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. 2023 May 22;17(11):10236–10251. doi: 10.1021/acsnano.3c00418

In Situ Vaccination Following Intratumoral Injection of IL2 and Poly-l-lysine/Iron Oxide/CpG Nanoparticles to a Radiated Tumor Site

Ying Zhang †,‡, Md Mahfuzur Rahman §, Paul A Clark §, Raghava N Sriramaneni §, Thomas Havighurst ∥, Caroline P Kerr §,⊥, Min Zhu †,‡,#, Jamie Jones †,‡, Xiuxiu Wang †,‡, KyungMann Kim ∥, Shaoqin Gong †,‡,#,*, Zachary S Morris §,*
PMCID: PMC10278176  PMID: 37216491

Abstract

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The in situ vaccine effect of radiation therapy (RT) has been shown to be limited in both preclinical and clinical settings, possibly due to the inadequacy of RT alone to stimulate in situ vaccination in immunologically “cold” tumor microenvironments (TMEs) and the mixed effects of RT in promoting tumor infiltration of both effector and suppressor immune cells. To address these limitations, we combined intratumoral injection of the radiated site with IL2 and a multifunctional nanoparticle (PIC). The local injection of these agents produced a cooperative effect that favorably immunomodulated the irradiated TME, enhancing the activation of tumor-infiltrating T cells and improving systemic anti-tumor T cell immunity. In syngeneic murine tumor models, the PIC+IL2+RT combination significantly improved the tumor response, surpassing the single or dual combinations of these treatments. Furthermore, this treatment led to the activation of tumor-specific immune memory and improved abscopal effects. Our findings suggest that this strategy can be used to augment the in situ vaccine effect of RT in clinical settings.

Keywords: cancer immunotherapy, in situ vaccine, nanoparticle, radiation therapy, abscopal effects

Cancer immunotherapy has revolutionized cancer treatment.1,2In situ vaccination is an immunotherapeutic strategy that seeks to convert a patient’s own tumor into a source for tumor antigen presentation and the development of adaptive anti-tumor immunity. This approach may stimulate response against tumors that exhibit limited de novo T cell infiltration and could overcome the challenges of tumor heterogeneity and limited availability of shared tumor-specific antigens that have historically hampered the development of conventional cancer vaccines.3−5 Radiation therapy (RT) has been reported in preclinical and clinical studies to activate an in situ vaccination response.6−8 By generating type I interferon responses and stimulating the release of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), RT can recruit immune cells to the inflamed tumor tissues, activate these immune cells, and stimulate antigen presentation, leading to a more diversified anti-tumor T cell response.9−11 Moreover, RT can also improve the immunogenicity of irradiated tumor cells and enhance their susceptibility to immune-mediated killing.12 These effects of RT may help to redress many of the features that can limit tumor response to cancer immunotherapies.13,14 However, RT can also exacerbate certain immunosuppressive features in tumors, including elevation of the tumor infiltration by regulatory T cells (Tregs) and M2 macrophages. These effects may constrain the efficacy of RT alone in eliciting in situ vaccine effect.15,16

Others and we have observed that the in situ vaccine effect of RT can be improved by combination with other therapies directly administered to the irradiated TME.9,17−19 We have previously evaluated two distinct approaches to augmenting the in situ vaccine effect of RT. The first aims to further stimulate inflammatory effects and immune cell recruitment and activation in the irradiated TME. The second aims to blunt the detrimental effects of RT in the TME. Among the former strategies, we have evaluated approaches to delivering interleukin-2 (IL2) in combination with RT.7,9 IL2 has been approved by U.S. Food and Drug Administration (FDA) and widely tested in various clinical settings for cancer.20,21 By binding its receptors (CD25, CD122, and CD132) on T cells and NK cells, IL2 plays a critical role in driving the proliferation and differentiation of these cells as well as their function and response to cancer immunotherapies.20,22 While toxicities of systemic IL2 administration have constrained its clinical use, intratumoral delivery has been investigated and may limit such risks.23,24 When combined with RT, IL2 can augment the adaptive T cell immune response and improve the in situ vaccine response.7,25,26 However, like RT, IL2 activates negative feedback activation of suppressive immune mechanisms in the TME, particularly infiltration by and activation of Tregs. Tregs constitutively express high-affinity IL2 receptors (CD25), making these highly sensitive to IL2. Treg activation can be further enhanced when IL2 is combined with RT.20 This may be overcome by an additional combination with anti-CTLA-4 immune checkpoint inhibition (ICI), which can deplete and functionally antagonize Tregs.27 Yet combining RT with locally immune stimulatory intratumoral IL2 does not directly address the detrimental effects of RT in the irradiated TME, which may continue to dampen the priming of T cell immunity and the in situ vaccine effect.

Recently, we designed a multifunctional nanoparticle (PIC) to specifically overcome the detrimental effects of RT in the TME and thereby augment the in situ vaccination of RT.16 PIC is engineered through a readily scalable and simple complexation of poly-l-lysine (PLL), CpG oligodeoxynucleotide (CpG), and iron oxide nanoparticle (ION).16 This design confers multiple functions including effects of ION enhanced RT killing potential and the activation of a type-I interferon (IFN-I) response in the TME following RT.16 We further demonstrated that, in the context of an irradiated TME, ION promotes M1 macrophage polarization and PLL stimulates antigen uptake in dendritic cells and CpG activates these to promote antigen presentation, thus improving the in situ vaccine effect of RT.16 Yet, like RT+IL2, the anti-tumor efficacy of RT+PIC was limited on its own and required combination with ICI to sustain systemic anti-tumor immunity.28 Here, we hypothesized that the in situ vaccine effect of RT could be further optimized by combining these approaches that aim to both locally stimulate an immune response in an irradiated TME and simultaneously antagonize potentially detrimental effects of RT in the TME. To achieve this, we have tested a combination of RT with intratumoral injection of the irradiated TME with both IL2 and PIC. We evaluated the interaction between PIC with IL2 in the irradiated tumors and found cooperative therapeutic efficacy, resulting in potent induction of systemic anti-tumor immunity and abscopal tumor response. The combination of PIC+IL2+RT may provide a simple strategy to use locally directed therapies at one tumor site to mount a clinically meaningful in situ vaccine response.

Results

PIC and IL2 Increase the Expression of Pro-Inflammatory Cytokines and Augment T Cell Activation

We prepared PIC with a particle size about 100–120 nm and a zeta potential about +30–+35 mV, as previously described (Figure 1a). We evaluated the effects of PIC and IL2 on whole splenocytes from naïve C57BL/6 mice, when these were cocultured with irradiated B78 melanoma cells and treated with PIC, IL2, or their combination (Figure 1b). By analyzing the mRNA expression of cytokines in these splenocytes using RT-qPCR, we found that PIC significantly enhanced the expression of Ifnγ when it was added to the IL2-treated irradiated cocultures, although it did not show any direct effect on the irradiated cocultures in the absence of IL2 (Figure 1c). We also found that PIC treatment reduced the expression of Il4, although IL2 and PIC+IL2 treatments did not influence this expression. We found that PIC promoted the expression ratios of Ifnγ/Il4 when combined with IL2 (Figure 1d). This could be consistent with PIC promoting Th1 over Th2 polarization of T cells within this splenocyte population.29,30

Figure 1.

Figure 1

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PIC and IL2 increase the expression of pro-inflammatory cytokines and augment T cell and NK cell activation. (a) Particle size (left) and zeta potential (right) of PIC. (b) Scheme of the studies (RT, 12Gy; PIC, 0.5 μg/mL; IL2, 67 IU/mL). Whole splenocytes were cocultured with B78 tumor cells and qPCR was used to determine the effects of PIC and IL2 on (c) Ifnγ and Il4 expression, (d) Ifnγ/Il4 expression ratios, (e) Fas expression, (f) Nos2 and Arg1 expression, (g) Nos2/Arg1 expression ratios, and (h) Il1α expression (n = 3 biologically independent samples, data are normalized relative to untreated controls). The percentages of CD69+ and CD44+ cells among (i) CD4 T cells (CD45+CD3+CD4+), (j) CD8 T cells (CD45+CD3+CD8+), and (k) NK cells (CD45+CD3–NK1.1+) after indicated treatment in B78 cocultures (n = 4 biologically independent samples). Statistical significance was calculated via one-way ANOVA test in c–k. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Panel (b) was created with Biorender.com.

Fas and Fas ligand (FasL) interactions are involved in immune-mediated tumor cell killing via activation of extrinsic apoptosis.31 We observed higher Fas expression in the splenocytes when the irradiated cocultures were treated with PIC+IL2 compared to those treated with IL2 alone (Figure 1e). Furthermore, as shown in Figure 1f,g, in the presence of IL2, the addition of PIC promoted a higher ratio in the expression of Nos2/Arg1 among splenocytes. In the absence of IL2, we previously demonstrated that PIC can promote a higher ratio in the expression of Nos2/Arg1 among macrophages, consistent with PIC promotion of M1 macrophage polarization.16 Higher expression of the pro-inflammatory cytokine, Il1α, was also observed among total splenocytes when PIC was added to the cocultures (Figure 1h).

Since IL2 has been reported to play a crucial role in modulating the functions of T cells and NK cells, we subsequently evaluated the effects of PIC on the activation state (CD69) and effector/memory state (CD44) of T cells and NK cells when it was combined with IL2 (Figure S1). In Figure 1i,j and Figure S2, IL2 promoted the expansion of T cells and NK cells, and the abundance of activated T cells (CD69+) and effector/memory T cells (CD44+) significantly increased among both CD4 T cells and CD8 T cells when PIC was added to the IL2-treated irradiated cocultures, although this effect of PIC was not observed in the absence of IL2. For NK cells, PIC did not influence the CD44+ proportion but slightly increased the CD69+ proportion, and these effects were significantly amplified in the presence of IL2 (Figure 1k). By analyzing regulatory T cells (Tregs), we found that IL2 treatment significantly increased the levels of CD25+FOXP3+ Tregs, while PIC showed negligible effects on Treg levels at the tested concentration (Figure S3). Although IL2+RT increased the PD-1 expression on CD8 T cells, PIC did not exhibit significant effects on PD-1 expression on either CD4 or CD8 T cells (Figure S4).

To determine whether the effects of PIC and PIC+IL2 on the splenocyte cytokine expression and T cell activation were direct effects of these therapies on immune cells, we treated splenocytes with PIC, IL2, or PIC+IL2 in the absence of irradiated tumor cells. Similar trends were found in the patterns of cytokine expression when PIC was added to IL2-treated splenocytes as compared to those observed in the splenocyte-irradiated tumor cell coculture system, leading to higher expression ratios of Ifnγ/Il4 and Nos2/Arg1 (Figure S5). This indicated that PIC+IL2 could directly regulate the cytokine expression of immune cells. In the context of our previously published studies,16 these data are consistent with a potential direct role for PIC+IL2 in promoting Th1 and M1 polarization of T cells and macrophages, respectively. When added to IL2, however, PIC did not affect the activation state (CD69+) or effector/memory state (CD44+) of T cells or NK cells in the absence of irradiated tumor cells (Figure S6). This suggested a critical role for irradiated tumor cells in promoting activation of T cells and NK cells, even in the presence of PIC+IL2.

PIC Enhances the Systemic Anti-Tumor Immune Response to RT+IL2 Resulting in Immune Memory

Given the coordination observed in vitro between PIC and IL2 in activating T cells and promoting the M1 and Th1 polarization of macrophages and T cells, we investigated the potential effects of PIC on the anti-tumor immunity of IL2+RT in the immunologically “cold” B78 melanoma model. We injected IL2 into the irradiated tumors on days 6–10 because our previous work demonstrated that the IL2-conjugates injected using this schedule were optimal for priming an anti-tumor T cell immune response after RT.7 PIC was injected on days 0, 3, 6, and 9 in accordance with our prior studies (Figure 2a).16 As shown in Figure 2b–d, both PIC+RT and IL2+RT treatments delayed the B78 melanoma growth and prolonged mouse survival, while these effects were further improved in the triple combination PIC+IL2+RT treatment group, resulting in 100% (7/7) mice tumor-free at day 60 following the initial treatment. These disease-free mice obtained after PIC+RT+IL2 treatment exhibited resistance to a second B78 rechallenge, with 6/7 of them not showing tumor development after rechallenge and 1/7 showing tumor growth but much slower than that on naïve mice (Figure 2e,f).

Figure 2.

Figure 2

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PIC enhanced the tumor therapeutic efficacy of IL2+RT and induced an abscopal effect and an immune memory effect in B78 melanoma bearing mice. (a) Scheme for the studies on B78 melanoma model. The treatment was started when the tumor size was about 100 mm3. (b) Average tumor growth curves and (c) survival rate of mice after the indicated treatments (PIC+IL2+RT, n = 7; others, n = 5 animals per cohort). (d) Individual tumor growth curves in (b). (e) Average tumor growth curves after the naïve mice and disease-free mice (mice tumor-free from B78 melanoma after PIC+IL2+RT treatment) were rechallenged with B78 melanoma cells (Naïve mice, n = 5; disease-free mice, n = 7 animals per cohort). (f) Individual tumor growth curves in (e). (g) Scheme for the studies on B78 melanoma two-tumor model. The treatment was started when the size of primary tumors was about 100 mm3, and treatment was delivered only to the primary tumors, whereas secondary tumors were shielded from radiation and were not injected. (h) Average tumor growth curves after the indicated primary tumor treatments. (i) Individual tumor growth curves in (h). (j) Survival rate and (k) body weight of mice after the indicated treatments (IL2+RT, n = 7; PIC+IL2+RT, n = 8; others, n = 5 animals per cohort). (l) Scheme for the splenocytes coculture studies. (m) Percentages of CD69+ among CD4 T cells and CD8 T cells and the CD44 MFI on these T cells in the splenocytes in the indicated cocultures. (n) Relative expression of Ifnγ and Il2 in the splenocytes in the indicated cocultures (n = 4 biologically independent samples). CR: complete response. DF: Disease-free mice. Statistical significance was calculated via linear mixed effects modeling in b, e, and h, log-rank test in c and j, and one-way ANOVA test in m and n. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

To assess systemic anti-tumor immunity resulting from PIC+IL2+RT treatment, we implanted C57BL/6 mice with two B78 tumors, the first on the right flank and the second on the left flank 1 week later to mimic a tumor metastasis. Only the right tumors received direct treatment with combinations of PIC, IL2, and/or RT, and the growth of both tumors was monitored (Figure 2g). As shown in Figure 2h–j, PIC+IL2+RT not only effectively eradicated the primary tumors but also significantly inhibited the progression of distant tumors and prolonged the survival of mice, when compared to the PIC+RT and IL2+RT treatments, leading to 25% (2/8) mice tumor-free at day 60 following the initial treatment. The mice showed negligible changes in body weight during the study (Figure 2k). At day 90 after the initiation of treatment, we collected splenocytes from the disease-free mice obtained after PIC+IL2+RT treatment and cocultured them with B16 melanoma cells to assess immune memory (Figure 2l and Figure S7). Using flow cytometry, we found that the expression of CD69 and CD44 on both CD4 T cells and CD8 T cells from disease-free mice was significantly higher compared to that on T cells from naïve control mice after cocultured with B16 cells (Figure 2m). When the B16 cells in the cocultures were preradiated, the upregulation of CD69 on CD4 T cells and CD8 T cells in the splenocytes of disease-free mice was further increased, which may be related to the enhanced immunogenicity of radiated tumor cells and/or the production of type I interferon by tumor cells following radiation.12 Gene expression profiling of splenocytes in this coculture demonstrated increased expression of Ifnγ and Il2 in the splenocytes from the disease-free mice compared to those from naïve mice after coculture, further confirming the generation of immune memory effects (Figure 2n).

Optimizing PIC+RT+IL2 Dosing for Clinical Translation

Although PIC+IL2 markedly improved the in situ vaccine effect of RT, the translational potential of this strategy could be limited by a requirement for multiple intratumoral injections.32 To ameliorate this, we tested whether comparable efficacy could be achieved by combining the four doses of PIC into a single injection and tested the effect of this single dosing approach on the in situ vaccination of RT at different time points (injection on day 0, 3, 6, or 9 after RT) (Figure S8a). We sought to separately evaluate the optimal timing of PIC and IL2 injections relative to the timing of RT. However, as we previously reported, PIC+RT has minimal efficacy on its own, so to enable an efficacy signal in this timing and dosing study, we combined PIC+RT with anti-CTLA-4, a regimen that we previously demonstrated to have anti-tumor response in the B78 melanoma model.16 We found that PIC injection at day 0 (1 day prior to RT) achieved similar tumor growth inhibition when compared to mice receiving multiple PIC injections (days 0, 3, 6, and 9) when combined with the immune checkpoint inhibitor anti-CTLA-4 (Figure S8b,c). This is consistent with our prior observation that intratumorally injected PIC is retained at tumor sites for many days16 and suggests that multiple PIC injections after RT may be less necessary.

Our previous work indicated that days 6–10 after RT may be a favorable time frame for the injection of IL2-conjugates to an irradiated TME in order to strengthen the in situ vaccine effect.7 To begin evaluating an optimal approach to IL2 administration using fewer injections in the context of PIC+RT, we combined PIC with IL2 and evaluated the injection on days 0 and 6. The anti-tumor and abscopal effects of PIC+IL2+RT were tested on mice bearing two B78 tumors. We compared our original PIC+IL2+RT treatment regimen (PIC injection at days 0, 3, 6, and 9; IL2 injection at days 6–10 daily) with two reduced injection regimens (PIC+IL2 injection on day 0 or day 0 + 6) (Figure 3a). (PIC+IL2)0+6+RT treatment was more effective than (PIC+IL2)0+RT with regards to inhibiting the growth of the radiation-targeted primary tumors and both regimens were less effective than our original multi-injection PIC+IL2+RT treatment. However, regarding the inhibition of distant tumor growth, we observed comparable efficacy for (PIC+IL2)0+6+RT compared to the original multi-injection PIC+IL2+RT regimen (Figure 3b,c). The survival curves for these treatments (Figure 3d) demonstrated a similar survival rate for mice receiving (PIC+IL2)0+6+RT and the original PIC+IL2+RT treatments with no significant changes in animal weight noted between these regimens (Figure 3e).

Figure 3.

Figure 3

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PIC and IL2 can be delivered using a reduced number of injections. (a) Scheme for the injection regimens tested on B78 melanoma two-tumor bearing mice. The treatment was started when the size of primary tumors was about 100 mm3, and direct treatment was only delivered to the primary tumor, whereas secondary tumors were shielded from radiation and were not injected. (b) Average tumor growth curves after the indicated treatments. (c) Individual tumor growth curves in (b). (d) Survival rate and (e) body weight of mice after the indicated treatments (n = 6 animals per cohort). Statistical significance was calculated via linear mixed effects modeling in b and log-rank test in d. The p values of b and d are shown in Table S1. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

PIC and IL2 Immunomodulate the Radiated TME and Stimulate Immune Response at Distant Tumors

To study the effects of (PIC+IL2)0+6+RT treatment on the tumor immune microenvironment and the specific roles of PIC in this immunomodulation, we analyzed treated tumors and their adjacent tumor-draining lymph nodes (TDLNs) at day 15 following (PIC+IL2)0+6+RT treatment and compared the results with those obtained from IL20+6+RT-treated or untreated control tumors (Figure 4a and Figures S9–S12). We found that (PIC+IL2)0+6+RT exhibited superior tumor control for both primary and distant tumors compared to IL20+6+RT treatment (Figure S13). H&E images of the tumor (Figure 4b) suggested marked destruction of tumor after (PIC+IL2)0+6+RT treatment. Using flow cytometry to analyze the populations of immune cells infiltrating in tumors, we found a similar proportion of CD45+ immune cells relative to total live cells in tumors from the (PIC+IL2)0+6+RT and IL20+6+RT treatment groups, both of which were significantly higher than the untreated control tumors (Figure 4c). Although neither the (PIC+IL2)0+6+RT nor the IL20+6+RT treatment changed the abundance of CD11b+F4/80+ macrophages in the tumor, PIC injection polarized these macrophages toward CD80+CD206– M1 phenotype, leading to higher M1/M2 macrophage ratios in (PIC+IL2)0+6+RT-treated tumors compared to IL20+6+RT-treated or untreated control tumors (Figure 4d,e and Figure S14a,b). This macrophage polarization effect of PIC was also observed in the primary TDLNs, which may be due to the accumulation of PIC in the TDLNs after intratumoral injection, as we observed previously (Figure 4k).16 We evaluated CD11c+MHCII+ dendritic cells (DCs) and found that (PIC+IL2)0+6+RT reduced the DC population in tumors but upregulated the expression of activation marker, CD80, on these DCs compared to IL20+6+RT treatment (Figure 4f). This downregulation of DCs population in tumor tissues may be associated with the migration of activated antigen-presenting cells (APCs) to the adjacent lymph nodes to prime T cells, as we observed an increased abundance of DCs and macrophages and an enhanced expression of CD69 on T cells in the TDLNs (Figure 4k–m).33,34

Figure 4.

Figure 4

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(PIC+IL2)0+6+RT favorably immunomodulated the TME and the adjacent TDLNs. (a) Scheme for the studies. The treatment was started when the size of primary tumors was about 100 mm3 and performed only on the primary tumors. At day 15, both primary tumors and distant tumors were collected for analyses. The analyses results for the primary tumors and distant tumors are provided in this and Figure 5, respectively. (b) Representative H&E and immunohistochemistry images of B78 melanomas (scale bar: 100 μm). (c) Percentages of CD45+ immune cells among live cells in B78 melanomas. (d) Percentages of CD80+CD206– M1 macrophages and CD206+CD80– M2 macrophages among CD11b+F4/80+ macrophages in B78 melanomas. (e) Ratios of M1/M2 macrophages in B78 melanomas. (f) Percentages of CD11c+MHCII+ DCs among CD45+ immune cells; the CD80 MFI on CD11c+MHCII+ DCs in B78 melanomas. (g) Percentages of CD4 T cells (CD4+CD3+) and CD8 T cells (CD8+CD3+) among CD45+ immune cells, activated T cells (CD69+) and Tregs (CD25+FOXP3+) among CD4 T cells and CD8 T cells, and PD-1 MFI on CD8 T cells in B78 melanomas. (h) Percentages of NK1.1+CD3– NK cells among CD45+ immune cells in B78 melanomas (n = 10 animals per cohort). (i) Relative expression of Ifnγ, Il4 and their ratios in the bulk B78 tumor samples. (j) Relative expression of Ifnβ1 in the bulk tumor samples (n = 6 animals per cohort). (k) Percentages of CD11b+F4/80+ macrophages among CD45+ immune cells, CD80+CD206– M1 macrophages, and CD206+CD80– M2 macrophages among CD11b+F4/80+ macrophages, and the M1/M2 macrophages ratios in the right TDLNs. (l) Percentages of CD11c+MHCII+ DCs among CD45+ immune cells in the right TDLNs (n = 6 animals per cohort). (m) Percentages of activated (CD69+) T cells among CD4 T cells and CD8 T cells. (n) Percentages of Tem (CD44+CD62L–) and Tcm (CD44+CD62L+) among CD4 T cells and CD8 T cells (n = 10 animals per cohort). G1, control; G2, IL20+6+RT; G3, (PIC+IL2)0+6+RT. Statistical significance was calculated via one-way ANOVA test in c–n. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

We analyzed tumor-infiltrating lymphocytes (TILs) in these B78 melanomas by flow cytometry and immunohistochemistry. We observed enhanced tumor infiltration by CD4 T cells and CD8 T cells after (PIC+IL2)0+6+RT treatment, when compared to untreated control tumors (Figure 4g). IL20+6+RT treatment improved the CD69 expression on both CD4 T cells and CD8 T cells, and this was further increased for CD8 T cells by the addition of PIC (Figure 4g). The effector/memory marker of T cells, CD44, also significantly increased when PIC was added to the IL20+6+RT treatment (Figure S14c). The PD-1 expression on CD8 T cells was significantly upregulated by IL20+6+RT, and this effect was reduced with (PIC+IL2)0+6+RT treatment and both of these regimens showed negligible effects on PD-1 expression in CD4 T cells (Figure 4g and Figure S14d). PIC injection also reduced the tumor infiltration by Tregs (CD25+FOXP3+) when added to IL20+6+RT treatment (Figure 4g). Moreover, we found that both the (PIC+IL2)0+6+RT and IL20+6+RT treatments significantly increased tumor infiltration by NK cells (NK1.1+CD3–), and these NK cells exhibited higher expression for CD69 and CD44 in (PIC+IL2)0+6+RT treatment tumors (Figure 4h and Figure S14e).

We used RT-qPCR to quantify the expression of cytokines in tumor samples following these treatments. We found that IL20+6+RT increased the levels of both Ifnγ and Il4. (PIC+IL2)0+6+RT treatment did not change the upregulation of Ifnγ but decreased the Il4 expression to a level that was similar to that observed in untreated control tumors, resulting in higher ratios of Ifnγ/Il4 expression in the (PIC+IL2)0+6+RT group compared to both the control and IL20+6+RT groups (Figure 4i). Further analysis demonstrated that (PIC+IL2)0+6+RT treatment did not change the expression of Ifnβ1 or Fas but enhanced the expression of the pro-inflammatory cytokine Il1α in the IL20+6+RT-treated tumors (Figure 4j and Figure S15a).

We evaluated memory T cells in the TDLNs from these mice using flow cytometry. As shown in Figure 4n, IL20+6+RT treatment significantly increased the levels of both effector memory T cells (Tem, CD44+CD62L–) and central memory T cells (Tcm, CD44+CD62L+) among CD4 T cells and CD8 T cells, while (PIC+IL2)0+6+RT treatment further enhanced the levels of Tcm, which have been reported to exhibit superior persistence and anti-tumor immunity compared to Tem but showed negligible effects on the generation of Tem.35 Importantly, histological analysis of vital organs from mice receiving these treatment regimens did not reveal any apparent toxic effect of these treatments on normal tissues including liver, kidney, spleen, and lung (Figure S16).

To study the systemic anti-tumor immunity generated by the (PIC+IL2)0+6+RT treatment, we collected blood from B78 melanoma two-tumor bearing mice at day 15 after RT and analyzed the circulating T cells (Figure S17). As shown in Figure 5a,b we found that (PIC+IL2)0+6+RT treatment significantly increased the abundance of CD8 T cells (CD8+CD3+) and exhibited a trend toward increasing CD4 T cell (CD4+CD3+) abundance in the blood. (PIC+IL2)0+6+RT treatment increased the expression of CD44 and NKG2D on the CD8 T cells, while no changes of these markers were observed for CD4 T cells (Figure 5a,b). NKG2D has been reported as one of the markers for circulating tumor-specific T cells, while CD44 is a marker for effector/memory T cells.36,37 These results indicated that (PIC+IL2)0+6+RT treatment can augment the systemic anti-tumor CD8 T cell response, consistent with our observation of effective suppression of distant tumor sites.

Figure 5.

Figure 5

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(PIC+IL2)0+6+RT treatment activated systemic anti-tumor T cell immunity and immunomodulated the distant tumors that were not treated directly. (a) Percentages of CD8+CD3+ cells among CD45+ immune cells, the CD44 MFI on CD8 T cells, and the percentages of NKG2D+ cells among CD8 T cells in the blood. (b) Percentages of CD4+CD3+ cells among CD45+ immune cells, the CD44 MFI on CD4 T cells, and the percentages of NKG2D+ cells among CD4 T cells in the blood (n = 6 animals per cohort). (c) Percentages of CD45+ immune cells among live cells in distant B78 melanomas. (d) Percentages of CD4+CD3+ cells and CD8+CD3+ cells among CD45+ immune cells in distant B78 melanomas. (e) Representative H&E and immunohistochemistry images of distant B78 melanomas (scale bar: 100 μm). (f) Percentages of CD69+ cells among CD8 T cells, and the PD-1 MFI and CD44 MFI on the CD8 T cells in distant B78 melanomas. (g) Percentages of CD80+CD206– M1 macrophages and CD206+CD80– M2 macrophages among CD11b+F4/80+ macrophages, and the M1/M2 macrophage ratios in distant B78 melanomas. (h) Percentages of CD11c+MHCII+ DCs among CD45+ immune cells; the CD80 MFI on CD11c+MHCII+ DCs in distant B78 melanomas (n = 10 animals per cohort). (f) G1, control; G2, IL20+6+RT; G3, (PIC+IL2)0+6+RT. The treatment was performed per Figure 4a and only directly targeted the B78 primary tumors. Statistical significance was calculated via one-way ANOVA test in a–f and g,h. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Since the (PIC+IL2)0+6+RT treatment was able to generate a systemic T cell response and exhibited considerable effects in inhibiting the growth of distant tumors (Figures 3 and 5), we analyzed the distant B78 melanomas to determine how the local treatment of (PIC+IL2)0+6+RT modulated the immune cells infiltration in distant tumors that did not receive direct treatment. As shown in Figure 5c, IL20+6+RT-treated mice exhibited similar CD45+ immune cell infiltration in distant tumors when compared to those tumors in untreated control mice, whereas (PIC+IL2)0+6+RT treatment significantly enhanced this tumor immune cell infiltration. A similar trend was observed for the tumor-infiltrating T cells, as both flow cytometry and immunohistochemistry suggested that the local IL20+6+RT treatment did not change the infiltration of CD4 T cells and CD8 T cells in distant tumors, but the infiltration of these cells significantly increased with (PIC+IL2)0+6+RT treatment (Figure 5d,e).

Given that analysis of the blood indicated a functional effect on circulating CD8 T cells after (PIC+IL2)0+6+RT treatment, we analyzed functional markers on CD8 T cells in the distant tumors. As shown in Figure 5f, (PIC+IL2)0+6+RT treatment significantly reduced the expression of the exhaustion marker, PD-1, on the CD8 T cells, although the treatment did not affect the expression of CD69 or CD44 on CD8 T cells when compared to IL20+6+RT treatment. Intriguingly, we found that (PIC+IL2)0+6+RT treatment downregulated the abundance of CD11b+F4/80+ macrophages and increased the M1/M2 macrophage ratios in distant tumors (Figure 5g and Figure S18a). (PIC+IL2)0+6+RT and IL20+6+RT treatments did not change the infiltration of the CD11c+MHCII+ DCs in distant tumors, while IL20+6+RT treatment increased CD80 expression on these DCs and (PIC+IL2)0+6+RT treatment further increased this (Figure 5h).

Quantification of gene expression from the bulk tumor by RT-qPCR (Figure S15b) demonstrated increased expression ratios of Ifnγ/Il4 and increased levels of pro-inflammatory cytokine Il1α in distant tumors from (PIC+IL2)0+6+RT-treated mice compared to those from untreated control mice, and this treatment showed negligible effects on the expression of Ifnβ1 and Fas at these distant tumor sites. To test whether this immunomodulation of distant tumors resulted from the biodistribution of PIC in distant tumors, we injected the Cy5-labeled PIC to the primary (right) tumor on B78 melanoma two-tumor bearing mice and studied its biodistribution in tumors and TDLNs. At day 15 after the first injection, we observed strong fluorescence signals of Cy5-PIC in the primary tumors and primary TDLNs, but not the distant tumor and distant TDLNs (Figure S19). Flow cytometry analysis of distant TDLNs also revealed no effects induced by contralateral PIC treatment (Figure S18b). These results indicated that the immunomodulation of distant tumors induced by (PIC+IL2)0+6+RT treatment may be mainly attributed to the generation of systemic anti-tumor immunity.

(PIC+IL2)0+6+RT Treatment in the Murine MOC2 Head and Neck Squamous Cell Carcinoma Model

One of the features of an in situ vaccine approach is that identification of tumor neoantigens is not required to achieve therapeutic efficacy and therefore the efficacy of such a regimen is not limited to a given disease type or by the expression of a given tumor antigen.38 We tested the (PIC+IL2)0+6+RT treatment on another immunologically “cold” and aggressive MOC2 head and neck squamous cell carcinoma (Figure 6a). Although the IL20+6+RT treatment delayed the MOC2 tumor growth, (PIC+IL2)0+6+RT-treated tumors exhibited much slower tumor growth, resulting in improved mouse survival compared to those treated with the doublet combinations of IL20+6+RT, PIC0+6+RT, or (PIC+IL2)0+6 (Figure 6b–d). This confirmed in a distinct tumor model that PIC played an important role in generating an effective anti-tumor immune response when delivered as a component of the (PIC+IL2)0+6+RT regimen.

Figure 6.

Figure 6

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(PIC+IL2)0+6+RT treatment delayed the MOC2 head and neck tumor growth and increased mice survival. (a) Scheme for the studies. The treatment was started when the tumor size was about 100 mm3. (b) Average tumor growth curves and (c) mice survival after the treatments indicated. (d) Individual tumor growth curves in (b) (n = 6 animals per cohort). Statistical significance was calculated via linear mixed effects modeling in b, and log-rank test in c. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

Cancer vaccines are promising methods for the treatment of solid tumors, yet traditional cancer vaccines are limited by the requirement for the identification and specific targeting of a known tumor-associated antigen (TAA).3,39 An in situ vaccine can bypass this limitation and enable cross presentation of TAAs in situ, without requiring identification of these, in order to activate an adaptive immune response.4,19 RT has been demonstrated to stimulate an in situ vaccine effect; however, this has not yet been potent enough to stimulate a clinically meaningful improvement in systemic anti-tumor immunity when added to immune checkpoint blockade.12,40 This may reflect inadequate priming of an adaptive T cell immunity and the stimulation of immunosuppressive mechanisms by RT.6,41 We introduced a multifunctional nanoparticle (PIC) to address these limitations. In the previous work, we have demonstrated that PIC sensitized tumor cells to RT, promoted antigen uptake by and activation of APCs in the injected tumor, and reduced immunosuppression in irradiated tumors by promoting M1 polarization of tumor-associated macrophages. Collectively, we previously reported that these mechanistic effects resulted in improvement of the in situ vaccine effect of RT.16 However, while PIC improved the in situ vaccine effect of RT when combined with immune checkpoint blockade, it was not sufficient to stimulate a de novo anti-tumor immune response in the absence of systemic immune checkpoint inhibition. We hypothesized that these findings may reflect a limitation of the PIC. Namely, while PIC enabled more effective adaptive immune priming and blunted potential suppressive effects of RT on the tumor microenvironment, it did not incorporate a stimulus to enhance clonal expansion of the stimulated immune response. To redress this, here, we combined PIC with intratumoral IL2 and RT.

We observed that the triple combination of PIC+IL2+RT was effective in stimulating anti-tumor immune response in the immunologically “cold” B78 tumor models, even in the absence of systemic immune checkpoint inhibition. In evaluating the mechanisms of cooperative therapeutic interaction between these locally administered therapies, we observed that PIC prevented specific detrimental effects caused by RT+IL2 and strengthened select favorable effects, resulting in a potent systemic anti-tumor immunity that improved tumor eradication and mouse survival (Figure 7). More specifically, we found that PIC coordinated with IL2 to promote Th1 polarization of the adaptive immune response and M1 polarization of macrophages (Figure 1). These effects of PIC relieved local immunosuppression and improved T cell activation following IL2+RT, facilitating the generation of potent adaptive anti-tumor immunity at tumors that did not directly receive any treatment. This may be related to not only the immunomodulation of the irradiated TME by PIC but also the enhanced antigen presentation by PIC, as we demonstrated previously.16

Figure 7.

Figure 7

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Proposed mechanisms of action for PIC+IL2+RT in tumor treatment. RT: RT can induce the immunogenic cell death of cancer cells and improve the susceptibility of cancer cells to immune-mediated killing while also increasing tumor-infiltrating by immunosuppressive Tregs and M2 macrophages. IL2 injection: when IL2 is injected into an irradiated tumor it can promote the clonal expansion and activation of effector T cells but also does this for Tregs, which can often exhibit increased infiltration in the irradiated TME. PIC injection: when PIC is injected to the irradiated tumors, it can relieve immunosuppression by decreasing tumor infiltration of M2 macrophages and Tregs, but its ability to prime and clonally expand T cells is limited. PIC+IL2 injection: when PIC+IL2 is injected into the irradiated tumors, PIC can dampen the immunosuppression caused by RT by decreasing tumor infiltration of Tregs and M2 macrophages; PIC combined with IL2 promotes the proliferation and function of effector T cells and primes systemic anti-tumor T cell immunity to eradicate distant tumors. This figure was created with Biorender.com.

By optimizing the PIC+IL2+RT treatment regimen, we found that injection of PIC+IL2 at days 0 and 6 relative to RT exhibited a similar induction of systemic anti-tumor immune response when compared with a more frequent injection schedule in B78 melanoma model (Figure 3). Due to the translational challenges posed by a need for repeated intratumoral injections, we then focused on the (PIC+IL2)0+6+RT treatment regimen and tested its efficacy in the MOC2 head and neck tumor model. Specifically, (PIC+IL2)0+6+RT suppressed the growth of MOC2 tumors (Figures 3 and 6).

Although PIC did not exhibit any direct effects on Tregs in vitro, in vivo studies demonstrated that PIC significantly lowered the abundance of Tregs in tumor tissues and downregulated PD-1 expression on CD8 T cells (Figure 4). Analyses of distant tumors that were not directly treated with RT or intratumoral injection revealed that PIC facilitated immunoregulation of these distant TMEs, leading to increased levels of tumor-infiltrating lymphocytes, higher M1/M2 macrophages ratios, and stimulation of a Th1 polarized adaptive immune response (Figure 5). This immunomodulation of distant untreated tumors was not directly stimulated by PIC, which exhibited biodistribution limited to the injected tumor and its TDLN, but may be related to the circulation of tumor-specific T cells after (PIC+IL2)0+6+RT treatment (Figure S19 and Figure 5). These results indicate that PIC is effective in both direct local tumor immunomodulation and indirect systemic modulation of tumors secondary to the generation of systemic T cell immunity.

In future studies, it will be important to further optimize the timing and dosing of PIC+IL2 injection to maximally improve systemic anti-tumor efficacy. In this work, we focused on studying the effects of PIC+IL2 on the hypofractionated radiation therapy using a single radiation dose (12 Gy) that we previously observed to be effective for stimulating IFN-I response and an in situ vaccine effect in the B78 model.7,12 It will be valuable in future studies to evaluate the effects of PIC+IL2 in a broader range of RT doses and in combination with fractionated RT. It will also be valuable to more precisely evaluate the tumor retention of PIC and IL2 following intratumoral injection. Although we demonstrated PIC retained in tumors for days after intratumoral injection (Figure S19), the short half-life time of the intratumorally injected IL2 may limit the efficacy of this combination or pose a need for repeated injections. Prior studies show that intratumoral injection of IL2 or IL2 derivatives can extend the half-life of these agents in the TME, enabling greater efficacy and reduced toxicity.24,42−44 In future studies it will be valuable to evaluate and optimize the retention of intratumoral IL2 when administered in the context of PIC coinjection. Therefore, it may be important to develop strategies to prolong the bioavailability of IL2 in tumors and to assess how the PIC nanoparticle affects the pharmacokinetics of intratumorally injected IL2. Moreover, it may be valuable in future studies to modify the PIC nanoparticle design and to investigate strategies that could enable safe and effective delivery of PIC and IL2 to the TME following intravenous rather than intratumoral injection. Despite these limitations, this work provides compelling evidence that the intratumoral injection of IL2 and a multifunctional PIC nanoparticle could cooperatively augment the in situ vaccine effect of RT and activate a systemic anti-tumor immune response. The capacity of this in situ vaccine to enable therapeutic efficacy across diverse tumor types confers the potential for considerable translational impact to this cancer treatment strategy.

Conclusion

Overall, our results demonstrated that PIC coordinated with IL2 to augment the in situ vaccine effects of RT, and PIC+IL2+RT combination therapy generated systemic anti-tumor T cell immunity that enabled therapeutic efficacy at distant untreated tumor sites following this combination of treatments delivered locally to a single tumor site.

Methods

Materials

Poly-l-lysine hydrobromide (PLL, molecular weight: 30–70 kDa) and ammonium hydroxide solution were purchased from Sigma-Aldrich. Ferric chloride hexahydrate (FeCl3·6H2O) and ferrous sulfate heptahydrate (FeSO4·7H2O) were purchased from Fisher Scientific. Citric acid was purchased from Acros Organics. CpG oligodeoxynucleotides 1826 (sequence: T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T) was customized from Integrated DNA Technologies (IDT). IL2 was obtained from National Cancer Institute (NCI). α-CTLA-4 (anti-CTLA-4, IgG2c, clone 9D9) was produced and purified by Neoclone. The information on Taqman probes used for RT-qPCR is listed in Table S2. The information on antibodies used for flow cytometry and immunohistochemistry is listed in Table S3.

Preparation of PIC

ION was synthesized via the same method as previously reported.16 PIC was prepared through the complexation of PLL, ION, and CpG according to the published method with minor modifications. Briefly, ION (7.2 mg/mL, 400 μL) was mixed with CpG (3 mg/mL, 200 μL), followed by the addition of PLL (10 mg/mL, 210 μL) and deionized water (190 μL). The mixture was vortexed for 15–20 s and incubated at room temperature for 20 min. PIC was diluted to a concentration of 0.1 mg/mL before being tested by a dynamic light scattering (DLS) spectrometer (Malvern Zetasizer Nano ZS).

Cell Culture and Animals

B78 (B78-D14, GD2+) melanoma cells were originated from B16 cells and obtained from Ralph Reisfeld (Scripps Research Institute). B16 melanoma cells were obtained from Memorial Sloan Kettering Cancer Center. MOC2 head and neck cancer cells were generously provided by Dr. Ravindra Uppaluri (Dana-Farber Cancer Institute). B78 cells, B16 cells, and MOC2 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium, which was supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. All animal studies in this research were approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison (protocol: M005670). All mice (C57BL/6, 7–8 weeks, male and female) were purchased from Taconic. To establish tumor-bearing mice, mice were intradermally injected with tumor cells (B78 melanoma model, 2 × 106 cells on the right flanks; B78 melanoma two-tumor model, 2 × 106 cells on the right flanks, and 1 week later 2 × 106 cells engrafted on left flanks; MOC2 head and neck tumor model, 2 × 106 cells on the right flanks). Once the tumor volumes (primary tumor volumes for the two-tumor bearing mice) were about 100 mm3, mice were randomized for the initiation of treatment. For the two-tumor bearing mice, the treatment was performed only on the right flank (primary) tumors. Tumors were measured twice weekly for at least 40 days unless mice died or were euthanized because of large tumor size, tumor necrosis, or evidence of pain or distress. Tumor diameters were measured with a Vernier caliper, and tumor volume was calculated through the equation: tumor volume = longer diameter × shorter diameter2 × 0.5.

In Vitro Coculture Studies

B78 cells were seeded in 6-well plates with 0.25 × 106/well and were radiated with an RS225 Cell Irradiator (Xstrahl) at a dose of 12Gy after culturing for 24 h. Fresh culture media was exchanged 1 h after the radiation. After culturing for another 4 days, splenocytes were collected from naïve C57BL/6 mice and added to the B78 cells with 4 × 106/well, and PIC and IL2 were added to the cocultures at concentrations of 0.5 μg/mL and 67 IU/mL, respectively. Cultures of splenocytes only were used as the control group. Twenty-four hours later, the splenocytes were collected for flow cytometry and RT-qPCR analyses. To study the direct impact of PIC+IL2 to the splenocytes, splenocytes were collected from the naïve C57BL/6 mice and seeded in 6-well plates with 4 × 106/well. PIC and IL2 were added to the cells at concentrations of 0.5 μg/mL and 67 IU/mL, respectively. Twenty-four hours later, the splenocytes were collected for flow cytometry and RT-qPCR analyses.

For flow cytometry analyses, the cells were stained with GhostRed 780, followed by incubating with anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained with anti-CD45 PE-Cy7, anti-CD3 BV510, anti-CD4 FITC, anti-CD8a Alexa 700, anti-NK1.1 PE-Cy5, anti-CD69 BV421, anti-CD44 BV605, and anti-CD25 APC. After the cells were fixed and permeabilized, they were stained with anti-FOXP3 PE. Flow cytometry was performed on an Attune cytometer (ThermoFisher), and UltraComp Beads eBeads (Invitrogen) were used for compensation. The flow cytometry data was analyzed using FlowJo 10.6.2.

For RT-qPCR analyses, the cells were washed with cold PBS, and then, 1 mL Trizol Reagent (Ambion) was added. RNA was isolated using an RNeasy Mini Kit (QIAGEN, Cat: 74106) according to the manufacturer’s instructions. cDNA was synthesized using a QuantiTect Reverse Transcription Kit (QIAGEN, Cat: 205314) according to the manufacturer’s standard protocol. Quantitative polymerase chain reaction (RT-qPCR) was performed using Taqman Fast Advanced Mix and Predesigned Taqman gene expression assays for Ifnγ, Il4, Fas, Nos2, Arg1, and Il1α. Thermal cycling conditions (QuantStudio 6, Applied Biosystems) included the UNG incubation stage at 50 °C for 2 min, followed by AmpliTaq Fast DNA polymerase activation stage at 95 °C for 2 min and 40 cycles of each PCR step (denaturation) 95 °C for 1 s and (annealing/extension) 60 °C for 20 s. For data analyses, Ct values were transferred to an Excel file and fold change was determined using the ΔΔCt method. Hprt was used as the endogenous control. The relative expression of genes was used to calculate the expression ratios for Ifnγ/Il4 and Nos2/Arg1.

Treatment

For PIC+RT, IL2+RT, and PIC+IL2+RT groups, PIC (1.4 mg/mL, 100 μL) was intratumorally injected on days 0, 3, 6, and 9. Radiation with a dose of 12Gy was delivered to the tumors on day 1 using an XPad 320 cabinet irradiator (Precision X-ray, Inc.). IL2 (75000 IU, 100 μL) was intratumorally injected daily on days 6–10. For PIC0+6+RT, IL20+6+RT, and (PIC+IL2)0+6+RT, PIC (5.6 mg/mL, 100 μL) was intratumorally injected on days 0 and 6. Radiation with a dose of 12Gy was delivered to the tumors on day 1 using an XPad 320 cabinet irradiator (Precision X-ray, Inc.). IL2 (75000 IU, 30 μL) was intratumorally injected daily on days 0 and 6. For (PIC+IL2)0+RT, PIC (5.6 mg/mL, 100 μL) was intratumorally injected on day 0. Radiation with a dose of 12Gy was delivered to the tumors on day 1 using an XRad 320 cabinet irradiator (Precision X-ray, Inc.). IL2 (75000 IU, 30 μL) was intratumorally injected on day 0.

Flow Cytometry Analysis of Tumors

Tumor tissues were collected at day 15 after the initiation of treatment from the B78 two-tumor bearing mice. The samples were enzymatically dissociated with DNase and collagenase on a Gentle MACS Octodissociator (Miltenyi Biotec) and then filtered through a 70 μm cell strainer and red blood cells were lysed using RBC lysis buffer. The single cell suspensions were divided for innate immune cell staining and adaptive immune cells staining separately. For innate immune cell staining, the cells were stained with GhostRed 780, followed by incubating with anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained with anti-CD45 PE-Cy7, anti-CD11b BV711, anti-F4/80 PE-Dazzle 594, anti-CD80 PE, anti-CD206 APC, anti-CD11c FITC, and anti-MHCII BV510. For adaptive immune cell staining, the cells were stained with GhostRed 780, followed by incubating with anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained with anti-CD45 PE-Cy7, anti-CD3 BV510, anti-CD4 FITC, anti-CD8a Alexa 700, anti-NK1.1 PE-Cy5, anti-CD69 PerCP-Cy5.5, and anti-CD25 APC. After the cells were fixed and permeabilized, they were stained with anti-FOXP3 PE. Flow cytometry was performed on an Attune Cytometer (ThermoFisher), and UltraComp Beads eBeads (Invitrogen) were used for compensation. The flow cytometry data was analyzed using FlowJo 10.6.2. The M1/M2 macrophage ratios were calculated with the counts of the cells.

Flow Cytometry Analysis of Tumor-Draining Lymph Nodes (TDLNs)

TDLNs were collected at day 15 after the initiation of treatment from the B78 two-tumor bearing mice. The samples were enzymatically dissociated with DNase and collagenase on a Gentle MACS Octodissociator (Miltenyi Biotec) and then filtered through a 70 μm cell strainer. The red blood cells were lysed using RBC lysis buffer. For innate immune cell staining, the cells were stained with GhostRed 780, followed by incubating with anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained with anti-CD45 PE-Cy7, anti-CD11b BV711, anti-F4/80 PE-Dazzle 594, anti-CD80 PE, anti-CD206 APC, anti-CD11c PerCP-Cy5.5, and anti-MHCII BV510. For adaptive immune cell staining, the cells were stained with GhostRed 780, followed by incubating with anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained with anti-CD45 PE-Cy7, anti-CD3 BV510, anti-CD4 FITC, anti-CD8a Alexa 700, anti-CD69 BV421, anti-CD44 PE, and anti-CD62L PE-Cy5. Flow cytometry was performed on an Attune cytometer (ThermoFisher), and UltraComp Beads eBeads (Invitrogen) were used for compensation. The flow cytometry data was analyzed using FlowJo 10.6.2.

Flow Cytometry Analysis of Blood

Blood was collected at day 15 after the initiation of treatment from the B78 two-tumor bearing mice. The red blood cells were lysed using RBC lysis buffer. The cells were stained with GhostRed 780, followed by incubating with anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained with anti-CD45 PE-Cy7, anti-CD3 BV510, anti-CD4 FITC, anti-CD8a Alexa 700, anti-PD-1 BV421, anti-CD44 PE, and anti-NKG2D PE-Dazzle 594. Flow cytometry was performed on an Attune cytometer (ThermoFisher), and UltraComp Beads eBeads (Invitrogen) were used for compensation. The flow cytometry data was analyzed using FlowJo 10.6.2.

qPCR Analysis of Tumors

Tumor tissues were collected at day 15 after the initiation of treatment from the B78 two-tumor bearing mice. Tumor tissues were homogenized using a bead mill homogenizer (Bead Ruptor Elite, Omni International). RNA was isolated using an RNeasy Mini Kit (QIAGEN, Cat: 74106) according to the manufacturer’s instructions. cDNA was synthesized using a QuantiTect Reverse Transcription Kit (QIAGEN, Cat: 205314) according to the standard protocol. Quantitative polymerase chain reaction (RT-qPCR) was performed using Taqman Fast Advanced Master Mix and predesigned Taqman gene expression assays for Ifnγ, Il4, Ifnβ, Il1α, and Fas. Thermal cycling conditions and data analysis were performed the same as indicated above. Hprt was used as the endogenous control.

Tumor Rechallenge

At day 60 after the initiation of treatment, tumor-free mice obtained from the PIC+IL2+RT treatment group in B78 melanoma model were rechallenged by an intradermal engraftment of 2 × 106 B78 melanoma cells in the left flank. A group of age-matched naïve mice was also engrafted with 2 × 106 B78 melanoma cells for tumor growth as control. Tumor growth was monitored for another 60 days.

Evaluation of Immune Memory

B16 cells were seeded in 12-well plates with 2 × 104/well and were irradiated with an RS225 Cell Irradiator (Xstrahl) at a dose of 12Gy after culturing for 24 h. Fresh culture media was exchanged 1 h after the radiation. After culturing for another 4 days, splenocytes were collected from the tumor-free mice rendered by the PIC+IL2+RT treatment and added to the B16 cells with 2 × 106/well. Twenty-four hours later, the splenocytes were collected for flow cytometry and RT-qPCR analyses.

For flow cytometry analyses, the cells were stained with GhostRed 780, followed by incubating with anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained with anti-CD45 PE-Cy7, anti-CD3 BV510, anti-CD4 FITC, anti-CD8a Alexa 700, anti-CD69 PE, and anti-CD44 BV605. Flow cytometry was performed on an Attune cytometer (ThermoFisher), and UltraComp Beads eBeads (Invitrogen) were used for compensation. The flow cytometry data was analyzed using FlowJo 10.6.2.

For RT-qPCR analyses, the cells were washed with cold PBS, and then, 1 mL of Trizol Reagent (Ambion) was added. RNA was isolated using an RNeasy Mini Kit (QIAGEN, Cat: 74106) according to the manufacturer’s instructions. cDNA was synthesized using a QuantiTect Reverse Transcription Kit (QIAGEN, Cat: 205314) according to the manufacturer’s standard protocol. Quantitative polymerase chain reaction (RT-qPCR) was performed using Taqman Fast Advanced Mix and predesigned Taqman gene expression assays for Ifnγ and Il2. Thermal cycling conditions and data analysis were performed the same as indicated above. Hprt was used as the endogenous control.

Immunohistochemistry

Tumor tissues were collected at day 15 after the initiation of treatment from the B78 two-tumor bearing mice. The tumor samples were embedded in paraffin, and the blocks were sectioned into 50 μm slices. The antibodies used for IHC included: CD4 Monoclonal Antibody (Invitrogen, Cat: 14-9766-37. Dilution: 1:500), CD8a Monoclonal Antibody (Invitrogen, Cat: 14-0808-82. Dilution: 1:1000). Standard IHC methods were performed as previously described.9,44 The samples stained without the primary antibody served as negative controls.

Biodistribution

Cy5 labeled PLL (Cy5-PLL) was synthesized as previously described.16 Cy5 labeled PIC (Cy5-PIC) was prepared through the complexation between Cy5-PLL, ION, and CpG as mentioned above. Cy5-PIC (200 μL, 560 μg) was intratumorally injected into the right tumors of B78 melanoma two-tumor bearing mice when the volume of right tumors was about 200 mm3 at days 0 and 6. At day 15, the mice were euthanized, and tumors and tumor-draining lymph nodes were collected for imaging.

Statistics

Prism 8 (GraphPad Software) and R (v 4.0.5) were used for all statistical analyses. One-way ANOVA was used for the analysis of gene expression and flow cytometry data both in vitro and in vivo. For tumor growth analysis, a linear mixed model after log transformation of tumor volume was fitted on treatment and day with the interaction between treatment and day, all three as fixed effects. A complete case analysis was used, which discards only the missing measurements, to handle the missing data. A log-rank test was conducted to compare the survival curves. All data presented are reported as mean ± SD unless otherwise noted. For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.

Acknowledgments

This work was supported by the UW2020 award and the Draper technology innovation fund from the University of Wisconsin-Madison and Wisconsin Alumni Research Foundation and in part by public health service grants P30CA014520, DP5OD024576, U01CA233102, and P01CA250972 from the National Cancer Institute. C.K. was supported by the UW-Madison Radiology MD-PhD Graduate Student Fellowship and NIH awards F30CA268780 and T32GM140935.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.3c00418.

  • Figures of gating strategies for all the flow cytometry studies, percents of Tregs or PD-1+ cells among CD4 T cells and CD8 T cells in the in vitro coculture studies, RT-qPCR and flow cytometry results for the splenocytes after treated with RT, IL2, or RT+IL2 in vitro, PIC injection schedule studies on B78 melanoma bearing mice, RT-qPCR and flow cytometry results for the B78 melanoma two-tumor bearing mice, H&E images, biodistribution of Cy5-labeled PIC, tables of p values for the B78 melanoma two-tumor bearing mice studies, information of Taqman genes for RT-qPCR, information for antibodies used for flow cytometry and immunohistochemistry (PDF)

Author Contributions

∇ Y.Z. and M.M.R. contributed equally. Conceptualization: Y.Z., S.G., and Z.S.M. Methodology: Y.Z. and M.M.R. Investigation: Y.Z., M.M.R., P.C., R.S., M.Z., J.J., C.K., and X.W. Visualization: Y.Z. and M.M.R. Statistical analysis: Y.Z., M.M.R., T.H., and K.K. Funding acquisition: S.G. and Z.S.M. Supervision: S.G. and Z.S.M. Writing-original draft: Y.Z. Writing-review and editing: Y.Z., M.M.R., K.K., S.G., and Z.S.M.

The authors declare the following competing financial interest(s): S.G., Z.S.M., Y.Z., and R.S. are inventors on a filed patent (title: nanoparticles for potentiating effects of radiation therapy on anticancer immunotherapy) managed by the Wisconsin Alumni Research Foundation relating to this work. Z.S.M. is a member of the scientific advisory board for Archeus Technologies, Northstar Medical Technologies and Seneca Therapeutics and received equity options for these companies. Z.S.M. is an inventor on patents or filed patents managed by the Wisconsin Alumni Research Foundation relating to the interaction of targeted radionuclide therapies and immunotherapies, nanoparticles designed to augment the anti-tumor immune response following radiation therapy, and the development of a brachytherapy catheter capable of delivering intra-tumor injectables. The remaining authors declare no competing interests.

Supplementary Material

nn3c00418_si_001.pdf (2.1MB, pdf)

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Associated Data

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Supplementary Materials

nn3c00418_si_001.pdf (2.1MB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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