Fibroblasts regulate tumor growth and immune surveillance. Here, we study FAP, PDGFβR and α-SMA fibroblast markers in a well-annotated clinical cohort of non-small-cell lung cancer (NSCLC) for analyses of associations with immune cell infiltration, mutation status and survival. Materials and Methods: A well-annotated NSCLC cohort was subjected to IHC analyses of stromal expression of FAP, PDGFβR and α-SMA and of stromal CD8 density. Fibroblast markers-related measurements were analyzed with regard to potential associations with CD8 density, cancer genetic driver mutations, survival and PD-L1 expression in the whole NSCLC cohort and in subsets of patients. Results: High stromal FAP expression was identified as an independent poor prognostic marker in the whole study population (HR 1.481; 95 % CI, 1.012-2.167, p = 0.023) and in the histological subset of adenocarcinoma (HR 1.720; 95 % CI, 1.126-2.627, p = 0.012). Among patients with adenocarcinoma, a particularly strong association of FAP with poor survival was detected in patients with low stromal CD8 infiltration, and in other subpopulations identified by specific clinical characteristics; elderly patients, females, non-smokers and patients with normal ECOG performance status. α-SMA expression was negatively associated with CD8 infiltration in non-smokers, but none of the fibroblast markers expression was associated with CD8 density in the whole study population.Significant associations were detected between presence of p53 mutations and high α-SMA (p = 0.003) and FAP expression (p < 0.001). Conclusion:The study identifies FAP intensity as a candidate independent NSCLC prognostic biomarker. The study also suggests continued analyses of the relationships between genetic driver mutations and the composition of tumor stroma, as well as continued probing of marker-defined fibroblasts as NSCLC subset-specific modifiers of immune surveillance and outcome.

Spermatogonial stem cells (SSCs) are pluripotent elements found in the adult seminiferous epithelium between Sertoli cells and a basal lamina which covers the multilayered external wall of peritubular myoid cells. The microenvironment of this pluripotent stem cell niche creates the complex and dynamic system that is necessary for the initiation of spermatogenesis, but this system also contains factors which can potentially collaborate in the progression of testicular germ cell tumors (TGCTs). In this review, we summarize our current knowledge about some important structural and molecular features related to the SSC niche, including growth factors, adhesion molecules, extracellular matrix, mechanical stress and vascularization. We discuss their possible collaborative effects on the generation and progression of TGCTs, which are a type of cancer representing the most frequent neoplasia among young men and whose incidence has grown very quickly during the past decades in North America and Europe. In this regard, a better understanding of the pluripotent stem cell niche where these malignancies arise will provide further insights into the origin of TGCTs and the mechanisms underlying their growth and invasion of adjacent and distant tissues.

The tumour surrounding stroma, known as reactive stroma, is a crucial factor to understand cancer cell growth and invasion. In the normal adult testis, the stroma contains extracellular matrix components, fibroblasts, infiltrating leucocytes, lymphocytes, macrophages and capillaries, as well as other specific cell populations, like Leydig cells and a thin myoepithelium surrounding the seminiferous tubules constituted by the peritubular cells. All these cells are an important source of proliferation and survival promoting signals, proteolytic enzymes, migratory cues and pro-angiogenic factors. Ascribable to this pro-invasive activity, the tumour reactive stroma cells, especially cancer-associated myofibroblasts, have emerged as a promising target for cancer therapy. This review is focused on the potential role of the peritubular myoid cells in the development of testicular germ cell tumours as the precursors of cancer-associated myofibroblast and on an experimental model for the study of testis germinal cancer stroma and on the differences between normal and tumour-associated stromal cells, including the molecular mechanisms that mediate the important cancer stroma crosstalk. Special attention will be paid to the cancer-associated myofibroblasts as possible therapeutic targets, because they are one of the main components of the reactive stroma and are known to secrete a variety of paracrine factors that stimulate tumour progression.

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We aimed to assess if the discrepant prognostic information between Programmed Death Ligand 1 (PD-L1) protein versus mRNA expression in early breast cancer (BC) could be attributed to heterogeneity in its expression. PD-L1 protein and mRNA expression in BC tissue microarrays from two clinical patient cohorts were evaluated (105 patients; cohort 1: untreated; cohort 2: neoadjuvant chemotherapy-treated). Immunohistochemistry (IHC) with SP142, SP263 was performed. PD-L1 mRNA was evaluated using bulk gene expression and RNA-FISH RNAscope®, the latter scored in a semi-quantitative manner and combined with immunofluorescence (IF) staining for the simultaneous detection of PD-L1 protein expression. PD-L1 expression was assessed in cores as a whole and in two regions of interest (ROI) from the same core. The cell origin of PD-L1 expression was evaluated using multiplex fluorescent IHC. IHC PD-L1 expression between SP142 and SP263 was concordant in 86.7% of cores (p < 0.001). PD-L1 IF/IHC was weakly correlated with spatial mRNA expression (concordance 54.6–71.2%). PD-L1 was mostly expressed by lymphocytes intra-tumorally, while its stromal expression was mostly observed in macrophages. Our results demonstrate only moderate concordance between the various methods of assessing PD-L1 expression at the protein and mRNA levels, which may be attributed to both analytical performance and spatial heterogeneity.

Background Cancer-associated fibroblasts (CAFs) are molecularly heterogeneous mesenchymal cells that interact with malignant cells and immune cells and confer both anti- and pro-tumorigenic functions. Prior in situ profiling studies of human CAFs have largely relied on scoring single markers, thus presenting a very 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 (mfIHC) was employed to spatially profile the CAF landscape in two population-based NSCLC cohorts (n = 636) using antibodies against four fibroblast markers: Platelet-derived growth factor receptor-alpha (PDGFRA) and -beta (PDGFRB), fibroblast activation protein (FAP), and alpha-smooth muscle actin (αSMA). The CAF subsets were analyzed for their correlations with mutations, immune characteristics, clinical variables as well as overall survival (OS). Results Two CAF subsets, CAF7 (PDGFRA-/PDGFRB+/FAP+/αSMA+) and CAF13 (PDGFRA+/PDGFRB+/FAP-/αSMA+), showed significant but opposite associations with tumor histology, driver mutations (TP53 and EGFR), immune features (PD-L1 and CD163), and prognosis. In patients with early-stage tumors (pTNM IA-IB), CAF7 and CAF13 acted as independent prognostic factors. Conclusions Multi-marker-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 mfIHC-based CAF profiling as a powerful tool for the discovery of clinically relevant CAF subsets.

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Emerging data indicate that genomic alterations can shape immune cell composition in early breast cancer. However, there is a need for complementary imaging and sequencing methods for the quantitative assessment of combined somatic copy number alteration (SCNA) and immune profiling in pathological samples. Here, we tested the feasibility of three approaches—CUTseq, for high-throughput low-input SCNA profiling, multiplexed fluorescent immunohistochemistry (mfIHC) and digital-image analysis (DIA) for quantitative immuno-profiling- in archival formalin-fixed paraffin-embedded (FFPE) tissue samples from patients enrolled in the randomized SBG-2004-1 phase II trial. CUTseq was able to reproducibly identify amplification and deletion events with a resolution of 100 kb using only 6 ng of DNA extracted from FFPE tissue and pooling together 77 samples into the same sequencing library. In the same samples, mfIHC revealed that CD4 + T-cells and CD68 + macrophages were the most abundant immune cells and they mostly expressed PD-L1 and PD-1. Combined analysis showed that the SCNA burden was inversely associated with lymphocytic infiltration. Our results set the basis for further applications of CUTseq, mfIHC and DIA to larger cohorts of early breast cancer patients.