Xiaodong Jia , Chengliang Yin, Shanshan Lu, Yan Chen, Qingyan Liu, Junfan Bai, Lei Zhi, Yinying Lu,5,6＊

Abstract The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is threatening public health. The human angiotensin-converting enzyme 2 (ACE2) has a remarkably high affinity binding to SARS-CoV-2. This study explored whether certain populations, including obese and cancer patients, are susceptible to SARS-CoV-2. The expression pattern of ACE2 in normal and tumor tissues of cancer patients was compared by the search for network database and re-analysis of pubic data.The level of ACE2 expression in normal adipose tissue was higher than that in normal lung tissue, which indicated the adipose tissue might be vulnerable to SARS-CoV-2; the levels of ACE2 expressed by adipocytes and adipose progenitor cells were similar between non-obese individuals and obese individuals (BMI ＞ 29), but obese individuals have more adiposes so as to increase the number of ACE2-expressing cells; the expression of ACE2 in tumor tissues posed by five different types of cancers increased significantly compared with that in adjacent tissues. Therefore, we proposed the following hypothesis: the obese individuals and five types of cancer patients might have a higher risk of SARS-CoV-2 infection, which might become the target population of SARS-CoV-2 infection in the future.

Keywords: COVID-19; adipose tissue; obesity; cancer; ACE2

Introduction

The coronavirus disease 2019 (COVID-19) is an infectious disease,which results in fever, respiratory symptoms, and intestinal symptoms [1]. It is threatening public health around the world, and kills countless people. Some diagnostic tools were developed to detect the early infectious patients effectively [2]. And some drugs including antibodies and traditional herbal medicine were used for treatment of COVID-19. However, the spread of COVID-19 has not stopped.

COVID-19 was caused by the serve acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [3]. SARS-CoV-2, respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) belong to a coronavirus family,which is an enveloped single-stranded RNA virus [4]. The spike protein 3-D structure of SARS-CoV-2 is similar to that of SARSCoV, and hence SARS-CoV-2 is also feasible to bind to angiotensinconverting enzyme 2 (ACE2) [5]. ACE2 regulates the levels of angiotensin II (Ang II) and Ang-(1-7) to exert its physiological functions [6]. The binding affinity between ACE2 and SARS-CoV-2 is approximately 10- to 20-fold higher than that between ACE2 and SARS-CoV [7]. Therefore, those ACE2-expressing cells and tissues,potentially act as targets of the novel coronavirus [8].

The adipose tissues could be infected by some virus, like H5N1,HIV [9, 10]. And obesity influence the mortality and transmission of influenza virus [11, 12]. Whether adipose is potentially infected by SARS-CoV-2 and whether obesity is associated with COVID-19 are unknown. In addition, although the COVID-19 patients with cancers have higher severe events [13], is the risk of different types of cancer patients facing with SARS-CoV-2 similar? The above problems were explored in this research.

Materials and methods

Tissue ACE2 expression analysis

Four network databases, HCCDB [22] (http://lifeome.net/database/hccdb/home.html), HPA [23,24](https://www.proteinatlas.org/),UALCAN[25] (http://ualcan.path.uab.edu/analysis.html) and GEPIA2 [26] (http://gepia.cancer-pku.cn/index.html), were used to search ACE2 gene. All data are available directly online. Enter the ACE2 gene directly into the search box of the database to get the results.

Cell ACE2 expression analysis

The cohort of obese and non-obese individuals was derived from public datasets (GEO: GSE80654), including RNA expression profiling by microarray of major white adipose tissue cell types(adipocytes from 7 non-obese and 7 obese human adipose tissue,progenitor cells from 10 non-obese and 9 obese human adipose tissue). Gene expression data was extracted and analyzed in R 3.4.4 statistical software.

Hypothesis

ACE2-expressing cells and tissues are vulnerable to be targets of SARS-CoV-2 [8]. We have compared the expression of ACE2 in different normal and tumor tissues through the network databases and re-analysis of pubic data. In consequence, we proposed the following hypothesis: the adipose tissues, and five types of tumor tissues were found to have a higher expression of ACE2, which suggests that the obese individuals and the patients with the indicated cancer should be more vigilant in response to SARSCoV-2 infection.

Adipose tissue might be a key target of SARS-Cov-2

We have searched the SARS-Cov-2 receptor ACE2 in normal tissues.As a result, we found the expression of ACE2 in gallbladder and adipose tissue (including subcutaneous and visceral) is significantly higher than that in lung (Figure 1).

Five types of cancer patients might be at higher risk of contracting SARS-Cov-

Figure 1. ACE2 expressions of different normal tissues were analyzed using two databases, HCCDB (a) and HPA (b). The red box indicates adipose tissue, and the blue box indicates gallbladder.

Through analyzing the ACE2 expression level in cancers, we found five types of cancers including CESC (Cervical squamous cell carcinoma and endocervical adenocarcinoma), PAAD (Pancreatic adenocarcinoma), READ (Rectum adenocarcinoma), KIRP (Kidney renal papillary cell carcinoma) and KIRC (Kidney renal clear cell carcinoma) in tumor tissues expressed more ACE2 than adjacent tissues (Figure 2).

Evaluation and empirical data

Adipose tissue might be a key target of SARS-Cov-2

We have employed two databases, HCCDB and HPA, to search the level, it shows that the expression of ACE2 in heart, kidney, testis,intestine and bladder is higher than that in lung, which is consistent with the previous report by Zin Zhou et al [8]. Moreover, we found the expression of ACE2 in gallbladder and adipose tissue (including subcutaneous and visceral) is significantly higher than that in lung(Figure 1). In addition, we have explored whether obesity could affect the expression of ACE2. On account of data deficiency, it is impossible to test ACE2 expression in the whole adipose tissue.Nevertheless, the level of ACE2 expression in adipocytes and adipocyte progenitor cells isolated from adipose tissues of non-obese and obese individuals was compared by re-analysis of public data from Anna Ehrlund et al [14]. The results of ACE2 expression levels between two kinds of cells from obese and non-obese individuals are similar (Figure 2).

Figure 2. ACE2 expressions of adipocytes and progenitor cells isolated from adipose tissues of non-obese and obese individuals were shown.

Five types of cancer patients might be at higher risk of contracting SARS-Cov-2

The ACE2 expression level in cancers was analyzed by three databases, HCCDB, UALCAN and GEPIA2. We found tumor tissues of five types of cancers including CESC, PAAD, READ, KIRP and KIRC expressed more ACE2 than adjacent tissues, especially the former three cancers (Figure 3). The expression level of ACE2 in tumor tissue of CESC was closed to that in lung tissue, whereas the expression level of ACE2 in tumor tissue of PAAD got higher than that in lung tissue. Therefore, these cancer sites are at greater risk of being infected by the SARS-Cov-2.

Figure 3. ACE2 expressions of different kinds of cancers were analyzed using three databases, HCCDB (a), GEPIA2 (b) and UALCAN (c). The blue box indicates that ACE2 expresses higher in tumor tissue than the normal tissue, i.e. adjacent tissue.

Discussion

Some organs (kidney, liver, bladder, testis et al.) were demonstrated to have the potential risk of SARS-Cov-2 infection by characterizing the expression patterns of ACE2 [15, 16]. However, it has not been reported whether the adipose tissue might be vulnerable to the novel virus. We used the public database to analyze the ACE2 expression in adipose tissue, and found a higher RNA level than lung tissue.It was reported that adipose tissue could serve as a reservoir for human adenovirus Ad-36, influenza A virus, HIV, cytomegalovirus Trypanosoma gondii, and Mycobacterium tuberculosis [17].Certainly, it’s further required to be determined whether SARSCov-2 could infect adipose tissue through nucleic acid detection and pathological diagnosis for adipose tissue.

We found obesity didn’t affect ACE2 expressions in adipocytes and adipocyte progenitor cells in human adipose tissues. In adipose tissues, the expression level of ACE2 in other cells (monocytes,macrophages, leukocytes and NK cells et al) was unknown. Thales de Almeida Pinheiro et al. reported that there was no difference in ACE2 expression between adipose tissues of eutrophic patients and obese patients [18]. However, epididymal adipose tissues of rats with HFD-induced nonalcoholic steatohepatitis and HFD-induced mice upregulated ACE2 expression [19, 20]. Obesity was reported to increase vulnerability to infection [21]. Obesity is an independent risk factor for hospitalization and death to H1N1 influenza virus,and HFD-induced and genetic-induced obese mice exhibited greater H1N1 mortality [11]. Symptomatic obese adults were shown to shed influenza A virus 42% longer than non-obese adults. Obesity increases the duration of influenza A virus shedding in adults,which suggests obesity may play an important role in influenza transmission [12, 19, 20]. Even though obesity really has no effect on ACE2 expression, the obese individuals have more adipose to increase the quantities of ACE2 proteins. Therefore, the role of obesity in SARS-Cov-2 infection shouldn’t be ignored.

Wenhua Liang et al. analyzed the risk for COVID-19 in patients with cancer for the first time, and they found cancer patients had more severe events [13]. As limited number of COVID-19 patients with cancer, they couldn’t compare the severe events between different types of cancers. We found five types of cancers (CESC,PAAD, READ, KIRP and KIRC) increased the ACE2 expression at the original organs, which might worsen the symptoms or outcome in these cancer patients with COVID-19. It’s also required more clinical data to further verify this find.

In conclusion, the above cancer patients and obese individuals sh ould pay great attention to protection from SARS-Cov-2 infection.Moreover, the more intensive surveillance or treatment should be delivered to the above cancer patients and obese individuals in the early stages of COVID-19.

12. Maier HE, Lopez R, Sanchez N, Ng S, Gresh L, Ojeda S,et al. Obesity Increases the Duration of Influenza A Virus Shedding in Adults. J Infect Dis. 2018;218(9):1378-82;doi: 10.1093/infdis/jiy370.

13. Liang W, Guan W, Chen R, Wang W, Li J, Xu K, et al.Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China.The Lancet Oncology.2020;doi: 10.1016/s1470-2045(20)30096-6.

14. Ehrlund A, Acosta JR, Bjork C, Heden P, Douagi I, Arner P, et al. The cell-type specific transcriptome in human adipose tissue and influence of obesity on adipocyte progenitors. Scientific data. 2017;4:170164;doi: 10.1038/sdata.2017.164.

15. Chai X, Hu L, Zhang Y, Han W, Lu Z, Ke A, et al. Specific ACE2 Expression in Cholangiocytes May Cause Liver Damage After 2019-nCoV Infection. 2020:2020.02.03.931766;doi: 10.1101/2020.02.03.931766 %J bioRxiv.

16. Lin W, Hu L, Zhang Y, Ooi JD, Meng T, Jin P, et al. Single-cell Analysis of ACE2 Expression in Human Kidneys and Bladders Reveals a Potential Route of 2019-nCoV Infection. 2020:2020.02.08.939892;doi: 10.1101/2020.02.08.939892 %J bioRxiv.

17. BourgeoisC,Gorwood J,Barrail-Tran A,Lagathu C, Capeau J, Desjardins D, et al. Specific Biological Features of Adipose Tissue, and Their Impact on HIV Persistence. Frontiers in microbiology. 2019;10:2837;doi: 10.3389/fmicb.2019.02837.

18. Pinheiro TA, Barcala-Jorge AS, Andrade JMO, Pinheiro TA,Ferreira ECN, Crespo TS, et al. Obesity and malnutrition similarly alter the renin-angiotensin system and inflammation in mice and human adipose. J Nutr Biochem. 2017;48:74-82;doi: 10.1016/j.jnutbio.2017.06.008.

19. Zhang W, Xu YZ, Liu B, Wu R, Yang YY, Xiao XQ, et al. Pioglitazone upregulates angiotensin converting enzyme 2 expression in insulinsensitive tissues in rats with high-fat diet-induced nonalcoholic steatohepatitis. ScientificWorldJournal. 2014;2014:603409;doi: 10.1155/2014/603409.

20. Gupte M,Boustany-Kari CM,Bharadwaj K, Police S, Thatcher S, Gong MC, et al. ACE2 is expressed in mouse adipocytes and regulated by a high-fat diet. Am J Physiol Regul Integr Comp Physiol. 2008;295(3):R781-8;doi: 10.1152/ajpregu.00183.2008.

21. Misumi I, Starmer J, Uchimura T, Beck MA, Magnuson T, Whitmire JK.Obesity Expands a Distinct Population of T Cells in Adipose Tissue and Increases Vulnerability to Infection. Cell reports. 2019;27(2):514-24 e5;doi: 10.1016/j.celrep.2019.03.030.

22. Lian Q, Wang S, Zhang G, Wang D, Luo G, Tang J, et al. HCCDB: a database of hepatocellular carcinoma expression atlas. 2018;16(4):269-75; doi: 10.1016/j.gpb.2018.07.003.

23. Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, et al. Towards a knowledge-based Human Protein Atlas. Nature biotechnology. 2010;28(12):1248-50;doi: 10.1038/nbt1210-1248.

24. Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P,Mardinoglu A, et al. Proteomics. Tissue-based map of the human proteome. Science (New York, NY). 2015;347(6220):1260419;doi: 10.1126/science.1260419.

25. Chandrashekar DS, Bashel B, Balasubramanya SAH, Creighton CJ, Ponce-Rodriguez I, Chakravarthi B, et al. UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses. Neoplasia (New York, NY). 2017;19(8):649-58;doi: 10.1016/j.neo.2017.05.002.

26. Tang Z, Kang B, Li C, Chen T, Zhang Z. GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic acids research. 2019;47(W1):W556-w60;doi: 10.1093/nar/gkz430.Abbreviations