Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel–forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors.

Significance:

We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation, providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression.

Canine hemangiosarcoma, a vasoformative tumor originating from bone marrow (BM)-derived progenitor cells, is a common occurrence in dogs, unlike the rare human angiosarcoma (1–4). Tumors from both species exhibit similar histology and natural history of disorganized, tortuous, and dilated blood vessels with high proliferative activity and metastatic potential (5, 6). Molecularly, convergent transcriptional programs involving angiogenesis and inflammation are observed in both canine hemangiosarcoma and human angiosarcoma (7, 8). While the tumor microenvironment (TME) plays a crucial role in tumor cell survival, disease progression, and metastasis through the niche it creates (9), its contribution to the molecular programs of hemangiosarcoma remains incompletely understood. Recent evidence suggests bidirectional interactions between cancer cells and niche cells, where cancer cells re-educate niche cells and vice versa, particularly in maintaining stemness and self-renewal of cancer stem cells in various tumors (9–11), including hematopoietic tumors (12–14).

The hematopoietic niche is composed of osteoblasts and various stromal cells including endosteal, endothelial cells, fibroblasts, nestin-positive mesenchymal stromal cells (MSC), leptin receptor-positive stromal cells, and CXCL12-abundant reticular cells. These cells support hematopoiesis by regulating the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPC) into lineage-committed cells (15). Dysregulation of HSPC function can result in blood disorders (16, 17) and hematopoietic malignancies such as leukemia and lymphoma (18–20).

In the current study, we aimed to elucidate the cellular and molecular properties of hemangiosarcoma. Our findings indicate that canine hemangiosarcoma cells not only have the capability to form vasoformative tumors but also create a niche for expansion and differentiation of blood cells, potentially contributing to the development of hematopoietic tumors.

Human Tissue Samples

The human angiosarcoma tissue samples used in this study were formalin-fixed and paraffin-embedded, as described previously (8). The samples were obtained from two sources: the University of Minnesota Biological Materials Procurement Network and the Cooperative Human Tissue Network. All sample acquisitions were performed under standardized patient consent protocols.

Canine Tissue Samples and Cell Lines

Previously established hemangiosarcoma cell lines (SB, COSB, Emma, DD1, JHE, and JLU; refs. 1, 2, 4, 21–23) were used in this study. Canine tissue samples were collected from surgical removals or tumor biopsies at the University of Minnesota (Minneapolis, MN) or private veterinary clinics. Additional hemangiosarcoma cell lines (DHSA-1401, DHSA-1420, and DHSA-1426) and nonmalignant endothelial cell lines from splenic hematomas (DHSA-0806, DHSA-1115, DHSA-1414, and DHSA-1501) were generated and cultured using established methods (1, 2, 22). Passage-6 of DHSA-1401, passage-5 of DHSA-1420, and passage-5 and -14 DHSA-1426 cell were used for xenografts. The passage numbers of the pre-established hemangiosarcoma cell lines were not available after long-term culture because their primary generation. Cell line authentication and Mycoplasma testing for canine cells were conducted by IDEXX BioAnalytics. All sample acquisition procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota (Minneapolis, MN) under protocols 0802A27363, 1101A94713, and 1312-31131A.

Human and Mouse Cell Lines

Human bone marrow–derived mesenchymal stromal cells (hBM-MSC) were isolated from whole BM purchased from AllCells, as described previously (24–27). M2-10B4 murine BM stromal cells were purchased from the ATCC and maintained following established protocols (25). Samples of human umbilical cord blood (hUCB) were obtained from the ClinImmune Stem Cell Laboratory at the University of Colorado Cord Blood Bank (28).

Mice and Xenotransplantation

We conducted multiple xenograft experiments using various cell lines and patient-derived tumor sections. First, we injected cultured tumor cells from three hemangiosarcoma cell lines (SB, Emma, and JHE) into NSG mice. Specifically, we injected 5 × 106 SB cells subcutaneously in four mice, and 5 or 10 × 106 SB cells intraperitoneally in 4 mice. In addition, we injected 2 × 106 Emma cells subcutaneously and intraperitoneally in 4 mice each, and 3 × 105 JHE cells subcutaneously in 4 mice.

Next, we injected tumor cells from five hemangiosarcoma cell lines (Emma, DD1, JLU, DHSA-1401, and COSB) into NSG neonates, 1 or 2 days after birth. We injected 5 × 105 Emma, DD1, JLU, or DHSA-1401 cells, or 6.25 × 105 COSB cells intraperitoneally in 50 µL of PBS.

We also injected 5 × 106 cells from three hemangiosarcoma cell lines (JLU, DHSA-1420, and DHSA-1426) in a mixture of 100 µL of PBS and 100 µL of BD Matrigel Basement Membrane Matrix into the subcutaneous space of beige-nude-xid (BNX) mice. For DHSA-1426, we injected tumor cells from passage-5 and passage-14 independently.

We used sections of viable tumors from 4 dogs affected with hemangiosarcoma and implanted them into subcutaneous pockets of 4 mice for each dog. In addition, we implanted sections of non-hemangiosarcoma splenic tissues from 7 dogs into 18 mice as controls. Finally, after visible tumors developed in mice, we serially transplanted the tumors by inoculation of cultured tumor cells in 3 mice or by direct implantation of single-cell suspensions of the tumor in 8 mice.

We monitored the mice for tumor development and sacrificed them when they reached a tumor endpoint, including a mass measuring 1.5 cm in the longest diameter or at the end of a 16-week period after xenotransplantation. Mice were euthanized by cervical dislocation according to the IACUC guidelines. All animal procedures were conducted in accordance with the Research Animal Resources husbandry and care protocols and reviewed and approved by the IACUC of the University of Minnesota (Minneapolis, MN; protocols 1006A84813, 1106A00649, 1306-30712A, and 1311-31104A). A total of 132 mice were used for xenotransplantation procedures, as described in Supplementary Table S1.

Flow Cytometric Analysis

We used the following antibodies for flow cytometry analysis of xenograft tumors: rat anti-dog CD45-APC (Clone YKIX716.13; AbD Serotec), rat anti-mouse CD45-APC (Clone 30-F11; eBioscience), and mouse anti-human αvβ3 (CD51/CD61)-FITC (Clone 23C6; BD Pharmingen). Isotype controls included rat IgG2b-APC (Clone eB149/10H5; eBioscience) and mouse IgG1 K-FITC (Clone P3.6.2.8.1; eBioscience). Data were acquired using a BD Acuri C6 flow cytometer (BD Biosciences) and analyzed with FlowJo software (version 10.1, Tree Star).

IHC

IHC staining was conducted at either the BioNet Histology Laboratory, Minnesota Veterinary Diagnostic Laboratory, or Comparative Pathology Shared Resource at the University of Minnesota (Minneapolis, MN). The following antibodies were used: CD3, PAX5, MAC387, CD163, CD204, Iba1, Ter-119, MPO, and CD45. Details of the antibodies used are provided in Supplementary Table S2.

RNA Sequencing for Gene Expression Profiling

To obtain transcriptomic profiles, we isolated total RNA from xenograft tumor samples using the TriPure Isolation Reagent (Roche Applied Science), followed by clean-up using the RNeasy Mini Kit (Qiagen). We generated RNA sequencing (RNA-seq) libraries with a targeted depth of 20 million paired-end reads (2 × 50 bp) at the UMGC, as described previously (4, 8). We conducted bioinformatic analyses for gene expression profiling as previously reported (4, 8, 29–31). RNA-seq data from human angiosarcoma tissues can be accessed via the Gene Expression Omnibus (GEO) under accession number GSE163359 (8). RNA-seq data from canine hemangiosarcoma tissues are available through the GEO (accession number GSE95183) and the NCBI Sequence Read Archive (accession number PRJNA562916; ref. 8).

Viral RNA Isolation and Library Generation

To detect RNA viruses, we isolated viral RNA from xenograft tumors using either a Qiagen viral RNA kit or TRIzol followed by the Qiagen viral RNA kit (Qiagen). The isolated RNA was then submitted to the University of Minnesota Genomics Center (UMGC) for viral RNA MiSeq. The Illumina MiSeq generated approximately 1 million paired-end reads (2 × 250) for each sample.

PathSeq Analysis

The PathSeq algorithm was used to determine transmissible agents in RNA-seq data from dog and mouse samples. The PathSeq algorithm (32) was used to perform computational subtraction of mouse and dog genomes followed by alignment of residual reads to mouse and dog reference genomes and microbial reference genomes (including bacterial, viral, archaeal, and fungal sequences downloaded from NCBI). These alignments resulted in the identification of microbial reads in the data.

Human reads were subtracted by first mapping reads to a database of mouse (mm9) and dog reference genomes (canFam3.1; GCF_000002285) using Burrows-Wheeler Aligner (BWA; Release 0.6.1, default settings; ref. 33), MegaBLAST (Release 2.2.25, cut-off E-value 10−7, word size 16) and BLASTN (Release 2.2.25, cut-off E-value 10−7, word size 7, nucleotide match reward 1, nucleotide mismatch score −3, gap open cost 5, gap extension cost 2; ref. 34). Only sequences with perfect or near perfect matches to the human genome were removed in the subtraction process. In addition, low complexity and highly repetitive reads were removed using RepeatMasker (version open-3.3.0; ref. 35).

To identify microbial reads, the residual reads were aligned with MegaBLAST to a database of microbial and dog reference genomes. Raw read counts were calculated using the reads that were mapped to Epstein-Barr virus (EBV) and Helicobacter pylori with at least 90% identity and 90% query coverage. Using the raw read counts, the abundance metric or normalized read count of a given microbe in a sample was calculated as

formula

Relative abundance in a given sample was calculated as abundance metric of taxa divided by the total abundance metric at kingdom level of the sample.

Long-term Culture Initiating Cell and Hematopoietic Colony-forming Unit Assays

We isolated hUCB CD34+ cells from 2 patients using the Miltenyi CD34 Microbead Kit and MACS separation column (Miltenyi Biotec), following the manufacturer's instructions. The purity of isolated hUCB CD34+ cells was determined by flow cytometry using the following antibodies (all anti-human): CD34-PECy7 (eBioscience), CD43-APC (BD Biosciences), and CD45-APC (BD Biosciences). Sorted hUCB CD34+ cells with >92% CD34+CD45+ were used for subsequent experiments. We seeded M2-10B4, hBM-MSCs, 1426, and Emma in gelatin-coated 24-well plates at a density of 105 cells/well overnight. After inactivation with 10 µg/mL Mitomycin C for 3 hours, 5,000 hUCB CD34+ cells were added to each well in Myelocult H5100 (StemCell Technologies) supplemented with 1 µmol/L dexamethasone (Sigma-Aldrich). Gelatin-coated wells without stroma were used as negative controls. Cells were conditioned for 6 weeks at 37°C and 5% CO2 with one-half media exchanges every week. At week 5, nonadherent cells from selected wells were harvested, counted, and analyzed for hematopoietic-specific surface antigens using the following antibodies (all anti-human): CD34-PECy7, CD33-APC (BD Biosciences), CD43-APC, and CD45-APC. At the end of the 6-week culture, both nonadherent and adherent cells were collected using 0.05% trypsin supplemented with 2% chicken serum for 5 minutes. Cells were filtered using a 70-µm cell strainer to generate single-cell suspensions and counted. We prepared triplicate CFU assays by seeding 50,000 cells/1.5 mL of semisolid Methocult H4435 Enriched media (StemCell Technologies) in 35 mm culture dishes (Greiner). After 2 additional weeks, the plates were manually scored for total number and phenotype of the CFUs, as described previously (36).

Statistical Analysis

Pearson and Spearman correlation coefficient was calculated for correlation between two variables. Statistical analysis was performed using GraphPad Prism 10 (GraphPad Software, Inc.) or Microsoft Excel.

Data Availability

The data generated in this study are publicly available in GEO at GSE163359 and GSE95183, as well as in NCBI Sequence Read Archive (PRJNA562916). Other relevant data generated in this study are available upon request from the corresponding author.

Canine Hemangiosarcoma Cells Can Recapitulate the Disease In Vivo

We used in vivo xenografts to investigate the biological behavior of canine hemangiosarcoma (21, 23, 37–39). Mice were inoculated with canine hemangiosarcoma cell lines or primary tissues, as detailed in Supplementary Table S1. The engraftment efficiency of canine hemangiosarcoma xenografts was found to be low, consistent with findings from previous studies (37, 38). Only cultured DHSA-1426 cells, when injected subcutaneously, consistently generated vasoformative tumors in immunodeficient BNX mice (Fig. 1A and B). The tumorigenic potential of this cell line was maintained over multiple passages, as demonstrated by serial passaging of cells cultured from tumor xenografts into new recipient mice (Supplementary Fig. S1).

FIGURE 1

Establishment of xenografts derived from canine hemangiosarcoma in immunodeficient mice. A, Schematic illustration depicts process of tumor xenografts in BNX mice. B, DHSA-1426 tumor cell line was established from a canine patient diagnosed with hemangiosarcoma (A). Cells from DHSA-1426 passages 5 (p5) and 14 (p14) formed tumors histologically classified as hemangiosarcoma (B and C). Cells cultured from xenograft-derived tumors developed histologically identical tumors after serial transplantation (D). Both the primary tumor (E) and the xenograft tumor (F) were positive for CD31 IHC staining. A–D, Hematoxylin and eosin (H&E) stain. E and F, IHC with an anti-CD31 antibody (alkaline phosphatase conjugates; counterstain = hematoxylin). Bar = 200 µm. C, FISH images using canine-specific (CXCL8, red) and mouse-specific (X chromosome, green) probes in a canine hemangiosarcoma xenograft transplanted into receptive immunodeficient female mouse hosts. Red and green arrows point to representative xenograft canine tumor cells and mouse stromal cells, respectively, to aid in identification. D, Individual points on graph represent relative quantity of donor (dog) and host (mouse) cells in each tumor type. 10–12 fields of pictures at high magnification (400X) per slide were acquired. A total of approximately 1,000 cells in individual xenograft tumor was counted, and the percentages for each species-specific cells are presented. E, Dot plot shows microenvironment scores for 76 primary hemangiosarcoma tissues, estimated using RNA-seq data and xCell algorithm. The red dot indicates a DHSA-1426 tumor tissue used for xenograft.

FIGURE 1

Establishment of xenografts derived from canine hemangiosarcoma in immunodeficient mice. A, Schematic illustration depicts process of tumor xenografts in BNX mice. B, DHSA-1426 tumor cell line was established from a canine patient diagnosed with hemangiosarcoma (A). Cells from DHSA-1426 passages 5 (p5) and 14 (p14) formed tumors histologically classified as hemangiosarcoma (B and C). Cells cultured from xenograft-derived tumors developed histologically identical tumors after serial transplantation (D). Both the primary tumor (E) and the xenograft tumor (F) were positive for CD31 IHC staining. A–D, Hematoxylin and eosin (H&E) stain. E and F, IHC with an anti-CD31 antibody (alkaline phosphatase conjugates; counterstain = hematoxylin). Bar = 200 µm. C, FISH images using canine-specific (CXCL8, red) and mouse-specific (X chromosome, green) probes in a canine hemangiosarcoma xenograft transplanted into receptive immunodeficient female mouse hosts. Red and green arrows point to representative xenograft canine tumor cells and mouse stromal cells, respectively, to aid in identification. D, Individual points on graph represent relative quantity of donor (dog) and host (mouse) cells in each tumor type. 10–12 fields of pictures at high magnification (400X) per slide were acquired. A total of approximately 1,000 cells in individual xenograft tumor was counted, and the percentages for each species-specific cells are presented. E, Dot plot shows microenvironment scores for 76 primary hemangiosarcoma tissues, estimated using RNA-seq data and xCell algorithm. The red dot indicates a DHSA-1426 tumor tissue used for xenograft.

Close modal

To establish the contribution of stromal elements to the formation of canine hemangiosarcoma, we utilized FISH to quantify the presence of both canine and mouse cells in the tumors. Specifically, we employed probes for canine CXCL8, as this gene is absent in the mouse genome, and a unique region of the mouse X chromosome, to differentiate between canine and mouse cells, respectively. Our data revealed that the hemangiosarcoma xenografts displayed a complex topological organization, with blood vessels lined by both donor and host cells (Fig. 1C; Supplementary Fig. S2). The hemangiosarcoma tumors were comprised of a mixture of 50%–70% malignant canine cells and 30%–50% mouse stromal cells (Fig. 1D). Furthermore, we sought to quantify the microenvironment components in original hemangiosarcoma tissues using RNA-seq data. Our cell type enrichment analysis revealed a mean microenvironment score of 42%, ranging between 14% and 70% across all 76 tumors. The DHSA-1426 tumor, which was used for the hemangiosarcoma xenograft, contained 37.5% of the estimated microenvironment score (Fig. 1E). These findings highlight the utility of xenografts as a valuable tool for quantifying the contribution of stromal elements to the TME.

To gain a deeper understanding of the involvement of stromal elements in canine hemangiosarcoma xenografts, we analyzed RNA-seq data obtained from cells and xenografts. The RNA-seq data were mapped to a reference genome that included both canine and mouse cells (29). Notably, we observed that the RNA-seq data from the cell lines predominantly mapped to the canine genome, whereas the xenografts exhibited mapping to both the canine and mouse genomes, which was consistent with the results obtained from FISH (Supplementary Fig. S3; Supplementary Table S3).

Canine Xenografts Generate Lymphomas of Mouse Origin

An unexpected series of findings shed light on the relationship between hemangiosarcoma and inflammation in the microenvironment. Previous studies, including our own, have demonstrated that canine hemangiosarcoma cells can successfully form hemangiosarcomas in athymic nude, NOD, and NSG mice (21, 23, 37–39). However, the studies are all indicative of low tumor take rates. For instance, in one series of transplantations, only two of 35 hemangiosarcoma cases achieved tumor formation in immunodeficient mice (38). In the current study, we noted instances where NSG mice, following inoculation with SB hemangiosarcoma cells or another cell line named Emma-brain (EFB), and BNX mice, following inoculation with tumor fragments derived from sample DHSA-1426 (freshly obtained from a dog with spontaneous hemangiosarcoma), developed exuberant myeloid hyperplasia and/or round cell tumors. Notably, 4 out of 5 mice that received 5 × 106 SB hemangiosarcoma cells intraperitoneally and 1 out of 4 mice that received 2 × 106 EFB cells subcutaneously, succumbed acutely 2 weeks after inoculation, displaying signs of anemia and splenomegaly. Histologic examination revealed that the spleens of these mice were expanded by monomorphic populations of hematopoietic cells (Fig. 2A and B). Upon further analysis, the cells were determined to be of mouse origin and represented erythroid progenitors (Ter-119+), with a few canine hemangiosarcoma cells admixed in the population (Fig. 2C–F). However, we were unable to definitively establish whether these cells had undergone malignant transformation.

FIGURE 2

Hematopoietic expansion derived from adoptive transplantation of canine hemangiosarcoma in immunodeficient mice. A and B, Xenotransplantation of canine hemangiosarcoma cell lines created exuberant myeloid hyperplasia in mouse spleens. Representative photomicrographs show histopathology by H&E staining of spleens from NSG mice transplanted with hemangiosarcoma cell lines SB (A) and EFB (B). C, Immunoreactivity of anti-mouse Ki-67 (Tec-3) antibody shows strongly positive signal in proliferating cells in the spleen. D, Immunostaining of anti-human (and canine cross-reactive) Ki-67 (MiB-1) antibody shows lack of positive staining among proliferating cells. E, The proliferating cells are immunoreactive with anti-mouse Ter-119 antibody. F, SB cells expressing Luciferase are detected in mouse spleen (arrows). Images shown in C, D, E, and F are from IHC staining done in mice inoculated with SB cells modified to express GFP and firefly Luciferase. Horseradish peroxidase (for Ki-67 stains) and alkaline phosphatase (for Ter-119 and Luciferase) conjugates were used. Counterstain = hematoxylin. A–D: Bar = 200 µm; E–F: Bar = 50 µm.

FIGURE 2

Hematopoietic expansion derived from adoptive transplantation of canine hemangiosarcoma in immunodeficient mice. A and B, Xenotransplantation of canine hemangiosarcoma cell lines created exuberant myeloid hyperplasia in mouse spleens. Representative photomicrographs show histopathology by H&E staining of spleens from NSG mice transplanted with hemangiosarcoma cell lines SB (A) and EFB (B). C, Immunoreactivity of anti-mouse Ki-67 (Tec-3) antibody shows strongly positive signal in proliferating cells in the spleen. D, Immunostaining of anti-human (and canine cross-reactive) Ki-67 (MiB-1) antibody shows lack of positive staining among proliferating cells. E, The proliferating cells are immunoreactive with anti-mouse Ter-119 antibody. F, SB cells expressing Luciferase are detected in mouse spleen (arrows). Images shown in C, D, E, and F are from IHC staining done in mice inoculated with SB cells modified to express GFP and firefly Luciferase. Horseradish peroxidase (for Ki-67 stains) and alkaline phosphatase (for Ter-119 and Luciferase) conjugates were used. Counterstain = hematoxylin. A–D: Bar = 200 µm; E–F: Bar = 50 µm.

Close modal

Three of 4 mice that received DHSA-1426 tumor fragments, representing first-generation canine patient-derived xenograft (CPDX), developed tumors in multiple organs, including spleen, lymph nodes, meninges, cerebrum, and mesentery, 12 weeks after implantation. However, the morphology of these tumors resembled that of round cell tumors (Supplementary Fig. S4A and S4B). IHC analysis showed that the tumor cells expressed CD45, B220, and Pax5, but did not express CD3, Ter-119, and MPO (Supplementary Fig. S4C–S4H), indicating a mouse B-cell origin. To further verify the mouse origin of these cells, flow cytometry was conducted, revealing positive staining for mouse CD45, but not for canine CD45 or human αVβ3-integrin (CD51/CD61), which exhibits cross-reactivity with canine, but not with mouse αVβ3-integrin (Supplementary Fig. S5). Furthermore, RNA-seq data from the mouse round cell tumors demonstrated a near-complete alignment with the mouse genome, consistent with the findings from flow cytometry (Supplementary Fig. S3), thereby indicating that the tumors were derived from mouse cells.

The malignant B cells demonstrated the ability to undergo serial passage and establish B-cell lymphomas in recipient BNX mice independently, without the requirement for canine hemangiosarcoma cell support (Supplementary Fig. S6). Intriguingly, similar results were obtained when single-cell suspensions derived from fresh tumor fragments of canine hemangiosarcoma xenografts were inoculated into BNX recipients, representing second-generation CPDX derived from a first-generation cell line tumor. The resulting tumors of mouse B-cell origin displayed comparable morphology and were likewise amenable to serial passage in BNX mice.

The potential etiology of these tumors was perplexing. To investigate the possibility of a transmissible, infectious agent as the cause of these tumors, we sequenced RNA from the xenografts and used the PathSeq platform for analysis. However, no bacterial or viral sequences with tumorigenic potential were identified in the xenografts or in the primary or metastatic canine hemangiosarcoma tumor samples or cell lines. While a recent study reported an association between Bartonella spp. and canine hemangiosarcoma (40, 41), only 10 of 24 dogs tested in our study had detectable Bartonella spp. sequences, and these were present in low abundance (Supplementary Table S4). Furthermore, four of the samples contained sequences from B. bacilliformis, B. grahamii, or B. tribocorum, which infect humans and rats, respectively, and are not known to infect dogs as primary or accidental hosts (42, 43). Three of the remaining 6 dogs had sequences for B. clarridgeiae, and 3 had sequences for multiple Bartonella spp., including human and rodent-specific types. The low abundance and the presence of sequences from organisms that do not infect dogs suggest that the Bartonella spp. sequences were contaminants.

Furthermore, we identified murine leukemia virus (MuLV) reads in mouse B-cell lymphomas that arose from the hemangiosarcoma xenografts through independent viral RNA-seq and PCR experiments (Supplementary Table S5). However, MuLV sequences were detected in normal mouse tissues (liver and spleen), as well as in two subcutaneous canine hemangiosarcoma xenograft tumors. MuLV was also found in SB hemangiosarcoma cells that had been previously passaged through mice as hemangiosarcomas. These findings indicate the transferability of MuLV sequences to xenografted tumors but are insufficient to support any conclusion regarding the role of a virus(es) as the transforming agent(s) responsible for the development of mouse lymphomas.

Human and Canine Hemangiosarcoma Tissues Show Evidence of Immune Cells

We reanalyzed RNA-seq data obtained from both canine hemangiosarcoma and human angiosarcoma samples, with the aim of investigating the presence of immune cells within the TME. To identify coexpressed genes, we used an unbiased approach called GCESS (Gene Cluster Expression Summary Score; ref. 30). Notably, the highest expression of these immune signature genes was observed in the inflammatory subtype of canine hemangiosarcoma (Supplementary Fig. S7). We next segregated human angiosarcomas into tumors with high and low immune scores (“immune-high” vs. “immune-low”) and identified 461 upregulated genes (Padjusted < 0.05) in the immune-high group compared with immune-low group (Fig. 3A). Fifty-eight of these genes were also found in canine inflammatory hemangiosarcomas, and they were associated with T- and B-cell activation (Fig. 3B and C). This approach allowed us to identify a common immune signature in both human and canine tumors.

FIGURE 3

Immune cell infiltration and comparative immune signatures between canine hemangiosarcoma and human angiosarcoma. A, A total of 461 upregulated genes were identified in immune-high (N = 8) compared with immune-low (N = 5) groups in human angiosarcomas (FDR P value < 0.05). B, 567 immune gene signatures were identified among three molecular subtypes of canine hemangiosarcomas (N = 76; FDR P value < 0.001; fold change > 3). The heat maps show upregulated (red) and downregulated (green) genes by unsupervised hierarchical clustering (average linkage; mean-centered; log2 transformed). C, Venn diagram shows 58 common genes associated with signaling pathways of immune cell functions between human and canine tumors. Representative photomicrographs of H&E and IHC staining showing histologic morphology and immune cell infiltration in canine hemangiosarcoma (D) and human angiosarcoma tissues (E) using anti-CD3, anti-PAX5, anti-MAC387, and anti-Iba1 (for canine) or anti-CD163 (for human) antibodies for detecting T cell, B cell, and macrophages. Horseradish peroxidase (counterstain = hematoxylin) or alkaline phosphate (for Iba1; counterstain = methylene blue). Bar = 50 µm. Scatter plots display correlation between transcriptional and IHC immune score in canine hemangiosarcoma (F) and human angiosarcoma (G). Spearman correlation coefficient (R) was calculated.

FIGURE 3

Immune cell infiltration and comparative immune signatures between canine hemangiosarcoma and human angiosarcoma. A, A total of 461 upregulated genes were identified in immune-high (N = 8) compared with immune-low (N = 5) groups in human angiosarcomas (FDR P value < 0.05). B, 567 immune gene signatures were identified among three molecular subtypes of canine hemangiosarcomas (N = 76; FDR P value < 0.001; fold change > 3). The heat maps show upregulated (red) and downregulated (green) genes by unsupervised hierarchical clustering (average linkage; mean-centered; log2 transformed). C, Venn diagram shows 58 common genes associated with signaling pathways of immune cell functions between human and canine tumors. Representative photomicrographs of H&E and IHC staining showing histologic morphology and immune cell infiltration in canine hemangiosarcoma (D) and human angiosarcoma tissues (E) using anti-CD3, anti-PAX5, anti-MAC387, and anti-Iba1 (for canine) or anti-CD163 (for human) antibodies for detecting T cell, B cell, and macrophages. Horseradish peroxidase (counterstain = hematoxylin) or alkaline phosphate (for Iba1; counterstain = methylene blue). Bar = 50 µm. Scatter plots display correlation between transcriptional and IHC immune score in canine hemangiosarcoma (F) and human angiosarcoma (G). Spearman correlation coefficient (R) was calculated.

Close modal

To confirm whether the inflammatory gene signatures were originating from the malignant cells themselves or from inflammatory cells within the tumors, we employed bioinformatics tools and immunostaining techniques. Tumor purity was assessed using ESTIMATE and immune scores were assigned to each tumor using xCell. Both tools yielded consistent scores (Supplementary Fig. S8A and S8B). As expected, we found a correlation between immune scores and the predominant transcriptional phenotype of the tumor in both canine and human samples (Supplementary Fig. S8C–S8F). Specifically, angiogenic tumors exhibited low immune scores, whereas inflammatory tumors exhibited high immune scores.

To further validate that the observed immune signatures were the result of immune and inflammatory cells present within the tumors, we performed IHC staining on sections from 11 canine hemangiosarcomas and 10 human angiosarcomas. Antibodies against T cells (CD3), B cells (Pax5), myeloid cells (Mac387), and macrophages (Iba1 for canine; CD163 for human) were used; CD3+ T cells, Pax5+ B cells, Mac387+ myeloid cells, and Iba1+ or CD163+ macrophages were present in both canine and human tumors (Fig. 3D and E). Myeloid cells were the most abundant, while T cells varied in frequency from rare to abundant, and B cells were infrequent. Importantly, the distribution of inflammatory cells was diffuse throughout the tumor tissue. Furthermore, there was a direct correlation between xCell immune scores and IHC scores for both canine (Spearman R = 0.38; P = 0.255) and human (Spearman R = 0.78; P = 0.011) samples that were examined (Fig. 3F and G). These findings provide evidence that the observed immune signatures in both canine and human tumors are likely attributed to the presence of immune and inflammatory cells within the TME.

Canine Hemangiosarcoma Cells Promote Hematopoiesis and Express Hematopoietic Cytokines

To determine whether hemangiosarcoma cells were functionally sufficient to expand hematopoietic progenitor cells (HPC) in vitro, we conducted long-term culture initiating cell assays using DHSA-1426 and EFB canine hemangiosarcoma cells, to assess their potential in promoting and maintaining hematopoiesis in CD34+ human umbilical cord blood HPCs. Mouse M2-10B4 and human BM-derived MSCs were used as positive controls, while HPCs cultured without feeder cells served as a negative control. Remarkably, DHSA-1426 cells were found to promote expansion of human CD34+ HPCs with at least comparable, if not superior, efficiency compared with conventional mouse or human feeder cells, and resulted in comparable proportions of hematopoietic cell differentiation in vitro across all lineages (Fig. 4; Supplementary Fig. S9). Similar results were observed with EFB cells, albeit with slightly more limited expansion and differentiation.

FIGURE 4

Hematopoietic support and stromal regulation of canine hemangiosarcoma cells. A, Flow cytometric data depict populations of cells expressing CD43 and CD45 differentiated from CD34+ hUCB cells. CD34+ hUCB cells were pooled from 2 patients. M2-10B4, hBM-MSCs, and canine hemangiosarcoma cells (DHSA-1426 and EFB) were seeded on gelatin-coated 24-well plates at a density of 1 × 105 cells/well. Gelatin-coated wells without stroma served as a negative control. Surface antigens CD34, CD43, and CD45 were analyzed at week 5. B and C, Bar graphs illustrate number of different colonies formed by hUCB CD34+ cells cocultured with feeder cells. Both DHSA-1426 and EFB canine hemangiosarcoma cell lines expanded hUCB CD34+ cells similarly to the M2-10B4 and hBM-MSC positive control lines, while gelatin-coated wells alone failed to support expansion. Burst-forming unit-erythroid (BFU-E), CFU (colony-forming unit)-Erythroid (CFU-E), CFU-granulocyte/macrophage (CFU-GM), CFU-macrophage (CFU-M), and CFU-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) were determined for CFU assay.

FIGURE 4

Hematopoietic support and stromal regulation of canine hemangiosarcoma cells. A, Flow cytometric data depict populations of cells expressing CD43 and CD45 differentiated from CD34+ hUCB cells. CD34+ hUCB cells were pooled from 2 patients. M2-10B4, hBM-MSCs, and canine hemangiosarcoma cells (DHSA-1426 and EFB) were seeded on gelatin-coated 24-well plates at a density of 1 × 105 cells/well. Gelatin-coated wells without stroma served as a negative control. Surface antigens CD34, CD43, and CD45 were analyzed at week 5. B and C, Bar graphs illustrate number of different colonies formed by hUCB CD34+ cells cocultured with feeder cells. Both DHSA-1426 and EFB canine hemangiosarcoma cell lines expanded hUCB CD34+ cells similarly to the M2-10B4 and hBM-MSC positive control lines, while gelatin-coated wells alone failed to support expansion. Burst-forming unit-erythroid (BFU-E), CFU (colony-forming unit)-Erythroid (CFU-E), CFU-granulocyte/macrophage (CFU-GM), CFU-macrophage (CFU-M), and CFU-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) were determined for CFU assay.

Close modal

To further explore the molecular properties that govern hematopoiesis, we analyze RNA-seq gene expression data from DHSA-1426 hemangiosarcoma cells compared with nonmalignant endothelial cells. Our analysis revealed 2,391 differentially expressed genes (DEG), with 1,034 genes upregulated and 1,357 genes downregulated in DHSA-1426 cells (Padjusted < 0.01; log2 fold change >|2|; Fig. 5A). Gene Ontology enrichment analysis highlighted the involvement of these genes in key biological processes including blood vessel morphogenesis, angiogenesis, cell differentiation, and extracellular matrix organization (Fig. 5B). Notably, DHSA-1426 cells exhibited an enrichment of marker genes for endothelial progenitors, such as PECAM1, TIE1, and KDR (Supplementary Fig. S10). Moreover, we observed a significant increase in cytokine genes associated with the regulation of HSPCs including CSF3, IL6, and IL8 in DHSA-1426 cells (Fig. 5C). We then investigated whether stromal cells in xenograft tumors express genes encoding receptors for these hemangiosarcoma-secreting cytokines. Our RNA-seq data enabled the identification of canine- and murine-specific genes in the xenograft tissues (Supplementary Fig. S3; Fig. 5D). Using these data, we discovered that hemangiosarcoma xenografts significantly increased murine-specific receptor genes, Csf3r, Il6ra, and Cxcr2, which bind to Csf3, Il6, and IL8 homologous genes (Cxcl1, Cxcl2, and Cxcl5), compared with mouse lymphomas (Fig. 5E). Furthermore, the expression of receptor genes was higher than that of their respective cytokine genes in the hemangiosarcoma xenografts. This indicates strong intercellular interactions and potential signal transduction in close proximity between donor canine hemangiosarcoma cells and host mouse stromal cells within the tumors, supporting the findings in our FISH data (Fig. 1C; Supplementary Fig. S2).

FIGURE 5

Gene expression analysis in DHSA-1426 hemangiosarcoma cells and xenograft tumors using RNA-seq data. A, The volcano plot visualizes 2,391 significant DEGs (1,034 upregulated and 1,357 downregulated) in DHSA-1426 cells (n = 3) compared with nonmalignant endothelial cells (n = 4; q-value < 0.01; log2 fold change > |2|). The DEGs are indicated by red dots. NS = not significant; log2FC = log2 fold change. B, Gene Ontology enrichment analysis depicts biological processes associated with significant DEGs. P, adjust = adjusted P-value. C, Bar graphs present normalized gene expression values of CSF3, IL6, and IL8 in RNA-seq data generated from canine nonmalignant endothelial cells (n = 4) and DHSA-1426 hemangiosarcoma cells (n = 3). D, Schematic illustration visualizes the experimental steps for species-specific gene expression analysis of xenograft tumors using RNA-seq data. E, Bar graphs show the expression of mouse-specific genes in RNA-seq data generated from xenograft tumor tissues of canine hemangiosarcoma (n = 4) and mouse lymphoma (n = 7). A two-way ANOVA test was conducted to compare the means between groups. ****, P < 0.0001

FIGURE 5

Gene expression analysis in DHSA-1426 hemangiosarcoma cells and xenograft tumors using RNA-seq data. A, The volcano plot visualizes 2,391 significant DEGs (1,034 upregulated and 1,357 downregulated) in DHSA-1426 cells (n = 3) compared with nonmalignant endothelial cells (n = 4; q-value < 0.01; log2 fold change > |2|). The DEGs are indicated by red dots. NS = not significant; log2FC = log2 fold change. B, Gene Ontology enrichment analysis depicts biological processes associated with significant DEGs. P, adjust = adjusted P-value. C, Bar graphs present normalized gene expression values of CSF3, IL6, and IL8 in RNA-seq data generated from canine nonmalignant endothelial cells (n = 4) and DHSA-1426 hemangiosarcoma cells (n = 3). D, Schematic illustration visualizes the experimental steps for species-specific gene expression analysis of xenograft tumors using RNA-seq data. E, Bar graphs show the expression of mouse-specific genes in RNA-seq data generated from xenograft tumor tissues of canine hemangiosarcoma (n = 4) and mouse lymphoma (n = 7). A two-way ANOVA test was conducted to compare the means between groups. ****, P < 0.0001

Close modal

Altogether, our data suggest that hemangiosarcoma cells regulate hematopoiesis and tumor stromal cells through the release of immune cytokines, potentially contributing to the creation of their distinctive vascular and immune niche.

Vasoformative sarcomas are aggressive tumors with uncertain cellular origin, proposed to arise from a multipotent BM cell or lineage-committed endothelial progenitor cells in humans, dogs, and mice (1–4, 8). While human angiosarcomas and canine hemangiosarcomas have a limited shared mutational spectrum, primarily in visceral forms of the disease and human breast angiosarcomas, convergent transcriptional programs characterized by deregulation of PI3K pathways are activated in tumors of both species, as well as in zebrafish (8, 44–50).

The transcriptional landscape of human angiosarcoma and canine hemangiosarcoma is strongly proangiogenic. However, a subset of tumors in both species exhibit robust transcriptional immune and inflammatory signatures that correlate with the presence of T cells and macrophages. The presence of immune and inflammatory infiltrates might be associated with longer survival outcomes, but further research is needed to determine their clinical significance regarding disease progression and their association with the approximately 15% of exceptional survivors reported in canine hemangiosarcoma (51, 52).

The findings from our xenograft experiments provide further evidence supporting the concept that the disordered vascular organization in these tumors is driven by the tumor cells (1, 21, 23). Our experiments reveal that the tumor vessels are comprised of both malignant tumor cells and nonmalignant host endothelial cells, a phenomenon that appears to be unique to hemangiosarcoma among the three types of xenografts we investigated. This observation underscores the ability of hemangiosarcoma cells to adopt endothelial functions and suggests that nonmalignant cells play a role in the formation of aberrant blood vessels in vasculogenic tumors. Interestingly, our experiments also demonstrate that the stromal cells in these xenografts contribute to the angiogenic and inflammatory transcriptional signatures. This highlights the possibility that the stromal cells themselves are also influenced or reprogrammed by the malignant cells and supports a complex interplay between the tumor and its microenvironment. These findings highlight a potential vulnerability in the formation of the hemangiosarcoma niche, where the interactions between the tumor cells and their microenvironment are tightly orchestrated.

The initial perplexity surrounding the development of exuberant myeloid and erythroid hyperplasia, as well as bona fide lymphomas, arising from mouse cells in animals with primary or secondary hemangiosarcoma xenografts has led us to investigate the underlying etiology. Our results suggest that a transmissible etiology from dog to mouse is unlikely to be the cause of these expanded hematopoietic cell populations. Instead, our data indicate that canine hemangiosarcomas have the capability to support robust expansion and differentiation of HPCs in vitro, which may account for the expansion of myeloid cells in vivo in mice and in primary canine and human tumors. This expansion of nonmalignant HPCs and mature leukocytes may also explain why clonal mutations (46) and fusions (8) are found less often in the inflammatory subtype of canine hemangiosarcoma. These findings are consistent with a previous report of angiosarcoma in the BM of a human patient with tumor-associated myeloid proliferation and extramedullary hematopoiesis (53). In the case of lymphomas that occurred in animals, MuLV might have driven the transformation of residual lymphoid elements within a hyperproliferative environment created by the hemangiosarcoma cells, but the precise etiology needs further investigation.

Other unexpected tumors have been reported in xenograft experiments and preclinical models of stem cell transplantation. For example, transplantation of murine MSCs has been shown to induce tumor formation and tissue malformation, possibly due to genetic instability and/or cellular transformation (54–56). Similarly, patient-derived xenografts of human solid cancers, such as breast, colon, pancreatic cancer, and rhabdomyosarcoma, have been reported to induce lymphomagenesis or lymphocytic tumors in immunodeficient mice, but in these cases, the tumors were derived from human tumor-infiltrating lymphocytes transformed by EBV (57–60). Importantly, these previously reported tumors were all of donor origin, while the tumors observed in our study originated from the mouse recipients and were distinct from the donor hemangiosarcomas.

We were unable to find any reports of hematopoietic tumors of recipient origin arising from xenotransplantation experiments using other types of canine cancers in the literature, and we have not observed such events in our own studies (29, 61, 62). In seeking feedback for our conclusions, we learned that Dr. Stuart Helfand had observed similar results when he transplanted the SB-HSA canine hemangiosarcoma cell line in NOD mice; however, he did not report these observations at the time (Dr. S. Helfand, personal communication). Thus, this finding appears to be unique to canine hemangiosarcoma and may be attributed to the ability of hemangiosarcoma cells to support hematopoietic expansion. Our findings also suggest that the normal counterparts of canine hemangiosarcoma cells might contribute to the development of hematopoietic malignancies through the creation of a permissive niche.

In this context, it is noteworthy that a shared region of the canine genome has been found to be significantly associated with B-cell lymphomas, hemangiosarcomas, and other blood-derived tumors in various breeds of dogs belonging to distinct genetic clades (63–65). This finding highlights the intriguing possibility of a molecular connection between these seemingly distinct tumors, suggesting that disruptions in hematopoiesis and cellular reprogramming may contribute to their development (66). It also sets the origin of this potential genomic vulnerability prior to the derivation of modern dog breeds.

The capacity of the hematopoietic niche to accommodate BM transplants and adoptive cell therapies has revealed its resilience, with BM stromal cells exhibiting high resistance to chemotherapy and radiation. These intrinsic properties may explain the relatively poor long-term responses of human patients with angiosarcoma and dogs with hemangiosarcoma to cytotoxic therapies, and they could provide opportunities for the development of more effective treatments. However, it is important to recognize that therapies targeting the hematopoietic niche may also carry the potential for high toxicity.

Our data present a novel model to elucidate the capability of canine hemangiosarcomas, and potentially human angiosarcomas, to regulate hematopoiesis. We propose that the malignant endothelial cells have the capability to create niches that promote angiogenic proliferation and hematopoietic expansion, contributing to distinct tumor immune and inflammatory phenotypes, as illustrated in Fig. 6. The robust inflammation observed in some of these tumors may therefore be intrinsic to the tumor itself, rather than solely due to extrinsic factors associated with tissue disruption. These paths of differentiation may also influence the biological behavior of the tumors, with those exhibiting strong angiogenic propensity displaying more aggressive behaviors (8).

FIGURE 6

Potential mechanisms that organize malignant vessels and establish distinct immune phenotypes in hemangiosarcoma. Hypothetical models illustrate that hemangiosarcoma cells may orchestrate with nonmalignant stromal cells to create malignant vascular channels through close intercellular cross-talk. Malignant vessels may facilitate the recruitment of hematopoietic progenitors, delivering of signaling molecules that promote lineage commitment of immune cells. This process enables hemangiosarcoma to create a niche for hematopoietic expansion and inflammation, establishing a vascular malignancy with a distinct immune phenotype or potentially giving rise to a hematopoietic tumor.

FIGURE 6

Potential mechanisms that organize malignant vessels and establish distinct immune phenotypes in hemangiosarcoma. Hypothetical models illustrate that hemangiosarcoma cells may orchestrate with nonmalignant stromal cells to create malignant vascular channels through close intercellular cross-talk. Malignant vessels may facilitate the recruitment of hematopoietic progenitors, delivering of signaling molecules that promote lineage commitment of immune cells. This process enables hemangiosarcoma to create a niche for hematopoietic expansion and inflammation, establishing a vascular malignancy with a distinct immune phenotype or potentially giving rise to a hematopoietic tumor.

Close modal

In conclusion, our study adds to the growing body of evidence demonstrating the complex interactions between tumor cells and the host microenvironment. Further investigations are warranted to unravel the underlying mechanisms that drive these observations and ascertain their significance in the development of human cancer.

M. Meyerson reports research funding from Bayer and Janssen; consulting for Bayer, Delve, Interline, Isabl; equity in Bayer, Delve, Interline, Isabl; patent licensing fees from Bayer, Labcorp. E.B. Dickerson reports grants from Canine Health Foundation and Morris Animal Foundation during the conduct of the study. D.S. Kaufman reports being a co-founder of Shoreline Biosciences and has an equity interest in the company. D.S. Kaufman also consults VisiCELL Medical and RedC Bio for which he receives income and/or equity. Studies in this work are not related to work of those companies. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. No disclosures were reported by the other authors.

J.H. Kim: Conceptualization, resources, data curation, formal analysis, funding acquisition, validation, investigation, visualization, methodology, writing-original draft, writing-review and editing. A.J. Schulte: Project administration, writing-review and editing. A.L. Sarver: Formal analysis, methodology, writing-review and editing. D. Lee: Data curation, formal analysis. M.G. Angelos: Data curation, formal analysis, methodology. A.M. Frantz: Data curation, formal analysis, methodology. C.L. Forster: Data curation, methodology. T.D. O'Brien: Methodology. I. Cornax: Data curation, formal analysis, methodology. M.G. O'Sullivan: Resources, formal analysis, investigation. N. Cheng: Data curation, formal analysis, methodology. M. Lewellen: Data curation, project administration. L. Oseth: Resources, data curation, methodology. S. Kumar: Formal analysis, methodology. S. Bullman: Data curation, formal analysis, methodology. C.S. Pedamallu: Methodology. S.M. Goyal: Methodology, writing-review and editing. M. Meyerson: Methodology, writing-review and editing. T.C. Lund: Methodology, writing-review and editing. M. Breen: Data curation, methodology. K. Lindblad-Toh: Formal analysis, methodology, writing-review and editing. E.B. Dickerson: Writing-review and editing. D.S. Kaufman: Validation, methodology, writing-review and editing. J.F. Modiano: Conceptualization, resources, data curation, supervision, funding acquisition, methodology, writing-original draft, project administration, writing-review and editing.

The authors acknowledge Keumsoon Im for assistance with experiments and data acquisition. The authors would also like to acknowledge Milcah Scott and Jessica Alfoldi for processing the next generation sequencing data and assistance with data analysis. Artistic design of Figs. 1A, 5D, and 6 was created with Biorender (biorender.com).

This work partially supported by a Career Development Award from the Department of Defense Peer Reviewed Cancer Research Program, CA191225 (to J.H. Kim). It was also supported by grants 1R03CA191713-01 (to J.F. Modiano, A.L. Sarver, and J.H. Kim) from the NCI of the NIH, grant nos. 02759 (to J.H. Kim), 422 (to J.F. Modiano), and 1889-G (to J.F. Modiano, M. Breen, and K. Lindblad-Toh) from the AKC Canine Health Foundation, grant JHK15MN-004 (to J.H. Kim, A.L. Sarver, J.F. Modiano) from the National Canine Cancer Foundation, grant D10-501 (to J.F. Modiano, M. Breen, and K. Lindblad-Toh) from Morris Animal Foundation, and grant from Swedish Cancerfonden (to K. Lindblad-Toh). This work was supported by an NIH NCI R50 grant, CA211249 (to A.L. Sarver).

The NIH Comprehensive Cancer Center Support Grant to the Masonic Cancer Center, University of Minnesota (P30 CA077598) provided support for the cytogenetic analyses performed in the Cytogenomics Shared Resource. J.H. Kim is supported by the Artificial Intelligence Initiative of the University of Florida. M. Breen is supported in part by the Oscar J. Fletcher Distinguished Professorship in Comparative Oncology Genetics at North Carolina State University. K. Lindblad-Toh is supported by a Distinguished Professor award from the Swedish Research Council. J.F. Modiano is supported by the Alvin and June Perlman Chair in Animal Oncology.

The authors gratefully acknowledge donations to the Animal Cancer Care and Research Program of the University of Minnesota that helped support this project. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of any of the funding agencies listed above.

Note: Supplementary data for this article are available at Cancer Research Communications Online (https://aacrjournals.org/cancerrescommun/).

1.
Fosmire
SP
,
Dickerson
EB
,
Scott
AM
,
Bianco
SR
,
Pettengill
MJ
,
Meylemans
H
, et al
.
Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium
.
Lab Invest
2004
;
84
:
562
72
.
2.
Lamerato-Kozicki
AR
,
Helm
KM
,
Jubala
CM
,
Cutter
GC
,
Modiano
JF
.
Canine hemangiosarcoma originates from hematopoietic precursors with potential for endothelial differentiation
.
Exp Hematol
2006
;
34
:
870
8
.
3.
Liu
L
,
Kakiuchi-Kiyota
S
,
Arnold
LL
,
Johansson
SL
,
Wert
D
,
Cohen
SM
.
Pathogenesis of human hemangiosarcomas and hemangiomas
.
Hum Pathol
2013
;
44
:
2302
11
.
4.
Gorden
BH
,
Kim
JH
,
Sarver
AL
,
Frantz
AM
,
Breen
M
,
Lindblad-Toh
K
, et al
.
Identification of three molecular and functional subtypes in canine hemangiosarcoma through gene expression profiling and progenitor cell characterization
.
Am J Pathol
2014
;
184
:
985
95
.
5.
Italiano
A
,
Thomas
R
,
Breen
M
,
Zhang
L
,
Crago
AM
,
Singer
S
, et al
.
The miR-17–92 cluster and its target THBS1 are differentially expressed in angiosarcomas dependent on MYC amplification
.
Genes Chromosomes Cancer
2012
;
51
:
569
78
.
6.
Kim
JH
,
Graef
AJ
,
Dickerson
EB
,
Modiano
JF
.
Pathobiology of hemangiosarcoma in dogs: research advances and future perspectives
.
Vet Sci
2015
;
2
:
388
405
.
7.
Tamburini
BA
,
Phang
TL
,
Fosmire
SP
,
Scott
MC
,
Trapp
SC
,
Duckett
MM
, et al
.
Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma
.
BMC Cancer
2010
;
10
:
619
.
8.
Kim
JH
,
Megquier
K
,
Thomas
R
,
Sarver
AL
,
Song
JM
,
Kim
YT
, et al
.
Genomically complex human angiosarcoma and canine hemangiosarcoma establish convergent angiogenic transcriptional programs driven by novel gene fusions
.
Mol Cancer Res
2021
;
19
:
847
61
.
9.
Quail
DF
,
Joyce
JA
.
Microenvironmental regulation of tumor progression and metastasis
.
Nat Med
2013
;
19
:
1423
37
.
10.
Colmone
A
,
Amorim
M
,
Pontier
AL
,
Wang
S
,
Jablonski
E
,
Sipkins
DA
.
Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells
.
Science
2008
;
322
:
1861
5
.
11.
Schreiber
RD
,
Old
LJ
,
Smyth
MJ
.
Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion
.
Science
2011
;
331
:
1565
70
.
12.
Lane
SW
,
Scadden
DT
,
Gilliland
DG
.
The leukemic stem cell niche: current concepts and therapeutic opportunities
.
Blood
2009
;
114
:
1150
7
.
13.
Charles
N
,
Ozawa
T
,
Squatrito
M
,
Bleau
AM
,
Brennan
CW
,
Hambardzumyan
D
, et al
.
Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells
.
Cell Stem Cell
2010
;
6
:
141
52
.
14.
Vermeulen
L
,
De Sousa
EMF
,
van der Heijden
M
,
Cameron
K
,
de Jong
JH
,
Borovski
T
, et al
.
Wnt activity defines colon cancer stem cells and is regulated by the microenvironment
.
Nat Cell Biol
2010
;
12
:
468
76
.
15.
Adams
GB
,
Scadden
DT
.
The hematopoietic stem cell in its place
.
Nat Immunol
2006
;
7
:
333
7
.
16.
de Haan
G
,
Lazare
SS
.
Aging of hematopoietic stem cells
.
Blood
2018
;
131
:
479
87
.
17.
Filippi
MD
,
Ghaffari
S
.
Mitochondria in the maintenance of hematopoietic stem cells: new perspectives and opportunities
.
Blood
2019
;
133
:
1943
52
.
18.
Wu
JY
,
Purton
LE
,
Rodda
SJ
,
Chen
M
,
Weinstein
LS
,
McMahon
AP
, et al
.
Osteoblastic regulation of B lymphopoiesis is mediated by Gs{alpha}-dependent signaling pathways
.
Proc Natl Acad Sci U S A
2008
;
105
:
16976
81
.
19.
Raaijmakers
MH
,
Mukherjee
S
,
Guo
S
,
Zhang
S
,
Kobayashi
T
,
Schoonmaker
JA
, et al
.
Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia
.
Nature
2010
;
464
:
852
7
.
20.
Kode
A
,
Manavalan
JS
,
Mosialou
I
,
Bhagat
G
,
Rathinam
CV
,
Luo
N
, et al
.
Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts
.
Nature
2014
;
506
:
240
4
.
21.
Akhtar
N
,
Padilla
ML
,
Dickerson
EB
,
Steinberg
H
,
Breen
M
,
Auerbach
R
, et al
.
Interleukin-12 inhibits tumor growth in a novel angiogenesis canine hemangiosarcoma xenograft model
.
Neoplasia
2004
;
6
:
106
16
.
22.
Tamburini
BA
,
Trapp
S
,
Phang
TL
,
Schappa
JT
,
Hunter
LE
,
Modiano
JF
.
Gene expression profiles of sporadic canine hemangiosarcoma are uniquely associated with breed
.
PLoS One
2009
;
4
:
e5549
.
23.
Kim
JH
,
Frantz
AM
,
Anderson
KL
,
Graef
AJ
,
Scott
MC
,
Robinson
S
, et al
.
Interleukin-8 promotes canine hemangiosarcoma growth by regulating the tumor microenvironment
.
Exp Cell Res
2014
;
323
:
155
64
.
24.
Hicok
KC
,
Thomas
T
,
Gori
F
,
Rickard
DJ
,
Spelsberg
TC
,
Riggs
BL
.
Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma
.
J Bone Miner Res
1998
;
13
:
205
17
.
25.
Kopher
RA
,
Penchev
VR
,
Islam
MS
,
Hill
KL
,
Khosla
S
,
Kaufman
DS
.
Human embryonic stem cell-derived CD34+ cells function as MSC progenitor cells
.
Bone
2010
;
47
:
718
28
.
26.
Zou
L
,
Kidwai
FK
,
Kopher
RA
,
Motl
J
,
Kellum
CA
,
Westendorf
JJ
, et al
.
Use of RUNX2 expression to identify osteogenic progenitor cells derived from human embryonic stem cells
.
Stem Cell Reports
2015
;
4
:
190
8
.
27.
Zou
L
,
Chen
Q
,
Quanbeck
Z
,
Bechtold
JE
,
Kaufman
DS
.
Angiogenic activity mediates bone repair from human pluripotent stem cell-derived osteogenic cells
.
Sci Rep
2016
;
6
:
22868
.
28.
Ferrell
PI
,
Hexum
MK
,
Kopher
RA
,
Lepley
MA
,
Gussiaas
A
,
Kaufman
DS
.
Functional assessment of hematopoietic niche cells derived from human embryonic stem cells
.
Stem Cells Dev
2014
;
23
:
1355
63
.
29.
Scott
MC
,
Tomiyasu
H
,
Garbe
JR
,
Cornax
I
,
Amaya
C
,
O'Sullivan
MG
, et al
.
Heterotypic mouse models of canine osteosarcoma recapitulate tumor heterogeneity and biological behavior
.
Dis Model Mech
2016
;
9
:
1435
44
.
30.
Scott
MC
,
Temiz
NA
,
Sarver
AE
,
LaRue
RS
,
Rathe
SK
,
Varshney
J
, et al
.
Comparative transcriptome analysis quantifies immune cell transcript levels, metastatic progression, and survival in osteosarcoma
.
Cancer Res
2018
;
78
:
326
37
.
31.
Cheng
N
,
Schulte
AJ
,
Santosa
F
,
Kim
JH
.
Machine learning application identifies novel gene signatures from transcriptomic data of spontaneous canine hemangiosarcoma
.
Brief Bioinform
2021
;
22
:
bbaa252
.
32.
Kostic
AD
,
Ojesina
AI
,
Pedamallu
CS
,
Jung
J
,
Verhaak
RG
,
Getz
G
, et al
.
PathSeq: software to identify or discover microbes by deep sequencing of human tissue
.
Nat Biotechnol
2011
;
29
:
393
6
.
33.
Li
H
,
Durbin
R
.
Fast and accurate short read alignment with Burrows-Wheeler transform
.
Bioinformatics
2009
;
25
:
1754
60
.
34.
Altschul
SF
,
Madden
TL
,
Schäffer
AA
,
Zhang
J
,
Zhang
Z
,
Miller
W
, et al
.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
.
Nucleic Acids Res
1997
;
25
:
3389
402
.
35.
Smit
A
,
Hubley
R
,
Green
P
.
RepeatMasker Open-3.0
. Available from: http://www.repeatmasker.org.
36.
Kaufman
DS
,
Hanson
ET
,
Lewis
RL
,
Auerbach
R
,
Thomson
JA
.
Hematopoietic colony-forming cells derived from human embryonic stem cells
.
Proc Natl Acad Sci U S A
2001
;
98
:
10716
21
.
37.
Kodama
A
,
Sakai
H
,
Matsuura
S
,
Murakami
M
,
Murai
A
,
Mori
T
, et al
.
Establishment of canine hemangiosarcoma xenograft models expressing endothelial growth factors, their receptors, and angiogenesis-associated homeobox genes
.
BMC Cancer
2009
;
9
:
363
.
38.
Andersen
NJ
,
Nickoloff
BJ
,
Dykema
KJ
,
Boguslawski
EA
,
Krivochenitser
RI
,
Froman
RE
, et al
.
Pharmacologic inhibition of MEK signaling prevents growth of canine hemangiosarcoma
.
Mol Cancer Ther
2013
;
12
:
1701
14
.
39.
Murai
A
,
Asa
SA
,
Kodama
A
,
Hirata
A
,
Yanai
T
,
Sakai
H
.
Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines
.
BMC Vet Res
2012
;
8
:
128
.
40.
Lashnits
E
,
Neupane
P
,
Bradley
JM
,
Richardson
T
,
Thomas
R
,
Linder
KE
, et al
.
Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States
.
PLoS One
2020
;
15
:
e0227234
.
41.
Varanat
M
,
Maggi
RG
,
Linder
KE
,
Breitschwerdt
EB
.
Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma sp. in dogs with splenic disease
.
J Vet Intern Med
2011
;
25
:
1284
91
.
42.
Chomel
BB
,
Boulouis
HJ
,
Maruyama
S
,
Breitschwerdt
EB
.
Bartonella spp. in pets and effect on human health
.
Emerg Infect Dis
2006
;
12
:
389
94
.
43.
Engel
P
,
Dehio
C
.
Genomics of host-restricted pathogens of the genus bartonella
.
Genome Dyn
2009
;
6
:
158
69
.
44.
Choorapoikayil
S
,
Weijts
B
,
Kers
R
,
de Bruin
A
,
den Hertog
J
.
Loss of Pten promotes angiogenesis and enhanced vegfaa expression in zebrafish
.
Dis Model Mech
2013
;
6
:
1159
66
.
45.
Wang
G
,
Wu
M
,
Maloneyhuss
MA
,
Wojcik
J
,
Durham
AC
,
Mason
NJ
, et al
.
Actionable mutations in canine hemangiosarcoma
.
PLoS One
2017
;
12
:
e0188667
.
46.
Megquier
K
,
Turner-Maier
J
,
Swofford
R
,
Kim
JH
,
Sarver
AL
,
Wang
C
, et al
.
Comparative genomics reveals shared mutational landscape in canine hemangiosarcoma and human angiosarcoma
.
Mol Cancer Res
2019
;
17
:
2410
21
.
47.
Wang
G
,
Wu
M
,
Durham
AC
,
Radaelli
E
,
Mason
NJ
,
Xu
X
, et al
.
Molecular subtypes in canine hemangiosarcoma reveal similarities with human angiosarcoma
.
PLoS One
2020
;
15
:
e0229728
.
48.
Beca
F
,
Krings
G
,
Chen
YY
,
Hosfield
EM
,
Vohra
P
,
Sibley
RK
, et al
.
Primary mammary angiosarcomas harbor frequent mutations in KDR and PIK3CA and show evidence of distinct pathogenesis
.
Mod Pathol
2020
;
33
:
1518
26
.
49.
Chan
JY
,
Lim
JQ
,
Yeong
J
,
Ravi
V
,
Guan
P
,
Boot
A
, et al
.
Multiomic analysis and immunoprofiling reveal distinct subtypes of human angiosarcoma
.
J Clin Invest
2020
;
130
:
5833
46
.
50.
Painter
CA
,
Jain
E
,
Tomson
BN
,
Dunphy
M
,
Stoddard
RE
,
Thomas
BS
, et al
.
The Angiosarcoma Project: enabling genomic and clinical discoveries in a rare cancer through patient-partnered research
.
Nat Med
2020
;
26
:
181
7
.
51.
Borgatti
A
,
Koopmeiners
JS
,
Sarver
AL
,
Winter
AL
,
Stuebner
K
,
Todhunter
D
, et al
.
Safe and effective sarcoma therapy through bispecific targeting of EGFR and uPAR
.
Mol Cancer Ther
2017
;
16
:
956
65
.
52.
Borgatti
A
,
Fieberg
A
,
Winter
AL
,
Stuebner
K
,
Taras
E
,
Todhunter
D
, et al
.
Impact of repeated cycles of EGF bispecific angiotoxin (eBAT) administered at a reduced interval from doxorubicin chemotherapy in dogs with splenic haemangiosarcoma
.
Vet Comp Oncol
2020
;
18
:
664
74
.
53.
Xie
W
,
Lin
P
,
Konoplev
S
.
An unexpected diagnosis: angiosarcoma with bone marrow involvement mimicking a myeloproliferative neoplasm
.
Br J Haematol
2019
;
184
:
495
.
54.
Miura
M
,
Miura
Y
,
Padilla-Nash
HM
,
Molinolo
AA
,
Fu
B
,
Patel
V
, et al
.
Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation
.
Stem Cells
2006
;
24
:
1095
103
.
55.
Foudah
D
,
Redaelli
S
,
Donzelli
E
,
Bentivegna
A
,
Miloso
M
,
Dalpra
L
, et al
.
Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells
.
Chromosome Res
2009
;
17
:
1025
39
.
56.
Jeong
JO
,
Han
JW
,
Kim
JM
,
Cho
HJ
,
Park
C
,
Lee
N
, et al
.
Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental myocardial infarction and diabetic neuropathy
.
Circ Res
2011
;
108
:
1340
7
.
57.
Chen
K
,
Ahmed
S
,
Adeyi
O
,
Dick
JE
,
Ghanekar
A
.
Human solid tumor xenografts in immunodeficient mice are vulnerable to lymphomagenesis associated with Epstein-Barr virus
.
PLoS One
2012
;
7
:
e39294
.
58.
Fujii
E
,
Kato
A
,
Chen
YJ
,
Matsubara
K
,
Ohnishi
Y
,
Suzuki
M
.
Characterization of EBV-related lymphoproliferative lesions arising in donor lymphocytes of transplanted human tumor tissues in the NOG mouse
.
Exp Anim
2014
;
63
:
289
96
.
59.
Bondarenko
G
,
Ugolkov
A
,
Rohan
S
,
Kulesza
P
,
Dubrovskyi
O
,
Gursel
D
, et al
.
Patient-derived tumor xenografts are susceptible to formation of human lymphocytic tumors
.
Neoplasia
2015
;
17
:
735
41
.
60.
Tillman
H
,
Vogel
P
,
Rogers
T
,
Akers
W
,
Rehg
JE
.
Spectrum of posttransplant lymphoproliferations in NSG mice and their association with EBV infection after engraftment of pediatric solid tumors
.
Vet Pathol
2020
;
57
:
445
56
.
61.
Weiskopf
K
,
Anderson
KL
,
Ito
D
,
Schnorr
PJ
,
Tomiyasu
H
,
Ring
AM
, et al
.
Eradication of canine diffuse large B-cell lymphoma in a murine xenograft model with CD47 blockade and anti-CD20
.
Cancer Immunol Res
2016
;
4
:
1072
87
.
62.
Ito
D
,
Endicott
MM
,
Jubala
CM
,
Helm
KM
,
Burnett
RC
,
Husbands
BD
, et al
.
A tumor-related lymphoid progenitor population supports hierarchical tumor organization in canine B-cell lymphoma
.
J Vet Intern Med
2011
;
25
:
890
6
.
63.
Tonomura
N
,
Elvers
I
,
Thomas
R
,
Megquier
K
,
Turner-Maier
J
,
Howald
C
, et al
.
Genome-wide association study identifies shared risk loci common to two malignancies in golden retrievers
.
PLoS Genet
2015
;
11
:
e1004922
.
64.
Hédan
B
,
Cadieu
É
,
Rimbault
M
,
Vaysse
A
,
Dufaure de Citres
C
,
Devauchelle
P
, et al
.
Identification of common predisposing loci to hematopoietic cancers in four dog breeds
.
PLoS Genet
2021
;
17
:
e1009395
.
65.
Evans
JM
,
Parker
HG
,
Rutteman
GR
,
Plassais
J
,
Grinwis
GCM
,
Harris
AC
, et al
.
Multi-omics approach identifies germline regulatory variants associated with hematopoietic malignancies in retriever dog breeds
.
PLoS Genet
2021
;
17
:
e1009543
.
66.
Zhang
Q
,
Orlando
EJ
,
Wang
HY
,
Bogusz
AM
,
Liu
X
,
Lacey
SF
, et al
.
Transdifferentiation of lymphoma into sarcoma associated with profound reprogramming of the epigenome
.
Blood
2020
;
136
:
1980
3
.
This open access article is distributed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.