Rapid Serial Immunoprofiling of the Tumor Immune Microenvironment by Fine Needle Sampling

There is increasing effort to discover and integrate predictive and/or prognostic biomarkers into treatment algorithms. While tissue-based methods can reveal tumor-immune cell compositions at a single time point, we propose that single-cell sampling via fine needle aspiration (FNA) can facilitate serial assessment of the tumor immune microenvironment (TME) with a favorable risk-benefit profile.

Primary antibodies directed against 20 murine and 25 human markers of interest were chemically modified via a custom linker-bio-orthogonal quencher (FAST) probe. A FAST-FNA cyclic imaging and analysis pipeline were developed to derive quantitative response scores. Single cells were harvested via FNA and characterized phenotypically and functionally both in preclinical and human samples using the newly developed FAST-FNA assay.

FAST-FNA samples analyzed manually versus the newly developed deep learning-assisted pipeline gave highly concordant results. Subsequently, an agreement analysis showed that FAST and flow cytometry of surgically resected tumors were positively correlated with an R² = 0.97 in preclinical samples and an R² = 0.86 in human samples with the detection of the relevant tumor and immune biomarkers of interest. Finally, the feasibility of applying this minimally invasive approach to analyze the TME during immunotherapy was assessed in patients with cancer revealing local antitumor immune programs.

The FAST-FNA is an innovative technology that combines bio-orthogonal chemistry coupled with a computational analysis pipeline for the comprehensive profiling of single cells obtained through FNA. This is the first demonstration that the complex and rapidly evolving TME during treatment can be accurately and serially measured by simple FNA.