Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry

Abstract

Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), novel highly multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimised and validated image segmentation strategies and automated the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. Incorporating user-specific plugins, imcyto can be flexibly tailored to a wide range of segmentation needs. With these methods we interrogated the dramatic remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, showcasing their potential as key discovery tools to enhance understanding of the interplay between tumour, stroma, and immune cells in the spatial context of the tissue.

Therapeutic KRASG12C inhibition drives effective interferon-mediated anti-tumour immunity in immunogenic lung cancers

“…However, similarly to other targeted therapies, early clinical results indicate that drug resistance frequently arises, resulting in clinical relapses (11,12). KRAS G12C inhibitors not only affect the survival of cancer cells but can also mediate immunomodulatory effects by reversing KRAS-driven immunosuppressive mechanisms and generate a TME that is more favorable for an anti-tumour immune response (8,19,21). This knowledge has served as a rationale to investigate clinical combinations of KRAS G12C inhibitors with anti-PD1 or PD-L1 antibodies (29).…”

“…KRAS inhibition in consequence leads to an increased sensitivity of tumour cells to type I and II IFNs, which translates to higher expression of IFN-induced genes such as T cell chemoattractants and antigen presentation genes that could positively affect anti-tumour immunity in vivo. Using Imaging Mass Cytometry (IMC) we recently showed that there was an inclusion of macrophages and neutrophils in the core of 3LL DNRAS lung tumours, while effector cells remained at the tumour periphery (21). Consistent with this apparent immunosuppressive TME, growth of these tumours was not affected by a lack of B and T cells in Rag1 showed that migration was significantly abrogated when bone marrow-derived monocytes were cultured in conditioned medium from MRTX-treated cells and to a similar extent when cultured in medium from Ccl2 -/cells (Fig 4H).…”

“…Tissue processing and antibody staining was performed as described in detail in (21). In short, 5µm cryosections of fresh frozen lungs were fixed (Image-iT™ Fixative Solution, ThermoFisher) and stained with the antibody panel listed in Table 3 and Cell-ID Intercalator-Ir (Fluidigm).…”

“…Fedele, et al showed that a combination of SHP2 and KRAS G12C inhibition led to good tumour control and increased T cell infiltration in an orthotopic model of lung cancer (20). Using the strongly immune evasive 3LL DNRAS lung cancer cell line (14), we recently developed an imaging mass cytometry analysis pipeline which showed that KRAS G12C inhibition was able to induce remodeling of the lung TME (21). Here, we use this 3LL DNRAS alongside other pre-clinical lung cancer models varying in degree of immunogenicity to perform an in-depth investigation of the impact of KRAS G12C inhibition on the TME and anti-tumour immunity and explore the mechanisms that underly the changes observed.…”

Drugging the Undruggable: Advances on RAS Targeting in Cancer

“…Further evidence for the potential of KRAS-G12C inhibition to reduce the immunosuppressive tumour microenvironment produced by oncogenic KRAS and potentially sensitise tumours to immune checkpoint blockade came from another study using the 3LL ∆NRAS murine lung carcinoma line [118]. Using imaging mass cytometry, significant changes in tumour immune contexture induced by KRAS inhibition were clearly evident.…”