Single-cell multi-omics reveals tumor microenvironment factors underlying poor immunotherapy responses in ALK-positive lung cancer

Anaplastic lymphoma kinase (ALK) rearrangement is a major oncogenic driver in non-small cell lung cancer (NSCLC). While ALK tyrosine kinase inhibitors have shown promising therapeutic effects, overcoming resistance with immunotherapy becomes necessary when resistance develops. However, various clinical trials have revealed that their efficacies remain limited. To investigate the tumor microenvironment (TME) factors contributing to poor immune checkpoint blockade responses in ALK-positive patients, we performed single-cell RNA and ATAC sequencing on lung adenocarcinoma (LUAD) tumors with and without ALK rearrangements. Integrative analysis with additional public LUAD cohorts revealed distinct immune landscapes in ALK-positive tumors, marked by enriched innate immunity and depleted adaptive immunity. ALK-positive malignant cells exhibit higher stemness and aggressive phenotype. Tumor-associated macrophages (TAMs) in these tumors predominantly maintain pro-tumoral M2-like states, reinforcing immune suppression. B cells show reduced immune reactivity and impaired tertiary lymphoid structure formation, while CD8+ T cells display bystander-like signatures and reduced tumor reactivity. Single-cell chromatin accessibility profiles combined with regulatory network analysis suggest that differences in transcription factor activities, rather than chromatin accessibility, may underlie T cell dysfunction. These findings provide insights into the immunosuppressive TME of ALK-positive LUAD, potentially explaining the failure of recent immunotherapy trials and highlighting targets for improving efficacy. Single-cell RNA and TCR libraries were generated using the Chromium controller in accordance with the 10x Chromium Next GEM Single Cell 5-v2_CellSurfaceProtein_UserGuide (CG000330). Cell suspensions were initially diluted with nuclease-free water to a target concentration of 10,000 cells. These suspensions were then combined with the master mix, Single Cell 5′ Gel Beads, and Partitioning Oil, and loaded into a Next GEM Chip K. Within the droplets, RNA transcripts from individual cells were barcoded and reverse-transcribed. The resulting cDNA was pooled and subjected to PCR enrichment. Following amplification, the cDNA was size-selected to generate three distinct library types: 5ʹ Gene Expression, V(D)J Enriched, and Cell Surface Protein. For the Cell Surface Protein Library, the cDNA pool underwent further enrichment using index PCR. The V(D)J Enriched Library required two rounds of amplification with specific primers. The 5’ Gene Expression Library was processed through end repair, addition of a single ‘A’ base, adapter ligation, and subsequent purification and PCR enrichment. Quantification of the purified libraries was performed using qPCR as outlined in the qPCR Quantification Protocol Guide (KAPA), and quality assessment was conducted using the Agilent Technologies 4200 TapeStation. Finally, sequencing was carried out on the HiSeq platform (Illumina) according to the read length specifications provided in the user guide. Single-cell ATAC Libraries were generated using the Chromium controller in accordance with the 10x Single Cell ATAC protocol (CG000168). Initially, nuclei were isolated and incubated with Transposase in a Transposition Mix. These transposed nuclei were then combined with master mix, Single Cell ATAC gel beads, and Partitioning Oil in a Chip E, where DNA fragments are barcoded in droplets during thermal incubation. The resulting barcoded DNA fragments were pooled and underwent sample index PCR. Quantification of the purified libraries was performed using qPCR as described in the qPCR Quantification Protocol Guide (KAPA), and quality assessment was conducted with the Agilent Technologies 4200 TapeStation. Finally, sequencing was carried out on the HiSeq platform (Illumina) following the read length specifications provided in the user guide.