IDO1+ Paneth cells promote immune escape of colorectal cancer.

Tumors have evolved mechanisms to escape anti-tumor immunosurveillance. They limit humoral and cellular immune activities in the stroma and render tumors resistant to immunotherapy. Sensitizing tumor cells to immune attack is an important strategy to revert immunosuppression. However, the underlying mechanisms of immune escape are still poorly understood. Here we discover Indoleamine-2,3-dioxygenase-1 (IDO1)+ Paneth cells in the stem cell niche of intestinal crypts and tumors, which promoted immune escape of colorectal cancer (CRC). Ido1 expression in Paneth cells was strictly Stat1 dependent. Loss of IDO1+ Paneth cells in murine intestinal adenomas with tumor cell-specific Stat1 deletion had profound effects on the intratumoral immune cell composition. Patient samples and TCGA expression data suggested corresponding cells in human colorectal tumors. Thus, our data uncovered an immune escape mechanism of CRC and identify IDO1+ Paneth cells as a target for immunotherapy.

Cancer-associated fibroblast-derived WNT2 increases tumor angiogenesis in colon cancer.

WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.

STAT1 is a sex-specific tumor suppressor in colitis-associated colorectal cancer.

The interferon-inducible transcription factor STAT1 is a tumor suppressor in various malignancies. We investigated sex-specific STAT1 functions in colitis and colitis-associated colorectal cancer (CRC) using mice with specific STAT1 deletion in intestinal epithelial cells (STAT1∆IEC ). Male but not female STAT1∆IEC mice were more resistant to DSS-induced colitis than sex-matched STAT1flox/flox controls and displayed reduced intraepithelial infiltration of CD8+ TCRαß+ granzyme B+ T cells. Moreover, DSS treatment failed to induce expression of T-cell-attracting chemokines in intestinal epithelial cells of male but not of female STAT1∆IEC mice. Application of the AOM-DSS protocol for induction of colitis-associated CRC resulted in increased intestinal tumor load in male but not in female STAT1∆IEC mice. A sex-specific stratification of human CRC patients corroborated the data obtained in mice and revealed that reduced tumor cell-intrinsic nuclear STAT1 protein expression is a poor prognostic factor in men but not in women. These data demonstrate that epithelial STAT1 is a male-specific tumor suppressor in CRC of mice and humans.

Myeloid STAT3 promotes formation of colitis-associated colorectal cancer in mice.

Myeloid cells lacking STAT3 promote antitumor responses of NK and T cells but it is unknown if this crosstalk affects development of autochthonous tumors. We deleted STAT3 in murine myeloid cells (STAT3Δm) and examined the effect on the development of autochthonous colorectal cancers (CRCs). Formation of Azoxymethane/Dextransulfate (AOM/DSS)-induced CRCs was strongly suppressed in STAT3Δm mice. Gene expression profiling showed strong activation of T cells in the stroma of STAT3Δm CRCs. Moreover, STAT3Δm host mice were better able to control the growth of transplanted MC38 colorectal tumor cells which are known to be killed in a T cell-dependent manner. These data suggest that myeloid cells lacking STAT3 control formation of CRCs mainly via cross activation of T cells. Interestingly, the few CRCs that formed in STAT3Δm mice displayed enhanced stromalization but appeared normal in size indicating that they have acquired ways to escape enhanced tumor surveillance. We found that CRCs in STAT3Δm mice consistently activate STAT3 signaling which is implicated in immune evasion and might be a target to prevent tumor relapse.

Induction of colorectal cancer in mice and histomorphometric evaluation of tumors.

Colorectal cancer (CRC) originates from the epithelial cells lining the colon or rectum of the gastrointestinal tract and represents the third most common form of cancer worldwide. CRC is frequently associated with Colitis Ulcerosa or Crohn's Disease demonstrating the tumor-promoting role of inflammation. Colorectal tumor cells establish heterotypic interactions with inflammatory cells and cancer-associated fibroblasts in the tumor stroma that support tumor angiogenesis and are essential for tumor progression. Therefore, establishment of suitable mouse models mimicking the inflammatory etiology of CRC is important. Here we describe methods to induce CRC in mice, to quantify tumor parameters (multiplicity, tumor load, mean tumor size), and to analyze the cellular composition of the CRC tumor stroma.