IL-2 stromal signatures dissect immunotherapy response groups in non-small cell lung cancer (NSCLC)

Abstract

IntroductionImmunotherapies, such as immune checkpoint inhibitors (ICI) have shown durable benefit in a subset of non-small cell lung cancer (NSCLC) patients. The mechanisms for this are not fully understood, however the composition and activation status of the cellular milieu contained within the tumour microenvironment (TME) is becomingly increasingly recognised as a driving factor in treatment-refractory disease.MethodsHere, we employed multiplex IHC (mIHC), and digital spatial profiling (DSP) to capture the targeted immune proteome and transcriptome of tumour and TME compartments of pre-treatment samples from a 2nd line NSCLC ICI-treated cohort (n=41 patients; n=25 responders, n=16 non-responders).ResultsWe demonstrate by mIHC that the interaction of CD68+ macrophages with PD1+, FoxP3+ cells is significantly enriched in ICI refractory tumours (p=0.012). Our study revealed that patients sensitive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p=0.028) within the tumour compartments, which corresponded with the increased expression of IL2 mRNA (p=0.001) within their stroma, indicative of key conditions for ICI efficacy prior to treatment. IL2 mRNA levels within the stroma positively correlated with the expression of pro-apoptotic markers cleaved caspase 9 (p=2e-5) and BAD (p=5.5e-4) and negatively correlated with levels of memory T cells (CD45RO) (p=7e-4). Immuno-inhibitory markers CTLA-4 (p=0.021) and IDO-1 (p=0.023) were also supressed in ICI-responsive patients. Of note, tumour CD44 (p=0.02) was depleted in the response group and corresponded inversely with significantly higher stromal expression of its ligand SPP1 (osteopontin, p=0.008). Analysis of differentially expressed transcripts indicated the potential inhibition of stromal interferon-gamma (IFNγ) activity, as well as estrogen-receptor and Wnt-1 signalling activity within the tumour cells of ICI responsive patients. Cox survival analysis indicated tumour CD44 expression was associated with poorer prognosis (HR=1.61, p=0.01), consistent with its depletion in ICI sensitive patients. Similarly, stromal CTLA-4 (HR=1.78, p=0.003) and MDSC/M2 macrophage marker ARG1 (HR=2.37, p=0.01) were associated with poorer outcome while levels of apoptotic marker BAD (HR=0.5, p=0.01) appeared protective. Interestingly, stromal mRNA for E-selectin (HR=652, p=0.001), CCL17 (HR=70, p=0.006) and MTOR (HR=1065, p=0.008) were highly associated with poorer outcome, indicating pro-tumourigenic features in the tumour microenvironment that may facilitate ICI resistance.ConclusionsThrough multi-modal approaches, we have dissected the characteristics of NSCLC and provide evidence for the role of IL2 and stromal activation by osteopontin in the efficacy of current generations of ICI therapy. The enrichment of SPP1 in the stroma of ICI sensitive patients in our data is a novel finding, indicative of stromal activation that may aid immune cell survival and activity despite no clear association with increased levels of immune infiltrate.

Spatial Profiling Identifies Prognostic Features of Response to Adjuvant Therapy in Triple Negative Breast Cancer (TNBC)

“…Moreover, our findings could be further examined in cohorts of neoadjuvant chemotherapy, an established therapy modality in TNBC, and associated with pathological complete response (pCR) ( 27 – 29 ). Future investigations should also utilise orthogonal approaches (immunohistochemistry) to qualify abundance of TIL infiltration and validate experimental findings from the DSP platform ( 30 ). Furthermore, biological characterisation and validation of putative targets within immunocompetent murine models is required to delineate their cellular roles in chemotherapeutic response.…”

Macrophages-based immune-related risk score model for relapse prediction in stage I–III non-small cell lung cancer assessed by multiplex immunofluorescence

“…The high heterogeneity of TAMs, characterized as the variability of their constituents depending on clinical stage, cancer types, and intratumor heterogeneity, has been studied by numerous researchers, revealing their vital role in cancer development, progression, and recurrence (24)(25)(26)(27). CD68, considered as the significant biomarker for quantifying macrophage amounts, is generally associated with prognosis and correlated with resistance to cancer treatment in solid tumors, especially lung cancer, which is the deadliest cancer worldwide (28).…”

Challenges and Opportunities for Immunoprofiling Using a Spatial High-Plex Technology: The NanoString GeoMx® Digital Spatial Profiler

“…After placing the ROIs, we can further divide them into different biological compartments based on the morphology markers’ expression. A segment profiling refers to phenotypic or distinct biological architectural compartments revealed by the morphology markers, with the most common compartments being “Tumor” and “Stroma” (tumor marker +) vs. “Stroma” (tumor marker -) ( 84 ), but we can also perform cell type-specific profiling for distinct cell populations with cell type-specific biomarkers, such as CD45+, CD3+, or CD68+ ( Figure 5 ).…”