Background: Soft tissue sarcomas (STS) are heterogeneous tumors which are morbid and lethal. G100 is under investigation in multiple clinical trials and contains a potent TLR4 agonist (oil-in-water emulsion of glucopyranosyl lipid A) that has been tested as vaccine adjuvant. We hypothesized IT G100 would induce robust local and potentially systemic anti-tumor immune response, leading to improved outcomes. Methods: 15 metastatic STS patients with superficial lesions were treated with weekly IT G100 for 8-12 wks; 12 patients received radiation (RT) for 2 wks to start, while 3 received IT G100 for 6 wks prior to RT. Biopsies and PBMC were collected pre and post treatment, and flow cytometry was performed on biopsies. TIL and PBMC were analyzed with TCR deep sequencing. PBMC were analyzed by single cell multiplex cytokine profiling. Results: No grade ≥ 3 toxicity was observed, and local tumor control was achieved in all evaluable tumors (14/14). Treated tumors tracked post-trial (mean 156 days) had persistent local control with 1 CR (7%), 1 PR (7%), and 11 SD (79%). In 3 patients with long term follow up, treated lesions remained controlled vs index lesions (-53% vs +31% at mean 235 days, p = 0.002). In all tumors after G100 alone, T cell infiltration increased. In P14, CD3 live cells in tumor rose from < 1% to 62%. PBMC clonality increased in 8/14 tested including P06, who had 4× increase in clonality and CR in the injected lesion; PBMC and TIL TCR overlap increased from 13.4% to 21.5%. P13 had a 2.3× rise in TIL clonality; the top clone (a CD4 T cell) expanded from 0.1% to 38% and expressed more TNFα than the rest (p < 0.0001). Single cell cytokine analysis of PBMC showed 7/13 (54%) increased in polyfunctionality (producing > 2 cytokines) in CD4 T cells; no consistent increase was seen in CD8 T cells. TNFα levels in pre-treatment monocytes correlated with PFS (R²= 0.5, p = 0.02). Conclusions: IT G100 is a viable agent for local control of metastatic STS lesions. With or without RT, G100 appears to cause CD4 T cell mediated local and systemic response. Combination of G100 with other immunomodulators could induce clinically significant systemic responses, as seen in follicular NHL treated with G100. Clinical trial information: NCT02180698