Transcriptomic Alterations of Canine Histiocytic Sarcoma Cells in Response to Different Stressors

Abstract

Canine histiocytic sarcoma (HS) is a rare tumor with a poor prognosis. Rapid tumor growth often causes central hypoxia and starvation, impacting tumor progression. In the present study, HS cells were cultured under hypoxia and starvation for 1 and 3 days, simulating intermediate and central tumor zones, respectively. Cells were counted at each time point, followed by RNAseq analysis. Only hypoxia significantly reduced the cell number (p < 0.05). Short-term hypoxia altered 1645 differentially expressed genes (DEGs). Upregulated genes belonged to vasculature development, and downregulated genes to cell cycle processes. Short-term starvation affected 157 genes, mainly involving responses to stimuli. Prolonged hypoxia and starvation induced 1301 and 836 DEGs, respectively. Prolonged hypoxia upregulated genes mainly involved in immune responses, response to stimulus, adhesion, and angiogenesis. Prolonged starvation upregulated genes associated with signaling, adhesion, circulatory system development, and response to stimulus. Lipid metabolism and cell cycle pathways were downregulated under prolonged hypoxia and starvation, respectively. KEGG "pathways in cancer" were enriched under all conditions (adjusted p-values < 0.05). These findings indicate that hypoxia and starvation significantly alter the expression of genes involved in tumor progression. Further studies, namely post-translational analyses, are needed to elucidate the functional impact of these changes and identify potential therapeutic targets.

Keywords: RNA sequencing; hypoxia; metabolism; starvation; stress; tumor zones.

Figure 1

Proliferation assay of DH82 cells cultured under control, hypoxic, and starving conditions. Depicted are minimum, 25% quartile, median, 75% quartile, and maximum. * p < 0.05, ** p < 0.01.

Figure 2

Principal component analysis plot of transcriptomic data, showing that all three replicates of all six groups clustered separately. DH82: canine histiocytic sarcoma cells, K: control, H: hypoxia, S: starvation, d1: day 1, d3: day 3.

Figure 3

Volcano plots (a,c) and heat maps (b,d) of DEGs in DH82 cells cultured under hypoxia for a short time frame (1d; (a,b)) and after prolonged exposure (3d; (c,d)) compared to control conditions. Volcano plots. Significantly up- and downregulated genes (adjusted p-value ≤ 0.05; log₁₀ adjusted p-value ≤ 1.3) are indicated by red (log₂FC > 1) and blue (log₂FC < −1), respectively. DH82: canine histiocytic sarcoma cells, K: control, H: hypoxia, S: starvation, d1: day 1, d3: day 3.

Figure 4

Venn diagram of common DEGs in DH82 cells cultured under hypoxic conditions for a short time frame (1d) and after prolonged exposure (3d). (a) Upregulated common DEGs. (b) Downregulated common DEGs.

Figure 5

Volcano plots (a,c) and heat maps (b,d) of DEGs in DH82 cells cultured under starvation for a short time frame (1d; (a,b)) and prolonged exposure (3d; (c,d)) compared to control conditions. Volcano plots. Significantly up- and downregulated genes (adjusted p-value ≤ 0.05; log₁₀ adjusted p-value ≤ 1.3) are indicated by red (log₂FC > 1) and blue (log₂FC < −1), respectively. DH82: canine histiocytic sarcoma cells, K: control, H: hypoxia, S: starvation, d1: day 1, d3: day 3.

Figure 6

Venn diagram of common DEGs in DH82 cells cultured under starvation conditions for short time frames (1d) and after prolonged exposure (3d). (a) Upregulated common DEGs. (b) Downregulated common DEGs.

Figure 7

Volcano plots (a,c,e) and heat maps (b,d,f) of DEGs in DH82 cells cultured under control (a,b), hypoxia (c,d), and starvation (e,f) after prolonged exposure (3d) compared to a short time exposure (1d). Volcano plots. Significantly up- and downregulated genes (adjusted p-value ≤ 0.05; log₁₀ adjusted p-value ≤ 1.3) are indicated by red (log₂FC > 1) and blue (log₂FC < −1), respectively. DH82: canine histiocytic sarcoma cells, K: control, H: hypoxia, S: starvation, d1: day 1, d3: day 3.

Figure 8

Venn diagram of common upregulated DEGs of hypoxia and starvation in terms of (a) circulatory system development and (b) cell adhesion.

Figure 9

Dot plots representing upregulated genes in the GO terms circulatory system development (a–d) and cell adhesion (c–g) in DH82 cells exposed to prolonged hypoxia and starvation. DH82: canine histiocytic sarcoma cells, K: control, H: hypoxia, S: starvation, d1: day 1, d3: day 3.