Oncogene

https://doi.org/10.1038/s41388-019-1128-4

REVIEW ARTICLE

Tumor propagating cells: drivers of tumor plasticity, heterogeneity,

and recurrence
Alexandre Teixeira Vessoni1 Eduardo Cremonese Filippi-Chiela2 Guido Lenz3
● ● ●

Luis Francisco Zirnberger Batista1,4,5

Received: 15 August 2019 / Revised: 15 November 2019 / Accepted: 20 November 2019

© The Author(s), under exclusive licence to Springer Nature Limited 2019

Abstract
Tumorigenesis is associated with the development of a highly variable pattern of cellular diversity, consequence of genetic
and epigenetic diversification, followed by clonal selection and expansion. This process is shaped by the microenvironment
and leads to intratumoral heterogeneity, which is characterized by differences between cancer cells in terms of gene
expression, phenotypic markers, growth dynamics, and resistance to treatment. Another relevant aspect in intratumor
heterogeneity is cell plasticity—the ability of a cell to switch to new identities. In this review, we focus on the mechanisms
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that regulate cancer cell plasticity within a tumor, and explore the concept of tumor propagating cells, or TPCs, a cancer cell
able to propagate/phenocopy the parental tumor and recapitulate tumor heterogeneity. We discuss the influence of the
microenvironment and driver mutations on TPCs formation and function, the existence of phenotypically distinct TPC
clones within a tumor, the evolution of TPCs with disease progression, and their implications for therapy.

Introduction microenvironment [1]. Different models have been pro-

A tumor is a complex admixture of phenotypically and
genetically distinct populations of transformed cells whose
behavior, including growth and resistance to therapy, is
shaped by the multiple interactions between them, the
extracellular matrix, and with other cells in the tumor
* Alexandre Teixeira Vessoni
alexandre.vessoni@wustl.edu
* Luis Francisco Zirnberger Batista
lbatista@wustl.edu
1
Department of Medicine, Washington University School of
Medicine, St. Louis, MO 63110, USA
2
Departmento de Ciências Morfológicas, Instituto de Ciências
Básicas da Saúde, Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil
3 ing disease relapse [3]. Comprehending intratumor hetero-
Departamento de Biofísica e Centro de Biotecnologia,
Universidade Federal do Rio Grande do Sul, Porto Alegre, RS,
Brazil
4
Department of Developmental Biology, Washington University
School of Medicine, St. Louis, MO 63110, USA
5
Center for Regenerative Medicine, Washington University School
of Medicine, St. Louis, MO 63110, USA
posed to explain how intratumor heterogeneity arises from a
a single cancer-initiating cell (CIC). Solid evidence supports
a non-linear, branched pattern of evolution, where cancer
cells share genetic alterations that occurred earlier in tumor
history, followed by a gradual accumulation of new driver
mutations at subclonal populations [2–11]. Evidence also
supports a punctuated form of evolution, where a large
number of genomic aberrations occur in short bursts of
time, generating high intratumor heterogeneity at very early
stages of cancer progression [12–15]. Epigenetic differences
add an additional layer of complexity to intratumor het-
erogeneity as they can induce significant alterations in the
gene expression landscape within a tumor [16–19].
Accordingly, gene expression profiles associated with
positive or negative prognosis can be detected in different
regions of the same tumor [3]. In this sense, a single biopsy
is likely to underestimate tumor complexity, leading to
difficulties in validating oncogenic markers and in predict-
geneity is thus critical to the design of more effective
therapies.
In addition to subclonal differences, another critical
contributor to intratumor heterogeneity is cell plasticity,
i.e., the ability of a given cell to change its identity and
acquire characteristics of other cell types. In this sense, two
 A. T. Vessoni et al.

models have been proposed to explain how a single cancer
cell can give rise to heterogenous progenies: (A) the sto-
chastic (or clonal evolution) and (B) the cancer stem cell
(or hierarchical) models. In the stochastic model, all tumor
cells are equipotent, i.e., they all can self-renew and give
rise to distinct progenies, and their behavior is stochasti-
cally determined by intrinsic and extrinsic variables. In the
cancer stem cell (CSC) model, only a fraction of the tumor
cells, termed CSCs, are endowed with self-renewal poten-
tial and able to give rise to phenotypically diverse cells,
thus sustaining tumor growth in a defined hierarchical
mode. In this model, only CSCs give rise to serially
transplantable tumors that phenocopy the parental tumor in
xenotransplantation assays using immunocompromised
animals [20, 21] (Fig. 1).
However, as we will explore in detail in the following
sections, the stochastic and CSC models are not necessarily
mutually exclusive, since: (i) cancer cells can interconvert
between states with high and low expression of tumor-
igenicity markers [16, 22, 23]; and (ii) genetically distinct
CSCs that share a common ancestral may exist in the same
patient and show biologically distinct growth properties
[9, 24]. In addition, CSCs do not necessarily originate from
a tissue stem cell, and tumorigenicity is not exclusively
found in cancer cells that express stem cell markers [25–
30]. Therefore, the term “tumor propagating cells” (TPCs)
[29, 31, 32] will be used hereafter to refer to cancer cells
responsible for tumor development and able to propagate
cancer upon serial transplantation assays. Tumors formed
by TPCs are histologically heterogeneous and recapitulate
critical histopathological features of the original tumor, such
as infiltration/migration and multilineage differentiation.
Although some authors also refer to these cells as CICs, we
reserve this term to the precancerous cell in which the
crucial hit that initiates cancer development occurred
[31, 32]. In this review, we consider CSCs a specific case in
which TPCs display stem cell markers, as opposed to the
existence of cancer cells able to propagate heterogeneous
tumors and that lack the expression of such markers (more
details in the following section). Please, refer to the glossary
for more information regarding TPC, CSC, and CIC
definitions.

Fig. 1 The stochastic,

hierarchical, and dynamic
models of tumor propagation. In
the stochastic model, all cancer
cells are equipotent in their
ability to propagate a tumor that
phenocopies the heterogeneity
of the primary tumor. In the
hierarchical model, only a subset
of cancer cells (also known as
cancer stem cells) are endowed
with the ability to propagate the
tumor. In the dynamic model,
stochastic state transitions allow
cancer cells to interchange
between states with high or null/
low tumor propagation capacity.
These state transitions are
influenced by the
microenvironment, genetic/
epigenetic signature, and
metabolism
Tumor propagating cells: drivers of tumor plasticity, heterogeneity, and recurrence
Assessing tumorigenic potential relies on observing
tumor formation in vivo. The inability of one given cancer
cell to induce tumor upon animal transplantation does not
necessarily mean that it lacks tumorigenic potential. It can
result from an inability to adapt to the host and/or survive
upon certain conditions that do not match the original niche
where the tumor was initially formed. Thus, these caveats
pose a hurdle to provide a definitive proof for the lack of
tumorigenicity in any given cancer cell. That said, the use of
cell surface markers to segregate and compare tumorigenic
potential between different cancer cells may be subject to
these hurdles. As it will be discussed in the following
sections, under specific circumstances, changing transplan-
tation conditions revealed high tumorigenic potential in
unappreciated cancer cell populations. Despite these con-
siderations, the use of cell surface markers to differentiate
TPCs from non-TPCs is still the most direct (and
approachable) method to compare the tumorigenicity
between different cancer cells derived from a tumor.
Therefore, we consider the use of surface markers to dis-
tinguish TPCs from non-TPCs in this review, although the
true TPC character can only be assessed by the growth of a
tumor, as stated previously.
TPCs display several strategies that increase their resis-
tance to therapy [33, 34] including increased checkpoint
activation and DNA repair capacity [35], enhanced anti-
oxidant defenses [36], autocrine production of anti-
apoptotic molecules [37], and expression of multidrug-
resistance efflux pumps [38–40]. In addition, evidences
suggest that the TPC phenotype is a dynamic state that
could be acquired by non-TPC tumor cells via state tran-
sition [23, 41–43]. In this review, we discuss (i) the phe-
notypic heterogeneity of TPC clones in solid tumors and
leukemias; (ii) how TPCs evolve with disease progression;
(iii) evidence of state transition enabling acquisition of high
tumorigenic potential; (iv) the role of the tumor micro-
environment in this process; (v) methodological challenges
associated to the study of TPCs, and finally, (vi) the
implications of TPCs for the development of improved
therapies.
Tumorigenic potential in cancer cells lacking
expression of stem cell markers
TPCs were first identified in acute myeloid leukemia (AML)
as a rare population of adult hematopoietic cells that
retained a stem cell signature profile (CD34+/CD38−) [44].
This initial finding was followed by the identification of
stem-like TPCs in several human solid tumors, including
breast [45], brain [46, 47], ovarian [48], and colon [49, 50].
In vivo lineage-tracing assays later provided evidence that
TPCs with stem cell signatures were responsible for the
long-term maintenance and recurrence of different tumor
types [51–53]. Those evidences provided support to the idea
that CSCs and TPCs were one and the same. However,
evidence shows that the early progeny of stem cells, the
progenitor cells, can also act as TPCs and sustain tumor
growth. For instance, during the late stage (blast crisis) of
human chronic myeloid leukemia associated to BCR-ABL
oncogenic protein, granulocytic/monocytic-restricted pro-
genitors can acquire self-renewal potential and the ability to
propagate leukemia through increased β-catenin activation
[54]. Progenitor-like TPCs were also described in solid
tumors. For instance, in mouse models of medulloblastoma,
the TPC population was restricted to immature granule
neuron precursor-like cells that do not express the stem cell
marker CD133 and cannot form neurospheres (an in vitro
test commonly used to detect stem cell potential), but
express the progenitor cell marker Math1 [27]. Likewise, in
a mouse model of high-grade glioma, Id1low tumor cells,
which have progenitor cells properties and limited self-
renewal capacity, display higher tumorigenicity than tumor
cells with higher self-renewal capacity and elevated
expression of the neural stem cell marker Id1 [28].
Collectively, these findings show that cancer cells with
high tumorigenic potential are not necessarily those that
display tissue stem cell markers and properties. In fact,
under specific circumstances, high stem-cell marker
expression can be a poor indicator of the tumor propagating
potential of a cancer cell [28]. Therefore, the term TPC can
be more generally used to describe cancer cells able to
propagate cancer regardless of the presence of stem cell
markers.
Reversible state transitions and the changes
in tumorigenic potential of cancer cells
TPCs can give rise to tumors containing cancer cells with
lower/null tumorigenic potential [44, 47–50]. Interestingly,
evidence also suggests the opposite way: that cancer cells
can interconvert between a low tumorigenic/non-TPC state
to a TPC state [23, 41, 42]. For instance, when basal,
luminal, and stem cells from breast cancer cell lines are
segregated and cultured separately in vitro, they all return to
equilibrium cell-state proportion over time via stochastic
state transitions [41]. More recently, the use of single cell
RNA sequencing and barcoding revealed the potential of
one single glioblastoma cell to generate the four states
(neural-progenitor-, oligodendrocyte-progenitor-, astrocyte-
, and mesenchymal-like) observed in this malignancy [55].
The ability of cells to transit between different states can be
significantly lowered via activation of oncogenes and/or
inactivation of tumor suppressor genes. For instance, pri-
mary human mammary epithelial cells immortalized with

human telomerase can spontaneously convert from a
CD44low (differentiated) to a CD44high (stem cell) pheno-
type [23]. Upon transformation with SV40 and HRAS
expression, the conversion of CD44low cells (low tumori-
genic) to CD44high (highly tumorigenic) phenotype is
greatly increased [23]. Similarly, tumorigenic p53KO
fibroblasts expressing Kras-G12D can convert from a less
tumorigenic/more differentiated (Thy1+) to a more
tumorigenic/less mature (Thy1−/Sca1−) phenotype. An
increase in oncogenic signaling with Myc overexpression
accentuates this process [42].
In addition, evidence linking oncogenic signaling to
dedifferentiation and tumorigenesis was also described in
mouse models of intestinal [30], breast [56], and squamous
cell carcinoma [57]. In the intestine, the Wnt pathway and
oncogenic KRAS-G12D have been shown to cooperate to
dedifferentiate villus cells into crypt stem cells (LGR5+) and
initiate tumorigenesis [30]. In the skin, cancer cells can
hijack a “lineage infidelity” mechanism that can take place
during normal processes of wound repair via HRAS/MAPK
signaling [57]. In breast tissue, expression of the oncogenic
mutant PIK3CA-H1047R conferred multipotency to uni-
potent mammary cells and ability to induce tumors in mouse,
which could be further potentiated by P53 ablation [56].
At last, activation of Epithelial-to-Mesenchymal Transi-
tion (EMT), a critical developmental program hijacked by
cancer cells that often allows them to disseminate and
metastasize [1], can induce the acquisition of TPC traits by
non-TPC cancer cells [16, 22, 58], and BRCA1 may be
involved in suppressing this process in mammary cells [59].
Combined, the evidence discussed above shows that cells
with low tumorigenic potential can transit to a state of
greater tumorigenicity in a dynamic manner. It also indi-
cates that the accumulation of genetic lesions that inactivate
tumor suppressor genes and/or that activate oncogene sig-
naling during disease progression can reduce the barrier for
a non-tumorigenic cancer cell to become tumorigenic,
which could account for the higher frequency of TPCs in
late-stage cancers [24, 60–62].
Phenotypically diverse clones of TPCs
contribute to intratumor heterogeneity
Evidence supports the existence of multiple TPCs popula-
tions in individual cancer samples [9, 24, 63–70]. In leu-
kemia, multiple -and genetically distinct- leukemia
propagating cell clones with different engraftment capacity
were identified [9, 11, 24, 71–75]. Subclones display unique
genetic alterations, yet shared copy number alterations with
the dominant clone at diagnosis, showing that these sub-
clones evolved from the initial diagnostic clone [11, 24, 71].
The genetic architecture of the TPC population can be
A. T. Vessoni et al.
relatively simple (i.e., presence of two to three clones that
evolved in a linear sequence) or very complex (presence of
up to ten clones related via a branching evolutionary pat-
tern). In addition, the clonal architecture at relapse could
vary greatly from that at diagnosis [9, 67, 69, 76, 77].
Intraclonal heterogeneity of TPCs also occurs in solid
tumors such as glioblastoma and colo-rectal cancer
[65, 66, 78, 79], and the phenotypic diversity among TPCs
can also arise between genetically identical multiple TPCs
lacking a hierarchical organization between them and dif-
fering in their cell cycle/proliferation abilities [68].
In the last few years, single cell analysis started to shed
more light on the extremely diverse genetics and types of
cells that cause cancer. For instance: single cell tran-
scriptomics revealed that subclasses of IDH-mutant glioma
share an architecture composed of malignant cells that
recapitulate glial and neural precursor programs but differ
considerably in microglia and macrophages (tumor micro-
environment cells) signature [80]. Single cell tran-
scriptomics also allowed the identitication of a
subpopulation of cells (primitive oligodendrocyte pro-
genitor cells) that drive gliomagenesis, and also revealed the
role played by a RNA-binding protein implicated in oligo-
dendrocyte fate specification (Zfp36l1) in this process [81].
In addition, recent work with single cell lineage tracing
using endogenous mitochondrial DNA variants in ATAC-
seq provides additional evidence that the human pre-
leukemic hematopoietic stem cell population is hetero-
genous, and that one subclone evolves to become the
leukemia propagating cell in AML [73].
Collectively, these data indicate that genetically distinct
TPC clones with biologically distinct growth properties can
exist in individual cancer samples, and that genetically
identical TPCs can interchange between phenotypically
diverse states. Use of single cell analysis will allow to
increase our understanding of this complexity, and its
clinical role in limiting clinical effectiveness and con-
tributing to tumor relapse.
Microenvironment and cancer therapy
regulate TPCs generation and function
Apart from genetic diversity, a plethora of microenviron-
mental stimuli contributes to intratumor heterogeneity by
regulating TPC generation and function. In this sense,
paracrine and juxtracrine signaling derived from stroma
cells, and changes in oxygen and nitric oxide levels are
some of the key players in the microenvironment–cancer
interaction. Some of these relations are depicted below and
illustrated in Fig. 2.
Immune cells, which are known to play a large and
central role in tumor progression and metastasis [82–85],
Tumor propagating cells: drivers of tumor plasticity, heterogeneity, and recurrence
Fig. 2 The influence of the microenvironment on the main pathways
regulating acquisition and maintenance of tumor propagating potential
in different cancer cells. Several pathways are depicted in this figure.
In gliomas, the TPC state is regulated by the Notch pathway (via nitric
oxide secreted by endothelial cells); by the Protein Tyrosine Phos-
phatase Receptor Type Z1 (PTPRZ1), whose activation is driven by
Pleiotropin secreted by macrophages; and by signal transducer and
activator of transcription 3 (STAT3) and Hedgehog pathways. In
addition, glioma TPCs can differentiate into pericytes that support a
pro-glioma vasculature. In breast cancer cells, the TPC state is regu-
lated by ADAM Metallopeptidase Domain 10 (ADAM10)-containing
exosomes, which are secreted by fibroblasts and activate the Notch and
RhoA (Ras homolog gene family, member A) pathway in the cancer
cells; and by monocytes/macrophages that physically interact with and
activate the EPH receptor A4 (EphA4), resulting in the activation of
NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B
can engage in a complex crosstalk with TPCs. For instance,
tumor-associated macrophages can support glioma TPCs
through activation of the Protein Tyrosine Phosphatase
Receptor Type Z1 (PTPRZ1) receptor via secretion of
pleiotropin [86]. Monocytes/macrophages can also physi-
cally interact with and activate the EPH receptor A4
(EphA4) in breast TPCs [87], resulting in the activation of
Nuclear Factor kappa-light-chain-enhancer of activated B
cells (NF-κB) and secretion of cytokines that maintain the
TPC state [87]. Similarly, lung and colon TPCs can induce
the secretion of milk fat globule-EGF factor 8 protein
(MFG-E8) by macrophages, which, in turn, increases the
tumorigenicity and the resistance of TPCs to therapy [88].
Myeloid cells at perivascular sites can enhance invasiveness
cells), and secretion of cytokines that maintain the TPC state. In lung
and colon cancer, the TPC state is regulated by milk fat globule-EGF
factor 8 protein (MFG-E8) secreted by macrophages. In addition,
colon cancer TPCs can be sustained by the activation of β-catenin/Wnt
pathway by hepatocyte growth factor (HGF) secreted by myofibro-
blasts. Colorectal cancer TPCs are sustained by NF-κB activation (via
Interleukin-17A (IL-17A) secreted by cancer associated fibroblasts, as
well as by the STAT3 and Hedgehog pathways. In squamous cell
carcinoma, the TPC state is regulated by transforming growth factor-β
(TGF-β) secreted by myeloid cells, which binds to its receptor (TbRI/
II) and activates Mothers Against DPP Homolog 2 (SMAD2) in the
cancer cell. Illustrated in this figure is also the ability of TPCs to
activate regulatory T-cells (T-reg) via Interleukin 4 (IL4), C-C Motif
Chemokine Ligand 5 (CCL5), and STAT3, thus inhibiting T-cells’
proliferation and activation, and promoting tumor immune tolerance
of mouse squamous cell carcinoma TPC via secretion of
transforming growth factor-β (TGF- β) and triggering of
EMT features, while TPCs that are distant to the peri-
vasculature adopt a faster cell cycle rate associated with
tumor growth and differentiation [43]. TGF-β activation can
also boost the resistance of TPCs to cisplatin via activation
of glutathione metabolism genes [43]. Finally, TPCs display
immunomodulatory features—for instance, TPCs can acti-
vate regulatory T-cells (T-reg) and inhibit T-cells' pro-
liferation and activation, thereby promoting tumor immune
tolerance [89–91].
In addition to immune cells, cancer-associated fibroblasts
can also regulate TPC function and their metastatic poten-
tial. Exosomes (cell-derived vesicles that transfer DNA,

RNA, and proteins to other cells; [92]) derived from
fibroblasts containing ADAM Metallopeptidase Domain 10
(ADAM10) activate Notch and Ras homolog gene family,
member A (RhoA) in breast cancer cells, resulting in
increased cell motility and expression of TPC markers [93].
Cancer-associated fibroblasts activated by chemotherapy
can also secrete Interleukin-17A (IL-17A), which augments
the tumorigenic potential, growth rates, migration, and
chemotherapy resistance of colorectal cancer TPCs in a NF-
κB-dependent manner [94]. Hepatocyte growth factor
derived from myofibroblasts can also induce a TPC phe-
notype in more differentiated colon cancer cells, causing
these cells to re-express TPC-associated markers such as
activation of β-catenin/Wnt pathway [95]. Similarly, mye-
lodysplastic syndrome cells can remodel their micro-
environment by instructing normal mesenchymal stem cells
to acquire a disease-associated phenotype that secrete fac-
tors such as LIF, VEGFA, IGFBP2, and ANGPTL4, sup-
porting engraftment and expansion of the myelodysplastic
propagating cells [96].
Another important component of the TPC regulation by
the microenvironment is the vasculature. Highly vascularized
brain tumors have higher incidence of CD133+/Nestin+ cells
closely associated to the tumor capillaries [97], suggesting
that the perivascular niche supports cells that have TPC
markers. In agreement, co-injection of primary human endo-
thelial cells with medulloblastoma cells accelerated tumor
growth and increased the amount of TPCs in the resulting
tumor, while pharmacological ablation of tumor vasculature
with Erlotinib and Bevacizumab caused tumor arrest and
complete loss of CD133+/Nestin+ tumor cells [97]. The
regulation of TPCs by endothelial cells involves the secretion
of soluble factors such as nitric oxide [98, 99]. Interestingly,
in a glioblastoma xenograft, endothelial cells were able to
induce the differentiation of the glioma TPCs into pericytes to
support vasculature formation and tumor growth [100].
Molecules released by dying cancer cells can also impact
the TPC population of the tumor. Upon chemotherapy,
dying cancer cells of a xenograft bladder tumor release
PGE2, inducing the mobilization of a small subpopulation
of quiescent cytokeratin 14-positive cells (CK14+), which
are highly tumorigenic and resistant to therapy. Blocking
PGE2 release prevented the expansion of these TPCs and
reduced lung metastasis [101]. Also, TPCs can protect the
non-TPC population from anoikis via a bystander effect
[102], revealing a cooperatively crosstalk between these
different cancer cell types.
Evidence also shows that commonly used chemother-
apeutic agents and radiotherapy can accelerate the inter-
conversion between non-TPC to TPC state. Temozolomide,
an alkylating agent used in the treatment of glioblastoma,
drives the acquisition of TPC characteristics by non-TPC
glioma cells, possibly via hypoxia-inducing factor 1-alpha
A. T. Vessoni et al.
(HIF1A) and HIF2A [103], while radiation induced repro-
gramming of breast cancer cells into breast TPCs via acti-
vation of the Notch pathway [104].
Finally, recent data showed a connection between neu-
ronal cell activity and glioma growth and invasion as well as
breast cancer metastasis to the brain [105–107]. In these
reports, glutamate released by neurons were able to activate
glutamate receptors expressed in the membrane of the cancer
cells, supporting tumor cell proliferation and/or metastasis.
Therefore, combined with other studies [27, 60, 68, 87],
data described in this section reveal that TPCs regulation
heavily depends on their interaction with the micro-
environment, which provides a plethora of inputs that are
critical to sustain their identity. Decoding the information
exchanged by TPCs and their surrounding microenviron-
ment might help develop new therapies aiming at differ-
entiating and “locking” cancer cells in a non-TPC stage.
Metabolism of TPCs
Cancer cells often display an altered energy metabolism that
favors glycolysis over oxidative phosphorylation even
under aerobic conditions, the so-called Warburg effect
[108, 109]. This is associated with a fast proliferation rate,
as glycolytic intermediates can be diverted into amino acid,
nucleotide, and lipid biosynthesis routes required for cell
proliferation [108, 109]. While the Warburg effect is well-
established in tumors, recent evidence shows that the
metabolism of cancer cells is often more complex. For
instance, lactate secreted by a population of cancer cells
under the Warburg effect can be used by another population
of cancer cells in oxygenated areas of the same tumor to
fuel their oxidative metabolism [110]. Significant metabolic
differences may also exist between primary and metastatic
tumor sites, as in the case of pancreatic cancer, where dis-
tant metastasis are dependent on the oxidative branch of the
pentose phosphate pathway to support an epigenetic sig-
nature that drives their tumorigenicity [111]. In this sense, it
is interesting to consider if the metabolism of TPCs differs
from non-TPC cancer cells, and how relevant this is for
their maintenance and function (Fig. 3).
A growing body of evidence indicates that TPCs from
different cancer types display increased dependence on
mitochondria oxidative phosphorylation (OXPHOS), when
compared with the more differentiated/non-TPCs cells of
the same tumor. This metabolic configuration is critical for
their maintenance and tumor growth [112–120]. However,
subpopulations of TPCs that are metabolically heterogenous
have also been described. For instance, a subset of TPCs
(CD36+) were found to reside in the gonadal adipose tissue
of a mouse model of blast-crisis chronic myeloid leukemia.
These cells are able to co-opt neighboring adipocytes to
Tumor propagating cells: drivers of tumor plasticity, heterogeneity, and recurrence
Fig. 3 Metabolism in the control of the acquisition and maintenance of
tumor propagating potential in cancer cells. Top: Cytochrome C and
Endonuclease G (EndoG) released by mitochondria to the cytosol
cause activation of caspase-activated DNAse and formation of geno-
mic DNA double strand breaks, which in turn activate Nuclear Factor
kappa-light-chain-enhancer of activated B cells (NF-KB) and signal
transducer and activator of transcription 3 (Stat3) pathways via ataxia
release free fatty acids and support the TPCs’ metabolic
demands and survival [121].
Mitochondria can also be involved in an intricate reg-
ulation of tumorigenic potential in different cancer cell lines
that involves self-inflicting DNA damage [122]. In this
sense, mitochondria releases cytochrome C and Endonu-
clease G (EndoG) to the cytosol, causing activation of
caspase-activated DNAse and formation of genomic DNA
double strand breaks. In response to this damage, NF-KB
and Signal transducer and activator of transcription 3 (Stat3)
pathways are activated (via ataxia telangiectasia mutated, or
ATM), supporting tumorigenic potential in cancer cells
[122]. Reactive oxygen species produced by mitochondria
OXPHOS can also sustain breast cancer TPCs via activation
of the hypoxia inducible factor 1α (HIF-1α) pathway [116].
Interestingly, while in breast cancer MYC positively reg-
ulates OXPHOS and TPCs maintenance, in pancreatic
cancer MYC decreased OXPHOS and reduced stemness
properties of TPCs [112], revealing a complex regulation of
metabolism by MYC in different cancers.
Intriguingly, elimination of mitochondria via mitophagy
(an autophagy-mediated pathway for mitochondria recy-
cling) sustains TPCs from liver and colorectal cancer
[123, 124]. In the case of liver TPCs, mitophagy eliminates
the mitochondria-associated PTEN-induced kinase 1
(PINK1) that can phosphorylate and activate P53, thus
telangiectasia mutated (ATM), thus supporting tumorigenic potential
in different cancer cells. Reactive oxygen species produced by mito-
chondria oxidative phosphorylation (OXPHOS) can also sustain breast
cancer TPCs via activation of the hypoxia inducible factor 1α (HIF-1α)
pathway. Bottom: mitophagy sustains TPCs in liver and colorectal
cancer cells via elimination of PTEN-induced kinase 1 (PINK1), thus
preventing P53-mediated inhibition of NANOG transcription
preventing its translocation to the nucleus where it binds
and represses activation of the NANOG promoter, a gene
that sustains TPC phenotype in these cancer cells [123]. In
ovarian TPCs, autophagy sustains tumorigenicity and pro-
tects these cells from chemotherapy-induced death, possibly
via elimination of reactive oxygen species-producing
mitochondria and/or by supporting metabolism during
nutrient/oxygen starvation [125].
Altogether, these studies highlight the important and the
diverse role played by mitochondria and OXPHOS in the
regulation of TPCs. Metabolic heterogeneity may also occur
between TPCs of different niches from the same tumor, and
these differences may account for therapy resistance and
tumor relapse [112], highligthing the relevance of the
metabolic regulation of TPCs on cancer development and
therapy response.
Methodology challenges to study TPCs
in vivo and in in vitro
Understanding methodology limitations and patient-to-
patient variations in TPCs populations is critical. In this
section, we highlight important studies that brought con-
siderable improvement in the methodologies used to study
TPCs, as well as important considerations regarding TPC

heterogeneity that arises during disease progression, and
during in vitro and in vivo propagation of these cells.
Xenotransplantation assays are the most stringent
method to detect and test TPC potential [21]. However, it
comes with caveats, as to whether the assay selects a
population of human cancer cells able to readily engraft and
grow in a foreign and immune-compromised environment
[126]. As mentioned before, the inability of a cancer cell to
grow a tumor ex vivo may not be a consequence of lack of
tumorigenicity, but a failure of the cell to adapt to certain
conditions that do not perfectly recapitulate the environment
where the tumor was originally formed. For instance,
xenotransplantation of human acute myeloid leukemia
samples into a severe combined immunodeficient (SCID)
mouse revealed very low TPC frequency (1 in every
250.000 transplanted cells) [44], while the transplantation of
mouse lymphoma or mouse leukemia cells into non-irra-
diated, congenic animals, showed that as few as ten
unsorted cells were enough to induce malignancy that
recapitulated features of the primary tumor [29].
A second caveat refers to the methods used to isolate and
transplant TPCs, and the degree of immunodeficiency of the
host. For instance, anti-CD38 antibodies used to sort human
leukemia TPCs had an inhibitory effect on the engraftment
of these cells as it targeted them to clearance by the innate
immune cells [127]. Combining the administration of
immunosuppressants with intrabone marrow injection of the
CD38-coated cells revealed the presence of TPCs in pre-
viously unappreciated CD34+/CD38+ populations in some
cases of AML [127]. Another important development was
the use of highly immunocompromised mice (IL2rg−/−
versus NOD/SCID) and the co-injection of cancer cells with
matrigel in xenotransplantation assays, which increased, by
orders of magnitude, the frequency of melanoma TPCs,
reaching up to one in every every unsorted melanoma cells
derived from patients [61, 128].
A third caveat stems from the fact that the TPC pheno-
type is not necessarily uniform across patients [129].
Moreover, TPC frequency increases with disease progres-
sion/malignancy [24, 60–62, 80]. For instance, in primary
serous ovarian cancer (SOC), not only TPC frequency
varies up to 100 times between patients, but also their
immunophenotype is different: CD133+ marks TPCs in
some patient’s samples, while in other samples TPCs are
CD133−; in other cases, CD133 status does not seem to be
related to the TPC phenotype of cells [129]. Also, the
growth of tumor samples in xenograft may change TPCs
phenotype. In this sense, matched primary SOC tumors and
xenografts showed opposite TPC phenotypes regarding
CD133 expression [129]. In the same line, it was shown in
freshly obtained samples of melanoma from different stages
of the disease that the CD271+ fraction of cells is enriched
for TPCs whereas in late stage samples (lymph node
A. T. Vessoni et al.
metastasis) or in samples passaged in xenografts, virtually
every tumor cell, regardless of its immunophenotype, seems
to have tumorigenic potential [61, 62, 128]. These results
suggest a continuous malignant progression of tumor cells
with disease progression and/or xenograft growth, arguing
caution in the comparison of samples obtained from dif-
ferent patients, at different stages of the disease, and/or
propagated in xenografts.
Isolation and propagation of cancer cells in vitro is an
essential tool for disease modeling and drug discovery.
Still, cancer cells may adapt to these artificial conditions,
with concomitant loss of characteristics relevant for the
disease in vivo. For instance, fetal bovine serum (FBS),
routinely used to grow cancer cell lines in vitro, can
severely impair growth and maintenance of certain TPCs,
such as those derived from human glioblastoma [130]. In
this case, the growth of freshly isolated cells in serum-free
neural basal media, which is used for the propagation of
neural stem cells, preserved the ability of these cultured
cells to give rise to highliy infiltrative tumors that phe-
nocopied key characteristics of the parental tumor. On the
other hand, cells grown in the presence of FBS acquired
several genetic and/or epigenetic alterations and could only
form intracranial tumors with very low infiltrative capacity
[130]. The serum-free neural basal media, however, does
not seem adequate to the in vitro growth of every type of
glioma TPCs, as it compromised the tumorigenic potential
of oligodendrocyte progenitor (OPC)-like mouse glioma
cells [131, 132]. As different media containing single or a
few growth factors were traditionally designed based on the
putative cell of origin of gliomas, they are prone to select
subpopulations which would otherwise be reduced either
in vivo or by a media containing a different combination of
growth factors [132].
Although serum can exert a drastic effect on the main-
tenance of glioma TPCs in vitro, it does not affect TPCs
from other cancers in the same manner. In the case of
melanoma, for instance, cell lines grown in media supple-
mented with FBS were still able to engraft at 100% effi-
ciency when only very few unsorted cells were transplanted
[62]. Combined, this indicates that the in vitro growth of
TPCs obtained from different tissues requires unique culture
conditions.
Future perspectives: the therapeutic
challenges to target TPCs
TPCs are influenced by a plethora of factors, including
interaction with the tumor microenvironment, acquisition of
cancer-associated mutations, and disease stage [22, 23, 60].
Understanding how these variables regulate TPCs dynamics
may reveal new therapeutic opportunities.
Tumor propagating cells: drivers of tumor plasticity, heterogeneity, and recurrence
As evidence suggests that non-TPC cancer cells may
change their phenotype and acquire tumorigenic potential
[23, 41, 42], targeting only TPCs may not be fruitful ther-
apeutically. One potential strategy is to try to differentiate and
“lock” cancer cells in a non-TPC state incapable of propa-
gating the tumor [16, 80, 133]. Such strategies may be
achieved pharmacologically [134], genetically [81, 135–139],
or by modulating microenvironment cues [17, 43, 68, 98,
133, 134]. This strategy, however, needs to take into con-
sideration the genetic and functional heterogeneity between
distinct TPC subpopulations [9, 24, 65–67], as well as
changes in clonal architecture during treatment and relapse
[134]. In this sense, spatially resolved omics analysis
[140, 141] and RNA sequencing at the single-cell [80] level
may provide personalized, deeper details regarding shared
and/or exclusive pathways that sustain tumorigenic potential
in distinct TPC subpopulations.
Therapeutic approaches that exclusively target TPCs,
but not normal stem/progenitor cells, would potentially
lower toxicity for patients. For instance, HIF2A is pre-
ferentially expressed in glioblastoma TPCs with stem cell
characteristics, but not in normal brain stem cells, and is
important for TPC proliferation, survival and tumor-
igenicity [142]. Similarly, leukemia propagating cells were
much more sensitive to the HIF1A inhibitor echinomycin
than normal hematopoietic progenitor cells [143]. Target-
ing CD44 can impair engraftment and induce differentia-
tion of some leukemia propagating cells, while sparing
normal hematopoietic stem cells [144, 145]. In the same
line, leukemia propagating cells have increased expression
of CD47 in comparison to normal HSCs, and the use of
anti-CD47 antibody preferentially induces phagocytosis of
the cancerous counterparts [146]. Leukemia propagating
cells also heavily rely on OXPHOS for ATP production, in
contrast to normal HSCs [120], making the use of
OXPHOS inhibitors, such as metformin [147], an attractive
approach. At last, the R882H mutated version of DNA
methyltransferase 3A (DNMT3A, detected in 20–30% of
human acute leukemia), but not the wild type version of
this gene, was found to activate expression of Mesi1, Mu1
and HOXA1 genes that block differentiation and induce
self-renewal; this mutated DNMT3A cooperates with
NRAS mutation to induce leukemogenesis, but renders the
TPCs sensitive to Dot1 inhibition, causing them to differ-
entiate and lose self-renewal potential [148]. Nevertheless,
it is important to decipher if a determined target pathway is
required for the maintenance of all the phenotypically
diverse TPCs of a given tumor, and if (and how) the
dependency of the TPCs on those pathways change with
disease progression. Targeting TPCs is an attractive ther-
apeutic approach, and comprehension of TPCs’ evolution
with disease progression/relapse, intratumor clonal diver-
sity, and their regulation by the microenvironment from a
single cell resolution will be critical for the success of such
therapeutic strategies.
Glossary
Tumor propagating cells (TPCs)
Cancer cells responsible for tumor development and able to
propagate cancer upon serial xenotransplantation assays.
The tumor formed by TPCs is histologically heterogeneous
and phenocopies critical histopathological features of the
original tumor, such as infiltration/migration and multi-
lineage differentiation (including into non-TPCs). TPCs
may or may not express markers that are characteristic of
normal stem cells.
Cancer stem cells (CSC)
A specific case in which TPCs share some characteristics
with normal stem cells (such as surface markers that are
widely used to purify these cells). CSCs may have origi-
nated from normal stem cells or acquired expression of stem
cell markers during cancer progression.
Cancer initiating cell (CIC)
Also known as tumor initiating cell, it is the cell of origin of
cancer, meaning the precancerous cell in which the earliest
and crucial hit that lead to the growth of cancer occurred.
Cell plasticity
The ability of one cell to change its functional
characteristics.
Stem cells
Unspecialized cells characterized by two main properties:
self-renewal and the ability to differentiate into specialized
cell types.
Progenitor cells
Early progeny of stem cells, able to differentiate into one or
more cell types. Progenitor cells are not able to self-renew
indefinitely.
Acknowledgements ATV was supported by the Philip Majerus Fel-
lowship Fund. LFZB is supported by the NHLBI (4R00HL114732;
1R01HL137793) and grants from the V Foundation for Cancer
Research, the Edward Mallinckrodt Jr. Foundation, the AA&MDS
International Foundation, the CONCERN Foundation, the Department
of Defense Bone Marrow Failure Research Program (BM160054) and

the American Cancer Society. We thank Sapiens Scientific Illustrations
for the artistic work on Figs. 1–3.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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