BACKGROUND: Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin β1 (CD29) or β4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy.
OBJECTIVE: Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion.
RESULTS: Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline.
CONCLUSIONS: BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

The lack of appropriate mesenchymal stem cells (MSCs) selection methods has given the challenges for standardized harvesting, processing, and phenotyping procedures of MSCs. Genetic engineering coupled with high-throughput proteomic studies of MSC surface markers arises as a promising strategy to identify stem cell-specific markers. However, the technical limitations are the key factors making it less suitable to provide an appropriate starting material for the screening platform. A more accurate, easily accessible approach is required to solve the issues.
This study established a high-throughput screening strategy with forward versus side scatter gating to identify the adipogenesis-associated markers of bone marrow-derived MSCs (BMSCs) and tonsil-derived MSCs (TMSCs). We classified the MSC-derived adipogenic differentiated cells into two clusters: lipid-rich cells as side scatter (SSC)-high population and lipid-poor cells as SSC-low population. By screening the expression of 242 cell surface proteins, we identified the surface markers which exclusively found in lipid-rich subpopulation as the specific markers for BMSCs and TMSCs.
High-throughput screening of the expression of 242 cell surface proteins indicated that CD49f and CD146 were specific for BMSCs and TMSCs. Subsequent immunostaining confirmed the consistent specific expression of CD49f and CD146 and in BMSCs and TMSCs. Enrichment of MSCs by CD49f and CD146 surface markers demonstrated that the simultaneous expression of CD49f and CD146 is required for adipogenesis and osteogenesis of mesenchymal stem cells. Furthermore, the fate decision of MSCs from different sources is regulated by distinct responses of cells to differentiation stimulations despite sharing a common CD49f+CD146+ immunophenotype.
We established an accurate, robust, transgene-free method for screening adipogenesis associated cell surface proteins. This provided a valuable tool to investigate MSC-specific markers. Additionally, we showed a possible crosstalk between CD49f and CD146 modulates the adipogenesis of MSCs.

Over the past decades, our knowledge of integrins has evolved from being understood as simple cell surface adhesion molecules to receptors that have a complex range of intracellular and extracellular functions, such as delivering chemical and mechanical signals to cells. Consequently, they actively control cellular proliferation, differentiation, and apoptosis. Dysregulation of integrin signaling is a major factor in the development and progression of many tumors. Many reviews have covered the broader integrin family in molecular and cellular studies and its roles in diseases. Nevertheless, further understanding of the mechanisms specific to an individual subunit of different heterodimers is more useful. Thus, we describe the current understanding of and exploratory investigations on the α6-integrin subunit (CD49f, VLA6; encoded by the gene itga6) in normal and cancer cells. The roles of ITGA6 in cell adhesion, stemness, metastasis, angiogenesis, and drug resistance, and as a diagnosis biomarker, are discussed. The role of ITGA6 differs based on several features, such as cell background, cancer type, and post-transcriptional alterations. In addition, exosomal ITGA6 also implies metastatic organotropism. The importance of ITGA6 in the progression of a number of cancers, including hematological malignancies, suggests its potential usage as a novel prognostic or diagnostic marker and useful therapeutic target for better clinical outcomes.

Epididymal development can be subdivided into three phases: undifferentiated, a period of differentiation, and expansion. The objectives of this study were (1) to assess gene expression profiles in epididymides, (2) predict signaling pathways, and (3) develop a novel 3D cell culture method to assess the regulation of epididymal development in vitro. Microarray analyses indicate that the largest changes in differential gene expression occurred between the 7- to 18-day period, in which 1452 genes were differentially expressed, while 671 differentially expressed genes were noted between days 18 and 28, and there were 560 differentially expressed genes between days 28 and 60. Multiple signaling pathways were predicted at different phases of development. Pathway associations indicated that in epididymides of 7- to 18-day old rats, there was a significant association of regulated genes implicated in stem cells, estrogens, thyroid hormones, and kidney development, while androgen- and estrogen-related pathways were enriched at other phases of development. Organoids were derived from CD49f + columnar cells from 7-day old rats, while no organoids developed from CD49f- cells. Cells cultured in an epididymal basal cell organoid medium versus a commercial kidney differentiation medium supplemented with DHT revealed that irrespective of the culture medium, cells within differentiating organoids expressed p63, AQP9, and V-ATPase after 14 days of culture. The commercial kidney medium resulted in an increase in the number of organoids positive for p63, AQP9, and V-ATPase. Together, these data indicate that columnar cells represent an epididymal stem/progenitor cell population.

Thymic epithelial cells (TECs) are essential in supporting the development of mature T cells from hematopoietic progenitor cells and facilitate their lineage-commitment, proliferation, T-cell receptor repertoire selection and maturation. While animal model systems have greatly aided in elucidating the contribution of stromal cells to these intricate processes, human tissue has been more difficult to study, partly due to a lack of suitable surface markers comprehensively defining human TECs. Here, we conducted a flow cytometry based surface marker screen to reliably identify and quantify human TECs and delineate medullary from cortical subsets. These findings were validated by transcriptomic and histologic means. The combination of EpCAM, podoplanin (pdpn), CD49f and CD200 comprehensively identified human TECs and not only allowed their reliable distinction in medullary and cortical subsets but also their detailed quantitation. Transcriptomic profiling of each subset in comparison to fibroblasts and endothelial cells confirmed the identity of the different stromal cell subsets sorted according to the proposed strategy. Our dataset not only demonstrated transcriptional similarities between TEC and cells of mesenchymal origin but furthermore revealed a subset-specific distribution of a specific set of extracellular matrix-related genes in TECs. This indicates that TECs significantly contribute to the distinct compartmentalization - and thus function - of the human thymus. We applied the strategy to quantify TEC subsets in 31 immunologically healthy children, which revealed sex-specific differences of TEC composition early in life. As the distribution of mature CD4- or CD8-single-positive thymocytes was correspondingly altered, the composition of the thymic epithelial compartment may directly impact on the CD4-CD8-lineage choice of thymocytes. We prove that the plain, reliable strategy proposed here to comprehensively identify human TEC subpopulations by flow cytometry based on surface marker expression is suitable to determine their frequency and phenotype in health and disease and allows sorting of live cells for downstream analysis. Its use reaches from a reliable diagnostic tool for thymic biopsies to improved phenotypic characterization of thymic grafts intended for therapeutic use.

OBJECTIVE: CD49f is expressed on a variety of stem cells and has certain effects on their cytological functions, such as proliferation and differentiation potential. However, whether CD49f is expressed on the surface of adipose tissue-derived mesenchymal stem cells (ADSCs) and its effect on ADSCs has not been clarified.
METHODS: The effects of in vitro culture passage and inflammatory factor treatment on CD49f expression and the adhesion ability of ADSCs from mice and rats were investigated. CD49f+ cells were selected from rat ADSCs (rADSCs) by magnetic-activated cell sorting (MACS), and the cellular functions of CD49f+ ADSCs and unsorted ADSCs, including their clonogenic, proliferation, adipogenic and osteogenic differentiation, migration and anti-apoptotic capacities, were compared.
RESULTS: CD49f expression and the adhesion ability of ADSCs decreased with increasing in vitro culture passage number. TNF-α and IFN-γ treatment decreased CD49f expression but increased the adhesion ability of ADSCs. After CD49f was blocked with an anti-CD49f antibody, the adhesion ability of ADSCs was decreased. No significant difference in clonogenic activity was observed between unsorted ADSCs and CD49f+ ADSCs. CD49f+ ADSCs had greater proliferation, adipogenic and osteogenic differentiation, migration and anti-apoptotic capacities than unsorted ADSCs.
CONCLUSIONS: In the current study, the expression of CD49f on ADSCs was identified for the first time. The expression of CD49f on ADSCs was influenced by in vitro culture passage number and inflammatory factor treatment. Compared with unsorted ADSCs, CD49f + ADSCs exhibited superior cellular functions, thus may have great application value in mesenchymal stem cell (MSC)-based therapies.

Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs\' self-renewal regulation, especially in dairy goats. We isolated and enriched the CD49f-positive and negative dairy goat testicular cells by magnetic-activated cell sorting strategies. The RNA- sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f-positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When the PAX7 gene was overexpressed in dairy goat immortal mGSCs-I-SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f+ spermatogonia, and PAX7 can maintain the self-renewal of CD49f-positive SSCs.

UNASSIGNED: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists.
UNASSIGNED: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation.
UNASSIGNED: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with β1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC.
UNASSIGNED: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.

New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f+ hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f+ hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f+ hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.

CD49f (integrin α6) is a useful marker for minimal residual disease (MRD) detection in B lymphoblastic leukemia and has recently been suggested to mediate infiltration of the central nervous system by leukemic B lymphoblasts. However, data regarding expression of CD49f protein in B lymphoblastic leukemia are limited, and whether CD49f protein expression varies among genetic subgroups of B lymphoblastic leukemia is unknown.
CD49f protein expression was characterized by flow cytometry in a series of 40 cases of B lymphoblastic leukemia, which included the genetic subgroups: KMT2A-rerranged, BCR-ABL1+, ETV6-RUNX1+, hypodiploidy, and hyperdiploidy.
Expression of CD49f differed significantly among the five genetic subgroups studied, whether assessed by percentage of blasts positive for the antigen (p = .0001, Kruskal-Wallis) or median fluorescence intensity (MFI) (p = .0001, Kruskal-Wallis). Moreover, the percentage of CD49f+ blasts and MFI of CD49f were significantly lower in KMT2A-rearranged cases than in cases without KMT2A rearrangement (p = .0002 for both, Mann-Whitney).
CD49f protein expression varies among genetic subgroups of B lymphoblastic leukemia, and is distinctly low in KMT2A-rearranged cases.