Spatial proteomics characterizes the transition of precancerous lesions of the fallopian tube to invasive cancer

Ovarian cancer is the deadliest gynecologic malignancy, affecting more than 300,000 women yearly. Most patients will be diagnosed at advanced stages, when the prognosis is poor. Earlier detection requires a better understanding of the precancerous and early stages of the disease. A prominent hypothesis on the origin of serous ovarian cancer is that it comes from early lesions in the fallopian tubes. These lesions are characterized by aberrant p53 expression and are divided into p53 signatures, Serous Tubal Intraepithelial Lesions (STILs) and Serous Tubal Intraepithelial Carcinomas (STICs), based on histopathological features. Such lesions are usually tiny in size, spanning only a few cells. This poses technical challenges, especially for input-limited approaches like mass spectrometry-based proteomics. Here we set out to characterize the transition of early lesions of the fallopian tubes to invasive tumors by applying Deep Visual Proteomics (DVP), an ultra-sensitive, cell-type resolved, spatial proteomics method that we developed.

We started with staining fallopian tube FFPE tissue sections for markers of interest. Based on these markers, we segmented single cells by employing deep learning and classified them in normal ciliated, normal secretory, p53 signature, STIL, STIC, invasive tumor and metastatic cells. From each of these categories, we laser microdissected epithelial cells (15,000 square um per sample) and matching stroma (25,000 square um per sample) and prepared the resulting samples in a semi-automatic fashion. Last, we measured the samples on a Thermo Orbitrap Astral coupled to an Evosep One liquid chromatography system running a 40 samples per day gradient.

We identified a median of 6562 and 5072 protein groups for epithelial and stromal samples, respectively. The corresponding PCAs demonstrated a partial separation between samples of different categories. Strikingly, the respective volcano plots uncovered hundreds of differentially regulated proteins in most of the pairwise comparisons. For example, members of the MCM complex, which is crucial for DNA replication, were upregulated in STIC epithelial cells vs normal secretory cells of the fallopian tube. On the stromal level, progression to STIC was associated with reduced amounts of collagens 1,3,5 and 6. Importantly, pseudotime analysis on single patients recapitulated and confirmed the hypothesized trajectory of early lesions to advanced disease. Along this progression, proteins such as ASPH and CNN2 gradually increase, while others like LAMB3 and WFS1 show the opposite trend, suggesting proteins with potential therapeutic or diagnostic implications. In conclusion, this first deep proteomic characterization of the transition of tiny early lesions of the fallopian tubes all the way to invasive and metastatic tumors, on a spatial and cell-type resolved manner, sheds unique insights into this deadly disease.