Most cancers stem cells (CSCs), the self-renewing drivers of tumorigenesis and therapeutic resistance, pose vital obstacles in oncology [1], [2], [3]. Analysis on CSCs is crucial for elucidating tumor biology and bettering therapeutic outcomes. Nevertheless, CSCs usually account for under 0.0001–1 % of the complete tumor inhabitants [4], making it troublesome to isolate ample numbers from bulk tumor tissues. Establishing a dependable in vitro CSC mannequin not solely presents an alternate technique for CSC acquisition but additionally performs a significant position in elucidating mechanisms of tumor recurrence, creating focused most cancers therapies, and evaluating drug efficacy. Serum-free suspension tradition, the usual methodology for CSC enrichment, usually yields sphere-forming efficiencies of lower than 5 %, even after 2–8 weeks of cultivation [5], [6], which considerably limits its applicability in CSC analysis. Equally, floor marker-dependent isolation utilizing circulation cytometry or immunomagnetic separation is cost-intensive and often ends in low yields of extremely pure and viable CSCs [7]. Because the creation of somatic cell nuclear switch and Yamanaka’s induced pluripotent stem cell (iPSC) expertise in 2006 [8], [9], quite a few reprogramming methods for controlling cell destiny have been proposed [10], [11], [12]. Nevertheless, CSCs’ inherent genomic mutations and epigenetic alterations often end in low reprogramming efficiencies (5–8 %) and should impair genomic integrity [13]. At the moment, no simple, cost-effective, and environment friendly strategies exist for reliably reprogramming tumor cells (TCs) into CSCs whereas monitoring stemness modifications. Due to this fact, various methods are urgently wanted to beat the restrictions of the present CSC tradition and characterization strategies.
Growing practical biomaterials that recapitulate the biochemical alerts and biophysical cues of the native extracellular matrix (ECM) to affect CSC conduct has grow to be a key focus in stem cell analysis [14], [15], [16]. Lately, each pure and artificial biomaterials, designed to imitate native cell-matrix interactions, have been developed to assist the enlargement of iPSCs and modulation of CSCs [17], [18], [19], [20]. In 2021, Tanaka et al. [21] positioned six forms of human most cancers cell strains right into a dual-network (DN) hydrogel composed of poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) and poly(N,N′-dimethyl acrylamide) (PDMAAm). This DN hydrogel, characterised by a excessive modulus (∼300 kPa) and unfavourable floor cost, efficiently reprogrammed differentiated TCs into CSCs inside 24 h and enabled purposes in drug screening. In 2023, the Tan’s group [17] employed a 3D-silica nanostructure matrix with twin performance to reprogram breast, colon, and lung most cancers cells, resulting in the formation of CSC spheroids inside 3 days. The platform additionally demonstrated direct applicability for drug and reagent screening. Regardless of these promising advances, in vitro reprogramming effectivity stays comparatively low as a result of transcriptional and epigenetic obstacles [22], [23], [24]. So far, the design of biomaterials that may effectively reprogram TCs into CSCs whereas sustaining their stemness stays a major problem.
Self-assembled peptide nanofibrous hydrogels, resembling 9-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF), have attracted growing consideration on account of their well-defined nanostructure, ultrafast gelation kinetics underneath physiological circumstances, inherent biocompatibility, satisfactory mechanical energy, and versatile performance [25]. These properties allow them to carefully mimic the native ECM and function a biomimetic microenvironment that influences cell conduct [26], [27], [28]. Upon self-assembly, Fmoc-FF molecules kind a 3D nanofibrous community, whose densely interwoven construction offers plentiful adhesion websites and enhanced mechanical assist for cells [25]. Earlier works [29], [30], [31] have demonstrated the utility of varied self-assembled peptide hydrogels in cell tradition and stem cell differentiation. For instance, Hung and associates [32] developed a supramolecular hydrogel by co-assembling peptides Fmoc-FF and Fmoc-arginine-glycine-aspartic acid (Fmoc-RGD). The ensuing ECM-mimetic scaffold supported each proliferation and multilineage differentiation of mesenchymal stem cells. Furthermore, Fmoc-FF can co-assemble with responsive molecules and electrodes for biosensor building, combining the fast response and excessive sensitivity of electrochemical strategies with the power to watch reside cells in actual time [17], [26], [33]. Nevertheless, using the Fmoc-FF hydrogels to each engineer the microenvironment for efficient cell reprogramming and function a sensing platform for in situ, non-destructive electrochemical monitoring of the reprogramming course of has but to be absolutely explored.
Herein, a self-assembled dipeptide hydrogel-based multifunctional platform (hereafter known as Fmoc-FF hydrogel) was engineered for 3D cell tradition and to robustly induce extremely environment friendly reprogramming of TCs into CSCs (Scheme 1) and direct in situ real-time cell monitoring. Three human TC strains—MCF-7 (breast), HeLa (cervical), and A549 (lung) have been encapsulated within the hydrogel. A dense nanofibrous community was fashioned by way of a self-assembly of Fmoc-FF monomers within the presence of TCs, which tightly enwrapped the cells and offered sturdy cell-matrix interactions. In contrast to different high-stiffness polymer hydrogels (usually ∼100–300 kPa) [21], [34], the Fmoc-FF hydrogel markedly enhanced stemness and achieved excessive reprogramming effectivity throughout all three TC strains, even underneath physiologically related delicate stiffness circumstances (∼10.08 kPa). Concurrently, a fast improve in cell quantity was additionally noticed throughout reprogramming, which can be attributed to low-density seeding circumstances that amplify mechanical cues skilled by particular person cells. As well as, the Fmoc-FF hydrogel, preloaded with TCs, was launched into the interspace of a 3D Au NPs/carbon foam (CF) electrode to ascertain an built-in platform. This technique allowed the profitable, in situ, non-destructive electrochemical monitoring of dynamic modifications within the stemness-associated biomarker aldehyde dehydrogenase 1 (ALDH1) throughout TC reprogramming. A progressive improve in ALDH1, electrochemical sign over time, indicated elevated enzymatic exercise, similar to a rise in stemness. This method circumvents the necessity for cell isolation, thereby minimizing the lack of essential organic knowledge. This research is the primary to show that Fmoc-FF hydrogel-based multifunctional platform quickly and successfully promotes the reprogramming of TCs to CSCs, concurrently enabling non-destructive electrochemical monitoring of TC stemness modifications. These findings present priceless insights for learning TC destiny regulation and monitoring advanced organic processes.
