Fig 2: Scatterplots of DEGs in gliospheres and treated gliospheres using a human cancer stem cell (CSC) arrayCSCs were determined with the RT2 Profiler PCR array (catalog no. PAHS-176Z, QIAGEN, USA). The upregulated genes are marked with red points, while the downregulated genes are marked with green points, and the unchanged genes are marked with black points. (A) Gliospheres comprised of glioma stem cells sorted from U87MGMG cells (ATCC HTB14) compared to control (cell line and –ve population). (B) Treated gliospheres with conditioned media of Wharton’s jelly mesenchymal stem cells (WJT) in comparison to gliospheres. (C) Treated gliospheres with conditioned media of bone marrow mesenchymal stem cells (BMT) in comparison to gliospheres.

Fig 3: Cytotoxic activity of Ganoderma aff. australe aqueous extract and Ag/Cu nanoparticles against cancer and non-cancerous cell lines evaluated by MTT assay. Cells (4,500 cells/well) were seeded in 96-well plates and allowed to adhere for 24 h before treatment. Concentrations tested were based on the amount of aqueous extract used to synthesize nanoparticles (see Table 1 ). All treatments were incubated with cells for 72 h at 37 °C with 5% CO₂. After incubation, cells were rinsed with PBS and incubated with 10 µl MTT solution (5 mg/ml) for 4 h, followed by addition of 100 µl DMSO. Absorbance was measured at 570 nm. Bar graphs show cell viability (expressed as IC₅₀ in mg/ml equivalent of extract) for five cancer cell lines and two non-cancerous control lines. Cancer cell lines: Caco-2 (colon cancer, ATCC HTB-37), HT-29 (colon cancer, ATCC HTN-38), MCF7 (breast cancer, ATCC HTB-22), A-172 (glioblastoma, ATCC CRL-1620), and U-87 MG (glioblastoma, ATCC HTB-14). Non-cancerous control lines: HDFn (human dermal fibroblasts, ATCC PCS-201-010) and Detroit 551 (normal skin fibroblasts, ATCC CCL-110). All cell lines were cultured in DMEM/F12 medium supplemented with 10% FBS, 1% antibiotic-antimycotic, 1% glutamine, 1% nonessential amino acids, and 1% sodium pyruvate. A Aqueous extract (0.5 g/50 ml) showing moderate cytotoxic activity with IC₅₀ values ranging from 1.61 ± 0.35 mg/ml (Caco-2) to 5.78 ± 1.48 mg/ml (Detroit 551), demonstrating baseline bioactivity of fungal metabolites. B M2-3-3 nanoparticles (2 ml extract + 3 ml AgNO₃ + 3 ml CuSO₄) exhibiting the highest cytotoxic efficacy across all cancer cell lines, with particularly remarkable activity against glioblastoma lines A-172 (IC₅₀: 0.26 ± 0.09 mg/ml) and U-87 MG (IC₅₀: 0.31 ± 0.12 mg/ml), and colorectal cancer lines Caco-2 (IC₅₀: 0.39 ± 0.12 mg/ml) and HT-29 (IC₅₀: 0.58 ± 0.28 mg/ml). Critically, M2-3-3 showed selective cytotoxicity with significantly higher IC₅₀ values in non-cancerous lines HDFn (2.87 ± 0.64 mg/ml) and Detroit 551 (3.45 ± 0.89 mg/ml), indicating preferential toxicity toward cancer cells. C M3-2-2 nanoparticles (3 ml extract + 2 ml AgNO₃ + 2 ml CuSO₄) demonstrating intermediate cytotoxic activity with IC₅₀ values consistently higher than M2-3-3 but lower than M5-3-3 across all cancer cell lines. D M5-3-3 nanoparticles (5 ml extract + 3 ml AgNO₃ + 3 ml CuSO₄) showing the lowest cytotoxic activity among the three nanoformulations, though still superior to the crude extract. Data represent mean ± SD of three independent experiments performed in triplicate. Statistical analysis performed using one-way ANOVA followed by Tukey’s HSD post-hoc test. Asterisks indicate significant differences: *p ≤ 0.050, **p ≤ 0.010, ***p ≤ 0.001. The hierarchical efficacy pattern (M2-3-3 > M3-2-2 > M5-3-3 > crude extract) demonstrates successful nanotechnological enhancement of anticancer properties and establishes M2-3-3 as the optimal formulation with superior therapeutic selectivity. The exceptional sensitivity of glioblastoma cell lines suggests potential application in treating brain cancers, which are notoriously difficult to treat due to blood-brain barrier penetration challenges. Complete IC₅₀ values and statistical comparisons are provided in Table 4