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Progression of carcinogen-induced fibrosarcomas is associated with the accumulation of naïve CD4+ T cells via blood vessels and lymphatics.

Ondondo B, Jones E, Hindley J, Cutting S, Smart K, Bridgeman H, Matthews KK, Ladell K, Price DA, Jackson DG, Godkin A, Ager A, Gallimore A - Int. J. Cancer (2013)

Bottom Line: In our study, we utilized a mouse model of carcinogen-induced fibrosarcoma to examine the composition of tumor-infiltrating lymphocytes during tumor progression.In particular, we sought to determine whether CD4(+) Foxp3(+) regulatory T cells (Tregs) became enriched during tumor progression thereby contributing to tumor-driven immunosuppression.However, we found no evidence for antigen-driven migration of these T cells or for their participation in an antitumor immune response.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom.

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Afferent lymphatic vessel function detected in MCA‐induced tumors. Evans Blue was injected into the base of the leg and approximately 60 min later the tumor and draining lymph nodes were removed. Frozen sections were prepared and examined for Evans Blue autofluorescence and also stained for LYVE‐1 and CD31 (a) and MOMA‐1 and LYVE‐1 (b). FITC–dextran (5,000 kDa) was injected into the base of the leg and approximately 60 min later the tumor and draining lymph nodes were removed. Frozen sections were prepared and stained for LYVE‐1 and were also counter stained with TOTO‐3 to detect DNA. Low‐power images are shown on the left and high‐power images of the area in the white box are shown on the right (c). Scale bars a, b, and c left: 100 µm. Scale bars c right: 20 µm.
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ijc28556-fig-0004: Afferent lymphatic vessel function detected in MCA‐induced tumors. Evans Blue was injected into the base of the leg and approximately 60 min later the tumor and draining lymph nodes were removed. Frozen sections were prepared and examined for Evans Blue autofluorescence and also stained for LYVE‐1 and CD31 (a) and MOMA‐1 and LYVE‐1 (b). FITC–dextran (5,000 kDa) was injected into the base of the leg and approximately 60 min later the tumor and draining lymph nodes were removed. Frozen sections were prepared and stained for LYVE‐1 and were also counter stained with TOTO‐3 to detect DNA. Low‐power images are shown on the left and high‐power images of the area in the white box are shown on the right (c). Scale bars a, b, and c left: 100 µm. Scale bars c right: 20 µm.

Mentions: Tumor development is also associated with lymphatic hyperplasia and the sprouting of new lymphatic vessels, processes that facilitate dissemination of cells from the primary tumor via peritumoral lymphatics, and metastasis to tumor draining lymph nodes (reviewed in Ref. 16). Here, we considered the novel possibility that the development of an expanded network of lymphatics in and around fibrosarcomas might facilitate entry of naïve T cells from neighboring tissues via afferent lymph. In support of this hypothesis, naïve T cells have been reported in afferent lymph, albeit in lower numbers than memory cells.17 Uptake of lymph into fibrosarcomas was assessed by monitoring the accumulation of Evans Blue Dye,18 after injection of the dye into the base of the leg of a tumor‐bearing mouse (Supporting Information Fig. 2). After approximately 1 hr, the primary tumor, as well as the draining ipsilateral and contralateral inguinal lymph nodes, was removed for examination (Supporting Information Fig. 2). Upon macroscopic inspection, tumors, as well as the draining lymph nodes, were visibly blue (Supporting Information Fig. 2). To confirm dye uptake by the tumor, Evans Blue Dye was extracted and measured by spectrometry (Supporting Information Fig. 2). Finally, as Evans Blue Dye is autofluorescent, sections were prepared from tumors and inguinal lymph nodes for examination by confocal microscopy. Uptake of dye in both tumors and draining lymph nodes was clearly observed (Figs. 4a and 4b). To further examine the tumors for the presence of a lymphatic network, tumors from Evans Blue‐injected mice were costained with LYVE‐1‐, CD31‐ and MOMA‐1‐specific antibodies. Colocalization of LYVE‐1 with both CD31 and MOMA‐1 was observed, indicating the development of lymphatic vessels and a subcapsular‐sinus‐like structure within the tumor. To further confirm that the lymphatic vessels can deliver lymph constituents to the tumor, we injected dextran–FITC into the base of the leg of a tumor‐bearing mouse and stained recovered tumors with LYVE‐1‐specific antibodies. As shown in Figure 4c, we observed colocalization of dextran–FITC and LYVE‐1 within both draining lymph nodes and tumors. Moreover, dextran–FITC could also be observed intratumorally, indicating that lymph constituents can enter the tumor body (Fig. 4c).


Progression of carcinogen-induced fibrosarcomas is associated with the accumulation of naïve CD4+ T cells via blood vessels and lymphatics.

Ondondo B, Jones E, Hindley J, Cutting S, Smart K, Bridgeman H, Matthews KK, Ladell K, Price DA, Jackson DG, Godkin A, Ager A, Gallimore A - Int. J. Cancer (2013)

Afferent lymphatic vessel function detected in MCA‐induced tumors. Evans Blue was injected into the base of the leg and approximately 60 min later the tumor and draining lymph nodes were removed. Frozen sections were prepared and examined for Evans Blue autofluorescence and also stained for LYVE‐1 and CD31 (a) and MOMA‐1 and LYVE‐1 (b). FITC–dextran (5,000 kDa) was injected into the base of the leg and approximately 60 min later the tumor and draining lymph nodes were removed. Frozen sections were prepared and stained for LYVE‐1 and were also counter stained with TOTO‐3 to detect DNA. Low‐power images are shown on the left and high‐power images of the area in the white box are shown on the right (c). Scale bars a, b, and c left: 100 µm. Scale bars c right: 20 µm.
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ijc28556-fig-0004: Afferent lymphatic vessel function detected in MCA‐induced tumors. Evans Blue was injected into the base of the leg and approximately 60 min later the tumor and draining lymph nodes were removed. Frozen sections were prepared and examined for Evans Blue autofluorescence and also stained for LYVE‐1 and CD31 (a) and MOMA‐1 and LYVE‐1 (b). FITC–dextran (5,000 kDa) was injected into the base of the leg and approximately 60 min later the tumor and draining lymph nodes were removed. Frozen sections were prepared and stained for LYVE‐1 and were also counter stained with TOTO‐3 to detect DNA. Low‐power images are shown on the left and high‐power images of the area in the white box are shown on the right (c). Scale bars a, b, and c left: 100 µm. Scale bars c right: 20 µm.
Mentions: Tumor development is also associated with lymphatic hyperplasia and the sprouting of new lymphatic vessels, processes that facilitate dissemination of cells from the primary tumor via peritumoral lymphatics, and metastasis to tumor draining lymph nodes (reviewed in Ref. 16). Here, we considered the novel possibility that the development of an expanded network of lymphatics in and around fibrosarcomas might facilitate entry of naïve T cells from neighboring tissues via afferent lymph. In support of this hypothesis, naïve T cells have been reported in afferent lymph, albeit in lower numbers than memory cells.17 Uptake of lymph into fibrosarcomas was assessed by monitoring the accumulation of Evans Blue Dye,18 after injection of the dye into the base of the leg of a tumor‐bearing mouse (Supporting Information Fig. 2). After approximately 1 hr, the primary tumor, as well as the draining ipsilateral and contralateral inguinal lymph nodes, was removed for examination (Supporting Information Fig. 2). Upon macroscopic inspection, tumors, as well as the draining lymph nodes, were visibly blue (Supporting Information Fig. 2). To confirm dye uptake by the tumor, Evans Blue Dye was extracted and measured by spectrometry (Supporting Information Fig. 2). Finally, as Evans Blue Dye is autofluorescent, sections were prepared from tumors and inguinal lymph nodes for examination by confocal microscopy. Uptake of dye in both tumors and draining lymph nodes was clearly observed (Figs. 4a and 4b). To further examine the tumors for the presence of a lymphatic network, tumors from Evans Blue‐injected mice were costained with LYVE‐1‐, CD31‐ and MOMA‐1‐specific antibodies. Colocalization of LYVE‐1 with both CD31 and MOMA‐1 was observed, indicating the development of lymphatic vessels and a subcapsular‐sinus‐like structure within the tumor. To further confirm that the lymphatic vessels can deliver lymph constituents to the tumor, we injected dextran–FITC into the base of the leg of a tumor‐bearing mouse and stained recovered tumors with LYVE‐1‐specific antibodies. As shown in Figure 4c, we observed colocalization of dextran–FITC and LYVE‐1 within both draining lymph nodes and tumors. Moreover, dextran–FITC could also be observed intratumorally, indicating that lymph constituents can enter the tumor body (Fig. 4c).

Bottom Line: In our study, we utilized a mouse model of carcinogen-induced fibrosarcoma to examine the composition of tumor-infiltrating lymphocytes during tumor progression.In particular, we sought to determine whether CD4(+) Foxp3(+) regulatory T cells (Tregs) became enriched during tumor progression thereby contributing to tumor-driven immunosuppression.However, we found no evidence for antigen-driven migration of these T cells or for their participation in an antitumor immune response.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom.

Show MeSH
Related in: MedlinePlus