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

Acta Parasitologica (2026) 71:117 https://doi.org/10.1007/s11686-026-01297-y BRIEF REPORT Differential Metabolic and Transcriptional Responses of PBMCs to Blastocystis sp. Subtypes in Chemotherapy-Treated Colorectal Cancer Patients Muhammad Mirza Halimi1 · Deviga Genasan1 · Arutchelvan Rajamanikam2 · Deepa Anbazhagan3 · Vetriselvan Subramaniyan1 · Suresh Kumar Govind2 · Umah Rani Kuppusamy4 · Rohit Gundamaraju5 · Vinoth Kumarasamy1 Received: 9 February 2026 / Accepted: 16 April 2026 © The Author(s) 2026 Abstract 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. Keywords Blastocystis sp. subtypes · Colorectal cancer · PBMCs · Effector transcripts · Transcriptional escape · Chemotherapy induced immunosuppression Vinoth Kumarasamy 1 Department of Parasitology & Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Cheras, 56000 Kuala Lumpur, Malaysia 2 Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia 3 International Medical School, Management and Science University (MSU), Shah Alam 40100, Selangor Darul Ehsan, Malaysia 4 Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia 5 Department of Pharmaceutical Engineering, B V Raju Institute of Technology, Narsapur, Medak, Telangana, India Introduction Blastocystis sp. (Blastocystis) is a ubiquitous enteric protist colonizing approximately one billion individuals globally, though its prevalence varies significantly based on geographic and clinical settings [7]. While traditionally regarded as a commensal, its pathophysiological role in colorectal carcinogenesis has recently gained significant attention [10]. The parasite is frequently detected in the faecal samples of colorectal cancer (CRC) patients, often emerging as an opportunistic infection during active chemotherapy cycles [9]. Beyond its association with 117 Page 2 of 10 gastrointestinal disorders like irritable bowel syndrome [11], the parasite’s immunomodulatory influence remains poorly characterized, particularly within the immunosuppressive milieu of patients undergoing cytotoxic therapy. Experimental evidence suggests that Blastocystis can elicit robust cellular immune responses through the upregulation of pro-inflammatory cytokines such as interferongamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) [8, 11]. Mechanistically, Blastocystis cysteine proteases are implicated in the activation of NF-κB signaling pathways, driving IL-8 secretion and localized inflammatory recruitment [20]. However, this pathogenic potential appears highly subtype-dependent. While ST7 has been noted for its potent pro-inflammatory effects, Subtype 3 (ST3) has emerged as the most prevalent genotype in CRC cohorts [5]. Previous in vitro studies have demonstrated that ST3 solubilized antigen significantly exacerbates the proliferation of malignant HCT116 cells and upregulates tumor-promoting factors like Cathepsin B [12]. The association between Blastocystis and CRC is further complicated by the parasite’s ability to modulate the tumor microenvironment. Chronic colonization may impair host immune surveillance by inducing oxidative stress evidenced by elevated lipid hydroperoxides (LHP) and advanced oxidation protein products (AOPP) potentially creating a niche conducive to tumor progression [2]. Crucially, emerging evidence indicates that Blastocystis may directly interfere with the efficacy of chemotherapy. Recent findings show that the presence of the parasite reduces the inhibitory potency of 5-fluorouracil (5-FU) by upregulating cytoprotective genes such as Nrf2 and TGF-β in cancer cells [13, 18]. Standard-of-care chemotherapy for CRC, including FOLFOX and Mayo regimens, induces profound systemic immunosuppression [17]. Antimetabolites like 5-FU and platinum-based agents cause DNA damage in peripheral blood mononuclear cells (PBMCs), leading to attenuated effector T-cell activity and a compromised innate response [4]. This systemic hyporesponsiveness likely alters the host’s ability to reach the antigenic threshold necessary to mount an effective defence, particularly during the treatment nadir. This study aims to evaluate the cellular modulations induced by Blastocystis solubilized antigen (BSA) on the metabolic activity and cytokine gene expression of PBMCs isolated from healthy donors and chemotherapy-treated CRC patients. By characterizing the metabolic thresholds and transcriptional profiles of these cohorts, we seek to elucidate how the chemotherapy-induced landscape alters the host’s defensive capacity. Specifically, we investigate whether certain subtypes, such as the clinically prevalent ST3, possess unique immunogenic properties that allow them to bypass systemic suppression, thereby clarifying 13 Acta Parasitologica (2026) 71:117 the opportunistic nature of Blastocystis in the oncological setting. Materials and Methods Cultivation and Subtyping of Blastocystis Blastocystis was isolated from stool samples collected from an aboriginal community in Kuala Langat, Malaysia. The isolates were cultured in Jones’ medium supplemented with 10% horse serum at 37 °C. Genomic DNA was extracted using the QIAamp DNA Stool Mini Kit, and subtyping was performed using sequenc (...truncated)