Robust expressions of stem cell markers, including leucine-rich repeat-containing G protein- coupled receptor 5, in association with early embryonic expression patterns of niche factors in untreated and chemoradiation-treated rectal cancer

Robust expressions of stem cell markers, including leucine-rich repeat-containing G protein- coupled receptor 5, in association with early embryonic expression patterns of niche factors in untreated and chemoradiation-treated rectal cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Robust expressions of stem cell markers, including leucine-rich repeat-containing G protein- coupled receptor 5, in association with early embryonic expression patterns of niche factors in untreated and chemoradiation-treated rectal cancer Kentaro Tsuji, Sachi Sekine, Hirotoshi Kawata, Tomoko Kamiakito, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3037112/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study aimed to ascertain the involvement of cancer stem cells (CSCs) in therapy resistance by estimating CSC markers, niche factors, and WNT/β-catenin-relating molecules in untreated and chemoradiation-treated (CRT) rectal cancer. Methods Expression ratios of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR), proto-oncogene and polycomb ring finger 1 (BMI1), yes-associated transcriptional regulator (YAP) and its paralog TAZ (hereafter; YAP/TAZ), and nuclear β-catenin were compared in untreated and CRT rectal cancer using in situ hybridization and immunostainings. Niche factors and WNT/β-catenin-relating molecules were also immunohistochemically compared in human rectal cancer specimens and with early embryonic intestine. Results The mean ratios were 15% and 14% in LGR5; 30% and 33% in BMI1; 2.7% and 7.6% in YAP/TAZ; 38% and 32% in nuclear β-catenin in untreated and CRT rectal cancer, respectively, suggesting their robust expressions after CRT. LGR5 and nuclear β-catenin expression was significantly correlated in the CRT cohort. High mobility group AT-hook 1, but not c-MYC and SRY-box transcription factor 9, was significantly upregulated in CRT tumors. WNT2B and GREM1 were uniformly expressed with similarity to the pattern of early embryonic intestine, whereas WNT3A and HES1 expressions were limited. Additionally, stromal GLI1 and YAP/TAZ were more expressed in the CRT cohort. Conclusions Stem cell markers of LGR5 and BMI1 as well as nuclear YAP/TAZ, a marker of stem cell regeneration, were robustly expressed in untreated and CRT rectal cancer. WNT2B and GREM1 were aberrantly and uniformly expressed in untreated and CRT rectal cancer with similarity to the early embryonic expression pattern. cancer stem cells therapy resistance rectal cancer LGR5 BMI1 YAP/TAZ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Therapy resistance is one of the most critical problems in managing patients with cancer. A growing body of evidence strongly suggests that cancer stem cells (CSCs) greatly contribute to therapy resistance [ 1 , 2 ]. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive cells are generally accepted as stem cells in human colorectal cancer as well as in normal colorectal crypts under the control of WNT/β-catenin signaling [ 3 , 4 ]. Meanwhile, cells that are located at the + 4 position and expressing proto-oncogene and polycomb ring finger 1 (BMI1) are shown to function as quiescent stem cells in the intestine [ 5 , 6 ] and these cells can compensate for intestinal homeostasis under complete loss of LGR5-positive cells [ 7 ]. Additionally, yes-associated transcriptional regulator 1 (YAP1) and its paralog TAZ (WW domain containing transcription regulator 1 [WWTR1]) (hereafter YAP/TAZ) are major factors to contribute to stem cell regeneration after mucosal injuries in the intestine [ 8 , 9 ]. An earlier report revealed YAP/TAZ as a marker of regenerative stem cells without LGR5 expression [ 10 ]. Histopathologic CSC status has been poorly analyzed in surgically resected specimens, in contrast to the expanded knowledge of cell biology on CSCs in colorectal cancer. This study compared LGR5, BMI1, and nuclear YAP/TAZ expression ratios, which cover most of CSCs, in untreated and chemoradiation-treated (CRT) rectal cancer, using a highly sensitive in situ hybridization (ISH) method for LGR5 and BMI1. Given the pivotal role of WNT/β-catenin signaling in rectal cancer, we also estimated ratios of nuclear β-catenin. In addition to these proteins, MYC proto-oncogene bHLH transcription factor (c-MYC), SRY-box transcription factor 9 (SOX9), and high mobility group AT-hook 1 (HMGA1) are involved in CSC activity in association with WNT/β-catenin signaling [ 11 – 13 ]. Meanwhile, niche factors contribute to stem cell maintenance. WNT family member 3A (WNT3A) is a prototypal epithelial niche factor secreted by Paneth cells and maintains LGR5-positive stem cells in the normal intestine [ 14 ]. Stromal WNT2B compensates for WNT3A in maintaining LGR5-positive stem cells under epithelial damage [ 15 ]. Moreover, gremlin 1 DAN family BMP antagonist (GREM 1), which is an endogenous BMP antagonist, generates a BMP signaling gradient to preserve the stem cell niche [ 16 ], and NOTCH signaling has been involved in stem cell regeneration [ 17 ]. Finally, GLI family zinc finger 1 (GLI1)-expressing mesenchymal cells are reported to contribute to the formation of stem cell niches in the intestine by secreting WNT proteins [ 18 , 19 ]. GLI1 is an effector of the hedgehog growth factor family and is involved in various biological functions, including organ morphogenesis and tumorigenesis [ 20 ]. These factors are likely involved in therapy resistance through CSC modulations. However, analysis of these molecules remains lacking at histopathologic levels. Therefore, the present study compared the expression levels of these factors in untreated and CRT rectal cancer. Finally, niche factors of WNT2B and GREM1 were also analyzed in murine embryonic intestine. In a series of histopathologic analyses, we demonstrated robust expressions of LGR5, BMI1, and nuclear YAP/TAZ and β-catenin in untreated and CRT rectal cancer. Additionally, HMGA1 as well as stromal YAP/TAZ and GLI1 are significantly upregulated in the CRT cohort. We also showed that niche factors of WNT2B and GREM1 are uniformly expressed with similarity to the expression pattern of early embryonic intestine, suggesting fetus-like niche formation in rectal cancer. Materials and Methods Cases Human rectal adenocarcinoma specimens resected from 29 patients receiving CRT (the sample from a 30th case was discarded due to the loss of cancer components during section preparation) and from 30 untreated patients were retrieved from the archives of the Pathology Department of Jichi Medical University Saitama Medical Center Hospital. All tumors demonstrated moderately to well-differentiated tubular adenocarcinoma with minimal foci of well-differentiated mucinous carcinoma. All samples exhibited a diffuse positive expression of cytokeratin 8 without obvious undifferentiated or sarcomatous components. Additionally, the lack of obvious neuroendocrine differentiation, which was estimated using synaptophysin expression, was confirmed. The patient characteristics are summarized in Table 1 . The CRT duration was 15–168 (median; 34.5) days. Immunohistochemistry and ISH were performed with the approval of the local ethics committee at Jichi Medical University Saitama Medical Center Hospital. Table 1 Patient characteristics and treatment regimens The number of cases is shown except for medial values with ranges in Age, Radiation, and CRT Duration untreated CRT (n = 30) (n = 29) Age (years) 69.6 (49ཞ84) 61.5 (38ཞ81) Sex (male/female) 23/7 25/5 Neoaduvant 5-FU/LV 0 28 others 0 2 Radiation (Gy) 0 43(40ཞ50) Duration of CRT (days) 0 36.1(15ཞ168) Animals Pregnant female mice were purchased from SLC (Shizuoka, Japan). Embryos were obtained at the indicated time, and whole paraffin-embedded sections of embryos were prepared using a routine histologic technique. An animal experiment was performed with the approval of the animal experiment committee at Jichi Medical University. Antibodies Mouse monoclonal antibody against beta-catenin (product ID: 610154) was purchased from BD Biosciences (Franklin Lakes, NJ). Rabbit polyclonal antibodies against WNT2B (product ID: ABO12151), GREM1 (product ID: PAB4716), GLI1 (product ID: NBP1-78259), and SHH (product ID: 06-1106) were purchased from abcepta (San Diego, CA), abnova (Taipei, Taiwan), Novus biologicals (Centennial, CO), and Millipore (Billerica, MA), respectively. Rabbit polyclonal antibodies against IHH (product ID: 13388-1-AP) and LIN28B (product ID: 16178-1-AP) were purchased from Proteintech (Rosemont, IL). Rabbit monoclonal antibody against YAP/TAZ (clone D24E4) (product ID: 8418S), HES1 (clone D6P2U) (product ID: 11988S), SOX9 (clone D8G8H) (product ID: 82630S), and HMGA1 (clone D4F8) (product ID: 12904S) were purchased from Cell Signaling Technologies. Rabbit monoclonal antibody against c-MYC (clone Y69) (product ID: ab32072) was purchased from abcam (Cambridge, UK). ISH and immunohistochemistry ISH was performed using specific probes, recognizing multiple mRNA sequences of human LGR5, BMI1, and WNT3A (Advanced Cell Diagnostics Inc. Hayward, CA). Slides were incubated in a dry oven for 1 h at 60°C and de-paraffinized. Then, they were boiled for 15 min, treated with the proteinase reagent provided by the kit, and incubated with the specific probes in an oven for 2 h at 40°C. Afterward, slides were incubated with the kit amplifying reagents and then with the kit detection reagents. Signals were visualized by an immunofluorescence microscope (BZ-X700, Keyence, Osaka, Japan). One or two representative sites were photographed by optic microscopy, and expression ratios were determined in each photo between the positive cells and the total cancer cells. Formalin-fixed and paraffin-embedded sections were pre-treated in a microwave oven or an autoclave for the indicated times for immunohistochemistry (Table 2 ). The pre-treated sections were incubated with each antibody at 4°C overnight, then with Envision (DAKO JAPAN, Tokyo, Japan), and stained with 3,3’-diaminobenzidine. Antibodies and methods used to retrieve and detect the antigens are summarized in Table 2 . Non-neoplastic mucosa and sections from mouse embryos were used as positive controls. Positive ratios of nuclear YAP/TAZ and β-catenin expression were determined by three representative photos. The immunostaining scores were defined by positive areas as follows: score 0, no positive staining or 10%. Table 2 Immunohistochemical protocols. Antibodies Dilution ratio Pretreatment b-catenin 1:50 Microwave 20 min YAP/TAZ 1:400 Autoclave 5 min HES1 1:1600 Autoclave 5 min WNT2B 1:3200 Autoclave 5 min GREM1 1:1600 Autoclave 5 min GLI1 1:400 Microwave 10 min SOX9 1:100 Autoclave 5 min HMGA1 1:400 Microwave 10 min c-MYC 1:100 Autoclave 5 min SHH 1:200 Microwave 10 min IHH 1:200 Microwave 10 min LIN28B 1:200 Microwave 10 min Statistical Analysis The statistical significance of the value changes was determined using the Mann-Whitney U test, and the correlation was estimated using Spearman’s correlation via rank testing with Statcel software, version 4 (OMS, Saitama, Japan). A p -value of < 0.05 was considered significant. Results Robust expressions of LGR5 and BMI1 as well as nuclear YAP/TAZ and β-catenin in CRT rectal cancer LGR5 transcripts were selectively detected in the crypt base using the specific probe in non-neoplastic mucosa as previously reported (supplementary Figs. 1A and 1B) [ 3 ]. In contrast, BMI1 transcripts were unrestricted to + 4 cells but scattered throughout the whole crypt, which is concordant with the previous description (supplementary Figs. 1C and D) [ 21 ]. We then compared the positive ratios of LGR5 and BMI1 in untreated and CRT rectal cancer using the highly sensitive ISH method. Additionally, nuclear YAP/TAZ and β-catenin expressions were investigated via immunohistochemistry to assess the nuclear localization. ISH revealed that LGR5-positive cells are often expressed in clusters, contrasting the diffusing and scattered patterns of BMI1 (Figs. 1 A–D). The expression ratios (mean ± standard deviation [SD]) were 15% ± 22% and 14% ± 27% for LGR5 and 30% ± 33% and 33% ± 33% for BMI1 in untreated and CRT tumors, respectively. No significant difference was found in LGR5 or BMI1 expression between untreated and CRT cohorts. As shown below, YAP/TAZ expression in cancer cells was limited in untreated and CRT rectal cancer, and scarce in normal mucosa (Figs. 4 D–F). Ratios of YAP/TAZ nuclear expressions (mean ± SD) were 2.7% ± 7.4% in untreated and 7.6% ± 16% in CRT rectal cancer, showing no significant YAP/TAZ upregulation in the CRT tumors. Meanwhile, β-catenin nuclear expression ratios (mean ± SD) were 38% ± 24% in untreated and 32% ± 20% in CRT tumors (Figs. 1 E and F). We scored LGR5, BMI1, β-catenin, and YAP/TAZ expression as described in the Materials and Methods section to further assess LGR5 expression (Table 3 ). A significant correlation was detected between LGR5 and β-catenin nuclear expression in the CRT tumors ( p < 0.05, rs = 0.43) using the Spearman’s correlation by rank test through this scoring (supplementary Table 1), suggesting that the LGR5/β-catenin axis is more evident in the CRT samples than in the untreated rectal cancer. No significant correlation was found between YAP/TAZ and β-catenin as well as BMI1 and β-catenin (data not shown). In summary, LGR5 and BMI1 as well as nuclear YAP/TAZ and β-catenin are robustly expressed in untreated and CRT rectal cancer. Table 3 Immunostaining results for LGR5 BMI1, β-catenin, and YAP/TAZ in untreated and CRT rectal cancer. The number of cases is shown by the scoring (score of 2, > 10%; score of 1, 1–10%; score of 0, no staining or < 1%). EXPRESSION SCORE LGR5 BMI1 b-catenin YAP/TAZ untreated CRT untreated CRT untreated CRT untreated CRT score 2 4 5 10 11 13 13 1 2 score 1 8 3 8 9 13 12 1 3 score 0 18 21 12 9 4 4 28 24 p = 0.469 p = 0.372 p = 0.941 p = 0.219 Upregulation of HMGA1, but not SOX9 and c-MYC, in CRT rectal cancer Next, we compared c-MYC, SOX9, and HMGA1 expressions due to their relation to WNT/β-catenin signaling [ 11 – 13 ]. C-MYC and HMGA1 expressions were scarce in non-neoplastic mucosa, whereas SOX9-positive cells were mainly located in lower portions of crypts (data not shown). C-MYC was expressed in 4 (13%) cases at a score of 1 in untreated tumors in rectal cancer specimens, whereas all cases revealed the expression of < 1% (score of 0) in the CRT cohort (Fig. 2 A, B and Table 4 ). C-MYC expression was at significantly higher levels in the untreated tumors than in the CRT rectal cancer, suggesting that its expression is correlated with proliferative activity. Meanwhile, SOX9 was expressed at > 10% (score of 2) in all cases of both untreated and CRT tumors (Figs. 2 C, 2 D and Table 4 ). HMGA1 was expressed in 2 (6.7%) cases at a score of 2 and 10 (33%) cases at a score of 1 out of 30 cases of the untreated tumors, whereas 11 (38%) were at a score of 2 and 9 (31%) at a score of 1 in the CRT cohort (Figs. 2 E, 2 F and Table 4 ). HMGA1 was expressed at significantly higher levels in the CRT cohort than in the untreated tumors ( p = 0.00273) (Table 4 ) though its expression was unrelated to nuclear β-catenin in the CRT tumors by the Spearman’s correlation by rank test (supplementary Table 2). We subsequently investigated expression of lin-28 homolog B (LIN28B) in untreated and CRT rectal cancer because HMGA proteins are regulated by LIN28 or the let-7 microRNA family [ 22 ]. LIN28B expression was scarce except for two cases with weak expression of < 1% (score of 0) in CRT rectal cancer (data not shown and Table 4 ). In summary, HMGA1, but not SOX9 and c-MYC, was significantly upregulated in CRT rectal cancer. Table 4 Immunostaining results for SOX9, c-MYC, ΗΜGΑ1, and LIN28B in cancer cells of untreated and CRT rectal cancer. EXPRESSION SCORE SOX9 c-MYC HMGA1 LIN28B untreated CRT untreated CRT untreated CRT untreated CRT score 2 30 29 0 0 2 11 0 0 score 1 0 0 4 0 10 9 0 0 score 0 0 0 26 29 18 9 30 29 not determined p = 0.006 p = 0.003 not determined The number of cases is shown by the scoring (score of 2, > 10%; score of 1, 1–10%; score of 0, no staining or < 1%). Uniform and aberrant expressions of mesenchymal niche factors, WNT2B, and GREM1 in cancer cells of untreated and CRT rectal cancer Next, we compared expressions of niche factors in untreated and CRT rectal cancer. Concordant with the reported data [ 14 , 15 ], WNT3A was expressed at crypt bases, whereas WNT2B and GREM1 were mainly expressed in mesenchymal cells in non-neoplastic mucosa (Figs. 3 A, 3 G, and 3 J). HES1 expression was scattered in non-neoplastic crypts (Fig. 3 D). WNT3A was detected at levels of > 10% (score of 2) in 3 (10%) out of 30 untreated tumors and 1 (3%) out of 29 CRT rectal cancer, respectively (Figs. 3 B, 3 C and Table 5 ). Nuclear expression of HES1 was detected at > 10% (score of 2) in 1 (3%) out of 30 untreated samples and 1 (3%) out of 29 cases of CRT rectal tumors (Figs. 3 E, 3 F and Table 5 ). The low expression of WNT3A and HES1 in rectal cancer suggests that both proteins have less significance for maintaining CSCs. In contrast, WNT2B and GREM1 were uniformly and aberrantly expressed in rectal cancer cells along with distinct expression in stroma cells (Figs. 3 H, 3 I, 3 K, and 3 L). WNT2B was detected at > 10% (score of 2) in all untreated rectal cancer and 27 (93%) out of 29 CRT tumors in the cancer cell expression (Table 5 ). GREM1 was detected at > 10% (score of 2) in all samples of untreated rectal cancer, whereas in 26 (90%) out of 29 cases of CRT rectal cancer (Table 5 ). Collectively, mesenchymal niche factors of WNT2B and GREM1 may support cancer survival by activation of WNT/β-catenin in association with suppression of BMP signals in CRT rectal cancer. Table 5 Immunostaining results for WNT3A, HES1, WNT2B, and GREM1 in untreated and CRT rectal cancer. The number of cases is shown by the scoring (score of 2, > 10%; score of 1, 1–10%; score of 0, no staining or < 1%). EXPRESSION SCORE WNT3A HES1 WNT2B GREM1 untreated CRT untreated CRT untreated CRT untreated CRT score 2 3 1 1 1 30 27 30 26 score 1 0 0 0 2 0 2 0 0 score 0 27 28 29 26 0 0 0 3 p = 0.321 p = 0.305 p = 0.147 p = 0.073 Upregulation of GLI1 and YAP/TAZ in stroma cells after CRT We next compared GLI1 nuclear expression in stroma cells between untreated and CRT rectal cancer because GLI1-expressing mesenchymal cells are reported to form stem cell niches in the colon [ 18 , 19 ]. GLI1 was detected in non-neoplastic and cancerous stomas (Figs. 4 A, 4 B, and 4 C). GLI1 nuclear expression in stroma cells was detected in 1 (3%) and 9 (30%) out of 30 untreated samples and 9 (31%) and 12 (41%) out of 29 CRT samples at scores of 2 and 1 in rectal cancer specimens, respectively (Table 6 ). GLI1 expression in CRT stroma was significantly higher than that in the untreated stroma of rectal cancers ( p = 0.003). This finding prompted us to re-analyze nuclear YAP/TAZ expression, specifically in stroma cells, because earlier reports revealed the involvement of YAP/TAZ in the generation of cancer-associated fibroblasts [ 23 , 24 ]. YAP/TAZ was expressed in stroma cells in 1 (3%) and 10 (33%) out of 30 untreated tumors, whereas 6 (21%) and 18 (62%) out of 29 CRT tumors at scores of 2 and 1, respectively (Figs. 4 E, 4 F and Table 6 ). YAP/TAZ expression in the nucleus of stroma cells in CRT tumors was significantly higher than that in untreated rectal cancer ( p = 0.002). We next compared GLI1 and YAP/TAZ stromal expressions with the β-catenin nuclear expression in cancer because GLI1-expressing stroma cells are shown to secrete WNT proteins [ 15 , 18 ]. This analysis revealed no significant correlation of GLI1 or YAP/TAZ expressions with β-catenin nuclear expression in both the untreated and CRT cohorts (data not shown). Additionally, we investigated the expressions of sonic hedgehog signaling molecule (SHH) and Indian hedgehog signaling molecule (IHH)) in untreated and CRT rectal cancer because GLI1 is a signal transducer of the hedgehog family growth factor. SHH was expressed in the surface epithelia and upper crypts in non-neoplastic mucosa, while IHH was diffusely expressed in crypts (Figs. 4 G and 4 J). SHH was expressed at > 10% in all rectal cancer specimens (Figs. 4 H, 4 I and Table 6 ). IHH was expressed in 17 (57%) and 9 (30%) cases in the untreated tumors, whereas 11 and 7 cases in the CRT rectal cancer at scores 2 and 1, respectively (Figs. 4 K, 4 L and Table 6 ). IHH and SHH expressions revealed no significant difference between the untreated and the CRT rectal cancer, suggesting that GLI1 upregulation is uncoupled to SHH or IHH expression. To summarize, GLI1 and YAP/TAZ expression levels were significantly upregulated in stroma cells in CRT rectal cancer, suggesting that stromal activation lasts for substantial time after CRT. Table 6 Immunostaining results for IHH, SHH, YAP/TAZ, and GLI1. The number of cases is shown by positive areas stained for IHH and SHH in cancer cells as well as YAP/TAZ and GLI1 in stroma cells of untreated and CRT rectal cancer. The numbers of cases were shown by the scoring (score of 2, > 10%; score of 1, 1–10%; score of 0, no staining or < 1%). EXPRESSION SCORE IHH SHH YAP/TAZ (stroma) GLI1(stroma) untreated CRT untreated CRT untreated CRT untreated CRT score 2 17 11 30 29 1 6 1 9 score 1 9 7 0 0 10 18 9 12 score 0 4 11 0 0 19 5 20 8 p = 0.055 not determined p = 0.0002 p = 0.0007 Expression patterns of WNT2B and GREM1 in murine embryonic intestine Fetal-like features of malignant cells, including stroma and immune cells, have been recently mentioned as “oncofetal reprogramming” [ 23 ]. Therefore, we investigated WNT2B and GREM1 expressions in murine embryonic intestine. In embryonic intestine at E12.5, WNT2B, and GREM1 were uniformly expressed in glandular and mesenchymal cells, whereas expressions of both proteins were weaker in glandular and mesenchymal cells at E16.5 (Figs. 5 C–F). As described above, WNT2B and GREM1 were limitedly expressed in mesenchymal cells of adult human rectum. Accordingly, expression patterns of WNT2B and GREM1 in rectal cancer mimic those of early embryonic intestine, suggesting the oncofetal reprogramming. Stem cell niches of rectal cancer might be similar to those of early embryonic intestine. Discussion We hypothesized that LGR5 or BMI1-positive cells are expanded in CRT rectal cancer based on the significance of CSCs in therapy resistance. Furthermore, YAP/TAZ, a regenerative CSC marker, is speculated to be abundant in CRT rectal cancer. The mean ratios of LGR5 were approximately 15% and 14% in untreated and CRT rectal cancer, respectively. Other CSC markers were 30% and 33% in BMI1, 3% and 8% in YAP/TAZ, and 38% and 32% in β-catenin in untreated and CRT rectal cancer, respectively. Against our hypothesis, overall expressions of LGR5 and BMI1 as well as nuclear YAP/TAZ and β-catenin are robust in untreated and CRT rectal cancer, suggesting the rigid CSC homeostasis in rectal cancer. Earlier reports evaluated CSC ratios at 2.5% ± 1.4% [ 24 ] and 12% (range: 1.8–24.5%) [ 25 ] in colorectal cancer though a recent report that estimated CSCs in a range from 0.0036–1.8% using organoid culture [ 19 ]. In contrast, a prior report reported a wide range of LGR5 expression from < 1–95% in colorectal cancer [ 26 ]. Other reports revealed approximately 20% LGR5 expression in adenoma of a murine model [ 27 ], 28% in adenoma and 54% in colorectal cancer [ 28 ], and 74% in colorectal cancer [ 29 ]. Additionally, BMI1 expression was reported as 47% [ 30 ] and 66.5% [ 31 ], while either YAP or TAZ-positive cells were described as 93.5% [ 32 ] in colorectal cancer. These values are considerably higher than the predicted CSCs as well as the present estimation for these proteins. It is important to mention that LGR5 is significantly correlated to nuclear β-catenin expression in CRT rectal cancer in this study. Accordingly, the present estimation of CSC markers, particularly LGR5, might provide standard values on CSC markers in rectal cancer though it is possible that nuclear expression of YAP/TAZ might be transient after CRT in rectal cancer. A series of immunohistochemical studies revealed that HMGA1 as well as stromal YAP/TAZ and GLI1 are significantly upregulated in the CRT cohort, compared to the untreated tumor. HMGA proteins preserve stem cell conditions in embryonic stem cells [ 33 ]. Additionally, HMGA1 induces intestinal polyposis [ 34 ] and amplifies WNT/β-catenin signaling in colorectal cancer [ 13 ]. These features indicate that HMGA1 supports the maintenance of CSCs to enhance the WNT/β-catenin signaling in CRT rectal cancer. Stromal activation of YAP/TAZ and GLI1 may also support maintenance of CSCs in CRT rectal cancer. In study of niche factors, we found that mesenchymal niche factors of WNT2B and GREM1 were uniformly and aberrantly expressed in rectal cancer while expressions of epithelial niche factors, WNT3A and HES1, were limited. Notably, similar aberrant and uniform expressions of WNT2B and GREM1 were found in early embryonic intestine. A prior report demonstrated that LGR5-positive cells are evenly distributed in glandular cells in embryonic intestine until the establishment of the mesenchymal BMP signaling [ 35 ]. Accordingly, CSCs are likely to be regulated in a manner like early embryonic intestine, but not like adult intestine, providing us with a novel insight into stem cell niches in rectal cancer. In conclusion, LGR5-positive cells are robust in CRT rectal cancer. Additionally, mesenchymal niche factors of WNT2B and GREM1 are aberrantly and uniformly expressed in untreated and CRT rectal cancer, suggesting early embryonic intestine-like regulation of CSCs in rectal cancer. Declarations Acknowledgments: We thank Dr. Michio Nakaya at the University of Kyushu for the helpful advice on this work. The authors would like to thank Enago (www.enago.jp) for the English language review. Statements and Declarations Competing Interests The authors declare no conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding Statement This work was supported, in part, by a Grant from the Community Health Science Laboratory. Author Contributions Conceived and designed the experiments: Akira Tanaka. Performed and analyzed immunohistochemistry and in situ hybridization: Hirotoshi Kawata and Kentaro Tsuji. Contributed reagents/ materials/ analysis tools: Sachi Sekine, Tomoko Kamiakito, and Takeo Nakaya. Analyzed clinical data: Yasuyuki Miyakura, Koichi Suzuki, and Toshiki Rikiyama. Informed Consent: Informed consent was obtained from all individual patients for histological study. References Batlle E, Clevers H (2017) Cancer stem cells revisited. Nat Med 23:1124–1134. https://doi.org/10.1038/nm.4409 Shibue T, Weinberg RA (2017) EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol 14:611–629. https://doi.org/10.1038/nrclinonc.2017.44 Barker N, van Es JH, Kuipers J et al (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003–1007. https://doi.org/10.1038/nature06196 Barker N, Ridgway RA, van Es JH et al (2009) Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457:608–611. https://doi.org/10.1038/nature07602 Sangiorgi E, Capecchi MR (2008) Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 40:915–920. https://doi.org/10.1038/ng.165 Yan KS, Chia LA, Li X et al (2012) The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Natl Acad Sci U S A 109:466–471. https://doi.org/10.1073/pnas.1118857109 Tetteh PW, Basak O, Farin HF et al (2016) Replacement of lost Lgr5-positive stem cells through plasticity of their enterocyte-lineage daughters. Cell Stem Cell 18:203–213. https://doi.org/10.1016/j.stem.2016.01.001 Gregorieff A, Liu Y, Inanlou MR, Khomchuk Y, Wrana JL (2015) Yap-dependent reporogramming of LGR5(+) stem cells drives intestinal regeneration and cancer. Nature 526:715–718. https://doi.org/10.1038/nature15382 Cai J, Zhang N, Zhen Y, de Wilde RF, Maitra A, Pan D (2010) The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program. Genes Dev 24:2383–2388. https://doi.org/10.1101/gad.1978810 Vazquez EG, Nasreddin N, Valbuena GN et al (2022) Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia. Cell Stem Cell 29:1213–1228.e8. https://doi.org/10.1016/j.stem.2022.07.008 Muncan V, Sansom OJ, Tertoolen L et al (2006) Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc. Mol Cell Biol 26:8418–8426. https://doi.org/10.1128/MCB.00821-06 Blache P, van de Wetering M, Duluc I et al (2004) SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes. J Cell Biol 166:37–47. https://doi.org/10.1083/jcb.200311021 Xian L, Georgess D, Huso T et al (2017) HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche. Nat Commun 8:15008. https://doi.org/10.1038/ncomms15008 Sato T, van Es JH, Snippert HJ et al (2011) Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469:415–418. https://doi.org/10.1038/nature09637 Valenta T, Degirmenci B, Moor AE et al (2016) Wnt ligands secreted by subepithelial mesenchymal cells are essential for the survival of intestinal stem cells and gut homeostasis. Cell Rep 15:911–918. https://doi.org/10.1016/j.celrep.2016.03.088 McCarthy N, Manieri E, Storm E et al (2020) Distinct mesenchymal cell populations generate the essential intestinal BMP signaling gradient. Cell Stem Cell 26:391–402.e5. https://doi.org/10.1016/j.stem.2020.01.008 Pellegrinet L, Rodilla V, Liu Z et al (2011) Dll1- and Dll4- mediated Notch signaling are required for homeostasis of intestinal stem cells. Gastroenterology 140:1230–1240.e1. https://doi.org/10.1053/j.gastro.2011.01.005 Degirmenci B, Valenta T, Dimitrieva S, Hausmann G, Basler K (2018) GLI1-expressing mesenchymal cells form the essential Wnt-secreting niche for colon stem cells. Nature 558:449–453. https://doi.org/10.1038/s41586-018-0190-3 Regan JL, Schumacher D, Staudte S et al (2017) Non-canonical hedgehog signaling is a positive regulator of the WNT pathway and is required for the survival of colon cancer stem cells. Cell Rep 21:2813–2828. https://doi.org/10.1016/j.celrep.2017.11.025 Doheny D, Manore SG, Wong GL, Lo HW (2020) Hedgehog signaling and truncated GLI1 in cancer. Cells 9:2114. https://doi.org/10.3390/cells9092114 Bankaitis ED, Ha A, Kuo CJ, Magness ST (2018) Reserve stem cells in intestinal homeostasis and injury. Gastroenterology 155:1348–1361. https://doi.org/10.1053/j.gastro.2018.08.016 Fusco A, Fedele M (2007) Roles of HMGA proteins in cancer. Nat Rev Cancer 7:899–910. https://doi.org/10.1038/nrc2271 Sharma A, Blériot C, Currenti J, Ginhoux F (2022) Oncofetal reprogramming in tumour development and progression. Nat Rev Cancer 22:593–602. https://doi.org/10.1038/s41568-022-00497-8 O’Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445:106–110. https://doi.org/10.1038/nature05372 Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115. https://doi.org/10.1038/nature05384 Merlos-Suárez A, Barriga FM, Jung P et al (2011) The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell 8:511–524. https://doi.org/10.1016/j.stem.2011.02.020 Kozar S, Morrissey E, Nicholson AM et al (2013) Continuous clonal labeling reveals small numbers of functional stem cells in intestinal crypts and adenomas. Cell Stem Cell 13:626–633. https://doi.org/10.1016/j.stem.2013.08.001 Fan XS, Wu HY, Yu HP, Zhou Q, Zhang YF Huang Q (2010) Expression of Lgr5 in human colorectal carcinogenesis and its potential correlation with beta-catenin. Int J Colorectal Dis 25:583–590. https://doi.org/10.1007/s00384-010-0903-z He S, Zhou H, Zhu X et al (2014) Expression of Lgr5, a marker of intestinal stem cells, in colorectal cancer and its clinicopathological significance. Biomed Pharmacother 68:507–513. https://doi.org/10.1016/j.biopha.2014.03.016 Ziskin J, Dunlap D, Yaylaoglu M et al (2013) In situ validation of an intestinal stem cell signature in colorectal cancer. Gut 62:1012–1023. https://doi.org/10.1136/gutjnl-2011-301195 Li DW, Tang H, Fan JW et al (2010) Expression level of Bmi-1 oncoprotein is associated with progression and prognosis in colon cancer. J Cancer Res Clin Oncol 136:997–1006. https://doi.org/10.1007/s00432-009-0745-7 Wang L, Shi S, Guo Z et al (2013) Overexpression of YAP and TAZ is an independent predictor of prognosis in colorectal cancer and related to the proliferation and metastasis of colon cancer cells. PLOS ONE 8:e65539. https://doi.org/10.1371/journal.pone.0065539 Shah SN, Kerr C, Cope L et al (2012) HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks. PLOS ONE 7:e48533. https://doi.org/10.1371/journal.pone.0048533 Belton A, Gabrovsky A, Bae YK et al (2012) HMGA1 induces intestinal polyposis in transgenic mice and drives tumor progression and stem cell properties in colon cancer cells. PLoS One 7:e30034. https://doi.org/10.1371/journal.pone.0030034 . Shyer AE, Huycke TR, Lee CH, Mhadevan L, Tabin CJ (2015) Bending gradients: how the intestinal stem cell gets its home. Cell 161:569–580. https://doi.org/10.1016/j.cell.2015.03.041 Additional Declarations No competing interests reported. Supplementary Files colonstemcellfinal230608copysuppl.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3037112","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":208891088,"identity":"a6f693cc-c9b9-4d3c-92d8-9c4fa60a2779","order_by":0,"name":"Kentaro Tsuji","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kentaro","middleName":"","lastName":"Tsuji","suffix":""},{"id":208891091,"identity":"bb72ea37-b167-4140-826b-1528f2d4e71b","order_by":1,"name":"Sachi Sekine","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sachi","middleName":"","lastName":"Sekine","suffix":""},{"id":208891092,"identity":"4f4161b0-0fc0-4e44-8ea5-c9f94da99984","order_by":2,"name":"Hirotoshi Kawata","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hirotoshi","middleName":"","lastName":"Kawata","suffix":""},{"id":208891093,"identity":"31738ef9-2201-4f98-be8d-52d652adbc15","order_by":3,"name":"Tomoko Kamiakito","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tomoko","middleName":"","lastName":"Kamiakito","suffix":""},{"id":208891094,"identity":"b6c26f0f-fd9f-461f-b0e8-958421f10fd0","order_by":4,"name":"Takeo Nakaya","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takeo","middleName":"","lastName":"Nakaya","suffix":""},{"id":208891095,"identity":"dfcc2fde-3597-4fb1-81e1-3ddc2aec0da5","order_by":5,"name":"Yasuyuki Miyakura","email":"","orcid":"","institution":"Jichi Medical University Saitama Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasuyuki","middleName":"","lastName":"Miyakura","suffix":""},{"id":208891096,"identity":"e1fca1cc-5fa3-415d-932f-492249f8dc72","order_by":6,"name":"Koichi Suzuki","email":"","orcid":"","institution":"Jichi Medical University Saitama Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Koichi","middleName":"","lastName":"Suzuki","suffix":""},{"id":208891097,"identity":"33408b13-afcf-4bff-9e00-b781d4638151","order_by":7,"name":"Toshiki Rikiyama","email":"","orcid":"","institution":"Jichi Medical University Saitama Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Toshiki","middleName":"","lastName":"Rikiyama","suffix":""},{"id":208891098,"identity":"922b12d8-a921-415c-8885-7f24dc9673b1","order_by":8,"name":"Akira Tanaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYNCCAwxyjM0MbAwJUB5+wAZRZEy6lsQGKJMw4J/f/OzBjzN26c3tzM8ePGCwk2dgPIvfGoljbOaGPTeScxub2cwNEhiSDRsYziXgt+YYg5kEzwdmoBYeNokEBmag8jMGeHXIH2P/JvnnQ306I0RLPWEtBsd4zKR5bhxOgGo5TFiL4bGcMmmZM8cNgX4xk0gwOG7YRsgvcoePb5N8c6xa3rD/8DPJHxXV8vwSBEIMYV0D2J3A2JE4Q5wOBnk4i7+HSC2jYBSMglEwUgAADalDBMpHLBkAAAAASUVORK5CYII=","orcid":"","institution":"Jichi Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Tanaka","suffix":""}],"badges":[],"createdAt":"2023-06-08 06:14:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3037112/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3037112/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":38555846,"identity":"28b87475-2ecb-4620-8142-7b188024710b","added_by":"auto","created_at":"2023-06-14 18:03:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":140159,"visible":true,"origin":"","legend":"\u003cp\u003eExpressions of LGR5, BMI1, and b-catenin in untreated and chemoradiation-treated rectal cancers (A, B) LGR5 transcripts are detected using the specific human LGR5 RNA probe as described in the Materials and Methods section in untreated and chemoradiation-treated (CRT) rectal cancer. (C, D) BMI1 transcripts are detected using the specific human BMI1 probe in untreated and CRT rectal cancer. (E, F) Representative pictures of b-catenin immunostaining in untreated and CRT rectal cancer. Scale bar: 50 mm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3037112/v1/d9e55e622785b5a2af449310.png"},{"id":38555848,"identity":"52235632-132b-40d5-9636-4b647fbca3ba","added_by":"auto","created_at":"2023-06-14 18:03:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105297,"visible":true,"origin":"","legend":"\u003cp\u003eExpressions of c-MYC, SOX9, and HMGA1 in untreated and CRT rectal cancers (A, B) Representative pictures of c-MYC immunostaining in untreated and in CRT rectal cancer. (C, D) Representative pictures of SOX9 immunostaining in untreated and CRT rectal cancer. (E, F) Representative pictures of HMGA1 immunostaining in untreated and CRT rectal cancer. Scale bar: 50 mm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3037112/v1/68a8f648caa8b914039785a5.png"},{"id":38557378,"identity":"58352cda-6b5e-4089-a21d-6dec9276019f","added_by":"auto","created_at":"2023-06-14 18:11:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":195555,"visible":true,"origin":"","legend":"\u003cp\u003eExpressions of WNT3A, HES1, WNT2B, and GREM1 in untreated and chemoradiation-treated rectal cancers (A, B, C) WNT3A transcripts are detected using the specific human WNT3A probe in non-neoplastic mucosa (Normal) and untreated and CRT rectal cancer. (D, E, F) Representative pictures of HES1 in non-neoplastic mucosa and untreated and CRT rectal cancer. (G, H, I) Representative pictures of WNT2B immunostaining in non-neoplastic mucosa and untreated and CRT rectal cancer. (J, K, L) Representative pictures of GREM1 in non-neoplastic mucosa and untreated and CRT rectal cancer. White arrows indicate WNT3A expression in crypt bases. Scale bar: 50 mm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3037112/v1/bb0cef909fc8ecdbeb5621ac.png"},{"id":38557377,"identity":"a6b047eb-38d7-4dc3-9146-c4188257e174","added_by":"auto","created_at":"2023-06-14 18:11:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":199174,"visible":true,"origin":"","legend":"\u003cp\u003eExpressions of YAP/TAZ, GLI1, SHH, and IHH in untreated and CRT rectal cancer (A, B, C) Representative pictures of YAP/TAZ (YAP/TAZ) immunostaining in non-neoplastic (Normal) mucosa and untreated and CRT rectal cancer. (D, E, F) Representative pictures of GLI1 immunostaining in non-neoplastic mucosa and untreated and CRT rectal cancer. (G, H, I) Representative pictures of SHH in non-neoplastic mucosa and untreated and CRT rectal cancer. (J, K, L) Representative pictures of IHH in non-neoplastic mucosa and untreated and CRT rectal cancer (K, L). Scale bar: 50 mm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3037112/v1/d36c7329518fb92bb7a67747.png"},{"id":38557379,"identity":"9152a346-23fc-4584-9936-c57ebaa91b29","added_by":"auto","created_at":"2023-06-14 18:11:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":139686,"visible":true,"origin":"","legend":"\u003cp\u003eExpressions of WNT2B and GREM1 in embryonic intestine (A, B) Hematoxylin \u0026amp; eosin staining (H \u0026amp; E) in intestine at E12.5 (E12.5) and E16.5 (E16.5). WNT2B (C) and GREM1 (E) immunostainings in the embryonic intestine at E12.5. Both proteins showed uniform expression in the intestine. WNT2B (D) and GREM1 (F) immunostainings in the intestine at E16.5. Both proteins were regionally expressed at E16.5. Scale bar: 20 mm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3037112/v1/3545c03cdabf6c73d343f4f2.png"},{"id":38623133,"identity":"d86c6695-ca06-47fe-a353-50474ee23b6f","added_by":"auto","created_at":"2023-06-15 19:29:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1413476,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3037112/v1/869ac2aa-839d-4785-b872-2bbcb0a0caa0.pdf"},{"id":38555850,"identity":"bac88fbd-847e-4d16-9152-5f405deaee6d","added_by":"auto","created_at":"2023-06-14 18:03:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":847034,"visible":true,"origin":"","legend":"","description":"","filename":"colonstemcellfinal230608copysuppl.docx","url":"https://assets-eu.researchsquare.com/files/rs-3037112/v1/710b11d55f647b62c719041b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Robust expressions of stem cell markers, including leucine-rich repeat-containing G protein- coupled receptor 5, in association with early embryonic expression patterns of niche factors in untreated and chemoradiation-treated rectal cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTherapy resistance is one of the most critical problems in managing patients with cancer. A growing body of evidence strongly suggests that cancer stem cells (CSCs) greatly contribute to therapy resistance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive cells are generally accepted as stem cells in human colorectal cancer as well as in normal colorectal crypts under the control of WNT/β-catenin signaling [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Meanwhile, cells that are located at the +\u0026thinsp;4 position and expressing proto-oncogene and polycomb ring finger 1 (BMI1) are shown to function as quiescent stem cells in the intestine [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and these cells can compensate for intestinal homeostasis under complete loss of LGR5-positive cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, yes-associated transcriptional regulator 1 (YAP1) and its paralog TAZ (WW domain containing transcription regulator 1 [WWTR1]) (hereafter YAP/TAZ) are major factors to contribute to stem cell regeneration after mucosal injuries in the intestine [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. An earlier report revealed YAP/TAZ as a marker of regenerative stem cells without LGR5 expression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Histopathologic CSC status has been poorly analyzed in surgically resected specimens, in contrast to the expanded knowledge of cell biology on CSCs in colorectal cancer. This study compared LGR5, BMI1, and nuclear YAP/TAZ expression ratios, which cover most of CSCs, in untreated and chemoradiation-treated (CRT) rectal cancer, using a highly sensitive \u003cem\u003ein situ\u003c/em\u003e hybridization (ISH) method for LGR5 and BMI1. Given the pivotal role of WNT/β-catenin signaling in rectal cancer, we also estimated ratios of nuclear β-catenin.\u003c/p\u003e \u003cp\u003eIn addition to these proteins, MYC proto-oncogene bHLH transcription factor (c-MYC), SRY-box transcription factor 9 (SOX9), and high mobility group AT-hook 1 (HMGA1) are involved in CSC activity in association with WNT/β-catenin signaling [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Meanwhile, niche factors contribute to stem cell maintenance. WNT family member 3A (WNT3A) is a prototypal epithelial niche factor secreted by Paneth cells and maintains LGR5-positive stem cells in the normal intestine [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Stromal WNT2B compensates for WNT3A in maintaining LGR5-positive stem cells under epithelial damage [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, gremlin 1 DAN family BMP antagonist (GREM 1), which is an endogenous BMP antagonist, generates a BMP signaling gradient to preserve the stem cell niche [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and NOTCH signaling has been involved in stem cell regeneration [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Finally, GLI family zinc finger 1 (GLI1)-expressing mesenchymal cells are reported to contribute to the formation of stem cell niches in the intestine by secreting WNT proteins [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. GLI1 is an effector of the hedgehog growth factor family and is involved in various biological functions, including organ morphogenesis and tumorigenesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These factors are likely involved in therapy resistance through CSC modulations. However, analysis of these molecules remains lacking at histopathologic levels. Therefore, the present study compared the expression levels of these factors in untreated and CRT rectal cancer. Finally, niche factors of WNT2B and GREM1 were also analyzed in murine embryonic intestine.\u003c/p\u003e \u003cp\u003eIn a series of histopathologic analyses, we demonstrated robust expressions of LGR5, BMI1, and nuclear YAP/TAZ and β-catenin in untreated and CRT rectal cancer. Additionally, HMGA1 as well as stromal YAP/TAZ and GLI1 are significantly upregulated in the CRT cohort. We also showed that niche factors of WNT2B and GREM1 are uniformly expressed with similarity to the expression pattern of early embryonic intestine, suggesting fetus-like niche formation in rectal cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cstrong\u003eCases\u003c/strong\u003e \u003cp\u003eHuman rectal adenocarcinoma specimens resected from 29 patients receiving CRT (the sample from a 30th case was discarded due to the loss of cancer components during section preparation) and from 30 untreated patients were retrieved from the archives of the Pathology Department of Jichi Medical University Saitama Medical Center Hospital. All tumors demonstrated moderately to well-differentiated tubular adenocarcinoma with minimal foci of well-differentiated mucinous carcinoma. All samples exhibited a diffuse positive expression of cytokeratin 8 without obvious undifferentiated or sarcomatous components. Additionally, the lack of obvious neuroendocrine differentiation, which was estimated using synaptophysin expression, was confirmed. The patient characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The CRT duration was 15\u0026ndash;168 (median; 34.5) days. Immunohistochemistry and ISH were performed with the approval of the local ethics committee at Jichi Medical University Saitama Medical Center Hospital.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient characteristics and treatment regimens The number of cases is shown except for medial values with ranges in Age, Radiation, and CRT Duration\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;29)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge (years)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.6 (49ཞ84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.5 (38ཞ81)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex (male/female)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25/5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNeoaduvant\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5-FU/LV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eothers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadiation (Gy)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43(40ཞ50)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of CRT (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.1(15ཞ168)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAnimals\u003c/em\u003e \u003c/p\u003e \u003cp\u003ePregnant female mice were purchased from SLC (Shizuoka, Japan). Embryos were obtained at the indicated time, and whole paraffin-embedded sections of embryos were prepared using a routine histologic technique. An animal experiment was performed with the approval of the animal experiment committee at Jichi Medical University.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAntibodies\u003c/em\u003e \u003c/p\u003e \u003cp\u003eMouse monoclonal antibody against beta-catenin (product ID: 610154) was purchased from BD Biosciences (Franklin Lakes, NJ). Rabbit polyclonal antibodies against WNT2B (product ID: ABO12151), GREM1 (product ID: PAB4716), GLI1 (product ID: NBP1-78259), and SHH (product ID: 06-1106) were purchased from abcepta (San Diego, CA), abnova (Taipei, Taiwan), Novus biologicals (Centennial, CO), and Millipore (Billerica, MA), respectively. Rabbit polyclonal antibodies against IHH (product ID: 13388-1-AP) and LIN28B (product ID: 16178-1-AP) were purchased from Proteintech (Rosemont, IL). Rabbit monoclonal antibody against YAP/TAZ (clone D24E4) (product ID: 8418S), HES1 (clone D6P2U) (product ID: 11988S), SOX9 (clone D8G8H) (product ID: 82630S), and HMGA1 (clone D4F8) (product ID: 12904S) were purchased from Cell Signaling Technologies. Rabbit monoclonal antibody against c-MYC (clone Y69) (product ID: ab32072) was purchased from abcam (Cambridge, UK).\u003c/p\u003e \u003cp\u003e \u003cem\u003eISH and immunohistochemistry\u003c/em\u003e \u003c/p\u003e \u003cp\u003eISH was performed using specific probes, recognizing multiple mRNA sequences of human LGR5, BMI1, and WNT3A (Advanced Cell Diagnostics Inc. Hayward, CA). Slides were incubated in a dry oven for 1 h at 60\u0026deg;C and de-paraffinized. Then, they were boiled for 15 min, treated with the proteinase reagent provided by the kit, and incubated with the specific probes in an oven for 2 h at 40\u0026deg;C. Afterward, slides were incubated with the kit amplifying reagents and then with the kit detection reagents. Signals were visualized by an immunofluorescence microscope (BZ-X700, Keyence, Osaka, Japan). One or two representative sites were photographed by optic microscopy, and expression ratios were determined in each photo between the positive cells and the total cancer cells.\u003c/p\u003e \u003cp\u003eFormalin-fixed and paraffin-embedded sections were pre-treated in a microwave oven or an autoclave for the indicated times for immunohistochemistry (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The pre-treated sections were incubated with each antibody at 4\u0026deg;C overnight, then with Envision (DAKO JAPAN, Tokyo, Japan), and stained with 3,3\u0026rsquo;-diaminobenzidine. Antibodies and methods used to retrieve and detect the antigens are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Non-neoplastic mucosa and sections from mouse embryos were used as positive controls. Positive ratios of nuclear YAP/TAZ and β-catenin expression were determined by three representative photos. The immunostaining scores were defined by positive areas as follows: score 0, no positive staining or \u0026lt;\u0026thinsp;1%; score 1, 1\u0026ndash;10%; score 2, \u0026gt;\u0026thinsp;10%.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmunohistochemical protocols.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibodies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDilution ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePretreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb-catenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicrowave 20 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYAP/TAZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAutoclave 5 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHES1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:1600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAutoclave 5 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWNT2B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAutoclave 5 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGREM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:1600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAutoclave 5 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGLI1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicrowave 10 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOX9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAutoclave 5 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHMGA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicrowave 10 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec-MYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAutoclave 5 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicrowave 10 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicrowave 10 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLIN28B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicrowave 10 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe statistical significance of the value changes was determined using the Mann-Whitney U test, and the correlation was estimated using Spearman\u0026rsquo;s correlation via rank testing with Statcel software, version 4 (OMS, Saitama, Japan). A \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eRobust expressions of LGR5 and BMI1 as well as nuclear YAP/TAZ and β-catenin in CRT rectal cancer\u003c/em\u003e \u003c/p\u003e \u003cp\u003eLGR5 transcripts were selectively detected in the crypt base using the specific probe in non-neoplastic mucosa as previously reported (supplementary Figs.\u0026nbsp;1A and 1B) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In contrast, BMI1 transcripts were unrestricted to +\u0026thinsp;4 cells but scattered throughout the whole crypt, which is concordant with the previous description (supplementary Figs.\u0026nbsp;1C and D) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We then compared the positive ratios of LGR5 and BMI1 in untreated and CRT rectal cancer using the highly sensitive ISH method. Additionally, nuclear YAP/TAZ and β-catenin expressions were investigated via immunohistochemistry to assess the nuclear localization. ISH revealed that LGR5-positive cells are often expressed in clusters, contrasting the diffusing and scattered patterns of BMI1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;D). The expression ratios (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation [SD]) were 15% \u0026plusmn; 22% and 14% \u0026plusmn; 27% for LGR5 and 30% \u0026plusmn; 33% and 33% \u0026plusmn; 33% for BMI1 in untreated and CRT tumors, respectively. No significant difference was found in LGR5 or BMI1 expression between untreated and CRT cohorts. As shown below, YAP/TAZ expression in cancer cells was limited in untreated and CRT rectal cancer, and scarce in normal mucosa (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;F). Ratios of YAP/TAZ nuclear expressions (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) were 2.7% \u0026plusmn; 7.4% in untreated and 7.6% \u0026plusmn; 16% in CRT rectal cancer, showing no significant YAP/TAZ upregulation in the CRT tumors. Meanwhile, β-catenin nuclear expression ratios (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) were 38% \u0026plusmn; 24% in untreated and 32% \u0026plusmn; 20% in CRT tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). We scored LGR5, BMI1, β-catenin, and YAP/TAZ expression as described in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eMaterials and Methods\u003c/span\u003e section to further assess LGR5 expression (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A significant correlation was detected between LGR5 and β-catenin nuclear expression in the CRT tumors (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, rs\u0026thinsp;=\u0026thinsp;0.43) using the Spearman\u0026rsquo;s correlation by rank test through this scoring (supplementary Table\u0026nbsp;1), suggesting that the LGR5/β-catenin axis is more evident in the CRT samples than in the untreated rectal cancer. No significant correlation was found between YAP/TAZ and β-catenin as well as BMI1 and β-catenin (data not shown). In summary, LGR5 and BMI1 as well as nuclear YAP/TAZ and β-catenin are robustly expressed in untreated and CRT rectal cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmunostaining results for LGR5 BMI1, β-catenin, and YAP/TAZ in untreated and CRT rectal cancer. The number of cases is shown by the scoring (score of 2, \u0026gt;\u0026thinsp;10%; score of 1, 1\u0026ndash;10%; score of 0, no staining or \u0026lt;\u0026thinsp;1%).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eEXPRESSION SCORE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLGR5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eBMI1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eb-catenin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eYAP/TAZ\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.469\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.372\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.941\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.219\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eUpregulation of HMGA1, but not SOX9 and c-MYC, in CRT rectal cancer\u003c/em\u003e \u003c/p\u003e \u003cp\u003eNext, we compared c-MYC, SOX9, and HMGA1 expressions due to their relation to WNT/β-catenin signaling [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. C-MYC and HMGA1 expressions were scarce in non-neoplastic mucosa, whereas SOX9-positive cells were mainly located in lower portions of crypts (data not shown). C-MYC was expressed in 4 (13%) cases at a score of 1 in untreated tumors in rectal cancer specimens, whereas all cases revealed the expression of \u0026lt;\u0026thinsp;1% (score of 0) in the CRT cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). C-MYC expression was at significantly higher levels in the untreated tumors than in the CRT rectal cancer, suggesting that its expression is correlated with proliferative activity. Meanwhile, SOX9 was expressed at \u0026gt;\u0026thinsp;10% (score of 2) in all cases of both untreated and CRT tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). HMGA1 was expressed in 2 (6.7%) cases at a score of 2 and 10 (33%) cases at a score of 1 out of 30 cases of the untreated tumors, whereas 11 (38%) were at a score of 2 and 9 (31%) at a score of 1 in the CRT cohort (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). HMGA1 was expressed at significantly higher levels in the CRT cohort than in the untreated tumors (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00273) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) though its expression was unrelated to nuclear β-catenin in the CRT tumors by the Spearman\u0026rsquo;s correlation by rank test (supplementary Table\u0026nbsp;2). We subsequently investigated expression of lin-28 homolog B (LIN28B) in untreated and CRT rectal cancer because HMGA proteins are regulated by LIN28 or the let-7 microRNA family [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. LIN28B expression was scarce except for two cases with weak expression of \u0026lt;\u0026thinsp;1% (score of 0) in CRT rectal cancer (data not shown and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In summary, HMGA1, but not SOX9 and c-MYC, was significantly upregulated in CRT rectal cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmunostaining results for SOX9, c-MYC, ΗΜGΑ1, and LIN28B in cancer cells of untreated and CRT rectal cancer.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eEXPRESSION SCORE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eSOX9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ec-MYC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eHMGA1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eLIN28B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003enot determined\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003enot determined\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe number of cases is shown by the scoring (score of 2, \u0026gt;\u0026thinsp;10%; score of 1, 1\u0026ndash;10%; score of 0, no staining or \u0026lt;\u0026thinsp;1%).\u003c/p\u003e \u003cp\u003e \u003cem\u003eUniform and aberrant expressions of mesenchymal niche factors, WNT2B, and GREM1 in cancer cells of untreated and CRT rectal cancer\u003c/em\u003e \u003c/p\u003e \u003cp\u003eNext, we compared expressions of niche factors in untreated and CRT rectal cancer. Concordant with the reported data [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], WNT3A was expressed at crypt bases, whereas WNT2B and GREM1 were mainly expressed in mesenchymal cells in non-neoplastic mucosa (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). HES1 expression was scattered in non-neoplastic crypts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). WNT3A was detected at levels of \u0026gt;\u0026thinsp;10% (score of 2) in 3 (10%) out of 30 untreated tumors and 1 (3%) out of 29 CRT rectal cancer, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Nuclear expression of HES1 was detected at \u0026gt;\u0026thinsp;10% (score of 2) in 1 (3%) out of 30 untreated samples and 1 (3%) out of 29 cases of CRT rectal tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The low expression of WNT3A and HES1 in rectal cancer suggests that both proteins have less significance for maintaining CSCs. In contrast, WNT2B and GREM1 were uniformly and aberrantly expressed in rectal cancer cells along with distinct expression in stroma cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). WNT2B was detected at \u0026gt;\u0026thinsp;10% (score of 2) in all untreated rectal cancer and 27 (93%) out of 29 CRT tumors in the cancer cell expression (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). GREM1 was detected at \u0026gt;\u0026thinsp;10% (score of 2) in all samples of untreated rectal cancer, whereas in 26 (90%) out of 29 cases of CRT rectal cancer (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Collectively, mesenchymal niche factors of WNT2B and GREM1 may support cancer survival by activation of WNT/β-catenin in association with suppression of BMP signals in CRT rectal cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmunostaining results for WNT3A, HES1, WNT2B, and GREM1 in untreated and CRT rectal cancer. The number of cases is shown by the scoring (score of 2, \u0026gt;\u0026thinsp;10%; score of 1, 1\u0026ndash;10%; score of 0, no staining or \u0026lt;\u0026thinsp;1%).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eEXPRESSION SCORE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eWNT3A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eHES1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eWNT2B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eGREM1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.321\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.305\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.147\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.073\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eUpregulation of GLI1 and YAP/TAZ in stroma cells after CRT\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe next compared GLI1 nuclear expression in stroma cells between untreated and CRT rectal cancer because GLI1-expressing mesenchymal cells are reported to form stem cell niches in the colon [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. GLI1 was detected in non-neoplastic and cancerous stomas (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). GLI1 nuclear expression in stroma cells was detected in 1 (3%) and 9 (30%) out of 30 untreated samples and 9 (31%) and 12 (41%) out of 29 CRT samples at scores of 2 and 1 in rectal cancer specimens, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). GLI1 expression in CRT stroma was significantly higher than that in the untreated stroma of rectal cancers (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). This finding prompted us to re-analyze nuclear YAP/TAZ expression, specifically in stroma cells, because earlier reports revealed the involvement of YAP/TAZ in the generation of cancer-associated fibroblasts [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. YAP/TAZ was expressed in stroma cells in 1 (3%) and 10 (33%) out of 30 untreated tumors, whereas 6 (21%) and 18 (62%) out of 29 CRT tumors at scores of 2 and 1, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). YAP/TAZ expression in the nucleus of stroma cells in CRT tumors was significantly higher than that in untreated rectal cancer (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). We next compared GLI1 and YAP/TAZ stromal expressions with the β-catenin nuclear expression in cancer because GLI1-expressing stroma cells are shown to secrete WNT proteins [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This analysis revealed no significant correlation of GLI1 or YAP/TAZ expressions with β-catenin nuclear expression in both the untreated and CRT cohorts (data not shown). Additionally, we investigated the expressions of sonic hedgehog signaling molecule (SHH) and Indian hedgehog signaling molecule (IHH)) in untreated and CRT rectal cancer because GLI1 is a signal transducer of the hedgehog family growth factor. SHH was expressed in the surface epithelia and upper crypts in non-neoplastic mucosa, while IHH was diffusely expressed in crypts (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). SHH was expressed at \u0026gt;\u0026thinsp;10% in all rectal cancer specimens (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI and Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). IHH was expressed in 17 (57%) and 9 (30%) cases in the untreated tumors, whereas 11 and 7 cases in the CRT rectal cancer at scores 2 and 1, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL and Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). IHH and SHH expressions revealed no significant difference between the untreated and the CRT rectal cancer, suggesting that GLI1 upregulation is uncoupled to SHH or IHH expression. To summarize, GLI1 and YAP/TAZ expression levels were significantly upregulated in stroma cells in CRT rectal cancer, suggesting that stromal activation lasts for substantial time after CRT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmunostaining results for IHH, SHH, YAP/TAZ, and GLI1. The number of cases is shown by positive areas stained for IHH and SHH in cancer cells as well as YAP/TAZ and GLI1 in stroma cells of untreated and CRT rectal cancer. The numbers of cases were shown by the scoring (score of 2, \u0026gt;\u0026thinsp;10%; score of 1, 1\u0026ndash;10%; score of 0, no staining or \u0026lt;\u0026thinsp;1%).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eEXPRESSION SCORE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eIHH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eSHH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eYAP/TAZ\u003c/p\u003e \u003cp\u003e(stroma)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eGLI1(stroma)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003euntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCRT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003escore 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.055\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003enot determined\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.0002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.0007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eExpression patterns of WNT2B and GREM1 in murine embryonic intestine\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFetal-like features of malignant cells, including stroma and immune cells, have been recently mentioned as \u0026ldquo;oncofetal reprogramming\u0026rdquo; [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, we investigated WNT2B and GREM1 expressions in murine embryonic intestine. In embryonic intestine at E12.5, WNT2B, and GREM1 were uniformly expressed in glandular and mesenchymal cells, whereas expressions of both proteins were weaker in glandular and mesenchymal cells at E16.5 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;F). As described above, WNT2B and GREM1 were limitedly expressed in mesenchymal cells of adult human rectum. Accordingly, expression patterns of WNT2B and GREM1 in rectal cancer mimic those of early embryonic intestine, suggesting the oncofetal reprogramming. Stem cell niches of rectal cancer might be similar to those of early embryonic intestine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe hypothesized that LGR5 or BMI1-positive cells are expanded in CRT rectal cancer based on the significance of CSCs in therapy resistance. Furthermore, YAP/TAZ, a regenerative CSC marker, is speculated to be abundant in CRT rectal cancer. The mean ratios of LGR5 were approximately 15% and 14% in untreated and CRT rectal cancer, respectively. Other CSC markers were 30% and 33% in BMI1, 3% and 8% in YAP/TAZ, and 38% and 32% in β-catenin in untreated and CRT rectal cancer, respectively. Against our hypothesis, overall expressions of LGR5 and BMI1 as well as nuclear YAP/TAZ and β-catenin are robust in untreated and CRT rectal cancer, suggesting the rigid CSC homeostasis in rectal cancer. Earlier reports evaluated CSC ratios at 2.5% \u0026plusmn; 1.4% [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and 12% (range: 1.8\u0026ndash;24.5%) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] in colorectal cancer though a recent report that estimated CSCs in a range from 0.0036\u0026ndash;1.8% using organoid culture [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In contrast, a prior report reported a wide range of LGR5 expression from \u0026lt;\u0026thinsp;1\u0026ndash;95% in colorectal cancer [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Other reports revealed approximately 20% LGR5 expression in adenoma of a murine model [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], 28% in adenoma and 54% in colorectal cancer [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and 74% in colorectal cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally, BMI1 expression was reported as 47% [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and 66.5% [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while either YAP or TAZ-positive cells were described as 93.5% [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] in colorectal cancer. These values are considerably higher than the predicted CSCs as well as the present estimation for these proteins. It is important to mention that LGR5 is significantly correlated to nuclear β-catenin expression in CRT rectal cancer in this study. Accordingly, the present estimation of CSC markers, particularly LGR5, might provide standard values on CSC markers in rectal cancer though it is possible that nuclear expression of YAP/TAZ might be transient after CRT in rectal cancer.\u003c/p\u003e \u003cp\u003eA series of immunohistochemical studies revealed that HMGA1 as well as stromal YAP/TAZ and GLI1 are significantly upregulated in the CRT cohort, compared to the untreated tumor. HMGA proteins preserve stem cell conditions in embryonic stem cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, HMGA1 induces intestinal polyposis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and amplifies WNT/β-catenin signaling in colorectal cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These features indicate that HMGA1 supports the maintenance of CSCs to enhance the WNT/β-catenin signaling in CRT rectal cancer. Stromal activation of YAP/TAZ and GLI1 may also support maintenance of CSCs in CRT rectal cancer.\u003c/p\u003e \u003cp\u003eIn study of niche factors, we found that mesenchymal niche factors of WNT2B and GREM1 were uniformly and aberrantly expressed in rectal cancer while expressions of epithelial niche factors, WNT3A and HES1, were limited. Notably, similar aberrant and uniform expressions of WNT2B and GREM1 were found in early embryonic intestine. A prior report demonstrated that LGR5-positive cells are evenly distributed in glandular cells in embryonic intestine until the establishment of the mesenchymal BMP signaling [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Accordingly, CSCs are likely to be regulated in a manner like early embryonic intestine, but not like adult intestine, providing us with a novel insight into stem cell niches in rectal cancer.\u003c/p\u003e \u003cp\u003eIn conclusion, LGR5-positive cells are robust in CRT rectal cancer. Additionally, mesenchymal niche factors of WNT2B and GREM1 are aberrantly and uniformly expressed in untreated and CRT rectal cancer, suggesting early embryonic intestine-like regulation of CSCs in rectal cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Dr. Michio Nakaya at the University of Kyushu for the helpful advice on this work. The authors would like to thank Enago (www.enago.jp) for the English language review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported, in part, by a Grant from the Community Health Science Laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the experiments: Akira Tanaka. Performed and analyzed immunohistochemistry and \u003cem\u003ein situ\u003c/em\u003e hybridization: Hirotoshi Kawata and Kentaro Tsuji. Contributed reagents/ materials/ analysis tools: Sachi Sekine, Tomoko Kamiakito, and Takeo Nakaya. Analyzed clinical data: Yasuyuki Miyakura, Koichi Suzuki, and Toshiki Rikiyama.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent:\u003c/strong\u003e Informed consent was obtained from all individual patients for histological study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBatlle E, Clevers H (2017) Cancer stem cells revisited. Nat Med 23:1124\u0026ndash;1134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nm.4409\u003c/span\u003e\u003cspan address=\"10.1038/nm.4409\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShibue T, Weinberg RA (2017) EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol 14:611\u0026ndash;629. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrclinonc.2017.44\u003c/span\u003e\u003cspan address=\"10.1038/nrclinonc.2017.44\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarker N, van Es JH, Kuipers J et al (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003\u0026ndash;1007. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature06196\u003c/span\u003e\u003cspan address=\"10.1038/nature06196\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarker N, Ridgway RA, van Es JH et al (2009) Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457:608\u0026ndash;611. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature07602\u003c/span\u003e\u003cspan address=\"10.1038/nature07602\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSangiorgi E, Capecchi MR (2008) Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 40:915\u0026ndash;920. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ng.165\u003c/span\u003e\u003cspan address=\"10.1038/ng.165\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan KS, Chia LA, Li X et al (2012) The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Natl Acad Sci U S A 109:466\u0026ndash;471. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1118857109\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1118857109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTetteh PW, Basak O, Farin HF et al (2016) Replacement of lost Lgr5-positive stem cells through plasticity of their enterocyte-lineage daughters. Cell Stem Cell 18:203\u0026ndash;213. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.stem.2016.01.001\u003c/span\u003e\u003cspan address=\"10.1016/j.stem.2016.01.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGregorieff A, Liu Y, Inanlou MR, Khomchuk Y, Wrana JL (2015) Yap-dependent reporogramming of LGR5(+) stem cells drives intestinal regeneration and cancer. Nature 526:715\u0026ndash;718. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature15382\u003c/span\u003e\u003cspan address=\"10.1038/nature15382\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai J, Zhang N, Zhen Y, de Wilde RF, Maitra A, Pan D (2010) The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program. Genes Dev 24:2383\u0026ndash;2388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/gad.1978810\u003c/span\u003e\u003cspan address=\"10.1101/gad.1978810\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVazquez EG, Nasreddin N, Valbuena GN et al (2022) Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia. Cell Stem Cell 29:1213\u0026ndash;1228.e8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.stem.2022.07.008\u003c/span\u003e\u003cspan address=\"10.1016/j.stem.2022.07.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuncan V, Sansom OJ, Tertoolen L et al (2006) Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc. Mol Cell Biol 26:8418\u0026ndash;8426. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/MCB.00821-06\u003c/span\u003e\u003cspan address=\"10.1128/MCB.00821-06\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlache P, van de Wetering M, Duluc I et al (2004) SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes. J Cell Biol 166:37\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1083/jcb.200311021\u003c/span\u003e\u003cspan address=\"10.1083/jcb.200311021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXian L, Georgess D, Huso T et al (2017) HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche. Nat Commun 8:15008. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms15008\u003c/span\u003e\u003cspan address=\"10.1038/ncomms15008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato T, van Es JH, Snippert HJ et al (2011) Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469:415\u0026ndash;418. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature09637\u003c/span\u003e\u003cspan address=\"10.1038/nature09637\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValenta T, Degirmenci B, Moor AE et al (2016) Wnt ligands secreted by subepithelial mesenchymal cells are essential for the survival of intestinal stem cells and gut homeostasis. Cell Rep 15:911\u0026ndash;918. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.celrep.2016.03.088\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2016.03.088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCarthy N, Manieri E, Storm E et al (2020) Distinct mesenchymal cell populations generate the essential intestinal BMP signaling gradient. Cell Stem Cell 26:391\u0026ndash;402.e5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.stem.2020.01.008\u003c/span\u003e\u003cspan address=\"10.1016/j.stem.2020.01.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePellegrinet L, Rodilla V, Liu Z et al (2011) Dll1- and Dll4- mediated Notch signaling are required for homeostasis of intestinal stem cells. Gastroenterology 140:1230\u0026ndash;1240.e1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1053/j.gastro.2011.01.005\u003c/span\u003e\u003cspan address=\"10.1053/j.gastro.2011.01.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDegirmenci B, Valenta T, Dimitrieva S, Hausmann G, Basler K (2018) GLI1-expressing mesenchymal cells form the essential Wnt-secreting niche for colon stem cells. Nature 558:449\u0026ndash;453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-018-0190-3\u003c/span\u003e\u003cspan address=\"10.1038/s41586-018-0190-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRegan JL, Schumacher D, Staudte S et al (2017) Non-canonical hedgehog signaling is a positive regulator of the WNT pathway and is required for the survival of colon cancer stem cells. Cell Rep 21:2813\u0026ndash;2828. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.celrep.2017.11.025\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2017.11.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoheny D, Manore SG, Wong GL, Lo HW (2020) Hedgehog signaling and truncated GLI1 in cancer. Cells 9:2114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells9092114\u003c/span\u003e\u003cspan address=\"10.3390/cells9092114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBankaitis ED, Ha A, Kuo CJ, Magness ST (2018) Reserve stem cells in intestinal homeostasis and injury. Gastroenterology 155:1348\u0026ndash;1361. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1053/j.gastro.2018.08.016\u003c/span\u003e\u003cspan address=\"10.1053/j.gastro.2018.08.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFusco A, Fedele M (2007) Roles of HMGA proteins in cancer. Nat Rev Cancer 7:899\u0026ndash;910. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrc2271\u003c/span\u003e\u003cspan address=\"10.1038/nrc2271\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma A, Bl\u0026eacute;riot C, Currenti J, Ginhoux F (2022) Oncofetal reprogramming in tumour development and progression. Nat Rev Cancer 22:593\u0026ndash;602. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41568-022-00497-8\u003c/span\u003e\u003cspan address=\"10.1038/s41568-022-00497-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445:106\u0026ndash;110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature05372\u003c/span\u003e\u003cspan address=\"10.1038/nature05372\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111\u0026ndash;115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature05384\u003c/span\u003e\u003cspan address=\"10.1038/nature05384\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerlos-Su\u0026aacute;rez A, Barriga FM, Jung P et al (2011) The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell 8:511\u0026ndash;524. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.stem.2011.02.020\u003c/span\u003e\u003cspan address=\"10.1016/j.stem.2011.02.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozar S, Morrissey E, Nicholson AM et al (2013) Continuous clonal labeling reveals small numbers of functional stem cells in intestinal crypts and adenomas. Cell Stem Cell 13:626\u0026ndash;633. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.stem.2013.08.001\u003c/span\u003e\u003cspan address=\"10.1016/j.stem.2013.08.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan XS, Wu HY, Yu HP, Zhou Q, Zhang YF Huang Q (2010) Expression of Lgr5 in human colorectal carcinogenesis and its potential correlation with beta-catenin. Int J Colorectal Dis 25:583\u0026ndash;590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00384-010-0903-z\u003c/span\u003e\u003cspan address=\"10.1007/s00384-010-0903-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe S, Zhou H, Zhu X et al (2014) Expression of Lgr5, a marker of intestinal stem cells, in colorectal cancer and its clinicopathological significance. Biomed Pharmacother 68:507\u0026ndash;513. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2014.03.016\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2014.03.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiskin J, Dunlap D, Yaylaoglu M et al (2013) In situ validation of an intestinal stem cell signature in colorectal cancer. Gut 62:1012\u0026ndash;1023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/gutjnl-2011-301195\u003c/span\u003e\u003cspan address=\"10.1136/gutjnl-2011-301195\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi DW, Tang H, Fan JW et al (2010) Expression level of Bmi-1 oncoprotein is associated with progression and prognosis in colon cancer. J Cancer Res Clin Oncol 136:997\u0026ndash;1006. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00432-009-0745-7\u003c/span\u003e\u003cspan address=\"10.1007/s00432-009-0745-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Shi S, Guo Z et al (2013) Overexpression of YAP and TAZ is an independent predictor of prognosis in colorectal cancer and related to the proliferation and metastasis of colon cancer cells. PLOS ONE 8:e65539. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0065539\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0065539\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah SN, Kerr C, Cope L et al (2012) HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks. PLOS ONE 7:e48533. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0048533\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0048533\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelton A, Gabrovsky A, Bae YK et al (2012) HMGA1 induces intestinal polyposis in transgenic mice and drives tumor progression and stem cell properties in colon cancer cells. PLoS One 7:e30034. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0030034\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0030034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShyer AE, Huycke TR, Lee CH, Mhadevan L, Tabin CJ (2015) Bending gradients: how the intestinal stem cell gets its home. Cell 161:569\u0026ndash;580. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2015.03.041\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2015.03.041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cancer stem cells, therapy resistance, rectal cancer, LGR5, BMI1, YAP/TAZ","lastPublishedDoi":"10.21203/rs.3.rs-3037112/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3037112/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study aimed to ascertain the involvement of cancer stem cells (CSCs) in therapy resistance by estimating CSC markers, niche factors, and WNT/β-catenin-relating molecules in untreated and chemoradiation-treated (CRT) rectal cancer.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eExpression ratios of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR), proto-oncogene and polycomb ring finger 1 (BMI1), yes-associated transcriptional regulator (YAP) and its paralog TAZ (hereafter; YAP/TAZ), and nuclear β-catenin were compared in untreated and CRT rectal cancer using \u003cem\u003ein situ\u003c/em\u003e hybridization and immunostainings. Niche factors and WNT/β-catenin-relating molecules were also immunohistochemically compared in human rectal cancer specimens and with early embryonic intestine.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe mean ratios were 15% and 14% in LGR5; 30% and 33% in BMI1; 2.7% and 7.6% in YAP/TAZ; 38% and 32% in nuclear β-catenin in untreated and CRT rectal cancer, respectively, suggesting their robust expressions after CRT. LGR5 and nuclear β-catenin expression was significantly correlated in the CRT cohort. High mobility group AT-hook 1, but not c-MYC and SRY-box transcription factor 9, was significantly upregulated in CRT tumors. WNT2B and GREM1 were uniformly expressed with similarity to the pattern of early embryonic intestine, whereas WNT3A and HES1 expressions were limited. Additionally, stromal GLI1 and YAP/TAZ were more expressed in the CRT cohort.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eStem cell markers of LGR5 and BMI1 as well as nuclear YAP/TAZ, a marker of stem cell regeneration, were robustly expressed in untreated and CRT rectal cancer. WNT2B and GREM1 were aberrantly and uniformly expressed in untreated and CRT rectal cancer with similarity to the early embryonic expression pattern.\u003c/p\u003e","manuscriptTitle":"Robust expressions of stem cell markers, including leucine-rich repeat-containing G protein- coupled receptor 5, in association with early embryonic expression patterns of niche factors in untreated and chemoradiation-treated rectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-14 18:03:40","doi":"10.21203/rs.3.rs-3037112/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7186acbd-24a3-48e7-bab1-916e99a92370","owner":[],"postedDate":"June 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-18T09:14:28+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-14 18:03:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3037112","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3037112","identity":"rs-3037112","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}