





































ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 

 

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. 
 

EXPLORING EPITHELIAL 

COMMUNICATION TO THE 

MESENCHYME AND ITS  

IMPACT ON THE EXPRES-

SION OF GENES RELATED 

TO TUMORIGENESIS 
 

JAY PATEL, MICHAEL VERZI (FACULTY ADVISOR) 

 

✵ ABSTRACT 
Homeostasis of the epithelium is depend-

ent on the Wnt/beta-catenin pathway, which regu-

lates the proliferation of intestinal stem cells. The 

gain of function mutations in the beta-catenin gene 

leads to rapid cell proliferation and malignant 

growth in the epithelium. In addition, the mainte-

nance of these stem cells appears to be linked to 

mesenchymal-derived factors. Although the com-

munication between epithelial and mesenchyme 

cell populations remains uncharacterized, under-

standing this mechanism will help us further under-

stand the various pathways involved in tumor initia-

tion processes. Our results show that the communi-

cation between the mesenchyme and epithelium 

during transformation is influenced by varying levels 

of protein-expressing genes including Wnt2b, 

Grem1, and Bmp6. 

 

1 INTRODUCTION 
The Wnt/beta-catenin pathway regulates 

the transcription of Wnt target genes—genes which 

are highly deregulated in solid tumors (Fevr et al., 

2007). In the absence of the Wnt ligand, beta-

catenin, a protein normally sequestered in the cyto-

plasm, is constantly degraded and is prevented 

from entering the nucleus.  

In the presence of the Wnt ligand, Wnt is 

able to bind to its receptor Frizzled, which activates 

downstream signaling and prevents the destruction 

of beta-catenin, thus enabling its entry into the nu-

cleus. In the nucleus, beta-catenin promotes the 

transcription of Wnt target genes, causing rapid cell 

proliferation (Cleavers and Nusse, 2012). Cells di-

viding at an accelerated rate can become cancerous 

and eventually lead to the formation of a tumor.   

 

The intestinal stem cell niche is primarily 

made up of several cell types, including: (1) differ-

entiated cells responsible for the transport and ab-

sorption of nutrients, and (2) mesenchymal cells 

which supply signals to the epithelial cells to main-

tain homeostasis. Increased Wnt signaling can be 

induced by activating the beta-catenin gene, which 

allows for abnormal epithelial growth. Due to the 

expression of tamoxifen inducible Cre recom-

binase, Villin-CreERT2 transgenic mice have the ability 

to delete exon 3 of the beta-catenin allele in the in-

testinal epithelium (Marjou et al., 2004). Mice ex-

pressing Villin-CreERT2 can delete a portion of the 

beta-catenin gene in the presence of tamoxifen, re-

sulting in a more transcriptionally active form of the 

gene. Mouse models have shown that mice with Vil-

lin-CreERT2 and injected with tamoxifen show visible 

malignant tissue transformation in the duodenum. 

But in organoid models, exposed to tamoxifen, 



ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 

 

rapid cell proliferation can be seen almost immedi-

ately. However, epithelial transformation only be-

comes visible around Day 13 in mouse models. Alt-

hough organoids mimic intestinal stem cells (crypts) 

in vitro, they do not provide a holistic view of a bio-

logical system. For example, they lack the presence 

of the mesenchyme. This suggests that the mesen-

chyme must play some role in the suppression of 

mutant growth. However, the way mesenchyme and 

epithelial cells influence one another is unknown. 

Yet, it is known that mesenchymal-derived factors 

are essential to maintain intestinal epithelial stem 

cells, so the reduction of these factors may be con-

tributing to the suppression of intestinal transfor-

mation in the transgenic mouse (Stzepourginski et 

al., 2017). 

AREG, a ligand of EGFR (epidermal growth 

factor receptor), can trigger signaling cascades that 

mediate cell survival, proliferation, and motility. 

Thus, AREG may be a candidate ligand that allows 

the epithelium to communicate with the mesen-

chyme (Wang et al., 2020). Transformed epithelium 

expresses high levels of AREG, which may be 

sensed by mesenchyme cells. Mesenchyme cells 

like PDGFRAhi are enriched near the crypt-villus 

junction where stem cells reside and promote BMP 

ligands (promotes differentiation/blocks prolifera-

tion). A subset of PDGFRAlo cells can be found at the 

bottom of crypts and provide growth factors for 

stem cells (McCarthy et al., 2020). If AREG is com-

municating with PDGFRAlo cells in the mesenchyme, 

the PDGFRAlo cells would express decreased levels 

of GREM1, a protein that functions to suppress BMP 

and permits proliferation. Subsequently, this may 

lead to an upregulation in expression levels of BMP. 

Early intestinal tumorigenesis reflects the deregula-

tion of Wnt and BMP signals (Davis et al; 2015). It has 

also been recognized that BMP signaling restricts in-

testinal epithelium hyperproliferation (Qi et al., 

2017). If we could understand how the mesenchyme 

is communicating with the epithelium, it would give 

insight to how it is able to suppress the abnormal 

growth, allowing for novel therapeutic interventions 

during initial tumor development. The goal of this 

project is to learn how epithelial cells are communi-

cating with mesenchyme cells in an elevated Wnt 

activity background. Elevated levels of Wnt prevents 

beta-catenin from being degraded, which pro-

motes expression of Wnt target genes, resulting in 

the mutant phenotype. Through signals (e.g. AREG) 

sent by the epithelium, the mesenchyme may detect 

the cellular transformation and respond in hopes of 

preventing the abnormal growth. If mesenchyme 

cells are treated with conditioned media from mu-

tant organoids (crypts), then the genes which are al-

tered are communicating with the mesenchyme. If 

epithelial cells undergo abnormal growth, the lig-

ands produced could be sensed by PDGFRAlo cells 

in the mesenchyme compartment, which may re-

spond by increasing Bmp genes and decreasing 

Grem1 and Wnt2b production. 

 

2 MATERIALS AND METHODS 
ANIMAL AND TISSUE PROCESSING 

Animal experiments are conducted in ac-

cordance with Rutgers University Institutional Ani-

mal Care and Use Committee (IACUC).. The trans-

genic mice in this study are engineered to condi-

tionally express the beta-catenin exon3 deletion. To 

induce recombination, tamoxifen (1mg/20g) is in-

jected intraperitoneally for 4 days daily. Day 1 is 

considered the beginning of treatment, and mice 

are sacrificed and investigated at Day 10. This 

model is ideal for studying tumorigenesis in-vivo be-

cause of similarities in mouse and human gastroin-

testinal tracts. At Day 10, mouse intestines (duode-

num) are collected and fixed in 4% paraformalde-

hyde solution overnight at 4°C. Tissues are then 

washed with phosphate-buffered saline (PBS) and 

dehydrated in increasing concentrations of ethanol. 

Tissues are then transferred to xylene and paraffin 

mixture for one hour, then 100% paraffin for another 

hour. Tissues are embedded for sectioning.  

 

ORGANOID FORMING ASSAY 

Crypt-derived organoids are isolated from 

mouse duodenum on Day 10 after 4 consecutive 

treatment days of tamoxifen administered at 

1mg/20g mice. Collected duodenum samples are 

washed in PBS, cut into ¼ inch pieces, and rotated 

in 3mM EDTA for 5, 10, and 20 minutes (each time 



ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 

 

replace the EDTA). Tissues are then agitated and fil-

tered through a 70μm filter for crypt enrichment. 

Crypts are then washed in PBS and the pellet is re-

suspended in BME-R1. Media for the crypts include 

advanced DMEM supplemented with GlutaMAX 

(auxiliary energy source for rapidly dividing cells), 

HEPES (used to maintain pH), EGF (epidermal 

growth factor: involved with cell signaling pathway 

controlling cell division), Noggin (prevents cell dif-

ferentiation), NAC (N-acetylcysteine: protects 

against rise of internal oxidant levels), N2 (promotes 

in vitro differentiation of stem cells), B27 (promotes 

growth and proliferation, but not differentiation), 

RSPO (positively regulates Wnt/beta-catenin signal-

ing, which induces proliferation), and Pen-Strep 

(prevents bacterial contamination). After 3 days of 

organoids being treated with this media, it becomes 

conditioned media (contains ligands excreted by 

epithelium). 

 

GENOTYPING 

Mice toes are clipped and used as a source 

for DNA genotyping analysis. DNA is extracted us-

ing the Kappa Buffer QIAgen Kit followed by PCR for 

amplification. Genotypes are confirmed by DNA 

electrophoresis.  

 

HEMATOXYLIN AND EOSIN STAINING 

To understand the changes in the epithelial 

architecture in terms of the formation of crypt pro-

genitor cell phenotype (CPC), we conducted H&E 

histological analysis. Tissues are initially washed in 

xylene twice (5 min each). Then tissues go through 

two consecutive 100% EtOH washes (5 min each), 

followed by a 3 min 95% EtOH wash, a 3 min 85% 

EtOH wash, a 3 min 70% EtOH wash, and a 5 min 

double-distilled water wash. Unstained sections of 

mouse duodenum are stained with Hematoxylin (30 

seconds) and dehydrated by dipping in water, fol-

lowed by increasing alcohol percentages. Tissues 

are then counterstained with eosin for 1 minute (eo-

sin in counterstain, which distinguishes between cy-

toplasm and nuclei).  

 

RNA/DNA PREPARATION AND QPCR 

Mesenchyme cells are dissolved in Trizol. 

RNA is prepared according to the manufacturer’s 

protocols. The RNA is then reverse transcribed to 

cDNA using SuperScript III First-Strand Synthesis 

System. SYBR Green is used to amplify the cDNA 

through qPCR analysis. Vimentin is used for normal-

ization. 

 

MESENCHYME TREATMENT WITH CONDITIONED MEDIA  

The main experiment consists of using mes-

enchyme cells and treating them with (1) condi-

tioned media from mutant organoids treated with 

CCM, (2) mutant organoids treated with mesen-

chyme media, (3) wild type organoids treated with 

CCM, and (4) mesenchyme media as a control. To 

demonstrate the response by the mesenchyme, 

changes in ligand expression are examined among 

treatment groups. After passaging mesenchyme 

cells, they are plated, and are treated with mesen-

chyme media on Day 1. Pictures of the cells are 

taken on Day 4, and the media is aspirated and re-

placed with conditioned media or mesenchyme me-

dia for control. The plate consists of 4 rows and 3 

columns; rows 1 and 2 are made up of a confluent 

wild-type mesenchyme cells, and row 3 consists of 

sub-confluent wild type mesenchyme cells. Samples 

in the first column serve as controls and are treated 

with mesenchyme media, samples in the second 

column are treated with conditioned CCM from mu-

tant organoids, samples in the third column are 

treated with conditioned mesenchyme media from 

mutant organoids, and samples in the fourth col-

umn are treated with conditioned CCM from wild 

type organoids. The fourth column is a control be-

cause the wild type organoids do not express mu-

tant growth, so we expect there to be no change in 

Grem1 and Wnt2b expression. On Day 6, the cells 

are harvested with Trizol for RNA isolation, which is 

then converted to cDNA. For this experiment we 

measure 11 protein coding genes (Bmp2, Bmp3, 

Bmp4, Bmp5, Bmp6, Grem1, Wnt2b, Rspo1, Rspo3, 

Wnt5a, and Wnt4) using qPCR and compare their 

relative abundances.  

 



ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 

 

3 RESULTS 
Tissue images are taken at Day 10 post-

tamoxifen treatment and stained using hema-

toxylin and eosin. FIGURE 1 shows the histology of 

the control and mutant epithelium. The control 

and mutant epithelium for replicates 1 and 2 

show little difference in terms of transformation. 

This demonstrates the delay stated before, sug-

gesting that the mesenchyme is playing a role 

to suppress mutant epithelial growth.  

To understand the mesenchyme re-

sponse to ligands secreted by mutant epithe-

lium, gene expression changes are examined 

following treatment with conditioned media de-

rived from organoid cultures. qPCR (FIGURE 2A 

and FIGURE 2B) analysis demonstrated that conflu-

ent and sub-confluent mesenchyme cells do not 

respond in the same way. For the confluent 

cells, Grem1 and Wnt2b tend to be downregu-

lated. Grem1 is a Bmp antagonist; therefore, by 

downregulating the gene Bmp can have a larger 

effect on the epithelium which would lead to a 

decrease in activity of the Wnt/beta-catenin 

pathway. For the sub-confluent mesenchyme 

cells, Grem1 is downregulated in all experi-

mental groups while Wnt2b is also downregu-

lated except in cells treated with conditioned 

CCM from wild type organoids. Again, the con-

ditioned media from mutant organoids are 

causing Grem1 and Wnt2b to be downregu-

lated in order to suppress mutant epithelial 

growth. 

 

 

FIGURE 1: H&E staining of two independent replicates 

showing the difference between the control and mu-

tant epithelium.

 

FIGURE 2A: The image shows the density of the cells; cells are confluent. 647 is the confluent mesenchyme cell line. 

The leftmost bars represent Grem1 and Wnt2b expression in the control: mesenchyme cells treated with mesen-

chyme media. To the right of that is mesenchyme cells treated with mesenchyme conditioned media from mutant 

organoids (647 MM-CM exon3f/f). To the right of that is mesenchyme cells treated with CCM condition media from 

wild type organoids (647 CCM-CM WT), and the rightmost graph shows mesenchyme cells treated with CCM con-

ditioned media from mutant organoids (647 CCM-CM Exon3f/f). The graph shows relative gene expression (n=3), 

compared to cells treated with mesenchyme media.  



ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 

 

 

FIGURE 2B: Image shows the density of cells; cells are sub-confluent. Graph shows relative gene expression of the 

three samples treated with conditioned media to the mesenchyme media for sub-confluent cells. Graph portrays 

same conditions as graph above except for sub-confluent cells.  

 

The graphs in FIGURE 3 demonstrate the 

relative gene expression seen across all 11 pro-

tein coding genes that were tested. The graph 

focuses on the mesenchyme conditioned media 

from mutant organoids vs. the control. The Bmp 

profile between the confluent and sub-conflu-

ent cells shows many similarities including 

Bmp2 and Bmp3 being downregulated, Bmp4 

staying constant, and Bmp6 being upregulated. 

The upregulation in sub-confluent cells is much 

more profound. The Wnt profile in confluent 

and sub-confluent cells contain some differ-

ences like Rspo3 being upregulated in conflu-

ent cells while being downregulated in sub-con-

fluent cells. While Wnt5a is upregulated in both 

confluent and sub-confluent, Wnt4 is downreg-

ulated in confluent, while profoundly upregu-

lated in sub-confluent cells. 

 

4 DISCUSSION 
The goal of the experiment is to identify 

possible signaling pathways that mesenchymal 

cells could use to communicate with epithelial 

cells. While the multitude of cells in the small in-

testine and their functions are known, like troph-

ocytes, telocytes, and enterocytes (McCarthy et 

al., 2020), their ability to communicate with the 

microenvironment is understudied. The signal-

ing crosstalk between the stroma cell popula-

tion and the epithelium remains unknown. Un-

derstanding how these two types of cells may 

communicate can provide novel therapeutic in-

terventions during initial tumor development. 

Histological review of H&E stained slides re-

ported a lack of altered or abnormal tissue 

structures, suggesting that the mesenchyme is 

responsible for suppressing mutant epithelial 

growth. Protein expressing genes such as 

Wnt2b, Grem1, and Bmp6 may be responsible 

for the cross talk between these two cell types. 

When conditioned media from mutant organ-

oids is introduced to mesenchyme cells Grem1 

and Wnt2b are downregulated. The downregu-

lation of Wnt prevents the expression of Wnt tar-

get genes, therefore controlling mutant trans-

formation. Grem1 in these cell lines is seen to be 

downregulated, which directly and indirectly 

(by inhibiting Bmp genes) prevents epithelial 

transformation. What is unusual is why most of 

the Bmp genes are either downregulated or 

stay constant while Bmp6 is upregulated. Bmp 

signaling can be seen as the primary suppressor 

of dedifferentiation in the intestinal epithelium, 

and better understanding of its ligands will pro-

vide important steps to how the epithelium pro-

tects against oncogenesis (Perekatt et al., 2018). 

Although the Bmp gene expression is found to



ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 

 

 

 

FIGURE 3: Graphs show relative gene expression of the aforementioned 11 genes in sub-confluent and confluent 

mesenchyme cells. Graph compares the major experimental group to the control group. Because CCM is the pri-

mary media used to grow and sustain organoids, its data would be most resembled in-vivo conditions. This group 

would have organoids function normally so that the conditioned media would mimic in-vivo conditions, and then 

the mesenchyme response could be observed. 

 

not increase, this could be because Grem1 ex-

pression is downregulated, leading to the trans-

lation of more Bmp proteins. The up and down 

regulation of Bmp, Grem1, and Wnt2b ligands 

may be, in part, how the mesenchyme is influ-

encing epithelial cells, preventing early tumor-

igenesis. 

The experiment demonstrated the dif-

ferences between confluent and sub-confluent 

mesenchyme cells, as well as how different con-

ditioned media affects mesenchyme cells. 

These differences are most profoundly seen in 

the genes Bmp6 and Wnt4. Confluent cells have 

the ability to sense when they are running out of 

space, which can cause the cells to become less 

proliferative. Sub-confluent cells are rather in a 

more active growth phase. This could be affect-

ing the signals the mesenchyme releases to the 



ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 

 

epithelium, and responsible for the difference 

seen in response between confluent and sub-

confluent cells. These results come from a single 

experimental design and provide a basis for fu-

ture experiments. 

Bmp, Grem1, and Wnt2b may be signals 

that allow the mesenchyme to communicate 

with the transformed epithelium. These genes 

could be responsible for early prevention of ini-

tial tumorigenesis. These genes are potential 

ligands that suppress mutant epithelial growth. 

From here, geneticists could design experi-

ments involving blocking these ligands with an-

tibodies or treatment with these ligands to see 

the epithelial response from the mesenchyme∎  

 

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ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 

 

 

 

Jay Patel is a junior undergraduate student conducting research in the Ge-

netics Department at Rutgers New Brunswick. His interest in scientific re-

search was sparked by his experience with the Rutgers Waksman pro-

gram, during his time in high school. He currently works with Dr. Verzi and 

his mentor, Oscar, where they investigate the pathway by which the intes-

tinal epithelium and mesenchymal cell populations are communicating. 

Along with being a researcher, Jay also enjoys riding as an EMT and the 

Highland Park First Aid Squad, volunteering as a part of the Rutgers Red 

Cross, and playing basketball with his friends.  


