Treatment of an Alveolar Rhabdomyosarcoma Allograft with Recombinant Myxoma Virus and Oclacitinib

Figures & data

Figure 1 Diagram of the timeline (in days) of oral (PO) and intratumoral (IT) treatments given to mice bearing subcutaneous (SQ) rhabdomyosarcomas (RMS). Day 0 was designated as the first day that IT treatment was administered. Subsets of mice were euthanized on Days 4 and 7 for sample collection. The shortest and longest survival times were Day 11 and Day 31, respectively.

Table 1 Primer Sequences and Specific PCR Parameters Used to Detect MYXVΔserp2 DNA in Tissues

Target Gene Segment(s)	Forward Primer Sequence	Reverse Primer Sequence	Annealing Temperature (°C)	Product Length (Base Pairs)
MYXV M034L (DNA polymerase)^a	5ʹ CGC CAT CCT TTA CCT AAC GA 3’	5ʹ ACC CGG TGT TAT TTT TGT CG 3’	60	87
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)^b	5ʹ CGA CTT CAA CAG CAA CTC CAA CTC TTC C 3’	5ʹ TGG GTG GTC CAG GGT TTC TTC CTC CTT 3’	60	285
MYXV M033R and M034L^c	5ʹ CAC CCT CTT TAG TAA AGT ATA CAC C 3’	5ʹ GAA ATG TTG TCG GAC GGG 3’	52	800

Notes: ^aPrimers were used for detection of MYXV∆serp2 using ddPCR. ^bPrimers were used to determine tissue volume for ddPCR reactions, Data from Liu et al.³⁹ cInterference within tissue type in ddPCR in was observed, therefore primers against MYXV M033R and M034L were used to perform confirmation of ddPCR positive MYXV ∆serp2 tissues using conventional PCR.

Abbreviations: PCR, polymerase chain reaction; MYXVΔserp2, myxoma virus deleted for serp2; °C, degrees Celsius.

Table 2 Tumor Histology Grading Schemes

	Grade 0	Grade 1	Grade 2	Grade 3	Grade 4
Percent necrosis	0	1–15	16–30	31–45	>45
Percent hemorrhage	0	1–15	16–30	31–45	>45
Neutrophils per 400× field*	0	1–5	5–10	10–20	>20
Histiocytes per 400× field	0	1–15	16–30	31–45	>45
Lymphocytes per 400× field	0	1–15	16–30	31–45	>45

Notes: *The grade was increased by 1 when microabscesses were observed.

Figure 2 (A) Photomicrographs showing cytopathic effects (phase) and recombinant protein expression (fluorescence) 48 h post-inoculation (hpi) of RK-13 cells and U21089 cells with recombinant MYXV expressing tomato red fluorescent protein [multiplicity of infection (moi) = 0.1 and 1 virus particle/cell]. (B) Multi-step viral growth curves. U21089 cells were inoculated with MYXV or MYXVΔserp2 (moi = 0.01). Samples were collected at 0, 12, 24, 48, 72, and 96 hpi then plaqued onto RK-13 cells. The number of viral plaque-forming units (pfu) per U21089 cell was calculated from the number of foci that formed in RK-13 cell monolayers. (C) Viability of U21089 cells after inoculation with myxoma virus (MYXV) or MYXVΔserp2 (moi = 1) expressed as a percentage of viable mock infected U21089 cells. Error bars indicate standard error of the mean.

Figure 3 Tumor growth. (A) Tumor volume on the day of the first intratumoral injection (Day 0) and (B) average tumor growth rate calculated from U21089 cell injection until euthanasia in mice treated with phosphate buffered saline (PBS), MYXVΔserp2, and oclacitinib with MYXVΔserp2 (O+MYXVΔserp2). Error bars indicate mean and standard deviation.

Figure 4 Representative images of subcutaneous rhabdomyosarcoma (RMS) tumors on Day 2 (A-C) and Day 19 (D-F) after the first intratumoral MYXVΔserp2 injection. Clinical signs associated with the tumors included erythema (A, B, D), swelling (B, E), ecchymosis (C, D, E), significant tumor development (D, E), and ulceration (F).

Figure 5 Contingency graphs indicating the number of mice with and without clinical signs of limb swelling, hematoma formation, erythema, and/or ecchymosis in each treatment group on (A) the day of the first intratumoral injection (Day 0) and (B) Day 4. On Day 0, the ratio of mice treated with oclacitinib with MYXVΔserp2 (O+MYXVΔserp2) that had clinical signs was higher than other treatment groups (*P-value = 0.02).

Table 3 Reason for Euthanasia of Mice with Rhabdomyosarcoma Allografts

Treatments	Total Number of Mice	Tumor Volume ≥ 3500 mm^3	Poor Clinical Condition	Ulceration
Placebo + PBS	12	2 (17%)	6 (50%)	4 (33%)
Placebo + MYXV∆serp2	12	6 (50%)	2 (17%)	4 (33%)
Oclacitinib + MYXV∆serp2	13	7 (54%)	2 (15%)	4 (31%)

Abbreviations: PBS, phosphate buffered saline. MYXVΔserp2, myxoma virus deleted for serp2.

Figure 6 Average histologic grades. (A) Necrosis, (B) hemorrhage, and (C) neutrophil grades were determined in histologic sections of tumors collected at the time of euthanasia. Error bars indicate standard error of the mean. *P-value of the t-test comparing phosphate buffered saline (PBS) to oclacitinib with MYXVΔserp2 (O+MYXVΔserp2) treatment = 0.02. †P-values of the t-tests comparing MYXVΔserp2 to O+MYXVΔserp2 treatment ≤ 0.05.

Table 4 Number of Samples in Which MYXV∆serp2 DNA Was Detected in Mouse Tissues by Droplet Digital PCR

	Day 4			Day 7			Survival
	PBS	MYXV Δserp2	O+MYXV Δserp2	PBS	MYXV Δserp2	O+MYXV Δserp2	PBS	MYXV Δserp2	O+MYXV Δserp2
Number of mice tested	12	12	12	12	12	12	12	12	13
Tumor	0	2	6	0	0	1	0	0	4*
Brain	0	0	0	0	0	0	0	0	0
Gonad	0	0	0	0	0	0	0	0	0
Heart	0	0	0	0	0	0	0	0	0
Kidney	0	0	0	0	0	0	0	0	0
Lung	0	0	0	0	0	0	0	0	0
Liver	0	0	0	0	0	0	0	0	0
Spleen	0	0	0	0	0	0	0	0	0
Salivary gland	0	0	0	0	0	0	0	0	0

Note: *Samples were collected 1, 6, 7, or 13 days after the second MYXV∆serp2 injection.

Abbreviations: MYXVΔserp2, myxoma virus deleted for serp2; DNA, deoxyribonucleic acid; PBS, phosphate buffered saline; O, oclacitinib.

Figure 7 Graphs of (A) age on the day of U21089 cell injection and (B and C) survival outcomes. (A) Mice treated with oclacitinib with MYXVΔserp2 (O+MYXVΔserp2) were significantly younger than mice in other treatment groups (*P-value < 0.02). However, there were no significant differences between (B) median survival times in treatment groups. (C) Likewise, no differences were calculated when median survival times of male and female mice in each treatment group were analyzed independently.

Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E. miR-147, a microRNA that is induced upon toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proc Natl Acad Sci U S A. 2009;106(37):15819–15824. doi:10.1073/pnas.090121610619721002

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