Molecular diagnosis of cancer using ambient ionization mass spectrometry

My dissertation focuses on advancing the development and application of ambient ionization mass spectrometry methodology and technology to the biomedical field. The primary ambient ionization method used in my studies is desorption electrospray ionization mass spectrometry (DESI-MS) imaging, which has been previously used to analyze and differentiate disease state (i.e. tumor and normal) and in some cases tumor subtype of human liver, kidney, bladder, testicular, prostate, and brain cancers. DESI-MS imaging is an ideal method for disease diagnosis, because it can be used to directly correlate disease state with histopathology to develop and validate MS libraries built using the molecular profiles that relate to tissue disease states. The goal of this research is to use ambient ionization mass spectrometry for intraoperative surgical-guidance to more accurately diagnose tissue and reduce surgical times. Technological developments during the course of research revolved around touch spray ambient ionization mass spectrometry (TS-MS). This method uses a small probe (e.g. teasing needle) to pick up a minuscule amount of material from a sample, transfer the probe to the front of a mass spectrometer, and, with the addition of high voltage and solvent, induce ESI-like mechanisms for ionization. An evaluation of TS for its use as a potential in vivo surgical tool for disease screening was performed by concurrently studying prostate cancer tissue obtained from surgery with DESI-MS imaging. DESI imaging was used to first establish the relationship between MS molecular profiles and pathology which were then targeted using TS. Further, TS was also evaluated as a non-targeted technique by analyzing prostate specimens with unknown disease states and comparing the unknown data to the previously built MS targeted library. Methodological developments include DESI-MS studies for preliminary diagnosis of disease state and tumor subtyping using fine needle aspirations (FNA) of canine lymphoma specimens. Lipid profiles obtained from FNA samples were tested against a MS library built from a matched set of surgical tissue sections with disease states confirmed by histopathology. DESI-MS imaging was also used to expand upon previously investigated human kidney cancer. Previous investigations included two subtypes and low sample numbers (~10 paired normal and tumor samples per subtype), this more recent study includes the top three most commonly diagnosed subtypes (clear cell, papillary, and chromophobe) and higher sample numbers (~20 paired normal and tumor samples per subtype). In summary, many methodological and technological advances were made during the course of my dissertation studies. These advances include the development of a novel ambient ionization method, an extension of current applications to include FNA samples for early diagnosis, and an expansion of previous work to build more complex and comprehensive MS libraries. Advances such as these continue to propel ambient ionization mass spectrometry deeper into the biomedical field and gives hope to the use of chemical profiling using these methods for biomedical applications in the near future

Ambient ionization mass spectrometric analysis of human surgical specimens to distinguish renal cell carcinoma from healthy renal tissue

Touch spray - mass spectrometry (TS-MS) is an ambient ionization technique (ionization of unprocessed samples in the open air) that may find intraoperative applications in quickly identifying the disease state of cancerous tissues and in defining surgical margins. In this study, TS-MS was performed on fresh kidney tissue (~1–5 cm3), within one hour of resection, from 21 human subjects afflicted by renal cell carcinoma (RCC). The preliminary diagnostic value of TS-MS data taken from freshly resected tissue was evaluated. Principal component analysis (PCA) of the negative ion mode (m/z 700–1000) data provided separation between RCC (16 samples) and healthy renal tissue (13 samples). Linear discriminant analysis (LDA) on the PCA compressed data estimated sensitivity (true positive rate) and specificity (true negative rate) of 98% and 95%, respectively, based on histopathological evaluation. The results indicate that TS-MS might provide rapid diagnostic information in spite of the complexity of unprocessed kidney tissue and the presence of interferences such as urine and blood. Desorption electrospray ionization imaging (DESI-MSI) in the negative ionization mode was performed on the tissue specimens after TS-MS analysis as a reference method. The DESI imaging experiments provided phospholipid profiles (m/z 700–1000) that also separated RCC and healthy tissue in the PCA space, with PCA-LDA sensitivity and specificity of 100% and 89%, respectively. The TS and DESI loading plots indicated that different ions contributed most to the separation of RCC from healthy renal tissue (m/z 794 [PC 34:1+Cl]− and 844 [PC 38:4+Cl]− for TS vs. m/z 788 [PS 36:1-H]− and 810 [PS 38:4-H]− for DESI), while m/z 885 ([PI 38:4-H]−) was important in both TS and DESI. The prospect, remaining hurdles, and future work required for translating TS-MS into a method of intraoperative tissue diagnosis is discussed.

Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer

Cholesterol accumulates in prostate lesions and has been linked to prostate cancer (PCa) incidence and progression. However, how accumulated cholesterol contributes to PCa development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared to normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, PCa cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical co-regulators that influence AR activity