Subset Of Tumor Cells Research Articles

A short-term chick embryo in vivo xenograft model to study retinoblastoma cancer stem cells.

Purpose:Cancer stem cells (CSCs) reported in various tumors play a crucial role in tumorigenesis and metastasis of retinoblastoma (Rb). Following the efforts to reduce, replace, and refine the use of mammalian models, we aimed to establish a short-term xenograft for Rb to evaluate the CSC properties of CD133- Rb Y79 cells, using the well-established chick embryo chorioallantoic membrane (CE-CAM) assay.Methods:Y79 cells were cultured, labeled with two different dyes (CM-Dil Y79 and enhanced green fluorescent protein (eGFP)) and sorted for CD133- and CD133 + subsets. Two million cells from each of the labeled groups were transplanted onto the abraded CAM on embryonic day 7 (E7). On E14, the tumor nodule formation on CAM and spontaneous metastasis to the embryos were evaluated by confocal microscopy, in vivo imaging, and histology.Results:Y79 cells formed pink–white raised perivascular nodules with feeder vessels on the CAM with both the types of labeled CD133- cells. CD133- cells, when compared to CD133 + cells, demonstrated significantly larger tumor volume (40.45 ± 7.744 mm3 vs 3.478 ± 0.69 mm3, P = 0.0014) and higher fluorescence intensity (CM-Dil: AUF = 6.37 × 107 ± 7.7 × 106 vs 1.08 × 107 ± 1.6 × 106; P < 0.0001; eGFP: AUF = 13.94 × 104 ± 2.54 × 104 vs AUF = 1.39 × 104 ± 0.4 × 104; P = 0.0003). The metastatic potential of CD133- cells was also observed to be higher as noted by in vivo imaging and histopathology.Conclusion:This study highlights that CE-CAM is a feasible alternative nonmammalian model for evaluating tumorigenicity and metastatic potential of Y79 CSCs. Increased tumorigenicity and metastatic potential of CD133- subset of tumor cells substantiate their CSC properties.

High CD44 Immunoexpression Correlates with Poor Overall Survival: Assessing the Role of Cancer Stem Cell Markers in Oral Squamous Cell Carcinoma Patients from the High-Risk Population of Pakistan.

Oral squamous cell carcinoma (OSCC) is a top-ranked cancer in the Pakistani population, and patient survival has remained unchanged at ∼50% for several decades. Recent advances have claimed that a subset of tumour cells, called cancer stem cells (CSCs), are responsible for tumour progression, treatment resistance, and metastasis, which leads to a poor prognosis. This study investigated the impact of CSC markers expression on overall survival (OS) and disease-free survival (DFS) of OSCC patients. Materials and Methods. Immunohistochemistry was used to evaluate CD44, CD133, L1CAM, and SOX2 expression in a well-characterized cohort of 100 Pakistani patients with primary treatment naïve OSCC. The immunoreactivity for each marker was correlated with patient clinicopathologic characteristics, oral cancer risk chewing habits, and survival. The minimum follow-up time for all patients was five years, and survival estimates were calculated using the Kaplan–Meier method and Cox proportional hazards model. Results. In this cohort of 100 patients, there were 57 males and 43 females. The median OS and DFS time durations observed were 64 and 52.5 months, respectively. Positive expression for CD44, CD133, L1CAM, and SOX2 was observed in 33%, 23%, 41%, and 63% of patients. High CD44 expression correlated with decreased OS (P=0.047) but did not influence DFS. However, CD133, L1CAM, and SOX2 had no effect on either OS or DFS. Tonsils, nodal involvement, and AJCC stage were independent predictors of worse OS and DFS both. Conclusion. Of the CSC markers investigated here, only CD44 was a predictor for poor OS. CD44 was also associated with advanced AJCC and T stages. Interestingly, CD133 was significantly lower in patients who habitually consumed oral cancer risk factors.

Single-Cell RNA Sequencing Uncovers Heterogeneous Circulating Tumor Cell Subsets in Breast Cancer

Metastasis remains the main cause of death for breast cancer (BC) patients, and conceivably, a huge effort has been directed toward the understanding of the metastatic process [...].

Tumor Cell Plasticity in Equine Papillomavirus-Positive Versus-Negative Squamous Cell Carcinoma of the Head and Neck.

Squamous cell carcinoma of the head and neck (HNSCC) is a common malignant tumor in humans and animals. In humans, papillomavirus (PV)-induced HNSCCs have a better prognosis than papillomavirus-unrelated HNSCCs. The ability of tumor cells to switch from epithelial to mesenchymal, endothelial, or therapy-resistant stem-cell-like phenotypes promotes disease progression and metastasis. In equine HNSCC, PV-association and tumor cell phenotype switching are poorly understood. We screened 49 equine HNSCCs for equine PV (EcPV) type 2, 3 and 5 infection. Subsequently, PV-positive versus -negative lesions were analyzed for expression of selected epithelial (keratins, β-catenin), mesenchymal (vimentin), endothelial (COX-2), and stem-cell markers (CD271, CD44) by immunohistochemistry (IHC) and immunofluorescence (IF; keratins/vimentin, CD44/CD271 double-staining) to address tumor cell plasticity in relation to PV infection. Only EcPV2 PCR scored positive for 11/49 equine HNSCCs. IHC and IF from 11 EcPV2-positive and 11 EcPV2-negative tumors revealed epithelial-to-mesenchymal transition events, with vimentin-positive cells ranging between <10 and >50%. CD44- and CD271-staining disclosed the intralesional presence of infiltrative tumor cell fronts and double-positive tumor cell subsets independently of the PV infection status. Our findings are indicative of (partial) epithelial–mesenchymal transition events giving rise to hybrid epithelial/mesenchymal and stem-cell-like tumor cell phenotypes in equine HNSCCs and suggest CD44 and CD271 as potential malignancy markers that merit to be further explored in the horse.

Abstract P3-09-08: An automated tool to determine optimal cluster numbers in single-cell RNA sequencing data identifies key prognostic subsets of T cells in breast tumors

Abstract Background: Single-cell RNA sequencing (scRNAseq) enables gene expression profiling of individual cells, thus providing new opportunities to identify and characterize cell types and tumor cell states, and which is typically accomplished through some type of clustering analysis. Determination of the optimal number of groups found is a crucial step in data interpretation, and one that is often ignored. Hence, there is still a great need to develop objective tools for estimation of the data driven optimal number of groups/clusters. Methods: We developed “MultiK”, a data-driven tool for objective selection of the optimal number of groups/clusters, which combines multiple resolution solutions together through a consensus clustering approach based upon repeated sub-sampling. MultiK gives multiple diagnostic plots to highlight the number of meaningful groups in the data, and makes objective group number suggestions, which encompasses both high and low-resolution parameters. Results: MultiK successfully identified the ground truth number of groups in a controlled data set of a mixture of 3 breast cell lines, and was sensitive in identification of classes and subclasses in a synthetic “spike in” experiment. We further applied MultiK to identify reproducible groups in complex tissue datasets, including mouse mammary glands and multiple T cell data sets. In both cases, we identified most of the previously known subsets/cell populations and did so without any prior knowledge needed. In the human T cell case, MultiK identified a total of 12 reproducible T cell subsets spanning 6 different data sets that represent multiple cancer types. Moreover, consistent with previous findings, some of these reproducible T cell signatures showed prognostic values in predicting breast cancer patient’s survival including Treg, and multiple CD8 subsets. In particular, we found that the CD4 T naïve signature was significantly associated with overall survival in multiple patient sets, including both HER2+ and TNBCs. We also found that the two CD4 T follicular helper subsets significantly correlated with survival in both HER2+ and TNBC samples. Furthermore, consistent with the previous finding that the CD8 Trm signature associated with good prognosis, we found that our CD8T resident memory signature was prognostic within the HER2+ and TNBC sets. Conclusion: MultiK improves current scRNAseq cluster/group number estimations using an objective data driven approach. This methodology should be important as using our T cell analyses as an example, it shows that many previous published analyses likely overestimated the true number of reproducible T cell subsets in the tumor immune microenvironment, which may lead to irreproducible findings across studies. Additional analyses on tumor cell subsets are also currently underway. Citation Format: Siyao Liu, Aatish Thennavan, J.S. Marron, Charles Perou. An automated tool to determine optimal cluster numbers in single-cell RNA sequencing data identifies key prognostic subsets of T cells in breast tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-08.

Exploring the Clinical Utility of Pancreatic Cancer Circulating Tumor Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most frequent pancreatic cancer type, characterized by a dismal prognosis due to late diagnosis, frequent metastases, and limited therapeutic response to standard chemotherapy. Circulating tumor cells (CTCs) are a rare subset of tumor cells found in the blood of cancer patients. CTCs has the potential utility for screening, early and definitive diagnosis, prognostic and predictive assessment, and offers the potential for personalized management. However, a gold-standard CTC detection and enrichment method remains elusive, hindering comprehensive comparisons between studies. In this review, we summarize data regarding the utility of CTCs at different stages of PDAC from early to metastatic disease and discuss the molecular profiling and culture of CTCs. The characterization of CTCs brings us closer to defining the specific CTC subpopulation responsible for metastasis with the potential to uncover new therapies and more effective management options for PDAC.

Identification of a unique tumor cell subset employing myeloid transcriptional circuits to create an immunomodulatory microenvironment in glioblastoma

ABSTRACT Glioblastoma (GBM) is an aggressive primary brain tumor with unique immunity predominated by myeloid cells. GBM cells have been implicated to evade immune attack through hijacking myeloid-affiliated transcriptional programs to establish an immunosuppressive microenvironment. However, molecular features of immune-evading GBM cells in heterogeneous GBMs and their interactions with immune cells remain unclear. Herein, we employed single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data to develop an in silico method for delineating GBM immune signature and identifying new molecular subsets for immunotherapy. We identified a new GBM cell subset, termed TC-6, that harbored immune-invading signature and actively interacted with tumor-associated macrophages (TAMs) to orchestrate an immune-suppressive niche. Proinflammatory transcriptional factors STAT1, STAT2, IRF1, IRF2, IRF3, and IRF7 were identified as the core regulons defining TC-6 subsets. Further immune transcriptome analyses revealed three immune subtypes (C1, C2, and C3). C3 subtype GBMs were enriched with TC-6 cells and immunosuppressive TAMs, and exhibited an immunomodulatory signature that associated with reduced efficacy of anti-PD-1 treatment. Interferon-related DNA damage resistance signaling was upregulated in C3 GBMs, predicting shortened survival of GBM patients who received chemo-radiation treatment. Treatment of OSI-930 as a molecular agent targeting c-kit and VEGFR2 tyrosine kinases may compromise the immunomodulatory signature of C3 GBMs and synergize with chemo-radiation therapy. We further developed a simplified 11-gene set for defining C3 GBMs. Our work identified TC-6 subset as an immune-evading hub that creates an immunomodulatory signature of C3 GBMs, gaining insights into the heterogeneity of GBM immune microenvironment and holding promise for optimized anti-GBM immunotherapy.

Utilizing Shared Big Data to Identify Liver Cancer Dedifferentiation Markers.

Big data reanalysis has the potential to generate novel comparative analyses which aim to generate novel hypotheses and knowledge. However, this approach is underutilized in the realm of cancer research, particularly for cancer stem cells (CSCs). CSCs are a rare subset of tumor cells, which dedifferentiate from healthy adult cells, and have the potential for self-renewal and treatment resistance. This analysis utilizes two publically available single-cell RNA-seq datasets of liver cancer and adult liver cell types to demonstrate how reanalysis of big data can lead to valuable new discoveries. We identify 519 differentially expressed genes between liver CSCs and healthy liver cell types. Here we report the potential novel liver CSC dedifferentiation factor, Msh Homeobox 2, which was significantly upregulated in liver CSCs by 1.36 fold (p-value < 1E-10). These findings have the potential to further advance our knowledge of genes governing the formation of CSCs.

O-Acetyl-GD2 as a Therapeutic Target for Breast Cancer Stem Cells.

SynopsisA sugar-lipid molecule called OAcGD2 is a novel marker for breast cancer stem cells. Treatment with anti-OAcGD2 mAb8B6 may have superior anticancer efficacy by targeting cancer stem cells, thereby reducing metastasis and recurrence of cancer.BackgroundCancer stem cells (CSCs) that drive tumor progression and disease recurrence are rare subsets of tumor cells. CSCs are relatively resistant to conventional chemotherapy and radiotherapy. Eradication of CSCs is thus essential to achieve durable responses. GD2 was reported to be a CSC marker in human triple-negative breast cancer, and anti-GD2 immunotherapy showed reduced tumor growth in cell lines. Using a specific anti-OAcGD2 antibody, mAb8D6, we set out to determine whether OAcGD2+ cells exhibit stem cell properties and mAb8D6 can inhibit tumor growth by targeting OAcGD2+CSCs.MethodOAcGD2 expression in patient-derived xenografts (PDXs) of breast cancer was determined by flow cytometric analyses using mAb8D6. The stemness of OAcGD2+ cells isolated by sorting and the effects of mAb8B6 were assessed by CSC growth and mammosphere formation in vitro and tumor growth in vivo using PDX models.ResultWe found that the OAcGD2 expression levels in six PDXs of various molecular subtypes of breast cancer highly correlated with their previously defined CSC markers in these PDXs. The sorted OAcGD2+ cells displayed a greater capacity for mammosphere formation in vitro and tumor initiation in vivo than OAcGD2− cells. In addition, the majority of OAcGD2+ cells were aldehyde dehydrogenase (ALDH+) or CD44hiCD24lo, the known CSC markers in breast cancer. Treatment of PDXs-bearing mice with mAb8B6, but not doxorubicin, suppressed the tumor growth, along with reduced CSCs as assessed by CSC markers and in vivo tumorigenicity. In vitro, mAb8B6 suppressed proliferation and mammosphere formation and induced apoptosis of OAcGD2+ breast cancer cells harvested from PDXs, in a dose-dependent manner. Finally, administration of mAb8B6 in vivo dramatically suppressed tumor growth of OAcGD2+ breast CSCs (BCSCs) with complete tumor abrogation in 3/6 mice.ConclusionOAcGD2 is a novel marker for CSC in various subtypes of breast cancer. Anti-OAcGD2 mAb8B6 directly eradicated OAcGD2+ cells and reduced tumor growth in PDX model. Our data demonstrate the potential of mAb8B6 as a promising immunotherapeutic agent to target BCSCs.

Hypoxia induction of SH2D3A triggers malignant progression of lung cancer.

Lung cancer is the most prevalent and aggressive cancer and is one of the leading causes of cancer-related death worldwide. Hypoxia in the tumor microenvironment is associated with poor patient survival and is a crucial characteristic of solid tumors. A subset of tumor cells, termed cancer stem cells (CSCs), with self-renewal and differentiation capabilities simultaneously, are regarded as responsible for cancer tumorigenesis, resistance to therapeutics, and cancer relapse. Recent advances have revealed that hypoxia plays an essential role in CSCs self-replication maintenance. Yet, the underlying mechanisms of hypoxia that trigger the stemness maintenance of CSCs are still poorly understood. Here, we provide evidence showing that SH2D3A expression level was increased in lung cancer and lung CSCs, and high expression of SH2D3A was associated with the overall survival of patients with lung cancer. Mechanistically, HIF-2α, which is a key transcription factor in response to hypoxia directly binds to the SH2D3A promoter and facilitates SH2D3A expression at the transcription level. SH2D3A was found to be functionally important for lung CSC malignant behaviors such as uncontrolled self-replication and proliferation. We demonstrated that pharmacological downregulation of SH2D3A expression by AM966, a small molecule compound, efficiently induces tumor regression in vitro and in vivo. Thus, this study highlights the biological implications of SH2D3A as a novel prognostic marker and therapeutic target in lung cancer in the future.

3186 – TRANSCRIPTOMIC ANALYSIS OF SYNTHETIC HUMAN CORD BLOOD LEUKEMIAS REVEALS DEVELOPMENT STAGE-SPECIFIC DEPENDENCE ON N-MYC

Descriptive sequencing of human leukemia samples has revealed the diversity and combinatorial complexity of oncogenic effector genes which can occur within all or just a subset of tumor cells. With the aim of deconvoluting the individual contributions of T-cell acute lymphoblastic leukemia (T-ALL) oncogenes, we employed our recently developed synthetic model of human T-ALL (Kusakabe et al, 2019) to perform a systematic analysis of transcriptional signatures associated with each of the four oncogenes used in our model. We identified synthetic leukemias to cluster based on two gene signatures which resembled distinct stages in T-cell development and had marked variation in MYCN expression, with its highest expression found on the more "immature" cluster. All synthetic leukemias carried activated NOTCH1, which is best known for upregulating MYC, but not MYCN. By analyzing publicly available RNA sequencing datasets, we found MYCN to be highly expressed in early T-cell progenitors (ETPs) and T-ALLs of more immature phenotype. To assess oncogene "addiction" to MYCN, cell lines and primary synthetic leukemias were transduced with shRNAs or sgRNAs to knock-down (KD) or knock-out (KO) MYCN, respectively. By both approaches, bulk cell growth and/or clonogenic activity were reduced in KD/KO cells as compared to controls, supporting their functional dependence on MYCN. MYCN overexpression resulted in increased clonogenic activity and differentiation delay of NOTCH1-transduced cells in vitro but was still insufficient to generate leukemia in vivo. We are currently exploring other genes with similar expression patterns to MYCN to identify upstream/downstream mediators of MYCN dependence in this context. Our results emphasize stage-specific oncogene dependencies and identify alternate therapeutic targets in T-ALLs with high MYCN expression.

Isolation of cancer stem cells from cultured breast cancer cells and xenografted breast tumors based on aldehyde dehydrogenase activity.

The heterogeneity of breast tumors is a major factor in the development, progression, and therapeutic response of breast cancer. In terms of therapy resistance, a subset of tumor cells commonly referred to as cancer stem cells (CSCs) or tumor initiating cells (TICs) have a prominent role. These cells have inherent increased tumorigenicity, self-renewal and differentiation capacity, and mechanisms for chemotherapy and radiation resistance. The importance of CSCs/TICs in cancer makes isolating and studying these cells via reliable methods critical. CSCs/TICs can be enriched for by discrete markers. Increased aldehyde dehydrogenase (ALDH) activity as detected by the AldefluorTM assay is a commonly used method. In this chapter, we describe the detailed methods for identification and isolation of putative CSCs/TICs from cultured cells and xenografted breast tumors using the AldefluorTM assay and describe the importance of the ALDH isoforms in breast cancer.

Targeting radiation-tolerant persister cells as a strategy for inhibiting radioresistance and recurrence in glioblastoma.

Compelling evidence suggests that glioblastoma (GBM) recurrence results from the expansion of a subset of tumor cells with robust intrinsic or therapy-induced radioresistance. However, the mechanisms underlying GBM radioresistance and recurrence remain elusive. To overcome obstacles in radioresistance research, we present a novel preclinical model ideally suited for radiobiological studies. With this model, we performed a screen and identified a radiation-tolerant persister (RTP) subpopulation. RNA sequencing was performed on RTP and parental cells to obtain mRNA and miRNA expression profiles. The regulatory mechanisms among NF-κB, YY1, miR-103a, XRCC3, and FGF2 were investigated by transcription factor activation profiling array analysis, chromatin immunoprecipitation, western blot analysis, luciferase reporter assays, and the MirTrap system. Transferrin-functionalized nanoparticles (Tf-NPs) were employed to improve blood-brain barrier permeability and RTP targeting. RTP cells drive radioresistance by preferentially activating DNA damage repair and promoting stemness. Mechanistic investigations showed that continual radiation activates the NF-κB signaling cascade and promotes nuclear translocation of p65, leading to enhanced expression of YY1, the transcription factor that directly suppresses miR-103a transcription. Restoring miR-103a expression under these conditions suppressed the FGF2-XRCC3 axis and decreased the radioresistance capability. Moreover, Tf-NPs improved radiosensitivity and provided a significant survival benefit. We suggest that the NF-κB-YY1-miR-103a regulatory axis is indispensable for the function of RTP cells in driving radioresistance and recurrence. Thus, our results identified a novel strategy for improving survival in patients with recurrent/refractory GBM.

Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability.On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

Suppression of Ribosome Biogenesis by Targeting WD Repeat Domain 12 (WDR12) Inhibits Glioma Stem-Like Cell Growth.

Glioma stem-like cells (GSCs) are a subset of tumor cells that initiate malignant growth and promote the therapeutic resistance of glioblastoma, the most lethal primary brain tumor. Ribosome biogenesis is an essential cellular process to maintain cell growth, but its regulatory mechanism in GSCs remains largely unknown. Here, we show that WD repeat domain 12 (WDR12), a component of the Pes1-Bop1 complex (PeBoW), is required for ribosome biogenesis in GSCs. WDR12 is preferentially expressed in GSCs compared to non-stem tumor cells and normal brain cells. High levels of WDR12 are associated with glioblastoma progression and poor prognosis. Silencing WDR12 results in the degradation of PeBoW complex components and prevents the maturation of 28S rRNA, thereby inhibiting ribosome biogenesis in GSCs. Subsequently, WDR12 depletion compromises GSC proliferation, inhibits GSC-derived orthotopic tumor growth, and extends animal survival. Together, our results suggest that WDR12 is crucial for ribosome biogenesis in GSCs, and is thus a potential target for GSC-directed therapy of glioblastoma.

CSIG-17. IMMUNE MODULATORY ROLE OF TYPE I INTERFERON IN SYNGENEIC MOUSE GLIOMA MODELS

Abstract Glioblastoma is characterized by a poor prognosis and a challenging phenotype for drug development. Although multimodal treatment, including surgery, radio- and chemotherapy is applied, the overall survival remains just above one year. Numerous clinical trials have studied targeted therapies against commonly deregulated pathways, but an efficient targeted drug is yet to be discovered. Likewise, immunotherapy has not been shown to be active. A subset of glioma tumor cells demonstrates stem-like properties; these cells are commonly referred to as glioma initiating cells (GIC). These types of cells are pluripotent and can by definition initiate and recapitulate glioma growth in experimental animals in vivo. Furthermore, these cells are often resistant to conventional therapies. Interferon β (IFN-β) is an immunomodulatory molecule with anti-cancer properties. We have previously shown that IFN-β greatly reduces sphere-formation capability of GIC. It was also confirmed that IFN-β sensitized resistant GIC to irradiation or the chemotherapeutic agent, temozolomide (TMZ). IFN-β treatment significantly prolonged survival in a xenograft model with GIC cells. In the current project, we want to use syngeneic mouse models to study the immunomodulatory effects of type I IFNs. Preliminary results indicate that abrogation of IFN signalling in tumor cells by CRISPR/Cas9 technology prolonged survival in mice only in cell lines which have substantial baseline autocrine IFN signalling. On the contrary, we did not observe a difference in survival when wild-type tumor cells were implanted in either IFNAR1 deficient or proficient hosts. Flow cytometry analysis will elucidate changes in immune cell recruitment and infiltration upon IFN signalling disruption. Moreover, we explore different treatments in combination with IFN-β as there are indications that TMZ or radiotherapy can have synergistic effects with stimulation of interferon type I signalling.

STEM-03. Oct4/Sox2 DRIVE AN IMMUNOSUPPRESSIVE GSC PHENOTYPE BY INDUCING T-REG EFFECTOR GENES VIA TGFBR2 SIGNALING

Abstract Solid malignancies, including GBM, contain small subsets of cells that display stem-like properties (i.e. glioma stem cells or GSCs) and act as key determinants of therapeutic resistance and tumor recurrence. Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. GBM cells escape antitumor immunity by modifying the tumor microenvironment by secreting immune-suppressive factors and recruiting anti-inflammatory/pro-oncogenic cells (e.g. T-regulatory cells, M2-like macrophages). However, whether subsets of GSCs can mimic T-reg-like functions by expressing proteins associated with T-reg immune-suppression remains unknown. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3- cell subpopulation with high expression of TGFb1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with T-reg cells and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with CD44 and TGFb type II receptor (TGFBR2) expression in both clinical GBM and primary GSCs. Mechanistically, Oct4 and Sox2 were found to directly induce TGFBR2 transcription concurrent with chromatin architectural changes (i.e. heterochromatin-to-euchromatin transition) involving the TGFBR2 gene. Transgenic TGFBR2 expression increased neurosphere cell growth capacity, upregulated expression of T-reg-like effector genes and enriched for CD44+ GSCs. Functionally, neurospheres expressing transgenic Oct4/Sox2 or transgenic TGFBR2 possess increased immunosuppressive capacity, and pharmacological inhibition of TGFBR2 depleted CD44+ GSCs and reduced immunosuppression in Jurkat cell co-cultures. Taken together, we show that reprogramming events initiated by Oct4 and Sox2 induce a CD44+/FOX3P- GSC cell subset with a T-reg-like immunosuppressive transcriptional profile via a TGFBR2-dependent mechanism. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies.

Antiproliferative activity of CD44 siRNA-PEI-PEG nanoparticles in glioblastoma: involvement of AKT signaling.

Background and purpose:Glioblastoma multiforme (GBM) is the most invasive type of cancer which starts inside the brain. GBM cells were found to have similar properties to glioblastoma cancer stem cells. CD44 can be used as a marker of the cancer stem cells in a subset of glioblastoma tumor cells. Recent studies showed that CD44 is involved in developing cancer cells via the protein kinase B (PKB or AKT) signaling pathway. Therefore, this study aimed to investigate the CD44 mRNA silencing effects on the glioblastoma cell cycle via AKT signaling pathway.Experimental approach:To determine CD44 expression in the samples of the patients with GBM, we used the analysis of data extracted from TCGA database. qRT-PCR and western blotting were used to evaluate the expression level of genes and proteins. Different cell cycles were evaluated by DAPI staining and flow cytometry.Findings/Results:Bioinformatics results showed that CD44 expression level in GBM tumor samples is higher than in normal samples. Effects of poly (ethylene imine)-polyethylene glycol (PEI-PEG)-loaded CD44 siRNA in cell cycle showed that CD44 silencing could inhibit cell cycle in G0-G1 phase by more than 20% compared to the negative control (P < 0.05). Furthermore, PEI-PEG-loaded CD44 siRNA reduces the expression of cyclin D1 and CKD-4. According to our findings, this structure also prevented AKT phosphorylation at Thr-308 and Ser-473.Conclusion and implications:Our results suggest that PEI-PEG-loaded CD44 siRNA may attenuate the cell cycle by suppressing AKT signaling pathway.

S-1 facilitates canerpaturev (C-REV)-induced antitumor efficacy in a triple-negative breast cancer model.

ABSTRACTCanerpaturev (C-REV) is a highly attenuated, replication-competent, mutant strain of oncolytic herpes simplex virus type 1 that may be an effective new cancer treatment option. S-1, an oral formulation containing the 5-fluorouracil (5-FU) prodrug tegafur and the two enzyme modulators gimeracil and oteracil, is used as a key chemotherapeutic agent for metastatic recurrent breast cancer. Although the antitumor effects of oncolytic viruses combined with 5-FU in vivo have been reported, the detailed mechanisms are unknown. Here, we investigated the antitumor mechanism of the combination of C-REV and S-1 in triple-negative breast cancer (TNBC) in the context of tumor immunity. The combined effect of C-REV and S-1 was evaluated in a bilateral tumor model of murine TNBC 4T1 in vivo. S-1 enhanced the TNBC growth inhibitory effects of C-REV, and decreased the number of tumor-infiltrating, myeloid-derived suppressor cells (MDSCs), which suppress both innate and adaptive immune responses. Moreover, C-REV alone and in combination with S-1 significantly increased the number of CD8+ T cells in the tumor and the production of interferon γ (IFNγ) from these cells. Our findings indicate that C-REV suppresses TNBC tumor growth by inducing the expansion of effector CD8+ T cell subsets in tumors in which S-1 can inhibit MDSC function. Our study suggests that MDSCs may be an important cellular target for breast cancer treatment. The combination of C-REV and S-1 is a new approach that might be directly translated into future clinical trials against TNBC.

Wee1 Kinase: A Potential Target to Overcome Tumor Resistance to Therapy.

During the cell cycle, DNA suffers several lesions that need to be repaired prior to entry into mitosis to preserve genome integrity in daughter cells. Toward this aim, cells have developed complex enzymatic machinery, the so-called DNA damage response (DDR), which is able to repair DNA, temporarily stopping the cell cycle to provide more time to repair, or if the damage is too severe, inducing apoptosis. This DDR mechanism is considered the main source of resistance to DNA-damaging therapeutic treatments in oncology. Recently, cancer stem cells (CSCs), which are a small subset of tumor cells, were identified as tumor-initiating cells. CSCs possess self-renewal potential and persistent tumorigenic capacity, allowing for tumor re-growth and relapse. Compared with cancer cells, CSCs are more resistant to therapeutic treatments. Wee1 is the principal gatekeeper for both G2/M and S-phase checkpoints, where it plays a key role in cell cycle regulation and DNA damage repair. From this perspective, Wee1 inhibition might increase the effectiveness of DNA-damaging treatments, such as radiotherapy, forcing tumor cells and CSCs to enter into mitosis, even with damaged DNA, leading to mitotic catastrophe and subsequent cell death.