H-1 Parvovirus-Induced Oncolysis and Tumor Microenvironment Immune Modulation in a Novel Heterotypic Spheroid Model of Cutaneous T-Cell Lymphoma

Abstract

The rat protoparvovirus H-1 (H-1PV) is an oncolytic virus known for its anticancer properties in laboratory models of various human tumors, including non-Hodgkin lymphomas (NHL) of B-cell origin. However, H-1PV therapeutic potential against hematological malignancies of T-cell origin remains underexplored. The aim of the present study was to conduct a pilot preclinical investigation of H-1PV-mediated oncolytic effects in cutaneous T-cell lymphoma (CTCL), a type of NHL that is urgently calling for innovative therapies. We demonstrated H-1PV productive infection and induction of oncolysis in both classically grown CTCL suspension cultures and in a novel, in vivo-relevant, heterotypic spheroid model, but not in healthy donor controls, including peripheral blood mononuclear cells (PBMCs). H-1PV-mediated oncolysis of CTCL cells was not prevented by Bcl-2 overexpression and was accompanied by increased extracellular ATP release. In CTCL spheroid co-cultures with PBMCs, increased spheroid infiltration with immune cells was detected upon co-culture treatment with the virus. In conclusion, our preclinical data show that H-1PV may hold significant potential as an ingenious viroimmunotherapeutic drug candidate against CTCL.

Keywords: cutaneous T-cell lymphoma (CTCL); heterotypic CTCL spheroid; oncolytic H-1 parvovirus; virotherapy.

Figure A1

Improved SeAx cell killing by combined H-1PV infection and Bcl-2 inhibition. Venetoclax (VCL, 100 nM)-mediated Bcl-2 inhibition combined with H-1PV infection (10 PFU/cell) of Bcl-2-overexpressing SeAx cells led to increased suppression of cell viability on both day three and day seven, in comparison with either virus or VCL treatment alone.

Figure A2

Surface P2X7 purinergic receptor and CD73 ectonucleotidase expression in H-1PV-infected (right panels) versus mock-infected (left panels) CTCL cells. In HH and MyLa cells, both P2X7 upregulation and CD73 downregulation was induced by H-1PV infection. In contrast, marked CD73 upregulation was observed in infected HuT 78 cells.

Figure 1

H-1PV entry and genomic DNA expression in CTCL cells. Positive fluorescence signals, indicative of virus P4 promoter activity, NS1 protein synthesis, and NS1-mediated virus P38 promoter activation, were detected on day three after infection with the recombinant H-1PV/EGFP virus (left panels, green) in all CTCL cell lines. Accordingly, H-1PV NS1 protein, the major inductor of virus-mediated oncotoxicity, was detected (right panels, red) in all cell lines after infection with the wt virus.

Figure 2

H-1PV-mediated induction of cytotoxicity in CTCL cells. On day three after infection, significant viability suppression was observed in HH, HuT 78, and MyLa cells, while in SeAx cells, in contrast, the 50% TCID could not be reached. On day seven after infection, progression of virus-induced oncolysis was seen in HH and, of note, in SeAx cultures, in contrast to the overgrowth of HuT 78 and MyLa cell fractions, which survived infection on day three.

Figure 3

H-1PV innocuousness for human primary healthy donor pan T-lymphocytes (panT) and peripheral blood mononuclear cells (PBMC). H-1PV infection of activated panT and PBMC cells failed to induce any significant toxicity, in contrast to the substantially impaired viability observed on day three in H-1PV-infected CTCL cultures (see above).

Figure 4

Induction of eATP release in H-1PV-infected CTCL cultures. In HH cells, which are prone to efficient quick oncolysis, increased eATP accumulation was detected twenty-four hours after infection with high (>10 PFU/cell) virus doses. In HuT 78, SeAx, and MyLa cells, which were found to display lower sensitivity to the virus, in comparison with HH, eATP secretion was induced up to seventy-two hours, and peaked forty-eight hours post infection. RLU, relative luminescence unit.

Figure 5

Spheroid formation in hanging-drop CTCL cultures. (a) In contrast to HH and MyLa cells, which displayed intrinsic spheroid-building ability, HuT 78 and SeAx cells were only capable of two-dimensional clustering under monoculture conditions; (b) under co-culture conditions, keratinocytes (left panels) or fibroblasts (right panels) provided the CTCL cells with substantial 3D growth support resulting in the reproducible formation of compact, regular-shape spheroids.

Figure 6

Immunofluorescence detection of cell type-specific markers in heterotypic CTCL spheroids. Immunofluorescence staining against CD4, vimentin (Vim), pancytokeratin (PCK), and CD31 (red) verified the incorporation into the heterotypic spheroid of lymphoma, fibroblast, keratinocyte, and endothelial cells, respectively.

Figure 7

Heterotypic CTCL spheroid structure. (a) The characteristic “core-periphery” structure displayed by all types of heterotypic CTCL spheroids is illustrated by the immunofluorescence labeling of keratinocyte-containing HuT 78 spheroid sections. The typical heterotypic CTCL spheroid consisted of: (i) CTCL core (*, CD4, green), (ii) TME zone (**, pancytokeratin, red), and (iii) an invasive CTCL periphery (***, CD4, green). (b) In fibroblast-containing HH and HuT 78 spheroids (left panels), the endothelial cells (CD31, red) populated the entire TME zone. In contrast, in the keratinocyte-containing counterparts (right panels), the CD31+ cells were shown to cluster around the CTCL core.

Figure 8

Reporter and viral gene expression in H-1PV-infected heterotypic CTCL spheroids. Reporter EGFP (green, left panels) and NS1 (red, right panels) protein expression generated positive immunofluorescence signals detected three days after spheroid infection with recombinant H-1PV/EGFP or wtH-1PV, respectively.

Figure 9

H-1PV-induced suppression of heterotypic CTCL spheroid viability. Virus treatment of HH, SeAx, and MyLa spheroids led to progressive reduction in spheroid viability, in comparison with mock-treated controls, which ensued from day three to day seven. In HuT 78 spheroids, the moderate H-1PV-induced cytotoxicity detected on day three was not detectable on day seven. RLU, relative luminescence unit.

Figure 10

H-1PV-induced shrinkage of heterotypic CTCL spheroids. The spheroids were treated, or not, with H-1PV and the reduction in brightfield object (BO) average area was evaluated using live-cell imaging analysis. Data obtained at 0 h (spheroid treatment and start of imaging) and 72 h are displayed and show significant shrinkage of all spheroids on day three after infection.

Figure 11

H-1PV-induced cytotoxicity in heterotypic CTCL spheroids. On day three after spheroid treatment with H-1PV (n = 6 per treatment condition), virus dose-dependent increase in the uptake of the dead cell tracker IncuCyte^® Cytotox Red Dye, in association with significant spheroid shrinkage, was observed.

Figure 12

Spheroid infiltration with CD8+ cells in co-cultures of keratinocyte-containing MyLa spheroids and PBMCs. H-1PV treatment (right panel) of the co-culture enhanced the spontaneous (left panel) spheroid infiltration with cytotoxic T-lymphocytes (CD8, red). Mean CD8+ cell counts per 500 cells were compared between H-1PV-infected and mock-treated co-cultures.

Figure 13

The multifaceted role of extracellular ATP in CTCL biology. CTCL cells release cytosolic ATP (cytATP) into the extracellular space (1). Extracellular ATP (eATP) may activate cellular purinergic P2X7 receptors (2), leading to Ca²⁺ influx, NFAT activation, IL-2 transcription (3), and, ultimately, CTCL cell growth and proliferation (4). On the other hand, H-1PV infection of CTCL cells (5) may additionally trigger eATP secretion, resulting in the induction of danger signaling in the CTCL TME (7).