Publications by authors named "Piero Dalerba"

32 Publications

A Trial of Lopinavir-Ritonavir in Covid-19.

N Engl J Med 2020 05 5;382(21):e68. Epub 2020 May 5.

Columbia University, New York, NY

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http://dx.doi.org/10.1056/NEJMc2008043DOI Listing
May 2020

Notch Signaling Mediates Differentiation in Barrett's Esophagus and Promotes Progression to Adenocarcinoma.

Gastroenterology 2020 08 20;159(2):575-590. Epub 2020 Apr 20.

Department of Medicine, Columbia University Irving Medical Center, New York, New York; Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York.

Background & Aims: Studies are needed to determine the mechanism by which Barrett's esophagus (BE) progresses to esophageal adenocarcinoma (EAC). Notch signaling maintains stem cells in the gastrointestinal tract and is dysregulated during carcinogenesis. We explored the relationship between Notch signaling and goblet cell maturation, a feature of BE, during EAC pathogenesis.

Methods: We measured goblet cell density and levels of Notch messenger RNAs in BE tissues from 164 patients, with and without dysplasia or EAC, enrolled in a multicenter study. We analyzed the effects of conditional expression of an activated form of NOTCH2 (pL2.Lgr5.N2IC), conditional deletion of NOTCH2 (pL2.Lgr5.N2fl/fl), or loss of nuclear factor κB (NF-κB) (pL2.Lgr5.p65fl/fl), in Lgr5 (progenitor) cells in L2-IL1B mice (which overexpress interleukin 1 beta in esophagus and squamous forestomach and are used as a model of BE). We collected esophageal and stomach tissues and performed histology, immunohistochemistry, flow cytometry, transcriptome, and real-time polymerase chain reaction analyses. Cardia and forestomach tissues from mice were cultured as organoids and incubated with inhibitors of Notch or NF-kB.

Results: Progression of BE to EAC was associated with a significant reduction in goblet cell density comparing nondysplastic regions of tissues from patients; there was an inverse correlation between goblet cell density and levels of NOTCH3 and JAG2 messenger RNA. In mice, expression of the activated intracellular form of NOTCH2 in Lgr5 cells reduced goblet-like cell maturation, increased crypt fission, and accelerated the development of tumors in the squamocolumnar junction. Mice with deletion of NOTCH2 from Lgr5 cells had increased maturation of goblet-like cells, reduced crypt fission, and developed fewer tumors. Esophageal tissues from in pL2.Lgr5.N2IC mice had increased levels of RelA (which encodes the p65 unit of NF-κB) compared to tissues from L2-IL1B mice, and we found evidence of increased NF-κB activity in Lgr5 cells. Esophageal tissues from pL2.Lgr5.p65fl/fl mice had lower inflammation and metaplasia scores than pL2.Lgr5.N2IC mice. In organoids derived from pL2-IL1B mice, the NF-κB inhibitor JSH-23 reduced cell survival and proliferation.

Conclusions: Notch signaling contributes to activation of NF-κB and regulates differentiation of gastric cardia progenitor cells in a mouse model of BE. In human esophageal tissues, progression of BE to EAC was associated with reduced goblet cell density and increased levels of Notch expression. Strategies to block this pathway might be developed to prevent EAC in patients with BE.
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http://dx.doi.org/10.1053/j.gastro.2020.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484392PMC
August 2020

miR-221 Targets QKI to Enhance the Tumorigenic Capacity of Human Colorectal Cancer Stem Cells.

Cancer Res 2019 10 15;79(20):5151-5158. Epub 2019 Aug 15.

Division of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.

miRNAs are key players in the integrated regulation of cellular processes and shape many of the functional properties that define the "cancer stem cell" (CSC) phenotype. Little is known, however, about miRNAs that regulate such properties in human colorectal carcinoma. In this study, we compared the expression levels of 754 miRNAs between paired samples of EpCAM/CD44 cancer cells (enriched in CSCs) and EpCAM/CD44 cancer cells (with CSC depletion) sorted in parallel from human primary colorectal carcinomas and identified miR-221 as the miRNA that displayed the highest level of preferential expression in EpCAM/CD44 cancer cells. High levels of miR-221 expression were associated with Lgr5 cells in mouse colon crypts and reduced survival in patients with colorectal carcinoma. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) . Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity and substantially reduced the tumorigenic capacity of CSC populations from PDX lines . Finally, the most abundant splicing isoform of the human () gene, , was identified as a functional target of miR-221; overexpression of miR-221-reduced QKI-5 protein levels in human colorectal carcinoma cells. As expected, overexpression of QKI-5 suppressed organoid-forming capacity and tumorigenic capacity of colorectal carcinoma PDX cells . Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer. SIGNIFICANCE: These findings uncover molecular mechanisms underlying the maintenance of cancer stem cell properties in colon cancer. http://cancerres.aacrjournals.org/content/canres/79/20/5151/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-3544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801097PMC
October 2019

promotes colorectal cancer by inducing Wnt/β-catenin modulator Annexin A1.

EMBO Rep 2019 04 4;20(4). Epub 2019 Mar 4.

Division of Periodontics, College of Dental Medicine, Columbia University, New York, NY, USA

, a Gram-negative oral anaerobe, is a significant contributor to colorectal cancer. Using an cancer progression model, we discover that stimulates the growth of colorectal cancer cells without affecting the pre-cancerous adenoma cells. Annexin A1, a previously unrecognized modulator of Wnt/β-catenin signaling, is a key component through which exerts its stimulatory effect. Annexin A1 is specifically expressed in proliferating colorectal cancer cells and involved in activation of Cyclin D1. Its expression level in colon cancer is a predictor of poor prognosis independent of cancer stage, grade, age, and sex. The FadA adhesin from up-regulates Annexin A1 expression through E-cadherin. A positive feedback loop between FadA and Annexin A1 is identified in the cancerous cells, absent in the non-cancerous cells. We therefore propose a "two-hit" model in colorectal carcinogenesis, with somatic mutation(s) serving as the first hit, and as the second hit exacerbating cancer progression after benign cells become cancerous. This model extends the "adenoma-carcinoma" model and identifies microbes such as as cancer "facilitators".
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http://dx.doi.org/10.15252/embr.201847638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446206PMC
April 2019

A cluster robustness score for identifying cell subpopulations in single cell gene expression datasets from heterogeneous tissues and tumors.

Bioinformatics 2019 03;35(6):962-971

Department of Bioengineering and Bar-Ilan Institute of Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, Israel.

Motivation: A major aim of single cell biology is to identify important cell types such as stem cells in heterogeneous tissues and tumors. This is typically done by isolating hundreds of individual cells and measuring expression levels of multiple genes simultaneously from each cell. Then, clustering algorithms are used to group together similar single-cell expression profiles into clusters, each representing a distinct cell type. However, many of these clusters result from overfitting, meaning that rather than representing biologically meaningful cell types, they describe the intrinsic 'noise' in gene expression levels due to limitations in experimental precision or the intrinsic randomness of biochemical cellular processes. Consequentially, these non-meaningful clusters are most sensitive to noise: a slight shift in gene expression levels due to a repeated measurement will rearrange the grouping of data points such that these clusters break up.

Results: To identify the biologically meaningful clusters we propose a 'cluster robustness score': We add increasing amounts of noise (zero mean and increasing variance) and check which clusters are most robust in the sense that they do not mix with their neighbors up to high levels of noise. We show that biologically meaningful cell clusters that were manually identified in previously published single cell expression datasets have high robustness scores. These scores are higher than what would be expected in corresponding randomized homogeneous datasets having the same expression level statistics. We believe that this scoring system provides a more automated way to identify cell types in heterogeneous tissues and tumors.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty708DOI Listing
March 2019

The Dynamic Identity of Intestinal Cancer Stem Cells.

Authors:
Piero Dalerba

Cell Stem Cell 2017 06;20(6):743-745

Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Medicine (Division of Digestive and Liver Diseases), Columbia University, New York, NY 10032, USA; Herbert Irving Comprehensive Cancer Center (HICCC), Columbia University, New York, NY 10032, USA. Electronic address:

Elimination of self-renewing cancer stem cells (CSCs) is necessary to permanently eradicate malignant tissues. In a recent article in Nature, de Sousa e Melo et al. (2017) reveal that intestinal tumors can contain dynamic pools of functionally distinct CSC populations, which seem to interconvert depending on the host tissue microenvironment.
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http://dx.doi.org/10.1016/j.stem.2017.05.018DOI Listing
June 2017

A Quiescent Bcl11b High Stem Cell Population Is Required for Maintenance of the Mammary Gland.

Cell Stem Cell 2017 02 29;20(2):247-260.e5. Epub 2016 Dec 29.

Institute for Stem Cell Biology and Regenerative Medicine, School of Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address:

Stem cells in many tissues sustain themselves by entering a quiescent state to avoid genomic insults and to prevent exhaustion caused by excessive proliferation. In the mammary gland, the identity and characteristics of quiescent epithelial stem cells are not clear. Here, we identify a quiescent mammary epithelial cell population expressing high levels of Bcl11b and located at the interface between luminal and basal cells. Bcl11b cells are enriched for cells that can regenerate mammary glands in secondary transplants. Loss of Bcl11b leads to a Cdkn2a-dependent exhaustion of ductal epithelium and loss of epithelial cell regenerative capacity. Gain- and loss-of-function studies show that Bcl11b induces cells to enter the G phase of the cell cycle and become quiescent. Taken together, these results suggest that Bcl11b acts as a central intrinsic regulator of mammary epithelial stem cell quiescence and exhaustion and is necessary for long-term maintenance of the mammary gland.
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http://dx.doi.org/10.1016/j.stem.2016.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341693PMC
February 2017

Organoid Culture of Human Cancer Stem Cells.

Methods Mol Biol 2019 ;1576:23-31

Division of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.

Organoid culture is a three-dimensional culture method that enables ex vivo analysis of stem cell behavior and differentiation. This method is also applicable to the studies on stem cell characters of human cancer stem cells. The components of organoid culture include Matrigel® and a culture medium containing growth factor cocktails that mimic the microenvironments of organ stem cell niches. Here, we describe the basic methods for the organoid culture of dissociated or FACS-sorted human cancer stem cells. Then, we introduce a method to dissociate the organoids for serial passage and propagation.
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http://dx.doi.org/10.1007/7651_2016_13DOI Listing
March 2020

CDX2 as a Prognostic Biomarker in Stage II and Stage III Colon Cancer.

N Engl J Med 2016 Jan;374(3):211-22

From the Herbert Irving Comprehensive Cancer Center and the Departments of Pathology and Cell Biology and Medicine, Columbia University, New York (P.D.); Institute for Stem Cell Biology and Regenerative Medicine (P.D., D.S., P.S.R., S.P.M., S.H., J.H., D.Q., M.F.C.) and the Departments of Pathology (X.G., E.F., M.R.), and Medicine, Division of Oncology (N.W.-F., G.A.F., M.F.C.), Stanford University, Stanford, and the Departments of Pediatrics and Computer Science and Engineering, University of California San Diego, San Diego (D.S.) - both in California; Faculty of Engineering, Bar-Ilan University, Ramat Gan, Israel (T.K.); the National Surgical Adjuvant Breast and Bowel Project, NRG Oncology (S.P., G.Y., N.S., N.W.) and the Allegheny Cancer Center at Allegheny General Hospital (N.W.) - both in Pittsburgh; Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea (S.P.); and the Department of Biochemistry and Molecular Biology, Medical School of Henan University, Kaifeng, China (X.G.).

Background The identification of high-risk stage II colon cancers is key to the selection of patients who require adjuvant treatment after surgery. Microarray-based multigene-expression signatures derived from stem cells and progenitor cells hold promise, but they are difficult to use in clinical practice. Methods We used a new bioinformatics approach to search for biomarkers of colon epithelial differentiation across gene-expression arrays and then ranked candidate genes according to the availability of clinical-grade diagnostic assays. With the use of subgroup analysis involving independent and retrospective cohorts of patients with stage II or stage III colon cancer, the top candidate gene was tested for its association with disease-free survival and a benefit from adjuvant chemotherapy. Results The transcription factor CDX2 ranked first in our screening test. A group of 87 of 2115 tumor samples (4.1%) lacked CDX2 expression. In the discovery data set, which included 466 patients, the rate of 5-year disease-free survival was lower among the 32 patients (6.9%) with CDX2-negative colon cancers than among the 434 (93.1%) with CDX2-positive colon cancers (hazard ratio for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). In the validation data set, which included 314 patients, the rate of 5-year disease-free survival was lower among the 38 patients (12.1%) with CDX2 protein-negative colon cancers than among the 276 (87.9%) with CDX2 protein-positive colon cancers (hazard ratio, 2.42; 95% CI, 1.36 to 4.29; P=0.003). In both these groups, these findings were independent of the patient's age, sex, and tumor stage and grade. Among patients with stage II cancer, the difference in 5-year disease-free survival was significant both in the discovery data set (49% among 15 patients with CDX2-negative tumors vs. 87% among 191 patients with CDX2-positive tumors, P=0.003) and in the validation data set (51% among 15 patients with CDX2-negative tumors vs. 80% among 106 patients with CDX2-positive tumors, P=0.004). In a pooled database of all patient cohorts, the rate of 5-year disease-free survival was higher among 23 patients with stage II CDX2-negative tumors who were treated with adjuvant chemotherapy than among 25 who were not treated with adjuvant chemotherapy (91% vs. 56%, P=0.006). Conclusions Lack of CDX2 expression identified a subgroup of patients with high-risk stage II colon cancer who appeared to benefit from adjuvant chemotherapy. (Funded by the National Comprehensive Cancer Network, the National Institutes of Health, and others.).
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http://dx.doi.org/10.1056/NEJMoa1506597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784450PMC
January 2016

EGFR amplified and overexpressing glioblastomas and association with better response to adjuvant metronomic temozolomide.

J Natl Cancer Inst 2015 May 3;107(5). Epub 2015 Mar 3.

Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).

Background: Lack of robust predictive biomarkers, other than MGMT promoter methylation, makes temozolomide responsiveness in newly diagnosed glioblastoma (GBM) patients difficult to predict. However, we identified patients with long-term survival (≥35 months) within a group of newly diagnosed GBM patients treated with standard or metronomic adjuvant temozolomide schedules. We thus investigated possible molecular profiles associated with longer survival following temozolomide treatment.

Methods: We investigated the association of molecular features with progression-free (PFS) and overall survival (OS). Human-derived GBM cancer stem cells (CSCs) were used to investigate in vitro molecular mechanisms associated with temozolomide responsiveness. Surgically removed recurrences allowed investigation of molecular changes occurring during therapy in vivo. Statistical analyses included one- and two-way analysis of variance, Student's t test, Cox proportional hazards, and the Kaplan-Meier method. All statistical tests were two-sided.

Results: No association was found between survival and gene classifiers associated with different molecular GBM subtypes in the standard-treated group, while in metronomic-treated patients robust association was found between EGFR amplification/overexpression and PFS and OS (OS, EGFR-high vs low: hazard ratiodeath = 0.22, 95% confidence interval = 0.09 to 0.55, P = .001). The result for OS remained statistically significant after Bonferroni correction (P interaction < .0005). Long-term survival following metronomic temozolomide was independent from MGMT and EGFRvIII status and was more pronounced in EGFR-overexpressing GBM patients with PTEN loss. In vitro findings confirmed a selective dose- and time-dependent decrease in survival of temozolomide-treated EGFR+ human-derived glioblastoma CSCs, which occurred through inhibition of NF-κB transcriptional activity. In addition, reduction in EGFR-amplified cells, along with a statistically significant decrease in NF-κB/p65 expression, were observed in specimens from recurrent metronomic-treated EGFR-overexpressing GBM patients.

Conclusions: EGFR-amplified/overexpressing glioblastomas strongly benefit from metronomic temozolomide-based therapies.
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http://dx.doi.org/10.1093/jnci/djv041DOI Listing
May 2015

miR-142 regulates the tumorigenicity of human breast cancer stem cells through the canonical WNT signaling pathway.

Elife 2014 Nov 18;3. Epub 2014 Nov 18.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, United States.

MicroRNAs (miRNAs) are important regulators of stem and progenitor cell functions. We previously reported that miR-142 and miR-150 are upregulated in human breast cancer stem cells (BCSCs) as compared to the non-tumorigenic breast cancer cells. In this study, we report that miR-142 efficiently recruits the APC mRNA to an RNA-induced silencing complex, activates the canonical WNT signaling pathway in an APC-suppression dependent manner, and activates the expression of miR-150. Enforced expression of miR-142 or miR-150 in normal mouse mammary stem cells resulted in the regeneration of hyperproliferative mammary glands in vivo. Knockdown of endogenous miR-142 effectively suppressed organoid formation by BCSCs and slowed tumor growth initiated by human BCSCs in vivo. These results suggest that in some tumors, miR-142 regulates the properties of BCSCs at least in part by activating the WNT signaling pathway and miR-150 expression.
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http://dx.doi.org/10.7554/eLife.01977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4235011PMC
November 2014

Quantitative assessment of single-cell RNA-sequencing methods.

Nat Methods 2014 Jan 20;11(1):41-6. Epub 2013 Oct 20.

1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Howard Hughes Medical Institute, Stanford, California, USA. [3] Department of Applied Physics, Stanford University, Stanford, California, USA.

Interest in single-cell whole-transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. We compared commercially available single-cell RNA amplification methods with both microliter and nanoliter volumes, using sequence from bulk total RNA and multiplexed quantitative PCR as benchmarks to systematically evaluate the sensitivity and accuracy of various single-cell RNA-seq approaches. We show that single-cell RNA-seq can be used to perform accurate quantitative transcriptome measurement in individual cells with a relatively small number of sequencing reads and that sequencing large numbers of single cells can recapitulate bulk transcriptome complexity.
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http://dx.doi.org/10.1038/nmeth.2694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022966PMC
January 2014

Oncogenic miRNAs and the perils of losing control of a stem cell's epigenetic identity.

Cell Stem Cell 2013 Jul;13(1):5-6

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA.

Pathways that regulate epigenetic control of stem cell identity are critical to the molecular etiology of cancer. In back-to-back articles in Cell and Cell Stem Cell, Song et al. identify miR-22 as both a repressor of TET proteins and a powerful oncogene in the mammary epithelium and hematopoietic system.
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http://dx.doi.org/10.1016/j.stem.2013.06.012DOI Listing
July 2013

The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.

Proc Natl Acad Sci U S A 2012 Apr 26;109(17):6662-7. Epub 2012 Mar 26.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Medical Center, Stanford, CA 94305, USA.

CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.
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http://dx.doi.org/10.1073/pnas.1121623109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340046PMC
April 2012

Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice.

Gastroenterology 2012 May 11;142(5):1195-1205.e6. Epub 2012 Feb 11.

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California 94305, USA.

Background & Aims: Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells.

Methods: We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types.

Results: Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased.

Conclusions: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.
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http://dx.doi.org/10.1053/j.gastro.2012.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911891PMC
May 2012

Single-cell dissection of transcriptional heterogeneity in human colon tumors.

Nat Biotechnol 2011 Nov 13;29(12):1120-7. Epub 2011 Nov 13.

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA.

Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.
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http://dx.doi.org/10.1038/nbt.2038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237928PMC
November 2011

Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.

Proc Natl Acad Sci U S A 2010 Oct 4;107(42):18115-20. Epub 2010 Oct 4.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Palo Alto, CA 94304, USA.

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.
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http://dx.doi.org/10.1073/pnas.1006732107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964232PMC
October 2010

Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells.

Cell 2009 Aug;138(3):592-603

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 1050 Arastradero Road, Palo Alto, CA 94304, USA.

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.
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http://dx.doi.org/10.1016/j.cell.2009.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731699PMC
August 2009

Cancer stem cells and tumor metastasis: first steps into uncharted territory.

Cell Stem Cell 2007 Sep;1(3):241-2

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Palo Alto, CA 94304, USA.

In several forms of human cancer, only a phenotypic subset of cancer cells, usually termed "cancer stem cells" (CSC), can initiate tumor growth when transplanted. In this issue of Cell Stem Cell, Hermann et al. (2007) analyze the relationship between CSC and tumor metastasis.
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http://dx.doi.org/10.1016/j.stem.2007.08.012DOI Listing
September 2007

The 5th International Society for Stem Cell Research (ISSCR) Annual Meeting, June 2007.

Stem Cells 2008 Jan 25;26(1):292-8. Epub 2007 Oct 25.

Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Rockefeller Research Laboratories, New York, New York 10021, USA.

This report presents highlights of discussions that focused on the biology of cancer stem cells as conducted at the fifth Annual Meeting of the International Society for Stem Cell Research, held in Cairns, Australia, June 17-20, 2007. The function of adult stem cells is believed to depend on their niches, that is, the microenvironment in which these stem cells reside. A similar concept applies to understanding the development of cancer, as it is becoming increasingly clear that only a small subset of cancer cell populations is capable of initiating/sustaining tumor formation. These tumorigenic cells, commonly referred to as cancer stem cells, also appear to reside in particular niches, and they bear the known, albeit dysfunctional, stem cell characteristics of self-renewal and differentiation. Dysregulation of stem cell niches is thought to contribute to tumorigenesis by affecting the complex network of signaling interactions that occur between stem cells and their neighboring cells, thus imbalancing the physiological controls on self-renewal and differentiation processes. This hypothesis was widely explored at the conference to shed new light on the mechanisms of tumor origin and progression and to unveil novel antitumor therapeutic approaches.
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http://dx.doi.org/10.1634/stemcells.2007-0647DOI Listing
January 2008

Phenotypic characterization of human colorectal cancer stem cells.

Proc Natl Acad Sci U S A 2007 Jun 4;104(24):10158-63. Epub 2007 Jun 4.

Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

Recent observations indicate that, in several types of human cancer, only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the "cancer stem cell" (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues, either primary tissues collected from surgical specimens or xenografts established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, were disaggregated into single-cell suspensions and analyzed by flow cytometry. Surface markers that displayed intratumor heterogeneous expression among epithelial cancer cells were selected for cell sorting and tumorigenicity experiments. Individual phenotypic cancer cell subsets were purified, and their tumor-initiating properties were investigated by injection in NOD/SCID mice. Our observations indicate that, in six of six human CRC tested, the ability to engraft in vivo in immunodeficient mice was restricted to a minority subpopulation of epithelial cell adhesion molecule (EpCAM)(high)/CD44+ epithelial cells. Tumors originated from EpCAM(high)/CD44+ cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Analysis of the surface molecule repertoire of EpCAM(high)/CD44+ cells led to the identification of CD166 as an additional differentially expressed marker, useful for CSC isolation in three of three CRC tested. These results validate the stem cell working model in human CRC and provide a highly robust surface marker profile for CRC stem cell isolation.
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http://dx.doi.org/10.1073/pnas.0703478104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891215PMC
June 2007

Identification of pancreatic cancer stem cells.

Cancer Res 2007 Feb;67(3):1030-7

Department of Surgery, University of Michigan Medical Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109, USA.

Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Although data have been provided to support this theory in human blood, brain, and breast cancers, the identity of pancreatic cancer stem cells has not been determined. Using a xenograft model in which primary human pancreatic adenocarcinomas were grown in immunocompromised mice, we identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD44, CD24, and epithelial-specific antigen (ESA). Pancreatic cancer cells with the CD44(+)CD24(+)ESA(+) phenotype (0.2-0.8% of pancreatic cancer cells) had a 100-fold increased tumorigenic potential compared with nontumorigenic cancer cells, with 50% of animals injected with as few as 100 CD44(+)CD24(+)ESA(+) cells forming tumors that were histologically indistinguishable from the human tumors from which they originated. The enhanced ability of CD44(+)CD24(+)ESA(+) pancreatic cancer cells to form tumors was confirmed in an orthotopic pancreatic tail injection model. The CD44(+)CD24(+)ESA(+) pancreatic cancer cells showed the stem cell properties of self-renewal, the ability to produce differentiated progeny, and increased expression of the developmental signaling molecule sonic hedgehog. Identification of pancreatic cancer stem cells and further elucidation of the signaling pathways that regulate their growth and survival may provide novel therapeutic approaches to treat pancreatic cancer, which is notoriously resistant to standard chemotherapy and radiation.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-2030DOI Listing
February 2007

The prognostic role of a gene signature from tumorigenic breast-cancer cells.

N Engl J Med 2007 Jan;356(3):217-26

Department of Internal Medicine, University of Michigan, Ann Arbor, USA.

Background: Breast cancers contain a minority population of cancer cells characterized by CD44 expression but low or undetectable levels of CD24 (CD44+CD24-/low) that have higher tumorigenic capacity than other subtypes of cancer cells.

Methods: We compared the gene-expression profile of CD44+CD24-/low tumorigenic breast-cancer cells with that of normal breast epithelium. Differentially expressed genes were used to generate a 186-gene "invasiveness" gene signature (IGS), which was evaluated for its association with overall survival and metastasis-free survival in patients with breast cancer or other types of cancer.

Results: There was a significant association between the IGS and both overall and metastasis-free survival (P<0.001, for both) in patients with breast cancer, which was independent of established clinical and pathological variables. When combined with the prognostic criteria of the National Institutes of Health, the IGS was used to stratify patients with high-risk early breast cancer into prognostic categories (good or poor); among patients with a good prognosis, the 10-year rate of metastasis-free survival was 81%, and among those with a poor prognosis, it was 57%. The IGS was also associated with the prognosis in medulloblastoma (P=0.004), lung cancer (P=0.03), and prostate cancer (P=0.01). The prognostic power of the IGS was increased when combined with the wound-response (WR) signature.

Conclusions: The IGS is strongly associated with metastasis-free survival and overall survival for four different types of tumors. This genetic signature of tumorigenic breast-cancer cells was even more strongly associated with clinical outcomes when combined with the WR signature in breast cancer.
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http://dx.doi.org/10.1056/NEJMoa063994DOI Listing
January 2007

Cancer stem cells: models and concepts.

Annu Rev Med 2007 ;58:267-84

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94304, USA.

Although monoclonal in origin, most tumors appear to contain a heterogeneous population of cancer cells. This observation is traditionally explained by postulating variations in tumor microenvironment and coexistence of multiple genetic subclones, created by progressive and divergent accumulation of independent somatic mutations. An additional explanation, however, envisages human tumors not as mere monoclonal expansions of transformed cells, but rather as complex tridimensional tissues where cancer cells become functionally heterogeneous as a result of differentiation. According to this second scenario, tumors act as caricatures of their corresponding normal tissues and are sustained in their growth by a pathological counterpart of normal adult stem cells, cancer stem cells. This model, first developed in human myeloid leukemias, is today being extended to solid tumors, such as breast and brain cancer. We review the biological basis and the therapeutic implications of the stem cell model of cancer.
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http://dx.doi.org/10.1146/annurev.med.58.062105.204854DOI Listing
April 2007

Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence: evidence against immortality as a constitutive trait of tumor cells.

Cancer Res 2005 Mar;65(6):2321-9

Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milan, Italy.

Although in vitro establishment of new colorectal carcinoma (CRC) cell lines is an infrequent event, we have observed that primary cultures of CRC can be repeatedly and reproducibly initiated following in vitro plating of tumor-derived epithelial cells. These cultures, however, usually display a short life span as they undergo a limited number of cell passages before entering a state of irreversible growth arrest. In this study, we show that short-lived CRC primary cultures lack constitutive telomerase activity and undergo a senescence process characterized by progressive telomere shortening. Moreover, transduction of these cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT) is sufficient to reconstitute telomerase activity and allow immortalization. Detailed molecular characterization of hTERT-immortalized CRC cell lines confirms their individual tumor origin by showing expression of colonic epithelial differentiation markers, such as cytokeratin-20 (CK20), full match with class I and class II human leukocyte antigen genotyping of autologous B-lymphoblastoid cells, and presence of somatic mutations in key cancer genes (KRAS2, APC) identical to those of the corresponding autologous original tumor tissues. Moreover, functional characterization of hTERT-immortalized CRC cell lines shows that they have a transformed phenotype, being able to form colonies in soft agar and tumors in severe combined immunodeficient mice. Most interestingly, immunohistochemical analysis of original tumor tissues indicates that short-lived CRC primary cultures, although hTERT-negative in vitro, derive from hTERT-positive tumors. Taken together, our data show that, in a least subset of CRC, biochemical pathways involved in maintenance of telomere length, such as telomerase, are not activated in a constitutive way in all tumor cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-3678DOI Listing
March 2005

Antigen-specific immunity in neuroblastoma patients: antibody and T-cell recognition of NY-ESO-1 tumor antigen.

Cancer Res 2003 Oct;63(20):6948-55

Units of Melanoma Genetics, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy.

Neuroblastoma cells have been shown to express molecularly defined tumor-associated antigens, which could represent potential targets of T and/or B cell-mediated immunity. However, the existence of a spontaneous immune response to such tumor antigens in neuroblastoma patients has yet to be investigated. In the present work we addressed the issue of whether NY-ESO-1, a germ cell antigen aberrantly expressed in different tumor types, is expressed by neuroblastoma cells and may represent a target for humoral and/or cellular immune responses in neuroblastoma patients. We found that a large fraction of neuroblastoma biopsies, independently from the clinical stage and degree of tumor cell differentiation, expressed significant levels of NY-ESO-1 as assessed by reverse transcription-PCR and immunohistochemistry. NY-ESO-1-specific IgG antibodies were detected in the sera of 10% of neuroblastoma patients with stage III or IV disease, but not in patients in clinical remission or with earlier stages. This suggests that antibody production occurred as a late event in the course of disease. NY-ESO-1-specific immune responses were observed for CD4(+) and CD8(+) T cells from peripheral blood lymphocytes in 4 of 8 neuroblastoma patients, as detected by IFN-gamma enzyme-linked immunospot assay after in vitro stimulation either with the NY-ESO-1 recombinant protein or with the HLA-A2-restricted peptide NY-ESO-1(157-167). NY-ESO-1-specific CD4(+) and CD8(+) T cells were also able to recognize NY-ESO-1 expressing neuroblastoma cells. The presence of T cells specific for NY-ESO-1 antigen was not associated with the stage of disease, or to the presence or absence of NY-ESO-1 specific antibodies. We conclude that NY-ESO-1 is an immunogenic antigen in neuroblastoma patients and represents a candidate target for immune-based therapy.
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October 2003

The apoptosis inhibitor protein survivin induces tumor-specific CD8+ and CD4+ T cells in colorectal cancer patients.

Cancer Res 2003 Aug;63(15):4507-15

Unit of Immunotherapy of Human Tumors, Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via G. Venezian 1, 20133 Milan, Italy.

The identification of tumor-associated antigens expressed by colorectal carcinoma remains one of the major goals for designing novel immunological treatments for this tumor. By using a reverse-immunology approach, we show here that the inhibitor of apoptosis protein, survivin, is immunogenic in colorectal cancer patients. In particular, we found that survivin elicited CD8(+) T cell-mediated responses in peripheral blood or in tumor-associated lymphocytes from patients at different disease stage. Colorectal carcinoma cells were recognized by survivin-specific T lymphocytes, and the survivin-specific, class-I HLA-restricted T lymphocytes were fully activated and released interleukin-2 in response to HLA/survivin-peptide complexes expressed by tumor cells. In addition to CD8-mediated responses, survivin specifically stimulated CD4+ T-cell reactivity in peripheral blood lymphocytes from the same patients, thus suggesting that a complete activation of the immune system may occur in response to this antiapoptotic protein. These findings indicate that survivin could be considered a valuable tumor-associated antigen for immune-based clinical approaches in colorectal cancer.
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August 2003

Immunology and immunotherapy of colorectal cancer.

Crit Rev Oncol Hematol 2003 Apr;46(1):33-57

Unit of Immunotherapy of Human Tumours, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milan, Italy.

This review critically discusses data on immunology of colorectal cancer, starting from pathology and molecular biology, and then considering the molecular characterisation of colon cancer antigens and the clinical trials of immunotherapy. A careful evaluation of histopathological studies on intra-epithelial infiltration by T cells in primary tumours, together with the analysis of HLA expression by colorectal cancer cells, suggest that anti-tumour T cell immune responses may take place in vivo in those patients, influencing prognosis and shaping the tumour immunological profile. Moreover, the molecular characterisation of tumour antigens expressed by colorectal carcinomas, together with improved understanding of mechanisms of the immune response and more sensitive methods for the in vivo detection of T cell responses, are now allowing researchers to design new and more effective vaccination protocols, with encouraging preliminary results. By drawing together the experimental evidence from different research fields, this review provides support for the concept that colorectal carcinoma is immunogenic and may reasonably be considered as a target for immunotherapy, and attempts to address critical issues and envisage future developments in this challenging research field.
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http://dx.doi.org/10.1016/s1040-8428(02)00159-2DOI Listing
April 2003
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