In Vivo Syngeneic Tumor Models with Acquired Resistance to Anti–PD-1/PD-L1 Therapies

Abstract

Antibodies targeting PD-1 and PD-L1 have produced durable responses in a subset of patients with cancer. However, a majority of these patients will ultimately relapse due to acquired resistance. To explore the underlying mechanisms of this secondary resistance, we developed five syngeneic murine tumor variants with acquired resistance to anti–PD-1 and/or PD-L1 antibodies in vivo. Resistant in vivo models were obtained by serial treatment/reimplantation cycles of the MC38 colorectal, MB49 and MBT2 bladder, and RENCA kidney and TyrNras melanoma models. Tumor immune infiltrates were characterized for wild type and resistant tumors using spectral cytometry and their molecular alterations analyzed using RNA sequencing analyses. Alterations in the tumor immune microenvironment were strongly heterogeneous among resistant models, involving select lymphoid and/or myeloid subpopulations. Molecular alterations in resistant models included previously identified pathways as well as novel candidate genes found to be deregulated in several resistant models. Among these, Serpinf1, coding for pigment epithelial-derived factor (PEDF) was further explored in the MC38 and the MBT2 models. Overexpression of Serpinf1 induced resistance to anti–PD-1 antibodies in the MC38 model, whereas knockdown of Serpinf1 sensitized this model as well as the primarily resistant MBT2 model. Serpinf1 overexpression was associated with increased production of free fatty acids and reduced activation of CD8+ cells, while orlistat, a compound that reduces the production of free fatty acids, reversed resistance to anti–PD-1 therapy. Our results suggest that a panel of syngeneic resistant models constitutes a useful tool to model the heterogeneity of resistance mechanisms encountered in the clinic.

Impact of mouse model tumor implantation site on acquired resistance to anti-PD-1 immune checkpoint therapy

“…However, PMN-MDSC become predominant in the SC resistant model compared to the sensitive model. This is in keeping with our previous observation that the combination anti-PD1 therapy with an anti-Ly6G antibody reverses resistance to anti-PD-1 ( 15 ). However, for the orthotopic model, the proportion of PMN-MDSC diminished after treatment in both site of implantation but the proportion remains higher in the resistant model compared to the sensitive model.…”

“…To understand whether tumor implantation had an impact on the TIME, we performed the immunophenotyping of MC38 sensitive tumor models at basal state and after treatment for both sensitive models implanted subcutaneously or orthotopically. For the SC model, we observed the same results reported in the literature, in particular at basal state in the MC38 sensitive model implanted subcutaneously, a large proportion of CD11b+ cells were detected and an equivalent proportion of lymphoid B and T cells and NK cells were identified ( Figure 3A ) ( 15 , 21 ). In the MC38 sensitive model implanted orthotopically we observed an extensive proportion of B cells and polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSC) ( Figure 3B ).…”

“…Firstly, at the basal state, as for the sensitive model, the TIME is extremely different depending on the implantation site (Figures 4A, B). In the SC MC38 resistant model, as previously described, we found a higher proportion of TAM M2-like cells compared to the sensitive model (15) but also compared to the orthotopic resistant model. In the orthotopic model, at the basal state, we…”

“…Cells were stained with the fluorescently labelled antibodies in the dark for 30 min at 4°C After surface staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD, 554714), then labeled with F4/80, FoxP3, Granzyme B, CD206 and T-bet in the dark for 30 min at 4°C ( Table 1 ). FlowJoV10 software (BD) was used for analyses as described previously ( 15 ) and GraphPad Prism software was used for statistical analysis (ANOVA with Bonferroni post-test). Experiments were performed twice for SC models, and once for orthotopic models.…”

“…However, there are very few models of secondary resistance to these compounds ( 14 ). To address this issue we have developed syngeneic models of resistance to ICI and found that the development of the resistant phenotype is associated with strong molecular and immunological heterogeneity ( 15 ). Another methodological difficulty is the fact that most tumor implantations in mice are performed subcutaneously rather than orthotopically.…”