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Disruption of the crypt niche promotes outgrowth of mutated colorectal tumor stem cells
Stefan Klingler, … , Anthony M.C. Brown, Richard Kolesnick
Stefan Klingler, … , Anthony M.C. Brown, Richard Kolesnick
Published March 8, 2022
Citation Information: JCI Insight. 2022;7(5):e153793. https://doi.org/10.1172/jci.insight.153793.
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Research Article Oncology Stem cells

Disruption of the crypt niche promotes outgrowth of mutated colorectal tumor stem cells

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Abstract

Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5–positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane–dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury–induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.

Authors

Stefan Klingler, Kuo-Shun Hsu, Guoqiang Hua, Maria Laura Martin, Mohammad Adileh, Timour Baslan, Zhigang Zhang, Philip B. Paty, Zvi Fuks, Anthony M.C. Brown, Richard Kolesnick

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Figure 1

DSS induces loss of Lgr5 expression in CESCs.

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DSS induces loss of Lgr5 expression in CESCs.
(A) Representative transve...
(A) Representative transverse section of distal colon from Lgr5-lacZ mice stained for lacZ. Scale bar = 10 μm. (B) Timing of loss of Lgr5+ stem cell lacZ signal after 1% DSS treatment. Left panel — H&E and lacZ staining of distal colons from Lgr5-lacZ mice at day 0, 2, and 7 after 1% DSS treatment, quantified in the right panel. Scale bar = 50 μm. For Lgr5 recovery, Lgr5-lacZ mice were treated with 1% DSS for 2 days followed by 2 days normal drinking water. Data (mean ± SEM) are from 6 mice/time point, evaluating 5 circumferences/mouse, except for Lgr5-lacZ recovery data, which are from 11 circumferences from 3 mice. ***P < 0.001 vs. day 0. Note, P < 0.001, 2-day recovery vs. day 2 DSS. (C) CESCs remain physically present at the crypt base after 2 days of 1% DSS treatment. White arrows identify CESCs between red fluorescent cKit+ cells in colonic crypts. Data (mean ± SD) are collated from 100 crypts (4 mice/time point), P > 0.05, 2-tailed Student’s t test. Scale bar = 10 μm. (D) Number of proliferating CESCs dramatically decreases at day 2 of 1% DSS treatment. S-phase cells were pulse-labeled with EdU for 2 hours before sacrifice. Scale bar = 50 μm. Data (mean ± SD) show representative results from 3 independent experiments. (E) Lineage tracing using Lgr5-mTmG transgenic mice demonstrates GFP+ CESCs at the crypt base after 2 days of 1% DSS treatment. Data (mean ± SD) are collated from 137 and 179 intact crypts for control and DSS groups, respectively, from 3 mice/group. P value, 2-tailed Student’s t test. Scale bar = 10 μm. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. 4-OHT, 4-hydroxytamoxifen.

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