Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-15-2870 Cancer Tumor and Stem Cell Biology Research Distinct Levels of Radioresistance in Lgr5þ Colonic Epithelial Stem Cells versus Lgr5þ Small Intestinal Stem Cells Guoqiang Hua1,2,3, Chu Wang1, Yan Pan1,2, Zhaoshi Zeng4, Sang Gyu Lee2, Maria Laura Martin2,4, Adriana Haimovitz-Friedman3, Zvi Fuks3, Philip B. Paty4, and Richard Kolesnick2 Abstract Although small and large intestines possess seemingly sim- DNA damage. Checkpoint recovery before complete double- ilar Wnt-driven leucine-rich repeat-containing G protein–cou- strand break (DSB) repair represents the sine qua non of a newly þ pled receptor 5 (Lgr5) adult epithelial stem cells, we report defined potentially lethal pathophysiology termed checkpoint here that the two organs exhibit distinct mechanisms of tissue adaptation. In the small intestinal mucosa, checkpoint adap- response to ionizing radiation. Employing Lgr5-lacZ transgenic tation resulted in CBCs succumbing to an 8-fold increase in the mice and Lgr5 in situ hybridization, we found colonic epithelial incidence of highly lethal chromosomal aberrations and mitot- þ stem cells (CESC) markedly more radioresistant in vivo than ic catastrophe by 48 hours postradiation. In contrast, Lgr5 small intestinal crypt base columnar stem cells (CBC; D0 ¼ 6.0 CESCs displayed delayed checkpoint recovery at 48 hours post- Æ 0.3 Gy vs. 1.3 Æ 0.1, respectively; P < 0.01). Accordingly, 19 Gy, coordinated with complete DSB repair and regeneration CESCs survived 30 Gy exposure, while CBCs were completely of colonic mucosa originating, at least in part, from surviving depleted after 15 Gy. EdU incorporation studies indicated that CESCs. The discovery that small intestinal CBCs succumb to after 19 Gy, CBCs exited growth arrest at 12 hours, resuming checkpoint adaptation is the first demonstration that this normal mitotic activity despite 60% of this population dis- aberrant cell-cycle response may drive mammalian tissue radio- playing residual gH2AX foci, indicative of persistent unrepaired sensitivity. Cancer Res; 77(8); 2124–33. Ó2017 AACR. Of currently studied adult stem cell populations in mammalian Introduction þ organs, response of the small intestinal Lgr5 crypt base columnar The differential response of mammalian tissues to high doses of cell (CBC) to genotoxic stress is perhaps the best studied. The ionizing radiation is posited in terms of differences in radiosen- small intestinal mucosa is a rapid turnover system, considered sitivity of tissue stem cell compartments (1–3), although no þ driven by mitotic activity of self-renewing Lgr5 CBCs (6, 7). Prior definitive study corroborating this widely held hypothesis has þ to discovery of the Lgr5 CBC, survival of ISCs to genotoxic stress ever been published. This is because until recently, validated solid was quantified indirectly using the microcolony assay (also tissue stem cells were not available for direct study. Although it is termed clonogenic assay) of Withers and Elkind (8) by counting well documented that the large intestine is more radioresistant regenerating crypts in histologic sections at 3.5 days postradia- than the small intestine, mechanisms underlying large intestine tion. The rationale of this assay, regarded as one of the best radioresistance remain poorly understood (4, 5). Here, we com- þ surrogate assays for adult mammalian stem cell responses for pare two highly similar Wnt-driven Lgr5 (also known as Gpr49; decades, is that it must have taken at least one stem cell to refs. 6, 7) intestinal stem cell (ISC) populations at the base of the regenerate a complete crypt. crypts of the small and large intestine in an attempt to elucidate The discovery that the CBC, most often located between Paneth mechanisms controlling radiosensitivity. cells at the base of the small intestinal crypt (9), is a legitimate ISC represents a milestone in the gastrointestinal field and has per- mitted detailed analysis of its response to genotoxic stress. 1Institute of Radiation Medicine, Fudan University, Shanghai, China. 2Laboratory Although many groups have confirmed that the threshold for of Signal Transduction, Memorial Sloan Kettering Cancer Center, New York, New survivability of the small intestine to ionizing radiation is approx- York. 3Department of Radiation Oncology, Memorial Sloan Kettering Cancer 4 imately 10% regenerative crypts at 3.5 days after irradiation as Center, New York, New York. Department of Surgery, Memorial Sloan Kettering determined by the microcolony assay (10–12), we determined Cancer Center, New York, New York. that the death of CBCs preceded crypt regeneration, detectable at 1 Note: Z. Fuks, P.B. Paty, and R. Kolesnick contributed equally to this article. day postirradiation and maximal at 2 days (13, 14). Furthermore, Corresponding Authors: Richard Kolesnick, Memorial Sloan-Kettering Cancer loss of CBCs predicted outcome of the microcolony assay as one Center, 1275 York Avenue, New York, NY 10065. Phone: 646-888-2174; Fax: 646- might anticipate if CBCs represented the relevant target for small 422-0281; E-mail: [email protected]; and Guoqiang Hua, intestinal survival. Loss of CBCs within the first 2 days postirra- [email protected] diation was biphasic, with about 30% of CBCs dying by apoptosis doi: 10.1158/0008-5472.CAN-15-2870 during the first 24 hours after 12 Gy, while cells are growth Ó2017 American Association for Cancer Research. arrested and repairing DNA damage, followed by mitotic death 2124 Cancer Res; 77(8) April 15, 2017 Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-15-2870 Lgr5þ CESCs Are Resistant to Mitotic Cell Death of approximately 60% of CBCs during the subsequent 24 hours, precise targeting of the distal colon. Field uniformity and dose leaving a residual of 10% of CBCs available for crypt regeneration. homogeneity were defined using a 16-mm thick Superflab phan- That CBCs were relevant ISCs for organ recovery was indicated by tom, with an IBA CC04 ionization chamber placed at 8 mm, and lineage-tracing studies postirradiation. At 15 Gy, a dose that by exposing Kodak XV film for density contour using a densi- reduces crypt counts by 95% to 99% in the Withers and Elkind tometer. Crypt and ISC survival curves were calculated by least analysis, and is not survivable, complete loss of CBCs was detected square regression analysis, with a modification of the FIT software by 48 hours postirradiation. Further study revealed that of all the program, as published previously (21). The program fits curves by þ cell populations of the small intestinal crypt/villus unit, the Lgr5 iteratively weighted least squares to each set of dose survival data, CBC was the most radioresistant due to efficient use of error-free estimates covariates of survival curve parameters and correspond- homologous recombination to repair double-strand breaks ing confidence regions, and plots the survival curve. It also derives (DSB). curve parameters, such as the D0, the reciprocal of the slope on the Like the small intestine, the large intestine is maintained by a exponential portion of the curve, representing the level of þ rapidly cycling population of Wnt-driven Lgr5 colonic epithe- radiosensitivity. lial stem cells (CESC) that reside at the crypt base in between a þ cKit population of niche cells. In the current study, we profile Tissue preparation þ these two highly similar Lgr5 stem cell populations in the large Intestinal tissue samples were fixed by 16- to 18-hour incuba- þ and small intestine. We show that Lgr5 CESCs in the distal large tion in 4% freshly prepared neutral-buffered formaldehyde (21) intestine are far more radiation resistant than their counterparts and embedded in paraffin blocks. Transverse sections of the full in the small intestine, differences attributable to substantive intestinal circumference were prepared and stained with hema- differences in cell cycle reentry and the propensity to undergo toxylin and eosin as described previously (13). mitotic death. Activation of cell-cycle checkpoints by DSBs that transiently arrest cell-cycle progression is critical in optimizing b-Galactosidase (lacZ) staining DSB repair and maintaining genomic integrity (15). Accordingly, Lgr5-lacZ mice were euthanized after radiation, and four 2.5-cm tight coordination between DSB repair and checkpoint recovery sequential segments of proximal jejunum from the ligament of constitutes a generic function of a high-fidelity DNA damage Treitz, or 2-cm segment of distal colon were obtained. Staining for response (DDR; ref. 16). Recently, however, a pathophysiologic the presence of b-galactosidase was as per ref. 6. alternative checkpoint dynamic has been described, termed checkpoint adaptation (reviewed in refs. 16, 17), an uncoupling Crypt microcolony assay of completion of DSB repair and checkpoint recovery that The microcolony assay was performed as described by Withers enables precocious cell-cycle progression in the presence of and Elkind (8). Briefly, at 3.5 days (small intestine) or 5 days residual unrepaired DNA (18). G2M transition in the presence (distal colon) after irradiation, intestines were obtained and of unrepaired DNA renders kinetochore/mitotic spindle dysfunc- hematoxylin and eosin stained as above. Surviving crypts were tion during segregation of damaged DNA strands (19), promot- defined as containing 10 or more adjacent chromophilic cells and