Expression Of Stemness Markers Research Articles

Abstract 3274: Trefoil factor 1 suppresses stemness and enhances chemosensitivity of pancreatic cancer

Abstract Background and Aims: Pancreatic cancer is one of the most lethal malignancies, partly due to resistance to conventional chemotherapy. The chemoresistance of malignant tumors is associated with epithelial-mesenchymal transition (EMT) and the stemness of cancer cells. The aim of this study is to investigate the availability and functional mechanisms of trefoil factor family 1 (TFF1), a tumor-suppressive protein in pancreatic carcinogenesis, to treat pancreatic cancer. Methods: To investigate the role of endogenous TFF1 in human and mice, specimens of human pancreatic cancer and genetically engineered mouse model of pancreatic cancer (KPC/TFF1KO; Pdx1-Cre/LSL-KRASG12D/LSL-p53R172H/TFF1−/−) were analyzed by immunohistochemistry (IHC). To explore the efficacy of extracellular administration of TFF1, recombinant and chemically synthesized TFF1 were administered to pancreatic cancer cell lines, a xenograft mouse model and a transgenic mouse model. Results: The deficiency of TFF1 was associated with increased EMT markers of cancer cells (Snail and Zeb1) in mouse models of pancreatic cancer, KPC. Analysis of the survival of patients who underwent chemotherapy for recurrent pancreatic cancer revealed that the expression of TFF1 in cancer cells was associated with better survival. Also, in the mouse model of pancreatic cancer, KPC mice showed improved survival when treated with gemcitabine, the KPC/TFF1KO mice did not. Pancreatic cancer cell lines were administered with gemcitabine and/or chemically synthesized TFF1, revealing that addition of TFF1 downregulated gemcitabine-induced EMT markers (Snail, Slug, ZEB1, and αSMA), Wnt pathway activation (β-catenin, TCF7, and EPHB3). In addition, TFF1 inhibited the expression of cancer stemness markers (CD133 and NANOG3) and the ability to form tumor sphere. Importantly, TFF1 accelerated the apoptosis of cancer cells induced by gemcitabine in vitro. In vivo, combined treatment of gemcitabine and subcutaneous administration of TFF1 arrested tumor growth in xenograft mouse model. Also, KPC mice with combined treatment showed the better survival rather than gemcitabine-alone treatment, with lower expression of CD133, high expression of ZEB1, and lower nuclear translocation of β-catenin. Conclusion: These results indicate that TFF1 can contribute to establishing a novel strategy to treat pancreatic cancer patients by enhancing chemosensitivity. Citation Format: Junpei Yamaguchi, Toshio Kokuryo, Yukihiro Yokoyama, Masaki Sunagawa, Taisuke Baba, Tomoki Ebata. Trefoil factor 1 suppresses stemness and enhances chemosensitivity of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3274.

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation. CECs were divided into central zone (8mm trephined boundary) and peripheral zone (8mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot. The results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate. Our findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.

Infrared diode laser enhances human periodontal ligament stem cells behaviour on titanium dental implants

Low level laser treatment (LLLT) is known for its photobiostimulatory and photobiomodulatory characteristics, which stimulate cell proliferation, increase cellular metabolism, and improve cellular regeneration. The objective of the present research was to assess the possible influence of infrared diode laser irradiation on the behaviour, attachment, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) seeded on different types of dental implants. Two distinct types of implants, one subjected to laser surface treatment and the other treated with acid etching, were longitudinally divided into two halves and submerged in six wells culture plates. Both implants were subjected to infrared diode laser treatment, and subsequently, the morphology and attachment of cells were examined using scanning electron microscopy (SEM) after 14 and 21 days. The behaviour of (hPDLSCs) towards two types of implants, when exposed to osteogenic medium and low-level laser therapy (LLLT), was assessed using quantitative real-time polymerase chain reaction to measure the expression of stemness markers and osteogenic markers. The scanning electron microscopy (SEM) demonstrated that the application of infrared diode laser irradiation substantially improved the attachment of cells to both types of implants. The stemness gene markers were significantly down regulated in cells seeded on both surfaces when challenged with osteogenic media in relation to control. At 14 days, early osteogenic markers, were upregulated, while late osteogenic markers, were downregulated in both challenged groups. At the 21-day mark, hPDLSCs seeded on an acid-etched implant exhibited increased expression of all osteogenic markers in response to stimulation with osteogenic media and infra-red diode laser, in contrast to hPDLSCs seeded on a laser surface treated implant under the same conditions. Finally, the findings of our research revealed that when subjected to infrared diode laser, human periodontal ligament stem cells cultured on both types of implants demonstrated improved cellular attachment and differentiation. This suggested that infrared diode laser enhanced the activity of the cells surrounding the implants. Hence, the use of infrared diode laser could be pivotal in improving and expediting the clinical osseointegration process around dental implants.

In vivo study of the effect of anlotinib on the stemness of the lenvatinib-resistant hepatocellular carcinoma cells and the underlying mechanisms

Abstract Background In vivo experiments were conducted to examine the effects of the targeted drug anlotinib on the stemness of hepatocellular carcinoma (HCC) cells and lenvatinib-resistant liver cancer cells and to explore the underlying molecular mechanisms. Methods A subcutaneous xenograft model of Hep3B-derived HCC was established in nude mice, which were randomly divided into 2 groups (n = 5 males per group): (1) intragastric administration of anlotinib (0.4 mg/kg) and (2) intragastric administration of normal saline. We constructed lenvatinib-resistant cell lines and randomly divided the mice into 3 groups (n = 5 males per group): (1) intragastric administration of anlotinib, (2) intragastric administration of lenvatinib, and (3) intragastric administration of normal saline. After 2 weeks of treatment, tumor tissues were harvested, and mRNA and proteins were isolated from the tissues. Changes in the expression of cancer stemness markers (epithelial cell adhesion molecule [EpCAM], CD13, CD90, aldehyde dehydrogenase 1 [ALDH1], CD44, and CD45), totipotency factors (sex-determining region Y-box 2 [Sox2], Nanog, octamer-binding transcription factor 4 [Oct4]), and genes related to the Notch signaling pathway were examined. Results Compared with that in the control group, tumor size and weight were reduced in nude mice treated with anlotinib. These differences were statistically significant in both the types of nude mice. Anlotinib affected stemness markers and totipotency factors by downregulating the expression of CD133, CD90, and G-protein–coupled receptor 5 (LGR5) and upregulating the expression of intercellular adhesion molecule 1 (ICAM-1) and Sox2. In addition, lenvatinib-resistant cell lines increased Notch signaling pathway, whereas anlotinib inhibited Notch signaling pathway. Conclusions The antitumor effect of anlotinib on HCC and lenvatinib-resistant HCC cells may occur through inhibition of the Notch signaling pathway. Anlotinib may be the drug of choice for sequential therapy in lenvatinib-resistant liver cancer.

Preparation and in vitro evaluation of cell adhesion and long-term proliferation of stem cells cultured on silibinin co-embedded PLGA/Collagen electrospun composite nanofibers

The present research aims to evaluate the efficacy of Silibinin-loaded mesoporous silica nanoparticles (Sil@MSNs) immobilized into polylactic-co-glycolic acid/Collagen (PLGA/Col) nanofibers on the in vitro proliferation of adipose-derived stem cells (ASCs) and cellular senescence. Here, the fabricated electrospun PLGA/Col composite scaffolds were coated with Sil@MSNs and their physicochemical properties were examined by FTIR, FE-SEM, and TGA. The growth, viability and proliferation of ASCs were investigated using various biological assays including PicoGreen, MTT, and RT-PCR after 21 days. The proliferation and adhesion of ASCs were supported by the biological and mechanical characteristics of the Sil@MSNs PLGA/Col composite scaffolds, according to FE- SEM. PicoGreen and cytotoxicity analysis showed an increase in the rate of proliferation and metabolic activity of hADSCs after 14 and 21 days, confirming the initial and controlled release of Sil from nanofibers. Gene expression analysis further confirmed the increased expression of stemness markers as well as hTERT and telomerase in ASCs seeded on Sil@MSNs PLGA/Col nanofibers compared to the control group. Ultimately, the findings of the present study introduced Sil@MSNs PLGA/Col composite scaffolds as an efficient platform for long-term proliferation of ASCs in tissue engineering.

LncRNA H19 Participates in Leukemia Inhibitory Factor Mediated Stemness Promotion in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common human malignancy and the third leading cause of cancer-related death worldwide. Cancer stem cells (CSCs) were considered to play important roles in the genesis and development of many tumors. In recent years, it has been observed that leukemia inhibitory factor (LIF) might be involved in the regulation of stemness in cancer cells. In this study, we observed that LIF could increase the spheroid formation and stemness marker expression (inculding Nanog and SOX2) in CRC cell lines, such as HCT116 and Caco2 cells. Meanwhile, we also observed that LIF could upregulate LncRNA H19 expression via PI3K/AKT pathway. Knockdown of the expression of LncRNA H19 could decrease the spheroid formation and SOX2 expression in LIF-treated HCT116 and Caco2 cells, and thereby LncRNA H19 knockdown could compensate for the stemness enhancement effects induced by LIF. Our results indicated that LncRNA H19 might participate in the stemness promotion of LIF in CRC cells.

Trk Inhibition Reduces Tumorsphere Formation and Changes Expression of Stemness Markers in SK-ES-1 Ewing Sarcoma Cells

Introduction: Ewing sarcoma (ES) is a highly aggressive type of childhood cancer characterized by a chromosomal translocation resulting in fusions between the gene encoding EWS RNA Binding Protein 1 (EWSR1) and one gene of the ETS family, most frequently FLI-1, resulting in the EWS-FLI1 aberrant transcription factor. ES tumors can contain a subpopulation of cells showing cancer stem cell (CSC) features, which express stemness markers including CD133, OCT4 (Octamer-binding transcription factor 4), and NANOG, and display capacity to form tumorspheres likely enriched in CSCs. Neurotrophin (NT) receptors of the tropomyosin receptor kinase (Trk) family (TrkA, TrkB, and TrkC) may play a role in stimulating ES progression, but their possible role in CSCs remains unknown. Objective: To verify the effect of Trks inhibition on the formation of tumorspheres as well as the gene expression of stem markers. Method: The cells were dissociated and the formation of spheres was induced with supplemented culture medium and the K252a treatment was performed. After RNA extraction, mRNA expression levels of target genes Prom1 (CD133), OCT4 (POU5F1), SOX2, and Musashi-1 (MSI1) were analyzed by qPCR. Results: The pan-Trk inhibitor K252a (100 or 500 mM) hindered tumorsphere formation in human SK-ES-1 ES cell cultures. K252a also reduced mRNA expression of Prom1 (CD133-coding gene) while enhancing expression of OCT4. No changes in mRNA levels of SOX2 or Musashi-1 were observed. Conclusion: These findings provide the first evidence suggesting that Trk activity can influence stemness in ES cells.

Transcriptomic analysis identifies B-lymphocyte kinase as a therapeutic target for desmoplastic small round cell tumor cancer stem cell-like cells

Desmoplastic small round cell tumor (DSRCT) is an aggressive pediatric cancer caused by the EWSR1-WT1 fusion oncoprotein. The tumor is refractory to treatment with a 5-year survival rate of only 15–25%, necessitating the development of novel therapeutics, especially those able to target chemoresistant subpopulations. Novel in vitro cancer stem cell-like (CSC-like) culture conditions increase the expression of stemness markers (SOX2, NANOG) and reduce DSRCT cell line susceptibility to chemotherapy while maintaining the ability of DSRCT cells to form xenografts. To gain insights into this chemoresistant model, RNA-seq was performed to elucidate transcriptional alterations between DSRCT cells grown in CSC-like spheres and normal 2-dimensional adherent state. Commonly upregulated and downregulated genes were identified and utilized in pathway analysis revealing upregulation of pathways related to chromatin assembly and disassembly and downregulation of pathways including cell junction assembly and extracellular matrix organization. Alterations in chromatin assembly suggest a role for epigenetics in the DSRCT CSC-like state, which was further investigated with ATAC-seq, identifying over 10,000 differentially accessible peaks, including 4444 sphere accessible peaks and 6,120 adherent accessible peaks. Accessible regions were associated with higher gene expression, including increased accessibility of the CSC marker SOX2 in CSC-like culture conditions. These analyses were further utilized to identify potential CSC therapeutic targets, leading to the identification of B-lymphocyte kinase (BLK) as a CSC-enriched, EWSR1-WT1-regulated, druggable target. BLK inhibition and knockdown reduced CSC-like properties, including abrogation of tumorsphere formation and stemness marker expression. Importantly, BLK knockdown reduced DSRCT CSC-like cell chemoresistance, making its inhibition a promising target for future combination therapy.

Comparative study of pluripotency-related gene expression in Umbilical Cord Mesenchymal Stem Cells (UC-MSC) from IUGR, SGA and normally grown fetuses

Background and Objectives: Intrauterine growth restriction (IUGR) is worldwide a public health problem. IUGR-born individuals acquire epigenetic “thrifty-phenotype” gene adaptations, according to the “Barker hypothesis” and are more susceptible to develop metabolic related diseases. Small for Gestational Age (SGA) fetuses consist of a transitional group between IUGR and normally grown fetuses (Appropriate for Gestational Age, AGA). Currently, there is limited information in the literature whether IUGR or SGA status affects the pluripotency of the Umbilical Cord Mesenchymal Stem Cell (UC-MSC) with respect to gene expression at various stages of cell culture. Materials and Methods: In this study, we investigated stemness marker expression in UC-MSCs derived from twenty-one AGA, IUGR, and SGA fetuses. The relative expression of key genes involved in stemness, pluripotency, and cell proliferation, namely IGF2BP1, CMYC, GLI1, P21, NANOG, OCT4, and SOX2, was assessed in UC-MSCs at passages P0 and P1 using flow cytometry and quantitative PCR. Results: We found lower median expression levels of IGF2BP1 in IUGR group (P=0.044) and P21 in SGA group (P=0.024) at P0 compared to control group. GLI1 and OCT4 exhibited reduced expression at P1 in the IUGR group (P=0.0434 and P=0.0343 respectively) compared to AGA. NANOG median expression differed statistically between CONTROL and IUGR group in P0 (P=0.034) and P1 (P=0.0205). No significant changes were noted for SOX2 and CMYC median expression among the various groups in P0 and P1. Conclusion: The comparative analysis of stemness marker expression among UC-MSCs from these three different sources is a novel descriptive aspect of our study, adding a new perspective to the existing literature.

OCT-4 and DAZL expression in benign and malignant breast lesions: a prospective case control study

Purpose: The impact of cancer stem cells on breast cancer tumor biology has been poorly investigated during the last years. In the present study we sought to identify the expression of OCT-4 and DAZL, two prominent stem cell biomarkers, on malignant breast lesions. Methods: A retrospective analysis of patients that underwent simple or radical mastectomy was performed. Immunohistochemical expression of OCT-4 and DAZL was sought, and real time PCR was also applied in deparaffinized tissue samples retrieved from the pathology bank of our hospital. Results: Eighteen women aged 43-84 years old (median 57 years) were included in the present study. Of those, 10 women had invasive ductal carcinoma, 4 women had in situ ductal carcinoma and 4 women had invasive lobular carcinoma. Immunohistochemistry of tissue samples did not reveal expression of OCT-4, whereas DAZL was expressed in only 3 samples. Real time PCR was available in 13 samples as the control gene (G6PD) was not expressed in 5 samples. The 13 studied breast cell samples showed positive expression for the Oct-4 gene. In the same population the expression of DAZL varied significantly with 9 samples showing complete absence of expression and 4 providing a small signal in the last cycles, indicating barely detectable expression. The expression was significant in lobular carcinoma cases compared to cases with invasive or in situ ductal carcinoma. Conclusion: The findings of our study indicate the difficulty of retrieving reliable tissue samples following the process of deparaffinization and points towards the need for prospective case control studies that will be based on fresh tissue samples for the analysis of stem cell markers. Nevertheless, it also indicates a potentially increased expression of stemness markers in lobular carcinoma samples compared to ductal cancer cases, an observation that requires further research in the future.

Multicellular Tumoroids for Investigating Cancer Stem-Like Cells in the Heterogeneous Tumor Microenvironment.

Cancer stem-like cells (CSC) are a major contributing factor to chemoresistance, tumor recurrence, and poor survival outcomes in patients across cancer types. Signaling from non-tumor cells in the tumor microenvironment (TME) enriches for and supports CSC. This complex cell-cell signaling in the heterogeneous TME presents a challenge for patient survival; however, it also presents an opportunity to develop new targeted therapies that can inhibit survival of CSC. In this chapter, we report a multicellular tumoroid model which can be used to investigate the interactions between cancer cells and non-tumor cells in the TME to better understand the contribution of various cell types to cancer cell phenotypes, as well as the underlying mechanisms involved. The following methods allow for each cell type to be distinguished using FACS and studied individually. Gene expression can be analyzed for cancer cells, as well as the other non-tumor cells using qPCR following sorting. The response to chemotherapeutic agents and expression of stem markers can be determined for cancer cells using flow cytometry, excluding the other cell types to get an accurate view of the cancer cells. Furthermore, the viability of non-tumor cells can be analyzed as well to determine if there are cytotoxic effects of the drugs on non-tumor cells. Thus, the multicellular tumoroid model will reveal the interactions between the CSC and non-tumor cells in the heterogenous TME, resulting in discoveries in the fields of cancer biology, novel targeted therapies, and personalized drug screening for precision medicine.

Erratum: Differential expression of hepatic cancer stemness and hypoxia markers in residual cancer after locoregional therapies for hepatocellular carcinoma.

Erratum: Differential expression of hepatic cancer stemness and hypoxia markers in residual cancer after locoregional therapies for hepatocellular carcinoma.

A Bioinspired Astrocyte-Derived Coating Promotes the In Vitro Proliferation of Human Neural Stem Cells While Maintaining Their Stemness.

The repair of neuronal tissue is a challenging process due to the limited proliferative capacity of neurons. Neural stem cells (NSCs) can aid in the regeneration process of neural tissue due to their high proliferation potential and capacity to differentiate into neurons. The therapeutic potential of these cells can only be achieved if sufficient cells are obtained without losing their differentiation potential. Toward this end, an astrocyte-derived coating (HAc) was evaluated as a promising substrate to promote the proliferation of NSCs. Mass spectroscopy and scanning electron microscopy were used to characterize the HAc. The proliferation rate and the expression of stemness and differentiation markers in NSCs cultured on the HAc were evaluated and compared to the responses of these cells to commonly used coating materials including Poly-L-Ornithine (PLO), and a Human Induced Pluripotent Stem Cell (HiPSC)-based coating. The use of the HAc promotes the in vitro cell growth of NSCs. The expression of the stemness markers Sox2 and Nestin, and the differentiation marker DCX in the HAc group was akin to the expression of these markers in the controls. In summary, HAc supported the proliferation of NSCs while maintaining their stemness and neural differentiation potential.

Enhancing mesenchymal stem cells cultivated on microcarriers in spinner flasks via impeller design optimization for aggregated suspensions

During the ex vivo expansion of umbilical cord-derived mesenchymal stem cells (hUCMSCs) in a stirred tank bioreactor, the formation of cell–microcarrier aggregates significantly affects cell proliferation and physiological activity, making it difficult to meet the quantity and quality requirements for in vitro research and clinical applications. In this study, computational fluid dynamic (CFD) simulations were used to investigate the effect of an impeller structure in a commercial spinner flask on flow field structure, aggregate formation, and cellular physiological activity. By designing a modified impeller, the aggregate size was reduced, which promoted cell proliferation and stemness maintenance. This study showed that increasing the stirring speed reduced the size of hUCMSC-microcarrier aggregates with the original impeller. However, it also inhibited cell proliferation, decreased activity, and led to spontaneous differentiation. Compared to low stirring speeds, high stirring speeds did not alter the radial flow characteristics and vortex distribution of the flow field, but did generate higher shear rates. The new impeller’s design changed the flow field from radial to axial. The use of the novel impeller with an increased axial pumping rate (Qz) at a similar shear rate compared to the original impeller resulted in a 43.7% reduction in aggregate size, a 37.4% increase in cell density, and a better preservation of the expression of stemness markers (SOX2, OCT4 and NANOG). Increasing the Qz was a key factor in promoting aggregate suspension and size reduction. The results of this study have significant implications for the design of reactors, the optimisation of operating parameters, and the regulation of cellular physiological activity during MSC expansion.Graphical

RFX2 promotes tumor cell stemness through epigenetic regulation of PAF1 in spinal ependymoma.

Spinal ependymoma (SE) is a rare tumor that is most commonly low-grade and tends to recur when complete tumor resection is not feasible. We investigated the molecular mechanism induces stem cell features in SE. Immunohistochemical staining was conducted to analyze the expression of RFX2 in tumor tissues of SE patients at different stages. The expression of tumor stemness markers (Netsin and CD133) was analyzed using western blot analysis and IF, and the efficiency of sphere formation in SE cells was analyzed. The biological activities of SE cells were analyzed by EdU proliferation assay, TUNEL, wound healing, and Transwell assays. The regulatory relationship of RFX2 on PAF1 was verified by ChIP-qPCR and the dual-luciferase assay. SE cells were injected into the spinal cord of nude mice for in vivo assays. RFX2 was higher in the tumor tissues of SE-III patients than in the tumor tissues of SE-I patients. RFX2 knockdown reduced the expression of tumor stemness markers in SE cells and inhibited the sphere formation efficiency. Moreover, RFX2 knockdown ameliorated the malignant progression of SE in nude mice, as manifested by prolonged survival and alleviated SE tumor infiltration. RFX2 bound to the PAF1 promoter to induce its transcription. Overexpression of PAF1 overturned the effects of RFX2 knockdown on stem cell features and biological activities of SE cells, thereby reducing survival in mice. RFX2 activates PAF1 transcription, which promotes tumor stemness of SE cells and leads to the malignant progression of SE.

Astragaloside IV enhances the sensitivity of breast cancer stem cells to paclitaxel by inhibiting stemness.

Chemotherapy is one of the common treatments for breast cancer. The induction of cancer stem cells (CSCs) is an important reason for chemotherapy failure and breast cancer recurrence. Astragaloside IV (ASIV) is one of the effective components of the traditional Chinese medicine (TCM) Astragalus membranaceus, which can improve the sensitivity of various tumors to chemotherapy drugs. Here, we explored the sensitization effect of ASIV to chemotherapy drug paclitaxel (PTX) in breast cancer from the perspective of CSCs. The study included both in vitro and in vivo experiments. CSCs from the breast cancer cell line MCF7 with stem cell characteristics were successfully induced in vitro. Cell viability and proliferation were detected using the Cell Counting Kit-8 (CCK-8) and colony formation assays, and flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) methods were performed to detect cell apoptosis. Stemness-related protein expression was determined by western blotting (WB) and immunohistochemistry (IHC). Body weight, histopathology, and visceral organ damage of mice were used to monitor drug toxicity. The expression of stemness markers including Sox2, Nanog, and ALDHA1 was stronger in MCF7-CSCs than in MCF7. PTX treatment inhibited the proliferation of tumor cells by promoting cell apoptosis, whereas the stemness of breast cancer stem cells (BCSCs) resisted the effects of PTX. ASIV decreased the stemness of BCSCs, increased the sensitivity of BCSCs to PTX, and synergistically promoted PTX-induced apoptosis of breast cancer cells. Our results showed that the total cell apoptosis rate increased by about 25% after adding ASIV compared with BCSCs treated with PTX alone. The in vivo experiments demonstrated that ASIV enhanced the ability of PTX to inhibit the growth of breast cancer. WB and IHC showed that ASIV reduced the stemness of CSCs. In this study, the resistance of breast cancer to PTX was attributed to the existence of CSCs; ASIV weakened the resistance of MCF7-CSCs to PTX by significantly attenuating the hallmarks of breast cancer stemness and improved the efficacy of PTX.

Endochondral Repair of Jawbone Defects Using Periosteal Cell Spheroids

Recapitulation of the natural healing process is receiving increasing recognition as a strategy to induce robust tissue regeneration. Endochondral ossification has been recognized as an essential reparative approach in natural jawbone defect healing. However, such an approach has been overlooked in the recent development of cell-based therapeutics for jawbone repair. Therefore, this study aimed to explore a bioinspired stem cell–based strategy for jawbone repair by mimicking the mesenchymal condensation of progenitor cells during the early endochondral ossification process. For this purpose, passage 3 of jawbone periosteum-derived cells (jb-PDCs) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 96.4 ± 5.8 µm after 48 h. Compared to monolayer culture, the jb-PDC spheroids showed a significant reduction of stemness marker expression evidenced by flow cytometry. Furthermore, a significant upregulation of chondrogenic transcription factor SOX9 in both gene and protein levels was observed in the jb-PDC spheroids after 48 h of chondrogenic induction. RNA sequencing and Western blotting analysis further suggested that the enhanced SOX9-mediated chondrogenic differentiation in jb-PDC spheroids was attributed to the activation of the p38 MAPK pathway. Impressively, inhibition of p38 kinase activity significantly attenuated chondrogenic differentiation jb-PDC spheroids, evidenced by a significant decline of SOX9 in both gene and protein levels. Strikingly, the jb-PDC spheroids implanted in 6- to 8-wk-old male C57BL/6 mice with critical-size jawbone defects (1.8 mm in diameter) showed an evident contribution to cartilaginous callus formation after 1 wk, evidenced by histological analysis. Furthermore, micro–computed tomography analysis showed that the jb-PDC spheroids significantly accelerated bone healing after 2 wk in the absence of exogenous growth factors. In sum, the presented findings represent the successful development of cell-based therapeutics to reengineer the endochondral bone repair process and illustrate the potential application to improve bone repair and regeneration in the craniofacial skeleton.

IL-38 regulates intestinal stem cell homeostasis by inducing WNT signaling and beneficial IL-1β secretion.

The IL-1 Family member IL-38 has been characterized primarily as an antiinflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL-38 in the murine small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL-38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL-38-deficient mice develop more slowly. This reduction in size is likely due to the downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, and Olfm4) expression compared with wild-type organoids. The IL-38 binding to IL-1R6 and IL-1R9 is still a matter of debate. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL-1R9-deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL-38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL-38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild-type mice, IL-38 stimulation induced low concentrations of IL-1β which contribute to organoid growth. However, high concentrations of IL-1β have detrimental effects on the cultures that were prevented by treatment with recombinant IL-38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an antiinflammatory molecule in the SI, maintaining homeostasis.

Low, medium, and high molecular weight hyaluronic acid effects on human dental pulp stem cells in vitro

Hyaluronic acid (HA), an extracellular biopolymer found throughout the human body, holds promise as a biocompatible and biodegradable scaffold material. High molecular weight (HMW) HA degrades, generating low molecular weight (LMW) fragments with distinct properties. These fragments can influence the behaviour of cells, including human dental pulp stem cells (hDPSCs) incorporated into HA-containing hydrogels or scaffolds. Therefore, a comprehensive examination of the impact of a range of HA molecular weights on hDPSCs is essential before designing HA-based scaffolds for these cells. hDPSC lines were cultured with LMW HA (800 Da, 1600 Da, 15 kDa), medium molecular weight HA (237 kDa), or HMW HA (1500 kDa) over six passages. The various molecular weights had negligible effects on hDPSCs viability, morphology, adhesion, or relative telomere length. Furthermore, the expression of key surface stemness markers (CD29, CD44, CD73, CD90) remained unaltered. HA did not induce osteogenic, chondrogenic, or adipogenic differentiation. Moreover, the potential for chondrogenic and osteogenic differentiation was not adversely affected by LMW or HMW HA. Various molecular weights of HA seem safe, biocompatible and therefore suitable components for hDPSCs-containing scaffolds. These findings affirm that the hDPCSs will not be negatively affected by HA fragments resulting from scaffold degradation.

The role of LOXL2 induced by glucose metabolism-activated NF-κB in maintaining drug resistance through EMT and cancer stemness in gemcitabine-resistant PDAC

Gemcitabine is considered a standard treatment for pancreatic cancer, but developing drug resistance greatly limits the effectiveness of chemotherapy and increases the rate of recurrence. Lysyl oxide-like 2 (LOXL2) is highly expressed in pancreatic cancer and is involved in carcinogenesis and EMT regulation. However, studies on the role of LOXL2 in drug resistance are limited. Here, we investigated the mechanism of LOXL2 induction and the effect of LOXL2 on EMT and CSC in gemcitabine-resistant pancreatic cancer. Glucose metabolism was activated in gemcitabine-resistant pancreatic cancer cells, and NF-κB signaling was regulated accordingly. Activated NF-κB directly induces transcription by binding to the promoters of LOXL2 and ZEB1. The EMT process was significantly inhibited by the coregulation of ZEB1 and LOXL2. In addition, LOXL2 inhibition reduced the expression of cancer stemness markers and stemness by regulating MAPK signaling activity. LOXL2 inhibits tumor growth of gemcitabine-resistant pancreatic cancer cells and increases the sensitivity to gemcitabine in mouse models.Key messagesWe identified a specific mechanism for inducing LOXL2 overexpression in gemcitabine-resistant pancreatic cancer. Taken together, our results suggest LOXL2 has an important regulatory role in maintaining gemcitabine resistance and may be an effective therapeutic target to treat pancreatic cancer.