Lysosomal-associated membrane protein 1 (LAMP1), typically localized to the lysosomal membrane, is increasingly implicated as a marker of cancer aggressiveness and metastasis when expressed on the cell surface. This study aimed to develop a LAMP1-targeted antibody-based PET tracer and assess its efficacy in mouse models of human breast and colon adenocarcinoma. : To determine the source of LAMP1 expression, we utilized human single-cell RNA sequencing and spatial transcriptomics, complemented by in-house flow cytometry on xenografted mouse models. Tissue microarrays of multiple epithelial cancers and normal tissue were stained for LAMP-1, and staining was quantified. An anti-LAMP1 monoclonal antibody was conjugated with desferrioxamine (DFO) and labeled with zirconium-89 (Zr). Human triple-negative breast cancer (MDA-MB-231) and colon cancer (Caco-2) cell lines were implanted in nude mice. PET/CT imaging was conducted at 24, 72, and 168 h post-intravenous injection of Zr-DFO-anti-LAMP1 and Zr-DFO-IgG (negative control), followed by organ-specific biodistribution analyses at the final imaging time point. : Integrated single-cell and spatial RNA sequencing demonstrated that LAMP1 expression was localized to myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs) in addition to the cancer cells. Tissue microarray showed significantly higher staining for LAMP-1 in tumor tissue compared to normal tissue (3986 ± 2635 vs. 1299 ± 1291, < 0.001). Additionally, xenograft models showed a significantly higher contribution of cancer cells than the immune cells to cell surface LAMP1 expression. In vivo, PET imaging with Zr-DFO-anti-LAMP1 PET/CT revealed detectable tumor uptake as early as 24 h post-injection. The Zr-DFO-anti-LAMP1 tracer demonstrated significantly higher uptake than the control Zr-DFO-IgG in both models across all time points (MDA-MB-231 SUV at 168 h: 12.9 ± 5.7 vs. 4.4 ± 2.4, = 0.003; Caco-2 SUV at 168 h: 8.53 ± 3.03 vs. 3.38 ± 1.25, < 0.01). : Imaging of cell surface LAMP-1 in breast and colon adenocarcinoma is feasible by immuno-PET. LAMP-1 imaging can be expanded to adenocarcinomas of other origins, such as prostate and pancreas.