Preliminary evaluation of the therapy approach using a subcutaneous lung cancer model

To evaluate a NP-enhanced RT approach we initially thought of using a subcutaneous tumor model that provides the advantage of directly injecting the BaNPs into the tumor and easy tumor measurements by caliper. For this purpose, A549-cell induced subcutaneous tumors were irradiated six times every second day using four consecutive CT scans of 2 mins at 90 kV and 200 µA resulting in a dose of about 5.5 Gy per session and a total dose of ~33 Gy. Mice were randomly assigned to four groups CTRL (no NPs, no irradiation, N=4), NP CTRL (NPs, no irradiation, N=5), RAD (no NPs, irradiation, N=5) and NP RAD (NPs and irradiation, N=5).

Tumor size was monitored by caliper measurements by two readers every second day. Body weight was also measured every second day.

The selected irradiation schedule revealed no significant adverse effect on the body weight, es demonstrated in Figure 21 A, which shows changes in body weight at the end of the experiment in comparison to the date of tumor cell implantation. Both non-irradiated groups (CTRL & NP CTRL) gained weight of approx. 5-7 %, suggesting no harmful side effects of the BaNPs on the wellbeing of the mice. Mice of both irradiated groups did not show any weight loss, indicating no severe side effects. The starting point of the experiment was defined as the day when the tumors reached a size of 300 mm³. Since the individual tumors showed very different growth kinetics, the animals were divided into 5 different cohorts. The treatment group of each mouse was randomly assigned independently of the cohort. Figure 21 B shows the measured tumor volumes for each mouse over time. Figure 21 C shows the relative change in tumor volume in comparison to the day of BaNP injection.

Figure 21: Results of the first irradiation experiment.

A) Mean weights over time for each treatment group. No significant weight loss was observed in any of the groups. B) Tumor volumes over time relative to the therapy start. C) Relative tumor volumes in comparison to the day of NP injection (day -1) over time.

Results 59 Interpretation of the graphs shown in Figure 21 B and C is rather difficult, because of the different growth kinetics, treatment dates and the large standard deviations of the caliper measurements. Therefore, Figure 22 shows the tumor volumes over time in the different treatment groups.

As shown in Figure 22 A and B the tumor volumes in both control groups (CTRL & NP CTRL) increased steadily over time. In contrast, we found that in both irradiated groups (RAD & NP RAD) the tumors shrunk, or the tumor growth was minimized (Figure 22 C and D). The effect can be observed 3-4 days after therapy start and is stronger in larger or faster growing tumors.

No apparent difference was detected in the tumor volume kinetics after treatment between the RAD and the NP RAD group.

Figure 22: Growth kinetics over time as a result of radiotherapy divided into the treatment groups.

Tumor volumes for individual animals over time measured by manual caliper measurements. A) CTRL group (no radiation, no BaNPs; N=4). B) NP CTRL group (no radiation, BaNPs; N=5). C) RAD group (radiation, no BaNPs; N=5). D) NP RAD group (radiation & BaNPs; N=5). Both non-irradiated groups show a steady tumor growth, while the irradiated tumors show a decrease in tumor volume due to the therapy.

In summary, this pilot experiment provided evidence that RT can be indeed performed successfully in a CT. However, it also showed experimental flaws, which most probably contributed to non-statistically significant results as tested by ANOVA. In particular, the measurement of the tumor by caliper is difficult to reproduce and especially challenging for small tumor volumes despite the superficial nature of the subcutaneous lung tumors. The current experimental numbers and set-up did not show a strong increase in the efficacy of RT based on the presence of NPs. We cannot exclude smaller differences in the tumor growth between RAD and NP RAD that were not measurable in these data sets.

In the following experiment, I optimized the treatment scheme and utilized the pH8N8 breast cancer model for TNBC.

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4.8.2 Radiation therapy effect in the orthotopically implanted breast cancer model

Since the main aim of my thesis was to evaluate if NP-enhanced RT can be used as a therapy for TNBC, I used an orthotopic breast cancer model for further experiments, which provides a more disease-relevant environment for the assessment of tumor growth. In addition, the pH8N8 breast cancer cells are of murine origin and allow the use of immunocompetent mice, which is important because of the role of the immune system in response to cancer treatment. Moreover, as the current route for administering the NPs was intratumoral injection an application in breast cancer therapy seemed more likely than in lung cancer. Since neither the cell viability assays nor the mice in the control group (no BaNPs, no irradiation) in the previous experiment showed any signs of toxicity or altered tumor growth compared to the NP CTRL group which received BaNPs, I only evaluated the concept in three groups: NP CTRL (NPs, no irradiation, N=2), RAD (no NPs, irradiation, N=4) and NP RAD (NPs and irradiation, N=5).

The preliminary irradiation experiment did not show a strong difference in the tumor volumes when BaNPs where used in combination with irradiation. I therefore adjusted the irradiation schedule to achieve an increase in the NP-based RT effect. For this purpose, the same radiation dose of 5.5 Gy was delivered per treatment, but the schedule was changed to daily irradiation instead of every second day to give the tumor cells less time to repair the damage. Thus, seven therapy sessions were performed with four 2 min CT scans at 90 kV and 200 µA per session.

This treatment schedule resulted in a strong weight loss of up to 20% within 10 days of treatment in both irradiated groups (Figure 23 A) and led to a premature termination of the experiment. Figure 23 B shows the relative volumes over time in comparison to the treatment start. While the mice in the non-irradiated NP CTRL group exhibited a tumor volume increase up to a factor of five, the irradiated groups (RAD & NP RAD) displayed a therapy effect. In the RAD group the tumors grew similarly to the NP CTRL group until day five and then the tumors receded in size. When the experiment was terminated the tumor volumes were approximately the same as at the beginning of the experiment. The NP RAD group showed a slower tumor growth from the start of the experiment. The tumors also began to shrink after day 5. However, these differences between the RAD and NP RAD group were not significant

Figure 23: Outcome of radiotherapy using the orthotopic breast cancer model.

Mean values for the following groups are depicted: NP CTRL (BaNPs, no irradiation; N=2), RAD (no BaNPs, irradiation; N=4) and NP RAD (BaNPs and irradiation; N=5). A) Mean relative weights over time for each treatment group. Significant weight loss in the treated groups. B) Tumor volume over time relative to the therapy start. Both treated groups show a reduction in tumor growth.

In summary, this experiment revealed a clear reduction in the pH8N8 tumor volumes in treated mice in comparison to the NP CTRL group, similar to the results we observed in the A594 subcutaneous tumors. However, a significant benefit of the BaNPs for reducing the tumor volumes was not detected. Due to the strong side effects of the irradiation dose, I decided to further optimize the treatment schedule by adding rest days with the aim to reduce side effects, while maintaining the effect of the RT.

Results 63

4.8.3 Improving the treatment schedule to reduce side effects for