T-cell-derived Hodgkin lymphoma has motility characteristics intermediate between Hodgkin and anaplastic large cell lymphoma

Abstract

Classic Hodgkin lymphoma (cHL) is usually characterized by a low tumour cell content, derived from crippled germinal centre B cells. Rare cases have been described in which the tumour cells show clonal T-cell receptor rearrangements. From a clinicopathological perspective, it is unclear if these cases should be classified as cHL or anaplastic large T-cell lymphoma (ALCL). Since we recently observed differences in the motility of ALCL and cHL tumour cells, here, we aimed to obtain a better understanding of T-cell-derived cHL by investigating their global proteomic profiles and their motility. In a proteomics analysis, when only motility-associated proteins were regarded, T-cell-derived cHL cell lines showed the highest similarity to ALK^- ALCL cell lines. In contrast, T-cell-derived cHL cell lines presented a very low overall motility, similar to that observed in conventional cHL. Whereas all ALCL cell lines, as well as T-cell-derived cHL, predominantly presented an amoeboid migration pattern with uropod at the rear, conventional cHL never presented with uropods. The migration of ALCL cell lines was strongly impaired upon application of different inhibitors. This effect was less pronounced in cHL cell lines and almost invisible in T-cell-derived cHL. In summary, our cell line-derived data suggest that based on proteomics and migration behaviour, T-cell-derived cHL is a neoplasm that shares features with both cHL and ALCL and is not an ALCL with low tumour cell content. Complementary clinical studies on this lymphoma are warranted.

Keywords: Hodgkin lymphoma; anaplastic large cell lymphoma; motility.

FIGURE 1

Unsupervised hierarchical clusters of ALCL and Hodgkin cell lines according to proteomics data. (A) Unsupervised hierarchical clustering based on all quantified protein groups. (B) Unsupervised hierarchical clustering after mapping of proteins related to cell movement

FIGURE 2

Baseline movement characteristics of ALCL and Hodgkin cell lines and movement after inhibitor treatment in straight microchannels with height 10 µm and width 8 µm. (A) Step based velocity of ALK⁺, ALK⁻ ALCL and Hodgkin cell lines. At least three independent experiments per cell line (***p < 0.001, **p < 0.01, one‐way ANOVA with Bonferroni´s post‐test for multiple comparisons). (B) Straightness of ALK⁺, ALK⁻ ALCL and Hodgkin cell lines. At least three independent experiments per cell line (***p < 0.001, **p < 0.01, one‐way ANOVA with Bonferroni´s post‐test for multiple comparisons). (C). Step based velocity of ALK⁺, ALK⁻ ALCL and Hodgkin cell lines after treatment with the ROCK inhibitor Y27632 (30 µM). At least three independent experiments per cell line (*p < 0.05, **p < 0.01, Mann–Whitney test). (D) Step based velocity of ALK⁺, ALK⁻ ALCL and Hodgkin cell lines after treatment with the myosin II inhibitor Blebbistatin (15 µM). At least three independent experiments per cell line (**p < 0.01, Mann–Whitney test). (E) Step based velocity of ALK⁺, ALK⁻ ALCL and Hodgkin cell lines after treatment with the actin inhibitor CK‐666 (50 µM). At least three independent experiments per cell line (**p < 0.01, Mann–Whitney test). (F) Straightness of ALK⁺, ALK⁻ ALCL and Hodgkin cell lines after treatment with the actin inhibitor CK‐666 (50 µM). At least three independent experiments per cell line (*p < 0.05, Mann–Whitney test). Labelled in grey: ALK⁺ ALCL: DEL, ALK⁻ ALCL: MAC2A, cHL: L‐1236, T‐cell‐derived cHL: L‐540. Labelled in black: ALK⁺ ALCL: SU‐DHL‐1, ALK⁻ ALCL: MAC1, cHL: L‐428, T‐cell‐derived cHL: HDLM‐2

FIGURE 3

Morphology of moving cells at baseline and after inhibitor treatment in straight microchannels with height 10 µm and width 8 µm. The ALK⁺ ALCL cell lines DEL and SU‐DHL‐1, ALK⁻ ALCL cell lines MAC1 and MAC2A, cHL cell lines L‐428 and L‐1236 and T‐cell‐derived cHL cell lines L‐540 and HDLM‐2 are displayed. First line, cells are shown at baseline, second line after ROCK inhibition with 30 µM Y27632, third line after myosin II inhibition with 15 µM Blebbistatin, last line after actin‐related protein Arp2/3 complex inhibition with 50 µM CK‐666 is shown. Representative examples in phase‐contrast microscopy at 10x magnification were chosen

FIGURE 4

Movement of Hodgkin and ALCL cell lines in microchannels with 12 µm diameter and 4 µm constrictions. (A) Passage of the first constriction by a DEL cell (ALK⁺ ALCL) within 28 min. The nucleus is highlighted in blue (Hoechst dye), 40× magnification. (B) Passage of the first constriction by an L‐1236 cell (cHL) within 266 min. The nucleus is highlighted in blue (Hoechst dye), 40× magnification. (C) Passage of the first constriction by an L‐540 cell (T‐cell‐derived cHL) within 35 min. The nucleus is highlighted in blue (Hoechst dye), 40× magnification. (D) Number of cells passing the first four constrictions according to number of constriction and type of lymphoma. ** p < 0.01, Kruskal–Wallis test with Dunn´s post‐test for multiple comparisons. 4–5 replicates for each cell line, two cell lines per lymphoma entity. (E) Time (min) required for the passage of the first constriction. Each dot represents one cell. Two cell lines per lymphoma entity. *p < 0.01, Kruskal–Wallis test with Dunn's post‐test for multiple comparisons

FIGURE 5

Lamin A/C expression in the nuclear laminae of Hodgkin and ALCL cell lines. (A) Lamin A protein expression in cHL and ALCL cell lines according to global proteomics data. (B) Lamin B1 protein expression in cHL and ALCL cell lines according to global proteomics data. (C) Lamin B2 protein expression in cHL and ALCL cell lines according to global proteomics data. Labelled in grey: ALK⁺ ALCL: DEL, ALK⁻ ALCL: MAC2A, cHL: L‐1236, T‐cell‐derived cHL: L‐540. Labelled in black: ALK⁺ ALCL: SU‐DHL‐1, ALK⁻ ALCL: MAC1, cHL: L‐428, T‐cell‐derived cHL: HDLM‐2. (D) 3D confocal image of cell nuclei in Lamin A/C immunofluorescence, nuclei counterstained with DAPI, 63× magnification, cell lines as indicated. (F) Cut section through cell nuclei in Lamin A/C immunofluorescence, nuclei counterstained with DAPI, 63× magnification, cell lines as indicated