Case Study 6: Reprogramming neutrophils to suppress metastasis and enable immunotherapy

Summary

• In vivo dissection of the roles played by neutrophils in metastatic niche priming and outgrowth

• Understanding the complex biology of neutrophil plasticity during primary tumour growth and metastasis

• Reprogramming neutrophil biology to facilitate immunotherapy

 Aggressive pancreatic and colorectal cancers are associated with increased neutrophils in the blood and at sites of metastasis. Tumours express growth factors and chemokines that drive neutrophil production and release from the bone marrow (G-CSF) (1) and attract neutrophils to metastatic sites (CXCL1, 2, 5 and TGFβ) (2). Tumour associated neutrophils are very heterogenous, often immature and can promote metastasis (3). Inhibition of CXCR2 through the small molecule AZD5069 can suppress metastasis in the liver and lungs through release of neutrophil mediated T-cell inhibition (4).

Work conducted at the CRUK Beatson Institute has been instrumental in establishing a role for neutrophils in the metastasis of pancreatic, colorectal and breast cancers. Mechanistically this is in part through tumour/metastasis associated neutrophils suppressing T-cells. Using a suite of mouse models we have shown that inhibition of CXCR2 (a key receptor on neutrophils), can promote the efficacy of checkpoint inhibition in cancers that are non or poorly responsive to immunotherapy (Pancreas, CRC and fatty liver associated HCC). Rather than ablating tumour associated neutrophils CXCR2 inhibition reprogrammes them so that they now oppose metastasis and are unable to suppress checkpoint inhibition. These findings have led to the establishment of two clinical trials to evaluate the effectiveness of CXCR2 inhibition in combination with immune checkpoint inhibition.

Neutrophils are the most abundant leukocyte in the peripheral blood of humans and are essential to anti-microbial defence. Recently, neutrophils have been associated with cancer development and spread. In the clinic, high neutrophil: lymphocyte ratios are a signature of poor prognosis in many cancers. Work led by groups at the Beatson has been critical to our fundamental understanding of how neutrophils contribute to cancer development and spread.  Given their rapid turnover, neutrophils had been somewhat overlooked and were thought to be relatively homogenous and inflexible. However, it is now appreciated that neutrophils, like other myeloid cells, are exquisitely sensitive to environmental signals. The work from the Institute has given key insights in the tumour promoting functions of neutrophils, the distinct phenotypes of neutrophils (and heterogeneity) in cancer and identified cancers of unmet need where co-targeting neutrophils with immunotherapy may be efficacious.

The pro-metastatic role of neutrophils

Neutrophils drive pancreatic cancer metastasis - The Sansom and Morton labs established that CXCR2, a chemokine receptor controlling neutrophil behaviour, drives tumour progression and metastasis in a KRAS and mutant p53 driven model (KPC) of pancreatic ductal adenocarcinoma (PDAC). This indicated that neutrophils accumulating at the site of metastasis inhibited anti-metastatic T-cell activity. Pharmacological blockade of CXCR2, with a drug called AZD5069, markedly decreased metastasis of PDAC to the liver and lungs.  Conditional knockout of the CXCR2 gene in the immune system (but not in the tumour) or whole-body depletion of neutrophils also opposed metastasis, confirming that CXCR2-mediated activation of neutrophils is a critical event in metastasis of PDAC. Importantly, this study then went on to demonstrate that combination of AZD5069 and an anti-PD1 antibody - a now standard immunotherapy - significantly opposed metastasis and extended survival (1).

Neutrophils drive colorectal cancer (CRC) metastasis – Poorly prognostic CRC (so called CMS4 subype) is associated with high neutrophil, myeloid infiltrate and Notch pathway activation. The Sansom lab developed a spontaneously metastatic model of CRC, which is driven by simultaneous activation of KRAS and NOTCH1 in combination with deletion of p53 in the gut epithelium (the KPN model). A careful mechanistic dissection showed that epithelial NOTCH1 drives metastasis by mobilising neutrophils via TGFβ and, crucially, this could be opposed by targeting neutrophils with AZD5069 (2) or by inhibiting TGFβ signalling. Importantly, patients that had high levels of neutrophils in their resected metastases had much poorer outcomes.

Further mechanistic insights into the crosstalk between tumour mutations,neutrophils and metastasis were revealed by Seth Coffelt and his collaborators.They demonstrated that loss of p53 drives secretion of WNT ligands and neutrophil-mediated inflammation that potentiates metastasis in breast cancer (3).

Understanding neutrophil plasticity

In a multi-lab effort, led by the Carlin group (with the Sansom, Morton, Norman, Murphy and Zanivan labs), we have extensively characterised mouse neutrophils that express intermediate and high levels of surface Ly6G – a neutrophil marker - as ‘immature’ and ‘mature’ populations, respectively across our suite of GEMM models. Using flow cytometry, single cell RNA sequencing, proteomic, and morphological analysis we have validated Ly6G surface expression as the only robust and consistent method for identification of neutrophil maturation in homeostasis, acute haematopoietic stress, and cancer. From this we have found that the site of neutrophil production (bone marrow or the spleen) influences neutrophil maturation through niche-specific cues. We identified that immature splenic-derived neutrophils undergo a more rapid maturation with altered transcriptomic, phenotypic, and functional capacity in the steady state and in cancer models compared to their bone marrow-derived counterparts and, importantly, that they associate with metastasis (Mackey J et al. https://www.biorxiv.org/content/10.1101/2021.07.21.453108v2).

Neutrophils oppose immunotherapy

Hepatocellular carcinoma (HCC) is strongly associated with non-alcoholic steatohepatitis (NASH), and NASH-HCC patients have a marked tumour-associated neutrophil signature. However, NASH-HCC is frustratingly resistant to standard immune therapies, such as immune checkpoint inhibition by anti-PDL1 antibodies. To address this, a multicentre team involving the Carlin, Bird and Sansom labs collaborated with Derek Mann’s group in Newcastle to establish state-of-the-art mouse models that recapitulate NASH-HCC and revealed the role of neutrophils in this disease. Recapitulating patients, these mouse models do not respond to immune checkpoint inhibition with anti-PD1 therapies. Excitingly though, and mirroring the findings above in metastasis, the CXCR2 inhibitor (AZD5069) could reprogramme neutrophils in NASH-HCC to enable therapeutic responses in mice where anti-PD1 alone was ineffective (4). The possibility of combining neutrophil reprogramming (AZD5069) with immune checkpoint blockade (Durvalumab) in advanced HCC is now being assessed in the Glasgow led phase I/II CUBIC clinical trial.

1. Steele CW et al. CXCR2 Inhibition profoundly suppresses metastases and augments immunotherapy in pancreatic ductal adenocarcinoma. Cancer Cell. 2016; 29: 832-45
2. Jackstadt R et al. Epithelial NOTCH signaling rewires the tumor microenvironment of colorectal cancer to drive poor-prognosis subtypes and metastasis. Cancer Cell. 2019; 36: 319-36 e317
3. Wellenstein MD et al. Loss of p53 triggers WNT-dependent systemic inflammation to drive breast cancer metastasis. Nature. 2019; 572: 538-42
4. Leslie J et al. CXCR2 inhibition enables NASH-HCC immunotherapy. Gut. 2022; 71: 2093-106