Decreasing the Susceptibility of Malignant Cells to Infection Does Not Impact the Overall Efficacy of Myxoma Virus-Based Oncolytic Virotherapy

Abstract

Oncolytic virotherapy relies on the induction of anti-tumor immune responses to achieve therapeutic efficacy. The factors that influence the induction of these responses, however, are not well understood. To begin to address this lack of knowledge, we asked how decreasing the susceptibility of malignant cells to direct viral infection would impact the induction of immune responses and therapeutic efficacy caused by oncolytic myxoma virus treatment. To accomplish this, we used CRISPR-Cas9 genome editing to remove the essential sulfation enzyme N-deacetylase/N-sulfotransferase-1 from B16/F10 murine melanoma cells. This eliminates the negative cell surface charges associated with glycosaminoglycan sulfation, which reduces a cell's susceptibility to infection with the myxoma virus by ∼3- to 10-fold. With the use of these cells as a model of reduced susceptibility to oncolytic infection, our data demonstrate that 3- to 10-fold reductions in in vivo infection do not hinder the ability of the oncolytic myxoma virus to induce anti-tumor immunity and do not lower the overall efficacy of localized treatment. Additionally, our data show that in mice bearing multiple distinct tumor masses, the choice to treat a less-susceptible tumor mass does not reduce the overall therapeutic impact against either the injected or noninjected lesion. Taken together, these data suggest that minor changes in the susceptibility of malignant cells to direct oncolytic infection do not necessarily influence the overall outcomes of treatment.

Keywords: immunotherapy; oncolytic virotherapy; viral dosing.

Graphical abstract

Figure 1

Loss of NDST1 Does Not Alter Melanoma Tumor Growth In Vivo Tumors were established by injecting either NDST1⁺ (WT) or NDST1^−/− (KO) B16/F10 cells subcutaneously into C57BL/6 mice. (A) Growth of individual tumors over time. Average tumor area of all tumors is marked by a darker line. (B) Average tumor area of all tumors 9 days after implantation (the day the first animal was euthanized). Significance was determined using Student’s t test. (C) Overall survival of animals. (A–C) Data represent the summation of three independent experiments (WT n = 21, KO n = 21). Significance was determined using log-rank analysis. (D and E) Tumors from a subset of animals were excised 12 days after implantation and immune infiltration analyzed using flow cytometry. (D) Example of gating strategy used. (E) Quantitation of individual immune subsets within each excised tumor. (E) Data represent the summation of two independent experiments (WT n = 9, KO n = 9). Significance was determined using Student’s t test (∗p < 0.05, ∗∗p < 0.01; N.S., not significant).

Figure 2

Tumors Lacking NDST1 Are Less Susceptible to MYXV Infection (A) Either NDST1⁺ (WT) or NDST1^−/− (KO) B16/F10 cells were incubated with Cy5-labeled MYXV particles. Direct binding of viral particles to the cell surface was then measured by flow cytometry. (B) WT or KO B16/F10 cells were infected with various MOIs of MYXV, ranging from 0.001 to 3. The rate of viral infection as then determined by quantitating the percent of GFP⁺ cells 24 h after infection using flow cytometry. Data are representative of three individual experiments. (C–E) WT or KO tumors of ~25–35 mm² were treated with a single intratumoral infection of 1 × 10⁷ FFU of MYXV (C). Tumors were excised 24 h after treatment. (D) Visualization of GFP⁺ region of infection in 8 μM sections of snap-frozen tumors. (E) Quantitation of the percent of GFP⁺ cells in each tumor analyzed using flow cytometry. (D and E) Data represent the summation of two independent experiments (WT n = 8, KO n = 5). Significance was determined using Student’s t test (∗∗p < 0.01). SQ, subcutaneous.

Figure 3

Reduced Direct Viral Infection Does Not Alter the Induction of Intratumoral Immune Responses (A) Schematic of experimental design. C57BL/6 mice were injected subcutaneously with either NDST1⁺ (WT) or NDST1^−/− (KO) B16/F10 cells and tumors allowed to establish until they reached ~25 mm². Tumors were then either mock treated or treated with three injections of 1 × 10⁷ FFU of MYXV over 5 days. On the 6th day, tumors were excised and analyzed. (B) Number of infectious viral particles in treated tumors. Data are normalized to tumor weight and represent the summation of two independent experiments (WT n = 8, KO n = 7). Significance was determined using Student’s t test (∗∗p < 0.01). (C) Quantitation of individual immune subsets within the indicated tumors. All data are pregated on single/living events. Data are representative of two independent experiments (mock n = 6, WT n = 8, KO n = 5). Significance was determined using Student’s t test (∗p < 0.05).

Figure 4

Reduced Direct Viral Infection Does Not Inhibit the Overall Efficacy of MYXV Therapy (A) Schematic of experimental design. C57BL/6 mice were injected subcutaneously with either NDST1⁺ (WT) or NDST1^−/− (KO) B16/F10 cells and tumors allowed to establish until they reached ~25 mm². Tumors were then either mock treated or treated with three injections of 1 × 10⁷ FFU of MYXV over 5 days. (B) Growth of individual tumors over time. Average tumor area of all tumors is marked by a darker line. (C) Average tumor area of all tumors. Significance was determined at day 11 post-treatment using Student’s t test (∗p < 0.05, ∗∗∗p < 0.001). (D) Overall survival of animals. Significance was determined using log-rank test (∗∗p < 0.01, ∗∗∗p < 0.001). (B–D) Data represent the summation of two independent experiments (WT mock n = 6, KO mock n = 8, WT MYXV n = 8, KO MYXV n = 10).

Figure 5

Efficacy of MYXV Treatment Is Based Primarily on Anti-tumor Immunity (A) Schematic of experimental design. NOD/SCID mice were injected subcutaneously with either NDST1⁺ (WT) or NDST1^−/− (KO) B16/F10 cells and tumors allowed to establish until they reached ~25 mm². Tumors were then either mock treated or treated with three injections of 1 × 10⁷ FFU of MYXV over 5 days. 10 days after the initiation of treatment, tumors were harvested and analyzed for viral burden. (B) Number of infectious viral particles in treated tumors. Data are normalized to tumor weight. Significance was determined using Student’s t test (∗∗p < 0.01). (C) Growth of individual tumors over time. Average tumor area of all tumors is marked by a darker line. (D) Average tumor area 10 days after the initiation of treatment. Significance was determined using Student’s t test. (E) Average tumor mass 10 days after the initiation of treatment. Significance was determined using Student’s t test (WT mock n = 6, KO mock n = 6, WT MYXV n = 7, KO MYXV n = 7).

Figure 6

Treatment of a Less-Susceptible Tumor Mass Does Not Reduce Overall Systemic Efficacy during Localized OV (A) Schematic of experimental design. C57BL/6 mice were injected on each flank with either PDL1^−/− (KO/KO, blue) or PDL1^−/−/NDST1^−/− (KO/DKO, red) cells, as indicated, and tumors allowed to establish until both tumors reached ~25 mm². The tumor on the left anatomical flank was then either mock treated or treated with three injections of 1 × 10⁷ FFU of MYXV over 5 days. Tumors on the right flank were left untreated. Growth of both tumors was then monitored, and mice euthanized with their total tumor burden exceeded 400 mm². (B) Overall survival of animals. Significance was determined using the log-rank test (∗p < 0.05). (C) Average area of both injected and noninjected tumors from all cohorts. (B and C) Data represent the summation of two independent experiments (KO/KO mock n = 6, KO/DKO mock n = 5, KO/KO MYXV n = 7, KO/DKO MYXV n = 5).