Table 1.
Characteristics of the patient cohort.
Figure 1.
SCLC cell line OH-1 binds to selectins and expresses CEA.
OH-1 cells, as a cell line of neuroectodermal origin, are positive for NCAM and EpCAM, which could be detected by flow cytometry analysis and can thus serve as a marker for these cells when grown in mice. Cells show binding to E- and P-selectin fusion protein. The glycosylation motif sialyl Lewis A, a binding partner recognized by selectins, could be detected with the CA19-9 antibody. The cell line OH-1 exhibits several known proteins which known to be protein backbones for selectin binding carbohydrate residues such as PSGL-1, MUC18, CD44 and CEA. Isotype controls are shown as dotted lines.
Figure 2.
OH-1-LUC/mCherry cells grown in vivo are CEA and E-/P- selectin binding positive.
Isolated OH-1-LUC/mCherry cells of primary tumors were stained in parallel against CEA and E- or P-selectin binding and FACS analyzed. (A) OH-1-LUC/mCherry cells showed no binding to isotype and FC-chimaera controls. (B) Almost all cells were CEA and E-selectin binding positive, by contrast (C) two thirds of the cells were P-selectin binding negative. Isotype controls are shown as dotted lines.
Figure 3.
SCLC cells behave similar to leukocytes in murine mesenteric veins.
Rolling behavior of injected fluorescent OH-1-LUC/mCherry cells (mCherry, red) in mesenteric veins (reflection mode, grey) were visualized in real time with high speed confocal intravital imaging. (A) The rolling behavior of one OH-1-LUC/mCherry cell were observed over 27 seconds. (B) Mean rolling velocity of injected OH-1-LUC/mCherry cells and endogenous leukocytes in mesenteric veins were determined with high speed intravital imaging (Nikon A1R confocal microscope). Scale bar: 500 µm for (F) and (H), 50 µm for (G).
Figure 4.
SCLC cell line OH-1 spontaneously metastasizes to lung and bone in immunodeficient mice.
OH-1-LUC/mCherry cells were subcutaneously implanted (A) and grown as a primary tumor (a) for 18 days at the site between the scapulae (b) with a hyper-intensive appearance in a two-dimensional turbo spin-echo (TSE) sequence (MR images in axial orientation) and a positive bioluminescence signal (B) of the corresponding OH-1-LUC/mCherry tumor as in panel A. The primary tumor was surgically removed 51 days after OH-1-LUC/mCherry cell injection. (F) Corresponding paraffin sections of the resected tumor revealed living tumor cells (e) around a central necrosis (f). (C) Luminescence signals of metastasizing OH-1-LUC/mCherry cells in vivo were detectable in the areas of snout, distal femur and chest 117 days after injection. Organs were removed and bioluminescent signals were confirmed ex vivo. (D) The lungs showed several luminescence spots (c), while the heart (d) did not show any sign of luminescence. (G) H&E staining of corresponding lung sections showed a a small metastatic deposit surrounded by normal lung tissue (g). (E) Luminescence from the tibia at the knee joint could be detected and (H) H&E staining of a corresponding bone paraffin section showed metastasized OH-1-LUC/mCherry cells (i) next to healthy bone marrow (h). Scale bars: 500 µm for (F) and (H), 50 µm for (G).
Figure 5.
Metastasis rate of SCLC cells is reduced in E-/P- selectin knockout mice.
OH-1-LUC/mCherry cells were subcutaneously injected in E-/P-selectin double knockout mice (select) or wild type litter mice (wt) and resulting tumors grown for 31–41 days. Tumors were resected and weighted. (A) No statistical difference concerning tumor weight could be determined (Students t test, p = 0.5025). Tumor resected mice lived until they reached end point criteria. (B) Kaplan Meier survival curve shows significantly decreased median survival of wt (n = 20) mice compared with select (n = 20) mice (log-rank test, p<0.0001). The organs were removed and a significant difference comparing wt (n = 12) and select mice (n = 18) were determined in terms of bioluminescence of the removed lungs (C) and legs (D) (Mann-Whitney test, p = 0.0003). In a parallel approach OH-1 cells were subcutaneously injected in select (n = 5) or wt (n = 3) mice and tumors grown for 36–65 days. Organs were removed and the tumor weight was determined. There was no statistically significant difference in the weight of the primary OH-1 tumors between mouse strains (E) (students t test, p = 0.6489). The numbers of spontaneous lung metastases in select and wt mice were determined. Note that the number of metastases was statistically significantly reduced by 50% in select mice in comparison with wild type mice (F) (students t test, p = 0.0181).
Table 2.
Summary of all cases included in the Kaplan-Meier analysis of wild type (wt) against E-/P-selectin knockout (select) mice.
Figure 6.
Spontaneous OH-1 metastases express CEA.
Primary OH-1 tumor as well as spontaneous OH-1 lung and bone metastases were positively stained for CD44, CEA, EpCAM and PGP9.5 (red = positive cells). Metastases are indicated by red asterisks. Scale bar: 50 µm for all panels.
Figure 7.
Decreased survival of SCLC patients with moderate CEA expression levels.
TMA of SCLC patients was immunohistochemically stained against CEA. Three patient groups were discriminated: (D) no CEA expression (CEA−), (E) moderate CEA expression (CEA+) and (F) strong CEA expression (CEA++). Kaplan Meier survival curves show significantly decreased median survival of patients with moderate CEA levels compared to patients with (A) negative (log-rank test, p = 0.0054) or (B) strong CEA levels (log-rank test, p = 0.0002), but no significant difference between median survival of patients (C) with negative compared to strong CEA levels (log-rank test, p = 0.1016). In parallel a TMA of SCLC was immunohistochemically stained against CD44. Two patient groups were discriminated: (H) CD44 negative (CD44−) and (I) CD44 positive (CD44+). Kaplan Meier survival curve shows no statistical different median survival of patients (G) with negative CD44 levels compared with patients with positive CD44 levels (log-rank test, p = 0.7). Scale bar = 200 µm.
Table 3.
Summary of all cases included in the Kaplan-Meier analysis of CEA and CD44.