Environmental enrichment (EE) replicates mind-body therapy by providing complex housing to laboratory animals to improve their activity levels, behavior, and social interactions. Using a Tcf4 Het/+ Apc Min/+ -mediated model of colon tumorigenesis, we found that EE vastly improved the survival of tumor-bearing animals, with differential effect on tumor load in male compared to female animals. Analysis of Tcf4 Het/+ Apc Min/+ males showed drastically reduced expression of circulating inflammatory cytokines and induced nuclear hormone receptor (NHR) signaling, both of which are common in the wound repair process. Interestingly, EE provoked tumor wound repair resolution through revascularization, plasma cell recruitment and IgA secretion, replacement of glandular tumor structures with pericytes in a process reminiscent of scarring, and normalization of microbiota. These EE-dependent changes likely underlie the profound improvement in survival of colon-tumor-bearing Tcf4 Het/+ Apc Min/+ males. Our studies highlight the exciting promise of EE in the design of future therapeutic strategies for colon cancer patients.

We previously developed a unique mouse model system in which heterozygous loss of the Tcf4 gene (TCF7L2) leads to unregulated proliferation of colonic epithelial cells and significantly enhances colon tumorigenesis when combined with an initiating mutation in the Apc gene (). Here, we show that EE profoundly improves the survival of Tcf4Apccolon-tumor-bearing animals. Although survival is improved in both male and female animals, we find that they respond differently to EE, because only females show significantly decreased tumor growth. We set out to explain the striking result by analyzing systemic changes, and changes within the tumor microenvironment and within the resident microbiome in male Tcf4Apcanimals. Our findings suggest that, in the tumors of Tcf4Apcmales exposed to EE, NHR activation promotes resolution of the wound repair process, which ultimately restores mucosal barrier function and microbial biodiversity. This EE-dependent restoration of barrier integrity likely contributes to the drastic improvement in the lifespan of tumor-bearing animals.

Lifestyle interventions, such as alterations in diet and physical activity, show promise for colon cancer prevention and treatment (). Mind-body medicine, in particular, focuses on reducing the physiological manifestations of stress and anxiety by improving social and cognitive stimulation as well as physical activities and is associated with profound improvements in overall health and disease (). Mind-body medicine is replicated in animal models using environmental enrichment (EE) housing conditions and has been shown to elicit anti-tumor effects in several mouse models of cancer, including cancers of the intestine (). Here, we further investigate the mechanisms that account for EE-mediated anti-tumor effects in a mouse genetic model of colon cancer.

A dysfunctional barrier can further reinforce the pro-inflammatory response because normal defenses against pathogenic intestinal microbiota are compromised (). Plasma cells produce immunoglobulin A (IgA), which helps maintain mutualism by fostering commensals while simultaneously neutralizing pathogenic bacteria (). The ability to efficiently discriminate between beneficial commensals and harmful bacteria thereby reduces the generation of pro-inflammatory signals (). During barrier dysfunction and infection, this mutualism is lost, and the immune response results in dysbiosis, infection, inflammation, and cancer ().

The tumor microenvironment shares many features with defective wound repair. The wound repair process is accomplished through several different phases characterized by vascular permeability, inflammation, formation of a tissue stroma, angiogenesis, tissue remodeling, revascularization, improved oxygen tension, and ultimately the formation of scar tissue (). Disruption of any phase results in chronic wounds that cannot heal. For instance, inflammation must be resolved before tissue remodeling and scar formation can proceed. The tumor microenvironment is similar to chronic wounding in that hypoxia and reactive oxygen species drive continuous angiogenesis and inflammation that is unable to be resolved, ultimately giving rise to barrier defects and associated tumor expansion (). In normal tissue, various nuclear hormone receptors (NHRs) play an important role in tissue homeostasis and wound repair (), and are often functionally altered in CRC ().

Colorectal cancer (CRC) is the third leading cause of cancer death in men and the second in women around the world (). There are several risk factors associated with CRC, including age, deficiencies in adenomatous polyposis coli (APC) and mismatch repair (MMR) proteins, inflammation, smoking, poor diet, sedentary lifestyle, alcohol consumption, and imbalance of intestinal microbiota (). Tumor and stromal cells, pericytes, inflammatory cells, immune cells, and tumor vasculature make up the heterogeneous tumor microenvironment, and tumor cell survival depends on the cells within the stromal microenvironment (). However, it is unclear how these factors contribute to CRC development, whether there is cross talk between risk factors, and which cell types are critical for tumorigenesis.

Our study suggests that EE promotes a resolution of the wound repair response in tumors of Tcf4Apcanimals ( Figures 7 A and 7B ). Here, EE provokes the activation of NHRs involved in the following wound repair process ( Figure 3 A) in tumors: (1–2) similar to revascularization in the wound repair process, pericyte recruitment to the perivascular region of tumors results in vessel normalization and reduced angiogenesis. (3) Re-epithelialization occurs within tumors through differentiation of proliferative cells into distinct and functional epithelial cell types. (4–5) Plasma cells are recruited to the tumor periphery and secrete IgA. (6) Inflammatory signaling is reduced, further supporting the final steps of a repair process. (1, 7) Glandular structures at the tumor periphery are replaced by IgA-bound pericytes, an effect that may be intended to “seal the wound” to prevent entry by pathogens. (8) EE drastically increases the prevalence of the symbiotic bacteria Sutterella within the colon.

Studies of CRC models have illuminated phyla level shifts in microbial populations that are apparent in diseased compared to normal tissue (). We therefore questioned whether EE alters the composition of the microbiome, but did not observe significant differences at the phyla level between NE WT and NE Tcf4Apcanimals. Surprisingly, however, we found drastic changes at the phyla level in tumor-bearing Tcf4Apcmales in response to EE, including an increase in relative abundance of proteobacteria ( Table S4 A). The EE-dependent increases in the phylum proteobacteria are highly significant, with a 2-fold increase in average relative abundance following EE (5.8% in NE Tcf4Apcand 11.3% in EE Tcf4Apcanimals; Figure 6 B; Table S4 ). Given the pathogenic role of proteobacteria in health (), it is surprising that an increase in proteobacteria was associated with improved survival of EE Tcf4Apcanimals. We found that the proteobacteria subclasses Alphaproteobacteria (2.8%) and Betaproteobacteria (6.7%) constituted the majority of proteobacteria that were significantly increased in EE Tcf4Apcanimals ( Figures 6 C and 6D; Table S4 B). Of the isolated Alphaproteobacteria in enriched conditions, 50% were classified at the genus level as RF32 (1.4%) ( Table S4 E). Isolated Betaproteobacteria were further classified as primarily the genus Sutterella ( Figure 6 E; Table S4 E). It has been suggested that Sutterella is likely a commensal () and may actually serve a protective role in inflammatory bowel syndrome (). The proteobacteria subclass Gammaproteobacteria includes the pathogenic Klebsiella, Enterobacter, and Escherichia, which are well-known lipopolysaccharide (LPS)-membrane-containing microbes that can trigger inflammation (). Although levels of the subclass Gammaproteobacteria are highly variable in NE Tcf4Apcanimals, they are severely diminished in the majority of EE Tcf4Apcanimals ( Figure 6 F; Table S4 B). The diverse phylum Proteobacteria may have either a protective or pathogenic role within the colons of Tcf4Apcanimals, yet the improved phenotype of EE Tcf4Apcanimals suggests that EE fosters colonization of commensal members and neutralizes colonization of pathogenic members of this phylum.

Epithelial barrier dysfunction in colon tumor tissue can lead to microbial invasion and associated inflammation (), and decreased microbial species biodiversity has been associated with inflamed gut tissues commonly found in patients with inflammatory bowel disease (). Because EE restores barrier function through the wound repair process and leads to an overall decrease in inflammatory signaling in colon-tumor-bearing mice, we questioned whether EE normalizes gut microbiota composition. To test this, we collected stool from the distal colons of both Tcf4Apcand WT animals exposed to NE and EE conditions, and examined the composition of the microbiome using 16S rRNA sequencing. Surprisingly, NE WT and Tcf4Apcanimals have similar biodiversity as demonstrated by similar number of unique sequences identified ( Figure 6 A). However, EE increased the biodiversity of Tcf4Apctumor-bearing animals beyond that observed in NE Tcf4Apcanimals and both NE and EE WT animals ( Figure 6 A). These results demonstrate an EE-dependent increase of biodiversity in Tcf4Apctumor-bearing males.

(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, Tcf4 Het/+ Apc +/+ , Tcf4 +/+ Apc Min/+ , and Tcf4 Het/+ Apc Min/+ animals. (B–E) R-ggplot2 generated box-whisker plots with outliers noted as filled circles. ∗∗ p = 0.005 using two-sample t test with Welch correction.

IgA exerts its action through IgA Fc receptors located on target cells, and activity of these receptors is important for the activation of mesangial cells (). In IgA nephropathy, IgA binds the Fc receptor Cd89 on mesangial cells (), which also express the Fc receptor Fcα/μR (FCAMR []). Mice lack the Fc receptor CD89 but express FCAMR (). To define whether colonic IgA binds FCAMR on pericytes, we co-stained tumor tissue from NE and EE animals for FCAMR, IgA, and the pericyte marker RGS5. In tumors isolated from EE mice, many pericytes that are RGS5and IgAhave lower expression levels of FCAMR ( Figure 5 E, yellow arrows). Surprisingly, RGS5pericytes that are IgAhighly express FCAMR ( Figure 5 E, cyan arrows). This result demonstrates that high FCAMR expression in RGS5pericytes correlates with IgA binding, and that FCAMR is the IgA receptor expressed on RGS5pericytes.

The final step in wound repair is the formation of the scar, and this process involves invasion by activated pericytes and myofibroblasts and subsequent scar formation (). In IgA nephropathy, a disease characterized by fibrosis, kidney pericytes (mesangial cells) are activated by IgA polymer binding (). Because EE promotes pericyte recruitment to colon tumor cells in Tcf4Apcanimals, we questioned whether the IgA-bound cells in glandular structures from these animals ( Figures 5 B and 5C) were indeed activated pericytes that migrate and contribute to wound repair. To test this directly, we stained tumor tissue isolated from EE and NE animals for IgA and the pericyte markers SMA and RGS5. We found RGS5SMAIgAintratumoral pericytes ( Figure 5 D, yellow arrows) in close contact with RGS5SMAIgAperivascular pericytes ( Figure 5 D, blue arrows), an observation that is consistent with invasion of glandular structures by pericytes that have migrated from the perivascular space. Moreover, the RGS5SMApericytes exhibited high levels of IgA staining ( Figure 5 D). This suggests that IgA-mediated pericyte invasion acts to restructure glandular tissue in a wound repair-like response.

CD138 is also a marker for epithelium within the glandular lumina (). When we analyzed glandular lumina from EE and NE Tcf4Apctumor sections, we found that NE tumors showed the expected epithelial expression of CD138 in glandular structures ( Figure 5 C, a and a′, black asterisks); however, EE tumors failed to show glandular CD138 expression ( Figure 5 C, b1′, red asterisks), and instead cells that were present were bound by IgA ( Figure 5 B, b1′, red arrows). IgA expression was not found in glandular cells from NE tumors ( Figure 5 B, a′). This suggested that, in EE tumors, IgA-bound CD138cells replace glandular epithelia structures.

Antibodies (IgA) secreted from plasma cells are actively engaged in wound repair in many tissue types (). By RNA-seq, we found that 20% of upregulated genes in the distal colon of EE compared to NE Tcf4Apcanimals are components of immunoglobulin (Ig) ( Table S2 ), including Ig constant heavy chain (IGHA) and many light chains (kappa, lambda). J chain is a component of the secreted IgA polymer and is significantly upregulated in EE Tcf4Apcanimals ( Table S2 ). These data suggest that EE stimulates IgA secretion from colonic plasma cells in male Tcf4Apcanimals. We next examined normal and tumor tissue isolated from EE and NE Tcf4Apcanimals by immunohistochemistry for plasma cell localization (anti-CD138) and IgA secretion (anti-IgA). Surprisingly, we found that IgA staining intensity and localization differed between NE and EE sections of normal tissue ( Figure 5 A). EE sections showed more intense IgA staining as well as localization to the luminal surface, in contrast to IgAcells from NE sections that were mainly concentrated at the crypt base. Similarly, in colon tumor sections, IgAstaining was mainly localized to the tumor periphery in EE animals ( Figure 5 B, b, b1′, b2′, red arrows), compared to a general lack of IgA staining in NE tumors, with the exception of nonspecific staining of necrotic debris in the glandular lumina found in both NE and EE animals ( Figure 5 B, a′, black arrows). To determine whether the IgAcells at the periphery of EE Tcf4Apctumors represented plasma cells, we analyzed expression of CD138. Indeed, many IgAcells in EE-tumor periphery ( Figure 5 B, b2′) were plasma cells based on anti-CD138 staining ( Figure 5 C, b2′). Taken together, our data suggest that EE increases migratory plasma cells within normal and tumor tissue, and these cells secrete higher levels of IgA than plasma cells in NE animals. The presence of EE plasma cells in this environment likely provides continuing immunity to barrier challenges.

(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.

(A and B) Colon-specific IgA immunohistochemical staining in NE and EE Tcf4 Het/+ Apc Min/+ 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.

The NPY peptide and its receptor NPY2R are important drivers of angiogenesis under ischemic conditions (). The NPY2R receptor is primarily expressed on endothelial cells in ischemic tissue or on pericytes when vessels are injured. By qPCR, we examined Npy and Npy2r expression in colon tumors from NE Tcf4Apccompared to NE Tcf4Apcmales. We found that, under NE conditions, the presence of the Apcallele in a Tcf4background upregulated colon expression of both Npy (25-fold) and Npy2r (5-fold) ( Figure 4 E, top panel). Interestingly, EE reduced these increases in expression in EE Tcf4Apccompared to EE Tcf4Apcmales ( Figure 4 E, middle panel) and for Npy in EE compared to NE Tcf4Apcanimals. We next examined colons from EE versus NE Tcf4Apcanimals and found a similar EE-mediated reduction in Npy and Npy2r expression ( Figure 4 E, bottom panel). Endothelial cells from NE, but not EE, Tcf4Apcanimals showed protein expression of NPY2R by immunohistochemistry ( Figure 4 F, yellow arrows). Instead, EE Tcf4Apcmales showed NPY2Rblood cells ( Figure 4 F, cyan arrows) and differentiated enterocytes ( Figure 4 F, double yellow asterisks), at the tumor surface, indicating that blood cells are recruited to the microenvironment and further validating EE-dependent differentiation of colonic mucosal cells. These results suggest that EE reduces angiogenesis, normalizes vessels, and improves circulation within the colon tumor microenvironment, and supports the role of EE in resolving the tumor-specific block of revascularization in the wound repair process.

Typically, angiogenesis within the tumor microenvironment results in tortuous and nonfunctional vessels, and ischemic and hypoxic tissue (), effectively blocking completion of the wound repair process. In wound repair, revascularization must have both an angiogenic component to form new vessels that are then stabilized by pericytes, and also the inhibition of additional angiogenesis (). IPA analysis of RNA-seq expression data from EE versus NE Tcf4Apcmales revealed decreased expression of genes involved in endothelial cell proliferation and movement, hypertension, vasculogenesis, and angiogenesis, and increased expression of genes involved in the regulation of blood pressure ( Figure 4 A; Table S3 C). Pericytes are multipotent cells that surround and stabilize blood vessels, which is important for wound repair (). Tortuous tumor vessels are highly angiogenic and lack pericytes. Interestingly, most of the top upregulated genes in the colons of EE Tcf4Apcmales are specific markers of the pericyte lineage ( Figure 4 B; Table S2 A), including Sma, desmin (Des), and regulator of G protein signaling 5 (Rgs5). Consistent with an EE-mediated increase in pericyte recruitment, functional analysis also revealed signs of improved vascular integrity through upregulation of several genes involved in smooth muscle contraction, circulatory system process, and blood circulation ( Table S2 C). We therefore hypothesized that improved vascular integrity through recruitment of pericytes reduces angiogenesis and increases normalized tumor vasculature. To test this hypothesis, we examined colon tumor tissue isolated from NE and EE Tcf4Apcanimals and stained with the blood vessel marker platelet endothelial cell adhesion molecule (PECAM) and SMA (a pericyte marker of vascular maturity). We found fewer PECAM-positive cells within the tumor stroma (outlined by yellow dotted lines) following EE ( Figure 4 C). We also examined the expression of a second pericyte marker, RGS5, which is expressed on activated pericytes (pericytes with mesenchymal potential) in tumors () and upregulated 2.5-fold in Tcf4Apccolons in response to EE ( Figure 4 B). Interestingly, RGS5pericytes are commonly SMAin both NE and EE colons of Tcf4Apcanimals, whereas there are more RGS5SMApericytes in response to EE ( Figure 4 D). The lack of RGS5/SMA co-staining is not unexpected as pericytes represent a heterogeneous population that does not uniformly express SMA (). As such, the RGS5/SMAsubset may represent a less mature pericyte that promotes a repair response, whereas the RGS5/SMAsubset may stabilize vessels in the wound repair process.

A second direct THRA transcriptional target is malic enzyme (ME1) (). Malic enzymes (cytoplasmic ME1 and mitochondrial ME2) catalyze the oxidative decarboxylation of malate to pyruvate and are essential for maintaining cellular NADPH levels (). Both ME1 and ME2 increase migration and invasion in wound repair assays, without effect on metastatic potential or animal survival (). Given the role of ME1 and ME2 in wound repair, we tested their expression patterns by immunohistochemical staining of EE and NE Tcf4Apctumors. ME1 is absent in tumors of NE Tcf4Apcanimals and is expressed primarily in stromal cells within the tumors of EE Tcf4Apcanimals ( Figure 3 D). Different from ME1 expression, ME2cells are largely found within the stroma of NE Tcf4Apctumors ( Figure 3 E, yellow arrows), whereas in EE Tcf4Apctumors, ME2cells are primarily localized to the tumor mucosal structures ( Figure 3 E, magenta arrows). Taken together, cell-type-specific ME1 and ME2 expression is altered following EE, which suggests that a metabolic switch within the tumor mucosal cells and stromal cells of EE animals is at least partially dependent on T3 and that this change in metabolism may be involved in cell migration, a requirement for wound repair.

The NHR THRA is essential for re-epithelialization in the wound repair process in mice (), suggesting that activation of THRA signaling may contribute to the improvement in barrier function observed in male EE Tcf4Apcanimals. To define whether EE animals have active thyroid hormone signaling within mucosal or stromal compartments of the tumor microenvironment, we stained tumors isolated from NE and EE animals for THRA, and downstream THRA transcriptional targets, and co-stained with the myofibroblast and pericyte marker smooth muscle actin (SMA) to delineate the stromal compartment. Immunohistochemical analysis of THRA revealed that staining was largely absent in NE tumors ( Figure 3 B), whereas THRA expression was found across mucosal and stromal compartments in EE tumors ( Figure 3 B). Transcription of iodothyronine deiodinase 1 (DIO1) is stimulated by Thormone and both THRA and THRB (). By RNA-seq, DIO1 is significantly upregulated in EE compared to NE Tcf4Apcanimals ( Table S2 A). By immunohistochemistry, we found that DIO1 expression is absent in NE Tcf4Apctumors and localized to membranes of both mucosal and stromal cell types in EE Tcf4Apctumors ( Figure 3 C). These results suggest that THRA is expressed in tumors isolated from EE Tcf4Apcanimals and that active Thormone is present and able to transcriptionally activate DIO1. Therefore, thyroid hormone signaling may restore the re-epithelialization process essential for wound repair.

GCs are involved in wound repair and exert an anti-inflammatory effect in the late wound repair process by binding to the glucocorticoid receptor (GR) that in turn elicits an anti-inflammatory transcriptional response. GR is a member of a large family of NHRs, and cross talk between family members is commonly observed in an array of diverse processes, including tissue development and wound repair (). To further define the contribution of the GR and other NHRs, we utilized IPA causal analysis () of RNA-seq data from NE and EE Tcf4Apccolons. As for the GC pathway, causal analysis revealed the activation of both the GR (NR3C1) and mineral corticoid receptor (NR3C2; MC; Figure 3 A; Table S3 B) signaling pathways. In addition to activation of these NHRs, common inhibitors of the GC pathway including breakpoint cluster region (BCR), 11-beta-dehydrogenase isozyme 2 (HSD11β2), and gamma secretase complex are inhibited ( Figure 3 A; Table S3 B), an activity that would further increase GC signaling. Interestingly, a subset of NHRs also involved in wound repair () are activated by EE in the colons of Tcf4Apcmice ( Figure 3 A; Tables S3 B and S3D), including peroxisome proliferator-activated receptor (PPARα; NR1C1; Figure 2 A []), thyroid hormone receptor (THRA; NR1A1 []), oxysterol receptors (liver X receptor; LXR; NR1H2, NR1H3, NR1H4 []), retinoic acid receptors (RARA and RXRA []), as well as the nuclear receptor coactivator (NCOA1) involved in co-activation of GR, RXR, THRA, and PPAR NHRs (). This finding suggests that NHRs commonly involved in the wound repair process may play a role in improving the lifespan of EE Tcf4Apcmale animals.

Elevated levels of inflammatory cytokines IL-6 and TNF-α help to initiate re-epithelialization, but levels decrease following re-epithelialization (). Therefore, this re-epithelialization in Tcf4Apcanimals may underlie the reduction in systemic IL-6 and TNF-α levels, and may be representative of an anti-inflammatory response necessary for the late wound repair response. Indeed, analysis of RNA-seq data for upstream regulators revealed that IL10RA, dexamethasone, and glucocorticoid (GC) anti-inflammatory pathways are highly active in response to EE in Tcf4Apcanimals, whereas the pro-inflammatory upstream regulators TNF-α and STAT3 are inhibited ( Table S3 A).

Next, we set out to define whether decreased expression of Wnt/β-catenin signaling pathway following EE promotes the differentiation of tumor cells, thereby improving epithelial barrier function. Indeed, we found by RNA-seq and qPCR that several markers for secretory and absorptive differentiated epithelial cell lineages were upregulated in response to EE in colons isolated from male Tcf4Apcanimals, including sucrase isomaltase (Sis) (enterocytes), chromogranin A (Chga) (enteroendocrine), chloride channel accessory 1 (Clca1) (goblet), and mucin 2 (Muc2) (goblet) ( Figure 2 C; Table S2 A). We also examined colon tissue isolated from NE and EE Tcf4Apcanimals for specific signs of differentiation. Peptide YY (PYY) can be used to directly visualize differentiated colonic enteroendocrine L cells, which have a distinctive size and shape ( Figure 2 D, “normal tissue,” green arrows). Importantly, these cells are not visible in tumors from NE Tcf4Apcanimals, and instead PYY binds to the NPY2R receptor on the cell surface of tumor cells ( Figure 2 D, cyan arrows). In response to EE, tumor cells internalize PYY ( Figure 2 D, red arrows), or L cells become visible throughout the tumor ( Figure 2 D, yellow arrows). Thus, tumors appear to have reduced proliferation and improved differentiation in male EE Tcf4Apcanimals. These findings demonstrate that EE promotes tumor re-epithelialization, which may define the first step toward resolution of the tumor wound repair process.

Barrier defects may arise through aberrant activation of the Wnt/β-catenin signaling pathway, which induces proliferation and prevents differentiation of epithelial cell lineages (). Many members of this pathway are more highly expressed in the colons of Tcf4Apcanimals compared to Apcanimals (). The canonical pathway of Wnt/β-catenin signaling was identified with a negative Z score in Tcf4Apcanimals in response to EE, suggesting reduced activation of this pathway ( Figure 2 A; Table S3 ). Indeed, several critical activating factors in this pathway were significantly reduced following EE of these animals (including Wif1, Axin2, Tcf7, Lef1, and Nkd1; Figure 2 B) by both Ingenuity Pathway Analysis (IPA) and functional analysis ( Figure 2 A; Tables S2 and S3 ). Importantly, these data demonstrate that EE reduces the expression of many genes involved in Wnt/β-catenin-mediated tumor cell proliferation.

(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.

Adenomas can cause a loss of barrier function, promoting microbial infiltration and inflammatory cytokine secretion, including IL-6 (). The EE-induced systemic reduction of IL-6 and TNF-α levels therefore suggests that Tcf4Apcmale animals may have restored barrier function. To define possible changes within the tumor microenvironment that could promote improved barrier function, we conducted RNA-sequencing (RNA-seq) analysis of distal colon samples isolated from male EE and NE Tcf4Apcanimals. We found many significant gene expression changes, including 205 upregulated and 105 downregulated genes in EE compared to NE animals ( Tables S2 A and S2B). Notably, when EE animals are compared to NE, several genes that are normally upregulated in adenomas are downregulated by EE, and those that are downregulated in adenomas are upregulated by EE, demonstrating that gene expression profiles in tumors are altered toward a normalized expression pattern in response to EE ( Table S2 C and S2D).

Severe inflammation is often associated with barrier dysfunction and colon cancer progression (). Both male and female Tcf4Apcanimals have elevated serum levels of the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) ( Table S1 ). Interestingly, we found that EE reduced serum IL-6 protein levels in male Tcf4Apcanimals by 45% and TNF-α levels by 70% ( Figure 1 G; Tables S1 A and S1B). This is an important finding because injection of IL-6 is sufficient to cause cancer cachexia in Apcmice (). However, EE had no significant effect on IL-6 and TNF-α levels in female Tcf4Apcanimals ( Table S1 B). These results suggest that EE improves the health and survival of male Tcf4Apcanimals, at least in part, through reduction of IL-6 and TNF-α-dependent cachexia symptoms. However, in female Tcf4Apcanimals, the effects of EE on improved health and survival are likely due to reduced tumorigenesis. However, it is unclear whether female-specific factors play a role, because estrogen has a protective role in tumorigenesis (). We therefore focus on addressing the mechanisms that underlie the EE-dependent alterations, including systemic inflammatory cytokine reduction, in the colons of male animals.

To assess the general health of the animals in the short-term study, animal weights were compared. Overall, female health was stable, with relatively no weight change in NE or EE animals when compared to WT ( Figure 1 F; Table S1 B). Interestingly, the NE male Tcf4Apcanimal weights were reduced by over 25% when compared to NE WT animals, but this reduction was mitigated by EE ( Figure 1 F; Table S1 A). However, EE was not sufficient to alleviate anemia, and both the absolute number and composition of leukocytes in peripheral blood remained unchanged ( Tables S1 A and S1B). These findings indicate that EE improves the health of male tumor-bearing Tcf4ApcEE animals without hematological effects.

One possible explanation for the lack of tumor load reduction following EE is that tumors develop more slowly with EE, but ultimately animals reach a tumor load threshold and succumb to the disease. To test this directly, we conducted a shorter-term experiment, in which at 4 months of age, NE and EE Tcf4Apcand Apcanimals were collected and tumor load was determined for each animal (EE2; Supplemental Experimental Procedures Figure S1 F; Figures 1 D and 1E; Table S1 ). Interestingly, we found that, under NE conditions, there were sex-specific differences in tumor initiation, but not necessarily tumor growth, in Tcf4Apcanimals (8.5 tumors in males compared to 4 tumors in females; p = 0.03, charted in Figure S1 G and Table S1 C). However, when comparing NE and EE tumors within female Tcf4Apcanimals, we found that, although the two groups develop similar numbers of colon tumors, colon tumor volume is significantly lower in response to EE (42% reduction in size; p = 0.016; Figures 1 D and 1E; Table S1 B), demonstrating that EE specifically limits tumor growth but not initiation in female Tcf4Apcmice. On the other hand, males show tumor count and volume (reduced by 27%; p = 0.299 and 32%; p = 0.413, respectively) that are widely variable and not significantly different between NE and EE ( Figures 1 D and 1E; Table S1 A). This indicates that a reduction in tumor load may underlie the EE-mediated improvement in survival of female, but not necessarily in male, Tcf4Apcanimals, and suggests that the lifespan extension in males and females in response to EE may arise from different mechanisms.

We next questioned whether EE altered tumor load (tumor number and weight) in NE and EE Tcf4Apcand Apcanimals. We found that tumor load was not significantly reduced in response to EE ( Figures 1 B and 1C; Figure S1 D). Small intestinal tumors from these animals also failed to respond to EE ( Figure S1 E). This suggested that EE improves the survival of Tcf4Apcanimals by a mechanism that does not decrease tumor number or size.

To determine the effects of long-term EE on tumorigenesis and survival of Tcf4Apccolon cancer mice, separately housed male and female wild-type (WT), Tcf4Apc, Tcf4Apc, and Tcf4Apcanimals were weaned into either an EE or non-enriched (NE) control environment ( Figure S1 A). Animal health was assessed daily, and animals were collected when they began to exhibit symptoms of cachexia, including decreased mobility, weight loss, and anemia. These phenotypes were used to define the end of lifespan for these animals ( Supplemental Experimental Procedures ; EE1). We found that EE drastically improved survival of male and female colon-tumor-bearing Tcf4Apcmice compared to NE cohorts ( Figure 1 A). Following enrichment, median survival days increased in Tcf4Apcmales from 173 to 228 days (55-day increase) and from 156 to 238 days (82-day increase) in females ( Figure 1 A; Figure S1 C). Importantly, we observed no significant increase in survival of EE compared to NE Apcanimals ( Figures S1 B and S1C), suggesting that this phenotype is Tcf4dependent and perhaps colon tumor specific.

(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 = 0.005 using two-sample t test with Welch correction.

(A–C) Lifespan EE. (A) Survival curves for male and female Tcf4 Het/+ Apc Min/+ 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 +/+ Apc Min/+ and Tcf4 Het/+ Apc Min/+ animals.

Discussion

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Rosenberg D. Sex differences in associations between psychosocial factors and aberrant crypt foci among patients at risk for colon cancer. Our studies reveal a remarkable, beneficial effect on the survival of both male and female mice with colon tumors in response to EE, a finding with profound clinical implications. Curiously, we observed clear phenotypic differences between male and female colon-tumor-bearing animals. The marked reduction in tumor growth in females alone would be expected to contribute to the extension of survival in females, but it clearly is not responsible for this EE-mediated health benefit in males. Moreover, it is possible that EE promotes health in each sex by different mechanisms. Sex-specific differences have previously been noted in epidemiological studies of psychosocial risk factors for colon cancer. Specifically, aberrant crypt foci (ACFs), a marker of colon cancer risk, are found to be specifically increased in women with depression and men with reduced social structure (). Importantly, our studies suggest that EE can reduce the adverse symptoms associated with colon cancer similarly in both sexes, to extend overall health and survival, albeit by different mechanisms.

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García-Serrano L.

Martínez-Fernández M.

Ruiz-Llorente L.

Paramio J.M.

Aranda A. Impaired hair growth and wound healing in mice lacking thyroid hormone receptors. 3 ( Bartalena et al., 1994 Bartalena L.

Brogioni S.

Grasso L.

Velluzzi F.

Martino E. Relationship of the increased serum interleukin-6 concentration to changes of thyroid function in nonthyroidal illness. NHRs are ligand-regulated transcription factors that have key roles in normal tissue development and homeostasis. As transcription factors, they often form heterodimers with other NHRs and also act synergistically to modulate target gene expression during wound repair. In EE colons, several different NHR signaling pathways are activated, suggesting that each may play a role in the tumor wound repair response. For example, in EE tumor-bearing animals, GC and MC signaling may play a role in reduced inflammation and revascularization (), whereas the reactivation of thyroid hormone signaling may effect both re-epithelialization and B cell recruitment and migration to the tumor periphery (). In our study, we have further characterized thyroid hormone signaling within the stromal and tumor cell compartments in EE tumor-bearing mice, and found activation of both hormone metabolism and the hormone receptor. However, the molecular mechanism that improves NHR signaling in EE colon-tumor-bearing mice is unclear. At least for thyroid hormone, it is possible that reduced IL-6 levels in EE animals play a role in the restoration of T3 metabolism, because local thyroid hormone metabolism is often inactivated as a direct effect of illness, and IL-6 cytokine levels are inversely correlated with the levels of activated thyroid hormone T). It remains to be tested whether IL-6 inhibitors alone or in combination with NHR agonists mimic the wound repair effects of EE in tumor-bearing animals.

+ SMA+ and RGS5+ SMA− pericyte subtypes both in revascularization and wound repair, respectively. It is possible that EE-dependent NHR ligands recruit and stabilize pericytes in the tumor microenvironment ( Hung et al., 2013 Hung C.

Linn G.

Chow Y.-H.

Kobayashi A.

Mittelsteadt K.

Altemeier W.A.

Gharib S.A.

Schnapp L.M.

Duffield J.S. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis. Ding et al., 2014 Ding L.

Cheng R.

Hu Y.

Takahashi Y.

Jenkins A.J.

Keech A.C.

Humphries K.M.

Gu X.

Elliott M.H.

Xia X.

Ma J.X. Peroxisome proliferator-activated receptor α protects capillary pericytes in the retina. Het/+ ApcMin/+ animals. Our data support a function for RGS5SMAand RGS5SMApericyte subtypes both in revascularization and wound repair, respectively. It is possible that EE-dependent NHR ligands recruit and stabilize pericytes in the tumor microenvironment (), as the NHR PPARα has been shown to protect pericytes in a mouse model of diabetic retinopathy (). Further studies will be required to define the EE-dependent mechanisms driving increased pericyte localization to the perivascular and intratumoral regions in EE Tcf4Apcanimals.

Mantis et al., 2011 Mantis N.J.

Rol N.

Corthésy B. Secretory IgA’s complex roles in immunity and mucosal homeostasis in the gut. Het/+ ApcMin/+ animals at least partially drives the EE-mediated increase in their health and survival. A review of the relatively limited mind-body-therapy studies available revealed that secretion of salivary IgA was the only significant outcome involving the immune system ( Wahbeh et al., 2009 Wahbeh H.

Haywood A.

Kaufman K.

Zwickey H. Mind-body medicine and immune system outcomes: a systematic review. Moon et al. (2015) Moon C.

Baldridge M.T.

Wallace M.A.

Burnham C.A.

Virgin H.W.

Stappenbeck T.S. Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation. Based on the well-known protective effect of IgA secretion in colon tissue (), it is likely that the comparative abundance of colonic IgA in EE Tcf4Apcanimals at least partially drives the EE-mediated increase in their health and survival. A review of the relatively limited mind-body-therapy studies available revealed that secretion of salivary IgA was the only significant outcome involving the immune system (). Our studies support and extend these findings to the colon. Recently,found a correlation between mice with low levels of secretory IgA (SIgA) on the luminal surface and the presence of Sutterella bacteria, suggesting that Sutterella may function in SIgA degradation. However, in our study, IgA levels increase in response to EE concomitantly with accumulation of Sutterella, suggesting that Sutterella may actually serve a protective function, possibly by normalizing SIgA levels at the mucosal surface.

In conclusion, we have described an NHR-, pericyte-, and IgA-dependent mechanism that supports the improvement of lifespan following EE in Tcf4Het/+ ApcMin/+ males. Our study indicates that EE delivers a tumor wound repair-centric therapeutic effect that improves the overall health of tumor-bearing animals. The reactivation of the NHR pathways in wound repair following EE has revealed potential therapeutic strategies for pharmaceutical interventions that mimic the beneficial effects of EE in wound repair in colon tumors.