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# A screening system to determine the effect of bacterial metabolites on MAdCAM-1 expression by transformed endothelial sinusoidal cells

Ai-Ling Tian, Marion Leduc, Marine Fidelle, Laurence Zitvogel, Guido Kroemer, Oliver Kepp

► To cite this version:

Ai-Ling Tian, Marion Leduc, Marine Fidelle, Laurence Zitvogel, Guido Kroemer, et al.. A screening system to determine the effect of bacterial metabolites on MAdCAM-1 expression by transformed endothelial sinusoidal cells. Methods in Cell Biology, 2024, 10.1016/bs.mcb.2024.01.007. hal-05143029

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https://hal.science/hal-05143029v1

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Distributed under a Creative Commons Attribution 4.0 International License
A screening system to determine the effect of bacterial
metabolites on MAdCAM-1 expression by transformed
endothelial sinusoidal cells

Ai-Ling Tian¹,², Marion Leduc¹,², Marine Fidelle³⁻⁴, Laurence Zitvogel³⁻⁶,

Guido Kroemer¹,²,⁷*, Oliver Kepp¹,²*

¹Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Center, Université Paris Saclay, Villejuif, France;

²Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université Paris Cité, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France;

³Gustave Roussy, Villejuif, France;

⁴Institut National de la Santé Et de la Recherche Médicale (INSERM) U1015, Equipe Labellisée - Ligue Nationale contre le Cancer, Villejuif, France;

⁵Faculté de Médecine, Université Paris-Saclay, Kremlin-Bicêtre, France;

⁶Center of Clinical Investigations for In Situ Biotherapies of Cancer (BIOTHERIS) INSERM, CIC1428, Villejuif, France;

⁷Institut du Cancer Paris CARPEM, Department of Biology, APHP, Hôpital Européen Georges Pompidou, Paris, France.

*Correspondence to: Dr. Oliver Kepp (captain.olsen@gmail.com) or Dr. Guido Kroemer (kroemer@orange.fr)

**Keywords:** Gut dysbiosis; regulatory T cells; immune checkpoint blockade; high content imaging;

**Word count:** 4232
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Abstract

Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expression in high endothelial venules is regulated by bacterial metabolites emanating from the gut and the interaction of MAdCAM-1 with α4β7 integrin mediates lymphocyte diapedesis into gut-associated secondary lymphoid tissues. MAdCAM-1 thus controls the abundance of circulating immunosuppressive T cells that can reach malignant tissue and compromise the therapeutic efficacy of anticancer immunotherapy. Here we describe a biosensor-based phenotypic assessment that facilitates the high throughput screening (HTS)-compatible assessment of MAdCAM-1 regulation in response to exposure to bacterial metabolites. This screening routine encompasses high endothelial venule cells expressing green fluorescent protein (GFP) under the control of the MAdCAM-1 promoter combined with robot-assisted bioimaging and a multistep image analysis pipeline. Altogether this system facilitates the discovery of bacterial composites that control anticancer immunity via the sequestration of Th17-specific regulatory T cells (Treg17) in the gut.
Introduction

The composition of the gut microbiome plays a crucial role in innate and adaptive
immunity as it maintains the homeostatic function of gut regulatory T (Treg) cells, thus
impacting the immune response to pathological insults and malignant transformation 1. In
consequence, intestinal dysbiosis has been recognized as general feature of declining
health and is often associated with advanced aging 2, 3. Gut microbial dysbiosis, which
can be caused by the use of broad-spectrum antibiotics, is also associated with the lack of
efficacy of anticancer immunotherapy across various types of malignancies not restricted
gastrointestinal cancers 4. The concomitant use of broad-spectrum antibiotics with
immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis has been shown to
reduce the therapeutic efficacy of the cancer therapy and is associated with disease
progression and poor outcome 5. Mechanistically, both in mice and in cancer patients, gut
dysbiosis resulting from the treatment with broad-spectrum antibiotics arises in part due
to the recolonization of the gut by *Enterocloster* spp. (i.e., *E. clostridioformis*, *E. bolteae*),
which negatively affects the outcome of immunotherapy 5, 6.

In this setting, bacterial composites trigger reprogramming of the tumor
microenvironment (TME) via the regulation of mucosal addressin cell adhesion molecule
1 (MAdCAM-1) expression on high endothelial venules (HEV) in the gut. The
interaction of MAdCAM-1 with α4β7 integrins expressed on T cells increases the
adhesion of lymphocytes to HEV and their consequent diapedesis across the vascular
endothelial barrier to gut-associated secondary lymphoid tissue (GALT). MAdCAM-1 is
constitutively expressed in high HEV. However, the level of expression of MAdCAM-1
can be regulated by inflammatory cytokines and bacterial metabolites 7. The
recolonization following antibiotic treatment of the gut by *E. clostridioformis* impacts the
metabolism of bile acids, which in turn leads to a downregulation of MAdCAM-1
expression 6. The resulting exodus of intestinal immunosuppressive Th17-specific
regulatory T cells (Treg17) cells towards the tumor microenvironment then interferes
with T cell-targeted immunotherapy. Altogether, the MAdCAM-1-α4β7 interaction keeps
Treg17 cells from migrating from the gut to tumors. For this reason, gut dysbiosis, which
decreases MAdCAM-1 expression, compromises cancer immunosurveillance, as well as
the anticancer effects of ICIs.

Here we generated a biological platform for the high throughput screening (HTS)-
compatible assessment of MAdCAM-1 expression that allows to screen bacterial
metabolites as well as pharmacological agents for their effects on the MAdCAM-1-α4β7
axis. This tool can be used for proof-of-concept studies identifying potentially harmful
bacterial composites. In addition, this method can be upscaled to high-throughput
screening campaigns that may lead to the identification of drugs that counteract the
adverse effects of gut dysbiosis.
# Materials and methods

## Cell lines and culture reagents

Culture media and supplements for cell culture were obtained from Life Technologies (Carlsbad, California, USA) and plastic material came from Greiner BioOne (Kremsmünster, Austria) and Corning (Corning, New York, USA). Mouse transformed endothelial sinusoidal cells (TSECs) were a generous gift from Professor B.P. Fennimore (University of Colorado, Aurora, CO, USA). The medium for this cell line is Dulbecco's modified Eagle medium / nutrient mixture F-12 (DMEM/F-12, Catalog No. 11320074, Gibco).

To complement growth medium, following components are added to the base medium:

* fetal bovine serum (FBS) to a final concentration of 10%,

* endothelial cell growth supplement (ECGS, Catalog No. 1052, ScienCell) diluted to 1x,

* 10 U/mL of penicillin sodium and 10 µg/mL of streptomycin sulfate.

TSECs were cultured in an air atmosphere enriched with 5% CO₂ at 37 °C.

## Cell culture procedures

1. Thaw the cells by gentle agitation of the cryovial in a 37 °C water bath. To reduce the possibility of contamination, keep the O-ring and cap from contacting the surface of the water. Thawing should be rapid (approximately 2 min).

2. Remove the vial from the water bath as soon as the contents are thawed and decontaminate by dipping in or spraying with 70% ethanol. All steps from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial's content to a centrifuge tube containing 9.0 mL complete growth medium, and spin at approximately 125 g for 5 to 7 min.

4. Resuspend the cell pellet with complete growth medium. It is important to avoid excessive alkalinity of the medium during the recovery of cells. It is suggested that, prior to the addition of the cells, the culture vessel containing the complete growth medium is placed in a cell culture incubator for at least 15 min to allow the medium to stabilize pH (7.0 to 7.6).

5. Incubate the culture in an air atmosphere enriched with 5% CO₂ at 37 °C.

## Sub-culture procedures

1. Remove and discard the culture medium.

2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.03% (w/v) EDTA solution to remove all traces of serum which contain trypsin inhibitor.

3. Add 1 to 2 mL of Trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer begins to detach.

4. Add 6 to 8 mL of complete growth medium and aspirate cells by gently pipetting.

5. Add appropriate aliquots of cell suspension to new culture vessels.

6. Every 2 to 3 days renew the medium.

## Establishment of Geneticin™ selection condition

1. Plate TSECs at a density of 2 x 10⁴ cells/mL in 0.5 mL complete growth medium per well in a 24-well tissue culture plate.

2. Culture overnight. Most cell types should be ≥80% confluent prior to adding the selection antibiotic.
3. Add increasing amounts of Geneticin™ selection antibiotic (G418 sulfate) (Catalog No. 10131027, Gibco) to duplicate wells of cells plated in complete growth medium. We suggest to use 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.5, and 2.0 mg/mL for establishing a killing curve and for identifying the optimal concentration of G418 sulfate for a given cell type. Include a control not containing selection antibiotic.

4. Replace the medium containing selection antibiotic every 2-3 days for up to a week. Examine the culture every day for signs of visual toxicity. Determine the optimal antibiotic dose which corresponds to the lowest antibiotic concentration at which all wild type cells die within 7 days of selection.

5. Proceed with stable cell line generation using the concentration determined in step 4 (in our case 0.5 mg/mL).

## MAdCAM-1 promoter plasmid

### Preparation of linearized vectors

1. Select appropriate cloning site to linearize pAcGFP1-1 vector (Catalog No. PT3846-5, Takara).

2. The linearized vector can be obtained by digesting the circular vector with EcoRI (Catalog No. R3101, NEB) and SacII (Catalog No. R0157, NEB). Set up reaction as follows:

<table><tr><td>Component</td><td>50 µL reaction</td></tr><tr><td>DNA</td><td>1 µg</td></tr><tr><td>10x rCutSmart buffer</td><td>5 µL (1X)</td></tr><tr><td>EcoRI-HF</td><td>1.0 µL (20 units)</td></tr><tr><td>SacII</td><td>1.0 µL (20 units)</td></tr></table>
<table><tr><td>Nuclease-free water</td><td>to 50 µL</td></tr></table>

3. Incubate at 37 °C overnight.

## Preparation of inserts

1. The general principle for the primer design: insertion of a homologous sequence (15-20 bp, excluding restriction sites) into the 5' ends of both forward & reverse primers.
Thereby, the ends of amplified insert and linearized vectors are complementary.

* Forward primer: 5'tcgagctcaagcttcgaattcTACCCCCACAGGCCTGCC -3'

* Reverse primer: 5'cggtggatcccgggcccgcggGGGCCGGCAGCTTCCTAC -3'

2. PCR amplification of MAdCAM-1 promoter sequence. Reaction setup:

<table><thead><tr><th>Component</th><th>50 µL<br>reaction</th><th>Final<br>concentration</th></tr></thead><tbody><tr><td>10 µM forward primer</td><td>1 µL</td><td>0.2 µM</td></tr><tr><td>10 µM reverse primer</td><td>1 µL</td><td>0.2 µM</td></tr><tr><td>Template DNA*</td><td>1 µg</td><td>1 µg</td></tr><tr><td>OneTaq® Hot Start Quick-Load®<br>2x Master Mix with Standard<br>Buffer (Catalog No. M0488, NEB)</td><td>25 µL</td><td>1x</td></tr><tr><td>Nuclease-free water</td><td>to 50 µL</td><td></td></tr></tbody></table>

* Template DNA: mMAdCAM-1 Promotor_pUC57-Kan (Catalog No. U135WGI270, synthesized by GenScript)

3. Transfer PCR tubes to a PCR machine and begin thermocycling. Thermocycling conditions for a routine PCR:

<table><thead><tr><th>Step</th><th>Temperature</th><th>Time</th></tr></thead><tbody><tr><td>Initial denaturation</td><td>94 °C</td><td>30 s</td></tr><tr><td rowspan="3">30 cycles of<br>denaturation,<br>annealing, extension</td><td>94 °C</td><td>30 s</td></tr><tr><td>68 °C</td><td>60 s</td></tr><tr><td>68 °C</td><td>2 min</td></tr><tr><td>Final extension</td><td>68 °C</td><td>5 min</td></tr></tbody></table>
Hold

4 °C

## Ratio of vector to insert

For ClonExpress MultiS recombination reaction system, the optimal amount is 0.03 pmol per fragments (including linearized vector). This mass can be roughly calculated according to the following formula:

* The optimal mass of vector required = [0.02 x number of base pairs] ng (0.03 pmol)

For example, our cloning inserts of 2 kb into the pAcGFP1-1 vector of 4.1 kb, the optimal mass of the vector and insert are as follows:

* The optimal mass of linearized vector required: 0.02 x 4,100 = 82 ng

* The optimal mass of insert of 2 kb required: 0.02 x 2,000 = 40 ng

## Recombination

1. The amount of DNA can be roughly calculated according to the above formula.

2. Dilute the pAcGFP1-1 vector and insert at an appropriate ratio to ensure the accuracy of pipetting before preparing the recombination reaction system, and the amount of each component is not less than 1 µL.

3. Prepare the following reaction on ice by using ClonExpress® MultiS One Step Cloning Kit (Catalog No. C113, Vazyme):

<table><thead><tr><th>Components</th><th>Recombination</th><th>Negative control-1<sup>b</sup></th><th>Negative control-2<sup>c</sup></th><th>Positive control<sup>d</sup></th></tr></thead><tbody><tr><td>Linearized vector<sup>a</sup></td><td>X µL</td><td>X µL</td><td>0 µL</td><td>1 µL</td></tr><tr><td>Insert<sup>a</sup></td><td>Y<sub>1</sub> Y<sub>n</sub> µL</td><td>0 µL</td><td>Y<sub>1</sub> Y<sub>n</sub> µL</td><td>1 µL</td></tr><tr><td>5 × CE MultiS</td><td>4 µL</td><td>0 µL</td><td>0 µL</td><td>4 µL</td></tr></tbody></table>
<table><thead><tr><th colspan="5">buffer</th></tr></thead><tbody><tr><td>Exnase MultiS</td><td>2 µL</td><td>0 µL</td><td>0 µL</td><td>2 µL</td></tr><tr><td>ddH<sub>2</sub>O</td><td>to 20 µL</td><td>to 20 µL</td><td>to 20 µL</td><td>to 20 µL</td></tr></tbody></table>

a X/Y is the amount of calculated vector/insert ratio.

b It is recommended to use negative control-1, which can confirm the residue of the cyclic plasmid template.

c It is recommended to use negative control-2, when the amplification template of the insert is a circular plasmid with the same antibiotic resistance as the cloning vector. It is recommended to perform the circular plasmid residue detection of the linearized vector and inserts independently.

d It is recommended to include positive controls to exclude technical problems.

4. Gently pipette up and down several times to mix thoroughly (DO NOT VORTEX!).

Briefly centrifuge to collect the reaction mix to the bottom of the tube.

5. Incubate at 37 °C for 30 min and immediately chill the tube at 4 °C or on ice.

## High efficiency transformation

1. Thaw a tube of NEB 5-alpha competent *E. coli* cells (Catalog No. C2987H, NEB) on ice for 10 min.

2. Pipette 5 µL of the recombination products to the cell mixture.

3. Carefully flick the tube wall 4-5 times to mix thoroughly (DO NOT VORTEX!).

4. Place the mixture on ice for 30 min.

5. Heat shock at exactly 42 °C for exactly 45 seconds and then immediately place on ice for 5 min.

6. Pipette 950 µL of room temperature SOC liquid medium (without antibiotics) into the mixture.

7. Shake vigorously at 37 °C for 60 min at 250 rpm.

8. Preheat the kanamycin (50 µg/mL) resistant LB solid medium plates in a 37 incubator.

9. Centrifuge the culture at 2,400 g for 5 min and discard 900 µL of supernatant.

10. Use the remaining medium to suspend the bacteria.
11. Use a sterile bent glass rod to gently spread onto the plate containing kanamycin (50 µg/mL) resistant.

12. Incubate at 37 °C for 12-16 h.

## Clone picking and DNA purification

1. After overnight incubation, hundreds of single clones will form on the plate of recombination reaction, whereas none of those will grow on the plate of the negative control(s).

2. Pick several clones from the plate of recombination reaction for colony PCR with at least one universal sequencing primer of the vector. If the colony is positive, a band slightly larger than the size of the inserts should appear.

3. Inoculate the remaining bacterial solution of positive clones into liquid LB medium containing kanamycin (50 µg/mL) for overnight incubation.

4. Purify the plasmid using the Monarch® plasmid miniprep kit (Catalog No. T1010, NEB) for restriction endonuclease digestion identification, and perform DNA sequencing to check for the correct insertion of the MAdCAM-1 promoter at the expected EcoRI and SacII restriction site in the pAcGFP1-1 plasmid using the following primers:

<table><thead><tr><td>Name</td><td>Sequence 5' to 3'</td></tr></thead><tbody><tr><td>mSeqPrimer1</td><td>GATTCTGTGGATAACCGTATTACC</td></tr><tr><td>mSeqPrimer2</td><td>CCTGTACATCCAGTCAGCAC</td></tr><tr><td>mSeqPrimer3</td><td>AGATTTCCATGCACTTGACC</td></tr></tbody></table>
5. Identify the correct pAcGFP1-1 construct and grow in ~200 mL of liquid LB medium containing kanamycin (50 µg/mL) resistant in order to prepare a larger amount of DNA.

6. Purify the plasmid DNA using the PureLink™ expi endotoxin-free maxi plasmid purification kit (Catalog No. A31231, Invitrogen) and determine the concentration using a Nanodrop device (Thermo Fisher Scientific).

MAdCAM-1 promoter reporter cell line

Cell seeding

1. To prepare cells, collect enough cells to complete the transfection experiment, and centrifuge for 5 min at 300 g in a swing-bucket rotor. Suspend the cell pellet to an appropriate concentration in medium.

2. Plate 0.5 x 10⁵ TSECs in 1 mL of complete growth medium per well of a 6-well plate.

Preparing the FuGENE® HD transfection reagent

1. Before use, allow the vial of FuGENE® HD transfection reagent (Catalog No. E2311, Promega) to reach room temperature.

2. Mix by inverting or vortexing briefly. No precipitate should be visible.

Transfection

1. The total volume of medium, DNA, and FuGENE® HD transfection reagent to add per well of a 6-well plate is 80 µL. To a sterile tube, add 920 µL of complete growth medium prewarmed to room temperature so that the final volume after addition of DNA is 1 mL. Add 20 µg of plasmid DNA (1 µg/µL) to the medium, and vortex. For a 3:1 FuGENE® HD transfection reagent:DNA ratio, add 60 µL of FuGENE® HD transfection reagent, and mix immediately.
2. Incubate the FuGENE® HD transfection reagent/DNA mixture for 15 min at room temperature.

3. Add 80 µL of the FuGENE® HD transfection reagent/DNA mixture per well to the 6-well plate containing 1 mL of cells in the complete growth medium. Mix well by shaking.

4. Return cells to an air atmosphere enriched with 5% CO₂ at 37 °C for 48 h.

5. Forty-eight hours post-transfection, harvest adherent cells and plate at several different dilutions (e.g., 1:2, 1:5, 1:10) in selection medium (complete growth medium containing 0.5 mg/mL of G418).

6. For the next 14 days, replace the selection medium every 3 to 4 days.

7. During the second week, monitor cells for distinct the formation of colonies. Complete cell death should occur in cultures transfected with the negative control plasmid.

8. The G418-resistant cells are sorted into single-cell clones in 96-well plates (one cell per well) to establish a TSEC::MAdCAM-1-GFP stable cell line expressing GFP under the control of the MAdCAM-1 promoter.

## Identification of positive clones

1. Once the clones start forming colonies visible by light microscopy, the exhausted medium is removed and 50 µL of Trypsin-EDTA solution is dropped into the well to detach the cells. Trypsin-mediated digestion is stopped by the addition of 150 µL fresh selective medium, and the cellular suspension is transferred to a new 96-well plate.
2. The plate that holds the selected clones is duplicated and one plate is kept as a maintenance culture whereas the other is cultured under the stimulation of recombinant murine TNF-α (20 ng/mL, Catalog No. 315-01A, PeproTech) and IL-1β (20 ng/mL, Catalog No. 211-11B, PeproTech) in the presence of 5% CO₂ at 37 °C for 48 h.

3. Clones that homogeneously express GFP are selected based on fluorescence microscopic assessment compared with the same but non-treated control clones. Selected clones are named TSEC::MAdCAM-1-GFP cells (Fig. 2A).

## Treatment with bacterial metabolites (Fig. 1B)

1. Recombinant murine TNF-α and IL-1β were diluted in culture media from a stock of 40 µg/mL at the time of treatment. Bile acids (lithocholic acid (LCA) (Catalog No. 700218P, Sigma-Aldrich), sodium glycodeoxycholate (GDCA) (Catalog No. G9910, Sigma-Aldrich), sodium chenodeoxycholate (CDCA) (Catalog No. C8261, Sigma-Aldrich), deoxycholic acid (DCA) (Catalog No. 30960, Sigma-Aldrich), tauroursodeoxycholic acid (TUDCA) (Catalog No. 580549, Sigma-Aldrich), glycoursodeoxycholic acid (GUDCA) (Catalog No. 06863, Sigma-Aldrich), taurodeoxycholic acid (TDCA) (Catalog No. 580221, Sigma-Aldrich) dissolved in DMSO were diluted 1:1,000 in culture media to a final concentration of 100 µM.

2. TSEC::MAdCAM-1-GFP cells were seeded in 96-well µclear imaging plates (Greiner BioOne) and allowed to adapt overnight.

3. Cells were treated with the indicated agents for 48 h at 37 °C with 5% CO₂, then fixed with 3.7% paraformaldehyde (PFA, w/v in PBS) (Catalog No. F8775,
Sigma-Aldrich) containing 2 µg/mL of Hoechst 33342 (Catalog No. H3570, Invitrogen) at 4°C overnight.

4. Subsequently, the fixative was exchanged with PBS for the further microscope scanning.

## Image acquisition and processing

Fixed plates underwent comprehensive analysis through automated microscopy. Image acquisition was conducted employing the ImageXpress Micro C automated confocal microscope (Molecular Devices, Sunnyvale, California, USA), equipped with a 20× PlanApo objective lens (Nikon, Tokyo, Japan). Subsequently, automated image processing was executed utilizing the R software, with both the EBImage package (accessible from the Bioconductor repository at https://www.bioconductor.org) and the MorphR package (available from the GitHub repository at https://github.com/kroemerlab/MorphR). Normalization of images was carried out on a plate-wise basis, employing the pixel intensity distribution of control samples. This normalization procedure aimed to eliminate extreme intensities and enhance image contrast. Nuclei were detected utilizing a combination of adaptive thresholding, followed by dimension reduction through maximum projection, succeeded by three morphological operations to refine the mask, and finally, a watershed transformation to segment individual objects. For FITC images, a normalization process was applied within an automatically computed range of intensities to enhance cytoplasmic signal. Cytoplasmic masks were generated through a sigmoid transformation in conjunction with Otsu thresholding. Subsequent morphological operations were applied to further refine the
cytoplasmic mask, resulting in object labeling. The extraction of cellular parameters was
performed utilizing distinct masks.

Data analysis

After exclusion of cellular debris and non-viable cells, the data underwent normalization,
followed by rigorous statistical evaluation and graphical representation. Also using R
software, representative images were extracted and pixel intensities scaled (to the same
extent for all images of a given experiment). A minimum of four distinct view fields were
systematically analyzed per well, and each experimental assessment was performed in
quadruplicate. The results obtained from the wells were subjected to comprehensive
statistical analysis employing one-way Mann-Withney U-test and were then graphically
displayed using GraphPad Prism 9.

Concluding remarks

The interplay between bacteria and the host organism has been largely explored 8-10.
Nevertheless, the mechanistic exploration of gut microbial dysbiosis is limited by the
lack of high-throughput assays compatible with large-scale screening efforts in
appropriate cells types. Here we developed a promoter reporter system in high
endothelial venule cells that allows for an automation friendly assessment of MAdCAM-
1 expression. Combined with robotized workflows, image-based analysis routines and
standardized data processing the described platform allows for the identification of
bacterial metabolites and pharmacological agents that reduce or enhance MAdCAM-1
expression. Metabolites and agents identified with this routine can impact innate and
adaptive immunity and might thus be of translational value in the area of immuno-
oncology.
## Notes

1. Replace cell culture medium every 2-3 days. Cells should be passaged after reaching 70-80% confluency.

2. Corning® T-75 flasks (Catalog No. 430641) are recommended for the culture of TSECs. A subcultivation ratio of 1:50 to 1:100 is recommended.

3. Gently mix the PCR amplification reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.

4. NEB 5-alpha Competent *E. coli* cells are best thawed on ice and DNA is added as soon as the last ice in the tube disappears. Cells can also be thawed by hand, but warming above 0 °C will decrease the transformation efficiency.

5. The volume of recombination products when needs high efficiency transformation should be ≤1/10 of the volume of competent cells.

6. Do not use cells after passage 9-10 as this may lead to reduced viabilities and transfection efficiencies.

7. Cells should be passaged 2-5 days before transfection depending on growth rate of cells.

8. Plate adherent cells one day before transfection so that cells are approximately 80% confluent on the day of transfection.

9. Cells transfected with a plasmid harboring the neomycin resistance (*neo*) gene should be grown in the complete growth medium for 48-72 h post-transfection before the selection antibiotic is applied.
10. Image quality of fluorescent intensity in the GFP channel can be ameliorated by using a maximum projection of a Z-stack.

11. The exposure time is adjusted according to the signal intensity of each channel.

## Disclosures

OK and GK have been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Tollys, and Vascage. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. OK is a scientific co-founder of Samsara Therapeutics. GK is in the scientific advisory boards of Hevolution, Institut Servier and Longevity Vision Funds. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK's brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim.

## Acknowledgments

MF is supported by the SEERAVE Foundation, OK receives funding from Institut National du Cancer (INCa); GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets blancs; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); European Research Council (ICD-Cancer), European Union Horizon 2020 Projects Oncobiome and Crimson; Fondation Carrefour; INCa; Institut Universitaire de France; LabEx Immuno-Oncology (ANR-18-IDEX-0001); a Cancer Research ASPIRE Award from the Mark Foundation; the RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM). This study contributes to the IdEx Université de Paris ANR-18-IDEX-0001.
<figure><img src="image_4.png" /></figure>
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**Figure legends**

**Fig. 1 Plasmid map and treatment scheme.** The MAdCAM-1-GFP promoter reporter plasmid (A) was used to generate TSEC::MAdCAM-1-GFP cells, which then were employed for high throughput screening campaigns according to the depicted schedule (B).

**Fig. 2 Effect of *Enterocloster clostridioformis*-induced perturbation of ileal biliary acids on MAdCAM-1 expression.** TSEC::MAdCAM-1-GFP cells exposed to multiple bile acids at a final concentration of 100 µM were used in high content confocal microscopy. Representative images are shown in (A). Scale bar equals 10 µm. Results are depicted as normalized cell fluorescent intensity of GFP (B). Mean±SD of triplicates from four independent experiments. Comparisons between groups were analyzed using one-way ANOVA.
<figure><img src="image_5.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>