Fibroblast subsets in non-small cell lung cancer : Associations with survival, mutations, and immune features

Background Cancer-associated fibroblasts (CAFs) are molecularly heterogeneous mesenchymal cells that interact with malignant cells and immune cells and confer anti- and protumorigenic functions. Prior in situ profiling studies of human CAFs have largely relied on scoring single markers, thus presenting a limited view of their molecular complexity. Our objective was to study the complex spatial tumor microenvironment of non-small cell lung cancer (NSCLC) with multiple CAF biomarkers, identify novel CAF subsets, and explore their associations with patient outcome. Methods Multiplex fluorescence immunohistochemistry was employed to spatially profile the CAF landscape in 2 population-based NSCLC cohorts (n = 636) using antibodies against 4 fibroblast markers: platelet-derived growth factor receptor-alpha (PDGFRA) and -beta (PDGFRB), fibroblast activation protein (FAP), and alpha-smooth muscle actin (alpha SMA). The CAF subsets were analyzed for their correlations with mutations, immune characteristics, and clinical variables as well as overall survival. Results Two CAF subsets, CAF7 (PDGFRA-/PDGFRB+/FAP+/alpha SMA+) and CAF13 (PDGFRA+/PDGFRB+/FAP-/alpha SMA+), showed statistically significant but opposite associations with tumor histology, driver mutations (tumor protein p53 [TP53] and epidermal growth factor receptor [EGFR]), immune features (programmed death-ligand 1 and CD163), and prognosis. In patients with early stage tumors (pathological tumor-node-metastasis IA-IB), CAF7 and CAF13 acted as independent prognostic factors. Conclusions Multimarker-defined CAF subsets were identified through high-content spatial profiling. The robust associations of CAFs with driver mutations, immune features, and outcome suggest CAFs as essential factors in NSCLC progression and warrant further studies to explore their potential as biomarkers or therapeutic targets. This study also highlights multiplex fluorescence immunohistochemistry-based CAF profiling as a powerful tool for the discovery of clinically relevant CAF subsets.Peer reviewe

A CD44(high)/EGFR(low) Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment

Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44(high)/EGFR(low), CD44(high)/EGFR(high) and CD44(low) cells, respectively, were collected by fluorescence-activated cell sorting. The CD44(high)/EGFR(low) population showed a spindle-shaped EMT-like morphology, while the CD44(low) population was dominated by cobblestone-shaped cells. The CD44(high)/EGFR(low) population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44(high)/EGFR(low) cells in comparison to CD44(low) cells. Moreover, CD44(high)/EGFR(low) cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44(high)/EGFR(high) and CD44(low) counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.Funding Agencies|Swedish Laryng Foundation||Foundation of Signhild Engkvist||Foundation of Ake Wiberg||Swedish Cancer Society|2008/5522010/545|County Council of Ostergotland||Linkoping University Hospital||</p

The CD44^high/EGFR^low population shows upregulation of cancer stem cell-associated genes.

<p>Relative mRNA expression levels of the stemness genes NANOG and SOX1 in LK0923, LK0827 and LK0863 subpopulations, here displayed as fold difference relative to the CD44^low population (GAPDH was used as an internal standard).</p

The CD44^high population shows upregulation of EMT-associated genes.

<p><b>A–C:</b> Relative mRNA expression levels of epithelial-mesenchymal transition-associated genes; E-cadherin, N-cadherin, vimentin, fibronectin1, Twist1, FOXC2 and matrix metalloproteinase 7 (MMP7) in (<b>A</b>) LK0923, (<b>B</b>) LK0827 and (<b>C</b>) LK0863 subpopulations. The expression levels in the CD44^high/EGFR^high and CD44^high/EGFR^low populations are displayed here as fold difference relative to the CD44^low population (GAPDH was used as an internal standard). <b>D:</b> Western blot analysis of N-cadherin, vimentin and fibronectin1 in LK0923 populations (β-actin was used as loading control).</p

<b>A:</b> Identification of subpopulations of cells in HNSCC cell lines.

<p>Dot plot analysis of LK0923, LK0827 and LK0863 cells co-stained with an APC-conjugated anti-CD44 antibody and a PE-conjugated anti-epidermal growth factor receptor (EGFR) antibody. The red colored areas represent the CD44^high cells, and the three boxes display the gating of the different subpopulations from which cells were isolated for further experiments. <b>B:</b> Light microscope images of CD44^low, CD44^high/EGFR^high and CD44^high/EGFR^low populations (3 days after sorting) and unsorted LK0923, LK0827 and LK0863 cell cultures.</p

Characterization of the CD44^low, CD44^high/EGFR^high and CD44^high/EGFR^low subpopulations within the LK0923 cell line.

<p><b>A:</b> Expression of CD44 and epidermal growth factor receptor (EGFR) in sorted subpopulations (β-actin was used as a loading control). <b>B:</b> Proliferation rate of subpopulations. Data represents mean ± SD from three experiments in triplicate, *p≤0.05. <b>C:</b> Cell cycle distribution comparison between CD44^low and CD44^high/EGFR^low cells. Data from one representative experiment out of two are shown. <b>D:</b> Plating efficiency, as assessed by colony formation in sorted subpopulations. Data represents mean ± SD from three experiments in duplicate, *p≤0.05.</p

Differentially expressed genes in LK0923 subpopulations.

*<p> =  Fold difference in gene expression relative to the CD44^low population.</p

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ.

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ