EZB-type diffuse large B-cell lymphoma cell lines have superior migration capabilities compared to MCD-type

Abstract

Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent aggressive B-cell lymphoma. The group is heterogeneous and the outcome is variable. A variety of approaches have been employed with the objective of improving the stratification of DLBCL patients according to their prognosis, based on the cell of origin. Recently, distinct genetic subtypes of DLBCL have been identified. Given the importance of cell migration in immune cells, the objective of this study was to ascertain whether different genetic subtypes of DLBCL exhibit disparate migration abilities. MCD- and EZB-type DLBCL cell lines were subjected to testing to ascertain their basal velocity in straight microchannels and their ability to overcome tight constrictions of 2 μm. The EZB-type cell lines showed superior basal migration velocity and constriction passage time, and a similar trend was observed in live cell imaging of native human DLBCL tissue. In addition, MCD-type DLBCL exhibited significantly elevated levels of nuclear lamin A/C, which is responsible for the stiffness of the nuclear envelope and could thus explain the disparate migration behaviours observed among these subtypes. Our study suggests that different genetic subtypes of DLBCL may not only influence the outcome after therapy but also the motility of the tumour cells.

Keywords: diffuse large B‐cell lymphoma; microchannel; migration.

FIGURE 1

Velocity of diffuse large B‐cell lymphoma (DLBCL) cell lines in straight microchannels. (A) Velocity of individual DLBCL cell lines (each data point means of one cell). (B) Velocity of DLBCL cell lines grouped by molecular subtype (each data point is the mean of one cell; 140 cells per each cell line; ****p < 0.0001, Mann‐Whitney‐test). (C) Example of a MCD‐type DLBCL cell line TMD8 with slow tumour cell velocity. (D) Example of an EZB‐type DLBCL cell line SU‐DHL‐4 with high tumour cell velocity.

FIGURE 2

Duration of constriction passage of diffuse large B‐cell lymphoma (DLBCL) cell lines in microchannels with constrictions. (A) Duration of constriction passage of individual DLBCL cell lines (each data point mean duration of one passage). (B) Duration of constriction passage of DLBCL cell lines grouped by molecular subtype (each data point mean duration of one passage, 15 passages per cell line; **p < 0.001, Mann‐Whitney‐test). (C) Example of a MCD‐type DLBCL cell (TMD8 cell line) with long duration of the constriction passage. (D) Example of an EZB‐type DLBCL cell (SU‐DHL‐4 cell line) with short duration of the constriction passage.

FIGURE 3

Nuclear Lamin A/C density in diffuse large B‐cell lymphoma (DLBCL) cell lines and primary DLBCL. (A) Nuclear Lamin A/C density of individual DLBCL cell lines is determined as % area lamin per nucleus (one data point per analysed nucleus). (B) Nuclear Lamin A/C density determined as % area lamin A/C per nucleus of DLBCL cell lines grouped by molecular subtype (one data point per analysed nucleus, 130 nuclei per cell line; ****p < 0.0001, Mann–Whitney test). (C) Nuclear Lamin A/C density determined as % area lamin A/C per nucleus of primary DLBCL cases grouped by molecular subtype (12 MCD‐like DLBCL from the central nervous system and one primary MCD‐type DLBCL from motility imaging; 7 EZB‐like transformations from follicular lymphoma and four primary EZB‐type DLBCL from motility imaging; one data point represents the mean per case; *p = 0.0461, unpaired t‐test). (D) Example of a Lamin A/C staining of the MCD‐type DLBCL cell line TMD8 (40× magnification). (E) Example of a Lamin A/C staining of the EZB‐type DLBCL cell line WSU‐DLCL2 (40× magnification). (F) Example of a Lamin A/C staining of a primary MCD‐like DLBCL case (40× magnification). (G) Example of a Lamin A/C staining of a primary EZB‐like DLBCL case (40× magnification).

FIGURE 4

Motility‐associated genes enriched in mutations in EZB‐type and MCD‐type diffuse large B‐cell lymphoma (DLBCL) from Wright et al. Motility‐associated genes were selected from the data of Wright et al. Mutation frequencies in EZB‐ and MCD‐type DLBCL were compared to the total dataset of all DLBCL. Fold change in mutation frequency compared to the total DLBCL cohort is shown and sorted by frequency (MCD‐type green, EZB‐type blue).

FIGURE 5

Velocity of tumour cells from primary human diffuse large B‐cell lymphoma (DLBCL) tissue, stained for CD20. (A) Velocity of the tumour cells from seven primary DLBCL cases (five movies per case). Four cases were classified EZB, one case ST2 and one case MCD subtype. In case 7, no information on molecular subtype was available due to poor DNA quality. According to Hans classifier, four cases were classified GCB and three cases non‐GCB. (B) Mean velocity of the CD20‐positive tumour cells in primary tissue according to DLBCL subtype, grouped according to Hans et al. (five movies per case). (C) Mean velocity of the CD20‐positive tumour cells in primary tissue according to molecular DLBCL subtype (five movies per case). (D) DLBCL case 1 of the EZB‐type (GCB‐type according to Hans et al. ¹⁹ ), tumour cells are stained for CD20 (green). Tracks display the velocity and path of the respective cells. (E) DLBCL case 2 of the EZB‐type (GCB‐type according to Hans et al. ¹⁹ ), tumour cells are stained for CD20 (green). Tracks display the velocity and path of the respective cells. (F) DLBCL case 3 of the EZB‐type (GCB‐type according to Hans et al. ¹⁹ ), tumour cells are stained for CD20 (green). Tracks display the velocity and path of the respective cells. (G) DLBCL case 4 of the EZB‐type (non‐GCB‐type according to Hans et al. ¹⁹ ), tumour cells are stained for CD20 (green). Tracks display the velocity and path of the respective cells. (H) DLBCL case 5 of the ST2‐type (GCB‐type according to Hans et al. ¹⁹ ), tumour cells are stained for CD20 (green). Tracks display the velocity and path of the respective cells. (I) DLBCL case 6 of the MCD‐type (non‐GCB‐type according to Hans et al. ¹⁹ ), tumour cells are stained for CD20 (green). Tracks display the velocity and path of the respective cells. (J) DLBCL case 7 lacking molecular information, non‐GCB‐type according to Hans et al., tumour cells are stained for CD20 (green). Tracks display the velocity and path of the respective cells. Speed scale bar in μm/s.