Perturbational single-cell RNA sequencing of patient tumors in Merkel cell and small cell lung carcinomas.

Abstract

2520 Background: Despite the transformational impact of immune checkpoint blockade, many cancer patients do not experience long-term survival. T cells with innate immune signatures can secrete inflammatory cytokines/chemokines and deliver potent cytotoxic signals potentially ideal for tumor immunity. The novel double-stranded RNA sensor RIG-I agonist SLR14 improved the control of murine melanoma. We tested the hypothesis that SLR14 transforms T cells to a cytotoxic state in immunologically “cold” human tumor specimens. Methods: We developed an approach, called PERCEPT, to directly test the response of patient tumor and immune samples to novel and established therapies ex vivo using perturbational single-cell RNA sequencing (Table). We obtained 9 surgical resections from primary or metastatic melanoma and Merkel Cell Carcinoma (MCC) tumors and lymph node metastases and made suspension replicates of tumor and infiltrating immune cell co-cultures. We stimulated for 42-48 hours (Table). We Flourescently Activated Cell Sorted live cells and then barcoded for multiplexed single-cell sequencing using 10x scRNAseq. We used CINEMA-OT to identify factors associated with response and resistance to the perturbations tested. We developed and validated CRISPR-KO MCC and small cell lung cancer (SCLC) cell lines, and co-cultured with CD14+ monocytes or monocyte-derived DCs. Results: Stimulation with RIG-I agonist SLR14 induced expression beyond canonical IFN-stimulated genes in tumor cells, NK cells, and T cells. SLR14 stimulates tumor-infiltrating T cells into antiviral states in tumor-immune co-cultures and primes in vitro T-cell production of IFNγ. However, MCC immune infiltrate responsiveness to IFN or SLR14 was notably decreased compared to the melanoma samples, and perturbational computational analyses with CINEMA-OT identified the cytokine midkine (MDK) associated with nonresponse in MCC. Knockout of MDK restored response to IFN and SLR14 by MCC and SCLC tumor cell lines, as well as co-cultured CD14+ monocytes or monocyte-derived DCs. Conclusions: Our approach revealed that midkine, a multifunctional cytokine, suppresses innate immune sensing of IFN and SLR14 in both tumor and immune cells, disrupting the tumor immunity cycle at multiple points. We show that this effect, while comparatively infrequent in melanoma, is pronounced in MCC and SCLC. Our study thus uses a direct assessment of patient tumor and immune samples to identify a novel resistance mechanism enriched in neuroendocrine tumors MCC and SCLC. Therapeutic class Stimulation Target Clinical development Immune checkpoint inhibitor αPD-1 PD-1 Standard of care Cytokine IFNγIFNβ IFNGRIFNAR Early-phase clinical trials orPre-clinical Innate immune agonist Poly(I:C)-NTPoly(I:C)-TADU-S100SLR14 TLR3MDA5/TIG-I/TLR3STINGRIG-I Combination αPD-1 + IFNβ PD-1/IFNAR