Small and big Hodgkin-Reed-Sternberg cells of Hodgkin lymphoma cell lines L-428 and L-1236 lack consistent differences in gene expression profiles and are capable to reconstitute each other

Abstract

The hallmark of classical Hodgkin lymphoma (cHL) is the presence of giant, mostly multinucleated Hodgkin-Reed-Sternberg (HRS) cells. Whereas it has recently been shown that giant HRS cells evolve from small Hodgkin cells by incomplete cytokinesis and re-fusion of tethered sister cells, it remains unsolved why this phenomenon particularly takes place in this lymphoma and what the differences between these cell types of variable sizes are. The aim of the present study was to characterize microdissected small and giant HRS cells by gene expression profiling and to assess differences of clonal growth behavior as well as susceptibility toward cytotoxic intervention between these different cell types to provide more insight into their distinct cellular potential. Applying stringent filter criteria, only two differentially expressed genes between small and giant HRS cells, SHFM1 and LDHB, were identified. With looser filter criteria, 13 genes were identified to be differentially overexpressed in small compared to giant HRS cells. These were mainly related to energy metabolism and protein synthesis, further suggesting that small Hodgkin cells resemble the proliferative compartment of cHL. SHFM1, which is known to be involved in the generation of giant cells, was downregulated in giant RS cells at the RNA level. However, reduced mRNA levels of SHFM1, LDHB and HSPA8 did not translate into decreased protein levels in giant HRS cells. In cell culture experiments it was observed that the fraction of small and big HRS cells was adjusted to the basic level several days after enrichment of these populations via cell sorting, indicating that small and big HRS cells can reconstitute the full spectrum of cells usually observed in the culture. However, assessment of clonal growth of HRS cells indicated a significantly reduced potential of big HRS cells to form single cell colonies. Taken together, our findings pinpoint to strong similarities but also some differences between small and big HRS cells.

Fig 1. Analysis of L-428 cells sorted by cell size or CD15/CD30 expression.

(A) L-428 cells were sorted by cell size (small, middle, big) or after staining for CD15 and CD30 into CD15+CD30+ double positive (DP) and CD15-CD30+ single positive (SP) cells. Frequency of (B) small- and (C) big-sized cells within the three different populations was recorded over time via fluorescence activated cell sorting (FACS).

Fig 2. Big DP L-428 cells have a reduced clonal growth potential.

L-428 cells were sorted by (A) cell size (small, middle, big) or (B) CD15/CD30 expression (SP, DP), and subsequently used for a colony formation assay (CFA). After one week, colony forming units (CFU) were counted compared to the bulk population. CFA were performed in duplicates and repeated three times.

Fig 3. Unsupervised hierarchical clustering of gene expression profiles obtained from microdissected small and giant HRS cells of the cHL cell lines L-428 and L-1236 shows a strong heterogeneity between the two cell lines.

Small HRS cells of both cell lines show more strongly expressed probesets (red color) than giant HRS cells. 958 differentially expressed probesets between the four samples representing a standard deviation of > 1 were considered for the analysis.

Fig 4. LDHB, SHFM1, and HSPA8 proteins are expressed in small and giant HRS cells.

a. LDHB protein expression in small and big HRS cells in a representative primary cHL case (LDHB-immunostaining, 400x). b. SHFM1 protein expression in small and big HRS cells in a representative primary cHL case (SHFM1-immunostaining, 400x). c. HSPA8 protein expression in small and big HRS cells in a representative primary cHL case (HSPA8-immunostaining, 400x). d. Representative example of LDHB expression in small and big HRS cells in the cHL cell line L-428 (LDHB-immunofluorescence staining, 400x). e. Representative example of SHFM1 expression in small and big HRS cells in the cHL cell line L-1236 (SHFM1-immunofluorescence staining, 400x). f. Representative example of HSPA8 expression in small and big HRS cells in the cHL cell line L-428 (HSPA8-immunofluorescence staining, 400x).

Fig 5. Effect of the anti-CD30 drug conjugate Brentuximab Vedotin to L-428 cells.

Cells from the cHL line L-428 were sorted according to FSC in subpopulations of small and big cells (each 20% of total live cell population). Total live gates were sorted as control (bulk). Thereafter, subpopulations were seeded at a density of 1 x 10⁵ cells per ml in triplicates and the indicated amount of Brentuximab Vedotin was added. After 48 h cell numbers were determined by FACS. * p = 0.0117, unpaired t-test.