Lung cancer is the malignancy with the highest morbidity and mortality rates worldwide . Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases, with a 5-year overall survival rate of approximately 15%. The majority of patients diagnosed with NSCLC have advanced-stage disease and are unsuitable for curative surgery . The development of immune checkpoint inhibitors (ICIs) has ushered in a new era in the treatment of NSCLC, following the era of chemotherapy and targeted therapy . ICIs are monoclonal antibodies targeting immune checkpoints, such as cytotoxic T lymphocyte-associated protein 4, programmed death 1 (PD-1), and programmed death ligand 1 (PD-L1) . ICIs are designed to optimise the host’s immune response against tumour cells by increasing T cell cytotoxicity and suppressing tumour growth. The clinical application of a variety of ICIs has led to dramatic changes in the treatment strategy for patients with NSCLC . However, the fact that only a small subset of patients with specific tumour types can benefit from ICIs limits their application. It has been reported that only tumours with pre-existing immunity (i.e., many tumour-infiltrating lymphocytes, dense CD8 + T cells, and PD-L1 expression) respond well to ICIs. Other phenotypes, such as immune excluded tumours (immune cells only present at the periphery) and ‘cold’ tumours (little or no immune cell infiltration), respond poorly to single-dose ICIs . However, the infiltration of most NSCLCs is characterised by immune exclusion . Therefore, a suitable combination therapy is needed for increase the infiltration of tumour antigen-specific T cells in malignant tumour tissues, and then combined with ICIs to reverse tumour-induced immunosuppression and destroy tumour cells .
Angiogenesis contributes to tumorigenesis, tumour progression, and metastasis in numerous human malignancies . In tumour tissues, a variety of transcription factors called hypoxia-inducible factors regulate the expression of angiogenic factors, including vascular epithelial growth factor (VEGF) and platelet-derived growth factor . VEGF is the main regulator of angiogenesis. It stimulates the proliferation, migration, and neovascularisation of vascular epithelial cells by binding to VEGF receptors (VEGFRs) . However, although the tumour has a rich blood supply, abnormal neovascularisation (stiffness, distortion, dilatation, and structural abnormalities) and low pericyte coverage leads to insufficient blood perfusion and increased vascular permeability. This reduces the supply of oxygen and nutrients, resulting in a microenvironment with high osmotic, hypoxic, acidic, and interstitial pressure . The VEGF pathway not only regulates tumour vascularisation but also contributes to an inhibitory immune microenvironment, thereby enabling tumour cells to evade host immune surveillance . Abnormal VEGF expression prevents the trafficking of tumour-reactive T cells to the tumour by inhibiting the expression of adhesion molecules in endothelial cells, specifically intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) .
Anti-angiogenic drugs are widely used clinically. The most targeted molecules in anti-angiogenic therapy fall within two broad categories: VEGF (e.g., bevacizumab targeting VEGFA) and VEGFRs (e.g., cediranib targeting VEGFRs). Bevacizumab, a monoclonal antibody against VEGF, was first approved by the United States Food and Drug Administration for the treatment of metastatic colorectal cancer in 2004. It has shown survival benefits in various solid tumour types, including NSCLC. Bevacizumab not only suppresses tumour growth by reducing neovascularisation and microvessel density (as demonstrated by reduced staining of vascular endothelial cell marker CD31 in tumours) but also activates ICAM-1 and VCAM-1 expression in endothelial cells, thereby recruiting immune cells to the tumour microenvironment . Therefore, we postulate that, in addition to the function of anti-angiogenic agents, the immunomodulatory properties of bevacizumab may also play a role in its clinical activity.
In view of the regulatory effect of the vasculature on the tumour microenvironment, research on the combination of the two has attracted much attention. Combination therapy with atezolizumab, carboplatin, paclitaxel, and bevacizumab has been approved as second-line treatment for patients with advanced NSCLC, and many anti-angiogenic agents and ICI combination therapies are in clinical trials . Preclinical trials of combination therapy have also been conducted in a variety of tumour models and have shown promise. One study showed that anti-VEGFR2 antibody-mediated vascular normalisation can improve immunotherapy by using a low-dose anti-VEGFR2 antibody (DC101) and ICI in a colon cancer model . Similarly, in a mouse model of hepatocellular carcinoma, anti-VEGFR2 antibody-mediated vascular normalisation improved ICI therapy by reprogramming the immune microenvironment . However, these preclinical models lack the human immune system, which is critical to fully recapitulate the human tumour immune microenvironment. Therefore, they are unable to use antibodies such as pembrolizumab for combination therapy. In recent years, the emergence of humanised immune system mice has brought new hope for preclinical immunotherapy research. Human peripheral blood mononuclear cell (PBMC) and human haematopoietic stem cell (HSC) mouse models were established by transplanting PBMCs or human cord blood-derived CD34 + HSCs into severe combined immunodeficiency mice . Tumour cell lines or patient-derived xenografts were also transplanted into mice. Humanised mouse models are essential for preclinical testing of immunotherapies as they provide insights into the interactions between the human immune system and tumours.
In this study, we hypothesised that bevacizumab may enhance the anti-tumour effect of pembrolizumab by inducing T cell infiltration into tumours without increasing toxicity. To this end, we established a human PBMC (Hu-PBMC) mouse model to conduct combination therapy experiments to investigate the changes in tumour vessels induced by bevacizumab, and determine what effect vascular changes have on tumour-infiltrating immune cells. Our preclinical findings suggest that combination therapy with bevacizumab and pembrolizumab has a synergistic anti-tumour effect, providing a theoretical basis for its first-line clinical application.