Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance

Supplementary data 3.docx

Digitization of the IHC slides and Image processing & analysis.</p

Supplementary data 2.docx

Antibody validation method of monoclonal anti-CD39, CD73, P2Y6 receptor and ENTPD5) and polyclonal anti-ENTPD4 antibodies.</p

Supplementary_data 5.xlsx

Page 1. Expression of gene of interest in RNA-Seq. Page 2. Gene set enrichment analysis: supporting the pyrimidine metabolism, P53 signaling and mismatch repair.</p

Supplementary_data 4.pdf

Data normalization for NMR data analysis.</p

Supplementary_data 6.pdf

Biological database and web resource (STRING) based protein–protein interactions prediction of ENTPD4 and ENTPD5.</p

Supplementary data 1.docx

Sample preparation from tissue sample for the NMR study.</p

Immune and genomic signatures in oral (head and neck) cancer

Head and neck squamous cell carcinoma (HNSCC) is responsible for a large number of deaths each year. Oral cancer is the most frequent subtype of HNSCC. Historically, oral cancer has been associated with an increase in the consumption of tobacco and alcohol products, seen especially in the Asian subcontinent. It has also been associated with infection by the human papilloma virus (HPV), particularly strain HPV16. Treatment usually involves a multidisciplinary approach of surgery combined with chemotherapy and radiation. The advent of immunotherapy has broadened the scope for treatment. A better immune response to the tumour can also elicit the action of other therapeutic approaches. A heightened immune response, on the other hand, can lead to resistant tumour formation through the process of immunoediting. Molecular profiling of the tumour microenvironment (TME) can provide us with better insight into the mechanism and progression of the disease, ultimately opening up new therapeutic options. High-throughput molecular profiling techniques over the past decade have enabled us to appreciate the heterogeneity of the TME. In this review, we will be describing the clinicopathological role of the immune and genomic landscape in oral cancer. This study will update readers on the several immunological and genetic factors that can play an important function as predictive and prognostic biomarkers in various forms of head and neck cancer, with a special emphasis on oral carcinoma