Heterogeneity Of Cancer Stem Cells Research Articles

Abstract 2580: Targeting chromatin in ovarian cancer stem cells

Abstract The dynamic organization of chromatin, governed by epigenetic modifiers, critically regulates cell plasticity and determines cell fate in both normal and malignant stem cells. We hypothesized that chromatin architecture plays a role in regulating transcriptional heterogeneity in ovarian cancer stem cells (OCSCs) to maintain stemness and chemoresistance. PWS microscopy showed that ALDH+ OCSCs derived displayed higher nuclear fractal dimension D signals (P<0.05) than ALDH- non-OCSCs, indicating that chromatin in OCSCs possesses higher packing compared to non-OCSCs. Cut & TAG Sequencing showed that of the 3606-histone repressive H3K27m3 enriched regions (P<0.05, fold change >2), 3208 were detected in OCSCs while 398 peaks were present in non-OCSCs, consistent with findings supporting high D features in OCSCs. The active mark H3K4me3 was enriched at the promoter of stemness-associated genes, including SOX2, ALDH1A3, and FZD7 in OCSCs; and the repressive histone mark H3K27me3 was enriched at the promoter of the epithelial cell marker CDH1 in OCSCs. These results support that the chromatin of OCSCs contains open genomic regions, corresponding to stemness-associated genes and repressed regions associated with genes related to differentiation. RNA sequencing indicated that the coefficient of variance (COV) reflecting transcriptional heterogeneity in OCSCs was higher than in non-OCSCs at baseline. In response to platinum, genes with low baseline expression in OCSCs were most upregulated, supporting increased transcriptional heterogeneity in response to chemotherapy. Inhibitors of epigenetic modifiers (DNMTi, EZH2i, and Dot1Li) induced chromatin re-organization in OCSCs and promoted OCSCs transition toward differentiation, supporting that stemness-associated chromatin structure is targetable. We demonstrated that a Dot1L inhibitor (Dot1Li) targeted OCSCs by decreasing chromatin packing D, resulting in decreased transcriptional heterogeneity. In conclusion, OCSCs harbor highly packed chromatin, contributing to chemoresistance and cell plasticity. Epigenetic modifiers (such as Dot1Li) inhibit stemness by regulating chromatin organization and transcriptional malleability. Citation Format: Yinu Wang, Jane Frederick, Yaqi Zhang, Elizabeth Thomas Bartom, Greta Bauer, Edward Tanner, Vadim Backman, Daniela Matei. Targeting chromatin in ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2580.

Abstract 540: Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development

Abstract Once cancer has metastasized, it is incurable because tumors evolve resistance to all systemically administered anti-cancer therapies. Resistance to therapy has classically been attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. Extracellular vesicles (EVs) are nano-sized (40-1000 nm) vesicles that are released by all cells into the extracellular space. EVs carry diverse cargo dependent on the cell of origin and have been studied as a source of novel cancer-specific liquid biopsy biomarkers and contributors to tumor progression and metastasis. However, the paracrine function of EVs in promoting therapy resistance is under-studied, and the impact of EVs released from therapy-resistant cancer cells on surrounding therapy-sensitive cancer cells is unknown. In this study, we develop an innovative microfluidic device, TREVCo, to enable simultaneous single-cell trap, EV detection, and co-culture modules to evaluate EVs-mediated transfer of cellular information between therapy-resistant cancer cells and adjacent cells, and to characterize EVs released from resistant cells and determine their impact on the resistance phenotype of adjacent therapy-sensitive cancer cells. We utilize resistant cells from multiple prostate (PCa) cell lines that have been selected for therapy resistance by culture with increasing dosages of cancer drugs such as cisplatin, docetaxel. We use the matched untreated therapy-sensitive parental cell lines as the sensitive cells. We hypothesize a novel model of therapy resistance in lethal PCa: EVs released from resistant cells contain pro-resistance factors that are transferred to adjacent sensitive cells upon EV uptake, thus directly enabling therapeutic resistance. We will assess the impact of resistant cell-EVs at a single cell level via a novel device, TREVCo, that enables comprehensive downstream analysis. This single-cell approach enables dissection of critical EV-associated therapy-resistant factors within the large heterogeneity of resistant PCa. Co-culture of a single resistant cell and a hundred adjacent sensitive cells mimicking intra-tumoral interactions is also critical to demonstrate evidence of resistant cell secreting factors inducing a pro-resistance phenotype. These studies will be the first-in-field to assess the impact of resistant PCa cell-derived EVs at single-cell resolution. An understanding of resistant cell-derived EVs and the impact of that EV cargo on adjacent sensitive cells will provide critical insight into the acquisition of therapy resistance in lethal PCa. The novel approach to apply resistant cell-derived EVs in co-culture assay with sensitive cells will enable the screening of transmittable resistance-associated factors and provides critical opportunities for accelerating precision medicine for advanced PCa patients. Citation Format: Chi-Ju Kim, Sarah Amend, Kenneth Pienta. Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 540.

Cell cycle heterogeneity and plasticity of colorectal cancer stem cells.

Cancer stem cells (CSCs) are a long-lived and self-renewing cancer cell population that drives tumor propagation and maintains cancer heterogeneity. They are also implicated in the therapeutic resistance of various types of cancer. Recent studies of CSCs in colorectal cancer (CRC) have uncovered fundamental paradigms that have increased understanding of CSC systems in solid tumors. Colorectal CSCs share multiple biological properties with normal intestinal stem cells (ISCs), including expression of the stem cell marker Lgr5. New evidence suggests that colorectal CSCs manifest substantial heterogeneity, as exemplified by the existence of both actively cycling Lgr5+ CSCs as well as quiescent Lgr5+ CSCs that are resistant to conventional anticancer therapies. The classical view of a rigid cell hierarchy and irreversible cell differentiation trajectory in normal and neoplastic tissues is now challenged by the finding that differentiated cells have the capacity to revert to stem cells through dynamic physiological reprogramming events. Such plasticity of CSC systems likely underlies both carcinogenesis and therapeutic resistance in CRC. Further characterization of the mechanisms underpinning the heterogeneity and plasticity of CSCs should inform future development of eradicative therapeutic strategies for CRC.

Prognostic clinical phenotypes associated with tumor stemness in the immune microenvironment of T-cell exhaustion for hepatocellular carcinoma

T-cell exhaustion (TEX) and high heterogeneity of cancer stem cells (CSCs) are associated with progression, metastasis, and treatment resistance in hepatocellular carcinoma (HCC). Here, we aim to characterize TEX-stemness-related genes (TEXSRGs) and screen for HCC patients who are more sensitive to immunotherapy. The immune cell abundance identifier (ImmuCellAI) was utilized to precisely evaluate the abundance of TEX and screen TEX-related genes. The stemness index (mRNAsi) of samples was analyzed through the one-class logistic regression (OCLR) algorithm. Application of the non-negative matrix decomposition algorithm (NMF) for subtype identification of HCC samples. The different subtypes were assessed for differences in prognosis, tumor microenvironment (TME) landscape, and immunotherapy treatment response. Then, the TEXSRGS-score, which can accurately forecast the survival outcome of HCC patients, was built by LASSO-Cox and multivariate Cox regression, and experimentally validated for the most important TEXSRGs. We also analyzed the expression of TEXSRGs and the infiltration of CD8+ T cells in clinical samples using qRT-PCR and immunohistochemistry (IHC). Based on 146 TEXSRGs, we found two distinct clinical phenotypes with different TEX infiltration abundance, tumor stemness index, enrichment pathways, mutational landscape, and immune cell infiltration through the non-negative matrix decomposition algorithm (NMF), which were confirmed in the ICGC dataset. Utilizing eight TEXSRGs linked to clinical outcome, we created a TEXSRGs-score model to further improve the clinical applicability. Patients can be divided into two groups with substantial differences in the characteristics of immune cell infiltration, TEX infiltration abundance, and survival outcomes. The results of qRT-PCR and IHC analysis showed that PAFAH1B3, ZIC2, and ESR1 were differentially expressed in HCC and normal tissues and that patients with high TEXSRGs-scores had higher TEX infiltration abundance and tumor stemness gene expression. Regarding immunotherapy reaction and immune cell infiltration, patients with various TEXSRGs-score levels had various clinical traits. The outcome and immunotherapy efficacy of patients with low TEXSRGs-score was favorable. In conclusion, we identified two clinical subtypes with different prognoses, TEX infiltration abundance, tumor cell stemness index, and immunotherapy response based on TEXSRGs, and developed and validated a TEXSRGs-score capable of accurately predicting survival outcomes in HCC patients by comprehensive bioinformatics analysis. We believe that the TEXSRGs-score has prospective clinical relevance for prognostic assessment and may help physicians select prospective responders in preference to current immune checkpoint inhibitors (ICIs).

Notch receptor/ligand diversity: contribution to colorectal cancer stem cell heterogeneity.

Cancer cell heterogeneity is a key contributor to therapeutic failure and post-treatment recurrence. Targeting cell subpopulations responsible for chemoresistance and recurrence seems to be an attractive approach to improve treatment outcome in cancer patients. However, this remains challenging due to the complexity and incomplete characterization of tumor cell subpopulations. The heterogeneity of cells exhibiting stemness-related features, such as self-renewal and chemoresistance, fuels this complexity. Notch signaling is a known regulator of cancer stem cell (CSC) features in colorectal cancer (CRC), though the effects of its heterogenous signaling on CRC cell stemness are only just emerging. In this review, we discuss how Notch ligand-receptor specificity contributes to regulating stemness, self-renewal, chemoresistance and cancer stem cells heterogeneity in CRC.

Fatty Acid Metabolites and the Tumor Microenvironment as Potent Regulators of Cancer Stem Cell Signaling.

Individual cancer cells are not equal but are organized into a cellular hierarchy in which only a rare few leukemia cells can self-renew in a manner reminiscent of the characteristic stem cell properties. The PI3K/AKT pathway functions in a variety of cancers and plays a critical role in the survival and proliferation of healthy cells under physiologic conditions. In addition, cancer stem cells might exhibit a variety of metabolic reprogramming phenotypes that cannot be completely attributed to the intrinsic heterogeneity of cancer. Given the heterogeneity of cancer stem cells, new strategies with single-cell resolution will become a powerful tool to eradicate the aggressive cell population harboring cancer stem cell phenotypes. Here, this article will provide an overview of the most important signaling pathways of cancer stem cells regarding their relevance to the tumor microenvironment and fatty acid metabolism, suggesting valuable strategies among cancer immunotherapies to inhibit the recurrence of tumors.

Abstract 3805: Revealing the role of microRNAs in the emergence of cancer stem cell heterogeneity during colorectal cancer progression

Abstract Our goal is to identify microRNAs (miRNAs) that target mRNAs involved in stem cell (SC) signaling pathways involved in colorectal cancer (CRC) tumorigenesis. SC overpopulation drives CRC progression but exact mechanism is unclear. Hypothesis: specific miRNAs target SC genes, which leads to the emergence of multiple cancer SC (CSC) sub-populations and tumor heterogeneity. We used flow cytometry to identify CSC sub-populations based on expression of SC markers including ALDH1, LGR5 and others in the HT-29 CRC cell line. CSC sub-populations were isolated and measured using FACS and expression of miRNAs in each CSC sub-population was quantified using NanoString profiling. The level of miRNA upregulation in each CSC sub-population was ranked based on differential expression of the miRNAs between different CSC sub-populations. We chose to further analyze the LGR5 and ALDH SC sub-populations because they showed: i) the largest difference between CSC sub-population sizes, ii) the smallest size (<1%) of the co-positive (LGR5+/ALDH+) cell sub-population, iii) the highest numbers of differentially expressed miRNAs. Note that WNT signaling mainly occurs via LGR5+ CSC and retinoic acid (RA) signaling mainly occurs via ALDH+ CSCs. We report herein miRNAs that are differentially expressed between ALDH+/LGR5- compared to ALDH-/LGR5- subpopulation and LGR5+/ALDH- versus ALDH-/LGR5-. The mRNAs expressed in CSC subpopulations identified from Nanostring profiling were analyzed using bioinformatics for their predicted binding by upregulated miRNAs. Our findings show that: 1) multiple CSC subpopulations exist in the HT-29 cell population; 2) each CSC subpopulation has a unique miRNA signature. Notably, RXRA mRNA is significantly upregulated in the ALDH+/LGR5- CSC sub-population but not in the LGR5+/ALDH- CSC subpopulation. We identified 5 miRs upregulated in the LGR5+/ALDH- CSC subpopulation that are predicted to target the 3’UTR of RXRA mRNA. One of these miRs, miR-660-3p, is specifically upregulated in the LGR5+/ALDH- CSC subpopulation and binding of miR-660-3p to RXRA mRNA was validated using luciferase assay in HT-29 cells. We previously reported that RXRA is selectively expressed in ALDH + CSC sub-populations. Also, RXRA is known to bind with β-catenin leading to its degradation and suppression of WNT-based transcriptional activity. Taken together, these results indicate that expression of miR-660-3p, by inhibiting RXRA expression, increases WNT signaling and decreases RA signaling in LGR5+/ALDH- CSCs. Thus, upregulated miR-660-3p targets a key gene in the RA signaling pathway (RXRA) which could contribute to the emergence of SC sub-populations during CRC development. Citation Format: Molly Lausten, Victoria Stark, Caroline Facey, Lynn Opdenaker, Bruce M. Boman. Revealing the role of microRNAs in the emergence of cancer stem cell heterogeneity during colorectal cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3805.

Are There Specific Cancer Stem Cell Markers?

The cancer stem cell (CSC) model states that heterogeneous tumor cell populations are organized in a hierarchical manner, with a small population of CSCs at the apex. These CSCs are capable of self-renewal and giving rise to other cancer cell populations, conceptually analogous to the function of normal adult stem cells present in almost all organs. However, there has been significant controversy regarding the existence and identification of CSCs. We argue that technical differences in experimentation and CSC assays, CSC niche-dependency and plasticity, and CSC heterogeneity itself may explain some of the differences observed.

Targeting redox regulation and autophagy systems in cancer stem cells.

Cancer is a dysregulated cellular level pathological condition that results in tumor formation followed by metastasis. In the heterogeneous tumor architecture, cancer stem cells (CSCs) are essential to push forward the progression of tumors due to their strong pro-tumor properties such as stemness, self-renewal, plasticity, metastasis, and being poorly responsive to radiotherapy and chemotherapeutic agents. Cancer stem cells have the ability to withstand various stress pressures by modulating transcriptional and translational mechanisms, and adaptable metabolic changes. Owing to CSCs heterogeneity and plasticity, these cells display varied metabolic and redox profiles across different types of cancers. It has been established that there is a disparity in the levels of Reactive Oxygen Species (ROS) generated in CSCs vs Non-CSC and these differential levels are detected across different tumors. CSCs have unique metabolic demands and are known to change plasticity during metastasis by passing through the interchangeable epithelial and mesenchymal-like phenotypes. During the metastatic process, tumor cells undergo epithelial to mesenchymal transition (EMT) thus attaining invasive properties while leaving the primary tumor site, similarly during the course of circulation and extravasation at a distant organ, these cells regain their epithelial characteristics through Mesenchymal to Epithelial Transition (MET) to initiate micrometastasis. It has been evidenced that levels of Reactive Oxygen Species (ROS) and associated metabolic activities vary between the epithelial and mesenchymal states of CSCs. Similarly, the levels of oxidative and metabolic states were observed to get altered in CSCs post-drug treatments. As oxidative and metabolic changes guide the onset of autophagy in cells, its role in self-renewal, quiescence, proliferation and response to drug treatment is well established. This review will highlight the molecular mechanisms useful for expanding therapeutic strategies based on modulating redox regulation and autophagy activation to targets. Specifically, we will account for the mounting data that focus on the role of ROS generated by different metabolic pathways and autophagy regulation in eradicating stem-like cells hereafter referred to as cancer stem cells (CSCs).

Genetic heterogeneity of liver cancer stem cells.

Cancer cell heterogeneity is a serious problem in the control of tumor progression because it can cause chemoresistance and metastasis. Heterogeneity can be generated by various mechanisms, including genetic evolution of cancer cells, cancer stem cells (CSCs), and niche heterogeneity. Because the genetic heterogeneity of CSCs has been poorly characterized, the genetic mutation status of CSCs was examined using Exome-Seq and RNA-Seq data of liver cancer. Here we show that different surface markers for liver cancer stem cells (LCSCs) showed a unique propensity for genetic mutations. Cluster of differentiation 133 (CD133)-positive cells showed frequent mutations in the IRF2, BAP1, and ERBB3 genes. However, leucine-rich repeat-containing G protein-coupled receptor 5-positive cells showed frequent mutations in the CTNNB1, RELN, and ROBO1 genes. In addition, some genetic mutations were frequently observed irrespective of the surface markers for LCSCs. BAP1 mutations was frequently observed in CD133-, CD24-, CD13-, CD90-, epithelial cell adhesion molecule-, or keratin 19-positive LCSCs. ASXL2, ERBB3, IRF2, TLX3, CPS1, and NFATC2 mutations were observed in more than three types of LCSCs, suggesting that common mechanisms for the development of these LCSCs. The present study provides genetic heterogeneity depending on the surface markers for LCSCs. The genetic heterogeneity of LCSCs should be considered in the development of LCSC-targeting therapeutics.

First person – Paul Ann Riya and Budhaditya Basu

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Paul Ann Riya and Budhaditya Basu are co-first authors on ‘ HES1 promoter activation dynamics reveal the plasticity, stemness and heterogeneity in neuroblastoma cancer stem cells’, published in JCS. Paul Ann Riya is a PhD student in the lab of Dr Jackson James at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India, investigating the key mechanisms that maintain stem cells. Budhaditya Basu is a PhD student in same lab, where he works on understanding the role of circular RNA in axon path finding.

HES1 promoter activation dynamics reveal the plasticity, stemness and heterogeneity in neuroblastoma cancer stem cells.

Notch signaling and its downstream gene target HES1 play a critical role in regulating and maintaining cancer stem cells (CSCs), similar to as they do during embryonic development. Here, we report a unique subclass of Notch-independent Hes-1 (NIHes-1)-expressing CSCs in neuroblastoma. These CSCs maintain sustained HES1 expression by activation of HES1 promoter region upstream of classical CBF-1 binding sites, thereby completely bypassing Notch receptor-mediated activation. These stem cells have self-renewal ability and potential to generate tumors. Interestingly, we observed that NIHes-1 CSCs could transition to Notch-dependent Hes-1-expressing (NDHes-1) CSCs where HES1 is expressed by Notch receptor-mediated promoter activation. We observed that NDHes-1-expressing CSCs also had the potential to transition to NIHes-1 CSCs and during this coordinated bidirectional transition, both CSCs gave rise to the majority of the bulk cancer cells, which had an inactive HES1 promoter (PIHes-1). A few of these PIHes-1 cells were capable of reverting into a CSC state. These findings explain the existence of a heterogenic mode of HES1 promoter activation within the IMR-32 neuroblastoma cell line and the potential to switch between them. This article has an associated First Person interview with the first authors of the paper.

Childhood Obesity: A Potential Key Factor in the Development of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a malignant primary tumor type of the central nervous system (CNS). This type of brain tumor is rare and is responsible for 12–15% of all brain tumors. The typical survival rate of GBM is only 12 to 14 months. GBM has a poor and unsatisfactory prognosis despite advances in research and therapeutic interventions via neurosurgery, radiation, and chemotherapy. The molecular heterogeneity, aggressive nature, and occurrence of drug-resistant cancer stem cells in GB restricts the therapeutic efficacy. Interestingly, the CNS tumors in children are the second most usual and persistent type of solid tumor. Since numerous research studies has shown the association between obesity and cancer, childhood obesity is one of the potential reasons behind the development of CNS tumors, including GBM. Obesity in children has almost reached epidemic rates in both developed and developing countries, harming children’s physical and mental health. Obese children are more likely to face obesity as adults and develop non-communicable diseases such as diabetes and cardiovascular disease as compared to adults with normal weight. However, the actual origin and cause of obesity are difficult to be pointed out, as it is assumed to be a disorder with numerous causes such as environmental factors, lifestyle, and cultural background. In this narrative review article, we discuss the various molecular and genetic drivers of obesity that can be targeted as potential contributing factors to fight the development of GBM in children.

Abstract 6066: Elucidating cancer stem cells heterogeneity in colorectal cancer by single-cell RNA sequencing

Abstract Cancer stem cells (CSCs) are a small subpopulation of cells with capabilities of self-renewal and differentiation potential and have been suggested to play a critical role in tumor-initiation, metastasis, and multidrug resistance. The marker genes used to identify CSCs in CRC and transcriptional state of these cell is not fully elucidated due to the plasticity and dynamic nature of CSCs. In this study, we perform single-cell RNA sequencing (scRNA-seq) on primary CRC and liver metastases from CRC to evaluate the CSC population. We profiled approximately 80,000 single cells from neoadjuvant chemotherapy (NAC) treated patients generated using 10X Chromium scRNA-seq and Illumina platforms. A total of 16 samples were analyzed (5 colon tumor, 3 colon matched normal, 3 liver metastasis, 5 liver matched normal), 4 samples failed quality control and were excluded. Following extensive quality control and batch effect correction, we performed dimensionality reduction and differential expression analysis with Seurat v3.0 and custom R packages. We used a set of 11 canonical colon CSC marker genes (CD44, PROM1, LGR5, LRIG1, ASCL2, EPHB2, OLFM4, AXIN2, SLC12A2, RNF43, LEFTY1) as CSCs signature to score the stemness of each cell by AddModuleScore. Established marker genes were used to identify cell type assignment including stem/TA, immature enterocytes, immature secretory, enterocytes, Best4 enterocytes, goblet, tuff, CD4, CD8, Treg, NK, B, plasma, macrophages, monocytes, dendritic cells, fibroblasts, endothelium, glia, pericytes and hepatocytes. In the 16,703 epithelial cells, the CSC signature genes were diffusely expressed in stem/TA and immature epithelium but not in a specific cell cluster. There was minimal overlap in expression of the 11 CSC marker genes at the single-cell level. By clustering the CSCs identified by high CSC signature score, we found CSCs grouped distinctly by patients, indicating significant patient to patient variability. Furthermore, CSCs did not cluster by positivity for any specific marker gene, suggesting that marker genes may just be markers, and do not define distinct transcriptional states. Gene set enrichment analysis (GSEA) showed intestinal stemness was significantly enriched (FDR < 0.01) in CSCs compared with non-CSCs. Comparing CSCs to stem cells from normal samples, suppression of the fatty acid metabolism pathway was the only common feature seen in CSCs from each tumor. The heterogeneity of CSC marker genes in CRC tumor cells suggests that defining CSCs by single or few marker genes is inadequate. GSEA verified that a CSC scoring system aggregating expression of 11 canonical colon CSC marker genes can identify CSCs from scRNA-seq. There is marked transcriptional heterogeneity in CSCs, with few common features from CSCs from different tumors. It is hoped that future study of how the diversity of CSCs states evolve under chemotherapy will shed light on overcoming chemo-resistance. Citation Format: Kangyu Lin, John Paul Shen. Elucidating cancer stem cells heterogeneity in colorectal cancer by single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6066.

Characterization of stem cell landscape and identification of stemness-relevant prognostic gene signature to aid immunotherapy in colorectal cancer

BackgroundIt is generally accepted that colorectal cancer (CRC) originates from cancer stem cells (CSCs), which are responsible for CRC progression, metastasis and therapy resistance. The high heterogeneity of CSCs has precluded clinical application of CSC-targeting therapy. Here, we aimed to characterize the stemness landscapes and screen for certain patients more responsive to immunotherapy.MethodsTwenty-six stem cell gene sets were acquired from StemChecker database. Consensus clustering algorithm was applied for stemness subtypes identification on 1,467 CRC samples from TCGA and GEO databases. The differences in prognosis, tumor microenvironment (TME) components, therapy responses were evaluated among subtypes. Then, the stemness-risk model was constructed by weighted gene correlation network analysis (WGCNA), Cox regression and random survival forest analyses, and the most important marker was experimentally verified.ResultsBased on single-sample gene set enrichment analysis (ssGSEA) enrichments scores, CRC patients were classified into three subtypes (C1, C2 and C3). C3 subtype exhibited the worst prognosis, highest macrophages M0 and M2 infiltrations, immune and stromal scores, and minimum sensitivity to immunotherapies, but was more sensitive to drugs like Bosutinib, Docetaxel, Elesclomol, Gefitinib, Lenalidomide, Methotrexate and Sunitinib. The turquoise module was identified by WGCNA that it was most positively correlated with C3 but most negatively with C2, and five hub genes in turquoise module were identified for stemness model construction. CRC patients with higher stemness scores exhibited worse prognosis, more immunosuppressive components in TME and lower immunotherapeutic responses. Additionally, the model’s immunotherapeutic prediction efficacy was further confirmed from two immunotherapy cohorts (anti-PD-L1 in IMvigor210 cohort and anti-PD-1 in GSE78220 cohort). Mechanistically, Gene Set Enrichment Analysis (GSEA) results revealed high stemness score group was enriched in interferon gamma response, interferon alpha response, P53 pathway, coagulation, apoptosis, KRAS signaling upregulation, complement, epithelial–mesenchymal transition (EMT) and IL6-mediated JAK-STAT signaling gene sets.ConclusionsOur study characterized three stemness-related subtypes with distinct prognosis and TME patterns in CRC patients, and a 5-gene stemness-risk model was constructed by comprehensive bioinformatic analyses. We suggest our stemness model has prospective clinical implications for prognosis evaluation and might facilitate physicians selecting prospective responders for preferential use of current immune checkpoint inhibitors.

Abstract B022: The polyaneuploid transition as a hedge against failures in resistance acquisition

Abstract Metastatic cancer is incurable because tumors evolve resistance to all classes of therapy, resulting the in the deaths of 10 million people each year. Therapy resistance is typically attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. However, therapeutic resistance can also emerge through a transient cell state: the Polyaneuploid Cancer Cell (PACC) state. The Polyaneuploid transition (PAT) is an evolutionary conserved polyploid program accessed by cancer cells in response to an external stress such as chemotherapy. Once the stress subsides, cells exit the PACC state and repopulate a non-polyploid population that is resistant to multiple classes of therapy. We hypothesize that the PAT facilitates inherited universal therapeutic resistance. We utilized the bacteria E. coli as a model system of the evolutionary conserved polyploid mechanism of resistance. The E. coli strain MG1655 was engineered to express an hupA::GFP reporter to monitor nucleoid DNA or a ibpA::GFP reporter to monitor the aggregation of misfolded proteins. We treated these cultures with ciprofloxacin at near lethal doses for 12 hours and recorded the dynamics with fluorescent time-lapse microscopy. Individual bacteria underwent a transient phase of polyploidization causing an elongated filamentous phenotype that is at least an order of magnitude bigger compared to the unstressed bacteria. During filamentation, we found that the misfolded protein aggregates are shuttled along the cell body to the tips. Eventually, the filaments bud resulting in a population of “typical” length cells. The buds that are produced by the filaments are filled with an excess of misfolded protein aggregate and, surprisingly, are commonly void of DNA. This response is unexpected in a cell that is already resistant due to the high fitness cost. However, if the cell is not resistant, then each misfolded protein may represent an attempt to solve the antibiotic puzzle and to achieve a resistant phenotype. In turn, the ability to efficiently collect and dispose of the misfolded proteins, i.e., the previous failed attempt, may be advantageous to reduce the need for protein degradation and ensure that future protein production is not impaired. Taken together, this finding suggests that antibiotic resistance might be dependent on the ability to endure repeated futile endeavors to explore the phenotypic landscape rather than selecting for individuals that already exist at an evolutionary optimum. As a result, the PAT may act as a buffer by which cancer cells “buy time” to search within the phenotypic landscape and look for a path to resistance. Future work will specifically test this model in cancer cells. If true, then this finding would suggest that therapeutic resistance can be actively delayed by inhibiting the PAT during first line treatment. Citation Format: George Butler, Julia Bos, Robert H. Austin, Sarah R. Amend, Kenneth J. Pienta. The polyaneuploid transition as a hedge against failures in resistance acquisition [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B022.

Heterogeneity of Cancer Stem Cells in Tumorigenesis, Metastasis, and Resistance to Antineoplastic Treatment of Head and Neck Tumours.

The discovery of a small subset of cancer cells with self-renewal properties that can give rise to phenotypically diverse tumour populations has shifted our understanding of cancer biology. Targeting cancer stem cells (CSCs) is becoming a promising therapeutic strategy in various malignancies, including head and neck squamous cell carcinoma (HNSCC). Diverse sub-populations of head and neck cancer stem cells (HNCSCs) have been identified previously using CSC specific markers, the most common being CD44, Aldehyde Dehydrogenase 1 (ALDH1), and CD133, or by side population assays. Interestingly, distinct HNCSC subsets play different roles in the generation and progression of tumours. This article aims to review the evidence for a role of specific CSCs in HNSCC tumorigenesis, invasion, and metastasis, together with resistance to treatment.

Cancer stem cells in hepatocellular carcinoma - from origin to clinical implications.

Hepatocellular carcinoma (HCC) is an aggressive disease with a poor clinical outcome. The cancer stem cell (CSC) model states that tumour growth is powered by a subset of tumour stem cells within cancers. This model explains several clinical observations in HCC (as well as in other cancers), including the almost inevitable recurrence of tumours after initial successful chemotherapy and/or radiotherapy, as well as the phenomena of tumour dormancy and treatment resistance. The past two decades have seen a marked increase in research on the identification and characterization of liver CSCs, which has encouraged the design of novel diagnostic and treatment strategies for HCC. These studies revealed novel aspects of liver CSCs, including their heterogeneity and unique immunobiology, which are suggestive of opportunities for new research directions and potential therapies. In this Review, we summarize the present knowledge of liver CSC markers and the regulators of stemness in HCC. We also comprehensively describe developments in the liver CSC field with emphasis on experiments utilizing single-cell transcriptomics to understand liver CSC heterogeneity, lineage-tracing and cell-ablation studies of liver CSCs, and the influence of the CSC niche and tumour microenvironment on liver cancer stemness, including interactions between CSCs and the immune system. We also discuss the potential application of liver CSC-based therapies for treatment of HCC.

A review of the racial heterogeneity of breast cancer stem cells.

Breast Cancer Stem Cells has become the toast of many breast cancer investigators in the past two decades owing to their crucial roles in tumourigenesis, progression, differentiation, survival and chemoresistance. Despite the growing list of research data in this field, racial or ethnic comparison studies on these stem cells remain scanty. This study is a comparative racial analysis of putative breast cancer stem cells. Research articles on the clinicopathological significance of breast cancer stem cells within a period of 17years (2003-2020) were reviewed across 5 major races (African/Black American, Asian, Caucasian/White, Hispanic/Latino, and American). The associations between the stem cells markers (CD44+/CD24-/low, BMI1, ALDH1, CD133, and GD2) and clinicopathological and clinical outcomes were analysed. A total of 40 studies were included in this study with 50% Asian, 25% Caucasian, 10% African, 5% American and 2.5% Hispanic/Latino, and 7.5% other mixed races. CD44+/CD24-/low has been associated with TNBC/Basal like phenotype across all races. It is generally associated with poor clinicopathological features such as age, tumour size, lymph node metastasis and lymphovascular invasion. In Asians, CD44+/CD24-/low was associated with DFS and OS but not in Caucasians. ALDH1 was the most studied breast CSC marker (40% of all studies on breast cancer stem cell markers) also associated with poor clinicopathological features including size, age, stage, lymph node metastasis and Nottingham Prognostic Index. ALDH1 was also associated with DFS and OS in Asians but not Caucasians. Racial variations exist in breast cancer stem cell pattern and functions but ill-defined due to multiple factors. Further research is required to better understand the role of breast CSC.

349P The amount of cancer stem cells and the sensitivity to anticancer drugs in glioblastoma primary cell culture

It is assumed that aggressiveness and therapy resistance of glioblastoma multiforme (GBM) is determined by its high heterogeneity and presence of cancer stem cells (CSC). The primary glioblastoma cell lines (pGBM lines) could better than the cultured cell lines reflect the tumor heterogeneity and, thus, provide the additional opportunities for personalized medicine. In our study, we compared the response of the pGBM lines and cell glioblastoma lines to chemotherapy drugs with the CSC content in cell cultures.