Migration Properties Distinguish Tumor Cells of Classical Hodgkin Lymphoma from Anaplastic Large Cell Lymphoma Cells

Abstract

Anaplastic large cell lymphoma (ALCL) and classical Hodgkin lymphoma (cHL) are lymphomas that contain CD30-expressing tumor cells and have numerous pathological similarities. Whereas ALCL is usually diagnosed at an advanced stage, cHL more frequently presents with localized disease. The aim of the present study was to elucidate the mechanisms underlying the different clinical presentation of ALCL and cHL. Chemokine and chemokine receptor expression were similar in primary ALCL and cHL cases apart from the known overexpression of the chemokines CCL17 and CCL22 in the Hodgkin and Reed-Sternberg (HRS) cells of cHL. Consistent with the overexpression of these chemokines, primary cHL cases encountered a significantly denser T cell microenvironment than ALCL. Additionally to differences in the interaction with their microenvironment, cHL cell lines presented a lower and less efficient intrinsic cell motility than ALCL cell lines, as assessed by time-lapse microscopy in a collagen gel and transwell migration assays. We thus propose that the combination of impaired basal cell motility and differences in the interaction with the microenvironment hamper the dissemination of HRS cells in cHL when compared with the tumor cells of ALCL.

Keywords: anaplastic large cell lymphoma; cell motility; chemokine receptors; classical Hodgkin lymphoma; dissemination; gene expression; image analysis; rosetting T cells; segmentation.

Figure 1

Chemokine receptor and ligand expression in primary cases of ALCL and cHL mixed cellularity as well as cell lines L-1236, L-428, DEL and SU-DHL-1. (A). Representative examples of chemokine receptor expression of CXCR3, CCR4, CCR5 and CCR1 in cases of ALCL and cHL. (B). Apart from CCL17 and CCL22, which are negative in ALCL, most corresponding chemokines are expressed in cases of ALCL. Inserts show the typical strong expression of CCL17 and CCL22 in cHL. (C). Examples of chemokine arrays showing strong secretion of CCL17 in cHL cell lines L-1236 and L-428 in contrast to ALCL cell lines DEL and SU-DHL-1.

Figure 2

Composition of the microenvironment differs between ALCL and cHL. (A,D,G) Examples of CD4-immunostainings (200× magnification) in cHL (mixed cellularity subtype), ALK⁻ and ALK⁺ ALCL with higher numbers of CD4-positive T cells in the cHL case. (B,E,H) Examples of CD8-immunostainings (200× magnification) in cHL (mixed cellularity subtype), ALK⁻ and ALK⁺ ALCL with the highest number of CD8-positive T cells in the cHL case. (C,F,I) Examples of CD163-immunostainings (200× magnification) in cHL (mixed cellularity subtype), ALK⁻ and ALK⁺ ALCL. In this example the ALK⁻ ALCL case has the highest number of CD163-positive macrophages. (J). Quantification of CD4-positive T cells/mm² revealing significantly higher numbers of CD4-positive T cells in the microenvironment of mixed cellularity cHL cases (n = 15) when compared with ALK⁺ (n = 10) and ALK⁻ ALCL (n = 12) (Mann-Whitney test, *** p < 0.001). Medians, minimums and maximums are plotted. (K). Quantification of CD8-positive T cells/mm² revealing significantly higher numbers of CD8-positive T cells in the microenvironment of mixed cellularity cHL cases (n = 15) when compared with ALK⁺ (n = 11) and ALK⁻ ALCL (n = 14) (Mann-Whitney test, *** p < 0.001). Medians, minimums and maximums are plotted. (L). Quantification of CD163-positive macrophages/mm². CD163-positive macrophages were significantly more abundant in the microenvironment of mixed cellularity cHL cases (n = 15) when compared with ALK⁻ ALCL (n = 13) (Mann-Whitney test, ** p < 0.01). The difference observed in ALK⁺ ALCL (n = 9) was not significant. Medians, minimums and maximums are plotted.

Figure 3

Clusters with CD4-positive T cells are more prominent with cHL cell lines L-1236 and L-428 when compared with ALCL cell lines DEL and SU-DHL-1. (A). Examples of clusters in the different cell lines on day 1 compared with monoculture of the corresponding cell lines. When CSFE-labeled tumor cells get in contact with red-labeled CD4⁺ T cells, a yellow color is created by overlay. The Burkitt lymphoma cell line Ramos was used as a negative control. (B). Mean cluster size on day 1. Clusters observed with the cHL cell lines L-428 and L-1236 were larger when compared with ALCL cell lines DEL and SU-DHL-1 (Mann-Whitney-test, * p < 0.05, *** p < 0.001). Means and standard error of means of four independent experiments with three to five images per experiment, 40× magnification. (C). Mean cluster size at day 4 with and without addition of blocking antibodies against CD2 and CD58. Clusters with the cHL cell lines L-1236 and L-428 are significantly larger than those seen with ALCL cell lines DEL and SU-DHL-1 at baseline conditions. Addition of either blocking antibody anti-CD2 or anti-CD58 resulted in a significantly reduced cluster size in cHL cell lines (one-way Anova with Bonferroni´s multiple comparison test. *** p < 0.001). Means and standard error of means of three images per experiment in three independent experiments, 10× magnification.

Figure 4

Filopodia-like structures are more frequent, longer and have a more orthogonal orientation in cHL cell lines L-1236 and L-428 compared with ALCL cell lines DEL and SU-DHL-1, which are embedded in a collagen gel. (A). Size distribution of the cell lines is wider in L-1236 and L-428 with on average larger cells compared with ALCL cell lines SU-DHL-1 and DEL. Cell sizes were determined in in 36–56 GFP-Life-Act-labeled cells in each of four independent experiments by spinning disk microscopy. Means and standard deviation. (B). The number of filopodia-like structures/perimeter of the main cell body is significantly higher in the cHL cell lines L-1236 and L-428 when compared with the ALCL cell lines SU-DHL-1 and DEL (Mann-Whitney-U-test, * p < 0.05). Small fragments < 10 µm were devoid of nuclear material, thus probably representing large oncosomes atypically large extracellular vesicles observed in cancer cell lines. A total of 36–56 GFP-Life-Act-labeled cells were analyzed each in four independent experiments by spinning disk microscopy. Means and standard error of means are plotted. (C). The average length of filopodia-like structures is significantly higher in both cHL cell lines L-1236 and L-428 when compared with ALCL cell lines SU-DHL-1 and DEL (Mann-Whitney-U-test, * p < 0.05). A total of 36–56 GFP-Life-Act-labeled cells were analyzed each in four independent experiments by spinning disk microscopy. Means and standard error of means are plotted. (D). Examples of the segmentation analysis of filopodia-like structures in the cell lines L-1236 and DEL. (E). The mean angle between filopodia-like structures and the orthogonal axis to the cell center is significantly lower in cHL cell lines L-1236 and L-428 when compared with with ALCL cell lines SU-DHL-1 and DEL, indicating a more straight orientation of filopodia-like structures in the cHL cells (Mann-Whitney-U-test, * p < 0.05). A total of 36–56 GFP-Life-Act-labeled cells were analyzed each in four independent experiments by spinning disk microscopy. Means and standard error of means are plotted.

Figure 5

Intrinsic migration properties of ALCL and cHL cell lines in collagen gel and transwell chambers with FCS gradient. (A). Basic velocity determined by tracking of time lapse microscopy of ALCL and cHL cell lines embedded in a collagen type I gel in Ibidi chemotaxis slides (* p < 0.05, ** p < 0.01, *** p < 0.001, one-way-Anova, with a Bonferroni-Holm p-value adjustment for multiple comparisons). Means and standard error of means of three independent experiments. (B). Straightness (euclidean distance/accumulated distance) of cell movements in a collagen type I gel in Ibidi chemotaxis slides determined by tracking of time lapse microscopy. Means and standard error of means of three independent experiments. (C). Example of tracks of the ALCL cell line DEL in bovine collagen type I gel in Ibidi chemotaxis slides determined by tracking of time lapse microscopy. (D). Example of tracks of the cHL cell line L-1236 in bovine collagen type I gel in Ibidi chemotaxis slides determined by tracking of time lapse microscopy. (E). Efficiency of migration versus an FCS gradient of ALCL and cHL cell lines in an uncoated transwell of 8 µm pore size. ALCL cell lines showed a higher migration efficiency, which was significantly higher for SU-DHL-1, MAC1 and DEL compared with L-1236 (* p < 0.05, ** p < 0.001, *** p < 0.0001, Kruskal-Wallis-test). Means and standard error of means of three independent experiments.