Allogeneic whole cell gene modified therapeutic melanoma vaccine (AGI-101H) comprising of two melanoma cell lines transduced with cDNA encoding fusion protein composed of IL-6 linked with the soluble IL-6 receptor (sIL-6R), referred to as H6 was developed. H6 served as a molecular adjuvant, however, it has altered vaccine cells phenotype towards melanoma stem cells (MSC)-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). AGI-101H was applied in advanced melanoma patients with non-resected and resected disease. In the adjuvant setting, it was combined with surgery in case of recurring metastases, which were surgically removed and vaccination continued. A significant fraction of AGI-101H treated melanoma patients is still alive (11–19 years). Out of 106 living patients, 39 were HLA-A2 positive and were the subject of the study. Immunization of melanoma patients resulted in the generation of cytotoxic CD8+ T cells specific for ALDH1A1, which were detected in circulation by HLA-A0201 MHC dextramers loaded with ALDH1A1_88-96(LLYKLADLI) peptide. Phenotypically they were central memory CD8⁺ T cells. Re-stimulation with ALDH1A1_88-96 ex vivo resulted in IFN-γ secretion and cells degranulation. Following each vaccine dose administration, the number of ALDH1A1-CD8⁺ T cells increased in circulation and returned to the previous level until next dose injection (one month). ALDH1A1-CD8⁺ T cells were also found, however in the lower number than in vaccinated patients, in the circulation of untreated melanoma with stage IV but were not found in stage II or III and healthy donors. Specific anti-ALDH1 antibodies were present in treated patients. Long-term survival suggests immuno-targeting of MSC in treated patients.

Therapeutic resistance remains a critical barrier in oncology, frequently leading to cancer relapse after initial treatment response. Growing evidence suggests the presence of drug-tolerant persisters (DTPs), a rare subpopulation of cancer cells that survives chemotherapy by entering a reversible specific adaptation. Unlike classical cell resistance, the DTP phenotype is independent of genetic changes and maintained through dynamic regulatory mechanisms. DTPs are phenotypically heterogeneous and can exhibit stem-like and quiescent cell phenotypes, non- or slow proliferation, and remarkable plasticity due to a di-pause-like state and executing epithelial–mesenchymal transition (EMT) or transdifferentiation processes. Despite advances in research, the molecular mechanisms underlying DTPs’ biology and their role in cancer relapse remain only partially understood. The review summarizes the current progress in processes that lead to the acquisition of cellular persistence status, which, in turn, constitute areas of vulnerability that can be exploited in cancer therapy. We highlight anti-DTP therapeutic strategies, including epigenetic modification, cell signaling and transcriptional regulation, metabolic reprogramming, and modification of cell interactions within the tumor microenvironment. Furthermore, we focus on the potential role of nanomaterials in the combat against DTPs. Nanoparticles not only act as part of the drug delivery process, enabling precise DTP targeting and enhancing intracellular drug accumulation, but their intrinsic properties can also be used to eradicate DTPs directly or by enhancing the effectiveness of other therapeutic strategies. The integrated approach offers strong potential to eliminate tumor persistence, prevent recurrence, and improve long-term patient outcomes beyond conventional therapies.

There is a growing pressure for further breast cancer treatment development to improve patient quality of life and clinical effectiveness while reducing drug resistance and side effects. Clinical research, however, remains both expensive and time-consuming, highlighting the importance of advancing preclinical studies. The ability of a model to accurately replicate the biological environment is essential for generating reliable data. Although research on two-dimensional in vitro cell culture systems has been conducted for over a century, their limited complexity hinders further development of cancer treatments. Consequently, the response to tested medications is artificial, since some drugs appear to be effective only in the two-dimensional model and not in clinical settings. A multicellular spheroid (MCS) is a three-dimensional assembly of cancer cells, used in oncology to study cancer biology and drug sensitivity. MCSs serve as valuable in vitro models in preclinical drug development. They can also be used in a clinical setting as a personalized tool for drug susceptibility screening. The objective of this review is to highlight new possibilities for improved anticancer drug screening and the development of personalized treatment regimens with multicellular spheroids.