In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis

Abstract

Identification of the early microscopic changes associated with ovarian cancer may lead to development of a diagnostic test for high-risk women. In this study we use optical coherence tomography (OCT) and multiphoton microscopy (MPM) (collecting both two photon excited fluorescence [TPEF] and second harmonic generation [SHG]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a long-term survival study and identify microscopic changes associated with early tumor development. These changes include alterations in tissue microstructure, as seen by OCT, alterations in cellular fluorescence and morphology, as seen by TPEF, and remodeling of collagen structure, as seen by SHG. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer.

Keywords: autofluorescence; cancer; multiphoton; non-linear microscopy; optical biopsy; optical coherence tomography; two-photon.

Figure 1. H&E stained sections for each diagnosis. Insets show higher magnification. (A) Normal cycling, (B) atrophic, (C) tubular hyperplasia, (D) cystic tumor, (E) fibrosarcoma, and (F) granulosa cell tumor.

Figure 2. Normal ovary OCT images. (A) Cross-section, (B) en face section. F, follicle; CL, corpus luteum; CT, connective tissue or fat; arrow points to bright edge.

Figure 3. Normal ovary MPM images. (A) TPEF from surface of the ovary. (B) TPEF image from deeper in the ovary. (C) SHG from the surface of the ovary. (D) SHG from deeper in ovary. E, epithelial cells; CL, corpus luteum; f , follicle; S, stroma; wide arrow points to bright cells in stroma; chevron points to theca cells; thin arrow points to granulosa cells; arrowhead points to thin net-like collagen structure; five-point stars indicate gaps in collagen structure in the stroma.

Figure 4. Normal ovary (CON/CON) time serial OCT images. (A) 6 mo, (B) 11 mo, and (C) 13 mo of age. F, follicle; CL, corpus luteum; CT, connective tissue or fat.

Figure 5. Normal ovary (CON/CON) time serial MPM maximum intensity projection images. (A–C) TPEF images. (D—F) SHG images. (A and D) from 6 mo, (B and E) from 11 mo, and (C and F) from 13 mo of age. Insets show detail from a single slice in stack.

Figure 6. Atrophy (CON/DMBA) development in time serial OCT images. (A) Six months (before DMBA dosing) and (B) at 13 mo of age (after DMBA dosing). F, follicle; CL, corpus luteum; CT, connective tissue; arrows point to small cysts.

Figure 7. Atrophy (CON/DMBA) development in time serial MPM maximum intensity projection images. (A and B) TPEF. (C and D) SHG. (A and C) are from 6 mo (before DMBA dosing) and (B and D) are from 13 mo of age (after DMBA dosing). Insets show detail from a single slice in the stack.

Figure 8. Tubular hyperplasia (VCD/CON) development in time serial OCT images. (A) 6 mo, (B) 11 mo, and (C) 13 mo of age. Cy, cyst; chevrons point to bursa; thin arrows point to small invaginations; thick arrows point to large invaginations.

Figure 9. Tubular hyperplasia (VCD/CON) development in time serial MPM maximum intensity projection images. (A–C) TPEF. (D–F) SHG. (A and D) are from the 6-mo time point, (B and E) are from the 11-mo time point, and (C and F) are from the 13-mo time point. Insets show detail from a single slice in the stack. Arrows point to hypointense tubules.

Figure 10. Cystic tumor (VCD/DMBA) development in time serial OCT images. (A) Six months, (B) 11 mo of age. CT, connective tissue; FT, fallopian tube; arrows point to small cysts.

Figure 11. Cystic tumor (VCD/DMBA) development in time serial MPM maximum intensity projection images. (A and B) TPEF. (C and D) SHG. (A and C) are from the 6-mo time point and (B and D) are from the 11-mo time point. Insets show detail from a single slice in the stack. Thin arrows point to blood vessels; wide arrow points to cells along the edge of a large follicle-like cyst.

Figure 12. Fibrosarcoma (CON/DMBA) development in time serial OCT images. (A) Six months (before DMBA dosing), (B) 13 mo of age (after DMBA dosing). CL, corpus luteum; f, follicle; thin arrows point to small cysts; wide arrow points to a deep invagination.

Figure 13. Fibrosarcoma (CON/DMBA) time serial development in MPM maximum intensity projection images. (A and B) TPEF. (C and D) SHG. (A and C) are from the 6-mo time point and (B and D) are from the 13-mo time point. Insets show detail from a single slice in the stack. Thin arrows point to spindle-shaped cells.

Figure 14. Granulosa cell tumor (VCD/DMBA) development in time serial OCT. (A) Six months, (B) 11 mo of age. Thin arrows point to punctate scatterers.

Figure 15. Granulosa cell tumor (VCD/DMBA) development in time serial MPM maximum intensity projection images. (A and B) TPEF. (C and D) SHG. (A and C) are from the 6-mo time point and (B and D) are from the 11-mo time point. Insets show detail. (B) Inset shows a cluster of granulosa cells.

Figure 16. Average fluorescent region area increases with age in this in vivo study. Results from a prior ex vivo study show a similar trend. Bars show the standard error of the mean.

Figure 17. Relative power in the low frequency band of the Fourier transform for each age. Bars indicate the standard error of the mean.

Figure 18. Dosing and imaging schedule.