The research team, led by Erwin Danil Yulian, Teguh Herdadi, and Karl Heinz Leonhardt Rowika, examined archived tumor tissue from 29 female TNBC patients treated between 2017 and 2022. All patients had histologically confirmed triple-negative disease, defined as estrogen receptor expression of 1 percent or less, progesterone receptor expression of 1 percent or less, and HER2 scores of 0 to 1+ or 2+ with negative in situ hybridization. Using immunohistochemistry on formalin-fixed paraffin-embedded blocks, the investigators scored vimentin expression semiquantitatively, from 0 for no staining to 3+ for strong cytoplasmic staining in more than 10 percent of tumor cells. Crucially, only cytoplasmic staining within invasive tumor epithelial cells was counted; staining in stromal fibroblasts, endothelial cells, and inflammatory cells was excluded, since vimentin is constitutively expressed in normal mesenchymal cells and would otherwise confound the analysis.

The results were striking. Vimentin expression was detected in 22 of 29 cases, or 75.9 percent of the cohort. Among these positive cases, 14 showed strong 3+ expression, 6 showed moderate 2+ expression, and only 2 showed weak 1+ expression. When the researchers correlated expression status with overall survival, defined as the time from diagnosis to death from any cause or last follow-up, the association was statistically significant at p = 0.019. Of the 22 patients with positive vimentin expression, 19 experienced mortality events, while only 3 remained alive at the end of the observation period. In contrast, among the 7 patients with negative vimentin expression, only 3 experienced mortality events and 4 were alive at final follow-up.

Kaplan-Meier survival curves told the same story visually. Patients with positive vimentin expression had a mean survival time of 20.86 plus or minus 2.01 months, with a 95 percent confidence interval of 16.90 to 24.82 months. Those with negative expression survived a mean of 30.42 plus or minus 4.36 months, with a 95 percent confidence interval of 21.87 to 38.98 months. The log-rank test confirmed a significant difference between the two curves at p = 0.004, indicating that vimentin expression status meaningfully affects patient prognosis in this population.

What makes vimentin such a potent prognostic signal? The protein is a mesenchymal intermediate filament that sits at the heart of the epithelial-mesenchymal transition, a developmental program hijacked by cancer cells that enhances motility, invasiveness, and metastatic potential. EMT activation is regulated by transcription factors such as Snail, Twist, and Slug, and by signaling pathways including TGF-beta/SMAD, STAT3, and Wnt/beta-catenin. Overexpression of vimentin reflects EMT activation, cytoskeletal remodeling, and increased therapeutic resistance. Previous studies support the Indonesian findings: Xu and colleagues reported that vimentin overexpression in TNBC enhances invasion and anthracycline resistance, while Fedele and colleagues found that high vimentin levels doubled mortality risk and were associated with distant metastasis, particularly to the lung and brain.

Beyond survival, the study explored whether vimentin correlated with conventional clinicopathological characteristics. No significant associations were observed with age, body mass index, tumor size, lymph node status, histopathological grade, or clinical stage, all p values exceeding 0.05. However, vimentin expression showed a significant correlation with Ki-67 expression at p = 0.045, suggesting a link between EMT activation and proliferative activity. This association implies that proliferation and EMT may occur in parallel, as both are regulated by TGF-beta and beta-catenin signaling, which induce vimentin, CCND1, and MYC transcription. Inflammatory cytokines such as IL-6 and TNF-alpha further enhance this process via the JAK/STAT pathway, supporting tumor cell survival under oxidative and hypoxic stress.

In Cox regression analysis, vimentin expression emerged as an independent predictor of overall survival with a hazard ratio of 5.64 and a 95 percent confidence interval of 1.39 to 22.7 at p = 0.015. Adequate therapy was also significantly associated with survival, with a hazard ratio of 4.05 and a 95 percent confidence interval of 1.37 to 11.9 at p = 0.011. In contrast, conventional variables including tumor size, lymph node status, histopathological grade, and Ki-67 expression showed no statistically significant association with survival outcomes. This suggests that biological factors may play a more dominant role than anatomical characteristics in determining prognosis in TNBC, and that conventional clinicopathological variables may underestimate true biological aggressiveness.

The patient profile itself reveals much about TNBC in developing countries. Most patients were aged 40 years or older at 82.8 percent, had a normal body mass index below 25 at 82.8 percent, and presented with locally advanced disease at 72.4 percent. Nearly two-thirds had Grade III tumors, and 72.4 percent showed a high Ki-67 index of 20 percent or greater, indicating elevated proliferative activity consistent with basal-like TNBC biology. Late-stage presentation reflects persistent barriers to early detection in Indonesia, including low health literacy and limited access to diagnostic services, contributing directly to poorer survival outcomes since advanced TNBC typically has a 5-year survival below 50 percent.

The study has limitations that warrant caution. The cohort was small, drawn from a single institution, and dominated by locally advanced disease, which may not fully represent the broader spectrum of TNBC. Statistical power to detect significant associations was limited, and generalization to early-stage populations requires validation. Nevertheless, the findings suggest that incorporating vimentin immunostaining into routine diagnostic panels could enhance risk stratification and personalized management, particularly in tertiary centers. Patients with high vimentin levels may benefit from EMT-inhibitory strategies such as TGF-beta or STAT3 blockade, which are being explored in TNBC trials. Future prospective multicenter studies with larger and more heterogeneous cohorts are needed to confirm the prognostic significance of vimentin expression and to translate these findings into clinical practice.

Subject of Research: Triple-negative breast cancer

Article Title: The Prognostic Role of Vimentin Expression in Triple‐Negative Breast Cancer: A Retrospective Cohort Study

Article References: Danil Yulian, E., Hellyanti, T., & Rowika, K. H. L. (2026). The Prognostic Role of Vimentin Expression in Triple‐Negative Breast Cancer: A Retrospective Cohort Study. Cancer Reports, 9(10), Article e70632. https://doi.org/10.1002/cnr2.70632

Image Credits: AI Generated

DOI: 10.1002/cnr2.70632

Keywords: vimentin, triple-negative breast cancer, TNBC, epithelial-mesenchymal transition, EMT, Ki-67, immunohistochemistry, prognosis, overall survival, biomarker, Prognostic, Role

The research team, led by Di Liu and Guo Deng of The People’s Hospital of the Qiandongnan Miao and Dong Autonomous Prefecture, together with Hanluo Yang and Qingyu Zhang of Jinping County People’s Hospital, began by asking a basic pharmacological question: how sensitive are bladder cancer cells to trifolirhizin? Using cell counting kit-8 assays on two widely studied human bladder carcinoma cell lines, T24 and UMUC3, they measured the concentration required to kill half of the cells. The answer was strikingly consistent across both lines, with IC50 values of 67.41 micromolar for T24 cells and 72.58 micromolar for UMUC3 cells. Those figures establish trifolirhizin as a genuine cytotoxic agent against these aggressive tumor cells rather than a marginal one, and they provided the dosing foundation for every experiment that followed.

Viability alone, however, tells only part of the story in oncology research. A compound can poison cells without touching the behaviors that actually make cancer dangerous: uncontrolled replication and tissue invasion. To probe these hallmarks, the researchers turned to colony formation assays, which measure the capacity of individual tumor cells to multiply into visible colonies over days and weeks. Trifolirhizin treatment markedly reduced the number of colonies the bladder cancer cells could establish, signaling a direct suppression of long-term proliferative potential. This kind of assay is widely regarded as a stringent test of a drug’s anti-tumor character, because surviving even a brief exposure is not enough; the cells must retain the full machinery for sustained growth, and trifolirhizin appears to compromise exactly that machinery.

Invasion was the next target on the team’s list. Malignant tumors earn their lethal reputation not from where they start but from where they spread, and bladder cancer is no exception. The investigators used transwell migration assays to quantify how many cells could push through a membrane barrier, a standard laboratory proxy for invasive behavior. Trifolirhizin treatment significantly decreased the number of invading cells. Crucially, the molecular correlates of that behavioral shift told a coherent story: levels of vimentin, an intermediate filament protein that supports cellular motility and is a classic marker of epithelial-mesenchymal transition, dropped after treatment, while E-cadherin, an adhesion molecule that helps hold epithelial cells together and restrains migration, increased. In other words, the compound appeared to pull the cells back from a mobile, invasive state toward a more settled, epithelial identity.

Perhaps the most forward-looking dimension of the study concerns immune evasion, the process by which tumors render themselves invisible or hostile to the body’s defensive cells. The researchers found that trifolirhizin-treated bladder cancer cells became more vulnerable to CD8-positive T cell-mediated cytotoxicity, the principal killing mechanism of cellular antitumor immunity. Alongside this, they measured elevated levels of interferon-gamma, a cytokine that serves as both a marker and a driver of an activated antitumor immune response. By quantifying cytokines in the culture environment, the team showed that the compound did not merely make tumor cells more fragile; it also shifted the signaling landscape in ways that favor immune recognition and attack. That dual action, direct toxicity plus immune reactivation, is precisely the profile that modern immunotherapy research seeks to amplify.

With the phenotypic effects established, the study moved to its mechanistic core: the AKT/NF-κB pathway. Protein kinase B, universally abbreviated as AKT, is a kinase that promotes survival and growth in countless cancers, while nuclear factor kappa-B is a transcription factor that switches on genes supporting inflammation, survival, invasion, and immune escape. The two are functionally intertwined, with AKT activity frequently reinforcing NF-κB signaling. Using immunoblotting, the researchers showed that trifolirhizin treatment attenuated the phosphorylation of both proteins, effectively dialing down the pathway’s activity. Because phosphorylation is the molecular switch that activates these signaling proteins, its reduction means the pathway was genuinely silenced, not merely perturbed.

Correlation is not causation, however, and the team designed an elegant validation experiment to close that gap. They co-treated the cells with SC79, a pharmacological activator of AKT that forces the pathway back into gear even in the presence of an upstream inhibitor. The result was decisive. When SC79 was added alongside trifolirhizin, the compound’s effects on proliferation, apoptosis, invasion, and immune escape indicators in T24 cells were substantially reversed. Markers that trifolirhizin had pushed in an antitumor direction drifted back toward their malignant baseline. This rescue experiment provides the strongest form of evidence short of genetic knockout that the AKT/NF-κB axis is not merely one of many targets but the central conduit through which trifolirhizin exerts its anticancer activity in bladder carcinoma cells.

Laboratory dishes are one thing; living tumors are another. To test whether the findings would survive the far more complex environment of an organism, the researchers turned to xenograft models, in which human bladder cancer cells are implanted into mice and allowed to form tumors. Animals treated with trifolirhizin carried a visibly reduced tumor burden compared with untreated controls, confirming that the compound’s cytotoxic and antiproliferative effects translate into slower tumor growth in vivo. Tissue analysis reinforced the cellular findings: hematoxylin and eosin staining, immunohistochemistry, and immunoblotting revealed reduced vimentin expression within the tumors, increased markers of apoptosis, and elevated interferon-gamma levels. Once again, the AKT/NF-κB pathway showed consistently inhibited expression in tumors harvested from trifolirhizin-treated mice, mirroring the in vitro mechanism at the level of intact tissue.

The convergence of these results paints trifolirhizin as a multimodal therapeutic candidate, a phrase the authors themselves use in their conclusion. By suppressing proliferation, blunting invasion, promoting apoptosis, and enhancing immune-mediated killing, all through downregulation of a single druggable signaling pathway, the compound occupies an unusually versatile position for a natural product. Pterocarpan flavonoids of this class have long attracted attention in chemoprevention and anticancer research, but their efficacy and molecular mechanisms in bladder carcinoma had remained unexplored until this study. The work was approved by the ethics committee of The People’s Hospital of the Qiandongnan Miao and Dong Autonomous Prefecture, and the authors declare no competing interests.

Cautious optimism is the appropriate stance. The IC50 values lie in the high micromolar range, and the journey from xenograft mice to human patients involves hurdles of pharmacokinetics, safety, and formulation that no cell culture or animal model can predict. Still, the study’s design, combining functional assays, cytokine quantification, flow cytometry, pathway rescue with a pharmacological activator, and in vivo validation, exemplifies the rigorous architecture that transforms a plant-derived molecule into a credible drug lead. For a disease that continues to challenge urologists with recurrence and immune evasion, trifolirhizin now offers a scientifically grounded reason for excitement, and a clear mechanistic road map for the therapies that may follow it.

The choice of T24 and UMUC3 as experimental platforms deserves brief elaboration, since these cell lines embody distinct molecular subtypes of bladder carcinoma. T24 cells are derived from a transitional cell carcinoma and display a highly aggressive, invasive phenotype, while UMUC3 carries activating mutations in the HRAS oncogene, a lesion that sits squarely upstream of the PI3K/AKT cascade. The fact that trifolirhizin suppressed AKT phosphorylation and produced comparable IC50 values in both lines suggests its activity does not depend on a single driver mutation, a property that would broaden its potential applicability across molecularly diverse tumors.

The immune findings also fit into a larger conceptual shift in oncology. For decades, cytotoxic chemotherapy was viewed as inherently immunosuppressive, but it is now understood that certain agents can provoke immunogenic cell death, releasing tumor antigens and inflammatory signals that recruit cytotoxic T lymphocytes. The observed rise in interferon-gamma and the heightened susceptibility of treated cells to CD8-positive T cell killing are consistent with this paradigm, raising the possibility that trifolirhizin could act synergistically with immune checkpoint inhibitors, though such combinations remain untested in this study.

Methodologically, the use of SC79 as a pathway rescue tool reflects a standard of mechanistic proof increasingly expected in natural product research, where compounds often have many off-target effects. By demonstrating that forced AKT reactivation reverses the drug’s phenotypic effects, the authors substantially narrow the causal interpretation. Nevertheless, the study does not identify the direct molecular target of trifolirhizin upstream of AKT, leaving open whether the compound binds AKT itself, an upstream activator, or some other regulator of the cascade. Future work with target-identification techniques such as affinity pull-down or thermal proteome profiling would help resolve this question and clarify whether the high micromolar potency can be improved through rational chemical optimization of the pterocarpan scaffold.

Subject of Research: The anticancer effects of the natural flavonoid trifolirhizin on bladder carcinoma via modulation of the AKT/NF-κB signaling pathway

Article Title: Trifolirhizin induces apoptosis and suppresses proliferation, invasion and immune evasion in bladder cancer via AKT/NF-κB signaling pathway

Article References: Liu, D., Deng, G., Yang, H., & Zhang, Q. (2026). Trifolirhizin induces apoptosis and suppresses proliferation, invasion and immune evasion in bladder cancer via AKT/NF-κB signaling pathway. BioMedical Engineering OnLine. https://doi.org/10.1186/s12938-026-01613-7

Image Credits: AI Generated

DOI: 10.1186/s12938-026-01613-7

Keywords: bladder cancer, trifolirhizin, apoptosis, AKT/NF-κB pathway, immune evasion, cell proliferation, invasion, vimentin, E-cadherin, CD8-positive T cells, interferon-gamma, xenograft models