Evaluation of a CD206-Targeted Peptide for PET Imaging of Macrophages in Syngeneic Mouse Models of Cancer.

Tumor-associated macrophages (TAMs) are large phagocytic cells that play numerous roles in cancer biology and are an important component of the relationship between immune system response and tumor progression. The peptide, RP832c, targets the Mannose Receptor (CD206) expressed on M2-like macrophages and is cross-reactive to both human and murine CD206. Additionally, it exhibits therapeutic properties through its ability to shift the population of TAMs from an M2-like (protumor) toward an M1-like phenotype (antitumor) and has demonstrated promise in inhibiting tumor resistance in PD-L1 unresponsive melanoma murine models. In addition, it has shown inhibition in bleomycin-induced pulmonary fibrosis through interactions with CD206 macrophages.1,2 Our work aims to develop a novel CD206 positron emission tomography (PET) imaging probe based on RP832c (Kd = 5.64 µM) as a direct, noninvasive method for the assessment of TAMs in mouse models of cancer. We adapted RP832c to incorporate the chelator DOTA to allow for radiolabeling with the PET isotope 68Ga (t1/2 = 68 min; ß+ = 89%). In vitro stability studies were conducted in mouse serum up to 3 h. The in vitro binding characteristics of [68Ga]RP832c to CD206 were determined by a protein plate binding assay and Surface Plasmon Resonance (SPR). PET imaging and biodistribution studies were conducted in syngeneic tumor models. Stability studies in mouse serum demonstrated that 68Ga remained complexed up to 3 h (less than 1% free 68Ga). Binding affinity studies demonstrated high binding of [68Ga]RP832c to mouse CD206 protein and that the binding of the tracer was able to be blocked significantly when incubated with a blocking solution of native RP832c. PET imaging and biodistribution studies in syngeneic tumor models demonstrated uptake in tumor and CD206 expressing organs of [68Ga]RP832c. A significant correlation was found between the percentage of CD206 present in each tumor imaged with [68Ga]RP832c and PET imaging mean standardized uptake values in a CT26 mouse model of cancer. The data shows that [68Ga]RP832c represents a promising candidate for macrophage imaging in cancer and other diseases.

In vivo Assessment of the Impact of Molecular Weight on Constructs of 68Ga-DOTA-Manocept in a Syngeneic Mouse Tumor Model.

PURPOSE: Manocept™ constructs are mannosylated amine dextrans (MADs) that bind with high affinity to the mannose receptor, CD206. Tumor-associated macrophages (TAMs) are the most numerous immune cells in the tumor microenvironment and a recognized target for tumor imaging and cancer immunotherapies. Most TAMs express CD206, suggesting utility of MADs to deliver imaging moieties or therapeutics to TAMs. The liver Kupffer cells also express CD206, making them an off-target localization site when targeting CD206 on TAMs. We evaluated TAM targeting strategies using two novel MADs differing in molecular weight in a syngeneic mouse tumor model to determine how varying MAD molecular weights would impact tumor localization. Increased mass dose of the non-labeled construct or a higher molecular weight (HMW) construct were also used to block liver localization and enhance tumor to liver ratios. PROCEDURES: Two MADs, 8.7 kDa and 22.6 kDa modified with DOTA chelators, were synthesized and radiolabeled with 68Ga. A HMW MAD (300 kDa) was also synthesized as a competitive blocking agent for Kupffer cell localization. Balb/c mice, with and without CT26 tumors, underwent dynamic PET imaging for 90 min followed by biodistribution analyses in selected tissues. RESULTS: The new constructs were readily synthesized and labeled with 68Ga with ≥ 95% radiochemical purity in 15 min at 65 °C. When injected at doses of 0.57 nmol, the 8.7 kDa MAD provided 7-fold higher 68Ga tumor uptake compared to the 22.6 kDa MAD (2.87 ± 0.73%ID/g vs. 0.41 ± 0.02%ID/g). Studies with increased mass of unlabeled competitors showed reduced liver localization of the [68Ga]MAD-8.7 to varying degrees without significant reductions in tumor localization, resulting in enhanced tumor to liver signal ratios. CONCLUSION: Novel [68Ga]Manocept constructs were synthesized and studied in in vivo applications, showing that the smaller MAD localized to CT26 tumors more effectively than the larger MAD and that the unlabeled HMW construct could selectively block liver binding of [68Ga]MAD-8.7 without diminishing the localization to tumors. Promising results using the [68Ga]MAD-8.7 show a potential path to clinical applications.

Sc-HOPO: A Potential Construct for Use in Radioscandium-Based Radiopharmaceuticals.

Three isotopes of scandium─43Sc, 44Sc, and 47Sc─have attracted increasing attention as potential candidates for use in imaging and therapy, respectively, as well as for possible theranostic use as an elementally matched pair. Here, we present the octadentate chelator 3,4,3-(LI-1,2-HOPO) (or HOPO), an effective chelator for hard cations, as a potential ligand for use in radioscandium constructs with simple radiolabeling under mild conditions. HOPO forms a 1:1 Sc-HOPO complex that was fully characterized, both experimentally and theoretically. [47Sc]Sc-HOPO exhibited good stability in chemical and biological challenges over 7 days. In healthy mice, [43,47Sc]Sc-HOPO cleared the body rapidly with no signs of demetalation. HOPO is a strong candidate for use in radioscandium-based radiopharmaceuticals.

Positron Emission Tomography Imaging of Macrophages in Cancer.

Macrophages are large phagocytic cells that can be classified as a type of white blood cell and may be either mobile or stationary in tissues. The presence of macrophages in essentially every major disease makes them attractive candidates to serve as therapeutic targets and diagnostic biomarkers. Macrophages that are found in the microenvironment of solid tumors are referred to as tumor-associated macrophages (TAMs) and have been shown to influence chemoresistance, immune regulation, tumor initiation and tumor growth. The imaging of TAMs through Positron Emission Tomography (PET) has the potential to provide valuable information on cancer biology, tumor progression, and response to therapy. This review will highlight the versatility of macrophage imaging in cancer through the use of PET.

Retinopathy and chronic kidney disease in the Chronic Renal Insufficiency Cohort (CRIC) study.

OBJECTIVE: To investigate the association between retinopathy and chronic kidney disease. METHODS: In this observational, cross-sectional study, 2605 patients of the Chronic Renal Insufficiency Cohort (CRIC) study, a multicenter study of chronic kidney disease, were offered participation. Nonmydriatic fundus photographs of the disc and macula in both eyes were obtained in 1936 of these subjects. The photographs were reviewed in a masked fashion at a central photograph reading center using standard protocols. Presence and severity of retinopathy (diabetic, hypertensive, or other) and vessel diameter caliber were assessed by trained graders and a retinal specialist using protocols developed for large epidemiologic studies. Kidney function measurements and information on traditional and nontraditional risk factors for decreased kidney function were obtained from the CRIC study. RESULTS: Greater severity of retinopathy was associated with lower estimated glomerular filtration rate after adjustment for traditional and nontraditional risk factors. The presence of vascular abnormalities usually associated with hypertension was also associated with lower estimated glomerular filtration rate. We found no strong direct relationship between estimated glomerular filtration rate and average arteriolar or venular calibers. CONCLUSIONS: Our findings show a strong association between severity of retinopathy and its features and level of kidney function after adjustment for traditional and nontraditional risk factors for chronic kidney disease, suggesting that retinovascular pathology reflects renal disease.

Association between retinopathy and cardiovascular disease in patients with chronic kidney disease (from the Chronic Renal Insufficiency Cohort [CRIC] Study).

Patients with chronic kidney disease experience co-morbid illnesses, including cardiovascular disease (CVD) and retinopathy. The purpose of the present study was to assess the association between retinopathy and self-reported CVD in a subgroup of the participants in the Chronic Renal Insufficiency Cohort study. For this observational, ancillary investigation, 2,605 Chronic Renal Insufficiency Cohort participants were invited to participate in the present study, and nonmydriatic fundus photographs in both eyes were obtained for 1,936 subjects. The photographs were reviewed in a masked fashion at a central photograph reading center. The presence and severity of retinopathy (diabetic, hypertensive, or other) and vessel diameter caliber were assessed using standard protocols by trained graders who were masked to the information about the study participants. A history of self-reported CVD was obtained using a medical history questionnaire. Kidney function measurements and traditional and nontraditional risk factors for CVD were obtained from the Chronic Renal Insufficiency Cohort study. A greater severity of retinopathy was associated with a greater prevalence of any CVD, and this association persisted after adjustment for the traditional risk factors for CVD. The presence of vascular abnormalities usually associated with hypertension was also associated with increased prevalence of CVD. We found a direct relation between CVD prevalence and mean venular caliber. In conclusion, the presence of retinopathy was associated with CVD, suggesting that retinovascular pathology might indicate macrovascular disease, even after adjustment for renal dysfunction and traditional CVD risk factors. This would make the assessment of retinal morphology a valuable tool in CKD studies of CVD outcomes.

Photographic assessment of baseline fundus morphologic features in the Comparison of Age-Related Macular Degeneration Treatments Trials.

OBJECTIVE: To describe the methods used for assessment of baseline fundus characteristics from color photography and fluorescein angiography (FA) in the Comparison of Age-Related Macular Degeneration Treatments Trials (CATT) and to describe the relationship between these characteristics and visual acuity. DESIGN: Randomized, masked, multicenter trial. PARTICIPANTS: This investigation included 1185 participants of the CATT study. METHODS: Baseline stereoscopic color fundus photographs and FAs of participants in the CATT study were assessed at a central fundus photograph reading center by masked readers. Replicate assessments of random samples of photographs were performed to assess intragrader and intergrader agreements. The association of the lesion characteristics with baseline visual acuity was assessed using analyses of variance and correlation coefficients. MAIN OUTCOME MEASURES: Intragrader and intergrader reproducibility, visual acuity, and lesion characteristics. RESULTS: Intragrader and intergrader reproducibility showed agreements ranging from 75% to 100% and weighted κ values ranging from 0.48 to 1.0 for qualitative determinations. The intraclass correlation coefficients were 0.96 to 0.97 for quantitative measurements of choroidal neovascularization (CNV) area and total area of CNV lesion. The mean visual acuity varied by the type of pathologic features in the foveal center: 64.5 letters (standard error, 0.7 letters) for fluid only, 59.0 letters (standard error, 0.5 letters) for CNV, and 58.7 letters (standard error, 1.3 letters) for hemorrhage (P<0.001). Fibrotic or atrophic scar present in the lesion, but not under the center of the fovea, also was associated with a markedly reduced visual acuity of 48.4 letters (standard error, 2.2 letters; P<0.0001). Although total area of CNV lesion was correlated weakly with visual acuity when all participants were assessed (Spearman correlation coefficient, ρ = -0.16; P<0.001), the correlation was stronger within patients with predominantly classic lesions (ρ = -0.42; P<0.001). CONCLUSIONS: These results show that the methodology used for grading CATT fundus images has good reproducibility. As expected, larger total CNV lesion area and pathologic findings such as hemorrhage, fibrosis, and atrophy at baseline are associated with decreased visual acuity.

Prevalence of ocular fundus pathology in patients with chronic kidney disease.

BACKGROUND AND OBJECTIVES: The objective of this study was to describe the prevalence of ocular fundus pathology in the Chronic Renal Insufficiency Cohort (CRIC) study, a multicenter, longitudinal study of individuals with varying stages of chronic kidney disease (CKD). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this cross-sectional study, 45 degree digital photos of the disc and macula in both eyes were obtained by nonophthalmologic personnel using a nonmydriatic Canon CR-DGI fundus camera in 1936 individuals who participated in the CRIC study. Photographs were assessed in a masked manner by graders and a retinal specialist at a central photograph reading center. The purpose of this review was to inform participants quickly of conditions that warranted a complete eye examination by an ophthalmologist. RESULTS: Among the 1936 participants who were photographed, 1904 (98%) had assessable photographs in at least one eye. Eye pathologies that required a follow-up examination by an ophthalmologist were identified in 864 (45%) of these 1904 participants. These eye pathologies included, among others, retinopathy (diabetic and/or hypertensive), a finding that was observed in 482 (25%) of these 1904 participants. Three percent (65 participants) of the 1904 participants had serious eye conditions that required urgent follow-up and treatment. Lower estimated GFR and cardiovascular disease were associated with greater eye pathology. Estimated GFR <30 ml/min per 1.73 m(2) was associated with a three times higher risk for retinopathy. CONCLUSIONS: We found a high prevalence of fundus pathology in participants with CKD. This finding supports recommendations for regular complete eye examinations in the CKD population.

Transforming growth factor beta3 regulates the versican variants in the extracellular matrix-rich uterine leiomyomas.

Uterine leiomyoma are common, benign tumors that are enriched in extracellular matrix. The tumors are characterized by a disoriented and loosely packed collagen fibril structure similar to other diseases with disrupted Transforming growth factor beta (TGF-beta) signaling. Here we characterized TGF-beta3 signaling and the expression patterns of the critical extracellular matrix component versican in leiomyoma and myometrial tissue and cell culture. We also demonstrate the regulation of the versican variants by TGF-beta3. Using leiomyoma and matched myometrium from 15 patients, messenger RNA (mRNA) from leiomyoma and myometrium was analyzed by semiquantitative real time reverse transcription-polymerase chain reaction (RT-PCR), while protein analysis was done by western blot. Transforming growth factor beta3 transcripts were increased 4-fold in leiomyoma versus matched myometrium. Phosphorylated-TGF-beta RII and phosphorylated-Smad 2/3 complex were greater in leiomyoma as documented by Western blot. The inhibitor Smad7 transcripts were decreased 0.44-fold. The glycosaminoglycan (GAG)-rich versican variants were elevated in leiomyoma versus myometrial tissue: specifically V0 (4.27 +/- 1.12) and V1 (2.01 +/- 0.27). Treatment of leiomyoma and myometrial cells with TGF-beta3 increased GAG-rich versican variant expression 7 to 12 fold. Neutralizing TGF-beta3 antibody decreased the expression of the GAG-rich versican variants 2 to 8 fold in leiomyoma cells. Taken together, the aberrant production of excessive and disorganized extracellular matrix that defines the leiomyoma phenotype involves the activation of the TGF-beta signaling pathway and excessive production of GAG-rich versican variants.