Cancer spheroids are spherical, three-dimensional (3D), in vitro assemblies of cancer cells, which are gaining importance as a useful model in cancer behavior studies. Designed to simulate key features of the in vivo tumor microenvironment, spheroids offer reliable insights for drug screening and testing applications. We observed contrasting phenotypes in 3D cervical cancer (CC) cultures. Thus, in this study, we compared the proteomes of 3D and traditional two-dimensional (2D) cultures of CC cell lines, HeLa, SiHa, and C33A. When cultured in in-house poly-(2-hydroxyethyl methacrylate)-coated plates under conditions suitable for 3D spheroid formation, these CC cell lines yielded spheroids exhibiting different features. Proteomic analysis of cells cultured in 2D and 3D cultures revealed similar protein profiles but remarkable differences in the expression levels of some proteins. In SiHa and C33A cells, the upregulation of key proteins required for spheroid formation was insufficient for the formation of compact spheroids. In contrast, HeLa cells could form compact spheroids because they upregulated the proteins, including cadherin-binding, cytoskeleton, and adhesion proteins, necessary for spheroid formation during the remodeling process. Overall, this study unravels the mechanisms underlying the formation of spheroids in the commonly used CC cell lines.

Cervical cancer (CC) remains a major global health challenge due to chemotherapy resistance and recurrence. Mesenchymal stem cell-derived exosomes (MSC-exosomes) have dual roles, as they can act as therapeutic agents and contribute to chemoresistance. However, their role in response to chemotherapy in CC remains unclear. Therefore, our study investigated the effects of MSC-exosome pretreatment on chemotherapy sensitivity using three-dimensional spheroid models generated from HeLa and SiHa CC cell lines. Proteomic profiling of MSC-exosomes identified key proteins, including ANXA1, ANXA2, EEF2, LGALS1, and PKM2, associated with tumor regeneration and chemotherapy response. MSC-exosomes exhibited context-dependent effects in both chemoresistance and chemosensitization by modulating drug efflux, metabolic reprogramming, stress adaptation, apoptosis, DNA damage response, and integrin-mediated signaling. MSC-exosome pretreatment altered spheroid responses to paclitaxel in combination with cisplatin or carboplatin. MSC-exosomes significantly enhanced chemotherapy-induced cytotoxicity in HeLa spheroids, as evidenced by reduced cell viability, increased caspase activity, and upregulation of the pro-apoptotic marker Bax. In contrast, SiHa spheroids represented selective responses: MSC-exosome pretreatment did not enhance sensitivity to paclitaxel–cisplatin but improved responsiveness to paclitaxel–carboplatin, particularly within the spheroid core. Overall, MSC-exosome pretreatment exerts cell type and drug-specific effects in CC spheroids, supporting their potential to modulate chemotherapy response.

MicroRNAs (miRNAs), derived from extracellular vesicles (EVs) are circulating intercellular communicators which influence pathogenesis and could be used as potential diagnostic markers. In this study, plasma-derived EVs from Plasmodium knowlesi-infected patients (n = 13) and healthy individuals (n = 10) were isolated using size exclusion chromatography and ultracentrifugation. The presence of EVs was confirmed by transmission electron microscopy and Western immunoblotting, and quantified by nanoparticle tracking analysis. The EVs isolated from patients exhibited a larger size, accompanied by an elevated concentration of EVs. The relative expression levels of 8 human miRNAs were quantified using reverse transcriptase quantitative polymerase chain reaction. Compared to uninfected groups, hsa-miR-223-5p (P-value = 0.0002) and hsa-miR-486-5p (P-value = 0.025) were upregulated in P. knowlesi-infected patients. Bioinformatic analysis revealed that these miRNAs are predicted to target both human host and parasite genes, and they were found to be enriched in various malaria-related pathways. The areas under the receiver operating characteristic curve of hsa-miR-223-5p and hsa-miR-486-5p were 0.9154 and 0.8231, respectively, suggesting the potential of EV-miRNAs as diagnostic markers. Results revealed that EV-miRNAs may play a significant role in the progression of P. knowlesi infection. Further investigations should explore their potential impact on gene expression regulation as diagnostic biomarkers or targets for therapeutic interventions.

Concurrent chemoradiation therapy (CCRT) is the standard treatment for locally advanced cervical cancer (LACC); however, approximately 30% of patients respond poorly, and alternative therapeutic options remain limited. We aimed to identify peripheral immune biomarkers associated with treatment response and characterize the immune mechanisms underlying clinical outcomes. Thirty patients with LACC undergoing CCRT were enrolled and classified three months after treatment as responders (n = 15) or non-responders (n = 15) according to Response Evaluation Criteria in Solid Tumors. Peripheral blood mononuclear cells were collected at baseline and at one and three months after treatment. Flow cytometry was used to analyze CD4⁺ and CD8⁺ T cells, CD4⁺ forkhead box P3 (FOXP3)⁺ regulatory T cells, interferon gamma (IFN-γ)- and interleukin-10 (IL-10)-producing T cells, and programmed cell death protein 1 (PD-1)-expressing exhausted T cells. Responders showed elevated baseline levels of CD4⁺ T cells, FOXP3⁺ regulatory T cells, and IL-10-producing CD4⁺ T cells, together with increased IFN-γ-producing CD8⁺ T cells during treatment. Non-responders exhibited elevated PD-1-expressing CD8⁺ T cells and reduced regulatory and effector T-cell activity. Ratios incorporating FOXP3⁺ regulatory T cells and PD-1 expression demonstrated strong predictive performance, with area under the curve values up to 0.98. These findings indicate that a balanced regulatory-to-effector T-cell profile is associated with favorable response to CCRT, whereas increased T-cell exhaustion is linked to treatment failure. Peripheral immune profiling may provide a minimally invasive approach for early prediction of treatment outcomes in LACC.