parameters and absorbed α-emission dosages from 225Ac and its daughters. For both tumor models, a significant tumor growth delay and time to experimental endpoint were observed following a single administration of [225Ac]Ac-DOTA-TATE relative to controls. CONCLUSIONS: These results suggest significant potential for the clinical translation of [225Ac]Ac-DOTA-TATE for lung NENs.


Subject(s)
Lung Neoplasms , Organometallic Compounds , Animals , Humans , Lung Neoplasms/drug therapy , Mice , Mice, Inbred BALB C , Mice, SCID , Octreotide/therapeutic use , Octreotide/toxicity , Organometallic Compounds/therapeutic use , Organometallic Compounds/toxicity , Radiopharmaceuticals/therapeutic use , Radiopharmaceuticals/toxicity , Tissue Distribution
3.
Mol Pharm ; 17(11): 4180-4188, 2020 11 02.
Article in English | MEDLINE | ID: mdl-32960613

ABSTRACT

Targeted α particle therapy (TAT) is ideal for treating disease while minimizing damage to surrounding nontargeted tissues due to short path length and high linear energy transfer (LET). We developed a TAT for metastatic uveal melanoma, targeting the melanocortin-1 receptor (MC1R), which is expressed in 94% of uveal melanomas. Two versions of the therapy are being investigated: 225Ac-DOTA-Ahx-MC1RL (225Ac-Ahx) and 225Ac-DOTA-di-d-Glu-MC1RL (225Ac-di-d-Glu). The biodistribution (BD) from each was studied and a multicompartment pharmacokinetic (PK) model was developed to describe drug distribution rates. Two groups of 16 severe combined immunodeficient (SCID) mice bearing high MC1R expressing tumors were intravenously injected with 225Ac-Ahx or 225Ac-di-d-Glu. After injection, four groups (n = 4) were euthanized at 24, 96, 144, and 288 h time points for each cohort. Tumors and 13 other organs were harvested at each time point. Isomeric γ spectra were measured in tissue samples using a scintillation γ detector and converted to α activity using factors for γ ray abundance per α decay. Time activity curves were calculated for each organ. A five-compartment PK model was built with the following compartments: blood, tumor, normal tissue, kidney, and liver. This model is characterized by a system of five ordinary differential equations using mass action kinetics, which describe uptake, intercompartmental transitions, and clearance rates. The ordinary differential equations were simultaneously solved and fit to experimental data using a genetic algorithm for optimization. The BD data show that both compounds have minimal distribution to organs at risk other than the kidney and liver. The PK parameter estimates had less than 5% error. From these data, 225Ac-Ahx showed larger and faster uptake in the liver. Both compounds had comparable uptake and clearance rates for other compartments. The BD and PK behavior for two targeted radiopharmaceuticals were investigated. The PK model fit the experimental data and provided insight into the kinetics of the compounds systematically.


Subject(s)
Alpha Particles/therapeutic use , Melanoma, Experimental/drug therapy , Melanoma/drug therapy , Radiopharmaceuticals/administration & dosage , Radiopharmaceuticals/pharmacokinetics , Uveal Neoplasms/drug therapy , alpha-MSH/administration & dosage , alpha-MSH/pharmacokinetics , Animals , Cell Line, Tumor , Ligands , Melanoma/metabolism , Melanoma/pathology , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Metabolic Clearance Rate , Mice , Mice, Inbred BALB C , Mice, SCID , Molecular Targeted Therapy/methods , Receptor, Melanocortin, Type 1/metabolism , Tissue Distribution , Treatment Outcome , Tumor Burden/drug effects , Uveal Neoplasms/metabolism , Uveal Neoplasms/pathology , Xenograft Model Antitumor Assays
4.
World J Clin Oncol ; 11(4): 169-179, 2020 Apr 24.
Article in English | MEDLINE | ID: mdl-32355639

ABSTRACT

Triple-negative breast cancer (TNBC) is defined as a type of breast cancer with lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor 2 protein. In comparison to other types of breast cancer, TNBC characterizes for its aggressive behavior, more prone to early recurrence and a disease with poor response to molecular target therapy. Although TNBC is identified in only 25%-30% of American breast cancer cases annually, these tumors continue to be a therapeutic challenge for clinicians for several reasons: Tumor heterogeneity, limited and toxic systemic therapy options, and often resistance to current standard therapy, characterized by progressive disease on treatment, residual tumor after cytotoxic chemotherapy, and early recurrence after complete surgical excision. Cell-surface targeted therapies have been successful for breast cancer in general, however there are currently no approved cell-surface targeted therapies specifically indicated for TNBC. Recently, several cell-surface targets have been identified as candidates for treatment of TNBC and associated targeted therapies are in development. The purpose of this work is to review the current clinical challenges posed by TNBC, the therapeutic approaches currently in use, and provide an overview of developing cell surface targeting approaches to improve outcomes for treatment resistant TNBC.

5.
Molecules ; 24(23)2019 Nov 26.
Article in English | MEDLINE | ID: mdl-31779154

ABSTRACT

Targeted alpha-particle therapy (TAT) aims to selectively deliver radionuclides emitting α-particles (cytotoxic payload) to tumors by chelation to monoclonal antibodies, peptides or small molecules that recognize tumor-associated antigens or cell-surface receptors. Because of the high linear energy transfer (LET) and short range of alpha (α) particles in tissue, cancer cells can be significantly damaged while causing minimal toxicity to surrounding healthy cells. Recent clinical studies have demonstrated the remarkable efficacy of TAT in the treatment of metastatic, castration-resistant prostate cancer. In this comprehensive review, we discuss the current consensus regarding the properties of the α-particle-emitting radionuclides that are potentially relevant for use in the clinic; the TAT-mediated mechanisms responsible for cell death; the different classes of targeting moieties and radiometal chelators available for TAT development; current approaches to calculating radiation dosimetry for TATs; and lead optimization via medicinal chemistry to improve the TAT radiopharmaceutical properties. We have also summarized the use of TATs in pre-clinical and clinical studies to date.


Subject(s)
Alpha Particles/therapeutic use , Neoplasms/radiotherapy , Radiopharmaceuticals/therapeutic use , Animals , Antibodies, Monoclonal/therapeutic use , Humans , Radioisotopes/therapeutic use , Radiometry/methods
6.
Molecules ; 24(18)2019 Sep 19.
Article in English | MEDLINE | ID: mdl-31546752

ABSTRACT

Using targeted ligands to deliver alpha-emitting radionuclides directly to tumor cells has become a promising therapeutic strategy. To calculate the radiation dose to patients, activities of parent and daughter radionuclides must be measured. Scintillation detectors can be used to quantify these activities; however, activities found in pre-clinical and clinical studies can exceed their optimal performance range. Therefore, a method of correcting scintillation detector measurements at higher activities was developed using Monte Carlo modeling. Because there are currently no National Institute of Standards and Technology traceable Actinium-225 (225Ac) standards available, a well-type ionization chamber was used to measure 70.3 ± 7.0, 144.3 ± 14.4, 222.0 ± 22.2, 299.7 ± 30.0, 370.0 ± 37.0, and 447.7 ± 44.7 kBq samples of 225Ac obtained from Oak Ridge National Lab. Samples were then placed in a well-type NaI(Tl) scintillation detector and spectra were obtained. Alpha particle activity for each species was calculated using gamma abundance per alpha decay. MCNP6 Monte Carlo software was used to simulate the 4π-geometry of the NaI(Tl) detector. Using the ionization chamber reading as activity input to the Monte Carlo model, spectra were obtained and compared to NaI(Tl) spectra. Successive simulations of different activities were run until a spectrum minimizing the mean percent difference between the two was identified. This was repeated for each sample activity. Ionization chamber calibration measurements showed increase in error from 3% to 10% as activities decreased, resulting from decreasing detection efficiency. Measurements of 225Ac using both detector types agreed within 7% of Oak Ridge stated activities. Simulated Monte Carlo spectra of 225Ac were successfully generated. Activities obtained from these spectra differed with ionization chamber readings up to 156% at 147.7 kBq. Simulated spectra were then adjusted to correct NaI(Tl) measurements to be within 1%. These were compared to ionization chamber readings and a response relationship was determined between the two instruments. Measurements of 225Ac and daughter activity were conducted using a NaI(Tl) scintillation detector calibrated for energy and efficiency and an ionization chamber calibrated for efficiency using a surrogate calibration reference. Corrections provided by Monte Carlo modeling improve the accuracy of activity quantification for alpha-particle emitting radiopharmaceuticals in pre-clinical and clinical studies.


Subject(s)
Alpha Particles , Monte Carlo Method , Radiation , Gamma Rays , Normal Distribution
7.
J Nucl Med ; 60(8): 1124-1133, 2019 08.
Article in English | MEDLINE | ID: mdl-30733316

ABSTRACT

New effective therapies are greatly needed for metastatic uveal melanoma, which has a very poor prognosis with a median survival of less than 1 y. The melanocortin 1 receptor (MC1R) is expressed in 94% of uveal melanoma metastases, and a MC1R-specific ligand (MC1RL) with high affinity and selectivity for MC1R was previously developed. Methods: The 225Ac-DOTA-MC1RL conjugate was synthesized in high radiochemical yield and purity and was tested in vitro for biostability and for MC1R-specific cytotoxicity in uveal melanoma cells, and the lanthanum-DOTA-MC1RL analog was tested for binding affinity. Non-tumor-bearing BALB/c mice were tested for maximum tolerated dose and biodistribution. Severe combined immunodeficient mice bearing uveal melanoma tumors or engineered MC1R-positive and -negative tumors were studied for biodistribution and efficacy. Radiation dosimetry was calculated using mouse biodistribution data and blood clearance kinetics from Sprague-Dawley rat data. Results: High biostability, MC1R-specific cytotoxicity, and high binding affinity were observed. Limiting toxicities were not observed at even the highest administered activities. Pharmacokinetics and biodistribution studies revealed rapid blood clearance (<15 min), renal and hepatobillary excretion, MC1R-specific tumor uptake, and minimal retention in other normal tissues. Radiation dosimetry calculations determined pharmacokinetics parameters and absorbed α-emission dosages from 225Ac and its daughters. Efficacy studies demonstrated significantly prolonged survival and decreased metastasis burden after a single administration of 225Ac-DOTA-MC1RL in treated mice relative to controls. Conclusion: These results suggest significant potential for the clinical translation of 225Ac-DOTA-MC1RL as a novel therapy for metastatic uveal melanoma.


Subject(s)
Melanoma/radiotherapy , Molecular Targeted Therapy , Receptor, Melanocortin, Type 1/chemistry , Uveal Neoplasms/radiotherapy , Alpha Particles , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Chelating Agents/chemistry , Female , Humans , Lanthanoid Series Elements/chemistry , Male , Maximum Tolerated Dose , Mice , Mice, Inbred BALB C , Mice, SCID , Neoplasm Metastasis , Neoplasm Transplantation , Prognosis , Radiometry , Radiopharmaceuticals/pharmacokinetics , Rats , Rats, Sprague-Dawley
8.
Medchemcomm ; 9(7): 1155-1163, 2018 Jul 01.
Article in English | MEDLINE | ID: mdl-30109003

ABSTRACT

The successful delivery of toxic cargo directly to tumor cells is of primary importance in targeted (α) particle therapy. Complexes of radioactive atoms with the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating agent are considered as effective materials for such delivery processes. The DOTA chelator displays high affinity to radioactive metal isotopes and retains this capability after conjugation to tumor targeting moieties. Although the α-decay chains are well defined for many isotopes, the stability of chelations during the decay process and the impact of released energy on their structures remain unknown. The radioactive isotope 225Ac is an α-particle emitter that can be easily chelated by DOTA. However, 225Ac has a complex decay chain with four α-particle emissions during decay of each radionuclide. To advance our fundamental understanding of the consequences of α-decay on the stability of tumor-targeted 225Ac-DOTA conjugate radiopharmaceuticals, we performed first principles calculations of the structure, stability, and electronic properties of the DOTA chelator to the 225Ac radioactive isotope, and the initial daughters in the decay chain, 225Ac, 221Fr, 217At and 213Bi. Our calculations show that the atomic positions, binding energies, and electron localization functions are affected by the interplay between spin-orbit coupling, weak dispersive interactions, and environmental factors. Future empirical measurements may be guided and interpreted in light of these results.

9.
Sci Rep ; 8(1): 3638, 2018 02 26.
Article in English | MEDLINE | ID: mdl-29483578

ABSTRACT

Targeted therapy has held promise to be a successful anticancer treatment due to its specificity towards tumor cells that express the target receptors. However, not all targeting drugs used in the clinic are equally effective in tumor eradication. To examine which biochemical and biophysical properties of targeted agents are pivotal for their effective distribution inside the tumor and their efficient cellular uptake, we combine mathematical micro-pharmacological modeling with in vivo imaging of targeted human xenograft tumors in SCID mice. The mathematical model calibrated to experimental data was used to explore properties of the targeting ligand (diffusion and affinity) and ligand release schemes (rates and concentrations) with a goal to identify the properties of cells and ligands that enable high receptor saturation. By accounting for heterogeneities typical of in vivo tumors, our model was able to identify cell- and tissue-level barriers to efficient drug uptake. This work provides a base for utilizing experimentally measurable properties of a ligand-targeted agent and patient-specific attributes of the tumor tissue to support the development of novel targeted imaging agents and for improvement in their delivery to individual tumor cells.


Subject(s)
Models, Theoretical , Animals , Cell Line, Tumor , Drug Delivery Systems , Humans , Mice , Mice, SCID , Microscopy, Fluorescence , Pancreatic Neoplasms/metabolism
10.
Cancer Res ; 77(18): 4763-4772, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28536279

ABSTRACT

Genomic instability and high mutation rates cause cancer to acquire numerous mutations and chromosomal alterations during its somatic evolution; most are termed passengers because they do not confer cancer phenotypes. Evolutionary simulations and cancer genomic studies suggest that mildly deleterious passengers accumulate and can collectively slow cancer progression. Clinical data also suggest an association between passenger load and response to therapeutics, yet no causal link between the effects of passengers and cancer progression has been established. To assess this, we introduced increasing passenger loads into human cell lines and immunocompromised mouse models. We found that passengers dramatically reduced proliferative fitness (∼3% per Mb), slowed tumor growth, and reduced metastatic progression. We developed new genomic measures of damaging passenger load that can accurately predict the fitness costs of passengers in cell lines and in human breast cancers. We conclude that genomic instability and an elevated load of DNA alterations in cancer is a double-edged sword: it accelerates the accumulation of adaptive drivers, but incurs a harmful passenger load that can outweigh driver benefit. The effects of passenger alterations on cancer fitness were unrelated to enhanced immunity, as our tests were performed either in cell culture or in immunocompromised animals. Our findings refute traditional paradigms of passengers as neutral events, suggesting that passenger load reduces the fitness of cancer cells and slows or prevents progression of both primary and metastatic disease. The antitumor effects of chemotherapies can in part be due to the induction of genomic instability and increased passenger load. Cancer Res; 77(18); 4763-72. ©2017 AACR.


Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/pathology , Breast/pathology , Cell Transformation, Neoplastic/pathology , Lung Neoplasms/secondary , Mutation , Animals , Breast/metabolism , Breast Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Disease Progression , Female , Humans , Lung Neoplasms/genetics , Mice , Mice, SCID
11.
Oncotarget ; 8(69): 113373-113402, 2017 Dec 26.
Article in English | MEDLINE | ID: mdl-29371917

ABSTRACT

Lung cancer is the leading cause of cancer deaths in the United States. Novel lung cancer targeted therapeutic and molecular imaging agents are needed to improve outcomes and enable personalized care. Since these agents typically cannot cross the plasma membrane while carrying cytotoxic payload or imaging contrast, discovery of cell-surface targets is a necessary initial step. Herein, we report the discovery and characterization of lung cancer cell-surface markers for use in development of targeted agents. To identify putative cell-surface markers, existing microarray gene expression data from patient specimens were analyzed to select markers with differential expression in lung cancer compared to normal lung. Greater than 200 putative cell-surface markers were identified as being overexpressed in lung cancers. Ten cell-surface markers (CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11 and TMPRSS4) were selected based on differential mRNA expression in lung tumors vs. non-neoplastic lung samples and other normal tissues, and other considerations involving known biology and targeting moieties. Protein expression was confirmed by immunohistochemistry (IHC) staining and scoring of patient tumor and normal tissue samples. As further validation, marker expression was determined in lung cancer cell lines using microarray data and Kaplan-Meier survival analyses were performed for each of the markers using patient clinical data. High expression for six of the markers (CA9, CA12, CXorf61, GPR87, LYPD3, and SLC7A11) was significantly associated with worse survival. These markers should be useful for the development of novel targeted imaging probes or therapeutics for use in personalized care of lung cancer patients.

12.
Theranostics ; 6(5): 698-709, 2016.
Article in English | MEDLINE | ID: mdl-27022417

ABSTRACT

The theranostic potential of (225)Ac-based radiopharmaceuticals continues to increase as researchers seek innovative ways to harness the nuclear decay of this radioisotope for therapeutic and imaging applications. This communication describes the evaluation of (225)Ac-DOTA-c(RGDyK) in both biodistribution and Cerenkov luminescence imaging (CLI) studies. Initially, La-DOTA-c(RGDyK) was prepared as a non-radioactive surrogate to evaluate methodologies that would contribute to an optimized radiochemical synthetic strategy and estimate the radioactive conjugate's affinity for αvß3, using surface plasmon resonance spectroscopy. Surface plasmon resonance spectroscopy studies revealed the IC50 and Ki of La-DOTA-c(RGDyK) to be 33 ± 13 nM and 26 ± 11 nM, respectively, and suggest that the complexation of the La(3+) ion to the conjugate did not significantly alter integrin binding. Furthermore, use of this surrogate allowed optimization of radiochemical synthesis strategies to prepare (225)Ac-DOTA-c(RGDyK) with high radiochemical purity and specific activity similar to other (225)Ac-based radiopharmaceuticals. This radiopharmaceutical was highly stable in vitro. In vivo biodistribution studies confirmed the radiotracer's ability to target αvß3 integrin with specificity; specificity was detected in tumor-bearing animals using Cerenkov luminescence imaging. Furthermore, tumor growth control was achieved using non-toxic doses of the radiopharmaceutical in U87mg tumor-bearing nude mice. To our knowledge, this is the first report to describe the CLI of αvß3 (+) tumors in live animals using the daughter products derived from (225)Ac decay in situ. This concept holds promise to further enhance development of targeted alpha particle therapy.


Subject(s)
Coordination Complexes/pharmacokinetics , Coordination Complexes/therapeutic use , Neoplasms/diagnostic imaging , Neoplasms/radiotherapy , Optical Imaging/methods , Radiopharmaceuticals/pharmacokinetics , Radiopharmaceuticals/therapeutic use , Animals , Integrin alphaVbeta3/metabolism , Mice, Nude , Treatment Outcome
13.
Oncotarget ; 7(12): 14605-15, 2016 Mar 22.
Article in English | MEDLINE | ID: mdl-26910279

ABSTRACT

The expression of cholecystokinin 2 receptor (CCK2R, CCKBR or gastrin receptor) has been reported on a diverse range of cancers such as colorectal, liver, lung, pancreatic, ovarian, stomach, thyroid and numerous neuroendocrine/carcinoid tumors. Some cancers of the colorectum, lung, pancreas and thyroid have been shown to overexpress CCK2R in relation to normal matched tissues of the same organ. This reported overexpression has led to the development of a number of CCK2R-ligand targeted imaging and therapeutic agents. However, no comprehensive study comparing the expression of CCK2R in multiple cancers to multiple normal tissues has been performed. Herein, we report the immunohistochemical analysis of cancer samples from gastrointestinal stromal tumor (GIST), hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), pancreatic adenocarcinoma, and thyroid cancer against multiple normal tissue samples from esophagus, liver, lung, pancreas, stomach, spleen and thyroid. These results show that CCK2R expression is present in nearly all cancer and normal samples tested and that none of the cancer samples had expression that was statistically greater than that of all of the normal samples.


Subject(s)
Biomarkers, Tumor/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Receptor, Cholecystokinin B/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Case-Control Studies , Gastrointestinal Stromal Tumors/metabolism , Gastrointestinal Stromal Tumors/pathology , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Staging , Neuroendocrine Tumors/metabolism , Neuroendocrine Tumors/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Prognosis , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , Pancreatic Neoplasms
14.
Oncotarget ; 7(14): 17773-89, 2016 Apr 05.
Article in English | MEDLINE | ID: mdl-26894861

ABSTRACT

Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC.


Subject(s)
Adenocarcinoma/genetics , Adenoma/genetics , Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adenoma/metabolism , Adenoma/pathology , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Early Detection of Cancer , Gene Expression Profiling , HT29 Cells , Humans , Immunohistochemistry , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics
15.
Bioconjug Chem ; 27(2): 427-38, 2016 Feb 17.
Article in English | MEDLINE | ID: mdl-26488422

ABSTRACT

In the United States, lung cancer is the leading cause of cancer death and ranks second in the number of new cases annually among all types of cancers. Better methods or tools for diagnosing and treating this disease are needed to improve patient outcomes. The delta-opioid receptor (δOR) is reported to be overexpressed in lung cancers and not expressed in normal lung. Thus, we decided to develop a lung cancer-specific imaging agent targeting this receptor. We have previously developed a δOR-targeted fluorescent imaging agent based on a synthetic peptide antagonist (Dmt-Tic) conjugated to a Cy5 fluorescent dye. In this work, we describe the synthesis of Dmt-Tic conjugated to a longer wavelength near-infrared fluorescent (NIRF) dye, Li-cor IR800CW. Binding affinity of Dmt-Tic-IR800 for the δOR was studied using lanthanide time-resolved fluorescence (LTRF) competitive binding assays in cells engineered to overexpress the δOR. In addition, we identified lung cancer cell lines with high and low endogenous expression of the δOR. We confirmed protein expression in these cell lines using confocal fluorescence microscopy imaging and used this technique to estimate the cell-surface receptor number in the endogenously expressing lung cancer cell lines. The selectivity of Dmt-Tic-IR800 for imaging of the δOR in vivo was shown using both engineered cell lines and endogenously expressing lung cancer cells in subcutaneous xenograft models in mice. In conclusion, the δOR-specific fluorescent probe developed in this study displays excellent potential for imaging of lung cancer.


Subject(s)
Carbocyanines/metabolism , Dipeptides/metabolism , Fluorescent Dyes/metabolism , Lung Neoplasms/diagnosis , Lung/metabolism , Optical Imaging , Receptors, Opioid, delta/metabolism , Tetrahydroisoquinolines/metabolism , Animals , Binding, Competitive , Carbocyanines/chemical synthesis , Carbocyanines/chemistry , Cell Line, Tumor , Dipeptides/chemical synthesis , Dipeptides/chemistry , Female , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Humans , Lung/pathology , Lung Neoplasms/metabolism , Mice , Mice, Nude , Receptors, Opioid, delta/analysis , Tetrahydroisoquinolines/chemical synthesis , Tetrahydroisoquinolines/chemistry
16.
Mol Pharm ; 13(2): 534-44, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26713599

ABSTRACT

Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist-fluorescent dye conjugate, as a tumor-targeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo, and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR nonexpressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long-lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation, and is not retained in non-δOR-expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent.


Subject(s)
Carbocyanines/pharmacokinetics , Colonic Neoplasms/drug therapy , Dipeptides/pharmacokinetics , Fluorescent Dyes/chemistry , Receptors, Opioid, delta/chemistry , Tetrahydroisoquinolines/pharmacokinetics , Animals , Apoptosis , Carbocyanines/administration & dosage , Cell Proliferation , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Dipeptides/administration & dosage , Female , Humans , Immunoenzyme Techniques , Kinetics , Mice , Mice, Nude , Narcotic Antagonists/administration & dosage , Narcotic Antagonists/pharmacokinetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Spectroscopy, Near-Infrared , Tetrahydroisoquinolines/administration & dosage , Tissue Distribution , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
17.
Mol Imaging Biol ; 18(2): 219-31, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26276155

ABSTRACT

PURPOSE: Hypoxia is commonly observed in regions of primary tumors and metastases, and is associated with resistance to treatment, more aggressive tumor phenotypes and poor prognosis. Reliable and validated imaging biomarkers of hypoxia are needed for pre-clinical studies and clinical use. Expression of cell-surface carbonic anhydrases IX and XII (CAIX and CAXII) in tumor cells has been associated with tumor hypoxia. CAIX and CAXII specific antibodies conjugated to fluorescent dye were evaluated for the non-invasive detection of hypoxia in vivo. PROCEDURES: Human breast cancer cell lines (MCF10A, DCIS, MCF7, ZR-75.1 and MDA-mb231) were characterized for CAIX and CAXII expression by real-time RT-PCR and immunocytochemistry (ICC) under normoxic and hypoxic conditions. Immunohistochemical (IHC) staining of CAIX, CAXII and the commercially available exogenous hypoxia marker, pimonidazole, was performed using sections of ZR-75.1 and MDA-mb-231 orthotopic breast cancer xenograft tumors from nude mice. In vivo fluorescence imaging of ZR-75.1 tumors in animals housed at varied levels of oxygen was used to quantify the relative uptake of the CAIX and CAXII agents and a commercially available sulfonamide-based agent. Corresponding tumor sections were IHC stained for CAIX, CAXII and pimonidazole. RESULTS: CAIX mRNA expression was significantly higher (p < 0.05) in hypoxia for all cell lines, which was in agreement with protein expression by ICC. CAXII expression was mixed, with a modest hypoxia-related increase in two cell lines (p < 0.05) and no change in others. Quantified IHC staining of ZR-75.1 and MDA-mb-231 tumor sections showed that CAIX and CAXII expression was elevated in regions with pimonidazole staining, but CAXII levels were lower than CAIX. Tumor uptake of the CAIX targeted agent, and IHC staining of CAIX and pimonidazole in corresponding tumor sections were correlated, and co-registered, and shown to be significantly elevated by level of oxygenation (p < 0.001): hypoxia > normoxia > hyperoxia. However, the CAXII and sulfonamide agents were not significantly correlated with hypoxia. CONCLUSION: These studies suggest that the fluorescently labeled CAIX-specific agent is a more robust indicator of hypoxia in vivo compared to the CAXII-specific agent or the agent specific to the CA active site.


Subject(s)
Antigens, Neoplasm/metabolism , Biomarkers, Tumor/metabolism , Breast Neoplasms/enzymology , Carbonic Anhydrase IX/metabolism , Carbonic Anhydrases/metabolism , Molecular Imaging/methods , Oxygen/pharmacology , Tumor Hypoxia/drug effects , Tumor Hypoxia/genetics , Analysis of Variance , Animals , Antigens, Neoplasm/genetics , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carbonic Anhydrase IX/genetics , Carbonic Anhydrases/genetics , Cell Line, Tumor , Female , Fluorescence , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice, Nude , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tomography , Xenograft Model Antitumor Assays
18.
Nat Commun ; 6: 8752, 2015 Dec 10.
Article in English | MEDLINE | ID: mdl-26658462

ABSTRACT

Early cancers are avascular and hence, profoundly acidic. Pre-malignant cells must adapt to acidosis to thrive in this hostile microenvironment. Here, we investigate MCF-7 cells that are adapted to grow in acidic conditions using SILAC proteomics and we reveal a significant upregulation of lysosomal proteins. Prominent among these is LAMP2 that functions to protect lysosomal membranes from acid proteolysis. LAMP2 upregulation by acidosis is confirmed both in vitro and in vivo. Furthermore, we show that the depletion of LAMP2 is sufficient to increase acidosis-mediated toxicity. In breast cancer patient samples, there is a high correlation of LAMP2 mRNA and protein expression with progression. We also observe that LAMP2 is located at the plasma membrane in clinical samples and this redistribution is acid-induced in vitro. Our findings suggest a potential adaptive mechanism, wherein cells chronically exposed to an acidic environment translocate lysosomal proteins to their surface, thus protecting the plasmalemma from acid-induced hydrolysis.


Subject(s)
Cell Membrane/metabolism , Gene Expression Regulation, Neoplastic/physiology , Lysosomal-Associated Membrane Protein 2/metabolism , Neoplasms/metabolism , Animals , Biomarkers, Tumor , Cell Line, Tumor , Female , Humans , Hydrogen-Ion Concentration , Lysosomal-Associated Membrane Protein 2/genetics , Mice , Mice, Nude , Neoplasms, Experimental/metabolism , Protein Array Analysis , Proteomics
19.
Front Immunol ; 5: 429, 2014.
Article in English | MEDLINE | ID: mdl-25368611

ABSTRACT

Toll-like receptors (TLRs) are expressed by immune cells, intestinal epithelium, and tumor cells. In the homeostatic setting, they help to regulate control over invading pathogens and maintain the epithelial lining of the large and small intestines. Aberrant expression of certain TLRs by tumor cells can induce growth inhibition while others contribute to tumorigenesis and progression. Activation of these TLRs can induce inflammation, tumor cell proliferation, immune evasion, local invasion, and distant metastasis. These TLR-influenced behaviors have similarities with properties observed in leukocytes, suggesting that tumors may be hijacking immune programs to become more aggressive. The concept of epithelial to leucocytic-transition (ELT) is proposed, akin to epithelial to mesenchymal transition, in which tumors develop the ability to activate leucocytic traits otherwise inaccessible to epithelial cells. Understanding the mechanisms of ELT could lead to novel therapeutic strategies for inhibiting tumor metastasis.

20.
Subcell Biochem ; 75: 221-54, 2014.
Article in English | MEDLINE | ID: mdl-24146382

ABSTRACT

Carbonic anhydrase IX (CAIX) which is a zinc containing metalloprotein, efficiently catalyzes the reversible hydration of carbon dioxide. It is constitutively up-regulated in several cancer types and has an important role in tumor progression, acidification and metastasis. High expression of CAIX generally correlates with poor prognosis and is related to a decrease in the disease-free interval following successful therapy. Therefore, it is considered as a prognostic indicator in oncology.In this review, we describe CAIX regulation and its role in tumor hypoxia, acidification and metastasis. In addition, the molecular imaging of CAIX and its potential for use in cancer detection, diagnosis, staging, and for use in following therapy response is discussed. Both antibodies and small molecular weight compounds have been used for targeted imaging of CAIX expression. The use of CAIX expression as an attractive and promising candidate marker for systemic anticancer therapy is also discussed.


Subject(s)
Antigens, Neoplasm/genetics , Carbonic Anhydrases/genetics , Gene Expression Regulation, Neoplastic , Neoplasms/drug therapy , Neoplasms/enzymology , Antigens, Neoplasm/biosynthesis , Carbon Dioxide/metabolism , Carbonic Anhydrase IX , Carbonic Anhydrases/biosynthesis , Cell Hypoxia/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Neoplasm Metastasis , Neoplasms/genetics , Neoplasms/pathology , Zinc/metabolism
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