Differential Metabolic and Transcriptional Responses of PBMCs to Blastocystis sp. Subtypes in Chemotherapy-Treated Colorectal Cancer Patients

PURPOSE: Blastocystis sp. is an opportunistic intestinal protist frequently associated with colorectal cancer (CRC). Its immunomodulatory influence within the immunosuppressive milieu of chemotherapy-treated CRC remains largely unexplored. This study investigated the effects of Blastocystis solubilized antigen (BSA) from subtypes ST1-ST5 on the metabolic activity and cytokine gene expression of peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs were isolated from healthy donors (HDs; n=5) and CRC patients undergoing 5-fluorouracil-based chemotherapy (CRCPs; n=5) sampled 7-14 days post-cycle to capture the treatment nadir. PBMCs were challenged with BSA (0.001-30 µg/ml) for 48 hours. Metabolic activity was assessed via MTT assay, and transcriptional profiles of cytokines (IL-6, IL-8, TGF-β, IFN-γ, TNF-α) were evaluated using real-time RT-PCR. RESULTS: HD PBMCs exhibited a biphasic metabolic response, shifting to significant stimulation at BSA concentrations exceeding 10 µg/ml. Conversely, CRCP PBMCs displayed a significantly attenuated, predominantly inhibitory metabolic profile across all concentrations (p < 0.001), despite maintaining viability to PHA. Transcriptionally, HDs showed balanced upregulation of effector and pro-tumorigenic transcripts. In contrast, the CRCP cohort exhibited widespread transcriptional blunting. Notably, subtype ST3 was a distinct outlier in CRCPs, uniquely inducing robust IFN-γ and TNF-α expression (p < 0.01) comparable to HDs. CONCLUSION: While chemotherapy induces systemic hyporesponsiveness, ST3 possesses unique immunogenic properties that bypass immune suppression. This transcriptional escape may facilitate an unbalanced inflammatory milieu conducive to CRC progression.