Nanomechanical Single-Cell Profiling Reveals Mechanical Dormancy Underlying Radiation Resistance in Polyploid Giant Cancer Cells.

Over a century ago, it was found that cancer cells often have extra chromosomes; that is, normal human cells contain 46 chromosomes, whereas cancer cells contain abnormal numbers of chromosomes with cell-to-cell variability. Polyploid giant cancer cells (PGCCs) refer to a special subpopulation of cancer cells with giant and multinuclei and contribute to solid tumor heterogeneity. PGCCs differ from normal cells and even other cancer cells in cell size, morphology, proliferation pattern, expression of cell differentiation markers, and chromosome numbers and contribute to tumor formation and chemoradioresistance. The shape of PGCC nuclei is usually irregular and the size is at least three to five times larger than those of regular diploid cancer cells. PGCCs are the key contributor to the heterogeneity of human solid cancers and chromosome structural abnormalities, such as inversions, deletions, duplications, and translocations. Mechanistically, PGCCs could be formed through end reduplication or cell fusion, reverting to regular cancer cells through splitting, budding, or burst-like mechanisms. PGCCs are divided asymmetrically and cycled slowly to form a dynamic population. However, these giant cells can also revert to regular-sized cancer cells through a reductive division, named as depolyploidization. Asymmetric cell division of giant cancer cells by meiosis-like depolyploidization had been previously proposed to explain the unexpected life cycle of these cells. In this special issue, D. Zhang et al. reported the asymmetric cell division in polyploid giant cancer cells and low eukaryotic cells and revealed the similarities in the budding process between yeast and PGCCs. This mechanism of PGCCs initialed the daughter cell generation which has also been reported in the normal growth of skeletal muscle and osteoclasts and in cells infected by virus or in vitro cell culture. Moreover, PGCCs were able to express certain normal and cancer stem cell markers and differentiate into the adipose tissue, cartilage, and bone. Single PGCC was able to form cancer spheroids in vitro and generate tumor xenograft in immunodeficient mice, indicating that these PGCCs had remarkable biologic features of cancer stem cells. Furthermore, PGCCs are able to generate erythrocytes in vitro and in vivo besides their cancer stem cell properties. The difference of erythrocytes generated by bone marrow and PGCCs is the different forms of hemoglobin (see below). In human body, erythrocytes are produced in the bone marrow with a process known as hematopoiesis. The bone marrow stroma contains mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), which give rise to erythrocytes, leukocytes, and platelets. In adults, bone marrow is generally considered the main source of erythrocytes. However, PGCCs have an ability to generate erythrocytes in vitro and in vivo. During cancer development, tumor cells undergo avascular growth. However, after a tumor mass reached a certain size, vasculogenic mimicry (VM) will connect with endothelium dependent vessels to obtain sufficient blood and oxygen supply to support further growth of tumor cells and support tumor invasion and metastasis. Accumulating evidence has demonstrated that different types of cancer utilize VM to form a blood supply network to support their growth, invasion, and metastasis and, clinically, such a tumor is usually associated with poor prognosis. However, the source of erythrocytes in VM remains unclear. PGCCs can be induced by treatment of cancer cells with cobalt chloride (a hypoxia mimic) in vitro and hypoxia will increase self-renewal of cancer stem cells and promote the stem cell-like phenotype besides induction of PGCCs formation. Moreover, hypoxia also promotes the formation of vasculogenic mimicry (VM). B. Sun et al. showed that hypoxia inducible factor-1α plays an important role in the VM formation, while L. Zhang et al. provided the evidence that erythroid cells were localized in the cytoplasm of or around the PGCCs in serous ovarian carcinoma tissues and cancer cells in the VM structures and that these erythroid cells expressed hemoglobin-β/γ/e/δ and hemoglobin-ζ detected by immunostaining. Thus, these VM structures can be formed by PGCCs or other cancer cells and their newly generated fetal erythrocytes with high O2 binding affinity. In addition, in this special issue, W. Wang et al. and L. Yao et al. demonstrated that epithelial-mesenchymal transition and Wnt signaling pathway could regulate the VM formation. Thus, elucidation of the molecular mechanisms of PGCC and VM formation could provide a novel insight into research in embryology, stem cells, and tumorigenesis. Identification of the PGCCs and tumor-derived erythrocytes could be a survival mechanism in hypoxia and targeting of PGCCs might be further developed as a potential therapeutic strategy for human cancers. Research focus on VM-targeted therapies could include dendritic cell vaccine and cytokine-induced killer cell therapy to conquer the recurrence and metastasis of aggressive cancers. Shiwu Zhang Xiaochun Xu Siwei Zhu Jun Liu

BackgroundGenomic instability and chemoresistance can arise in cancer due to a unique form of plasticity: that of polyploid giant cancer cells (PGCCs). These cells form under the stress of chemotherapy and have higher than diploid chromosome content. PGCCs are able to then repopulate tumors through an asymmetric daughter cell budding process. PGCCs have been observed in ovarian cancer histology, including the deadly and common form high-grade serous ovarian carcinoma (HGSC). We previously discovered that drugs which disrupt the cellular recycling process of autophagy are uniquely efficacious in pre-clinical HGSC models. While autophagy induction has been associated with PGCCs, it has never been previously investigated if autophagy modulation interacts with the PGCC life cycle and this form of tumor cell plasticity.MethodsCAOV3 and OVCAR3 ovarian cancer cell lines were treated with carboplatin or docetaxel to induce PGCC formation. Microscopy was used to characterize and quantify PGCCs formed by chemotherapy. Two clinically available drugs that inhibit autophagy, hydroxychloroquine and nelfinavir, and a clinically available activator of autophagy, rapamycin, were employed to test the effect of these autophagy modulators on PGCC induction and subsequent colony formation from PGCCs. Crystal violet-stained colony formation assays were used to quantify the tumor-repopulating stage of the PGCC life cycle.ResultsAutophagy inhibitors did not prevent PGCC formation in OVCAR3 or CAOV3 cells. Rapamycin did not induce PGCC formation on its own nor did it exacerbate PGCC formation by chemotherapy. However, hydroxychloroquine prevented efficient colony formation in CAOV3 PGCCs induced by carboplatin (27% inhibition) or docetaxel (41% inhibition), as well as in OVCAR3 cells (95% and 77%, respectively). Nelfinavir similarly prevented colony formation in CAOV3 PGCCs induced by carboplatin (64% inhibition) or docetaxel (94% inhibition) as well as in OVCAR3 cells (89% and 80%, respectively). Rapamycin surprisingly also prevented PGCC colony outgrowth (52–84% inhibition).ConclusionsWhile the autophagy previously observed to correlate with PGCC formation is unlikely necessary for PGCCs to form, autophagy modulating drugs severely impair the ability of HGSC PGCCs to form colonies. Clinical trials which utilize hydroxychloroquine, nelfinavir, and/or rapamycin after chemotherapy may be of future interest.

Introduction/Objective Polyploid giant cancer cells (PGCCs) are large, mono- or multinucleated tumor cells implicated in cancer initiation, metastasis, and therapy resistance. First described in 1914, PGCCs have been observed across various solid tumors, including breast, prostate, and ovarian cancers. They are believed to arise through endoreplication in response to environmental stressors such as hypoxia, inflammation, and anticancer therapies. Despite their clinical relevance, PGCCs remain underrecognized due to their morphological heterogeneity and the absence of standardized detection methods. Their identification is further complicated by the lack of computational tools capable of reliably detecting and characterizing them in tumor samples. In ovarian cancer, particularly high-grade serous ovarian carcinoma (HGSOC), PGCCs may contribute to treatment failure and poor prognosis. This study aimed to address this gap by integrating immunohistochemistry (IHC) with digital pathology to develop a reproducible method for PGCC detection. Using membrane-targeted markers, we sought to delineate tumor cell boundaries and identify PGCCs in formalin-fixed paraffin-embedded (FFPE) samples. The study also evaluated the association between PGCC burden and clinical, histopathological, and molecular features in ovarian cancer. By establishing a standardized detection method, this research aims to enhance our understanding of PGCCs and their potential prognostic value in ovarian cancer management. Methods/Case Report This study evaluated four membrane markers—EpCAM, Na+/K+ ATPase, cadherins, and PMCA1—via immunohistochemistry (IHC) in 40 aggressive tumor cases, including high-grade serous ovarian carcinoma (HGSOC), glioblastoma, triple-negative breast cancer, and pancreatic adenocarcinoma. Marker performance was assessed based on specificity, intensity, uniformity, reproducibility, and compatibility with other IHC reagents. EpCAM was selected for its superior membrane delineation and applied to 26 FFPE ovarian cancer samples collected between 2015 and 2022. Slides were digitized using the Aperio ScanScope CS System and analyzed with Aperio ImageScope software. PGCCs were identified and scored (0–3) using both digital pathology and conventional light microscopy. EpCAM staining intensity was evaluated visually and digitally, and differences were statistically analyzed using the Wilcoxon signed-rank test. Clinical data (age, stage, treatment type), histological parameters (mitotic index, morphology), and molecular findings (BRCA, TP53, ATM, CHEK2) were collected. TP53 mutations were assessed via next-generation sequencing (NGS) in 25 patients. Treatment sequence (primary debulking vs. neoadjuvant chemotherapy) and survival outcomes were also analyzed. The study aimed to correlate PGCC burden with clinical and molecular features to assess its prognostic significance. All procedures were conducted under institutional review board approval and followed standard pathology protocols. Results All four membrane markers delineated tumor cell membranes effectively, enabling PGCC identification. EpCAM, Na+/K+ ATPase, and PMCA1 outperformed cadherins in staining quality. EpCAM was selected for further analysis due to its superior performance. In the 26 HGSOC cases, EpCAM staining revealed significant intra- and intertumoral heterogeneity. PGCCs were more frequently identified using digital pathology than visual assessment, with statistically significant differences in scoring (Wilcoxon signed-rank test, p < 0.05). Patients with low PGCC scores (0–1) had a longer median overall survival (62 months) compared to those with high scores (2–3; 39 months), though this difference did not reach statistical significance (Mann–Whitney U test: p = 0.066). At the time of analysis, 50% of low-score patients were alive versus 17% in the high-score group (Fisher’s Exact Test: p = 0.149). All sequenced tumors harbored TP53 mutations, including frameshifts, missense substitutions, and deletions, with strong concordance between mutation type and p53 IHC patterns. BRCA mutations were present in ∼18% of cases; ATM and CHEK2 mutations were less frequent. Patients who underwent primary debulking surgery showed a trend toward improved survival compared to those receiving neoadjuvant chemotherapy, although this was not statistically significant (p = 0.096). Conclusion This study establishes a reproducible method for detecting polyploid giant cancer cells (PGCCs) in high-grade serous ovarian carcinoma using EpCAM-based immunohistochemistry and digital pathology. Digital analysis proved more sensitive than visual assessment, enabling consistent identification of PGCCs and revealing a trend toward poorer survival in patients with higher PGCC scores. Although not statistically significant, this trend suggests a potential prognostic role for PGCCs. The universal presence of TP53 mutations and the observed variability in treatment outcomes underscore the biological and clinical complexity of HGSOC. These findings support further investigation into PGCCs as prognostic biomarkers and therapeutic targets in ovarian cancer.

Whole genomic duplications (WGD) have been reported in 37% to 56% of high-grade cancers. WGD leads to formation of polyploid giant cancer cells (PGCCs) with salient and atypical nuclear morphology noted by light microscopy. However, PGCCs are generally considered nonviable due to their inability to execute mitosis. We have recently reported that PGCCs are capable of generating mitosis-competent progeny cells via primitive amitotic division including budding, splitting, and burst. We have further demonstrated that PGCCs are part of the process of somatic reprogramming named the giant cell life cycle composed of four distinct but overlapping phases including initiation, self-renewal, termination, and stability. The giant cell life cycle resets the somatic cell from the mitotic cycle to stress-induced endoreplication cell cycle via WGD to form PGCCs. The PGCCs activate endogenous embryonic factors for the reprogramming, which leads to the birth of drug-resistant cells by clonal selection from newly generated heterogeneous cancer stem cells. However, the underlying genetic and epigenetic mechanisms during WGD remain unclear. By mapping DNA methylation and whole-genome sequencing, monitoring the telomere dynamics, and tracking the cellular and nuclear morphology using microscopic time-lapse imaging observations during the time course of giant cell life cycle, we hereby provide new evidence that the daughters cells derived from PGCCs have massive alterations at the whole-genome and epigenome levels. The reprogrammed resistant daughter cells are achieved, at least in part, by genomic instability via shortened telomeres. Targeting PGCCs and promoting their differentiation during the course of giant cell life cycle may represent a novel approach to intercept drug resistance. Citation Format: Na Niu, Xiaoran Li, Jun Yao, Anil Sood, Jinsong Liu. Polyploid giant cancer cells lead to evolution of drug resistance in high-grade ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A17.

Simple SummaryRecently, cells of large size called PGCC (Polyploid Giant Cancer Cells) have emerged as a pillar in cancer development and progression, possibly being the “first cells” from which the cancer starts. PGCC have been studied in cancer tissues from patients and in laboratory models. They have also been found in the blood, occasionally. By applying a method able to detect rare cells in urine, we found these PGCC in the urine of patients with prostate cancer. No study has ever published this finding. Our work is preliminary but deserves to be shared with the scientific community as it opens the way for more studies targeting the role of these PGCC and their possible use as an early and non-invasive marker of prostate cancer development.Prostate cancer is the third cause of cancer-related deaths in men. Its early and reliable diagnosis is still a public health issue, generating many useless prostate biopsies. Prostate cancer cells detected in urine could be the target of a powerful test but they are considered too rare. By using an approach targeting rare cells, we have analyzed urine from 45 patients with prostate cancer and 43 healthy subjects under 50 y.o. We observed a relevant number of giant cells in patients with cancer. Giant cells, named Polyploid Giant Cancer Cells (PGCC), are thought to be involved in tumorigenesis and treatment resistance. We thus performed immune-morphological studies with cancer-related markers such as α-methylacyl-CoA racemase (AMACR), prostate-specific membrane antigen (PSMA), and telomerase reverse transcriptase (TERT) to understand if the giant cells we found are PGCC or other urinary cells. We found PGCC in the urine of 22 patients, including those with early-stage prostate cancer, and one healthy subject. Although these results are preliminary, they provide, for the first time, clinical evidence that prostate cancers release PGCC into the urine. They are expected to stimulate further studies aimed at understanding the role of urinary PGCC and their possible use as a diagnostic tool and therapeutic target.

Polyploid giant cancer cells (PGCCs) are a distinct subpopulation of tumor cells characterized by enlarged morphology, increased nuclear content, and stem cell-like plasticity. Once considered senescent or non-functional, PGCCs are now recognized as critical drivers of tumor progression, metastasis, therapeutic resistance, and relapse. Their formation can be triggered by various stresses, including chemotherapy, radiotherapy, targeted therapies, as well as by other conditions such as endoplasmic reticulum (ER) stress or hypoxia. Mechanistically, PGCCs arise through processes such as endoreplication, mitotic slippage, cell fusion, and failed cytokinesis, which enable cells to escape mitotic catastrophe and transition into a polyploid state. Under therapeutic stress, PGCCs can persist by adopting a dormant or quiescent phenotype and later resume proliferation through neosis, characterized by asymmetric cytokinesis, generating daughter cells with enhanced migratory, invasive, and tumor-initiating capabilities. These progenies, along with the PGCCs themselves, frequently exhibit cancer stem cell (CSC)-like traits and undergo epithelial-mesenchymal transition (EMT), contributing to tumor heterogeneity and plasticity. Key signaling pathways implicated in PGCC biology include IL-6/IL-6R signaling, unfolded protein response (UPR), impaired p53 pathway, Aurora kinase B (AURKB) inhibition, and activation of the PLK4/CDC25C axis. PGCCs have also been shown to promote angiogenesis, induce therapy resistance, and evade immune surveillance. Clinically, elevated PGCC levels correlate with poor prognosis and resistance across multiple cancer types, including breast, colorectal, lung, ovarian, and so on. Given their unique properties and clinical relevance, PGCCs represent a promising frontier in cancer biology with the potential to overcome therapeutic resistance and prevent tumor recurrence through targeted interventions. This review seeks to elucidate the role of PGCCs across multiple cancer types and highlights their emerging potential as novel targets for future cancer therapies.Graphical

Purpose: We previously reported that polyploid giant cancer cells (PGCCs) induced by cobalt chloride (CoCl2) exhibit cancer stem cell properties. Daughter cells generated by PGCCs possess epithelial mesenchymal transition (EMT) phenotype changes and EMT plays an important role in cancer development and progression. This study investigated the characteristics of PGCCs from LoVo and HCT116 induced by CoCl2 and the clinicopathological significances of PGCCs in colorectal cancer (CRC).Materials and Methods: Western blotting and immunocytochemical staining were used to compare the expression levels of EMT-related proteins between PGCCs with budding daughter cells and the control cells. In addition, tissue samples were collected from 159 patients with CRC for analysis of PGCCs, vasculogenic mimicry (VM), and single stromal PGCCs with budding, as well as immunohistochemical staining for cathepsin B, vimentin, and hemoglobin A.Results: Single PGCCs induced by CoCl2 formed spheroids in vitro. Poorly differentiated CRCs showed the highest numbers of PGCCs and VM, and expression of cathepsin B. There was greater expression of EMT-related proteins in PGCCs with budding daughter cells than in control cells. The expression of vimentin located in PGCC nuclei. Single stomal PGCCs with budding were detected in 27.45% of well differentiated, 50% of moderately differentiated, and 90.20% of poorly differentiated CRC samples. PGCCs can generate erythroid cells that express delta-hemoglobin to form VM. Erythroid cells generated by PGCCs were positive for hemoglobin A immunocytochemical staining.Conclusion: PGCCs from LoVo and HCT116 treated by CoCl2 exhibited cancer stem cell properties. The number of PGCCs and VM were associated with CRC differentiation and daughter cells budded from PGCCs may promote the lymph node metastasis via expression of EMT-related proteins. PGCCs and their newly generated erythroid cells form VM structures.

Despite decades of progress in ovarian cancer treatment, therapeutic resistance remains a persistent and lethal challenge. Emerging evidence suggests polyploid giant cancer cells (PGCCs) as key drivers of this resistance. PGCCs are distinguished by multinucleation or a single oversized nucleus containing multiple chromosome sets. Polyploid cell populations are present in premalignant lesions and expand with disease progression and therapeutic stress. PGCCs demonstrate heightened resistance to treatment relative to non-PGCCs and can regenerate non-PGCCs via budding once therapeutic pressure is alleviated, leading to relapse. Supported by extensive preclinical and clinical evidence, we hypothesize that elimination of PGCCs may reduce therapeutic resistance in ovarian cancer. Given their significant role in resistance and relapse, PGCCs remain largely untargeted due to a lack of scalable detection methods. Standard viability assays such as MTT, XTT, and ATP-based screens fail to capture the rare but critical PGCC subpopulation. Current gold-standard methods, fluorescence-activated cell sorting (FACS) coupled with manual microscopy, while accurate, are time-intensive and unsuitable for high-throughput drug screening. Thus, a rapid and accurate technique for studying PGCCs is essential. In this pilot study, we have developed a high-throughput single-cell morphological analysis pipeline capable of precisely quantifying PGCC numbers and proportions. The pipeline accurately quantifies PGCCs based on nuclear and cellular features, including size and DNA content. Leveraging this platform, we screened a library of 2,726 FDA-approved compounds to identify agents that effectively kill PGCCs. Hits from this screen were validated in 3D spheroid cultures, revealing compounds with potent anti-PGCC activity. Complementary transcriptomic analysis (RNA-Seq) of flow-sorted PGCCs versus non-PGCCs unveiled profound dysregulation of the FOXM1 signaling axis, implicating this pathway as a key regulator of PGCC formation and survival. Consistent with this, the high-throughput screen identified Thiostrepton, a known FOXM1 inhibitor, as a selective anti-PGCC agent. Across multiple ovarian cancer cell lines, FOXM1 inhibitors outperformed standard-of-care agents (Paclitaxel, Cisplatin, Olaparib) in eliminating PGCCs. siRNA knockdown of FOXM1 significantly reduced PGCC numbers, particularly under Paclitaxel stress. Moreover, FOXM1 inhibition triggered apoptosis in PGCCs, an effect reversible with the pan-caspase inhibitor Z-VAD-FMK. Together, these findings underscore PGCCs as a therapeutically actionable cell state driving resistance in ovarian cancer. Our integrative single-cell morphological and transcriptomic platform enables rapid discovery of anti-PGCC agents and establishes FOXM1 inhibition as a compelling strategy to eradicate this resilient subpopulation. This pipeline offers broad utility for overcoming therapy resistance across diverse malignancies. Citation Format: Hsiao-Chun Chen, Yuan Zhang, Yushu Ma, Yu-Chih Chen. Combating ovarian cancer resistance via polyploid cancer cell targeted screening [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A023.

Our recent perplexing findings that polyploid giant cancer cells (PGCCs) acquired embryonic-like stemness and were capable of tumor initiation raised two important unanswered questions: how do PGCCs acquire such stemness, and to which stage of normal development do PGCCs correspond. Intriguingly, formation of giant cells due to failed mitosis/cytokinesis is common in the blastomere stage of the preimplantation embryo. However, the relationship between PGCCs and giant blastomeres has never been studied. Here, we tracked the fate of single PGCCs following paclitaxel-induced mitotic failure. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the blastomere, polyploid blastomere, compaction, morula and blastocyst-like stages by light, scanning electron or three-dimensional confocal scanning microscopy. Formation of PGCCs was associated with activation of senescence, while budding of daughter cells was associated with senescence escape. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2 and SSEA1 and lacked expression of Xist. PGCCs acquired mesenchymal phenotype and were capable of differentiation into all three germ layers in vitro. The embryonic-like stemness of PGCCs was associated with nuclear accumulation of YAP, a key mediator of the Hippo pathway. Spheroids derived from single PGCCs grew into a wide spectrum of human neoplasms, including germ cell tumors, high-grade and low-grade carcinomas and benign tissues. Daughter cells derived from PGCCs showed attenuated capacity for invasion and increased resistance to paclitaxel. We also observed formation of PGCCs and dedifferentiation in ovarian cancer specimens from patients treated with chemotherapy. Taken together, our findings demonstrate that PGCCs represent somatic equivalents of blastomeres, the most primitive cancer stem cells reported to date. Thus, our studies reveal an evolutionarily conserved archaic embryonic program in somatic cells that can be de-repressed for oncogenesis. Our work offers a new paradigm for cancer origin and disease relapse.

We have recently shown that polyploid giant cancer cells (PGCCs) are capable of tumor initiation and acquisition of embryonic-like stemness and thus represent a novel type of cancer stem cells. However, two important questions remain to be answered from this surprising finding: (1) how PGCCs acquire such stemness; (2) on which stage of normal development PGCCs correspond to. Here, we tracked the fate of single PGCCs induced via mitotic failure by paclitaxel. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the polyploid blastomere, compaction, morula, and blastocyst-like stages by scanning electron microscopy. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2, and SSEA-1 and lacked expression of Xist. PGCCs also showed time-dependent activation of expression of the germ layer-specific markers alpha-fetoprotein, smooth muscle actin, and β3-tubulin and were capable of redifferentiation into three germ layers in vitro. PGCCs-derived daughter cells showed attenuated invasive ability and increased resistance to paclitaxel. PGCCs-derived spheroids grew into a wide spectrum of human neoplasms, including malignant dysgerminoma and embryonic carcinoma, poorly differentiated or well-differentiated carcinomas, and benign squamous tissue. We also observed PGCCs in ovarian cancer from patients treated with chemotherapy. Thus, our data demonstrated that PGCCs acquired novel cancer stem cell properties and capability to redifferentiate into different tumors including the germ cell tumors, which are at the topmost developmental hierarchy. Our studies, for the first time, link PGCCs to the polyploid blastomere-like cancer stem cells and thus offer a new paradigm for the origin of cancer. Citation Format: Na Niu, Jinsong Liu. Dedifferentiation into polyploid blastomere-like cancer stem cells via formation of polyploid giant cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 924. doi:10.1158/1538-7445.AM2017-924

Are polyploid giant cancer cells in high grade serous carcinoma of the ovary blastomere-like cancer stem cells?

Polyploid giant cancer cells (PGCCs), characterised by multinucleation and atypical nuclear morphology, are a common feature of undifferentiated pleomorphic sarcomas. While PGCCs may be a critical substrate for cancer evolution, their formation pathways and genomic consequences remain underexplored. In this study, we characterise PGCCs in ten pleomorphic sarcomas and use topographic single-cell DNA sequencing (scDNA-seq) to investigate their genomic landscape. We selected PGCCs based on their nuclear morphology, including mononucleated or multinucleated bizarre, misshapen nuclei, and analysed them at single-cell resolution. Histopathological analysis showed that PGCCs were often randomly distributed throughout the tumour and did not appear in clusters, suggesting that they arise de novo rather than through clonal expansion. scDNA-seq revealed that PGCCs originate from the dominant tumour population and exhibit extensive copy number heterogeneity, either due to subsequent or ongoing chromosomal instability. Both clonal and subclonal chromothripsis-like events were identified in PGCCs, indicating that chromothripsis is a key driver of heterogeneity in these cells and is linked to multinucleation rather than mononuclear PGCC formation. FACS-based ploidy analysis of one undifferentiated pleomorphic sarcoma (UPS) revealed a twice whole-genome-duplicated population (6.2n) distinct from the bulk tumour (3.3n). This population contained all clonal, but none of the subclonal chromothripsis-like events observed in PGCCs. Our findings highlight PGCCs as a highly heterogeneous and evolutionarily dynamic component of UPSs. The recurrent chromothripsis-like events observed in PGCCs suggest ongoing genomic reshaping that may drive tumour progression and the poor clinical outcomes observed for these tumours.

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 differentiatedcells.

Cytoplasmic SIRT1 enhances the stemness of polyploid giant cancer cells by promoting β-catenin protein stability and nuclear accumulation in ovarian carcinoma upon neoadjuvant chemotherapy.

Radiation and chemotherapy are highly effective at killing cancer cells but cells that survive treatment often develop therapy-induced senescence (TIS). Since growth is arrested in TIS, this has been considered a positive treatment outcome; however, senescent cells that remain metabolically active and develop a senescence associated secretory phenotype (SASP) can also promote cancer progression. In addition, a small number of cancer cells are able to escape this dormant state and contribute to cancer progression. Polyploid giant cancer cells (PGCCs) represent a small subpopulation of non-mitotic cancer cells that evades treatment through periods of transient dormancy and then relapses into full-blown disease through amitotic budding into chemoresistant progenitor cells. The PGCC phenotype mimics senescent cells in multiple ways, including enlarged size, increased beta-galactosidase expression, increased metabolic activity, and pro-inflammatory SASP; yet, there is a significant gap in our understanding of how PGCCs contribute to TIS escape and subsequent chemoresistance. In addition, increased numbers of large PGCCs are seen in late stage and metastatic cancers; yet, their role in tumor recurrence and metastasis has not been established. We previously showed PGCCs have a unique actin cytoskeletal organization, giving rise to elevated stiffness and migratory persistence. These findings prompted us to further examine vimentin intermediate filaments (VIFs) that act as shock absorbers in the cell, protecting cells from compressive loads. Based on this, we postulated that PGCCs have unique adaptations in their vimentin structure to help support their enlarged morphology and drive their persistent migration. Indeed, we showed that PGCCs have increased levels of cytoplasmic vimentin and evenly distributed VIFs compared to non-PGCCs. This dispersed network of VIFs often polarized at the leading-edge during migration, and was necessary for PGCCs phenotype, as disruption of VIFs decreased PGCC volume and blocked migratory persistence. The VIF network also scaffolds lysosomes and autophagosomes in the cytosol to regulate their fusion during autophagy. Our new data clearly shows that inhibiting autophagy with Bafilomycin limits PGCC migration. In addition, targeting VIF structure also blocks autophagy. Thus, the structure of VIFs and autophagic flux are both critical in directing PGCCs migratory persistence. We have developed a novel 3D tumor microenvironment model that allows us to monitor cancer cell behavior over 4-6 weeks, which allows us to monitor cell recovery from TIS and dormancy. Using this model, we show that PGCC’s unique biophysical properties are directly linked to their dysregulated metabolism and altered cell structure. These studies provide critical information about how aging, TIS, and polyploidy affect evolving tumor microenvironments. Citation Format: Michelle R. Dawson, Deepraj Ghosh. Physical and metabolic aspects of therapy induced senescence and polyploidy in an evolving tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR007.