Nanomechanical Characteristics of Cervical Cancer and Cervical Intraepithelial Neoplasia Revealed by Atomic Force Microscopy

Abstract

BACKGROUND Understanding the biological features and developmental progress of cervical cancer is crucial for disease prevention. This study aimed to determine the nanomechanical signatures of cervical samples, ranging from cervicitis to cervical carcinomas, and to investigate the underlying mechanisms. MATERIAL AND METHODS Forty-five cervical biopsies at various pathological stages were subjected to atomic force microscopy (AFM) measurements. Cdc42 and collagen I were quantified using immunohistochemical staining to investigate their relationship with nanomechanical properties of cervical cancers and premalignant lesions. RESULTS We found that the lower elasticity peaks (LEPs) in the high-grade squamous intraepithelial lesion (HSIL) group (21.24±3.83 kPa) and higher elasticity peaks (HEPs) in the cancer group (81.23±8.82 kPa) were upshifted compared with the control group (LEP at 8.51±0.18 kPa and HEP at 44.07±3.54 kPa). Furthermore, compared with the control [29.51±13.61 for cell division cycle 42 (Cdc42) expression and 28.61±17.65 for collagen I expression], immunohistochemical staining verified a significant increase of Cdc42 in the HSIL group (50.57±23.85) and collagen I (56.09±25.70) in the cancer group. In addition, using the Pearson correlation coefficient, Cdc42 expression tended to be positively correlated with LEP locations (r=0.63, P=0.012), while collagen I expression displayed a strong and positive correlation with HEP positions (r=0.88, P<0.001). CONCLUSIONS The nanomechanical properties of HSIL and cancer biopsies show unique features compared with controls, and these alterations are probably due to changes in cytoskeleton and extracellular matrix contents.

Figure 1

Measurement of the nanomechanical properties of cervical biopsies using AFM. (A) Top view of an immobilized cervical biopsy in Ringer’s solution for AFM measurement. (B) Image of Bioscope™ Catalyst™ AFM. (C) Schematic illustration of AFM measurement of cervical tissue samples.

Figure 2

Elasticity distribution of cervical biopsies. (A) Representative images of various stages of cervical lesions and the right panel is the corresponding stiffness distribution (Scale bars: 100 μm). (B) Two distinguishing peaks were identified at 8.51±0.18 kPa (LEP) and 44.07±3.54 kPa (LEP) in the control group. (C) LEP in the HSIL group shifted up to 21.24±3.83 kPa, while its HEP location (43.60±9.42 kPa) was approximately the same as in the controls. (D) LEP in the cancer group (8.82±2.05 kPa) did not show visible differences from the controls, while its HEP shifted upward (81.23±8.82 kPa) compared with the controls.

Figure 3

IHC staining of P16ink4A, Cdc42, and collagen I in cervical biopsies. (A) Representative IHC images of P16, Cdc42, and collagen I in control, HSIL, and cancer groups. (Scale bars: 100 μm). From left to right: P16, Cdc42, and collagen I; from top to bottom: control, HSIL, and cancer groups. (B) Quantification of P16, Cdc42, and collagen I expression in each group. From left to right: P16, Cdc42, and collagen I (* P<0.05, ** P<0.01).

Figure 4

Correlation analysis between LEP and Cdc42 expression, and between HEP and collagen I expression. (A) Pearson’s correlation indicated that LEP and Cdc42 IOD/area values showed a significant positive tendency of correlation (r=0.63, P=0.012). (B) Correlation analysis verified a close positive correlation between HEP and collagen I IOD/area values (r=0.88, P<0.001).