While mass spectrometry (MS) imaging is widely used to investigate the molecular composition of ex vivo slices of cancerous tumors, little is known about how variations in the cellular properties of cancer tissue can influence cancer biomarker ion images. To better understand the basis for variations in the abundances of cancer biomarker ions seen in MS images of relatively homogeneous ex vivo tumor samples, sections of snap frozen human breast cancer murine xenografts were subjected to desorption electrospray ionization mass spectrometry (DESI-MS) imaging. Serial sections were then stained with hematoxylin and eosin (H&E) and subjected to detailed morphometric cellular analysis, using a commercial digital pathology platform augmented with custom-tailored image analysis algorithms developed in-house. Gross morphological heterogeneities due to stroma, vasculature, and noncancer cells were mapped in the tumor and found to not correlate with the areas of suppressed cancer biomarker abundance. Instead, the ion abundances of major breast cancer biomarkers were found to correlate with the cytoplasmic area of cancer cells that comprised the tumor tissue. Therefore, detailed cellular analyses can be used to rationalize subtle heterogeneities in ion abundance in MS images, not explained by the presence of gross morphological heterogeneities such as stroma.88241209912107Spatio-Temporal Targeting and Amplification of Radiation Response (STTARR) program