LDN 193189 br that the mice were divided into six
that, the mice were divided into six groups and treated respectively as we described previously (Fig. 3).
3.4. Inhibition of lymphatic metastasis by lymphatic chemotherapy
Abundant lymphatic vessels are located in the footpad of nude mouse, which form unidirectional lymphatic drainage. Thus, there ex-ists a good anatomic basis of lymphatic metastasis and lymph che-motherapy. In our preliminary experiments, we injected Indian ink that could simulate and color lymph node into the footpad of nude mouse subcutaneously and found black colored lymph node located similar to the previous report . Given the characteristics of lymphatic me-tastasis in CRC, we mainly observed the subiliac lymph node, medial iliac lymph node, and renal lymph node in the mouse.
After the 18-day treatment, we dissected the mice, counted the number of lymph node and confirmed the metastatic lymph node with H&E staining. The results indicated that HD-E and MD-E produced a significantly greater inhibition eﬀect on LNM (P < 0.05) (Fig. 5). No diﬀerence was found between CI and NS. The concrete regional LNM rate of each group was shown in Table 2.
In addition, we also discovered the order and time of LNM. The HCT116 LDN 193189 first move to subiliac lymph node, and then to medial iliac lymph node, finally to the renal lymph node. 5 days after in-oculation, granule size of subiliac lymph node was palpated in each side of inguinal region (Fig. 6A) and another 5 days later, the metastatic ones were confirmed by H&E staining. At the end day of the experi-ment, 2 to 5 medial iliac lymph nodes were observed in iliac angio-somes (Fig. 6B) and 0 to 2 renal lymph nodes were observed in renal hilum after nephrectomy (Fig. 6C). However, the metastatic lymph node was only confirmed in minority of detected medial iliac lymph nodes. No renal lymph node metastasized was found until 6 weeks after inoculation. Histological analysis showed a massive tumor cells in-filtrated within the lymph nodes (Fig. 6D and E).
3.5. The eﬀects of HELT fraction emulsions on immune function
To assess the eﬀects of F18 emulsions at diﬀerent concentrations on immune function, we analyzed the SI value, NK cells and B cells. HD-E and MD-E promoted the proliferation of spleen cells as shown in Fig. 7. Moreover, the proportion of NK cells among peripheral blood was sig-nificantly higher in HD-E and MD-E group than others (P < 0.01,
Fig. 5. Number of lymph node metastases per mouse of each group. Closed circles indicate one mouse with histologically-positive lymphatic metastasis. Opened circles indicate one mouse with histologically-negative lymphatic me-tastasis. The lymph node metastasis rate was significantly reduced in HD-E group (P = 0.006) and MD-E group (P = 0.012). NS: normal saline; CI: Cinobufacini Injection; CE: control emulsion; LD-E: low-dose emulsion; MD-E: middle-dose emulsion; HD-E: high-dose emulsion.
Table 2 Regional lymph node metastases in diﬀerent groups.
17.65 Fig. 7. The spleen index (SI) of each group. The spleen index was significantly
9.80 saline; CI: Cinobufacini Injection; CE: control emulsion; LD-E: low-dose emul-
8.33 sion; MD-E: middle-dose emulsion; HD-E: high-dose emulsion.
Ln.: lymph node; m/d: metastatic/detected; NS: normal saline; CI: Cinobufacini
Injection; CE: control emulsion; LD-E: low-dose emulsion; MD-E: middle-dose
emulsion; HD-E: high-dose emulsion.
Fig. 8A). The proportion of NK cells among spleen cells was also sig-nificantly higher in comparison of LD-E, CE, and NS group (P < 0.05, Fig. 8B). The proportion of B cells among peripheral blood and spleen cells was accordingly reduced.
To evaluate the secrete function of NK cells and B cells, the content
of IFN-γ and IgG1 in serum and the splenic cell supernatant were also respectively detected (Table 3). The results suggested that HD-E and MD-E significantly enhanced the secretion of IFN-γ in serum and the splenic cell supernatant as well as IgG1 in serum.
3.6. The potential toxicity of lymphatic chemotherapy with HELT fraction emulsions
We performed the hematological examination and evaluation of liver and renal function. As shown in Fig. 9, HD-E and MD-E
Fig. 6. Regional lymph nodes in mouse. A. Enlarged subiliac lymph nodes (red dotted circles) 5 days after tumor challenging. B. Enlarged medial iliac lymph nodes (yellow dotted circle) 21 days after tumor challenging and iliac vessels (yellow arrows). C. Renal lymph nodes (yellow dotted circles) 21 days after tumor challenging. D and E. H&E staining of metastasized lymph node sections showed numerous lymphocytes accumulating in lymph node. D. ×100 magnification. E. ×400 magnification. (For inter-pretation of the references to color in this figure le-gend, the reader is referred to the web version of this article.)