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. 2024 Aug 3;32(9):3145–3162. doi: 10.1016/j.ymthe.2024.07.029

Reprogramming tumor immune microenvironment by milbemycin oxime results in pancreatic tumor growth suppression and enhanced anti-PD-1 efficacy

Shreyas Gaikwad 1, Sanjay K Srivastava 1,∗
PMCID: PMC11403213  PMID: 39097773

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has a survival rate of 12%, and multiple clinical trials testing anti-PD-1 therapies against PDAC have failed, suggesting a need for a novel therapeutic strategy. In this study, we evaluated the potential of milbemycin oxime (MBO), an antiparasitic compound, as an immunomodulatory agent in PDAC. Our results show that MBO inhibited the growth of multiple PDAC cell lines by inducing apoptosis. In vivo studies showed that the oral administration of 5 mg/kg MBO inhibited PDAC tumor growth in both subcutaneous and orthotopic models by 49% and 56%, respectively. Additionally, MBO treatment significantly increased the survival of tumor-bearing mice by 27 days as compared to the control group. Interestingly, tumors from MBO-treated mice had increased infiltration of CD8+ T cells. Notably, depletion of CD8+ T cells significantly reduced the anti-tumor efficacy of MBO in mice. Furthermore, MBO significantly augmented the efficacy of anti-PD-1 therapy, and the combination treatment resulted in a greater proportion of active cytotoxic T cells within the tumor microenvironment. MBO was safe and well tolerated in all our preclinical toxicological studies. Overall, our study provides a new direction for the use of MBO against PDAC and highlights the potential of repurposing MBO for enhancing anti-PD-1 immunotherapy.

Keywords: drug repurposing, anti-helminthic drug, pancreatic tumor, tumor suppression, immunomodulation, CD8+ T-cells, immune checkpoint inhibitors, ICI, anti-PD-1, STING, immunogenic cell death, ICD, chemokines

Graphical abstract

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This study for the first time identifies anti-cancer and immunomodulatory activity of milbemycin oxime (MBO), an anti-parasitic compound. Results from the study performed by Gaikwad and Srivastava provide pre-clinical evidence of the efficacy of MBO in enhancing the effects of anti-PD-1 therapy and improving the survival of PDAC tumor-bearing mice.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) represents a formidable challenge in the field of oncology, characterized by its late-stage diagnosis, aggressive tumor biology, and limited treatment options.1 The complex microenvironment, marked by immunosuppression and desmoplasia, further complicates therapeutic interventions in this malignancy.2 While chemotherapy offers some degree of survival advantage to both resectable and non-resectable PDAC patients, the benefits remain poor due to the development of drug resistance and grade 3–4 adverse effects. Among the approved chemotherapies, the combination of gemcitabine and nab-paclitaxel is the most commonly prescribed regimen. However, the PDAC tumor microenvironment (TME) confers resistance to both of these compounds in most of the patients through various mechanisms such as modulation of the metabolic activity of gemcitabine while simultaneously disrupting drug delivery within the tumors.3 Considering the chemotherapeutic roadblocks in PDAC, the search for alternative treatments to combat this deadly cancer remains a priority. One of these treatments is immunotherapy, which has shown significant progress in various cancer types.4

Although remarkable progress has been made in the field of cancer immunotherapy in the past few years, PDAC remains a particularly resistant solid malignancy to multiple immunotherapies. In particular, immune checkpoint inhibitors (ICIs), either single or dual, have demonstrated limited benefit to PDAC patients.5,6 Among multiple factors, the scarcity of specific and effective antigens and low abundance of lymphocytes in PDAC makes it challenging to induce a robust antitumor immune response.7 These factors, combined with the rapid metastasis of the disease after local advancement, contributes to the disappointing outcomes of immunotherapies in PDAC.8 The dense fibrotic reaction and immunosuppressive TME presents a formidable barrier to effective T cell infiltration and function. This barrier can be overcome by increasing the infiltration and proliferation of T cells in the tumors and stimulating their antitumor activity.9 This strategy opens doors to testing more ICIs, personalized treatment approaches, therapeutic vaccines, and localized treatment methods that leverage the potential of T cells.10 Some of the ICIs studied in PDAC include the anti-CTLA-4 antibody ipilimumab and the anti-programmed cell death protein 1 (PD-1) antibody pembrolizumab. Pembrolizumab offered an objective response in a small subset of PDAC patients with microsatellite instability-high tumors.11 To widen the application of anti-PD-1 therapy to the remaining PDAC population, new approaches such as combination chemotherapy or repurposed drugs need to be explored.12 Few anti-parasitic compounds have shown the ability to enhance the anti-tumor efficacy of ICIs.13 Specifically, macrolides that contain avermectins have shown immunomodulatory effects; however, the anti-cancer and immunomodulatory effects of the other class of macrolides called milbemycins have not yet been studied.

Milbemycin oxime (MBO), an anti-parasitic compound, belongs to the class of milbemycins. Pre-clinical studies suggest that milbemycin compounds are not associated with side effects and relatively safe.14,15 So far, only one study has reported the anti-cancer effects of MBO.

In this study, we evaluated the immunomodulatory effect of MBO in PDAC. Our results clearly show that MBO treatment suppressed PDAC tumor growth by enhancing CD8+ T cell infiltration in the tumors of MBO-treated mice and significantly increased the anti-tumor efficacy of anti-PD-1, indicating promise for PDAC treatment. Furthermore, our mechanistic study using reverse-phase protein array (RPPA) analysis showed suppression of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway with an increase in the activity of the stimulator of interferon genes (STING) pathway.

Results

MBO suppresses PDAC cell growth in vitro

To evaluate the anti-proliferative effects, human PDAC cell lines AsPC1, BxPC3, MiaPaCa-2, PANC1, SUIT-2, CAPAN-2, and MiaPaCa-2 GR (gemcitabine resistant), and murine pancreatic cancer cell line PO2 transfected with luciferase (PO2-Luc) were treated with varying concentrations of MBO for different time intervals. The viability of cells was evaluated by sulforhodamine B (SRB) assay. Our results show that MBO treatment suppressed the growth of all the PDAC cell lines. MBO suppressed the growth of AsPC-1 cells at 24-, 48-, and 72-h time points. However, MBO reduced the growth of other PDAC cells at 48- and 72-h time points (Figures 1A–1H). The half-maximal inhibitory concentration (IC50) of MBO ranged from 4.4 to 17 μM after 24, 48, and 72 h of treatment in all the cell lines tested.

Figure 1.

Figure 1

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MBO suppresses the growth of PDAC cell lines and suppresses their colony-formation ability

(A–H) Growth-suppressive effects of MBO in (A) MiaPaCa-2, (B) AsPC-1, (C) BXPC3, (D) Panc-1, (E) SUIT-2, (F) PO2-Luc cells, (G) MiaPaCa-2 GR, and (H) Capan-2. The cells were treated with varying concentrations of MBO for 24, 48, and 72 h. Cell viability was analyzed using the SRB assay and the data were plotted using GraphPad Prism 8. (I–L) Effects of MBO on colony-formation ability of (I) AsPC1, (J) SUIT-2, (K) MiaPaCa-2, and (L) PO2. The cells were treated with various concentrations of MBO, and the colonies were stained using SRB dye. Each experiment was repeated three times with at least three replicates in each experiment. Statistically significant when compared with control. ∗p < 0.05, ∗∗p < 0.01.

MBO inhibits the colony-formation ability of PDAC cells

Since MBO was able to suppress the growth of various PDAC cells, we next explored its ability to inhibit the colony formation of PDAC cells. AsPC1, SUIT-2, MiaPaCa-2, and PO2 cells were treated with 2, 4, and 6 μM MBO for 48 h and allowed to form colonies for 14 days. Significant reduction in colony formation was observed by MBO treatment starting with sub-toxic concentrations in all the cell lines, while no colonies were observed at all with 6 μM MBO treatment (Figures 1I–1L). To test whether this effect is time dependent, we exposed the AsPC1 and SUIT-2 cells to MBO at IC50 concentrations for 24, 48, and 72 h. MBO inhibited AsPC-1 cells in an almost similar manner at all three time points. However, significant inhibition of SUIT-2 cell colonies was observed at 48 and 72 h by MBO treatment, with modest effects at the 24-h time point.

Apoptotic cell death induction by MBO

After determining the growth suppressive effects of MBO in various PDAC cells, we analyzed the mode of cell death induced by MBO in MiaPaCa-2, AsPC1, BXPC3, and SUIT-2 cell lines. The cells were stained with PI and annexin/allophycocyanin (APC) dyes following 48-h treatment with 4, 6, and 8 μM MBO. As shown in Figures 2A–2D, a significant apoptosis was observed following MBO treatment. MiaPaCa-2 and AsPC1 cells mostly exhibited early apoptosis, while BXPC3 and SUIT-2 cells showed early to late apoptosis, respectively.

Figure 2.

Figure 2

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MBO induces apoptotic cell death in PDAC cells

(A) MiaPaCa-2, (B) AsPC1, (C) BXPC3, and (D) SUIT-2 cells were treated with various concentrations of MBO for 48 h, and the cells were processed for annexin-V/APC apoptosis assay. Results were analyzed using FlowJo software and plotted using GraphPad Prism 8. Each experiment was repeated three times with at least three replicates in each experiment. Statistically significant when compared with control. ∗p < 0.05, ∗∗p < 0.01.

MBO treatment causes G1 phase cell-cycle arrest

We next explored the effect of MBO on cell-cycle distribution using flow cytometry. As shown in Figures S1A–S1C, MBO treatment induced G1 phase arrest in all three cell lines (AsPC1, MiaPaCa-2, and SUIT-2). The G1 phase arrest was observed in a concentration-dependent manner, with the highest number of cells in G1 phase observed with 8 μM MBO treatment.

MBO suppresses growth of subcutaneously implanted PDAC tumors

We evaluated the efficacy of MBO in an in vivo xenograft model using female athymic nude mice. About 5 × 106 MiaPaCa-2 cells were injected subcutaneously in the left flank of mice, and the tumors were measured twice per week. Once the tumors reached approximately 100 mm3, mice were randomly divided into four groups, and MBO was administered at various doses ranging from 2.5 to 10 mg/kg/day by oral gavage. Our results showed a significant tumor growth suppression at 5 and 10 mg/kg MBO treatment (Figure 3A). The percent suppression of tumor growth was approximately 49% for both the treatments. Similarly, reduction in tumor weights by MBO was observed at the end of the study (Figure 3B). No significant difference was observed in the body weights of mice treated with MBO versus the vehicle-treated group (Figure S2A). Furthermore, no difference was observed in the weight of various organs of the mice treated with MBO as compared with the vehicle group (Figure S2B), indicating no toxicity due to MBO treatment. To determine whether a sufficient amount of MBO reached inside the tumors, MBO concentration was evaluated using the previously described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in the tumors of 5 mg/kg treated mice, since this dose showed maximum tumor growth suppression.16 The average concentration of MBO in the tumors was around 2,456 ng/g, while the highest concentration was 5,160 ng/g (Figure 3C).

Figure 3.

Figure 3

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MBO suppresses growth of PDAC tumors in subcutaneous and orthotopic models by inducing apoptosis

(A) Tumor curve representing the growth of subcutaneously implanted MiaPaCa-2 tumors in control, 2.5 mg/kg MBO, 5 mg/kg MBO, and 10 mg/kg MBO. (B) Average tumor weight recorded on the final day of the experiment. (C) Average concentration of MBO in tumors analyzed by LC-MS/MS method. (D) Average luminescence curve representing the growth of orthotopically implanted PO2-Luc tumors in control versus 5 mg/kg MBO. (E) Average tumor weight recorded on the final day of the experiment. (F) Representative images of orthotopically implanted tumors from control versus MBO groups. (G) Representative tumor sections depicting apoptosis in orthotopically implanted tumors from control and MBO-treated groups. (H) Quantitative analysis of apoptosis induction in tumor sections (n = 3). Statistically significant differences between groups are reported as p values in the panels. Statistically significant when compared with control. ∗p < 0.05, ∗∗p < 0.01.

MBO suppresses growth of orthotopically implanted PDAC tumors

To further validate the anti-tumor effects of MBO we observed in the xenograft model, the efficacy of MBO was evaluated in an orthotopic PDAC model using murine PO2-Luc cells. About 1 × 106 PO2-Luc cells were implanted in the pancreas of C57/BL6 mice by performing surgery as described by us previously.17 After 20 days of tumor cell implantation, once each mouse showed significant luminescence, the mice were randomized into two groups and the experimental group of mice received 5 mg/kg/day MBO by oral gavage. The growth of tumors was monitored using an in vivo imaging system (IVIS) imager. MBO treatment suppressed tumor growth in mice by approximately 56% compared to vehicle-treated control mice (Figures 3D–3F). The body weight of mice did not show any significant change between control and treatment groups throughout the treatment period (Figure S2C). No significant change in the weight of critical organs was observed in the MBO-treated group (Figure S2D). Furthermore, blood chemistry and other organ function parameters showed no significant differences between treatment and control groups (Figure S3), indicating no side effect or toxicity by MBO treatment. Significantly high apoptosis was observed in the tumors of mice treated with MBO compared with the tumors from control mice as analyzed by TUNEL assay (Figures 3G and 3H).

Treatment with MBO generated a greater pool of CD8+ T cells

Fluorescence-activated cell sorting (FACS) analysis of the tumor samples was performed to explore any possible effect of MBO on the tumor immune microenvironment. While there was no effect on the total number of T cells, we observed an increased number of CD8+ T cells in the tumors of MBO-treated mice. A modest increase in the number of CD4+ T cells was observed (Figure 4A). However, the number of CD8+ T cells and CD4+ T cells remained unchanged in the spleens of mice from both groups (Figure 4B). The increased number of CD8+ T cells in the tumors was also confirmed by immunohistochemistry (IHC), wherein the MBO-treated tumors showed a 66% higher number of CD8+ T cells as compared to the vehicle-treated group (Figures 5A and 5B).

Figure 4.

Figure 4

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MBO treatment increases CD8+ T cell infiltration in the TME

Frequencies of CD8+ T cells and CD4+ T cells within tumors (A) and spleens (B) as a percentage of viable cells. FACS analysis was performed as described in materials and methods. Immune cell frequencies were analyzed using FlowJo software and plotted using GraphPad Prism 8. Statistically significant differences between groups are reported as p values in the panels. Statistically significant when compared with control. ∗p < 0.05, ∗∗p < 0.01.

Figure 5.

Figure 5

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Tumor-suppressive effect of MBO is CD8 T cell dependent, and MBO increases the survival of tumor-bearing mice

(A) Representative tumor sections depicting tumor-infiltrating CD8+ T cells after MBO treatment as confirmed by IHC. (B) Quantitation of CD8+ T infiltrates by IHC. (C) Tumor curve representing growth of subcutaneously implanted PO2-Luc tumors following treatment with control, MBO, anti-CD8α alone, and anti-CD8α + MBO groups. (D) Average tumor weight recorded on the final day of the experiment. (E) Survival proportions of control and MBO-treated groups. Statistically significant differences between groups are reported as p values in the panels. Statistically significant when compared with control. ∗p < 0.05, ∗∗p < 0.01.

Tumor-suppressive effect of MBO is CD8+ T cell dependent

Since we observed a greater pool of CD8+ T cells in the tumors of MBO-treated mice, we sought to determine whether the anti-tumor effect of MBO was CD8+ T cell dependent. An in vivo experiment using a PO2 subcutaneous model was thus performed. When the tumors reached 70–100 mm3, mice were randomized into four groups—vehicle alone, MBO (5 mg/kg), anti-CD8α antibody, and anti-CD8α + MBO (5 mg/kg)—and the tumor growth was monitored. It is important to note that the depletion of CD8+ T cells significantly reduced the anti-tumor efficacy of MBO, indicating the dependency of MBO on CD8+ T cells (Figures 5C and 5D).

MBO increases survival of tumor-bearing mice

In a separate experiment, about 1 × 106 PO2-Luc cells were orthotopically implanted in C57/BL6 mice. After 20 days of tumor cell implantation and once each mouse showed significant luminescence, mice were randomized into two groups, and the experimental group of mice received 5 mg/kg/day MBO by oral gavage. Animals were monitored for signs of morbidity and decline in body condition score. Throughout the study, MBO showed a significant tumor growth-suppressive effect, and consequently increased the survival of mice by 27 days as compared to untreated controls (Figure 5E).

MBO augments the anti-tumor efficacy of immunotherapy

Based on our observations, we hypothesized that MBO might augment the efficacy of immunotherapy in immunologically cold pancreatic tumors. To test this hypothesis, we combined MBO with anti-PD-1 antibody in the syngeneic PO2 pancreatic tumor mice model. About 1 × 106 PO2 murine pancreatic cancer cells were implanted in C57/BL6 mice subcutaneously. Once the tumors reached around 70–100 mm3, mice were randomized into 4 groups—vehicle alone, 5 mg/kg MBO, anti-PD-1, and MBO+anti-PD-1. As shown in Figures 6A and 6B, the combination of MBO with anti-PD-1 treatment showed almost 80% tumor growth suppression as compared to almost no tumor-suppressive effect of anti-PD-1 treatment alone. In addition, our results showed a significant increase in overall T cell infiltration in the tumors of mice that received combination treatment (Figures 6C and 6D). Specifically, the CD8+ T cell population was higher in the combination group as compared to both vehicle or anti-PD-1 treatment group (Figures 6C and 6D). The active effector population (CD8+ interferon-positive [IFN+] T cells, CD8+ CD25+ T cells, and CD8+ CD62L− T cells) increased as well in the combination group. We further evaluated the secretion of chemokines to understand the reason behind increased effector T cell infiltration. Our results indicated higher levels of C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 9 (CXCL9) and IFN-γ in the tumor cell lysates from MBO- and anti-PD-1 combination-treated mice as compared to vehicle-treated or anti-PD-1-treated mice (Figure 6E). Overall, our results indicated that MBO treatment enhanced the efficacy of anti-PD-1 in PDAC tumor growth suppression due to the ability of MBO to attract more T cells in the tumors.

Figure 6.

Figure 6

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MBO synergizes with anti-PD-1 immunotherapy

(A) Tumor curve representing growth of orthotopically implanted PO2-Luc tumors following treatment with control, MBO, anti-PD-1 alone, and anti-PD-1 + MBO. (B) Average tumor weight recorded on the final day of the experiment. (C) Quantification of various CD8+ T cell populations. (D) Quantification of various CD4+ T cell populations. (E) Quantification of IFN-γ, CXCL10, and CXCL9 in tumor lysates. Statistically significant differences between groups are reported as p values in the panels. Statistically significant when compared with control. ∗p < 0.05, ∗∗p < 0.01.

MBO induces immunogenic cell death in PDAC cells

Since immunogenic cell death is a well-known characteristic responsible for immunomodulation, we analyzed the surface expression of heat-shock protein-90 (HSP90), calreticulin (CRT), and major histocompatibility complex class I (MHC class I) by flow cytometry in AsPC1, PO2, and SUIT-2 cells. As shown in Figures 7A–7E, we observed significantly higher surface expression of these markers. The increase in the surface expression of HSP90 and CRT was observed to be concentration dependent, with maximum effect observed with 6 and 8 μM MBO (Figures 7A–7E).

Figure 7.

Figure 7

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MBO induces immunogenic cell death marker expression

(A) Expression of HSP90 in AsPC1, PO2, and SUIT-2 cells. (B) Quantification of HSP90 in respective cell lines. (C) Expression of CRT in AsPC1, PO2, and SUIT-2 cells. (D) Quantification of CRT in respective cell lines. (E) Expression of MHC-1 in PO2 and SUIT-2 cells. Statistically significant differences between groups are reported as p values in the figures. Each experiment was repeated three times with at least three replicates in each experiment. Statistically significant when compared with control. ∗p < 0.05, ∗∗p < 0.01.

Immunogenic and apoptotic cell death markers correlate with increased effector T cell infiltration in the clinical samples

To understand the clinical relevance of our results, we analyzed the Tumor Immune Estimation Resource (TIMER) 2.0 database for CD8+ T cell infiltration in PDAC tumors. The TIMER database showed a positive correlation of CD8+ T cells inside the tumors with that of immunogenic cell death (ICD) markers HSP90 and CRT, along with apoptotic markers such as annexin and caspase-3 (Figures S5A–S5E). Furthermore, an increase in CXCL9 and CXCL10 correlated with a higher pool of CD8+ T cells in PDAC tumors (Figures S5F and S5G). Thus, The Cancer Genome Atlas (TCGA) patient data from the TIMER database showed that increased expression of various cellular death markers and chemokines was associated with the increased infiltration of CD8+ T cells in the tumors, corroborating the observations made in our current study with MBO.

MBO inhibits PI3K/AKT pathway while activating STING signaling in PDAC cells

We performed RPPA to delineate the mechanism of action of MBO in PDAC cells. The results of RPPA analysis showed changes in the expression of various signaling pathways as depicted by heatmaps in Figure 8. The heatmap data were further analyzed using Ingenuity Pathway Analysis (IPA) to understand the most downregulated and upregulated pathways by MBO treatment. The two most modulated pathways observed were PI3K/Akt and STING signaling pathways. Our results indicated a statistically significant downregulation of the PI3K/Akt signaling pathway by MBO treatment with a fold change of −log (p value) 6.1. However, MBO treatment resulted in the activation or upregulation of the STING pathway with a fold change value of −log (p value) 5.2 (Figure 8).

Figure 8.

Figure 8

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MBO downregulates PI3K/Akt signaling while activating STING signaling in PDAC cells

(A) Heatmaps depicting the up- and downregulated proteins in MiaPaCa-2 cells following treatment with MBO. The red color signifies upregulated proteins, while the green color denotes downregulation. The values generated from RPPA were analyzed further by IPA. PI3K/Akt was the most downregulated pathway, while STING signaling was the most upregulated signaling pathway. (B) Graphical summary depicting the mechanism of action of MBO in PDAC. Statistically significant when compared with control. ∗p < 0.05, ∗∗p < 0.01.

Discussion

Treating PDAC presents a formidable challenge due to its typically aggressive nature and a propensity to invade and metastasize, limited treatment options, and high resistance to chemotherapy.18 Moreover, PDAC tumors are typically unresponsive to immunotherapy due to the strikingly immune privileged nature of these tumors. In various clinical trials, chemotherapeutic drugs are being tested to sensitize the TME to immunotherapy.19,20 Drug repurposing remains an effective strategy for the development of novel therapies, including novel combinations for several diseases including cancer. Various studies, including ours, have successfully evaluated this strategy in different cancer models.21,22,23,24,25,26,27,28

In this study, for the first time, we evaluated the anti-cancer effects of MBO against PDAC. Our results demonstrated that MBO treatment exerts significant cytotoxicity against a broad range of PDAC cell lines. It is interesting to note that the IC50 of MBO in Capan-2 cells is ∼13 μM, whereas the IC50 of gemcitabine (first line of PDAC treatment) is ∼244 μM per reported studies. Patients with pancreatic cancer experience many side effects due to high doses of gemcitabine, and moreover become resistant to this therapy.29 Notably, our results show that MBO significantly suppressed the growth of gemcitabine-resistant Mia-Paca-2 (MiaPaCa-2 GR) cells. MBO does not show steep dose-response curves (window between inactive state and complete inhibition of cell viability), a desired trait for any anti-cancer drug.30 Similar to MBO, a few other anthelmintic drugs such as mebendazole, parbendazole, and pyrvinium pamoate have also shown similar activity against PDAC cell lines.30,31,32 However, some compounds such as pyrvinium show preferential toxicity. Moreover, pyrvinium was effective only under glucose-deprived conditions against PANC-1 cells, but it loses activity under nutrient-rich conditions. Such preferential activity is attributed to the presence of stem cell populations in cancer cells.33 In our study, the strong anti-clonogenic activity of MBO indicates its capacity to suppress the growth of cancer stem cells.34,35,36 However, further evaluation of MBO on various PDAC stem cells is warranted.

The arrest of PDAC cells by MBO treatment in G0/G1 phase confirms the anti-proliferative effects of MBO wherein MBO prevents the cells from moving past the restriction point, making the cells undergo apoptosis.37 The arrest in G0/G1 phase could also be an effect of cyclin D1 degradation by MBO.38 It has been observed that CDK4/6 inhibitors increase the compensatory anti-apoptotic mechanism in cells; therefore, combination with drugs such as MBO could synergistically enhance the therapeutic effects of CDK4/6 inhibitors in the PDAC model.39

ICD has recently gained prominence in cancer therapy. ICD activates the immune system to recognize and target cancer cells, presenting opportunities for combination with immunotherapies and personalized medicine while minimizing side effects.40,41 ICD is particularly characterized by the expression of damage-associated molecular pattern (DAMP) proteins.42 In ICD, proteins such as HSP90, calreticulin, and HSP70 are expressed on the cell surface along with secreted DAMPs such as HMGB1, ATP, annexin A1, and CXCL10 following endoplasmic reticulum stress.43 In our study, MBO treatment increased the expression of ICD markers HSP90 and calreticulin along with CXCL10 and apoptotic markers in PDAC cells. In congruence, other anthelmintic drugs such as rafoxanide and ivermectin have shown induction of ICD.44,45 In a study on colorectal cancer, rafoxanide showed induction of ICD followed by an immunological memory when re-challenged in the in vivo model.46 However, the effect of rafoxanide on immune cell infiltration was not reported in this study. Among the standard therapies in PDAC, oxaliplatin is a known ICD inducer. A study reported ICD induction in PDAC tumors by oxaliplatin nanoparticles along with higher dendritic cell maturation and CD8+ T cell infiltration.47 Only a limited number of US Food and Drug Administration (FDA)-approved anti-cancer agents induce ICD. This makes our study with MBO highly relevant for PDAC.

Increasing infiltration or proliferation of effector T cells within PDAC tumors is a critical therapeutic modality being explored clinically due to the fact that PDAC tumors are immunologically cold tumors.48,49 In our orthotopic tumor model, an increased pool of CD8+ T cells was observed in the tumors of MBO-treated mice. The increased number of CD8+ T cells might be due to the enhanced proliferation of tissue-resident T cells or to increased tumor infiltration. Additionally, the changes in intracellular signaling by MBO might have modulated the T cell infiltration. A study by Sivaram et al. has shown the role of PI3K signaling in PDAC immunogenicity.50 This study observed an increase in CD8 T cell infiltration in tumors following suppression of the PI3K/Akt pathway. Our RPPA analysis revealed the downregulation of PI3K/Akt signaling by MBO treatment. Various studies have established the role of STING in increasing immunogenicity in tumors. STING agonists have been shown to inflame the cold PDAC microenvironment and activate the anti-tumor immunity in mouse models.51 STING agonists are also known to generate a chemokine gradient such as CXCL9 and CXCL10 increase.52,53,54 Our results not only have shown the activation of the STING pathway but also increase in CXCL9 and CXCL10 by MBO treatment. Thus, the effect of MBO on signaling pathways and chemokine expression is similar to the observations made by other groups on increased CD8 T cell infiltration. MBO may provide advantage in therapy since the majority of the STING agonists need intratumoral injection, which is not feasible in the case of PDAC.

To establish the dependency of MBO on CD8+ T cells for tumor growth suppression, anti-CD8α antibody was used to deplete CD8+ T cells from the systemic circulation of tumor-bearing mice. The depletion of CD8+ T cells resulted in lower efficacy of MBO, indicating that the anti-tumor effects of MBO were due to the enhanced presence of CD8+ T cells. Similar to our studies, the anti-cancer activity of acarbose, an anti-diabetic drug, dependence on CD8+ T cells was observed.55

To understand the translational aspect of CD8+ T cell infiltration in clinic, the TIMER database was analyzed to correlate ICD with apoptosis. Analysis showed a strong positive correlation between the DAMP and apoptosis markers with CD8+ T cells within human PDAC tumors. These clinical data suggest that chemotherapy-induced immunogenic apoptosis might be beneficial as an adjuvant for immunotherapy since the dearth of immune cells within PDAC tumors has been a primary roadblock.

Considering the increased infiltration of CD8+ T cells by MBO treatment, anti-PD-1 therapy with MBO was evaluated in vivo. MBO treatment in combination substantially enhanced the tumor growth-suppressive effects of anti-PD-1 as compared to anti-PD-1 therapy alone, along with an enhanced population of active CD8+ T cells. Combination strategies are critical for PDAC, considering the disappointing results obtained in various clinical trials involving immune checkpoint proteins.56 Similar to our findings, drugs such as azelnidipine, niclosamide, albendazole, and metformin have increased the efficacy of ICI therapies.57,58,59 Ivermectin was shown to convert breast tumors from the cold to the hot immunophenotype, leading to an increased efficacy of anti-PD-1 therapy.13 Our results are in agreement with these studies and clearly show that MBO treatment converts the cold PDAC TME into a hot TME, leading to an increased abundance of CD8+ T cells.

Oral administration of 5 mg/kg MBO led to a significant suppression of pancreatic tumor growth in both subcutaneous and orthotopic models. MBO treatment extended the median survival of tumor-bearing mice by 27 days (36%) as compared to the vehicle-treated group, which translates to a survival advantage of 3 human years.60 Our findings are important considering the lower survival rate (11%) of patients with metastatic PDAC.18 Ivermectin, when administered in combination with anti-PD-1, increased the survival of 40% of mice13; however, it failed to increase the survival as a single agent. On the contrary, MBO treatment alone in our studies showed increased survival. In another study, 10 mg/kg ivermectin showed approximately 33% tumor suppression in subcutaneous models of PDAC,61 compared to 5 mg/kg MBO treatment in our studies with superior tumor growth inhibition. Overall, MBO might be a better option since we observed better therapeutic effects at a lower dose.

Following chronic administration of 5 mg/kg MBO for approximately 33 days in both subcutaneous and orthotopic models, the side effects of MBO were evaluated. MBO was found to be safe and without any signs of toxicity, compared to gemcitabine, which shows severe grade 3–4 adverse effects in PDAC patients. MBO in our studies was given at a dose of 5 mg/kg to mice, which is equivalent to 2.7 mg/kg in humans, using FDA-approved guidelines for calculating human equivalent dose. A recent study demonstrated 5 mg/kg MBO to be completely safe in dogs, which is equivalent to 33 mg/kg MBO in mice.62 Side effects such as platelet reduction, effects on aspartate aminotransferase/alanine aminotransferase levels are commonly observed with standard therapies; however, our toxicity studies on MBO did not exhibit any of these side effects in mice, indicating that it could also be relatively safe in humans, but more studies are required to substantiate this claim. MBO is not approved for human use yet, so our study is a proof-of-concept work and indicates its significant potential to be used in humans.

In summary, the immunomodulatory effects of MBO could be the consequence of suppression of the PI3K/Akt pathway along with STING activation and ICD resulting in increased tumor infiltration with CD8+ cells and reduced PDAC tumor growth. Nonetheless, more work is required to conclusively establish this assumption, and will remain the focus of our future studies.

Conclusion

Overall, our results demonstrated that MBO is a promising anti-cancer agent with potent efficacy against PDAC while also serving as a new immune adjuvant for improving the anti-tumor efficacy of anti-PD-1 therapy. The outcome of this study warrants clinical investigation with this combination therapy in patients with PDAC.

Materials and methods

Cell lines

Human PDAC cell lines AsPC1, BxPC3, PANC-1, MiaPaCa-22, SUIT-2, Capan-2 and MiaPaCa-2 GR, and murine pancreatic cancer cell line PO2 were procured and cultured as mentioned in our previous study.63 The PO2-Luc cell line was generated by us using purified hLUC-Lv105 lentiviral particles (GeneCopoeia) according to the manufacturer’s instructions. AsPC1 and BXPC3 cell lines were maintained in RPMI 1640, while all other cell lines were maintained in DMEM. Cell lines were supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin antibiotic mixture, 2 mM l-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 2.2 g/L sodium bicarbonate, and 10 mL/L glucose. All cell lines were maintained at 37°C and 5% CO2 in incubators.

Cytotoxicity assay

Cells were plated at a density of 3,000–4,000 cells per well in 96-well plates and allowed to attach overnight. After overnight incubation, cells were treated with different concentrations of MBO (MedChemExpress Chemicals) for 24, 48, and 72 h. Cells were then analyzed by SRB assay as described by us previously.64,65 All of the experiments were repeated independently at least three times.

Clonogenic assay

Briefly, 500 cells per well were seeded in a 6-well plate and incubated overnight. The cells were then treated with various concentrations (0–6 μM) of MBO for 48 h. After 48 h, the media was replaced with 2 mL fresh medium and incubated at 37°C with 5% CO2 for 14 days or until the cells in the control group formed large clones. The cells were then fixed with 10% trichloroacetic acid overnight and stained with SRB for 2 h.

Annexin-V/APC apoptosis assay

The assay was performed with an annexin V/APC-PI apoptosis detection kit (BioLegend). Briefly, 0.2 × 106 cells were treated with varying concentrations of MBO (0–10 μM) for 48 h. Cells were then evaluated for apoptosis according to the manufacturer’s instructions and analyzed by flow cytometer (BD Fortessa). All the experiments were repeated independently at least three times.

Cell-cycle analysis

Approximately 0.2 × 106 cells/well were seeded in a 6-well plate. After 24 h, cells were treated with different concentrations of MBO (0–8 μM). After 48 h, cells were collected and fixed with ice-cold ethanol (70%) for 24 h at 4°C and then stained with propidium iodide (PI) and analyzed using flow cytometry (BD Fortessa) as described by us previously.66 Approximately 2 × 104 cells were analyzed from each sample. Cell debris and clumps were excluded from the analysis in all samples.

ELISA

ELISA assays were performed for CXCL10, CXCL9, and IFN-γ, using the ELISA kit from BioLegend, according to the manufacturer’s instructions. PO2-Luc tumors were collected from control and MBO-treated groups at the end of the experiment (day 51). Tumors were lysed by using radioimmunoprecipitation assay buffer, and protein was estimated by using Bradford reagent (Bio-Rad). Equal amounts of protein (60–70 μg) from control and MBO-treated tumor lysate samples were used to perform the ELISA assays.

Animal studies

C57/BL6 male mice (4–5 weeks old) were purchased from Charles River Laboratories and Envigo RMS. Athymic nude male mice (5 weeks old) were purchased from Charles River Laboratories. All animals were housed at the animal core facility at the Texas Tech University Health Sciences Center (TTUHSC) Laboratory Animal Resources Center under Biosafety Level 2 (BSL2) conditions. All mice experiments were performed in compliance with the regulations of the Institutional Animal Care and Use Committee (IACUC) at the TTUHSC.

Subcutaneous implantation of pancreatic tumor xenografts

Briefly, 2 × 106 MiaPaCa-2 cells were implanted subcutaneously in the right flank of 6-week-old male athymic nude mice. Once the tumors reached 70 mm3 in size, mice were randomized into 4 groups—control (group 1), 2.5 mg/kg MBO (group 2), 5 mg/kg MBO (group 3), and 10 mg/kg MBO (group 4). Overall, 20 male athymic nude mice were used in this study, with each group containing 5 mice. MBO was administered daily through oral gavage until day 42. All the authors were aware of the group allocation in all stages of the experiment. Tumor volume was measured periodically using a Vernier caliper. Tumor measurements were calculated using the formula [V = ½ (Length × Width2)]. Mice were humanely sacrificed at day 42 due to tumor ulceration, mainly in the control group.

Orthotopic implantation of pancreatic tumors

C57/BL6 mice (4–6 weeks old) were used for the orthotopic implantation of pancreatic cancer cells. Mice were anesthetized by using isoflurane, and a minor incision was made in the left abdomen. Stably transfected luciferase expressing PO2 (PO2-Luc) cells were orthotopically implanted in the pancreas. A 20-μL PBS suspension containing 1 × 106 exponentially growing PO2-Luc cells was injected into the subcapsular region of the pancreas using a 30G sterile needle. The peritoneum and skin incisions were closed sequentially with absorbable sutures. Buprenorphine was administered to mice as a pain killer every 8 h for 2 days. The growth of orthotopically implanted tumors was monitored by measuring luminescence via IVIS (Caliper Life Sciences). To determine the basal luminescence value, mice were imaged on the same day after injecting luciferin (3 mg/kg per mouse, intraperitoneally [i.p.]). After 20 days post-tumor cell implantation, mice were randomly divided into 2 groups (control and treatment). The control group received vehicle only, whereas the treatment group received 5 mg/kg MBO by oral gavage every day. Mice weight was measured periodically. The experiment was terminated at day 51 by humanely euthanizing the mice using CO2 overdose. The pancreas/tumors from the control and treatment groups were aseptically excised out, weighed, and snap frozen for further analysis. A part of the tumor samples was used to evaluate MBO concentration using LC-MS/MS. Another portion of tumors from both control and MBO-treated mice was fixed in formalin for IHC and TUNEL analysis. The weight of excised organs such as liver, spleen, kidney, pancreas, lungs, and brain were recorded.

TIMER database analysis

Data on T cell infiltration was obtained from the TIMER database. The TIMER website (https://cistrome.shinyapps.io/timer/) is divided into seven modules. The first six modules present TCGA data and the last module provides quantitative analysis of the infiltration level of immune cells. Here, we investigated the correlations of HSP90, CALR, ANAX1, ANAX5, CASP3, CXCL9, and CXCL10 with CD8+ T cells using the TIMER algorithm.

Lymphocyte-depletion experiment

CD8+ T cells were depleted in PO2-Luc tumor-bearing mice by i.p. injecting 100 μL anti-CD8α antibody (clone 2.43, 10 mg/kg, Bio X Cell) on days 1, 3, 5, 8, and 12 post-tumor implantations. Four groups of mice were included in the analysis: group 1 (control) received PBS, group 2 received MBO, group 3 received anti-CD8α, and group 4 received MBO + anti-CD8α.

Combination study with anti-PD-1

The combination study of MBO and anti-PD-1 was performed by administering InVivoMAb anti-mouse PD-1 (CD279) from Bio X Cell. Anti-PD-1 antibody (clone RMP1-14, 10 mg/kg) was administered by i.p. injections 3 days after starting MBO administration and every seventh day until the end of the study.

Multi-color flow cytometry

For multi-color FACS, tumor tissues were harvested and minced. Single-cell suspensions were prepared by passing through a 70-μm cell strainer, washing twice with PBS, resuspending in FACS buffer, and counting by using the Vi-CELL XR Cell Viability Analyzer (Beckman Coulter). The cells were pre-incubated with purified anti-CD16/32 unconjugated antibody (clone 93) to block Fc receptors prior to surface staining with fluorochrome-conjugated anti-mouse monoclonal antibodies, which include APC/Cyanine7 anti-mouse T cell receptor β (TCR-β) chain (clone H57-597), PerCP/Cyanine 5.5 anti-mouse CD4 (clone GK1.5), PE (phycoerythrin)/Cy7 anti-mouse CD8α (clone 53-6.7), PE anti-mouse IFN-γ (clone XMG1.2), PE anti-mouse/rat/human FOXP3 (clone 150D), APC/Cyanine7 anti-mouse/human CD11b (clone M1/70), PE anti-mouse CD69 (clone H1.2F3), Alexa Fluor 700 anti-mouse CD62L (clone MEL-14), and Pacific Blue CD25 (clone PC61). Zombie Aqua Dye was used to stain dead cells as per the manufacturer’s instructions. Intracellular staining (FOXP3 and IFN-γ) was performed by following the FOXP3 intracellular staining protocol (eBioscience). To identify the population of functional T cells, single cells dissociated from tumor were seeded in a 96-well plate and stimulated with a Cell Activation Cocktail kit (catalog no. 423303, BioLegend) at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 50 U/mL penicillin, and 50 μg/mL streptomycin. After 5–6 h, stimulated cells were washed twice with PBS and processed. UltraComp eBeads (eBioscience) were used to prepare single-color compensation controls for each fluorescent conjugated antibody according to the manufacturer’s instructions. Data were acquired on BD Fortessa and analyzed with FlowJo software version 10.7.0. Representative gating strategies are provided in Figure S4. Briefly, following stimulation and staining, viable cells were separated using Amcyan gating. This was followed by sub-gating with TCR-β. TCR-β+ cells were further sub-gated to CD4 and CD8+ T cells. Following this, CD8 T cells were sub-gated into IFN-γ, CD69, CD25, PD-1, and IFN-γ+ populations. The CD4+ cell population was also analyzed similarly with the addition of a CD25+Foxp3+ population for identifying regulatory T cells.

IHC

Tumors were collected and perfused with 4% paraformaldehyde followed by dehydration to embed in paraffin. Paraffin-embedded tissues were sliced into 5-μm sections, deparaffinized in xylene (10 min × 2), followed by gradual rehydration using various concentrations of ethanol (100%, 90%, 80%, and 70%). Sections were left in distilled water for 5 min, followed by treatment with boiling sodium citrate for 20 min. After cooling down, sections were subjected to hydrogen peroxide to block the endogenous peroxidase for 5 min at room temperature. Sections were washed with PBS twice, then blocked with 5% BSA/PBS for 30 min, and incubated with primary antibodies against mouse CD8α (Cell Signaling Technologies) overnight at 4°C. The next day, primary antibodies were washed out with 0.05% Tween 20 in PBS and incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature. Sections were washed again with PBS, followed by dipping in a hematoxylin container for 1 min, washing with tap water for 5 min, and dehydrating with various ethanol concentrations (70%, 80%, 90%, and 100%). Finally, sections were treated with xylene and mounted in xylene-based media (Cytoseal XYL, Thermo Scientific).

Statistical analyses

All statistical analyses were done with Prism 8 GraphPad software version 9.4.1. To compare tumor growth kinetics, the level of multi-cytokines produced by stimulated T cells, and the difference in the organs’ weight in safety analysis, a two-way ANOVA test with an alpha level of 0.05 was used. To compare the difference in the immune cells’ composition of the TME and spleen between groups by multi-color flow cytometry, a one-way ANOVA test with an alpha level of 0.05 was applied. Survival data were analyzed by Kaplan-Meier survival curves, and comparisons were performed using the log-rank test. Statistical significance was considered at different p values and depicted as follows: ns, not significant; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001.

Data and code availability

Data are available upon reasonable request.

Acknowledgments

This work was supported in part by the Syngenta Fellowship Award in collaboration with the Society of Toxicology to S.G. The authors also appreciate the funding from the Dodge Jones Foundation, Abilene, TX, USA. We also thank the TTUHSC IACUC for their suggestions. All mouse experiments were performed in accordance with the regulations of the IACUC at the TTUHSC. We would like to thank Dr. Rajareddy Kallem from the LC-MS/MS lab at TTUHSC Dallas for conducting the LC/MS quantification of MBO.

Author contributions

Conception and design: S.G. and S.K.S.; development of methodology: S.G. and S.K.S.; acquisition of data: S.G.; analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.G. and S.K.S.; writing, review, and/or revision of the manuscript: S.G. and S.K.S.; administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.K.S.; study supervision: S.K.S. The authors have read and agreed to the published version of the manuscript.

Declaration of interests

The authors declare no competing interests.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2024.07.029.

Supplemental information

Document S1. Figures S1–S5
mmc1.pdf (1.8MB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (8.3MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S5
mmc1.pdf (1.8MB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (8.3MB, pdf)

Data Availability Statement

Data are available upon reasonable request.

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

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