Background: Hepatocellular carcinoma (HCC) and colorectal cancer liver metastasis (CRLM) represent the two most occurring liver cancer. Aim of the project was to analyze the impact of TME on HCC versus CRLM. In particular, Tregs were evaluated in tumor (TT), peritumoral (PT) tissue and peripheral blood (PB) of liver tumor to identify immune mechanisms and therapeutic targets.

Methods: Tregs were characterized on PB/PT/TT cell suspension from 40 HCC and 34 CRLM by flow cytometry (FOXP3, CD25, CD4, CD45RA, CTLA-4, CXCR4, PD-1, ICOS and ENTPD1). PB-, PT- and TT-Tregs were isolated by Dynabeads Regulatory T-cell kit and tested for the capability to inhibit CFSE-labeled autologous T effector (Teff) proliferation. Tregs were also treated with Pep R29, a new CXCR4 antagonists, before coculturing with Teffs. Quantitative real-time PCR was performed to assess the expression of cyto-chemokines in PT and TT samples (IL-15, Arginase, CCL5, CXCL12).

Results: In HCC/CRLM-PB higher number of CXCR4 (p<0,001/p<0,05), CTLA-4 (p<0,001/p<0,05), PD-1 (p<0,001/p<0,05), ICOS (p<0,05/0,06% vs 0,03%) and ENTPD-1 (p<0,001/p<0,05) expressing Tregs was detected as compared to healthy donors (HD). In HCC/CRLM-TT higher number of FOXP3hiCD45RA-Activated Tregs subpopulation (p<0,01/p<0,05) and higher CXCR4-positive Tregs (p<0,05) were observed as compared to PT. Moreover, Tregs from HCC showed higher expression of ICOS (p<0,05) and ENTPD1 (p<0,05) as compared to CRLM. In terms of function, TT-Tregs exert a stronger immunosuppressive effect on Teffs proliferation than PT-Tregs (p<0,05) as shown by IL-35 cytokine secretion (150 vs 15 pg/mL/p<0,05). Interestingly, HCC-Tregs more powerfully inhibit Teffs proliferation at the tumor site and also in periphery. Expression of TME cyto-chemokines responsible of immunity promotion (IL-15) or inhibition (CXCL12, CCL5, Arginase) and tumor features, such as epithelial to mesenchymal transition (EMT) are under evaluation. Preliminary data showed higher CCL5, Tregs recruiting, in HCC as compared to CRLM suggesting a more suppressive TME in HCC. Based on high % of CXCR4-positive Tregs in HCC and CRLM (PB-HCC/CRLM: p<0,001 and TT-HCC/CRLM: p<0,01), ex vivo PB-Tregs were treated with Pep R29. CXCR4 targeting re-induced Teffs proliferation (PB-HCC -/+ Pep R29:12% vs 32%, p<0,001; PB-CRLM-/+ Pep R29:18,3% vs 49%, p<0,01) and decreased Tregs-derived IL-35 secretion (PB-HCC -/+ Pep R29: 106vs 16,5pg/mL, p<0,05; -/+ Pep R29:70 vs 0,6pg/mL, p<0,01).

Conclusions: These data suggest a more suppressive TME in HCC compared to CRLM and highlighted CXCR4 antagonism as a potential strategy to overcome Tregs-mediated TME immunosuppressive function in HCC and CRLM patients.

Citation Format: Daniela Castaldo, Sara Santagata, Giuseppina Rea, Maria Napolitano, Crescenzo D'Alterio, Rita Guarino, Vittorio Albino, Andrea Belli, Carmen Cutolo, Francesco Izzo, Stefania Scala. Tumor microenvironment (TME) is more suppressive in hepatocellular carcinoma (HCC) than in colorectal cancer liver metastasis (CRLM): CXCR4 antagonism as strategy to revert T regulatory cells (Tregs) suppressive activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2550.