Exosomes loaded with circPARD3 promotes EBV-miR-BART4-induced stemness and cisplatin resistance in nasopharyngeal carcinoma side population cells through the miR-579-3p/SIRT1/SSRP1 axis.

OBJECTIVE: To explore the effects of exosomes loaded with circular RNA PARD3 on EBV-miR-BART4-induced stemness and resistance of cisplatin in nasopharyngeal carcinoma side population (NPC-SP) cells through the miR-579-3p/SIRT1/SSRP1 axis. METHODS: Sixty-five cancer tissues and 65 noncancerous tissues were collected from NPC patients or patients with rhinitis. The expressions of circPARD3, miR-579-3p, SIRT1, and SSRP1 were detected by qRT-PCR, western blot, or immunohistochemistry. In vivo tumor formation assay was performed in nude mice. Immunofluorescence and qRT-PCR were conducted for the determination of CD44 and CD133 expressions, and flow cytometry combined with Hoechst 33,342 dye efflux for identifying SP cells, CCK-8 and EdU assays for cell proliferation, and Transwell assay for migration and invasion. RESULTS: CircPARD3, SIRT1, and SSRP1 were upregulated while miR-579-3p was downregulated in NPC tissues and cells. CircPARD3 was positively correlated with the expressions of SIRT1 and SSRP1, and miR-579-3p was negatively correlated with circPARD3, SIRT1, and SSRP1. Exosomes loaded with circPARD3 promoted EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells, while miR-579-3p reversed the effect of exosomal circPARD3 on EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells. Additionally, miR-579-3p suppressed EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells by regulating SIRT1. SIRT1 upregulated SSRP1 expression by catalyzing H3K4 methylation and down-regulation of SSRP1 reversed the effect of SIRT1 on EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells. CONCLUSION: Exosomes loaded with circPARD3 promoted EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells through the miR-579-3p/SIRT1/SSRP1 axis. Graphical Headlights • EBV-miR-BART4 induces the stemness and resistance of NPC-SP cells. • CircPARD3 regulates SIRT1 by miR-579-3p. • SIRT1 regulates SSRP1 expression by histone methylation. • Exosomes loaded with circPARD3 promotes EBV-miR-BART4-induced NPC-SP cell stemness and resistance by the miR-579-3p/SIRT1/SSRP1 axis.

[SENP1 induced protein deSUMO modification increased the chemotherapy sensitivity of endometrial cancer side population cells].

Objective: To inhibit the stemness maintenance potential of endometrial cancer and increase the sensitivity of endometrial cancer side population cells to chemotherapy drugs by inducing extensive deSUMOylation modification of proteins. Methods: Flow cytometry was used to sort and culture CD133(+) CD44(+) KLE endometrial cancer cell clone spheres. Protein expression level of small ubiquitin-related modifier 1 (SUMO1) and two stemness maintenance genes of tumor side population cells, octamer binding transcription factor-4 (Oct4) and sex determining region Y-box2 (Sox2), were detected by western blotting method. Lentivirus-mediated Sentrin/SUMO-specific proteases 1 (SENP1) gene was stably transfected into KLE side population cells. Western blotting was used to detect the protein expressions of SENP1, SUMO1, Oct4 and Sox2. The clone formation rate was compared between KLE side population cells with or without SENP1 overexpression. Flow cytometry was applied to detect cell cycle changes. 3-(4, 5-Dimethylthiazole-2)-2, 5-diphenyl-tetrazolium bromide (MTT) experiment and flow cytometry apoptosis method were used to detect the chemosensitivity of the side population of endometrial cancer cells to cisplatin. Tumor-bearing mouse models of endometrial cancer were established to detect the effect of SENP1 overexpression on the chemotherapy sensitivity of cisplatin. Results: Compared with CD133(-)CD44(-) KLE cells, CD133(+) CD44(+) KLE side population cells could form clonal spheres and express higher levels of SUMO1, Oct4 and Sox2 proteins (P<0.05). Compared with KLE side population cells that were not transfected with SENP1 gene, the expression level of SENP1 protein in KLE side population cells overexpressing SUMO1、Oct4 and Sox2 were lower. The clonal sphere formation rate was reduced from (25.67±5.44)% to (7.46±1.42)%, and cell cycle shifted from G(0)/G(1) phase to G(2) phase. IC(50) of cisplatin decreased from (55.46±6.14) µg/ml to (11.55±3.12) µg/ml, and cell apoptosis rate increased from (9.76±2.09)% to (16.79±3.44)%. Overexpression of SENP1 could reduce the tumorigenesis rate of KLE side population cells in vivo and increase their chemotherapy sensitivity to cisplatin (P<0.05). Conclusion: Overexpression of SENP1 can induce protein deSUMOylation modification, inhibit the stemness maintenance potential of endometrial cancer side population cells, and enhance their chemotherapy sensitivity, which provides a new reference for gene therapy of endometrial cancer.

Sinomenine Inhibits Vasculogenic Mimicry and Migration of Breast Cancer Side Population Cells via Regulating miR-340-5p/SIAH2 Axis.

Hypoxia and its induced vasculogenic mimicry (VM) formation, which both closely related with stem-like side population (SP) cells, are the main culprits leading to tumor invasion and metastasis. Sinomenine exhibits excellent anticancer activity in breast cancer, but whether and how it affects hypoxia-triggered VM formation in breast cancer SP cells remains unclear. In this study, breast cancer SP cells were sorted from MDA-MB-231 cells and cultured with sinomenine under hypoxic conditions. Sinomenine obviously repressed the migration and VM formation of breast cancer SP cells. Through downregulating SIAH2 and HIF-1α, sinomenine can inhibit epithelial-mesenchymal transition process of breast cancer SP cells. SIAH2 was identified as a target of miR-340-5p and was downregulated by it, and sinomenine can upregulate miR-340-5p. Hypoxia-induced downregulation of miR-340-5p and activation of SIAH2/HIF-1α pathway can be both counteracted by the sinomenine. Moreover, miR-340-5p inhibition and SIAH2 overexpression can partly counteract the anticancer effects of sinomenine. Taken together, sinomenine inhibits hypoxia-caused VM formation and metastasis of breast cancer SP cells by regulating the miR-340-5p/SIAH2 axis.

Impact of transforming growth factor beta 1 on normal and thyroid cancer side population cells.

PURPOSE: To determine the impact of exogenous transforming growth factor beta 1 (TGF-ß1) on side population (SP) cells isolated from normal, papillary thyroid cancer and anaplastic thyroid cancer cell lines and from human thyroid tissues. METHODS: All cell populations were stained with Hoechst 33342 and analysed using dual wavelength flow cytometry to identify SP cells. This SP assay was used to assess the impact of TGF-ß1 treatment and withdrawal of treatment on SP percentages. Semi-quantitative and quantitative PCR were used for molecular analysis of cells pre and post TGF-ß1 treatment. RESULTS: All cell lines expressed mRNA for both TGFB1 and its receptors, as well as showing variable expression of CDH1 and CDH2, with expressing of CDH1 being highest and CDH2 being lowest in the normal cell line. Exposure to exogenous TGF-ß1 resulted in a reduction in mRNA expression of ABCG2 compared to controls which was significant between control and treated cancer cell lines. SP cells were isolated from primary human thyroid tissues, with numbers being significantly higher in papillary thyroid cancers. Exposure to TGF-ß1 decreased the SP percentage in both thyroid cancer cell lines and completely abrogated these cells in the primary papillary thyroid cancer cultures. On withdrawal of TGF-ß1 the SP phenotype was restored in the cancer cell lines and SP percentages increased to above that of untreated cells. CONCLUSIONS: TGF-ß1 exposure transiently regulates thyroid cancer SP cells, leading to a reduction in SP percentages, while withdrawal of TGF-ß1 results in restoration of the SP phenotype.

Fms-like tyrosine kinase 3 is a regulator of the cardiac side population in mice.

Fms-like tyrosine kinase 3 (Flt3) is a regulator of hematopoietic progenitor cells and a target of tyrosine kinase inhibitors. Flt3-targeting tyrosine kinase inhibitors can have cardiovascular side effects. Flt3 and its ligand (Flt3L) are expressed in the heart, but little is known about their physiological functions. Here, we show that cardiac side population progenitor cells (SP-CPCs) from mice produce and are responsive to Flt3L. Compared with wild-type, flt3L-/- mice have less SP-CPCs with less contribution of CD45-CD34+ cells and lower expression of genes related to epithelial-to-mesenchymal transition, cardiovascular development and stem cell differentiation. Upon culturing, flt3L-/- SP-CPCs show increased proliferation and less vasculogenic commitment, whereas Akt phosphorylation is lower. Notably, proliferation and differentiation can be partially restored towards wild-type levels in the presence of alternative receptor tyrosine kinase-activating growth factors signaling through Akt. The lower vasculogenic potential of flt3L-/- SP-CPCs reflects in decreased microvascularisation and lower systolic function of flt3L-/- hearts. Thus, Flt3 regulates phenotype and function of murine SP-CPCs and contributes to cellular and molecular properties that are relevant for their cardiovasculogenic potential.

Side population analysis in clear cell renal cell carcinoma.

Cancer stem cells have an important role in tumour biology. While their identity in haematological malignancies is clearly defined, stem cell identity remains elusive in some solid tumours. Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer, but the identity or existence of ccRCC stem cells remains unknown. We aimed to discern their existence using the widely utilised side population approach in ccRCC cell lines. In all cells tested, a well-defined side population was identified, and cell-based assays suggested stem-like properties. However, limiting dilution assays revealed comparable tumour initiating abilities and tumour histology of side and non-side populations, and single cell RNA-sequencing revealed minimal differences between these populations. The results indicate that the side population approach is not sufficient for cancer stem cell discovery in ccRCC.

Tumor-propagating side population cells are a dynamic subpopulation in undifferentiated pleomorphic sarcoma.

Sarcomas contain a subpopulation of tumor-propagating cells (TPCs) with enhanced tumor-initiating and self-renewal properties. However, it is unclear whether the TPC phenotype in sarcomas is stable or a dynamic cell state that can derive from non-TPCs. In this study, we utilized a mouse model of undifferentiated pleomorphic sarcoma (UPS) to trace the lineage relationship between sarcoma side population (SP) cells that are enriched for TPCs and non-SP cells. By cotransplanting SP and non-SP cells expressing different endogenous fluorescent reporters, we show that non-SP cells can give rise to SP cells with enhanced tumor-propagating potential in vivo. Lineage trajectory analysis using single-cell RNA sequencing from SP and non-SP cells supports the notion that non-SP cells can assume the SP cell phenotype de novo. To test the effect of eradicating SP cells on tumor growth and self-renewal, we generated mouse sarcomas in which the diphtheria toxin receptor is expressed in the SP cells and their progeny. Ablation of the SP population using diphtheria toxin did not impede tumor growth or self-renewal. Altogether, we show that the sarcoma SP represent a dynamic cell state and targeting TPCs alone is insufficient to eliminate tumor progression.

Abnormal expression of FAK and paxillin correlates with oral cancer invasion and metastasis.

Globally, the tenth most common cancer is the oral squamous cell carcinoma (OSCC) and the treatment strategy for improving of OSCC patients survival rate still remains a challenging one. Aberrant regulation of cell to extracellular matrix protein interactions leads to progression of human cancers. The focal adhesion kinase (FAK) and its downstream target paxillin have been implicated in cancer growth, migration, invasion and metastasis of different cancers. However, the clinical significance of FAK and paxillin in OSCC is not well characterized so far. In the present work, we showed that relative mRNA and protein expressions of FAK and paxillin are significantly higher in side population (SP) cells of OSCC cell line SCC-55. Concomitantly, the matrix metalloproteinase-11 (MMP-11) level is also significantly elevated in SP cells. The enhanced expression of paxillin is strongly correlated with increased chemoresistance, proliferation rate, migration and invasion potential of SP cells. In addition, inhibition of paxillin expression by RNAi makes SP cells more sensitive to chemotherapy drugs. Therefore, our results suggest that paxillin over expression might play a significant role in cancer progression, invasion and chemoresistance of OSCC.

Clinical significance of cancer stem cell markers in lung carcinoma.

The identification of side population (SP) cells in several cancer studies has been proved to be involved in the treatment failure (chemotherapy) and tumor relapse. Here we have sorted 7% of side population (SP) cells from lung adenocarcinoma by Hoechst 33342 dye expulsion method. Further, the characterization of sorted SP cells showed cancer stem like properties such as transcriptional upregulation of stemness genes (OCT-4, SOX2 and NANOG), ATP binding cassette (ABC) transporter protein (ABCG2) and enhanced level of stem cell surface markers such as CD133 and CD44. Therefore, the aforesaid properties of lung adenocarcinoma SP cells play a significant and functional role in tumor invasion, metastasis, chemotherapeutic drug resistance and tumor recurrence in lung cancer.

Analysis of Side Population in Solid Tumor Cell Lines.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

In vitro and ex vivo anti­tumor effect and mechanism of Tucatinib in leukemia stem cells and ABCG2­overexpressing leukemia cells.

Leukemia stem cells (LSCs), which evade standard chemotherapy, may lead to chemoresistance and disease relapse. The overexpression of ATP­binding cassette subfamily G member 2 (ABCG2) is an important determinant of drug resistance in LSCs and it can serve as a marker for LSCs. Targeting ABCG2 is a potential strategy to selectively treat and eradicate LSCs, and, hence, improve leukemia therapy. Tucatinib (Irbinitinib) is a novel tyrosine kinase inhibitor, targeting ErbB family member HER2, and was approved by the Food and Drug Administration in April 2020, and in Switzerland in May 2020 for the treatment of HER2­positive breast cancer. In the present study, the results demonstrated that tucatinib significantly improved the efficacy of conventional chemotherapeutic agents in ABCG2­overexpressing leukemia cells and primary leukemia blast cells, derived from patients with leukemia. In addition, tucatinib markedly decreased the proportion of leukemia stem cell­like side population (SP) cells. In SP cells, isolated from leukemia cells, the intracellular accumulation of Hoechst 33342, which is an ABCG2 substrate, was significantly elevated by tucatinib. Furthermore, tucatinib notably inhibited the efflux of [3H]­mitoxantrone and, hence, there was a higher level of [3H]­mitoxantrone in the HL60/ABCG2 cell line. The result from the ATPase assay revealed that tucatinib may interact with the drug substrate­binding site and stimulated ATPase activity of ABCG2. However, the protein expression level and cellular location of ABCG2 were not affected by tucatinib treatment. Taken together, these data suggested that tucatinib could sensitize conventional chemotherapeutic agents, in ABCG2­overexpressing leukemia cells and LSCs, by blocking the pump function of the ABCG2 protein. The present study revealed that combined treatment with tucatinib and conventional cytotoxic agents could be a potential therapeutic strategy in ABCG2­positive leukemia.

Aberrant activation of Wnt/catenin signaling and overexpression of ABCG2 contributes to apoptosis down regulation and tumor progression of high grade ovarian cancer.

Side Population (SP) cells are the small pool of CSC like progenitor cells, which are drug resistant and recapitulate tumor generation. The occurrence of SP cells is the major inference for attaining a better treatment and improved patient survival. In this work, we have isolated 6% SP cells from a high grade ovarian carcinoma. Our functional characterization of SP cells revealed that elevated ABCG2 and anti-apoptotic factors contribute to chemoresistance and increased life span of SP cells. Further, the overexpression of surface antigens, such as CD133 and CD117 in SP cells, are the key driving forces for high clonogenic and invasion properties of SP cells. More importantly, we found by RT-PCR aberrant activation and upregulation of Wnt/ ß-catenin and its downstream targeting genes, such as DKK1 and AXIN2 in SP cells. These findings suggest that development of new anticancer drugs which target Wnt/ß-catenin signaling might effectively exterminate the SP cells and aid in disease free survival.

Diallyl thiosulfinate enhanced the anti-cancer activity of dexamethasone in the side population cells of multiple myeloma by promoting miR-127-3p and deactivating the PI3K/AKT signaling pathway.

BACKGROUND: Side population (SP) cells, which have similar features to those of cancer stem cells, show resistance to dexamethasone (Dex) treatment. Thus, new drugs that can be used in combination with Dex to reduce the population of SP cells in multiple myeloma (MM) are required. Diallyl thiosulfinate (DATS, allicin), a natural organosulfur compound derived from garlic, has been shown to inhibit the proliferation of SP cells in MM cell lines. Therefore, we investigated the effect of a combination of DATS and Dex (DAT + Dex) on MM SP cells. METHODS: SP cells were sorted from MM RPMI-8226 and NCI-H929 cell lines using Hoechst 33342-labeled fluorescence-activated cell sorting. The growth of SP cells was evaluated using the cell counting kit-8 assay. Cell cycle and apoptosis assays were conducted using a BD Calibur flow cytometer. miRNA expression was measured using quantitative reverse transcription-polymerase chain reaction. Phosphoinositide 3-kinase (PI3K), phosphorylated AKT (p-AKT), AKT, p-mechanistic target of rapamycin (mTOR), and mTOR levels were measured using western blot analysis. RESULTS: Our results showed that the combination of DATS+Dex inhibited sphere formation, colony formation, and proliferation of MM SP cells by inducing apoptosis and cell cycle arrest in the G1/S phase. In addition, the combination of DATS+Dex promoted miR-127-3p expression and inhibited PI3K, p-AKT, and p-mTOR expression in SP cells. Knockdown of miR-127-3p expression weakened the effect of DATS+Dex on cell proliferation, colony formation, apoptosis, and cell cycle of MM SP cells. Additionally, knockdown of miR-127-3p activated the PI3K/AKT/mTOR signaling pathway in MM SP cells cotreated with DATS+Dex. CONCLUSION: We demonstrated that cotreatment with DATS+Dex reduced cell proliferation, promoted apoptosis, and caused cell cycle arrest of MM SP cells by promoting miR-127-3p expression and deactivating the PI3K/AKT/mTOR signaling pathway.

Gene-expression profile and postpartum transition of bovine endometrial side population cells†.

The mechanism of bovine endometrial regeneration after parturition remains unclear. Here, we hypothesized that bovine endometrial stem/progenitor cells participate in the postpartum regeneration of the endometrium. Flow cytometry analysis identified the presence of side population (SP) cells among endometrial stromal cells. Endometrial SP cells were shown to differentiate into osteoblasts and adipocytes. RNA-seq data showed that the gene expression pattern was different between bovine endometrial SP cells and main population cells. Gene Set Enrichment Analysis identified the enrichment of stemness genes in SP cells. Significantly (false discovery rate < 0.01) upregulated genes in SP cells contained several stem cell marker genes. Gene ontology (GO) analysis of the upregulated genes in SP cells showed enrichment of terms related to RNA metabolic process and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of upregulated genes in SP cells revealed enrichment of signaling pathways associated with maintenance and differentiation of stem/progenitor cells. The terms involved in TCA cycles were enriched in GO and KEGG pathway analysis of downregulated genes in SP cells. These results support the assumption that bovine endometrial SP cells exhibit characteristics of somatic stem/progenitor cells. The ratio of SP cells to endometrial cells was lowest on days 9-11 after parturition, which gradually increased thereafter. SP cells were shown to differentiate into epithelial cells. Collectively, these results suggest that bovine endometrial SP cells were temporarily reduced immediately after calving possibly due to their differentiation to provide new endometrial cells.

Verteporfin induces apoptosis and reduces the stem cell-like properties in Neuroblastoma tumour-initiating cells through inhibition of the YAP/TAZ pathway.

Neuroblastoma is an embryonal malignancy of early childhood arising from the embryonic sympatho-adrenal lineage of the neural crest. About half of all cases are currently classified as high-risk of disease recurrence, with an overall survival rate of less than 40% at 5 years despite intensive therapy. Recent studies on matched primary tumours and at the relapse revealed downregulation of genes transcriptionally silenced by YAP as significant association with neuroblastoma relapse. Here, we evaluated the pharmacological targeting of YAP/TAZ with the YAP/TAZ-TEAD inhibitor Verteporfin (VP) in Tumour Initiating Cells (TICs) derived from High-Risk Neuroblastoma patients. VP treatment suppresses YAP/TAZ expression, induces apoptosis and causes the re-organization of the cytoskeleton reducing cells migration and clonogenic ability. Moreover, VP reduces the percentage of side population cells and ABC transporters involved in drug resistance, and the percentage of stem cell subpopulations CD133+ and CD44+ of TICs. Finally, we demonstrated that VP sensitizes TICs to the standard drugs used for neuroblastoma therapy etoposide and cis-platin opening the way to use VP as drug repositioning candidate for recurrent neuroblastoma.

4,4'­Bond secalonic acid D targets SP cells and inhibits metastasis in hepatocellular carcinoma.

The existence of cancer stem cells (CSCs) is considered to be the main reason for chemoresistance, metastasis and the ultimate failure of treatment in hepatocellular carcinoma (HCC). However, there are a few chemical agents that may inhibit CSCs. The present study identified that 4,4'­bond secalonic acid D (4,4'­SAD), a compound isolated from the marine­derived fungus Penicillium oxalicum, inhibited the growth of side population (SP) cells isolated from human liver cancer cell lines PLC/PRF/5 and HuH­7 by attenuating the expression of ATP­binding cassette superfamily G member 2. Furthermore, the results of wound healing, Transwell, western blotting and reverse transcription­quantitative PCR assays demonstrated that 4,4'­SAD suppressed the invasion and migration of SP cells by downregulating matrix metallopeptidase 9 (MMP­9) and upregulating the antagonist tissue inhibitor of metalloproteinases 1 in vitro. Moreover, in vivo study results found that 4,4'­SAD had anti­lung metastasis efficacy via the decrease of MMP­9 expression in the H22 HCC model of Kunming mice. Therefore, the present study identified the potential of 4,4'­SAD as a promising candidate for the treatment of advanced liver cancer.

Identification, characterization and microRNA expression profiling of side population cells in human oral squamous cell carcinoma Tca8113 cell lines.

The present study aimed to evaluate the stem cell markers, characteristics and biological functions of cancer stem­like side population (SP) cells in human oral cancer. SP cells were isolated from the human oral squamous cell carcinoma Tca8113 cell line by Hoechst 33342 fluorescence dye and flow cytometry. The colony forming and proliferative capability of SP and non­SP cells were detected using a live­cell analysis system in vitro. The number of cells expressing stem cell markers was compared between SP cells and non­SP cells by flow cytometry. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of stem cell genes, respectively. Differential expression of microRNAs (miRNAs) in SP and non­SP cells was determined by microarray hybridization and an miRNA regulation network was produced. With regard to the proliferation capability, SP cells reached 60.0% confluence after 40 h of growth compared with 35.1% confluence for non­SP cells (P<0.05). The number of colonies in SP cells was 43.1±9.2 compared with 33.0±8.2 of non­SP cells (P<0.05). The aldehyde dehydrogenase­1 (ALDH1)­positive cell number in the SP cells was increased by 10 times compared with the non­SP cells (P<0.01). The mRNA and protein expression levels of ALDH1, SRY­box 2, POU class 5 homeobox 1 and Nanog homeobox in SP cells were significantly higher compared with non­SP cells (P<0.05). Microarray hybridization demonstrated that 21 miRNAs were upregulated and 13 miRNAs were downregulated in SP cells compared with non­SP cells. SP cells in Tca8113 demonstrated greater capability of proliferation and colony formation compared with non­SP cells in vitro. Stem cell markers were overexpressed in SP cells compared with non­SP cells.

Abcg2-expressing side population cells contribute to cardiomyocyte renewal through fusion.

The adult mammalian heart has a limited regenerative capacity. Therefore, identification of endogenous cells and mechanisms that contribute to cardiac regeneration is essential for the development of targeted therapies. The side population (SP) phenotype has been used to enrich for stem cells throughout the body; however, SP cells isolated from the heart have been studied exclusively in cell culture or after transplantation, limiting our understanding of their function in vivo. We generated a new Abcg2-driven lineage-tracing mouse model with efficient labeling of SP cells. Labeled SP cells give rise to terminally differentiated cells in bone marrow and intestines. In the heart, labeled SP cells give rise to lineage-traced cardiomyocytes under homeostatic conditions with an increase in this contribution following cardiac injury. Instead of differentiating into cardiomyocytes like proposed cardiac progenitor cells, cardiac SP cells fuse with preexisting cardiomyocytes to stimulate cardiomyocyte cell cycle reentry. Our study is the first to show that fusion between cardiomyocytes and non-cardiomyocytes, identified by the SP phenotype, contribute to endogenous cardiac regeneration by triggering cardiomyocyte cell cycle reentry in the adult mammalian heart.

Do Different Stemness Markers Identify Different Pools of Cancer Stem Cells in Malignancies: A Study on ER+ and ER-Breast Cancer Cell Lines.

In view of popularity of cancer stem cell (CSC) model all events in evolution of cancer are being explained in that context. Breast cancer is first solid tumor in which CSCs were identified. We aimed to compare stemness profile of two major subtypes [Estrogen receptor positive (ER+) and negative (ER-)] breast cancer using different sets of markers. Expression of CD44/CD24, CK/Vimentin, E-Cadherin/Fibronectin and percentage of side population (SP) was studied in ER+ (T47D) and ER- (MDA-MB-231) cell lines by flow cytometry. Breast CSCs (BCSCs) were sorted using CD44+/CD24-/low expression and SP analysis and cultured. BCSCs were then compared with Non-CSCs (NCSCs) for response to drugs (Paclitaxel and Cisplatin), Ki67 and ER expression. Results showed higher expression of stemness markers (CD44+/CD24-/low, CK+/Vimentin+ and E-Cadherin-/FibrinectinF+) in MDA-MB-231 cells. Percentage SP representing BCSCs was found to be significantly more in later (3.20 ± 0.002 cf. T47D 1.25% ± 0.0007). BCSCs were found to be more resistant to drugs as compared to NCSCs in both cell lines. ER expression was weak in BCSCs sorted from T47D as compared to NCSCs. Ki67 was expressed in both BCSCs and NCSCs. Differences in expression of stemness markers help to explain aggressive behavior, higher recurrence rate and metastatic potential of MDA-MB-231 cells. However, no correlation amongst different markers used suggests that they may be identifying varied populations of cells in tumor hierarchy. A weak ER expression in BCSCs may be strategy used by BCSCs to escape effect of hormone therapy in ER+ breast cancers.

SOX2 upregulates side population cells and enhances their chemoresistant ability by transactivating ABCC1 expression contributing to intrinsic resistance to paclitaxel in melanoma.

Paclitaxel is the last choice for the treatment of advanced melanoma as a second-line chemotherapeutic agent, but there are still many cases of intrinsic resistance to paclitaxel in melanoma and the reasons that cause paclitaxel resistance remain unclear. Here, we identified that high expression of SRY-box transcription factor 2 (SOX2) and high ratio of side population (SP) cells reduced the sensitivity to paclitaxel in melanoma cells. The knockout and the induction of SOX2 completely depleted and significantly upregulated the ratios of melanoma SP cells, respectively. These data suggest that SOX2, a pluripotent transcription factor for inducing cancer stem cells in melanoma, is also sufficient and necessary for the induction of melanoma SP cells. ATP-binding cassette (ABC) subfamily C member 1 (ABCC1) is one of ABC transporters which causes SP cells to be resistance to chemotherapeutic agents by efficiently pumping drugs out of cells. The knockout and the induction of ABCC1 significantly increased and decreased the sensitivity of melanoma cells to paclitaxel. High expression of ABCC1 was identified in melanoma cell lines with high expression of SOX2 and in their SP cells. SOX2 was identified to induce ABCC1 transcription. Taken together, SOX2 upregulates SP cells and enhances their chemoresistant ability by increasing ABCC1 expression, which contributes to intrinsic resistance to paclitaxel in melanoma. Our findings will lead to new insights into melanoma biology and therapy resistance, and eventually to new therapeutic targets.