Understanding Primary Metastasis of Ovarian Cancer via Imaging Mass Spectrometry of a Novel 3D Tissue Explant and Cellular Coculture

Significance of the Topic

High-grade serous ovarian cancer (HGSOC) often initiates in the fallopian tube epithelium before metastasizing to the ovary, yet the molecular cues driving this primary spread remain poorly characterized. Conventional analytical methods lack spatial resolution and dynamic context, underscoring the need for advanced imaging approaches. MALDI-TOF imaging mass spectrometry (IMS) combined with 3D coculture models offers a powerful platform to map chemical signaling in situ, enhancing our understanding of tumor–tissue interactions that facilitate metastasis.

Objectives and Study Overview

This study sought to establish and validate a novel agarose‐based 3D coculture system of murine fallopian tube epithelial (FTE) cells and healthy ovarian explants. The main aims were:

• To optimize a homogenous 3D culture that permits diffusion of small signaling molecules while immobilizing cells.
• To refine matrix application techniques for consistent MALDI-TOF imaging of soft agarose plugs.
• To identify and localize specific m/z signals associated with tumorigenic FTE cells interacting with ovarian tissue, with a focus on metastatic drivers.

Methodology and Instrumentation

3D Coculture Model

• Primary ovarian explants were harvested from postnatal CD-1 mice (day 16–18) and maintained in warm media prior to plating.
• Four murine cell lines were used: MOE parental, MOE SCRshRNA control, MOE PTENshRNA tumorigenic, and MOSE ovarian surface epithelial cells.
• Cells were suspended at 166 cells/µL in 1% agarose prepared by mixing equal volumes of cell‐laden DMEM and 2% agarose.
• Cultures were assembled in eight‐well Permanox chambers on ITO‐coated slides, using 300 µL plugs, and incubated at 37 °C, 5% CO₂ for 4 days.

Matrix Application and MALDI-TOF Imaging

• Precoat matrix layer applied to slides prior to sample plating to enhance adherence.
• Post‐desiccation spraying of 50:50 CHCA:DHB matrix (5 mg/mL in 90:10 ACN:H₂O + 0.1% TFA) using HTX TM-Sprayer: 0.2 mL/min flow, 110 mm/min velocity, 10 psi nitrogen, 48 passes.
• Slides dried at 37 °C for 4 h (rotated hourly) to yield flat agarose plugs.
• MALDI-TOF acquisition on Bruker autoflex speed LRF (m/z 100–2000 Da, positive mode): 40% laser power, 2 reflector gain, 500 shots/location, 50 µm pixel pitch.
• Data processed with flexImaging v4.1 and SCiLS Lab 2015b, normalizing spectra to total ion count.

Main Findings and Discussion

Comparison of four conditions (agarose/media, MOE PTENshRNA alone, explant alone, and coculture) revealed 44 statistically significant m/z signals (p<0.05). A broader analysis across eight conditions isolated 33 signals uniquely elevated in the ovarian explant + MOE PTENshRNA coculture, indicating tumor‐specific cross-talk. Notably, m/z 170 was assigned to norepinephrine, implicating it as a putative metastatic signaling molecule. A refined spatial experiment, segregating tissue and cells within divided wells, confirmed the ovary as the source of norepinephrine, diffusing through agarose toward tumorigenic cells.

Benefits and Practical Applications

This integrated 3D IMS approach enables:

• Spatial mapping of intercellular metabolites and signaling compounds in a near‐physiological context.
• Identification of metastasis‐associated biomarkers such as norepinephrine, guiding targeted therapeutic strategies.
• Rapid screening of tumor–tissue interactions for drug discovery and biomarker validation in preclinical research.

Future Trends and Potential Uses

Advancements may include:

• Integration of multi-modal imaging (e.g., ion mobility, high-resolution MS) for deeper chemical characterization.
• Extension to human organoid models and patient-derived explants for personalized cancer profiling.
• Automated workflows and AI-driven data analysis to accelerate biomarker discovery.

Conclusion

This work demonstrates a robust MALDI-TOF IMS workflow for novel 3D tissue–cell cocultures, revealing key molecular drivers of ovarian cancer metastasis. The identification of ovary-derived norepinephrine highlights the utility of spatially resolved mass spectrometry in decoding dynamic tumor–tissue signaling.

Reference

• Zink KE, Dean M, Burdette JE, Sanchez LM. Imaging Mass Spectrometry Reveals Crosstalk between the Fallopian Tube and the Ovary that Drives Primary Metastasis of Ovarian Cancer. ACS Cent Sci. 2018;4(10):1360–1370. DOI: 10.1021/acscentsci.8b00405.