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Methods in
Molecular Biology 2777

Federica Papaccio
Gianpaolo Papaccio Editors

Cancer
Stem Cells
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Cancer Stem Cells

Methods and Protocols

Second Edition

Edited by

Federica Papaccio
Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno,
Baronissi, Italy

Gianpaolo Papaccio
Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”, Naples, Italy
Editors
Federica Papaccio Gianpaolo Papaccio
Department of Medicine, Surgery and Department of Experimental Medicine
Dentistry “Scuola Medica Salernitana” University of Campania “Luigi Vanvitelli”
University of Salerno Naples, Italy
Baronissi, Italy

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-3729-6 ISBN 978-1-0716-3730-2 (eBook)
https://doi.org/10.1007/978-1-0716-3730-2
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Preface

This second edition of the book Cancer Stem Cells: Methods and Protocols follows the
great success of its first edition, published in 2018.
In this second edition, a great effort has been made to provide readers with all the
scientific advances in methods and protocols related to cancer stem cells. They will, there-
fore, find both the most important updated chapters from the previous edition and new
chapters on hot topics.
The aim of the book is always to try to be a comprehensive collection of all methods,
protocols, and procedures used for the identification, characterization, and selection of
cancer stem cells. The new chapters focus on the latest technologies that have improved
our knowledge in this field. Among the newly included chapters, we feature contributions
on organoids, machine learning, nanoparticles, and other recent advances.
Regarding the topics of the new chapters, we believe that organoids, in particular,
represent a great challenge, and the related techniques are of great importance for cancer
stem cells thanks to the many applications they can have. At this point, it is very important to
be rigorous and understand the difference between spheroids and organoids.
We hope that the hard work we put into producing this book will be useful to scientists
who want to read it.

Baronissi, Italy Federica Papaccio

Naples, Italy Gianpaolo Papaccio

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Cancer Stem Cells: Current Challenges and Future Perspectives . . . . . . . . . . . . . . 1
Muhammad Vaseem Shaikh, Stefan Custers, Alisha Anand,
Petar Miletic, Chitra Venugopal, and Sheila K. Singh
2 Immunohistochemistry for Cancer Stem Cell Detection: Principles
and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Giuseppa Zannini, Renato Franco, and Federica Zito Marino
3 Isolating Cancer Stem Cells from Solid Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Vitale Del Vecchio, Marcella La Noce, and Virginia Tirino
4 Surface Markers for the Identification of Cancer Stem Cells . . . . . . . . . . . . . . . . . . 51
Tasfik Ul Haque Pronoy, Farhadul Islam, Vinod Gopalan,
and Alfred King-yin Lam
5 CD44-Based Detection of CSCs: CD44 Immunodetection
by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Lornella Seeneevassen, Anissa Zaafour, and Christine Varon
6 ALDH Activity Assay: A Method for Cancer Stem Cell (CSC)
Identification and Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Vitale Del Vecchio, Marcella La Noce, and Virginia Tirino
7 In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer
Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Amani Yehya, Hisham Bahmad, and Wassim Abou-Kheir
8 Multicellular Tumoroids for Investigating Cancer Stem-Like
Cells in the Heterogeneous Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . 99
Kathleen M. Burkhard and Geeta Mehta
9 Generation, Expansion, and Biobanking of Gastrointestinal
Patient-Derived Organoids from Tumor and Normal Tissues. . . . . . . . . . . . . . . . . 123
Manuel Cabeza-Segura, Blanca Garcia-Mico, Andrés Cervantes,
and Josefa Castillo
10 Prostate Cancer Organoids for Tumor Modeling and Drug Screening. . . . . . . . . 135
Amani Yehya, Fatima Ghamlouche, Sana Hachem, and Wassim Abou-
Kheir
11 Mimicking the Tumor Niche: Methods for Isolation, Culture,
and Characterization of Cancer Stem Cells and Multicellular Spheroids. . . . . . . . 145
Laura De Lara-Peña, Cristiano Farace, Andrea Pisano,
Julia Lopez de Andrés, Grazia Fenu, Federica Etzi,
Carmen Griñán-Lison, Juan Antonio Marchal, and Roberto Madeddu

vii
viii Contents

12 Detection of Cancer Stem Cells in Normal and Dysplastic/Leukemic

Human Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Alessia De Stefano, Alessandra Cappellini, Irene Casalin, Stefania Paolini,
Sarah Parisi, Maria Vittoria Marvi, Antonietta Fazio, Irene Neri,
Foteini-Dionysia Koufi, Stefano Ratti, Carlo Finelli, Antonio Curti,
Lucia Manzoli, Lucio Cocco, and Matilde Y. Follo
13 Methods to Study the Role of Mechanical Signals in the Induction
of Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Alessandro Gandin, Paolo Contessotto, and Tito Panciera
14 Co-Delivery Polymeric Poly(Lactic-Co-Glycolic Acid) (PLGA)
Nanoparticles to Target Cancer Stem-Like Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Catherine S. Snyder, Taylor Repetto, Kathleen M. Burkhard,
Anish Tuteja, and Geeta Mehta
15 Isolating Circulating Cancer Stem Cells (CCSCs) from Human
Whole Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Carla Kantara and Pomila Singh
16 Generation of Cancer Stem Cells by Co-Culture Methods . . . . . . . . . . . . . . . . . . . 219
Biswajit Das and Chanakya Nath Kundu
17 Deep Learning of Cancer Stem Cell Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Hiroyuki Kameda, Hiroaki Ishihata, and Tomoyasu Sugiyama

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Contributors

WASSIM ABOU-KHEIR • Department of Anatomy, Cell Biology, and Physiological Sciences,

Faculty of Medicine, American University of Beirut, Beirut, Lebanon
ALISHA ANAND • Department of Biochemistry and Biomedical Sciences, McMaster University,
Hamilton, ON, Canada
HISHAM BAHMAD • Arkadi M. Rywlin M.D. Department of Pathology and Laboratory
Medicine, Mount Sinai Medical Center, Miami Beach, FL, USA
KATHLEEN M. BURKHARD • Department of Biomedical Engineering, University of Michigan,
Ann Arbor, MI, USA
MANUEL CABEZA-SEGURA • Department of Medical Oncology, Hospital Clı́nico Universitario
de Valencia, INCLIVA, Biomedical Research Institute, University of Valencia, Valencia,
Spain
ALESSANDRA CAPPELLINI • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
IRENE CASALIN • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
JOSEFA CASTILLO • Department of Medical Oncology, Hospital Clı́nico Universitario de
Valencia, INCLIVA, Biomedical Research Institute, University of Valencia, Valencia,
Spain; Centro de Investigacion Biomédica en Red (CIBERONC), Instituto de Salud
Carlos III, Madrid, Spain; Department of Biochemistry and Molecular Biology,
Universitat de Valencia, Burjassot, Spain
ANDRÉS CERVANTES • Department of Medical Oncology, Hospital Clı́nico Universitario de
Valencia, INCLIVA, Biomedical Research Institute, University of Valencia, Valencia,
Spain; Centro de Investigacion Biomédica en Red (CIBERONC), Instituto de Salud
Carlos III, Madrid, Spain
LUCIO COCCO • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
PAOLO CONTESSOTTO • Department of Molecular Medicine, University of Padua, Padua,
Italy
ANTONIO CURTI • IRCCS – Azienda Ospedaliero-Universitaria di Bologna, Istituto di
Ematologia “Sera ` gnoli”, Bologna, Italy
STEFAN CUSTERS • Department of Biochemistry and Biomedical Sciences, McMaster
University, Hamilton, ON, Canada
BISWAJIT DAS • Cancer Biology Division, School of Biotechnology, Kalinga Institute of
Industrial Technology (KIIT)-Deemed to be University, Bhubaneswar, Odisha, India
JULIA LÓPEZ DE ANDRÉS • Biopathology and Regenerative Medicine Institute (IBIMER),
Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain;
Instituto de Investigacion Biosanitaria ibs.GRANADA, University Hospitals of Granada-
University of Granada, Granada, Spain; Excellence Research Unit ‘Modeling Nature’
(MNat), University of Granada, Granada, Spain; Department of Human Anatomy and
Embryology, Faculty of Medicine, University of Granada, Granada, Spain; BioFab i3D
Lab- Biofabrication and 3D (bio)printing Singular Laboratory, University of Granada,
Granada, Spain

ix
x Contributors

LAURA DE LARA-PEÑA • Biopathology and Regenerative Medicine Institute (IBIMER),

Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain;
Instituto de Investigacion Biosanitaria ibs.GRANADA, University Hospitals of Granada-
University of Granada, Granada, Spain; Excellence Research Unit ‘Modeling Nature’
(MNat), University of Granada, Granada, Spain; Department of Human Anatomy and
Embryology, Faculty of Medicine, University of Granada, Granada, Spain; BioFab i3D
Lab- Biofabrication and 3D (bio)printing Singular Laboratory, University of Granada,
Granada, Spain
ALESSIA DE STEFANO • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
VITALE DEL VECCHIO • Department of Experimental Medicine, Histology and Embryology
Section, University of Campania “L. Vanvitelli”, Naples, Italy
FEDERICA ETZI • Department of Biomedical Sciences-Histology, University of Sassari, Sassari,
Italy; I.N.B.B. National Institute of Biostructure and Biosystem, Rome, Italy
CRISTIANO FARACE • Department of Biomedical Sciences-Histology, University of Sassari,
Sassari, Italy; I.N.B.B. National Institute of Biostructure and Biosystem, Rome, Italy
ANTONIETTA FAZIO • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
GRAZIA FENU • Department of Biomedical Sciences-Histology, University of Sassari, Sassari,
Italy
CARLO FINELLI • IRCCS – Azienda Ospedaliero-Universitaria di Bologna, Istituto di
Ematologia “Sera ` gnoli”, Bologna, Italy
MATILDE Y. FOLLO • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
RENATO FRANCO • Pathology Unit, Dipartimento di Salute Mentale, Fisica e Medicina
Preventiva, Università degli Studi della Campania ‘Luigi Vanvitelli’, Naples, Italy
ALESSANDRO GANDIN • Department of Industrial Engineering (DII), University of Padua,
Padua, Italy
BLANCA GARCIA-MICÓ • Department of Medical Oncology, Hospital Clı́nico Universitario de
Valencia, INCLIVA, Biomedical Research Institute, University of Valencia, Valencia,
Spain
FATIMA GHAMLOUCHE • Department of Anatomy, Cell Biology, and Physiological Sciences,
Faculty of Medicine, American University of Beirut, Beirut, Lebanon
VINOD GOPALAN • Cancer Molecular Pathology, School of Medicine and Dentistry, Griffith
University, Gold Coast, QLD, Australia
CARMEN GRIÑÁN-LISÓN • Biopathology and Regenerative Medicine Institute (IBIMER),
Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain;
Instituto de Investigacion Biosanitaria ibs.GRANADA, University Hospitals of Granada-
University of Granada, Granada, Spain; Excellence Research Unit ‘Modeling Nature’
(MNat), University of Granada, Granada, Spain; BioFab i3D Lab- Biofabrication and
3D (bio)printing Singular Laboratory, University of Granada, Granada, Spain;
Department of Biochemistry and Molecular Biology 2, School of Pharmacy, University of
Granada, Granada, Spain; GENYO, Centre for Genomics and Oncological Research,
Pfizer/University of Granada/Andalusian Regional Government, Granada, Spain
SANA HACHEM • Department of Anatomy, Cell Biology, and Physiological Sciences, Faculty of
Medicine, American University of Beirut, Beirut, Lebanon
HIROAKI ISHIHATA • School of Computer Science, Tokyo University of Technology, Tokyo, Japan
 Contributors xi

FARHADUL ISLAM • Department of Biochemistry and Molecular Biology, University of

Rajshahi, Rajshahi, Bangladesh
HIROYUKI KAMEDA • School of Bioscience and Biotechnology, Tokyo University of Technology,
Tokyo, Japan; School of Computer Science, Tokyo University of Technology, Tokyo, Japan
CARLA KANTARA • Departments of Neuroscience and Cell Biology and Biochemistry and
Molecular Biology, The University of Texas Medical Branch, Galveston, TX, USA
FOTEINI-DIONYSIA KOUFI • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
CHANAKYA NATH KUNDU • Cancer Biology Division, School of Biotechnology, Kalinga
Institute of Industrial Technology (KIIT)-Deemed to be University, Bhubaneswar, Odisha,
India
MARCELLA LA NOCE • Department of Experimental Medicine, Histology and Embryology
Section, University of Campania “L. Vanvitelli”, Naples, Italy
ALFRED KING-YIN LAM • Cancer Molecular Pathology, School of Medicine and Dentistry,
Griffith University, Gold Coast, QLD, Australia
ROBERTO MADEDDU • Department of Biomedical Sciences-Histology, University of Sassari,
Sassari, Italy; I.N.B.B. National Institute of Biostructure and Biosystem, Rome, Italy
LUCIA MANZOLI • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
JUAN ANTONIO MARCHAL • Biopathology and Regenerative Medicine Institute (IBIMER),
Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain;
Instituto de Investigacion Biosanitaria ibs.GRANADA, University Hospitals of Granada-
University of Granada, Granada, Spain; Excellence Research Unit ‘Modeling Nature’
(MNat), University of Granada, Granada, Spain; Department of Human Anatomy and
Embryology, Faculty of Medicine, University of Granada, Granada, Spain; BioFab i3D
Lab- Biofabrication and 3D (bio)printing Singular Laboratory, University of Granada,
Granada, Spain
MARIA VITTORIA MARVI • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
GEETA MEHTA • Department of Biomedical Engineering, University of Michigan, Ann
Arbor, MI, USA; Department of Materials Science and Engineering, University of
Michigan, Ann Arbor, MI, USA; Macromolecular Science and Engineering, University of
Michigan, Ann Arbor, MI, USA; Rogel Cancer Center, University of Michigan, Ann
Arbor, MI, USA; Precision Health, University of Michigan, Ann Arbor, MI, USA
GEETA MEHTA • Department of Materials Science and Engineering, University of Michigan,
Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan,
Ann Arbor, MI, USA; Macromolecular Sciences and Engineering, University of Michigan,
Ann Arbor, MI, USA; Rogel Cancer Center, University of Michigan, Ann Arbor, MI,
USA; Precision Health, University of Michigan, Ann Arbor, MI, USA
PETAR MILETIC • Department of Surgery, McMaster University, Hamilton, ON, Canada
IRENE NERI • Cellular Signalling Laboratory, Department of Biomedical and Neuromotor
Sciences, University of Bologna, Bologna, Italy
TITO PANCIERA • Department of Molecular Medicine, University of Padua, Padua, Italy
STEFANIA PAOLINI • IRCCS – Azienda Ospedaliero-Universitaria di Bologna, Istituto di
Ematologia “Sera ` gnoli”, Bologna, Italy
SARAH PARISI • IRCCS – Azienda Ospedaliero-Universitaria di Bologna, Istituto di
Ematologia “Sera ` gnoli”, Bologna, Italy
xii Contributors

ANDREA PISANO • Department of Biomedical Sciences-Histology, University of Sassari,

Sassari, Italy
TASFIK UL HAQUE PRONOY • Department of Biochemistry and Molecular Biology, University
of Rajshahi, Rajshahi, Bangladesh
STEFANO RATTI • Cellular Signalling Laboratory, Department of Biomedical and
Neuromotor Sciences, University of Bologna, Bologna, Italy
TAYLOR REPETTO • Department of Materials Science and Engineering, University of
Michigan, Ann Arbor, MI, USA
LORNELLA SEENEEVASSEN • INSERM U1312 BRIC Bordeaux Institute of Oncology,
University of Bordeaux, Bordeaux, France
MUHAMMAD VASEEM SHAIKH • Department of Surgery, McMaster University, Hamilton,
ON, Canada
POMILA SINGH • Departments of Neuroscience and Cell Biology and Biochemistry and
Molecular Biology, The University of Texas Medical Branch, Galveston, TX, USA
SHEILA K. SINGH • Department of Surgery, McMaster University, Hamilton, ON, Canada;
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton,
ON, Canada
CATHERINE S. SNYDER • Department of Materials Science and Engineering, University of
Michigan, Ann Arbor, MI, USA
TOMOYASU SUGIYAMA • School of Bioscience and Biotechnology, Tokyo University of Technology,
Tokyo, Japan
VIRGINIA TIRINO • Department of Experimental Medicine, Histology and Embryology
Section, University of Campania “L. Vanvitelli”, Naples, Italy
ANISH TUTEJA • Department of Materials Science and Engineering, University of Michigan,
Ann Arbor, MI, USA; Macromolecular Sciences and Engineering, University of Michigan,
Ann Arbor, MI, USA; Department of Chemical Engineering, University of Michigan, Ann
Arbor, MI, USA; Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA
CHRISTINE VARON • INSERM U1312 BRIC Bordeaux Institute of Oncology, University of
Bordeaux, Bordeaux, France
CHITRA VENUGOPAL • Department of Surgery, McMaster University, Hamilton, ON,
Canada
AMANI YEHYA • Department of Anatomy, Cell Biology, and Physiological Sciences, Faculty of
Medicine, American University of Beirut, Beirut, Lebanon
ANISSA ZAAFOUR • INSERM U1312 BRIC Bordeaux Institute of Oncology, University of
Bordeaux, Bordeaux, France
GIUSEPPA ZANNINI • Pathology Unit, Dipartimento di Salute Mentale, Fisica e Medicina
Preventiva, Università degli Studi della Campania ‘Luigi Vanvitelli’, Naples, Italy
FEDERICA ZITO MARINO • Pathology Unit, Dipartimento di Salute Mentale, Fisica
e Medicina Preventiva, Universita` degli Studi della Campania ‘Luigi Vanvitelli’, Naples,
Italy
 Chapter 1

Cancer Stem Cells: Current Challenges and Future

Perspectives
Muhammad Vaseem Shaikh, Stefan Custers, Alisha Anand, Petar Miletic,
Chitra Venugopal, and Sheila K. Singh

Abstract
Despite major advances in health care including improved diagnostic tools, robust chemotherapeutic
regimens, advent of precision, adjuvant and multimodal therapies, there is a major proportion of patients
that still go on to experience tumor progression and recurrence. Cancer stem cells (CSCs) are shown to be
responsible for tumor persistence and relapse. This subpopulation of cancer cells possess normal stem
cell like traits of self-renewal, proliferation, and multilineage differentiation. Currently, they are isolated and
enriched based on the cell surface markers that can be detected and sorted through fluorescence and
magnetic-based cell sorting. In this chapter, we review the current challenges and limitations often
encountered in CSC research, including the identification of universal markers, therapy resistance, and
new drug development. Current and future perspectives are discussed to address these challenges including
utilization of cutting-edge technologies such as next-generation sequencing to elucidate the genome,
epigenome, and transcriptome on a single-cell level and genome-wide CRISPR-Cas9 screens to identify
novel pathway-based targeted therapies. Further, we discuss the future of precision medicine and the need
for the improvement of clinical trial designs.

Key words Cancer stem cells, Tumor initiating cells, Standard of care, Intratumoral heterogeneity,
Cell surface makers, Fluorescent activated cell sorting, CRISPR-Cas9 system, Single cell RNA
sequencing, Minimal residual disease.

1 Introduction

Cancer is the second leading cause of death worldwide, accounting

for nearly 10 million deaths in 2020 [1]. Although there have been
significant improvements in the prognosis of patients due to
advancements in diagnostic tools, intensive chemotherapeutic
regimens, and the advent of precision and multimodal therapies
[2–4], most patients are faced with relapse and disease

Muhammad Vaseem Shaikh and Stefan Custers contributed equally with all other contributors.

Federica Papaccio and Gianpaolo Papaccio (eds.), Cancer Stem Cells: Methods and Protocols, Methods in Molecular Biology,
vol. 2777, https://doi.org/10.1007/978-1-0716-3730-2_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

1
2 Muhammad Vaseem Shaikh et al.

progression and ultimately death. Moreover, there have been little

to no improvements in prognosis for some advanced and aggressive
cancers [5].
This therapeutic failure can be partly attributed to extensive
intratumoral heterogeneity (ITH) present at the genetic, transcrip-
tomic, and functional levels [6, 7]. Several studies have shown
correlation between tumor heterogeneity and poorer clinical out-
comes [8–10]. ITH was traditionally explained by the clonal evolu-
tion model, wherein carcinogenesis was believed to be a result of
sequential accumulation of mutations and the subsequent gradual
selection of clones under various attractor states [11]. Recently,
several studies indicate heterogeneity to be driven by distinct sub-
populations of cells commonly referred as cancer stem cells (CSCs)
that possess traits of normal stem cells, such as multilineage differ-
entiation, self-renewal, and therapy evasion [12]. CSCs are believed
to adapt and survive the intensive standard of care (SoC) regimen,
become chemoradiotherapy-resistant, and seed the formation of
therapy-resistant fulminant relapse [13]. CSCs are also referred to
as tumor-initiating cells (TICs), as they are believed to initiate
tumorigenesis in a hierarchical manner, with CSCs at the apex and
rapidly proliferating cells forming the bulk of the tumor [14].
In 1997, Bonnet and Dick were the first to isolate and charac-
terize cancer stem cell subpopulations from acute myeloid leukemia
(AML) patients [15]. They demonstrated that a finite number of
cells termed leukemic stem cells (LSCs), characterized by the pres-
ence of cell surface phenotype CD34+CD38-, when injected into
immunodeficient mice were able to proliferate and differentiate,
giving rise to disease similar to the patient donor. They were able to
demonstrate the self-renewal capacity of CSCs by serial transplan-
tation of cells into secondary recipient mice, resulting in
re-establishment of AML. Moreover, they demonstrated an
enhanced tumorigenic ability of these cells by limiting dilution
assay. Later, Al-Hajj et al. demonstrated the presence of CSCs in
solid tumors, when they demonstrated CD44+/CD24- cells in
breast cancer tumors had enhanced tumorigenic capacity
[16]. CSCs have since then been characterized in several cancers,
viz., brain, lung, liver, pancreatic, and melanoma [17–21].
Understanding the biology of CSCs is instrumental for the
development of novel therapeutics that can target CSCs. In doing
so we recognize the challenges that accompany CSC research which
include the origins of CSCs, inconsistent markers, drug resistance,
and drug development. To circumvent these barriers, novel tools
and techniques such as single-cell RNA sequencing, genome-wide
CRISPR-Cas9 screens, and modifications in precision medicine and
clinical trial designs are being incorporated into CSC research.
 Cancer Stem Cells: Current Challenges and Future Perspectives 3

2 Challenges in CSC Research

The concept of the CSC has progressed from being a highly specu-
lative and intensively debated concept to a fairly defined and better
understood biological entity in many tumor types, including AML,
breast, brain, colon, lung, and other cancers. Despite the advances
made in understanding CSCs, many challenges lie ahead and will be
discussed below.

2.1 The CSC CSCs by definition are a subset of tumor cells that contain both
Definition Is a self-renewal and multilineage differentiation capacities that fuel
Functional Trait Rather tumor initiation, treatment resistance, and recurrence [22]. This
than a Specific Cell definition is based on the shared functional tumor-initiation trait
among CSCs. Considering that CSC phenotypes are complex, vary
between tumors, and are affected by abnormalities arising from
neoplastic transformation, defining CSCs outside the scope of the
shared functional trait of tumor initiation can be difficult
[23]. Importantly, this functional CSC definition can make data
interpretation complex as the gold standard assay for CSC identifi-
cation is in vivo serial transplantation, a functional read-out which
can underestimate total CSC numbers and has a bias towards more
malignant CSC types. Moreover, serial implantation risks isolating
artifacts and may limit true heterogeneity of the original tumor.
Due to this limiting effect a more refined definition has been
proposed: CSCs are tumor cells that can be proven to generate
long-term progeny, either by direct engraftment or genetic
tracing [22].

2.2 Molecular Historically, CSCs have been isolated by Hoechst staining, utilizing
Markers Used to the ATP-binding cassette (ABC) transporter protein family, which
Identify the CSC Do Not was later replaced and standardized by fluorescence-activated cell
Specifically Nor sorting (FACS) based on cell surface markers. Although marker-
Uniformly Mark CSCs based CSC isolation is easier to perform, the markers used are not
always reliable, leading to challenges in CSC identification and data
interpretation [22].
First, the markers currently used do not specifically identify
CSCs, as these markers play central roles in normal stem cell biol-
ogy and are also expressed in other normal cell types. For some
markers, CSC specificity can be achieved by targeting splice var-
iants. For example, full-length CD144 is widely expressed, while
the CD44v6 splice variant is more indicative of CSCs [24–26]. The
use of marker combinations has alleviated this problem to some
extent; however, the lack of specificity remains a hurdle for reliable
CSC detection [22, 23]. On top of this, markers may have roles in
other mechanisms, which may be favorably selected for during
in vivo serial transplantation. For example, CD44, CD90, and
CD34 are involved in cell adhesion and attachment and are thus
4 Muhammad Vaseem Shaikh et al.

favorably selected for during in vivo engraftment. Together these

observations indicate that markers that support the CSC phenotype
need to be identified [27].
Second, CSC markers are not universal for CSC identification
due to inter- and intratumoral heterogeneity, as CSC biology dif-
fers substantially both within tumors and among cancer types
[22, 23]. Proper use of existing markers is highly tumor dependent;
for example, ALDH1 seems to be a reliable CSC marker in certain
cancers such as breast cancer [28], but its validity is debated in
other cancer types such as liver and pancreatic cancer [29]. How-
ever, there is evidence indicating that deletion of ALDH1 enhances
stemness in breast cancer, making its validity as a CSC marker
highly contested only in certain contexts [30]. The identification
of consensus markers, capable of uniformly detecting CSCs across
and within tumors, is warranted to improve CSC research.
Third, CSC markers are not universally applicable, as antibodies
targeting CSC markers may not be suitable for every research
method [22]. This is the case for CD133, which can be used to
sort CSCs by FACS, but its suitability for mRNA- or immunohis-
tochemistry (IHC)-based applications is limited [31, 32].
Fourth, current CSC markers may not be constitutively
expressed. CD133 is an example of a marker that is downregulated
during the G0/G1 phase of the cell cycle [33]. Additionally, inter-
and intratumor heterogeneity may make markers unreliable as they
might be downregulated, potentially leaving certain subclones
undetectable [22], as is the case for CD133 inactivation by CpG
methylation in colorectal cancer and glioblastoma (GBM) [34].
In conclusion, consensus markers are needed that are function-
ally linked to stemness mechanisms and assure reliable and uniform
expression on CSCs. Research into enzymatic and signaling
mechanisms is a starting point on the functional CSC road of
discovery. Additional work should be performed to identify cell
surface markers that are selectively induced by signaling pathways
involved in normal stem cell biology (Wnt, TGF-b, Hedgehog).
Stringent validation in human primary tumor samples is warranted
for these potential CSC markers. It is expected that these markers
will be different between tumor types and potentially between
clones [22].

2.3 The Origins of One of the most debated topics of the CSC is its cell of origin.
CSCs Tumors are the result of genetic and epigenetic alterations accu-
mulated over many years. The presence of stem cell markers seems
indicative of a stem cell origin. Another possibility is that CSCs arise
from transit-amplifying/progenitor cells that increase mutational
burden during multistep tumorigenesis and introduce their
acquired mutations into corresponding stem cell pools via a process
of spontaneous dedifferentiation [23, 35, 36].
 Cancer Stem Cells: Current Challenges and Future Perspectives 5

Realizing these two hypotheses may not exclude one another,

the rigid hierarchical CSC model has progressed into the more
flexible model of phenotypic reversion. Phenotypic reversion indi-
cates that there is a balance between CSCs and differentiated cell
states which can interconvert and hereby link stochastic and hierar-
chical properties [22].
Stemness is acquired via CSC intrinsic changes in gene expres-
sion programs and underlying regulatory mechanisms of self-
renewal and differentiation. On top of these intrinsic mechanisms,
studies propose a prominent role of the cancer microenvironment,
or CSC niche, generating feed-forward and feed-backward
mechanisms. For example, gliomas can be initiated from terminally
differentiated neurons by reverting back into the CSC state, a
mechanism stimulated by Notch secreting endothelial cells
[37, 38]. Additionally, studies suggest that epithelial-to-mesenchy-
mal transitioning (EMT) signaling can induce CSCs from differ-
entiated tumor cells [39]. Not all CSCs express EMT markers [40]
and EMT signaling factors in some cases can reduce CSC
qualities [41].
Molecular understanding of how CSC-intrinsic and environ-
mental signaling dictate CSC hierarchies and how they govern
effective interconversion between tumor states remains limited.
In summary, current models capable of capturing microenvi-
ronmental interactions with CSCs remain suboptimal. Molecular
understanding of these mechanisms will provide further insights in
self-renewal and differentiation cues and thus tumor cell hierarchy
dependencies on the CSC niche.
Single cells would need to be followed in vitro and in vivo using
genetic tracing and live imaging. Lineage tracing animal models are
needed to determine whether CSC reversion occurs in vivo and to
assess its frequency of occurrence. Moreover, this would help
understand to what extent differentiation becomes irreversible.

2.4 Drug Resistance A major hurdle in cancer treatment is CSC-mediated treatment

resistance to SoC and initiation of relapse. These cells are causa-
tively correlated with poor prognosis, treatment resistance, and
subsequent relapse. Unraveling these mechanisms and effective
targeting of CSCs will be a decisive victory in the treatment of
cancer as a whole [42].
CSCs are mostly quiescent and abundantly express ABC2 trans-
porter proteins and anti-apoptotic proteins. Together these proper-
ties aid in evading apoptosis and drug-induced cytotoxicity when
compared to differentiated tumor cells [23, 43–45]. As described
above, research has indicated the involvement of (partial) EMT
programs in the initiation and sustainment of CSCs, and secreted
EMT signaling molecules from stromal cells have been shown to
induce chemo- and radiotherapy resistance [46–48]. Further
insights into treatment resistance are warranted for more efficient
therapeutic targeting of CSCs.
6 Muhammad Vaseem Shaikh et al.

2.5 Drug Another challenge is the development of drugs targeting CSCs.

Development Currently, there are two approaches considered: the direct target-
ing of CSCs and the targeting of the CSC niche.
Targeting CSCs is dependent on its genetics and environment,
resulting in context-dependent signaling pathways and unique
challenges per tumor type. Drug screens can be technically difficult
due to the rarity of CSCs in most tumors and limited means of
propagating them in vitro. Means to circumvent these issues
include the use of enrichment media used for brain cancers such
as medulloblastoma and GBM [17, 49]. Another method that has
been employed is genetically enforcing an EMT-like state in epithe-
lial tumors [23].
Heterotypical signals arising from tumor-associated stroma are
often responsible for activating EMT. Targeting paracrine signaling
pathways that activate and sustain CSC programs should provide
insights for targeting CSCs. There have been successes with anti-
bodies targeting tropical signals. For example, macrophage M-CSF
receptor antibodies have been developed and effectively reduced
macrophage homing tumors in syngeneic mouse models [50]. In
addition, stimulating paracrine signaling molecules favoring
non-CSCs could be used to push the CSC/non-CSC balance
towards the non-CSC side. Derivates of molecules such as secreted
frizzled-related protein 1 (SFRP) and Dickkopf WNT signaling
pathway inhibitor 1 (DKK) could be developed and employed to
induce SoC susceptibility [23, 40].
CSCs exist in a niche formed by multiple cell types and distinct
signaling molecules (growth factors, cytokines, extracellular matrix
components). Studying biological properties of CSCs in isolation
or performing screens in the absence of a surrounding relevant
niche is unlikely to yield translatable results in vivo. Currently,
in vivo models depend on immunodeficient mice or syngeneic
mouse models. Immunodeficient models lack a full microenviron-
ment as the adaptive immune system is lacking and syngeneic
settings are not fully translational as they are artificially induced
by knocking out one or two genes. Neither model recapitulates the
clinical setting to a full extent and are therefore limited in translat-
ing therapies to the clinic.

3 Future Perspectives

To overcome the challenges encountered in CSC research, an

enormous amount of work is being done to better characterize
the CSCs, understand resistance mechanisms, develop precision
medicines and multimodal therapy regimens, and enhance clinical
trial design (Fig. 1).
 Cancer Stem Cells: Current Challenges and Future Perspectives 7

Fig. 1 Schematic representation of target discovery for the development of CSC-specific therapies

3.1 Single-Cell RNA Indeed, next-generation sequencing (NGS) technologies have

Sequencing helped us to sequence large amounts of DNA and RNA and have
led to a better understanding of cancer biology and intertumoral
heterogeneity [51]. However, cancer is a heterogenous ecosystem
comprising bulk cancer cells, tumor immune microenviron-
ment (TIME), and CSCs [52], and bulk sequencing obscures the
accurate understanding of all the different cellular subpopulations
[53]. Rapidly evolving methods of single-cell RNA sequencing
(scRNA seq), which can profile genome, epigenome, and transcrip-
tome of an individual cell, can help us better elucidate the drivers of
tumor progression, metastasis, and therapy resistance [54, 55].
scRNA seq technology not only illuminates intratumoral het-
erogeneity, but also highlights the plasticity and dynamic nature of
8 Muhammad Vaseem Shaikh et al.

various cellular states. Neftel et al. profiled glioma stem cells

(GSCs) using scRNA seq and classified GBM into four major sub-
types: oligodendrocyte progenitor cells (OPC-like), astrocytes
(AC-like), neural progenitor cells (NPC-like), and mesenchymal
(MES-like) [7]. They discovered that a single GBM tumor com-
prises multiple cellular states in stark contrast to a singular cellular
state as initially believed [7]. Through lineage tracing experiments,
they further discovered that when cells of a particular type are
engrafted in mice, they not only form the tumor of that specific
state but re-establish the heterogeneity seen in the primary tumor
through lineage tracing experiments [7]. Additionally, when they
investigated the commonly known GSC marker and transcription
factors, they found each of the four states was enriched for specific
markers; for example, CD133 was enriched in OPC-like and CD44
was enriched in MES-like state [7].
Additionally, scRNA seq has aided in profiling circulating
tumor cells, identifying prognostic markers, understanding the
TIME, and studying tumor resistance mechanisms, thereby
providing a new perspective in CSC research. Aceto et al. [56]
identified oligoclonal circulating tumor clusters to have increased
propensity for breast-to-lung metastasis, and abundance of circu-
lating tumor cells (CTC) correlated with poorer outcomes. scRNA
seq helped identify the cell junction component plakoglobin as
differentially expressed, and knockdown of plakoglobin led to sig-
nificant reduction in metastasis. Lei et al. [57] employed scRNA seq
to profile breast cancer stem cells (BCSCs) isolated from two
HER2+ breast cancer patients and compared the transcriptomic
profile with breast cancer cells, mammary cells, and CD44+ mam-
mary cells. They were able to identify 404 genes differentially
expressed in BCSCs, and additionally, they identified CA12 as a
novel prognostic marker in HER2+ breast cancer patients. Chung
et al. [58] performed scRNA seq in 11 patients with primary breast
cancer. At a single-cell resolution, the immune population was
characterized in three distinct clusters of T lymphocytes, B lym-
phocytes, and macrophages. T lymphocyte and macrophages dis-
played T cell regulatory and M2 phenotype, respectively, which
promotes an immunosuppressive and pro-tumorigenic environ-
ment. To understand resistance, Zhang et al. carried out scRNA
seq in samples before and after treatment in high-grade serous
ovarian cancer. They identified a stress-associated genetic signature
present before treatment which was subsequently enriched post-
treatment and is correlated with poor patient prognosis. In addition
to stress-associated genetic signature genes associated with stem-
ness, pro-survival and pro-inflammation were also enriched, further
protecting the cells from chemotherapy [59].
Research over the past decade has concentrated on characteriz-
ing CSCs as their elimination might translate to clinical benefit.
scRNA seq has helped to deconvolute the CSC theory, ITH, drug
 Cancer Stem Cells: Current Challenges and Future Perspectives 9

resistance mechanisms, and identify prognostic markers. The vast

amount of data generated can be used to identify vulnerabilities of
CSCs and develop targeted therapies or multimodal therapies to
synchronously attack the susceptible targets.

3.2 Genome-Wide Comprehensive genetic studies in the past two decades have
CRISPR Screens emphasized and helped us understand correlations between the
disease phenotype and the mutations seen in cancers; however,
gene editing technologies that could allow us to study the driving
forces of cancer and correct mutations in the genome were needed
to drive further breakthroughs in precision medicine. Discovery of
zinc finger nucleases (ZFNs) [60], TALE domains in transcription
activator-like effector nucleases (TALENs) [61], and CRISPR/Cas
(clustered regularly interspaced palindromic repeats/CRISPR-
associated) technology have allowed for the precise editing of the
eukaryotic genome. The CRISPR/Cas system has revolutionized
the gene editing technology due to its ability to precisely manipu-
late genes even if they are silenced or in a dense location and have
allowed for ease of design, affordability, and scalability compared to
other techniques [62, 63]. With the advent of genome-wide loss-
of-function (LOF) and gain-of-function (GOF) single-guide RNA
(sgRNA) libraries, it is now possible to screen and discover new
therapeutic vulnerabilities in CSCs.
LOF screens are carried out by using genome-wide knockdown
libraries, in the presence or absence of therapies in order to identify
the fitness genes and biological processes that make the cells resis-
tant. Shalem et al. [64] constructed a genome-wide CRISPR/Cas9
knockout library (GecKO) and employed it to mice for genes that
confer resistance to a human melanoma cell line when treated with
vemurafenib. Hart et al. [65] developed an improved CRISPR loss-
of-function library known as the Toronto KnockOut (TKO) library
targeting over 18,000 genes, identifying ~2000 fitness genes which
is 4–5 times more than RNAi screens. Chen et al. [66] employed a
genome-wide CRISPR LOF screen (mGeCKOa) comprising
67,405 sgRNAs targeting 20,611 protein-coding genes and 1175
microRNA precursors to study the evolution of cancer and metas-
tasis in a mouse model.
LOF screens have been routinely used to discover novel and
adjuvant therapeutic targets as well as studying the TIME. CRISPR
LOF screen conducted on CSCs from patient-derived colon cancer
lines identified 44 essential genes including genes involved in the
cholesterol biosynthesis pathway. The authors validated these find-
ings by demonstrating enhanced antitumor efficacy in vivo when
treated with combinatorial treatment with cholesterol biosynthesis
inhibitors and 5-FU [67]. Wei et al. [68] discovered loss of
Regnase-1 led to increased fitness in CD8 + Tcells, prolonging
their persistence and their ability to infiltrate and fight cancer
cells. Recently, CRISPR Cas9 screens carried out on GSCs
10 Muhammad Vaseem Shaikh et al.

identified SOX transcription family, SOCS3, USP8, and DOT1L,

and protein UFMylation as essential genes responsible for temozo-
lomide resistance [69]. Xu et al. [70] identified that NMDAR as an
adjuvant therapeutic target with tyrosine kinase inhibitors (TKIs) in
hepatocellular carcinoma (HCC), co-treatment with an NMDAR
antagonist, ifenprodil, with TKI, sorafenib, suppressed genes asso-
ciated with the WNT signaling pathway and stemness and reduced
self-renewal ability and proliferation of HCC cells.
In contrast CRISPR activation (CRISPRa) screens are a potent
tool for testing gain of function of endogenous genes by transcrip-
tional upregulation of target genes through the selective activation
using transcriptional activators targeting the promoter sites of tar-
get genes guided by sgRNA and a dead CAS9 system
[71, 72]. CRISPRa screens have tremendous potential in the eluci-
dation of drug resistance mechanisms, which are normally believed
to be gain-of-function events in response to therapy. Recently, a
reciprocal CRISPRa and CRIPR LOF screen was carried out on
GSCs and chimeric antigen receptor-T (CAR-T) revealing RELA
and NPLOC4 to be essential in GSCs for resistance against immu-
notherapy and also elucidated that the knockout of TLE4 and
IKZF2 led to superior effector function and reduced the exhaus-
tion of CAR-Ts [73]. Moreover, CRISPRa screens carried out on
pancreatic ductal adenocarcinoma lines identified a cellular resis-
tance mechanism to gemcitabine, 5-fluorouracil, irinotecan, and
oxaliplatin, revealing overexpression of ABCG2, a drug efflux
pump, as a common drug resistance mechanism to all the four
drugs [74].
Taken together these studies suggest the immense promise for
CRISPR technology in the development of new precision oncoly-
tics by identifying novel targets and facilitating existing therapies
through the elucidation of resistance mechanisms.

3.3 Precision Pathway-based medicines have been developed using our deeper
Medicine understanding of cancer biology from the genomic, proteomic, and
functional data generated from CRISPR screens and single-cell
sequencing. These therapies could not only target aberrant cellular
populations but also help to spare healthy cells. Imatinib, which
targets gain-of-function mutations in the genes encoding platelet-
derived growth factor receptor-α (PDGFRα) in gastrointestinal
stromal tumors, was the first clinical success story for targeted
cancer treatment [75].
Since then, several targeted therapies have been designed
against CSCs owing to the increased clinical significance of target-
ing these functionally important cells. Hedgehog signaling pathway
expression is correlated with chemoresistance, metastatic spread,
and CSC maintenance. Hh regulates the target gene expression
through the transmembrane protein Smoothened (Smo)
[76]. Glasdegib, vismodegib, and sonidegib showed significant
Cancer Stem Cells: Current Challenges and Future Perspectives 11

activity in locally advanced and metastatic basal cell carcinoma and

AML and were approved by the US Food and Drug Administration
(FDA) [77, 78]. MK-0752 a γ-secretase inhibitor that targets the
Notch signaling pathway, which is implicated for maintenance of
CSCs [79], showed clinical benefit in refractory central nervous
tumors in pediatric patients [80]. Gao et al. used CRISPR LOF
screens on CSCs that identified 44 genes in the cholesterol biosyn-
thesis pathway to be essential for stemness and chemoresistance in
colon cancer. They also further demonstrated that lovastatin and
5-flurouracil combination treatment showed significant preclinical
efficacy [67].
However, clinical approval of new drugs is a lengthy and an
expensive process, on average taking 13 years of research and 2–-
3 billion USD for a drug to reach from bench to market
[81]. Hence, recently, drug repurposing (“teaching old dogs new
tricks”) is receiving lot of traction in the research community,
wherein the strategy is to use next-generation biological data and
leverage it for the usage of inhibitors that have already received
regulatory approval. This pathway greatly reduces development
costs and time as well as alleviates business risks as safety, toxicity,
and pharmacokinetic profiles of the investigational drug have
already been established, allowing drugs to be directly fast-tracked
to Phase II and III of the clinical trial process [82].
Machine learning and data mining are vital to tap into the
unprecedented resources of approved drugs. Connectivity
Mapping (CMap) and the Library of Integrated Network-based
Cellular Signatures (LINCS) are extensive databases of aberrant
gene expression signatures and putative drugs that could poten-
tially have an inhibitory or potentiating effect on these genetic
profiles [83, 84]. These tools have greatly facilitated and acceler-
ated the process for identification of new molecules. Using CMap,
Singh et al. identified apomorphine, a dopamine antagonist, to
prevent brain metastasis in mouse models by targeting pre-
metastasis-associated genes KIF16B, SEPW1, and TESK2 in
BTICs [85]. Recently, a clinical trial conducted in patients with
pancreatic cancer showed significant survival benefit in patients
treated with metformin, a common antidiabetic drug [86]. This
strategy has also shown clinical benefit in elderly non-fit AML
patients, and the addition of all-trans retinoic acid to decitabine
showed significant higher remission rates as compared to the single
treatment arm [87]. These studies highlight the window of oppor-
tunity that drug repurposing has opened.
Carcinogenesis is believed to be strongly related to a dysfunc-
tional immune system as well as immune surveillance [88]. Cancer’s
cellular landscape evolves with cancer progression and cancer cells
remodel the TIME to their advantage [89], while the dysregulated
immune system activates stem cell signaling pathways responsible
for therapy resistance, antigen escape, and recurrence
12 Muhammad Vaseem Shaikh et al.

[89, 90]. Based on our understanding of the TIME, over the past
decade, novel immunotherapeutic strategies such as immune
checkpoint inhibitors and CAR-T cell therapy have emerged. In a
first of its kind study, Vora et al. carried out a head-to-head com-
parison of three immunotherapeutic modalities (antibodies, bispe-
cific T cell engagers [BITEs], and CAR-T) targeting CD133 on
GSCs and found the CAR-T-based treatment modality to be most
efficacious [91]. Several immune checkpoint inhibitors including
CTLA4 (ipilimumab) [92], PD-1 (cemiplimab) [93], and PDL-1
(avelumab) [94] have been evaluated clinically in various cancers
and have shown efficacy. CD19 and CD22 CAR-T cell therapy have
shown clinical success in liquid tumors [95–97] and several trials
testing their efficacy are underway for solid tumors
(NCT02915445, NCT02465983, NCT03585764).

3.4 Clinical Trial Improved therapeutic regimens have allowed complete remission in
Design patients with most cancers and thereby improving prognosis
[98, 99]. Complete remission is defined as reduction in the tumor
burden by 99% as measured by advanced radiological techniques
(PET, CT, or MRI) [100]. However, small therapy-resistant sub-
populations of tumor cells survive aggressive treatment regimens to
drive tumor recurrence [101, 102]. These cells are traditionally
called “minimal residual disease” (MRD) [103]. Recent studies
indicate that tumor recurrence is not driven by innate mutational
mechanisms [104, 105], rather by enrichment of a small population
of cells (CSCs) that are inherently resistant to anticancer therapy
[102, 106].
There are several conceptual advantages of treating patients at
MRD rather than waiting for clinical relapse to initiate further
therapy: first, the fact that cancer which is characterized by exten-
sive ITH will be at its lowest subclonal complexity and most homo-
geneous state which would increase the likelihood of designing a
tailored drug, targeting a rare minority of CSCs enriched at MRD
[107]. Additionally, due to the decrease in tumor burden there is
also reduction in the ability of the cells to remodel the surrounding
environment, or reprogram the TIME leading to a chemoprotec-
tive milieu [108]. Moreover, at MRD patients are clinically well and
generally off SoC therapy and hence more likely to tolerate any
potential therapeutic side effects compared to being treated at
relapse [109].
Identification, characterization, validation, and developing tar-
geted therapies against MRD will allow for the development of
precision treatment modalities that would most likely improve
patient outcomes and may lead to a potential cure [110]. Unfortu-
nately, it comes with a new set of clinical and technical challenges in
sampling, especially in solid tumors wherein repeated invasive pro-
cedures would lead to patient discomfort and reduce clinical appli-
cability [111]. To overcome this situation there is a need to develop
 Cancer Stem Cells: Current Challenges and Future Perspectives 13

in vitro and in vivo models that would recapitulate human disease.

Recently, Qazi et al. [109] used a unique model of human GBM
recurrence along with barcoding technology to track the BTIC
clonal population through SoC revealing that MRD can be driven
by either prior fitness of the subclones or prior equipotency result-
ing in selection of subclones under therapeutic selection pressure.
Additionally, they profiled GBM temporally by bulk sequencing
and scRNA-seq which could be used for potential target mining
as well as predicting and anticipating recurrence. Also, several
patient-derived xenograft models have been used to study MRD
for breast cancer, AML, ALL, and melanomas.
Monitoring patients at MRD using iterative sampling especially
in liquid cancers is being used as a standard practice to tailor the
anticipation of relapse and to refine clinical decisions [112]. Detec-
tion of MRD above or below a threshold is used as a tool for
escalating or tapering off therapy, though still it is not being used
to select targeted therapies to truly exploit the vulnerabilities of
MRD. Additionally, there are several studies indicating the use of
adjuvant therapies after complete remission resulting in improved
clinical outcomes [113–115]. Clinical trials in colorectal cancer are
underway wherein MRD is being used to inform which patients
should be enrolled for an adjuvant therapy. Targeting CSCs at
MRD appears to be a tractable strategy for efficiently eliminating
the surviving CSCs after SoC and preventing relapse.

References

1. Sung H, Ferlay J, Siegel RL, Laversanne M, 6. El-Sayes N, Vito A, Mossman K (2021)

Soerjomataram I, Jemal A et al (2021) Global
cancer statistics 2020: GLOBOCAN esti-
mates of incidence and mortality worldwide
for 36 cancers in 185 countries. CA Cancer J
Clin 71(3):209–249
2. Skaane P, Gullien R, Bjørndal H, Eben EB,
Ekseth U, Haakenaasen U et al (2012) Digital
breast tomosynthesis (DBT): initial experi-
ence in a clinical setting. Acta Radiol 53(5):
524–529
3. Romond EH, Perez EA, Bryant J, Suman VJ,
Geyer CE, Davidson NE et al (2005) Trastu-
zumab plus adjuvant chemotherapy for oper-
able HER2-positive breast cancer. N Engl J
Med 353(16):1673–1684
4. Baselga J, Cortés J, Kim SB, Im SA, Hegg R,
Im YH et al (2012) Pertuzumab plus Trastu-
zumab plus Docetaxel for metastatic breast
cancer. N Engl J Med 366(2):109–119
5. Oberstein PE, Olive KP (2013) Pancreatic
cancer: why is it so hard to treat? Ther Adv
Gastroenterol 6(4):321–337
Tumor heterogeneity: a great barrier in the
age of cancer immunotherapy. Cancers
13(4):806
7. Neftel C, Laffy J, Filbin MG, Hara T, Shore
ME, Rahme GJ et al (2019) An integrative
model of cellular states, plasticity and genetics
for glioblastoma. Cell 178(4):835–849.e21
8. Jamal-Hanjani M, Quezada SA, Larkin J,
Swanton C (2015) Translational implications
of tumor heterogeneity. Clin Cancer Res
21(6):1258–1266
9. Patel AP, Tirosh I, Trombetta JJ, Shalek AK,
Gillespie SM, Wakimoto H et al (2014)
Single-cell RNA-seq highlights intratumoral
heterogeneity in primary glioblastoma. Sci-
ence 344(6190):1396–1401
10. Mroz EA, Rocco JW (2013) MATH, a novel
measure of intratumor genetic heterogeneity,
is high in poor-outcome classes of head and
neck squamous cell carcinoma. Oral Oncol
49(3):211–215
14 Muhammad Vaseem Shaikh et al.
11. Greaves M, Maley CC (2012) Clonal evolu-
tion in cancer. Nature 481(7381):306–313
12. Steinbichler TB, Dudás J, Skvortsov S,
Ganswindt U, Riechelmann H, Skvortsova II
(2018) Therapy resistance mediated by cancer
stem cells. Semin Cancer Biol 53:156–167
13. Baisiwala S, Hall RR, Saathoff MR,
Shireman J, Park C, Budhiraja S et al (2020)
LNX1 modulates Notch1 signaling to pro-
mote expansion of the glioma stem cell popu-
lation during Temozolomide therapy in
glioblastoma. Cancers 12(12):E3505
14. Walcher L, Kistenmacher AK, Suo H, Kitte R,
Dluczek S, Strauß A et al (2020) Cancer stem
cells—origins and biomarkers: perspectives
for targeted personalized therapies. Front
Immunol 11:1280
15. Bonnet D, Dick JE (1997) Human acute
myeloid leukemia is organized as a hierarchy
that originates from a primitive hematopoietic
cell. Nat Med 3(7):730–737
16. Al-Hajj M, Wicha MS, Benito-Hernandez A,
Morrison SJ, Clarke MF (2003) Prospective
identification of tumorigenic breast cancer
cells. Proc Natl Acad Sci 100(7):3983–3988
17. Singh SK, Hawkins C, Clarke ID, Squire JA,
Bayani J, Hide T et al (2004) Identification of
human brain tumour initiating cells. Nature
432(7015):396–401
18. Eramo A, Lotti F, Sette G, Pilozzi E,
Biffoni M, Di Virgilio A et al (2008) Identifi-
cation and expansion of the tumorigenic lung
cancer stem cell population. Cell Death Differ
15(3):504–514
19. Ma S, Chan KW, Hu L, Lee TKW, Wo JYH,
Ng IOL et al (2007) Identification and char-
acterization of tumorigenic liver cancer stem/
progenitor cells. Gastroenterology 132(7):
2542–2556
20. Li C, Heidt DG, Dalerba P, Burant CF,
Zhang L, Adsay V et al (2007) Identification
of pancreatic cancer stem cells. Cancer Res
67(3):1030–1037
21. Schatton T, Murphy GF, Frank NY,
Yamaura K, Waaga-Gasser AM, Gasser M
et al (2008) Identification of cells initiating
human melanomas. Nature 451(7176):
345–349
22. Medema JP (2013) Cancer stem cells: the
challenges ahead. Nat Cell Biol 15(4):
338–344
23. Pattabiraman DR, Weinberg RA (2014) Tack-
ling the cancer stem cells – what challenges do
they pose? Nat Rev Drug Discov 13(7):
497–512
24. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X,
Cho RW et al (2007) Phenotypic
characterization of human colorectal cancer
stem cells. Proc Natl Acad Sci U S A
104(24):10158–10163
25. Al-Hajj M, Wicha MS, Benito-Hernandez A,
Morrison SJ, Clarke MF (2003) Prospective
identification of tumorigenic breast cancer
cells. Proc Natl Acad Sci U S A 100(7):
3983–3988
26. Snyder EL, Bailey D, Shipitsin M, Polyak K,
Loda M (2009) Identification of CD44v6+/
CD24- breast carcinoma cells in primary
human tumors by quantum dot-conjugated
antibodies. Lab Investig 89(8):857–866
27. Kemper K, Grandela C, Medema JP (2010)
Molecular identification and targeting of
colorectal cancer stem cells. Oncotarget
1(6):387–395
28. Ginestier C, Hur MH, Charafe-Jauffret E,
Monville F, Dutcher J, Brown M et al
(2007) ALDH1 is a marker of normal and
malignant human mammary stem cells and a
predictor of poor clinical outcome. Cell Stem
Cell 1(5):555–567
29. Deng S, Yang X, Lassus H, Liang S, Kaur S, Ye
Q et al (2010) Distinct expression levels and
patterns of stem cell marker, aldehyde dehy-
drogenase isoform 1 (ALDH1), in human
epithelial cancers. PLoS One 5(4):e10277
30. Ginestier C, Wicinski J, Cervera N,
Monville F, Finetti P, Bertucci F et al (2009)
Retinoid signaling regulates breast cancer
stem cell differentiation. Cell Cycle 8(20):
3297–3302
31. Kemper K, Sprick MR, de Bree M,
Scopelliti A, Vermeulen L, Hoek M et al
(2010) The AC133 epitope, but not the
CD133 protein, is lost upon cancer stem cell
differentiation. Cancer Res 70(2):719–729
32. Mak AB, Blakely KM, Williams RA, Penttil€a
PA, Shukalyuk AI, Osman KT et al (2011)
CD133 protein N-glycosylation processing
contributes to cell surface recognition of the
primitive cell marker AC133 epitope. J Biol
Chem 286(47):41046–41056
33. Sun Y, Kong W, Falk A, Hu J, Zhou L, Pollard
S et al (2009) CD133 (Prominin) negative
human neural stem cells are clonogenic and
tripotent. PLoS One 4(5):e5498
34. Yi JM, Tsai HC, Glöckner SC, Lin S, Ohm JE,
Easwaran H et al (2008) Abnormal DNA
methylation of CD133 in colorectal and glio-
blastoma tumors. Cancer Res 68(19):
8094–8103
35. Chaffer CL, Brueckmann I, Scheel C, Kaestli
AJ, Wiggins PA, Rodrigues LO et al (2011)
Normal and neoplastic nonstem cells can
spontaneously convert to a stem-like state.
 Cancer Stem Cells: Current Challenges and Future Perspectives
Proc Natl Acad Sci U S A 108(19):
7950–7955
36. Gupta PB, Fillmore CM, Jiang G, Shapira SD,
Tao K, Kuperwasser C et al (2011) Stochastic
state transitions give rise to phenotypic equi-
librium in populations of cancer cells. Cell
146(4):633–644
37. Borovski T, Verhoeff JJC, ten Cate R,
Cameron K, de Vries NA, van Tellingen O
et al (2009) Tumor microvasculature sup-
ports proliferation and expansion of glioma-
propagating cells. Int J Cancer 125(5):
1222–1230
38. Friedmann-Morvinski D, Bushong EA, Ke E,
Soda Y, Marumoto T, Singer O et al (2012)
Dedifferentiation of neurons and astrocytes
by oncogenes can induce gliomas in mice.
Science 338(6110):1080–1084
39. Vermeulen L, De Sousa E, Melo F, van der
Heijden M, Cameron K, de Jong JH, Bor-
ovski T et al (2010) Wnt activity defines
colon cancer stem cells and is regulated by
the microenvironment. Nat Cell Biol 12(5):
468–476
40. Scheel C, Eaton EN, Li SHJ, Chaffer CL,
Reinhardt F, Kah KJ et al (2011) Paracrine
and autocrine signals induce and maintain
mesenchymal and stem cell states in the
breast. Cell 145(6):926–940
41. Bruna A, Greenwood W, Le Quesne J,
Teschendorff A, Miranda-Saavedra D, Rueda
OM et al (2012) TGFβ induces the formation
of tumour-initiating cells in claudinlow breast
cancer. Nat Commun 3(1):1055
42. Chen J, Li Y, Yu TS, McKay RM, Burns DK,
Kernie SG et al (2012) A restricted cell popu-
lation propagates glioblastoma growth after
chemotherapy. Nature 488(7412):522–526
43. Roesch A, Fukunaga-Kalabis M, Schmidt EC,
Zabierowski SE, Brafford PA, Vultur A et al
(2010) A temporarily distinct subpopulation
of slow-cycling melanoma cells is required for
continuous tumor growth. Cell 141(4):
583–594
44. Grimm M, Krimmel M, Polligkeit J,
Alexander D, Munz A, Kluba S et al (2012)
ABCB5 expression and cancer stem cell
hypothesis in oral squamous cell carcinoma.
Eur J Cancer 48(17):3186–3197
45. Moitra K, Lou H, Dean M (2011) Multidrug
efflux pumps and cancer stem cells: insights
into multidrug resistance and therapeutic
development. Clin Pharmacol Ther 89(4):
491–502
46. Gupta PB, Onder TT, Jiang G, Tao K,
Kuperwasser C, Weinberg RA et al (2009)
Identification of selective inhibitors of cancer
15
stem cells by high-throughput screening. Cell
138(4):645–659
47. Farmer P, Bonnefoi H, Anderle P,
Cameron D, Wirapati P, Becette V et al
(2009) A stroma-related gene signature pre-
dicts resistance to neoadjuvant chemotherapy
in breast cancer. Nat Med 15(1):68–74
48. Kurrey NK, Jalgaonkar SP, Joglekar AV, Gha-
nate AD, Chaskar PD, Doiphode RY et al
(2009) Snail and slug mediate radioresistance
and chemoresistance by antagonizing
p53-mediated apoptosis and acquiring a
stem-like phenotype in ovarian cancer cells.
Stem Cells 27(9):2059–2068
49. Venugopal C, Hallett R, Vora P,
Manoranjan B, Mahendram S, Qazi MA et al
(2015) Pyrvinium targets CD133 in human
glioblastoma brain tumor–initiating cells.
Clin Cancer Res 21(23):5324–5337
50. Sica A, Allavena P, Mantovani A (2008) Can-
cer related inflammation: the macrophage
connection. Cancer Lett 267(2):204–215
51. Garziera M, Roncato R, Montico M, De
Mattia E, Gagno S, Poletto E et al (2019)
New challenges in tumor mutation heteroge-
neity in advanced ovarian cancer by a targeted
next-generation sequencing (NGS) approach.
Cell 8(6):E584
52. Sathe A, Grimes SM, Lau BT, Chen J,
Suarez C, Huang RJ et al (2020) Single-cell
genomic characterization reveals the cellular
reprogramming of the gastric tumor microen-
vironment. Clin Cancer Res 26(11):
2640–2653
53. Sun G, Li Z, Rong D, Zhang H, Shi X, Yang
W et al (2021) Single-cell RNA sequencing in
cancer: applications, advances, and emerging
challenges. Mol Ther Oncolytics 21:183–206
54. Tang F, Barbacioru C, Wang Y, Nordman E,
Lee C, Xu N et al (2009) mRNA-Seq whole-
transcriptome analysis of a single cell. Nat
Methods 6(5):377–382
55. Navin N, Kendall J, Troge J, Andrews P,
Rodgers L, McIndoo J et al (2011) Tumour
evolution inferred by single-cell sequencing.
Nature 472(7341):90–94
56. Aceto N, Bardia A, Miyamoto DT, Donald-
son MC, Wittner BS, Spencer JA et al (2014)
Circulating tumor cell clusters are oligoclonal
precursors of breast cancer metastasis. Cell
158(5):1110–1122
57. Lei B, Zhang XY, Zhou JP, Mu GN, Li YW,
Zhang YX et al (2016) Transcriptome
sequencing of HER2-positive breast cancer
stem cells identifies potential prognostic
marker. Tumour Biol 37(11):14757–14764
16 Muhammad Vaseem Shaikh et al.
58. Chung W, Eum HH, Lee HO, Lee KM, Lee
HB, Kim KT et al (2017) Single-cell RNA-seq
enables comprehensive tumour and immune
cell profiling in primary breast cancer. Nat
Commun 8:15081
59. Zhang K, Erkan EP, Jamalzadeh S, Dai J,
Andersson N, Kaipio K et al (2022) Longitu-
dinal single-cell RNA-seq analysis reveals
stress-promoted chemoresistance in meta-
static ovarian cancer. Sci Adv 8(8):eabm1831
60. Bibikova M, Beumer K, Trautman JK, Carroll
D (2003) Enhancing gene targeting with
designed zinc finger nucleases. Science
300(5620):764–764
61. Boch J, Scholze H, Schornack S, Landgraf A,
Hahn S, Kay S et al (2009) Breaking the code
of DNA binding specificity of TAL-type III
effectors. Science 326(5959):1509–1512
62. Knight SC, Xie L, Deng W, Guglielmi B, Wit-
kowsky LB, Bosanac L et al (2015) Dynamics
of CRISPR-Cas9 genome interrogation in liv-
ing cells. Science 350(6262):823–826
63. Zhang F, Wen Y, Guo X (2014) CRISPR/
Cas9 for genome editing: progress, implica-
tions and challenges. Hum Mol Genet 23
(R1):R40–R46
64. Shalem O, Sanjana NE, Hartenian E, Shi X,
Scott DA, Mikkelson T et al (2014) Genome-
scale CRISPR-Cas9 knockout screening in
human cells. Science 343(6166):84–87
65. Hart T, Chandrashekhar M, Aregger M,
Steinhart Z, Brown KR, MacLeod G et al
(2015) High-resolution CRISPR screens
reveal fitness genes and genotype-specific can-
cer liabilities. Cell 163(6):1515–1526
66. Chen S, Sanjana NE, Zheng K, Shalem O,
Lee K, Shi X et al (2015) Genome-wide
CRISPR screen in a mouse model of tumor
growth and metastasis. Cell 160(6):
1246–1260
67. Gao S, Soares F, Wang S, Wong CC, Chen H,
Yang Z et al (2021) CRISPR screens identify
cholesterol biosynthesis as a therapeutic target
on stemness and drug resistance of colon can-
cer. Oncogene 40(48):6601–6613
68. Wei J, Long L, Zheng W, Dhungana Y, Lim
SA, Guy C et al (2019) Targeting REGNASE-
1 programs long-lived effector T cells for can-
cer therapy. Nature 576(7787):471–476
69. MacLeod G, Bozek DA, Rajakulendran N,
Monteiro V, Ahmadi M, Steinhart Z et al
(2019) Genome-wide CRISPR-Cas9 screens
expose genetic vulnerabilities and mechanisms
of Temozolomide sensitivity in glioblastoma
stem cells. Cell Rep 27(3):971–986.e9
70. Xu F, Tong M, Tong CSW, Chan BKC, Chu
HY, Wong TL et al (2021) A combinatorial
CRISPR–Cas9 screen identifies ifenprodil as
an adjunct to sorafenib for liver cancer treat-
ment. Cancer Res 81(24):6219–6232
71. Decker CE, Young T, Pasnikowski E, Chiu J,
Song H, Wei Y et al (2019) Genome-scale
CRISPR activation screen uncovers tumor-
intrinsic modulators of CD3 bispecific anti-
body efficacy. Sci Rep 9(1):20068
72. Joung J, Kirchgatterer PC, Singh A, Cho JH,
Nety SP, Larson RC et al (2022) CRISPR
activation screen identifies BCL-2 proteins
and B3GNT2 as drivers of cancer resistance
to T cell-mediated cytotoxicity. Nat Commun
13(1):1606
73. Wang D, Prager BC, Gimple RC, Aguilar B,
Alizadeh D, Tang H et al (2021) CRISPR
screening of CAR T cells and cancer stem
cells reveals critical dependencies for cell-
based therapies. Cancer Discov 11(5):
1192–1211
74. Ramaker RC, Hardigan AA, Gordon ER,
Wright CA, Myers RM, Cooper SJ (2021)
Pooled CRISPR screening in pancreatic can-
cer cells implicates co-repressor complexes as a
cause of multiple drug resistance via regula-
tion of epithelial-to-mesenchymal transition.
BMC Cancer 21(1):632
75. Dagher R, Cohen M, Williams G,
Rothmann M, Gobburu J, Robbie G et al
(2002) Approval summary: imatinib mesylate
in the treatment of metastatic and/or unre-
sectable malignant gastrointestinal stromal
tumors. Clin Cancer Res 8(10):3034–3038
76. Zhao Y, Tong C, Jiang J (2007) Hedgehog
regulates smoothened activity by inducing a
conformational switch. Nature 450(7167):
252–258
77. Sekulic A, Migden MR, Oro AE, Dirix L,
Lewis KD, Hainsworth JD et al (2012) Effi-
cacy and safety of vismodegib in advanced
basal-cell carcinoma. N Engl J Med 366(23):
2171–2179
78. Norsworthy KJ, By K, Subramaniam S,
Zhuang L, Del Valle PL, Przepiorka D et al
(2019) FDA approval summary: glasdegib for
newly diagnosed acute myeloid leukemia. Clin
Cancer Res 25(20):6021–6025
79. Quail DF, Taylor MJ, Postovit LM (2012)
Microenvironmental regulation of cancer
stem cell phenotypes. Curr Stem Cell Res
Ther 7(3):197–216
80. Hoffman LM, Fouladi M, Olson J, Daryani
VM, Stewart CF, Wetmore C et al (2015)
Phase I trial of weekly MK-0752 in children
with refractory central nervous system malig-
nancies: a pediatric brain tumor consortium
study. Childs Nerv Syst 31(8):1283–1289
 Cancer Stem Cells: Current Challenges and Future Perspectives
81. Su M, Zhang Q, Bai X, Wu C, Li Y, Mossialos
E et al (2017) Availability, cost, and prescrip-
tion patterns of antihypertensive medications
in primary health care in China: a nationwide
cross-sectional survey. Lancet 390(10112):
2559–2568
82. Maxmen A (2016) Busting the billion-dollar-
myth: how to slash the cost of drug develop-
ment. Nature 536(7617):388–390
83. Lamb J, Crawford ED, Peck D, Modell JW,
Blat IC, Wrobel MJ et al (2006) The
connectivity map: using gene-expression sig-
natures to connect small molecules, genes,
and disease. Science 313(5795):1929–1935
84. Vidović D, Koleti A, Schürer SC (2014)
Large-scale integration of small molecule-
induced genome-wide transcriptional
responses, Kinome-wide binding affinities
and cell-growth inhibition profiles reveal
global trends characterizing systems-level
drug action. Front Genet 5:342
85. Singh M, Venugopal C, Tokar T,
McFarlane N, Subapanditha MK, Qazi M
et al (2018) Therapeutic targeting of the pre-
metastatic stage in human lung-to-brain
metastasis. Cancer Res 78(17):5124–5134
86. Chaiteerakij R, Petersen GM, Bamlet WR,
Chaffee KG, Zhen DB, Burch PA et al
(2016) Metformin use and survival of patients
with pancreatic cancer: a cautionary lesson.
JCO 34(16):1898–1904
87. Lübbert M, Grishina O, Schmoor C, Schlenk
RF, Jost E, Crysandt M et al (2020) Valproate
and retinoic acid in combination with Decita-
bine in elderly nonfit patients with acute mye-
loid leukemia: results of a multicenter,
randomized, 2 × 2. Phase II Trial J Clin
Oncol 38(3):257–270
88. Hiam-Galvez KJ, Allen BM, Spitzer MH
(2021) Systemic immunity in cancer. Nat
Rev Cancer 21(6):345–359
89. Prager BC, Xie Q, Bao S, Rich JN (2019)
Cancer stem cells: the architects of the tumor
ecosystem. Cell Stem Cell 24(1):41–53
90. Lytle NK, Barber AG, Reya T (2018) Stem
cell fate in cancer growth, progression and
therapy resistance. Nat Rev Cancer 18(11):
669–680
91. Vora P, Venugopal C, Salim SK, Tatari N,
Bakhshinyan D, Singh M et al (2020) The
rational development of CD133-targeting
immunotherapies for glioblastoma. Cell
Stem Cell 26(6):832–844.e6
92. Sakamuri D, Glitza IC, Betancourt Cuellar
SL, Subbiah V, Fu S, Tsimberidou AM et al
(2018) Phase I dose-escalation study of anti-
CTLA-4 antibody Ipilimumab and
17
lenalidomide in patients with advanced can-
cers. Mol Cancer Ther 17(3):671–676
93. Migden MR, Rischin D, Schmults CD,
Guminski A, Hauschild A, Lewis KD et al
(2018) PD-1 blockade with cemiplimab in
advanced cutaneous squamous-cell carci-
noma. N Engl J Med 379(4):341–351
94. Motzer RJ, Penkov K, Haanen J, Rini B,
Albiges L, Campbell MT et al (2019) Avelu-
mab plus axitinib versus sunitinib for
advanced renal-cell carcinoma. N Engl J Med
380(12):1103–1115
95. Park JH, Rivière I, Gonen M, Wang X,
Sénéchal B, Curran KJ et al (2018) Long-
term follow-up of CD19 CAR therapy in
acute lymphoblastic leukemia. N Engl J Med
378(5):449–459
96. Maude SL, Frey N, Shaw PA, Aplenc R, Bar-
rett DM, Bunin NJ et al (2014) Chimeric
antigen receptor T cells for sustained remis-
sions in leukemia. N Engl J Med 371(16):
1507–1517
97. Fry TJ, Shah NN, Orentas RJ, Stetler-
Stevenson M, Yuan CM, Ramakrishna S et al
(2018) CD22-targeted CAR T cells induce
remission in B-ALL that is naive or resistant
to CD19-targeted CAR immunotherapy. Nat
Med 24(1):20–28
98. Kahl B (2008) Chemotherapy combinations
with monoclonal antibodies in non-Hodg-
kin’s lymphoma. Semin Hematol 45(2):
90–94
99. Stupp R, Mason WP, van den Bent MJ,
Weller M, Fisher B, Taphoorn MJB et al
(2005) Radiotherapy plus concomitant and
adjuvant temozolomide for glioblastoma. N
Engl J Med 352(10):987–996
100. Adler S, Seidel J, Choyke P, Knopp MV,
Binzel K, Zhang J et al (2017) Minimum
lesion detectability as a measure of PET sys-
tem performance. EJNMMI Phys 4(1):13
101. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q,
Hjelmeland AB et al (2006) Glioma stem cells
promote radioresistance by preferential acti-
vation of the DNA damage response. Nature
444(7120):756–760
102. Liu G, Yuan X, Zeng Z, Tunici P, Ng H,
Abdulkadir IR et al (2006) Analysis of gene
expression and chemoresistance of CD133+
cancer stem cells in glioblastoma. Mol Cancer
5(1):67
103. Rambow F, Rogiers A, Marin-Bejar O,
Aibar S, Femel J, Dewaele M et al (2018)
Toward minimal residual disease-directed
therapy in melanoma. Cell 174(4):843–855.
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 He was soon at the head of a large and powerful army. With this
he marched forward, defeated the English troops that advanced to
meet him, and, in three months after his arrival, he took Edinburgh,
the capital of Scotland.—​France now sent him aid, and, with a force
of 7000 men, he marched southward into England, and took the
town of Carlisle. At Preston Pans, he defeated an English army of
4000 strong; and such was his success, that the English
government, under King William, of Orange, trembled for their safety.
They therefore made great efforts, and in April, 1746, they sent a
large army against him, under the Duke of Cumberland. At Culloden,
the two armies met, and a terrible battle followed; Prince Edward
was defeated, and his army entirely dispersed. He was scarce able
to save his life by flight; and, indeed, he wandered about, from place
to place, among the wilds of Scotland, being every day in danger of
being seized and given up to the English government, who offered
$150,000 to anybody who would bring him to them. It seems strange
that so large a bribe could be resisted; but, such was the love that
the Scottish people bore him, and such their fidelity, that no one was
found to betray him, though many people were entrusted with the
secret of his being among them. Even the poor mountaineers
refused to give him up, though offered a sum of money that would
have made them very rich.
At last, a faithful Scottish nobleman, by the name of O’Neil, took
him in charge, and after wandering along the sea-shore in a skiff,
flying from island to island, and experiencing the greatest sufferings
and dangers, he was put on board a French frigate, that had been
sent for his rescue. He was now taken to France, and soon after,
giving up all hopes of seeing his family restored to the throne, he
settled in Italy, where he died in 1788, in the 68th year of his age. He
was the last of the Stuart line, and was called the Pretender, on
account of his pretending to set up claims to the throne of England.
 Winter.

December has come! Winter is here! These are common-place

words, but they mean more, perhaps, than we are apt to consider.
Winter, then, means that the myriad leaves of the forest are
shrivelled and torn from the trees, and scattered in the valley: it
means that the sap of the trees has ceased to flow, and that these
giants of the vegetable world have passed into a state of stupor, in
which they must remain till spring again returns.
Winter means that the myriad races of annual weeds and plants
are dead, to revive again no more; that myriads of blossoms have
faded forever from the view; that the verdure of the forest has
passed away; that the gemmed garment of the meadow is
exchanged for the thin, brown mantle of leanness and poverty; that
the velvet of the lawn has given place to the scanty covering of dried
and faded grass.
Winter means that the minstrelsy of the birds is gone, and that the
field and forest, so lately cheered by a thousand forms and sounds
of happy existence are now silent, or rendered more dreary and
desolate by the moaning winds. It means that the birds are gone to
their southern retreats; that the myriad races of insects are dead;
that the whole generation of butterflies has perished; that the
grasshoppers have sung their last song; that even the pensive
cricket has gone to his long home. It means that death has breathed
on our portion of the world, and that nature herself, as if weary of her
efforts, has fallen into a cold and fearful slumber.
Winter means all these melancholy things; but it also means
something more. It means that the granary of the farmer is full; that
his barn is supplied; that there is good and ample store for the
beasts that look to man for support, and for man himself. It means,
too, that the comfortable fire will be kindled, around which the family
will assemble, and where, secure from the bitter blast without, there
will still be peace, comfort, and content. It means, too, that there is
such a thing as poverty, shivering, without fire, without food—
perhaps, without sufficient shelter; and it means that charity should
seek and save those who are suffering in such a condition.
And winter means something more than all this: it means, by its
examples of decay and death, to teach us that we, too, must pass
away; and that it is well for us to make preparation for the great
event. Winter also brings us to the end of the year, and suggests a
serious self-inquiry, and self-examination. It would ask us if the last
year has been one of profit or loss? Are we better, and wiser, than
when it began? Are we more kind, more just, more patient, more
faithful, more fond of truth?—Summer is the season for the harvest
of the field; winter is the season for the moral harvest of the heart.
Let it not pass with any of us as a barren and unproductive season,
in which we neither sow nor reap the fruits of wisdom and peace.
 The Hand.

Every limb and member of the body is made for some good
purpose.
The eye is made to see with; the ear is made to hear with; the
nose is made to smell with; the mouth is made to eat and speak with.
The feet are made to run and walk with; the hands are made to
work with, to write with, and to do many other things.
But do you think children’s hands were ever made to strike their
brothers, or sisters, or playmates? Were your little hands ever made
to snatch away things from each other?
Who gave you hands? God gave them. Did he give you hands to
steal with? Did God give you hands that you might throw stones at
geese, or dogs, or hens, or cows, or any other innocent animals?
Did God give you hands to injure or wound any of the creatures
he has made?
Take care of your little hands, then, my children! Take care that
the hands God has given, do nothing that God disapproves.
 Nuts to Crack.

The Word “Fast.”—This is as great a contradiction as we have in

the language. The river is fast, because the ice is immoveable; and
then the ice disappears fast for the contrary reason—it is loose. A
clock is called fast when it goes quicker than time; but a man is told
to stand fast, when he is desired to remain stationary. People fast
when they have nothing to eat, and eat fast when opportunity offers.

Military Courtesy.—Gen. Meadows, equally renowned for his

wit and bravery, being on a reconnoitring party, in the Mysore
country, a twenty-four pound shot struck the ground at some
distance from the General, and was passing in such a direction as
would have exposed him to danger had he continued on his route;
quick as lightning he stopped his horse, and, pulling off his hat very
gracefully, as the shot rolled on, good-humoredly said: “I beg you to
proceed, sir; I never dispute precedence with any gentleman of your
family.”

A doctor, in Scotland, was employed by a poor man to attend

his wife, who was dangerously ill. The doctor gave a hint, amounting
to the suspicion that he would not be paid. “I have,” says the man,
“five pounds; and if you kill, or cure her, you shall have it.” The
woman died, under the hands of the doctor, and, after a reasonable
time, he called for his five pounds. The man then said: “Did you kill
my wife?—did you cure her?” “No.” “Then,” said the poor man, “you
have no legal demand,” and turned upon his heel.
 How to shake off Trouble.—Set about doing good to
somebody: put on your hat, and go and visit the sick and poor—
inquire into their wants, and minister to them; seek out the desolate
and oppressed, and tell them of the consolations of religion. I have
often tried this method, and have always found it the best medicine
for a heavy heart.

A Father’s Impulse.—When Lord Erskine made his debut at the

bar, his agitation almost overpowered him, and he was just going to
sit down: “At that moment,” said he, “I thought I felt my little children
tugging at my gown, and the idea roused me to an exertion, of which
I did not think myself capable.”

The Sublime.—Over the stall of a public writer, in Rue de Bac, at

Paris, is the following inscription: “M. Renard, public writer and
compiler—translates the tongues, explains the language of flowers,
and sells fried potatoes.”

Feeling for Another.—A Quaker, once hearing a person tell

how much he felt for a friend who needed his assistance, dryly
observed: “Friend, hast thou ever felt in thy pocket for him?”

“What are you writing such a thundering big hand for, Patrick?”
“Why, do you see, my grandmother is deaf, and I am writing a loud
lether to her.”

A Knotty Case.—Not many years ago, a man appeared in court,

whether as plaintiff, defendant, or witness, tradition does not inform
us. Be this as it may, the following dialogue ensued:—Court—“What
is your name, sir?” “My name is Knott Martin, your honor.” “Well,
what is it?” “It is Knott Martin.” “Not Martin, again! We do not ask you
what your name is not, but what it is. No contempt of court, sir.” “If
your honor will give me leave, I will spell my name.” “Well, spell it.”
“K-n-o-tt, Knott, M-a-r, Mar, t-i-n, tin—Knott Martin.” “O, well, Mr.
Martin, we see through it now; but it is one of the most knotty cases
we have had before us for some time.”

Good.—It was a judicious resolution of a father, as well as a most

pleasing compliment to his wife, when, on being asked by a friend
what he intended to do with his daughters, he replied: “I intend to
apprentice them to their mother, that they may become like her—
good wives, mothers, heads of families, and useful members of
society.”

A Learned Character.—“Give me ‘Venice Preserved,’” said a

gentleman, last week, on going to a celebrated bookseller’s at the
West-end. “We don’t sell preserves,” said an apprentice, newly-
imported from the country; “but you will get them next door, at Mr.
Brown’s, the confectioner.”

Ten To One.—Strict attention to office hours is a duty incumbent

upon every public officer. We heard of a case of an American consul,
in a foreign country, who was not remarkable for his attention to duty.
A gentleman, calling one day, found his office shut, and a label
sticking upon the door, with these words: “In from ten to one.” Having
called again several times within those hours, without finding him, he
wrote at the bottom of the label—“Ten to one he’s not in.”
 To the Black-ey’d and Blue-ey’d Friends of
Robert Merry.

It is now about a twelvemonth since our acquaintance

commenced; and I hope the feeling is such between us, that there is
a mutual desire to continue it. I know that the young, the happy, and
the gay-hearted, are apt to think that we old fellows are sour and sad
—disposed to look with an evil eye upon childhood and its sports;
and more ready to preach than practise charity.
I will not pretend to deny that, now and then, a person gets cross
and crabbed as he grows old, and like cider too long kept, turns to
vinegar: but this is not my case, or, if it be, my ill-humor never
displays itself toward the young. They are to me the buds and
blossoms of life, and their presence ever brings the welcome
feelings that belong to sunshine and summer.
Old age has been often compared to winter—the close of the
year; the season of desolation; the period of storms and tempests;
the funeral-time of the vegetable world; the time when the leaves,
the fruits, and the flowers are laid in their tomb, and covered over
with a winding-sheet of snow. This is a sad picture at first view; and I
believe many a child is led to avoid old people from the habit of
regarding them in this light—from the idea that they are shrivelled,
frost-bitten, bitter, and disagreeable.
Now, I will not deny that there is some resemblance between
winter and old age: an old man has not the warm blood of youth; his
pulses are, perhaps, like the river, chilled and obstructed by ice; his
temper is sometimes capricious and gusty, like the winds of
December; and his head, bald, or covered with a few silvery hairs, is
like the oak, stripped of its covering, and having its boughs
powdered with snow.
 All this may be true enough; but it is not good reason why the old
should be deserted by the young. I remember very well, that, when I
was a boy, there was a fine old walnut-tree, upon a hillside, not far
from where I lived. Now, I never thought or cared about this tree, till
the time when winter approached. Then, when the leaves were
scattered, the nuts were all ripe, then it was that the tree became an
object of interest to me. Then it was that I loved to visit it; to climb its
limbs and give it a shake, and hear the fruit rattle down like hail.
Never, in all my boyhood days, did I meet with anything more
delightful than this!
And let me tell you, my black-ey’d and blue-ey’d friends, that this
old walnut-tree was like many an old person you may meet with. You
will remark that, in this case, it was when winter had come, or was
near at hand, that the fruit was ripe, and ready for those who would
climb up for it and gather it. And let me tell you, that old people, like
this tree, have many a good nut to crack, many a good story to tell,
to those who will climb up in the lap and ask for it.
This is my view of the matter; and I hope that young people,
instead of running away from me, as a crusty, crabbed, one-legged
old chap, will treat me as I did the old walnut-tree—give it a shake,
and see if the nuts don’t rattle down!
I am not fond of making great promises; but, as I am anxious to
have my readers, who have set out on a journey with me, still keep
me company—at least for one year more—I am ready to engage to
do my best to please them. I shall, if I live, tell the rest of my own
story, and bring the history of Brusque to a close. The tale of the
Sable-Hunters, the travels of Thomas Trotter, the stories of the
Indians, will be continued and completed; and a variety of other
things are in store.
I can promise one thing more—and that is, some tales from the
pen of Peter Parley. That pleasant, kind-hearted old man is no more;
but I knew him better than anybody else, and all his papers are in my
hands. Among them are several tales, and I intend to publish them in
my magazine. My young readers, perhaps, do not know how
shabbily poor old Peter was treated. The fact was, that several
people in this country, as well as in others, wrote stories, and put his
name to them; thus pretending that they were actually his! Some of
these were very silly, and some were very improper. This cut Peter to
the heart, and it served greatly to shorten his days. I am sorry that,
even now, people are palming off trumpery works of their own as
Peter Parley’s.
But the tales that I propose to give, are genuine; there is no
mistake. They are by the same hand that wrote the tales about
Europe, Asia, Africa, and America; and I hope they may be as
acceptable as those were.
I return a thousand thanks to my many young friends, who have
written me letters, whether of criticism, advice, or commendation. I
am glad to know that so many of them like Bill Keeler: let them be
assured his whole story will come out in due time. I shall be very
glad to get the bear story, which L. S., of Vermont, offers to tell. The
Indiana legend of the Wolf and the Wild-cat, is received, and will
appear soon. Jane R—— will accept my thanks for—she knows
what! If she were not so many hundred miles off, I should ask her to
let me see whether she is a blue-eyed or black-eyed friend. The
basket of chestnuts were duly received from Alice D——, and were
very welcome. Ralph H—— will see that I have done as he
requested; I have given a portrait of the fine gray squirrel he sent
me, in this number. He is well, and as lively as ever.
Robert Merry.
 WINTER—A SONG.
the words and music composed for
merry’s museum.

“Tell me what does winter mean!”

’Tis a drea-ry change of scene—
When the meadow yields its bloom,
And the blo-soms seek their tomb.
Winter is the time of storms,
When the cloud in angry forms,
O’er the land in terror sweeps,
And the sighing forest weeps.
’Tis the funeral time of flowers,
Withered in their lovely bowers;
While the zephyr sings in grief,
O’er each shrivelled stem and leaf.
’Tis the dreary time of snow,
Falling chill on all below,
As a winding-sheet it weaves
O’er the graves of myriad leaves.

Winter is a time of tears,

For the poor, in youth or years,—
Where the storm drives keenly in,
And the blanket’s brief and thin.
Winter is the time of wreck,
When the billow cleaves the deck,
And the mariners go down
Where the battling surges frown.
 Transcriber’s Note:
This book was written in a period when many words had not
become standardized in their spelling. Words may have multiple
spelling variations or inconsistent hyphenation in the text. These
have been left unchanged unless indicated below. Obsolete and
alternative spellings were left unchanged. Misspelled words were not
corrected.
One Footnote was moved to the end of the chapter. Obvious
printing errors, such as backwards, upside down, or partially printed
letters and punctuation, were corrected. Final stops missing at the
end of sentences and abbreviations were added. Duplicate letters at
line endings or page breaks were removed. Quotation marks were
adjusted to common usage. Page numbers in the Table of Contents
were corrected to match book pages.
Links to audio files were added for music. The music files are the
music transcriber’s interpretation of the printed notation and are
placed in the public domain. At the time of this writing, music file
links will not work in mobile e-book formats like epub or Kindle/mobi.
Users who are reading the e-book in one of these formats can listen
to the music or download music files in the HTML version. Lyrics to
musical scores are presented as poetry following the illustration of
the music.
*** END OF THE PROJECT GUTENBERG EBOOK ROBERT
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