Our study aims to address the methodological challenges frequently encountered in RNA-Seq data analysis within cancer studies. Specifically, it enhances the identification of key genes involved in axillary lymph node metastasis (ALNM) in breast cancer. We employ Generalized Linear Models with Quasi-Likelihood (GLMQLs) to manage the inherently discrete and overdispersed nature of RNA-Seq data, marking a significant improvement over conventional methods such as the t-test, which assumes a normal distribution and equal variances across samples. We utilize the Trimmed Mean of M-values (TMMs) method for normalization to address library-specific compositional differences effectively. Our study focuses on a distinct cohort of 104 untreated patients from the TCGA Breast Invasive Carcinoma (BRCA) dataset to maintain an untainted genetic profile, thereby providing more accurate insights into the genetic underpinnings of lymph node metastasis. This strategic selection paves the way for developing early intervention strategies and targeted therapies. Our analysis is exclusively dedicated to protein-coding genes, enriched by the Magnitude Altitude Scoring (MAS) system, which rigorously identifies key genes that could serve as predictors in developing an ALNM predictive model. Our novel approach has pinpointed several genes significantly linked to ALNM in breast cancer, offering vital insights into the molecular dynamics of cancer development and metastasis. These genes, including ERBB2, CCNA1, FOXC2, LEFTY2, VTN, ACKR3, and PTGS2, are involved in key processes like apoptosis, epithelial-mesenchymal transition, angiogenesis, response to hypoxia, and KRAS signaling pathways, which are crucial for tumor virulence and the spread of metastases. Moreover, the approach has also emphasized the importance of the small proline-rich protein family (SPRR), including SPRR2B, SPRR2E, and SPRR2D, recognized for their significant involvement in cancer-related pathways and their potential as therapeutic targets. Important transcripts such as H3C10, H1-2, PADI4, and others have been highlighted as critical in modulating the chromatin structure and gene expression, fundamental for the progression and spread of cancer.

The biological effects of ionizing radiation are exploited in the clinical practice of radiotherapy to destroy tumour cells while sparing the surrounding normal tissue. While most of the radiotherapy research focused on DNA damage and repair, recently a great attention is going to cells' interactions with the mechanical microenvironment of both malignant and healthy tissues after exposure. In fact, the stiffness of the extracellular matrix can modify cells' motility and spreading through the modulation of transmembrane proteins and surface receptors' expression, such as CD-44. CD-44 receptor has held much interest also in targeted-therapy due to its affinity with hyaluronic acid, which can be used to functionalize biodegradable nanoparticles loaded with chemotherapy drugs for targeted therapy. We evaluated changes in CD-44 expression in two mammary carcinoma cell lines (MCF10A and MDA-MB-231) after exposure to X-ray (2 or 10 Gy). To explore the role of the mechanical microenvironment, we mimicked tissues' stiffness with polyacrylamide's substrates producing two different elastic modulus values (0.5 and 15 kPa). We measured a dose dependent increase in CD-44 relative expression in tumour cells cultured in a stiffer microenvironment. These findings highlight a crucial connection between the mechanical properties of the cell's surroundings and the post-radiotherapy expression of surface receptors.

Since last two decades, the major cancer research has focused on understanding the characteristic properties and mechanism of formation of Cancer stem cells (CSCs), due to their ability to initiate tumor growth, self-renewal property and multi-drug resistance. The discovery of the mechanism of acquisition of stem-like properties by carcinoma cells via epithelial-mesenchymal transition (EMT) has paved a way towards a deeper understanding of CSCs and presented a possible avenue for the development of therapeutic strategies. In spite of years of research, various challenges, such as identification of CSC subpopulation, lack of appropriate experimental models, targeting cancer cells and CSCs specifically without harming normal cells, are being faced while dealing with CSCs. Here, we discuss the biology and characteristics of CSCs, mode of acquisition of stemness (via EMT) and development of multi-drug resistance, the role of tumor niche, the process of dissemination and metastasis, therapeutic implications of CSCs and necessity of targeting them. We emphasise various strategies being developed to specifically target CSCs, including those targeting biomarkers, key pathways and microenvironment. Finally, we focus on the challenges that need to be subdued and propose the aspects that need to be addressed in future studies in order to broaden the understanding of CSCs and develop novel strategies to eradicate them in clinical applications. Graphical Abstract Cancer Stem Cells(CSCs) have gained much attention in the last few decades due to their ability to initiate tumor growth and, self-renewal property and multi-drug resistance. Here, we represent the CSC model of cancer, Characteristics of CSCs, acquisition of stemness and metastatic dissemination of cancer, Therapeutic implications of CSCs and Various strategies being employed to target and eradicate CSCs.

Detection and analysis of circulating tumor cells (CTCs) from patients with metastatic malignancies have become active areas of research in recent years. CTC enumeration has already proven useful in establishing prognosis for patients with metastatic breast, colon, and prostate cancer. More recently, studies are going beyond enumeration, exploring the CTCs as a means to better understand the mechanisms of tumorigenesis, invasion, and metastasis and the value of CTC characterization for prognosis and tailoring of treatment. Analysis of CTC subpopulations, for example, is highlighting the importance of the epithelial to mesenchymal transition (EMT), a process which may be crucial for allowing tumors to invade into and grow at sites distant from the original tumor site. Similarly, the detection of CTCs expressing markers of stemness may also have important implications for treatment resistance. Genomic analysis of CTC and CTC subpopulations may allow for selection of novel therapeutic targets to combat treatment resistance. CTCs become a particularly valuable biospecimen resource when tissue biopsies are unavailable or not feasible and liquid biopsies allow for serial monitoring. Lastly, cultures of patient-derived CTCs may allow for an evaluation of therapeutic strategies performed ex vivo and in real time. This review article will focus on these developments, starting with the CTC pathogenesis, going on to discuss the different platforms available for CTC isolation and their use to date in these arenas, then will explore multiple topics including the existing data concerning CTC subpopulations and their clinical relevance, genomic characterization, and lastly, avenues for future research.