Effects of Short-term Stress on Tumor Emergence and Development
shows the effects of short-term stress on the time course of tumor counts () and percentage of mice with tumors (). Sample means (symbols) as well as mean predictions from the regression model (lines) are shown. Since tumor progression is known to consist of different biological phases, we performed a Bayesian changepoint analysis to identify empirically if tumor progression is phased. This analysis revealed that changes in tumor counts and areas over time occurred in three phases: Weeks 11-16, weeks 17-26, and weeks 27-31 which roughly overlap with the phases of papilloma emergence and regression, transition from papilloma to SCC, and SCC progression, respectively. Changes in % of mice with tumors occurred in two phases: Weeks 11-20 and weeks 21-31. To identify whether there were differences in tumor burden between control and short-term stressed mice, we compared the mean of the per-mouse averages between treatments using one-tailed two-sample t-tests for each of the three outcomes (tumor count, tumor area, and presence of tumor) during each of the empirically determined phases. Compared to controls, acutely stressed mice showed a lower mean tumor count during weeks 11-16 (p < 0.001) and weeks 17-26 (p < 0.005) but not weeks 27-31 (p > 0.23). The percentage of mice with tumors in the short-term stress group was lower than that in controls during weeks 11-20 (p < 0.005) but not after week 21 (p > 0.21).
Effects of short-term stress on tumor emergence
shows Kaplan-Meier estimates of the distribution of tumor-free survival by group. Tumors are estimated to have appeared earlier in the control group which reached 50% incidence in 12 weeks versus 17.5 weeks for the short-term stress group. Because of the “crossing” of the tumor-free survival curves, the distribution of time to first tumor did not differ significantly between the two arms (log-rank p > 0.3). However, application of Peto’s test, which is weighted toward detecting short-term risks, gave p < 0.07 for the difference between the two curves suggesting that short-term stress affected early but not late phases of tumor development. Both groups reached approximately 90% incidence at 22 weeks.
Kaplan-Meier estimates of distribution of proportions of tumor-free mice
The quantitative assessment of tumors described above was complimented with histopathological examination and identification of tissue characteristics. At week 7, among the control group, skin sections from 3 of 3 mice were characterized as having mild dysplastic epidermal hyperplasia (DEH); among the short-term stress group, 1 mouse had mild DEH, 1 had mild multifocal hyperplasia, and 1 had moderate multifocal hyperplasia. Additionally, all lesions from all animals showed leukocyte infiltration. At week 20, among the control group, 1 mouse had mild DEH, and 2 mice showed microinvasive squamous cell carcinoma (MICA); among the short-term stress group, 1 mouse had mild DEH, 1 mouse had a well-differentiated SCC, and 1 lesion could not be positively identified as an SCC but had moderate diffuse hyperplasia. Additionally, all lesions from all animals showed leukocyte infiltration. At week 32, among the control group, 2 mice had papillomas and 1 had MICA; among the short-term stress group, 2 mice had SCC, and 1 had a very small area of MICA.
Effects of Short-term Stress on Chemokine and Cytokine Gene Expression
Gene expression of CTACK, RANTES, IL-12, IFN-γ, IL-10 and IL-4 was measured in skin of the control and short-term stress groups, at weeks 7, 20, and 32 (). At week 7, acutely stressed mice showed higher levels of RANTES (P < 0.05) and IL-12 (P < 0.01) gene expression. At week 20, acutely stressed mice showed higher levels of CTACK/CCL27 (P < 0.05), RANTES (P < 0.01), IL-12 (P < 0.0001), and IFN-γ (P < 0.0001) gene expression. The magnitude of the increase in gene expression induced by short-term stress was greater at week 20 than week 7, which indicates greater activation of specific cytokines and chemokines at this midpoint of tumor development and growth. At week 32, acutely stressed mice continued to show higher levels of CTACK (P < 0.05), RANTES (P < 0.05), IL-12 (P < 0.05), and IFN-γ (P < 0.05) gene expression in skin containing pre-malignant lesions compared to similar lesions from controls. Although estimates for average IL-4 and IL-10 levels were higher in acutely stressed mice at week 20, differences in means between groups were not statistically significant at any time point. In order to examine whether the presence or absence of tumors affected gene expression data at week 20, we estimated the partial point-biserial correlation between treatment group and gene expression in skin, adjusting for presence of tumor, and observed a statistically significant effect of short-term stress for each of the parameters examined (P < 0.05) except for IL-10. We did not run a similar analysis at week 7, because none of the animals had tumors before that time, and week 32, because all animals had tumors at some point prior to week 32.
Effects of short-term stress on chemokine and cytokine gene expression
Effects of Short-term Stress on Circulating and Skin Infiltrating T cells
It is likely that the changes in gene expression described above would shift the Type 1/Type 2 cytokine balance towards Type 1 cytokine driven responses and promote cell-mediated immunity. Since T cells are crucial mediators of cell-mediated immunity, the observed stress-induced increase in T cell attracting chemokines and Type 1 cytokines suggested that short-term stress may induce an increase in T cell number/activity. Therefore, we quantified absolute numbers of peripheral blood T cells. At week 20, acutely stressed mice showed significantly higher numbers of circulating helper (CD3+CD4+, P<0.0001) and cytolytic (CD3+CD8+, P<0.0001) T cells compared to controls (). No differences in circulating T cell numbers were observed between the two groups at weeks 7 or 32 ( and ).
Helper (CD3+CD4+) and cytolytic (CD3+CD8+) T cells in peripheral blood
We also quantified CD4+ and CD8+ T cell infiltration into UV-exposed skin at weeks 7, 20 and 32 in order to examine whether there were differences in T cell infiltration in control versus acutely stressed mice. Photomicrographs of representative sections and quantified cell numbers are shown in (week 7) and (week 20). At week 7 and week 20, respectively, acutely stressed mice showed significantly higher numbers of CD4+ (P < 0.0001) and CD8+ (P = 0.003 and 0.016) T cell infiltration compared to controls. At week 32, acutely stressed mice showed only slightly higher CD4+ T cell numbers, and no statistically significant differences CD8+ T cell numbers (data not shown). In order to examine whether the presence or absence of tumors affected the observed effects on CD4 or CD8 numbers at week 20, we estimated the partial point-biserial correlation between treatment group and cell numbers, adjusting for presence of tumor. We observed a statistically significant effect of short-term stress for each of the parameters examined (P < 0.05). We did not run a similar analysis at week 7, because none of the animals had tumors before that time, and week 32, because all animals had tumors at some point prior to week 32.
CD4+ and CD8+ T cell infiltration at week 7
CD4+ and CD8+ T cell infiltration at week 20