Inverse correlation between PD-L1 expression and LGR5 expression in tumor budding of stage II/III colorectal cancer
Annals of Diagnostic Pathology
Sato, K;Uehara, T;Nakajima, T;Iwaya, M;Miyagawa, Y;Watanabe, T;Ota, H;
| DOI: 10.1016/j.anndiagpath.2021.151739
We investigated the expression of LGR5, the most robust and reliable known cancer stem cell (CSC) marker of colorectal cancer, and PD-L1 in tumor budding (TB), as well as clinicopathological features. Tissue microarrays (TMAs) were generated from TB samples from 32 stage II/III colorectal adenocarcinoma patients, and LGR5 expression in TMAs was evaluated by RNAscope, an extremely sensitive RNA in situ hybridization technique. LGR5 expression was significantly lower in the PD-L1-positive group than in the PD-L1-negative group (P = 0.0256). In the PD-L1-positive group, the tumor-infiltrating lymphocytes (TILs) score tended to be higher while the TNM stage was lower compared with the PD-L1 negative group (P = 0.0822 and P = 0.0765, respectively). There was no significant difference in Overall Survival between the PD-L1-positive and PD-L1-negative groups (log-rank test, P = 0.8218). This study showed that PD-L1-positive patients are a unique population with low LGR5 expression, and that LGR5-positive cells may be a promising therapeutic target in PD-L1-negative patients.
Expression profile of intestinal stem cell and cancer stem cell markers in gastric cancers with submucosal invasion
Pathology, research and practice
Kim, HS;Song, HJ;Kim, HU;Jeong, IH;Koh, HM;Shin, JH;Jang, BG;
PMID: 33450435 | DOI: 10.1016/j.prp.2020.153336
Cancer stem cells (CSCs) are believed to be responsible for tumor growth, invasion, and metastasis. Submucosal invasion, which greatly enhances metastasis risk, is a critical step in gastric cancer (GC) progression. To identify stem cell-related markers associated with submucosal invasion and lymph node (LN) metastasis in GCs, we investigated the expression of candidate CSC markers (CD133, CD44, and ALDH1A) and intestinal stem cell (ISC) markers (EPHB2, OLFM4, and LGR5) in early GCs that manifested submucosal invasion. We discovered that EPHB2 and LGR5 expression was frequently confined to the basal area of the lamina propria (basal pattern) in mucosal cancer, and the proportion of stem cell marker-positive cells substantially increased during submucosal invasion. CD44 expression showed a focal pattern, ALDH1A was predominantly expressed diffusely, and there was no expansion of CD44 or ALDH1A expression in the submucosal cancer cells. Unexpectedly, no CSC markers showed any associations with LN metastasis, and only loss of EPHB2 expression was associated with increased LN metastasis. Treatment of RSPO2, a niche factor, along with Wnt 3a, to GC cells led to increased EPHB2 and LGR5 mRNA levels. RNA in situ hybridization confirmed specific RSPO2 expression in the smooth muscle cells of the muscularis mucosa, suggesting that RSPO2 is responsible for the increased expression of ISC markers in GC cells at the basal areas. In summary, no stem cell markers were associated with increased LN metastasis in early GCs. Conversely, isolated EPHB2 expression was associated with lower LN metastasis. EPHB2 and LGR5 showed a basal distribution pattern along with enhanced expression in submucosal invading cells in early GCs, which was induced by a niche factor, RSPO2, from the muscularis mucosa.
Overexpression of Cancer-Associated Stem Cell Gene OLFM4 in the Colonic Epithelium of Patients With Primary Sclerosing Cholangitis
Inflammatory bowel diseases
Neyazi, M;Bharadwaj, SS;Bullers, S;Varenyiova, Z;Oxford IBD Cohort Study Investigators, ;Travis, S;Arancibia-Cárcamo, CV;Powrie, F;Geremia, A;
PMID: 33570127 | DOI: 10.1093/ibd/izab025
To examine immune-epithelial interactions and their impact on epithelial transformation in primary sclerosing cholangitis-associated ulcerative colitis (PSC-UC) using patient-derived colonic epithelial organoid cultures (EpOCs). The EpOCs were originated from colonic biopsies from patients with PSC-UC (n = 12), patients with UC (n = 14), and control patients (n = 10) and stimulated with cytokines previously associated with intestinal inflammation (interferon (IFN) γ and interleukin (IL)-22). Markers of cytokine downstream pathways, stemness, and pluripotency were analyzed by real-time quantitative polymerase chain reaction and immunofluorescence. The OLFM4 expression in situ was assessed by RNAscope and immunohistochemistry. A distinct expression of stem cell-associated genes was observed in EpOCs derived from patients with PSC-UC, with lower expression of the classical stem-cell marker LGR5 and overexpression of OLFM4, previously associated with pluripotency and early stages of neoplastic transformation in the gastrointestinal and biliary tracts. High levels of OLFM4 were also found ex vivo in colonic biopsies from patients with PSC-UC. In addition, IFNγ stimulation resulted in the downregulation of LGR5 in EpOCs, whereas higher expression of OLFM4 was observed after IL-22 stimulation. Interestingly, expression of the IL-22 receptor, IL22RA1, was induced by IFNγ, suggesting that a complex interplay between these cytokines may contribute to carcinogenesis in PSC-UC. Higher expression of OLFM4, a cancer stemness gene induced by IL-22, is present in PSC-UC, suggesting that IL-22 responses may result in alterations of the intestinal stem-cell niche in these patients.
A constant pool of Lgr5+ intestinal stem cells is required for intestinal homeostasis
Tan, SH;Phuah, P;Tan, LT;Yada, S;Goh, J;Tomaz, LB;Chua, M;Wong, E;Lee, B;Barker, N;
PMID: 33503423 | DOI: 10.1016/j.celrep.2020.108633
Lgr5+ crypt base columnar cells, the operational intestinal stem cells (ISCs), are thought to be dispensable for small intestinal (SI) homeostasis. Using a Lgr5-2A-DTR (diphtheria toxin receptor) model, which ablates Lgr5+ cells with near-complete efficiency and retains endogenous levels of Lgr5 expression, we show that persistent depletion of Lgr5+ ISCs in fact compromises SI epithelial integrity and reduces epithelial turnover in vivo. In vitro, Lgr5-2A-DTR SI organoids are unable to establish or survive when Lgr5+ ISCs are continuously eliminated by adding DT to the media. However, transient exposure to DT at the start of culture allows organoids to form, and the rate of outgrowth reduces with the increasing length of DT presence. Our results indicate that intestinal homeostasis requires a constant pool of Lgr5+ ISCs, which is supplied by rapidly reprogrammed non-Lgr5+ crypt populations when preexisting Lgr5+ ISCs are ablated.
ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation
Sun, T;Annunziato, S;Bergling, S;Sheng, C;Orsini, V;Forcella, P;Pikiolek, M;Kancherla, V;Holwerda, S;Imanci, D;Wu, F;Meylan, LC;Puehringer, LF;Waldt, A;Oertli, M;Schuierer, S;Terracciano, LM;Reinker, S;Ruffner, H;Bouwmeester, T;Sailer, AW;George, E;Roma, G;de Weck, A;Piscuoglio, S;Lohmann, F;Naumann, U;Liberali, P;Cong, F;Tchorz, JS;
PMID: 34129813 | DOI: 10.1016/j.stem.2021.05.013
AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.
Epithelium-derived Indian Hedgehog restricts stromal expression of ErbB family members that drive colonic tumor cell proliferation
Westendorp, F;Karpus, ON;Koelink, PJ;Vermeulen, JLM;Meisner, S;Koster, J;Büller, NVJA;Wildenberg, ME;Muncan, V;van den Brink, GR;
PMID: 33479497 | DOI: 10.1038/s41388-020-01633-0
Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc-/-Ihh-/- double mutant mice. We found that recombinant EREG, BTC, and NRG1 but not Lgr5 ligand R-Spondin promoted growth and proliferation of Apc double mutant colonic organoids. Thus, the loss of Ihh enhances Apc-driven colonic adenomagenesis via upregulation of ErbB pathway family members in colonic stromal cells. Our findings highlight the critical role of epithelium-derived Indian Hedgehog as a stromal tumor suppressor in the intestine.
Induction of gastric cancer by successive oncogenic activation in the corpus
Douchi, D;Yamamura, A;Matsuo, J;Melissa Lim, YH;Nuttonmanit, N;Shimura, M;Suda, K;Chen, S;ShuChin, P;Kohu, K;Abe, T;Shioi, G;Kim, G;Shabbir, A;Srivastava, S;Unno, M;Bok-Yan So, J;Teh, M;Yeoh, KG;Huey Chuang, LS;Ito, Y;
PMID: 34391772 | DOI: 10.1053/j.gastro.2021.08.013
Metaplasia and dysplasia in the corpus are reportedly derived from dedifferentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen C-transcript expressing cells (PGC-transcript expressing cells) represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model.We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate Pgc-CreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments, histological and immunofluorescence staining. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether PGC-transcript expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC-transcript expressing cells with activated Kras, inactivated Apc and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice.Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;KrasG12D/+ mice, PGC-transcript expressing cells with KrasG12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven KrasG12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal dysplasia/carcinoma, while Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic gastric carcinoma.The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.
Hayakawa Y, Tsuboi M, Asfaha S, Kinoshita H, Niikura R, Konishi M, Hata M, Oya Y, Kim W, Middelhoff M, Hikiba Y, Higashijima N, Ihara S, Ushiku T, Fukayama M, Tailor Y, Hirata Y, Guha C, Yan KS, Koike K, Wang TC.
PMID: 30448068 | DOI: 10.1053/j.gastro.2018.11.024
Abstract
BACKGROUND & AIMS:
The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated.
METHODS:
We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apcflox/flox, LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5 mRNA-positive cells, irradiated, or given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues we analyzed the fate of progeny that expressed Bhlha15 mRNA. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using the TCGA dataset.
RESULTS:
Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15+ cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15+ secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15+ secretory precursors to long-lived enterocyte progenitors (EPs). Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with EPs, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15+ precursors, towards that of ISCs. Bhlha15+ EPs with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells following dextran sodium sulfate-induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon.
CONCLUSION:
In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation, in addition to traditional ISCs.
