Identification of a head and neck cancer cell line with lowest efficacy to intratumoral treatment with Tigilanol tiglate

Intratumoral treatment with 30 μg of TT successfully ablated FaDu and CAL-27 xenografts in BALB/c Foxn1nu [4]. In an attempt to identify a cell line that displayed a weaker response to TT treatment, three additional tongue SCC cell lines were obtained and tested: SCC-9, SCC-15 and SCC-25. Xenografts of the three cell lines were established by subcutaneously injecting ten five-week old BALB/c Foxn1nu mice for each group with 2 × 106 cells at two sites. SCC-15 xenografts required 8 days of growth to reach the appropriate treatment volume of approximately 100 mm3. SCC-9 and SCC-25 xenografts were slower growing and reached treatment size at 17 days post-injection. Of the ten mice in each group, five were treated with 30 μg of TT (50 μl of 600 μg/ml TT in 40% propylene glycol (PG)) at both tumor sites, whilst the remaining five mice were injected with 50 μl of 40% PG only (Fig. 1).

Fig. 1 Treatment of HNSCC with TT in BALB/c Foxn1nu mice. Kaplan-Meier plots comparing the differences in survival of BALB/c Foxn1nu mice treated with 30 μg TT (red) or 40% PG vehicle control (blue). a SCC-9, b SCC-15, c SCC-25 tumors. Data represents 5 mice per group, 2 tumors per mouse. ***, P < 0.001; ****, P < 0.0001; Log-rank (Mantel-Cox) test Full size image

In all three xenograft groups treated with TT, the previously described localized haemorrhagic response and subsequent eschar formation occurred. This was not seen in the groups treated with the vehicle control. Average tumor volumes showed a steady decline following treatment with TT. SCC-15 xenografts were completely ablated by day six post treatment with TT, compared to day 14 post treatment for SCC-9 and SCC-25. In contrast, the control groups for all three xenografts continued to have tumor growth following intratumoral injection with the vehicle control. Mice were euthanized once total tumor burden had reached approximately 1000 mm3 per mouse. TT treatment led to a statistically significant increase in survival time in all three xenograft models (SCC-9, P < 0.001; SCC-15, P < 0.0001; SCC-25, P < 0.0001; Fig. 1a-c).

Recurrences were seen at two tumor sites (2/10; 20%) for SCC-9 xenografts (Fig. 1a), and one tumor site (1/10; 10%) for SCC-25 xenografts (Fig. 1c). Three recurrences (3/10; 30%) in the SCC-15 group were identified by day 21 post-initial treatment with TT (Fig. 1b). Of the total six recurrences seen across the groups, three tumors (one per group) were re-treated with 30 μg TT resulting in complete ablation. Mice were monitored for ten months and no further recurrences were seen. Given the greatest proportion of recurrences, SCC-15 was identified as the cell line with lowest efficacy to treatment with TT and therefore selected to be the cell line utilized for further optimisation of TT administration.

The role of the host in Tigilanol tiglate efficacy

Ten five-week old male NOD/SCID mice were subcutaneously inoculated with 2 × 106 SCC-15 cells/site, with two sites per mouse. Tumor growth occurred very quickly in comparison to the BALB/c Foxn1nu mice xenografts, with an average tumor volume of 159 mm3 (± 20 mm3 SEM) seven days following initial tumor cell injection. On day seven, five mice were intratumorally injected with the previously established treatment of 30 μg TT and five mice with 50 μl of the vehicle control 40% PG. The mice injected with the vehicle control continued to display exponential tumor growth, as seen in the BALB/c Foxn1nu cohort (Fig. 2a). In comparison, tumor growth in the group treated with the previously established dosing regimen of 30 μg TT did not mirror that seen in the nude mice. On average, tumor growth was suppressed by approximately 50%, and no tumors were successfully ablated (0/10). Mice reached maximum tumor burden and were all culled by day 14 and day 21, for the control and treated groups, respectively (Fig. 2b). The survival time difference between the control and treatment groups was however statistically significant (P = 0.0028).

Fig. 2 Treatment of SCC-15 tumors in NOD/SCID mice. a Tumor volumes of large (>150 mm3) SCC-15 tumors in NOD/SCID mice treated with 30 μg TT and compared to treatment with 40% PG vehicle control. Error bars represent ± SEM. b Kaplan-Meier plot comparing the differences in survival of NOD/SCID mice treated for large (>150 mm3) SCC-15 tumors with 30 μg TT (red) or 40% PG vehicle control (blue). Data represents five mice per treatment group, 2 tumors per mouse. Dashed line represents point of treatment (day 7). ***, P < 0.001, Log-rank (Mantel-Cox) test Full size image

Intratumoral injection of Tigilanol tiglate to small SCC xenografts in NOD/SCID mice

The reduced efficacy of TT treatment seen in the larger SCC-15 xenografts in NOD/SCID mice potentially could have been attributed to the larger average tumor size at the time of treatment (159 mm3 ± 20 mm3 SEM). The dose of TT may have been inadequate for the number of tumor cells, and/or local spread of tumor cells may have already occurred to a site outside the treatment field. In order to understand the reduced efficacy of TT treatment, NOD/SCID mice were inoculated with 2 × 105 cells/site and treated at a reduced mean tumor volume (67 mm3 ± 3 mm3 SEM). Within the mice with smaller tumors, ten tumors were treated with 50 μl of 40% PG vehicle control and ten treated with 30 μg TT in a single bolus injection (Fig. 3a). Of the ten tumors treated with TT in 40% PG, 7 were successfully ablated at day 35 post treatment. The remaining tumors showed initial clearance but developed local recurrence two weeks following TT treatment.

Fig. 3 Effect of fractionated doses or alternative excipient on TT efficacy. a Tumor volume of small SCC-15 tumors in NOD/SCID mice treated with single or multiple doses of TT and with 40% PG or 4% 2-hydroxypropyl-β-cyclodextrin as a vehicle control. Error bars represent ± SEM. b Kaplan-Meier plot comparing the differences in survival of NOD/SCID mice with SCC-15 tumors treated with single or multiple doses of TT and with 40% PG or 2-hydroxypropyl-β-cyclodextrin as a vehicle control. 40% PG vehicle control (blue line), 4% 2-hydroxypropyl-β-cyclodextrin (black dashes), 30 μg bolus TT in 40% PG vehicle (red line), 30 μg bolus TT in 4% 2-hydroxypropyl-β-cyclodextrin vehicle (green line), 30 μg fractionated TT in 40% PG vehicle (purple line). Data represents 5 mice per group, 2 tumors per mouse. Dashed line represents point of treatment (day 14). ns, not significant; ****, P < 0.0001, Log-rank (Mantel-Cox) test Full size image

Single versus divided dose administration of Tigilanol tiglate

To investigate whether more tumor cells could be targeted with a dose given in each tumor quadrant, compared to the single dose, five mice were treated with a total 30 μg TT given in four divided doses (7.5 μg TT × 4). No tumors were successfully ablated as seen in Fig. 3a. Mice treated with divided doses rather than the single dose of TT had significantly reduced survival (P = 0.0001; Fig. 3b). Upon administration of the divided doses, there was a significant amount of leakage of the solution out of the preceding needle puncture sites, reducing the overall concentration of TT the tumor tissue would have otherwise retained. Solution leakage was also seen in tumors that appeared to be particularly necrotic/ulcerated.

Propylene glycol compared to 2-hydroxypropyl-β-cyclodextrin as an excipient for Tigilanol tiglate administration

We wished to assess a different exipient for TT, and its effect on treatment efficacy. Cyclodextrins are a family of cyclic oligosaccharides used widely in the food, agricultural, and pharmaceutical industries. The ring-like structure of these compounds provides a hydrophobic inner and a hydrophilic outer region, facilitating the inclusion of hydrophobic/lipophilic compounds and potentially increasing their bioavailability when used as a drug excipient [5]. We therefore used 4% 2-hydroxypropyl-β-cyclodextrin as the vehicle for TT (Fig. 3a). There was no difference in the survival of mice with tumors treated with 40% PG or 4% 2-hydroxypropyl-β-cyclodextrin alone (Fig. 3b). All tumors continued to grow after injection of either excipient with all reaching endpoint at day 13 post treatment. Treatment with TT lead to significant increases in survival of mice with either excipiant (TT in 40% PG versus 40% PG alone, P = 0.0003; TT in 4% 2-hydroxypropyl-β-cyclodextrin versus 4% 2-hydroxypropyl-β-cyclodextrin alone, P < 0.0001). Further, TT in 4% 2-hydroxypropyl-β-cyclodextrin did not significantly improve number of recurrences compared to TT in 40% PG (P = 0.9603, Fig. 3b). A total of 6 sites had no recurrence with TT in 4% 2-hydroxypropyl-β-cyclodextrin while treatment with TT in 40% PG showed no recurrence in 7 sites. These results suggest that 2-hydroxypropyl-β-cyclodextrin was no more effective as an excipient compared to 40% PG, and importantly show that the anti-tumor activity of TT is maintained in different vehicles.

Impact of delivery volume on efficacy of Tigilanol tiglate

As described above, during administration of 30 μg of TT in 50 μl of vehicle to some xenografts in NOD/SCID mice, significant leakage of the solution from necrotic areas of the tumor occurred, resulting in the observed reduction of tumor regression.. Those particular tumors often were not successfully ablated likely due to the overall reduced concentration of TT at the tumor site or reduced treatment field area. It was therefore proposed that increasing the solution volume, with the same dose (30 μg) could potentially minimize dose wastage from leaking and improve tumor infiltration. SCC-15 xenografts were established in NOD/SCID mice and treated with 40% PG alone (n = 5), 30 μg TT in 50 μl with 40% PG (n = 5), or 30 μg TT in 100 μl with 40% PG (n = 5) (Fig. 4a). Following TT treatment, the resulting haemorrhagic region surrounding the tumor appeared moderately more extensive for those treated with 30 μg TT in 100 μl with 40% PG. No additional adverse effects were noted during routine mouse and tumor monitoring. No significant difference in tumor growth or survival was apparent between the two TT treated groups (Fig. 4b).

Fig. 4 Variation of injection volume on TT efficacy. a Tumor volume of SCC-15 tumors in NOD/SCID mice treated with 30 μg bolus TT in 100 μl of 40% PG vehicle (green) compared to 30 μg bolus TT in 50 μl of 40% PG (red). Injection of 50 μl of 40% PG alone is shown as a control (blue). Error bars represent ± SEM. b Kaplan-Meier plot comparing the differences in survival of NOD/SCID mice with SCC-15 tumors treated with 30 μg bolus TT in 100 μl of 40% PG vehicle control compared to 30 μg bolus TT in 50 μl of 40% PG. Data represents 5 mice per group, 2 tumors per mouse. Dashed line represents point of treatment (day 10). ns, not significant; ***, P < 0.001; ****, P < 0.0001; Log-rank (Mantel-Cox) test Full size image

Immunohistochemistry on tumors from different mouse hosts show variation in neutrophil recruitment

To investigate the differences in response that occurred following treatment of SCC-15 xenografts in BALB/c Foxn1nu and NOD/SCID mice, and whether a recruitment of macrophages and/or neutrophils also occurred, immunohistochemistry was performed for general morphology and salient markers following TT treatment. Immunohistochemical staining was performed for LyG6, a myeloid differentiation antigen present on peripheral neutrophils in addition to myeloperoxidase (MPO), a lysosomal protein expressed in neutrophil granules. Both stains identified the presence of neutrophils in the tumor; however, MPO could also potentially identify sites of recent neutrophil degranulation. A substantial infiltration of neutrophils into tumor tissue was apparent 24 h after intratumoral treatment with TT in BALB/c Foxn1nu (Fig. 5). Evidence of this infiltration was supported with a similar response seen in MPO staining at 24 h (data not shown). No neutrophil infiltrations or areas of degranulation were demonstrated in tumor tissue treated with 40% PG. In contrast to the patterns seen in the BALB/c Foxn1nu mice, no infiltration of neutrophils or evidence of increased sites of neutrophil degranulation was identified in NOD/SCID mice at any time point following treatment with TT (Fig. 5). LyG6 and MPO staining across all time points were similar to that seen in the untreated tumor tissue. No differences in staining patterns for CD-31 or F4/80 were observed (data not shown).