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Despite the success of immunotherapy, many cancer patients do not respond to this therapy. The aim of this pre-clinical study was to try and convert non-responding tumours into responders using radiation that primarily kills tumour cells directly, but also has an indirect effect through the induction of vascular damage. In addition, we also used OXi4503, a drug that primarily induces vascular damage, but also has a secondary direct cell killing effect.

Experiments were performed using a C3H mammary carcinoma grown in the right rear foot of CDF1 mice and treatments started when tumours reached 200 mm³ (day 0). Treatments included local tumour irradiation with single proton doses (5-20 Gy on day 0; given in an 83-107MeV pencil scanning proton beam in the center of a 3 cm spread out Bragg peak), or the vascular disrupting agent, OXi4503 (5-50 mg/kg injected i.p. on days 0, 3, 7 and 10). The radiation and drug treatments were also combined with an antibody to the checkpoint inhibitor CTLA-4 (10 mg/kg; i.p. injected on days 1, 4, 8, and 11). The endpoint was tumour growth time (TGT3; time for tumours to grow to 3 times the treatment volume). A Student’s T-test was used for statistical comparisons (significance level of p<0.05).

Untreated control tumours had a mean (+ 1 S.E.) TGT3 of 4.3 days (+ 0.5). This was increased in a dose dependent fashion when mice were treated with either protons or OXi4503. Anti-CTLA-4 alone had no significant effect on tumour growth, the TGT3 being 3.9 days (+ 0.1). It also failed to enhance the response to proton irradiation or OXi4503 until the highest doses were reached. With protons, a TGT3 value of 17.7 days (+ 0.7) was obtained with 20 Gy and this was significantly increased to 22.4 days (+ 1.2) when combined with anti-CTLA-4. For OXi4503 the TGT3 at the 50 mg/kg dose was 15.8 days (+ 0.4), which significantly increased to 24.2 days (+ 4.0) with anti-CTLA-4. Interestingly, larger effects were seen when smaller tumours were used. Despite the significant increase in TGT3 when mice bearing 200 mm³ tumours were treated with OXi4503 and anti-CTLA-4, the number of tumours that were completely controlled was 0/10. However, applying the treatments when tumours were 100 mm³ in size resulted in 2/9 tumours being controlled, and this level of control further increased to 5/9 tumours when the tumours were at 50 mm³ when the treatments started.

Tumours that are unresponsive to checkpoint inhibitors can become responsive after treatment with other therapies. While this effect appears to be independent of how tumor damage is induced, it does seem to depend on the level of damage caused and the size of the tumours at the start of treatment. 

Supported by grants from the Danish Cancer Society, the Danish Council for Independent Research: Medical Sciences, and The Danish National Board of Health.