Triple-negative breast cancer is characterized by heterogeneity that influences treatment response and patient outcome. This can be attributed to distinct “cells of origin”, unique differentiation blockades, and different repertoires of mutations [1]. TNBC tumors exhibit a high degree of intra and inter-tumoral heterogeneity, thereby rendering the existing treatment modalities inefficacious. Given the inherent limitations of bulk-cell sequencing, which averages and masks information from individual cells, cancer genomics should preferably be approached on a single-cell basis. The introduction of single-cell technology has permitted an unprecedented comprehension of cellular heterogeneity. This is especially pertinent to tumors, which function as ecosystems made up of immune, stromal, and malignant cells [2] (see Scheme 1).

Numerous signaling pathways have been identified to be responsible for the induction and maintenance of EMT and CSC characteristics. Among these signaling pathways, Wnt/β-catenin signaling has been reported to be a prominent contributor of EMT, stemness, and CSC properties of TNBC. The Nuclear accumulation of β-catenin promotes EMT, cell motility, invasion, colony formation, stem cell-like properties, and chemoresistance [3]. Similarly, the Wnt/PCP (planar cell polarity) pathway regulates cancer cell motility and invasion, thereby facilitating cancer progression [4]. Altogether, emphasizes a pivotal role of canonical and non-canonical Wnt signaling as one of the driving forces of TNBC tumorigenesis and metastasis.

The prevalence of cells with mesenchymal features is one of TNBCs' well-known characteristics; where cells have undergone EMT and are found to be poorly differentiated. In fact, EMT is known to generate minimally differentiated cells, which give birth to cancer stem cells (CSCs). CSCs have self-renewal potential and these cells can lead to different clonal populations, which results in intratumoral heterogeneity [5]. Intratumoral heterogeneity contributes to chemoresistance and subsequently tumor recurrence. Therefore, targeting CSCs may be instrumental in treating EMT and CSC-rich TNBCs.

Concurrently, studies have highlighted the correlation between stress-induced autophagy and survival and maintenance of MSCs/CSCs in TNBC. SQSTM1/P62 (Sequestosome-1; also known as p62) is a ubiquitin-binding scaffolding protein capable of functioning in diverse cellular processes [6]. SQSTM1/P62 predominantly participates in cell proliferation, survival, EMT, cell death signaling programs and autophagy regulation via its versatile protein adaptor functions [7]. Interestingly, experimental evidence suggests SQSTM1 mediated stabilization of the key EMT regulators and transcription factors [8]. These findings suggest that SQSTM1/P62 might be used as a therapeutic target for TNBC. An intriguing correlation between Wnt signaling and SQSTM1/P62 is that SQSTM1/P62 cooperates with the components of the non-canonical Wnt/PCP signaling pathway and helps in EMT and oncogenesis [9].

In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize the single-cell transcriptome dynamics of individual cells from TNBC tumors. It acts as a proof-of-concept study, demonstrating the viability and benefits of single-cell genomics in defining intra-tumoral heterogeneity and locating rare cell subclones in TNBC tumors. Through combining the inferCNV analysis and tumor markers, the mesenchymal stem cells were identified. The differential gene expression analysis suggested the role of SQSTM1 in maintaining the MSC population. Recognizing the centrality of both Wnt signaling and selective autophagy in the molecular pathogenesis of TNBC via EMT and MDR, we postulated that modulating these pathways might be a method for achieving dramatic therapeutic results in TNBC.