Understanding the myeloid compartment within the tumour microenvironment

Abstract

Tumour-associated macrophages (TAMs) are one of the most abundant populations in the tumour microenvironment (TME) and can contribute to cancer progression. The behaviour of TAMs is shaped by environmental stimuli within tumours, including hypoxia (oxygen deprivation), to further promote therapy resistance and cancer relapse. Whilst there is an interest in targeting TAMs to improve anti-tumour efforts, only one drug has thus far been approved as TAMs represent a challenging heterogeneous population to target. Thus, further study is needed to identify markers for TAMs before developing therapies.

To address this, we first sought to identify possible target molecules of interest by performing RNA-sequencing on human monocytes (macrophage precursors) treated with dimethyloxalylglycine (DMOG), a hypoxia mimetic. To identify genes encoding cell surface molecules, we filtered the data using a published surfaceome dataset. We then evaluated the top 100 upregulated genes based on their known expression profile in various tissues; a subsequent literature review confirmed 9 genes of interest suitable for further investigation. These 9 targets were evaluated on human monocyte-derived macrophages (MDMs) incubated under physiological hypoxia (1% O2) for 48 hours, revealing 3 targets, CD87, TREM1, and TNFR2 whose expression was significantly increased at both the mRNA and protein level. These targets were subsequently evaluated and shown to be expressed in 5-45% of macrophages in primary human tumour samples from different cancer types.

To investigate the expression and possible regulation of these markers in more clinically relevant models, their expression was assessed on macrophages generated in the presence of cancer cell line conditioned media (CM). Notably, treating MDMs with CM from a colorectal cancer cell line, DLD-1, and a renal cancer cell line, RCC12, yielded opposing outcomes, inducing pro- and anti-inflammatory MDM markers, respectively. We then combined hypoxia with CM to better reflect the TME; treating MDMs with hypoxic CM at 1% O2 was shown to enhance the expression of our candidate markers compared to each stimulus alone. In addition, we found that DLD-1 CM significantly increased the phagocytic capacity of macrophages to engulf cancer cells, whilst RCC12 CM reduced it.

Next, we sought to identify potential mediators of these processes. It was found that extracellular vesicles (EVs), important mediators of intracellular crosstalk within the TME, isolated from DLD-1 CM were responsible for mimicking its inflammatory effects. Interrogating the profile of these EVs using RNA-sequencing several miRNAs were identified, including miR-429/141/200. These miRNAs are predicted to target the PTEN/PI3K/Akt pathway which is known to play a role in macrophage function, specifically phagocytosis. Thus, the role of EV-associated miRNAs in modulating TAM phenotype warrants further investigation.

Ultimately, expanding on these findings will yield an improved understanding of the tumour:TAM interface and regulators of the candidate markers, thereby providing us with clear directions towards developing improved treatment modalities in the future.

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Identifiers

ORCID for Rosa Gomes Alves Martins: orcid.org/0000-0003-2098-5268

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

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