Mesenchymal stromal cells in healthy and melanoma infiltrated human lymph nodes

Abstract

Mesenchymal stromal cells (MCs) in the tumour micro environment (TME) are receiving increasing interest, as they can support tumourigenesis. The analysis of MCs in lymph node (LN) is important as what happens in this organ may support early tumour metastasis and may regulate generation of anti-tumour immune responses. However, very little is known about the phenotype and function MCs in both healthy LNs and tumour infiltrated LNs, which hinders targeting of these cells in cancer therapy. In this study, we present molecular markers that enable a new level of discrimination between the different subtypes of MCs in healthy LNs. We characterised a population of CD90+ CD34+ cells residing in the capsule, trabeculae and hilum and overall showed a similar phenotypic profile to adult mesenchymal stem/stromal cells (MSCs) identified in other tissues. We showed that we could distinguish pericytes (CD34- podoplanin- CD146+ cells) from other LN MCs. These cells showed interesting dichotomy of NG2 and CD36 expression on arterial versus venous vasculature. This finding is likely to be helpful in distinguishing pericyte populations in other human tissues as well. We next sought to examine melanoma infiltrated LNs (MILNs) surgically resected from patients. We identified CD90+ CD34+ MCs in regions containing dense collagen fibres. These cells expressed FAP and CD26, suggesting they have become activated. CD34- reticular cells showed dim or no expression of immune cell adhesion molecules, ICAM1 and VCAM1. Despite this, we observed tight association of T cells with the stromal network, which suggested that CD34- reticular cells may be preventing T cells from migrating into melanoma nests. These results suggest that both CD90+ CD34+ cells and CD34- reticular cells were altered by infiltrating melanoma cells to acquire different phenotypic and functional properties. We subsequently examined the transcriptome profile of CD90+ CD34+ cells and CD34- reticular cells using microarray. CD90+ CD34+ cells expressed several genes that are part of ECM or related to ECM modulation, as well as genes related to adipogenic, osteogenic and chondrogenic lineages. CD34- reticular cells expressed genes that promote migration of immune cells, as well as number of other genes associated with immune system. Overall, the gene signature of CD90+ CD34+ cells were similar to adult MSCs, and the gene signature of CD34- reticular cells were similar to reticular stromal cells in healthy LNs. CD90+ CD34+ cells also expressed genes associated with chemokine degradation, suggesting that interplay of 2 types of MCs may play key roles in modulating migration of T cells. The revealed markers now offer new ways to further discriminate melanoma-associated MCs. Next, we sought to analyse the function of MCs in vitro. However, the MCs in vitro showed different phenotypic profiles to MCs in tissues and we were not able to observe any functional differences between MCs cultured from healthy LNs and MILNs. These results highlighted the difficulties in establishing in vitro analysis of MCs that properly represents their function in vivo. Collectively, we now have better understanding about the diversity of MCs in both healthy and melanoma LNs. For the first time, we described the presence of CD34+ cells in MILNs and we have divided melanoma-associated MCs into distinct subtypes; CD90+ CD34+ cells producing thick ECM, CD34- reticular cells producing chemokines and CD34- podoplanin- CD146+ pericytes surrounding vasculature. These results will enable deeper analysis of melanoma-associated MCs and aid discovery of new therapeutic targets.