Fig 1.
Kaplan-Meier analysis of updated event-free and overall survival for ten dogs on trial.
Kaplan-Meier survival curves for lung-metastasis free survival (dashed line) and overall survival (solid line) for dogs with locally advanced, non-metastatic osteosarcoma treated with palliative radiotherapy and intra-tumoral NK transfer x2. Median survival was not reached for lung-metastasis free survival during the follow up period.
Table 1.
Clinical characteristics and vital status of canine patients on clinical trial.
Fig 2.
Canine NK cells are enriched in NKp46 and Granzyme B expression with time-dependent changes in cytotoxic function.
(A) Canine NK cells were isolated from peripheral blood mononuclear cells via CD5 antibody depletion (left), thereby enriching for a CD3-NKp46+ population of cells (right). (B) Ex vivo expansion with an irradiated feeder cell line (human K562 leukemia line transduced with 4-1BBL and membrane bound IL-21) yields a CD3 population of cells that are markedly positive for NKp46+ (left) and Granzyme B+ (right) after 14 days of stimulation. For panels A and B, representative flow cytometry plots from 10 study dogs and 6 healthy beagles are shown. (C) The cytotoxicity of NK cells was assayed at multiple time points (using CTAC cells as targets) and compared to fresh PBMCs (incubated with 100 IU/mL rhIL-2) in 12–16 hour killing assays. Data from one experiment performed in triplicate are shown. Mean values ± SD are shown. This experiment was repeated with 3 different beagle donors. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 via one-way ANOVA with Tukey’s post-test.
Fig 3.
Baseline serum cytokine expression in trial patients.
Baseline expression of serum cytokines was compared between dogs who were alive at 6 months versus those who died. There was no significant different in serum IL-2 (A) or serum TNF-α (C) between survivors and non-survivors. Baseline serum IL-6 (B) was notably higher in non- survivors (32.1 pg/mL ±19.2) compared to survivors (not detectable), although this difference was not statistically significant (P = 0.08).
Fig 4.
Expression of Granzyme B and IFN-γ in isolated peripheral blood mononuclear cells.
PBMCs were isolated from dog patients on trial and analyzed via flow cytometry for the expression of activation markers. Baseline PBMC expression between dogs who were alive at 6 months versus those who died showed no significant difference in percent granzyme B positive (A) or percent IFN-γ positive PBMCs (D). Representative flow cytometry staining for granzyme B (B) with FMO control (C) and IFN-γ (E) with FMO control (F) is shown.
Fig 5.
Intra-Tumoral immune cell infiltration and gene expression in dog sarcomas.
(A) Lymph node from a dog necropsy specimen was compared to tissue from untreated dog soft tissue sarcoma and osteosarcoma cases. Immunohistochemical staining for CD3+ cells revealed high infiltration in normal lymph node (left) versus rare to absent lymphocyte presence in soft tissue sarcoma (middle) or osteosarcoma (right) tumors. Scale bar = 200 μm. RNA analysis by PCR of PBMCs from healthy dogs (A–bottom left) demonstrated greatest expression of CD3 compared to CD8 or NK marker NKG2D. Expression of these genes in PBMCs was approximately 100-fold greater than expression in osteosarcoma tumor tissue (A–bottom right), and intra-tumoral expression of NKG2D was significantly lower than both CD3 and CD8. Technical replicates from one of 3 experiments are shown. * P<0.05, *** P<0.001 via one-way ANOVA with Tukey’s post-test. (B) Tumor tissue from 8 study subjects was obtained at baseline and after palliative RT and intra-tumoral NK and analyzed for change in intra-tumoral gene expression after therapy. qPCR results demonstrated marked heterogeneity in changes in gene expression of key immune-related genes. Interestingly, the patient who lived the longest (17.9 months) showed the greatest fold-change in the expression of CD3, CD8, and IDO1 genes. On univariate analysis, there was no significant difference (P>0.05) in intra-tumoral changes in gene expression and survival for CD3 (C), CD8 (D), IDO1 (E), IL-10 (F), IL-6 (G), or TGF-β (H). Symbols represent fold change in gene expression from before therapy to after using pairwise comparisons from individual treated dogs. Mean fold change with standard deviation is shown. Groups were compared using an unpaired t-test.
Fig 6.
Intra-tumoral gene expression and survival outcomes in human sarcomas from the cancer genome atlas.
The Cancer Genome Atlas (TCGA) was queried for gene expression data with immunoregulatory function from human sarcoma patients with non-metastatic disease. Patients were categorized into high and low expression by quartiles, and survival differences were analyzed between low expression (quartile 1) and high expression (quartile 4). Significant survival differences were identified between high and low gene expression for (A) CD3e, (B) CD8a, (C) IFN-γ (E) PRF1 (perforin), (F) CD122/ interleukin 2 (and interleukin 15) receptor subunit beta, (G) IL-6, and (H) IL-6R. Notably, and somewhat paradoxically, greater intra-tumoral expression of IL-6 and IL-6R were associated with superior survival in human sarcomas. Sarcoma patients with higher intra-tumoral GZMB (granzyme B) expression (D) had longer survival, but this difference was not statistically significant (HR 0.65, 95% CI 0.37–1.13, P = 0.13).