biomedicines Review How Review Lineage Tracing Studies Can Unveil Tumor How Lineage Tracing Heterogeneity Studies in Breast CancerCan Unveil Tumor Heterogeneity in Breast Cancer Elena Vinuesa-Pitarch †, Daniel Ortega-Álvarez † and Verónica Rodilla * Elena Vinuesa-Pitarch † , Daniel Ortega-Álvarez † and Verónica Rodilla * Cancer Heterogeneity and Hierarchies Group, Josep Carreras Leukaemia Research Institute (IJC), 08916 Bada- Cancer Heterogeneity and Hierarchies Group, Josep Carreras Leukaemia Research Institute (IJC), lona, Spain; evinuesa@carrerasresearch.org (E.V.-P.); dortega@carrerasresearch.org (D.O.-Á.) 08916 Badalona, Spain; evinuesa@carrerasresearch.org (E.V.-P.); dortega@carrerasresearch.org (D.O.-Á.) * Correspondence: vrodilla@carrerasresearch.org * Correspondence: vrodilla@carrerasresearch.org † Equal contribution. † Equal contribution. Abstract: Lineage tracing studies have become a well-suited approach to reveal cellular hierarchies Abstract: Lineage tracing studies have become a well-suited approach to reveal cellular hierarchies and tumor heterogeneity. Cellular heterogeneity, particularly in breast cancer, is still one of the main and tumor heterogeneity. Cellular heterogeneity, particularly in breast cancer, is still one of the main concerns regarding tumor progression and resistance to anti-cancer therapies. Here, we review the concerns regarding tumor progression and resistance to anti-cancer therapies. Here, we review the current knowledge about lineage tracing analyses that have contributed to an improved compre- current knowledge about lineage tracing analyses that have contributed to an improved compre- hension of the complexity of mammary tumors, highlighting how targeting different mammary ep- hension of the complexity of mammary tumors, highlighting how targeting different mammary ithelial cells and tracing their progeny can be useful to explore the intra- and inter-heterogeneity epithelial cells and tracing their progeny can be useful to explore the intra- and inter-heterogeneity observed in breast cancer. In addition, we examine the strategies used to identify the cell of origin observed in breast cancer. In addition, we examine the strategies used to identify the cell of origin in different breast cancer subtypes and summarize how cellular plasticity plays an important role in different breast cancer subtypes and summarize how cellular plasticity plays an important role during tumorigenesis. Finally, we evaluate the clinical implications of lineage tracing studies and during tumorigenesis. Finally, we evaluate the clinical implications of lineage tracing studies and the the challenges remaining to address tumor heterogeneity in breast cancer. challenges remaining to address tumor heterogeneity in breast cancer. Keywords: breast cancer; lineage tracing; heterogeneity; cellular plasticity Keywords: breast cancer; lineage tracing; heterogeneity; cellular plasticity Citation: Vinuesa-Pitarch, E.; Ortega-Álvarez, D.; Rodilla, V. How  Lineage Tracing Studies Can Unveil 1. Tumor Heterogeneity in Breast 1.Introduction Introduction Citation: Vinuesa-Pitarch, E.; Cancer. Biomedicines Breast Breast cancer cancer isis the the most most common common cancercancer inin women women worldwide worldwideand andisis the the second second Ortega-Álvarez, D.; 2021, 9, x.V. How Rodilla, https://doi.org/10.3390/xxxxx leading leading cause cause ofof cancer cancer death death in in women, women, exceeded exceededonly onlyby bylung lungcancer cancer(based (basedon ondata data Lineage Tracing Studies Can Unveil from from the World Health the World HealthOrganization, Organization,2021).2021).This Thistype typeof of cancer cancer originates originates in the in the mam- mammary Tumor Heterogeneity in Breast Academic Editor(s): Mariangela mary gland,gland, which which is a ductal is a ductal tree composed tree composed of two of two epithelial epithelial compartments: compartments: cellscells facing facing the Cancer. Biomedicines 2022, 10, 3. Russo https://doi.org/10.3390/ the ductal lumen called luminal cells (LCs), and basal cells (BCs) found in the outer layerlayer ductal lumen called luminal cells (LCs), and basal cells (BCs) found in the outer with biomedicines10010003 Received: 08 November 2021 with a capacity a capacity to contract, to contract, which which includes includes basalbasal progenitor progenitor cellscells andand terminal terminal differenti- differentiated Accepted: 17 December 2021 ated myoepithelial myoepithelial cells. cells. Luminal Luminal cellsbecan cells can be further further subdivided subdivided into two into two independent independent subpop- Academic Editor: Mariangela Russo Published: date subpopulations based on the expression of the hormone receptor, estrogen ulations based on the expression of the hormone receptor, estrogen receptor alpha (ERα) receptor alpha [1] Received: 8 November 2021 (ERα) [1] (Figure 1).(Figure 1). Accepted: 17Note: Publisher’s December MDPI2021 stays neu- Published: tral 21 December with regard 2021 to jurisdictional claims in published maps and institu- Publisher’s Note: MDPI stays neutral tional affiliations. with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Submitted for possible open access publication under the terms and con- Copyright: © 2021 by the authors. ditions of the Creative Commons At- Licensee MDPI, Basel, Switzerland. tribution (CC BY) license This article is an open access article (https://creativecommons.org/licen- distributed under the terms and Figure1. Figure Modelof 1. Model ofnormal normalmammary mammarygland glandstructure. structure.This Thistissue tissueisiscomposed composedof ofducts, ducts,which whichareare ses/by/4.0/). conditions of the Creative Commons formedby formed by three three epithelial epithelial populations: populations:basal basalcells, cells,in incontact contactwith withthe thebasal basal membrane; membrane;estrogen estrogen Attribution (CC BY) license (https:// receptor-positive receptor-positive (ERα pos (ERαpos ) )luminal luminalcells; cells;and andestrogen estrogenreceptor-negative receptor-negative (ERα neg) )luminal (ERαneg luminalcells. cells.The The creativecommons.org/licenses/by/ dotted black line indicates the cross-section of the mammary duct represented in the magnified 4.0/). scheme on the right. This figure was created with Biorender.com (accessed on 12 December 2021). Biomedicines 2021, 9, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/biomedicines Biomedicines 2022, 10, 3. https://doi.org/10.3390/biomedicines10010003 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, x FOR PEER REVIEW 2 of 16 Biomedicines 2022, 10, 3 2 of 15 dotted black line indicates the cross-section of the mammary duct represented in the magnified scheme on the right. This figure was created with Biorender.com (accessed on 12 December 2021). In In clinical clinicalpractice, practice,breast breasttumors tumors areare classified based classified on on based the the histological expression histological expres- of ERα, progesterone receptor (PR), receptor tyrosine-protein kinase ErbB-2 sion of ERα, progesterone receptor (PR), receptor tyrosine-protein kinase ErbB-2 (HER2), (HER2), and the proliferation marker Ki-67. They are divided into three main groups: and the proliferation marker Ki-67. They are divided into three main groups: hormone hormone recep- tor-positive subtype, receptor-positive whichwhich subtype, includes tumors includes expressing tumors ERα and/or expressing PR, which ERα and/or are sub- PR, which are classified as luminal A or B depending on the percentage of Ki-67; HER2-positive subclassified as luminal A or B depending on the percentage of Ki-67; HER2-positive tumors,tu- defined by theby mors, defined presence of ERBB2/HER2 the presence of ERBB2/HER2amplifications and loss amplifications andofloss ERαof expression; and ERα expression; triple-negative breastbreast and triple-negative cancercancer (TNBC), characterized (TNBC), by theby characterized lack theoflack expression of the afore- of expression of the mentioned molecular markers [2] (Figure aforementioned molecular markers [2] (Figure 2). 2). Figure 2. Molecular classification of breast cancer subtypes. Breast cancer can be divided into three Figure 2. Molecular classification of breast cancer subtypes. Breast cancer can be divided into three main subtypes depending on the histological expression of four markers (ERα, PR, HER2, and Ki-67): main subtypes depending on the histological expression of four markers (ERα, PR, HER2, and Ki- hormone 67): receptor hormone (HR)-positive, receptor HER2-positive, (HR)-positive, and and HER2-positive, triple-negative. ThisThis triple-negative. figure was created figure with was created Biorender.com with (accessed Biorender.com on 12on (accessed December 2021).2021). 12 December At the At the cellular cellular level, level, breast breast cancer cancer is is aa heterogeneous heterogeneous disease disease [3], [3], thereby thereby evoking evoking disparate phenotypes not only between patients, but also within the tumor disparate phenotypes not only between patients, but also within the tumor itself, known itself, known as inter- and intratumor heterogeneity, respectively [4]. It was suggested that this intratumor as inter- and intratumor heterogeneity, respectively [4]. It was suggested that this intra- heterogeneity is perpetuated by tumor stem-like cells, leading many research groups to tumor heterogeneity is perpetuated by tumor stem-like cells, leading many research focus on the identification of biomarkers that allow them to target those cells responsible for groups to focus on the identification of biomarkers that allow them to target those cells tumor maintenance, including CD44 [5], CD29 and CD49f, [6], CD133 [7], Lgr5 [8], Procr [9], responsible for tumor maintenance, including CD44 [5], CD29 and CD49f, [6], CD133 [7], ALDH [10], or CD61 [11]. Although the origin of breast cancer, as many other types of Lgr5 [8], Procr [9], ALDH [10], or CD61 [11]. Although the origin of breast cancer, as many cancer, remains largely elusive, a plausible theory is that adult mammary stem/progenitor other types of cancer, remains largely elusive, a plausible theory is that adult mammary cells, which are very long-lived compared with differentiated cells, are better targets for stem/progenitor cells, which are very long-lived compared with differentiated cells, are accumulating the multiple genetic mutations necessary for malignant transformation [12]; better targets for accumulating the multiple genetic mutations necessary for malignant however, accumulating evidence has demonstrated that specific mutations in differentiated transformation [12]; however, accumulating evidence has demonstrated that specific mu- cells are also able to initiate a tumor [13–16]. Importantly, both luminal and basal cells are tations possibleintargets differentiated cells are for malignant also able to initiate transformation [17,18]. a tumor [13–16]. Importantly, both luminal and basal cells are possible targets During the last decade, lineage tracing studiesfor malignant transformation have provided deeper [17,18]. insights into the cellular heterogeneity and molecular mechanisms underlying cellular deeper During the last decade, lineage tracing studies have provided insights plasticity into in different the cellular heterogeneity and molecular mechanisms underlying cellular subtypes of breast cancer. Accordingly, this review outlines the main milestones concerningplasticity in dif- ferent subtypes in this technology of the breast cancer. breast cancerAccordingly, thisasreview context, as well outlines underlining the main unsolved milestones questions and concerning this technology future prospects in the field. in the breast cancer context, as well as underlining unsolved questions and future prospects in the field. 2. Lineage Tracing Is the Gold-Standard Approach for Exploring Cellular Hierarchies and 2. TumorTracing Lineage Heterogeneity in Breast Cancer Is the Gold-Standard Approach for Exploring Cellular Hierarchies and Tumor Heterogeneity in Breast Cancer Genetic fate mapping studies commonly use a lineage-specific promoter followed by an inducible Genetic fateform of Cre mapping recombinase studies commonly fusedusetoa lineage-specific the mutant ligand-binding domainby promoter followed of the human estrogen receptor (CreER, CreERT, or CreERT2) [19–21], an inducible form of Cre recombinase fused to the mutant ligand-binding domain of the which does not bind to endogenous human estrogenestradiol, receptor but can be (CreER, activated CreERT, by the administration or CreERT2) [19–21], which of synthetic does not ligands, bind to such as tamoxifen or 4-hydroxytamoxifen (4-OHT). Furthermore, this Cre line needs to be mated with a reporter line carrying the β-galactosidase enzyme [22], one or several fluorescent protein(s) [23–27], or barcoding sequences [28,29], in order to monitor the (Figure 3A). The use of this particular system has been controversial, since high doses of tamoxifen can delay mammary gland development [30]; however, the use of low doses (0.1 mg/g of mouse body weight) is a good balance between correct mammary epithelial development and sufficient labeling efficiency [31]. Alternatively, other studies have Biomedicines 2022, 10, 3 opted for tetracycline-inducible systems, where tetracycline administration, or its3 ana- of 15 logue doxycycline (Dox), enables the expression of a reverse tetracycline-controlled trans- activator (rtTA). Activated rtTA will bind to tetracycline response elements (TRE), which trigger the expression of Cre recombinase to mediate the recombination of loxP sequences targeted cells. In this way, tamoxifen or 4-OHT drive the inducible Cre to the nucleus, where located at the reporter transgene [32,33] (Figure 3B). Advantageously, this system allows it promotes the recombination of loxP sequences, allowing the expression of the reporter us to perform lineage tracing at saturation [34], relying on the long-term administration (Figure 3A). The use of this particular system has been controversial, since high doses of of Dox and inducing reporter recombination in every single cell of a given lineage, over- tamoxifen can delay mammary gland development [30]; however, the use of low doses coming the low recombination efficiency obtained by classic lineage tracing experiments (0.1 mg/g of mouse body weight) is a good balance between correct mammary epithelial using ER-dependent Cre lines. development and sufficient labeling efficiency [31]. Alternatively, other studies have A new genetically engineered mouse model using the Flippase (Flp)/flippase recog- opted for tetracycline-inducible systems, where tetracycline administration, or its analogue nition target (Frt) system was developed to specifically study the mammary gland [35]. doxycycline (Dox), enables the expression of a reverse tetracycline-controlled transactivator The authors generated a transgenic mouse line expressing a mouse codon-optimized Flp (rtTA). Activated rtTA will bind to tetracycline response elements (TRE), which trigger under the control of the mouse mammary tumor virus (MMTV) promoter, which recom- the expression of Cre recombinase to mediate the recombination of loxP sequences located bines Frt sequences exclusively in mammary epithelial cells. Similar to the doxycycline- at the reporter transgene [32,33] (Figure 3B). Advantageously, this system allows us to dependent system, perform lineage this in tracing vivo strategy at saturation does [34], not require relying the use ofadministration on the long-term tamoxifen for activat- of Dox ing Flp enzyme. Importantly, this model consists of a Cre alternative and inducing reporter recombination in every single cell of a given lineage, recombinase (Flp), overcoming which allows the combination of multiple site-specific recombination systems, the low recombination efficiency obtained by classic lineage tracing experiments using such as Cre/loxP and Flp/Frt ER-dependent [35,36]. Cre lines. Figure 3. Inducible Cre/Lox systems. (A) Tamoxifen-inducible Cre/Lox model, where, upon tamox- Figure 3. Inducible Cre/Lox systems. (A) Tamoxifen-inducible Cre/Lox model, where, upon tamox- ifen (Tam) administration, the cells expressing CreER, CreERT, or CreERT2 (determined by a specific ifen (Tam) administration, the cells expressing CreER, CreERT, or CreERT2 (determined by a spe- promoter) will be cific promoter) labeled will with β-galactosidase, be labeled with β-galactosidase,fluorescent protein(s), fluorescent or barcode protein(s), sequence(s) or barcode due to sequence(s) the Cre-mediated recombination of the loxP sites, excising the STOP codon in the due to the Cre-mediated recombination of the loxP sites, excising the STOP codon in the reporterreporter transgene. (B) Tetracycline-inducible transgene. Tet/Lox system, (B) Tetracycline-inducible Tet/Loxwhere, uponwhere, system, doxycycline (Dox) administration, upon doxycycline the cells (Dox) administra- tion, the cells expressing rtTAexpressing rtTA will will be labeled be labeled by the chosen by the chosen reporter due toreporter due to the binding of the the binding of the rtTA rtTA transactivator transactivator to TRE sequences to TRE (suchsequences (such asactivation as transcriptional transcriptional elements activation elementsin(TetO)), (TetO)), resulting resulting Cre expression in and Cre expression consequent and consequent excision excisionSTOP of the loxP-flanked of the loxP-flanked codon upstreamSTOP to thecodon upstream reporter to the transgene. reporter This figure transgene. was createdThis withfigure was created Biorender.com with Biorender.com. (accessed on 12 December 2021). AOne newof genetically the most suitable approaches engineered for lineage mouse model usingtracing is pulse the Flippase experiments,recogni- (Flp)/flippase where 16–24 tion h after target Cre (Frt) recombination, system the targeted was developed cells are to specifically analyzed; study and chase the mammary experiments, gland [35]. The which allow authors us to visualize generated the mouse a transgenic progeny of expressing line the targeteda cells, mousegenetically labeled regardless codon-optimized Flp under of the the expression control of the specific of the mouse mammarygenetumor used for lineage virus (MMTV)tracing. Recently, promoter, some which groups have recombines Frt sequences exclusively in mammary epithelial cells. Similar to the doxycycline-dependent system, this in vivo strategy does not require the use of tamoxifen for activating Flp en- zyme. Importantly, this model consists of a Cre alternative recombinase (Flp), which allows the combination of multiple site-specific recombination systems, such as Cre/loxP and Flp/Frt [35,36]. One of the most suitable approaches for lineage tracing is pulse experiments, where 16–24 h after Cre recombination, the targeted cells are analyzed; and chase experiments, which allow us to visualize the progeny of the targeted cells, genetically labeled regardless of the expression of the specific gene used for lineage tracing. Recently, some groups have used this technology to explore the capacity of different mammary epithelial cells to initiate tumors or metastasize, as well as to understand the potential mechanisms underlying cellular plasticity within breast tumors, which could ultimately lead to new therapeutic strategies for treating heterogeneous tumors in the near future. Biomedicines 2022, 10, 3 4 of 15 2.1. Searching for the Tumor Cell of Origin in Different Breast Cancer Subtypes Gene expression analyses have provided a new sub-classification of breast cancer patients based on their transcriptomic profile, which allowed them to be originally subclas- sified into five different molecular breast cancer subtypes: luminal A, luminal B, HER2- positive, normal-like, and basal-like [37]. Further gene expression studies identified a new breast cancer subtype known as claudin-low or mesenchymal-like [38]; and later, clustering analysis of genomic and transcriptomic data of breast tumors revealed 10 novel integrative clusters, which displayed different copy number alterations and gene expression profiles associated with distinct clinical outcomes [39]. Importantly, several studies compared the gene expression signature of different healthy mammary epithelial cells (MECs) with transcriptional profiles of different tumor subtypes [17,40,41]. Interestingly, it was found that the luminal progenitor cell signature resembled that of basal-like tumors, suggesting that the cell of origin in this breast cancer subtype could be an LC, both in mouse and human [17,40,41]. Conversely, the BC signature was upregulated in the claudin-low subtype, and the mature luminal gene signature was closely aligned with the luminal A and B subtypes [17,41]. Overall, these results introduced comparative expression profiling as a powerful tool to elucidate the cell of origin in different cancer subtypes, which could serve as a cellular target for oncogenic events; however, only genetic studies at the single-cell level or lineage tracing experiments are the current definitive approaches to identify the cell of origin in each breast cancer subtype. So far, only a few studies have explored this particular issue using lineage tracing tools. A good example is a recent study that proved that cells positive for leucine-rich repeat- containing G-protein coupled receptor 6 (Lgr6), which is expressed in LCs and BCs during the early stages of tumorigenesis, contributed to mammary tumor progression [42]. Specifi- cally, when Lgr6pos cells were genetically labeled at the hyperplasia stage (P12, 12-day-old) in a mouse mammary tumor virus promoter-driven Polyomavirus middle T antigen breast cancer mouse model (MMTV-PyMT), which mostly generates luminal tumors, these cells clonally expanded, contributing to the formation of carcinomas [42]. Surprisingly, when they used the Medroxyprogesterone Acetate (MPA) plus 7,12-Dimethylbenz[a]anthracene (DMBA)-dependent model to generate mixed luminal and basal tumors [42], their lineage tracing analysis showed that neither basal nor luminal Lgr6pos cells were involved in the formation of basal-like tumors. These results suggest that Lgr6pos cells could be the cell of origin in the luminal breast cancer subtype exclusively; however, the fact that the Lgr6 promoter is activated in LCs and BCs at the start point of the lineage tracing makes it complicated to draw strong conclusions regarding the true cell of origin in each breast cancer subtype. Remarkably, when Lgr5-expressing cells (exclusively expressed in BCs [43]) were traced in C3(1)Tag mice, a murine model that spontaneously develops TNBC [44,45], the hyperplastic lesions generated were mostly Lgr5pos -derived progeny, denoting Lgr5pos cells as the cellular origin of TNBCs [46]; nevertheless, an exhaustive histological character- ization of the resulting tumors was missing in this work, making it difficult to conclude which specific types of tumors were formed from these cells. Fascinatingly, a recent work combined lineage tracing analysis and the RCAS-TVA system. This consists of expressing the TVA cell surface receptor under the control of a specific target promoter recognized by an avian leukosis virus-derived vector (RCAS) [47] to elucidate the contribution of ERα during cancer progression and metastasis of HER2- positive tumors [48]. By using Esr1-Cre/MMTV-TVA/Rosa26-tdRFP mice infected with RCAS-Erbb2 (to generate HER2-positive tumors), the authors could demonstrate that ERαpos tumor cells with an overexpression of HER2 have to progressively lose their ERα expression in order to clonally expand and metastasize [48]. Importantly, HER2-positive cells originated from ERαpos cells were more aggressive than those originated from ERαneg cells, suggesting that the cell of origin plays an important role in the clinical outcome of breast cancer patients. Biomedicines 2022, 10, 3 5 of 15 Undoubtedly, more studies are required to figure out which mammary epithelial cell type is the cell of origin for each specific tumor type. The lack of good preclinical breast cancer mouse models that truly recapitulate human subtypes, and the selection of suitable inducible Cre lines, which exclusively label one mammary epithelial cell type at a time, are the major concerns that the research community currently face to address this important issue. 2.2. Importance of the Epithelial-to-Mesenchymal Transition during Tumor Progression The epithelial-to-mesenchymal transition (EMT) is a biologic process by which ep- ithelial cells lose their cell polarity and cell–cell adhesion to undergo multiple biochemical changes that enable them to assume mesenchymal attributes, such as an elongated shape, fibroblast-like morphology, enhanced mobility, invasiveness, resistance to apoptotic stimuli, and production of extracellular matrix components [49]. Crucially, this process could be exploited by tumor cells, allowing them to detach from each other within the primary tumor and metastasize to distant organs [50]. In vivo monitorization of different mesenchymal markers (FSP1 and Vimentin) using different breast cancer models demonstrated that the EMT process does not contribute to lung metastasis development [51]. These studies evaluated the appearance of EMT in two breast cancer models (MMTV-PyMT and MMTV-Neu), being a biological process that can be observed in vivo; however, the metastases developed were neither FSP1- nor Vimentin- derived progenies [51–53]. Notwithstanding, FSP1-derived mesenchymal cells can undergo mesenchymal-to-epithelial transition (MET) and contribute to tumor recurrence, although this was addressed using serial transplantations as a rough way of recapitulating this tumor process [54]. The main concern regarding these studies was that these murine lines were targeting a small fraction of the total cells that undergo EMT, having a non- negligible difference between the total percentage of mesenchymal E-Cadherin (Ecad)low cells and FSP1pos -cells (5% and 0.3%, respectively) [52]. Crucially, when Ecadlow tumor cells were injected into the circulation, these cells generated metastases [52]; however, these experiments did not address the fact that this process occurs naturally in vivo during the progression of this disease. The fact that many different cells can undergo EMT and the lack of a universal marker encouraged the combinatorial design of Cre and Dre systems to study this biological pro- cess. Recently, Li and colleagues generated EMTracer, a triple transgenic mouse model carrying Kit-CreER, EMTgene-LSL-Dre, and NR1-reporter. Thus, this EMTracer was crossed with the MMTV-PyMT model to monitor EMT during tumor progression [55]. With this strategy, after tamoxifen administration, luminal-Kitpos cells recombined LoxP sequences expressing ZsGreen fluorescent protein, inducing Dre expression exclusively in mesenchy- mal cells which were positive for Vim or Cdh2/N-cadherin, turning tdTomato positive [55]. In their functional assays, they found that Vimentin was not functionally required to metas- tasize (as previously reported); however, the activation of N-cadherin was critical for lung colonization [55]. In addition, the authors demonstrated that breast cancer cells underwent the EMT program during primary tumor growth rather than during dissemination or lung colonization [55], indicating the importance of studying EMT at different stages of the metastatic cascade to draw reliable conclusions. The heterogeneity underlying the EMT process could be explained by the emergence of a partial rather than a full EMT [56], resulting in the appearance of intermediate hybrid states that share epithelial and mesenchymal features. In fact, combining lineage tracing strategies with single cell RNA-sequencing (scRNA-seq) analyses at different time points of the metastatic cascade could shed light on the characterization of the different EMT transitioning states that breast cancer cells undergo. 2.3. Differential Clonal Expansion during Tumor Progression and the Metastatic Process New microscopy technologies, such as intravital or 3D-whole mount imaging, have allowed the visualization of mammary tumor progression from adenoma to carcinoma Biomedicines 2022, 10, 3 6 of 15 in vivo and at large-scale single-cell resolution, respectively [57,58]. Combining intravital imaging with random multi-labeling of the mammary gland (using Rosa26-CreERT2), Zomer and colleagues observed that only a small subset of tumor cells clonally expanded during tumor progression, and the vast majority of cells within the primary tumor either disappear, grow slowly, or partially expand to finally regress [57]. The combination of these technologies with cell-specific lineage tracing analysis to further study the potential of each MEC to metastasize still represents a gap in our field of knowledge. Performing multicolor fluorescent lineage tracing in the MMTV-PyMT model demon- strated that the metastatic process is produced by the collective dissemination of cancer cells forming cohesive clusters rather than the serial seeding of single tumor cells [59]. Concretely, after the orthotopic implantation of mammary tumors formed of cells randomly labeled with different fluorescent proteins, the resulting lung metastases were formed of multicolored cells, signifying multicellular seeds [59]. Moreover, these lineage tracing stud- ies showed multicolored tumor cell clusters at five different stages of metastasis: collective invasion, locally disseminated clusters in the adjacent stroma, extravasated tumor emboli, circulating tumor cell clusters, and distant micro- and macro-metastases [59]. A powerful approach is combining lineage tracing with scRNA-seq by introducing genetic barcodes. Indeed, using this strategy, Ginzel and colleagues measured the tu- morigenic capacity of different oncogenic HER2 isoforms (HER2, d16HER2 and p95HER2) within the same mammary gland [60]. Specifically, the authors mated MMTV-Cre mice with HER2-Crainbow (HER2BOW) mice, which encoded for the three HER2 variants, fluo- rescently barcoded and flanked by LoxP sites. Using fluorescence and RNA sequencing, they could characterize the tumor phenotype associated with each barcoded isoform [60]. Although wild-type HER2 rarely induced indolent tumors, d16HER2 generated luminal- like proliferative in situ lesions, which eventually progressed, and p95HER2 prompted the early appearance of invasive cancers characterized by double-positive luminal and basal epithelial cells. From a clinical standpoint, these results underscore the importance of subclassifying HER2-positive breast cancer patients based on their HER2 isoform [60]. 3. In Vivo Models to Study Mammary Gland Tumorigenesis Many groups have tried to recapitulate the wide diversity of breast cancer subtypes detected in the clinics, designing preclinical mouse models that overexpress oncogenes or silence tumor suppressor genes. Indeed, recent studies have relied on Cre/lox systems for defining novel drivers of mammary tumorigenesis and assessing their consequences in different cellular contexts. 3.1. Classic Preclinical Models to Recapitulate Different Breast Cancer Subtypes It was reported that specific murine strains can spontaneously develop breast tu- mors, such as CH3, that produces adenocarcinomas with a latency of 6–10 months [61]; BALB/c that also generates adenocarcinomas at 12 months of age [61]; and the Kunming strain that can develop invasive ductal carcinomas in 13.5 months [62]. In addition, there are inducible models that can be sorted by chemical treatments (DMBA or N-nitroso-N- methylurea (NMU)) [63]; physically, by the effects of radiation [64]; or biologically, by lentiviral infection [65]. However, the transcriptomic profiles of most of these models have not been fully analyzed. In order to shed light on this important issue, different research groups analyzed in depth tumors derived from different animal models by comparing their gene expression profiles with different human public databases, and calculating to what extent these models resembled the human disease [45,66]. Thus, murine models were divided into mesenchymal (also known as claudin-low subtype), basal, luminal, or HER2-enriched tumors. Among the animal models with tumors presenting mesenchy- mal features, mainly characterized by the expression of Vim and Snai1, they found the MMTV-Cre/Brca1Co/Co /p53+/− [67]; DMBA-induced [68], few C3(1)-Tag [44], MMTV- Lpa [69], WAP-T121 [70], and p53+/− irradiated [71] models. Recapitulating human basal- like tumors, they included MMTV-Cre/Brca1Co/Co /p53+/− [67], DMBA-induced [68], Biomedicines 2022, 10, 3 7 of 15 C3(1)-Tag [44], MMTV-Myc [72], WAP-Myc [73], WAP-Tag [74], MMTV-Aib1 [69], and MMTV-Wnt1 [75] mice. Resembling luminal tumors, they detected MMTV-Neu [76], MMTV-PyMT [77], WAP-Myc [73], MMTV-Myc [72], MMTV-Aib1 [69], MMTV-Hras [72], and WAP-Int3 [78]. Additionally, recapitulating HER2-enriched tumors, they found MMTV- Neu [76], Bgr1+/− [79], p18−/− [80], Rb−/− [81], MMTV-Aib1 [69], WAP-Cre/Etv6 [82], WAP-T121 [70], and MMTV-Fgf3 [83]. (Table 1). Table 1. Murine models that recapitulate different human breast cancer subtypes. This table summa- rizes the transcriptomic analysis performed in tumors derived from different murine in vivo models that are able to resemble specific human breast cancer subtypes. Human Breast Cancer Mouse Model Oncogenic Expression Ref. Subtype MMTV-Wnt1 Wnt1 overexpression Basal-like [75] WAP-Tag SV40 large T antigen Basal-Like [74] WAP-Int3 Notch 4 overexpression Luminal-Like [78] MMTV-Hras Hras overexpression Luminal A [72] Activation of Src, PI3K, and MMTV-PyMT Luminal B [77] Shc Inactivated rat ErbB2 MMTV-Neu HER2-enriched [76] overexpression in MECs Etv6-Ntrk3 fusion gene WAP-Cre/Etv6 HER2-enriched [82] overexpression Brg1+/− Brg1 heterozygous HER2-enriched [79] p18−/− Cdkn2c homozygous null HER2-enriched [80] Rb−/− Rb homozygous null HER2-enriched [80] MMTV-Fgf3 Fgf3 overexpression HER2-enriched [83] Lpa1, Lpa2, or Lpa3 MMTV-Lpa Mesenchymal-like [69] overexpression Trp53 heterozygous, p53+/− irradiated Mesenchymal-like [71] irradiated Basal-like DMBA-induced Random DMBA induction [68] Mesenchymal-like pRb, p107, p130, p53, p300 Basal-like C3(1)-Tag inactivation and others in [44] Mesenchymal-like MECs Basal-like WAP-Myc Myc overexpression in LCs [73] Luminal B Basal-like MMTV-Myc Myc overexpression [72] Luminal B MMTV- Brca1 truncation in MECs, Basal-like [67] Cre/Brca1Co/Co /p53+/− Tp53 heterozygous null Mesenchymal-like Basal-Like MMTV-Aib1 Aib1 overexpression Luminal B [69] HER2-enriched pRb, p107 and p130 HER2-enriched WAP-T121 [70] inactivation in LCs Mesenchymal-like Some of these models have been the in vivo approaches of reference to study breast cancer for decades; however, they are not sufficient to understand the wide spectrum of human breast tumors. Moreover, the major differences in the composition of the stroma Biomedicines 2022, 10, 3 8 of 15 between the murine and human mammary gland, being more adipocytic and less fibrotic in mice than in humans [84], might play an important role during tumor progression, as well as the colonization of different metastatic organs, since mouse models only develop distant lung metastases, whereas humans are able to metastasize into lung, liver, bone, or brain [85]. 3.2. Cellular Plasticity Plays an Important Role in the Development of Mammary Tumors Numerous lineage tracing studies in healthy mammary glands have demonstrated that there are no multipotent stem cells in adult mice, but distinct pools of unipotent stem cells, which self-sustain the lineage restriction of each mammary epithelial population [86–88]. However, adult MECs have been shown to be extremely plastic under different stress situa- tions, such as transplantation [86], oncogene activation [18,43,89,90], cellular ablation [91], or the ectopic expression of key cell fate determinants [92,93], interconverting their cellular potency and destiny. This cellular plasticity observed in normal epithelial cells may be conceivably magnified in tumors, thus contributing to the cellular heterogeneity observed in breast cancer. In the cancer context, two independent groups have shown that luminal ERαpos tumors can arise from BCs and LCs due to the expression of the oncogenic form PIK3CAH1047R [43,89]. In fact, PIK3CAH1047R was enough to induce cell plasticity and the acquisition of multi- lineage features, defined by the expression of both luminal and basal gene signatures at the same time [43]. Interestingly, when the same oncogenic hit was overexpressed in LCs (Krt8-expressing cells) or BCs (Lgr5-expressing cells), the resulting tumors were basal-like, HER2-positive, and luminal B (Krt8-derived tumor), or mainly luminal A and B (Lgr5- derived tumors) [43] (Figure 4). Similar results were found using the Krt5 promoter to target BCs [89]; concretely, overexpression of PIK3CAH1047R in BCs led to the formation of luminal B tumors (Figure 4), while Krt8-expressing cells generated luminal B and basal-like tumors [89]. All these results suggest that ERα-positive tumors with mutations in PI3K could originate from LCs and also BCs, whereas luminal ERα-negative, basal-like, and HER2-enriched tumors may exclusively arise from LCs. Indeed, these studies show how different the cellular plasticity is in LCs and BCs, and how the activation of a specific oncogene can give rise to different latency and tumor types depending on the cell of origin. In the same direction, the loss of Brca1 and Tp53 in different cellular compartments resulted in the development of different breast cancer subtypes [18]. In this particular work, the authors used Krt14-Cre or Blg-Cre lines to monitor BCs or ERαneg LCs, respectively, and mated them with Brca1fl/fl /p53+/− mice. Importantly, BRCA1/p53 deficiency generated different types of tumors that histologically expressed basal markers (Keratin-14 or p63) and luminal markers (Keratin-18 and/or ERα), with similar but not identical gene expression profiles, which closely resembled that of human basal-like tumors, regardless of the cell of origin (Figure 4). Importantly, only Bgl-derived tumors histologically resembled human BRCA1 loss-of-function [18]. Strikingly, using the luminal Cre line Wap-Cre, different groups were able to ob- serve a luminal-to-basal conversion, either overexpressing NTRK3 [94], active NOTCH1 (N1ICD) [95], or KRASG12D [96]. NTRK3 was able to induce mixed tumors bearing both basal and luminal cells, as well as hybrid tumors with cells expressing basal and luminal markers simultaneously [94]; N1ICD specifically generated tumors that transcriptionally matched with distinct luminal subtypes, but also with a mixed subtype containing lu- minal and basal identities [95], and the exogenous expression of mutant KRASG12D led to metastatic claudin-low mammary tumors with a mesenchymal-like phenotype [96] (Figure 4). and infected in Wap-TVA transgenic mice that specifically express the TVA receptor in LCs [90]. Similar to the aforementioned studies, the activation of certain oncogenes in the Biomedicines 2022, 10, 3 9 of 15 luminal compartment was sufficient to develop tumors with either luminal, basal, or mixed (Keratin-8 and Keratin-5-positive) features [90]. Figure 4.4. Schematic representation of the the mammary mammary cellularcellular plasticity plasticity during during tumorigenesis. tumorigenesis. The use of different murine Cre lines (Lgr5-Cre ERT2 (Lgr5-Cre , ,Krt5-Cre ERT2 ERT2 Krt5-Cre or or ERT2 Krt14-Cre Krt14-Cre to to target target BCs; BCs; Bgl-Cre Bgl-Cre or Wap-Cre to trace ERα neg neg LCs;LCs; andand Esr1-Cre to monitor ERαERα pos LCs) pos LCs) to induce the expression of spe- or Wap-Cre to trace ERα Esr1-Cre to monitor to induce the expression of cific mutations specific mutations in different in differentmammary mammary epithelial epithelialcell celltypes typesresulted resultedininthe thegeneration generation of of tumors that tumors that resembled some resembled some ofof the the human human subtypes: subtypes: luminal luminal A A [43], [43], luminal luminal B B [43,89], HER2-enriched [48,66], [43,89], HER2-enriched [48,66], claudin-low (or mesenchymal-like) [96], and basal-like [18,73] tumors. Notes: this figure only in- claudin-low (or mesenchymal-like) [96], and basal-like [18,73] tumors. Notes: this figure only includes cludes studies that have used comparisons with human breast cancer transcriptomic datasets studies that have used comparisons with human breast cancer transcriptomic datasets (PAM50, Hu306 (PAM50, Hu306 or similar); studies performed with non-specific Cre lines, such as MMTV-Cre or ERT2 (which or similar); Krt8-Cre studies ERT2 (whichperformed labels all with MECs,non-specific or both LC Crepopulations, MMTV-Cre orwere lines, such asrespectively), Krt8-Cre discarded. This labels figure all was MECs, or both created with LC populations,(accessed Biorender.com respectively), on 12were discarded. December This figure was created with 2021). Biorender.com (accessed on 12 December 2021). Collectively, these studies have demonstrated that cellular plasticity could be respon- sible Another interesting for the intra- strategy to heterogeneity and inter-tumor study cellular found plasticity is the use in breast of the cancer andRCAS-TVA pose new system [47]. Using this retrovirus-mediated in vivo lineage tracing, Hein and colleagues questions such as whether any mammary epithelial cell has the potential to become a tu- engineered RCAS vectors bearing either PyMT or HER2 constructs, which were produced mor-initiating cell, or, by contrast, only a suitable combination of oncogenic hits is the and infected in Wap-TVA transgenic mice that specifically express the TVA receptor in crucial determinant in developing a tumor regardless of the cell of origin. Hence, these LCs [90]. Similar to the aforementioned studies, the activation of certain oncogenes in the findings emphasize the importance of searching for key factors underlying cellular plas- luminal compartment was sufficient to develop tumors with either luminal, basal, or mixed ticity, which could have significant implications for cancer therapeutics. (Keratin-8 and Keratin-5-positive) features [90]. Collectively, these studies have demonstrated that cellular plasticity could be respon- 4. Clinical Implications of Lineage Tracing Studies and Future Perspectives sible for the intra- and inter-tumor heterogeneity found in breast cancer and pose new Clinical questions decisions such as whetherare made dependingepithelial any mammary on the molecular cell has subtype diagnosed, the potential which to become a includes anti-hormone therapies for those patients with ERα pos cells, anti-HER2 treat- tumor-initiating cell, or, by contrast, only a suitable combination of oncogenic hits is the ments for crucial HER2-positive determinant patients,aand in developing tumorchemotherapy regardless offor thepatients diagnosed cell of origin. Hence,with TNBC, these find- due to the lack of specific targets [2]. These therapeutic strategies do not take ings emphasize the importance of searching for key factors underlying cellular plasticity, into account that tumors which couldare heterogeneous, have meaning that significant implications for they aretherapeutics. cancer composed of different types of cells, which could be the main cause of clinical failures. In this sense, the main handicap that 4. Clinical Implications researchers are currentlyof Lineage facing is theTracing lack of Studies reliable and Futuremodels preclinical Perspectives that resemble this human intratumor Clinical heterogeneity. decisions Currently,onthe are made depending thevast majoritysubtype molecular of in vivo mammary diagnosed, tu- which morigenesis includes models available anti-hormone therapiesdepend for thoseon patients the expression with ERαof pos a specific driver, which cells, anti-HER2 even- treatments tually for contributes topatients, HER2-positive the formation of practically for and chemotherapy (intra)homogeneous patients diagnosed tumors. The combi- with TNBC, due nation of multiple in vivo systems (Cre/lox, RCAS/TVA, Dre/rox, and Flp/Frt) to the lack of specific targets [2]. These therapeutic strategies do not take into account could be the key tumors that to designing a model ablemeaning are heterogeneous, to recapitulate that theytheare tumor evolution composed observed of different typesin of human cells, breast cancer. which could be the main cause of clinical failures. In this sense, the main handicap that researchers are currently facing is the lack of reliable preclinical models that resemble this human intratumor heterogeneity. Currently, the vast majority of in vivo mammary tumorigenesis models available depend on the expression of a specific driver, which eventually contributes to the formation of practically (intra)homogeneous tumors. The combination of multiple in vivo systems (Cre/lox, RCAS/TVA, Dre/rox, and Flp/Frt) could Biomedicines 2022, 10, 3 10 of 15 be the key to designing a model able to recapitulate the tumor evolution observed in human breast cancer. Remarkably, lineage tracing studies have disclosed that the cell of origin matters. Here, we have presented several good examples, such as the work of Ding and colleagues that proved that HER2-positive tumors with an ERαpos or ERαneg cellular origin will determine their aggressiveness and metastatic capacity [48]. Moreover, these in vivo approaches have also revealed an inherent cellular plasticity in MECs; along this line, different groups have demonstrated that upon the activation of a specific oncogenic hit, MECs can ac- quire new transcriptomic features and expand to develop tumors with different cell fate signatures [18,43,89,94–96]. Some of these studies tried to understand whether the cell of origin or the oncogenic activation were playing a major role during tumorigenesis, and concluded that the cell of origin was crucial for determining the tumor subtype and/or ag- gressiveness; however, in all cases, those specific oncogenes (PI3KCA [43,89] or HER2 [48]) were able to generate tumors, suggesting that both facts (cell of origin and oncogenic hit) are equally important. Numerous studies on the normal mammary gland have demonstrated the high plasticity of BCs upon different stressors (transplantation or cell ablation [86,91]) compared with LCs; nevertheless, in the cancer context, all these studies demonstrated that LCs are more plastic than BCs, being able to generate a wider range of breast tumor subtypes [18,43,89,94–96], representing a new field to be explored. Human cells contain somatic mutations that have served as genetic barcodes to per- form retrospective lineage tracing analysis in healthy and diseased human tissues [97]. For example, topographic single-cell sequencing from laser-capture microdissected breast cancer samples at different tumorigenic stages, analysis of copy number alterations and clonal dynamics of different areas suggested a multiclonal invasion model for breast can- cer [98]. Another strategy is the use of mitochondrial DNA mutations as a natural barcode, which McDonald and colleagues used to find common mutations that would indicate a common cell of origin in both normal and premalignant breast sections [99]. Alternatively, the use of DNA barcodes, introduced by infection in isolated normal human mammary cells, revealed a complex clonal landscape within heterogeneous breast tumors expressing KRASG12D [100]. Notably, over the last years, single-cell RNA sequencing has emerged as a new tool to replace lineage tracing studies, since it provides a recapitulation of the clonal dynamics of different cell populations within a tumor in a retrospective manner; however, the question of the identity of the tumor-initiating cell still remains to be solved with this type of technology. Combining both approaches would allow us to genetically and phenotypically trace each individual cell, redefining the phylogenetic trees, cell trajectories, and cell–cell interactions. In keeping with recent breakthroughs, the development of barcode systems has enabled us to target individual cells with unique nucleic acid sequences [101]. This technology could be used for lineage tracing together with the sc-RNAseq technique to reveal the transcriptomics of each cell population within heterogeneous mammary tumors, and also to perform high- throughput genetic screening to discover key plasticity factors and tumor drivers, being potential druggable targets for breast cancer therapeutics [102]. For example, Ying and Beronja employed long-term lineage tracing using stable barcodes to study mammary tissue hierarchy, ensuring each progenitor was labeled with a single barcode [102], and designed a large-scale genetic screening with a barcoded lentivirus library that targeted multiple clinically relevant mutations [102]. Definitively, the combination of multiple technologies will allow us to identify new crucial biomarkers and novel therapeutic targets, especially for TNBC patients who have no targeted therapies available. Beyond lineage tracing, multi-omic and high-throughput methods will help us to understand the complex genomics that underlie the human cancer disease. Indeed, single- cell genomics has enabled us to associate specific genetic mutations with different molecular subtypes [103], and single-cell transcriptomics has provided cancer-specific gene signatures that could help clinicians to determine the prognosis of patients [104]. For instance, there are currently different gene expression profiling tests for breast cancer in clinics, such Biomedicines 2022, 10, 3 11 of 15 as MammaPrint® and Oncotype DX® , both of which predict the risk of distant disease recurrence. Moreover, among some spatial transcriptomic technologies, fluorescence in situ hybridization (FISH) methods are commonly used in cancer diagnosis as they allow the detection and chromosomal location of specific genes which are aberrantly expressed or harbor rare mutations in tumors [105]. On the other hand, there are other techniques such as laser capture microdissection and photoactivatable transcriptome in vivo analysis that could be combined with sequencing-based approaches to decipher the genetic information of the desired area of the tissue [105]. The integration of all these spatial data promises the identification of multiple reliable molecular biomarkers that would be helpful for the diagnostics and therapeutics of breast cancer patients in the near future. Author Contributions: E.V.-P. and D.O.-Á. performed the literature search and drafted the work. V.R. had the idea for the article and critically revised the work. All authors have read and agreed to the published version of the manuscript. Funding: The research leading to this work received funding from the Spanish Ministry of Science abd Innovation (MCI), PID2020-117212RB-I00/AEI/10.13039/501100011033 and RYC2018-024099-I. 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