Immune Evasion by Murine Melanoma Mediated through CC Chemokine Receptor-10

Human melanoma cells frequently express CC chemokine receptor (CCR)10, a receptor whose ligand (CCL27) is constitutively produced by keratinocytes. Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors. In vitro, exposure of tumor cells to CCL27 led to rapid activation of Akt, resistance to cell death induced by melanoma antigen-specific cytotoxic T cells, and phosphatidylinositol-3-kinase (PI3K)–dependent protection from apoptosis induced by Fas cross-linking. In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells. We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression

Rat model of metastatic breast cancer monitored by MRI at 3 tesla and bioluminescence imaging with histological correlation

<p>Abstract</p> <p>Background</p> <p>Establishing a large rodent model of brain metastasis that can be monitored using clinically relevant magnetic resonance imaging (MRI) techniques is challenging. Non-invasive imaging of brain metastasis in mice usually requires high field strength MR units and long imaging acquisition times. Using the brain seeking MDA-MB-231BR transfected with luciferase gene, a metastatic breast cancer brain tumor model was investigated in the nude rat. Serial MRI and bioluminescence imaging (BLI) was performed and findings were correlated with histology. Results demonstrated the utility of multimodality imaging in identifying unexpected sights of metastasis and monitoring the progression of disease in the nude rat.</p> <p>Methods</p> <p>Brain seeking breast cancer cells MDA-MB-231BR transfected with firefly luciferase (231BRL) were labeled with ferumoxides-protamine sulfate (FEPro) and 1-3 × 10⁶cells were intracardiac (IC) injected. MRI and BLI were performed up to 4 weeks to monitor the early breast cancer cell infiltration into the brain and formation of metastases. Rats were euthanized at different time points and the imaging findings were correlated with histological analysis to validate the presence of metastases in tissues.</p> <p>Results</p> <p>Early metastasis of the FEPro labeled 231BRL were demonstrated onT2*-weighted MRI and BLI within 1 week post IC injection of cells. Micro-metastatic tumors were detected in the brain on T2-weighted MRI as early as 2 weeks post-injection in greater than 85% of rats. Unexpected skeletal metastases from the 231BRL cells were demonstrated and validated by multimodal imaging. Brain metastases were clearly visible on T2 weighted MRI by 3-4 weeks post infusion of 231BRL cells, however BLI did not demonstrate photon flux activity originating from the brain in all animals due to scattering of the photons from tumors.</p> <p>Conclusion</p> <p>A model of metastatic breast cancer in the nude rat was successfully developed and evaluated using multimodal imaging including MRI and BLI providing the ability to study the temporal and spatial distribution of metastases in the brain and skeleton.</p

Comparing the Effects of Isoflurane and Alpha Chloralose upon Mouse Physiology.

Functional magnetic resonance imaging of mice requires that the physiology of the mouse (body temperature, respiration and heart rates, blood pH level) be maintained in order to prevent changes affecting the outcomes of functional scanning, namely blood oxygenation level dependent (BOLD) measures and cerebral blood flow (CBF). The anesthetic used to sedate mice for scanning can have major effects on physiology. While alpha chloralose has been commonly used for functional imaging of rats, its effects on physiology are not well characterized in the literature for any species. In this study, we anesthetized or sedated mice with isoflurane or alpha chloralose for up to two hours, and monitored physiological parameters and arterial blood gasses. We found that, when normal body temperature is maintained, breathing rates for both drugs decrease over the course of two hours. In addition, alpha chloralose causes a substantial drop in heart rate and blood pH with severe hypercapnia (elevated blood CO2) that is not seen in isoflurane-treated animals. We suggest that alpha chloralose does not maintain normal mouse physiology adequately for functional brain imaging outcome measures

Heart Rate drops under alpha chloralose sedation.

<p>Mice retain a steady heart rate under isoflurane anesthesia, ranging from 463±20 beats/min to 489±20 beats/min. However, under alpha chloralose sedation, heart rate rapidly drops to below 350 beats/min, leading to a significant difference in heart rate between the treatment groups (F_(1,384) = 516.2,p<0.0001, n = 9–23 per time point). The shaded region indicates the normal heart rate for an awake mouse at rest (450–500 beats/min).</p

Breathing Rate decreases over time during prolonged anesthesia.

<p>There were no group differences between isoflurane and alpha chloralose-treated mice at any time (F_(1,386) = 0.001, p = 0.97), but there was a significant overall drop in breathing rates for both groups (F_(11,386) = 5.68, p<0.0001, n = 7–24 per time point).</p

Blood carbon dioxide levels increase significantly under alpha chloralose sedation over the course of 120 minutes.

<p>A) Carbon dioxide levels in arterial blood were significantly higher under alpha chloralose sedation (F_(1,82) = 38.06, p<0.0001, n = 4–19 per time point). Although there was an overall effect of time (F_(5,82) = 4.06, p = 0.002), reflecting increasing CO₂ levels over time, these levels did not differ significantly from baseline over the course of 120 minutes under isoflurane anesthesia (p>0.05). B) After 120 minutes, carbon dioxide levels are significantly higher in alpha chloralose-sedated animals (p = 0.002).</p

Alpha chloralose sedation causes clear blood acidosis.

<p>A) While pH drops under both types of anesthesia (F_(5,86) = 8.97, p<0.0001, n = 3–19 per time point), the drop is much more pronounced under alpha chloralose sedation (F_(1,86) = 35.72, p<0.0001), dropping from a normal reading of pH 7.39±0.05 during the first 15 minutes, to 7.13±0.07 by 120 minutes. In contrast, blood pH under isoflurane drops from 7.34±0.04 to 7.27±0.06 after two hours, which is still within a normal physiological range for the mouse. The shaded region indicates non-acidotic blood pH for the mouse (15). B) Blood is acidotic after 120 minutes of alpha chloralose sedation (p = 0.0006).</p

Blood oxygenation levels do not differ between isoflurane and alpha chloralose.

<p>A) Over the course of 120 minutes of sedation/anesthesia, there were no group differences in blood oxygenation levels (F_(1,81) = 0.6, p = 0.44, n = 4–19 per time point), but there was a significant effect of time in the isoflurane group, where blood oxygenation levels spiked between 15 and 30 minutes (F_(5,82) = 4.06, p = 0.002), then returned to baseline levels. B) Blood oxygenation is not different between isoflurane and alpha chloralose after 120 minutes of anesthesia/sedation (p = 0.67).</p