Figure 1.
Formation of tumors and lymph node metastases after subcutaneous implantation of 5×106 C33A-RFP cells.
Tumors and lymph nodes of six to seven mice per time point were analyzed (7 and 21 dpti: n = 7; 14, 28, 35 and 49 dpti: n = 6). a Bright field (BF), RFP and overlay images of C33A-RFP cells, scale bar represents 100 µm. b Time curve of C33A-RFP tumor growth. c Volume of lumbar and renal lymph nodes over time. d Representative images of tumors (upper row) and corresponding lymph nodes (lower row) of C33A-RFP tumor-bearing mice at the indicated days post tumor cell implantation. Images of tumors (T) were taken of living mice using the Maestro EX Imaging System. Imaging of lumbar (LN1, LN2) and renal (RN1, RN2) lymph node metastases in the abdomen of tumor-bearing mice was performed post mortem, after opening the abdomen and removing organs. Scale bars represent 2 mm. e Percentage of all lymph nodes positive for RFP over time. f Percentage of LN1, LN2, RN1 and RN2, respectively, positive for RFP per time point.
Figure 2.
Metastatic migration of the C33A-RFP cells in tumor-bearing nude mice.
a Migration of C33A-RFP cells in a lymph vessel connecting LN1 and RN1 42 dpti. Scale bars represent 2 mm. b LYVE-1 staining of 100 µm cross sections of the part between LN1 and RN1. Scale bars represent 200 µm. c Migration of C33A-RFP cells in a lymph (filled arrowhead) and in an erythrocyte containing blood vessel (open arrowhead) 32 dpti. Scale bars represent 2 mm (left) and 500 µm (right). d RFP signals in lungs of C33A-RFP tumor-bearing mice. Above: representative image of a lung 42 dpti. Scale bar represents 2 mm. Below: percentage of lungs tested positive for RFP spots over time. Lungs of six to seven mice per time point were examined (7 and 21 dpti: n = 7; 14, 28, 35 and 49 dpti: n = 6).
Figure 3.
RFP signals in kidneys of C33A-RFP tumor-bearing mice 7, 14, 21, 28 and 35 dpti.
Six to seven mice were analyzed per time point (7 and 21 dpti: n = 7; 14, 28 and 35 dpti: n = 6). a Representative images of RFP signal in kidneys. Upper row: overlay of bright field and RFP images. Lower row: images of RFP signal. Scale bars represent 2 mm. b Mean fluorescence intensity of the RFP signal in kidneys of C33A-RFP tumor-bearing mice over time.
Figure 4.
Histological analysis of tumors, LNs, RNs and kidneys of C33A-RFP tumor-bearing mice.
Nuclei in a, b and c were stained with Hoechst dye. a Overlays of bright field, RFP and Hoechst images of 10 µm sections of a tumor, LN, RN and kidney 31 dpti. Scale bars represent 50 µm. b Images of 100 µm sections of a kidney 31 dpti, stained with anti-CD31 antibody and Hoechst dye. Scale bar represents 2 mm. c 10 µm kidney section stained with Hoechst dye. Right: confocal image. Scale bars represent 100 µm (left) and 10 µm (right). d CD31 and Hoechst staining of 100 µm lung sections 42 dpti. Filled arrowhead: in a blood vessel migrating C33A-RFP cell; empty arrowhead: RFP positive fragments. Scale bars represent 250 µm (left) and 50 µm (right). All images are representative examples.
Figure 5.
Analysis of tumor, LN, RN, lung and kidney single cell suspensions from C33A-RFP tumor-bearing mice 49 dpti.
Tissues of all 5 mice were analyzed (n = 5). a Amount of RFP positive (RFP+) and negative (RFP-) cells in single cell suspensions of tumor, LN, RN, lung and kidneys. 10,000 events were analyzed by FACS. b Growth of different dilutions (10−1–10−4) of C33A-RFP single cell suspensions in wells of 24-well plates after one week of blasticidin selection. Left image: media consumption in wells containing 2 days old media. Right image: wells after staining with crystal violet. c Numbers of colony forming C33A-RFP cells per gram tumor, LN, RN, lung and kidney, respectively. Cell colonies were counted for each organ after blasticidin-selection and crystal violet staining.
Figure 6.
Influence of oncolytic virotherapy on C33A-RFP tumors and metastases.
a Time curve of C33A-RFP tumor growth in 12 mice after administration of the oncolytic vaccinia virus GLV-1h68 and PBS, respectively (n = 6). Analyses of C33A-RFP tumors and metastases in b-g were done 32 dpti and 21 days after administration of GLV-1h68 or PBS (dpi). Six mice were examined per group (n = 6). b Left: C33A-RFP tumor-bearing mice 21 dpi. Right: 100 µm sections of a PBS and a GLV-1h68 treated tumor. Nuclei were stained with Hoechst dye, GFP is expressed by GLV-1h68 and RFP by C33A cells. c Volume of lumbar and renal lymph nodes and d percentage of RFP positive lymph nodes in PBS and GLV-1h68 treated C33A-RFP tumor-bearing mice (left). Right: Images of lumbar and renal lymph nodes in PBS and GLV-1h68 treated mice. e 100 µm sections of a PBS (left) and a GLV-1h68 (right) treated lumbar lymph node metastasis. f Left: Percentage of kidneys positive for RFP in PBS and GLV-1h68 treated mice. Right: Images of kidneys in the PBS and virus group. g Percentage of lungs positive for RFP in PBS and GLV-1h68 group. All images are representative examples. All scale bars represent 2 mm.