Treatment with M51R VSV slows CT26 tumor growth.
We sought to determine the therapeutic effects of two M51R VSV doses in a subcutaneous CT26 CRC tumor model. We found a significant reduction in tumor volume at both treatment doses relative to mock-treated controls. While mice in the control group harbored tumors with an average volume of 919.85 ± 150.79 mm3, those treated with M51R VSV at 1 and 10 MOI had tumor volumes that averaged 259.67 ± 15.85 mm3 and 410.64 ± 30.90 mm3, respectively (p < 0.001). (Fig. 1B). These results demonstrate that both M51R VSV doses effectively treated CT26 CRC.
Gene expression analysis demonstrates that treatment with M51R VSV produces strong anti-tumor immune responses.
We employed NanoString's PanCancer Immune Profiling RNA Panel to investigate differential immune gene expression in control and M51R VSV-treated CT26 tumors. Based on our criteria, we identified 34 differentially upregulated genes that were shared by both treatment groups. We also identified nine uniquely upregulated genes in tumors treated with 1 MOI of M51R VSV and 35 such genes in tumors treated with 10 MOI of the virus. (Figs. 2A, 2B, 2C). Our analysis also identified seven genes that were downregulated after treatment with either dose of the M51R VSV. Interestingly, 16 genes were downregulated after treatment at MOI 1, and no unique genes were identified after treatment at MOI 10 (Fig. 2C).
The top 10 differentially upregulated genes in both treatment groups were primarily associated with T (Cd8a, Pdcd1, Cd40, Ifng) and NK (Gzmm, IFa4, Klra7, Klrc1) cell functions (Figs. 2D and 2E). Importantly, Cd8a, the most upregulated gene, showed a 40.9 (p < 0.001) and 48.8 (p < 0.001) in the MOI 1 and MOI 10 groups, respectively (Figs. 2D and 2E). Effector cell cytotoxic function genes, such as Grzmm (6.88, p < 0.01; 8.44, p < 0.01) and Ifng (4.33,p < 0.01; 5.14,p < 0.01), were also significantly upregulated in the treatment groups compared to the control. The PD-1 (Pdcd1) gene was also overexpressed in the experimental groups by 15.95 (p < 0.05) and 19.56 (p < 0.05) fold after treatment with MOI 1 and MOI 10, respectively. Interestingly, Ctsg (20.6, p < 0.05) and Tnfrsf4 (3.12,p < 0.05), genes associated with cancer progression and T cell stimulation, respectively, were upregulated in tumors treated with 1 MOI of M51R VSV, whereas genes related to cytotoxicity (Fasl, 6.37, p < 0.05), T cells (Slamf7, 5.48, p < 0.05), and a negative regulator of PD-1 signaling (Sh2d1a, 5.10, p < 0.05) were overexpressed in tumors treated with 10 MOI of M51R VSV.
In contrast, the top 10 differentially downregulated genes that were shared among both treatment groups were primarily associated with cancer progression (Mmp9), M2 polarized tumor-associated macrophages (F13a1, Cxcl3, Ppbp), and Treg recruitment (Maf) (Figs. 2D and 2E). In addition, five additional genes related to M2 TAMS (Cd163), Tregs (Ccl22 and Ccl11), MDSCs recruitment (S100a8), and cancer progression (Ccl6) were uniquely downregulated in tumors treated at 1 MOI (Fig. 2D). In addition, two collagen-associated genes (Col3A1 and Col4A1) were downregulated in tumors treated with 10 MOIs (Fig. 2E).
As expected, immune cell infiltration increased in tumors treated with the M51R VSV. The abundance of various cell populations was calculated on ROSALIND using the cell-type profiling module. This method quantifies cell populations using marker genes that are stably expressed in the given cell types. This analysis showed increased immune cell infiltration in the treated groups compared with that in the control group (Fig. 2F). However, treatment with 10 MOI M51R VSV resulted in a significant increase in the total cytotoxic cells (p < 0.005), NK cells (p < 0.05), and T cells (p < 0.005) (Fig. 2G).
We evaluated the differential gene expression regarding their effect on cellular pathways using the ROSALIND gene set analysis tool. The differentially expressed genes shared between the two treatment groups belonged to T-, B-, and NK-cell functions, antigen processing, innate immunity, basic cell functions, cytokines and receptors, and cancer progression pathways (Supplementary Figure S4 A).
Evaluation of genes differentially expressed after treatment with 1 MOI of M51R VSV revealed differential over- and under-expression of additional genes associated with immune pathways (Supplementary Figure S4 B). Genes associated with interferon (Ifit1, Ifih1), apoptosis (Jun), cytokine and receptor (Cxcl10), and innate (Elk1, Itga5, Serping1) pathways were differentially expressed. In contrast, genes associated with T-cell functions (Ccl11 and Itgam), apoptosis (Clec5a), cancer progression (Ccl11, Cd163), basic cell functions (S100a8, Pdgf-c), adaptive immune response (Ccl11, Cxcl14), cytokines and receptors (Ccl22, Ccl6, C5ar1, Clec4n, Ccl9), and innate immunity (Colec12) were differentially under-expressed.
A broader immune response against CT26 tumors was observed in the MOI 10 group, which showed 35 uniquely overexpressed genes (Supplementary Figure S4 C). Two genes belonging to the TNF superfamily, Tnfrsf9, and FasL, were upregulated in the MOI 10 group. Tnfrsf9 is expressed by T cells that recognize cognate antigens and promote the survival, expansion, and enhanced effector function of activated T cells. Fasl is present in the membranes of activated cytotoxic T cells and TH1 cells, and induces apoptosis in target cells expressing Fas in their membranes.
Twelve of the 35 overexpressed genes in the MOI 10 group belonged to the T-cell function pathway (Supplementary Figure S4 C). IL-15 stimulates the proliferation and activation of NK, NKT, and CD8+ T cells, especially memory phenotype CD8+ T cells, leading to increased cytotoxicity and the production of IFN-γ and IFN-α. Both Il-15 and Il-15ra were overexpressed exclusively in the MOI10 group. In addition, two pro-inflammatory and interrelated cytokines with important functions, Cxcl9 and Ccl5, were found to be overexpressed in MOI10. Cxcl9 expression enhances T cell infiltration and positions activated T cells near antigen-presenting cells within the TME. Ccl5 is required for Cxcl9 expression and actively recruits T cells, macrophages, eosinophils, and basophils into inflammatory sites. Among this group, Il18r1, which encodes the IL-18 receptor (IL-18R), was significantly more expressed by a minority of cells with a functional (T-bet+Eomes+) phenotype, although we did not find that these two genes were overexpressed in our samples. In line with the high expression of IFNγ, Irf1, an early downstream gene target of IFNγ signaling, was also upregulated. In addition, high levels of Stat1, which enhances immune surveillance and increases the tumoricidal effect of natural killer (NK) cells, were also observed. Another important effector gene for T cells, Prf1, which encodes the pore-forming protein perforin, was found to be overexpressed in tumors treated with 10 MOI M51R VSV. Similarly, negative regulators of T-cell functions, including Pdcd1lg2 and Socs1, and Treg markers, including Fcgr4 and CD36, were upregulated.
Strong activation of NK cells was demonstrated in the MOI10 treatment group based on the upregulation of seven additional genes (Il15, Sh2d1b1, Klra2, Slamf7, Ccl5, Sh2d1a, Il15ra) associated with NK cell functions. Interestingly, SLAMF7 is a highly expressed marker on the surface of suppressive CD8+ T cells, and its expression correlates with an exhausted T cell phenotype. In addition, SH2D1A has a dual function in T cells. Research has shown a connection between SH2D1A expression and the functioning of NK cells, as well as an opposite relationship between SH2D1A expression and PD-1 capacity to hinder T cell function.
Our analysis identified upregulation of IFNγ-induced genes, including Gbp5, Gbp2b, Nlrc5, and Irgm2. In addition, Nlrc5, which transcriptionally regulates MHC class I gene activation via a CITA enhanceosome complex that associates explicitly with MHC class I gene promoters, was upregulated after treatment with M51R VSV.
Activation of the innate immune response was evidenced by the overexpression of 14 of the 35 genes in the MOI 10 group. (Supplementary Figure S4 C). Nod1 encodes a pattern recognition receptor (PRR) that recognizes both pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Ripk2 is a potent activator of nuclear factor κB, Ly86 constitutes a TAMs marker, Zbp1 expression is dramatically elevated in necrotic tumors, and C1s1 and Cfb are associated with the complement pathway. An indicator of the presence of M1 TAMs is the up-regulation of Slc11a1. In addition, Prdm1 regulates B- and T-cell differentiation and is critical for T cell-mediated immunosuppression.
Overall, the treatment of CT26 tumors with M51R VSV at an MOI of 10 showed an increased innate and adaptive response with the expression of numerous genes associated with cytotoxic CD8+ T and NK cells. In addition, the differential expression of genes associated with TAMs indicates polarization from the M2 subtype towards the M1 subtype with proinflammatory and anti-tumor properties. However, the higher dose administered at an MOI of 10 likely leads to T cell exhaustion due to chronic exposure to tumor antigens.
Treatment with M51R VSV results in an anti-tumor immune microenvironment.
A nonlinear dimensionality reduction technique, uniform manifold approximation and projection (UMAP) (Fig. 3A), followed by the FlowSOM clustering algorithm (Fig. 3B), along with heat maps of median marker expression in each cluster (Fig. 3C), was used to visualize immune cell phenotypes. First, we manually annotated the major cell types based on the median marker intensities observed in the clusters (Fig. 3C). We subsequently examined the differences in the frequencies of the identified clusters between the M51R VSV-treated and control groups (Figs. 3D and 3E). The FlowSOM algorithm yielded ten meta-clusters, of which two showed significant differences. (Fig. 3D). Cluster 3 represents G-MDSCs. In this cluster, the M51R VSV-treated groups showed a significant decrease of almost 3-fold in comparison to the control group (MOI 1 (p < 0.05) and MOI 10 (p < 0.05)) (Fig. 3D). Cluster 9 was primarily composed of CD8+ T cells. Treatment with M51R VSV at both MOI 1 and MOI 10 resulted in a 9-fold increase (p < 0.01) in these cells, accounting for almost 12% of the total cell count.
The Astrolabe Cytometry Platform was used to analyze the CyTOF data and confirm the results. This platform utilizes a customized hierarchical method to identify the main subsets of immune cells. (Supplementary Table S5). According to the findings of NanoString and UMAP/FlowSOM, the use of M51R VSV to treat CT26 tumors led to an increase in CD8+ T cells and a reduction in G-MDSC cells. (Fig. 4A). In addition, we found a decrease in CD4+ T and Treg cells in the M51R VSV-treated groups compared to the control group (Fig. 4A).
Cytotoxic CD8+ T cells of the adaptive immune system are the most potent effectors of anticancer immune response and constitute the backbone of cancer immunotherapy. Treatment with M51R VSV resulted in an approximately 8-fold increase in the frequency of CD8+ T cells (MOI 1:8.94%, p < 0.01; MOI 10:7.97%, p < 0.01) compared to that in the control group (1.01%) (Fig. 4B). CD8+ T cells from both experimental treatment groups also showed higher expression of CTLA4, Granzyme B, ICOS, Ki-67, LAG-3, and PD-1 relative to PBS-treated control tumors (Fig. 4C). Figure 4D shows the confirmed levels of CD8+ T cells expressing six specific markers, which were subjected to manual analysis. All markers, except for CTLA4+, were found to be significantly increased in CD8+ T cells in both treatment groups. Regarding the differential expression analysis run by Astrolabe, we found a significant downregulation of TNF alfa in the CD8+ T cells present in the treated groups (MOI 1 (p < 0.01) and MOI 10 (p < 0.05)) (Fig. 4E).
Four CD8+ T cell subsets were identified based on Granzyme B, LAG-3, and IFNg expression. CD8+ T cells expressing low levels of these three markers were the most abundant subpopulation in the treatment groups, with a frequency of approximately 2.6%, more than twice the number found in the control group (p < 0.05) (Fig. 5A). CD8+ T cells in this subpopulation express high levels of the Ki67 proliferative and PD-1 exhaustion markers. They also expressed CTLA4 and ICOS (Fig. 5A). In contrast, the second most abundant CD8+ T cell subpopulation in the treated groups expressed high levels of Granzyme B and LAG-3 (MOI 1,2.05%, p < 0.01; MOI 10,2.38%, p < 0.01) (Fig. 5B). This population was barely present in the control group, indicating that the phenotype was highly related to treatment with M51R VSV. The third subpopulation of CD8+ T cells expressing high levels of LAG-3, along with low levels of granzyme B and IFN-γ, was also significantly increased in both treated groups (MOI 1:1.26%, p < 0.05; MOI 10:1.52% p < 0.01) (Fig. 5C). The fourth CD8+ T cell subset expressed low levels of Granzyme B and LAG-3 but high levels of IFNg. Only the MOI 10 group showed a significant increase in these cells (1.04%, p < 0.05) (Fig. 5D). These cells expressed low levels of TNFα compared to those in the control group (MOI 1:4.1, p < 0.01; MOI 10:4.8, p < 0.05) (Fig. 5E). These CD8+ T cell subtypes could represent exhausted CD8+ T cells that progressively acquire immune checkpoint markers (ICOS, PD-1, LAG-3, and CTLA4), along with reduced secretion of effector proteins, including granzyme B and IFN-γ.
We found a 3-fold decrease in the frequency of G-MDSCs in both M51R VSV-treated groups (MOI 1:2.91%, p < 0.01; MOI 10:3.65%, p < 0.01) compared to the control group (10.09%) (Fig. 6A). Importantly, PD-1 was detected in these cells, indicating its involvement in cell growth (Fig. 6A).
We found a significant decrease in FoxP3+CD25− (MOI 1:0.43%, p < 0.05; MOI 10:0.50%, p < 0.05 vs. Control:0.91%) Treg cells in the treated groups compared to that in the control group (Fig. 6B).
Treatment with M51R VSV results in CD8 + responses against CT26 tumor-associated antigens.
Based on the above results, it can be concluded that CD8+ cytotoxic T-cells play a central role in the immune response triggered by M51R VSV against CT26 tumors. Thus, bulk TCR sequencing was performed on CD8+ TILs to determine whether clonal expansion of anti-tumor T cells occurs due to M51R VSV therapy. We employed novel bioinformatic techniques to analyze TCR sequencing data to define the comparative abundance and specificity of TCR clonotypes. We observed that treatment with M51R VSV resulted in five high-frequency TIL TCR clonotypes, which represented over 10% of the TCR clonotype population (Fig. 7A). We also found that M51R VSV treatment decreased clonotype diversity, as we identified a lower clonotype count (Fig. 7B) and a higher clone fraction in the M51R VSV-treated groups (Fig. 7C).
The five TIL CDR3 sequences, their respective epitopes, and clone fraction data predicted to have a high frequency by TCRMatch are shown in Supplementary Table S6. CDR3 sequences of T-cell clones in tumors treated with 1 MOI of M51R VSV were analyzed. The clones that comprised 11–25% of the total clone fraction matched epitopes from myelin basic protein, insulin-2, cytomegalovirus, and MuLV. However, when tumors were treated with 10 MOI of VSV, the two most common clone fractions contained CDR3 sequences that did not match any murine or MuLV proteins. This information is provided in Supplementary Table S6.
Consequently, many stable cancer cell lines express endogenous viral peptides recognized by the immune system as tumor-associated antigens (TAAs) (26–28). In this study, the CT26 murine colon carcinoma cell line expressed the murine leukemia retrovirus (MuLV) antigen. This antigen contains two immunodominant epitopes: AH-1(peptide sequence SPSYVYHQF) and KSP (peptide sequence KSPWFTTL). These epitopes are recognized in the context of the major histocompatibility complex class I molecules H-2Ld and H-2Kb (29–31). In one tumor treated with 1 MOI of M51R VSV, we discovered that 11% of the CD8+ TIL were directed at the immunodominant KSP epitope. In addition, we observed an increased frequency of TCRs specific to the AH1 epitope. Although clone fractions specific to this epitope were less than 10%, CD3 sequences matching AH1 were present in five out of six M51R VSV-treated samples, but not in the control samples (Supplementary Table S7). These results indicated that CD8+ T cells undergo clonal expansion against epitopes for MuLV TAA.
We then evaluated the MHC binding affinity. We first identified unique CD8+ TCR clonotypes in each treatment group, and then predicted their cognate epitopes using TCRMatch. We then predicted MHC class I epitope-binding affinity using TepiTool. Epitopes with predicted IC50 values < 50 nM were considered to have high affinity, peptides with predicted IC50 < 500 nM were considered to have intermediate affinity, and those with predicted IC50 < 5000 nM had low MHC I affinity (32). Comparative analysis revealed that treatment with M51R VSV resulted in the production of TCRs for low-affinity epitopes (Wilcoxon test, p = 0.0003) (Fig. 7D). We then predicted 314 epitopes in M51R-treated tumors with IC50 values below 1000 nM and 312 epitopes in tumors treated with 1 MOI of M51R VSV, forecasted to have IC50 values below 500 nM. When analyzing murine and MuLV epitopes, there was no notable difference in IC50 values between the control and M51R VSV groups (Fig. 7C). Interestingly, TCR sequences for epitopes with IC50 values below 500 were present only in tumors treated with 1 MOI of M51R VSV (Supplementary Table S8). The results showed that M51R VSV treatment caused the expansion of T cells that targeted tumor antigens, resulting in positive therapeutic outcomes.