DS1_JVDI_10.1177_1040638718770239 – Supplemental material for Immunohistochemical detection of p53, PTEN, Rb, and p16 in canine osteosarcoma using tissue microarray

<p>Supplemental material, DS1_JVDI_10.1177_1040638718770239 for Immunohistochemical detection of p53, PTEN, Rb, and p16 in canine osteosarcoma using tissue microarray by Duncan S. Russell, Lauren Jaworski, William C. Kisseberth in Journal of Veterinary Diagnostic Investigation</p

DS2_JVDI_10.1177_1040638718770239 – Supplemental material for Immunohistochemical detection of p53, PTEN, Rb, and p16 in canine osteosarcoma using tissue microarray

<p>Supplemental material, DS2_JVDI_10.1177_1040638718770239 for Immunohistochemical detection of p53, PTEN, Rb, and p16 in canine osteosarcoma using tissue microarray by Duncan S. Russell, Lauren Jaworski, William C. Kisseberth in Journal of Veterinary Diagnostic Investigation</p

Human Genetic Relevance and Potent Antitumor Activity of Heat Shock Protein 90 Inhibition in Canine Lung Adenocarcinoma Cell Lines

<div><p>Background</p><p>It has been an open question how similar human and canine lung cancers are. This has major implications in availability of human treatments for dogs and in establishing translational models to test new therapies in pet dogs. The prognosis for canine advanced lung cancer is poor and new treatments are needed. Heat shock protein 90 (HSP90) is an ATPase-dependent molecular chaperone ubiquitously expressed in eukaryotic cells. HSP90 is essential for posttranslational conformational maturation and stability of client proteins including protein kinases and transcription factors, many of which are important for the proliferation and survival of cancer cells. We investigated the activity of STA-1474, a HSP90 inhibitor, in two canine lung cancer cell lines, BACA and CLAC.</p><p>Results</p><p>Comparative genomic hybridization analysis of both cell lines revealed genetic relevance to human non-small cell lung cancer. STA-1474 inhibited growth and induced apoptosis of both cell lines in a dose- and time-dependent manner. The ICs₅₀ after 72 h treatment with STA-1474 were 0.08 and 0.11 μM for BACA and CLAC, respectively. When grown as spheroids, the IC₅₀ of STA-1474 for BACA cells was approximately two-fold higher than when grown as a monolayer (0.348 μM <i>vs</i>. 0.168 μM), whereas CLAC spheroids were relatively drug resistant. Treatment of tumor-stromal fibroblasts with STA-1474 resulted in a dose-dependent decrease in their relative cell viability with a low IC₅₀ of 0.28 μM.</p><p>Conclusions</p><p>Here we first established that lung adenocarcinoma in people and dogs are genetically and biochemically similar. STA1474 demonstrated biological activity in both canine lung cancer cell lines and tumor-stromal fibroblasts. As significant decreases in relative cell viability can be achieved with nanomolar concentrations of STA-1474, investigation into the clinical efficacy of this drug in canine lung cancer patients is warranted.</p></div

Viability and half-maximal inhibitory concentration (ICs₅₀) of STA-1474 treated canine lung cancer cell-line derived tumor spheroids, monolayers and tumor-stromal fibroblasts.

<p>(A) Tumor spheroids derived from both cell lines were allowed to form for 72 h after seeding cells in the ultra-low attachment wells. Images taken of one field of view/well on an inverted microscope at 40X magnification, scale bar represents 200 μM (B) Immediately after formation, tumor spheroids were treated with DMSO or increasing concentrations of STA-1474 for 72 h. Tumor spheroids and monolayers from each cell line were grown and treated identically. Cultured cells were treated with STA-1474 for an additional 72 h and ICs₅₀ were calculated. (C) Tumor-stromal fibroblasts were seeded, allowed to form a monolayer for 24 h then incubated with STA-1474 for 72 h and viability determined. Experiments were performed in four replicates and repeated twice. Each graph shows mean ± SEM and each group was compared to DMSO. The dotted line in the y-axis represents the 50% relative viability. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Viability assays and half-maximal inhibitory concentrations (ICs₅₀) for canine lung cancer cell lines.

<p>Cells were treated with increasing concentrations cytotoxic drugs (A) or small molecules inhibitors (B) and proliferation was evaluated after 72 h. Treatment effects were normalized to the drug vehicle-treated control group. Each graph shows mean±SEM. ICs₅₀ were calculated for each experiment. The dotted line in the y-axis represents the 50% relative viability. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Canine RT-PCR Primers.

<p>Canine RT-PCR Primers.</p

Evaluation of apoptosis in canine lung cancer cell lines treated with STA-1474.

<p>Apoptosis was assessed by annexin V/PI staining flow cytometry and detection of caspase 3/7 enzymatic activity. (A) Cells were treated with dimethyl sulfoxyide (DMSO, control) or 0.005–1 μM of STA-1474 for 24 h. Staining with annexin V and the vital dye, propidium iodide (PI), were used to evaluate early and late apoptosis. Cells that are considered viable are both annexin V and PI negative, while cells that are in early apoptosis are annexin V positive and PI negative, and cells that are in late apoptosis or already dead are both annexin V and PI positive. (B) Both cell lines were treated as above and evaluated for executioner caspase-mediated apoptosis. Activated caspases 3 and 7 were assessed 24 and 48 h after treatment. Experiments were performed in triplicate and repeated three times. Each graph shows mean ± SEM. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Relative cell viability assays, half-maximal inhibitory concentrations (ICs₅₀) after treatment with an HSP70 inhibitor (VER155008) or a combination of STA-1474 and VER155008.

<p>(A) Cells were treated with HSP70 inhibitor, (VER155008) for 72 h. The relative viability and ICs₅₀ were determined for both cell lines after treatment. (B) BACA or CLAC cells were plated in DMEM media for 24 h and then treated for 72 h with VER155008 only (gray squares), STA-1474 only (black circles) or a combination of VER155008 and STA-1474 concentrations (red triangles) ranging from 0.0625X to 16X their IC₅₀ concentrations. Each graph shows mean ± SEM. Each group was compared to the DMSO control. The dotted line in the y-axis represents the 50% relative viability. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001. (C) Multi-drug combination dose-effect analysis for the doublet combination of VER155008 and STA-1474 on BACA and CLAC cell lines as measured by the combination index (CI). The CI value definitions are represented in the description column.</p

Comparative genomic hybridization analysis of canine BACA and CLAC lung adenocarcinoma cell lines.

<p>A custom 1M feature array was used to measure the DNA copy number across the genome relative to germline DNA from a normal dog. The zero line corresponds to a ratio of 1:1 between test DNA and a male reference DNA, or generally a copy number of 2. Gains are above the line and losses below. (A) Whole genome copy number analysis is shown with BACA on top (red) and CLAC on bottom (blue). Among the important findings, both cell lines have a 2-copy loss of <i>CDKN2A/B/B-AS1</i> (encoding tumor suppressors p14-p19/ARF and p16/INK4; also shown in close-up in B panels) and a 2-copy gain of chr13 (which contains the oncogene <i>MYC</i>). Other large alterations are a 1-copy loss of most of chr2 and chr16 in BACA and a 1-copy gain and loss of chr17 and chr11, respectively in CLAC. Most of all other variants shared by the two cell lines represent Copy Number Variation (CNV) in the reference DNA. (B) Copy number analysis of complete chr11 shows BACA has a 1-copy gain over 2/3rds of its length and a 1-copy loss over the remainder and CLAC has a 1-copy loss over its full length. Both lines have a relatively small 2-copy loss (focal in BACA) that overlaps only <i>CDKN2A/B/B-AS1</i> between the two cell lines.</p

Gene annotation summary of focal copy number alterations in BACA and CLAC.

<p>Gene annotation summary of focal copy number alterations in BACA and CLAC.</p