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.

Distinct 3D Structural Patterns of Lamin A/C Expression in Hodgkin and Reed-Sternberg Cells

“…To investigate the above hypothesis, we divided the H and RS cells from three independent experiments (90 cells total for each cell type) into different groups according to which specific pattern they showed. The obtained percentages are consistent with the hypothesis that a significant proportion of mono-nuclear H-cells is still capable of progression to RS-cells [ 30 ]. However, considering that likely not every mono-nuclear H cell will complete the transition to a RS cell [ 6 ], lamin A/C may be a useful tool in predicting the future behavior of an H cell and, therefore, of the neoplastic population of any given patient on the whole.…”

3D image analysis reveals differences of CD30 positive cells and network formation in reactive and malignant human lymphoid tissue (classical Hodgkin Lymphoma)

“…Our results are consistent with previous research on development of HRS-cells by incomplete cytokinesis and re-fusion of daughter cells [34], as well as increased gene expression causing proliferation [35]. Single cell analysis resulted in small sized cells in NScHL which leads to the assumption that HRS-cells tend to cluster and form networks after gaining multinuclearity since a big cell size comes along with significantly less clonal growth [36].…”

Fibroblasts in Nodular Sclerosing Classical Hodgkin Lymphoma Are Defined by a Specific Phenotype and Protect Tumor Cells from Brentuximab-Vedotin Induced Injury

“…However, no effect on viability or proliferation was observed (data not shown). Next, L-428 cells were cocultured with NS cHL fibroblasts in a ratio of 5:1 for 12 h. In this setting, 50 µg/mL cell culture volume Brentuximab-Vedotin, a CD30-specific antibody drug conjugate, was applied as previously published [31]. Cells were stained for Annexin V and 7-amino-actinomycin D (7-AAD) and the proportion of positive cells was determined by flow cytometry after 48 h. For comparison, the same procedure was performed with the CD30-negative Burkitt lymphoma cell line Raji.…”

“…Brentuximab-Vedotin was applied in a dosage of 50 µg/mL as described before [31] and apoptosis rates were determined in both the interacting adherent coculture fraction as well as the non-interacting floating fraction of suspension cells by APC-Annexin V Apoptosis Detection Kit (Thermo Fisher Scientific) including Annexin V and 7-Aminoactinomycin (7-AAD) staining at 48 h. Apoptosis rates of single cHL cell line culture supplemented with fibroblast conditioned media supernatant were also determined after identical Brentuximab-Vedotin treatment. Since L-1236 cells show some ability to adhere on their own to cell culture dishes and KM-H2 HRS cells express CD30 to a lesser extent they have not been selected for this experimental set-up.…”