CTGF/CCN2 Promotes Invasive Growth in Cervical Cancer Spheroids and Is Associated With Metastatic Cervical Cancer...

Metastatic spread defines the lethality of cervical cancer (CC). Connective tissue growth factor (CTGF/CCN2) regulates cell- extracellular matrix interactions but its role in CC is not well-defined. This study investigates the role of CTGF in driving CC invasive growth and its prevalence in patient tissues.

CC spheroids (C33A, HT3) were treated with recombinant human CTGF (rhCTGF) or a function-blocking antibody (IgG CTGF). Invasive growth was assessed via 3D spheroid assay using a Celigo imaging cytometer. Cancer stem cell (CD133, CD44) and epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin) were analyzed by immunofluorescence. CTGF expression was evaluated using a tissue microarray containing 69 cases in triplicate from pre-invasive, invasive (FIGO I-III), and metastatic cervical lesions, quantified via immunofluorescence scoring.

Functional blockade of CTGF significantly reduced 3D spheroid invasive growth in C33A and HT3 cells (p<0.0001). Immunofluorescence revealed that CTGF modulation altered spatial distribution of key proteins: rhCTGF induced surface clustering of CD133 and peripheral N-cadherin enrichment, while CTGF blockade was associated with apparent nuclear/perinuclear enrichment of CD133 and E-cadherin and reduced N-cadherin signal. In patient tissue cores, metastatic samples exhibited the highest CTGF fluorescence intensity. High CTGF expression [immunoreactivity score (IRS) ≥ 6] was most prevalent in FIGO stage I (35.5%) compared to stage III (10.0%). Kaplan-Meier analysis revealed that high CTGF mRNA expression was associated with significantly reduced recurrence-free survival (log-rank p=0.0032).

In 3D models of CC, CTGF appears to regulate an invasive phenotype, presumably by controlling aberrant localization of stemness and EMT markers. Its apparently elevated expression in early-stage cervical carcinomas and metastases, combined with its prognostic value for recurrence-free survival, suggests that CTGF may be involved in triggering the potential for metastasis and could therefore serve as an early prognostic biomarker.