The induction of senescence/polyploidization and their role in cancer recurrence is still a poorly explored issue. We showed that MDA-MB-231 and MCF-7 breast cancer cells underwent reversible senescence/polyploidization upon pulse treatment with doxorubicin (dox). Subsequently, senescent/polyploid cells produced progeny (escapers) that possessed the same amount of DNA as parental cells. In a dox-induced senescence/polyploidization state, the accumulation of autophagy protein markers, such as LC3B II and p62/SQSTM1, was observed. However, the senescent cells were characterized by a very low rate of new autophagosome formation and degradation, estimated by autophagic index. In contrast to senescent cells, escapers had a substantially increased autophagic index and transcription factor EB activation, but a decreased level of an autophagy inhibitor, Rubicon, and autophagic vesicles with non-degraded cargo. These results strongly suggested that autophagy in escapers was improved, especially in MDA-MB-231 cells. The escapers of both cell lines were also susceptible to dox-induced senescence. However, MDA-MB-231 cells which escaped from senescence were characterized by a lower number of γH2AX foci and a different pattern of interleukin synthesis than senescent cells. Thus, our studies showed that breast cancer cells can undergo senescence uncoupled from autophagy status, but autophagic flux resumption may be indispensable in cancer cell escape from senescence/polyploidy.

The p21^WAF1/Cip1 protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers.

Purpose: Small extracellular vesicles (sEVs) are nanoscale biomaterial-like structures involved in intercellular communication and cancer progression. Aberrant surface glycosylation may serve as a diagnostic marker for malignancy. This study aimed to compare the size, glycosylation, and biophysical properties of sEVs secreted by primary and metastatic melanoma cells, and to evaluate a novel analytical technique for glycoprofiling. Methods: sEVs were isolated from the primary (WM115) and metastatic (WM266-4) melanoma cell lines. Their size and concentration were assessed via Nanoparticle Tracking Analysis (NTA), and exosomal identity was confirmed using Western blotting. Glycosylation profiling was performed using a multimodal strategy: Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Nanoplasmonic Sensing (NPS), and, for the first time, Flow-Induced Dispersion Analysis (FIDA). Concanavalin A (Con A) was used as the probe for high-mannose glycans. Results: WM266-4-derived sEVs were significantly larger, whereas WM115 cells secreted more vesicles. Western blotting confirmed the presence of exosomal markers and absence of organelle contaminants. QCM-D and NPS showed stronger Con A binding and higher glycan viscoelasticity index (gVI) in metastatic sEVs, indicating altered glycan architecture. FIDA further confirmed these differences by quantifying a lower dissociation constant (Kd) and multivalent binding behavior in WM266-4-derived sEVs, consistent with a denser glycan coat. Conclusion: Metastatic melanoma–derived sEVs exhibited distinct Con A–detectable high-mannose glycosylation patterns that may represent malignancy-associated features. This study demonstrates the utility of multimodal nanobiophysical methods, particularly FIDA, as sensitive tools for EV glycoprofiling. While the present findings are based on cell line–derived sEVs, they support the translational potential of glycan-based signatures for future liquid biopsy platforms and expand the analytical capabilities of cancer nanodiagnostics. Plain Language Summary: Melanoma is a dangerous type of skin cancer that can spread quickly. Doctors need better ways to tell how aggressive a melanoma is, ideally using simple blood tests. One promising source of information is small extracellular vesicles (sEVs). These nanosized particles are released by all cells, including cancer cells, and carry biological signals that may reveal disease status. In this study, researchers compared sEVs released by two melanoma cell lines: one from an early-stage tumor (WM115) and one from a later, metastatic tumor (WM266-4). Both came from the same patient, making the comparison more reliable. The team found that metastatic cells produced fewer but larger vesicles, with clear differences in the sugar molecules (glycans) displayed on their surfaces. To study these sugar patterns, the researchers combined advanced nanoscale tools: Quartz Crystal Microbalance with Dissipation (QCM-D), NanoPlasmonic Sensing (NPS), and, for the first time in this context, Flow-Induced Dispersion Analysis (FIDA). Using a plant protein called Concanavalin A (Con A), which specifically binds to high-mannose glycans, they showed that vesicles from metastatic cells carried denser and more accessible sugar motifs. These findings suggest that vesicle surface sugars could provide useful clues about melanoma progression. However, the results come from cell line–derived vesicles and have not yet been validated in patient samples. Future studies using broader lectin panels and clinical material will be needed. Even so, this research highlights the potential of glycan signatures and FIDA as part of future non-invasive, nanotechnology-enabled blood tests to monitor cancer.

Background: A number of late‐onset Alzheimer’s disease (AD) risk genes have been discovered, among which the most studied but not fully explored is the apolipoprotein E gene (APOE). PICALM (coding Phosphatidylinositol Binding Clathrin Assembly Protein) is another well‐recognized risk‐gene, shown to interact with the APOE gene in conferring AD risk. MicroRNAs (miRNAs) are post‐transcriptional regulators of mRNAs, some of which are known to be involved in AD pathology. The purpose of this study was to investigate in middle‐aged, healthy participants the association between AD risk‐genes (APOE and PICALM) and circulating miRNAs that were previously identified as altered in AD patients.Method: Blood samples were collected from 76 healthy individuals (36 F, 36 M; age: 56.38±3.16), assigned to three groups: A+P+ (APOE/PICALM risk), A+P‐ (APOE risk, PICALM neutral alleles) and N group (APOE/PICALM neutral alleles). RT‐qPCR was used to compare the levels of 11 microRNA identified by us in previous studies as candidate early AD biomarkers in blood plasma (hsa‐miR‐29b‐3p, hsa‐miR‐34a‐5p, hsa‐miR‐125b, hsa‐miR‐146a, hsa‐miR‐135a, hsa‐miR‐30b‐5p, hsa‐miR‐142‐3p, hsa‐miR‐200a‐3p, hsa‐miR‐483‐5p, hsa‐miR‐486‐5p, hsa‐miR‐502‐3p) [1, 2]. We followed standard procedures of blood assays.Result: Single gene risk (APOE) carriers had lower levels of miR‐29b‐3p than non‐carriers and double risk carriers (F(2,73) = 12.75, p < 0.001 with Welch homogeneity correction; post‐hoc with Tukey correction: p < 0.01 for A+P‐ versus N, p < 0.001 for A+P‐ versus A+P+). No other changes in miRNA panels were discovered.Conclusion: Downregulation of miR‐29b‐3p in blood was detected repeatedly before in patients with AD, in 4 independent studies [1]. Here we show the same effect in a subpopulation of middle‐age, healthy individuals with APOE related genetic risk for AD. Further research is needed to understand the interaction reversing this effect in PICALM risk carriers. Longitudinal follow‐up may validate this blood‐based miRNA not only as an easy‐to‐access diagnostic biomarker, but also as a risk‐assessment biomarker for AD prevention.Funding: This work was funded by the Polish National Science Centre (NCN) grant no. 2018/31/N/HS6/03551, 2016/20/W/NZ4/00354 and 2018/29/B/NZ7/02757.[1], [2] https://doi.org/10.1016/j.arr.2018.10.008; 10.18632/oncotarget.15109.