BCN057 induces intestinal stem cell repair and mitigates radiation-induced intestinal injury
Bhanja et al. Stem Cell Research & Therapy
BCN057 induces intestinal stem cell repair and mitigates radiation-induced intestinal injury
Payel Bhanja 1
Andrew Norris 2
Pooja Gupta-Saraf 1
Andrew Hoover 1
Subhrajit Saha 0 1
0 Department of Cancer Biology, The University of Kansas Medical Center , MS 4033, 3901 Rainbow Boulevard, Kansas City, Kansas 66160 , USA
1 Department of Radiation Oncology, The University of Kansas Medical Center , MS 4033, 3901 Rainbow Boulevard, Kansas City, Kansas 66160 , USA
2 BCN Bio Sciences , Pasadena, CA , USA
Background: Radiation-induced gastrointestinal syndrome (RIGS) results from the acute loss of intestinal stem cells (ISC), impaired epithelial regeneration, and subsequent loss of the mucosal barrier, resulting in electrolyte imbalance, diarrhea, weight loss, sepsis, and mortality. The high radiosensitivity of the intestinal epithelium limits effective radiotherapy against abdominal malignancies and limits the survival of victims of nuclear accidents or terrorism. Currently, there is no approved therapy to mitigate radiation toxicity in the intestine. Here we demonstrate that BCN057, an anti-neoplastic small molecular agent, induces ISC proliferation and promotes intestinal epithelial repair against radiation injury. Methods: BCN057 (90 mg/kg body weight, subcutaneously) was injected into C57Bl6 male mice (JAX) at 24 h following abdominal irradiation (AIR) and was continued for 8 days post-irradiation. BCN057-mediated rescue of Lgr5-positive ISC was validated in Lgr5-EGFP-Cre-ERT2 mice exposed to AIR. The regenerative response of Lgr5-positive ISC was examined by lineage tracing assay using Lgr5-EGFP-ires-CreERT2-TdT mice with tamoxifen administration to activate Cre recombinase and thereby marking the ISC and their respective progeny. Ex vivo three-dimensional organoid cultures were developed from surgical specimens of human colon or from mice jejunum and were used to examine the radio-mitigating role of BCN057 on ISC ex vivo. Organoid growth was determined by quantifying the budding crypt/total crypt ratio. Statistical analysis was performed using Log-rank (Mantel-Cox) test and paired two-tail t test. Results: Treatment with BCN057 24 h after a lethal dose of AIR rescues ISC, promotes regeneration of the intestinal epithelium, and thereby mitigates RIGS. Irradiated mice without BCN057 treatment suffered from RIGS, resulting in 100% mortality within 15 days post-radiation. Intestinal organoids developed from mice jejunum or human colon demonstrated a regenerative response with BCN057 treatment and mitigated radiation toxicity. However, BCN057 did not deliver radio-protection to mouse or human colon tumor tissue. Conclusion: BCN057 is a potential mitigator against RIGS and may be useful for improving the therapeutic ratio of abdominal radiotherapy. This is the first report demonstrating that a small molecular agent mitigates radiation-induced intestinal injury by inducing ISC self-renewal and proliferation.
Intestinal stem cell; RIGS; Abdominal radiation; Radiotherapy; Tumor
Intestinal injury is a limiting factor for definitive
chemoradiation therapy of abdominal malignancies such as gastric,
pancreatic, and colorectal cancer. Thus, tumoricidal doses
of radiotherapy and/or chemotherapy often cannot be
administered for the treatment of abdominal tumors
resulting in poor survival and early metastatic spread.
Moreover, radiation-induced gastrointestinal syndrome
(RIGS) limits the survival of victims in a mass casualty
setting from nuclear accidents or terrorism. While
supportive care with antibiotics, hydration, and bone
marrow transplantation can avoid death due to the
hematopoietic syndrome, currently there is no approved
therapy for protecting or mitigating against RIGS.
Radiation doses more than 10 Gy primarily lead to
gastrointestinal injury, resulting in diarrhea, dehydration, sepsis,
and intestinal bleeding with eventual mortality within 10
to 15 days post-exposure [
]. A high dose of radiation
induces the loss of intestinal stem cells (ISC) [
thereby impairs epithelial regeneration. The damaged
intestinal epithelium significantly reduces the mucosal
integrity and promotes systemic influx of bacterial
pathogens resulting in sepsis and death [
]. These lethal
gastrointestinal symptoms after radiation exposure are
collectively known as radiation-induced gastrointestinal
syndrome (RIGS), or clinically known as radiation
enteritis. So far there are no Food and Drug Administration
(FDA)-approved agents available to mitigate
radiationinduced intestinal injury [
]. Considering the logistical
barrier and unavoidable delay in treating victims in large
casualty settings there is a tremendous need for
therapeutic measures which can be effective even if started days
after the radiation incident.
Dose-dependent radiation damage to the ISC is the
primary cause of RIGS. We have reported previously
that inhibition of radiation-induced ISC loss will
mitigate RIGS [
]. Our recent study demonstrated that
extracellular vesicle (EV)-mediated delivery of Wnt
rescues ISC from radiation toxicity and induces intestinal
epithelial repair with the activation of Wnt-β-catenin
signaling. ISC self-renewal and proliferation, and thereby
maintenance of intestinal epithelial homeostasis and
repair, is primarily dependent on Wnt-β-catenin
]. ISC growth factors, such as R-spondin 1
(RSPO1), activate the Wnt-β-catenin pathway to repair
and regenerate the intestine following
chemoradiationinduced injury [
]. DKK1, a negative regulator of the
Wnt-β-catenin pathway, impairs the RSPO1-induced
intestinal regeneration [
]. RSPO1 binds to the Lgr5
] which is associated with the Frizzled/Lrp
Wnt receptor complex [
]. Genetic deletion of Lgr5 in
mouse intestine inhibits the regenerative role of Rspo1,
but epithelial regeneration can be rescued by Wnt
In this study we demonstrated that a small molecular
(3-[(Furan-2-ylmethyl)-amino]-2-(7methoxy-2-oxo-1,2-dihydro-quinolin-3-yl)-6-methyl-imidazo[1,2-a]pyridin-1-ium) activates canonical
Wnt-βcatenin signaling, mitigates RIGS, and improves survival
when applied 24 h after a lethal dose of radiation
exposure. BCN057 induces strong Wnt activity as
demonstrated by TCF/LEF reporter assay. In an ex-vivo
crypt organoid model developed from human and mice
intestinal epithelium, we demonstrated that BCN057 rescued
ISC from radiation toxicity and induced epithelial repair
with the activation of Wnt-β-catenin signaling. However,
BCN057 did not show any radioprotective effect in tumor
tissue. Taken together, these observations indicate that
BCN057 is an agonist of canonical Wnt-β-catenin
signaling and mitigates radiation-induced intestinal injury by
accelerating the repair and regeneration of ISC.
BCN057 is a novel small molecule designed with
moieties targeting G protein-coupled receptors (GPCRs),
and 12 mg/mL BCN057 in 30% Captisol® (β-cyclodextrin
sulfobutyl ether sodium) has been formulated for
subcutaneous (s.c.) administration. This formulation has
shown excellent stability up to 1 year and has been well
tolerated in both cell and animal use. BCN057 (mass
401.16) was administered via a single subcutaneous (s.c.)
injection at the designated dose in 200 μL. Time points
(post-dose) were collected by cardiac puncture in
euthanized C57BL/6 mice at 0, 1, 2, 4, 6, 16, and 24 h, with
three mice per time point for a total of 21 animals.
Plasma samples (20 μL) were processed by a protein
precipitation method. All samples were analyzed using a
triple quadrupole mass spectrometer (Agilent® 6460)
coupled to an HPLC system (Agilent® 1290) using a
reverse-phase analytical column (Agilent® Poro Shell
300SB, C-8, 5 mm, 2.1 × 75 mm). For the analysis of
BCN057, RT = 12.5 min is measured against an internal
with an exact mass of 387.15 and RT = 12.1 min. BCN057
is monitored with the transition from m/z 401 → 320 and
quantitation is performed with the use of the internal
standard yielding a linear regression least-squares fit from
2 fmol to 20 pmol with R2 = 0.99. Pharmacokinetics (PK)
data were processed using PK Solutions© 2.0 (Summit
Research Services Montrose, CO, USA).
Five- to 6-week-old male C57BL6/J mice,
Gt(ROSA)26Sortm4(ACTB-tdTomatoEGFP)Luo/J mice, and
B6.Cg-Gt(ROSA)26Sortm9(CAGtdTomato)Hze/J mice (Jackson laboratories) were
maintained ad libitum and all studies were performed
under the guidelines and protocols of the Institutional
Animal Care and Use Committee of the University of
Kansas Medical Center. All the animal experimental
protocols were approved by the Institutional Animal Care
and Use Committee of the University of Kansas Medical
Center (ACUP number 2016-2316).
Development of subcutaneous tumor in mouse flank
Mice were injected subcutaneously with 1 × 105 MC38
(colon carcinoma cell line) cells on the flank. About
10 days later, the tumor became palpable (3–5 mm in
diameter), whereupon abdominal irradiation (AIR) of
16 Gy was delivered. Mice were divided into four groups
(n = 10 per group): those receiving no treatment; those
with AIR; those receiving BCN057; and those receiving
BCN057 plus AIR. Animals received BCN057 eight
times starting 24 h after AIR. Tumor measurements
were performed thrice weekly using Vernier calipers
along with simultaneous physical assessment of signs of
systemic toxicity (malaise and diarrhea).
AIR was performed on anesthetized mice (with a
continuous flow of 1.5 mL/min 1.5% isoflurane in pure
oxygen) using the small animal radiation research
platform (SARRP; XStrahl, Surrey, UK). A 3-cm area of the
mice containing the gastrointestinal tract (GI) was
irradiated (Fig. 1c), thus shielding the upper thorax, head,
and neck, as well as the lower and upper extremities,
and protecting a significant portion of the bone marrow,
thus predominantly inducing RIGS. A radiation dose of
14–15 Gy was delivered to the midline of the GI,
ensuring homogeneous delivery by performing half of the total
irradiation from the anterior-posterior direction and the
second half from the posterior-anterior direction. Partial
body irradiation (PBI) was delivered to mice after
shielding the head and fore limbs where 40% of the total bone
marrow was exposed (BM40) to irradiation (Fig. 1e)
]. The total irradiation time to deliver the intended
dose was calculated with respect to dose rate, radiation
field size, and fractional depth dose to ensure accurate
TCF/LEF (TOPFLASH) reporter assay
To determine the canonical Wnt activity of BCN057,
HEK293 cells (Signosis, Santa Clara, CA, USA) with a
TCF/LEF luciferase reporter construct were treated with
BCN057 or vehicle control or phosphate-buffered saline
(PBS). Lithium chloride (LiCl; 10 mM) treatment was used
as a positive control for luciferase activity. Luciferase
activity was determined after 24 h using the
Dual-Luciferase Reporter Assay System (Promega) as per the
manufacturer’s protocol. HEK293 cells with a FOPFLASH
construct (mutated TCF/LEF-binding site), were used as a
negative control. The HEK293 (human embryonic kidney)
cell line was routinely characterized in the laboratory
based on morphology and gene-expression patterns. Cells
were confirmed to be free of mycoplasma contamination.
Since radiation doses > 8 Gy induce cell cycle arrest
and apoptosis of the crypt epithelial cells within day
1 post-radiation, resulting in a decrease in
regenerating crypt colonies by day 3.5 and ultimately villi
denudation by day 7 post-radiation exposure, animals
were euthanized when moribund or at 3.5 days after
AIR for time-course experiments, and intestines were
collected for histology (Additional file 1: Supplement
Crypt proliferation rate
To visualize the villous cell proliferation, mid-jejunum was
collected for paraffin embedding and Ki67
immunohistochemistry. Tissue sections were routinely deparaffinized
and rehydrated through graded alcohols and incubated
overnight at room temperature with a monoclonal
antiKi67 antibody (M7240 mib1; Dako). Nuclear staining was
visualized using streptavidin-peroxidase and
diaminobenzidine (DAB) and samples were lightly counterstained with
hematoxylin. Murine crypts were identified histologically
according to the criteria reported previously [
(Additional file 1: Supplement method). To detect the
presence of Ki67 in Lgr5-positive ISC, jejunal sections
from Lgr5-eGFP-IRES-CreERT2 mice were stained with
rabbit polyclonal antibody to Ki67 (Abcam, #ab15580;
dilution 1:250) followed by secondary antibody donkey
anti-Rabbit Alexa fluor 647 (Life technologies, #A31573;
dilution 1:1000). Nuclei was counterstained with DAPI.
Determination of villi length and crypt depth
The crypt depth was independently and objectively
analyzed and quantitated in a blinded manner from coded
digital photographs of crypts from hematoxylin and eosin
(H&E) stained slides using ImageJ 1.37 software to
measure the height in pixels from the bottom of the crypt to
the crypt villus junction. Villi length was determined by
measuring the length from the crypt villus junction to the
villous tip. This measurement (in pixels) was converted to
length (in μm) by dividing with the following a conversion
factor (1.46 pixels/μm).
Detection of apoptosis in situ
Apoptotic cells were detected in situ by performing
TdTmediated digoxigenin-labeled dUTP nick-end labeling
(TUNEL) staining. Briefly, paraffin embedded sections were
de-paraffinized, rehydrated through graded alcohols, and
stained using an ApopTag kit (Intregen Co., Norcross,
(See figure on previous page.)
Fig. 1 BCN057 treatment at 24 h post-irradiation mitigates RIGS and improves survival in mice. a Chemical structure of BCN057
(3-[(Furan-2-ylmethyl)amino]-2-(7-methoxy-2-oxo-1,2-dihydro-quinolin-3-yl)-6-methylimidazo[1,2-a]pyridin-1-ium). b Pharmacokinetics of a single injection of BCN057 90 mg/
kg via s.c. administration in C57BL/6 mice. Cmax (obs) 1130.5 ng/mL, Tmax (obs) 2.0 h, Vss (expo) 15935.8 mL, CL (obs area) 700.075 mL/h. c Portal camera
image demonstrating abdominal irradiation (AIR) exposure field (i) and BCN057 treatment schema (ii). A 3-cm area (indicated by the rectangular box)
of the mouse containing the gastrointestinal tract was irradiated (irradiation field), while shielding the upper thorax, head, and neck, as well as the
lower and upper extremities, and protecting a significant portion of the bone marrow, thus predominantly inducing radiation-induced gastrointestinal
syndrome (RIGS). Mice exposed to AIR were treated with BCN057 (s.c.) (90 mg/kg body weight) at 24 h following irradiation and continued up to day
8 (single dose/day). d Kaplan-Meier survival analysis of C57BL/6 mice (n = 25/group) receiving vehicle, BCN057, or no treatment at 24 h after AIR
(14 Gy, 15 Gy, or 16 Gy) and continued up to day 8. Mice receiving BCN057 after 14 Gy, 15 Gy, or 16 Gy AIR demonstrated 80%, 60%, and 40% survival,
respectively, and they continued to survive beyond 60 days without any symptoms of gastroenteritis or any other health complications, whereas mice
receiving vehicle or no treatment following AIR died within 15 days (p < 0.0003, p < 0.0004, and p < 0.0007, respectively, log-rank (Mantel-Cox) test).
BCN057 or vehicle do not confer any toxicity to normal mice. e (i) Kaplan-Meier survival analysis of C57BL/6 mice (n = 25/group) exposed to partial
body irradiation (PBI). Head, neck, and upper extremities were shielded to spare 40% of bone marrow (BM40%). The part of the body exposed to
irradiation is indicated by a rectangular box. (ii) Mice receiving BCN057 at 24 h post-PBI demonstrated 70% survival compared with untreated controls
(p < 0.0001 log-rank (Mantel-Cox) test). f H&E stained representative cross section of jejunum from C57BL/6 mice treated with BCN057 at 24 h post-AIR
(upper panels). Note restitution of the epithelium in mice receiving BCN057 compared with irradiated controls. H&E stained representative transverse
section of jejunum from C57BL/6 mice treated with BCN057 at 24 h post-AIR (middle panels). Note restitution of crypt villus structure in
BCN057treated mice. However, irradiated, untreated mice showed significant loss of crypts along with villi denudation. Representative Ki67
immunohistochemistry of mice jejunal sections (lower panels). Note the increase in Ki67-positive crypt cells in mice receiving BCN057 at 24 h after AIR
(iv) compared with AIR controls (ii). g–i Histogram showing crypt depth and villi length (g), percentage of Ki67-positive crypt cells (h), and number of
crypts per mm (i) in the jejunum. Irradiated mice receiving BCN057 at 24 h after AIR demonstrated an increase in crypt depth and villi length (*p <
0.0006), number of crypts (*p < 0.0004), and percentage of Ki67-positive crypt cells (*p < 0.0005) compared with irradiated controls. j Histogram
demonstrating serum dextran level in C57BL/6 mice exposed to AIR and then treated with or without BCN057. Mice receiving BCN057 treatment
demonstrated lower serum dextran levels, thereby suggesting restitution of epithelial integrity compared with irradiated untreated controls (*p < 0.004,
n = 3 per group, unpaired t test, two-tailed). Unirradiated control mice and unirradiated BCN057 treated mice also showed lower serum dextran level
compared with irradiated controls (*p < 0.006, *p < 0.005, unpaired t test, two-tailed)
Georgia, USA). The apoptotic rate in crypt cells was
quantified by counting the percentage of apoptotic cells in each
crypt with analysis restricted to “intact” longitudinal crypt
sections in which the base of the crypt was aligned with all
the other crypt bases and the lumen.
β-catenin immunohistochemistry of mouse jejunum
β-Catenin immunohistochemistry was performed in
paraffin-embedded sections of mouse jejunum [
brief, tissue was stained using the anti-β-catenin antibody
(1:100 dilution; BD Transduction Laboratories, Franklin
Lakes, NJ; #610154) at room temperature for 2 h followed
by staining with horseradish peroxidase-conjugated
antimouse antibody (Dako, Denmark) at room temperature
for 1 h. Nuclei were counter-stained with hematoxylin
(blue). β-Catenin-positive nuclei (stained dark brown)
were calculated from 15 crypts per field, and five fields per
mice (Additional file 1: Supplement method).
Real-time polymerase chain reaction to determine the
expression of β-catenin target genes and intestinal stem
cell markers in crypt epithelium
To compare the mRNA levels of β-catenin target
genes in intestinal crypt cells from irradiated mice
treated with BCN057 or PBS, real-time polymerase
chain reaction (PCR) was performed for the genes
Ephb2, Ascl2, Olf, Tcf-4, Lef1, Sox9, and Axin2 using
the Wnt target gene quantitative PCR (qPCR) primers
(Additional file 2: Table S1). The expression of the
intestinal stem cell markers LGR5, K19, HES-1, and
CD44 were determined by real-time PCR using the
primer pairs listed in Additional file 3 (Table S2).
Total RNA was extracted using TRIzol kit (Invitrogen,
CA, USA). A detailed protocol is described in the
supplementary methods section (Additional file 1:
FITC-dextran permeability assay
At day 5 post-AIR, animals were gavaged with 0.6 mg/g
body weight of an FITC-dextran solution (4000 kD size;
Sigma). Four hours after gavage, mice were killed and
serum was obtained by cardiac puncture [
]. Samples were
measured in a 96-well plate using a Flexstation II 384
multiwell fluorometer (Molecular Devices). A standard curve
was constructed using mouse serum having increasing
amounts of FITC-dextran to determine the serum levels of
FITC-dextran in different treatment groups.
In vitro culture of intestinal crypt organoids
Small intestine from Lgr5-eGFP-IRES-CreERT2 and
R26-ACTB-tdT-EGFP mice, or their littermate control
mice, and malignant/non-malignant colon tissue from
human surgical specimens was used for Crypt isolation
and development of ex vivo organoid culture [
Lgr5-eGFP-IRES-CreERT2 mice were crossed with
(Jackson Laboratories) [
Gt(ROSA)26Sortm4(ACTBtdTomato-EGFP)Luo/J mice tdTomato is constitutively
expressed (independent of Cre recombination) in the
membrane of all cells, and therefore allows better
visualization of cellular morphology. Human tissues were
received from the University of Kansas Medical Center
Biorepository (HSC #5929). A detail protocol is described
in the supplementary methods section (Additional file 1:
In vivo lineage tracing assay
Lgr5-eGFP-IRES-CreERT2 mice were crossed with
(Jackson Laboratories) [
] to generate the
Lgr5-eGFPIRES-CreERT2; Rosa26-CAG-tdTomato heterozygote.
To examine the contribution of Lgr5 ISC to tissue
regeneration under steady-state conditions, lineage tracing
was induced by tamoxifen administration in Cre reporter
mice to mark the ISC and their respective tdT-positive
progeny. Adult mice were injected with tamoxifen (Sigma;
9 mg per 40 g body weight, intraperitoneally) to label Lgr5
+ lineages. For irradiation injury studies, mice were given
14–15 Gy AIR, and tissue was harvested on day 8
NCI 60 Cancer Cell Line Screen
The NCI 60 Cancer Cell Line Screen was performed
according to the protocol described previously [
Briefly, 100 μL of each cell preparation was tested in
accordance with its particular type and density, ranging
from 5000–40,000 cells per well in a 96-well microtiter
plate, corresponding to their own growth rate. BCN057
was evaluated at 10 μM with incubation for 48 h in a 5%
CO2 atmosphere with 100% humidity. Proliferation was
assayed using the sulforhodamine B assay [
] with a
plate reader to read the optical densities.
Mice survival/mortality in the different treatment group
was analyzed by Kaplan-Meier statistics as a function of
radiation dose using Graphpad Prism 6.0 software for
Mac. Mice were sorted randomly after genotyping to
each experimental and control group. The minimum
number of mice used for survival/mortality study was n
= 25 per group. For histopathological analysis, jejunal
sampling regions were chosen at random for digital
acquisition for quantitation. Digital image data were
evaluated in a blinded manner as to treatment. A
two-sided student’s t test was used to determine
significant differences between experimental cohorts
(P < 0.05) with representative standard errors of the
BCN057 mitigates RIGS and improves survival following a lethal dose of radiation
Lethality from acute radiation syndrome (ARS) depends
upon dose-dependent injury to various organs. Total
body exposure to a radiation dose higher than 10 Gy
results in mortality within 15 days post-exposure
primarily due to RIGS. Intestinal epithelium is highly
radiosensitive because of its rapid self-renewal rate compared
with any other organ. Every 4–5 days a new epithelium
takes charge of mucosal defense under very strict
epithelial homeostasis. A high dose of radiation disrupts this
homeostatic balance, kills ISC, and impairs the repair
process resulting in complete loss of the mucosal barrier
within 5–10 days post-exposure.
In this study, we have demonstrated that BCN057
mitigates RIGS and improves survival of mice exposed to a
lethal dose of irradiation. Initially, pharmacokinetic
studies of BCN057 were performed to examine
timedependent plasma exposure parameters for the
subcutaneous route of administration (Fig. 1b). BCN 057
showed plasma exposure over 24 h with a rapid Cmax at
approximately 2 h and complete clearance over 24 h.
Consequently, one dose was given every 24 h for 8 days
(Fig. 1c) as a dose regimen. To examine the
radiomitigating role of BCN057 against RIGS, C57BL/6 mice
were exposed to graded doses of AIR (14–16 Gy) after
shielding the thorax, head, neck, and extremities, thus
protecting the bone marrow (Fig. 1c) [
]. A single
fraction of 14, 15 or 16 Gy AIR induces RIGS and
lethality in 100% of animals within 7–14 days post-exposure.
Mice receiving BCN057 at 24 h post-AIR continued to
survive beyond 30 days post-exposure without showing
any symptoms of RIGS (Fig. 1d). These results clearly
indicate that BCN057 mitigates the lethal radiation
injury in the intestine.
In the event of accidental radiation, it is highly probable
that many other organ systems will also be exposed and
their differential responses to various doses of irradiation
will impact the gastrointestinal acute radiation syndrome
(GI-ARS) dose response. Involvement of bone marrow
will have a major impact on GI-ARS, primarily regarding
intestinal inflammation and mucosal immunity to mitigate
infection resulting from bacterial translocation through an
impaired intestinal mucosal barrier. To understand the
involvement of bone marrow in survival outcome on
BCN057 treatment, C57BL/6 mice were exposed to partial
body irradiation (PBI) where 40% of the total bone
marrow was exposed (BM40) to irradiation after shielding the
head and forelimbs (Fig. 1e) [
]. Treatment with
BCN057 at 24 h post-exposure 14.5 Gy PBI rescued 60%
of mice from radiation lethality (p < 0.0001). However, all
the untreated mice were dead within 12 days
postexposure (Fig. 1e). These data indicate that BCN057 can
rescue GI epithelium from radiation lethality even in the
absence of a protective function of bone marrow.
We continued to observe these BCN057-treated mice
following AIR/PBI up to day 60 post-exposure. These mice
did not develop any clinical conditions, indicating
complete cure with BCN057 treatment. Histopathological
analysis of mice jejunum at 3.5 days post-AIR clearly
demonstrated a loss of crypts with significant denudation of
villus length, indicating that RIGS is the primary cause of
death. Mice receiving BCN057 treatment demonstrated
normal crypt villus structure with an increase in the
number of crypts and preserved villous length (Fig. 1f, g, i).
The percentage of Ki67 crypt epithelial cells was
significantly higher in BCN057-treated mice compared with
untreated irradiated controls (Fig. 1f, h; p < 0.0005). However,
treatment with BCN057 in non-irradiated mice did not
induce any changes in crypt villus morphology or
Ki67positive cells (Fig. 1f, h) compared with unirradiated
Since dextran is unable to cross the GI epithelia
unless it is compromised, dextran in the blood is a good
indicator of epithelial damage [
]. Blood FITC-dextran
levels were measured at 4 h after gavage. Treatment
with BCN057 significantly reduced the FITC-dextran
uptake in the blood stream in irradiated mice compared
with untreated irradiated control mice (p < 0.004,
unpaired t test, two-tailed; Fig. 1j). These data indicate
restitution of intestinal epithelial integrity by BCN057
BCN057 activates β-catenin in irradiated jejunum
Intestinal epithelial self-renewal, homeostasis, and repair
are dependent upon Wnt-β-catenin signaling. Activation
of Wnt-β-catenin signaling translocates β-catenin to the
nucleus to switch on a series of gene expressions that
support ISC maintenance and proliferation [
]. The Wnt
activity of BCN057 was first examined by TCF/LEF
reporter assay. Graded doses of BCN057 demonstrated a
significant increase in the luciferase signal compared
with vehicle controls, indicating Wnt activity of BCN057
(p < 0.001; Fig. 2a).
We then analyzed the effect of BCN057 on crypt
epithelial β-catenin activation. Immunohistochemical
analysis of jejunal sections from non-irradiated mice
showed characteristic β-catenin with 40 ± 5 cells being
positive for nuclear β-catenin per 75 crypts (Fig. 2b, c).
Mice exposed to AIR (16 ± 2) had significantly fewer
nuclear β-catenin-positive cells compared with unirradiated
controls at day 3.5 post-AIR. However, mice receiving
BCN057 at 24 h post-AIR demonstrated a significant
increase in nuclear β-catenin-positive cells compared with
irradiated untreated animals (Fig. 2b, c). Nuclear
β-catenin-positive cells were primarily observed in the crypt
bottom which is also the location for ISC, indicating
activation of Wnt-β-catenin signaling in ISC. PCR array
analysis of β-catenin target genes in crypt epithelial cells
also showed several fold increases in the mRNA levels in
irradiated mice treated with BCN057 compared with
irradiated controls (Table 1). In summary, these data
suggest that BCN057 activates the Wnt-β-catenin signaling
in the irradiated crypt to induce crypt stem cell
proliferation and regeneration.
BCN057 rescues Lgr5+ ISC from radiation toxicity
To study this effect in vivo we examined the role of
BCN057 on ISC survival by exposing
Lgr5/GFP-IRESCre-ERT2 knock-in mice to 15 Gy AIR and then
treatment with BCN057. A time-course study demonstrated
that Lgr5+GFP+ ISC were present up to 24 h
postirradiation but disappeared thereafter from the crypt
base (Fig. 3a). TUNEL staining demonstrated that most
of the crypt base columnar cells were apoptotic at 48 h
post-AIR (Fig. 3c). Irradiated mice receiving BCN057 at
24 h post-irradiation showed significant preservation of
Lgr5-positive ISC (p < 0.001; Fig. 3b) with a significant
reduction in radiation-induced apoptosis in crypt base
columnar cells (Fig. 3c). However, mice receiving a first
dose of BCN057 at 72 h post-irradiation could not
induce repair of the intestinal epithelium (Additional file
4: Figure S1), possibly due to the absence of ISC. This
result suggests a potential window of opportunity up to
24 h post-irradiation to mitigate radiation-induced
damage in the intestine.
Treatment with BCN057 at 24 h post-irradiation
activates proliferation of Lgr5-positive ISC in irradiated
intestinal epithelium. Ki67 staining on jejunal sections
from Lgr5-EGFP-ires-CreERT2 mice demonstrated that
Lgr5-positive ISC are also positive for Ki67 in response
to BCN057 treatment (Fig. 3b). Lineage tracing assay
using Lgr5-EGFP-ires-CreERT2-R26-CAG-tdT mice
] demonstrated that BCN057 induces the
regenerative response of Lgr5-positive ISC. In this mouse model,
tamoxifen-mediated activation of cre-recombinase
(Fig. 3d) under the Lgr5 promoter expresses tdTomato
in epithelial cells derived from Lgr5-positive ISC.
Therefore, quantification of these tdTomato (tdT)-positive
cells in irradiated epithelium with or without BCN057
determines the regenerative response of Lgr5-positive
ISC. Tamoxifen treatment in the AIR + BCN057 group
demonstrated the presence of tdT-positive cells in the
crypt epithelium (Fig. 3d; Additional file 5: Figure S2).
However, in irradiated untreated mice, tdT-positive
cells are absent, suggesting the loss of regenerative
capacity of Lgr5-positive ISC (Fig. 3d). We have
quantified the number of villi containing tdT-positive
red cells (regenerative villi) for a comparison between
the AIR and AIR + BCN057 treatment groups. BCN
treatment after AIR results in a significant increase in
regenerative villi compared with untreated irradiated
controls (*p < 0.002; Fig. 3d). All this evidence clearly
demonstrates that BCN057 induces the repair process
of the intestinal epithelium by inducing the growth
and proliferation of intestinal stem cells.
To further analyze the specific effect of BCN057 on
the ISC population, we developed an ex vivo intestinal
organoid culture system [
] exposed to graded doses of
irradiation. Treatment with BCN057 (10 μM) at 1 h after
irradiation rescued the organoids from radiation toxicity
and improved the ratio of budding crypt/total crypt
(Fig. 4a, b). Intestinal crypts were isolated from Lgr5/
mice to allow the visualization of the ISC. At a dose
level of 8 Gy most of the Lgr5-positive ISC had
disappeared within 48 h resulting in a significant loss in
budding crypts with changes in existing crypt
morphology indicating inhibition of ISC growth and
proliferation in response to radiation exposure (Fig. 4c).
Treatment with BCN057 (10 μM) at 1 h after
irradiation rescued the organoids from radiation toxicity
(See figure on previous page.)
Fig. 3 BCN057 rescued Lgr5+ ISC and induced a regenerative response in vivo. a (i) Time-course study on the effect of abdominal irradiation (AIR)
on Lgr5-positive ISC. Representative images of jejunal sections demonstrating the presence of green fluorescent protein (GFP)+Lgr5+ ISC
(indicated with arrow) in Lgr5/GFP-IRES-Cre-ERT2 knock-in mice up to 24 h post-AIR. All the ISC at the crypt base disappeared at 72 h post-AIR. (ii)
The number of Lgr5+GFP+ ISC per crypt in jejunal sections from Lgr5/GFP-IRES-Cre-ERT2 knock-in mice at different time points post-AIR. The
number of Lgr5+GFP+ ISC per crypt reduced at 24 h post-irradiation (*p < 0.04). At 72 h post-AIR, most of the Lgr5+GFP+ ISC disappeared (p <
0.0001). b (i) Representative images of jejunal sections at 3.5 days post-AIR demonstrating the presence of GFP+Lgr5+ ISC (indicated with arrow)
in Lgr5/GFP-IRES-Cre-ERT2 knock-in mice receiving BCN057 at 24 h post-AIR. Note the absence of GFP+ cells in mice receiving only AIR. (ii) The
number of Lgr5+GFP+ ISC per crypt in jejunal sections from Lgr5/GFP-IRES-Cre-ERT2 knock-in mice exposed to irradiation and then treated with
BCN057. The number of Lgr5+ cells are significantly higher in BCN057-treated irradiated mice compared with AIR controls (*p < 0.0001).
Unirradiated mice receiving BCN057 also demonstrated a higher number of Lgr5+ cells at the crypt base compared with AIR controls (*p < 0.0002;
unpaired t test, two-tailed). (iii) Representative images of jejunal sections demonstrating the presence of Ki67 in Lgr5+GFP+ ISC localized in the
crypt base. Representative images from the single fluorescence channel showed localization of Lgr5+GFP+ cells (green, indicated with yellow
arrow head) and Ki67+ cells (red, indicated with green arrow head). Cells that are double-positive for Ki67 and GFP are indicated with white
arrows in both the single fluorescence channel and in the merged image. (iv) The percentage of Lgr5+GFP+/Ki67+ in jejunal sections from Lgr5/
GFP-IRES-Cre-ERT2 knock-in mice exposed to irradiation and then treated with BCN057. The percentage of Lgr5+GFP+/Ki67+ cells are significantly
higher in BCN057-treated irradiated mice compared with AIR controls (*p < 0.0001). Unirradiated mice receiving BCN057 also demonstrated a
higher percentage of Lgr5+GFP+/Ki67+ cells at the crypt base compared with AIR controls (*p < 0.0003; unpaired t test, two-tailed). c (i)
Representative image of jejunal sections at 48 h post-AIR demonstrating the presence of TUNEL-positive apoptotic cells at the crypt base in mice
exposed to AIR. However, mice receiving the BCN057 treatment at 24 h post-AIR did not show any TUNEL-positive cells. (ii) Percentage of
TUNELpositive apoptotic cells in jejunal sections from mice exposed to AIR. The percentage of TUNEL-positive cells are significantly higher in the AIR
group compared with mice receiving BCN057 at 24 h post-AIR (p < 0.0002). d (i) Schematic representation of the treatment schema for lineage
tracing assay in Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice. (ii) Confocal microscopic images (×40) of the jejunal section from
Lgr5eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice. tdTomato (tdT)-positive cells are shown in red; Lgr5+GFP+ cells are shown in green. Nuclei are
stained with DAPI (blue). Marked expansion of tdT-positive red cells above the +4 position (representing transit amplifying cells) were noted with
BCN057 treatment. Please note the presence of yellow cells at the bottom of the crypt representing tdT-positive and GFP+Lgr5+ ISC (yellow due
to red + green). (iii) Confocal microscopic images (×10) of the jejunal section from Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice. Please
note the presence of villi containing red tdT-positive cells (regenerative villi) in unirradiated controls or BCN057-treated mice. In the absence of
BCN057 treatment, no tdT-positive cells were noted in irradiated mice jejunum. (iv) The number of regenerative villi. Irradiated mice receiving
BCN057 showed a significantly higher number of regenerative villi compared with irradiated controls (*p < 0.002). Un-irradiated mice receiving
BCN057 also demonstrated a higher number of regenerative villi compared with irradiated controls (*p < 0.0006)
and improved the
positive ISC (Fig. 4c).
BCN057 mitigates radiation injury in human colonic epithelium-derived organoids
To examine the effect of BCN057 on human intestinal
epithelial tissue, surgical specimens collected from
normal colon at least 10 cm apart from the malignant site
were used to develop an ex vivo crypt organoid. At a
dose level of 8 Gy all the budding crypts have
disappeared in the organoids. However, organoids treated
with BCN057 at 1 h post-irradiation had budding crypts
with complete restitution of organoid structure (Fig. 4d).
Quantification of the budding crypt-like structure
demonstrated a higher number of budding crypts/total crypt
ratio with BCN057 treatment compared with irradiated
controls, suggesting improvement in growth and
proliferation in organoids receiving BCN057 (Fig. 4e).
BCN057-treated organoids demonstrated an increase in
mRNA levels of intestinal stem cell-specific markers
such as LGR5, K19, CD44, and HES1 (p < 0.001, p <
0.005, p < 0.001, and p < 0.004, respectively) compared
with irradiated untreated organoids (Table 2). We have
also evaluated the effect of BCN057 on mRNA levels of
β-catenin target genes in human colonic organoids.
Organoids exposed to irradiation and then treated with
BCN057 demonstrated a several-fold increase in
expression of β-catenin target genes, indicating activation of
Wnt-β-catenin signaling (Fig. 4f ).
BCN057 does not protect malignant tissue from radiation
BCN057 was first examined in the National Cancer
Institute (NCI) 60 Cancer Cell Line platform [
includes the colon cancer cell lines HCT166, HCT15,
COLO205, KM12, HT29, SW-620, and HCC-2998.
Several of these cell lines are known to be positive for
dysregulation of the Wnt/β-catenin signaling pathway.
None of these cells showed any proliferative response to
BCN057 treatment in our study (Additional file 6: Table
S3). We also examined the effect of BCN057 on human
colonic tumor-derived organoids exposed to irradiation.
Surgical specimens of malignant colonic tissue were
obtained from the same individual from whom we
collected non-malignant tissue. Treatment with BCN057
(10 μM) at 1 h post-radiation exposure (8 Gy) did not
rescue organoids from radiation toxicity. All the budding
crypts disappeared within 72–96 h post-irradiation in
both BCN057-treated and untreated organoids (Fig. 5a).
Moreover, there was no difference in budding crypts/
total crypt ratio in BCN057-treated and untreated
organoids (Fig. 5a), suggesting that BCN057 does not
Table 2 Intestinal stem cell-specific marker gene expression
have a radioprotective effect in malignant tissue-derived
Subcutaneous tumors were developed by injecting
MC38 colon cancer cells into the mice flanks (Fig. 5b).
Mice with palpable subcutaneous tumor were exposed
to AIR (15 Gy) followed by treatment with or without
eight doses of BCN057. AIR alone produced 100%
mortality of animals within 12 days (Fig. 5b) of radiation
exposure. Therefore, the tumor growth could not be
studied in these mice beyond day 12. Compared with
AIR alone, mice receiving BCN057 post-AIR showed a
significant improvement in survival time (Fig. 5b). In the
AIR + BCN057 treated group, 60% of mice survived
beyond 20 days post-radiation exposure (Fig. 5b) and
showed significant tumor growth retardation compared
with untreated and non-irradiated controls (p < 0.0004;
n = 10; Fig. 5b). MC38 colon cancer cells are positive for
Wnt-β-catenin signaling [
analysis of MC38 tumor tissue from untreated
nonirradiated mice showed β-catenin-positive nuclei
(Additional file 7: Figure S3). However, tumor tissue
from BCN057 and AIR + BCN057 did not show the
presence of β-catenin-positive nuclei (Additional file 7:
Figure S3) indicating that BCN057 failed to activate
β-catenin signaling in the MC38 colon tumor.
These results clearly suggest that BCN057 has no
protective effect on tumors during radiation therapy and
therefore BCN057 may have use in combination therapy to
minimize the toxic side-effects of abdominal radiotherapy.
A higher self-renewal rate of ISC makes intestinal
epithelium very sensitive to high doses of irradiation.
Therefore, it is critical to mitigate radiation-induced
gastrointestinal injury to overcome acute radiation
syndrome. The present study indicates that treatment with
BCN057 starting at 24 h post-abdominal irradiation
induces repair and regeneration of intestinal epithelium
and improves survival against lethal doses of irradiation.
Moreover, BCN057 also rescued mice from RIGS when
40% BM was exposed along with radiation to the
intestine, which indicates that BCN057 can partially
substitute the radioprotective role of the BM in GI injury.
BCN057 promotes the regenerative response of
Lgr5-positive ISC to mitigate RIGS. These data have been replicated
in intestinal organoid cultures from Lgr5-EGFP-Cre-ERT2
mice designed to examine the role of Lgr5-positive ISC in
stem cell regeneration. This study along with the intestinal
organoid culture developed from patient-derived
nonmalignant colonic epithelium demonstrated that BCN057
induces ISC regeneration. Intestinal epithelial homeostasis
and regeneration depends upon Wnt-β-catenin signaling.
BCN057 is a small molecular agent which activates
Wnt-βcatenin signaling as demonstrated in TCF/LEF luciferase
assay as well as in irradiated crypt where it induces the
nuclear localization of β catenin. These observations clearly
indicate that BCN057 is an agonist of Wnt-β-catenin
signaling, and it can rescue the normal epithelial pathology
with a resultant survival of mice; this suggests that BCN057
might be an effective mitigator of RIGS.
Intestinal crypts have two types of stem cells.
Bmi1positive ISC are long-lived, label-retaining stem cells
present at the +4 position of the crypt base [
Bmi1-positive ISC interconvert with more rapidly
proliferating Lgr5-positive stem cells known as crypt base
columnar cells (CBCs) [
] that express markers including
Lgr5, Olfm4, Lrig1, and Ascl2 [
]. These CBCs are
also active stem cells and are primarily involved in
selfrenewal and differentiation. Our previous observation
demonstrated that activation of these stem cells
postradiotherapy is critical for repair and regeneration of
intestinal epithelium [
]. We have also demonstrated that
supplementation of Wnt ligands is critical for activating
Wnt-β-catenin signaling and rescuing these stem cells
following radiation injury [
]. In the present study, we
have demonstrated that BCN057 as a small molecular
agent activates Wnt-β-catenin signaling and rescues
these ISC from radiation toxicity.
Identification of a suitable animal model to study RIGS
and test the candidate agents as mitigators is still a major
challenge as the mechanisms underlying this symptom may
vary between models. Thus far, multiple animal models
have been used to study RIGS, including mice, mini-pigs,
canine and non-human primates (NHPs). However, we are
not aware of reports describing the testing of
radiomitigators in healthy human tissues to re-affirm the
translational relevance of the mechanism from animal studies. In
this study, we have used ex-vivo organoids developed from
colonic epithelium from human donors and demonstrated
that BCN057 induces human colonic stem cell growth and
proliferation following radiation. Intestinal organoids retain
the crypt villus structure along with all the major cell types
of the intestinal epithelium, including ISC, paneth cells,
enteroendocrine cells, and enterocytes [
organoid growth primarily depends on the presence of stem
]. Therefore, this organoid system provides a
perfect platform to examine and validate the efficacy of any
potential GI radio-mitigators in human tissue and to
validate mechanistically the relationship between animal
data and their translational value to human tissue.
BCN057 does not have any radioprotective effect on
organoids derived from human colon tumors or in
mouse subcutaneous tumors. This is consistent with an
NCI 60 Cancer Cell Line screen of colon, breast, prostate,
ovary, lung, kidney, central nervous system, pancreas, skin
and blood, several of which are Wnt-positive cell lines
where no enhancement of growth was noted with the
application of BCN057. However, some cell lines showed
significantly inhibited growth in the presence of the drug.
We have observed that BCN057 treatment reduces the
proliferation of colon cancer cell lines HCT166, HCT15,
COLO205, KM12, HT29, SW-620, and HCC-2998
(Additional file 6: Table S3) where several of these cell
lines have Wnt signaling upregulated [
Cancer cells in general are more resistant to apoptosis
by acquiring mutations in genes, such as p53, or inducing
anti-apoptotic genes [
]. Upon genotoxic stress, normal
cells with intact p53 undergo apoptotic cell death and can
be rescued by inhibition of apoptosis, whereas tumor cells
are non-responsive to inhibition of apoptosis and more
prone to senescence [
]. Thus, the anti-apoptotic role of
BCN057 could protect the ISC by reducing
radiationinduced apoptosis but it does not appear to affect the
colon cancer cells, which undergo cell death, or
senescence, after radiation exposure. Therefore, during
radiation therapy, systemic use of BCN057 may be useful in
patients undergoing abdominal irradiation for GI
malignancies. Clinically, radiation enteritis is a response to the
damage to the small and large bowel that occurs with
radiation therapy to the pelvic, abdominal, or rectal areas, with
about 15–20% of patients requiring an altered therapeutic
]. Chronic conditions due to radiation enteritis
can present within 1.5 to 6 years after radiotherapy, with
some reported up to 30 years later [
], and upwards of
90% of the patients who receive pelvic radiotherapy
develop a permanent change in their bowel habit [
to half of these patients describe their quality of life as
being adversely affected by a variety of GI symptoms [
] with a significant portion scoring the effect as
moderate or severe [
]. Loss of ISC due to acute radiation injury
impairs the repair process and promotes the radiation
enteritis at later time points [
]. Our data suggest that
BCN057 may inhibit radiation enteritis by mitigating the
acute effects of radiation with activation of the repair
Several growth factors and cytokines, such as KGF,
transforming growth factor (TGF)β, tumor necrosis
factor (TNF)α, prostaglandin (PG)E2, and interleukin
], including Wnt agonist R-spondin1, protect
the intestine from radiation injury when applied before
radiation exposure. However, so far there are no reports
on growth factors that can mitigate intestinal injury
when applied after radiation exposure. To our
knowledge, this is the first demonstration of the salutary
effect of BCN057 in the context of radiation injury of the
intestine where it mitigates RIGS when applied 24 h
after exposure to lethal doses of radiation.
The present study demonstrates that BCN057 treatment
at 24 h post-irradiation exposure rescues Lgr5-positive
ISC from radiation-induced loss and promotes epithelial
repair and regeneration to mitigate RIGS. However,
BCN057 did not have any radioprotective effect in
abdominal tumors and therefore could improve the
therapeutic efficacy of abdominal radiotherapy.
Additional file 1: Supplement method. Detailed methods of
histopathology, immunohistochemistry to determine crypt proliferation
rate, β-catenin immunohistochemistry of mouse jejunum, and real-time
PCR are described. (DOC 27 kb)
Additional file 2: Table S1. β-catenin target gene specific real-time PCR
primers (mouse). (DOC 31 kb)
Additional file 3: Table S2. Stem cell marker genes and primer
sequences (human). (DOC 29 kb)
Additional file 4: Figure S1. BCN057 treatment at 72 h post-irradiation
could not induce repair of intestinal epithelium. H&E stained
representative section of jejunum from C57BL/6 mice treated with BCN057 at 72 h
post-AIR. Note the significant damage to intestinal epithelium in both
BCN057-treated and untreated mice. (DOC 306 kb)
Additional file 5: Figure S2. Confocal microscopic images (×40) of the
jejunal section from Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice.
tdTomato (tdT)-positive cells are shown in red; Lgr5-positive/GFP-positive
cells are shown in green. Nuclei are stained with DAPI (blue). (DOC 388 kb)
Additional file 6: Table S3. Cancer cell proliferation in the presence of
BCN057 10 μM. Table of cells tested at 10 μM BCN057 in neat DMSO on
the indicated cell lines representing various cancer types. Values are
represented as a percentage of control growth which is the vehicle alone
(DMSO). (DOC 26 kb)
Additional file 7: Figure S3. Representative microscopic images (×20
magnification) of MC38 colon tumor sections immunostained with
antiβ-catenin antibody to determine β-catenin nuclear localization. Please
note the absence of β-catenin-positive nuclei in the AIR + BCN057 group.
BCN057 treatment in non-irradiated tumors also did not demonstrate
βcateninpositive nucleis. (DOC 461 kb)
AIR: Abdominal irradiation; ARS: Acute radiation syndrome; BM: Bone
marrow; CBC: Crypt base columnar cell; EV: Extracellular vesicle;
GI: Gastrointestinal tract; H&E: Hematoxylin and eosin; ISC: Intestinal stem
cells; LiCl: Lithium chloride; NHP: Non-human primate; PBI: Partial body
irradiation; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction;
PK: Pharmacokinetics; qPCR: Quantitative polymerase chain reaction;
RIGS: Radiation-induced gastrointestinal syndrome; RSPO1: R-spondin 1;
s.c.: Subcutaneous; TUNEL: TdT-mediated digoxigenin-labeled dUTP
The authors acknowledge the Biorepository core facility, histopathology
core facility and confocal microscopy core facility of KUMC for their help and
This work was supported by grants from K01DK096032, ACS IRG Pilot, KUCC
support grant, and KUCC pilot (all to SS). The authors thank the Biomedical
Advanced Research and Development Authority (BARDA) for their support
under T01EP130002-01-00 for BCN057 formulations and development.
Availability of data and materials
The authors declare that all data supporting the findings of this study are
available within the article and its Supplementary Information files
(Additional files) or from the corresponding author on reasonable request.
PB: Conceived and designed the experiments, performed the experiments,
analyzed the data, wrote the paper; AN: Contributed reagents, analyzed the
data, wrote the paper; AH: Wrote the paper; SS: Contributed reagents/
materials/analysis tools, conceived and designed the experiments, wrote the
paper. All authors read and approved the final manuscript.
The present study is not considered as human subject research under HHS
regulations at 45 CFR Part 46 and University of Kansas Human Subject
Committee as we have used de-identified specimens and no living individual
is involved as a study subject.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Leibowitz BJ , Wei L , Zhang L , Ping X , Epperly M , Greenberger J , Cheng T , Yu J . Ionizing irradiation induces acute haematopoietic syndrome and gastrointestinal syndrome independently in mice . Nat Commun . 2014 ; 5 : 3494 .
2. Saha S , Bhanja P , Liu L , Alfieri AA , Yu D , Kandimalla ER , Agrawal S , Guha C. TLR9 agonist protects mice from radiation-induced gastrointestinal syndrome . PLoS ONE . 2012 ; 7 ( 1 ): e29357 .
3. Saha S , Aranda E , Hayakawa Y , Bhanja P , Atay S , Brodin NP , Li J , Asfaha S , Liu L , Tailor Y , et al. Macrophage-derived extracellular vesicle-packaged Wnts rescue intestinal stem cells and enhance survival after radiation injury . Nat Commun . 2016 ; 7 : 13096 .
4. Saha S , Bhanja P , Kabarriti R , Liu L , Alfieri AA , Guha C . Bone marrow stromal cell transplantation mitigates radiation-induced gastrointestinal syndrome in mice . PLoS ONE . 2011 ; 6 ( 9 ): e24072 .
5. Rios CI , Cassatt DR , Dicarlo AL , Macchiarini F , Ramakrishnan N , Norman MK , Maidment BW . Building the strategic national stockpile through the NIAID Radiation Nuclear Countermeasures Program . Drug Dev Res . 2014 ; 75 ( 1 ): 23 - 8 .
6. Clevers H , Nusse R. Wnt /beta-catenin signaling and disease . Cell . 2012 ; 149 ( 6 ): 1192 - 205 .
7. Bhanja P , Saha S , Kabarriti R , Liu L , Roy-Chowdhury N , Roy-Chowdhury J , Sellers RS , Alfieri AA , Guha C . Protective role of R-spondin1, an intestinal stem cell growth factor, against radiation-induced gastrointestinal syndrome in mice . PLoS ONE . 2009 ; 4 ( 11 ): e8014 .
8. Kim KA , Kakitani M , Zhao J , Oshima T , Tang T , Binnerts M , Liu Y , Boyle B , Park E , Emtage P , et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium . Science . 2005 ; 309 ( 5738 ): 1256 - 9 .
9. Zhao J , Kim KA , De Vera J , Palencia S , Wagle M , Abo A . R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/beta-catenin pathway . Proc Natl Acad Sci U S A . 2009 ; 106 ( 7 ): 2331 - 6 .
10. Zhao J , de Vera J , Narushima S , Beck EX , Palencia S , Shinkawa P , Kim KA , Liu Y , Levy MD , Berg DJ , et al. R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice . Gastroenterology . 2007 ; 132 ( 4 ): 1331 - 43 .
11. Yan KS , Chia LA , Li X , Ootani A , Su J , Lee JY , Su N , Luo Y , Heilshorn SC , Amieva MR , et al. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations . Proc Natl Acad Sci U S A . 2012 ; 109 ( 2 ): 466 - 71 .
12. de Lau W , Peng WC , Gros P , Clevers H. The R-spondin/ Lgr5/Rnf43 module: regulator of Wnt signal strength . Genes Dev . 2014 ; 28 ( 4 ): 305 - 16 .
13. Janda CY , Waghray D , Levin AM , Thomas C , Garcia KC . Structural basis of Wnt recognition by Frizzled . Science . 2012 ; 337 ( 6090 ): 59 - 64 .
14. MacVittie TJ , Farese AM , Bennett A , Gelfond D , Shea-Donohue T , Tudor G , Booth C , McFarland E , Jackson 3rd W. The acute gastrointestinal subsyndrome of the acute radiation syndrome: a rhesus macaque model . Health Phys . 2012 ; 103 ( 4 ): 411 - 26 .
15. Potten CS , Booth C , Pritchard DM . The intestinal epithelial stem cell: the mucosal governor . Int J Exp Pathol . 1997 ; 78 ( 4 ): 219 - 43 .
16. Barker N , van den Born M. Detection of beta-catenin localization by immunohistochemistry . Methods Mol Biol . 2008 ; 468 : 91 - 8 .
17. Karhausen J , Furuta GT , Tomaszewski JE , Johnson RS , Colgan SP , Haase VH . Epithelial hypoxia-inducible factor-1 is protective in murine experimental colitis . J Clin Invest . 2004 ; 114 ( 8 ): 1098 - 106 .
18. Sato T , van Es JH , Snippert HJ , Stange DE , Vries RG , van den Born M , Barker N , Shroyer NF , van de Wetering M , Clevers H . Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts . Nature . 2011 ; 469 ( 7330 ): 415 - 8 .
19. Sato T , Vries RG , Snippert HJ , van de Wetering M , Barker N , Stange DE , van Es JH , Abo A , Kujala P , Peters PJ , et al. Single Lgr5 stem cells build cryptvillus structures in vitro without a mesenchymal niche . Nature . 2009 ; 459 ( 7244 ): 262 - 5 .
20. Barker N , Huch M , Kujala P , van de Wetering M , Snippert HJ , van Es JH , Sato T , Stange DE , Begthel H , van den Born M , et al. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro . Cell Stem Cell . 2010 ; 6 ( 1 ): 25 - 36 .
21. Muzumdar MD , Tasic B , Miyamichi K , Li L , Luo L . A global doublefluorescent Cre reporter mouse . Genesis . 2007 ; 45 ( 9 ): 593 - 605 .
22. Madisen L , Zwingman TA , Sunkin SM , Oh SW , Zariwala HA , Gu H , Ng LL , Palmiter RD , Hawrylycz MJ , Jones AR , et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain . Nat Neurosci . 2010 ; 13 ( 1 ): 133 - 40 .
23. Park ES , Rabinovsky R , Carey M , Hennessy BT , Agarwal R , Liu W , Ju Z , Deng W , Lu Y , Woo HG , et al. Integrative analysis of proteomic signatures, mutations, and drug responsiveness in the NCI 60 cancer cell line set . Mol Cancer Ther . 2010 ; 9 ( 2 ): 257 - 67 .
24. Monks A , Scudiero D , Skehan P , Shoemaker R , Paull K , Vistica D , Hose C , Langley J , Cronise P , Vaigro-Wolff A , et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines . J Natl Cancer Inst . 1991 ; 83 ( 11 ): 757 - 66 .
25. Alley MC , Scudiero DA , Monks A , Hursey ML , Czerwinski MJ , Fine DL , Abbott BJ , Mayo JG , Shoemaker RH , Boyd MR . Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay . Cancer Res . 1988 ; 48 ( 3 ): 589 - 601 .
26. Rubinstein LV , Shoemaker RH , Paull KD , Simon RM , Tosini S , Skehan P , Scudiero DA , Monks A , Boyd MR . Comparison of in vitro anticancer-drugscreening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines . J Natl Cancer Inst . 1990 ; 82 ( 13 ): 1113 - 8 .
27. Skehan P , Storeng R , Scudiero D , Monks A , McMahon J , Vistica D , Warren JT , Bokesch H , Kenney S , Boyd MR . New colorimetric cytotoxicity assay for anticancer-drug screening . J Natl Cancer Inst . 1990 ; 82 ( 13 ): 1107 - 12 .
28. Dawson PA , Huxley S , Gardiner B , Tran T , McAuley JL , Grimmond S , McGuckin MA , Markovich D. Reduced mucin sulfonation and impaired intestinal barrier function in the hyposulfataemic NaS1 null mouse . Gut . 2009 ; 58 ( 7 ): 910 - 9 .
29. Asfaha S , Hayakawa Y , Muley A , Stokes S , Graham TA , Ericksen RE , Westphalen CB , von Burstin J , Mastracci TL , Worthley DL , et al. Krt19(+ )/Lgr5(-) cells are radioresistant cancer-initiating stem cells in the colon and intestine . Cell Stem Cell . 2015 ; 16 ( 6 ): 627 - 38 .
30. Klose J , Eissele J , Volz C , Schmitt S , Ritter A , Ying S , Schmidt T , Heger U , Schneider M , Ulrich A . Salinomycin inhibits metastatic colorectal cancer growth and interferes with Wnt/beta-catenin signaling in CD133+ human colorectal cancer cells . BMC Cancer . 2016 ; 16 ( 1 ): 896 .
31. Barker N , van de Wetering M , Clevers H. The intestinal stem cell . Genes Dev . 2008 ; 22 ( 14 ): 1856 - 64 .
32. Wong VW , Stange DE , Page ME , Buczacki S , Wabik A , Itami S , van de Wetering M , Poulsom R , Wright NA , Trotter MW , et al. Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling . Nat Cell Biol . 2012 ; 14 ( 4 ): 401 - 8 .
33. Munoz J , Stange DE , Schepers AG , van de Wetering M , Koo BK , Itzkovitz S , Volckmann R , Kung KS , Koster J , Radulescu S , et al. The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cell markers . EMBO J . 2012 ; 31 ( 14 ): 3079 - 91 .
34. van der Flier LG , Haegebarth A , Stange DE , van de Wetering M , Clevers H. OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells . Gastroenterology . 2009 ; 137 ( 1 ): 15 - 7 .
35. Holcombe RF , Marsh JL , Waterman ML , Lin F , Milovanovic T , Truong T. Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma . Mol Pathol . 2002 ; 55 ( 4 ): 220 - 6 .
36. Tavana O , Benjamin CL , Puebla-Osorio N , Sang M , Ullrich SE , Ananthaswamy HN , Zhu C . Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity, enhanced immunosuppression and cellular senescence . Cell Cycle . 2010 ; 9 ( 16 ): 3328 - 36 .
37. Demidenko ZN , Vivo C , Halicka HD , Li CJ , Bhalla K , Broude EV , Blagosklonny MV . Pharmacological induction of Hsp70 protects apoptosis-prone cells from doxorubicin: comparison with caspase-inhibitor- and cycle-arrestmediated cytoprotection . Cell Death Differ . 2006 ; 13 ( 9 ): 1434 - 41 .
38. Do NL , Nagle D , Poylin VY . Radiation proctitis: current strategies in management . Gastroenterol Res Pract . 2011 ; 2011 : 917941 .
39. Kountouras J , Zavos C . Recent advances in the management of radiation colitis . World J Gastroenterol . 2008 ; 14 ( 48 ): 7289 - 301 .
40. Olopade FA , Norman A , Blake P , Dearnaley DP , Harrington KJ , Khoo V , Tait D , Hackett C , Andreyev HJ . A modified Inflammatory Bowel Disease questionnaire and the Vaizey Incontinence questionnaire are simple ways to identify patients with significant gastrointestinal symptoms after pelvic radiotherapy . Br J Cancer . 2005 ; 92 ( 9 ): 1663 - 70 .
41. Widmark A , Fransson P , Tavelin B . Self-assessment questionnaire for evaluating urinary and intestinal late side effects after pelvic radiotherapy in patients with prostate cancer compared with an age-matched control population . Cancer . 1994 ; 74 ( 9 ): 2520 - 32 .
42. Crook J , Esche B , Futter N. Effect of pelvic radiotherapy for prostate cancer on bowel, bladder, and sexual function: the patient's perspective . Urology . 1996 ; 47 ( 3 ): 387 - 94 .
43. Gami B , Harrington K , Blake P , Dearnaley D , Tait D , Davies J , Norman AR , Andreyev HJ . How patients manage gastrointestinal symptoms after pelvic radiotherapy . Aliment Pharmacol Ther . 2003 ; 18 ( 10 ): 987 - 94 .
44. Andreyev HJ . Gastrointestinal problems after pelvic radiotherapy: the past, the present and the future . Clin Oncol (R Coll Radiol ) . 2007 ; 19 ( 10 ): 790 - 9 .
45. Booth C , Tudor G , Tonge N , Shea-Donohue T , MacVittie TJ. Evidence of delayed gastrointestinal syndrome in high-dose irradiated mice . Health Phys . 2012 ; 103 ( 4 ): 400 - 10 .
46. Hauer-Jensen M , Denham JW , Andreyev HJ . Radiation enteropathy-pathogenesis, treatment and prevention . Nat Rev Gastroenterol Hepatol . 2014 ; 11 ( 8 ): 470 - 9 .
47. Hanson WR , Thomas C. 16 ,16 -dimethyl prostaglandin E2 increases survival of murine intestinal stem cells when given before photon radiation . Radiat Res . 1983 ; 96 ( 2 ): 393 - 8 .
48. Khan WB , Shui C , Ning S , Knox SJ . Enhancement of murine intestinal stem cell survival after irradiation by keratinocyte growth factor . Radiat Res . 1997 ; 148 ( 3 ): 248 - 53 .
49. Potten CS , Booth D , Haley JD . Pretreatment with transforming growth factor beta-3 protects small intestinal stem cells against radiation damage in vivo . Br J Cancer . 1997 ; 75 ( 10 ): 1454 - 9 .