Background: Colorectal cancer (CRC) is one of the deadliest malignancies worldwide. Though immune checkpoint inhibition has proven effective for a number of other tumors, it offers benefits in only a small group of CRC. In general, heterogenous cell groups in the tumor microenvironment (TME) are considered as the major barrier for unveiling the causes of low immune response. Therefore, deconvolution of cellular components in highly heterogeneous microenvironments is crucial for understanding those mechanisms. Single cell sequencing technology revolutionized TME research enabling profiling cells in high resolution. Methods: We have analyzed scRNA-seq data from 23 CRC patients with pre-treatment primary tumors using Seurat V3 pipeline. To investigate intercellular ligand-receptor interactions, we used CellPhoneDB and CellChat methods. The results of two independent analyses showed 4 CRC samples with no SPP1-CD44 interaction. It is known, that OPN which is the protein encoded by SPP1 gene, binds to CD44 and can cause cell survival, proliferation, and angiogenesis. Interestingly, analysis of the cellular composition of all 23 samples did not reveal differences in SPP1+ macrophages’ content for those 4 “no SPP1-CD44” samples. To investigate the mechanisms that could cause differences in SPP1-CD44 expression across the samples, we analyzed developmental trajectories of single cells using Slingshot trajectory inference method. Results: Ligand-receptor interactions analysis revealed 4 CRC samples that lacked SPP1-CD44 interaction that is known to be responsible for tumor progression in CRC. But the proportion of SPP1+ cells was not significantly different in those 4 samples compared to other samples. Trajectory inference analysis showed that the cells from “no SPP1-CD44” samples had high expression of anti-inflammatory macrophage markers in the end of the trajectory. While cells from “high SPP1-CD44” samples had high expression of pro-inflammatory macrophage markers at the same point. Conclusions: Based on our data-driven study, we suggest that SPP1+ macrophages’ heterogeneity may affect SPP1-CD44 interaction. Thus, targeting SPP1+ macrophages that have anti-inflammatory phenotype can potentially interrupt SPP1-CD44 interaction and therefore reduce tumor progression and immune suppression.