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BACKGROUND Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, therapy resistance and poor blood-brain-barrier penetration hinder the amelioration of the standard-of-care. Included in the GBM mass are the tumor initiating cells (TICs), representing the driver of GBM growth and relapse in virtue of their stem-like traits and therapy-resistance. Being constantly exposed to environmental stress, including nutrients deficiency, hypoxia and therapeutic insults, all GBM cells -and TICs in particular- have to be highly adaptive in order to survive. Thus, their ability to cope with stress could be targeted to curtail TICs maintenance and the whole GBM aggressiveness. The key of TICs adaptation relies, among the others, on their epigenetic plasticity, hence encouraging epigenetic drugs testing. MATERIAL AND METHODS By exploiting patient-derived GBM TICs and orthotopic xenograft models, we tested the antitumorigenic features of a novel, selective, orally bioavailable and brain-penetrant Lysine-specific histone demethylase 1 inhibitor (LSD1i). We confirmed the specificity of its effects by LSD1 genetic targeting. A combination of RNA-seq, Chromatin Immunoprecipitation(ChIP)-seq, Mass Spectrometry and reverse genetic experiments unraveled LSD1 molecular players in GBM TICs. RESULTS We identified LSD1 as a druggable target in human GBM: LSD1i treatment, mirrored by LSD1 genetic targeting, impairs growth, viability, stem-like traits and in vivo tumorigenicity of GBM TICs. Mechanistically, LSD1 is crucial for the expression of the activating transcription factor 4 (ATF4), which coordinates the integrated stress response (ISR) to manage stressful stimuli as nutrient deprivation and endoplasmic reticulum stress. By mimicking these stress cues in vitro, we found that LSD1i triggers a delayed but unabated ATF4 translation which provokes an over-lasting ISR, eventually culminating in GBM TICs apoptosis. Lastly, LSD1 demethylase activity is dispensable for ATF4 induction. Rather, LSD1i exerts its anti-tumorigenic potential by interfering with LSD1 scaffolding function in GBM TICs. CONCLUSION LSD1-directed therapy is likely a promising strategy to hinder GBM. By sensitizing GBM TICs to stress, LSD1i endangers the GBM TICs pool. The effectiveness of LSD1i administration in different patient-derived GBM TICs and xenografts, regardless of their molecular profile, places a strong rationale toward the clinical translation of this approach for GBM management. FUNDING Italian association for Cancer Research (AIRC) and Italian Ministry of Health

Functional analysis of 167 genes overexpressed in Krebs-2 tumor initiating cells was performed. Genes were assigned to the three functional groups that determine the malignant phenotype of cancer cells. These groups represent the following features of tumor cells: proliferative self-sufficiency, invasive growth and metastasis, and multiple drug resistance. Malignancy of cancer stem cells was found to be provided by the same genes that provide the stemness of normal pluri-/multipotent stem cells. These results suggest that the malignancy is just the ability to maintain the stem cell specific genes expression profile, and, as a consequence, the stemness itself regardless of the controlling effect of stem niches.

Summary: Colorectal cancer (CRC) is one of the most common cancers worldwide, in which adenomatous polyposis coli (APC) mutations are frequently and uniquely observed. Here we find that cis-HOX (circular RNA stabilizing HOXC10) is robustly expressed in colorectal tumor-initiating cells (TICs). cis-HOX knockout decreases colorectal TIC numbers and impairs the self-renewal, tumorigenesis, and metastatic capacities of TICs, whereas cis-HOX overexpression drives colorectal TIC self-renewal and metastasis. Mechanistically, cis-HOX binds to HOXC10 mRNA to attenuate its decay through blocking the K-homology splicing regulatory protein (KSRP)-binding sequence of HOXC10 3′ UTR. HOXC10 is highly expressed in colorectal tumors and TICs and triggers Wnt/β-catenin activation by activating FZD3 expression. HOXC10 inhibitor salinomycin exerts efficient therapeutic effects in APC-wild-type colorectal tumors, but not in tumors with APC nonsense mutations. Therefore, the cis-HOX-HOXC10 pathway drives colorectal tumorigenesis, stemness, and metastasis and serves as a potential therapeutic target for APC-wild-type colorectal tumors.

Kamphuis et al . argue that macrophages accumulated in the proximity of tumor-initiating cells do not express the high-affinity immunoglobulin E receptor FcεRIα. Although we cannot exclude the possibility of nonspecific binding of anti-FcεRIα antibody (clone MAR-1), we provide evidence that macrophages in squamous cell carcinomas express FcεRIα and that IL-33 induces FcεRIα expression in bone marrow cell–derived macrophages.

Simple Summary Different types of cells with tumor-initiating cell (TIC) activity contribute to colorectal cancer (CRC) progression and resistance to anti-cancer treatment. In this study, we aimed to understand whether different cell types exist within a patient-derived tumor culture, distinguishable by different patterns of their gene expression. By mRNA sequencing of patient-derived CRC cultures at the single-cell level, we defined expression programs that closely resemble differentiated cell populations of the normal intestine. Here, cell type-associated subpopulations showed differences in functional properties such as cell growth and energy metabolism. Subsequent functional analyses in vitro and in vivo demonstrated that metabolic states are linked to TIC activity in primary CRC cultures. We also show that TIC activity is dependent on oxidative phosphorylation, which may therefore represent a target for novel therapies. Abstract Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC.

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In-depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.

Osteosarcoma (OS) is the most frequent primary bone cancer in children and adolescents and the third most frequent in adults. Many inherited germline mutations are responsible for syndromes that predispose to osteosarcomas including Li Fraumeni syndrome, retinoblastoma syndrome, Werner syndrome, Bloom syndrome or Diamond–Blackfan anemia. TP53 is the most frequently altered gene in osteosarcoma. Among other genes mutated in more than 10% of OS cases, c-Myc plays a role in OS development and promotes cell invasion by activating MEK–ERK pathways. Several genomic studies showed frequent alterations in the RB gene in pediatric OS patients. Osteosarcoma driver mutations have been reported in NOTCH1, FOS, NF2, WIF1, BRCA2, APC, PTCH1 and PRKAR1A genes. Some miRNAs such as miR-21, -34a, -143, -148a, -195a, -199a-3p and -382 regulate the pathogenic activity of MAPK and PI3K/Akt-signaling pathways in osteosarcoma. CD133+ osteosarcoma cells have been shown to exhibit stem-like gene expression and can be tumor-initiating cells and play a role in metastasis and development of drug resistance. Although currently osteosarcoma treatment is based on adriamycin chemoregimens and surgery, there are several potential targeted therapies in development. First of all, activity and safety of cabozantinib in osteosarcoma were studied, as well as sorafenib and pazopanib. Finally, novel bifunctional molecules, of potential imaging and osteosarcoma targeting applications may be used in the future.

© 2020 by the authors., Poly(ADP-ribose) polymerases (PARPs), represent a family of 17 proteins implicated in a variety of cell functions; some of them possess the enzymatic ability to synthesize and attach poly (ADP-ribose) (also known as PAR) to different protein substrates by a post-translational modification; PARPs are key components in the cellular response to stress with consequences for different physiological and pathological events, especially during neoplasia. In recent years, using PARP inhibitors as antitumor agents has raised new challenges in understanding their role in tumor biology. Notably, the function of PARPs and PAR in the dynamic of tumor microenvironment is only starting to be understood. In this review, we summarized the conclusions arising from recent studies on the interaction between PARPs, PAR and key features of tumor microenvironment such as hypoxia, autophagy, tumor initiating cells, angiogenesis and cancer-associated immune response., This work was supported by Ministerio de Economía y Competividad, Spanish Ministry of Economy and Competitiveness SAF2012-40011-C02-01, SAF2015-70520-R, RTICC RD12/0036/0026, RTI2018-098968-B-I00, CIBER Cáncer ISCIII CB16/12/00421 and Fundación Domingo Martínez to FJO. JMMC, is recipient of a FPI fellowship from Spanish Ministerio de Ciencia; MFC and EZM are recipients of FPU fellowships from the Spanish Ministerio de Ciencia.

Lung cancer is the main cause of cancer death worldwide. Rapid accurate diagnosis, recognition of risk factors and improvement of treatment efficacy represent the main challenges. An advanced stage of the disease at the time of diagnosis, observed in the majority of cases, makes the introduction of radical treatment impossible or ineffective. Immunotherapy was a breakthrough in achieving long-term survival in recent years. The reason for developing this type of treatment is to know the interaction between the host cells and tumor cells. Cancer stem cells theory is increasingly gaining greater significance in the world. The biological heterogeneity of lung cancer may be due to the presence of a small percentage of cancer stem cells (CSCs). CSCs are the results of genetic and epigenetic changes in normal stem cells, progenitor cells or differentiated cells. CSCs are functionally defined by their unlimited self-renewal capacity, multiple differentiation and their ability to imitate tumors. It is believed that CSCs determine unrestricted growth of tumors and their different morphology. Their ability to self-renew can be the cause of relapses even after long periods of remission. CSCs reside in niches, where they receive signals for differentiation and proliferation process. The identification of CSCs is based on the presence of specific molecules (CD133, CD44, CD90, ALDH, EpCAM). An important feature of CSCs is their resistance to conventional cancer treatment: radio therapy and chemotherapy. Presumably, CSCs are responsible for the synthesis of immunosuppressive particles, and recruiting other molecules, which have immunosuppressive properties. The paper presents the current state of knowledge on CSCs in lung cancer detailing adenocarcinoma.

Ovarian cancer is a lethal malignancy that has not seen a major therapeutic advance in over 30 years. We demonstrate that ovarian cancer exhibits a targetable alteration in iron metabolism. Ferroportin (FPN), the iron efflux pump, is decreased, and transferrin receptor (TFR1), the iron importer, is increased in tumor tissue from patients with high grade but not low grade serous ovarian cancer. A similar profile of decreased FPN and increased TFR1 is observed in a genetic model of ovarian cancer tumor initiating cells (TICs). The net result of these changes is an accumulation of excess intracellular iron and an augmented dependence on iron for proliferation. A forced reduction in intracellular iron reduces the proliferation of ovarian cancer TICs in vitro, and inhibits both tumor growth and intraperitoneal dissemination of tumor cells in vivo. Mechanistic studies demonstrate that iron increases metastatic spread by facilitating invasion through expression of matrix metalloproteases and synthesis of IL6. We show that the iron dependence of ovarian cancer tumor initiating cells renders them exquisitely sensitive in vivo to agents that induce iron-dependent cell death (ferroptosis) as well as iron chelators, and thus creates a metabolic vulnerability that can be exploited therapeutically.

// Xin-Xing Zhu 1, 2, * , Ya-Wei Yan 1, 2, * , Chun-Zhi Ai 1 , Shan Jiang 1 , Shan-Shan Xu 1 , Min Niu 3 , Xiang-Zhen Wang 4 , Gen-Shen Zhong 5 , Xi-Feng Lu 6 , Yu Xue 7 , Shaoqi Tian 8 , Guangyao Li 9 , Shaojun Tang 10 , Yi-Zhou Jiang 1 1 Institute for Advanced Study, Shenzhen University, Shenzhen, Guangdong, China 2 Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, China 3 Department of Statics, University of Wisconsin-Madison, Madison, WI, USA 4 Maternal and Child Health Hospital of Nanshan District, Shenzhen, Guangdong, China 5 The First Affiliated Hospital of Xinxiang Medical University, Weihui, Henan, China 6 Department of Physiology, Center for Diabetes, Obesity and Metabolism, Shenzhen University, Shenzhen, Guangdong, China 7 Minnan Normal University, Zhangzhou, Fujian, China 8 The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China 9 Department of Health Outcomes and Policy, College of Medicine, University of Florida, Gainesville, FL, USA 10 Innovation Center for Biomedical Informatics, Georgetown University Medical Center, Washington, DC, USA * These authors contributed equally to this work Correspondence to: Yi-Zhou Jiang, email: jiangyz@szu.edu.cn Keywords: Jarid2, bladder tumors, tumor-initiating cells, p16, histone modification Received: November 24, 2016 Accepted: February 07, 2017 Published: February 20, 2017 ABSTRACT Bladder cancer is the most common urologic malignancy in China, with an increase of the incidence and mortality rates over past decades. Recent studies suggest that bladder tumors are maintained by a rare fraction of cells with stem cell proprieties. Targeting these bladder tumor initiating cell (TICs) population can overcome the drug-resistance of bladder cancer. However, the molecular and genetic mechanisms regulating TICs in bladder cancer remain poorly defined. Jarid2 is implicated in signaling pathways regulating cancer cell epithelial-mesenchymal transition, and stem cell maintenance. The goal of our study was to examine whether Jarid2 plays a role in the regulation of TICs in bladder cancer. We found that knockdown of Jarid2 was able to inhibit the invasive ability and sphere-forming capacity in bladder cancer cells. Moreover, knockdown of Jarid2 reduced the proportion of TICs and impaired the tumorigenicity of bladder cancer TICs in vivo . Conversely, ectopic overexpression of Jarid2 promoted the invasive ability and sphere-forming capacity in bladder cancer cells. Mechanistically, reduced Jarid2 expression led to the upregulation of p16 and H3K27me3 level at p16 promoter region. Collectively, we provided evidence that Jarid2 via modulation of p16 is a putative novel therapeutic target for treating malignant bladder cancer.

In article https://doi.org/10.1002/advs.201901728, Suling Liu and co‐workers report that the “decoy receptor” IL‐1 receptor 2 (IL1R2) is highly expressed on the most tumorigenic intermediate‐state breast tumor initiation cells (BTICs), which fall in between the epithelial‐like state and mesenchymal‐like state. IL‐1β in the tumor microenvironment activates the IL1R2 signaling pathway and then increases intermediate‐state BTIC self‐renewal and tumor progression, which can be specifically blocked by IL1R2 neutralizing antibody.

Highlights • Secretion of progastrin is a signature event of early malignant transformation in the colon. • In the healthy epithelium, progastrin is produced by a subset of enteroendocrine cells expressing both Bmi1 and Prox1. • LGR5-high intestinal stem cells are a primary source of progastrin production in early mouse and human intestinal adenomas., Progastrin is an unprocessed soluble peptide precursor with a well-described tumor-promoting role in colorectal cancer. It is expressed at small levels in the healthy intestinal mucosa, and its expression is enhanced at early stages of intestinal tumor development, with high levels of this peptide in hyperplastic intestinal polyps being associated with poor neoplasm-free survival in patients. Yet, the precise type of progastrin-producing cells in the healthy intestinal mucosa and in early adenomas remains unclear. Here, we used a combination of immunostaining, RNAscope labelling and retrospective analysis of single cell RNAseq results to demonstrate that progastrin is produced within intestinal crypts by a subset of Bmi1+/Prox1+/LGR5low endocrine cells, previously shown to act as replacement stem cells in case of mucosal injury. In contrast, our findings indicate that intestinal stem cells, specified by expression of the Wnt signaling target LGR5, become the main source of progastrin production in early mouse and human intestinal adenomas. Collectively our results suggest that the previously identified feed-forward mechanisms between progastrin and Wnt signaling is a hallmark of early neoplastic transformation in mouse and human colonic adenomas.

Breast tumor initiating cells (BTICs) with ALDH+CD24−CD44+ phenotype are the most tumorigenic and invasive cell population in breast cancer. However, the molecular mechanisms are still unclear. Here, it is found that a negative immune regulator interleukin‐1 receptor type 2 (IL1R2) is upregulated in breast cancer (BC) tissues and especially in BTICs. BC patients with high IL1R2 expression have a poorer overall survival and relapse‐free survival. High IL1R2 promotes BTIC self‐renewal and BC cell proliferation and invasion. Mechanistically, IL1R2 is activated by IL1β, as demonstrated by the fact that IL1β induces the release of IL1R2 intracellular domain (icd‐IL1R2) and icd‐IL1R2 then interacts with the deubiquitinase USP15 at the UBL2 domain and promotes its activity, which finally induces BMI1 deubiquitination at lysine 81 and stabilizes BMI1 protein. In addition, IL1R2 neutralizing antibody can suppress the protein expression of both IL1R2 and BMI1, and significantly abrogates the promoting effect of IL1R2 on BTIC self‐renewal and BC cell growth both in vitro and in vivo. The current results indicate that blocking IL1R2 with neutralizing antibody provides a therapeutic approach to inhibit BC progression by targeting BTICs., Interleukin‐1 receptor type 2 (IL1R2) increases BMI1 deubiquitination and stability via binding and enhancing the activity of ubiquitin‐specific protease 15 (USP15) in cell nuclei, intrinsically promoting the self‐renewal of breast tumor initiating cells (BTICs) as well as breast cancer cell proliferation and invasion. Targeting IL1R2 with its neutralizing antibody inhibits the self‐renewal of BTICs, breast tumorigenesis, and cancer resistance to docetaxel.

Summary Aberrant NOTCH3 signaling and overexpression is oncogenic, associated with cancer stem cells and drug resistance, yet therapeutic targeting remains elusive. Here, we develop NOTCH3-targeted antibody drug conjugates (NOTCH3-ADCs) by bioconjugation of an auristatin microtubule inhibitor through a protease cleavable linker to two antibodies with differential abilities to inhibit signaling. The signaling inhibitory antibody rapidly induces ligand-independent receptor clustering and internalization through both caveolin and clathrin-mediated pathways. The non-inhibitory antibody also efficiently endocytoses via clathrin without inducing receptor clustering but with slower lysosomal co-localization kinetics. In addition, DLL4 ligand binding to the NOTCH3 receptor mediates transendocytosis of NOTCH3-ADCs into ligand-expressing cells. NOTCH3-ADCs internalize into receptor and ligand cells independent of signaling and induce cell death in both cell types representing an atypical mechanism of ADC cytotoxicity. Treatment of xenografts with NOTCH3-ADCs leads to sustained tumor regressions, outperforms standard-of-care chemotherapy, and allows targeting of tumors that overexpress NOTCH3 independent of signaling inhibition., Graphical abstract, Highlights NOTCH3 receptor is overexpressed in breast, lung, and ovarian tumors Newly generated NOTCH3-targeted antibody drug conjugates are efficacious and safe NOTCH3 antibodies internalize through different routes depending on signaling status NOTCH3 antibody intercellular trafficking occurs by transendocytosis into ligand cells, Inhibition of Notch signaling in cancer is challenging due to dose-limiting, on-target toxicities associated with inhibition of its normal function. Geles et al. demonstrate that a NOTCH3-targeted antibody drug conjugate without signaling inhibition is efficacious by killing receptor and ligand expressing cells, and thus has a lower potential for side effects.

Tumor-initiating cells are a diminutive subpopulation of stem cells that have ability of long term self-renewal and generation of varied traits of tumor cell population. Understanding the concept of tumor-initiating cells may have a great implicative intimation for our comprehension of cancer pathobiology and for the delineation of new therapies directed towards these stem cells. The present review is an endeavor to conceptualize the role of tumor-initiating cells in the Squamous Cell Cancers (SCC) of head and neck, their role in tumorigenesis and the possible supplementary approach in the latest treatment modalities.

// Alice Nomura 1, 2 , Patricia Dauer 1, 2 , Vineet Gupta 1, 2 , Olivia McGinn 1 , Nivedita Arora 1 , Kaustav Majumdar 1 , Charles Uhlrich III 1 , Joseph Dalluge 3 , Vikas Dudeja 1, 2 , Ashok Saluja 1, 2 , Sulagna Banerjee 1, 2 1 Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN, USA 2 Division of Surgical Oncology, Department of Surgery, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA 3 Department of Chemistry, Mass Spectrometry Laboratory, University of Minnesota, Minneapolis, MN, USA Correspondence to: Sulagna Banerjee, email: Sulagna.Banerjee@med.miami.edu Keywords: CD133, tumor initiating cells, metabolism, hypoxia, ROS Received: April 25, 2016 Accepted: July 10, 2016 Published: July 26, 2016 ABSTRACT Chemoresistance in pancreatic cancer has been attributed to tumor-initiating cells (TICs), a minor sub-population of tumor cells. However, the mechanism of chemo-resistance in these cells is still unclear. In the current study, immunohistochemical analysis of LSL-Kras G12D ; LSL-Trp53 R172H; PdxCre (KPC) murine tumors indicated that hypoxic regions developed through tumor progression. This hypoxic “niche” correlated with increased CD133 + population that had an increased HIF1A activity. Consistent with this observation, CD133 + cells had increased glucose uptake and activity of glycolytic pathway enzymes compared to CD133 − cells. Mass spectrometric analysis (UPLC-TQD) following metabolic labeling of CD133 + cells with [ 13 C]-U6 glucose confirmed this observation. Furthermore, although both populations had functionally active mitochondria, CD133 + cells had low mitochondrial complex I and complex IV activity and lesser accumulation of ROS in response to standard chemotherapeutic compounds like paclitaxel, 5FU and gemcitabine. CD133 + cells also showed increased resistance to all three chemotherapeutic compounds and treatment with Glut1 inhibitor (STF31) reversed this resistance, promoting apoptotic death in these cells similar to CD133 − cells. Our study indicates that the altered metabolic profile of CD133 + pancreatic TIC protects them against apoptosis, by reducing accumulation of ROS induced by standard chemotherapeutic agents, thereby confering chemoresistance. Since resistance to existing chemotherapy contributes to the poor prognosis in pancreatic cancer, our study paves the way for identifying novel therapeutic targets for managing chemoresistance and tumor recurrence in pancreatic cancer.

Drug resistance remains an obstacle hindering the success of chemotherapy. Cancer stem cells (CSCs) have been recently found to confer resistance to chemotherapy. Therefore functional markers of CSCs should be discovered and specific therapies targeting these cells should be developed. In our investigation, a small population of B16F10 cells which was positive for ATP-binding cassette sub-family B member 5 (ABCB5) was isolated. This population displayed characteristics similar to those of CSCs and ABCB5 was identified to confer tumor growth and drug resistance in B16F10 cell line. Although targeting ABCB5 by small short interfering RNA delivered by VNP20009 failed to inhibit tumor growth, the combined treatment of VNP-shABCB5 and chemotherapy can act synergistically to delay tumor growth and enhance survival time in a primary B16F10 mice model. Results suggest that the combined treatment of VNP-shABCB5 and chemotherapy can improve the efficacy of chemotherapeutic drugs. Therefore, this combination therapy is of potential significance for melanoma treatment.

Ovarian cancer (OC) is a global health burden with the poorest 5-year survival rate of the gynecological malignancies. Disease recurrence is the major cause of morbidity and mortality of OC, likely driven by the survival of chemoresistant, self-renewing tumor-initiating cells (TICs). The TIC population in OC acquires features to survive cellular stress and avoid apoptosis but these can also be targeted as vulnerabilities specific to the TIC population of OC to prevent recurrence. To reveal these vulnerabilities, a high-throughput drug screen was performed to identify drugs that showed increased efficacy against OC cells grown in TIC-enriching spheroid (TES) conditions compared to adherently grown cells. Four drugs were selected for further investigation. Disulfiram, Bardoxolone Methyl, Elesclomol and Salinomycin were tested in vitro to confirm the effects on viability of OC cells in adherent or TES conditions. The drugs' efficacy against sphere formation and expression of markers of stemness as high ALDH activity and CD133 expression (ALDH+CD133+) as single agents or in combination with carboplatin was also investigated in vitro. Disulfiram showed the greatest efficacy against spheroid viability and decreased the ALDH+CD133+ population of several OC cell lines. This effect was enhanced when combined with carboplatin. Bardoxolone Methyl, Elesclomol and Salinomycin showed similar efficacy against cell viability in OC cells grown adherently or in TES conditions; Elesclomol significantly inhibited the ALDH+CD133+ population of the OC cells which was again enhanced in combination with carboplatin. RNAseq analysis revealed a strong enrichment of genes involved in oxidative phosphorylation and reactive oxygen species (ROS) pathways in TES conditions compared to adherent conditions. The drugs were investigated for their ability to promote oxidative stress and OC cells were measured for accumulation of intracellular ROS and mitochondrial superoxide after exposure to the drugs. OC cells grown in TES conditions had higher basal intracellular ROS levels than adherently grown cells and the level of ROS was significantly enhanced in TES cells treated with Elesclomol or Disulfiram but no differences were seen in adherently grown cells. Salinomycin and Bardoxolone Methyl increased mitochondrial superoxide accumulation in some OC cell lines under TES conditions. In a model of OC relapse in vitro, Disulfiram and Elesclomol following carboplatin treatment significantly increased cell death compared to carboplatin alone. Disulfiram and Salinomycin were incorporated into a mouse model to test their ability to prevent OC relapse after chemotherapy in vivo. These results demonstrate that targeting key pathways mediating increased oxidative stress in TICs can eliminate this population and provide a means to prevent OC recurrence. Citation Format: Brittney S. Harrington, Christina M. Annunziata. Drugs targeting ovarian cancer tumor-initiating cells enhance oxidative stress and prevent disease recurrence [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3582.

Steady-state mitochondrial structure or morphology is primarily maintained by a balance of opposing fission and fusion events between individual mitochondria, which is collectively referred to as mitochondrial dynamics. The details of the bidirectional relationship between the status of mitochondrial dynamics (structure) and energetics (function) require methods to integrate these mitochondrial aspects. To study the quantitative relationship between the status of mitochondrial dynamics (fission, fusion, matrix continuity and diameter) and energetics (ATP and redox), we have developed an analytical approach called mito-SinCe(2). After validating and providing proof of principle, we applied mito-SinCe(2) on ovarian tumor-initiating cells (ovTICs). Mito-SinCe(2) analyses led to the hypothesis that mitochondria-dependent ovTICs interconvert between three states, that have distinct relationships between mitochondrial energetics and dynamics. Interestingly, fusion and ATP increase linearly with each other only once a certain level of fusion is attained. Moreover, mitochondrial dynamics status changes linearly with ATP or with redox, but not simultaneously with both. Furthermore, mito-SinCe(2) analyses can potentially predict new quantitative features of the opposing fission versus fusion relationship and classify cells into functional classes based on their mito-SinCe(2) states. This article has an associated First Person interview with the first author of the paper.

This is the peer reviewed version of the following article: Hsu, W.‐H., Sánchez‐Gómez, P., Gomez‐Ibarlucea, E., Ivanov, D.P., Rahman, R., Grabowska, A.M., Csaba, N., Alexander, C. and Garcia‐Fuentes, M. (2019), Structure‐Optimized Interpolymer Polyphosphazene Complexes for Effective Gene Delivery against Glioblastoma. Adv. Therap., 2: 1800126. doi:10.1002/adtp.201800126, which has been published in final form at https://doi.org/10.1002/adtp.201800126. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions Safe and efficient gene delivery vectors would enhance the prospects for polynucleotide-based therapies. Herein we describe a new approach towards structurally-optimized gene vector design based on the preparation of clickable poly(allylamino-phosphazene)s that can be converted to several cationic and anionic derivatives via thiol-ene addition. Simultaneous co-incubation of alkylamine- and alkylcarboxylate-poly(phosphazenes) with polynucleotides generated binary-polyelectrolyte nanoparticles. Screening of a series of these complexes for transfection in glioblastoma cells showed that the inclusion of 6-mercaptohexanoic acid substituted poly(phosphazene)s in the complexes resulted in 6-fold and 19-fold higher luciferase expression in U87MG cells and primary GBM1 cell- line, respectively. This effect was attributed to the specific ionization properties of this materials that improved polyplex intracellular trafficking. Transfection in 3D-spheroid models and subcutaneous xenograft U87MG tumors confirmed higher transgene expression for the binary cationic/anionic poly(phosphazene) complexes compared to the related polycation-pDNA complexes and to PEI- pDNA complexes. The data also indicated a notable capacity of the mixed complexes to deliver genes to the inner cores of tumor spheroids. Extension of this approach to siRNA delivery showed that the mixed poly(phosphazene) complexes were able to silence DYRK1A, a gene implicated in tumor initiation and progression, reducing U87MG cell renewal in vitro and delaying tumor growth in vivo. This work has been funded by Ministerio de Economía y Competitividad (MINECO-RETOS, Grant MAT2017-84361-R, Feder Funds) and Xunta de Galicia (Grupos de Referencia Competitiva, Feder Funds) to MGF and Engineering and Physical Sciences Research Council Grants EP/N006615/1, EP/ N03371X/1 to CA. WHH was a recipient of a contract from EU Erasmus Mundus NanoFar joint doctorate program SI

Background: Breast cancer is a heterogeneous disease. Based on gene expression profiling, it can be classified into at least 5 subtypes. They are luminal A and B, normal breast-like, Her2-positive, basal-like, and newly described claudin-low. Among all these subtypes, basal-like and claudin-low tumors have relatively poor prognosis. Claudin-low tumors are characterized by the low expression of cell-cell junction proteins like claudin 3 and E-cadherin along with an enrichment of EMT markers such as SNAI1 and SNAI2 and high levels of mesenchymal markers such as vimentin. Claudin-low tumors are generally more aggressive and the rate of response to primary chemotherapy is around 32%. Initial data have shown that p-53 null claudin-low tumors are sensitive to cyclophosphamide treatment. Cyclophosphamide (CTX) is a chemotherapy drug that has been used in lymphoma, leukemia and some solid tumors. At low and continuous doses, it shows immunostimulatory properties. The overall goal of this proposal is to test the therapeutic response of claudin-low tumors to cyclophosphamide treatment and define the functional role of the immune system in a mouse model of claudin-low breast cancer. Preliminary data have shown that tumor cells in a p53 null claudin-low murine model (T12) possess tumor-initiating abilities and have STAT3 activation. Initial data in claudin-low T12 tumors also suggest that CTX induces CD4+ and CD8+ T-cell homing to the primary tumor. Additionally, wild-type Balb/c mice implanted with T12 tumors respond favorably to CTX treatment as compared to T cell-deficient NSG mice. Our studies have shown that treatment-resistant claudin-low tumors have a high tumor-associated macrophage (TAM) infiltrate. Tumors with poor response to CTX show higher levels of vimentin staining as compared to responsive tumors. Depletion of specific T-cell subsets to determine their functional importance in the response to CTX shows that while CD8+ T-cell depletion does not have a significant effect on treatment response, depletion of CD4+ T cells results in impaired tumor response to CTX treatment. Additionally, TAMs increase upon CD4+ T-cell depletion. We hypothesize that CTX treatment can sensitize T12 primary tumors to CD4+ T-cell mediated apoptosis. We further hypothesize that TAMS can suppress T-cell function and this, along with changes in the tumor EMT status, can promote chemoresistance. TAMs can also interact with tumor-initiating cells (TIC) within claudin-low tumors via the STAT3 pathway to promote survival and proliferation of chemotherapy resistant TICs. Furthermore, depleting immunosuppressive cells such as TAMs may provide us with an additional therapeutic modality for chemotherapy-resistant breast cancer in combination with checkpoint immunotherapy. Reference: 1. Prat A et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 2010;12:R68. Citation Format: Swarnima Singh, Se-Jin Kim, Xiang Zhang, Jeffrey Rosen. Immune cells, tumor-initiating cells, and drug sensitivity in claudin-low TNBC: A delicate balance [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B13.