Mesenchymal stem cells are enriched in head neck squamous cell carcinoma, correlates with tumour size and inhibit T-cell proliferation

Abstract

Background: Cancer is a multifactorial disease not only restricted to transformed epithelium, but also involving cells of the immune system and cells of mesenchymal origin, particularly mesenchymal stem cells (MSCs). Mesenchymal stem cells contribute to blood- and lymph- neoangiogenesis, generate myofibroblasts, with pro-invasive activity and may suppress anti-tumour immunity.

Methods: In this paper, we evaluated the presence and features of MSCs isolated from human head neck squamous cell carcinoma (HNSCC).

Results: Fresh specimens of HNSCC showed higher proportions of CD90+ cells compared with normal tissue; these cells co-expressed CD29, CD105, and CD73, but not CD31, CD45, CD133, and human epithelial antigen similarly to bone marrow-derived MSCs (BM-MSCs). Adherent stromal cells isolated from tumour shared also differentiation potential with BM-MSCs, thus we named them as tumour-MSCs. Interestingly, tumour-MSCs showed a clear immunosuppressive activity on in vitro stimulated T lymphocytes, mainly mediated by indoelamine 2,3 dioxygenase activity, like BM-MSCs. To evaluate their possible role in tumour growth in vivo, we correlated tumour-MSC proportions with neoplasm size. Tumour-MSCs frequency directly correlated with tumour volume and inversely with the frequency of tumour-infiltrating leukocytes.

Conclusions: These data support the concept that tumour-MSCs may favour tumour growth not only through their effect on stromal development, but also by inhibiting the anti-tumour immune response.

Figure 1

CD90+ cells are enriched in HNSCC. (A) The relative proportions of HEA+, CD90+, CD31+, and CD45+ cells were assessed by flow cytometry on fresh single-cell suspensions derived from HNSCC specimens (black columns, n=13 for HEA, CD90, and CD45; n=8 for CD31), or control healthy tissue (white columns, n=6). Columns represent mean values±s.e. of independent experiments. P-value is calculated comparing tumoral tissue vs healthy tissue. (B) Tumour-derived stromal cells and control BM-MSCs share the same immunophenotype. Adherent homogeneous stromal cells obtained from seven different patients were collected at list after 3 weeks of culture and assessed by flow cytometry for MSCs markers (black columns), and compared with control BM-MSCs obtained from three different healthy donors (white columns). Columns represent mean values±s.e.

Figure 2

Adherent stromal cells derived from tumours specimens acquires typical adipoblast or osteoblast staining after adequate in vitro conditioning. (A) Adherent cells derived from HNSCC specimens of six different patients or control BM-MSCs, were cultured in normal culture medium (CM), in the presence of osteogenic medium (OM) or adipogenic medium (AM) for 21 days. After this period, tumour-derived stromal cells were stained with haematoxylin and eosin plus Oil Red O or with Alizarin Red, and compared with BM-MSCs to assess their in vitro cell differentiation. Phase contrast images were obtained by an optic microscope (Axiovert 40C Carl Zeiss) with a × 20 magnification. A representative case out of six is reported (tumour-MSCs) and compared with a representative control case out of three (BM-MSCs). (B, C) Adherent stromal cells derived from HNSCC specimens acquires typical adipoblast or osteoblast mRNAs after in vitro culture in the presence of appropriate conditioning medium. mRNA levels for adipoblast or osteoblast markers were evaluated in HNSCC-derived adherent stromal cells (black columns, n=6) or in BM-MSCs (white columns, n=3) at the beginning of the experiment (time 0 – T0), after 7 and 21 days of culture in the presence of normal culture medium (CM), adipogenic medium (AM), or osteogenic medium (OM). B reports mean values±s.e. of the specific mRNAs for the adipose markers ppar-γ and FABp4 at the different time points. C reports the mRNAs mean values±s.e. for the osteogenic markers alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX 2) and osteocalcin. P-values are evaluated for HNSCC-derived adherent stromal cells vs own control condition (CM) at each time point.

Figure 3

Tumour-MSCs inhibit in vitro cell proliferation and cytokines production by polyclonally stimulated CD4+ and CD8+ T lymphocytes. (A) Proliferation of polyclonally stimulated (anti-CD3 plus anti-CD28) T lymphocytes was assessed after 5 days of culture on the basis of 3H thymidine incorporation in the absence (T) or in the presence of tumour-MSCs (black columns) or of BM-MSCs (white columns) as control condition. Histograms represent mean values±s.e. of six different experiments. P-values are evaluated vs T cells alone. (B) Cytokine production by polyclonally stimulated T lymphocytes was assessed in supernatants by ELISA, after 3 days of culture in the absence (T) or in the presence of tumour-MSCs. Histograms represent mean values±s.e. of three different experiments. P-values are evaluated vs T cells alone.

Figure 4

Indoelamine 2,3 dioxygenase (IDO) activity is on the basis of tumour-MSCs immunoregulatory activity. (A) Cell proliferation of polyclonally stimulated CD4+ T cells was assessed as previously indicated in the absence or in the presence of tumour-MSCs with or without the IDO1 inhibitor methyl-L-triptophane in scalar doses. Histograms represent mean values±s.e. of four different experiments. P-values are evaluated vs T cells alone. (B) Analogous experiments were conducted in the absence or in the presence of the COX inhibitor indomethacin in scalar concentration. Histograms represent mean values±s.e. of four different experiments; P-value was calculated vs T cells alone and resulted <0.01 at MSC/T-cell ratio 1/5 and 1/10 in the control condition (medium alone) and also in the presence of the different concentrations of indomethacin.

Figure 5

Tumour-MSC actively attract CD4+ and CD8+ T cells. In vitro chemotaxis of CD4+ or CD8+ T cells was evaluated in transwell chambers in the presence of normal culture medium (negative control), in the presence of CXCL10 (positive control), or in the presence of medium conditioned by tumour-MSC derived from two different patients (tumour-MSC1 and 2). As further control, the conditioned medium obtained from BM-MSC also was evaluated. Histograms represent the mean values±s.e. of triplicates of two separated experiments.

Figure 6

Frequency of CD90-positive cells in HNSCC directly correlates with tumour volume and inversely with leukocyte infiltrate. (A) The frequency of CD90+ cells present at level of HNSCC fresh samples, as assessed by flow cytometry on single-cell suspensions, was correlated with tumour size (longitudinal × lateral × transversal diameter (cm) × 0.52), n=13. (B) The frequency of CD90+ cells determined as previously described, was correlated with the frequency of tumour-infiltrating leukocyte (CD45+) n=13. (C) The frequency of CD90+ cells was correlated with the frequency of endothelial cells (CD45−CD31+ cells) n=8. R² and P-values are indicated in each panel.