The tumor microenvironment is heterogeneous, exhibiting severe structural and functional vascular abnormalities^{1, 2}. Specifically, tumor hypoxia has been associated with aggressive tumor progression as well as resistance to radiation and/or chemotherapy, resulting in poor clinical outcome and prognosis^1-5. Thus, visualization of the extent of hypoxia in tumors would provide critical information for strategic treatment planning. Conventionally, dynamic contrast enhanced (DCE)-MRI is used to investigate tumor perfusion by administration of Gd-DTPA with pharmacokinetic modeling. Recently, features of the tumor microenvironment were classified by pattern recognition approaches, such as Gaussian mixture model (GMM)⁶ or constrained non-negative matrix factorization (cNMF)⁷. These approaches minimize biases or errors from purely model-based fitting. However to-date, the choice of number of classifiers has been predefined manually. Here, we focus on developing and validating a robust algorithm for the automated determination of the number of classifiers, characterizing heterogeneity of tumor vascular features without subjective user intervention.Different approaches of identifying the number of classifiers automatically were tested on DCE-MRI data from 5 different preclinical tumor models – HEK, LAPC-4⁸, Myc-CaP⁹, PC-3¹⁰, and RM-1¹¹ flank tumors in Nod/SCID mice. Experimental parameters for the DCE-MRI data acquisition: T₁-weighted fast low-angle shot (FLASH) sequence with 3.2 ms echo time, minimum repetition time (TR_min), 256 repetitions (NR₂₅₆, time points), 1 average, 15 mm x 15 mm FOV, 128x128 matrix, 1 mm slice thickness, and 5-7 slices to cover the entire tumor. For 5, 6, and 7 slices respectively, the resulting TR_min were 42.875 ms, 51.450 ms, and 60.025 ms with a corresponding temporal resolution of 5.487 s, 6.585 s, and 7.683 s. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. As the principal component analysis (PCA, 1st analysis step) identifies the sources of largest variations, the number of classifiers (NC) was evaluated by three different criteria: signal enhancement, SNR, and half area under the curve (HAUC) of principal components (PCs). Signal enhancement was calculated as (max(PCs) – mean(PCs))/mean(PCs)×100 and a threshold for selecting significant PCs was set manually to 6000. SNR was calculated as max(PCs)/(2×std(PCs of pre contrast agent injection)) and a threshold set to 5 (SNR_Th5). Additionally, a threshold of 2 (SNR_Th2)was considered for tumors with low SNR. HAUC was calculated by adding the signal intensity during the initial half of total acquisition time (~10 mins) with a threshold set to 0.5×first HAUC. To assess the performance of the approaches to automatically determine NC, NC was also determined manually by an expert for each tumor slice. After selecting NC, cNMF was accordingly performed as described previously⁷. To generate pattern masks of tumor microenvironments, the representative curve showing the maximum value of normalized weights in a given pixel is allocated to each pattern mask. Also, pattern masks of pixels associated with a mixture of features are generated for pixels where the difference between maximum weight to other pattern weight is lower than 25%.Three criteria – signal enhancement, SNR, and HAUC - for determining NC automatically were applied to five different preclinical tumor models with a total of 104 slices (HEK n = 6, LAPC-4 n = 6, MycCaP n = 2, PC-3 n = 2, RM-1 n = 4). The NCs obtained from these three criteria were compared to the NC determined by manual expert inspection and the corresponding accuracy is shown in Fig. 1. The accuracy was 75.00%, 86.54%, 97.12%, and 49.04% for enhancement, SNR_Th5, SNR_Th2 (considering necrotic tumor), and HAUC, respectively. The cNMF-derived representative pattern curves and corresponding weight maps are shown in Fig. 2. Based on vascular features characterizing specific tumor microenvironments⁷, red, green, and blue plots represent well-perfused, hypoxic, and necrotic tumor areas. Two different types of pattern masks characterizing the tumor slice were generated: “Decision map 1” was generated by finding maximum weight and allocating index as 1, 2, and 3 for necrotic, hypoxic, and well-perfused, respectively; “Decision map 2” was generated by adding the additional condition for mixtures. Of the three approaches tested, the SNR approach demonstrated to be the most useful strategy to automatically determine NC and could be optimized to account for DCE-MRI data with low contrast agent enhancement.The visualization of tumor heterogeneity (perfusion, hypoxia, necrosis) with automated analysis of DCE-MRI can reduce the need for manual expert intervention, extensive pharmacokinetic modeling and could provide critical information for treatment planning.