REVIEW

published: 27 May 2021

doi: 10.3389/fonc.2021.687594

The Use of Zebrafish Xenotransplant

Assays to Analyze the Role of
lncRNAs in Breast Cancer
Cecilia Zampedri 1, Williams Arony Martı́nez-Flores 2 and Jorge Melendez-Zajgla 1*
1 Functional Genomics Laboratories, Instituto Nacional de Medicina Genomica, Mexico City, Mexico, 2 Departamento de

Ecologı´a de Agentes Patógenos, Hospital General “Dr. Manuel Gea González, Mexico City, Mexico

Breast cancer represents a great challenge since it is the first cause of death by cancer in
women worldwide. LncRNAs are a newly described class of non-coding RNAs that
participate in cancer progression. Their use as cancer markers and possible therapeutic
targets has recently gained strength. Animal xenotransplants allows for in vivo monitoring
of disease development, molecular elucidation of pathogenesis and the design of new
therapeutic strategies. Nevertheless, the cost and complexities of mice husbandry makes
medium to high throughput assays difficult. Zebrafishes (Danio rerio) represent a novel
Edited by: model for these assays, given the ease with which xenotransplantation trials can be
Wenwen Zhang,
Nanjing Medical University, China
performed and the economic and experimental advantages it offers. In this review we
Reviewed by:
propose the use of xenotransplants in zebrafish to study the role of breast cancer lncRNAs
Hamed Shoorei, using low to medium high throughput assays.
Birjand University of Medical Sciences,
Iran Keywords: lncRNAs, breast cancer, xenotransplant, zebrafish, non-coding RNAs
Evelien Schaafsma,
Dartmouth College, United States
Yasuhito Shimada,
Mie University, Japan INTRODUCTION
*Correspondence:
Jorge Melendez-Zajgla
Breast Cancer
jmelendez@[Link] Breast cancer is the most frequent malignancy in women worldwide and the leading cause of
malignancy-related death (1). Whereas early breast cancer is considered curable in 70 to 80% of
Specialty section: patients, metastatic disease is considered incurable with our current therapeutic options. The tumor
This article was submitted to characteristics that lead a breast cancer to become metastatic are not fully understood; however,
Cancer Genetics, great efforts are currently being made to elucidate the early mechanisms involved in metastasis, and
a section of the journal find early molecular markers and new therapeutic targets related to progression. This malignancy is
Frontiers in Oncology a heterogeneous group of diseases that deserves our attention and focus on finding new markers that
Received: 29 March 2021 can better discriminate among different subtypes and/or to individualize molecular characteristics
Accepted: 04 May 2021 of these tumors, allowing for a more reliable prognosis and precise treatments (2). Gene expression
Published: 27 May 2021 profiling of breast cancer has improved the understanding of breast cancer’s heterogeneity on the
Citation: genomic level, challenged the current classification of breast cancer, served as an important
Zampedri C, Martínez-Flores WA prognostic indicator; and most important, begun to guide the treatment in women with early
and Melendez-Zajgla J (2021)
breast cancer (2). It is of pivotal importance to find noninvasive biomarkers with high sensitivity
The Use of Zebrafish Xenotransplant
Assays to Analyze the Role
and specificity, which can be used for breast cancer detection at an early stage and monitor of
of lncRNAs in Breast Cancer. response to therapy (3). Recent advances in technologies, such as microarray and high-throughput
Front. Oncol. 11:687594. sequencing, represented a deeper understanding of molecular biology, especially long noncoding
doi: 10.3389/fonc.2021.687594 RNA (lncRNA).

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Zampedri et al. Zebrafish Models for lncRNAs Studies

THE ROLE OF lncRNAs IN proteins, DNA and several RNA species (mRNA, small RNA
CANCER PROGRESSION and even other lncRNAs).
LncRNAs mediate the interaction between proteins, RNAs,
Through a varied repertoire of interactions, lncRNAs are and lipids, not only in physiological situations but also during
involved in health and disease, through a diverse array of cancer progression (9). These interactions regulate two key
processes such as differentiation and embryonic development cancer processes: Cancer Stem Cells (CSCs) maintenance and
(4–6), innate immunity (7, 8) and cancer progression (9, 10). The the tumor cells´ interaction with their microenvironment. These
world of lncRNAs is constantly growing; today, several databases characteristics give lncRNAs important features as molecular
have information on hundreds of thousands of lncRNAs from markers for the diagnosis, prognosis (27), and prediction (28) of
human and other species (11, 12). Several studies revealed that cancer. Additionally, circulating lncRNAs have great potential as
lncRNAs are key to cancer initiation and progression. Although molecular markers for non-invasive detection since variations in
the biological function and molecular mechanisms of lncRNAs lncRNA expression can be detected in a serum or body fluid
are not known in detail, many lncRNAs are expressed sample, avoiding invasive approaches such as tumor tissue
abnormally in cancer. biopsies (29). This advantage makes lncRNAs a promising tool
The expression dynamics of lncRNAs are finely controlled by on the road to early cancer detection and drug design.
epigenetic, transcriptional and post-transcriptional regulation.
The characteristic tissue-specific expression and low transcription lncRNAs Involved in Breast Cancer
levels of lncRNAS are epigenetically regulated. Transcription of There is currently an extensive list of lncRNAs associated with
non-coding RNA genes is regulated by central transcription breast cancer with either oncogenic or tumor suppressor
factors that also regulate nearby coding genes; however, some functions, according to their roles in promoting or inhibiting
lncRNA may allow their transcription to be unsynchronized proliferation, metastasis, invasion, apoptosis, autophagy,
with their near mRNAs. Moreover, lncRNAs are also regulated inflammation, stemness, and drug resistance (30).
at the post-transcriptional level, including modulation by
miRNAs (13). lncRNAs in Breast Cancer Metastasis
lncRNAs are involved in a large number of molecular regulatory Plenty information has recently been generated for some
mechanisms such as chromatin dynamics, gene expression, growth, lncRNAs such as MALAT1, HOTAIR (31) and NEAT1 (32)
differentiation, and development. Consequently, they participate in describing their breast cancer progression and metastasis roles;
the maintenance of homeostasis, and thus, in several pathologic and although knowledge about lncRNAs and their association
states. These molecules are transcribed at sizes ranging from 200 with breast cancer metastasis is constantly growing (Table 1),
nucleotides to several thousand base pairs with little or no much remain to be elucidated. In particular, there is a paucity of
translation potential (14). lncRNAs comprise non-coding RNAs information regarding the molecular mechanisms by which
(lncRNAs) previously annotated as antisense transcripts, intronic lncRNAs exert their function and clinical relevance. One of the
transcripts, processed pseudogenes, lncRNAs (long intergenic non- first mechanisms required to initiate metastasis is the epithelial-
coding RNAs), and coding-transcript isoforms that do not translate mesenchymal transition (EMT) (48), which paves the way for the
to a functional protein (15–18). These RNAs are transcribed in the migration and invasion of cancer cells from the primary tumor
cell nucleus and then transported to the cytoplasm to be edited and site to distant secondary sites (49). More than a dozen lncRNAs
directed to their final destination to fulfill the function, either in the are known to be involved in the EMT of breast cancer cells.
cytoplasm, nucleus, local organelles (cell-autonomous function) or
outside the cell (non-cell-autonomous function) (18). lncRNAs Are Involved in Apoptosis
On vertebrates, lncRNAs are transported from cells into Avoidance During Breast
interstitial spaces and body fluid through exosomes, similar to Cancer Progression
lipids, proteins, DNA, and mRNA (19, 20). Secreted exosomes It is clear that lncRNAs are involved in a wide range of biological
circulate in different fluids and can be internalized by and physiological processes during breast cancer progression.
neighboring cells (in autocrine and paracrine communication) One of these is regulated cell death, in particular apoptosis.
or distant cells (in endocrine communication). They can also lncRNA-Zfas1 is an antisense of the 5′end of the gene encoding
be transferred from one organism to another, thus facilitating the Zfas1 protein, which is localized to the ducts and alveoli of
genetic and epigenetic information exchange between the mammary gland. Deletion of lncRNA-Zfas1 in breast cancer
organisms (21). cells resulted in increased cell proliferation with a concomitant
LncRNAs act at various gene regulation levels, e.g., reduction of Zfas1 expression (50). Thus, Zfas1 is a novel and
modulating methylation at the chromatin level (22, 23) or potential suppressor of breast cancer.
regulating genes through association with activator or repressor LncRNA-Smad has recently been identified as adjacent to the
complexes at the transcriptional level (23, 24). They also mouse Smad7 gene (51). LncRNA-Smad7 expression is induced
participate in processes of splicing, transport, translation and by TGF-beta in all mammary gland epithelial cells and breast
mRNA decay, as is the case of the versatile lncRNAs MALAT1 cancer cell lines (51). Deletion of this lncRNA neutralized the
(17, 25, 26). In summary, lncRNAs fulfill the functions by their antiapoptotic function of TGF-b. This finding suggests a
molecular interaction with other biomolecules, including tumorigenic role of this lncRNA. LOC554202 is an additional

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TABLE 1 | lncRNAs involved in breast cancer metastasis.

lncRNA Function Cancer Reference

ANCR Tumor suppressor (33)

NKILA Tumor suppressor (34)
XIST Tumor suppressor (35)
Linc00052 Tumor suppressor (36)
NEAT1 Oncogenic (37)
Linc-ROR Oncogenic (38)
UCA1 Oncogenic (39)
TINCR Oncogenic (24)
BORG Oncogenic (22)
LincIN Oncogenic (40)
Lnc015192 Oncogenic (23)
LINC01638 Oncogenic (41)
ARNILA Oncogenic (42)
Lnc-BM Oncogenic (43)
MALAT1 Tumor suppressor and oncogenic (44, 45)
HOTAIR Tumor suppressor and oncogenic (31, 46, 47)

lncRNAs that have been linked to apoptosis repression through suppressing the TGF-b-induced tumor metastasis in breast
interaction with mir-31 in triple negative breast cancer (52). We cancer (34).
envision that there are still much to learn about the role of STAT3 is a component of another important pathway that
lncRNAs in the regulation of cell death of breast cancer cells. plays a role in inflammation during breast cancer progression,
and several lncRNAs (e.g., HOTAIR and Lnc-BM) participate in
lncRNAs and Autophagy in Breast Cancer this process (43, 46). In breast cancer cells, Lnc-BM increased the
Recent studies have shown that the regulation of autophagy is STAT3-dependent expression of ICAM1 and CCL2, which
involved in the progression and recurrence of cancer (53), and in regulated vascular co-option and recruitment of macrophages
the resistance of breast tumors to chemotherapy drugs (54). So, it in the brain, respectively (43).
is not surprising that lncRNA could play a role in the regulation
of autophagy in breast cancer cells (55). For example, recent The Role of lncRNAs in the Tumor
work has identified an autophagy-related lncRNA prognostic Microenvironment Crosstalk in
signature (ALPS) model composed of five autophagy-related Breast Cancer
lncRNAs (MAPT-AS1, LINC01871, AL122010.1, AC090912.1, The tumoral microenvironment (TME) is a complex biochemical
AC061992.1). These results suggested that the autophagy-related and physiological system involved in tumorigenesis and
lncRNAs are clinically valuable prognostic biomarkers in breast metastasis (61–63). It comprises the cancer cells, extracellular
cancer (56). matrix, vasculature, non-cancer cells and the tumor’s acidic and
hypoxic microenvironment. The cellular component consists of
Role of lncRNAs in Inflammation During cancer-associated fibroblasts (CAFs), adipose cells, endothelial
Breast Cancer cells, cancer stem cells (CSCs), infiltrated immune cells such as T
It has recently become widely accepted that the immune system lymphocytes and natural killer cells (NKs), myeloid-derived
can prevent tumor growth and promote it, through processes suppressor cells (MDSCs), and tumor-associated macrophages
grouped in three phases: elimination, equilibrium and escape (TAMs) (64–66). There is evidence that lncRNAs are involved in
(57, 58). Elimination is achieved through the identification and the communication between tumor and non-tumoral cells
destruction of transformed cells by tumor-inhibiting required to induce or maintain cancer hallmarks such as
inflammation. This phase is characterized by the infiltration of proliferation, migration, and metastasis.
cells of the innate and adaptive immune system. The escape One of the best-known examples of the relationship of lncRNAs
phase is maintained by tumor-promoting chronic inflammation, in the communication between tumor microenvironment and
mainly involving immunosuppressive cells (58). tumor cells is the HOTAIR lncRNA. In breast cancer, TGF-b1
NF-kB is a family of proinflammatory inducible transcription secreted by CAFs up-regulates HOTAIR expression to promote
factors that are involved in breast cancer progression (59). epithelium- mesenchyme transition (EMT) and metastasis (67).
Several lncRNAs play pivotal regulatory roles in the NF-kB HOTAIR inhibits miR-7 in CSCs of MCF-7 and MDA-MB-231
pathway. LncRNA NKILA was first found up-regulated by the breast cancer cell lines and thus promote the overexpression of
inflammatory cytokine TNF-a through the NF-kB pathway in SETDB1, STAT3, c-Myc, twist, and miR-9 (46) and repression of
breast cancer. NKILA could directly bind to the NF-kB/IkB E-cadherin (46, 68) to the benefit of the EMT process. HOTAIR
complex and inhibit NF-kB signaling from suppressing breast also contributes to EMT through regulation of VEGF, MMP-9, b-
cancer metastasis (60). In another report, NKILA was shown to cantenin and Vimentin (69). Also, in breast cancer HOTAIR up-
be up-regulated by TGF-b to block NF-kB signaling, thereby regulates SNAIL expression, as a master regulator of the EMT

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Zampedri et al. Zebrafish Models for lncRNAs Studies

pathway (31). HOTAIR also mediates the establishment of the CCL2 expression, and mediate macrophage recruitment to the
SNAIL/HOTAIR/EZH2 tripartite complex by inhibiting the brain and consequently metastasis. lnc-BM and JAK2 promote
expression of epithelial genes (such as HNF4a, HNF1a, and E- BCBMs by mediating communication between breast cancer
cadherin) through chromatin remodeling in favor of EMT (70). cells and the brain microenvironment. Thus, lnc-BM could be
Another essential component of the stroma required for EMT a promising therapeutic target for invasive breast cancer (43).
and subsequent metastasis is tumor vasculature. There is clear LncRNAs are a diverse set of molecules that can perform their
evidence that dysregulation of a group of lncRNAs can trigger functions intracellularly, travel free in the extracellular matrix,
changes in endothelial cells that favor angiogenesis and affect distant cells’ function and even be transported by exosomes
metastasis of breast cancer cells. For example, the lncRNA during intercellular communication. Tumor-derived exosomal
NR2F1-AS1 promotes breast cancer angiogenesis by activating lncRNAs affect the TME by generating changes in the transferred
the IGF-1/IGF-1R/ERK pathway (71). Similarly, overexpression cells, E.g. stromal cells, endothelial cells, macrophages, and
of MEG3 suppresses breast cancer angiogenesis through the mesenchymal stem cells, leading to induction of proliferation,
AKT pathway (72). M2 macrophage-induced lncRNA PCAT6 angiogenesis and metastasis (85, 86).
facilitates angiogenesis of triple-negative breast tumors through
modulation of VEGFR2 (73). lncRNAs in Breast Cancer
CSCs are a subpopulation of cancer cells that can self-renew Drug Resistance
and proliferate limitlessly. They may be responsible for cancer Besides its involvement with classic cancer hallmarks, a group of
initiation, progression, and even treatment resistance (74). lncRNAs has also been linked to drug treatment resistance. The
Several lncRNAs act by modulating the self-renewal and expression of a diverse array of lncRNAs changes dynamically in
differentiation of CSCs, such as lncH19 and HOTAIR. LncH19 response to various drugs contributing to anti-tumor drug
acts as a lncRNA sponge for miRNA let-7, inhibiting its function resistance through various mechanisms, such as cell cycle
and favoring the maintenance of CSCs in breast cancer (75). arrest, inhibition of apoptosis, DNA damage repair (87–89),
HOTAIR, on the other hand, also regulates the self-renewal of EMT (90), transport and internalization of drugs by cancer
CSCs in breast cancer, inhibiting miR-34a and thus positively cells (91, 92), and drug metabolism. lncRNAs involved in
regulating Sox2 (76). Interestingly, lncRNAs can also modulate breast cancer cell drug-resistance are UCA1 in doxorubicin
the development, activation and differentiation of T cells, which resistance (93), PANDA in anthracycline resistance (94), ARA
have both tumor-promoting and tumor-suppressive functions in adriamycin resistance (95), CCAT2 in 5-fluorouracil (96), and
(77). Regulatory T cells (Treg) are a subset of CD4+ T BCAR4, HOTAIR, and M41 in tamoxifen resistance (97–99).
lymphocytes that contribute to the inhibition of anti-tumor Since lncRNAs aberrant expression is a marker of drug resistance
immunity of the TME (78, 79). The lncRNA SNHG1 promotes (100), they are potential targets of new therapeutic strategies.
Treg differentiation, and the knockdown of this long noncoding
lncRNA inhibits Treg differentiation through increased
expression of miR-448 and indoleamine 2,3-dioxygenase (IDO)
inhibition, preventing immune escape in breast cancer (80). ZEBRAFISH XENOTRANSPLANTS TO
LncRNAs also modulate immunosuppression and cancer STUDY THE ROLE OF lncRNAs IN
progression through the regulation of ROS (reactive oxygen BREAST CANCER
species), NO (nitric oxide), and ARG1 (arginase 1) production
in MDSCs. MDSCs are generated in the bone marrow and have Although studies in cancer cell lines have advanced our
been shown to promote EMT and play an important role in knowledge of lncRNAs functions at the molecular level, the use
cancer progression by suppressing the immune response (81, 82). of animal models provides a rich context in which to investigate
TAMs are also key players in cancer progression through the phenotypic impact of these molecules in the breast cancer.
invasion and metastasis regulation. Two functional types of The involvement of lncRNAs in the breast cancer tumor
macrophages have been identified, classically activated phenotype can be modeled in vivo by genetic modifications in an
macrophages (M1) and alternatively activated macrophages animal, altering the expression of a lncRNA and studying the
(M2) (65). M1 macrophages participate in the Th1-type effects on cancer development. However, breast cancer´s
inflammatory response and have anti-tumor activity, and M2 multigenic and multifactorial nature requires an integrative
macrophages are anti-inflammatory macrophages and have a approach in which the genetic landscape that drives the
proto-oncogenic role (65, 83, 84). Recently, several studies have development of the disease is present.
shown that lncRNAs can modulate M2 macrophage polarization Xenotransplantation, which is generated by implanting
and by this induce tumor cell migration and invasion in several human tumor cells into an animal host, allows the study of the
types of cancer. The lncRNA associated with breast cancer brain effects of altering a particular gene in the development of breast
metastases (BCBMs), lnc-BM, was found to be overexpressed in cancer through genetic manipulation of human cell lines before
breast cancer cells, and associated with the induction of brain transplantation. Mouse xenotransplants were the first to be used,
metastasis in murine models (43). In breast cancer, lnc-BM but zebrafish have recently emerged due to the experimental,
increased JAK2 kinase activity to mediate oncostatin M- and economic, and visualization advantages they offer. In recent
IL-6-triggered STAT3 phosphorylation, promote ICAM1 and years, several breast cancer cell lines have been successfully

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Zampedri et al. Zebrafish Models for lncRNAs Studies

xenotransplanted in zebrafish, such as MDA-MB-468 (74), temperatures close to human physiological ones. These
MDA-MB-231 (101–104), MDA-MB-435 (105, 106), MDA- characteristics make the zebrafish an excellent in vivo model to
MB-23 (102), HCC1806 (101), MCF-7 (36, 107–109) and BT- visualize the tumor cell behavior and interactions with the
474 (106) among others. These experiments allowed the host microenvironment.
exploration of the participation of various genes in pathways In addition to facilitating in vivo assays related to the breast
related to proliferation-tumorigenesis, apoptosis (109), tumor itself, zebrafish help study functional aspect related to
macrophage-mediated tumor cell invasion (104), migration- particular molecules such as lncRNAs in breast cancer hallmarks.
metastasis, angiogenesis, drug resistance, stem cell maintenance Xenotransplantation of breast cancer cell lines in zebrafish makes
(106) and tumor microenvironment crosstalk (102, 103). it possible to study human lncRNAs’ role in the tumor
Zebrafish xenotransplantation represents a step forward in phenotype and microenvironment, giving a comprehensive in
modeling the complexity of breast cancer tumors, and the vivo perspective of the functions of this molecule.
involvement of a particular gene in each of the events that Zebrafish xenotransplant facilitates the study of signaling
accompany cancer, as cells are implanted into a living mechanisms involved at the whole organism level during cancer
organism in which many types of dynamic interactions can initiation and progression. Furthermore, there is significant
occur. Xenotransplanted cancer cells do not depend on the conservation of oncogenes and tumor suppressor genes between
artificial addition of nutrients, serum, cytokines, and growth zebrafish and humans, so the data obtained from zebrafish are
factors. In zebrafish, with all functional organs, tumors can relevant to humans (115). The xenotransplantation platform in
engage in both local and systemic cell-cell interactions, shaping zebrafish is also helpful for drug discovery in the context of
tumor progression. These interactions occur between tumor and breast cancer research (116). Zebrafish cell xenotransplantation
host and vice versa, with long-distance communication, allowing studies have the advantage of maintaining the effects of the
recapitulation of cancer features such as cell migration, invasion, microenvironment in cell communication and cancer progression,
metastasis, angiogenesis, and immune evasion that are not even when there are inter species differences.
possible to observe in vitro. When breast cancer tumor cells Zebrafish present ideal characteristics that allow multiple
are implanted, many different zebrafish cells are recruited to the statistically robust experiments to be performed simultaneously;
tumor site following tumor instructions (102, 103). The zebrafish however, the zebrafish xenotransplantation platform is not
xenotransplantation model allows simultaneous single-cell without limitations. On the one hand, the lack of an adaptive
resolution monitoring of tumorigenesis at various steps in vivo, immune response is beneficial for initial transplantation and
including tumor vascularization, localized tumor growth, injection, but could become a limitation for translation of
tumor invasion, and micrometastasis formation. Zebrafish findings, as adaptive immune cells may play vital roles in
xenotransplantation of breast cancer cells enabled the discovery promoting or inhibiting breast cancer progression and the
of a new mechanism of metastatic niche formation, and the roles effects of some treatments (117, 118).
of macrophages in this process were described. The experimental The zebrafish and human genomes are 70% similar based on the
advantages offered by zebrafish also allowed the discovery that conservation of individual genes, including cancer-related coding
physiological migration of neutrophils controls tumor invasion and non-coding genes. However, zebrafish are not mammalian, so
by conditioning the collagen matrix to facilitate the metastatic some important pathways in breast cancer tumor development are
niche (102). absent, including BRCA1, p16 (CDKN2A), Leukemia Inhibitory
Finally, drug sensitivity profiling of breast cancer cells using Factor (LIF), oncostation M (OSM) and interleukin 6 (IL6) (119).
the zebrafish xenotransplantation model allows the assessment of These absent pathways pose several challenges when studying the
pharmacokinetics, pharmacodynamics and toxicity in a whole functions of these “missing” genes or the pathways in which they
living organism, and in a short time. In vivo testing has great play a role. Furthermore, when foreign tissues and cells are
advantages over in vitro assays. E.g., to produce in vivo introduced into fishes, there is no guarantee that all molecular
phenotypes, compounds must be absorbed, reach targets, mechanisms linking the recipient tissue and xenograft are fully
circumvent elimination, and cannot be too toxic, otherwise the conserved, which could affect interactions between host cells and the
animal will not survive. The complexity of in vitro models is cancerous xenograft. This issue is especially relevant to the study of
given by the experience of the investigator, whereas in in vivo breast cancer as there is no orthotopic site in fish. However, it may
models, the complexity is built according to the dynamic be possible to “add” the necessary cells or growth signals to mitigate
instructions and signals of the tumor itself. Zebrafish this problem during xenotransplantation, or to “humanize” the fish
xenotransplantation also allows in vivo evaluation at the single by creating transgenic animals that express appropriate human
cell level of the cell autonomous and non-cell autonomous effects growth factors, receptors and/or cytokines, as has been done in
of a drug on the different hallmarks of cancer (110). mice (120).
There are several methodological advantages for using zebrafish, Zebrafish offers two options for cancer modeling by
such as their rapid and external development, the transparency of xenotransplantation of breast cancer cell lines or patient-derived
their embryos (111), the availability of fluorescent cell reporter lines tumor cells by microinjection, (Figure 1). In 48 hpf (hours post-
(112), the ease of genetic manipulation (113), and pharmacological fertilization) embryos, into the yolk sac, duct of Cuvier (common
approaches (114). Moreover, its wide range of growth temperatures cardinal vein), caudal vein, or perivitelline space. In adults, into the
that allows xenotransplantation experiments to be carried out at intraperitoneal cavity, (Figures 1A–C). Either option may result in

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FIGURE 1 | Comparison between xenotransplantation assays in zebrafish embryos and adult animals. (A) Common sites of injection. Shown are the most
commonly used injection sites for xenotransplantation of a zebrafish in two different stages of development. Left: 48 hpf Stage. The yolk sac is the most common site
of injection, but hindbrain ventricle, caudal vein; previteline space and duct of cuvier can be used also. Right: Juvenile Adult. The majority of xenografts occur within
the intraperitoneal cavity, and hinbrain ventricle can be used also. (B) Xenotransplantation of cancer cell lines and (C) patient-derived cells can be performed in both
embryos and adults. (D) Xenotransplantation allows evaluating the rate of tumor formation, metastasis and angiogenesis. (E–J) Advantages and disadvantages of
xenotransplantation in embryos versus adult fish assays.

tumor masses, which induce a neo-vascular response around the compatibility for microscopic imaging, and drug screening potential
tumor and consequent migration and metastasis, (Figure 1D). taking advantage of their small size, (Figures 1E–G) (121).
Embryo assays are the top choice in most work, given their On the other hand, adult animal assays are ideal for studying
methodological advantages for fast results, low-cost experiments, human physiological temperature-dependent characteristics

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Zampedri et al. Zebrafish Models for lncRNAs Studies

such as tumor growth rate or determining the dose using oral expression of lncRNAs in xenotransplanted cells, the zebrafish
gavage, (Figures 1H–J) (121, 122). xenotransplantation model could provide valuable information
The use of fishes with fluorescent vasculatures such as Tg(Fli : on the participation of lncRNAs in this complex process. As
EGFP) or VEGFR2:G-RCFP allows the visualization of previously stated, zebrafish xenotransplantation models are
angiogenesis in vivo (123–125). In xenotransplantation, the efficient for providing information about several breast cancer
zebrafish provides the necessary signals for the transplanted hallmarks, e.g. tumor progression, angiogenesis, spread,
cells to integrate into the organs, migrate, proliferate and metastasis or drugs response, revealing the existence of
interact with the zebrafish microenvironment (126, 127). interactions between cancer cells and cellular and non-cellular
Xenotransplantation has evolved as one of the most valuable components of the host inter-species microenvironment.
strategies to study breast cancer’s discrete aspects, as evidenced Xenotransplantation assays in zebrafish have shown that it is
by the growing volume of scientific publications on this subject possible to investigate the mechanisms and biological
over the last 15 years, (Supplementary Table 1). Xenotransplants implications of tumor-host cell crosstalk. A clear example is the
of breast cancer cell lines in zebrafish allow rapid in vivo testing molecular interaction between breast cancer cells and zebrafish
of coding and non-coding genes, pathways involved in host cells. They allow the recapitulation of cancer hallmarks such
tumorigenesis, migration, angiogenesis, or screening for new as angiogenesis in CXCR4 chemokine signaling across zebrafish
drugs. Moreover, in recent years, in vivo modeling by zebrafish and humans in xenotransplantation experiments. Tullota et al.
xenotransplantation revealed important information on the role showed that human cancer cells expressing CXCR4 responded
of some lncRNAs in breast cancer hallmarks (36, 107, 128, 129) to the zebrafish Cxcl12 ligand, and zebrafish cells expressing
(Supplementary Table 1, blue data). We recently uncovered the Cxcr4 migrated to the human CXCL12 ligands (130). On the
role of lncRNA-HAL in promoting the stemness in breast cancer other hand, substantial evidence supporting the molecular
cells; the action of lncRNA LINC00052 in the suppression of interrelationship between human and zebrafish, and involving a
migration, as well as the role of LncMat2B in the induction of lncRNA, is the resistance to tumor formation of a zebrafish
breast cancer cell invasiveness using in vivo xenotransplantation knockout of Thor (THOR-/-) (an oncogenic lncRNA conserved
assays in zebrafish (36, 107, 129). Likewise, Peperstrate et al. between zebrafish and human) after xenotransplantation with
showed that lncRNA H19 increases breast cancer cells’ invasive NRAS61K melanoma cells (131).
capacities in xenografted transgenic zebrafish models (128). In The interaction between cancer cells and zebrafish immune
this work, breast cancer cell lines were modified to alter the cells was discovered by experiments transplanting cancer cells
expression of lncRNAs, then stained or labeled with reporter directly into the blood circulation through the duct of Cuvier or
genes and transplanted into zebrafish embryos. Tumor cells that the perivitelline space. Neutrophil and macrophage infiltration
migrated to distant sites within the fish embryos, and the growth surrounding the tumor was observed by using transgenic
of the transplanted mass, or the development of tumors at zebrafish strains with labeling in immune system cells (Tg
secondary sites, were related to the different hallmarks of cancer (mpx:GFP)i114 (132) in neutrophils, Tg(mpeg1:eGFP)gl22 (133)
to infer the involvement of the lncRNA ones in these events. and Tg(mpeg1: mCherry)UMSF001 (134) for macrophages (135);
In conclusion, zebrafish xenotransplants allow the in vivo and the interaction of cancer cells with endothelium using vessel-
functional study of the involvement of lncRNAs in breast cancer tagged strains such as Tg(fli:eGFP)y1 (112), Tg(flk1:eGFP)s843
in short timescales. (136) and Tg(flk1:mCherry) (137). The zebrafish immune cells
are recruited and localized near the breast cancer cells at the
primary tumor growth and secondary micrometastasis sites.
ZEBRAFISH XENOTRASPLANT FOR THE Also, it was observed that the non-disseminated tumor cells
STUDY OF lncRNAs IN BREAST CANCER associated with the endothelium of the duct of Cuvier and
TUMOR MICROENVIRONMENT remodeled it, forming new vessel-like structures and then
forming functional vasculature. Subsequently, by knocking
One of the most attractive advantages of using an in vivo model down the expression of myeloid differentiation transcription
for the study of breast cancer as a complement of an in vitro factors in zebrafish, the suppression of tumor vascularization,
model is the possibility of representing the complex context of invasion and micrometastasis was observed (102), showing the
the tumor microenvironment. dynamic interaction of zebrafish immune cells with human
As discussed above, lncRNAs are involved in a various breast cancer cells.
molecular pathways related to communication from the tumor On the other hand, using vasculature-tagged reporter strains,
microenvironment to the tumor cells themselves to promote cancer cells injected into the yolk of zebrafish embryos were
cancer establishment and progression. The zebrafish shown to interact with the endothelium of the embryos blood
xenotransplantation platform for breast cancer will facilitate vessels, migrate through them and form secondary tumors (138).
the discovery of functional information of lncRNAs in the The induction of angiogenesis mediated by the interaction
complex process of communication with the tumor between zebrafish immune cells and transplanted human
microenvironment. Zebrafish xenotransplantation allows breast cancer cells was also confirmed through the positive
visualization of in vivo events in a real time and cellular level, correlation between the expression levels of vascular
such as cell-cell interaction. Together with assays to alter the endothelial growth factor A (VEGFA) secreted by transplanted

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Zampedri et al. Zebrafish Models for lncRNAs Studies

breast cancer cell line, the number of immune cells recruited modeling using PDX, such as the embryo transparency, the
around the tumor, the interaction of macrophages with the possibility of real time visualization, and the short time to obtain
vessels, and the induction of new vessel formation around the results (149). Patient derived xenotransplantation in zebrafish
tumor in zebrafish (103). (zPDX) consists of obtaining a very small fraction of the patient’s
In recent work, it was observed that one of the mechanisms by tumor by biopsy and transplanting it into zebrafish directly or after
which the interaction between host cells and tumor cells occurs is obtaining a primary culture of cells, (Figures 2A, C).
through the transfer of cytoplasm from zebrafish macrophages to The combination of zPDX with the lncRNAs based
the transplanted tumor cells. Although it is unknown what transcriptomic analysis-guided drug screening assays would
components are exchanged, it is presumed that it could be enable the finding of efficient and personalized anti-breast
RNA molecules (139). cancer treatments, (Figures 2B–D). The zebrafish xenograft
Zebrafish xenotransplantation assays of breast cancer cells, model allows rapid sensitivity profiling to new anticancer drugs
coupled with single cell transcriptional analyses, could facilitate but is also ideal for determining the effects of different therapeutic
the elucidation of the molecular mechanisms and lncRNAs combinations on tumorigenesis, metastasis, and angiogenesis, in a
involved in the communication between the tumor micro timeframe compatible with the clinical decision-making process
environment and cancer cells. (110), (Figures 2D–F). More important, these assays maximize
the use of the small amount of breast cancer tissue available after a
biopsy, which can be a limiting factor in precision medicine.

lncRNAs AND ZEBRAFISH PATIENT-

DERIVED XENOTRANSPLANTATION ZEBRAFISH CONSERVED lncRNAs IN
(zPDX) IN THE SEARCH FOR THE FUTURE DIRECTIONS FOR
PERSONALIZED BREAST BREAST CANCER MODELING
CANCER TREATMENTS BY XENOTRANSPLANTATION
Due to breast cancer’s genomic advances, one of the greatest LncRNAs are key players in the communication between tumor
challenges in translational and personalized medicine is quick, cells and the surrounding microenvironment, actively
cheap and reproducible in vivo disease modeling. Although participating in cancer progression. These non-coding RNAs
molecular breast cancer markers and pharmacogenomics can travel free or via exosomes to neighbor cells in the tumor
analyses help to predict the best treatment option, many microenvironment and carry out their function in a cell-non-
patients do not respond as expected. This event is probably autonomous manner (19). Breast cancer xenotransplantation
due to the heterogeneity of breast tumors, in which there are assays in zebrafish showed that there is interspecies molecular
non-responding cells immersed in a large group of responding communication that allows the development and progression of
cells, which will escape treatment. cancer in the animal model. Despite the low conservation of
LncRNAs have been associated to breast cancer progression by interspecies sequences of lncRNAs, the knowledge of those that
modulating a large number of oncogenic processes. These results are conserved between human and zebrafish will allow the study
point toward the possibility that they could be useful as future of their cell-non-autonomous function, and to test their potential
targets for therapeutic intervention against breast cancer (129, 140, as therapeutic targets.
141). In addition, Several studies have proposed lncRNA LncRNAs can be evolutionarily conserved through sequence,
signatures that could potentially be used for predictive and structure, function, and expression of the locus of synthesis. In
prognostic value in response to breast cancer treatments general, lncRNAs do not have high sequence conservation across
(142–144). the full-length sequence because partial sequences or local spatial
LncRNAs can be targeted for inhibition through multiple structures mainly mediate their biological functions. The speed
mechanisms, such as antisense oligonucleotides (ASOs), short of base change in lncRNA sequences exceeds the evolutionary
hairpin RNAs (shRNAs), short interfering RNAs (siRNAs), time scale. It follows that lncRNAs evolve faster than protein
aptamers, CRISPR-Cas approaches and small molecule coding genes, suggesting that nucleotide sequence conservation
inhibitors (145, 146). There is evidence that ASOs could be a is not essential for preserving lncRNAs functionality (150, 151).
potential targeted therapy for cancer-associated lncRNAs (147). lncRNAs follow different conservation criteria than those of
It was recently reported that ASOSs directed against the breast protein coding genes (150, 152). Identity concentrates on short
cancer-associated lncRNA MALAT1 effectively suppressed sections and the secondary structure, unlike coding genes that
cancer spread to the lung in a murine model of breast cancer focus on conservation in all their length to preserve the open
xenotransplantation (148). reading frame and ensure similarity in amino acid sequence
The use of avatars or patient derived xenotransplantation (150). In order to find these conserved segments, diverse groups
(PDX) breast cancer models may help evaluate in vivo the global have generated tools that allow us to study their evolution and to
response of tumor cells, detect those that escape the drug, and estimate the functional conservation of lncRNAs across species,
find and decide the most appropriate treatment for that patient. for example PLAR (153), Gencode V7 (154), Lncipedia (12), and
Zebrafishes offer suitable characteristics for breast cancer ZFLNC (11). Currently, there are databases focused mainly on

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Zampedri et al. Zebrafish Models for lncRNAs Studies

E
F

FIGURE 2 | Workflow of zAVATARS in the context of personalized medicine. (A–C) Experimental setup for generating zebrafish xenotransplant models. Cells
derived from itumor biopsy are analyzed by RNAseq, subsequently labeled and microinjected in the 2dpf larvae. (D) One day after injection, larvae are screened for
successful injection and distributed in groups for testing chemo-, and/or biological therapies. (E) Three days after treatment, larvae are processed for in vivo
microscopy for analysis of proliferation, cell death, angiogenesis, and metastatic potential. (F) Treatment decision of breast cancer patients based on response of
zebrafish cancer hallmarks in drug screening. hpf: hours post fertilization. dpi: days post injection. BRCA zPDX trials: Trial of patient-derived xenotransplants in
zebrafish for the study of breast cancer.

zebrafish lncRNAs annotation and expression profiles, such as involved in the development of the nervous system) were
ZFLNC and zflncRNApedia, for a total of 13,604 genes that rescued with mature RNA from their corresponding human
transcribe 21,128 lncRNAs (11), of which 1,890 are conserved orthologous (157). Similarly, Tuna knockdown resulted in fish
in human. with motor and locomotion defects revealing functional
One of the most important characteristics observed in the conservation with their human counterpart, known as
expression patterns of zebrafish lncRNAs is the strong dynamics lncRNAs involved in Huntington’s disease (158). These results
of temporal expression compared to protein-coding genes (155). suggest that despite the evolutionary distance between zebrafish
This feature is undoubtedly relevant to consider these molecules and humans, and the discrete conservation of these molecules in
as indicators or markers in disease processes. Also, it has been sequence, lncRNAs are essential in homeostasis and health
found that not only specific gene regions of the lncRNAs are maintenance throughout evolution.
conserved, but there is also significant conservation into the Given the functional and expression conservation of zebrafish
upstream regulatory regions (156), and the epigenetic regulatory and human lncRNAs, these molecules are likely to play a crucial
mechanisms in the lncRNAs between zebrafish and human role in developing cancer in zebrafish. We found 15 lncRNAs
(155), suggesting that additional conserved non-coding RNAs annotated in the zebrafish genome, orthologous to 18 human
have not been identified. Knockdown assays have revealed lncRNAs associated with breast cancer, using the Gencode (154),
functional conservation between zebrafish and human Lnc2Cancer v2.0 (30), LNCipedia (12), and ZFLNC (11)
lncRNAs. For example, morphological defects generated by databases. The 18 human lncRNAs conserved in the zebrafish
Cyrano and Megamind knockdown in zebrafish (lncRNAs genome participate in oncogenic processes such as cell cycle

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Zampedri et al. Zebrafish Models for lncRNAs Studies

control, proliferation, differentiation, migration, invasion, CONCLUSION

metastasis, angiogenesis and maintenance of cancer stem cells,
according to CancerSEA ([Link] Addressing breast cancer in a comprehensive manner, which
CancerSEA), LncTarD and Lnc2Cancer v2.0 data (Table 2). involves early diagnosis through sensitive tumor markers and
Zebrafish offer experimental advantages for manipulating of advancing personalized treatment design, are two of the most
gene expression, facilitating the study of functional aspects of critical challenges in breast cancer medicine and biomedical
genes. Altering the expression of conserved lncRNAs in zebrafish research. Breast cancer study through xenotransplantation in
by knockdown with morpholinos or genomic editing by CRISPR zebrafish is a valuable tool given the speed with which tumors are
cas9, will allow the study of the non-autonomous functions of obtained and experiments are concluded. Transplantation of human
lncRNAs in the tumor microenvironment during breast cancer breast cancer cells in fish allows the discriminated and efficient study
xenotransplantation trials, as previously carried out in the of aspects related to disease development, such as tumorigenesis,
melanoma cell xenotransplants (131). migration, metastasis, angiogenesis and response to drugs.

TABLE 2 | Functional relationship between conserved lncRNAs and breast cancer hallmarks.

Zebrafish name Human lncRNA type Activate Inhibited O or Expression

lncRNA S
name

ZFLNCT16634 (LOC103909273 un-characterized) DLX6-AS1 Antisense P, I, EMT, Mi, T Ap, CellC, D, DNAd, O Up-
DNAr, H, Inf, Q regulated
ZFLNCT02505 (ENSDART00000153684.2, HAGLR Antisense, An, D, Inf, Me DNA r, I, Q O _
CR751227.1-201) (HOXD-AS1)
ZFLNCT08532 (NONDRET012204.1) HOTAIR Antisense, D, Mi, Me, I, T DNAr, EMT, I, Ap O Up-
lincRNA regulated
ZFLNCT02498 (ENSDART00000155072.3, HOXA11-AS Antisense, CellC, Inf, Q, P, I, Me, Mi DNAd, DNAr, I, Ap O/S Up-
ENSDART00000155419.2, ENSDART00000155896.2, sponge in regulated
LOC103910246) OC
HOXA-AS2 Antisense, Ch, P, I, Mi, T, Prog, EMT D, Ap O –
ceRNA, sponge
HOXA-AS3 Antisense, P, Mi, I, S, Me EMT, Inf, Me, An, Ap O Up-
sponge regulated
HOXB-AS1 Antisense, P, CellC, I, Mi Ap O Up-
ceRNA regulated
ZFLNCT19656 (si:dkey-81p22.11) HOXC-AS3 Sponge P, H, T, I, Mi, Me Q O Up-
regulated
ZFLNCT01281 LINC00649 Antisense An, Ap, cellC, D, _ Down-
EMT, H, I, Me, P, Q regulated
ZFLNCT17432 (ENSDART00000153409.2) LINC00324 lincRNA P, I, Me, Mi, S Ap O Up-
regulated
ZFLNCT14004 (ENSDART00000149569.3) LINC00461 lincRNA, ceRNA, P, cellC, DNAd, S, Mi, I, Me, An, H, Q, Ap O Up-
Sponge T, EMT regulated
ZFLNCT12716 (lnc2_zgc:194285, lnc1_zgc:194285) MALAT1 Sponge, ceRNA, Mi, I, EMT, An, H, S, P, Me, An, CellC, DNAd, O _
lincRNA Ap DNAr, EMT, Inf, I, and
Me, Q, P S
ZFLNCT01941 (lnc_ghrhra) MAPT-AS1 Antisense CellC, S. In ER negative Ap, DNAd, DNAr, S _
BRCA induce P and Mi. EMT, H, I, Me, P, Q
ZFLNCT11804 (oip5-as1-202, Cyrano) OIP5-AS1 ceRNA P, Mi, I, T, CellC, DNAd, S, P, RR (CRC) O Up-
(Linc-OIP5, Me, EMT ans regulated
Cyrano) S
ZFLNCT11748 (NONDRET002400.1) PAX8-AS1 Processed Ap Ap, CellC, DNAd, S Down-
transcript DNAr, H, Inf, I, Me, regulated
P, Q, S
ZFLNCT19020 (PF102167.1-201, SOX2-OT Sense P, I, Me Ap, CellC, D, DNAd, O Up-
ENSDART00000149948.3, sox2ot, si:ch73-334e23.1, overlapping DNAr, H, Q regulated
LOC101883930)
ZFLNCT09180 (BX571737.1-201) TTN-AS1 Antisense, DNAd, P and Me in ESCC. P, Ap, CellC, DNAd, O Up-
ceRNA, sponge, I, EMT and Mi in BRCA and DNAr, EMT, H, Inf, I, regulated
lincRNA CRC Me
PTENP1 PTENP1 ceRNA, sponge Ap Mi, P, I S Down-
regulated

P, proliferation; An, angiogenesis; Me, metastasis; CellC, Cell cycle; D, differentiation; DNAd, DNA damage; H, Hypoxia; Inf, Inflamation; Q, quiescence; I, Invasion; Mi, Migration; EMT,
Epithelial-Mesenchymal transition; T, Tumorigenesis; S, Stemness; Ch, Chemoresitance; Prog, Progression; DR, Drug resistance; RR, Radio resistance; BRCA, Breast cancer; O,
oncogenic roll; S, Tumor suppressor roll.

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Zampedri et al. Zebrafish Models for lncRNAs Studies

Today, these assays are mainly used to validate in vitro assays; that the zebrafish transparency will elucidate the relationship of
however, the significant finding of conservation of zebrafish’s each of the cancer cellular phenotypes (migratory, proliferative,
lncRNAs and their expression change during the zebrafish angiogenic) with specific zebrafish lncRNAs expression, facilitating
cancer process broadens the perspective. We propose that this the interpretation and analysis of the results.
in vivo model will allow the study of the functional impact of
lncRNA dysregulation in the host microenvironment allowing a
simultaneous search for new breast cancer-associated lncRNAs
through transcriptional studies. The fine dynamics of lncRNAs AUTHOR CONTRIBUTIONS
expression and their relationship with the alteration of the tumor
CZ reviewed the literature, wrote the initial abstract, drafted the
microenvironment show that these molecules are excellent
manuscript, figures, and tables, and revised the second draft
candidates for the prediction and prognosis assays and possible
following feedback from JM-Z. WAM-F reviewed the lncRNAs
therapeutic targets in the area of drug development.
databases, and contributed to the second draft review. JM-Z
The advantages of xenotransplantation experiments in
reviewed the literature, proposed an article outline, contributed
zebrafish compared to other models suggest their potential in
sections of the initial draft, and made editorial suggestions for the
the personalized approach to the breast cancer treatment. One of
second draft. All authors contributed to the article and approved
the main strategies is zPDXs modeling, and subsequent drug
the submitted version.
screening. Subtle variations in lncRNAs expression could
effectively predict the response of cancer cells to drugs and may
in turn serve as new targets in the development of new treatments.
The combination of the ease of performing drug screening, FUNDING
including lncRNAs targeted drugs, on zebrafish embryos after
xenotransplantation, and the possibility of evaluating the CZ was supported by a post-doctoral fellowship from the
functional in vivo response through the real time microscopy CONACYT grant A1-S-8462, and this study is part of her
study, increases the robustness of xenotransplantation models. postdoctoral work. The work at JM-Z laboratory is supported
In addition, the conservation in cancer-related pathways by CONACYT grant A1-S-8462.
between humans and zebrafish and the existence of interspecies
molecular crosstalk during xenotransplantation support the use of
knockdown and knockout zebrafish for conserved lncRNAs to
determine the nature of the molecular pathways that respond to SUPPLEMENTARY MATERIAL
lncRNAs signaling. In the same way, xenotransplantation assays on
The Supplementary Material for this article can be found online
a knockout or overexpressing cancer-related lncRNAs in zebrafish
at: [Link]
could reveal the non-autonomous function of lncRNAs in the
687594/full#supplementary-material
tumor microenvironment. Besides, drug-screening trials targeting
zebrafish lncRNAs or related pathways are another area that could Supplementary Table 1 | Growing scientific output of work on breast cancer
benefit from xenotransplantation assays. An additional benefit is modeling by xenotransplantation in zebrafish.

8. Zhang Y, Cao X. Long Noncoding RNAs in Innate Immunity. Cell Mol

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Frontiers in Oncology | [Link] 15 May 2021 | Volume 11 | Article 687594