The role of interactive states of imnune checkpoint regulators in cancer: determined by quantitative imaging

Abstract

Immune checkpoint blockade therapies have enhanced cancer therapy in lung and renal cancers aswell as melanoma. Most patients are stratified for anti-PD-1 or anti-CTLA-4 treatments based ontheir PD-L1 ligand expression that is currently assessed by traditional immunohistochemistry (IHC)approaches, which are subjective, lack quantitation and a dynamic range. We have developed a novelquantitative imaging platform, underpinned by time-resolved Förster resonance energy transfer(FRET) determined by frequency-domain fluorescence lifetime imaging microscopy (FLIM) tospatially quantitate these immune checkpoint interactions at a nanoscopic (<10nm) resolution. Thisassay is termed immune-FRET (iFRET). We validated the ability of iFRET to measure differentimmune checkpoints in patients with malignant melanoma and malignant non-small cell lungcarcinoma (NSCLC). Here, iFRET was predictive of patient outcome whereas IHC approaches werenot. This was then applied to a subset of colorectal cancer patients with bilateral lung metastases whofailed to respond to classical treatments and therefore had their metastases treated by radiofrequencyablation (RFA). iFRET could detect differential PD-1/PD-L1 and CTLA-4/CD80 interaction stateswithin patients and between patients. It was shown that neither CD80 nor PD-L1 expressioncorrelated with checkpoint interaction in these patients. Moreover, a negative correlation was seenbetween PD-1/PD-L1 interactive states and intratumoral CD3+ density. Taken together, these resultsindicate a novel technique by which to assess immune checkpoint engagement in patient samples.The results from this multi-disciplinary investigation and thus the implementation of iFRET to carryout quantitative immune surveyance may change the way patients are selected for immunotherapiesand may provide a mechanism by which to monitor their response to treatment.