Validation of DaRT technology, In-Vitro and in Animal Studies
Preclinical studies were performed in-vitro and in vivo with eight mouse tumor models and ten human-derived tumors, implanted in athymic mice, to establish the therapeutic effect of Diffusing Alpha-Emitters Radioactive Therapy (DaRT). In addition, we performed an experiment on a goat to determine the effect of the radioactive wires on normal tissue and on blood vessels. DaRT was applied by intratumoral insertion of Ra-224 embedded stainless steel wires.
The results of the preclinical studies were published in 14-refereed papers.
The report includes the following results:
Killing of tumor cells by alpha radiation in vitro.
Destruction of mouse tumors by DaRT seed(s) and effect on lung metastases and survival.
Determining the effect of a DaRT wire inserted human derived tumors implanted in athymic mice.
Activation of anti tumor immunity after tumor ablation by DaRT.
Radioactivity distribution and tissue damage patterns in DaRT treated tumors.
Distribution of radioactive material released from DaRT wires in various organs and tissues.
Effect of DaRT wires on normal skin, cartilage, and blood vessel tissues in a goat.
Experimental models of mouse and human-derived tumors
DaRT achieves a high degree of tumor ablation (destruction) of mouse and human-derived tumor cells of various histological types.
DaRT can be combined with chemotherapy to achieve better control of local and metastatic cancer.
1. Killing of tumor cells by alpha radiation in vitro.
In vitro studies were performed to measure the effect of alpha particles on tumor cell viability. Experiments demonstrated that SQ2 cells exposed to alpha particles at a total dose of the order of 1-5 Gy die within 48 hours (Cooks et al., 2008; Lazarov et al., 2012). Similar results were obtained with tumor cells from mouse and human pancreatic (Horev et al., 2012; Lazarov et al., 2012), lung carcinoma (Cooks et al 2009b), cervical carcinoma (Milrot et al., 2012), colon carcinoma (Lazarov et al., 2012), melanoma and B lymphoma.
2. Destruction of mouse tumors by DaRT wire(s) and effect on lung metastases and survival.
In order to establish the effect of DaRT against several tumor models, experiments were performed on mice, whereby one or two DaRT wires having a typical Ra-224 activity in the range of 300-1200 nano-curies per wire, were inserted into an induced subcutaneous tumor, 5-10 millimeters in diameter. Animals were monitored over time to determine the local tumor response and survival. Control animals bearing similar tumors were either treated with placebo wires or left untouched. Tumor size development was monitored and recorded over several weeks. Tumor diameters were measured using a digital caliper and tumor volume was calculated.
Experiments performed on experimental cancer mouse models of various histotypes showed that DaRT wires effectively destroyed the tumors and achieved a high degree of local control of squamous cell carcinoma (SCC) (Arazi et al., 2007; Cooks et al., 2008, Cooks et al., 2009) colon carcinoma (Reitkopf et al., 2015), pancreas adenocarcinoma (Horev et al., 2012), lung carcinoma (Cooks et al., 2009b), and breast carcinoma (Confino et al., 2015).
Since chemotherapy is a major treatment modality for cancer, we examined the effect of intra-tumoral Ra-224 wires combined with chemotherapy on the development of several carcinoma derived tumors.
DaRT in combination with cisplatin was superior to each treatment alone in inhibiting tumor growth and prolongation of survival in an SCC derived tumor (Cooks et al., 2009). A similar effect was observed when pancreatic (Panc02) tumors were treated with Ra-224 wires and Gemcitabine (Horev-Drori 2012) and colon (CT26) tumors were treated with DaRT and 5-fluorouracil (Reitkopf et al., 2015).
3. The efficiency of one DaRT wire inserted to human derived tumors implanted in athymic mice.
We examined DaRT ability to destroy and control the development of several human-derived tumors implanted in athymic mice. The experiments were performed on athymic mice bearing malignant human head and neck squamous cell carcinoma derived cell line (Cal-27), and lung SCC (NCI-H520) derived tumors, prostate (PC-3), glioblastoma (GBM, U87-MG), colon (HCT15), squamous cell carcinoma (FaDu) and melanoma (C32). One or more 224Ra-loaded wires were inserted into the tumors, and the mice were assessed for tumor growth rate and survival. In vivo studies showed that DaRT can effectively destroy the tumors, and in vitro tests confirmed the sensitivity of the studied cells to alpha particles. While the C32 cells were relatively resistant, other tumor types (e.g. HCT15) exhibited sensitivity in both measured aspects
(Cooks et al., 2008; 2009b). Treatment of human prostate cancer cell derived tumors in nude mice resulted in strong tumor growth retardation (Cooks et al., 2012).
4. Activation of anti tumor immunity
Ablation treatments such as DaRT carry the option to expose the body to large amounts of tumor antigens and danger signals and thus trigger anti-tumor immunity. Tumor ablation eliminates the need to identify specific tumor antigens, and takes care of the heterogeneity of the cells and the possibility that their antigenicity is changing with time. We examined the possibility to induce anti tumor immunity by destroying the tumor by alpha radiation and reinforcing this activity by immuno-manipulation.
Our studies revealed that tumor ablation by DaRT rendered the animals resistant to a second tumor challenge in two tumor models, colon carcinoma and breast carcinoma (Confino et al., 2015).
We were able to achieve improved tumor control by a combined treatment with DaRT and the immunoadjuvant, CpG (Confino et al., 2015). Further studies proved that the use of DaRT in combination with an immunoadjuvant and inhibitors of immune suppressor cells can achieve complete elimination of primary tumors, reduction in metastatic load and extension of survival (Confino et al., 2016)
5. Radioactivity distribution and tissue damage patterns in DaRT treated tumors.
Understanding the transport mechanisms governing the distribution of Ra-224 progeny (namely - Rn-220, Po-216, Pb-212 and Bi-212) within the tumor and throughout the body is a key issue. Experimental work addressing the distribution of alpha-emitting atoms throughout the tumor has been done in several tumors in mice. Because of the relatively long half-life of Pb-212 (10.64 h), significant Pb-212 and Bi-212 activities are found several millimeters away from the source. A high degree of consistency was observed between the radioactivity distribution pattern and the region of necrotic tumor tissue. Only minor cell damage was observed in tumors treated with placebo wires (Arazi et al., 2007; Cooks et al., 2008; Cooks et al., 2009b; Horev-Drori et al., 2012).
6. Distribution of radioactive material released from DaRT wires in various organs and tissues.
Our experimental work in SCC bearing mice also addressed the issue of bio-distribution – i.e., the distribution of alpha-emitters throughout the body. This issue is obviously important from patient safety considerations. Because of their short half-lives, Rn-220 and Po-216 decay entirely within the tumor. Pb-212, however, has a sufficiently long half-life to be partially taken by the blood stream outside of the tumor and distribute throughout the body. The fraction of Pb-212 atoms ‘leaking’ out of the tumor depends on its size (or equivalently – on the distance from the source to the periphery of the tumor): the larger the tumor, the less likely it is for a Pb-212 atom to escape from it.
7. Effect of DaRT wires on normal skin, cartilage, and blood vessel tissues in a goat.
Ra-224 wires with radioactivity levels of 400 (14.6 kBq) to 1600 nCi (59.3 kBq) were inserted in the legs and ear of a goat at distances of 1-5 mm from local major blood vessel. After 9 days the tissues were harvested and fixed and histological analysis was performed by an expert veterinary pathologist. Preliminary inspection showed no major damage to normal tissues in the vicinity of the radioactive wires.