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Quantitative assessment of STAT3 and HPV16 E6 transcripts using Flow-FISH approach for early detection of progressive cervical lesions
Arun Chhokar, Udit Joshi, Tanya Tripathi, Bindiya Gupta, Madeeha Mudassir, Divya Janjua, Apoorva Chaudhary, Joni Yadav, Nikita Aggarwal, Vinita K Jaggi, Alok C Bharti
Arun Chhokar, Udit Joshi, Tanya Tripathi, Divya Janjua, Apoorva Chaudhary, Joni Yadav, Nikita Aggarwal, Alok C Bharti, Department of Zoology, University of Delhi, Delhi 110007, India
Arun Chhokar, Department of Zoology, Deshbandhu College, University of Delhi, New Delhi 110019, Delhi, India
Bindiya Gupta, Madeeha Mudassir, Department of Obstetrics and Gynecology, University College of Medical Sciences and Guru Teg Bahadur Hospital, New Delhi 110095, Delhi, India
Vinita K Jaggi, Department of Gynaecological Oncology, Delhi State Canc Inst, Delhi 110095, India
Co-first authors: Arun Chhokar and Udit Joshi.
Author contributions: Chhokar A and Joshi U performed the research, curated the data, conducted the formal analysis, they contributed equally to this article, they are the co-first authors of this manuscript; Chhokar A, Joshi U, Tripathi T wrote the first draft of the manuscript; Tripathi T, Mudassir M, Janjua D, and Chaudhary A contributed to the investigation and methodology; Gupta B and Jaggi VK supervised the study and validated the data; Bharti AC conceptualized and designed the research study, supervised and administered the project, validated the data, and provided the necessary resources; and all authors critically reviewed the manuscript and approved the final version prior to submission.
Supported by Indian Council of Medical Research, No. 5/13/4/ACB/ICRC/2020/NCD-III and No. SG/Dev. Res/05750/2025-2028 (261835); Indian Council of Medical Research AdHOC, No. 2021-10573/GENOMIC/ADHOC-BMS and No. IG/Dev.Res/00265/2029-2025 (256963); Central Council for Research in Homoeopathy, Ministry of AYUSH, Government of India, No. 17-30/2023-24/CCRH/Tech./Coll./DO-Cervical Cancer Phase-II/898; Institution of Eminence, University of Delhi, No. /IoE/2025-26/12/FRP; ANRF, No. ANRF/PAIR/2025/000003/PAIR-B and No. 73(CSIR-UGC NET JUNE 2017); University Grants Commission, No. 764/(CSIR-UGC NET JUNE 2019); and Council of Scientific and Industrial Research, No. 09/0045/(11635)/2021-EMR-1, No. 09/0045(12901)/2022-EMR-1, No. 09/045(1629)/2019-EMR-I and No. 09/045(1622)/2018-EMR-I.
Institutional review board statement: This study was approved by the Medical Ethics Committee of Department of Zoology, University of Delhi, approval No. IHEC/DU/NP-1/2020.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: All the data sets used in the present study have been provided in
Supplementary material and there is no additional data to disclose.
Corresponding author: Alok C Bharti, PhD, Professor, Department of Zoology, University of Delhi, Gate No. 3 Chaatra Marg, University of Delhi North Campus, Delhi 110007, India.
alokchandrab@yahoo.com
Received: December 28, 2025
Revised: January 28, 2026
Accepted: March 2, 2026
Published online: June 20, 2026
Processing time: 167 Days and 17.8 Hours
BACKGROUND
Dysregulated signal transducer and activator of transcription 3 (STAT3) signaling is a key feature of human papillomavirus (HPV)-driven cervical carcinogenesis. Our earlier work demonstrated elevated STAT3 expression plays a regulatory role in oncogenic transcription of HPV16 E6/E7. A combined analysis of STAT3 and HPV E6/E7 mRNA by fluorescence in situ hybridization (FISH) showed diagnostic clinical relevance in screening for pre-cancerous cervical lesions. However, use of FISH does not provide clear objective diagnostic threshold to establish accurate signal positivity.
AIM
To assess the feasibility of FISH for quantitative detection of STAT3 and HPV E6/E7 transcripts using a flow cytometry-based approach.
METHODS
HPV-negative (C33a), HPV16-positive (SiHa) and HPV18-positive (HeLa) cervical cancer cell lines were analyzed for STAT3 and HPV E6/E7 transcript expression using FISH approach. Signal specificity and distribution were assessed by fluorescence microscopy using target-specific and scrambled probes. The same probes were further evaluated using flow cytometry-based FISH to determine their suitability for quantitative transcript detection.
RESULTS
Fluorescence microscopy revealed strong and discrete STAT3-associated signals in SiHa and HeLa cells, whereas C33a cells exhibited comparatively diffuse fluorescence. Scrambled control probes produced minimal background staining, supporting the specificity of STAT3 probe. HPV16 E6 probe also produced detectable signals but comparable fluorescence intensity was observed across all cell lines irrespective of HPV status and was similar to scrambled probe controls. Moreover, when assessed by flow cytometry-based FISH, the same probes displayed limited performance. STAT3-positive populations were low and accounted for approximately 10% of cells, while no clearly distinguishable HPV16 E6-positive population could be identified in any of the tested cell lines. These findings indicate that optimized microscopy-based FISH assay may not be directly compatible with flow-based transcript detection platforms.
CONCLUSION
Therefore, re-optimization of assay is required for quantitative transcript detection for HPV-associated cervical cancer screening using flow cytometry-based FISH. The manuscript addresses the bottlenecks and potential strategies to mitigate the issues.
Core Tip: This study evaluates the feasibility of translating a microscopy-optimized fluorescence in situ hybridization (FISH) assay for signal transducer and activator of transcription 3 and human papillomavirus (HPV) 16 E6 transcripts into a flow cytometry-based FISH (Flow-FISH). While microscopy-based FISH reliably detected transcripts in cervical cancer cell lines, pre-published probes demonstrated limited sensitivity and specificity in Flow-FISH, particularly for HPV16 E6. These findings highlight critical technical bottlenecks in adapting slide-based FISH assays to flow-cytometry and underscore the need for platform-specific probe design and assay re-optimization before Flow-FISH can be applied as a scalable and objective screening tool for HPV-associated cervical cancer.
