Novel In Vivo Mouse Cryoablation Model to Explore Unique Therapeutic Approaches for Premalignant Columnar Lesions

Patients with epithelial metaplasias have an increased risk of developing malignancies. In Barrett’s esophagus, neo-columnar epithelium develops proximal to the squamous-columnar junction (SCJ) in the esophagus as the result of prolonged exposure to bile and acid reflux. Patients require lifetime periodic surveillance, due to lack of effective eradication therapies. The shortage of innovative treatment options is mostly attributable to the paucity of adequate in vivo models of neo-columnar epithelium regeneration. This protocol describes the generation of a cryoablation model to study regeneration of neo-epithelia at the SCJ. Cryoablation of the columnar and squamous mucosa at the SCJ was achieved through local application of liquid N2O in wild-type and reporter mice in combination with acid suppression. Acid suppression alone, showed restoration of the SCJ with normal histological features of both the neo-columnar and neo-squamous epithelium within 14 days. As a proof of principle, mice were treated with mNoggin, an inhibitor of bone morphogenetic proteins (BMPs), which are involved in the development of columnar epithelia. Local application of mNoggin to the ablated area at the SCJ significantly reduced the development of the neo-columnar mucosa. Although this model does not faithfully recapitulate the exact characteristics of Barrett’s esophagus, it is a well-suited tool to study the mechanisms of therapeutic inhibition of neo-columnar regeneration. It therefore represents an efficient and easy platform to test novel pharmacological therapies for treatment of neo-epithelial lesions at the SCJ.


Introduction
Metaplasia is the replacement of one tissue by a neo-tissue which is better adapted to its novel environment [1]. Metaplasia results from environmental stimuli, such as pH change, alcohol, smoking, and hormones, which cause chronic tissue inflammation and consequently the release of growth factors, cytokines, and trophic factors such as bone morphogenetic proteins (BMPs) [1][2][3]. Several of these factors promote tissue regeneration, progenitor cells may be activated and give rise to neo-epithelia, which are better adapted to the new environment [4]. Although metaplastic lesions are associated with an increased risk for malignant degeneration, at baseline, these lesions have almost normal physiological functions and benign histological features [1,5]. Due to persistent environmental conditions and ongoing inflammation, metaplastic lesions may become dysplastic and eventually progress to cancer [1,4,5].
Columnar metaplasia can be found in the distal esophagus at the squamo-columnar junction (SCJ) or in the glandular stomach [1]. Gastric intestinal metaplasia (GIM) and Barrett's esophagus (BE) are examples of metaplastic lesions in the stomach and esophagus, respectively. GIM occurs mainly through atrophic gastritis caused by H. pylori infection and autoimmune gastritis [6]. These inflammatory processes induce a neo-epithelium (intestinal

Experimental Design
Cryoablation is performed at the SCJ in the mice stomach where the neo-epithelia develop. The mouse cryoablation model here described was established in CB6F1 wild-type mice and further validated in lineage tracing K5-GFP mice.

Animals
All animal experiments were approved by the Animal Experimental Committee of the Amsterdam University Medical Center (Amsterdam UMC) and in compliance with the Animal Welfare Body (IvD), under the protocol number LEX159. All animals were kept at the Animal Research Institute of the Amsterdam UMC (ARIA), and procedures were performed under ARIA standard operating procedures (SOP).
All mice strains were housed in individual ventilated cages (+/+ IVC) in ventilated racks before, during, and after the cryoablation procedure. All mice used were older than 8 weeks with initial weights above 20 g.
The development of the cryoablation model was performed using wild-type CB6F1 mice purchased from Charles River (CB6F1/Crl strain code 176). Cryoablation protocol was validated by using K5-Cre-Rosa26-Tomato-GFP mice (K5-GFP).

1.
Remove any solid pellet food from the +/+ IVC cages 3 days before the cryoablation procedure.

2.
Replace the pellet food for liquid food (commercial baby milk prepared according to the supplier in drinking water).

3.
Day prior to the cryoablation procedure (approximately 18 h prior), remove liquid food for overnight fasting ( Figure 1A). Leave drinking water available ad libitum.

1.
In the non-sterile area, place the mouse in the induction chamber with 2% isoflurane. Under anesthesia, weigh the mouse and register his weight. Shave the abdominal area and transfer the mouse to the sterile field.

2.
Mobilize the mouse in the sterile field with medical tape, place the anesthesia cone on the mouse nose, and apply eye ointment to prevent corneal damage ( Figure 2A).

Cryoablation Procedure. Time for Completion: 30 min Per Mouse
1. In the non-sterile area, place the mouse in the induction chamber with 2% isoflurane. Under anesthesia, weigh the mouse and register his weight. Shave the abdominal area and transfer the mouse to the sterile field. 2. Mobilize the mouse in the sterile field with medical tape, place the anesthesia cone on the mouse nose, and apply eye ointment to prevent corneal damage ( Figure 2A).

CRITICAL STEP:
It is important to prepare the sterile field onto a heating pad (37 °C) to be able to maintain the mouse body temperature throughout the procedure.
3. Administer pain killer (Meloxicam, 5 mg/kg body weight) with a single subcutaneous (s.c.) injection with a 26 G needle syringe (Figure 2A). 4. Clean the skin with betadine solution ( Figure 2B). 5. Start the surgical procedure by lifting the mouse skin with forceps and making a midline vertical incision on the abdomen, of about 2 cm ( Figure 2C,D). 6. Use the round atraumatic forceps to reach the stomach inside the intraperitoneal cavity. 7. Pull the stomach out from the intraperitoneal cavity for better visibility and performance ( Figure 2E). 8. Embed a sterile medical gauze pad with warm PBS and place it between the stomach and the mouse skin ( Figure 2F).

CRITICAL STEP:
The use of sterile gauze pad is important to avoid direct contact between the two distinguish surfaces, stomach and skin. In addition, it is essential when removing the stomach content in step 10. This step will minimize possible infection post-surgery. 9. Using a fine scissor, make an incision of 0.75 to 1 cm long in the middle of the greater curvature of the mouse stomach including the SCJ as shown in Figure 2F,G.

CRITICAL STEP:
It is important to prepare the sterile field onto a heating pad (37 • C) to be able to maintain the mouse body temperature throughout the procedure.
Clean the skin with betadine solution ( Figure 2B).

5.
Start the surgical procedure by lifting the mouse skin with forceps and making a midline vertical incision on the abdomen, of about 2 cm ( Figure 2C,D). 6.
Use the round atraumatic forceps to reach the stomach inside the intraperitoneal cavity. 7.
Pull the stomach out from the intraperitoneal cavity for better visibility and performance ( Figure 2E). 8.
Embed a sterile medical gauze pad with warm PBS and place it between the stomach and the mouse skin ( Figure 2F).

Cryoablation Procedure. Time for Completion: 30 min Per Mouse
1. In the non-sterile area, place the mouse in the induction chamber with 2% isoflurane. Under anesthesia, weigh the mouse and register his weight. Shave the abdominal area and transfer the mouse to the sterile field. 2. Mobilize the mouse in the sterile field with medical tape, place the anesthesia cone on the mouse nose, and apply eye ointment to prevent corneal damage ( Figure 2A).

CRITICAL STEP:
It is important to prepare the sterile field onto a heating pad (37 °C) to be able to maintain the mouse body temperature throughout the procedure.
3. Administer pain killer (Meloxicam, 5 mg/kg body weight) with a single subcutaneous (s.c.) injection with a 26 G needle syringe (Figure 2A). 4. Clean the skin with betadine solution ( Figure 2B). 5. Start the surgical procedure by lifting the mouse skin with forceps and making a midline vertical incision on the abdomen, of about 2 cm ( Figure 2C,D). 6. Use the round atraumatic forceps to reach the stomach inside the intraperitoneal cavity. 7. Pull the stomach out from the intraperitoneal cavity for better visibility and performance ( Figure 2E). 8. Embed a sterile medical gauze pad with warm PBS and place it between the stomach and the mouse skin ( Figure 2F).

CRITICAL STEP:
The use of sterile gauze pad is important to avoid direct contact between the two distinguish surfaces, stomach and skin. In addition, it is essential when removing the stomach content in step 10. This step will minimize possible infection post-surgery. 9. Using a fine scissor, make an incision of 0.75 to 1 cm long in the middle of the greater curvature of the mouse stomach including the SCJ as shown in Figure 2F,G.

CRITICAL STEP:
The use of sterile gauze pad is important to avoid direct contact between the two distinguish surfaces, stomach and skin. In addition, it is essential when removing the stomach content in step 10. This step will minimize possible infection post-surgery.

9.
Using a fine scissor, make an incision of 0.75 to 1 cm long in the middle of the greater curvature of the mouse stomach including the SCJ as shown in Figure 2F,G. 10. Once opened, remove the stomach content with the help of round-tip forceps and cotton swaps until the area at the SCJ for the ablation is clean ( Figure 2H-J). 11. For better visualization of the stomach interior, make a stich with a 5/0 non-absorbable suture and hold it with a needle holder and set it aside ( Figure 2K).

Cryoablation Procedure. Time for Completion: 30 min Per Mouse
1. In the non-sterile area, place the mouse in the induction chamber with 2% isoflurane. Under anesthesia, weigh the mouse and register his weight. Shave the abdominal area and transfer the mouse to the sterile field. 2. Mobilize the mouse in the sterile field with medical tape, place the anesthesia cone on the mouse nose, and apply eye ointment to prevent corneal damage ( Figure 2A).

CRITICAL STEP:
It is important to prepare the sterile field onto a heating pad (37 °C) to be able to maintain the mouse body temperature throughout the procedure.
3. Administer pain killer (Meloxicam, 5 mg/kg body weight) with a single subcutaneous (s.c.) injection with a 26 G needle syringe (Figure 2A). 4. Clean the skin with betadine solution ( Figure 2B). 5. Start the surgical procedure by lifting the mouse skin with forceps and making a midline vertical incision on the abdomen, of about 2 cm ( Figure 2C,D). 6. Use the round atraumatic forceps to reach the stomach inside the intraperitoneal cavity. 7. Pull the stomach out from the intraperitoneal cavity for better visibility and performance ( Figure 2E). 8. Embed a sterile medical gauze pad with warm PBS and place it between the stomach and the mouse skin ( Figure 2F).

CRITICAL STEP:
The use of sterile gauze pad is important to avoid direct contact between the two distinguish surfaces, stomach and skin. In addition, it is essential when removing the stomach content in step 10. This step will minimize possible infection post-surgery. 9. Using a fine scissor, make an incision of 0.75 to 1 cm long in the middle of the greater curvature of the mouse stomach including the SCJ as shown in Figure 2F,G.

CRITICAL STEP:
The time of each liquid N 2 O application depends on how stretched the mouse stomach is, i.e., how thin the stomach wall is. It is important not to exceed the duration of 10 s and pause briefly between applications to assess the damage and avoid perforation of the stomach wall.

Cryoablation Procedure. Time for Completion: 30 min Per Mouse
1. In the non-sterile area, place the mouse in the induction chamber with 2% isoflurane. Under anesthesia, weigh the mouse and register his weight. Shave the abdominal area and transfer the mouse to the sterile field. 2. Mobilize the mouse in the sterile field with medical tape, place the anesthesia cone on the mouse nose, and apply eye ointment to prevent corneal damage (Figure 2A).

CRITICAL STEP:
It is important to prepare the sterile field onto a heating pad (37 °C) to be able to maintain the mouse body temperature throughout the procedure.
3. Administer pain killer (Meloxicam, 5 mg/kg body weight) with a single subcutaneous (s.c.) injection with a 26 G needle syringe (Figure 2A). 4. Clean the skin with betadine solution ( Figure 2B). 5. Start the surgical procedure by lifting the mouse skin with forceps and making a midline vertical incision on the abdomen, of about 2 cm ( Figure 2C,D). 6. Use the round atraumatic forceps to reach the stomach inside the intraperitoneal cavity. 7. Pull the stomach out from the intraperitoneal cavity for better visibility and performance ( Figure 2E). 8. Embed a sterile medical gauze pad with warm PBS and place it between the stomach and the mouse skin ( Figure 2F).

CRITICAL STEP:
The use of sterile gauze pad is important to avoid direct contact between the two distinguish surfaces, stomach and skin. In addition, it is essential when removing the stomach content in step 10. This step will WARNING: Cryoalpha pen allows for precise and accurate applications of liquid N 2 O, which can produce temperatures of −89 • C. Cryoalpha pen is a safe instrument and should be held like a pencil when in use, only aiming the applicator to the area of interest.
14. Close the stomach using an 8/0 non-absorbable suture with continuous stiches ( Figure 2O). The ablated area can be observed on the exterior of stomach by a darker red color of the wall ( Figure 2P, dotted line).

Cryoablation Procedure. Time for Completion: 30 min Per Mouse
1. In the non-sterile area, place the mouse in the induction chamber with 2% isoflurane. Under anesthesia, weigh the mouse and register his weight. Shave the abdominal area and transfer the mouse to the sterile field. 2. Mobilize the mouse in the sterile field with medical tape, place the anesthesia cone on the mouse nose, and apply eye ointment to prevent corneal damage (Figure 2A).

CRITICAL STEP:
It is important to prepare the sterile field onto a heating pad (37 °C) to be able to maintain the mouse body temperature throughout the procedure.
3. Administer pain killer (Meloxicam, 5 mg/kg body weight) with a single subcutaneous (s.c.) injection with a 26 G needle syringe (Figure 2A). 4. Clean the skin with betadine solution ( Figure 2B). 5. Start the surgical procedure by lifting the mouse skin with forceps and making a midline vertical incision on the abdomen, of about 2 cm ( Figure 2C,D). 6. Use the round atraumatic forceps to reach the stomach inside the intraperitoneal cavity.
CRITICAL STEP: Carefully inspect if the stomach is completely closed. Small openings in the mouse stomach can lead to leakage of the stomach content into the peritoneal cavity. 15. Place the ablated stomach back inside the intraperitoneal cavity and close the abdominal wall and skin separately by using and a 5/0 vicryl absorbable suture ( Figure 2Q,R).

NOTE:
Troubleshooting of different problems, reasoning, and solutions, that can be encountered during and after the cryoablation procedure can be found in Table 1.
applicator to the area of interest.
14. Close the stomach using an 8/0 non-absorbable suture with continuous stiches ( Figure 2O). The ablated area can be observed on the exterior of stomach by a darker red color of the wall ( Figure 2P, dotted line).

CRITICAL STEP:
Carefully inspect if the stomach is completely closed. Small openings in the mouse stomach can lead to leakage of the stomach content into the peritoneal cavity.
15. Place the ablated stomach back inside the intraperitoneal cavity and close the abdominal wall and skin separately by using and a 5/0 vicryl absorbable suture ( Figure 2Q,R).

NOTE:
Troubleshooting of different problems, reasoning, and solutions, that can be encountered during and after the cryoablation procedure can be found in Table 1.

Problem Procedure Step Possible Reason Solution
Stomach perforation Figure 2M Extensive exposure to liquid N 2 O This step needs to be adjusted depending on the animal model used. K5-GFP mice are more fragile than CB6F1, therefore the time of exposure needs to be slightly less than with the CB6F1 mice (2× 5 to 8 s for K5-GFP and 2 × 8 to 10 s for CB6F1 mice). Make sure that the stomach is outside the abdominal cavity to avoid exposure of other vital organs to liquid N 2 O.
Bleeding from the stomach Figure 2O Cryoablation of the stomach It is normal to observe some minimal bleeding after cryoablation.
Bleeding from the stomach Figure 2G Damage of the stomach vasculature Make sure to open the stomach at the greater curvature, where there is less vasculature. Apply pressure using a cotton swab for approximately 1 min on the bleeding spot or blood vessel.
Bleeding from the stomach Figure 2I-O Forced grabbing of the stomach wall during ablation Use appropriate forceps to hold the stomach wall during cryoablation. Holding too tight will damage the stomach wall.
Leakage from the stomach incision Figure 2P The stomach is not sutured properly Additional stitches are required.

1.
Remove the mouse from the sterile field and place the mouse back to the +/+IVC cage in the ventilated rack onto a warm pad for the first 24 h postoperative hours.
Observe the mouse recovery from the anesthesia and register any signs of discomfort.

NOTE:
On day 1 after the cryoablation procedure, administer a single s.c. injection of pain killer (5 mg/kg body weight) in case of visible signs of discomfort and a single s.c. injection of saline solution (200-300 µL) when signs of dehydration are observed.
NOTE: Animals need to be euthanized immediately if a humane endpoint is reached after surgery or during the follow up period. In our institution, humane endpoints include severe weight loss (>15% body weight loss compared to the initial weight, an extended period of weight loss without improvement after 48 h) or severe dehydration for more than 72 h without improvement after s.c. saline injections.
On day 5 post-cryoablation replace antibiotic water by tap water ad libitum.

1.
Cull the mice at the pre-determined experimental endpoints (t = 7, 14, and 21 days) by placing the mice in a CO 2 chamber. 2.
Immediately after culling, remove mice from the CO 2 chamber, shave the abdominal area, and open using a round tips scissor by making an upper-midline vertical incision of the abdomen.
Holding the isolated stomach, with the stomach curvature upwards, open the stomach from the duodenum opening to the forestomach part along the greater curvature ( Figure 3A).

5.
Remove the stomach content and clean with PBS.

6.
Pin the open stomach to a paraffin block to flatten the stomach for histological preparation.    ) overnight. 5. Transfer the cassettes to an embedding station and cut the fixed tissue in four pieces as represented in Figure 3D,F. 6. Place tissue pieces in a mold and embed it in paraffin. 7. Cut the tissue paraffin blocks as 5 µm sections and place onto a slide. 8. Dry the tissue slides at 37 °C for at least 16 h. Fix the pinned stomach into 10% neutral buffered formalin solution for 24 h.

2.
Place the tissue pieces flat inside a tinned-metal sample container or a cryovial.

3.
Snap frozen in liquid nitrogen and store the samples at −80 • C until further analysis. In a CryoStat station, remove the cryopreserved tissue and place it onto a cold base.

3.
Place the mold in the Peltier fast-freezing platform of the cryostat to ensure the tissue is frozen before sectioning. 4.
Cut the frozen tissue block into 6 µm sections and place onto a Superfrost Plus adhesion glass slide.

5.
Dry the slides at room temperature and store them in a slide box at −20 • C for later use. 6.
Stain the slides with DAPI (1:1000) for nuclear staining and visualize under a fluorescent microscope.

Establishment of the Cryoablation Mouse Model
To establish the cryoablation model, the SCJ of the stomach in wild-type CB6F1 mice was ablated and histologically analyzed at day 7, 14, and 21 post-cryoablation on hematoxylin and eosin (HE) staining tissue slides. The mouse stomach consists of two main compartments, the forestomach, lined with multilayered keratinizing squamous epithelium and the glandular stomach, lined with single layered columnar epithelium. The two compartments are delineated by the SCJ as represented in Figure 3B,C. All mice received proton pump inhibition after the ablation and daily during the healing process. When compared to the not ablated side of the stomach, we observed that 7 days after the ablation the SCJ was damaged and inflamed, and both the columnar and squamous cells were absent, indicating the success of the cryoablation to eliminate both type of epithelia at the SCJ ( Figure 3D). Fourteen days post-cryoablation, we found regeneration of normally appearing neo-squamous and neo-columnar proximal and distal to the SCJ in approximately 60% of the cryoablated mice. Representative HE stainings of nearly completed regenerated cryoablated mouse stomach at day 14 is demonstrated in Figure  3E. After 21 days, the ablated epithelia were completely regenerated with neo-epithelia and their histology was comparable to that of the not ablated stomach side ( Figure 3F). Macroscopic analysis of surrounded tissues after culling, didn't show any tissue injury was observed at 14 and 21 days post-cryoablation.

Intervening with the Regenerative Process of the Neo-Columnar Epithelium at the SCJ
We performed a proof-of-principle study to validate the cryoablation model by testing an unselective inhibitor for bone morphogenetic proteins (BMPs), Noggin, which inhibits BMP2 and BMP4, among other BMPs [27]. BMP proteins are critical for columnar cell regeneration and the homeostasis of columnar epithelia [28]. For this purpose, K5-GFP lineage-tracing mice were used to trace the proliferation of K5 positive cells while mice were treated with either vehicle (control) or with vehicle plus mouse Noggin (mNoggin) ( Figure 4A). We observed that in cryoablated vehicle-treated K5-GFP mice, the renewal of epithelium in the SCJ is similar to the not cryoablated untreated K5-GFP mice. The number of K5 positive cells was higher and cells more intensely expressed K5-GFP in the cryoablated vehicle-treated K5-GFP mice. This is probably due to the rapid proliferation of K5 positive squamous (stem) cells of the cryoablated region of the stomach to regenerate the squamous mucosa ( Figure 4B,C). In the mNoggin treated K5-GFP mice, regeneration of the columnar region failed and instead a neo-squamous tissue was observed similar to the squamous region in control mice. In addition, the squamous region extended to the ablated columnar zone distally of the SCJ. The regeneration of columnar epithelium was therefore inhibited and was replaced by squamous epithelium. Thus, it seems that Noggin inhibits the regeneration of the columnar epithelium by inhibiting several BMPs, while the regeneration of the squamous epithelium is not affected ( Figure 4D).

Intervening with the Regenerative Process of the Neo-Columnar Epithelium at the SCJ
We performed a proof-of-principle study to validate the cryoablation model by testing an unselective inhibitor for bone morphogenetic proteins (BMPs), Noggin, which inhibits BMP2 and BMP4, among other BMPs [27]. BMP proteins are critical for columnar cell regeneration and the homeostasis of columnar epithelia [28]. For this purpose, K5-GFP lineage-tracing mice were used to trace the proliferation of K5 positive cells while mice were treated with either vehicle (control) or with vehicle plus mouse Noggin (mNoggin) ( Figure 4A). We observed that in cryoablated vehicle-treated K5-GFP mice, the renewal of epithelium in the SCJ is similar to the not cryoablated untreated K5-GFP mice. The number of K5 positive cells was higher and cells more intensely expressed K5-GFP in the cryoablated vehicle-treated K5-GFP mice. This is probably due to the rapid proliferation of K5 positive squamous (stem) cells of the cryoablated region of the stomach to regenerate the squamous mucosa ( Figure 4B,C). In the mNoggin treated K5-GFP mice, regeneration of the columnar region failed and instead a neo-squamous tissue was observed similar to the squamous region in control mice. In addition, the squamous region extended to the ablated columnar zone distally of the SCJ. The regeneration of columnar epithelium was therefore inhibited and was replaced by squamous epithelium. Thus, it seems that Noggin inhibits the regeneration of the columnar epithelium by inhibiting several BMPs, while the regeneration of the squamous epithelium is not affected. ( Figure  4D).

Conclusions
To date, due to the lack of good pre-clinical models, no promising molecular targeting therapies were developed for premalignant columnar lesions. Here, we developed a novel mouse model using wild-type and genetically modified mice. This model allows to study the cellular changes and regeneration process that occur upon cryoablation of epithelial tissue at the SCJ. We developed this model as a platform to be

Conclusions
To date, due to the lack of good pre-clinical models, no promising molecular targeting therapies were developed for premalignant columnar lesions. Here, we developed a novel mouse model using wild-type and genetically modified mice. This model allows to study the cellular changes and regeneration process that occur upon cryoablation of epithelial tissue at the SCJ. We developed this model as a platform to be able to study potential therapies for the treatment of neo-epithelia that occurs at the SCJ in stomach and esophagus. We believe that the model can also be translated to other organs in which neo-epithelia develop in transitional zones of squamous to columnar mucosa, but further validation will be necessary. Ablative therapies are frequently used for treatment of epithelial lesions, for instance, as applied in the esophagus for treatment of Barrett's esophagus. In our set up, we complemented the ablative procedure with an inhibitor of BMPs to illustrate the effects on the regeneration of columnar epithelium at the SCJ. In our protocol, this inhibitor (mNoggin) was locally applied to prevent systemic off target effects. The cryoablation surgical model described here is a viable model, where data material can be obtained in a reasonable timeline of about 2 to 3 weeks, after starting the experiment. This procedure was successful in two different mouse models, and we believe that it can also be used in other mouse models and applied to other organs. Both CB6F1 and lineage tracing K5-GFP mice showed similar recovery time and recuperating to their normal histology after cryoablation.
In our protocol, mice were treated with acid suppression medication (PPIs-proton pump inhibitors) to reduce the acidic environment in the stomach of the mice. We believe that this approach, together with a targeted molecular therapy, will be required in the clinic for a better and efficient regeneration of the neo-epithelium, as shown in our animal model.
Targeting BMP signaling revealed to be an effective molecular candidate for the treatment of columnar metaplasia at the SCJ. To our knowledge, there are no other targeted therapies specifically developed for columnar pre-neoplastic lesions. Noggin is a nonselective BMP inhibitor, and given its pleiotropic effects, it is not suitable for systemic treatment in humans. Recently, we developed novel highly selective inhibitors against BMP2 and BMP4 [29,30]. Future studies will demonstrate the efficacy and safety of these novel anti-BMP therapies and their use for treatment of BE.
In sum, this novel in vivo model shows to be a potential tool to test promising targeting drugs, for instance, to complement the ablative therapies used for the treatment of metaplasia and neo-epithelia, including Barrett's esophagus.