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. 2023 Sep 22;21(1):653.
doi: 10.1186/s12967-023-04517-5.

Transcriptional landscape of Kaposi sarcoma tumors identifies unique immunologic signatures and key determinants of angiogenesis

Ramya Ramaswami  1 , Takanobu Tagawa  1 , Guruswamy Mahesh  1 , Anna Serquina  1 , Vishal Koparde  2 , Kathryn Lurain  1 , Sarah Dremel  1 , Xiaofan Li  1 , Ameera Mungale  1 , Alex Beran  1 , Zoe Weaver Ohler  2 , Laura Bassel  2 , Andrew Warner  2 , Ralph Mangusan  1 , Anaida Widell  1 , Irene Ekwede  1 , Laurie T Krug  1 , Thomas S Uldrick  1 , Robert Yarchoan  1 , Joseph M Ziegelbauer  3
Affiliations

Transcriptional landscape of Kaposi sarcoma tumors identifies unique immunologic signatures and key determinants of angiogenesis

Ramya Ramaswami et al. J Transl Med. .

Abstract

Background: Kaposi sarcoma (KS) is a multicentric tumor caused by Kaposi sarcoma herpesvirus (KSHV) that leads to morbidity and mortality among people with HIV worldwide. KS commonly involves the skin but can occur in the gastrointestinal tract (GI) in severe cases.

Methods: RNA sequencing was used to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions in 44 paired samples from 19 participants with KS alone or with concurrent KSHV-associated diseases. Analyses of KSHV expression from KS lesions identified transcriptionally active areas of the viral genome.

Results: The transcript of an essential viral lytic gene, ORF75, was detected in 91% of KS lesions. Analyses of host genes identified 370 differentially expressed genes (DEGs) unique to skin KS and 58 DEGs unique to GI KS lesions as compared to normal tissue. Interleukin (IL)-6 and IL-10 gene expression were higher in skin lesions as compared to normal skin but not in GI KS lesions. Twenty-six cellular genes were differentially expressed in both skin and GI KS tissues: these included Fms-related tyrosine kinase 4 (FLT4), encoding an angiogenic receptor, and Stanniocalcin 1 (STC1), a secreted glycoprotein. FLT4 and STC1 were further investigated in functional studies using primary lymphatic endothelial cells (LECs). In these models, KSHV infection of LECs led to increased tubule formation that was impaired upon knock-down of STC1 or FLT4.

Conclusions: This study of transcriptional profiling of KS tissue provides novel insights into the characteristics and pathogenesis of this unique virus-driven neoplasm.

Trial registration: ClinicalTrials.gov NCT00006518 NCT03300830.

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Conflict of interest statement

R. Yarchoan reports receiving research support from Celgene (now Bristol Myers Squibb) through CRADAs with the NCI. Dr. Yarchoan also reports receiving drugs for clinical trials from Merck, EMD-Serano, Eli Lilly, and CTI BioPharma through CRADAs with the NCI, and he has received drug supply for laboratory research from Janssen Pharmaceuticals. R. Yarchoan is a co-inventor on US Patent 10,001,483 entitled "Methods for the treatment of Kaposi's sarcoma or KSHV-induced lymphoma using immunomodulatory compounds and uses of biomarkers." An immediate family member of R. Yarchoan is a co-inventor on patents or patent applications related to internalization of target receptors, epigenetic analysis, and ephrin tyrosine kinase inhibitors. All rights, title, and interest to these patents have been assigned to the U.S. Department of Health and Human Services; the government conveys a portion of the royalties it receives to its employee inventors under the Federal Technology Transfer Act of 1986 (P.L. 99-502). Other authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Sample types and RNA expression profiles. A Diagram of the normal and KS matched samples used in this study. Differentially expressed genes from GI (KS tumor versus normal) samples and differentially expressed genes from skin (tumor vs normal in green) samples. The differential gene expression cutoff of log2 Fold Change > or < 2.0 and padj. < 0.05 between tumor vs normal in both GI and skin samples was used. B Venn diagram shows unique and shared differentially expressed genes. C Heatmap showing shared differentially expressed genes using average Log2 fold change values
Fig. 2
Fig. 2
Selected genes associated with viral pathogenesis or immune responses are plotted. A, C, D Asterisks (blue* for GI and orange * for skin) represent statistically significant (Student’s t test, p < 0.05) genes, in individual GI (tumor vs normal, log2 Fold Change, blue squares) and skin (tumor vs normal, log2 Fold Change, orange dots) samples. B For only the KS tumor samples: transcripts per million (TPM) were collected for all KSHV genes on the horizontal axis and TPM for IL6 is shown on the vertical axis (orange for KS skin, blue for KS GI)
Fig. 3
Fig. 3
RNA expression samples were separated into samples from participants with only KS or with KS and additional KSHV associated diseases. A-B. Selected cytokines and expression changes between normal and tumor samples are shown. C The CCBE1 expression patterns in skin samples are shown
Fig. 4
Fig. 4
Heatmaps of KSHV gene expression KS tumors in GI (A) and skin (B) samples. Samples were either from participants untreated for KS (blue) or treated (red). Heatmap shows TPM expression from black (low) to medium (light blue) to high (red). ‘Class’ represent different stages of KSHV genes classification based on previous studies as Immediate Early (brown), Early (purple), Delayed Early (green), and Late (blue). The latency genes were represented as not latent (N -white) and latent (Y-pink). C KSHV gene expression heatmaps from the same matched participants. KSHV viral genes expression (Transcript per million (TPM) values (low-black, medium-blue, high-red) in GI and skin tumor samples from the same participants
Fig. 5
Fig. 5
Expression of KSHV and human genes in KS lesions. A Sections from KS skin tumor sample S9T were stained with H&E for pathology or stained by immunohistochemistry for KSHV LANA protein or human endothelial marker CD31. Scale bars = 100 µm. B The same tissue sample was analyzed by RNA in situ hybridization with probes to the KSHV RNAs ORF75 and vIL6. Scale bars = 100 µm for left and 50 µm for right image. C KS GI tumor sample G2T was stained with H&E or for KSHV LANA protein. Scale bars = 100 µm for left two images and 20 µm for image on right. D KS GI tumor sample G2T was analyzed for expression of KSHV ORF75 and vIL6 RNAs. Scale bar = 100 µm for left image and 50 µm for right image
Fig. 6
Fig. 6
Gene expression in KS samples and cell culture assays. A Total KSHV transcript per million (TPM) values versus STC1 and FLT4 TPM values were plotted. Spearman correlation analysis was performed for KS tumor samples. B Human dermal lymphatic endothelial primary cells (LEC) were infected with KSHV and RNA expression was analyzed 4 to 72 h post-infection using qPCR. C KSHV-infected iSLK cells were lytically induced and RNA was harvested 8 to 72 h after induction. D, E Viral transcripts (immediate early, IE; early, E; delayed-early, DE; late, L) were measured using genomic standard curves. F iSLK cells without KSHV infection were treated with doxycycline (Dox) to induce RTA expression. G Control SLK cells lacking inducible RTA were treated with combinations of Dox and sodium butyrate (NaB)
Fig. 7
Fig. 7
Repression of STC1 and FLT4 in primary human dermal lymphatic endothelial cells. A Cells were transfected with siRNA non-targeting control (siNTC) or siRNAs targeting STC1 (siSTC1). One day post-transfection, cells were infected with KSHV strain BAC16 (contains a GFP reporter). The percentage of GFP-positive cells was determined by flow cytometry (left). DNA harvested by cells was used in qPCR assays to measure viral genomes per cell at 1 day post-infection. B New viral particles were measured from conditioned media from cells infected and transfected. Samples were collected 5 days after infection. Each line represents a separate biological replicate. C RNA was purified from cells after transfection and infection and KSHV transcript levels were compared between siSTC1-transfected cells and siNTC cells. D Endothelial cells were transfected with siRNAs, infected (MOI = 0.25), then seeded on basement membrane extract at 2 dpi, and imaged by brightfield microscopy at 3 dpi. Tubule formation (Nodes, Junctions, Meshes, Segments) was assessed by image analysis software. Paired t-test was performed for statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001)
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