A novel therapeutic strategy for lung cancer treatment: Priming a favorable tumor microenvironment by chemotherapeutic agents for cancer immunotherapy with immune checkpoint inhibitor
Immunotherapy is the use of methods to improve human immune system by inducing the body’s own immune system to re-identify cancer cells, thereby adjusting the tumor microenvironment to effectively inhibit the survival of cancer cells. There are different types of immunotherapy, among which immune checkpoint blockade (ICB) treatment has recently been considered as a way to improve the overall response rate in clinical cancer treatment.
When cancer cells appear in the body, it should be recognized by the immune system and attacked by cytotoxic T cells. However, cancer cells developed defensive mechanisms that can suppress immune cells, causing cytotoxic T cells to lose their ability to attack cancer cells. This mechanism is due to a specific protein receptor (PD-L1) produced on the surface of cancer cells, which can bind to the “immune checkpoint protein PD-1” on the surface of cytotoxic T cells to activate immune checkpoints and make T cells inactive. Therefore, as long as inhibitors that block their binding is applied, we can keep the immune checkpoints inactivated and make cytotoxic T cells aggressive towards cancer cells. This method is called "immune checkpoint blockade (ICB)" therapy. Among them, monoclonal antibodies against PD-1 or PD-L1 have become a commonly used immune checkpoint inhibitor around the world. However, not all ICB therapies are beneficial to lung cancer patients, partly due to the low or no expression of PD-L1 on lung tumors.
In the past, patients with advanced lung cancer could not use targeted therapies and can only rely on chemotherapy if the genetic test results of cancer cells showed no gene mutations such as EGFR or ALK mutations. However, when tumor immunotherapy emerged, whether it is combined with chemotherapy or used alone in patients with high PD-L1 expression, the survival time is significantly better than that of chemotherapy alone, which has become a new treatment option for lung cancer patients. However, there are many kinds of chemotherapeutic drugs but not all combinations are beneficial to patients with lung cancer. Some ineffective combinations of treatments bring more side effects and pain on cancer patients. Therefore, finding a chemo drug that can be perfectly combined with ICB treatment has become a new opportunity for the treatment of lung cancer.
Recently, the combination of Pemetrexed and platinum-based chemotherapeutics with ICB therapy has demonstrated convincing clinical activity in non-small cell lung cancer (NSCLC), and clinical results have led to the accelerated approval of this immunochemotherapy combination for advanced NSCLC patients. Although this novel combination therapy has become the first-line treatment for advanced NSCLC, little is known about the potential anti-tumor mechanism of the combination between Pemetrexed and immunotherapy.
Experimental Designs & Research Findings
In this study, we assessed that the most effective first-line NSCLC chemotherapeutic agents, including antimetabolic agents and antimitotic agents, to check whether they can regulate PD-L1 expression and prime a favorable tumor microenvironment for immune check point blockade (ICB) therapy. The long-term objective of this study is to investigate the effect and mechanism of chemotherapeutics on the priming of immune tumor microenvironment in NSCLCs, to provide a better molecular basis for chemoimmunotherapy.
First, we administered different first-line NSCLC chemotherapeutic agents, including anti-metabolic agents and anti-mitotic agents, and tested their PD-L1 prime activities to NSCLC cells. The results showed that compared with other chemotherapeutic agents, such as cisplatin and paclitaxel, the low-dose antimetabolitic chemotherapy drugs Pemetrexed and Fluorouracil (5-FU) significantly increased the expression of PD-L1 in lung cancer cells, priming a tumor microenvironment conducive to ICB treatment.
Second, we used the cancer/T-cell co-culture system to test whether using first-line NSCLC chemotherapeutic agents really have the function of regulating immune cell activation in tumor microenvironment. We found that Pemetrexed and anti-PD-L1 antibody synergistically induced T cell activation in vitro, mainly through restoring the exhausted T-cell activities. In addition, to dissect the crosstalk mechanism between chemotherapeutic agent and ICB, we selected CL1-5-derived subclones that displayed different Pemetrexed (PEM) responses and classified them into two groups, PEM responder (PEM-R) and PEM non-responder (PEM-NR). RNA-sequencing (RNA-seq) for each CL1-5 subclone were also performed. Gene Ontology (GO) enrichment analysis was applied for identification of the candidate regulatory pathways. Through cell-based experiments, we have revealed a novel molecular mechanism of how Pemetrexed induces the up-regulation of PD-L1 in lung cancer cells. We found that Pemetrexed suppresses the activity of thymidylate synthase (TS), and in turn increases the intracellular level of ROS, which further triggers the NF-κB signaling pathway followed by the activation of PD-L1 gene transcription in NSCLC cells.
Finally, we confirmed the combined therapeutic effect of the chemotherapeutic agent Pemetrexed and anti-PDL1 antibodies by using syngeneic mouse models. Compared with monotherapy, we observed that the combined treatment of Pemetrexed and anti-PD-L1 antibody can induce the accumulation of tumor-infiltrating T lymphocytes, synergistically inhibiting tumor growth; followed by increased PD-L1 expression, the recruitment of tumor infiltrating T lymphocytes, and reverse exhaustion of T cell activities by inducing antitumor cytokine IL2, TNF-α, and IFN-γ’s expression, rendering the tumor microenvironment more favorable for immunotherapy. In contrast, data from the syngeneic mouse lung tumor model indicates that platinum-based chemotherapy, e.g., cisplatin, cannot induce the expression of PD-L1 in NSCLC cells and provides no advantage in the combinatory therapy of Pemetrexed and anti-PD-L1 antibody for lung cancer treatment. This strongly suggests that Pemetrexed can be combined with ICB therapy without platinum-based chemotherapy.
Summary & Therapeutic Implications
Our findings showed that Pemetrexed increases the expression of PD-L1 in lung cancer cells, thereby creating a tumor microenvironment that is more suitable for immunotherapy. We revealed a novel mechanism that Pemetrexed can activate and increase PD-L1 gene transcription in cancer cells by inducing the TS−ROS−NF-κB signaling pathway, which provides a mechanistic basis for the FDA-approval combinatory chemoimmunotherapy in NSCLC treatment. Our results also provide more detailed instructions to avoid unnecessary treatment (for example, the use of Platinum drugs) and to improve the therapeutic value of cancer patients. We believe that our findings will put forward more specific concepts and clinical therapeutic significance for the direction of cancer immunotherapy.