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General Health Characteristics

In terms of body weight, there were no significant differences between animals given an ITI of W256 and HK W256 cells in experiment 1 [F_{(7,17,119/238)} = 1.2, 1125, 1.5; p > 0.05], experiment 2 [F_{(1,43,43/387)} = 2.2, 619.7, 0.9; p > 0.05] (Figure ​Figure2A2A), experiment 3 (p > 0.05) (Figure ​Figure2B2B), experiment 5 (p > 0.05) (Figure ​Figure2C2C), experiment 7 [F_(1,14,14/98) = 0.7, 766.6, 1.3; p > 0.05], experiment 8 [F_(1,8,18/64) = 0.4, 400.8, 2.7; p > 0.05], experiment 9 [F_(1,11,11/88) = 1.1, 364, 0.05; p > 0.05], experiment 10 [F_(1,8,8/64) = 0.3, 180.6, 2.8; p > 0.05], experiment 11 [F_(1,8,8/64) = 1.0, 314.8, 1.6; p > 0.05], experiment 12 [F_(1,7,7/63) = 0.5, 66.0, 0.2; p > 0.05], experiment 13 [F_(1,7,7/49) = 1.1, 195.4, 0.8; p > 0.05] and experiment 14 [F_(1,9,9/63) = 0.7, 141.5, 1.6; p > 0.05]. Additionally, there were no significant differences in between body weights of animals given an ITI of DPBS and age-matched control female Wistar Han rats in experiment 4 (p > 0.05). Although there were slight differences between body weights of rats administered W256 and HK W256 cells in experiment 6 [F_{(1,12,12/108)} = 8.9, 508.1, 3.1; p ≤ 0.05], experiment 15 [F_(1,6,6/18)= 11.9, 84.1, 1.3; p ≤ 0.05] and experiment 16 [F_(1,11,11/33)= 62.6, 314.2, 0.6; p ≤ 0.05] at some time points, these were either due to differences in initial body weights or due to non-cancer factors. Importantly, there were consistent gains in the body weights of all animals throughout the experiments (Supplementary Figure 2).

Body weight of rats from individual experiments. Panels in the figure show mean (±SEM) body weight of rats from (A) experiment 2, (B) experiment 3 and (C) experiment 5. HK, heat-killed; W256, Walker 256. There were no statistically significant differences in body weight between any treatment groups (p > 0.05; experiment 2, Two-way ANOVA, post hoc Bonferroni test; experiment 3 and 5, Mann–Whitney test).

A total of 173 rats were used in this study, out of which 9 rats (5%) were found to have health-related issues and of these 3 (1.7%) were euthanized for ethical reasons as outlined below. One rat from experiment 1 given an ITI of 4 × 10³ W256 cells had a slight tumor like appearance in the bone near the injection site. Nevertheless, the overall health of the animal was satisfactory and no other complications were observed. Another rat from experiment 1 given an ITI of 1.5 × 10⁵ W256 cells had a mild infection at the sight of surgery with visible pus exudate. Under anesthesia, the infected site was cleaned and topical antibiotic powder was applied, followed by a s.c. injection of benzylpenicillin at a dose of 60 mg. Subsequently, the infection resolved. One rat given an ITI of 4 × 10³ W256 cells, two rats given an ITI of 1.5 × 10⁵ W256 cells and one rat given an ITI of 4 × 10⁵ HK W256 cells from experiment 1 had mild swelling at the injection site, which resolved spontaneously. One rat from experiment 4 given an ITI of DPBS was excluded from the study and euthanized due to lack of normal body weight gain due to a crooked incisor. One rat given an ITI of 4 × 10⁵ W256 cells from experiment 5 was excluded and euthanized due to the presence of a large external tumor growth on the injected tibia. One rat from experiment 6 given an ITI of 4 × 10⁵ W256 cells was excluded and euthanized due to lethargic behavior and low body weight; upon post-mortem investigation, a large metastatic lesion was observed on the intestine.

Assessment of Mechanical Allodynia in the Hindpaws

Experiment 2

Unilateral ITI of 4 × 10⁴ W256 cells in rats did not significantly reduce PWTs in either the ipsilateral [F_(1,9,9/81)= 0.8, 2.1, 1.3; p > 0.05] or the contralateral [F_(1,9,9/81) = 4.2, 2.9, 1.2; p > 0.05] hindpaws throughout the experiment c.f. rats administered a unilateral ITI of 4 × 10⁴ HK W256 cells (Figure ​Figure3A3A).

Paw withdrawal thresholds (PWTs) of ipsilateral and contralateral hindpaws of rats. Panels in the figure show mean (±SEM) PWTs of rats from (A) experiment 2, (B) experiment 3 and (C) experiment 5. Rats with PWTs ≤6 g in the ipsilateral hindpaw were considered to have fully developed mechanical allodynia as indicated by the dotted line. HK, heat-killed; W256, Walker 256. p ≤ 0.05 (Two-way ANOVA, post hoc Bonferroni test) c.f. rats given an ITI of HK W256 cells.

Assessment of Mechanical Hyperalgesia in the Hindpaws

Experiment 2

Unilateral ITI of 4 × 10⁴ W256 cells in rats did not significantly reduce PPTs in either the ipsilateral [F_(1,9,9/81)= 0.1, 0.5, 0.7; p > 0.05] or the contralateral [F_(1,9,9/81) = 0.1, 1.5, 0.5; p > 0.05] hindpaws throughout the experiment c.f. rats administered a unilateral ITI of 4 × 10⁴ HK W256 cells (Figure ​Figure4A4A).

Paw pressure thresholds (PPTs) of ipsilateral and contralateral hindpaws of rats. Panels in the figure show mean (±SEM) PPTs of rats from (A) experiment 2, (B) experiment 3 and (C) experiment 5. Rats with PPTs ≤80 g in the ipsilateral hindpaw were considered to have fully developed mechanical hyperalgesia as indicated by the dotted line. HK, heat-killed; W256, Walker 256. p ≤ 0.05 (Two-way ANOVA, post hoc Bonferroni test) c.f. rats given an ITI of HK W256 cells.