Exploring the Role of Liver Cancer Stem Cells in Hepatocellular Carcinoma Metastasis

Abstract

Metastasis refers to disseminating cancerous tumors from their primary site to distant locations inside the body. Cancer cells must go through a sequence of events called the “metastatic cascade” to develop metastases. Each stage necessitates a unique functional alteration. Cancer stem cells (CSCs) play a crucial role in tumor metastasis, but understanding their dynamic behavior and regulating mechanisms remains incomplete. This review explores the influence of liver CSCs on the biological processes that drive the spread and growth of cancer cells, as described by the “metastatic cascade” concept. Liver CSCs can spread to other organs by undergoing epithelial-mesenchymal transition (EMT). This alteration in the microenvironment facilitates cellular dissemination, immune surveillance evasion, dormancy induction, and subsequent reactivation. To effectively prevent and treat advanced hepatocellular carcinoma (HCC) metastases, it is crucial to understand the heterogeneity and features of liver CSCs involved in these processes.

Full text

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Proceedings of Ancancer Research, 2024, Volume 8, Issue 6
hp://ojs.bbwpublisher.com/index.php/PAR
ISSN Online: 2208-3553
ISSN Print: 2208-3545
Exploring the Role of Liver Cancer Stem Cells in
Hepatocellular Carcinoma Metastasis
Maria Fatima1*, Maria Riaz2, Muhammad Amjad1, Muhammad Waqas2, Muhammad Hashim Raza2
1Institute of Chemistry, The Islamia University of Bahawalpur, Pakistan
2Institute of Biological Sciences, Khwaja Fareed University of Engineering and Information Technology, Rahim Yar Khan,
Punjab, Pakistan
*Corresponding author: Maria Fatima, mariafatima14112@gmail.com
Copyright: © 2024 Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), permitting distribution and reproduction in any medium, provided the original work is cited.
Abstract: Metastasis refers to disseminating cancerous tumors from their primary site to distant locations inside the
body. Cancer cells must go through a sequence of events called the “metastatic cascade” to develop metastases. Each
stage necessitates a unique functional alteration. Cancer stem cells (CSCs) play a crucial role in tumor metastasis, but
understanding their dynamic behavior and regulating mechanisms remains incomplete. This review explores the influence
of liver CSCs on the biological processes that drive the spread and growth of cancer cells, as described by the “metastatic
cascade” concept. Liver CSCs can spread to other organs by undergoing epithelial-mesenchymal transition (EMT). This
alteration in the microenvironment facilitates cellular dissemination, immune surveillance evasion, dormancy induction,
and subsequent reactivation. To effectively prevent and treat advanced hepatocellular carcinoma (HCC) metastases, it is
crucial to understand the heterogeneity and features of liver CSCs involved in these processes.
Keywords: Liver cancer; Stem cells; Hepatocellular carcinoma; Signaling pathway; Tumor microenvironment; Metastasis
Online publication: November 22, 2024
1. Introduction
Hepatocellular carcinoma is a cancer of the liver. Hepatocellular carcinoma (HCC) ranks as the sixth most
common form of cancer and is the third-highest contributor to cancer-related deaths [1]. For individuals with
early-stage solitary tumors, surgical intervention is the primary therapy for their treatment [2]. A study indicates
that patients who undergo resection have a 5-year survival rate of over 60% [3]. However, after five years, up
to 70% of these patients experience the tumor’s return [4]. Liver transplantation is often the optimal surgical
treatment option for patients with early-stage tumors of HCC [5] due to the high prevalence of liver disease [6]
and cirrhosis in these cases [7]. Globally, approximately 20%–40% of liver transplant recipients receive this form
of medication [8]. According to Asrani et al.’s study, the occurrence of recurrent HCC after liver transplantation

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is between 10% and 20% [9]. This rate may increase if the patient selection criteria are broadened [10]. After liver
transplantation, tumor recurrence is frequently associated with the spread of cancer to other organs outside
the liver [11,12], especially the lungs and bones [13]. Due to its high metastatic incidence, HCC’s poor prognosis
continues to be a significant concern [14]. Thus, there is an urgent need to gain greater knowledge of the
mechanisms behind HCC metastasis [15].
A range of cellular processes known as the “metastatic cascade” is necessary to develop metastases [16,17].
These processes include being able to spread from where they started, encouraging vascular invasion [18],
staying alive while moving through the bloodstream, going into and coming out of dormancy [19], making new
cell environments in secondary sites, starting angiogenesis, and avoiding the immune system of the host [20]. The
complicated process of tumor metastasis takes years to complete [21]. A tiny percentage of cells can spread to
other locations [22]. For these cells to become adept at starting the spread of metastatic illness [22], they must go
through a difficult metamorphosis and acquire a range of phenotypic features [23]. This particular subpopulation
of cells, which is incurable, still causes the majority of tumor metastasis [24]. Research indicates that cancer cells
with stem cell-like phenotypes are the ones that start the metastatic colonization of distant organs [25].
Cancer stem cells (CSCs) are a small group of stem-like tumor cells that can self-renew, differentiate,
and initiate tumor formation [26]. In the last two decades, scientific research has significantly increased focus
on identifying and understanding CSCs, particularly in the liver [27]. This has brought renewed optimism
for the diagnosis and treatment of HCC [28]. Scientists have discovered multiple markers for liver CSCs [29],
including OV6, CD24, ICAM-1, CD133, CD90, EpCAM, CD13, CD44, and K19 [30]. Multiple studies have
substantiated the correlation between liver CSCs [31] and the spread of HCC despite the precise mechanisms
remaining unidentified [32]. Gaining insight into the function of liver CSCs in advancing HCC cell metastasis
can potentially enhance the detection and treatment of metastatic HCC [33,34]. Liver CSCs are involved in all
stages of liver cancer is spread. This was done to learn more about liver CSCs’ critical role in the metastasis of
HCC and to develop new ways to treat the disease that work better.
2. The interplay of liver CSCs and EMT in HCC
Investigating and comprehending the molecular pathways involved in tumor metastasis consists of examinin
g the epithelial-mesenchymal transition (EMT) [5]. Epithelial cells undergo a biological process called EMT [35],
where they lose their characteristics as epithelial cells and acquire the characteristics of mesenchymal stem
cells [36]. Erroneously activating this transformation process in malignancies can have devastating effects as
the differentiated epithelial cells gain the ability to migrate and invade, thereby initiating the tumor metastasis
process [37]. Researchers have discovered that EMT drives many biological processes, extending beyond the
early stages of cancer cell invasion [36]. A growing body of research suggests that giving epithelial tumor cells
experimental EMT activation results in the transfer of stemness characteristics, including CSCs [38].
EMT in HCC cells invariably accompanies the acquisition of stemness, according to an increasing body
of evidence [39]. There is still some disagreement about the exact connection between EMT and HCC stem
cells, but most scientists agree that β1-induced EMT helps tumor cells, including HCC cells, change into CSC
phenotypes [40]. Different reports say that turning on Notch1 started the EMT process [41], which turned HCC
cells into cells that behaved like CSCs, which were controlled by HIF-1α [41]. It is noteworthy that the process
of inducing stemness promotes both stemness and the EMT. MHCC97-L cells’ CSC properties by artificially

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expressing the Oct4 and Nanog genes, which are linked to stemness [43]. After that, they went through EMT,
which made it easier for the tumor to move, invade, and spread, both in living things and in the lab setting [44].
Another study found that culturing CSC spheres in a specialized medium containing neural survival factor-1
enhanced their ability to undergo metastatic development [45], thereby promoting the process of EMT [46].
Nevertheless, specific experimental results question the correlation between stemness and EMT. For example,
cells that are negative for vimentin and positive for CD133 (vimentin-CD133+) [36] demonstrate a higher level of
aggressive metastasis compared to cells that are positive for both vimentin and CD133 (vimentin+CD133+). On
the other hand, HCC cells that are positive for vimentin and negative for CD133 (vimentin+CD133-) show EMT
traits but stem cells that are negative for vimentin and positive for CD133 (vimentin-CD133+) do not [47].
2.1. Liver CSC niche required for CSC maintenance and survival
The interaction between tumor cells and the tumor microenvironment (TME) leads to the development,
invasion, and spread of many malignancies, such as HCC [48,49]. The structure of the adjacent extracellular
matrix (ECM) plays a critical role in tumor advancement [50]. Various cytokines or growth factors, in addition
to alterations in tissue oxygen levels, facilitate communication between cancer cells, endothelial cells, stromal
fibroblasts, and immune cells [51]. The gathering of additional data reveals the presence of environmental factors
causing inflammation, such as TGF-1, chemokine, IL-17 lipopolysaccharide, IL-6 [52], and HGF, in the tumor
microenvironment of HCC, which also play a role in regulating stemness [53]. Either CSCs can sustain this
stemness or non-CSC cells can acquire it. Normal liver stem cells have the potential to transform into metastatic
liver CSCs in reaction to the inflammatory cytokine IL-6 [54].
Specific regions within the microenvironment that harbor CSCs as “CSC niches” [55]. Several malignancies
have observed the importance of interactions between CSCs [56] and their surrounding environments, or niches, in
facilitating cancer metastasis [57]. One type of extracellular protein complex called laminin-332 helps liver CSCs
keep their ability to grow new cells and protect them from chemotherapy when they’re not working [58]. Solid
tumors, particularly HCC, commonly exhibit a low-oxygen environment [59]. This environment is important
in the area where stem cells are located, as it increases HCC stemness by activating HIF-1α [60]. Hypoxia can
cause metastases to spread and invade more quickly [61]. This is related to the fast change of tumor cells into
liver CSCs, which can create their environment such as a scaffold [62] by releasing proteins interacting with
macrophages [63]. Reports suggest that HCC cells in low oxygen conditions generate the protein MYDGF, which
could potentially enhance the ability of liver CSCs to self-renew.
Table 1. Signaling pathways activated in hepatic stem cells [64]
Signaling pathway Role in normal liver development (species) Role in liver cancer development (source)
BMP Liver specification from endoderm (mouse) Differentiation of CD133+ CSC, (PLC/PRL/5, Huh7,
MHCC-97L)
FGF Liver specification from endoderm (mouse) Cell proliferation and angiogenesis, (HCC-1.2, HepG2,
Hep3B)
OSM Hepatocytic differentiation of hepatoblast (mouse) Differentiation of EpCAM+ CSC, (Huh1, Huh7,
primary HCC)
Wnt
Liver specification from endoderm (mouse), inhibition
of hepatic maturation (mouse),
biliary differentiation of hepatoblast (mouse),
Hepatocytic differentiation of hepatoblast (mouse)
Liver CSC self-renewal (Huh1, Huh7, SMMC7721),
cell proliferation (Hep3B)

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Table 1 (Continued)
Signaling pathway Role in normal liver development (species) Role in liver cancer development (source)
TGF-b Biliary differentiation of hepatoblast (mouse) Controversial (mouse, rat, Huh7, primary HCC)
JAG1/Notch Biliary differentiation of hepatoblast (mouse) Controversial (mouse, primary HCC)
IL-6/STAT3 Liver regeneration (mouse) Liver CSC maintenance (mouse)
HGF/c-Met Liver regeneration (mouse), hepatocyte, trans-
differentiation into biliary epithelium (mouse) Epithelial-mesenchymal transition (mouse)
2.2. Effect of liver CSCs-mediated ECM remodeling on metastasis of HCC
CSCs make lysyl oxidase (LOX), which creates an ECM that is dense and stiff. This stiff ECM is essential for
developing and preserving HCC’s CSC properties [65]. Changes in the stiffness of the hepatic CSC environment
may affect how CSCs renew themselves and what they are like as HCC metastasis spreads [66]. The ECM
experiences substantial alterations throughout the advancement of cancer [67] and plays a pivotal role in the
metastatic process [68]. The mechanical properties of the ECM [69] surrounding tumor cells undergo significant
alterations, particularly in terms of stiffness, as the cancer cells proliferate, infiltrate, and metastasize [70].
Metastatic HCC had tumor tissues that were stiffer than those of people who did not have metastatic HCC [71,72].
However, the correlation between CSC stemness and extracellular matrix stiffness in HCC is still a subject
of debate [73]. In HCC cells, enhanced matrix rigidity promotes stem cell growth. CD133+ liver CSCs create
a specialized matrix by altering the ECM [74], resulting in a soft spot. This soft spot matrix has the ability to
increase stemness maintenance, treatment resistance, and metastatic HCC cell spread. Still, studies have shown
that a stiffer matrix improves the features of CSCs [75] and lowers the number of times sorafenib-induced cell
death happens. Using a stiffer matrix significantly improved the characteristics of liver cancer stem cells (LCSCs)
compared to a softer matrix.
3. Relationship between liver CSCs and circulating tumor cells
Circulating tumor cells (CTCs) are cancerous cells that get out of primary tumors and into the bloodstream,
where they start metastases that spread to other parts of the body [76]. Previous studies have demonstrated
that CTCs possess the traits of CSCs [77]. However, there is a limited understanding of the characteristics of
circulating CSCs in HCC [78]. The CSC marker CD44 revealed the presence of CTCs in 71.4% of patients
diagnosed with HCC [45]. As a result, these patients had a significant number of CTCs that exhibited
characteristics of CSCs [79]. Most current methods for capturing CTCs depend on the detection of epithelial
cell adhesion molecule (EpCAM), which is also applicable to HCC [80]. People commonly use EpCAM as
a biomarker to detect liver CSCs. The stem cell-like properties of HCC EpCAM+ CTCs have been proven
by studying the presence of stem cell-related markers and their important ability to form tumors [81]. Several
studies have shown that monitoring and predicting the outcomes of patients with HCC relies on the quantity of
circulating CSCs.
4. Quiescence/dormancy liver CSCs in HCC metastasis
Clinical manifestations of HCC metastases typically occur several years after the removal of the initial tumor

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through resection or liver transplantation [6]. The metastasis originates from a specific group of cancer cells that
have spread and can remain inactive until becoming active again [21]. Upon immunological examination, LCC cells
exhibit characteristics similar to those of stem cells that are critical for their survival, ultimately leading to the
development of metastatic expansion under favorable conditions [82]. CSCs can evade immune system recognition
and attack through the release of diverse proteins or cytokines that regulate the immune response [83]. This enables
them to maintain a condition of quiescence and dormancy. As a result, the immune system is unable to identify
CSCs until they have spread and returned due to their transition into a dormant state.
Gaining insight into the molecular properties of dormant CSCs [84] and the mechanisms responsible for
their reactivation is crucial for developing effective therapeutic approaches to prevent cancer relapse and
metastasis [21]. Researchers have identified CD13 as a partially inactive marker for CSC in human liver cancer
cell lines and clinical samples [85]. It protects cells from apoptosis caused by genotoxic chemo/radiation stress
by stopping the cell cycle in the G0 phase and lowering the damage that reactive oxygen species (ROS) do to
DNA [86]. A subsequent study demonstrated that liver CSCs expressing CD13 could maintain a state of inactivity
and resistance to chemotherapy drugs by using tyrosine through aerobic metabolism [87]. A specific CSC niche
containing a high concentration of laminin-332 encloses side population (SP) CSCs [74]. This niche plays a
crucial role in keeping liver CSCs in a dormant condition.
5. The effect of liver CSCs on immune evasion
Immune evasion in HCC begins early and progresses gradually and consistently, peaking in middle-stage
II tumors [88]. Cancer cells, once isolated from the immunosuppressive milieu of the primary tumor, become
vulnerable to immune surveillance. In order to produce metastases, they must successfully avoid being destroyed
by the immune system [83]. CSCs elude the immune system, a crucial factor in preserving their capacity to generate
tumors [89]. This is accomplished by modifying the molecular expression of CSCs and reprogramming the immune
response. Research has shown the essential roles of natural killer (NK) cells in the primary immune response to the
development of cancer, specifically HCC [90]. Scientists have demonstrated that liver CSCs that express EpCAM
are resistant to the harmful effects of NK cells by increasing the expression of the CEACAM1 gene [91]. CD133+
liver CSCs possess a high proficiency in evading immune surveillance [79]. They achieve this by establishing a
connection with lymphatic endothelial cells and initiating IL-17A signaling [92]. Scientists have found that liver
CSCs can evade the body’s adaptive immune response by interacting with activated regulatory CD4+, CD25+,
and FoxP3+ T cells (Tregs) through a process known as paracrine signaling [93]. Additional investigation is
necessary to understand the immune evasion mechanism of liver CSC.
Table 2. Presents the surface markers that impact the signaling pathways, characteristics, and ability to resist
therapeutic medicines in LCSCs [85]
LCSs Phenotype of LCSCs Signaling involved in LCSCs Resistance to clinical drug
EpCAM
Cell signaling, metabolism, differentiation, cell
adhesion, metastasis, tumorigenesis, chemoresistance,
organogenesis, regeneration, self-renewal of Hep3B,
HepG2, Huh7, Huh1, and Dt1, Hepa1-6 cells
Activation of the Wnt
signaling pathway
Sorafenib
CD47 Self-renewal, tumor-initiating, tumorigenicity, and
chemoresistance (MHCC97L, PLC, and Huh7 cells)
Activation of the IL-6/STAT3
signaling pathway, and NF
Doxorubicin, Sorafenib

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Table 2 (Continued)
LCSs Phenotype of LCSCs Signaling involved in LCSCs Resistance to clinical drug
CD13+CD133+
Tumor initiation, chemoresistance, and anti-apoptosis
(Huh7 and PLC cells)
Reduction of ROS-induced
DNA damage and inhibition of
apoptosis
Doxorubicin, Fluorouracil
(5-FU)
CD13+CD90+
Tumor initiation, chemoresistance, and anti-apoptosis
(Huh7 and PLC cells)
Reduction of ROS-induced
DNA damage and inhibition of
apoptosis
Doxorubicin, Fluorouracil
(5-FU)
CD133
Tumorigenic, cell cycle progression, differentiation,
chemoresistance, and self-renewal (Huh7,
SMMC7721, PLC8024, PLC8024, HepG2, and
HCCLM3 cells)
Activation of AKT/PKB Doxorubicin, Fluorouracil
(5-FU) and Sorafenib
CD24
Metastasis, differentiation, self-renewal and
chemoresistance (MHCC97H, HCCLM3, PLC/PRF/5,
Huh7, and Hep3B cells)
Autophagy activation,
activation of AKT/mTOR
signaling pathway, and Notch1
signaling pathway
Cisplatin, Sorafenib
CD90
Tumorigenesis, metastasis, self-renewal and
chemoresistance (MHCC97L, PLC, HepG2, Hep3B,
primary HCC, and JHH-6 cells)
Activation of the mTOR
signaling pathway
Doxorubicin
CD13 Chemoresistance, tumorigenesis and self-renewal
(Huh7, PLC, and HepG2 cells)
Activation of ERK1/2
signaling pathway
Sorafenib, Doxorubicin, and
Fluorouracil (5-FU)
6. Metabolism reprogramming is crucial to maintain liver CSC stemness
Tumor development widely recognizes metabolic reprogramming as a significant characteristic [94]. This
reprogramming provides additional energy and essential components for cellular growth and compensates for
redox imbalances to ensure their eventual survival and spread to other parts of the body. CSCs change their
metabolism and energy regulation to adapt to a hostile tumor microenvironment (TME) [95] and ensure their
survival and maintenance of stem cell properties. Researchers have demonstrated that HCC cells can undergo
retro-differentiation into CSCs [27]. Metabolic reprogramming follows this process, involving alterations in
mitochondrial activity that lead to decreased membrane potential, reduced ATP generation [96], and increased
lactate synthesis. Liver CSCs control the characteristics of stemness by controlling the function of the
mitochondria involved in respiration [97].
For CSC stemness preservation, reprogramming amino acid metabolism is essential [98]. The research found
that HCC cells that were not responding to chemotherapy had a CSC phenotype [99], which means they had
a changed metabolism and were not metabolically active [100]. Unlike chemo-sensitive HCC cells, these cells
demonstrated independence from glucose and reliance on glutamine metabolism. Furthermore, maintaining liver
CSCs heavily relies on glutamine metabolism [101]. Specifically, targeting glutamine metabolism or the enzyme
glutaminase 1 (GLS1) can decrease the characteristics of CSCs in HCC [39]. These investigations demonstrated
that the viability and ability of liver CSCs to regenerate themselves relied on glutamine.
7. Liver CSCs promote angiogenesis
Solid tumors necessitate the formation of novel blood vessels to facilitate their growth and spread to other
body parts [102]. There is strong evidence that CSCs are linked to the growth of new blood vessels (tumor

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angiogenesis) in the area around the tumor [103]. The development and spread of cancer directly correlate
with these connections. Research has demonstrated that CSCs from various types of cancer, including
glioblastoma, ovarian cancer, lung cancer, and breast cancer [104], can transform into endothelial cells and
subsequently create fully functional new blood vessels. Researchers found an unfavorable prognosis for
HCC and elevated expression levels of liver CSC biomarkers associated with tumor angiogenesis [85].
Previous studies have shown that cancer stem-like sphere cells derived from HCC cells can transform into
endothelial cells. This transformation occurs regardless of VEGF and NOTCH signaling but relies on Akt
and IKK activation [105]. Liver CSCs that are positive for CD90 can affect endothelial cells and help make new
blood vessels (angiogenesis) in a way that allows the cancer to spread (pro-metastatic) [79]. Researchers have
demonstrated that CD133+ liver CSCs stimulate tumor angiogenesis through the signaling pathway involving
neurotensin, interleukin-8, and CXCL1 [53]. In liver cancer, new research has shown that Oct4, a transcription
factor linked to stem cells, helps control how CSCs change into cells that look like endothelial cells [106].
The results of this study indicate that liver CSCs primarily stimulate the formation of new blood vessels
(angiogenesis) by releasing signaling molecules to neighboring cells or transforming them into endothelial
cells. This process is believed to contribute to recurrence, spread to other organs (metastasis), and resistance to
treatments that target angiogenesis.
8. Current approaches for dissecting HCC heterogeneity
Prior methodologies for investigating tumor heterogeneity included microscopic analysis of tumor tissues
[107], utilization of cancer cell lines with diverse genetic and pathological profiles [108], immunohistochemistry
staining, and bulk RNA sequencing to scrutinize distinct cell populations within the tumor. By combining
cutting-edge technologies with clinically relevant translational models [109], it is important to effectively study
how different types of cells in the tumor bulk interact and behave [110]. These technologies include scRNA-seq,
spatial transcriptomic, pathway enrichment analysis, and whole genome sequencing [111]. This approach has the
potential to replicate the actual pathogenesis of HCC accurately.
8.1. Organoids
Organoids are three-dimensional tissue models created in a laboratory using stem cells [112]. These models resemble
the natural complexity of organs, including their biological, structural, and genetic characteristics [113]. Researchers
have used organoids in recent decades to explore the intricate nature of tumors [114], assess the efficacy of drugs,
and explore the mechanisms behind tumor formation [115]. This 3D model can be used to explore the cellular
heterogeneity within tumors, leading to a comprehensive understanding of the interactions among different
subpopulations in tumors [116]. The diverse population of LCSCs poses a significant challenge to achieving
a successful therapeutic outcome and contributes to the development of medication resistance [85]. Multiple
differentiation factors can be employed, such as dexamethasone, a Notch signaling inhibitor [117], and BMP
without Rspo1, to create healthy liver organoids from liver stem cells [118]. These factors are used to guide the
stem cells toward growing into organoids based on hepatocytes [119]. These organoids can be used to investigate
the transfer of stemness to non-stem-like, highly specialized hepatocytes. This enables us to gain insights into
the functions of stem cells and CSCs in the development of diverse tumors [120], as well as the mechanisms
underlying drug resistance in populations of diverse cells [121]. Recent research demonstrates the significant

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capacity of using organoids to investigate tumor heterogeneity [45], examining organoids derived from primary
and metastatic colorectal cancer (CRC) through transcriptome analysis and histology [122]. This investigation
showed the presence of both intra- and inter-tumoral heterogeneity in CRC [123]. These organoids made from
HCC patient tissues with different histories will be a useful way to study the processes that cause tumor
heterogeneity, especially the role of LCSCs in HCC.
8.2. Precision-cut liver slice
The Precision-Cut Liver Slice (PCLS) [124] is an ex vivo model created by slicing human liver tissues. PCLS
stands out due to its ability to preserve and maintain the multicellular histoarchitecture, spatial structural
relations [125], and genetic characteristics of the original cell populations and organs for a certain period in
a laboratory setting [126]. This makes it an excellent model for studying diverse subpopulations of HCC [127].
Researchers have widely employed phenotypic cell-based profiling to investigate medication response and
toxicity [128], elucidate fibrosis stages, and assess the effectiveness of anti-fibrotic medicines [129]. The PCLS
comprising both HCC and nearby normal liver tissues is the best way to test how well and selectively anti-
cancer drugs work on a group of cells with different histological features [130]. Intratumoral heterogeneity (ITH)
can be studied on its effects on treatment outcomes by looking at primary circulating tumor cells [131] from other
parts of the same tumor. Similarly, pre- and post-chemotherapy primary circulating tumor cells can serve as a
valuable model for investigating spatial and temporal variations and inferring treatment effectiveness [132].
8.3. Liquid biopsy
Liquid biopsy is becoming a viable substitute for tumor tissue biopsy [133]. The technology enables the
examination of CTCs [134], as well as nucleic acids such as circulating tumor RNA (ctRNA) and circulating
tumor DNA (ctDNA) [135], together with other tumor biomarkers present in the bloodstream and other bodily
fluids [136]. The main and/or secondary tumors release these materials, displaying their diversity [137]. Specifically,
the existence of a diverse population of CTCs [138] in the bloodstream suggests that there is variation in both the
physical characteristics and genetic makeup of the tumor [139]. Indeed, the analysis of the physical traits of CTCs
can provide valuable information for selecting appropriate treatments [140]. Furthermore, the number of CTCs [141]
and their observable traits can contribute to our understanding of the biology of metastasis and the reasons
behind medication resistance [142]. Patients with HCC can use spatial examination of CTCs [143] from various
blood vessels to predict the occurrence of metastases [144]. The result can provide important information about
the differences within a tumor and can potentially anticipate the spread of cancer to other parts of the body by
analyzing circulating tumor cells in their specific locations.
9. Conclusion
Metastasis is a complex tumor process that is cancer’s most lethal characteristic. Scientists have found that
a small group of treatment-resistant cancer cells called phenotypically CSCs, is responsible for the tumor
spreading to other organs, even though primary cancer has been painless for years. Therefore, it is imperative to
develop more effective treatments that can entirely eradicate these residual cells. Understanding the principles
that underpin the entire metastatic process, however, is critical for the development of effective metastase
therapeutics [145]. Metastasis is the process by which only a small number of cells inside a tumor can migrate

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from their initial position and disseminate to different areas of the body. Researchers have discovered a small
number of blood cells, known as CSCs, involved in this process. Various studies have demonstrated that
liver CSCs have a substantial influence on various aspects of HCC metastasis. These factors encompass the
dissemination of cancer cells through the circulatory system, the capacity to endure adverse circumstances,
and the establishment of metastatic locations. Liver CSCs can change into metastasis-initiating cells (MICs)
through various biological processes, such as EMT, interactions within the CSC niche, and changes to the ECM.
Subsequently, they can infiltrate the circulatory system while retaining their stem cell characteristics. Liver
CSCs can survive in the bloodstream or other challenging environments by entering a dormant state, remaining
inactive, and undergoing metabolic reprogramming. Liver CSCs exert a substantial influence on angiogenesis,
the biological mechanism responsible for the formation of metastatic lesions. This article presents a detailed
summary of the role of liver CSCs in different stages of the metastatic process. Understanding the role of liver
CSCs in regulating the dissemination of HCC lays the groundwork for developing innovative approaches and
therapies to prevent or control metastatic HCC.
Author contribution
Conceptualization: Maria Fatima
Validation: Maria Riaz, Muhammad Amjad, Muhammad Waqas
Investigation: Maria Fatima, Maria Riaz, Muhammad Amjad
Writing – original draft: Maria Fatima, Muhammad Waqas, Muhammad Hashim Raza
Writing – review & editing: all authors
Disclosure statement
The authors declare no conflict of interest.
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