Cancer Stem Cells In Tumors Research Articles

CD133 expressionand clinicopathologic significance in benign and malignant breast lesions.

CD133 is the molecular marker of normal stem cells and progenitor cells and also confirmed as a marker for cancer stem cells in various tumors. The aim of this study is to examine the expression of CD133 and assess its clinicopathologic significance in benign and malignant breast lesions. We analyzed the distribution of CD133 positive cells in breast usual ductal hyperplasia, atypical ductal hyperplasia (ADH), breast ductal carcinoma in situ (DCIS), and invasive breast carcinomas. We then explored the relationship between the CD133 expression and clinicopathologic features using immuno-histochemical staining. We found that CD133 is not expressed in the cells of normal breast tissue, but the expression rate increased with progression of lesions from usual hyperplasia, through atypical ductal hyperplasia, ductal carcinoma in situ and invasive carcinoma. The positive expression rate of CD133 in breast invasive ductal carcinoma correlated to histological grade, cancer stage, nodal status, metastasis, recurrence, event-free survival and overall survival. There was no significant correlation between CD133 expression and factors such as age, postmenopausal status, histological type, tumor size, estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 expression. CD133 may play an important role in the occurrence and development of breast cancer. CD133 positive breast cancer cells are closely related to invasiveness and its expression may predict a poor prognosis.

Inhibition of Tumor Cell Growth and Cancer Stem Cell Expansion by a Bispecific Antibody Targeting EGFR and HER3.

The frequent activation of HER3 signaling as a resistance mechanism to EGFR-targeted therapy has motivated the development of combination therapies that block more than one receptor tyrosine kinase. Here, we have developed a novel tetravalent, bispecific single-chain diabody-Fc fusion protein targeting EGFR and HER3 (also known as ErbB3) that integrates the antigen-binding sites of a humanized version of cetuximab as well as a recently developed anti-HER3 antibody, IgG 3-43. This bispecific antibody combines the binding and neutralizing properties of the parental antibodies, as observed in biochemical and in vitro two-dimensional and three-dimensional cell culture assays, and gave rise to long-lasting growth suppression in a subcutaneous xenograft head and neck tumor model. In triple-negative breast cancer (TNBC) cell lines, treatment with the bispecific antibody inhibited the proliferation and oncosphere formation efficiency driven by HER3 signaling. In an orthotopic MDA-MB-468 tumor model, this translated into antitumor effects superior to those obtained by the parental antibodies alone or in combination and was associated with a reduced number of cells with stem-like properties. These findings demonstrate that the bispecific antibody efficiently blocks not only TNBC proliferation, but also the survival and expansion of the cancer stem cell population, holding promise for further preclinical development.

Dual-mTOR Inhibitor Rapalink-1 Reduces Prostate Cancer Patient-Derived Xenograft Growth and Alters Tumor Heterogeneity.

Bone metastasis is the leading cause of prostate cancer (PCa) mortality, frequently marking the progression to castration-resistant PCa. Dysregulation of the androgen receptor pathway is a common feature of castration-resistant PCa, frequently appearing in association with mTOR pathway deregulations. Advanced PCa is also characterized by increased tumor heterogeneity and cancer stem cell (CSC) frequency. CSC-targeted therapy is currently being explored in advanced PCa, with the aim of reducing cancer clonal divergence and preventing disease progression. In this study, we compared the molecular pathways enriched in a set of bone metastasis from breast and prostate cancer from snap-frozen tissue. To further model PCa drug resistance mechanisms, we used two patient-derived xenografts (PDX) models of bone-metastatic PCa, BM18, and LAPC9. We developed in vitro organoids assay and ex vivo tumor slice drug assays to investigate the effects of mTOR- and CSC-targeting compounds. We found that both PDXs could be effectively targeted by treatment with the bivalent mTORC1/2 inhibitor Rapalink-1. Exposure of LAPC9 to Rapalink-1 but not to the CSC-targeting drug disulfiram blocked mTORC1/2 signaling, diminished expression of metabolic enzymes involved in glutamine and lipid metabolism and reduced the fraction of CD44+ and ALDEFluorhigh cells, in vitro. Mice treated with Rapalink-1 showed a significantly delayed tumor growth compared to control and cells recovered from the tumors of treated animals showed a marked decrease of CD44 expression. Taken together these results highlight the increased dependence of advanced PCa on the mTOR pathway, supporting the development of a targeted approach for advanced, bone metastatic PCa.

Deep Learning of Cancer Stem Cell Morphology Using Conditional Generative Adversarial Networks.

Deep-learning workflows of microscopic image analysis are sufficient for handling the contextual variations because they employ biological samples and have numerous tasks. The use of well-defined annotated images is important for the workflow. Cancer stem cells (CSCs) are identified by specific cell markers. These CSCs were extensively characterized by the stem cell (SC)-like gene expression and proliferation mechanisms for the development of tumors. In contrast, the morphological characterization remains elusive. This study aims to investigate the segmentation of CSCs in phase contrast imaging using conditional generative adversarial networks (CGAN). Artificial intelligence (AI) was trained using fluorescence images of the Nanog-Green fluorescence protein, the expression of which was maintained in CSCs, and the phase contrast images. The AI model segmented the CSC region in the phase contrast image of the CSC cultures and tumor model. By selecting images for training, several values for measuring segmentation quality increased. Moreover, nucleus fluorescence overlaid-phase contrast was effective for increasing the values. We show the possibility of mapping CSC morphology to the condition of undifferentiation using deep-learning CGAN workflows.

Abstract B10: A fluorescent cancer stem cell sensor reveals dynamic induction of a stem phenotype in non-stem tumor cells on contact with macrophages in vitro and in vivo

Abstract Many tumors are hierarchically organized, with a minor population of cancer stem cells (CSCs)/tumor-initiating cells at the apex of the hierarchy. These CSCs are highly enriched for the ability to drive tumor initiation, metastasis, and disease recurrence after therapy. Thus, a better understanding of the CSC population will be critical to the design of more effective anticancer therapeutics. To address this challenge, we have generated a lentiviral-based cancer stem cell sensor that reports with high temporal resolution on the cancer stem cell phenotype as a dynamic state. Expression of a destabilized fluorescent protein is driven by activation of an artificial enhancer element (SORE6) that responds to the master stem cell transcription factors Oct4 and Sox2 or their paralogs. We have extensively validated this reporter as marking cells with the expected characteristics of cancer stem cells, including the ability to self-renew, initiate, and sustain tumorigenesis and metastasis and exhibit chemoresistance (Tang et al., Stem Cell Reports 4:155-159). Intravital videomicroscopy and ex vivo imaging of orthotopically-implanted MDAMB231 breast tumors carrying the SORE6 stem cell sensor revealed that 60% of the CSCs in the primary tumor are found in association with macrophages. Macrophage depletion with clodronate liposomes in vivo reduced the number of CSCs in the tumor, indicating a role for macrophages in supporting or expanding the CSC population. Importantly, intravital imaging showed instances in which contact with intratumoral macrophages appeared to cause an induction of the stem phenotype in non-stem tumor cells and that this induction is concentrated at TMEM intravasation doorways where macrophages are enriched. Modeling this phenomenon in heterotypic cocultures in vitro, we showed that coculture of tumor cells with M2-like macrophages but not endothelial cells could increase the proportion of CSCs. This increase was contact dependent and involved Notch signaling. Single cell fate-mapping experiments in the coculture model revealed that the macrophages caused a >4x increase in the rate of direct conversion of non-stem to stem cells, without affecting CSC proliferation. The induced CSCs were capable of initiating tumorsphere formation in vitro, thus confirming their functionality. In summary, this novel imaging approach allowed us to make the important finding that macrophage contact can induce phenotypic plasticity in more differentiated tumor cells, thereby allowing them to acquire a stem phenotype during intravasation and dissemination. These results suggest that targeting the interaction between macrophages and tumor cells may be a new strategy to reduce the cancer stem cell population both locally and systemically. Citation Format: Bingwu Tang, Ved Sharma, Yarong Wang, Yuval Raviv, David Entenberg, Maja Oktay, John Condeelis, Lalage Wakefield. A fluorescent cancer stem cell sensor reveals dynamic induction of a stem phenotype in non-stem tumor cells on contact with macrophages in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B10.

Blood and Cancer: Cancer Stem Cells as Origin of Hematopoietic Cells in Solid Tumor Microenvironments.

The concepts of hematopoiesis and the generation of blood and immune cells from hematopoietic stem cells are some steady concepts in the field of hematology. However, the knowledge of hematopoietic cells arising from solid tumor cancer stem cells is novel. In the solid tumor microenvironment, hematopoietic cells play pivotal roles in tumor growth and progression. Recent studies have reported that solid tumor cancer cells or cancer stem cells could differentiate into hematopoietic cells. Here, we discuss efforts and research that focused on the presence of hematopoietic cells in tumor microenvironments. We also discuss hematopoiesis from solid tumor cancer stem cells and clarify the notion of differentiation of solid tumor cancer stem cells into non-cancer hematopoietic stem cells.

Establishment of surface marker expression profiles for colorectal cancer stem cells under different conditions.

BackgroundPositive expression of CD44 and CD133 and high expression levels of an epithelial cell adhesion molecule are reliable markers for colorectal cancer stem cells in tumors or among circulating tumor cells. However, the heterogeneity of circulating tumor cells makes it very difficult to detect these stem cells. In this study, we investigated the expression of CD44 and CD133 of colorectal cancer stem cells under different treatments in order to understand the expression profile of these markers when cancer cells grow in different conditions.MethodsCells from a colorectal cancer stem cell line, Caco-2, were seeded at four different initial concentrations and cultured for 3 days. We observed for changes in cell morphology and analyzed the expression of CD44 and CD133 by flow cytometry. In addition, Caco-2 cells were treated with eicosapentaenoic acid (EPA), dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA) for 3 days followed by flow cytometry analysis.ResultsWe demonstrated that the single and combined expression of CD44 and CD133 decreased when the initial seeding concentration was reduced. The expression of both CD44 and CD133 was reduced dramatically when Caco-2 cells were treated with EPA, DMSO, or EDTA. The single expression of CD44 or CD133 was not affected dramatically when cells were treated with DMSO or EDTA, but there was no expression of either markers when treated with EPA.ConclusionsBoth single and combined expressions of CD44 and CD133 were critical for establishing the profiles of colorectal cancer stem cells under different conditions.

Abstract B50: Tissue factor-targeting CAR-NK cells for immunotherapy of triple-negative breast cancer

Abstract The objective of the study is to develop tissue factor (TF)-targeted chimeric antigen receptor-natural killer (TF-CAR-NK) cell immunotherapy for the treatment of triple-negative breast cancer (TNBC). TNBC represents nearly 15% of globally diagnosed breast cancer, which is also the most frequently diagnosed cancer in women in the United States. Due to the lack of validated therapeutic target molecules, TNBC is one of the most difficult-to-treat malignancies, and, in most cases, is considered an incurable malignancy. CAR-expressing NK and T-cell therapy represents a novel cancer immunotherapy approach. To address the unmet issue, we first identified that TF is a novel, common, yet selective membrane-bound surface protein receptor and oncotarget for TNBC on the TNBC cancer cells, tumor neovascular endothelial cells and cancer stem cells. It is worth noting that under physiologic conditions, TF is not expressed on normal peripheral lymphocytes such as NK, B, T cells and monocytes, nor on the cells (quiescent vascular endothelial cells) forming the inner layer of normal vasculature, but is solely restricted on some cells (such as pericytes and smooth muscle cells) that are located outside the blood circulation and inner layer of blood vessel walls. Then we developed and tested a novel TF-targeting CAR-NK cell line (NK92MI, a human NK line derived from NK92 after being transfected for endogenous production of interleukin-2) expressing factor VII light chain, natural ligand for TF, as TF-targeting domain followed by CD28-4-1BB-CD3zeta with or without a hinge region of human IgG1 as TF-CAR1 dimer or monomer for CAR-NK immunotherapy of TNBC. Our preclinical results demonstrate that both the TF-CAR1 dimer and monomer-NK cells can kill TNBC cells in vitro and are effective and safe for the treatment of TNBC in mouse models of TNBC cell line-derived and patient’s tumor-derived xenografts (CDX and PDX). Thus, this study established the proof of concept of targeting TF for CAR-NK immunotherapy of TNBC and may warrant further investigation for TNBC patients. Accumulating evidence suggested that TF is selectively expressed in pathologic angiogenesis-dependent as well as macrophage-associated human diseases. Pathologic angiogenesis, the formation of neovasculature, is involved in many clinically significant human diseases, notably cancer (including solid cancer, acute leukemia and sarcoma), age-related macular degeneration, endometriosis and rheumatoid arthritis, whereas macrophage is involved in the progression of a variety of human diseases, such as atherosclerosis and viral infections (HIV and Ebola). If successful, TF-CAR immunotherapy may have potential to treat a broad range of angiogenesis-dependent or macrophage-associated human diseases. Acknowledgments: This work was supported by the Dr. Ralph and Marian Falk Medical Research Trust Awards Programs and partly by an NCI grant (R21CA216697). Z.H. is the inventor of PCT/U.S. Pending Patents on “Tissue Factor-Targeting CAR-NK and CAR-T Cell Therapy.” Citation Format: Zhiwei Hu. Tissue factor-targeting CAR-NK cells for immunotherapy of triple-negative breast cancer [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 B50.

Immunomodulatory Molecules On Lung Cancer Stem Cells From Lymph Nodes Aspirates.

Over the past decade, immune checkpoint inhibitors have revolutionized the treatment of non-small cell lung cancer (NSCLC). Unfortunately, not all patients benefit from PD-(L)1 blockade, yet, the PD-L1 tumor cell expression is the only approved biomarker, and other biomarkers have been investigated. In the present study, we analyzed the presence of immunomodulatory molecules: PD-L1, CD47, CD73, Fas, and FasL on mature tumor cells (MTCs) and cancer stem cells (CSCs) in lymph nodes (LNs) aspirates and refer it to the lymphocyte subpopulation in peripheral blood (PB). PB samples and LNs aspirates obtained during the endobronchial ultrasound-guided transbronchial needle aspiration (EBUS/TBNA) procedure of 20 patients at different stages of NSCLC. The cells were analyzed by multiparameter flow cytometry. We reported the higher frequency of MTCs and CSCs expressing the investigated immunomodulating molecules in metastatic LNs than in nonmetastatic. The expression of CD47 and PD-L1 was significantly higher on CSCs than on MTCs. Among the lymphocyte subpopulation in PB, we observed a higher frequency of PD-1+ CD8 T cells and Fas+ CD8 T cells in patients with confirmed metastases than in nonmetastatic. Next, we found that the percentage of FasL+ MTCs correlated with the frequency of Fas+ CD3 T cells in LNs aspirates and Fas+ CD8 T cells in PB. Finally, we found that patients with metastatic disease had a significantly higher FasL+/Fas+ MTCs ratio than patients with nonmetastatic disease. Both MTCs and CSCs express different immunomodulatory molecules on their surface. The frequency of FasL+ MTCs associates with altered distribution of Fas+ lymphocyte subpopulations in LNs and PB.

Tumor-infiltrating regulatory T cells, CD8/Treg ratio, and cancer stem cells are correlated with lymph node metastasis in patients with early breast cancer.

Tumor-infiltrating lymphocytes are an important component of the tumor microenvironment (TME) in breast cancer. They have been linked with tumor pathogenesis in advanced stages. However, little is known about their contribution in early phases. In this study, we analyzed the infiltration of leukocytes and cancer stem cells (CSC) in tumors from patients with early breast cancer. Samples of blood and tumor tissue from 30 patients with breast cancer were collected, and the number of dendritic cells (DC), T cells, and CSC were analyzed by flow cytometry. Tumor-infiltrating CD4 and CD8 T cells expressed higher levels of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) compared with peripheral T cells. Regulatory T cells (Treg) were enriched in tumors and overexpressed glucocorticoid-induced TNFR-related protein and CTLA-4. Tumor Treg had a positive correlation with the amount of myeloid DC (mDC) and disease progression. The CD8/Treg ratio was associated with lymph node metastasis and tumor stages. The main subset of DC in early breast tumors was mDC, while plasmacytoid DC were almost absent. CSC were present in most tumors with higher frequencies in patients with lymph node metastasis. CSC were also associated with the amount of tumor-infiltrating Treg. Early breast cancer has an inflammatory milieu characterized by mDC, Treg, and CSC infiltration. The frequencies of Treg, CSC and CD8/Treg ratio were associated with disease progression. The composition of leukocytes and the presence of CSC in early breast tumors should be considered for the development of new therapeutic approaches.

Ovarian Cancer, Cancer Stem Cells and Current Treatment Strategies: A Potential Role of Magmas in the Current Treatment Methods.

Epithelial ovarian cancer (EOC) constitutes 90% of ovarian cancers (OC) and is the eighth most common cause of cancer-related death in women. The cancer histologically and genetically is very complex having a high degree of tumour heterogeneity. The pathogenic variability in OC causes significant impediments in effectively treating patients, resulting in a dismal prognosis. Disease progression is predominantly influenced by the peritoneal tumour microenvironment rather than properties of the tumor and is the major contributor to prognosis. Standard treatment of OC patients consists of debulking surgery, followed by chemotherapy, which in most cases end in recurrent chemoresistant disease. This review discusses the different origins of high-grade serous ovarian cancer (HGSOC), the major sub-type of EOC. Tumour heterogeneity, genetic/epigenetic changes, and cancer stem cells (CSC) in facilitating HGSOC progression and their contribution in the circumvention of therapy treatments are included. Several new treatment strategies are discussed including our preliminary proof of concept study describing the role of mitochondria-associated granulocyte macrophage colony-stimulating factor signaling protein (Magmas) in HGSOC and its unique potential role in chemotherapy-resistant disease.

Interactions Between Tumor Biology and Targeted Nanoplatforms for Imaging Applications.

Although considerable efforts have been conducted to diagnose, improve, and treat cancer in the past few decades, existing therapeutic options are insufficient, as mortality and morbidity rates remain high. Perhaps the best hope for substantial improvement lies in early detection. Recent advances in nanotechnology are expected to increase the current understanding of tumor biology, and will allow nanomaterials to be used for targeting and imaging both in vitro and in vivo experimental models. Owing to their intrinsic physicochemical characteristics, nanostructures (NSs) are valuable tools that have received much attention in nanoimaging. Consequently, rationally designed NSs have been successfully employed in cancer imaging for targeting cancer-specific or cancer-associated molecules and pathways. This review categorizes imaging and targeting approaches according to cancer type, and also highlights some new safe approaches involving membrane-coated nanoparticles, tumor cell-derived extracellular vesicles, circulating tumor cells, cell-free DNAs, and cancer stem cells in the hope of developing more precise targeting and multifunctional nanotechnology-based imaging probes in the future.

Methylation Status of the Nanog Promoter Determines the Switch between Cancer Cells and Cancer Stem Cells.

Cancer stem cells (CSCs) are the main cause of tumor development, metastasis, and relapse. CSCs are thus considered promising targets for cancer therapy. However, it is hard to eradicate CSCs due to their inherent plasticity and heterogeneity, and the underlying mechanism of the switch between non‐CSCs and CSCs remains unclear. Here, it is shown that miR‐135a combined with SMYD4 activates Nanog expression and induces the switch of non‐CSCs into CSCs. The miR‐135a level, once elevated, lowers the methylation level of the CG5 site in the Nanog promoter by directly targeting DNMT1. SMYD4 binds to the unmethylated Nanog promoter to activate Nanog expression in Nanog‐negative tumor cells. The in vivo regulation of miR‐135a levels could significantly affect both the CSCs proportion and tumor progression. These findings indicate that DNA methylation of the Nanog promoter modulates the switch of non‐CSCs into CSCs under the control of the miRNA‐135 level. In addition, the related pathways, miR‐135a/DNMT1 and SMYD4, involved in these processes are potential targets for CSC‐targeted therapy.

Drug repurposing towards targeting cancer stem cells in pediatric brain tumors.

In the pediatric population, brain tumors represent the most commonly diagnosed solid neoplasms and the leading cause of cancer-related deaths globally. They include low-grade gliomas (LGGs), medulloblastomas (MBs), and other embryonal, ependymal, and neuroectodermal tumors. The mainstay of treatment for most brain tumors includes surgical intervention, radiation therapy, and chemotherapy. However, resistance to conventional therapy is widespread, which contributes to the high mortality rates reported and lack of improvement in patient survival despite advancement in therapeutic research. This has been attributed to the presence of a subpopulation of cells, known as cancer stem cells (CSCs), which reside within the tumor bulk and maintain self-renewal and recurrence potential of the tumor. An emerging promising approach that enables identifying novel therapeutic strategies to target CSCs and overcome therapy resistance is drug repurposing or repositioning. This is based on using previously approved drugs with known pharmacokinetic and pharmacodynamic characteristics for indications other than their traditional ones, like cancer. In this review, we provide a synopsis of the drug repurposing methodologies that have been used in pediatric brain tumors, and we argue how this selective compilation of approaches, with a focus on CSC targeting, could elevate drug repurposing to the next level.

SFRP1 in Skin Tumor Initiation and Cancer Stem Cell Regulation with Potential Implications in Epithelial Cancers.

SummaryWnt signaling is involved in the regulation of cancer stem cells (CSCs); however, the molecular mechanism involved is still obscure. SFRP1, a Wnt inhibitor, is downregulated in various human cancers; however, its role in tumor initiation and CSC regulation remains unexplored. Here, we used a skin carcinogenesis model, which showed early tumor initiation in Sfrp1−/− (Sfrp1 knockout) mice and increased tumorigenic potential of Sfrp1−/− CSCs. Expression profiling on Sfrp1−/− CSCs showed upregulation of genes involved in epithelial to mesenchymal transition, stemness, proliferation, and metastasis. Further, SOX-2 and SFRP1 expression was validated in human skin cutaneous squamous cell carcinoma, head and neck squamous cell carcinoma, and breast cancer. The data showed downregulation of SFRP1 and upregulation of SOX-2, establishing their inverse correlation. Importantly, we broadly uncover an inverse correlation of SFRP1 and SOX-2 in epithelial cancers that may be used as a potential prognostic marker in the management of cancer.

Targeting the medulloblastoma: a molecular-based approach.

Background:The lack of success of standard therapies for medulloblastoma has highlighted the need to plan a new therapeutic approach. The purpose of this article is to provide an overview of the novel treatment strategies based on the molecular characterization and risk categories of the medulloblastoma, also focusing on up-to-date relevant clinical trials and the challenges in translating tailored approaches into clinical practice.Methods:An online search of the literature was carried out on the PubMed/MEDLINE and ClinicalTrials.gov websites about molecular classification of medulloblastomas, ongoing clinical trials and new treatment strategies. Only articles in the English language and published in the last five years were selected. The research was refined based on the best match and relevance.Results:A total 58 articles and 51 clinical trials were analyzed. Trials were of phase I, II, and I/II in 55%, 33% and 12% of the cases, respectively. Target and adoptive immunotherapies were the treatment strategies for newly diagnosed and recurrent medulloblastoma in 71% and 29% of the cases, respectively.Conclusion:Efforts are focused on the fine-tuning of target therapies and immunotherapies, including agents directed to specific pathways, engineered T-cells and oncoviruses. The blood-brain barrier, chemoresistance, the tumor microenvironment and cancer stem cells are the main translational challenges to be overcome in order to optimize medulloblastoma treatment, reduce the long-term morbidity and increase the overall survival. (www.actabiomedica.it)

TMIC-47. INHIBITION OF GLIOBLASTOMA GROWTH THROUGH TUMOR-MICROENVIRONMENT CROSSTALK USING CLINICALLY SUITABLE NANOBIOCONJUGATE

Abstract Tumor environment in glioblastoma (GBM) is a dynamic interactive complex between tumor, immune and stem cells and extracellular matrix (ECM). In 226 patient glioma samples, we found a clinical correlation between expression of tumor vascular laminin-411 (α4β1γ1) and cancer stem cell (CSC) markers: Notch pathway members, CD133, Nestin, and c-Myc, with faster tumor recurrence and shorter survival of patients (Tao S, et al, Cancer Res., 2019). Novel nanotechnology approach to block trimeric ECM laminin-411 was developed for GBM treatment in experimental and preclinical models on human U87MG and LN229, and patient-derived TS543 and TS576 GBM cell lines. Nanobioconjugates (NBC) based on natural polymer, poly(β-L-malic acid), with antisense against laminin-411 α4 and β-chains, endosome escape unit (Ding H, et al, PNAS, USA, 2010), antibodies for blood-brain barrier (BBB) crossing and tumor targeting was characterized for toxicity and biodistribution according to FDA guidelines. Ex vivo depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of intracranial tumors in mice, and significantly increased survival in hosts compared to mice with untreated cells. A nanobioconjugate potentially suitable for clinical use and capable of crossing BBB was designed to block laminin-411 expression. In biodistribution studies the NBC labeled with 125I on tyrosines of attached antibodies was accumulated in GBM but not in healthy brain tissue. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, Notch signaling system through the b1 integrin-Dll4 (Notch ligand) pathway. No toxicity revealed in 4 naïve Cynomolgus macaques after administration of three therapeutic 1X and acute 10X dosages of NBC. Conclusion: An efficient strategy of GBM treatment was developed via targeting a critical component of the tumor microenvironment, laminin-411, which is independent of heterogeneous genetic mutations in glioblastomas. Support: NIH grants U01CA151815, R01CA188743, R01CA206220, R01CA209921.

Autophagy is essential for the endothelial differentiation of breast cancer stem‑like cells.

Blood vessels serve an important role in tumor growth and metastasis, and recent studies have shown that certain tumor cancer stem cells may differentiate into endothelial cells and contribute to angiogenesis. In the present study, vascular endothelial growth factor (VEGF) was used to induce endothelial differentiation of breast cancer stem‑like cells (BCSLCs), and methods including flow cytometry, western blotting and immunofluorescence were used to study the relationship between autophagy and the endothelial differentiation of BCSLCs. The results showed that BCSLCs could differentiate into endothelial cells under the induction of VEGF invitro. Subsequently, the role of autophagy in the endothelial differentiation of BCSLCs was examined. Autophagic activity was measured during endothelial differentiation of BCSLCs, and the association between autophagy and endothelial differentiation was investigated using autophagy activators, autophagy inhibitors and autophagy related 5 (Atg5)‑knockdown BCSLCs. Autophagy was increased during endothelial differentiation of BCSLCs, and there was a positive association between autophagy and endothelial differentiation. The ability of cells to undergo endothelial differentiation was reduced in BCSLCs with Atg5 knockdown. Therefore, autophagy was essential for endothelial differentiation of BCSLCs, and the findings of the present study may highlight novel potential avenues for reducing angiogenesis and improving treatment of breast cancer.

Oncolytic Effect of Adenoviruses Serotypes 5 and 6 Against U87 Glioblastoma Cancer Stem Cells.

Oncolytic adenoviruses are promising therapeutic agents against both the bulk of tumor cells and cancer stem cells. The present study intended to test the oncolytic capability of adenovirus serotype 6 (Ad6), which has a lower seroprevalence and hepatotoxicity relatively to adenovirus 5 (Ad5), against the glioblastoma and its cancer stem cells. Oncolytic efficacy of Ad6 was compared to widespread Ad5 both in vitro and in vivo, using the U87 and U251 human glioblastoma cell lines and subcutaneously transplanted U87 cells in SCID mice, respectively. Ad6 had a dose-dependent cytotoxicity toward glioblastoma cells in vitro and its intratumoral injections lead to a significant (p<0.05) decrease in volume of U87 xenografts, similarly to Ad5. Based on the innate capability of glioblastoma cancer stem cells to internalize a fluorescent-labeled double-stranded DNA probe, the spatial localization of these cells was estimated and it was shown that the number of cancer stem cells tended to decrease under adenovirus therapy as compared to the control group. Ad6 was shown to be a promising agent for treating glioblastomas.

Co-targeting Bulk Tumor and CSCs in Clinically Translatable TNBC Patient-Derived Xenografts via Combination Nanotherapy.

Triple-negative breast cancer (TNBC) accounts disproportionally for the majority of breast cancer-related deaths throughout the world. This is largely attributed to lack of a specific therapy capable of targeting both bulk tumor mass and cancer stem cells (CSC), as well as appropriate animal models to accurately evaluate treatment efficacy for clinical translation. Thus, development of effective and clinically translatable targeted therapies for TNBC is an unmet medical need. We developed a hybrid nanoparticles-based co-delivery platform containing both paclitaxel and verteporfin (PV-NP) to target TNBC patient-derived xenograft (PDX) tumor and CSCs. MRI and IVIS imaging were performed on mice containing PDX tumors to assess tumor vascularity and accumulation of NPs. NF-κB, Wnt, and YAP activities were measured by reporter assays. Mice bearing TNBC PDX tumor were treated with PV-NPs and controls, and tumors progression and CSC subpopulations were analyzed. MRI imaging indicated high vascularization of PDX tumors. IVIS imaging showed accumulation of NPs in PDX tumors. In comparison with control-NPs and free-drug combination, PV-NPs significantly retarded tumor growth of TNBC PDX. PV-NPs simultaneously repressed NF-κB, Wnt, and YAP that have been shown to be crucial for cancer growth, CSC development, and tumorigenesis. In conclusion, NPs containing two clinically used drugs concurrently inhibited NF-κB, Wnt, and YAP pathways and exhibited synergic effects on killing TNBC bulk tumor and CSCs. This combination nanotherapy evaluated with a PDX model may lead to an effective treatment of patients with TNBC.