A subpopulation within cancer, known as cancer stem cells (CSCs), regulates tumor initiation, chemoresistance, and metastasis. At a closer look, CSCs show functional heterogeneity and hierarchical organization. The present review is an attempt to assign marker profiles to define the functional heterogeneity and hierarchical organization of CSCs, based on a series of single-cell analyses. The evidences show that analogous to stem cell hierarchy, self-renewing Quiescent CSCs give rise to the Progenitor CSCs with limited proliferative capacity, and later to a Progenitor-like CSCs, which differentiates to Proliferating non-CSCs. Functionally, the CSCs can be tumor-initiating cells (TICs), drug-resistant CSCs, or metastasis initiating cells (MICs). Although there are certain marker profiles used to identify CSCs of different cancers, molecules like CD44, CD133, ALDH1A1, ABCG2, and pluripotency markers [Octamer binding transcriptional factor 4 (OCT4), SOX2, and NANOG] are used to mark CSCs of a wide range of cancers, ranging from hematological malignancies to solid tumors. Our analysis of the recent reports showed that a combination of these markers can demarcate the heterogeneous CSCs in solid tumors. Reporter constructs are widely used for easy identification and quantification of marker molecules. In this review, we discuss the suitability of reporters for the widely used CSC markers that can define the heterogeneous CSCs. Since the CSC-specific functions of CD44 and CD133 are regulated at the post-translational level, we do not recommend the reporters for these molecules for the detection of CSCs. A promoter-based reporter for ABCG2 may also be not relevant in CSCs, as the expression of the molecule in cancer is mainly regulated by promoter demethylation. In this context, a dual reporter consisting of one of the pluripotency markers and ALDH1A1 will be useful in marking the heterogeneous CSCs. This system can be easily adapted to high-throughput platforms to screen drugs for eliminating CSCs.

Although the role of microtubule dynamics in cancer progression is well-established, the roles of tubulin isotypes, their cargos and their specific function in the induction and sustenance of cancer stem cells (CSCs) were poorly explored. But emerging reports urge to focus on the transport function of tubulin isotypes in defining orchestrated expression of functionally critical molecules in establishing a stem cell niche, which is the key for CSC regulation. In this review, we summarize the role of specific tubulin isotypes in the transport of functional molecules that regulate metabolic reprogramming, which leads to the induction of CSCs and immune evasion. Recently, the surface expression of GLUT1 and GRP78 as well as voltage-dependent anion channel (VDAC) permeability, regulated by specific isotypes of β-tubulins have been shown to impart CSC properties to cancer cells, by implementing a metabolic reprogramming. Moreover, βIVb tubulin is shown to be critical in modulating EphrinB1signaling to sustain CSCs in oral carcinoma. These tubulin-interacting molecules, Ephrins, GLUT1 and GRP78, are also important regulators of immune evasion, by evoking PD-L1 mediated T-cell suppression. Thus, the recent advances in the field implicate that tubulins play a role in the controlled transport of molecules involved in CSC niche. The indication of tubulin isotypes in the regulation of CSCs offers a strategy to specifically target those tubulin isotypes to eliminate CSCs, rather than the general inhibition of microtubules, which usually leads to therapy resistance.

In view of the importance of cancer stem cells (CSCs) in chemoresistance, metastasis and recurrence, the biology of CSCs were explored in detail. Based on that, several modalities were proposed to target them. In spite of the several clinical trials, a successful CSC-targeting drug is yet to be identified. The number of molecules screened and entered for clinical trial for CSC-targeting is comparatively low, compared to other drugs. The bottle neck is the lack of a high-throughput adaptable screening strategy for CSCs. This review is aimed to identify suitable reporters for CSCs that can be used to identify the heterogeneous CSC populations, including quiescent CSCs, proliferative CSCs, drug resistant CSCs and metastatic CSCs. Analysis of the tumor microenvironment regulating CSCs revealed that the factors in CSC-niche activates effector molecules that function as CSC markers, including pluripotency markers, CD133, ABCG2 and ALDH1A1. Among these factors OCT4, SOX2, NANOG, ABCG2 and ALDH1A1 are ideal for making reporters for CSCs. The pluripotency molecules, like OCT4, SOX2 and NANOG, regulate self-renewal, chemoresistance and metastasis. ABCG2 is a known regulator of drug resistance while ALDH1A1 modulates self-renewal, chemoresistance and metastasis. Considering the heterogeneity of CSCs, including a quiescent population and a proliferative population with metastatic ability, we propose the use of a combination of reporters. A dual reporter consisting of a pluripotency marker and a marker like ALDH1A1 will be useful in screening drugs that target CSCs.

Recent advancements in cancer research have shown that cancer stem cell (CSC) niche is a crucial factor modulating tumor progression and treatment outcomes. It sustains CSCs by orchestrated regulation of several cytokines, growth factors, and signaling pathways. Although the features defining adult stem cell niches are well-explored, the CSC niche is poorly characterized. Since membrane trafficking proteins have been shown to be essential for the localization of critical proteins supporting CSCs, we investigated the role of TUBB4B, a probable membrane trafficking protein that was found to be overexpressed in the membranes of stem cell enriched cultures, in sustaining CSCs in oral cancer. Here, we show that the knockdown of TUBB4B downregulates the expression of pluripotency markers, depletes ALDH1A1⁺ population, decreases in vitro sphere formation, and diminishes the tumor initiation potential in vivo. As TUBB4B is not known to have any role in transcriptional regulation nor cell signaling, we suspected that its membrane trafficking function plays a role in constituting a CSC niche. The pattern of its expression in tissue sections, forming a gradient in and around the CSCs, reinforced the notion. Later, we explored its possible cooperation with a signaling protein, Ephrin-B1, the abrogation of which reduces the self-renewal of oral cancer stem cells. Expression and survival analyses based on the TCGA dataset of head and neck squamous cell carcinoma (HNSCC) samples indicated that the functional cooperation of TUBB4 and EFNB1 results in a poor prognosis. We also show that TUBB4B and Ephrin-B1 cohabit in the CSC niche. Moreover, depletion of TUBB4B downregulates the membrane expression of Ephrin-B1 and reduces the CSC population. Our results imply that the dynamics of TUBB4B is decisive for the surface localization of proteins, like Ephrin-B1, that sustain CSCs by their concerted signaling.

Treatment outcome after surgical removal in oral carcinoma is poor due to inadequate methodologies available for marking surgical margins. Even though some methodologies for intraoperative margin assessment are under clinical and preclinical trials for other solid tumours, a promising modality for oral cancer surgery is not developed. Fluorescent-based optical imaging using Near Infrared (NIR) dyes tagged to tumour specific target will be an optimal tool for this purpose. One such target, Gastrin Releasing Peptide Receptor (GRPR) was selected for the study, and its binding peptide, TM1-IR680, was tested for its efficacy for surgical margin prediction in murine orthotopic model of oral cancer, derived from primary samples. Here, for the first time in a preclinical analysis, we show that the size and margin of oral cancer can be predicted, as revealed by 3D-imaging. Interestingly, the peptide was sensitive enough to detect lymph nodes that harboured dispersed tumour cells before colonization, which was impossible to identify by conventional histopathology. We recommend the use of TM1-NIR dyes alone or in combination with other technologies to improve the clinical outcome of oral cancer surgery.