(T1430-09-54) A Targeted LC-MS/MS Method for Quantitative Tissue Distribution Analysis (QTDA) of Metabolomics in Tumors using Laser Microdissection

Purpose: Tumor cells require a large quantity of various nutrients to support their high rate of cellular proliferation. An understanding of the different sources of energy and their utilization by cancer cells will provide an insight into cancer cell environment and growth. Additionally, an understanding of the differences within the tumor micro-environment will provide further understanding about tumor growth. To accomplish this goal, we have developed an approach to collect small samples from different regions of the tumor and quantitatively determine a number of metabolites. This approach allows us to construct an image of the tumor using absolute concentrations of numerous metabolites across different regions of the tumor.

Methods: Uniformly labeled Carbon-13 glucose (¹³C₆-glucose) was infused into mice bearing murine melanoma cell line mediated tumor. The tumor was surgically dissected, flash frozen, sectioned into ~ 60-micron thick sections, and fixed onto polyethylene terephthalate membrane slides. Laser microdissection (LMD) was used to sequentially cut approximately 447 square micron sections. This yields numerous, small (approximately 0.012 mm³ or 16,000 cells) pieces of tumor. It is notable that a typical small tumor size of 5 ´ 5 mm will yield approximately 125 individual samples. These small samples are subjected for extraction and the resultant solution analyzed using UHPLC-MS/MS targeted metabolomics method.

Results: A specific and sensitive targeted bioanalytical method was developed and validated to quantitate the carbon-12 and carbon-13 forms of glucose, lactate, pyruvate, 3-phosphoglycerate (3-PG), succinate, fumarate, citrate, 2 hydroxy glutarate and glutamic acid in order to reconstruct a detailed image of the tumor and the absolute concentration of target analytes. This method was validated over the calibration range of 0.5-500 ng/mL for both ¹²C and ¹³C forms of all the listed analytes. The lower limit of quantitation is 0.5 pg/mm³ for all the target analytes. The determined method accuracy, precision, and linearity were within the specifications of the FDA’s guidance for industry for bioanalytical method validation. This method yields an image of the tumor microenvironment with the absolute concentration of specific analytes across the tumor, as well as other target tissues.

Conclusion: A number of tumors and other tissues like spleen and liver were resected and analyzed. Analysis of within-tumor samples demonstrates that the distribution of the analytes is heterogenous in nature across the tumor environment; therefore, metabolism is not uniform across a single tumor. Moreover, analysis of samples from multiple tumors demonstrates that the glucose metabolism is even more heterogenous between different tumors. The distribution of the metabolites is relatively more uniform in other tissues such as the spleen and liver; therefore, metabolism is more non-homogenous in cancer tissues when compared to non-cancerous tissues.