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Organoids and metastatic
orthotopic mouse model for
mismatch repair-deficient
colorectal cancer
Yurong Song
1
*, Travis D. Kerr
1
, Chelsea Sanders
2
, Lisheng Dai
1
,
Shaneen S. Baxter
1
, Brandon Somerville
1
, Ryan N. Baugher
3
,
Stephanie D. Mellott
3
, Todd B. Young
3
, Heidi E. Lawhorn
3
,
Teri M. Plona
3
, Bingfang Xu
4
, Lei Wei
5
, Qiang Hu
5
, Song Liu
5
,
Alan Hutson
5
, Baktiar Karim
6
, Sandra Burkett
7
,
Simone Difilippantonio
2
, Ligia Pinto
1
, Johannes Gebert
8
,
Matthias Kloor
8
, Steven M. Lipkin
9
, Shizuko Sei
10
and Robert H. Shoemaker
10
1
Frederick National Laboratory for Cancer Research, Vaccine, Immunity, and Cancer Directorate,
Frederick, MD, United States,
2
Frederick National Laboratory for Cancer Research, Laboratory Animal
Sciences Program, Frederick, MD, United States,
3
Frederick National Laboratory for Cancer Research,
Clinical Laboratory Improvement Amendments (CLIA) Molecular Diagnostics Laboratory, Frederick,
MD, United States,
4
Frederick National Laboratory for Cancer Research, Genomics Laboratory, Frederick,
MD, United States,
5
Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer
Center, Buffalo, NY, United States,
6
Molecular Histopathology Laboratory, Frederick National Laboratory
for Cancer Research, Frederick, MD, United States,
7
Molecular Cytogenetics Core Facility, National
Cancer Institute, Frederick, MD, United States,
8
Department of Applied Tumor Biology, Institute of
Pathology, University of Heidelberg, Heidelberg, Germany,
9
Department of Medicine, Weill Cornell
Medical College, Cornell University, New York, NY, United States,
10
Chemopreventive Agent
Development Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda,
MD, United States
Background: Genome integrity is essential for the survival of an organism. DNA
mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical
role in the DNA damage response pathway for genome integrity maintenance.
Germline mutations of MMR genes can lead to Lynch syndrome or constitutional
mismatch repair deficiency syndrome, resulting in an increased lifetime risk of
developing cancer characterized by high microsatellite instability (MSI-H) and
high mutation burden. Although immunotherapy has been approved for MMR-
deficient (MMRd) cancer patients, the overall response rate needs to be improved
and other management options are needed.
Methods: To better understand the biology of MMRd cancers, elucidate the
resistance mechanisms to immune modulation, and develop vaccines and
therapeutic testing platforms for this high-risk population, we generated
organoids and an orthotopic mouse model from intestine tumors developed in
a Msh2-deficient mouse model, and followed with a detailed characterization.
Results: The organoids were shown to be of epithelial origin with stem cell features,
to have a high frameshift mutation frequency with MSI-H and chromosome
Frontiers in Oncology frontiersin.org01
OPEN ACCESS
EDITED BY
Kumar Sanjiv,
Karolinska Institutet (KI), Sweden
REVIEWED BY
Michela Pozzobon,
University of Padua, Italy
Weilin Li,
NIH, United States
Todd M. Pitts,
University of Colorado Anschutz Medical
Campus, United States
Qingfei Pan,
St. Jude Children’s Research Hospital,
United States
*CORRESPONDENCE
Yurong Song
songy3@mail.nih.gov
RECEIVED 18 May 2023
ACCEPTED 21 August 2023
PUBLISHED 08 September 2023
CITATION
Song Y, Kerr TD, Sanders C, Dai L,
Baxter SS, Somerville B, Baugher RN,
Mellott SD, Young TB, Lawhorn HE,
Plona TM, Xu B, Wei L, Hu Q, Liu S,
Hutson A, Karim B, Burkett S,
Difilippantonio S, Pinto L, Gebert J,
Kloor M, Lipkin SM, Sei S and
Shoemaker RH (2023) Organoids
and metastatic orthotopic mouse
model for mismatch repair-deficient
colorectal cancer.
Front. Oncol. 13:1223915.
doi: 10.3389/fonc.2023.1223915
COPYRIGHT
© 2023 Song, Kerr, Sanders, Dai, Baxter,
Somerville, Baugher,Mellott,Young,
Lawhorn, Plona, Xu, Wei, Hu, Liu, Hutson,
Karim, Burkett, Difilippantonio, Pinto, Gebert,
Kloor, Lipkin, Sei and Shoemaker. This is an
open-access article distributed under the
terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that
the original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
TYPE Original Research
PUBLISHED 08 September 2023
DOI 10.3389/fonc.2023.1223915
instability, and intra- and inter-tumor heterogeneity. An orthotopic model using
intra-cecal implantation of tumor fragments derived from organoids showed
progressive tumor growth, resulting in the development of adenocarcinomas
mixed with mucinous features and distant metastasis in liver and lymph node.
Conclusions: The established organoids with characteristics of MSI-H cancers
can be used to study MMRd cancer biology. The orthotopic model, with its
distant metastasis and expressing frameshift peptides, is suitable for evaluating
the efficacy of neoantigen-based vaccines or anticancer drugs in combination
with other therapies.
KEYWORDS
mismatch repair deficiency, Lynch syndrome, microsatellite instability, chromosome
instability, MSH2, organoid, mouse model, colorectal cancer
1 Introduction
Genome integrity is maintained by multiple pathways. Among
these, the accuracy of DNA replication relies on DNA mismatch
repair (MMR) genes, including MLH1, MSH2, MSH6, and PMS2.
Germline deficiency in MMR genes (MMRd) leads to Lynch
syndrome (LS) via monoallelic mutation or constitutional
mismatch repair deficiency syndrome (CMMRD) via biallelic
mutation. Individuals with MMRd usually have a high mutation
burden due to an inability to repair errors that occur during DNA
replication, especially in coding microsatellite regions. The
accumulation of these mutations in the genome leads to a
genomic state of high microsatellite instability (MSI-H). Thus,
carriers with pathogenic MMR variants usually have an increased
lifetime risk for developing various cancers with early cancer onset
(e.g., colorectal cancer [CRC] or hereditary non-polyposis
colorectal cancer [HNPCC], endometrial cancer, gastric cancer,
and ovarian cancer mainly for LS, and brain cancer,
gastrointestinal cancer, and lymphomas for CMMRD) (1,2). In
addition, somatic mutations in MMR genes can also lead to MSI-H
tumors (3–5). The disease penetrance and age of onset vary among
the four deficient MMR genes. MLH1 and MSH2 mutations are
mainly implicated in LS, while PMS2 and MSH6 mutations are
predominantly responsible for CMMRD. The incomplete disease
penetrance suggests that other genetic or epigenetic factors are also
involved in the disease etiology.
Recent advances in immuno-oncology have supported
tremendous progress in the treatment of MMRd/MSI-H cancers
by engaging patients’own immune system. To date, FDA has
approved three immune checkpoint inhibitors for treatment of
MSI-H metastatic CRC (6–8). In studies using the anti-PD-1
antibody pembrolizumab in heavily pretreated MMRd/MSI-H
patients, the overall response rate was 40% and 71% in MSI-H
CRC and non-CRC cancers, respectively (9), while a 53% objective
response rate was observed across tumor types (10). In a pooled
phase 2 (KEYNOTE-158, KEYNOTE-164, and KEYNOTE-051)
trials analysis, the objective response rate was 33.3%. Treatment
with another anti-PD-1 antibody, nivolumab, showed a similar
objective response rate (31.3%) in chemotherapy-refractory
MMRd/MSI-H CRC patients, with 14.3 months of median
progression-free survival. The most recent phase 3 study showed
an objective response of 45% with pembrolizumab used as a first-
line therapy in MMRd/MSI-H/metastatic CRC patients, vs. 33%
with chemotherapy (11). The combination therapy using
nivolumab and the anti-CTLA4 antibody ipilimumab showed an
increased overall response rate of 55% in advanced MMRd/MSI-H
CRC patients (12). Responders had marked expansion of T cells
targeting frameshift neopeptides (10), which contributes to overall
disease control and tumor suppression. These frameshift
neoantigens have been the basis for immune-based therapies and
have been actively explored as preventative cancer vaccine antigens
for individuals at risk for MMRd/MSI-H cancers (13–16). With the
best overall response rate of 55% in advanced MMRd/MSI-H CRC
patients (12), a model system that recapitulates human disease is
urgently needed to better understand the disease biology and
heterogeneity, elucidate resistance mechanisms, and test new
treatment strategies and preventive approaches preclinically.
Established cell lines, organoids, and patient-derived xenograft
(PDX) models from LS CRC patients are of great value to the
research community. However, they cannot be used to develop a
Abbreviations: B2M, b-2 microglobulin; ChgA, Chromogranin A; CI, confidence
interval; CIN, chromosome instability; CMMRD, constitutional mismatch repair
deficiency; CRC, colorectal cancer; dpi, days post implantation; FFPE, formalin-
fixed, paraffin-embedded; FSM, frameshift mutation; FSP, frameshift peptides;
H&E, hematoxylin and eosin; ICB, immune checkpoint blockade; IHC,
immunohistochemical staining; LS, Lynch syndrome; MMRd, mismatch repair
deficiency; MNR, mononucleotide repeat; MRI, magnetic resonance imaging;
MSI-H, microsatellite instability high; MSS, microsatellite-stable; NBF, neutral-
buffered formalin; NGS, next-generation sequencing; NSG, NOD SCID gamma;
SFM, serum-free media; SKY, spectral karyotyping; s.c., subcutaneously; TA,
transit amplifying; TUNEL, terminal deoxynucleotidyl transferase dUTP nick
end labeling; US, ultrasound imaging; VAF, variant allele frequency; VCMsh2,
Msh2
LoxP/LoxP
;Villin-Cre; WT, wildtype.
Song et al. 10.3389/fonc.2023.1223915
Frontiers in Oncology frontiersin.org02
mouse model for immunotherapy testing due to the
immunodeficiency in NSG mice. Organoids and orthotopic
models derived from MMR proficient tumors from genetically
engineered mouse models (GEMMs) (e.g., Apc
Min
model) are
great tools for CRC studies. However, there have not been
organoid-based models for MMRd/MSI-H intestinal tumors.
While GEMMs for MMRd/MSI-H CRC have been generated and
widely used in cancer biology and therapeutic treatment studies (17,
18), intestinal tumor development in these models usually takes
many months (19). To accelerate the discovery process for this
high-risk population and provide a feasible model system for in
vitro and in vivo screening of vaccine and therapeutic candidates,
we aimed to generate organoids and develop an orthotopic mouse
model for MMRd/MSI-H CRC. The Msh2
LoxP/LoxP
;Villin-Cre
(VCMsh2) mouse model is particularly useful because these mice
are predisposed to develop intestine tumors (mainly from the small
intestine) without metastasis (19). We generated organoids from
small intestine and colon tumors from this model and developed an
intra-cecal implantation model using organoid-derived tumor
fragments. The established tumor organoids, driven by Msh2-
deficiency, can be used to better understand MMRd/MSI-H
cancer biology and tumor heterogeneity. The orthotopic model,
with distant metastasis and frameshift (FS) neoantigen expression,
may be suitable for testing neoantigen-based vaccines and
developing new approaches for effective combination treatments.
2 Materials and methods
2.1 Mice
VCMsh2 mice (19) were provided by Dr. Winfried Edelmann at
Albert Einstein College of Medicine and maintained individually by
crossing to C57BL/6J (the Jackson Laboratory) mice. The
experimental mice were generated by crossing Msh2
LoxP/+
;Villin-
Cre to Msh2
LoxP/LoxP
mice. Animals were fed ad libitum on a normal
chow diet (Purina 5L79 –regular). Genotyping was performed as
described previously (19). C57BL/6J mice and NOD SCID gamma
(NSG™) mice (the Jackson Laboratory) were used as recipients for
in vivo tumorigenicity studies.
NCI Frederick is accredited by AAALAC International and
follows the Public Health Service Policy for the Care and Use of
Laboratory Animals. Animal care was provided in accordance with
the procedures outlined in the “Guide for Care and Use of
Laboratory Animals”(National Research Council, 1996; National
Academy Press; Washington, D.C.).
2.2 Tumor and wildtype
organoid generation
Tumor organoids were generated from tumor-bearing VCMsh2
mice following published protocols (20,21). Briefly, tumors were
excised and sliced into small pieces from tumor periphery. Tumor
pieces were not assessed for tumor cell content for organoid
generation. They were incubated in EDTA chelation buffer for 1
hour on ice, then digestion buffer for 2 hours at 37°C. After washing
with cold PBS, supernatant was collected by allowing tumor
fragments to settle under normal gravity for 1 minute, then single
cells in the supernatant were pelleted and washed with PBS. Cells
were resuspended in growth factor-reduced and phenol red-free
Matrigel (Corning #356231) on ice and plated in 24-well plates at
15,000 cells/50 µL. The plate was then incubated at 37°C to
polymerize the Matrigel. After 15 minutes, 500 µL basal culture
medium (BCM; Supplementary Material) containing 50 ng/mL
murine EGF was added to each well, which was refreshed every
2–3 days. WT organoids were generated from a WT littermate
(Msh2
+/+
;Villin-Cre
+/+
) following a modified protocol (22,23).
Briefly, intestinal villi were gently scraped off and discarded, and
the tissue was cut into 1-cm pieces and incubated in a 50 mL Falcon
tube containing PBS with 5 mM EDTA for 45 minutes at 4°C in a
HulaMixer set to 30 rpm for orbital rotation with a 60° turning
angle for reciprocal rotation. After vigorously shaking by hand,
tissue fragments were collected on a sieve and discarded. The flow-
through was then centrifuged and the supernatant was washed by
adding cold RPMI 1640 (Gibco). After centrifugation, the resulting
pellet containing detached crypts was washed and resuspended in
RPMI 1640, then purified by filtration through 70 µm mesh. The
resulting pellet was resuspended in complete culture medium
(CCM; Supplementary Material). To each well of a 24-well plate,
100 µL of crypts with 40 µL of additional CCM and 60 µL of
Matrigel was added to a pre-warmed 24-well plate. After
polymerizing the Matrigel, 300 µL of prewarmed CCM was added
to each well. Organoid growth was monitored daily using an
inverted microscope. CCM was gently pipetted off and fresh
CCM with fresh Matrigel was replenished every 2 days. For
passaging, organoids and Matrigel were mechanically disrupted
using a P1000 pipet tip and collected and washed with BCM or
CCM, then resuspended and plated at 50 µL Matrigel/well. To
freeze in liquid nitrogen, organoids were disrupted using a P1000
pipette with cut-off tips and transferred into a 15 mL Falcon tube,
then washed with 5 mL of BCM or CCM and centrifuged at 200 × g
for 2 minutes. The pellet was resuspended in BCM with 20% fetal
bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). Aliquots
of 1 mL per cryovial were placed in a Corning CoolCell®container
(Corning, NY) in −80°C freezer, then transferred to vapor-phase
liquid nitrogen for long-term storage.
2.3 Organoid injection and tumor
fragment implantation
Mechanically disrupted organoids and Matrigel were collected
and pipetted up and down ten times to disassociate the organoids.
After resuspending in 30 mL of PBS, organoids were further
disassociated by pipetting ten times, and resuspended in 2 mL
BCM containing 50% Matrigel after removing the supernatant and
a majority of the Matrigel using a P1000 pipet. A small volume of
organoid suspension was further digested with TrypLE at 37°C for
10 minutes to obtain a single-cell suspension, then FBS was added
Song et al. 10.3389/fonc.2023.1223915
Frontiers in Oncology frontiersin.org03
to stop the TrypLE and clumps were resuspended by pipetting up
and down. Cells were counted using a hemocytometer under a
microscope, then the number of cells in each volume was calculated
for the original organoid suspension. The appropriate number of
cells was diluted in a final volume containing 50% Matrigel for
subcutaneous injection. Under anesthesia, organoids in 100 µL with
50% Matrigel were injected subcutaneously (s.c.) to gender-
matched, syngeneic recipients by making a small incision at the
injection site and injecting using 20G needles. Animals were
monitored daily for tumor growth. Tumor volumes were
measured using calipers and calculated using the formula
L*W*H* p/6 [3D]. For serial passaging, s.c. organoid tumors were
harvested using sterile technique once they reached 1 cm. Briefly,
tumors were excised and pieces, which appeared to be solid (to
avoid thick mucinous or cystic region), were dissected. To avoid the
necrotic center of a tumor mass, tumor pieces from the periphery
were sliced into 2x2-mm tumor fragments (approximately 30 mg
each) and implanted s.c. into recipients. Tumor fragments were not
assessed for tumor cell content. Fragments were also viably frozen
in vapor-phase liquid nitrogen for implantation later. While
holding the small incision with forceps in a tented fashion, a
tumor fragment was placed gently into the right flank by pushing
the fragment into the pocket. One or two drops of 0.25%
Bupivacaine were applied to the incision site before closing the
incision with one surgical staple.
Intra-cecal tumor fragment implantation was performed in
syngeneic mice by following established procedures (24,25).
Briefly, the blind-ended pouch of the cecum was exteriorized and
supported on a sterile pre-cut gauze after a small midline abdominal
incision was made. Warmed sterile saline was used to keep the
cecum moist. A figure-eight stitch was placed onto the cecum using
absorbable suture material. A small, approximately 3x3-mm area of
the cecal wall under the suture was lightly damaged by grasping and
pulling with serrated forceps to facilitate the infiltration of cancer
cells. A fresh tumor fragment generated from s.c. tumor tissue (see
above) was positioned under the suture on the cecal wall. The stitch
was then tied down. After returning the cecum to the abdominal
cavity, the peritoneal layer was closed with a 5-0, absorbable suture
and the skin incision was closed with 1–2 wound clips after applying
1–2 drops of 0.25% Bupivacaine to the incision. Wound clips were
removed after 10–14 days. Animals were closely monitored during
post-surgery recovery.
2.4 Histopathology and immunostaining
Tumors were removed and fixed in 10% neutral buffered
formalin overnight, transferred to 70% ethanol, and then
routinely processed and embedded in paraffin. Hematoxylin and
eosin (H&E) and immunohistochemical (IHC) staining were
carried out on 4-mm sections as previously described (26)
(Supplementary Table 1). Slides were digitally imaged using an
Aperio ScanScope Scanner (Leica Biosystems). Staining was
quantified using a HALO®image analysis platform (Indica labs,
Albuquerque, NM).
2.5 Microsatellite instability detection
For MSI detection, primers were designed (Supplementary
Table 2; Integrated DNA Technologies, Coralville, IA, USA) for
mouse target regions of MSI markers (L24372, U12235, Bat64,
Bat30, Bat37, Bat59, and Bat67) on mouse build GRCm38.p6 as
reported previously (27–31). PCR amplification was carried out
with the Platinum™SuperFi™PCR Master Mix using cycling
conditions in Supplementary Table 3. Fragment analysis and Sanger
sequencing were performed as previously described (31). Bat67
fragment data were validated by Sanger sequencing (31). WT tail
and organoid DNA were used as controls. Positivity was scored
when at least a 1-bp shift of the repeat was observed or if new peaks
appeared that were not present in the control tissue.
2.6 Frameshift mutation detection
DNA from organoids was extracted using the Qiagen DNeasy
Blood & Tissue kit (Qiagen, Valencia, CA). Targeted sequencing was
performed using the rhAmpSeq amplicon sequencing system
(Integrated DNA Technologies IDT, Coralville, IA) with a dual-
indexing strategy. Approximately 55 ng of purified DNA was
combined with rhAmpSeq primers and cycled using the following
conditions: 1 cycle of 95°C for 10 minutes, followed by 10 cycles of
95°C for 15 seconds and 61°C for 4 minutes, enzyme deactivation by
1 cycle of 99.5°C for 15 minutes and 4°C hold at the end. After bead-
based cleanup using AMPure XP beads (Beckman Coulter, Brea, CA),
amplicons were combined with IDT indexes for Illumina sequencing.
PCR was performed using the following index PCR conditions: 1
cycle of 95°C for 3 minutes, followed by 18 cycles of 95°C for 15
seconds, 60°C for 30 seconds, and 72°C for 30 seconds; extension by 1
cycle of 72°C for 1 minute and 4C hold at the end. After a second
bead-based cleanup, amplicon libraries were sized and quantified
using a D1000 ScreenTape on TapeStation (Agilent Technologies,
Santa Clara, CA) and a KAPA q-PCR system (Roche, Frederick,
MD).Sequencinglibrariesweredilutedto4nM,combined,and
denatured for loading into the sequencing system. The final loading
concentration was 12 pM. Sequencing was performed on an Illumina
MiSeq system (Illumina, San Diego, CA) using a V3 reagent kit with a
1 x 151 bp length for the amplicon and 2 x 8 bp for the indexes.
For data analysis, index primers on raw reads from FASTQ files
were trimmed and aligned to mouse reference genome
Mus_musculus.GRCm39.dna.toplevel.fa using in-house-developed
pipeline (available upon request). For each target FSM detection,
the genomic site in all samples was revisited to extract the reads
supporting the mutant or WT allele. The numbers of mutant and
WT reads were used to calculate the insertion/deletion (indel)
variant allele frequency (VAF) across all samples. To distinguish
mutations from background errors, each indel’s background error
rate distributions were modeled by fitting its VAF from all WT
control samples into a Weibull distribution, then each tumor
sample’sVAFwascomparedtothefitted distribution as
previously described (32). A sample was defined as positive when
the sample’s VAF of a mutation was significantly above background
Song et al. 10.3389/fonc.2023.1223915
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(p< 0.05, after multiple testing correction using the false discovery
rate [FDR] method).
2.7 Ctnnb1,Apc, and Trp53
mutation detection
For Ctnnb1 mutation detection, 18 sets of Sanger sequencing
primers were designed to cover all coding exons (Supplementary
Table 4). For Apc mutation detection, seven hotspot mutations
reported in VCMsh2 tumors (19) were assessed by fragment
analysis or Sanger sequencing as described previously (31).
Briefly, for fragment analysis, all primers were ordered
(Supplementary Table 2; Integrated DNA Technologies) and
amplified on ProFlex PCR System (Thermo Fisher Scientific,
Waltham, MA) using PCR conditions stated in Supplementary
Table 3. The resulting products were then checked for quality and
concentration with Agilent’s 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA) and Agilent’s DNA 1000 kit,
using Bioanalyzer software version 2100 Expert B.02.11 SI824.
Amplified samples were diluted with molecular grade water up to
a 1:15 ratio as needed. A master mix combining Hi-Di™
Formamide (8.5 µL per reaction; Thermo Fisher Scientific),
GeneScan™500 LIZ™dye size standard (0.5 µL per reaction;
Thermo Fisher Scientific), and 1 µL of diluted sample was created
and incubated at 95°C for 5 minutes followed by 4°C for 2 minutes
in the ProFlex™PCR System. Samples were then processed on
3730xl DNA Analyzer (Thermo Fisher Scientific), using 96 capillary
50 cm array, a DS-33 Matrix Standard Kit (Dye Set 5; Thermo
Fisher Scientific), and 3730XL Data Collection Software (version
5.0; Thermo Fisher Scientific). Data were then analyzed and
overlayed with GeneMapper™software (version 5.0; Thermo
Fisher Scientific). Length of the PCR product in the testing
sample was compared to the WT sample and the altered length
was defined as instability. MSI status was classified as MSI-high
(MSI-H) (instability at 2 or more microsatellite loci) and
microsatellite stable (MSS) (instability at 1 locus or none).
For Sanger sequencing, primers were designed and amplified
(Supplementary Tables 2,3). PCR products were purified using
exonuclease I (GE Healthcare, Pittsburgh, PA) and shrimp alkaline
phosphatase (SAP; Affymetrix USB) by incubating the sample
mixture at 37°C for 15 minutes, then 80°C for 15 minutes,
followed by a 4°C hold in the ProFlex™PCR System. The
purified amplicon then proceeded into cycle sequencing with
BigDye™Terminator v3.1 Cycle Sequencing Kit and M13
forward and M13 reverse primers (Thermo Fisher Scientific)
using the following conditions in the ProFlex™PCR System: 96°
C for 1 minute, 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds,
60°C for 1 minute and 15 seconds; followed by a hold at 4°C.
Samples were then processed on 3730xl DNA Analyzer using 96
capillary 50cm array, Sequencing Standards, BigDye™Terminator
v3.1 Kit (Thermo Fisher Scientific), and 3730XL Data Collection
Software. Data were then analyzed using Mutation Surveyor
(version 5.1.2; SoftGenetics, State College, PA).
For Trp53 mutation detection, 10 sets of PCR reactions were
run for each sample using the primers described in Supplementary
Table 5.Trp53-coding exon regions and splicing junctions were
amplified using the following conditions: 95°C for 2 minutes, 36
cycles of 94°C for 15 seconds, 58°C for 15 seconds, and 68°C for 1
minute; and a hold at 68°C for 1 minute. After quantifying using
TapeStation, amplicons from each sample were pooled by equal
molarity and barcoded using the following conditions: 95°C for 2
minutes, 15 cycles of 94°C for 15 seconds, 56°C for 15 seconds, and
68°C for 1 minute, and a hold at 68°C for 1 minute. Barcoded PCR
products were treated with Exonuclease I (New England Biolabs,
Ipswich, MA) at 37°C for 15 minutes and purified using the
Ampure XP protocol (Thermo Fisher Scientific). Purified
amplicons were pooled together based on equal molarity, then
pooled libraries were quantified using TapeStation and the final
concentration was determined using a Qubit assay (Thermo Fisher
Scientific). Paired-end sequencing was performed on an Illumina
MiSeq sequencer using MiSeq V2 reagent kits 500-cycles (Illumina).
The sequencing quality was monitored and the raw FASTQ files
were downloaded for data analysis. Paired-end reads were trimmed
using Trimmomatic (33). Top 100-300 most common reads for
each sample were identified and sequence reads containing
mutations with a frequency greater than 4% were detected, and
the mutation frequency was calculated using custom-developed
Python scripts. The sequence reads with mutations were
visualized by aligning them with the WT mouse Trp53 sequence
using SnapGene software (Insightful Science; available
at snapgene.com).
2.8 Spectral karyotyping
To evaluate chromosome instability (CIN), SKY analysis was
performed as described previously (31). Briefly, organoids were
disassociated from Matrigel by incubating with Dispase II, and
single-cell suspensions were generated by incubating with TrypLE
(ThermoFisher Scientific). Cells were then arrested in metaphase by
incubating with Colcemid™(10 µg/mL; 15210-040, KaryoMAX ®
Colcemid Solution, Invitrogen, Carlsbad, CA) for 3 hours, treated
with hypotonic solution (KCl 0.075M, 6858-04, Macron Chemical)
for 15 minutes at 37°C, and then fixed with methanol:acetic acid 3:1.
The prepared metaphase spread slides were aged overnight and
then hybridized with the 21-color mouse SKY paint kit
(FPRPR0030, ASI) in a humidity chamber at 37°C for 16 hours
(34). Spectral images were acquired using a Hyper Spectral Imaging
System (Applied Spectral Imaging Inc., CA) mounted on top of an
epi-fluorescence microscope (Imager Z2, Zeiss) and analyzed using
HiSKY 7.2 acquisition software(GenASIs,AppliedSpectral
Imaging Inc., CA). An average of 10–15 mitoses of comparable
staining intensity and quality were examined per organoid line and
evaluated for chromosomal abnormalities.
2.9 Multiplex in situ hybridization staining
The expression of WT Lgr5 and WT and mutant Asxl1 were
detected by staining 5 mmformalin-fixed, paraffin-embedded
(FFPE) tissue sections with the following probes: Mm-Lgr5-O2-
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C1 (ACD, Cat# 1237631-C1), RNAscope®2.5 LS Probes Mm-
Asxl1-C1 (ACD, Cat# 421968), and Mm-Asxl1-O1-C2 (ACD, Cat#
1149108-C2), and analyzing with the LS Multiplex Fluorescent
Assay (ACD, Cat# 322800) using the Bond RX autostainer (Leica
Biosystems). Tissue sections were pretreated with Bond Epitope
Retrieval Solution 2 (Leica Biosystems) at 95°C for 15 minutes,
Protease III (ACD) at 40°C for 15 minutes, and then a 1:750 dilution
of TSA-Cyanine 3 Plus and TSA-Cyanine 5 Plus (Akoya
Biosciences). The RNAscope®3-plex LS multiplex negative
control probe (Bacillus subtilis dihydrodipicolinate reductase
(dapB) gene in channels C1, C2, and C3, Cat# 320878) and the
RNAscope®LS 2.5 3-plex positive control probe-Hs were used as
controls. Slides were digitally imaged using an Aperio ScanScope FL
Scanner (Leica Biosystems). Images were analyzed and quantified
using a HALO®image analysis platform (Indica labs,
Albuquerque, NM).
2.10 Statistical analysis
All graphs and statistical analyses were made using GraphPad
Prism 9 (GraphPad Software, San Diego, CA). All statistical tests
were two-sided and p< 0.05 was considered significant unless
otherwise stated. Kaplan–Meier survival curves were plotted, and
the log-rank test was used for the median overall
survival comparisons.
3 Results
3.1 Organoid generation and
characterization by markers
Organoids were successfully generated from small intestine and
colon tumors developed in VCMsh2 mice with 59% and 100% success
rate at the animal level, respectively (Supplementary Table 6). Cells
seeded in one well were treated as one organoid line. In total, 125
small intestine and 29 colon tumor organoid lines were established
(passaged up to P3 in vitro) with some from the same tumor (20%
and 57% success rate at the line level, respectively). One healthy
organoid line from intestinal crypt cells from a WT littermate control
was generated. Organoids were grown in Matrigel supplemented with
EGF. Morphologically, most tumor organoids were very similar
(round or round with small protrusions) except organoid line
586T2A4, which had convoluted, tube-like structures in vitro
(Supplementary Figure 1A). H&E staining showed that these
organoids were composed of either single or multiple cell layers
with an open lumen filled with dead cells (Supplementary Figure 1C).
To confirm the Msh2 deletion by Villin-Cre in vivo,Msh2IHC
staining was performed on FFPE sections prepared from organoid
pellets and de novo VCMsh2 tumors. As expected, Msh2 was not
expressed in VCMsh2 tumors or organoids (Figure 1A;
Supplementary Figures 1D,2A), while strong expression was
detected in WT intestine (Supplementary Figure 1D).
BC
A
FIGURE 1
Characterization of organoid line 586T2A4 by (A) IHC staining, (B) mRNA in situ hybridization, and (C) TUNEL staining. Scale bar: 50 µm.
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To evaluate the cell type of the organoids, a panel of markers
were assessed using IHC on FFPE sections made from 10 organoid
lines. All organoids tested had very high expression of epithelial
markers (e.g., Epcam and E-cadherin), but didn’t express Muc2
(goblet cell marker), chromogranin A (ChgA; enteroendocrine
marker), or lysozyme (Paneth cell marker) (Figure 1A;
Supplementary Figure 2A). One organoid line (961T3C3) had
very low expression of Muc2, ChgA, and lysozyme. This indicates
that the organoids were of epithelial cell origin. To determine if
tumor organoid cells were of progenitor/stem cell origin, crypt cell
markers Klf5, Ephb2, and Ascl2 expression were assessed using
IHC, revealing high expression of these markers (Figure 1A;
Supplementary Figure 2A). Lgr5 is a well-accepted intestine stem
cell marker. Lgr5 expression was further assessed by mRNA in situ
hybridization, showing strong but heterogenous positivity
(Figure 1B;Supplementary Figures 2B,3). Some organoids
showed high Lgr5 expression, but others low or no Lgr5
expression within the same organoid line. As expected, Ki67 IHC
showed a high proliferation rate regardless of the Lgr5 expression
level, along with a low apoptosis rate as assessed by the TUNEL
assay (Figure 1C;Supplementary Figure 2C). These tumor
organoids also showed strong membrane staining of b-catenin
(Figure 1A;Supplementary Figure 2A). Supplementary Table 7
summarizes the staining quantification used for Figure 1.
To determine if the antigen presentation complex was
functional in tumor organoids, flow cytometry analysis of MHC
class I and B2M was performed, revealing that the organoids had
very low basal levels of H-2Db, H-2Kb, and B2M (Supplementary
Figure 4). However, MHC class I expression could be induced by
IFNgtreatment. H-2Db, H-2Kb, and B2M expression were
significantly increased compared to non-treated controls,
indicating that the MHC class I antigen processing and
presentation machinery was functional in these tumor organoids.
3.2 Microsatellite and
chromosome stability
MMRd cells characteristically show MSI-H. To confirm the MSI
status in the tumor organoids, up to seven MSI markers were
assessed in 27 tumor organoids and WT organoids via DNA
fragment length analysis. As expected, all tumor organoids
showed MSI-H, while WT organoids did not (Figure 2;
Supplementary Table 8). This is consistent with the MSI status in
de novo tumors from VCMsh2 mice (data not shown). Organoids
derived from different tumors from the same VCMsh2 mouse
showed the similar MSI profiles (Supplementary Table 8). For
example, 960T2A1 and 960T3D3 were derived from tumors T2
and T3, respectively, from mouse #960. Their MSI profile for three
markers mU12235-A24, mL24372-A27 and mBat64 was m1 and
m1, m7 and m4, and m23 and m21, respectively. Moreover,
organoids generated from serially transplanted tumors in vivo
showed higher instability compared to parental 586T2A4 and
961T3C3 organoids (Supplementary Table 8). For example,
parental 586T2A4 had m4, m6, and m25, while P8 organoid from
a serially transplanted tumor from this parental line (586T2A4-P8-
T41) had m6, m11, and m37 for markers mU12235-A24, mL24372-
A27 and mBat64, respectively. This indicates progressive instability
during in vivo tumor progression.
A mixed genomic state of MSI and CIN has been reported in
colorectal cancer (35,36). To determine if CIN also occurred in
VCMsh2 MMRd tumors, tumor organoids were assessed via SKY.
586T2A4 showed high levels of chromosomal abnormality due to
whole-chromosome amplification (e.g., chr1, 3, 9, and 12), deletion
(e.g., chr2, 8, 10, 15, and 16), or translocation (e.g., T(3:4),+9(T6))
(Figure 3;Supplementary Table 9). However, the instability was
variable and heterogenous within each organoid and among
different organoid lines. For example, some cells from 586T2A4
and most cells from 357T2B2 had normal chromosomes (e.g., 40,
XY for 586T2A4 and 40,XX for 357T2B2), while 546T2A2 and
961T3C3 showed high instability (e.g., amplification of
chromosome 19, translocation of chromosomes 3, 4, and 6, and
tetrasomy of chromosomes 1, 3, 4, 6, 8, 9, 10, 11, 17, 18, and X for
546T2A2, and loss of chromosome Y, deletion of chromosomes 13
and 18, and trisomy of chromosomes 1, 2, 3, 5, 8, 13, 14, and 17 for
961T2C3; Supplementary Figure 5,Supplementary Table 9).
3.3 Mutations in tumor organoids
Many recurrent FSMs have been reported in coding
microsatellite repeat regions (e.g., mononucleotide repeats) in
MMRd/MSI-H tumors (16,28,37,38). Clinically, multiplex
PCR and capillary electrophoresis (CE) (fragment analysis) and
next-generation sequencing (NGS)-based assays have been
approved by the Food and Drug Administration (FDA) for MSI
detection. To determine whether tumor organoids from VCMsh2
mice harbored the FSMs reported by Gebert et al. (14), targeted
sequencing was performed via NGS. As expected, in all organoids,
FSMs were detected with variable mutation frequency (e.g., 100%
for Asxl1(-1), Xirp1(-1), and Senp6(-1), and 75% for Nacad(-1))
(Table 1) and high VAF, except for Maz(-1) (Supplementary
Table 10). Consistently, FSMs were also detected in de novo
end-stage tumors and tumor-matched mucosa tissues from
VCMsh2 mice. Interestingly, the similar mutation frequency was
observed between tumors and tumor-matched mucosa, indicating
that these FSMs driven by Msh2 deletion may not be sufficient to
drive the tumor development and that secondary mutations may
be required.
Somatic mutation of Apc has been reported in VCMsh2 tumors
(19). Fragment analysis or Sanger sequencing for seven hotspot loci
(reported by Kucherlapati et al. (19)) was performed in 43 organoid
lines derived from 27 VCMsh2 tumors (several organoid lines for
some tumors). The Apc mutations were detected at four codons
(c854, c956, c1211, and c1464), with the highest frequency at c1464
(44.4%) (Supplementary Table 11). The same mutation was
detected in several organoids derived from the same tumor. Some
organoids had more than one Apc mutation. For example, organoid
357T3A3 had R956X and c1464 delAG, and 979T2A2 had c1211
delTC and c1464 delAG.b-catenin is downstream of Wnt/Apc
signaling. Ctnnb1 mutation in tumor organoids was further
examined via Sanger sequencing. Primers for 18 amplicons were
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designed to cover all the exons of Ctnnb1, except exon 1. Two
Ctnnb1 mutations were found in 27 organoids (7.4%): a missense
mutation (S37P) and a coding-silent mutation (F560)
(Supplementary Table 12). The same Ctnnb1 S37P mutation was
found in five organoid lines derived from the same tumor (586T2),
indicating this mutation may be an early clonal event. Interestingly,
there was no Apc mutation in organoids from the 586T2 tumor.
Tumor suppressor p53 has been shown to play a critical role in
tumorigenesis of most cancer types (39). Trp53 mutation was
assessed in 26 organoid lines from 15 tumors by targeted
sequencing using 10 amplicons to cover all the coding exons of
Trp53. Three mutations (G242ATer*,R280C, and T380Q) were
detected in parental 586T2A4 and its second- and third-generation
organoids, while T380Q was also detected in the 968T1 organoid
line (Supplementary Table 12). Trp53 G242D mutation was only
detected in one organoid line (961T3C3) with low VAF.
Interestingly, VAF was lower in the parental line compared to
organoid lines generated from serially passaged tumors (6.5% vs.
50%), indicating in vivo biologically relevant natural selection of
pre-existing clones. Consistently, nuclear accumulation of p53 was
observed in 586T2A3 and 961T3C3 organoids via IHC staining
(Figure 1A;Supplementary Figure 2A). In general, Trp53 mutation
frequency was low in Msh2-deficient mouse tumor organoids.
Notably, some organoids had neither Apc/Ctnnb1 nor Trp53
mutations, suggesting that other secondary mutations may drive
the tumor development.
FIGURE 2
Fragment analysis of seven MSI markers in 586T2A4-P8-T11 and WT organoids. Blue peaks are 586T2A4-P8-T11 and WT organoids, pink or green
peaks are WT tail, and yellow peaks are size markers. Two vertical red lines align the WT fragment peaks in WT tail.
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3.4 Frameshift peptide expression in
tumor organoids
FSP neoantigens have been used as vaccine targets for MMRd
cancers (40). To determine whether FSPs were expressed in tumor
organoids, targeted mRNA sequencing was performed via MiSeq.
Frameshift-mutant Senp6 and Asxl1 mRNA were detected in both
586T2A4 and 961T3C3 organoids tested, and mutant Maz mRNA
was detected only in 961T3C3 (Supplementary Table 13), while
mutant Nacad and Xirp1 mRNA were not detectable. This may be
due to poor sequencing coverage (8 and 134 reads count,
respectively) or low expression of these two targets. To further
confirm the expression of Asxl1 since there was no mutant-specific
anti-Asxl1 antibody available, mRNA in situ hybridization via
RNAScope®was employed. Two customized probes were
designed, with one probe targeting upstream and the other
targeting downstream of a mononucleotide repeat region. The
sequence against the downstream probe is not present in the
frameshift-mutant transcript due to the frameshift causing early
termination of the transcript. Asxl1-mutant mRNA, along with WT
Asxl1 mRNA, was found to be highly expressed in both tumor
organoids (Figure 4;Supplementary Table 14).
3.5 Tumorigenicity of organoids via
subcutaneous injection in syngeneic
and NSG mice
To determine the tumorigenicity of organoids derived from
VCMsh2 tumors, low-passage organoids (< P10) were injected s.c.
into gender-matched syngeneic recipients with two different
inocula. To our surprise, s.c. tumors did not grow well
(Figure 5A;Supplementary Figure 6A). After initial growth,
tumors plateaued and then, for some tumors, regressed to become
non-palpable. An additional eight organoid lines were tested s.c.,
and a similar growth pattern was observed (data not shown). To
TABLE 1 FSM frequency in organoids, and matched end-stage tumor and mucosa from VCMsh2 mice.
Gene Organoids (n = 10) End-stage tumors (n = 18) Tumor-matched mucosa (n = 16)
Asxl1(-1) 100% 94.4% 100%
Maz(-1) 20% 12.5% 7.1%
Xirp1(-1) 100% 100% 100%
Senp6(-1) 100% 94.4% 100%
Nacad(-1) 75% 94.4% 100%
-1: deletion of one nucleotide in MNR region.
B
A
FIGURE 3
Karyotype by SKY analysis in (A) 586T2A4: 40,XY,+1,-2,+3,T(3;4),-8,+9(T6),-10,+12,-15,-16. T: translocation. One copy of chromosome 15 was bigger
than that in (B) a WT normal cell.
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determine whether this was due to immune editing, these organoids
were injected s.c. into immune-compromised NSG mice, resulting
in a similar growth pattern (Supplementary Figure 6B). Histological
analysis showed that s.c. tumors in C57BL/6 or NSG recipients had
heterogenous pathology, with adenocarcinomas mixed with cystic/
mucinous features (Supplementary Figure 7A,B). To determine
whether the regression of s.c. tumors was due to low proliferation or
high apoptosis rate, Ki-67 IHC was used to assess proliferation, and
TUNEL or Caspase-3 IHC was used to assess apoptosis. Results
showed that these tumors had strong Ki-67 expression and a low
apoptosis rate (Supplementary Figures 7C,D).
3.6 Serial in vivo passaging of
tumor organoids
Not all s.c. tumors from organoids had growth plateau and
regression. To determine whether they could grow continually
without plateau or regression, tumors were serially passaged s.c.
to establish a syngraft model for preclinical use. After nine passages
of 586T2A4 in vivo, tumors grew continuously without regression
(Figure 5B). Consistent with the parental 586T2A4 s.c. tumor
histology (Supplementary Figure 8A, upper panel), serially
passaged tumors showed classic adenocarcinoma with low
B
A
FIGURE 5
In vivo growth of organoids. (A) Growth of parental organoids 586T2A4 and 961T3C3 by s.c. injection of 1 × 10
6
cells in C57BL/6J recipients (n = 10
per group). This was in vivo passage 0 (P0). *Tumor fragments from P0 tumor #3268 (left panel) and #3274 (right panel) were serially passaged s.c.
in vivo.(B) Growth of 586T2A4 serially passaged tumor fragments at P9 from two different donor tumors (n = 5 per group). The median time for a
tumor to reach 500 mm
3
was 26.4 days (95% CI, 21.5 to 31.3) (left panel) and 28.3 days (95% CI, 26.4 to 35) (right panel). TV, tumor volume.
FIGURE 4
Asxl1 expression by RNA in situ hybridization in organoids 586T2A4 and 961T3C3. Two RNAScope®probes were designed upstream (red; left panel)
and downstream (yellow; the panel next to the left) of the mononucleotide repeat region in Asxl1 mRNA. 3rd panel: merged images of two probes;
right panel: merged images of two probes and DAPI for nuclei counter staining. WT: both yellow and red signals (merged as orange, red arrow).
Mutant: red only (blue arrow). Scale bar: 50 µm.
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mucinous features (Supplementary Figure 8A, middle and bottom
panels). The median time for a tumor to reach 500 mm
3
was 26.4
days (95% CI, 21.5 to 31.3; Figure 5B, left panel) and 28.3 days (95%
CI, 26.4 to 35; Figure 5B, right panel). 961T3C3 was passaged to P2
in vivo, and the resultant tumors showed more cystic/mucinous
tumor growth (Supplementary Figure 8B) compared to 586T2A4
(Supplementary Figure 8A). This is consistent with the pathology
observed in de novo 586 and 961 tumors (Supplementary
Figure 1B), which recapitulates different histopathological types of
LS CRC tumors (41–44). P2 s.c. tumors of 961T3C3 were not
further passaged in vivo. As shown above, Lgr5 was highly
expressed in some organoids, but not others from the same
organoid line (Figure 1B,Supplementary Figures 2B,3,
Supplementary Table 7). To determine whether these s.c. tumor
cells expressed Lgr5, we performed Lgr5 mRNA in situ
hybridization. For 586T2A4 P0 parental s.c. tumors, strong Lgr5
expression was observed in the solid tumor mass region but not in
the mucinous tumor (Supplementary Figure 9A left panel). Serially
implanted s.c. tumors also expressed Lgr5, but cells in the inner
mass had weak expression compared to those at the periphery
(Supplementary Figure 9A, middle and right panels). Mucinous
tumors had either heterogenous Lgr5 expression (546T2A4 and
577T2A4; Supplementary Figure 9B, left and middle panels), or no
Lgr5 expression (961T3C3; Supplementary Figure 9B, right panel,
bottom). This may be because 961T3C3 s.c. tumors were filled with
thick mucus in the lumen without any live tumor cells lining the
lumen (Supplementary Figure 9B, right upper panel).
3.7 An orthotopic model for MMRd/MSI-H
tumors in syngeneic mice
The impact of microenvironment on tumor growth and immune
modulation has been well studied. To generate an orthotopic model
and determine whether tumor cells could grow well in their native
microenvironment, 2x2-mm tumor fragments were generated from
three s.c. tumors derived from organoids, then implanted one
fragment per animal to the wall of the cecum (n = 5 per donor).
Thirteen out of 15 animals had cecal tumor growth (87% success rate;
Supplementary Table 15) with median survival of 67, 78, and 60 days
post implantation (dpi) for each donor group, respectively
(Figure 6A). The tumors were much larger compared to de novo
B
C
DE
F
A
FIGURE 6
Intra-cecal implantation model developed using P8 tumor fragments from 586T2A4 organoids (n = 5 per group). (A) Kaplan-Meier survival curve
using tumor fragments from three different donor tumors originating from 586T2A4 organoids. The median survival was 60, 67, and 78 days post
implantation (dpi). (B) Gross primary cecal tumor and liver metastasis (red arrows). (C) H&E staining of cecal tumors and liver metastasis. The red star
indicates the invasion of tumors into intestinal serosa. Scale bar: 200 µm (left panel) and 25 µm (middle and right panels). (D) H&E staining of lymph
node metastasis. Scale bar: 50 µm. (E) Images of ultrasound (US; left) and MRI (right, red arrow points to tumor mass). (F) Tumor volume (TV)
measured by MRI.
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tumors (Figures 6B,F). Histopathological analysis showed orthotopic
tumors growing on the serosal surface of the large intestine. The
tumor cells formed ductal structures as adenocarcinoma with a high
mitotic index (Figure 6C), recapitulating the de novo tumor
pathology (Supplementary Figure 1B). Tumor cells also invaded the
intestinal wall (Figure 6C) and metastasized to the liver (4/13, 31%;
Figures 6B,C) and lymph node (4/13, 31%; Figure 6D), which are
clinically relevant metastatic sites for LS CRC (45). Three animals had
both liver and lymph node metastases. Two animals did not have
cecal tumors, which might be due to surgical failure. To determine
whether orthotopic tumors can be monitored by imaging, ultrasound
(US) and magnetic resonance imaging (MRI) were performed. While
cecal tumors were detectable by both MRI and US (Figure 6E),
metastatic tumors could not be imaged,possibly due to the small size.
A good correlation was observed between tumor volume measured by
US and MRI (Supplementary Figure 10B).
4 Discussion
Organoids have been extensively used to study cancer biology
and drug sensitivities (46–48). Intra- and interpatient heterogeneity
in ovarian cancer (49) and heterogeneity of breast cancer subtypes
(50) were investigated using organoids. A progressive association
between niche-independent growth and the adenoma-carcinoma
transition was observed using a comprehensive organoid library
from CRC (51). A distinct mutational signature in CRC was
uncovered using intestinal organoids, which reveals an underlying
mutational process potentially resulting directly from past exposure
to a specific type of bacteria (52). Moreover, organoids have been
used in high throughput drug screening, which allows the detection
of gene-drug associations (53). Although the etiology of MMRd
CRC has been known for decades, it is not clear what impact
thousands of FSMs throughout the genome have on tumor
initiation and progression and which secondary mutations drive
the tumorigenesis. It is evident that the penetrance and frequency of
MMRd CRC were different among 4 MMR genes. However, it is not
known whether FSMs are the same in tumors with different MMR
gene deficiency. Shared frameshift neoantigens are ideal for vaccine
development for MMRd cancers. Moreover, the mechanisms of
resistance to immunotherapies are under-investigated in MMRd
CRC. As described in this report, a panel of mouse intestine
organoids from MMRd/MSI-H tumors has been generated and
characterized and is well suited for mechanistic studies and in vitro
drug screening for MMRd CRC.
Several intra-cecal syngeneic CRC models have been reported
using either MSS cell lines in syngeneic mice (e.g., MC38 (54,55)or
CT26 (56–59)) or human MSI-H cell lines (e.g., HCT116) in
immune-deficient mice (60,61). Herein, we described an intra-
cecal syngeneic model using tumor fragments derived from MMRd/
MSI-H mice. There are several advantages of this orthotopic model
over the existing ones. 1. A study cohort can be generated in a
relatively short period of time compared to GEMMs (e.g., VCMsh2
model); 2. It can be used for preclinical testing in immune
competent mice thus allowing the modeling of the host immune-
tumor interactions, as compared to xenograft models in
immunodeficient mice using cell lines derived from human
patients; 3. Compared to subcutaneous models, tumors developed
in this model have relevant organ site, which is required for
emergence of metastasis and studying host responses and
interactions with growing tumors; 4. It can be used to test site-
specific dependence of therapies, assess the efficacy of therapies on
metastasis, and evaluate drugs that modulate the tumor-host
interaction; and 5. It can be used to study the pathogenesis of
metastases for MMRd/MSI-H tumors. The implantation success
rate was high (87% vs. 67% by Greenlee et al. (54) and 65% by Evans
et al. (58)) and the distant metastasis was biologically relevant in
this model (liver (15%) and intra-abdominal lymph node (20%)
metastases in human MSI-H CRC) (45). Orthotopic CRC models
have been serving as valuable tools for studying genes involved in
metastasis (56) and evaluating the efficacy of immune checkpoint
blockade therapies (59) and other therapies (e.g., recombinant
methioninase to target the methionine (MET)-dependent cancers
(62)). The model described here is the first orthotopic model of
MMRd/MSI-H intestine cancer, and well suited for studying
MMRd/MSI-H cancer biology and preclinically testing vaccines
and immunotherapies in combination with targeted therapies for
MSI-H tumors. We are currently assessing the efficacy of frameshift
peptide and mRNA vaccines in this intra-cecal model.
It is evident that the microenvironment is critical for tumor
growth and metastasis (63–67). In this study, we found that MMRd/
MSI-H organoid s.c. tumors usually plateaued or regressed after
initial growth in both syngeneic and immune-compromised mice,
indicating that intrinsic factors or the microenvironment play an
important role in organoid growth in vivo.Indeed,Lietal.reported
that the tumor microenvironment can be shaped by a chemokine,
CXCL1, intrinsically produced by tumor cells, which acts as a
molecular “switch”(68), indicating that intrinsic factors can be
responsible for the adaptability of tumor cells in the
microenvironment and outcome of a therapy. Interestingly, after
tumors were serially passaged s.c. in vivo, the organoids derived from
these tumors showed good growth without regression or plateau
when they were injected s.c. in syngeneic mice, suggesting that the
most fit clones, presumably with driver mutations, were selected or
evolved in vivo. Notably, distant metastasis was observed only in the
intra-cecal model, not in the s.c. model or VCMsh2 model,
demonstrating the importance of the native microenvironment in
metastasis. It seems that there is a requirement for further evolution
of tumor cells in a more native environment for execution of a
metastatic propensity during tumor progression. This was supported
by evidence that the tumor-immune microenvironment of CRC
cannot be modeled in s.c. models (69) and that the stroma can
control tumor aggressiveness (70). In addition, past work in a
melanoma model indicates that tumor location determines tissue-
specific recruitment of tumor-associated macrophages (71). Thus, it
is plausible to assume that tissue-specific microenvironmental factors,
including the immune system, are required for tumor progression
and metastasis. More research is needed to determine the factors
involved in pro-metastatic tumor growth and mechanisms of
immune evasion in MMRd/MSI-H tumors.
Ki-67 is expressed during all active phases of the cell cycle (G1,
S, G2, and M) but absent in resting cells (G0). Thus, it has been
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widely used as a proliferation marker. However, Ki-67 positivity
does not always correlate with proliferation. Cancer cells usually
have a deregulated cell cycle and, in some cases, can be stuck at an
active phase without proliferation but show positivity for Ki-67 (72,
73). It has been reported that Ki-67 was concomitantly expressed
with p16, a senescence marker, in cervical epithelial cells in HPV+
women (74), indicating an abnormal cell cycle caused by HPV
oncoproteins. Ki-67 positivity in s.c. tumors with plateaued growth
indicates that the cell cycle of these tumor cells may be dysregulated,
which can happen due to several reasons (e.g., DNA damage,
mutations in certain genes related to the cell cycle, or an invoked
checkpoint). To assess the proliferation status in these s.c. tumors,
one can perform bromodeoxyuridine (5-bromo-2’-deoxyuridine,
BrdU) labeling in vivo, which only incorporates into newly
synthesized DNA during the S phase, and subsequently use a
specific anti-BrdU antibody for detection in tumors. Interestingly,
these s.c. tumors were mucinous. High mucin production may play
a role in dysregulating the cell cycle and tumor growth. Past work
has reported that mucins can serve as signaling molecules that alter
the proliferation, differentiation, or cell-adhesion status of the
tumor cells/epithelial cells (75). It remains to be determined
whether blocking mucin production can increase the proliferation
rate of these s.c. tumors.
The cell of origin has been studied extensively but remains
elusive for most cancers. Two hypotheses have been proposed (76)
and supported by experimental evidence: the stem cell origin, that
stem/progenitor cells undergo tumorigenic transformation that
eventually leads to tumor formation (77), and the non-stem-cell
origin, that fully differentiated mature cells can undergo
reprograming and dedifferentiate into progenitor-like cells with
stem cell properties, which eventually give rise to tumors (78).
Both stem cells and differentiated cells have been implicated as the
cell of origin for CRC (77,79–81). In this study, we found that
organoids derived from Msh2-deficient tumors were of epithelial
cell origin with very low expression of differentiated cell markers
(e.g., Muc2, ChgA, and lysozyme). These organoids strongly
expressed crypt cell markers Ephb2, Ascl2, and Klf5 but
heterogeneously expressed stem cell marker Lgr5. This is
consistent with the finding that about 50% of MMRd/MSI-H
CRC had Lgr5 expression (82) and indicates that tumor cells were
initiated either solely from Lgr5+ cells (with some of them
differentiating to Lgr5−cells during tumor progression) or from
both Lgr5+ stem cells and Lgr5−progenitor cells in the transit
amplifying (TA) zone (since Villin-Cre is expressed in intestine
epithelial cells all along the crypt-villus axis) (83). A lineage-tracing
study using a multicolor Cre-reporter model showed that the Lgr5+
stem cells, only representing 5–10% of the adenoma cells, generated
additional Lgr5+ cells and other adenoma cell types (84), indicating
that tumors are initiated in Lgr5+ cells. However, microadenoma
could be initiated in TA cells in a mouse model, although the
authors concluded that it was highly unlikely that large adenomas
were derived from short-lived TA cells (77). Interestingly, mice with
one allele of Msh2 deleted throughout the body and another allele
specifically deleted in Lgr5+ cells (Lgr5-CreER
T2
;Msh2
flox/-
) survived
longer (average 19 months) and had low penetrance of intestinal
tumors (40%) (85) compared to VCMsh2 mice with both alleles of
Msh2 deleted in Lgr5+ and TA cells (19) (median survival of 11.6
months, with 100% penetrance; data not shown). This strongly
argues that intestinal tumors might be initiated in both Lgr5+ stem
cells and Lgr5−TA cells. Thus, the cell of origin may not be
stemness-dependent (although stem cells are much more efficient in
tumor formation) but rather dependent on a serial event (e.g., initial
Msh2 deletion followed by accumulation of other mutations or
initial Apc deletion followed by inflammation (86)). It remains to be
determined whether deletion of Msh2 only in intestinal TA cells can
lead to tumor formation.
One of the hallmarks of cancer cells is genomic instability (87),
which is usually manifested through CpG island methylator
phenotype (CIMP) or variations at the nucleotide level (e.g., base
pair mutation and MSI) or chromosome level (CIN) (88–90).
MMRd typically leads to MSI-H phenotype. As expected, all the
tumor organoids derived from VCMsh2 tumors showed MSI-H.
Interestingly, we observed increased instability in organoids derived
from syngraft tumors serially passaged in vivo compared to parental
organoids, indicating genome instability and continuous tumor
evolution in vivo. In addition to the MSI, heterogeneous
instability at the chromosome level was also detected in these
organoid tumor cells. Some cells showed significant abnormality,
including deletion, translocation, and amplification of whole
chromosomes, while others were normal. This is consistent with
the recent finding of mixed genomic states of MSI and CIN in CRC
(35) and mesothelioma (31). Heterogeneity has also been reported
at the genomic and transcriptomic level in LS CRC (91), which may
account for the heterogenous response to immunotherapy
treatment. Our data indicate that CIN is not a direct result of
MMRd since all the tumor cells lacked Msh2 expression. It is not
clear whether the heterogeneity of CIN is related to the MSI status
in each cell or secondary mutations accumulated in these cells
during tumor progression.
MMRd/MSI-H tumors usually have high mutation frequencies
in microsatellite repeat regions (e.g., mono-, di- or tetra-nucleotide
repeats) due to errors during DNA replication. Thus, MSI-H
patients with a high mutation burden and neoantigens respond
well to immunotherapy and have better survival than MSS patients.
FSMs in coding mononucleotide repeat regions have been reported
in MMRd/MSI-H patients and mouse models (16,28,37,38,92).
The resulting FSPs can serve as neoantigens when they are
expressed. Frameshift-neoantigen-based vaccines with different
formulations have been tested and showed promising activities
clinically and preclinically for cancer prevention and treatment
(14,15,93–97). In this study, we confirmed that these tumor
organoids and intra-cecal implanted tumors expressed
characteristic FSMs/FSPs with variable mutation frequency and
VAF. The significance of these FSMs/FSPs in tumor progression
and immunoediting is unknown. It has been shown that FSP
neoantigens can induce a T-cell response in vitro (98). With so
many FSMs/FSPs in MMRd/MSI-H cancers, more studies are
needed to assess the quality of neoantigens with high
immunogenic potential for neoantigen-based vaccine
development (99). Interestingly, we found that Apc and Ctnnb1
mutations were mutually exclusive in organoids and some harbored
Trp53 mutation, presumably functioning as drivers for tumor
Song et al. 10.3389/fonc.2023.1223915
Frontiers in Oncology frontiersin.org13
development. However, some organoids had neither Apc/Ctnnb1
nor Trp53 mutations. This highlights the importance of secondary
driver mutations during tumor progression (100–104) and the need
to identify other secondary drivers in MMRd/MSI-H tumors.
In summary, the intra-cecal implantation model described here,
driven by Msh2 deficiency, had the characteristics of MMRd/MSI-
H tumors and recapitulated the de novo tumors developed in
VCMsh2 mice, with the potential for tumor growth monitoring
by US or MRI. This is the first syngeneic model of MMRd/MSI-H
intestine cancer. Expressing characteristic frameshift neoantigens in
tumors enables studies to better understand the sequence and
significance of FSMs and to test targeted interventions. Moreover,
this model with distant metastasis allows us to study progressive
genome instability and tumor evolution with heterogeneity. In
addition, organoids derived from Msh2-deficient small intestine
and colon tumors can be used to investigate MMRd/MSI-H cell
biology and the tumorigenic process in depth (105).
Data availability statement
The data and Python scripts used for Trp53 mutation
analysis presented here are available upon request from the
corresponding author.
Ethics statement
The animal study was approved by Animal Care and Use
Committee (ACUC) at the National Cancer Institute (NCI) at
Frederick. The study was conducted in accordance with the local
legislation and institutional requirements.
Author contributions
YS, SS, and RHS contributed to the concept and study design.
TK, CS, LD, SSB, BS, RNB, SDM, TBY, HEL, TMP, SB, and JG
contributed to the experimental planning and execution, and data
acquisition, analysis, and interpretation. BK contributed to the
histology analysis. BX, LW, QH, and SL contributed to the
sequencing data analysis. YS drafted the manuscript. YS, SS,
and RHS contributed to the critical revision of the manuscript.
All authors contributed to the article and approved the
submitted version.
Funding
This research was supported in part with federal funds from the
National Cancer Institute, National Institutes of Health, under
Contract No. HHSN261201500003I; IOTN Moonshot grant
U24CA232979-01 (LW, QH, SL, and AH); and NIH grants U54
CA 272688U01 and U01 CA233056 and contracts from NCI
PREVENT program (HHSN2612015000391) (SML, JG, and MK).
The content of this publication does not necessarily reflect the views
or policies of the Department of Health and Human Services, nor
does mention of trade names, commercial products, or
organizations imply endorsement by the U.S. government.
Acknowledgments
We thank Dr. Winfried Edelmann at Albert Einstein College of
Medicine for providing Msh2
LoxP/LoxP
mice; the staff at Animal
Research Technical Support (ARTS) for conducting the animal
studies; Dr. Joseph Kalen, Lisa Riffle, and Lilia Ileva at the Small
Animal Imaging Program for MRI and ultrasound imaging; Dr.
Wang-Ting Hsieh and his staff at the Animal Diagnostic Laboratory
(ADL) for genotyping and pathogen testing; Andrew Warner, Donna
Butcher, Brad Gouker, and staff at Molecular Histotechnology
Laboratory (MHL) for histology, immunohistochemistry, and in
situ hybridization RNAScope®work; Kristen M. Pike, Dr. Daniel
R. Soppet, and the late Dr. Gordon R. Whiteley at the CLIA
Molecular Diagnostics Laboratory for project support; Dr. Holli
Loomans-Kropp at NCI, DCP for technical assistance, and CIPL
members and scientists at the Chemopreventive Agent Development
Research Group at NCI, DCP, for data discussions.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
The reviewer WL declared a shared affiliation with the authors
SB, SS, and RS to the handling editor at the time of review.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fonc.2023.1223915/
full#supplementary-material
Song et al. 10.3389/fonc.2023.1223915
Frontiers in Oncology frontiersin.org14
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