1. Volume 19, Issue 4, Pages 760-773 (April 2017)
Environmental Enrichment Induces Pericyte and IgA-Dependent Wound Repair and Lifespan Extension in a Colon Tumor Model 
Benjamin D. Bice, Megan R. Stephens, Stephanie J. Georges, Ashlee R. Venancio, Peter C. Bermant, Annika V. Warncke, Kajsa E. Affolter, Julio R. Hidalgo, Melinda L. Angus-Hill 
Cell Reports 
Volume 19, Issue 4, Pages (April 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 760-773DOI: (10.1016/j.celrep.2017.04.006)
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3. Figure 1
EE Increases the Lifespan of Male and Female Tcf4Het/+ ApcMin/+ Mice without Effect on Tumor Number or Weight
Animals were collected when cachexia symptoms became apparent (see text for details).
(A–C) Lifespan EE. (A) Survival curves for male and female Tcf4Het/+ ApcMin/+ mice. N = number of animals per sex/genotype. The p values by chi-square statistical analysis were calculated using the log-rank Mantel-Cox tests for greater than three groups.(B and C) Tumor count (B) and tumor weight (C) in NE and EE Tcf4+/+ ApcMin/+ and Tcf4Het/+ ApcMin/+ animals.
(D–G) Short-term EE, 4 months at collection. (D and E) Colon tumor count and volume. (F) Animal weight comparisons in NE and EE animals. (G) Systemic changes in inflammatory cytokine levels of male animals, as indicated. Lower limit of detection: IL-6, 4.5 pg/μL; and TNF-α, 0.85 pg/μL. ∗p ≤ 0.05, ∗∗p = using two-sample t test with Welch correction.
Error bars were calculated using SEM.
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4. Figure 2
EE Reduces Expression of Tumor Growth Genes and Improves Tumor Cell Differentiation in Tcf4Het/+ ApcMin/+ Colons
(A) IPA canonical pathway analysis of RNA-seq data.
(B) RNA-seq analysis revealed significantly decreased Wnt-signaling components and downstream targets.
(C) qPCR analysis of genes involved in differentiation.
(D) Differentiation of enteroendocrine L cells (PYY) in EE and NE animals. The green and yellow arrows mark enteroendocrine cells in normal and tumor tissue, respectively. The cyan and red arrows mark PYY bound to NPY2R receptor in NE and EE, respectively, with internalization of PYY following EE. The dashed lines delineate crypts in normal colon tissue. Scale bar, 100 μm.
Error bars were calculated using SEM.
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5. Figure 3 EE Restores NHR Signaling in Tcf4Het/+ApcMin/+ Colons
(A) IPA Causal Network analysis depicting significant changes in NHRs and NHR signaling pathways. See text for references regarding NHR in wound repair.
(B–E) Immunohistochemical staining of (B) THRA, (C) DIO1, (D) ME1, and (E) ME2 in NE or EE tumors. Yellow arrows mark some positive stromal cells, and magenta arrows mark some positive tumor glandular cells. Scale bar, 100 μm.
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6. Figure 4
EE Reduces Angiogenesis, Improves Pericyte Recruitment, and Normalizes Vessels in Tcf4Het/+ ApcMin/+ Colons
(A) IPA diseases and functional analysis of RNA-seq data in EE compared to NE animals.
(B) qPCR analysis of pericyte markers in EE compared to NE mice.
(C) PECAM and SMA immunofluorescence staining of tumor vessels. The dashed yellow lines outline stromal cells.
(D) RGS5 and SMA immunofluorescence staining of tumor pericytes.
(E) qPCR of Npy and Npy2r.
(F) NPY2R immunofluorescence staining of tumor vessels. The dashed yellow lines outline stromal cells. The yellow arrows mark endothelial cells. The green arrows mark blood cells, including red blood cells. The p values were calculated using two-sample t test with Welch correction. Scale bar, 100 μm.
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7. Figure 5
EE Increases IgA Levels, Plasma Cell Recruitment, and Pericyte Activation and Migration into Glandular Structures in Tcf4Het/+ ApcMin/+ Males
(A and B) Colon-specific IgA immunohistochemical staining in NE and EE Tcf4Het/+ ApcMin/+ male normal (A) and tumor (B) tissue. a′ and b′ denote higher-magnification views of boxed regions. The red arrows mark IgA+ tumor regions, whereas the black arrows mark necrotic debris.
(C) Immunohistochemical staining of tumor tissue for the glandular epithelial and plasma cell marker CD138. The black asterisks denote the presence of CD138+ glandular structures, whereas the red asterisks denote the replacement of glandular structures with CD138− cells.
(D) The blue arrows mark some SMA+ RGS5+ IgA-low vascular pericytes, whereas the yellow arrows mark SMA− RGS5+ IgA-high cells within the tumor glandular structures.
(E) The blue arrows mark cells with high FCAMR and IgA expression, whereas the yellow arrows mark cells with low FCAMR and IgA expression. The red arrows denote necrotic debris. Scale bar, 100 μm.
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8. Figure 6 EE-Mediated Microbiota Changes in Tcf4Het/+ ApcMin/+ Mice
(A) Alpha diversity of NE and EE of WT (20,000 reads, p = 0.64) and NE and EE Tcf4Het/+ ApcMin/+ (20,000 reads, p = 0.03) animals.
(B–F) Specific contributions to the proteome within the (B) phylum Proteobacteria and the classes (C) Alphaproteobacteria, (D) Betaproteobacteria, the (E) genus Sutterella, and (F) the phylum Gammaproteobacteria in response to EE in WT, Tcf4Het/+ Apc+/+, Tcf4+/+ ApcMin/+, and Tcf4Het/+ ApcMin/+ animals. (B–E) R-ggplot2 generated box-whisker plots with outliers noted as filled circles. ∗∗p = using two-sample t test with Welch correction.
Error bars were calculated using SEM.
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9. Figure 7
Model Depicting the Mechanism of EE-Dependent Improvements in Survival of Tcf4Het/+ ApcMin/+ Males
(A) Diagram and (B) cartoon models of tumor wound repair following EE. See text for details.
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