Aim: Tumor cells can prime distant microenvironment building a pre-metastatic niche (PMN) where circulating tumor cells (CTCs) will home. 0.01% of CTCs, endowed with the capability to extravasate and infiltrate distant sites, will develop distant metastasis. In this process, a crucial role is played by chemokine gradients. Highly tumorigenic CTCs express the chemokine receptor CXCR4. CXCR4 ligand, CXCL12, attracts bone marrow-derived cells and CXCR4+ cancer cells in pre-metastatic sites to form a PMN. A CXCL12-loaded-hydrogel (CLG) was developed with the commercially available Belotero Hyaluronic gel dermal filler to develop a “pseudo niche” attracting immune and CTCs-CXCR4+cells. Aims: 1. Biological characterization of CTCs, 2. CTC/tumor microenvironment interplay 3. feasibility for clinical CTC isolation and characterization. Materials and methods: Human renal cancer cells SN12C (100 and 500) and A498 (500 and 1000) were seeded on 150ul Empty Gels (EG) or CLG [300ng/ml CXCL12], and allowed to migrate within gels, then fixed and DAPI-stained for enumeration or recovered by gel digestion and grown in complete media for 6 days. CXCR4 expression and colonies formation capability were evaluated. 100 SN12C or HT29, human colon cancer cells, were spiked in 7cc HD blood, ficoll-paqued and the mixture seeded on CLG. Isolated cells were fixed and stained with anti-hCD45-FITC, panCK-594 and DAPI (cancer cells identified as DAPI+/pan-CK+/CD45-). 7cc blood derived from 15 metastatic cancer patient’s (colon, lung, ovary, endometrial, kidney and glioblastoma) will be processed as previously described (CTCs identified as DAPI+/pan-CK+/CD45-); as comparison, CTCs will be isolated and enumerated using the commercially available Screen Cell™ filters according to manufacturer’s instructions. Results: A498 and SN12C cells efficiently infiltrate CLG as compared to empty gel (EG) (CLG/EG fold increase 1.6-1.9). SN12C and HT29 EG/CLG-recovered cells developed colonies after 6 days of culture. HT29-CLG recovered cells overexpressed CXCR4 compared to plastic grown cells and to EG cells and originates a higher number of colonies compared to plastic grown cells (171±21 for CLG VS 131±8 for plastic grown). SN12C and HT29 cells were successfully recovered in 7cc HD blood (recovery rate 20 and 14% respectively). Clinical trial on feasibility in isolating CTC cells on CLG recently started patient’s recruitment in ovarian, colon, lung and renal cancer metastatic patients. Conclusion: CLG-device mimics a PMN for capturing CTCs able to extravasate and infiltrate distant tissue, and potentially, more metastatic. CLG-isolated cells highly express CXCR4, develop higher number of colonies and migrate toward CXCL12. Moreover, the device allows cancer cell recovery from HD blood being potentially clinically useful for CTCs identification, characterization and trapping in cancer patients. Citation Format: Luigi Portella, Caterina Ieranò, Giulia Bertolini, Crescenzo D'Alterio, Giuseppina Rea, Sara Santagata, Anna Maria Trotta, Gelsomina Monaco, Roberto Pacelli, Stefania Scala. CXCL12 embedded-Hyaluronate based 'pseudo niche': a new device for CTCs/DTCs capturing and characterization [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO036.