Subpopulation Of Tumor Cells Research Articles

Abstract 1487: Cytotoxic CD4+ T cells contribute to anti-tumor immune responses in NSCLC

Abstract Immune checkpoint blockade (ICB) is widely used to treat non-small cell lung cancer (NSCLC) patients and works by inhibiting the PD-1/PDL1 axis to reinvigorate exhausted T cells. In the prevailing model, the primary mechanism for direct tumor cell killing occurs through cytotoxic CD8+ T cells. Here, we use single-cell multi-omic profiling (a combination of single-cell RNA sequencing, TCR sequencing, and surface protein profiling) in 10 NSCLC patients to identify a population of CD4+ T cells that are tumor-infiltrating, clonally expanded, and express a cytotoxic gene program. Concordantly, we found in the same patients a subpopulation of tumor cells with elevated HLA class II expression, suggesting a mechanism for tumor-mediated antigen presentation to CD4+ T cells. Finally, we show that a cytotoxic CD4 gene signature is associated with improved progression-free survival in a cohort of 180 NSCLC patients treated with ICB regimens, including those with loss of heterozygosity at the HLA class I locus. Overall, these results suggest a model where cytotoxic CD4+ T cells can perform direct tumor cell killing in a class II restricted manner and that their presence is associated with favorable ICB outcomes in NSCLC. Citation Format: Denise Lau, Sonal Khare, Derek Reiman, Tim Rand, Ameen A. Salahudeen, Aly Khan. Cytotoxic CD4+ T cells contribute to anti-tumor immune responses in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1487.

Abstract 1387: MET inhibition radiosensitizes KRAS-mutant rectal cancer

Abstract Rectal carcinoma, representing about a third of all newly diagnosed colorectal cancers, is one of the most common malignant tumors. The standard of care for locally advanced rectal cancer (LARC), consisting of neoadjuvant chemo/radiotherapy prior to surgical resection, is poorly effective, leading to complete tumor regression only in 10-30% of the cases. Approximately 40% of LARC harbor activating KRAS mutations, which have been extensively associated with primary resistance to targeted EGFR therapy and with radioresistance as well. Previous work showed that the MET receptor tyrosine kinase supports radioresistance and can be inhibited to radiosensitize tumor cell subpopulations retaining stem-like properties. Here, we show that LARC often express high levels of MET and can be successfully radiosensitized by MET inhibition. This was assessed in rectal stem-like cells isolated from human tumors (rectospheres), and transplanted in the mouse to regenerate tumors that faithfully reproduce the phenotype, the genotype and the therapeutic response of the original tumor. Mechanistically, we found that radioresistant KRAS-mutant rectospheres display significantly higher basal levels of RAD51, a master regulator of DNA homologous recombination repair, and increased RAD51 recruitment to irradiation-induced DNA double-strand breaks, as compared with KRAS-wild type rectospheres. Importantly, we showed that MET pharmacological inhibition by small-molecule kinase inhibitors, combined with radiotherapy, impairs RAD51 expression and function. This leads to DNA damage accumulation and results in effective radiosensitization of K-RAS-mutant rectal stem-like cells in vitro and in vivo, and significant inhibition of experimental tumors. Therefore, preclinical evidence is provided that MET can be exploited as a therapeutic target to radiosensitize KRAS-mutant rectal cancer at stem-like cell level. Citation Format: Antonio D'Ambrosio, Federica Verginelli, Francesca Orzan, Raffaella Albano, Elena Casanova, Paolo Luraghi, Francesca De Bacco, Andrea Bertotti, Livio Trusolino, Rebecca Senetta, Anna Sapino, Elena Del Mastro, Marco Gatti, Paolo M. Comoglio, Carla Boccaccio. MET inhibition radiosensitizes KRAS-mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1387.

Starvation and Climate Change—How to Constrain Cancer Cell Epigenetic Diversity and Adaptability to Enhance Treatment Efficacy

Advanced metastatic cancer is currently not curable and the major barrier to eliminating the disease in patients is the resistance of subpopulations of tumor cells to drug treatments. These resistant subpopulations can arise stochastically among the billions of tumor cells in a patient or emerge over time during therapy due to adaptive mechanisms and the selective pressures of drug therapies. Epigenetic mechanisms play important roles in tumor cell diversity and adaptability, and are regulated by metabolic pathways. Here, I discuss knowledge from ecology, evolution, infectious disease, species extinction, metabolism and epigenetics to synthesize a roadmap to a clinically feasible approach to help homogenize tumor cells and, in combination with drug treatments, drive their extinction. Specifically, cycles of starvation and hyperthermia could help synchronize tumor cells and constrain epigenetic diversity and adaptability by limiting substrates and impairing the activity of chromatin modifying enzymes. Hyperthermia could also help prevent cancer cells from entering dangerous hibernation-like states. I propose steps to a treatment paradigm to help drive cancer extinction that builds on the successes of fasting, hyperthermia and immunotherapy and is achievable in patients. Finally, I highlight the many unknowns, opportunities for discovery and that stochastic gene and allele level epigenetic mechanisms pose a major barrier to cancer extinction that warrants deeper investigation.

Identification of TAZ-Dependent Breast Cancer Vulnerabilities Using a Chemical Genomics Screening Approach.

Breast cancer stem cells (BCSCs) represent a subpopulation of tumor cells that can self-renew and generate tumor heterogeneity. Targeting BCSCs may ameliorate therapy resistance, tumor growth, and metastatic progression. However, the origin and molecular mechanisms underlying their cellular properties are poorly understood. The transcriptional coactivator with PDZ-binding motif (TAZ) promotes mammary stem/progenitor cell (MaSC) expansion and maintenance but also confers stem-like traits to differentiated tumor cells. Here, we describe the rapid generation of experimentally induced BCSCs by TAZ-mediated reprogramming of human mammary epithelial cells, hence allowing for the direct analysis of BCSC phenotypes. Specifically, we establish genetically well-defined TAZ-dependent (TAZDEP) and -independent (TAZIND) cell lines with cancer stem cell (CSC) traits, such as self-renewal, variable resistance to chemotherapeutic agents, and tumor seeding potential. TAZDEP cells were associated with the epithelial to mesenchymal transition, embryonic, and MaSC signature genes. In contrast, TAZIND cells were characterized by a neuroendocrine transdifferentiation transcriptional program associated with Polycomb repressive complex 2 (PRC2). Mechanistically, we identify Cyclin D1 (CCND1) as a critical downstream effector for TAZ-driven tumorigenesis. Overall, our results reveal a critical TAZ-CCND1-CDK4/CDK6 signaling axis, suggesting novel therapeutic approaches to eliminate both BCSCs and therapy-resistant cancer cells.

Cancer Stem Cells as a Source of Drug Resistance in Bone Sarcomas.

Bone sarcomas are commonly characterized by a high degree of intra-tumor heterogeneity, which in part is due to the presence of subpopulations of tumor cells presenting stem cell properties. Similar to normal stem cells, these cancer stem cells (CSCs) display a drug resistant phenotype and therefore are responsible for relapses and tumor dissemination. Drug resistance in bone sarcomas could be enhanced/modulated during tumor evolution though the acquisition of (epi)-genetic alterations and the adaptation to changing microenvironments, including drug treatments. Here we summarize findings supporting the involvement of pro-stemness signaling in the development of drug resistance in bone sarcomas. This include the activation of well-known pro-stemness pathways (Wnt/β-Cat, NOTCH or JAT/STAT pathways), changes in the metabolic and autophagic activities, the alteration of epigenetic pathways, the upregulation of specific non-coding RNAs and the crosstalk with different microenvironmental factors. This altered signaling is expected to be translated to the clinic in the form of biomarkers of response and new therapies able to overcome drug resistance.

Association of Circadian Clock Gene Expression with Glioma Tumor Microenvironment and Patient Survival.

Simple SummaryGliomas are the most common type of malignant primary brain tumors and are classified according to the cell of origin and genetic features, which can help predict the prognosis and treatment sensitivity. Improving the prognosis remains a challenge; however, chronobiology is a promising field for future works, as circadian clock genes are linked to the tumor biology and outcomes in multiple cancers, including glioma. Here, we examined the relationship of circadian clock genes, IDH mutational status, and prognosis in glioma patients by using unsupervised clustering of the expression of 13 clock genes. We further explored the expression of the clock genes across the tumor regions and cell subpopulations, highlighting the importance of the tumor microenvironment in researching circadian rhythms in cancer. Our research is important for understanding how best to target circadian rhythms to improve patient outcomes in neuro-oncology.Circadian clock genes have been linked to clinical outcomes in cancer, including gliomas. However, these studies have not accounted for established markers that predict the prognosis, including mutations in Isocitrate Dehydrogenase (IDH), which characterize the majority of lower-grade gliomas and secondary high-grade gliomas. To demonstrate the connection between circadian clock genes and glioma outcomes while accounting for the IDH mutational status, we analyzed multiple publicly available gene expression datasets. The unsupervised clustering of 13 clock gene transcriptomic signatures from The Cancer Genome Atlas showed distinct molecular subtypes representing different disease states and showed the differential prognosis of these groups by a Kaplan–Meier analysis. Further analyses of these groups showed that a low period (PER) gene expression was associated with the negative prognosis and enrichment of the immune signaling pathways. These findings prompted the exploration of the relationship between the microenvironment and clock genes in additional datasets. Circadian clock gene expression was found to be differentially expressed across the anatomical tumor location and cell type. Thus, the circadian clock expression is a potential predictive biomarker in glioma, and further mechanistic studies to elucidate the connections between the circadian clock and microenvironment are warranted.

Fluorescence-Based Quantitative and Spatial Analysis of Tumour Spheroids: A Proposed Tool to Predict Patient-Specific Therapy Response.

Tumour spheroids are widely used to pre-clinically assess anti-cancer treatments. They are an excellent compromise between the lack of microenvironment encountered in adherent cell culture conditions and the great complexity of in vivo animal models. Spheroids recapitulate intra-tumour microenvironment-driven heterogeneity, a pivotal aspect for therapy outcome that is, however, often overlooked. Likely due to their ease, most assays measure overall spheroid size and/or cell death as a readout. However, as different tumour cell subpopulations may show a different biology and therapy response, it is paramount to obtain information from these distinct regions within the spheroid. We describe here a methodology to quantitatively and spatially assess fluorescence-based microscopy spheroid images by semi-automated software-based analysis. This provides a fast assay that accounts for spatial biological differences that are driven by the tumour microenvironment. We outline the methodology using detection of hypoxia, cell death and PBMC infiltration as examples, and we propose this procedure as an exploratory approach to assist therapy response prediction for personalised medicine.

Mini-Review: PDPK1 (3-phosphoinositide dependent protein kinase-1), An Emerging Cancer Stem Cell Target.

Cancer stem cells (CSCs) are subpopulations of tumor cells that possess abilities for self-renewal, differentiation, and tumor initiation. These rare but therapy-recalcitrant cells are assumed to repopulate tumors following administration of systemic chemotherapy driving therapy failure, tumor recurrence, and disease progression. In early clinical trials, anti-CSC therapies have found limited success to-date possibly due to the inherent heterogeneity and plasticity of CSCs and the incomplete characterization of essential CSC targets. Here, we review the role of 3-phosphoinositide dependent protein kinase-1 (PDPK1) as an emerging CSC target. While most previous studies have relied on CSC models which are based on lineage and tissue-specific marker profiles to define the relationships between putative target and CSC traits, this review discusses PDPK1 and its role in CSC biology with an emphasis on CSC systems which are based on proposed function like label-retaining cancer cells (LRCCs).

Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines

BackgroundRelapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics.MethodsCRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-cadherin, E-cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated.ResultsAlthough with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value < 0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value < 0.05). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 (p-value = 0.02). The significant up-regulation of promoting EMT genes, ZEB1, TWIST1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value = 0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value < 0.05) and N-cadherin down-regulation in both spheroids were observed.ConclusionEnrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.

The Cancer Cell Dissemination Machinery as an Immunosuppressive Niche: A New Obstacle Towards the Era of Cancer Immunotherapy.

Although cancer immunotherapy has resulted in unpreceded survival benefits to subsets of oncology patients, accumulating evidence from preclinical animal models suggests that the immunosuppressive tumor microenvironment remains a detrimental factor limiting benefit for many patient subgroups. Recent efforts on lymphocyte-mediated immunotherapies are primarily focused on eliminating cancer foci at primary and metastatic sites, but few studies have investigated the impact of these therapies on the highly complex process of cancer cell dissemination. The metastatic cascade involves the directional streaming of invasive/migratory tumor cells toward specialized blood vessel intravasation gateways, called TMEM doorways, to the peripheral circulation. Importantly, this process occurs under the auspices of a specialized tumor microenvironment, herewith referred to as “Dissemination Trajectory”, which is supported by an ample array of tumor-associated macrophages (TAMs), skewed towards an M2-like polarization spectrum, and which is also vital for providing microenvironmental cues for cancer cell invasion, migration and stemness. Based on pre-existing evidence from preclinical animal models, this article outlines the hypothesis that dissemination trajectories do not only support the metastatic cascade, but also embody immunosuppressive niches, capable of providing transient and localized immunosubversion cues to the migratory/invasive cancer cell subpopulation while in the act of departing from a primary tumor. So long as these dissemination trajectories function as “immune deserts”, the migratory tumor cell subpopulation remains efficient in evading immunological destruction and seeding metastatic sites, despite administration of cancer immunotherapy and/or other cytotoxic treatments. A deeper understanding of the molecular and cellular composition, as well as the signaling circuitries governing the function of these dissemination trajectories will further our overall understanding on TAM-mediated immunosuppression and will be paramount for the development of new therapeutic strategies for the advancement of optimal cancer chemotherapies, immunotherapies, and targeted therapies.

A theoretical analysis of tumour containment.

Recent studies have shown that a strategy aiming for containment, not elimination, can control tumour burden more effectively in vitro, in mouse models and in the clinic. These outcomes are consistent with the hypothesis that emergence of resistance to cancer therapy may be prevented or delayed by exploiting competitive ecological interactions between drug-sensitive and drug-resistant tumour cell subpopulations. However, although various mathematical and computational models have been proposed to explain the superiority of particular containment strategies, this evolutionary approach to cancer therapy lacks a rigorous theoretical foundation. Here we combine extensive mathematical analysis and numerical simulations to establish general conditions under which a containment strategy is expected to control tumour burden more effectively than applying the maximum tolerated dose. We show that containment may substantially outperform more aggressive treatment strategies even if resistance incurs no cellular fitness cost. We further provide formulas for predicting the clinical benefits attributable to containment strategies in a wide range of scenarios and compare the outcomes of theoretically optimal treatments with those of more practical protocols. Our results strengthen the rationale for clinical trials of evolutionarily informed cancer therapy, while also clarifying conditions under which containment might fail to outperform standard of care.

Single-cell transcriptomics reveals the landscape of intra-tumoral heterogeneity and transcriptional activities of ECs in CC

Cervical cancer (CC) is the fourth leading cause of deaths in gynecological malignancies. Although the etiology of CC has been extensively investigated, the exact pathogenesis of CC remains incomplete. Recently, single-cell technologies demonstrated advantages in exploring intra-tumoral diversification among various tumor cells. However, single-cell transcriptome analysis (single-cell RNA sequencing [scRNA-seq]) of CC cells and microenvironment has not been conducted. In this study, a total of 20,938 cells from CC and adjacent normal tissues were examined by scRNA-seq. We identified four tumor cell subpopulations in tumor cells, which had specific signature genes with different biological functions and presented different prognoses. Among them, we identified a subset of cancer stem cells (CSCs) that was related to the developmental hierarchy of tumor progression. Then, we compared the expressive differences between tumor-derived endothelial cells (TECs) and normal ECs (NECs) and revealed higher expression of several metabolism-related genes in TECs. Then, we explored the potential biological function of ECs in vascularization and found several marker genes, which played a prior role in connections between cancer cells and ECs. Our findings provide valuable resources for deciphering the intra-tumoral heterogeneity of CC and uncover the developmental procedure of ECs, which paves the way for CC therapy.

Magnetic Nanoparticle-Based Hyperthermia Mediates Drug Delivery and Impairs the Tumorigenic Capacity of Quiescent Colorectal Cancer Stem Cells.

Cancer stem cells (CSCs) are the tumor cell subpopulation responsible for resistance to chemotherapy, tumor recurrence, and metastasis. An efficient therapy must act on low proliferating quiescent-CSCs (q-CSCs). We here investigate the effect of magnetic hyperthermia (MHT) in combination with local chemotherapy as a dual therapy to inhibit patient-derived colorectal qCR-CSCs. We apply iron oxide nanocubes as MHT heat mediators, coated with a thermoresponsive polymer (TR-Cubes) and loaded with DOXO (TR-DOXO) as a chemotherapeutic agent. The thermoresponsive polymer releases DOXO only at a temperature above 44 °C. In colony-forming assays, the cells exposed to TR-Cubes with MHT reveal that qCR-CSCs struggle to survive the heat damage and, with a due delay, restart the division of dormant cells. The eradication of qCR-CSCs with a complete stop of the colony formation was achieved only with TR-DOXO when exposed to MHT. The in vivo tumor formation study confirms the combined effects of MHT with heat-mediated drug release: only the group of animals that received the CR-CSCs pretreated, in vitro, with TR-DOXO and MHT lacked the formation of tumor even after several months. For DOXO-resistant CR-CSCs cells, the same results were shown, in vitro, when choosing the drug oxaliplatin rather than DOXO and applying MHT. These findings emphasize the potential of our nanoplatforms as an effective patient-personalized cancer treatment against qCR-CSCs.

MicroRNA Expression Profile Distinguishes Glioblastoma Stem Cells from Differentiated Tumor Cells.

Glioblastoma (GBM) represents the most common and aggressive tumor of the brain. Despite the fact that several studies have recently addressed the molecular mechanisms underlying the disease, its etiology and pathogenesis are still poorly understood. GBM displays poor prognosis and its resistance to common therapeutic approaches makes it a highly recurrent tumor. Several studies have identified a subpopulation of tumor cells, known as GBM cancer stem cells (CSCs) characterized by the ability of self-renewal, tumor initiation and propagation. GBM CSCs have been shown to survive GBM chemotherapy and radiotherapy. Thus, targeting CSCs represents a promising approach to treat GBM. Recent evidence has shown that GBM is characterized by a dysregulated expression of microRNA (miRNAs). In this study we have investigated the difference between human GBM CSCs and their paired autologous differentiated tumor cells. Array-based profiling and quantitative Real-Time PCR (qRT-PCR) were performed to identify miRNAs differentially expressed in CSCs. The Cancer Genome Atlas (TCGA) data were also interrogated, and functional interpretation analysis was performed. We have identified 14 miRNAs significantly differentially expressed in GBM CSCs (p < 0.005). MiR-21 and miR-95 were among the most significantly deregulated miRNAs, and their expression was also associated to patient survival. We believe that the data provided here carry important implications for future studies aiming at elucidating the molecular mechanisms underlying GBM.

Therapeutic and prognostic implications of NOTCH and MAPK signaling in bladder cancer.

Signaling pathways that drive bladder cancer (BC) progression may be promising and specific targets for systemic therapy. Here, we investigated the clinical significance and targetability of NOTCH and mitogen‐activated protein kinase (MAPK) signaling for this aggressive malignancy. We assessed NOTCH1 and MAPK activity in 222 stage III and IV BC specimens of patients that had undergone radical cystectomy, and tested for clinical associations including cancer‐specific and overall survival. We examined therapeutic effects of NOTCH and MAPK repression in a murine xenograft model of human bladder cancer cells and evaluated tumor growth and tumor cell plasticity. In BC, NOTCH1 and MAPK signaling marked two distinct tumor cell subpopulations. The combination of high NOTCH1 and high MAPK activity indicated poor cancer‐specific and overall survival in univariate and multivariate analyses. Inhibition of NOTCH and MAPK in BC xenografts in vivo depleted targeted tumor cell subpopulations and revealed strong plasticity in signaling pathway activity. Combinatorial inhibition of NOTCH and MAPK signaling most strongly suppressed tumor growth. Our findings indicate that tumor cell subpopulations with high NOTCH and MAPK activity both contribute to tumor progression. Furthermore, we propose a new concept for BC therapy, which advocates specific and simultaneous targeting of these different tumor cell subpopulations through combined NOTCH and MAPK inhibition.

Effects of the Anti-Tumorigenic Agent AT101 on Human Glioblastoma Cells in the Microenvironmental Glioma Stem Cell Niche.

Glioblastoma (GBM) is a barely treatable disease due to its profound chemoresistance. A distinct inter- and intratumoral heterogeneity reflected by specialized microenvironmental niches and different tumor cell subpopulations allows GBMs to evade therapy regimens. Thus, there is an urgent need to develop alternative treatment strategies. A promising candidate for the treatment of GBMs is AT101, the R(-) enantiomer of gossypol. The present study evaluates the effects of AT101, alone or in combination with temozolomide (TMZ), in a microenvironmental glioma stem cell niche model of two GBM cell lines (U251MG and U87MG). AT101 was found to induce strong cytotoxic effects on U251MG and U87MG stem-like cells in comparison to the respective native cells. Moreover, a higher sensitivity against treatment with AT101 was observed upon incubation of native cells with a stem-like cell-conditioned medium. This higher sensitivity was reflected by a specific inhibitory influence on the p-p42/44 signaling pathway. Further, the expression of CXCR7 and the interleukin-6 receptor was significantly regulated upon these stimulatory conditions. Since tumor stem-like cells are known to mediate the development of tumor recurrences and were observed to strongly respond to the AT101 treatment, this might represent a promising approach to prevent the development of GBM recurrences.

Role of synergy and antagonism in designing multidrug adaptive chemotherapy schedules.

Chemotherapeutic resistance via the mechanism of competitive release of resistant tumor cell subpopulations is a major problem associated with cancer treatments and one of the main causes of tumor recurrence. Often, chemoresistance is mitigated by using multidrug schedules (two or more combination therapies) that can act synergistically, additively, or antagonistically on the heterogeneous population of cells as they evolve. In this paper, we develop a three-component evolutionary game theory model to design two-drug adaptive schedules that mitigate chemoresistance and delay tumor recurrence in an evolving collection of tumor cells with two resistant subpopulations and one chemosensitive population that has a higher baseline fitness but is not resistant to either drug. Using the nonlinear replicator dynamical system with a payoff matrix of Prisoner's Dilemma (PD) type (enforcing a cost to resistance), we investigate the nonlinear dynamics of this three-component system along with an additional tumor growth model whose growth rate is a function of the fitness landscape of the tumor cell populations. A key parameter determines whether the two drugs interact synergistically, additively, or antagonistically. We show that antagonistic drug interactions generally result in slower rates of adaptation of the resistant cells than synergistic ones, making them more effective in combating the evolution of resistance. We then design evolutionary cycles (closed loops) in the three-component phase space by shaping the fitness landscape of the cell populations (i.e., altering the evolutionary stable states of the game) using appropriately designed time-dependent schedules (adaptive therapy), altering the dosages and timing of the two drugs. We describe two key bifurcations associated with our drug interaction parameter which help explain why antagonistic interactions are more effective at controlling competitive release of the resistant population than synergistic interactions in the context of an evolving tumor.

A2 KRT15+ TUMOR CELLS AS PUTATIVE CANCER STEM CELLS IN ESOPHAGEAL CANCER.

Abstract Background Esophageal cancer is a particularly deadly cancer with a 5-year survival rate of only 14% in Canada. Treatment resistance ascribed for at least 30% of the death. The acquisition of resistance to radio- and chemotherapy is mostly attributed to the presence of cancer stem cells (CSCs) and their persistence following classical treatments. CSCs are a subpopulation of tumor cells with high self-renewal and multipotent capacity which amongst others contribute to tumor heterogeneity. Our previous work identified Krt15+ esophageal cells as a rare and long-lived subpopulation of basal cells with higher self-renewal and multipotent capacities than other basal cells. Furthermore, preliminary observations suggest that Krt15+ cells could act as the cell-of-origin for ESCC, the most prevalent type of esophageal cancer worldwide. Though, we still ignore the role of Krt15+ cells in later stages of esophageal cancer such as treatment resistance and if therefore, they could act as CSC. Aims Determine if Krt15+ cells act as CSCs in ESCC patients and if they could contribute to treatment resistance. Methods To do so, we used Krt15-CrePR1;R26mT/mG mice treated with the carcinogen 4 Nitroquinoline-1-oxide (4NQO) in their drinking water for 16 weeks to induce ESCC. Twelve weeks following the beginning of 4NQO treatment, we induced Cre recombination with RU486, a PR1 agonist, leading to GFP expression specifically in Krt15+ cells. Following 4NQO treatment, mice were put back on normal water for 8 to 12 weeks allowing tumors to grow. At euthanasia, esophageal tumor cells were FACS sorted to isolate Krt15+ (GFP+) and Krt15- (GFP-) cells, which were then grown as tumoroids. Results We first validated that 4NQO successfully induced the formation of esophageal lesions in our model, which comprises Krt15+ and Krt15- tumor cells. Tumoroids were then successfully derived from these FACS-sorted cell populations. We demonstrated the increase of CSC-like cells within Krt15+ tumoroids compared to Krt15- tumoroids by measuring the presence of CD44highCD24high cells, two well-known CSC markers, by flow cytometry. Interestingly, Krt15+ and Krt15- tumoroids are histologically distinct. As observed for normal cells, Krt15+ tumoroids appeared as more multipotent and heterogenous than Krt15- tumoroids. Furthermore, Krt15+ tumoroids display higher hyperplasia than Krt15- tumoroids suggesting that Krt15+ tumor cells are functionally distinct from Krt15- tumor cells. Conclusions Krt15+ tumoroids display higher CSC content and hyperplastic capacity suggesting their potential role in esophageal cancer. With this project, we aim to highlight the role of Krt15+ cells in treatment resistance and put forward new targets to overcome this deadly issue in ESCC patients. Funding Agencies CAGCanada research chair TIER 2

Gradient Magnetic Separation and Fluorescent Imaging‐Based Heterogeneous Circulating Tumor Cell Subpopulations Assay with Biomimetic Multifunctional Nanoprobes

AbstractThe heterogeneity of circulating tumor cells (CTCs) greatly impacts cancer metastasis and malignancy, contributing to increased mortality. Therefore, the analysis of such heterogeneity is essential to clinical diagnosis, treatment, and monitoring of patients. Though varied, the types and expression levels of proteins on heterogeneous CTCs enable specific recognition and separation. Accordingly, herein the sequential magnetic separation and fluorescence visualization‐based assay of heterogeneous CTCs after the model tumor cells of BT474Her2+++, LNCaPPSMA++, and MDA‐MB‐231Vim+ binding with corresponding antibody‐modified biomimetic fluorescent‐magnetic nanoprobes with different magnetic sensitivities is reported. These nanoprobes‐bound cell subpopulations are specifically recognized and magnetically separated at different time points under an external magnetic field with good capture performance within several minutes. Moreover, a low detection limit of 1 cell mL−1 in blood sample could be achieved. Consequently, the as‐proposed strategy based on gradient magnetic separation, fluorescent imaging, and biomimetic membrane, shows promise for clinical applications.

An immature inhibin-α-expressing subpopulation of ovarian clear cell carcinoma cells is related to an unfavorable prognosis.

Inhibin‐α, a member of transforming growth factor‐β, is elevated in multiple tumors, but its specific roles are poorly understood. Here, we examined the feature of inhibin‐α‐expressing cells in ovarian tumors. Immunohistochemically, inhibin‐α‐expressing tumor cells were detected only in ovarian clear cell carcinoma (OCCC) among various types of ovarian tumors. By comparing the expression of inhibin‐α and Ki‐67, inhibin‐α‐expressing tumor cells were revealed to be less proliferative. When spheroids and chemoresistant cells were derived from OCCC cell lines, the expression level of inhibin‐α was elevated, and that of an immature marker, aldehyde dehydrogenase, was also elevated. In consistent with this, inhibin‐α expression was correlated with other immature markers, such as OCT3/4 and SOX2, and inversely correlated with proliferative marker MKI67 in public database on OCCC. Knockdown of inhibin‐α in OCCC cell decreased chemoresistance. Moreover, prognostic analysis with 69 surgically resected OCCC cases revealed that the increased inhibin‐α expression was an independent unfavorable prognostic factor. These findings suggested that inhibin‐α‐expressing subpopulation of OCCC tumor cells appeared to be less proliferative, immature, and angiogenic and to be related to acceleration of malignant progression.