Stromal impact on tumor growth and lymphangiogenesis in human carcinoma xenografts

Squamous cell carcinomas (SCCs) arising in the oral cavity are associated with poor survival, mainly due to metastatic disease. In contrast, skin SCCs rarely metastasize and are usually curable. To study influence of tongue and skin stroma on cancer growth and induction of lymphangiogenesis, xenograft tumors of human carcinoma cells were established either in tongue or skin of BALB/c nude mice. Two oral and two skin SCC cell lines were used, as well as an endometrial adenocarcinoma cell line. Tongue tumors established from all cell lines were larger than corresponding skin tumors. Peritumoral lymphatic vessel density was up to five times higher in tongue than in corresponding skin tumors, and mRNA level of the lymphangiogenic growth factor vascular endothelial growth factor (VEGF)-C was twice as high in tongue tumors compared with corresponding skin tumors. Contrary to lymphatic vessel density, blood vessel density was higher in skin tumors than in tongue tumors. In a cohort of patient samples, lymphatic vessel density was found to be higher in tongue SCCs compared with skin SCCs, supporting a clinical relevance of our findings. Our results show that the tumor stroma has a profound impact on cancer growth and induction of lymphangiogenesis and angiogenesis. The difference in lymphatic vessel density between tongue and skin tumors may be important in directing metastatic potential of tumors arising in these organs

Kidney tertiary lymphoid structures in Lupus Nephritis develop into large interconnected networks and resemble lymph nodes in gene signature

Immune aggregates organized as tertiary lymphoid structures (TLS) are observed within the kidneys of patients with systemic lupus erythematosus and lupus nephritis (LN). Renal TLS was characterized in lupus-prone New Zealand black × New Zealand white F1 mice analyzing cell composition and vessel formation. RNA sequencing was performed on transcriptomes isolated from lymph nodes, macrodissected TLS from kidneys, and total kidneys of mice at different disease stages by using a personal genome machine and RNA sequencing. Formation of TLS was found in anti–double-stranded DNA antibody–positive mice, and the structures were organized as interconnected large networks with distinct T/B cell zones with adjacent dendritic cells, macrophages, plasma cells, high endothelial venules, supporting follicular dendritic cells network, and functional germinal centers. Comparison of gene profiles of whole kidney, renal TLS, and lymph nodes revealed a similar gene signature of TLS and lymph nodes. The up-regulated genes within the kidneys of lupus-prone mice during LN development reflected TLS formation, whereas the down-regulated genes were involved in metabolic processes of the kidney cells. A comparison with human LN gene expression revealed similar up-regulated genes as observed during the development of murine LN and TLS. In conclusion, kidney TLS have a similar cell composition, structure, and gene signature as lymph nodes and therefore may function as a kidney-specific type of lymph node

LMW Heparin Prevents Increased Kidney Expression of Proinflammatory Mediators in (NZBxNZW)F1 Mice

We have previously demonstrated that continuous infusion of low molecular weight (LMW) heparin delays autoantibody production and development of lupus nephritis in (NZBxNZW)F1 (B/W) mice. In this study we investigated the effect of LMW heparin on renal cytokine and chemokine expression and on nucleosome-mediated activation of nucleosome-specific splenocytes. Total mRNA extracted from kidneys of heparin-treated or -untreated B/W mice was analysed by qPCR for the expression of several cytokines, chemokines, and Toll-like receptors. Splenocytes taken from B/W mice were stimulated with nucleosomes with or without the presence of heparin. Splenocyte cell proliferation as thymidine incorporation and the expression of costimulatory molecules and cell activation markers were measured. Heparin treatment of B/W mice reduced the in vivo expression of CCR2, IL1�, and TLR7 compared to untreated B/W mice. Nucleosome-induced cell proliferation of splenocytes was not influenced by heparin. The expression of CD80, CD86, CD69, CD25, CTLA-4, and TLR 2, 7, 8, and 9 was upregulated upon stimulation by nucleosomes, irrespective of whether heparin was added to the cell culture or not. In conclusion, treatment with heparin lowers the kidney expression of proinflammatory mediators in B/W mice but does not affect nucleosomal activation of splenocytes

Mesenchymal stem cells and T cells in the formation of Tertiary Lymphoid Structures in Lupus Nephritis

Tertiary lymphoid structures (TLS) develop in the kidneys of lupus-prone mice and systemic lupus erythematosus (SLE) patients with lupus nephritis (LN). Here we investigated the presence of mesenchymal stem cells (MSCs) in the development of TLS in murine LN, as well as the role of human MSCs as lymphoid tissue organizer (LTo) cells on the activation of CD4+ T cells from three groups of donors including Healthy, SLE and LN patients. Mesenchymal stem like cells were detected within the pelvic wall and TLS in kidneys of lupus-prone mice. An increase in LTβ, CXCL13, CCL19, VCAM1 and ICAM1 gene expressions were detected during the development of murine LN. Human MSCs stimulated with the pro-inflammatory cytokines TNF-α and IL-1β significantly increased the expression of CCL19, VCAM1, ICAM1, TNF-α, and IL-1β. Stimulated MSCs induced proliferation of CD4+ T cells, but an inhibitory effect was observed when in co-culture with non-stimulated MSCs. A contact dependent increase in Th2 and Th17 subsets were observed for T cells from the Healthy group after co-culture with stimulated MSCs. Our data suggest that tissue-specific or/and migratory MSCs could have pivotal roles as LTo cells in accelerating early inflammatory processes and initiating the formation of kidney specific TLS in chronic inflammatory conditions

Positron emission tomography and single photon emission computed tomography imaging of tertiary lymphoid structures during the development of lupus nephritis

Lymphoid neogenesis occurs in tissues targeted by chronic inflammatory processes, such as infection and autoimmunity. In systemic lupus erythematosus (SLE), such structures develop within the kidneys of lupus-prone mice ((NZBXNZW)F1) and are observed in kidney biopsies taken from SLE patients with lupus nephritis (LN). The purpose of this prospective longitudinal animal study was to detect early kidney changes and tertiary lymphoid structures (TLS) using in vivo imaging. Positron emission tomography (PET) by tail vein injection of 18-F-fluoro-2-deoxy-D-glucose (18F-FDG)(PET/FDG) combined with computed tomography (CT) for anatomical localization and single photon emission computed tomography (SPECT) by intraperitoneal injection of 99mTC labeled Albumin Nanocoll (99mTC-Nanocoll) were performed on different disease stages of NZB/W mice (n = 40) and on aged matched control mice (BALB/c) (n = 20). By using one-way ANOVA analyses, we compared two different compartmental models for the quantitative measure of 18F-FDG uptake within the kidneys. Using a new five-compartment model, we observed that glomerular filtration of 18FFDG in lupus-prone mice decreased significantly by disease progression measured by anti-dsDNA Ab production and before onset of proteinuria. We could not visualize TLS within the kidneys, but we were able to visualize pancreatic TLS using 99mTC Nanocoll SPECT. Based on our findings, we conclude that the five-compartment model can be used to measure changes of FDG uptake within the kidney. However, new optimal PET/SPECT tracer administration sites together with more specific tracers in combination with magnetic resonance imaging (MRI) may make it possible to detect formation of TLS and LN before clinical manifestations