Development of a squamous cell carcinoma mouse model for immunotoxicity testing

Figures & data

Table 1. Experimental design.

Model	Tumor cells	Dose (mg/kg)	Dosing	Tumor cell injection	Termination
Invasion	SCC VII, 1 × 10^6, IM	Azathioprine (25), Etanercept (10), CTX (20), Prednisone (10), Abatacept (20)	Every 2–3 days starting on Day 1	Day 5	Day 24
Colonization	SCC VII, 3 × 10^5, IV	Azathioprine (100), Etanercept (125), CTX (200), Prednisone (10), Abatacept (250)	Days 1, 3	Day 5	Day 24
Progression	SCC VII, 3 × 10^5, IV	Azathioprine (25), Etanercept (10), CTX (20), Prednisone (10), Abatacept (20)	Days 7, 10, 14, 17, 21, 23	Day 1	Day 24

Figure 1. Drug effects on SCC VII cells in vitro. Azathioprine and Prednisone show dose responsive effects on SCC VII cells in vitro at high concentrations. Data shown are mean ± SD. * Cell count was significantly less than control (p < 0.05).

Figure 2. Primary tumor volume increases over time with and without treatment. Data shown are mean ± SD. *At Day 19, Prednisone is significantly less than vehicle control (p < 0.05); n = 12/group. **At Day 24, Prednisone is statistically less than vehicle control and Abatacept is significantly greater than control (p < 0.05); n = 12/group.

Figure 3. Fluorescence traffic to popliteal lymph node. Abatacept and Prednisone decrease fluorescence traffic to the popliteal lymph nodes, while Azathioprine, Etanercept and CTX have no effect on traffic. Line at mean ± SD is shown. *Significantly less than vehicle control (p < 0.05); n = 12/group. Treatment group always compared to respective control. Red lymph nodes are positive for growth (histological assessment).

Figure 4. Experimental metastases. CTX increases experimental metastases. Line at median. *Significantly less than vehicle control (p < 0.05); n = 12/group. Treatment group always compared back to respective control.

Figure 5. Representative IHC images of SCC VII cells in lung, (a) IHC for BrdU, (b) IHC for vWF. 20X Magnification.

Figure 6. Lesion area for established lung colonies. Abatacept decreases lesion area in established lung colonies. Box and whiskers, Min to Max. *Significantly different from control (p < 0.05); n = 12/group. Treatment group always compared back to respective control.

Figure 7. Growth fraction in established colonies. CTX and Azathioprine increase growth fraction in established colonies. Box and whiskers, Min to Max. *Significantly different from control (p < 0.05); n = 12/group. Treatment group always compared back to respective control.

Figure 8. Immunosuppressive drug effects on angiogenesis in established lung colonies. Box and whiskers, Min to Max. *Significantly different from control (p < 0.05); n = 12/group. Treatment group always compared back to respective control.

Table 2. Immunosuppressive drugs tested had varying effects on models of invasion, colonization and progression: Summary of results.

	Invasion			Colonization	Progression
Validation set	Primary tumor volume	Histological assessment	Fluorescence trafficking	Number of colonies	Lesion size	Growth fraction	Angiogenesis
Cyclosporin**	−	NE	+	−	−	−	−
Cyclophosphamide	NE	+/NE*	NE	+	NE	+	+
Prednisone	−	NE	−	NE	NE	NE	−
Azathioprine	NE	NE	NE	NE	NE	+	+
Etanercept	NE	NE	NE	NE	NE	NE	+
Abatacept	+	NE	NE	NE	−	NE	−

NE, No effect; +, Increase; −, decrease.

*CTX has shown both an increase (Egenolf et al., 2011) and no effect (Figure 3) on growth during the histological assessment. The dosing and timing of administration for these two experiments were different.

**The data for CSA was reported in Rafferty et al. (2012).

Egenolf, D. D., Rafferty, P., Brosnan, K., et al. 2011. Development of a murine model of lymph node metastases suitable for immunotoxicity studies. J. Pharmacol. Toxicol. Meth. 63:236–249

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Rafferty, P., Egenolf, D., Brosnan, K., et al. 2012. Immunotoxicologic effects of cyclosporine on tumor progression in models of squamous cell carcinoma and B-cell lymphoma in C3H mice. J. Immunotoxicol. 9:43–55

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