Macrophage polarization in ALK+ non-small cell lung cancer: Implications for treatment targeting

Background: Anaplastic lymphoma kinase-positive non-small cell lung cancer (ALK+ NSCLC) comprises 5-6% of all lung cancers with a median survival rate of 6.8 years. The primary treatment is tyrosine kinase inhibitors (TKI) which have a 30-80% response rate but acquired resistance is inevitable. One area of potential intervention is within the tumor-immune microenvironment (TIME) but the standard immunotherapies, such as immune checkpoint inhibitors, have resulted in low overall response rate (10-15%), toxicity, or inconclusive therapeutic effects in ALK+ NSCLC as a single agent or combined with TKIs in clinical trials. Therefore, it is imperative to gain a more in-depth understanding of the TIME evolution. In particular, there has been increasing evidence showing that macrophage polarization is involved in NSCLC tumorigenesis and drug resistance. The timing, phenotype, and functional state of macrophages within ALK+ NSCLC may offer opportunities for therapeutic targeting. Methods :Three investigations were carried out to investigate ALK+ NSCLC macrophage evolution: (1) a de novo and treatment-naïve study setting where intratracheal instillation of a CRISPR/Cas9 adenoviral system was administered in C57BL/6 mice for chromosomal rearrangement of alk and eml4 leading to spontaneous formation of tumors in the lungs, (2) a heterotopic TKI treatment study setting where ALK+ NSCLC cells syngeneic to C57BL/6 mice were injected subcutaneously, and (3) analysis of ALK+ NSCLC patient legacy and local cohorts. Tumors from the animal studies were harvested at early, intermediate, and late time points for analysis of macrophage phenotypes. Legacy and local patient cohorts were analyzed to investigate and validate macrophage results in a broad and heterogenous population of ALK+ NSCLC patients. Results :Macrophage frequency in ALK+ NSCLC increased during tumor progression with localization occurring primarily at the periphery of the tumor. The addition of Lorlatinib further increased macrophage frequency. Macrophage polarization was also altered by specific TKI treatments. Alectinib increased pro-inflammatory:anti-inflammatory macrophage ratios over time, reaching 0.55 at the late time point, which was significantly higher than Lorlatinib at 0.13. Compared with other oncogene-driven NSCLC, ALK+ patients in legacy and local RNAseq datasets demonstrated a lower frequency of pro-inflammatory macrophages. Conclusions :Differential macrophage polarization was demonstrated using de novo untreated and subcutaneous TKI treated ALK+ NSCLC animal models. Macrophage polarization appears to be TKI-specific with Alectinib treatment increasing pro-inflammatory macrophage subsets and decreasing anti-inflammatory subsets while Lorlatinib shows the opposite despite a smaller tumor size. Macrophage polarization has potential to be a promising avenue for therapeutic intervention.