# Preclinical Study of Plasmodium Immunotherapy Combined with Radiotherapy for Solid Tumors

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## Abstract

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^{+}T cells and downregulating the myeloid-derived suppressor cells (MDSCs), and was thus more effective in the treatment of cancer. The clinical transformation of PI combined with RT in the treatment of solid tumors, especially glioma, is worthy of expectation.

## 1. Introduction

^{+}leukocytes, including innate immune cells and effector T cells) into the tumor tissues [15]. In this way, the activated innate immune cells kill some of the tumor cells, and then the dying tumor cells release tumor antigens or tumor-associated antigens that activate the effector T cells [15]. Through some undefined mechanisms, PI down-regulates the expression level of PD-1 on CD8

^{+}T cells within tumor tissues [16] so that these effector T cells can effectively kill tumor cells, whilst the expression level of the PD-1 on CD8

^{+}T cells in peripheral blood is up-regulated [17]. At the same time, PI can significantly down-regulate the number and function of immune suppressor cells, such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) in tumor tissues, and therefore systematically remove the immunosuppressive tumor microenvironment [13,16,18]. PI also inhibits tumor angiogenesis through multiple pathways and targets [18,19,20], as well as inhibiting the epithelial-mesenchymal transition (EMT) of tumor cells by suppressing the CCR10-mediated PI3K/Akt/GSK-3β/Snail signaling pathway. PI therefore inhibits the growth and metastasis of solid tumors in mice, prevents tumor recurrence after surgical resection [21], and significantly prolongs the survival time of tumor-bearing mice [13]. Based on these studies, clinical trials of PI for advanced solid tumors have been approved and are ongoing in China (NCT02786589, NCT03474822 and NCT03375983) [13]. However, PI also has an obvious insufficiency; that is, the specific killing effect of tumor cells is relatively weak and needs to be combined with existing therapies to further enhance its efficacy.

## 2. Materials and Methods

#### 2.1. Mice

#### 2.2. Cell Culture and Parasites

^{TM}, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS, Cat# 10270, Invitrogen Gibco

^{TM}) and 1% penicillin-streptomycin (Cat# 15140122, ThermoFisher, Waltham, MA, USA). LLC cells were maintained in Roswell Park Memorial Institute media 1640 (RPMI-1640, Cat# SH30809.01B, Hyclone), supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were grown in a humidified atmosphere of 5% CO

_{2}at 37 °C.

#### 2.3. Tumor Models and Animal Grouping

^{5}LLC or 2 × 10

^{6}GL261 cells were subcutaneously (s.c.) inoculated into the right flank of C57BL/6J mice. The inoculation volume was 100 μL for each mouse.

^{5}GL261-Luc cells in a volume of 1 μL using a 5 μL Hamilton micro-syringe (26-gauge needle) were stereotactically injected into a 1 mm diameter drill hole in the left striatum that was defined by the following coordinates: 1.8 mm lateral to bregma, 1 mm posterior to the coronal suture, 3.4 mm deep to the cortical surface. The tumor burden was monitored by bioluminescent imaging after tumor cell inoculation.

^{2})/2, where V was the tumor volume, a was the long diameter, and b was the short diameter.

^{3}), and so on.

#### 2.4. Treatment Regimens

^{5}Plasmodium-infected erythrocytes or the same number of uninfected erythrocytes. The mice that were inoculated with GL261 (or GL261-Luc) cells or LLC cells and injected with non-infected erythrocytes were used as the control group.

#### 2.5. Luciferase In Vivo Imaging

#### 2.6. Flank Tumor Rechallenge Experiments

^{6}syngeneic GL261 cells in the right flank and the inoculation of 5 × 10

^{5}non-syngeneic LLC cells in the left flank. Both cells were suspended in a sterile 0.9% sodium chloride solution. The inoculation volume was 100 μL for each mouse. Naïve mice were also injected with the same number of GL261 cells and LLC cells as the controls. Tumor volume was measured every 3 days.

#### 2.7. Western Blotting

^{TM}, Nanjing, China), and then transferred to PVDF membranes (Cat# ISEQ00010, Millipore, Burlington, MA, USA). Following incubation with an appropriate secondary antibody, bands were detected with ECL reagents (Cat# WBULS0500, Millipore) and visualized using the Chemiluminescent Image Analyzer (Tanon 5200, Tanon Science and Technology, Shanghai, China). The densitometry and semi-quantitative of the bands’ signals were quantified using ImageJ 1.38 (NIH, https://imagej.nih.gov/ij/, accessed on 26 October 2022). Results were presented as the ratio of two target proteins’ densitometry. The following antibodies were used in this section: anti-caspase 3 (Cat# ab184787, Abcam, Cambridge, UK); anti-GAPDH antibody (Cat# ab8245, Abcam); HRP-linked goat anti-rabbit IgG H&L antibody (cat# ab97051, Abcam); HRP-linked anti-mouse IgG H&L antibody (Cat# 7076, CST, Louisville, KY, USA).

#### 2.8. Single Cell Sample Preparation and Isolation

^{TM}tube (Cat# 340334, BD Biosciences, Franklin Lakes, NJ, USA) for measuring the absolute number of leukocytes, according to the manufacturer’s instructions. Ammonium chloride potassium (ACK) lysis buffer was used to lyse the erythrocytes. The cells were washed and resuspended with phosphate buffered saline (PBS), following flow cytometry staining.

^{TM}, Glendale, AZ, USA), into 50 mL conical tubes (Jet Biofil, Guangzhou, China). ACK lysis buffer was used to lyse erythrocytes. Trypan blue exclusion was then used to count the cells, using a Countstar FL automatic cell counter (Ruiyu, Shanghai, China), according to the manufacturer’s instructions. The cells were washed and resuspended with PBS following flow cytometry staining.

_{2}, for 30~60 min, at 37 °C. ACK lysis buffer was used to lyse erythrocytes. Subsequently, all of the cells were filtered through a 70-µm nylon cell strainer (Cat# 352350, Corning Falcon

^{TM}), washed with 10 mL RPMI 1640 containing 2% FBS, and collected by centrifugation (300× g, 5 min). Pelleted cells were added with Precision Count Beads™ (Cat# 424902, Biolegend, San Diego, CA, USA) to obtain the absolute number of cells, and resuspended in PBS following flow cytometry staining.

#### 2.9. Flow Cytometry Analysis

^{6}cells, in 100 µL cell staining buffer, for 10 min in the dark, on ice. The cells were washed with 2 mL cell staining buffer. For surface markers staining, the cells were incubated with the appropriate dilution of fluorescence-conjugated surface antibodies (anti-CD45, Cat# 103147, Biolegend; anti-CD3, Cat# 100204, Biolegend; anti-CD4, Cat# 100538, Biolegend; anti-CD8a, Cat# 100752, Biolegend; anti-CD11b, Cat# 101261, Biolegend; anti-CD25, Cat# 102051, Biolegend; anti-Ly6C, Cat# 128032, Biolegend, and anti-Ly6G, Cat# 127641, Biolegend), for 30 min in the dark, on ice. For intracellular and nuclear staining, a FoxP3/Transcription factor staining buffer set (Cat# 00-5523, Invitrogen eBioscience

^{TM}, Waltham, MA, USA) was used for fixation and permeabilization procedures, according to the manufacturer’s instructions. Following the permeabilization procedures, the cells were stained with an intracellular antibody (anti-perforin, Cat# 154303, Biolegend) and nuclear antibody (anti-FoxP3, Cat# 126404, Biolegend), in 1× permeabilization buffer, for 30 min in the dark, on ice. The cells were washed, twice, using 1× permeabilization buffer. The cells were resuspended in staining buffer. The cells were filtered using a 200 mesh-filter before analysis by Cytek Aurora spectrum flow cytometry (Cytek Bioscience, Fremont, CA, USA). The data were analyzed using FlowJo software V10.6 (Tree Star Inc., https://www.flowjo.com/, accessed on 26 October 2022).

#### 2.10. Immunohistochemical Analysis

#### 2.11. Statistical Analysis

## 3. Results

#### 3.1. Antitumor Effect of the Combination of Plasmodium Immunotherapy (PI) and Radiotherapy (RT) in Orthotopic Glioma Model

#### 3.2. Antitumor Effect of the Combination of PI and RT in Subcutaneous Glioma Model

#### 3.3. Antitumor Effect of the Combination of PI and RT in Lung Cancer Model

#### 3.4. The Effect of PI and RT Combination on Lung Cancer Cell Proliferation and Apoptosis

#### 3.5. The Effect of PI and RT Combination on the Immune Profiles in Tumor Microenvironment

^{+}T cells infiltrating into the tumor in orthotopic glioma model was observed by immunohistochemical analysis, using an anti-CD3 antibody. The PI group, RT group and PI+RT group all had higher CD3

^{+}T cells infiltrating into the brain tumors compared with the control group (Figure 5A,B). The PI+RT group had the highest CD3

^{+}T cells infiltration (Figure 5A,B). These results suggested that both PI and RT could transform a cold tumor into a hot tumor, and PI combined with RT had a more significant effect on such a transformation.

^{+}T cells calculation, as mentioned above, the LLC model was selected to further investigate the immune profiles in tumor microenvironment. The tumor tissues were harvested on day 19 after tumor inoculation and the single cells were separated in order to analyze the immunophenotypes. The gating strategy of flow cytometry is presented in Figure S5.

^{+}T cells, play a key role in anticancer immunity [41]. As shown in Figure S6A, a higher level (absolute number) of CD45

^{+}leukocytes in the PI group (p = 0.04), and a lower level of these cells in the RT group (p = 0.04), were observed in comparison to the control group; there was no difference in the level of leukocytes between the PI+RT group and control group. The PI group had a higher CD3

^{+}T/CD45

^{+}cell proportion (p = 0.0009) and a higher CD3

^{+}T cell absolute number (p = 0.0009); similarly, the RT group also had a higher CD3

^{+}T/CD45

^{+}cell proportion (p = 0.0012) and a higher CD3

^{+}T cell absolute number (p = 0.04) compared to the control group (Figure S6B,C). The PI+RT group had a higher CD3

^{+}T/CD45

^{+}cell proportion (Figure S6B) and a higher CD3

^{+}T cell absolute number (Figure S6C) than the PI group and the RT group (all p < 0.05).

^{+}T/CD45

^{+}cell proportion (Figure 6A), CD8

^{+}T cell absolute number (Figure 6B), CD4

^{+}T/CD45

^{+}cell proportion (Figure S6D), and CD4

^{+}T cell absolute number (Figure S6E) in the PI+RT group was the highest among all of the groups (all p < 0.05). The data concerning the cell number, shown in Figure 6B, Figure S6C,E, suggests that PI was better than RT for promoting the T cells (both CD8

^{+}T and CD4

^{+}T) infiltration into tumors, and the combination of both was better than each single therapy. The PI group (p = 0.04) had a lower CD4

^{+}T/CD8

^{+}T cell ratio than the control group, and the RT group (p = 0.02) had a higher CD4

^{+}T/CD8

^{+}T cell ratio than the control group; there were no significant differences in this ratio between the PI+RT group and control group (Figure S6F).

^{+}T cells that can induce tumor cells apoptosis or directly kill the tumor cells [42]. Our previous study showed that Plasmodium infection upregulates the expression levels of perforin in CD8

^{+}T cells within lung cancer tissues [16]. As shown in Figure 6C,D, the PI+RT group had the highest perforin

^{+}CD8

^{+}T/CD45

^{+}cell proportion and perforin

^{+}CD8

^{+}T cell absolute number within the tumors, among all of the groups (all p < 0.05). Accordingly, the PI group and RT group had a higher perforin

^{+}CD8

^{+}T/CD45

^{+}cell proportion (Figure 6C), and a higher perforin

^{+}CD8

^{+}T cell absolute number (Figure 6D) (PI was more significant than RT (both p < 0.05)), compared to the control group (all p < 0.05). These results suggested that PI was better than RT in promoting effector CD8

^{+}T cells infiltration into tumor tissues, and that the combination of PI and RT exerted a synergistic effect on enhancing tumor-specific immune response.

^{+}T cell proportion (p = 0.02) and a lower MDSCs/CD45

^{+}cell proportion (p = 0.02), while the RT group had a higher Tregs/CD4

^{+}T cell proportion (p = 0.008) and a lower MDSCs/CD45

^{+}cell proportion (p = 0.0003), compared to the control group (Figure 6E,F). The Tregs/CD4

^{+}T cell proportion in the PI+RT group was at a level between the PI group and RT group (Figure 6E), which suggests that the down-regulation of PI on Tregs could cancel the up-regulation of RT on Tregs. The proportion of MDSCs in CD45

^{+}cells in the PI+RT group was the lowest among all groups (all p < 0.05, Figure 6F), which suggests that the combination of PI and RT played a synergistic role in down-regulating MDSCs. MDSCs can be mainly classified into CD11b

^{+}Ly6C

^{low}Ly6G

^{+}(PMN-MDSCs) and CD11b

^{+}Ly6C

^{high}Ly6G

^{−}(Mo-MDSCs) subpopulations [45]. Upon further analysis of the subgroups of MDSCs, the results suggested that the combination of PI and RT down-regulated PMN-MDSCs more significantly (Figure S6G,H).

^{+}T cells to Tregs is a marker of treatment outcome in various cancers [46,47,48,49], and the ratio of CD8

^{+}T cells to MDSCs may have a similar significance. Our results indicated that the PI group had a higher ratio of perforin

^{+}CD8

^{+}T cells to Tregs (p = 0.0002) and a higher ratio of perforin

^{+}CD8

^{+}T cells to MDSCs (p = 0.005), and the RT group had a lower ratio of perforin

^{+}CD8

^{+}T cells to Tregs (p = 0.02), but a higher ratio of perforin

^{+}CD8

^{+}T cells to MDSCs (p = 0.04), compared to the control group (Figure 6G,H). The ratio of perforin

^{+}CD8

^{+}T cells to Tregs in the PI+RT group was at a level between the PI group and RT group (both p < 0.01) (Figure 6G), which suggests that PI could make up for the deficiency of RT in this respect. The ratio of perforin

^{+}CD8

^{+}T cells to MDSCs in the PI+RT group was the highest among all groups (all p < 0.01, Figure 6H), suggesting that the combination of PI and RT could play a synergistic role in this regard.

#### 3.6. The Effect of PI and RT Combination on the Immune Profiles in the Spleens

^{+}cell absolute number (p = 0.0003), a higher CD3

^{+}T/CD45

^{+}cell proportion (p < 0.0001), and a higher CD3

^{+}T cell absolute number; and the RT group had a lower CD45

^{+}cell absolute number (p = 0.007) and a lower CD3

^{+}T cell absolute number (p = 0.03), but a higher CD3

^{+}T/CD45

^{+}cell proportion (p = 0.006), in comparison to the control group (Figure S8A–C). The CD45

^{+}cell absolute number (Figure S8A) and CD3

^{+}T cell absolute number (Figure S8C) in the PI+RT group were at the levels between the PI group and RT group (all p < 0.05), suggesting that the defect of RT in reducing the numbers of leukocytes and T cells in the spleens could be compensated by PI. However, the CD3

^{+}T/CD45

^{+}cell proportion in the PI+RT group was the highest among all of the groups (all p < 0.05, Figure S8B).

^{+}T/CD45

^{+}cell proportion (all p < 0.05, Figure S8D) and CD4

^{+}T/CD45

^{+}cell proportion (Figure S8E) were the highest in the PI+RT group among all of the groups. The ratio of CD4

^{+}T to CD8

^{+}T cells in the PI+RT group was the highest among all of the groups (Figure S8F). The PI group had a higher CD8

^{+}T cell absolute number (p = 0.002) and a higher CD4

^{+}T cell absolute number (p < 0.001), and the RT group had a lower CD8

^{+}T cell absolute number (p = 0.04), a lower CD4

^{+}T cell absolute number (p = 0.04), compared to the control group (Figure S8G,H). The CD8

^{+}T cell absolute number and CD4

^{+}T cell absolute number in PI+RT group were both at a level between the PI and RT group (all p < 0.05, Figure S8G,H).

^{+}CD8

^{+}T/CD45

^{+}cell proportion (Figure S8I) and perforin

^{+}CD8

^{+}T cell absolute number (Figure S8J) in the PI+RT group was the highest among all groups (all p < 0.05). The PI group had a higher perforin

^{+}CD8

^{+}T/CD45

^{+}cell proportion and a higher perforin

^{+}CD8

^{+}T cell absolute number (both p < 0.0001), but the RT group had lower levels in both the proportion and absolute number (p = 0.05), in comparison to the control group (Figure S8I,J). The PI+RT group had a higher perforin

^{+}CD8

^{+}T/CD45

^{+}cell proportion and a higher perforin

^{+}CD8

^{+}T cell absolute number compared with those of the PI group or RT group (all p < 0.05, Figure S8I,J). These results suggested that the combination of PI and RT significantly increased the storage of perforin

^{+}CD8

^{+}T cells in the spleens, which is the primary anticancer force.

^{+}T cell proportion (Figure S8K) and a lower MDSCs/CD45

^{+}cell proportion (Figure S8L) in the spleens in comparison to the control group (all p < 0.05). When further analyzing the subgroups of MDSCs in the spleens, we found that all of the treatment (PI, RT and PI+RT) groups had a lower level of PMN-MDSCs (Figure S8M), but not Mo-MDSCs (Figure S8N), compared to the control group.

^{+}CD8

^{+}T cells to Tregs (Figure S8O) and the ratio of perforin

^{+}CD8

^{+}T cells to MDSCs (Figure S8P) in the PI+RT group was the highest among all of the groups (all p < 0.05). The PI group had a higher ratio of perforin

^{+}CD8

^{+}T cells/Tregs (p < 0.0001) and a higher ratio of perforin

^{+}CD8

^{+}T cells to MDSCs (p < 0.0001), while the RT group had a lower ratio of perforin

^{+}CD8

^{+}T cells to Tregs (p = 0.007) and a higher ratio of perforin

^{+}CD8

^{+}T cells to MDSCs (p = 0.03), compared to the control group (Figure S8O,P).

#### 3.7. The Effect of PI and RT Combination on the Peripheral Blood

^{+}cell absolute number than the control group (Figure S9A). The PI+RT group had the lowest CD45

^{+}cell absolute number among all of the groups (all p < 0.05, Figure S9A). The PI group (p = 0.002) and RT group (p = 0.0002) both had a higher CD3

^{+}T/CD45

^{+}cell proportion than that of the control group (Figure S9B). The PI+RT group had a higher CD3

^{+}T/CD45

^{+}cell proportion than that of the PI group (p = 0.002) and the RT group (p < 0.0001) (Figure S9B). The RT group had the lowest CD3

^{+}T cell absolute number, and the PI+RT group had a number between that of the PI group and RT group (Figure S9C).

^{+}T/CD45

^{+}cell proportion (Figure S9D) and CD4

^{+}T/CD45

^{+}cell proportion (Figure S9E) among all of the groups. The PI group and RT group both had a higher CD8

^{+}T/CD45

^{+}cell proportion (Figure S9D) and a higher CD4

^{+}T/CD45

^{+}cell proportion (Figure S9E) than those of the control group (all p < 0.05). The CD4

^{+}T/CD8

^{+}T cell ratio in the PI group and PI+RT group was significantly lower than that of the RT group and the control group (all p < 0.05, Figure S9F). There was no significant difference in the CD4

^{+}T/CD8

^{+}T cell ratio between the RT group and the control group (Figure S9F). The PI group had a higher CD8

^{+}T cell absolute number (p = 0.04) and a lower CD4

^{+}T cell absolute number (p = 0.04), and the RT group had a lower CD8

^{+}T and CD4

^{+}T cell absolute number (both p = 0.02) compared to the control group (Figure S9G,H). The CD8

^{+}T cell absolute number in the PI+RT group was at a level between that of the PI group and RT group (all p < 0.05, Figure S9G). The CD4

^{+}T cell absolute number in the PI+RT group was the lowest among groups (Figure S9H).

^{+}T cell proportion (p = 0.02) and a lower MDSCs/CD45

^{+}cell proportion (p = 0.002), and the RT group had a higher Tregs/CD4

^{+}T cell proportion (p = 0.005) and a lower MDSCs/CD45

^{+}cell proportion (p = 0.03), in comparison to the control group (Figure S9I,J). The Tregs/CD4

^{+}T cell proportion in the PI+RT group was at a level between that of the PI group and RT group (Figure S9I), suggesting that the defect of RT in increasing this proportion was compensated by PI. The PI+RT group had the lowest MDSCs/CD45

^{+}cell proportion among all of the groups (Figure S9J), suggesting that the combination of both therapies played a synergistic role in this regard. In the further analysis of the subgroups of MDSCs, the results suggested that the combination of PI and RT down-regulated the PMN-MDSCs more significantly than Mo-MDSCs in the peripheral blood (Figure S9K,L).

^{+}T cells to Tregs (p = 0.004) and a higher ratio of CD8

^{+}T cells to MDSCs (p = 0.005), and the RT group had a lower ratio of CD8

^{+}T cells/Tregs (p = 0.03) and a higher ratio of CD8

^{+}T cells to MDSCs (p = 0.007), compared to the control group (Figure S9M,N). The ratio of CD8

^{+}T cells to Tregs in the PI+RT group was at a level between the PI group and RT group (all p < 0.05, Figure S9M), suggesting that the defect of RT in reducing this ratio was compensated by PI. The ratio of CD8

^{+}T cells to MDSCs in the PI+RT group was the highest among all of the groups (all p < 0.05, Figure S9N), suggesting that the combination of both therapies played a synergistic role in this respect.

## 4. Discussion

^{+}T cells into the tumor tissues of orthotopic GL261 glioma, suggesting that the cold tumor could be transformed into a hot tumor, and the combination of both had a synergistic effect, which was more significant than that of any single therapy (Figure 5). As a result, the tumor of GL261 glioma in the combination group was too small, and approximately 70% of the tumor-bearing mice (as mentioned above, regardless of orthotopic or subcutaneous model) were cured; we were unable to obtain enough samples for systemic analysis of immunophenotypes (with the exception of the simple count of CD3

^{+}T cells, as mentioned above). Therefore, we could only use lung cancer specimens for a series of immune assays. The results based on lung cancer tissues showed that RT down-regulated the number of CD45

^{+}leukocytes, while PI was able to compensate for this defect of RT (Figure S6A). Both PI and RT significantly up-regulated the proportion of CD8

^{+}T cells in CD45

^{+}leukocytes, and the combination of both had a synergistic effect (Figure 6A). The combination treatment group had the highest number of CD8

^{+}T cells among all of the groups (Figure 6B). For effector CD8

^{+}T cells expressing perforin, both PI and RT up-regulated its proportion and number (PI was more significant than RT), and the combination of both had a synergistic effect (Figure 6C,D). In addition, we also found that PI significantly down-regulated the proportion of Tregs in CD4

^{+}T cells, while RT significantly up-regulated this proportion (Figure 6E). The combination of both compensated for this defect of RT, resulting in a significantly decreased proportion in the combination therapy group, compared with the control group (Figure 6E). Both PI and RT down-regulated the proportion of MDSCs in CD45

^{+}leukocytes, and the combination of both had a synergistic effect (Figure 6F). We observed that PI significantly up-regulated the ratio of perforin-expressing effector CD8

^{+}T cells to Tregs, and, thus, was able to compensate for the deficiency of RT that down-regulated this ratio; the ratio in the combined treatment group was therefore significantly higher than that in the control group (Figure 6G). Both PI and RT up-regulated the ratio of perforin-expressing effector CD8

^{+}T cells to MDSCs, and the combination of both was synergistic (Figure 6H). These immunophenotypic results based on tumor tissues have demonstrated that the combination of PI and RT significantly enhances the antitumor immune response in tumor-bearing mice, showing a synergistic or complementary effect. In addition to directly killing tumor cells by interrupting DNA of tumor cells, RT also enhances the efficacy of immunotherapy by inducing immunogenic death of tumor cells [54].

^{+}T cells in tumor tissues (Figure 6E), and significantly down-regulate the number of CD45

^{+}leukocytes in tumor tissues (Figure S6A) and the spleen (Figure S8A). ICB may not be able to compensate for these defects of RT, however, PI can produce complementary effects. In addition to facilitating the entry of effector T cells into tumor tissues, PI also promotes the entry of other immune cells, such as CD45

^{+}leukocytes (observed in the current study), including NK cells and DCs (observed in our previous study [15], into tumor tissues. Therefore, PI can transform cold tumors into hot tumors, which is more effective than RT, as shown in the current study, and is thus also effective against cold tumors. Therefore, its indications should be wider than ICB. Based on these results, we proposed that PI should be more suitable than ICB as a basic treatment in combination therapy. However, as mentioned before, the specific killing ability of PI on tumor cells may be relatively weak, and RT has the advantage in this respect, so the combination of PI and RT could produce synergistic and complementary effects.

^{+}T cells in the peripheral blood of tumor-bearing mice infected with a Plasmodium parasite, without affecting the secretion of effector molecules perforin and granzyme B by these T cells [17]. These results suggest that, after the activation of the immune system by Plasmodium infection, the immune system itself initiates the immune balancing mechanism to prevent overactive immune response. For example, in tumor-bearing mice infected with a Plasmodium parasite, the PD-1 levels on CD8

^{+}T cells in peripheral blood increase [17], but the levels of this molecule on CD8

^{+}T cells in tumor tissues decrease significantly [16]. This suggests that a high expression of PD-1 in peripheral blood can avoid excessive systemic immune response, while a low expression of PD-1 in tumor tissues is conducive to antitumor immune response, not to mention that Plasmodium infection also undoes the immunosuppressive microenvironment of tumor. Therefore, based on the principle of immune equilibrium (balance) [78], the observed increase in MDSCs/Tregs and the simultaneous expression of coinhibitory molecules on T cells in peripheral blood cannot be attributed to the suppression of the immune system by the Plasmodium infection itself; instead, they can serve as the markers of immune activation. Interestingly, our recent study indicates that subsequent Plasmodium infection induces a high proportion of CD4

^{+}CD28

^{high}CD95

^{high}central memory T cells and a strong SIV (simian immunodeficiency virus)-specific T cell response drives the hosts to maintain the diversity of SIV-specific T cell receptor repertoire, generating new SIV-specific T cell clones to track the antigenic variations of SIV, and thus extending the life span of rhesus monkeys infected with SIV [79]. This suggests that Plasmodium infection enhances immune response to different pathogen and drives T cells to track its variations, and thus may also drive T cells in tumor-bearing hosts to trace the antigenic variations of tumor cells. In brief, our series of studies show no conflicting results that either high or low densities of parasitemia Plasmodium infection activates the immune system in tumor-bearing mice [15,16,17,18] [and unpublished data]. Nevertheless, based on the activation of the immune system, at least by the low grade of Plasmodium infection mentioned above, our clinical trial protocol of Plasmodium immunotherapy for advanced solid tumors requires the use of antimalarial drug (artesunate) to control parasitemia at a reasonably low level (below 0.1% or 0.05%), which has been shown to activate the immune systems of cancer patients [13]. In theory, the immune system of the tumor-bearing host has already been suppressed by cancer cells, and Plasmodium parasite, as a foreign pathogen, has strong danger signals, including a large amount of pathogen-associated molecular patterns (PAMPs) [69] and more than 5000 heterologous proteins [80]; therefore, the overall effect of Plasmodium infection on the immune system should be wake-up and activation rather than inhibition.

## 5. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

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**Figure 1.**Therapeutic efficacy of PI in combination with RT in orthotopic GL261-Luc glioma model. (

**A**) Schematic representation of combination therapy in orthotopic intracerebral GL261-Luc tumor-bearing mice. (

**B**) Living imaging for all groups. (

**C**) Tumor growth curves based on the data on days 7, 14, 21, 28, 35 and 42 after inoculation of the GL261-Luc cells (n = 7 per group). Photon-density heat maps were shown for all groups at the same bioluminescence signals. The statistical differences at the experiment endpoint between groups were analyzed with an unpaired two-tailed Student’s t-test. (

**D**) The Kaplan-Meier survival curves of mice (n = 7) were shown and analyzed by a log-rank test. “Cured” mice (n = 6) were rechallenged with 2 × 10

^{6}syngeneic GL261-Luc cells (

**E**) in the right flank and non-syngeneic 5 × 10

^{5}LLC cells (

**F**) in the left flank. Naïve mice (n = 6) received the same rechallenges as control. The data showed the mean ± SEM. Statistical differences were indicated by the p values, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

**Figure 2.**Therapeutic efficacy of PI in combination with RT in subcutaneous GL261 tumor model. (

**A**) Schematic representation of combination therapy in subcutaneous GL261 tumor-bearing mice. (

**B**) Tumor growth curves (n = 10 per group). At the endpoint, the statistical differences between groups were analyzed with an unpaired two-tailed Student’s t-test. (

**C**) Tumor sizes and (

**D**) weight of the s.c. tumor mass on day 29 after tumor cell inoculation (n = 5). The statistical differences between groups were analyzed with an unpaired two-tailed Student’s t-test. (

**E**) The Kaplan-Meier survival curves of mice (n = 10). Survival curves were analyzed by a log-rank test. Cured mice were rechallenged with 2 × 10

^{6}syngeneic GL261 cells (

**F**) in the right flank and non-syngeneic 5 × 10

^{5}LLC cells (

**G**) in the left flank (n = 6) after 120 days post inoculation and compared with 6 naïve mice. The data showed the mean ± SEM. Statistical differences were indicated by the p values, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

**Figure 3.**Therapeutic efficacy of PI in combination with RT in subcutaneous LLC lung cancer model. (

**A**) Schematic representation of combination therapy in subcutaneous LLC tumor mice. (

**B**) Tumor growth curves (n = 10 per group). The statistical differences between groups were analyzed with an unpaired two-tailed Student’s t-test. (

**C**) Comparisons by visual observation of tumor sizes between groups. (

**D**) Weight of the s.c. tumor mass on day 19 after tumor cell implantation (n = 5). The statistical differences between groups were analyzed with an unpaired two-tailed Student’s t-test. (

**E**) The Kaplan-Meier survival curves of mice (n = 10). Survival curves were analyzed by a log-rank test. The data showed the mean ± SEM. Statistical differences were indicated by the p values, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

**Figure 4.**The effect of PI in combination with RT on cell proliferation and apoptosis in subcutaneous LLC lung cancer model. (

**A**) Immunohistochemical staining: Arrows showed Ki67-expressing cells. (

**B**) Assay quantification for Ki67 at 400× magnification (n = 6). (

**C**) Western blotting assay results of pro-caspase 3 and cleaved caspase 3. (

**D**) Assay quantification for caspase 3: Ratio of cleaved caspase 3/pro-caspase 3 (n = 3). GAPDH was used as a loading control for western blotting. The statistical differences between groups were analyzed with an unpaired two-tailed Student’s t-test. The data showed the mean ± SEM. Statistical differences were indicated by the p values, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

**Figure 5.**The effect of PI in combination with RT on the infiltration of CD3

^{+}T cells in tumor tissues in orthotopic GL261-Luc glioma-bearing mice. (

**A**) Immunohistochemical staining and quantification of CD3 at 400× magnification. Black arrows showed CD3-expressing T cells. (

**B**) The percentage of CD3

^{+}T cells among all calculated cells in tumor tissues (n = 4). The statistical differences between groups were analyzed with an unpaired two-tailed Student’s t-test. The data showed the mean ± SEM. Statistical differences were indicated by the p values, *, p ≤ 0.05; **, p ≤ 0.01.

**Figure 6.**The effect of PI in combination with RT on the immune profiles in tumor tissues in subcutaneous lung cancer-bearing mice. Lymphocytes were isolated from tumors on day 19 post inoculation (n = 5). (

**A**) Proportion of CD8

^{+}T cells in CD45

^{+}cells. (

**B**) Absolute number of CD8

^{+}T cells. (

**C**) Proportion of perforin

^{+}CD8

^{+}T cells in CD45

^{+}cells. (

**D**) Absolute number of perforin

^{+}CD8

^{+}T cells. (

**E**) Proportion of Tregs in CD4

^{+}T cells. (

**F**) Proportion of MDSCs in CD45

^{+}cells. (

**G**) Ratio of perforin

^{+}CD8

^{+}T cells to Tregs. (

**H**) Ratio of perforin

^{+}CD8

^{+}T cells to MDSCs. Absolute number was presented as the number of cells per gram of tumor. The statistical differences between groups were analyzed with an unpaired two-tailed Student’s t-test. The data showed the mean ± SEM. Statistical differences were indicated by the p values, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

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Tao, Z.; Ding, W.; Cheng, Z.; Feng, Y.; Kang, Z.; Qiu, R.; Zhao, S.; Hu, W.; Zhou, F.; Wu, D.; Duan, Z.; Qin, L.; Chen, X. Preclinical Study of *Plasmodium* Immunotherapy Combined with Radiotherapy for Solid Tumors. *Cells* **2022**, *11*, 3600.
https://doi.org/10.3390/cells11223600

**AMA Style**

Tao Z, Ding W, Cheng Z, Feng Y, Kang Z, Qiu R, Zhao S, Hu W, Zhou F, Wu D, Duan Z, Qin L, Chen X. Preclinical Study of *Plasmodium* Immunotherapy Combined with Radiotherapy for Solid Tumors. *Cells*. 2022; 11(22):3600.
https://doi.org/10.3390/cells11223600

**Chicago/Turabian Style**

Tao, Zhu, Wenting Ding, Zhipeng Cheng, Yinfang Feng, Zhongkui Kang, Runmin Qiu, Siting Zhao, Wen Hu, Fang Zhou, Donghai Wu, Ziyuan Duan, Li Qin, and Xiaoping Chen. 2022. "Preclinical Study of *Plasmodium* Immunotherapy Combined with Radiotherapy for Solid Tumors" *Cells* 11, no. 22: 3600.
https://doi.org/10.3390/cells11223600