• Immunotherapy

Netrin-1 is highly expressed in many cancers, such as endometrial cancer and lung cancer. Recently, Cassier et al from Université de Lyon1 and Lengrand and her colleagues from Université Libre de Bruxelles2 both reported that the therapeutic monoclonal netrin-1 antibody NP137 can inhibit the epithelial-mesenchymal transition (EMT) and cancer progression, and these results were published online in Nature side by side.

EMT is a cellular process in which cells lose their epithelial characteristics and acquire mesenchymal features. The EMT process is fundamental for driving plasticity during embryonic development and in adult tissue homeostasis but is an unintentional behavior of cells during cancer progression.3 4 In cancer, this process has been associated with tumorigenesis, including tumor initiation, tumor stemness, metastasis, and therapy resistance.4 Moreover, many works have revealed that the EMT is not a binary process, can be transient and reversible and represents a continuous flux between two endpoint states (epithelial and mesenchymal), which occur in a gradual manner with an intermediate state between the epithelial and mesenchymal states referred to as partial or hybrid EMT states.3

Netrin-1 was described in the early 1990s as a secreted chemotropic cue that guided neuron or axon direction. Netrin-1 interacts with its main receptors, deleted in colorectal carcinoma and UNC5-homologues, which belong to the dependence receptor family.5 These receptors are characterized by triggering apoptosis in the absence of ligands while activating signaling in the presence of ligands.6 Many aggressive tumors achieved their survival advantage by loss of netrin-1 receptor expression or upregulation of netrin-1 expression.5 Netrin-1 dependence receptors could serve as conditional tumor suppressors depending on the presence of netrin-1. Upregulated netrin-1 has been shown to inhibit apoptosis and promote angiogenesis in tumor development, which could be an appealing therapeutic strategy.5 7 Thus, a monoclonal antibody (mAb) that neutralizes netrin-1 (NP137) was developed, and preliminary safety and efficacy assessments were conducted in patients with advanced solid tumors in a phase 1 trial (NCT02977195). Several clinical trials of NP137 therapy for cancer treatment are currently underway, the content of clinical trials is summarized in table 1 (source from https://www.clinicaltrials.gov/).

Recently, two research teams reported the important roles of NP137 in EMT inhibition and in triggering tumor microenvironment changes, as described in figure 1.1 2 In the clinical trial of NP137 for endometrial carcinoma (EC), 14 patients with advanced EC were recruited for NP137 treatment, and the results showed an antitumor response in nine patients after injection of NP137, with disease stabilization in eight patients and liver metastases shrinking by more than 50% in one patient.1 More importantly, NP137 has been shown to be safe, with no significant adverse effects in patients with EC. In addition, in a preclinical mouse model, the therapeutic efficacy of NP137 was improved when combined with conventional chemotherapy drugs (carboplatin and paclitaxel), which suggests that NP137 can alleviate the resistance of tumor cells to chemotherapy.

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Figure 1

Pharmacological targeting of netrin-1 by NP137 inhibits the EMT and cancer progression. NP137 administration in patients with endometrial cancer or a primary mouse model decreased EMT scores and increased tumour cell apoptosis, as well as changing immune cell infiltration and increasing the strength of the interaction between antigen-presenting cells and tumor cells, which led to tumor inhibition. Moreover, NP137 can improve the tumor-killing effects of chemotherapy drugs such as carboplatin-paclitaxel. CAF, cancer-associated fibroblast; EMT, epithelial-mesenchymal transition; NK, natural killer; SCC, skin squamous cell carcinoma; Treg, regulatory T cell.

What is the potential mechanism by which NP137 inhibits tumor growth? In addition to the well-known effects of NP137 on tumor cell apoptosis,7 the authors also used RNA sequencing (RNA-seq) and found EMT feature inhibition by NP137 in a preclinical mouse model and in patients with EC. Although some studies have suggested that netrin-1 may be implicated in the EMT in vitro,8 9 strong supportive evidence about the effects of netrin-1 on the EMT in vivo is lacking. Cassier et al supplied direct clinical data for fully elucidating the effects of netrin-1 inhibition on tumor EMT features and cancer progression.

Meanwhile, Lengrand and her colleagues also demonstrated a vital link between netrin-1 and EMT states.2 They found differential expression of netrin-1 in cells with epithelial and mesenchymal characteristics, and the netrin-1 gene was overexpressed in EpCAM⁻ tumor cells compared with EpCAM⁺ tumor cells in a primary mouse model of skin squamous cell carcinoma (SCC) with spontaneous EMT. Furthermore, in this primary model of skin SCC, overexpression of netrin-1 promoted tumor initiation and increased the proportions of tumor cells with full EMT, while NP137-mediated netrin-1 inhibition reduced the proportion of tumor cells that underwent EMT, reduced the number of lung metastases, and increased the chemotherapy sensitivity of tumor cells. Single-cell RNA-seq (scRNA-seq) revealed different EMT states in control versus NP137-treated squamous cell carcinomas. The EMT states in the control group included epithelial cells, early and late hybrid EMTs, and complete EMT states. In contrast, NP137 treatment prevented the progression of tumor cells to an advanced EMT state and kept a persistent tumor epithelial state. Researchers also used NP137 to treat the human cancer cell lines A549 and Ishikawa, confirming that netrin-1 blockade suppressed the EMT process in these tumor xenografts. NP137 has been shown to specifically block the interaction of netrin-1 with its receptor UNC5B.7 Thus, the authors used short hairpin RNA (shRNA) to knockdown netrin-1 or its receptor UNC5B in EpCAM⁺ tumor cells and found that knockdown of netrin-1 or UNC5B inhibits the EMT in vitro and regulates a common genetic signature that promotes tumor epithelial status and limits the EMT in the absence of stromal cells. Bioinformatics analysis results showed that UNC5B expression has a good correlation with EMT scores in the Cancer Genome Atlas (TCGA) data sets (lung SCC, lung adenocarcinoma and skin cutaneous melanoma). Correspondingly, UNC5B overexpression could be a selective advantage for tumorigenesis and the EMT process. UNC5B may be a promising diagnostic and prognostic biomarker and a potential target in cancer treatment.

Of note, both studies performed scRNA-seq to comprehensively assess the effect of netrin-1 inhibition on the composition of the tumor stroma. The results showed the differential changes in diverse cell populations, including tumor cells, immune cells, cancer-associated fibroblasts (CAFs), and endothelial cells. NP137 treatment resulted in a statistically significant reduction in tumor cell compartments, especially tumor cell clusters with high EMT scores.1 Another study also demonstrated that NP137 administration was associated with an increase in the proportion of cells in the epithelial tumor state, a decreased proportion of late hybrid EMT cells and a strong decrease in late EMT cells compared with the control.2 In addition, lineage trajectory analysis revealed that distinct lineage trajectories could be identified in the control and NP137-treated tumors, including control tumors with a trajectory from epithelial cells towards hybrid EMT or a late full EMT state. Meanwhile, NP137-treated tumor cells had two new trajectories towards epithelial states (epithelial-B1 and epithelial-B2) and two hybrid EMT trajectories. Together, Cassier et al attributed the clinical treatment effects of NP137 to EMT inhibition, and Lengrand et al demonstrated the different EMT tumor states and differentiation trajectories by pharmacological inhibition of netrin-1.

However, there were significant differences in the proportion of CAFs that changed between the two studies. The scRNA-seq data from Cassier’s report showed a decrease in the proportion of CAFs, but data from Lengrand et al observed the opposite phenomenon, with results showing that the CAF proportion was relatively increased. Following NP137 treatment, the identification of CAF subtypes showed that the proportions of two inflammatory CAFs (iCAFs.1 and iCAFs.2) were decreased, while the proportions of matrix CAFs and vascular CAFs were increased.1 Another study used single-cell compositional data analysis to perform differential abundance analysis, and there were no significant differences in the proportions of the different CAF clusters.2 The reasons for the opposite changes in the proportions of CAF and its subgroups have not been explained in these two articles. The possible reasons are different tumor types (EC vs skin SCC), different sample sources (EC patients vs primary mouse model), etc. Future studies should investigate the association between netrin-1 and the dynamic change in CAF clusters.

Of interest, NP137 treatment clearly appeared to affect immune cell infiltration.1 More specifically, following NP137 treatment, the authors noted an increase in lymphocytes endowed with cytotoxic functions, such as CD8⁺ T cells, natural killer cells, and neutrophils. scRNA-seq data and spatial transcriptomic data showed an increase in the number and strength of interactions between CD8⁺ T cells and tumor cells in patients with EC following NP137 administration. Moreover, the authors also verified the decrease in M2 macrophage proportion, together with an increase in the antigen presentation molecules MHC I/II, which suggests that NP137 led to the formation of an antitumor immune landscape in the tumor microenvironment. Correlatively, netrin-1 has been reported to be involved in the infiltration of macrophages and neutrophils in inflammatory responses and has been shown to promote the immunosuppressive activity of myeloid-derived suppressive cells in colorectal cancer.10 11 Here, due to the complexity and heterogeneity of the tumor microenvironment, the authors have not yet confirmed the direct or indirect effect of NP137 on immune cell changes, and they consider that NP137 may have dual effects on the EMT and stromal cells. Thus, the safety and efficacy of NP137 in combination with carboplatin-paclitaxel and/or pembrolizumab (anti-PD-1 mAb) in patients with endometrial or cervical cancer are currently being tested in a phase 2 GYNET trial (table 1). Although clinical trials of NP137 are progressing rapidly, the effects of netrin-1 on immune cell infiltration and interaction are issues for future research to explore.

Taken together, these two timely and innovative studies identified a pharmacological strategy for targeting EMT progression in preclinical primary mouse models and human patients with cancer, demonstrating that a monoclonal antibody against netrin-1 (NP137) can inhibit the EMT, reduce tumor cell metastasis, enhance tumor cell sensitivity to chemotherapy, and eventually block the progression of cancers. These cutting-edge research projects have important implications for the development of anticancer therapy, as well as the novel biomarkers discovery for designing anti-netrin-1 therapy in responding patients.