VDA-1275 leads to cell apoptosis
The chemical structure and formula name of VDA-1275 is shown in figure 1a. The effect of VDA-1275 on apoptosis induction was tested by measuring the increase in cellular levels of cleaved caspase-3/7 activity within 6 hours of treatment, showing over 50% and 300% elevation of activity using low VDA-1275 concentrations of 0.03 and 0.1 µM, respectively (Figure 1b). This early event is in line with a caspase-dependent apoptotic mechanism.
VDA-1275 inhibits cancer cell proliferation
The effect of VDA-1275 on cellular proliferation was tested using the XTT cell viability assay. Cells were incubated with VDA-1275 for 72 hours. Two human cell lines were tested, the non-small cell lung cancer (NSCLC) H358 and the prostate cancer PC-3 cell lines, resulting in IC50 values of 0.009 and 0.053 µM, respectively. In addition, two murine Cancer cell lines, the colorectal CT26 and the colon adenocarcinoma cells MC38 cell lines, were also tested resulting in IC50 values of 0.049 and 0.133µM, respectively (Figure 1c).
VDA-1275 reduces glycolysis that leads to decreased proliferation of cancer cells.
To test the effect of VDA-1275 on Glycolysis, the Extra-Cellular Acidification Rate (ECAR) was measured to study the lactic acid levels, formed during the conversion of glucose to lactate using the B16 mouse melanoma cancer cell line. Glucose was added to cells and cells were treated with 0.1% DMSO as vehicle control and with 1 and 10 µM VDA-1275. A clear dose response of VDA-1275 on glycolysis rate decrease as well as on the glycolytic capacity of the cells was observed (Figure 2).
Glycolysis levels measured via the extra-cellular acidification rate (ECAR) following treatment of the B16 mouse melanoma cancer cells with VDA-1275. Sequentially, glucose (GLUC) is added to start glycolysis, oligomycin (OM) is added for maximum glycolytic capacity, and 2DG is added to block glycolysis. The inserts show the effect of VDA-1275 on glycolysis and on glycolytic capacity.
VDA-1275 effects on immune cells.
VDA-1275 induces M1 phenotype and inhibits M2 phenotype in macrophages.
The effect of VDA-1275 on macrophage activity was studied. Metabolic changes of cancer cells, mainly the high levels of aerobic glycolysis, affect the tumor microenvironment and impose constraints on immune cell metabolism that can favor immunosuppressive phenotypes and block antitumor responses. However, it also inhibits the function of tumor-associated macrophages (TAMs), that are ubiquitously present in solid tumors, thereby facilitating the immune evasion of malignant tumor cells. TAMs are generally divided into the pro-inflammatory M1 phenotype and the pro-tumor M2 phenotype. Large excess of lactate leads TAMs to adopt an immunosuppressive phenotype and collaborate with tumor cells to promote angiogenesis. [19].
In order to study the direct effect of VDA-1275 on macrophages, we first used primed macrophages, in which activation of the NLRP3 inflammasome leads to secretion of the cytokines IL-1ß and IL-18. IL-1ß is a major pro-inflammatory cytokine, while IL-18 acts together with IL-12 to induce a Th1 immunological response that characterizes the M1 phenotype of macrophages. Bone marrow-derived macrophage (BMDM) cells were grown using recombinant macrophage colony stimulating factor (M-CSF). Macrophages were treated with VDA-1275, followed by priming with LPS, and peptidoglycan. The effect on NLRP3 inflammasome was tested by measuring the levels of IL-1ß and IL-18. A significant elevation of IL-1ß and IL-18 was observed when VDA-1275 was added to the cells at concentrations of 0.03-1 µM and 0.1-0.3 µM, respectively (Figure 3a). An additional study with BMDM was performed to study the effect of VDA-1275 on macrophages that were prone to become M2 macrophages by treatment with the Th2 cytokine IL-4. Cells were treated with VDA-1275 for 24 hrs. and the effect on the expression of the M2-phenotype genes Fizz1, Chi313 and Cdh-1 was studied using real-time PCR. Results demonstrated a strong and significant reduction in the expression of these three M2 type genes following VDA-1275 treatment, suggesting that VDA-1275 affects the metabolism of TAMs by shifting pro-tumor M2 macrophages to the pro-immunologic M1 type macrophages (Figure 3b).
VDA-1275 increases Central Memory CD8 T-cell population ex-vivo.
To study the effect of VDA-1275 on the activity of CD8 T-cells that are major players in the immune response against tumor cells, we isolated splenocytes from a transgenic mouse strain that carries a T-cell receptor transgene specific for the mouse homologue pmel-17, that can be stimulated by a specific peptide. Mouse splenocytes were primed with the peptide for 24 hours in the absence or presence of VDA-1275 (0.1µM). Cells were analyzed on FACS for their activation state using CD44 and for the Central Memory biomarker CD62L. While VDA-1275 did not affect CD8 T-cell activation or survival, a clear increase (about 49%) in the central memory CD8 T-cells population was observed (Figure 3c). These results may indicate a stronger immune memory acquired as a result of VDA-1275 treatment, that may reduce reoccurrence of metastases after treatment completion.
VDA-1275 treatment significantly reduces tumor growth in-vivo.
In order to study the effect of VDA-1275 on cancer inhibition in-vivo, we used the MC38 syngeneic mouse model. C57BL/6 immuno-competent mice were inoculated subcutaneously at the right flank with MC38 cells for tumor development. Five days after tumor inoculation, 20 mice were selected and assigned into 2 groups using stratified randomization with 10 mice in each group based upon their tumor volumes. The treatments were started from the day of randomization (defined as D0) and included the following: (1) Vehicle group (25%PEG400+75% Labrafac Lipophile WL 1349) PO, QD x 25 days (2) treatment group VDA-1275 300mpk PO, QD x 25 days. The tumor sizes were measured during the treatment period. Survival was monitored with tumor volume exceeding 2000mm3 as endpoint. The entire study was terminated on day 25. Syngeneic models include immunocompetent mice and are known to lead to divergent responses. Tumor growth inhibition (TGI) was measured for both groups at day 11, the last day on which all were animals still alive (Figure 4a). The overall TGI on day 11 for the treatment arm vs. vehicle was 47% (p=0.02). Observing the individual curves for the VDA-1275 300mpk group suggests that the group could be divided into ‘responders’ (n=7; tumor size <400 mm3 on day 11) and ‘non-responders’ (n=3; tumor size >1000 mm3 on day 11). The TGI for the 70% of the mice in the ‘responders’ sub-group was 80% tumor growth inhibition (TGI) (p<0.0001). Treatment was well-tolerated without any adverse effect observed in the MC-38 tumor-bearing mice. Time-to-endpoint Kaplan-Meier survival analyses shows that Vehicle group median survival was 15 days while treatment group median survival was 21 days (survival prolongation of 6 days), p=0.0147 (Figure 4b)
VDA-1275 demonstrate a synergistic effect in combination with other anti-cancer drugs in 3D human in-vitro model on BIOMIMESYS® hydroscaffold.
To study and quantitate the effect of VDA-1275 in combination with existing first-line treatments for solid tumor on cancer cells, the human hepatocellular carcinoma cell line HepG2 was used in a 3D organoids model using BIOMIMESYS® matrix [20]. As shown in Figure 5a, VDA-1275 shows a dose-dependent decrease of proliferation rate, as well as of viability (as determined by viability rate or number of live cells/plate).
Next, two anti-cancer drugs were chosen to study their combination effect with VDA-1275. The first drug, Sorafenib, is a kinase inhibitor antineoplastic drug that blocks cell growth. The second is the chemotherapy drug Cisplatin. Preliminary experiment was performed to determine the IC50 of each drug using HEPG2 cells. The cells were incubated for 72 hours with increasing concentrations of the two anti-cancer drugs, followed by EdU staining for proliferation and DRAQ7 for viability tests. Viability Results show IC50 of 9.7µM for Sorafenib and no effect for Cisplatin, whereas IC50 for proliferation were about 2.4 µM and 15 µM for Sorafenib and Cisplatin, respectively. IC50 for number of live cells/plate was 0.6 µM for Cisplatin and 2.2 µM for Sorafenib.
In order to study the combined effect of VDA-1275 with the two anti-cancer drugs, we used Sorafenib at 3µM and Cisplatin at 5µM. Cell media with no drug was added as vehicle control. For each treatment, increasing concentrations of VDA-1275 (3-1000 nM) or vehicle were added. Cells were incubated for 3 days followed by viability and proliferation tests, as mentioned above.
Synergistic effect was defined using the Response additivity method. This reference model is also known as Linear Interaction Effect [18] and assumes that a positive interaction occurs when the combination of drug A and drug B elicits a greater effect than the sum of the individual drug’s effects (EAB > EA + EB), termed potentiating synergistic effect. In addition, when the drug combination is greater than each of the individual drug’s effects, but smaller than their sum, a partial additive effect is observed.
Proliferation results demonstrated no potentiating synergistic effect. However, surprisingly, when the combination effect of Sorafenib or Cisplatin with VDA-1275 was tested on Percent of viability or number of live cells per plate, respectively, a clear potentiating synergistic effect was detected. Table 1. and Table 2. show the synergism effect of VDA-1275 with Sorafenib and with Cisplatin on tumor cell viability, and number of live cells, respectively.
Table 1. Potentiating Synergistic effect of VDA-1275 and Sorafenib.
|
Viability rate (%)
|
% inhibition
|
|
|
|
Vehicle control
|
100.0%
|
0.0%
|
|
|
|
VDA-1275 1000nM
|
72.1%
|
27.9%
|
|
|
|
Sorafenib 3µM
|
91.3%
|
8.7%
|
|
|
|
VDA-1275 1000nM + Sorafenib 3µM
|
46.8%
|
53.2% *
|
|
|
|
SUM VDA-1275 1000nM alone and Sorafenib 3µM alone
|
|
36.6%
|
* Potentiating synergistic effect (ΣE > EA + EB)
Table 2. Potentiating Synergistic effect of VDA-1275 and Cisplatin.
|
number of live cells (norm to vehicle ctrl)
|
% inhibition
|
|
|
|
Vehicle control
|
100.0%
|
0.0%
|
|
|
|
VDA-1275 300nM
|
57.4%
|
42.6%
|
|
|
|
Cisplatin 5µM
|
96.5%
|
3.5%
|
|
|
|
VDA-1275 300nM + Cisplatin 5µM
|
25.6%
|
74.4% *
|
|
|
|
SUM VDA-1275 300nM alone and Cisplatin 5µM alone
|
|
46.1%
|
* Potentiating synergistic effect (ΣE > EA + EB)
In addition, partial additive effects were found for the combination of Sorafenib and VDA-1275 on number of live cells/well, and for the combination of Cisplatin and VDA-1275 on proliferation rate (Table 3. and Table 4., respectively).
Table 3. Partial additive Synergistic effect of VDA-1275 and Sorafenib.
|
number of live cells (norm to vehicle ctrl)
|
% inhibition
|
|
|
|
Vehicle control
|
100.0%
|
0
|
|
|
|
VDA 1000nM
|
42.8%
|
57.2%
|
|
|
|
Sorafenib 10µM
|
26.3%
|
73.7%
|
|
|
|
VDA 1000nM + Sorafenib 10µM
|
16.1%
|
83.9% *
|
|
|
|
SUM VDA 1000nM alone and Sorafenib 10µM alone
|
|
131%
|
* Partial additive synergy (ΣE < EA + EB)
Table 4. Partial additive Synergistic effect of VDA-1275 and Cisplatin.
|
Proliferation rate
|
% inhibition
|
|
|
|
Vehicle control
|
100%
|
0
|
|
|
|
VDA 300nM
|
20.7%
|
79.3%
|
|
|
|
Cisplatin 5µM
|
80.5%
|
19.5%
|
|
|
|
VDA 300nM + Cisplatin 5µM
|
8.6%
|
91.4% *
|
|
|
|
SUM VDA 300nM alone and Cisplatin 5µM alone
|
|
99%
|
* Partial additive synergy (ΣE < EA + EB)
An additional important aspect of the synergistic effect is the ability to reduce the drug doses in combination. Sorafenib was used at 3µM with VDA-1275 (1000 nM) to reach IC50 effect on viability rate, while when tested alone a dose of 9.7µM was required for the same effect. Cisplatin was used at 5µM with VDA-1275 (300 nM) to reach 74.4% reduction of live cells number per plate, while even a dose of 100µM was not enough to reach such an effect when Cisplatin was used alone (Figure 5b).
Our data suggests that VDA-1275 can be combined with Cisplatin or Sorafenib at lower concentrations and lead to a synergistic effect that results in a stronger antineoplastic outcome with less potential for adverse effects due to lower concentration of Cisplatin or Sorafenib as examples for chemotherapy and targeted therapy drugs.