Tractable mouse TNBC models capture the heterogeneous tumor immune microenvironment and adaptation to PD-L1 blockade

Triple-negative breast cancer (TNBC) patients exhibit variable responses to programmed death (PD)-ligand (L)1 blockade, largely determined by the 'hot' versus 'cold' state of the tumor immune microenvironment (TIME). We here characterized 9 mouse TNBC models, relying on intraductal mammary gland inoculation of established mouse TNBC cell lines, with a heterogeneous TIME to study anti-PD-L1 resistance mechanisms. Complementary in vitro and in vivo screening classified the 4T1-hot-based model, a highly inflamed control through its immunogenic luciferase tag expression compared to the untagged 4T1-cold-based model, as displaying the 'hottest' TIME. However, both 4T1-based counterparts did not respond to anti-PD-L1, which was attributed to their immunosuppressive myeloid cell content as well as upregulation of cancer-associated fibroblasts in the 4T1-hot and high PD-L1-expressing CXCL10+ tumor-associated macrophages in 4T1-cold primary tumors. These anti-PD-L1 adaptation mechanisms across TIME states as captured by mouse TNBC models highlight specific cellular targets for future studies. Overall design: RNA was isolated from 4T1-hot and 4T1-cold primary tumors at 3 w p.i. using Rneasy Mini Kit (QiAgen, Valencia, CA, USA). All analyses were done on 4 samples or 3 samples per treatment group (IgG control- or anti-PD-L1-treated) for 4T1-cold and 4T1-hot TNBC models respectively. Bulk RNA sequencing was done in collaboration with Azenta Life Sciences and performed on Illumina Novaseq system (paired end, length of 150 bp), aiming for 10 million reads per sample.