Sean Smart

Recruitment of a myeloid cell subset (CD11b/Gr1mid) via CCL2/CCR2 promotes the development of colorectal cancer liver metastasis

Liver metastasis from colorectal cancer is a leading cause of cancer mortality. Myeloid cells play pivotal roles in the metastatic process, but their prometastatic functions in liver metastasis remain incompletely understood. To investigate their role, we simulated liver metastasis in C57BL/6 mice through intrasplenic inoculation of MC38 colon carcinoma cells. Among the heterogeneous myeloid infiltrate, we identified a distinct population of CD11b/Gr1mid cells different from other myeloid populations previously associated with liver metastasis. These cells increased in number dramatically during establishment of liver metastases and were recruited from bone marrow by tumor‐derived CCL2. Liver metastasis of Lewis lung carcinoma cells followed this pattern but this mechanism is not universal as liver colonization by B16F1 melanoma cells did not recruit similar subsets. Inhibition of CCL2 signaling and absence of its cognate receptor CCR2 reduced CD11b/Gr1mid recruitment and decreased tumor burden. Depletion of the CD11b/Gr1mid subset in a transgenic CD11b‐diphtheria toxin receptor mouse model markedly reduced tumor cell proliferation. There was no evidence for involvement of an adaptive immune response in the prometastatic effects of CD11b/Gr1mid cells. Additionally, an analogous myeloid subset was found in liver metastases of some colorectal cancer patients. Conclusion: Collectively, our findings highlight the importance of myeloid cells—in this case a selective CD11b/Gr1mid subset—in sustaining development of colorectal cancer liver metastasis and identify a potential target for antimetastatic therapy. (HEPATOLOGY 2013)

A temporal MRI assessment of neuropathology after transient middle cerebral artery occlusion in the rat: correlations with behavior.

The purpose of this study was to evaluate the temporal and spatial pathological alterations within ischemic tissue using serial magnetic resonance imaging (MRI) and to determine the extent and duration of functional impairment using objective behavioral tests after transient middle cerebral artery occlusion (tMCAO) in the rat. MRI signatures derived from specific anatomical regions of interest (ROI) were then appropriately correlated to the behavioral measures over the time course of the study (up to 28 days post-tMCAO). Sprague-Dawley rats (n = 12) were initially trained on the following behavioral tasks before surgery: bilateral sticky label test (for contralateral neglect); beam walking (for hindlimb coordination); staircase test (for skilled forelimb paw-reaching). Rats were then randomly assigned to receive either tMCAO (90 minutes, n = 6), by means of the intraluminal thread technique, or sham-control surgery (n = 6). Proton density, T2- and T2-diffusion-weighted MR images were acquired at 1, 7, 14, and 28 days post-tMCAO that were then smoothed into respective proton density, T2 relaxation, and apparent diffusion coefficient (ADC) maps. Apparent percent total lesion volume was assessed using T2W imaging. MR signatures were evaluated using the tissue maps by defining ROI for MCAO and sham-control groups, which corresponded to the caudate-putamen, forelimb, hindlimb, and lower parietal cortices both ipsilateral and contralateral to the occlusion site. Behavioral tests were undertaken daily from 1 to 28 days post-tMCAO. Results demonstrate that apparent percent lesion volume reduced from 1 to 7 days (P < 0.05) but then remained constant up to 28 days for the MCAO group. Pathological changes in the temporal profile of T2 and ADC tissue signatures were significantly altered in specific ROI across the time course of the study (P < 0.05 to < 0.001), reflecting the progression of edema to necrosis and cavitation. Both T2 and ADC measures of ischemic pathology correlated with parameters defined by each of the functional tests (r ≥0.5, P < 0.05) across the time course. The staircase test revealed bilateral impairments for the MCAO group (P < 0.001), which were best predicted by damage to the ipsilateral lower parietal cortex by means of hierarchical multiple regression analyses (R2 changes ≥0.21, P < 0.03). Behavioral recovery was apparent on the beam walking test at 14 to 28 days post-MCAO, which was mirrored by MRI signatures within the hindlimb cortex returning to sham-control levels. This long-term study is the first of its kind in tracing the dynamic pathologic and functional consequences of tMCAO in the rat. Both serial MRI and objective behavioral assessment provide highly suitable outcome measures that can be effectively used to evaluate promising new antiischemic agents targeted for the clinic.

A comparison of the behavior of (64)Cu-acetate and (64)Cu-ATSM in vitro and in vivo.

64Cu-diacetyl-bis(N4-methylthiosemicarbazonate), 64Cu-ATSM, continues to be investigated clinically as a PET agent both for delineation of tumor hypoxia and as an effective indicator of patient prognosis, but there are still aspects of the mechanism of action that are not fully understood. Methods: The retention of radioactivity in tumors after administration of 64Cu-ATSM in vivo is substantially higher for tumors with a significant hypoxic fraction. This hypoxia-dependent retention is believed to involve the reduction of Cu-ATSM, followed by the loss of copper to cellular copper processing. To shed light on a possible role of copper metabolism in hypoxia targeting, we have compared 64Cu retention in vitro and in vivo in CaNT and EMT6 cells or cancers after the administration of 64Cu-ATSM or 64Cu-acetate. Results: In vivo in mice bearing CaNT or EMT6 tumors, biodistributions and dynamic PET data are broadly similar for 64Cu-ATSM and 64Cu-acetate. Copper retention in tumors at 15 min is higher after injection of 64Cu-acetate than 64Cu-ATSM, but similar values result at 2 and 16 h for both. Colocalization with hypoxia as measured by EF5 immunohistochemistry is evident for both at 16 h after administration but not at 15 min or 2 h. Interestingly, at 2 h tumor retention for 64Cu-acetate and 64Cu-ATSM, although not colocalizing with hypoxia, is reduced by similar amounts by increased tumor oxygenation due to inhalation of increased O2. In vitro, substantially less uptake is observed for 64Cu-acetate, although this uptake had some hypoxia selectivity. Although 64Cu-ATSM is stable in mouse serum alone, there is rapid disappearance of intact complex from the blood in vivo and comparable amounts of serum bound activity for both 64Cu-ATSM and 64Cu-acetate. Conclusion: That in vivo, in the EMT6 and CaNT tumors studied, the distribution of radiocopper from 64Cu-ATSM in tumors essentially mirrors that of 64Cu-acetate suggests that copper metabolism may also play a role in the mechanism of selectivity of Cu-ATSM.

Micro-CT for Anatomic Referencing in PET and SPECT: Radiation Dose, Biologic Damage, and Image Quality

CT is widely used for anatomic referencing of PET and SPECT images of small animals but requires sufficiently high radiation doses capable of causing significant DNA damage. Therefore, we described the relationship between radiation dose, biologic damage, and image quality to determine whether CT can be used without significantly compromising radiotherapy and tumor development studies. Methods: The CT dose index generated by the nanoSPECT/CT system was compared with measurements using EBT2 gafchromic film. The effects of micro-CT were evaluated in 2 mouse strains that differ in sensitivity to radiation. γH2AX foci analysis to determine leukocyte, liver, and jejunum DNA damage and hematoxylin and eosin staining to investigate macroscopic jejunum damage were performed. Signal-to-noise ratio, contrast-to-noise ratio, and scanner linearity were determined to assess image quality. Results: For the standard settings, that is, as set by the manufacturers, EBT2 gafchromic film dosimetry showed that the nanoSPECT/CT system underestimated the absorbed dose. Moreover, significant doses were obtained, resulting in a significant increase in γH2AX formation in leukocytes, liver, and jejunum 40 min after CT, using preset parameters when compared with nonimaged controls. The jejenum response was more pronounced for the more radiosensitive strain. In contrast to leukocytes, the liver and jejunum still showed evidence of DNA damage 3 d after CT. Contrast-to-noise ratio, signal-to-noise ratio, and scanner linearity were sufficient to allow for anatomic referencing for both imaging protocols tested. Conclusion: Anatomic reference images can be produced with no observable DNA damage or compromising image quality using low radiographic voltage, flux, and duration.

Ultra-fast and accurate assessment of cardiac function in rats using accelerated MRI at 9.4 Tesla

MRI can accurately and reproducibly assess cardiac function in rodents but requires relatively long imaging times. Therefore, parallel imaging techniques using a 4‐element RF‐coil array and MR sequences for cardiac MRI in rats were implemented at ultra‐high magnetic fields (9.4 Tesla [T]). The hypothesis that these developments would result in a major reduction in imaging time without loss of accuracy was tested on female Wistar rats under isoflurane anesthesia. High‐resolution, contiguous short‐axis slices (thickness 1.5 mm) were acquired covering the entire heart. Two interleaved data sets (i) with the volume coil (eight averages) and (ii) with the four‐element coil array (one average) were obtained. In addition, two‐, three‐, and fourfold accelerated data sets were generated through postprocessing of the coil array data, followed by a TGRAPPA reconstruction, resulting in five data sets per rat (in‐plane voxel size 100 × 100 μm). Using a single blinded operator, excellent agreement was obtained between volume coil (acquisition time: 88 min) and the fourfold accelerated (<3 min) data sets (e.g., LV mass 436 ± 21 mg vs 433 ± 19 mg; ejection fraction 74 ± 5% vs 75 ± 4%). This finding demonstrates that it is possible to complete a rat cine‐MRI study under 3 min with low variability and without losing temporal or spatial resolution, making high throughput screening programs feasible. Magn Reson Med 59:636–641, 2008.

Robust and high resolution hyperpolarized metabolic imaging of the rat heart at 7 T with 3D spectral-spatial EPI.

Hyperpolarized metabolic imaging has the potential to revolutionize the diagnosis and management of diseases where metabolism is dysregulated, such as heart disease. We investigated the feasibility of imaging rodent myocardial metabolism at high resolution at 7 T.

Hypoxia Imaging Using PET and SPECT: The Effects of Anesthetic and Carrier Gas on [64Cu]-ATSM, [99mTc]-HL91 and [18F]-FMISO Tumor Hypoxia Accumulation

Background Preclinical imaging requires anaesthesia to reduce motion-related artefacts. For direct translational relevance, anaesthesia must not significantly alter experimental outcome. This study reports on the effects of both anaesthetic and carrier gas upon the uptake of [64Cu]-CuATSM, [99mTc]-HL91 and [18F]-FMISO in a preclinical model of tumor hypoxia. Methodology/Principal Findings The effect of carrier gas and anaesthetic was studied in 6 groups of CaNT-bearing CBA mice using [64Cu]-CuATSM, [99mTc]-HL91 or [18F]-FMISO. Mice were anaesthetised with isoflurane in air, isoflurane in pure oxygen, with ketamine/xylazine or hypnorm/hypnovel whilst breathing air, or in the awake state whilst breathing air or pure oxygen. PET or SPECT imaging was performed after which the mice were killed for organ/tumor tracer quantitation. Tumor hypoxia was confirmed. Arterial blood gas analysis was performed for the different anaesthetic regimes. The results demonstrate marked influences on tumor uptake of both carrier gas and anaesthetic, and show differences between [99mTc]-HL91, [18F]-FMISO and [64Cu]-CuATSM. [99mTc]-HL91 tumor uptake was only altered significantly by administration of 100% oxygen. The latter was not the case for [18F]-FMISO and [64Cu]-CuATSM. Tumor-to-muscle ratio (TMR) for both compounds was reduced significantly when either oxygen or anaesthetics (isoflurane in air, ketamine/xylazine or hypnorm/hypnovel) were introduced. For [18F]-FMISO no further decrease was measured when both isoflurane and oxygen were administered, [64Cu]-CuATSM did show an additional significant decrease in TMR. When using the same anaesthetic regimes, the extent of TMR reduction was less pronounced for [64Cu]-CuATSM than for [18F]-FMISO (40–60% versus 70% reduction as compared to awake animals breathing air). Conclusions/Significance The use of anaesthesia can have profound effects on the experimental outcome. More importantly, all tested anaesthetics reduced tumor-hypoxia uptake. Anaesthesia cannot be avoided in preclinical studies but great care has to be taken in preclinical models of hypoxia as anaesthesia effects cannot be generalised across applications, nor disease states.

Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

Neutrophils promote hepatic metastasis growth through fibroblast growth factor 2–dependent angiogenesis in mice

Hepatic metastases are amenable to ablation; however, many patients are not suitable candidates for such therapy and recurrence is common. The tumor microenvironment is known to be essential for metastatic growth, yet identification of plausible targets for cancer therapy in the microenvironment has proven elusive. We found that human colorectal cancer liver metastases and murine gastrointestinal experimental liver metastases are infiltrated by neutrophils. Plasticity in neutrophils has recently been shown to lead to both protumor and antitumor effects. Here, neutrophils promoted the growth of hepatic metastases, given that depletion of neutrophils in already established, experimental, murine liver metastases led to diminished metastatic growth. Decreased growth was associated with reductions in vascular density and branching suggestive of vessel normalization. Metastasis‐associated neutrophils expressed substantially more fibroblast growth factor 2 (FGF2) than naïve neutrophils, indicating neutrophil polarization by the tumor microenvironment. Administration of FGF2 neutralizing antibody to mice bearing experimental liver metastases phenocopied neutrophil depletion by reducing liver metastatic colony growth, vascular density, and branching. Conclusion: Here, we show, using FGF2 as an example, that identification of factors responsible for the protumoral effects of infiltrating myeloid cells can be used to target established liver metastases. Such therapies could be utilized to limit disease progression and potentiate the effects of standard ablative therapies. (Hepatology 2017;65:1920‐1935).

Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical.

UNLABELLED (111)In-DTPA-anti-γH2AX-Tat, which combines an anti-γH2AX antibody with a cell-penetrating peptide, Tat, and the Auger electron-emitting radioisotope, (111)In, targets the DNA damage signalling protein, γH2AX, and has potential as a probe for imaging DNA damage in vivo. The goal of this study was to investigate whether (111)In-DTPA-anti-γH2AX-Tat labelled to high specific activity (6MBq/μg) can amplify treatment-related DNA damage for therapeutic gain. METHODS MDA-MB-468 and MDA-MB-231/H2N (231-H2N) breast cancer cells were incubated with (111)In-DTPA-anti-γH2AX-Tat (3MBq, 6MBq/μg) or a control radioimmunoconjugate, (111)In-DTPA-mIgG-Tat, and exposed to IR or bleomycin. DNA damage was studied by counting γH2AX foci and by neutral comet assay. Cytotoxicity was evaluated using clonogenic assays. (111)In-DTPA-anti-γH2AX-Tat was administered intravenously to 231-H2N-xenograft-bearing Balb/c nu/nu mice in tumor growth inhibition studies. RESULTS The number of γH2AX foci was greater after exposure of cells to IR (10Gy) plus (111)In-DTPA-anti-γH2AX-Tat compared to IR alone (20.6±2.5 versus 10.4±2.3 foci/cell; P<.001).(111)In-DTPA-anti-γH2AX-Tat resulted in a reduced surviving fraction in cells co-treated with IR (4Gy) versus IR alone (5.2%±0.9% versus 47.8%±2.8%; P<.001). Similarly, bleomycin (25-200μg/mL) plus (111)In-DTPA-anti-γH2AX-Tat resulted in a lower SF compared to bleomycin alone. The combination of a single exposure to IR (10Gy) plus (111)In-DTPA-anti-γH2AX-Tat significantly decreased the growth rate of 231-H2N xenografts in vivo compared to either (111)In-DTPA-anti-γH2AX-Tat or IR alone (-0.002±0.004 versus 0.036±0.011 and 0.031±0.014mm(3)/day, respectively, P<.001). CONCLUSION (111)In-DTPA-anti-γH2AX-Tat amplifies anticancer treatment-related DNA damage in vitro and has a potent anti-tumor effect when combined with IR in vivo.