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Early diagnosis of lymph node metastasis: Importance of intranodal pressures.

Miura Y, Mikada M, Ouchi T, Horie S, Takeda K, Yamaki T, Sakamoto M, Mori S, Kodama T - Cancer Sci. (2016)

Bottom Line: We found that intranodal pressure in the subiliac lymph node increased at the stage when metastasis was detected by in vivo bioluminescence, but when proper axillary lymph node volume (measured by high-frequency ultrasound imaging) had not increased significantly.Intravenously injected liposomes, encapsulating indocyanine green, were detected in solid tumors by in vivo bioluminescence, but not in the proper axillary lymph node.Basic blood vessel and lymphatic channel structures were maintained in the proper axillary lymph node, although sinus histiocytosis was detected.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.

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Leakage of indocyanine green (ICG) liposomes. Bioluminescence and fluorescence images of mice inoculated with KM‐Luc/GFP (A,C,E) or FM3A‐Luc (B,D,F) cells. Representative bioluminescence images: A(i), A(iii), B(i), and B(iii). Representative fluorescence images: A(ii), A(iv), B(ii), and B(iv). Solid tumor groups: A(i), A(ii), B(i), and B(ii). Metastasis groups: A(iii), A(iv), B(iii), and B(iv). Bioluminescence images were obtained 6 days after inoculation of KM‐Luc/GFP cells, and 14 days after inoculation of FM3A‐Luc cells. ICG liposomes were then injected i.v., and fluorescence images obtained 24 h later, that is, days 7 and 15 after inoculation of KM‐Luc/GFP and FM3A‐Luc cells, respectively. Quantification of luciferase activity (averaged values): KM‐Luc/GFP (C) and FM3A‐Luc (D). Luciferase activities were obtained 6 days after inoculation of KM‐Luc/GFP cells (**P < 0.01, solid tumor versus proper axillary lymph node [PALN]), and 14 days after inoculation of FM3A‐Luc cells (**P < 0.01, solid tumor versus PALN). E(i), E(ii), F(i), and F(ii): Following imaging, the subiliac lymph node (SiLN), PALN, and solid tumor were homogenized, and the fluorescence intensity of the supernatant measured by IVIS. E(i), F(i): Fluorescence images of individual wells of a 48‐well plate. E(ii), F(ii): Averaged values for the fluorescence/weight ratio. Mean ± SEM values are shown. **P < 0.01 versus solid tumor (one‐way anova and Tukey's test).
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cas12873-fig-0004: Leakage of indocyanine green (ICG) liposomes. Bioluminescence and fluorescence images of mice inoculated with KM‐Luc/GFP (A,C,E) or FM3A‐Luc (B,D,F) cells. Representative bioluminescence images: A(i), A(iii), B(i), and B(iii). Representative fluorescence images: A(ii), A(iv), B(ii), and B(iv). Solid tumor groups: A(i), A(ii), B(i), and B(ii). Metastasis groups: A(iii), A(iv), B(iii), and B(iv). Bioluminescence images were obtained 6 days after inoculation of KM‐Luc/GFP cells, and 14 days after inoculation of FM3A‐Luc cells. ICG liposomes were then injected i.v., and fluorescence images obtained 24 h later, that is, days 7 and 15 after inoculation of KM‐Luc/GFP and FM3A‐Luc cells, respectively. Quantification of luciferase activity (averaged values): KM‐Luc/GFP (C) and FM3A‐Luc (D). Luciferase activities were obtained 6 days after inoculation of KM‐Luc/GFP cells (**P < 0.01, solid tumor versus proper axillary lymph node [PALN]), and 14 days after inoculation of FM3A‐Luc cells (**P < 0.01, solid tumor versus PALN). E(i), E(ii), F(i), and F(ii): Following imaging, the subiliac lymph node (SiLN), PALN, and solid tumor were homogenized, and the fluorescence intensity of the supernatant measured by IVIS. E(i), F(i): Fluorescence images of individual wells of a 48‐well plate. E(ii), F(ii): Averaged values for the fluorescence/weight ratio. Mean ± SEM values are shown. **P < 0.01 versus solid tumor (one‐way anova and Tukey's test).

Mentions: Angiogenesis, a hallmark of cancer,22, 23 leads to immature, hyperpermeable tumor vessels and an elevation of interstitial fluid pressure in a solid tumor. To examine the enhanced permeability and retention (EPR) effect24 during the early stages of LN metastasis, we injected ICG liposomes i.v. and compared their leakages from the SiLN, PALN, and solid tumor. For the KM‐Luc/GFP group at day 6, the average sizes of the solid tumor, SiLN, and PALN were 182.81 ± 49.15 mm3 (n = 5), 358.76 ± 73.69 mm3 (n = 5), and 195.49 ± 34.78 mm3 (n = 5), respectively. For the FM3A‐Luc group at day 14, the average sizes of the solid tumor, SiLN, and PALN were 165.18 ± 87.74 mm3 (n = 5), 411.41 ± 33.00 mm3 (n = 5), and 157.90 ± 27.45 mm3 (n = 5), respectively. There were no significant differences in LN size between the KM‐Luc/GFP and FM3A‐Luc groups (one‐way anova). First, we used in vivo bioluminescence imaging to confirm solid tumor growth (Fig. 4A(i),B(i)) and metastasis progression (Fig. 4A(iii),B(iii)). Twenty four hours after injection, ICG liposomes were detected around the solid tumor (Fig. 4A(ii),B(ii)), but did not accumulate in the SiLN and PALN (Fig. 4A(iv),B(iv)). Figure 4(C,D) shows the luciferase activities of solid tumor (Fig. 4A(i),B(i)), SiLN, and PALN (Fig. 4A(iii),B(iii)). There were no significant differences in luciferase activities between solid tumor and SiLN (a site of tumor injection), whereas the luciferase activity of the solid tumor was significantly higher than that of the PALN (a site of metastasis) (P < 0.01, solid tumor versus PALN). Measurement of fluorescence / weight ratios in homogenized tissues revealed significant differences between solid tumor, SiLN, and PALN (Fig. 4E,F; P < 0.01, versus solid tumor). Thus, under the present conditions, the EPR effect that accompanies angiogenesis in solid tumors was not detected in the SiLN or PALN.


Early diagnosis of lymph node metastasis: Importance of intranodal pressures.

Miura Y, Mikada M, Ouchi T, Horie S, Takeda K, Yamaki T, Sakamoto M, Mori S, Kodama T - Cancer Sci. (2016)

Leakage of indocyanine green (ICG) liposomes. Bioluminescence and fluorescence images of mice inoculated with KM‐Luc/GFP (A,C,E) or FM3A‐Luc (B,D,F) cells. Representative bioluminescence images: A(i), A(iii), B(i), and B(iii). Representative fluorescence images: A(ii), A(iv), B(ii), and B(iv). Solid tumor groups: A(i), A(ii), B(i), and B(ii). Metastasis groups: A(iii), A(iv), B(iii), and B(iv). Bioluminescence images were obtained 6 days after inoculation of KM‐Luc/GFP cells, and 14 days after inoculation of FM3A‐Luc cells. ICG liposomes were then injected i.v., and fluorescence images obtained 24 h later, that is, days 7 and 15 after inoculation of KM‐Luc/GFP and FM3A‐Luc cells, respectively. Quantification of luciferase activity (averaged values): KM‐Luc/GFP (C) and FM3A‐Luc (D). Luciferase activities were obtained 6 days after inoculation of KM‐Luc/GFP cells (**P < 0.01, solid tumor versus proper axillary lymph node [PALN]), and 14 days after inoculation of FM3A‐Luc cells (**P < 0.01, solid tumor versus PALN). E(i), E(ii), F(i), and F(ii): Following imaging, the subiliac lymph node (SiLN), PALN, and solid tumor were homogenized, and the fluorescence intensity of the supernatant measured by IVIS. E(i), F(i): Fluorescence images of individual wells of a 48‐well plate. E(ii), F(ii): Averaged values for the fluorescence/weight ratio. Mean ± SEM values are shown. **P < 0.01 versus solid tumor (one‐way anova and Tukey's test).
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cas12873-fig-0004: Leakage of indocyanine green (ICG) liposomes. Bioluminescence and fluorescence images of mice inoculated with KM‐Luc/GFP (A,C,E) or FM3A‐Luc (B,D,F) cells. Representative bioluminescence images: A(i), A(iii), B(i), and B(iii). Representative fluorescence images: A(ii), A(iv), B(ii), and B(iv). Solid tumor groups: A(i), A(ii), B(i), and B(ii). Metastasis groups: A(iii), A(iv), B(iii), and B(iv). Bioluminescence images were obtained 6 days after inoculation of KM‐Luc/GFP cells, and 14 days after inoculation of FM3A‐Luc cells. ICG liposomes were then injected i.v., and fluorescence images obtained 24 h later, that is, days 7 and 15 after inoculation of KM‐Luc/GFP and FM3A‐Luc cells, respectively. Quantification of luciferase activity (averaged values): KM‐Luc/GFP (C) and FM3A‐Luc (D). Luciferase activities were obtained 6 days after inoculation of KM‐Luc/GFP cells (**P < 0.01, solid tumor versus proper axillary lymph node [PALN]), and 14 days after inoculation of FM3A‐Luc cells (**P < 0.01, solid tumor versus PALN). E(i), E(ii), F(i), and F(ii): Following imaging, the subiliac lymph node (SiLN), PALN, and solid tumor were homogenized, and the fluorescence intensity of the supernatant measured by IVIS. E(i), F(i): Fluorescence images of individual wells of a 48‐well plate. E(ii), F(ii): Averaged values for the fluorescence/weight ratio. Mean ± SEM values are shown. **P < 0.01 versus solid tumor (one‐way anova and Tukey's test).
Mentions: Angiogenesis, a hallmark of cancer,22, 23 leads to immature, hyperpermeable tumor vessels and an elevation of interstitial fluid pressure in a solid tumor. To examine the enhanced permeability and retention (EPR) effect24 during the early stages of LN metastasis, we injected ICG liposomes i.v. and compared their leakages from the SiLN, PALN, and solid tumor. For the KM‐Luc/GFP group at day 6, the average sizes of the solid tumor, SiLN, and PALN were 182.81 ± 49.15 mm3 (n = 5), 358.76 ± 73.69 mm3 (n = 5), and 195.49 ± 34.78 mm3 (n = 5), respectively. For the FM3A‐Luc group at day 14, the average sizes of the solid tumor, SiLN, and PALN were 165.18 ± 87.74 mm3 (n = 5), 411.41 ± 33.00 mm3 (n = 5), and 157.90 ± 27.45 mm3 (n = 5), respectively. There were no significant differences in LN size between the KM‐Luc/GFP and FM3A‐Luc groups (one‐way anova). First, we used in vivo bioluminescence imaging to confirm solid tumor growth (Fig. 4A(i),B(i)) and metastasis progression (Fig. 4A(iii),B(iii)). Twenty four hours after injection, ICG liposomes were detected around the solid tumor (Fig. 4A(ii),B(ii)), but did not accumulate in the SiLN and PALN (Fig. 4A(iv),B(iv)). Figure 4(C,D) shows the luciferase activities of solid tumor (Fig. 4A(i),B(i)), SiLN, and PALN (Fig. 4A(iii),B(iii)). There were no significant differences in luciferase activities between solid tumor and SiLN (a site of tumor injection), whereas the luciferase activity of the solid tumor was significantly higher than that of the PALN (a site of metastasis) (P < 0.01, solid tumor versus PALN). Measurement of fluorescence / weight ratios in homogenized tissues revealed significant differences between solid tumor, SiLN, and PALN (Fig. 4E,F; P < 0.01, versus solid tumor). Thus, under the present conditions, the EPR effect that accompanies angiogenesis in solid tumors was not detected in the SiLN or PALN.

Bottom Line: We found that intranodal pressure in the subiliac lymph node increased at the stage when metastasis was detected by in vivo bioluminescence, but when proper axillary lymph node volume (measured by high-frequency ultrasound imaging) had not increased significantly.Intravenously injected liposomes, encapsulating indocyanine green, were detected in solid tumors by in vivo bioluminescence, but not in the proper axillary lymph node.Basic blood vessel and lymphatic channel structures were maintained in the proper axillary lymph node, although sinus histiocytosis was detected.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.

Show MeSH
Related in: MedlinePlus