Expression of intestinal stem cell and cancer stem cell markers in submucosal invasion and its prognostic significance in gastric cancers

(Running head: stem cell markers in gastric cancer) Hye Sung Kim1, Hyun Joo Song2, In Ho Jeong3, Bo Gun Jang1 1Department of Pathology, Jeju National University School of Medicine and Jeju National University Hospital, 63241, Korea 2Department of Internal Medicine, Jeju National University School of Medicine and Jeju National University Hospital, 63241, Jeju, Korea 3Department of Surgery, Jeju National University School of Medicine and Jeju National University Hospital, 63241, Jeju, Korea


Introduction
The cancer stem cell (CSC) model suggests that tumor growth is induced by a small group of selfsustaining cells with longevity, infinite proliferation, and the ability to differentiate into a heterogeneous population of tumors [1]. The subpopulation at the top of the hierarchy, responsible for tumor formation, maintenance, and sustained growth, is essential to the CSC model [2]. The expression of distinct combinations of cell surface markers is an important tool in the identification and isolation of CSCs. Several candidate CSC markers have been identified for gastric cancer (GC), including CD133, CD44, and aldehyde dehydrogenase 1 (ALDH1) [3]. CD133 is a transmembrane glycoprotein that is believed to be a CSC marker in various solid tumors. Although research has demonstrated CSC properties in the CD133 subpopulation, there is controversy regarding the utility of CD133 as a CSC marker [4][5][6]. CD44 is also a transmembrane glycoprotein with various biological roles, and the first evidence of gastric CSCs was the self-renewal and heterogeneous lineage of a CD44+ subpopulation [7].
CSC populations in GC have been identified using a combination of CD44+ and other markers, including EPCAM [8,9], CD54+ [10], and CD24 [11]. Additionally, ALDH1 functions as a regulator of cell proliferation and stem cell differentiation and a marker of CSCs in a variety of cancers [3]. ALDH1+ cells isolated from gastric cancers have CSC properties [12,13].
The epithelial lining of the small intestine represents a prototype example of a mammalian stem cell-driven self-renewal tissue. A lineage-tracing experiment identified a leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) as a potent intestinal stem cell (ISC) marker [14], and ISCs were subsequently identified as the cellular origin of intestinal tumors [15]. Combinatorial microarray and proteomic approaches have discovered additional markers of LGR5+ stem cells, such as the ephrin type-B receptor 2 (EPHB2), Olfactomedin 4 (OLFM4), SPARC-related modular calcium-binding 2 (SMOC2), Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 July 2020 doi:10.20944/preprints202007.0484.v1 and ring finger protein 43 (RNF43) [16,17]. In addition, LGR5+ cells act as CSCs to induce tumor growth in human colorectal cancers [18,19]. Most gastric adenomas and adenocarcinomas occur in the background of intestinal metaplasia (IM). We have previously demonstrated the appearance of cells expressing ISC markers, LGR5, ASCL2, EPHB2, and OLFM4 in IM and gastric adenoma samples, suggesting these markers have a functional relevance in early gastric carcinogenesis [20,21]. Indeed, LGR5+ cells in the gastric antrum are of cellular origin in gastric adenomas and intestinal-type GCs in mice [22]. It is reasonable to consider these ISC markers as candidate CSC markers in human GCs.
An increasing number of studies suggest that CSCs are also responsible for tumor migration and invasion. Metastatic cancer cells of most carcinomas tend to recapitulate the organization of their primary tumors, indicating the cells with stem cell properties in the migrating or invading cancer cells [23]. In GC, crossing the muscularis mucosa (MM), a thin muscular layer that separates the mucosa from the submucosa, is a critical invasive step. This increases the risk of metastases, as the submucosal space contains substantial lymphatic and venous vessels. Submucosal invasion in GC is frequently detected by identifying small cancer cell clusters with intact MM, allowing for the comparison of SC marker expression between mucosal and submucosal cancer cells. However, no one has investigated the differential SC marker expression during submucosal invasion in GC. In this study, we examined the expression of ISC and CSC markers in GC cells infiltrating the submucosal space and investigated the prognostic significance of these SC markers in a large cohort of GC patients. Laboratories, Seoul, Korea).

Immunohistochemistry interpretation
Immunohistochemistry was carried out on TMA sections using a BOND-MAX automated immunostainer and a Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany) according to the manufacturer's instructions [25].

RNA in situ hybridization and interpretation
In situ hybridization (ISH) for LGR5, RSPO2, and RSPO3 was performed using RNAscope FFPE assay kit (Advanced Cell Diagnostics, Inc., Hayward, CA, USA) as described previously [20]. Briefly, 4μm tissue sections of TMA are pretreated with heat and protease digestion followed by hybridization with the LGR5, RSPO2, and RSPO3 probe. Then, an HRP-based signal amplification system is hybridized to the probe before color development with 3,3′-diaminobenzeidine tetrahydrochloride

RNA extraction and quantitative real-time PCR
Total RNA was isolated from the 37 paired fresh-frozen GCs and corresponding non-cancerous gastric tissue samples using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). Total RNA (1 -2μg) was subjected to reverse transcription with the GoScript reverse transcription system (Promega, Madison, Wisconsin, USA). Quantitative RT-PCR reactions were performed with Premix EX Taq (Takara bio, Shiga, Japan) according to the manufacturer's instructions. Cycling conditions were followed: initial denaturation for 30 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 5 s in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) [25].

Transfection of CDX2
Transfection was performed previously described [25]. Cells were plated at 1 × 10 6 cells per well of manufacturer. pCMV empty vector was used as a control vector. One day after transfection, cells were subjected to real-time PCR.

Western blot analysis
Cellular proteins were isolated using lysis buffer (iNtRON Biotechnology, Seongnam, Korea) and were quantitated with BCA protein assay kits (Pierce, Rockford, IL, USA). Cell lysates were run on a Alliance-Mini.HD9 chemiluminescence documentation system (UVItec Cambridge, UK) was used to visualize the target proteins.

Statistical analysis
Statistical analyses were performed using the PASW 18.0 statistical software program (IBM SPSS

Distribution of intestinal stem cell (ISC) and cancer stem cell (CSC) markers in early gastric cancers
We hypothesized that cancer cells with stem cell features might be responsible for submucosal  Figure S2). Among all ISC and CSC markers, only EPHB2 showed a significant association with lymph node metastasis; early GCs with diffuse EPHB2 expression had no lymph node metastasis. This finding suggests that EPHB2 might be a predictive marker for lymph node metastasis Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 July 2020 doi:10.20944/preprints202007.0484.v1 in early GCs (Table 1).

Expression of ISC and CSC markers in submucosal gastric cancers
Next, we examined the proportion of SC marker-positive cells and the intensity of marker expression in submucosal cancer cells in comparison with mucosal cancer cells. In GCs with frequently expressing ISC markers (EPHB2, LGR5, and OLFM4) and CD133, the proportion of SC marker-positive cancer cells in the submucosa was significantly higher compared to that in the lamina propria ( Figure   3). For instance, as seen in Figure 3a

Wnt3a/RSPO2-induced EPHB2 expression in gastric cancers
Recently, stromal R-spondin3 has been shown to be essential for the stem cell maintenance in the normal gastric mucosa of the mice [29]. In addition, GREM1, an antagonist of BMP signaling, also has been suggested as an important molecule secreted from MM in the colon [30]. Therefore, we hypothesized that these niche factors from MM may lead to upregulation of ISC markers, such as

Association of ISC and CSC markers with CDX2 in gastric cancers
ISC markers were originally defined in the intestinal and colonic epithelium, and have been recently found in the intestinal metaplasia of the stomach, indicating their close relationship with intestinal phenotype and differentiation. To examine whether these ISC markers are implicated in the intestinal phenotype of GCs, we investigated the association between mRNA expression of CDX2 and ISC markers in GCs. We performed a real-time PCR with 37 pairs of fresh-frozen human GC samples and matched non-cancerous gastric mucosa (NCM), and we noted that ISC marker expression was higher in GCs than in adjacent NCM, whereas CSC marker expression in GCs either showed no significant difference (CD133) or was lower compared to that of the NCM (CD44 and ALDH1A) ( Figure   5a). As expected, CDX2 expression was significantly correlated with EPHB2 (r 2 = 0.31, p < 0.001) and OLFM4 (r 2 = 0.18, p < 0.01).

Prognostic significance of intestinal stem cell and cancer stem cell markers in gastric cancer patients
We examined the prognostic value of ISC (EPHB2 and OLFM4) and CSC (CD133, CD44, ALDH1A) markers in a large number of GC patients (n = 706) and found that EPHB2, OLFM4, and CD133 expression was significantly associated with better overall survival (p = 0.000, p = 0.000, p = 0.039, respectively) ( Figure 6). However, EPHB2, OLFM4, and CD133were not independent prognostic markers in a multivariate analysis (Supplementary Table S3). Intestinal-and diffuse-type GCs involve different molecular pathways. We therefore separately analyzed the prognostic significance of ISC and CSC markers in intestinal-and diffuse-type GCs. Representative images of EPHB2 expression in the normal glands, intestinal metaplasia, and GCs are shown in Figure 7a-b. Notably, the prognostic value of EPHB2 was significant in intestinal-type GCs (p < 0.001), whereas in diffuse-type GCs, EPHB2 expression was not associated with improved survival (p = 0.176) (Figure 7c). In addition, multivariate analysis revealed that EPHB2 expression is an independent prognostic factor in intestinal-type GCs (HR: 0.520, p = 0.022) (Figure 7d). EPHB2 positivity was significantly higher in GCs with papillary or well-differentiated GCs than in poorly differentiated or signet-ring cell carcinoma (p < 0.001) ( Table 2).

Discussion
Accumulating evidence suggests that CSCs are responsible for cancer invasion and metastasis. In early GCs, submucosal invasion is a common and critical step for cancer progression that is easily recognized by histologic examination. These features make GCs with submucosal invasion a valuable model to explore CSCs' involvement in the invasion process. However, no study has investigated CSC marker expression in early GCs with submucosal invasion. In this study, we chose three ISC markers and three candidate CSC markers to identify SC markers associated with submucosal invasion in GCs.
We found three distinct distribution patterns of SC markers. A basal pattern was predominant for all ISC markers and CD133. A restricted pattern of ISC marker expression is normally found in the intestinal epithelium [31] and at the bottom of tumor structures in colon cancers [32], representing a stem cell hierarchy. Therefore, the observed basal pattern of ISC markers in early GCs suggests that a hierarchical organization fueled by cancer stem cells may be present in the very early stages of cancer progression.
We localization. We must clarify whether these SC marker-positive cells have any functional advantages over other cancer cells in the submucosal invasion. In CRC, cells expressing high levels of nuclear βcatenin are frequently observed at the invasive fronts and comprised potential migrating CSCs [34]. We therefore sought to examine nuclear β-catenin in the submucosal gastric cancer cells. However, there was no increase in the number of nuclear β-catenin-positive gastric cancer cells at the invasive front.
Retained stem cell-like hierarchy in GCs, as determined by the basal expression of ISC markers, suggests that the differentiation potential is likely to be maintained. As ectopic CDX2 expression plays a key role in the intestinal phenotype of GCs [35], we investigated the correlation between ISC markers and CDX2 expression and noted that all ISC markers had a strong positive correlation with CDX2 expression. However, none of the CSC markers were significantly correlated with CDX2. Additionally, we found that CDX2 overexpression is involved in EPHB2 transcription induction, but not OLFM4 or LGR5 induction. These findings link intestinal differentiation and ISC marker expression in GCs and suggest that ISC marker expression might depend on CDX2 transcription factors. Further research may elucidate the underlying mechanism by which CDX2 regulates the expression of EPHB2, particularly in the cancer stem cell population.
CSC marker expression in cancer tissue is emerging as a clinically-relevant prognostic biomarker in GC management. Its clinical implications are controversial, however, due to variation in experimental procedures and patient populations [3]. In this study, we found that EPHB2, OLFM4, and CD133 are associated with better overall survival, whereas CD44 and ALDH1A expression had no significant impact on clinical outcomes. We previously reported that LGR5 had no prognostic significance in the same cohort of GC patients when we evaluated LGR5 expression by RNA in situ Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 July 2020 doi:10.20944/preprints202007.0484.v1 hybridization and found the positive rate to be low (7%) due to RNA degradation in FFPE samples [36].
It is therefore necessary to perform survival analysis for LGR5 expression when adequate antibodies to human LGR5 for immunohistochemistry becomes available. Interestingly, all markers that have a favorable prognostic significance are SC markers which predominantly exhibited a basal expression pattern in early GCs, and the proportion of marker-positive cancer cells increased in submucosal invasion. Therefore, it seems less likely that SC marker expression has any functional implication in the invasion process or aggressive tumor behavior. Instead, we could hypothesize that although ISC  Figure S8b), whereas in the mouse stomach and colon only RSPO3 was shown to be expressed in the MM and to play essential roles for stem cell reprogramming [29,37].
Considering the fact that RSPO2 and RSPO3 have common biological activities [38], it is possible that RSPO2 in human stomach plays the same role as RSPO3 in murine stomach.
An EPHB2 reduction accelerates tumorigenesis in the colon of Apc Min/+ mice [39], and a loss of EPHB2 expression is a strong indicator of poor overall survival in colorectal cancer (CRC) patients [26,40]. This suggests that EPHB2 has a tumor suppressor role in CRC progression. However, we noted higher EPHB2 expression in GCs compared to matched non-cancerous gastric mucosa, consistent with previous work [41]. Even though this appears to contradict its tumor-suppressive role in CRC, EPHB2 is not normally expressed in the gastric mucosa, but rather it appears as the intestinal metaplasia develops, and substantially increases in gastric adenomas [20]. Both univariate and multivariate analyses have demonstrated that a reduction in EPHB2 expression is significantly correlated with poor overall survival in GC patients [42]. In this study, we also found that a reduction in EPHB2 expression is closely correlated with lymph node metastasis in early GCs and poor clinical outcome in advanced GCs. The prognostic significance of EPHB2 remained in intestinal-type GCs, but not in diffuse-type GCs. This indicates a close relationship between EPHB2 and intestinal differentiation, similar to its correlation with CDX2. Therefore, it appears that EPHB2 expression from precancerous lesions, such as intestinal metaplasia to early gastric cancers, represents a phase during which hierarchical structure and intestinal differentiation are retained and loss of EPHB2 expression occurs as the cancer acquires more oncogenic mutations and progresses to advanced stage.

Conclusion
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 July 2020 doi:10.20944/preprints202007.0484.v1 In summary, the present study identified the distributions of CSC and ISC markers in early-stage GC progression. All ISC markers (EPHB2, OLFM4, and LGR5) and CD133 predominantly demonstrated a basal pattern, confined to the bottom of the mucosa in early GCs, and their expression continued during submucosal invasion. ISC marker expression was strongly correlated with CDX2 and was induced by CDX2 overexpression in GCs. These findings suggest that a significant proportion of GCs with ISC markers retain hierarchical structure with intestinal differentiation potential. These ISC markers and CD133 were associated with improved overall survival in GC patients, and EPHB2 was an independent prognostic marker in intestinal GCs.