Retinoic Acid Induces an IFN-Driven Inflammatory Tumour Microenvironment, Sensitizing to Immune Checkpoint Therapy.

With immune checkpoint therapy (ICT) having reshaped the treatment of many cancers, the next frontier is to identify and develop novel combination therapies to improve efficacy. Previously, we and others identified beneficial immunological effects of the vitamin A derivative tretinoin on anti-tumour immunity. Although it is known that tretinoin preferentially depletes myeloid derived suppressor cells in blood, little is known about the effects of tretinoin on the tumour microenvironment, hampering the rational design of clinical trials using tretinoin in combination with ICT. Here, we aimed to identify how tretinoin changed the tumour microenvironment in mouse tumour models, using flow cytometry and RNAseq, and we sought to use that information to establish optimal dosing and scheduling of tretinoin in combination with several ICT antibodies in multiple cancer models. We found that tretinoin rapidly induced an interferon dominated inflammatory tumour microenvironment, characterised by increased CD8+ T cell infiltration. This phenotype completely overlapped with the phenotype that was induced by ICT itself, and we confirmed that the combination further amplified this inflammatory milieu. The addition of tretinoin significantly improved the efficacy of anti-CTLA4/anti-PD-L1 combination therapy, and staggered scheduling was more efficacious than concomitant scheduling, in a dose-dependent manner. The positive effects of tretinoin could be extended to ICT antibodies targeting OX40, GITR and CTLA4 monotherapy in multiple cancer models. These data show that tretinoin induces an interferon driven, CD8+ T cell tumour microenvironment that is responsive to ICT.

Temporally restricted activation of IFNß signaling underlies response to immune checkpoint therapy in mice.

The biological determinants of the response to immune checkpoint blockade (ICB) in cancer remain incompletely understood. Little is known about dynamic biological events that underpin therapeutic efficacy due to the inability to frequently sample tumours in patients. Here, we map the transcriptional profiles of 144 responding and non-responding tumours within two mouse models at four time points during ICB. We find that responding tumours display on/fast-off kinetics of type-I-interferon (IFN) signaling. Phenocopying of this kinetics using time-dependent sequential dosing of recombinant IFNs and neutralizing antibodies markedly improves ICB efficacy, but only when IFNß is targeted, not IFNα. We identify Ly6C+/CD11b+ inflammatory monocytes as the primary source of IFNß and find that active type-I-IFN signaling in tumour-infiltrating inflammatory monocytes is associated with T cell expansion in patients treated with ICB. Together, our results suggest that on/fast-off modulation of IFNß signaling is critical to the therapeutic response to ICB, which can be exploited to drive clinical outcomes towards response.

PPARα and PPARγ activation is associated with pleural mesothelioma invasion but therapeutic inhibition is ineffective.

Mesothelioma is a cancer that typically originates in the pleura of the lungs. It rapidly invades the surrounding tissues, causing pain and shortness of breath. We compared cell lines injected either subcutaneously or intrapleurally and found that only the latter resulted in invasive and rapid growth. Pleural tumors displayed a transcriptional signature consistent with increased activity of nuclear receptors PPARα and PPARγ and with an increased abundance of endogenous PPAR-activating ligands. We found that chemical probe GW6471 is a potent, dual PPARα/γ antagonist with anti-invasive and anti-proliferative activity in vitro. However, administration of GW6471 at doses that provided sustained plasma exposure levels sufficient for inhibition of PPARα/γ transcriptional activity did not result in significant anti-mesothelioma activity in mice. Lastly, we demonstrate that the in vitro anti-tumor effect of GW6471 is off-target. We conclude that dual PPARα/γ antagonism alone is not a viable treatment modality for mesothelioma.

Bilateral murine tumor models for characterizing the response to immune checkpoint blockade.

The therapeutic response to immune checkpoint blockade (ICB) is highly variable, not only between different cancers but also between patients with the same cancer type. The biological mechanisms underlying these differences in response are incompletely understood. Identifying correlates in patient tumor samples is challenging because of genetic and environmental variability. Murine studies usually compare different tumor models or treatments, introducing potential confounding variables. This protocol describes bilateral murine tumor models, derived from syngeneic cancer cell lines, that display a symmetrical yet dichotomous response to ICB. These models enable detailed analysis of whole tumors in a highly homogeneous background, combined with knowledge of the therapeutic outcome within a few weeks, and could potentially be used for mechanistic studies using other (immuno-)therapies. We discuss key considerations and describe how to use two cell lines as fully optimized models. We discuss experimental details, including proper inoculation technique to achieve symmetry and one-sided surgical tumor removal, which takes only 5 min per mouse. Furthermore, we outline the preparation of bulk tissue or single-cell suspensions for downstream analyses such as bulk RNA-seq, immunohistochemistry, single-cell RNA-seq and flow cytometry.

Sensitization to immune checkpoint blockade through activation of a STAT1/NK axis in the tumor microenvironment.

Cancer immunotherapy using antibodies that target immune checkpoints has delivered outstanding results. However, responses only occur in a subset of patients, and it is not fully understood what biological processes determine an effective outcome. This lack of understanding hinders the development of rational combination treatments. We set out to define the pretreatment microenvironment associated with an effective outcome by using the fact that inbred mouse strains bearing monoclonal cancer cell line-derived tumors respond in a dichotomous manner to immune checkpoint blockade (ICB). We compared the cellular composition and gene expression profiles of responsive and nonresponsive tumors from mice before ICB and validated the findings in cohorts of patients with cancer treated with ICB antibodies. We found that responsive tumors were characterized by an inflammatory gene expression signature consistent with up-regulation of signal transducer and activator of transcription 1 (STAT1) and Toll-like receptor 3 (TLR3) signaling and down-regulation of interleukin-10 (IL-10) signaling. In addition, responsive tumors had more infiltrating-activated natural killer (NK) cells, which were necessary for response. Pretreatment of mice with large established tumors using the STAT1-activating cytokine interferon-γ (IFNγ), the TLR3 ligand poly(I:C), and an anti-IL-10 antibody sensitized tumors to ICB by attracting IFNγ-producing NK cells into the tumor, resulting in increased cure rates. Our results identify a pretreatment tumor microenvironment that predicts response to ICB, which can be therapeutically attained. These data suggest a biomarker-driven approach to patient management to establish whether a patient would benefit from treatment with sensitizing therapeutics before ICB.