A molecular subtype of colorectal cancers initiates independently of epidermal growth factor receptor and has an accelerated growth rate mediated by IL10-dependent anergy

Abstract

Although epidermal growth factor receptor (EGFR)-targeted therapies are approved for colorectal cancer (CRC) treatment, only 15% of CRC patients respond to EGFR inhibition. Here, we show that colorectal cancers (CRC) can initiate and grow faster through an EGFR-independent mechanism, irrespective of the presence of EGFR, in two different mouse models using tissue-specific ablation of Egfr. The growth benefit in the absence of EGFR is also independent of Kras status. An EGFR-independent gene expression signature, also observed in human CRCs, revealed that anergy-inducing genes are overexpressed in EGFR-independent polyps, suggesting increased infiltration of anergic lymphocytes promotes an accelerated growth rate that is partially caused by escape from cell-mediated immune responses. Many genes in the EGFR-independent gene expression signature are downstream targets of interleukin 10 receptor alpha (IL10RA). We further show that IL10 is detectable in serum from mice with EGFR-independent colon polyps. Using organoids in vitro and Src ablation in vivo, we show that IL10 contributes to growth of EGFR-independent CRCs, potentially mediated by the well-documented role of SRC in IL10 signaling. Based on these data, we show that the combination of an EGFR inhibitor with an anti-IL10 neutralizing antibody results in decreased cell proliferation in organoids and in decreased polyp size in pre-clinical models harboring EGFR-independent CRCs, providing a new therapeutic intervention for CRCs resistant to EGFR inhibitor therapies.

Fig. 1. EGFR-independent intestinal and colonic adenoma development.

a Multiplicity of intestinal polyps is decreased in Apc^Min/+, Egfr^f/f (Tg(Vil-Cre) mice. Dots represents polyp number in each 100-day-old mouse. b Distribution of polyp sizes show Apc^Min/+, Egfr^f/f (Tg(Vil-Cre) polyps are larger than Apc^Min/+, Egfr^+/+. c Colonic polyp penetrance 18 weeks after AdCre induction. d Polyp number by endoscopic analysis after AdCre induction. e Serial endoscopic images after AdCre administration to the distal colon. f Growth curves of AdCre-induced polyps. Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 2. IL10 signaling activation, in EGFR-independent colon polyps, increased infiltrating immune cells, especially M2-type macrophages.

Transcript levels of a Il10ra, b Socs3, and c Il10 measured by qPCR. White bars, mean transcript levels in the adjacent normal tissue; colored bars, mean transcript levels in colon polyps. d IL10 levels in serum from mice. White bars, animals without polyps; black bars, animals with polyps. e Immunohistochemical staining of macrophage markers. Immunohistochemistry quantification of f total, g iNOS-positive, and h CD163⁺-positive macrophages in induced colon polyps. Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All scale bars represent 100um.

Fig. 3. Effect of IL10 in cell proliferation in colon organoids.

a Left panel shows number of cells (solid bars, left axis) and IL10 levels in media (white bars, right axis) 24h after seeding. Right panel shows correlation of cell proliferation and IL10 levels in media at 24h after seeding. b-e Representative organoid images at 72h post treatment (left panel) and cell proliferation assay (right panel) when organoids were treated with b mouse recombinant IL10, c IL10 neutralizing antibody, d EGFR inhibitor AG1478 and e combinatory treatment of AG1478 (1mM) and IL10 antibody (2ug/ml). Equal number of cells (2000 cell/well) were seeded at time 0. Data are represented as mean ± SEM of three experiments performed under the same conditions. Blue bars represent Egfr^+/+, Apc^f/f, Kras^LSL/+ and green bars represent Egfr^f/f, Apc^f/f, Kras^LSL/+. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All scale bars represent 100um.

Fig. 4. Anti-IL10 neutralizing antibody treatment.

a Colonoscopic images of colon polyps. Polyp locations are outlined. b Polyp sizes measured at necropsy. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 5. Proposed EGFR-independent mechanism of colorectal cancer progression.

Black arrows, proven links; grey arrows, proposed interactions from literature.