Relative Abundance of SARS-CoV-2 Entry Genes in the Enterocytes of the Lower Gastrointestinal Tract Jaewon J. Lee,1,2,3 Scott Kopetz,4 Eduardo Vilar,4,5 John Paul Shen,4 Ken Chen,6 Anirban Maitra1,2 1Department of Translational Molecular Pathology 2Sheikh Ahmed Center for Pancreatic Cancer Research 3Department of Surgical Oncology 4Department of Gastrointestinal Medical Oncology 5Department of Clinical Cancer Prevention 6Department of Bioinformatics and Computational Biology The University of Texas MD Anderson Cancer Center Correspondence: Anirban Maitra, M.B.B.S. Sheikh Ahmed Center for Pancreatic Cancer Research Department of Translational Molecular Pathology The University of Texas MD Anderson Cancer Center 6565 MD Anderson Blvd Houston, TX 77030 Email: amaitra@mdanderson.org Conflict of Interest: A.M. receives royalties for a pancreatic cancer biomarker test from Cosmos Wisdom Biotechnology, and this financial relationship is managed and monitored by the UTMDACC Conflict of Interest Committee. A.M. is also listed as an inventor on a patent that has been licensed by Johns Hopkins University to Thrive Earlier Detection. E.V. has a consulting and advisory role with Janssen research and Development. The remaining authors disclose no conflicts.

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COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2

(SARS-CoV-2), has rapidly spread throughout the world and was declared a pandemic by

the World Health Organization, thus leading to a rapid surge in the efforts to understand

the mechanisms of transmission, methods of prevention, and potential therapies. While

COVID-19 frequently manifests as a respiratory infection,1 there is evidence for infection of

the gastrointestinal (GI) tract1-4 with documented viral RNA shedding in the stool of

infected patients.2,4 In this study, we aimed to investigate the expression of ACE2 and

TMPRSS2, which are required for SARS-CoV-2 entry into mammalian cells,5 from single-

cell RNA sequencing (scRNA-seq) datasets of five different parts of the GI tract:

esophagus, stomach, pancreas, small intestine, and colon/rectum.

Methods

We utilized previously published scRNA-seq data, which are summarized in

Supplementary Table 1. For in-house normal colon samples, a total of 7 patients were

recruited at the University of Texas MD Anderson Cancer Center through written informed

consent following Institutional Review Board approval. Single cell dissociation, library

generation, and sequencing were performed following standard protocols described in

Supplementary Materials.

Results

Analysis of scRNA-seq was performed separately for each GI segment and

available cell identities from the original studies were assigned to the new clusters after

confirming the expression of relevant marker genes (Figure 1). Overall, there were 82,422

cells from the esophagus, 18,405 cells from the stomach, 13,407 cells from the pancreas,

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9,858 cells from the small intestine, and 20,161 cells from the colon and rectum

(Supplementary Table 1). The proportions of cells expressing ACE2 were approximately

16-fold lower in the upper GI tract (esophagus, stomach; 1% or 1,004/100,827 cells) than

in the lower GI tract (ileum, colon, rectum; 16% or 4,777/30,019 cells), and overall, higher

proportions of cells expressed TMPRSS2 than ACE2 throughout the GI tract

(Supplementary Figures 1A-E).

Of note, higher proportions of esophageal columnar cells – which were mostly from

Barrett’s esophagus samples – co-expressed ACE2 and TMPRSS2, compared to the

native squamous epithelium (Figure 1A). Pancreatic ductal and acinar cells co-expressed

ACE2 and TMPRSS2 but endocrine cells did not show detectable co-expression (Figure

1C). Parenthetically, the expression of TMPRSS2 in acinar cells underscores the rationale

for using a TMPRSS2 inhibitor (camostat mesylate) in acute pancreatitis. In fact, this agent

is now undergoing early phase clinical trials in COVID-19 patients.5

Taken together, within the GI tract, the co-expression of ACE2 and TMPRSS2

transcripts was highest in the small intestine and colon. Approximately 20% of enterocytes

from the small intestine and 5% of colonocytes (enterocytes from colon/rectum) co-

expressed ACE2 and TMPRSS2 (Figure 1D-E). Analysis of 308 genes that were positively

correlated (Pearson’s r > 0.1) with ACE2 in the small intestine revealed the enrichment of

functional Gene Ontology (GO) terms such as metabolic and transport pathways in the

small intestine (Supplementary Figure 1F), thus confirming the inherent digestive and

absorptive functions of enterocytes. Interestingly, 251 genes positively correlated with

ACE2 in the colon and rectum showed enrichment of not only secretion-associated

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pathways but also immune-related processes (Supplementary Figure 1G), which may be

upregulated in functional colonocytes due to the microbiome.

Discussion

To summarize, our results confirm the co-expression of SARS-CoV-2 entry genes

ACE2 and TMPRSS2 in a subset of epithelial cells in the GI tract, especially functional

enterocytes from the lower GI tract (small intestine and colon), and provide potential

insights into why entero-colitic symptoms may arise in acute COVID-19 infection. These

findings parallel the findings from recent studies that revealed the co-expression of ACE2

and TMPRSS2 in olfactory epithelium6 and intrahepatic cholangiocytes,7 which may

explain atypical COVID-19 symptoms and laboratory findings such as anosmia/ageusia

and transaminitis. Our results further support the feasibility of SARS-CoV-2 entry into the

GI tract with implications for GI infection and transmission.

Acknowledgements

We thank Charles Bowen for performing single cell dissociations from colonic

samples, Kangyu Lin and Jinzhuang Dou for assistance in data acquisition and transfer,

and the Single Cell Core at MD Anderson Cancer Center.

Funding

This work was supported by the MD Anderson Pancreatic Cancer Moon Shot

Program and the Khalifa Bin Zayed Foundation to A.M.; the Human Breast Cell Atlas Seed

Network Grant HCA3-0000000147 from the Chan Zuckerberg Initiative to K.C.; the NIH

K22 CA234406-01 and the Cancer Prevention and Research Institute of Texas (CPRIT)

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Scholar in Cancer Research to J.P.S.; a gift from the Feinberg Family and MD Anderson

Cancer Center Pre-Cancer Atlas Project to E.V.; the MD Anderson Colorectal Cancer

Moon Shot Program to S.K. and E.V.; the NIH T32 CA009599 to J.J.L.; the NIH/NCI P30

CA016672 to the MD Anderson Cancer Center Core Support Grant; and the CPRIT

SINGLE CORE Facilities Grant RP180684.

References

1. Guan WJ, Ni ZY, Hu Y, et al. N Engl J Med 2020 [E-pub ahead of print].

2. Holshue ML, et al. N Engl J Med 2020;382:929-936.

3. Xiao F, Tang M, Zheng X, et al. Gastroenterology 2020 [E-pub ahead of print].

4. Wolfel R, Corman VM, Guggemos W, et al. Nature 2020 [E-pub ahead of print].

5. Hoffmann M, Kleine-Weber H, et al. Cell 2020 [E-pub ahead of print].

6. Brann DH, Tsukahara T, Weinreb C, et al. bioRxiv 2020.

7. Wen Seow JJ, et al. bioRxiv 2020.

Figure Legends

Figure 1. Uniform manifold approximation and projection plots (left) of scRNA-seq data

from esophagus (A), stomach (B), pancreas (C), small intestine (D), and colon and rectum

(E); bubble plots showing expression levels of cell-type marker genes (middle); and bar

plots depicting proportions of cell types co-expressing ACE2 and TMPRSS2 (right). EEC:

enteroendocrine cell; GMC: antral basal gland mucous cell; PMC: pit mucous cell; TA:

transit-amplifying cell.

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Figure 1

Percentexpressed 25 50 75 100 Mean

expression Low High

A

B

C

D

E

Proportion co-expressingACE2 and TMPRSS2

KRT4ECM1

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EnterocyteProgenitorStemTAGobletPanethEEC

EnterocyteProgenitorPaneth−likeGobletTAStemEECTuft

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