Amitotic Cell Division, Malignancy, and Resistance to Anticancer Agents: A Tribute to Drs. Walen and Rajaraman.

Cell division is crucial for the survival of living organisms. Human cells undergo three types of cell division: mitosis, meiosis, and amitosis. The former two types occur in somatic cells and germ cells, respectively. Amitosis involves nuclear budding and occurs in cells that exhibit abnormal nuclear morphology (e.g., polyploidy) with increased cell size. In the early 2000s, Kirsten Walen and Rengaswami Rajaraman and his associates independently reported that polyploid human cells are capable of producing progeny via amitotic cell division, and that a subset of emerging daughter cells proliferate rapidly, exhibit stem cell-like properties, and can contribute to tumorigenesis. Polyploid cells that arise in solid tumors/tumor-derived cell lines are referred to as polyploid giant cancer cells (PGCCs) and are known to contribute to therapy resistance and disease recurrence following anticancer treatment. This commentary provides an update on some of these intriguing discoveries as a tribute to Drs. Walen and Rajaraman.

Novel centromeric repetitive DNA elements reveal karyotype dynamics in polyploid sainfoin (Onobrychis viciifolia).

Polyploidy is a common feature in eukaryotes with one of paramount consequences leading to better environmental adaptation. Heterochromatin is often located at telomeres and centromeres and contains repetitive DNA sequences. Sainfoin (Onobrychis viciifolia) is an important perennial forage legume for sustainable agriculture. However, there are only a few studies on the sainfoin genome and chromosomes. In this study, novel tandem repetitive DNA sequences of the sainfoin genome (OnVi180, OnVi169, OnVi176 and OnVidimer) were characterized using bioinformatics, molecular and cytogenetic approaches. The OnVi180 and OnVi169 elements colocalized within functional centromeres. The OnVi176 and OnVidimer elements were localized in centromeric, subtelomeric and interstitial regions. We constructed a sainfoin karyotype that distinguishes the seven basic chromosome groups. Our study provides the first detailed description of heterochromatin and chromosome structure of sainfoin and proposes an origin of heterozygous ancestral genomes, possibly from the same ancestral diploid species, not necessarily from different species, or for chromosome rearrangements after polyploidy. Overall, we discuss our novel and complementary findings in a polyploid crop with unique and complex chromosomal features.

Polyploid cancer cells reveal signatures of chemotherapy resistance.

Therapeutic resistance in cancer significantly contributes to mortality, with many patients eventually experiencing recurrence after initial treatment responses. Recent studies have identified therapy-resistant large polyploid cancer cells in patient tissues, particularly in late-stage prostate cancer, linking them to advanced disease and relapse. Here, we analyzed bone marrow aspirates from 44 advanced prostate cancer patients and found the presence of circulating tumor cells with increased genomic content (CTC-IGC) was significantly associated with poorer progression-free survival. Single cell copy number profiling of CTC-IGC displayed clonal origins with typical CTCs, suggesting complete polyploidization. Induced polyploid cancer cells from PC3 and MDA-MB-231 cell lines treated with docetaxel or cisplatin were examined through single cell DNA sequencing, RNA sequencing, and protein immunofluorescence. Novel RNA and protein markers, including HOMER1, TNFRSF9, and LRP1, were identified as linked to chemotherapy resistance. These markers were also present in a subset of patient CTCs and associated with recurrence in public gene expression data. This study highlights the prognostic significance of large polyploid tumor cells, their role in chemotherapy resistance, and their expression of markers tied to cancer relapse, offering new potential avenues for therapeutic development.

Homoeologous crossovers are distally biased and underlie genomic instability in first-generation neo-allopolyploid Arabidopsis suecica.

First-generation polyploids often suffer from more meiotic errors and lower fertility than established wild polyploid populations. One such example is the allopolyploid model species Arabidopsis suecica which originated c. 16 000 generations ago. We present here a comparison of meiosis and its outcomes in naturally evolved and first-generation 'synthetic' A. suecica using a combination of cytological and genomic approaches. We show that while meiosis in natural lines is largely diploid-like, synthetic lines have high levels of meiotic errors including incomplete synapsis and nonhomologous crossover formation. Whole-genome re-sequencing of progeny revealed 20-fold higher levels of homoeologous exchange and eightfold higher aneuploidy originating from synthetic parents. Homoeologous exchanges showed a strong distal bias and occurred predominantly in genes, regularly generating novel protein variants. We also observed that homoeologous exchanges can generate megabase scale INDELs when occurring in regions of inverted synteny. Finally, we observed evidence of sex-specific differences in adaptation to polyploidy with higher success in reciprocal crosses to natural lines when synthetic plants were used as the female parent. Our results directly link cytological phenotypes in A. suecica with their genomic outcomes, demonstrating that homoeologous crossovers underlie genomic instability in neo-allopolyploids and are more distally biased than homologous crossovers.

Integrative analyses of long and short-read RNA sequencing reveal the spliced isoform regulatory network of seedling growth dynamics in upland cotton.

The polyploid genome of cotton has significantly increased the transcript complexity. Recent advances in full-length transcript sequencing are now widely used to characterize the complete landscape of transcriptional events. Such studies in cotton can help us to explore the genetic mechanisms of the cotton seedling growth. Through long-read single-molecule RNA sequencing, this study compared the transcriptomes of three yield contrasting genotypes of upland cotton. Our analysis identified different numbers of spliced isoforms from 31,166, 28,716, and 28,713 genes in SJ48, Z98, and DT8 cotton genotypes, respectively, most of which were novel compared to previous cotton reference transcriptomes, and showed significant differences in the number of exon structures and coding sequence length due to intron retention. Quantification of isoform expression revealed significant differences in expression in the root and leaf of each genotype. An array of key isoform target genes showed protein kinase or phosphorylation functions, and their protein interaction network contained most of the circadian oscillator proteins. Spliced isoforms from the GIGANTEA (GI) protien were differentially regulated in each genotype and might be expected to regulate translational activities, including the sequence and function of target proteins. In addition, these spliced isoforms generate diurnal expression profiles in cotton leaves, which may alter the transcriptional regulatory network of seedling growth. Silencing of the novel spliced GI isoform Gh_A02G0645_N17 significantly affected biomass traits, contributed to variable growth, and increased transcription of the early flowering pathway gene ELF in cotton. Our high-throughput hybrid sequencing results will be useful to dissect functional differences among spliced isoforms in the polyploid cotton genome.

Centromere drive may propel the evolution of chromosome and genome size in plants.

BACKGROUND: Genome size is influenced by natural selection and genetic drift acting on variations from polyploidy and repetitive DNA sequences. We hypothesized that centromere drive, where centromeres compete for inclusion in the functional gamete during meiosis, may also affect genome and chromosome size. This competition occurs in asymmetric meiosis, where only one of the four meiotic products becomes a gamete. If centromere drive influences chromosome size evolution, it may also impact post-polyploid diploidization, where a polyploid genome is restructured to function more like a diploid through chromosomal rearrangements, including fusions. We tested if plant lineages with asymmetric meiosis exhibit faster chromosome size evolution compared to those with only symmetric meiosis, which lack centromere drive as all four meiotic products become gametes. We also examined if positive selection on centromeric histone H3 (CENH3), a protein that can suppress centromere drive, is more frequent in these asymmetric lineages. METHODS: We analyzed plant groups with different meiotic modes: asymmetric in gymnosperms and angiosperms, and symmetric in bryophytes, lycophytes, and ferns. We selected species based on available CENH3 gene sequences and chromosome size data. Using Ornstein-Uhlenbeck evolutionary models and phylogenetic regressions, we assessed the rates of chromosome size evolution and the frequency of positive selection on CENH3 in these clades. RESULTS: Our analyses showed that clades with asymmetric meiosis have a higher frequency of positive selection on CENH3 and increased rates of chromosome size evolution compared to symmetric clades. CONCLUSIONS: Our findings support the hypothesis that centromere drive accelerates chromosome and genome size evolution, potenatially also influencing the process of post-polyploid diploidization. We propose a model which in a single famework helps explain the stability of chromosome size in symmetric lineages (bryophytes, lycophytes, and ferns) and its variability in asymmetric lineages (gymnosperms and angiosperms), providing a foundation for future research in plant genome evolution.

Doubling down on polyploid discoveries: Global advances in genomics and ecological impacts of polyploidy.

All flowering plants are now recognized as diploidized paleopolyploids (Jiao et al., 2011; One Thousand Plant Transcriptomes Initiative, 2019), and polyploid species comprise approximately 30% of contemporary plant species (Wood et al., 2009; Barker et al., 2016a). A major implication of these discoveries is that, to appreciate the evolution of plant diversity, we need to understand the fundamental biology of polyploids and diploidization. This need is broadly recognized by our community as there is a continued, growing interest in polyploidy as a research topic. Over the past 25 years, the sequencing and analysis of plant genomes has revolutionized our understanding of the importance of polyploid speciation to the evolution of land plants.

Towards the conservation of the crucian carp in Europe: Prolific hybridization but no evidence for introgression between native and non-native taxa.

Hybridization plays a pivotal role in evolution, influencing local adaptation and speciation. However, it can also reduce biodiversity, which is especially damaging when native and non-native species meet. Hybridization can threaten native species via competition (with vigorous hybrids), reproductive resource wastage and gene introgression. The latter, in particular, could result in increased fitness in invasive species, decreased fitness of natives and compromise reintroduction or recovery conservation practices. In this study, we use a combination of RAD sequencing and microsatellites for a range-wide sample set of 1366 fish to evaluate the potential for hybridization and introgression between native crucian carp (Carassius carassius) and three non-native taxa (Carassius auratus auratus, Carassius auratus gibelio and Cyprinus carpio) in European water bodies. We found hybridization between native and non-native taxa in 82% of populations with non-natives present, highlighting the potential for substantial ecological impacts from hybrids on crucian carp populations. However, despite such high rates of hybridization, we could find no evidence of introgression between these taxa. The presence of triploid backcrosses in at least two populations suggests that the lack of introgression among these taxa is likely due to meiotic dysfunction in hybrids, leading to the production of polyploid offspring which are unable to reproduce sexually. This result is promising for crucian reintroduction programs, as it implies limited risk to the genetic integrity of source populations. Future research should investigate the reproductive potential of triploid hybrids and the ecological pressures hybrids impose on C. carassius.

Polyploids of Brassicaceae: Genomic Insights and Assembly Strategies.

The Brassicaceae family is distinguished by its inclusion of high-value crops such as cabbage, broccoli, mustard, and wasabi, all noted for their glucosinolates. In this family, many polyploidy species are distributed and shaped by numerous whole-genome duplications, independent genome doublings, and hybridization events. The evolutionary trajectory of the family is marked by enhanced diversification and lineage splitting after paleo- and meso-polyploidization, with discernible remnants of whole-genome duplications within their genomes. The recent neopolyploidization events notably increased the proportion of polyploid species within the family. Although sequencing efforts for the Brassicaceae genome have been robust, accurately distinguishing sub-genomes remains a significant challenge, frequently complicating the assembly process. Assembly strategies include comparative analyses with ancestral species and examining k-mers, long terminal repeat retrotransposons, and pollen sequencing. This review comprehensively explores the unique genomic characteristics of the Brassicaceae family, with a particular emphasis on polyploidization events and the latest strategies for sequencing and assembly. This review will significantly improve our understanding of polyploidy in the Brassicaceae family and assist in future genome assembly methods.

A Fungicide, Fludioxonil, Formed the Polyploid Giant Cancer Cells and Induced Metastasis and Stemness in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Fludioxonil, an antifungal agent used as a pesticide, leaves a measurable residue in fruits and vegetables. It has been identified to cause endocrine disruption, interrupt normal development, and cause various diseases such as cancers. In this study, fludioxonil was examined for its effects on the development and metastasis of breast cancer cells. On fludioxonil exposure (10-5 M) for 72 h, mutant p53 (mutp53) MDA-MB-231 triple-negative breast cancer (TNBC) cells significantly inhibited cell viability and developed into polyploid giant cancer cells (PGCCs), with an increase in the number of nuclei and expansion in the cell body size. Fludioxonil exposure disrupted the normal cell cycle phase ratio, resulting in a new peak. In addition, PGCCs showed greater motility than the control and were resistant to anticancer drugs, i.e., doxorubicin, cisplatin, and 5-fluorouracil. Cyclin E1, nuclear factor kappa B (NF-κB), and p53 expressions were remarkably increased, and the expression of cell cycle-, epithelial-mesenchymal-transition (EMT)-, and cancer stemness-related proteins were increased in the PGCCs. The daughter cells obtained from PGCCs had the single nucleus but maintained their enlarged cell size and showed greater cell migration ability and resistance to the anticancer agents. Consequently, fludioxonil accumulated Cyclin E1 and promoted the inflammatory cytokine-enriched microenvironment through the up-regulation of TNF and NF-κB which led to the transformation to PGCCs via abnormal cell cycles such as mitotic delay and mitotic slippage in mutp53 TNBC MDA-MB-231 cells. PGCCs and their daughter cells exhibited significant migration ability, chemo-resistance, and cancer stemness. These results strongly suggest that fludioxonil, as an inducer of potential genotoxicity, may induce the formation of PGCCs, leading to the formation of metastatic and stem cell-like breast cancer cells.

Plant chromosome polytenization contributes to suppression of the root growth in high-polyploids.

Autopolyploidization, which refers to a polyploidization via genome duplication without a hybridization, promotes growth in autotetraploids, but suppresses growth in high-polyploids (autohexaploids or autooctoploids). The mechanism underlying this growth suppression (i.e., "high-ploidy syndrome") has not been comprehensively characterized. In this study, we conducted a kinematic analysis of the root apical meristem cells in Arabidopsis thaliana autopolyploids (diploid, tetraploid, hexaploid, and octoploid) to determine the effects of the progression of genome duplication on root growth. The results of the root growth analysis showed that tetraploidization increases the cell volume, but decreases cell proliferation. However, cell proliferation and volume growth are suppressed in high-polyploids. The whole-mount fluorescence in situ hybridization analysis revealed extensive chromosome polytenization in the region where cell proliferation does not usually occur in the high-polyploid roots, which is likely at least partly correlated with the suppression of endoreduplication. The study findings suggest that chromosome polytenization is important for the suppressed growth of high-polyploids.

Cells in the Polyaneuploid Cancer Cell State are Pro-Metastatic.

There remains a large need for a greater understanding of the metastatic process within the prostate cancer field. Our research aims to understand the adaptive - ergo potentially metastatic - responses of cancer to changing microenvironments. Emerging evidence has implicated a role of the Polyaneuploid Cancer Cell (PACC) state in metastasis, positing the PACC state as capable of conferring metastatic competency. Mounting in vitro evidence supports increased metastatic potential of cells in the PACC state. Additionally, our recent retrospective study of prostate cancer patients revealed that PACC presence in the prostate at the time of radical prostatectomy was predictive of future metastatic progression. To test for a causative relationship between PACC state biology and metastasis, we leveraged a novel method designed for flow-cytometric detection of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in subcutaneous, caudal artery, and intracardiac mouse models of metastasis. This approach provides both quantitative and qualitative information about the number and PACC-status of recovered CTCs and DTCs. Collating data from all models, we found that 74% of recovered CTCs and DTCs were in the PACC state. In vivo colonization assays proved PACC populations can regain proliferative capacity at metastatic sites following dormancy. Additional direct and indirect mechanistic in vitro analyses revealed a PACC-specific partial Epithelial-to-Mesenchymal-Transition phenotype and a pro-metastatic secretory profile, together providing preliminary evidence that PACCs are mechanistically linked to metastasis.

Expansions and contractions of repetitive DNA elements reveal contrasting evolutionary responses to the polyploid genome shock hypothesis in Brachypodium model grasses.

Brachypodium grass species have been selected as model plants for functional genomics of grass crops, and to elucidate the origins of allopolyploidy and perenniality in monocots, due to their small genome sizes and feasibility of cultivation. However, genome sizes differ greatly between diploid or polyploid Brachypodium lineages. We have used genome skimming sequencing data to uncover the composition, abundance, and phylogenetic value of repetitive elements in 44 representatives of the major Brachypodium lineages and cytotypes. We also aimed to test the possible mechanisms and consequences of the "polyploid genome shock hypothesis" (PGSH) under three different evolutionary scenarios of variation in repeats and genome sizes of Brachypodium allopolyploids. Our data indicated that the proportion of the genome covered by the repeatome in the Brachypodium species showed a 3.3-fold difference between the highest content of B. mexicanum-4x (67.97%) and the lowest of B. stacei-2x (20.77%), and that changes in the sizes of their genomes were a consequence of gains or losses in their repeat elements. LTR-Retand and Tekay retrotransposons were the most frequent repeat elements in the Brachypodium genomes, while Ogre retrotransposons were found exclusively in B. mexicanum. The repeatome phylogenetic network showed a high topological congruence with plastome and nuclear rDNA and transcriptome trees, differentiating the ancestral outcore lineages from the recently evolved core-perennial lineages. The 5S rDNA graph topologies had a strong match with the ploidy levels and nature of the subgenomes of the Brachypodium polyploids. The core-perennial B. sylvaticum presents a large repeatome and characteristics of a potential post-polyploid diploidized origin. Our study evidenced that expansions and contractions in the repeatome were responsible for the three contrasting responses to the PGSH. The exacerbated genome expansion of the ancestral allotetraploid B. mexicanum was a consequence of chromosome-wide proliferation of TEs and not of WGD, the additive repeatome pattern of young allotetraploid B. hybridum of stabilized post-WGD genome evolution, and the genomecontraction of recent core-perennials polyploids (B. pinnatum, B. phoenicoides) of repeat losses through recombination of these highly hybridizing lineages. Our analyses have contributed to unraveling the evolution of the repeatome and the genome size variation in model Brachypodium grasses.

Polyploid giant cancer cells: origin, possible pathways of formation, characteristics, and mechanisms of regulation.

Polyploid giant cancer cells (PGCCs) are characterized by the presence of either a single enlarged nucleus or multiple nuclei and are closely associated with tumor progression and treatment resistance. These cells contribute significantly to cellular heterogeneity and can arise from various stressors, including radiation, chemotherapy, hypoxia, and environmental factors. The formation of PGCCs can occur through mechanisms such as endoreplication, cell fusion, cytokinesis failure, mitotic slippage, or cell cannibalism. Notably, PGCCs exhibit traits similar to cancer stem cells (CSCs) and generate highly invasive progeny through asymmetric division. The presence of PGCCs and their progeny is pivotal in conferring resistance to chemotherapy and radiation, as well as facilitating tumor recurrence and metastasis. This review provides a comprehensive analysis of the origins, potential formation mechanisms, stressors, unique characteristics, and regulatory pathways of PGCCs, alongside therapeutic strategies targeting these cells. The objective is to enhance the understanding of PGCC initiation and progression, offering novel insights into tumor biology.

Disentangling sources of gene tree discordance for Hordeum species via target-enriched sequencing assays.

Hordeum is an economically and evolutionarily important genus within the Triticeae tribe of the family Poaceae, and contains 33 widely distributed and diverse species which cytologically represent four subgenomes (H, Xa, Xu and I). These wild species (except Hordeum spontaneum, which is the primary gene pool of barley) are secondary or tertiary gene-pool germplasms for barley and wheat improvement, and uncovering their complicated evolutionary relationships would benefit for future breeding programs. Here, we developed a complexity-reduced pipeline via capturing genome-wide distributed fragments via two novel target-enriched assays (HorCap v1.0 and BarPlex v1.0) in conjugation with high-throughput sequencing of the enrichments. Both assays were tested for genotyping 40 species from three genera (Hordeum, Triticum, and Aegilops) containing 82 samples 67 accessions. Either of both assays worked efficiently in genotyping, while integration of both assays can significantly improve the robustness and resolution of the Hordeum phylogenetic trees. Interestingly, the incomplete lineage sorting (ILS) was inferred for the first time as the major factor causing phylogenetic discordance among the four subgenomes, whereas in New World species (carrying I genome) post-speciation introgression events were revealed. Through revising the evolutionary relationships of the Hordeum species based on an ancestral state reconstruction for the diploids and parental donor inference for the polyploids, our results raised new queries about the Hordeum phylogeny. Moreover, both newly-developed assays are applicable in genotyping and phylogenetic analysis of Hordeum and other Triticeae wild species.

Cytological Observation and RNA-Seq Analyses Reveal miR9564 and Its Target Associated with Pollen Sterility in Autotetraploid Rice.

Understanding the regulation of autotetraploid sterility is essential for harnessing the strong advantages in genomic buffer capacity, biodiversity, and heterosis of autotetraploid rice. miRNAs play crucial roles in fertility regulation, yet information about their reproductive roles and target genes in tetraploid rice remains limited. Here, we used three tetraploid lines, H1 (fertile), HF (fertile), and LF (sterile), to investigate cytological features and identify factors associated with autotetraploid sterility. LF showed abnormal meiosis, resulting in low pollen fertility and viability, ultimately leading to scarce fertilization and a low-seed setting compared to H1 and HF. RNA-seq revealed 30 miRNA-candidate target pairs related to autotetraploid pollen sterility. These pairs showed opposite expression patterns, with differential expression between fertile lines (H1 and HF) and the sterile line (LF). qRT-PCR confirmed that miR9564, miR528, and miR27874 were highly expressed in the anthers of H1 and HF but not in LF, while opposite results were obtained in their targets (ARPS, M2T, and OsRPC53). Haplotype and expression pattern analyses revealed that ARPS was specifically expressed in lines with the same haplotype of MIR9564 (the precursor of miR9564) as LF. Furthermore, the Dual-GFP assay verified that miR9564 inhibited the fluorescence signal of ARPS-GFP. The over-expression of ARPS significantly decreased the seed setting rate (59.10%) and pollen fertility (50.44%) of neo-tetraploid rice, suggesting that ARPS plays important roles in autotetraploid pollen sterility. This study provides insights into the cytological characteristic and miRNA expression profiles of tetraploid lines with different fertility, shedding light on the role of miRNAs in polyploid rice.

Intersectional Hybrids between Darrow's Blueberry (V. darrowii Camp) and Lingonberry (V. vitis-idaea L.).

An initial cross of V. darrowii 'Johnblue' (Darrow's blueberry) × V. vitis-idaea 'Red Sunset' (lingonberry) produced more than 30 true intersectional diploid hybrids as confirmed by molecular markers. The most vigorous of these hybrids was extensively evaluated. This hybrid, US 2535-A, was floriferous and morphologically intermediate to the respective parents. Examination of pollen suggested low male fertility. Numerous crosses using the hybrid as a female reflected similarly low fertility and potential crossing barriers. Stylar examination suggested blockage of pollen tube growth in self-pollinations and significantly retarded growth in backcross pollinations. Nonetheless, two confirmed hybrid offspring were produced using the F1 hybrid as a female in crosses with V. vitis-idaea and V. darrowii, respectively. In a second set of crosses utilizing additional V. darrowii and V. vitis-idaea genotypes, another 23 verified hybrids in seven parental combinations were produced. Hybrids such as the ones presented offer the potential for generating de novo interspecific fruit types in blueberry and/or broadening the adaptation of lingonberry.

Origin and diversity of the wild cottons (Gossypium hirsutum) of Mound Key, Florida.

Elucidating genetic diversity within wild forms of modern crops is essential for understanding domestication and the possibilities of wild germplasm utilization. Gossypium hirsutum is a predominant source of natural plant fibers and the most widely cultivated cotton species. Wild forms of G. hirsutum are challenging to distinguish from feral derivatives, and truly wild populations are uncommon. Here we characterize a population from Mound Key Archaeological State Park, Florida using genome-wide SNPs extracted from 25 individuals over three sites. Our results reveal that this population is genetically dissimilar from other known wild, landrace, and domesticated cottons, and likely represents a pocket of previously unrecognized wild genetic diversity. The unexpected level of divergence between the Mound Key population and other wild cotton populations suggests that the species may harbor other remnant and genetically distinct populations that are geographically scattered in suitable habitats throughout the Caribbean. Our work thus has broader conservation genetic implications and suggests that further exploration of natural diversity in this species is warranted.

Generation of Polyploid Giant Cancer Cells (PGCCs) from Hey Ovarian Cancer Cells and Analysis of Embryonic Properties.

Polyploid giant cancer cells (PGCCs) play a fundamental role in tumor initiation, dormancy, drug resistance, and metastasis, although the detailed biology of PGCCs remains poorly understood. The lack of literature on establishing a reproducible in vitro system for generating PGCCs is the leading technological obstacle to studying the biology of PGCCs. Here we provide a detailed protocol for generating stable PGCCs from Hey cancer cells and studying the PGCCs' embryonic stemness. This protocol includes (1) generating PGCCs of high purity in 2D culture by exposing Hey cells to paclitaxel, monitoring the cell cycle and amitotic budding of daughter cells from PGCCs, and collecting and studying the daughter cells; (2) inducing PGCCs to form spheroids expressing embryonic stemness markers and observing the spheroids' cleavage and blastocyst-like structure; and (3) inducing redifferentiation of PGCCs into different lineages of differentiated cells.

Single-Cell MTT: A Simple and Sensitive Assay for Determining the Viability and Metabolic Activity of Polyploid Giant Cancer Cells (PGCCs).

Solid tumors and tumor-derived cell lines commonly contain highly enlarged (giant) cancer cells that enter a state of transient dormancy (active sleep) after they are formed, but retain viability, secrete growth promoting factors, and exhibit the ability to generate rapidly proliferating progeny with stem cell-like properties. Giant cells with a highly enlarged nucleus or multiple nuclei are often called polyploid giant cancer cells (PGCCs). Although PGCCs constitute only a subset of cells within a solid tumor/tumor-derived cell line, their frequency can increase markedly following exposure to ionizing radiation or chemotherapeutic drugs. In this chapter we outline a simple and yet highly sensitive cell-based assay, called single-cell MTT, that we have optimized for determining the viability and metabolic activity of PGCCs before and after exposure to anticancer agents. The assay measures the ability of individual PGCCs to convert the MTT tetrazolium salt to its water insoluble formazan metabolite. In addition to evaluating PGCCs, this assay is also a powerful tool for determining the viability and metabolic activity of cancer cells undergoing premature senescence following treatment with anticancer agents, as well as for distinguishing dead cancer cells and dying cells (e.g., exhibiting features of apoptosis, ferroptosis, etc.) that have the potential to resume proliferation through a process called anastasis.