Environmental factors, including exposure to stress, are known to contribute to the propensity to consume ethanol. However, stress produces inconsistent effects on ethanol drinking in rodent models. Therefore, the present study examined the impact of different stressors on limited access ethanol consumption and determined whether there were sex-dependent differences in response to stress. To this end, male and female C57BL/6J mice had 2-hr access to a water and 10% ethanol solution, beginning 30 minutes before onset of the dark cycle. Once ethanol intake was stable, the effect of restraint, tail suspension, predator odor, foot shock, and tail pinch on subsequent intake was explored. Both plasma corticosterone (CORT) and allopregnanolone (ALLO) were assessed as indices of hypothalamic-pituitary-adrenal (HPA) axis activity and of endogenous neurosteroid levels respectively, following restraint, tail suspension and predator odor. Ethanol intake was decreased following restraint, tail suspension, foot shock, and tail pinch in both sexes, with stressor-related differences in the duration of the suppression. The effect of predator odor on ethanol intake was biphasic in females; ethanol consumption was significantly reduced on the day of stress but significantly increased on the following two days. In males, predator odor produced a delayed significant increase in ethanol intake on the second day after stress. All three stressors increased plasma CORT, with higher CORT levels in females when compared with males. Notably, there was a significant positive correlation between CORT levels immediately after predator odor stress and ethanol intake on the following day as well as a significant positive linear relationship between CORT levels immediately after restraint stress and ethanol intake on the following day in females. Furthermore, the three stressors produced a greater increase in ALLO levels in female versus male mice, but ALLO levels following predator odor were not correlated with subsequent ethanol intake. In summary, the type of stressor administered had a profound impact on subsequent ethanol consumption, with subtle sex differences in the magnitude and persistence of the effect. These findings are the first to demonstrate that a single, acute exposure to restraint, tail suspension, and predator odor stress increased plasma CORT and ALLO levels in animals with a history of ethanol consumption and that female mice were more responsive than males to the ability of stress to increase CORT levels as well as to the ability of predator odor stress to produce a delayed increase in ethanol intake. Because predator odor stress is a model of posttraumatic stress disorder, the present sex differences have important implications for future preclinical studies modeling the comorbidity of posttraumatic stress disorder and alcohol use disorders.

Polymeric nanocarriers such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers deliver drugs to solid tumors and avoid the systemic toxicity of conventional chemotherapy. Because HPMA copolymers can target sites of inflammation and accumulate within innate immune cells, we hypothesized that HPMA copolymers could target tumor-associated macrophages (TAMs) in both primary and metastatic tumor microenvironments. We verified this hypothesis, first in preliminary experiments with isolated bone marrow macrophage cultures in vitro, and subsequently in a spontaneously metastatic murine breast cancer model generated from a well-established, cytogenetically characterized 4T1 breast cancer cell line. Using our standardized experimental conditions, we detected primary orthotopic tumor growth at 7 days and metastatic tumors at 28 days after orthotopic transplantation of 4T1 cells into the mammary fat pad. We investigated the uptake of HPMA copolymer conjugated with Alexa Fluor 647 and folic acid (P-Alexa647-FA) and HPMA copolymer conjugated with IRDye 800CW (P-IRDye), following their retroorbital injection into the primary and metastatic tumor-bearing mice. A significant uptake of P-IRDye was observed at all primary and metastatic tumor sites in these mice, and the P-Alexa647-FA signal was found specifically within CD11b+TAMs co-stained with pan macrophage marker CD68. These findings demonstrate, for the first time, a novel capacity of a P-Alexa647-FA conjugate to colocalize to CD11b+CD68+ TAMs in both primary and metastatic breast tumors. This underscores the potential of this HPMA nanocarrier to deliver functional therapeutics that specifically target tumor-promoting macrophage activation and/or polarization during tumor development.

<p>Distribution of clonal abnormalities in cell line 4T1</p>

<p>Copolymer composition</p>

<p>Representative karyotype from A, 4T1 (MSKCC) and B, 4T1 (ATCC).</p>