4. Discussion
The rationale for reducing the expression of key immune checkpoint receptors, such as CTLA-4 and PD-1 with siRNA, is based on the success of the CTLA-4 and PD-1 targeting immunoglobulin therapies [20]-[22].
We hypothesized that reducing the expression of CTLA-4 and PD-1 should result in a more effective immune response that produces a slow-down in tumor growth.
The rationale for running Western blots on tumor proteins with 10 mice per group was done to reduce cost and to produce an even sampling of the protein measures in tumors. Randomly selecting tumors from both groups allowed for unbiased detection of total protein change with balanced arms.
The exposure of the A2 signal, which is a magnetic field recording of the siRNAs targeting the murine CTLA-4 and PD-1 mRNA, resulted in the statistically significant reduction of tumor volume and reduction of CTLA-4 Pcdc-1 receptor protein expression in the tumors (Figure 4(c)). The differential contribution of the sources of the CTLA-4 Pcdc-1 receptor protein is unknown, as both tumor and WBCs express CTLA-4 Pcdc-1 receptor. In either case, an observed decrease in CTLA-4 Pcdc-1 receptor expression was measured in the A2 exposed group, similar to the reported reduction of EGFR mRNA expression using the same technology [10]. The CTLA-4 protein levels were trending towards lower expression in the A2 exposure group.
The PD-1 expression profile was mixed, with half of the tumors that were ex posed to the A2 signal expressing a lower amount of PD-1 versus control (Figure 4(b), right). The other tumors showed the opposite effect, with greater PD-1 ex pression than controls. The difference in the subsets of Figure 4(b), left (between the controls and A2 groups) are statistically significant (data not shown), but when looked in aggregate, there is no significant difference. We are unsure as to why this occurred, as CTLA-4 expression was reduced significantly, in all tumors analyzed.
The lack of consistent PD-1 CTLA-4 receptor protein reduction may explain the differences observed between the absolute tumor volume changes reported by our mouse study and the result from Mukthavaram et al.’s article [7]. The Kesari study demonstrated a stronger inhibition of tumor growth using the A2 signal than ours. Although both were statistically significant in effect, the magnitude was less in our hands. Genetic background differences may play a role, as the mouse strains used in the UCSD study [7] and the IDRI study (ours) came from two different vivariums. Unfortunately, Mukthavaram et al. do not report on the expression of CTLA-4, PD-1 or CBCs in their publication.
Both CTLA-4 and PD-1 are expressed in a wide variety of tumors [23] [24], but the exact mechanism by which the immune system induces tumor cell death or tumor cell growth arrest (apoptosis, necrosis or cell cycle inhibition) appears to be dependent on multiple factors.
Total tumor protein expression, specifically Caspase 3 and Ki67 (Figure 5), suggests that the observed reduction in tumor growth was not due primarily to apoptosis, but and not by a reduction in the rate of tumor cell division and potentially increased necrosis. Although the The proteins levels did not reach statistical significance between the A2 and Control groups for Caspase 3, with the A2 groups showing an increase in expression, Ki67, the protein expression differences of Caspase 3 was trending to significance. This trend suggesting a lower level of apoptosis in the A2 exposure group than the control group. indicating an increase in apoptosis. The levels of Ki67 expression were equivalent across both A2 and control, indicating no significant differences in the rate of cell division between tumors.
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In our study, we demonstrated that exposure to the A2 signal significantly (Figure 3, Lymph #) lowers lymphocyte levels when compared to the Control group. When an analysis of the ratio between polymorphonucleocytes to lymphocytes (PLR), eosinophils to lymphocytes (ELR) and platelets to lymphocytes (PlaLR) was done, there was a trend towards significance (Table 1). Increases in the ratios between these sub-populations suggest that decreasing the expression of CTLA-4 and Pcdc-1 produce changes in the immune cell numbers.
These alterations may be specific to our mechanism of action, a reduction in CTLA-4 and Pcdc-1 expression, as compared to the infusion of immunoglobulins that target CTLA-4 or Pcdc-1. The change in lymphocyte counts in our study could be indicative of an effective tumor response when CTLA-4 and Pcdc-1 receptor expression is reduced. Studies in CTLA-4 conditional KO mice (tamoxifen suppressor) demonstrate a reduction of lymphocytes [27] after tamoxifen induced KO after several weeks. The report by Paterson et al. parallels the response we measure when CTLA-4 knock-down occurs with our A2 signal.
The reduced lymphocyte cell counts (Figure 3) correspond with a trend towards a lower an increased expression of the CD4+ and CD8+ antigen cell-surface markers, as measured in Western blot analysis (Figure 6(c)) of the tumors analyzed. CD4+ and CD8+ T-cells are primary effector cells involved in tumor infiltration and the primary targets of the co-inhibitory and co-stimulatory axis of current immune therapies [28]. Reduction in blood lymphocyte counts coincides with the trend observed in the CD4+ and CD8+ protein expression in the tumor and is supported by reports of GL261 mouse models treated with an anti-PD-1 antibody [1] . Although we did not directly measure the relative number of CD4+ and CD8+ T-lymphocytes between the A2 and control group, the reduction in lymphocytes could indicate an effect produced by the CTLA-4 and Pcdc-1 reduction via the signal, indicating increased infiltration of T-cells into the tumors.