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. Author manuscript; available in PMC: 2024 Apr 1.
Published in final edited form as: Biomaterials. 2023 Feb 8;295:122037. doi: 10.1016/j.biomaterials.2023.122037

Pharmacological ascorbate potentiates combination nanomedicines and reduces cancer cell stemness to prevent post-surgery recurrence and systemic metastasis

Xiaomin Jiang 1,†, Jianqiao Liu 1,†, Jianming Mao 1, Wenbo Han 1, Yingjie Fan 1, Taokun Luo 1, Junjie Xia 1, Morten Lee 1, Wenbin Lin 1,2,*
PMCID: PMC9998353  NIHMSID: NIHMS1875293  PMID: 36773429

Abstract

Conventional chemotherapy targets proliferative cancer cells to halt tumor progression or regress tumors. However, the plasticity of tumor cells enables their phenotypical changes to acquire chemo-resistance, leading to treatment failure or tumor recurrence after a successful treatment course. Here, we report the use of high-dose pharmacologic ascorbate to potentiate treatment efficacy of nanoscale coordination polymers (NCPs) delivering two clinical combinations of chemotherapeutics, carboplatin/docetaxel and oxaliplatin/SN38, and to target metabolic plasticity of tumor cells. Combination treatments of high-dose ascorbate and NCPs overcome multi-drug resistance by significantly reducing the abundance of cancer stem cells (CSCs) in solid tumors, as evidenced by reduced expression of tumor pluripotency factors. The clearance of CSCs inhibits post-surgery recurrence and systemic metastasis in multiple mouse models of cancer.

INTRODUCTION

Increasing evidence suggests that a small population of cancer cells can exhibit stem cell-like properties including expression of characteristic genes and the capacities for long-term self-renewal and repopulation. This population of tumor cells is termed cancer stem cells (CSCs) and has been identified in a variety of cancer types(1, 2). The existence of CSCs provides compelling explanations for many clinical observations, including chemo- and radio-resistance, metastasis and post-surgery recurrence(3–5). These stem cell-associated features are referred to as stemness, which is a fundamentally important indicator of malignancy and has strong association with poor therapeutic outcomes in multiple cancer types(6, 7).

Although traditional chemotherapy can efficiently eliminate bulk tumor cells and inhibit tumor progression, its selective stress often enriches resistant CSCs to mediate tumor regrowth and metastasis, leading to treatment failure. CSCs become resistant to conventional chemotherapy via their plastic phenotype, which adapts them to therapy through overexpression of anti-apoptotic factors(8), defense against oxidative stress(9), and efficient repair of DNA damages(10). Failure to eradicate these resistant CSCs significantly limits the response rate to chemotherapy in clinical practice and shortens the overall survival of cancer patients(3, 11). Thus, re-sensitizing CSCs to chemotherapy provides a promising approach to improve treatment outcomes.

Pharmacological ascorbate (AA) by intravenous injection of high-dose AA increases plasma concentration of AA to the millimolar level, and has been shown to kill cancer cells through disrupting reactive oxygen species (ROS) metabolism and inhibiting fermentative glycolysis(12). Notably, CSCs are intriguingly vulnerable to disruption of metabolic homeostasis as they rely on delicate balance of glycolysis and oxidative phosphorylation to maintain stemness(13). Modulating the energy supply in CSCs can disrupt the metabolism equilibrium and reduce self-renewal ability and resistance to chemotherapy(14, 15). In addition, we recently discovered that core-shell nanoscale coordination polymer (NCP) particles formed by coordination polymerization between Zn2+ and phosphate groups of a carboplatin (Carb) or oxaliplatin (OX) prodrug could be preferentially activated to generate Carb and OX active drugs inside cancer cells via reduction of the Pt(IV)-bis(carbamate) intermediates by ascorbate and other intracellular reductants(16, 17). Therefore, we hypothesized that high-dose AA could preferentially activate combination chemotherapies delivered by NCPs in cancer cells and promote metabolic status change of CSCs to overcome resistance to NCP combination chemotherapies and improve anticancer efficacy over current chemotherapy regimens.

Herein, we report tumor-responsive core-shell NCP particles which are versatilely engineered to deliver standard-of-care combinations of chemotherapeutics. The NCP core with a coordination polymer of Zn2+ and carboplatin (Carb) or oxaliplatin (OX) prodrug was further decorated with a lipid bilayer containing a cholesterol-docetaxel or cholesterol-SN38 conjugate to afford core-shell Carb/DTX or OX/SN38 NCP particles(18). We found that high-dose AA significantly potentiated the cytotoxicity of Carb/DTX or OX/SN38 due to enhanced reduction of the Pt(IV)-bis(carbamate) intermediates inside cancer cells. The combination of AA and NCP particles also reprogrammed CSC metabolism state by shifting from glycolysis to oxidative phosphorylation, causing disruption of mitochondrial dynamics and loss of self-renewal. The metabolic change conferred CSC vulnerability to chemotherapy, thus enhancing potency of Carb/DTX and OX/SN38 against resistant CSCs. Moreover, combination treatment of AA and NCP particles prohibited enrichment of CSCs caused by NCPs alone, as evidenced by significantly reduced expression of cancer pluripotency factors. As a result of CSC clearance, combination treatment of AA and NCP particles prevented post-surgery recurrence in a colon cancer model and inhibited systemic metastasis in an orthotopic breast cancer model.

RESULTS

Design and characterization of Carb/DTX and OX/SN38

Core-shell NCP nanoparticles Carb/DTX or OX/SN38 were designed for the co-delivery of Carb and DTX or OX and SN38 combination chemotherapies (Fig. 1A). The NCP core, Carb-bare or OX-bare, was synthesized by coordination polymerization between Zn2+ ions and Carb-biphosphate or OX-biphosphate prodrug in reverse emulsions in the presence of DOPA(17, 18). Zn2+ ions were used to coordinate to the Carb-biphosphate or OX-biphosphate prodrug because of their biocompatibility and minimal effects on mitochondrial function. The bare NCP particle was then coated with a lipid mixture of DOPC, cholesterol, DSPE-PEG2000 and the cholesterol-drug conjugate Chol-DTX or Chol-SN38 to afford core-shell Carb/DTX or OX/SN38 particle.

Figure 1. Synthesis and characterization of NCPs.

Figure 1.

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(A) Carb/DTX and OX/SN38 were synthesized via coordination polymerization in reverse emulsions and coating with lipid mixtures containing cholesterol-conjugated prodrugs. (B) Hydrodynamic diameters measured by DLS. (C) TEM images of Carb/DTX and OX/SN38. Scale bar, 50 nm. (D) Release of Carb from Carb/DTX at different pH values. (E) Release of DTX from Carb/DTX at different pH values.

Both Carb/DTX and OX/SN38 were monodispersed with a hydrodynamic diameter (Dh) of 64.3±0.5 nm and 77.4±0.8 nm, respectively, by Dynamic Light Scattering (DLS) (Fig. 1B). The monodispersed spherical morphologies of Carb/DTX and OX/SN38 were confirmed by transmission electron microscopy (TEM, Fig. 1C). Because of the low electron density of the lipid bilayer, only the electron-dense core is visible under TEM for Carb/DTX and OX/SN38. We then determined the drug loading ratios of Carb/DTX and OX/SN38 to be 1:1 and 1:2, respectively, by liquid chromatography-mass spectrometry (LC-MS) and inductively coupled plasma-mass spectrometry (ICP-MS).

To enable tumor-responsive controlled release, we used a pH-sensitive linker to conjugate DTX and SN38 to cholesterol (Fig. S1)(17). The pH-triggered release of Carb and DTX from Carb/DTX was quantified at pH=7.4 and pH=4.7 that correspond to blood circulation and acidic environment in late endosomes and lysosomes, respectively (Fig. 1D and 1E). NCP particles were shown to enter cancer cells via endocytosis (17, 19). Under acidic condition, the carbonate linkage of Chol-DTX was hydrolyzed to release DTX and carb-bisphosphate prodrug was hydrolyzed to form carb-biscarbamate (Fig. S2–3)(20). Carb-biscarbamate was reduced to Carb by intracellular reducing agents in the tumor environment.

Chol-DTX in Carb/DTX exhibited a long circulation half-life and excellent stability in plasma with a DTX exposure of only 0.6% of Chol-DTX (Fig. S4 and Table S1). Unlike a quick clearance of DTX in the bloodstream with a half-life of < 2.2 h(21), Carb/DTX prolonged the DTX half-life to 6.6±0.6 h as a prodrug form, Chol-DTX, which would be triggered to release DTX in the tumor. Similarly, free Carb was mainly cleared through kidney with a half-life < 3.2 h in the blood(22), while Carb/DTX enhanced the Pt prodrug circulation with a half-life 23.6±3.6 h. C arb/DTX also enhanced the AUC0-inf for >26 times and >170 times over free Carb and DTX, respectively(17). We previously reported the triggered release of OX and SN38 from OX/SN38 in tumor environments and prolonged blood circulation of both Pt and Chol-SN38 by OX/SN38(17). This prolonged prodrug circulation strategy with a tumor cleavable release linker design enhanced tumor deposition of drugs by 4–6 times to elicit better antitumor effects.

AA potentiates Carb/DTX and OX/SN38 against CSCs in vitro

As AA was previously reported to potentiate chemotherapy on bulk tumor cells(23), we first tested if AA could potentiate our combination NCPs on bulk CT26 and 4T1 cells. AA reduced the IC50 of Carb/DTX from 0.93±0.19 μM to 0.51±0.11 μM against bulk 4T1 cells and the IC50 of OX/SN38 from 0.27±0.04 μM to 0.11±0.01 μM against bulk CT26 cells (Fig. S5A, 5B). We then tested whether AA could enhance the cytotoxicity of NCPs against CSCs in vitro. We pretreated 4T1-CSCs and CT26-CSCs with 5 mM AA for 2 h, which did not cause cytotoxicity as confirmed by cell viability assays (Fig. S5C). We then treated 4T1-CSCs and CT26-CSCs with Carb/DTX and OX/SN38, respectively. We found that combination therapy of AA and Carb/DTX decreased the IC50 against 4T1-CSCs to 0.79±0.1 μM from 7.2±2.0 μM for Carb/DTX (Fig. 2A). The combination of AA and OX/SN38 decreased the IC50 against CT26-CSCs from 0.88±0.1 μM to 53.6±6.0 nM for OX/SN38 (Fig. 2B). To understand how AA affects the synergy of our combination NCPs, we quantified the combination indices of NCPs at various effective levels. We found that AA reduced the combination indices of Carb/DTX and OX/SN38 on 4T1-CSCs and CT26-CSCs, respectively, indicating stronger synergy between NCP-delivered chemotherapeutics in the presence of high-dose AA (Fig. S5D, 5E). In addition, combination treatments with 1 μM Carb/DTX and 5 mM AA induced stronger apoptosis over Carb/DTX alone, as evidenced by higher expression of Annexin V and increased permeability to propidium iodide (PI, Fig. 2C). 32.4% 4T1-CSCs treated with AA and Carb/DTX were Annexin V+ and PI+, while only 4.8% of Carb/DTX-treated 4T1-CSCs were Annexin V+ and PI+. These results suggest that AA can sensitize resistant CSCs to chemotherapy-induced apoptosis in vitro. We believe that high-dose AA potentiates the cytotoxicity of NCPs against CSCs via two different mechanisms. First, AA restores vulnerability of CSCs to chemoresistance by changing their metabolic phenotype. This was supported by the enhancement of free Carb cytotoxicity against 4T1-CSCs by AA (Fig. S5F). Pretreatment of 4T1-CSCs with 5 mM AA reduced the IC50 of free carb against 4T1-CSCs from 0.67±0.11 μM to 0.32±0.06 μM. Second, high concentrations of intracellular AA enhanced reduction of Pt(IV)-biscarbamate prodrugs to Pt(II) active drugs to potentiate the cytotoxicity of NCPs against CSCs(17).

Figure 2. AA enhances cytotoxicity of NCPs against CSCs.

Figure 2.

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(A) AA potentiates OX/SN38 against CT26-CSCs. (B) AA potentiates Carb/DTX against 4T1-CSCs. (C) AA and OX/SN38 combination induces stronger apoptosis of CT26-CSCs. (D) AA signficantly reduces stemness factors of CT26-CSCs upon treatment with OX/SN38. (E) AA signficantly reduces stemness factors of 4T1-CSCs upon treatment with Carb/DTX.

We then measured the expression of’Yamanaka’ factors Sox2 and Oct4, and the downstream marker Nanog, after treatments. These transcription factors are considered strong indicators for cancer cell stemness(24). We found that, Carb/DTX or OX/SN38 treatment significantly increased expression of these factors over PBS control; Carb/DTX increased Sox2, Oct4, and Nanog expressions of 4T1 cells by 12.3±1.1%, 21.2±2.9%, and 7.2±0.3%, respectively, whereas OX/SN38 treatment increased Sox2, Oct4, and Nanog expressions of CT26 cells by 5.0±0.8%, 9.4±1.9%, and 15.0±2.8%, respectively. These results indicate that our NCPs show strong cytotoxicity against bulk tumor cells, but also lead to the enrichment of CSCs after chemotherapy.

We next determined if the combination strategy could avoid CSC enrichment after NCP treatment. Interestingly, combination treatment of AA and Carb/DTX reduced the abundance of Sox2, Oct4, and Nanog in 4T1 cells by 62.8±7.4%, 48.9±8.4%, and 42.8±1.1%, respectively, when compared to treatment with Carb/DTX (Fig. 2D). Similarly, combination treatment of AA and OX/SN38 decreased the expressions of Sox2, Oct4, and Nanog in CT26 cells by 34.2±4.1%, 55.3±13.6%, and 41.0±13.0%, respectively, when compared to treatment with OX/SN38 (Fig. 2E). In comparison, AA treatment only slightly decreased the expressions of these factors over PBS control; AA decreased the expressions of Sox2, Oct4, and Nanog in 4T1 cells by 4.4±0.3%, 9.7±1.8%, and 17.5±0.7%, respectively, whereas AA decreased the expressions of Sox2, Oct4, and Nanog in CT26 cells by 8.7±1.3%, 9.7±1.9%, and 18.1±3.8%, respectively. These results confirm that combination treatments of AA and NCPs significantly reduce the abundance of CSCs in vitro. Taken together, our results show that chemotherapy by NCPs enriches CSCs and acquires stemness, while AA disrupts CSC homeostasis and decreases pluripotency. As a result, AA significantly potentiates the cytotoxicity of NCPs by preventing cancer cells from acquiring stemness and chemo-resistance.

AA promotes transition from glycolysis-addicted CSCs to oxidative phosphorylation phenotype

We then explored how AA selectively sensitized CSCs to chemotherapy. Given the pervasive relationship between metabolism and resistance, we investigated the metabolic phenotype of CSCs. As AA was suggested to deplete glutathione and raise intracellular ROS level(12), we first measured the intracellular ROS after AA treatment. We found that AA treatment increased the ROS level in both cultured cells (Fig. 3A) and solid tumors (Fig. S6). The intracellular ROS was found to oxidize and inactivate GAPDH (Fig. 3B), thereby inhibiting glycolysis(12). Glycolysis inhibition induced cell apoptosis in bulk tumor cells, which are predominantly dependent on glycolysis for energy as described by the Warburg effect. However, considering metabolic plasticity of CSCs, we then probed how CSCs would respond to glycolysis inhibition by AA. As the supplementary energy source, we hypothesized that oxidative phosphorylation (OXPHOS) would most likely be involved. To quantitatively analyze the OXPHOS rate, we measured the fluorescence lifetime of an endogenous fluorophore, flavin adenine dinucleotide (FAD), by fluorescence lifetime imaging microscopy (FLIM). When bound to enzymes (active OXPHOS), FAD exhibits shorter lifetime during decay (<0.1 ns). In contrast, in free state, FAD has longer lifetime of 2.3-2.9 ns and a lower OXPHOS rate. Interestingly, we found that the mean lifetime was shortened from 1.89 ns to 0.67 ns after AA treatment. This result suggests that CSCs efficiently transit to OXPHOS for energy supply (Fig. 3C and 3D). In contrast, when the glycolysis was inhibited in bulk tumor cells, they were not actively promoted to OXPHOS (Fig. S7), likely due to their low plasticity to metabolism inhibition.

Figure 3. AA transitions CSCs from glycolysis to OXPHOS and inhibits CSC self-renewal.

Figure 3.

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(A) AA increases ROS level in CT26-CSCs. (B) AA inhibits CT-CSCs glycolysis via decreasing GAPDH activity. (C,D) AA transitions glycolysis-addicted CT26-CSCs to OXPHOS phenotype as confirmed by shorter FAD fluorescence lifetime. Scale bar, 20 μm. (E) AA induces mitochondria elongation in CT26-CSCs. Scale bar, 20 μm. (F-G) CT26-CSCs lose self-renewal ability after disruption of mitochondria dynamics by AA.

CSC transition to OXPHOS suppresses self-renewal

We then evaluated how transition to OXPHOS affected CSCs’ response to chemotherapy. OXPHOS primarily occurs in mitochondria, a highly dynamic organelle that synchronizes with cell cycle and state. Specifically, chemoresistance can be mediated through dynamic mitochondrial fission and fusion(25). Thus, we first examined the mitochondrial morphology by confocal microscopy. We found that CSCs in their native state preferentially adopted a dynamic state with fragmented mitochondria, suggestive of an OXPHOS-quiescence state (Fig. 3E). However, transition to OXPHOS led to elongation of mitochondria, suggesting mitochondrial hyperactivity (Fig. 3E). We further determined if mitochondria over-fusion was companied by a mitochondrial membrane potential change. We measured the mitochondrial membrane potential via JC-1 staining. We found the mitochondrial membrane potential did not change (Fig. S8), suggesting the normal function of mitochondria. These results indicate that deprivation of glycolysis by AA does not affect the OXPHOS activity but changes mitochondria dynamics. As dynamic fission and fusion of mitochondria are closely linked to CSC fate and CSC-mediated chemoresistance, we determined the self-renewal ability of CSCs using spheroid assays. We found that treatment with 5mM AA for 2 h strongly inhibited the formation of tumor spheroids by CSCs, indicating their loss of self-renewal ability (Fig. 3F and 3G). These results suggest that AA reshapes CSC metabolism and confers CSC vulnerability to chemotherapy through disrupting mitochondrial dynamics. However, the molecular pathways by which AA affects mitochondria dynamics remain to be elucidated.

AA potentiates antitumor efficacy of NCPs and reduces tumor cell stemness in vivo

As CSCs have been considered as the progenitor cells in tumor progression, their eradication can lead to stronger inhibition of tumor growth. We determined whether AA could enhance chemotherapeutic effects of NCPs in vivo. To evaluate their anticancer efficacy, combination therapy strategy was designed for elicit synergy from multiple chemotherapeutics. As Carb and DTX are clinically used to treat triple negative breast cancer, we tested the efficacy of Carb/DTX and AA against on murine triple negative breast cancer model 4T1. Mice with subcutaneous 4T1 tumors were treated by intraperitoneal (i.p.) injection of high-dose AA (4 g/kg) twice daily, intravenous (i.v.) injection of Carb/DTX (5 mg/kg Carb equivalent) once every week, or a combination of AA (4 g/kg) and Carb/DTX (3 mg/kg Carb equivalent). Combination treatment of AA and Carb/DTX produced the greatest inhibition of 4T1 growth with a tumor growth inhibition index (TGI) of 94.1±3.0%, compared to a TGI of 82.5±3.0% for Carb/DTX alone. AA treatment alone slightly inhibited tumor growth with a TGI of 42.1±6.0%. This result demonstrates enhanced anticancer efficacy from combination treatment of pharmacological AA and Carb/DTX (Fig. 4A).

Figure 4. AA enhances the anticancer efficacy of NCPs in vivo.

Figure 4.

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(A) AA and Carb/DTX combination treatment against 4T1 tumors. n=6 (B) AA and OX/SN38 combination treatment against MC38 tumors. n=6 (C) AA and OX/SN38 combination treatment against CT26 tumors. n=6 (D, F, H) Immunofluorescence staining revealed decreased stemness markers after treatment with AA and Carb/DTX. Scale bar, 100 μm. (E, G, I) Reduction of 4T1 pluripotency factors in vivo after AA and Carb/DTX treatments assayed by flow cytometry.

As the combination of OX and irinotecan (which releases active SN38 in vivo) is a stand-of-care regimen for the treatment of colorectal cancer, we used OX/SN38 to treat subcutaneous CT26 and MC38 murine colorectal adenocarcinomas. Mice bearing subcutaneous tumors of CT26 and MC38 were treated by i.p. injection of high-dose AA (4 g/kg) twice daily, i.v. injection of OX/SN38 (3.5 mg/kg) once every three days, or a combination of AA (4 g/kg) and OX/SN38 (1.75 mg/kg) for 3-4 weeks, at which time the control group had to be euthanized due to its tumor size. We found OX/SN38 significantly inhibited the growth of CT26 and MC38 with TGIs of 77.1±6.0% and 69.0±11.0%, respectively (Fig. 4B, 4C and Fig. S9–10). AA treatment had modest inhibitory effects with TGI values of 35.0±9.0% and 43.9±13.0%, respectively. Combination treatment with AA and OX/SN38 showed significantly stronger anticancer efficacy against CT26 and MC38 even at a lower dose of OX/SN38, leading to TGI values of 95.9±3.0% and 87.3±4.0%, respectively. These results confirm the ability of pharmacological AA to significantly potentiate the antitumor efficacy of OX/SN38 against murine colorectal cancer.

We next determined the expression of Sox2, Oct4, and Nanog CSC pluripotency factors in 4T1 tumors by flow cytometry and immunofluorescence staining (Fig. 4D–I). We found a significant decrease of CSC pluripotency factors in tumors treated with AA and Carb/DTX (Fig. S11). While treatment by Carb/DTX increased Sox2, Oct4, and Nanog factors by 44.8±11.4%, 12.2±1.8%, and 6.2±1.0%, respectively, over PBS control, combination treatment with AA and Carb/DTX reduced the expressions of Sox2, Oct4, and Nanog factors by 63.0±13.8%, 63.7±23.0%, and 47.5±12.1%, respectively, compared with Carb/DTX. These results show that AA and NCP combination treatments effectively eliminate CSCs to enhance the antitumor effects of NCPs.

We also evaluated NCP delivery efficiency of therapeutics to the CSCs (Fig. S12). We used fluorescently labelled NCP with cholesterol-pyropheophytin (Chol-Pyro) as a surrogate for Chol-SN38 or Chol-DTX(17). We used flow cytometry to determine Pyro fluorescence signals (the APC channel) in tumor CSCs (Sox2+, Oct4+, or Nanog+ cells) from CT26 tumor-bearing BALB/c mice 24 h after intravenous injection of 100 μg of Pyro or Pyro NCP. Pyro NCP showed significantly higher Pyro mean fluorescent intensity (MFI) in CSCs than free Pyro, with 10.1-, 5.1-, and 4.6-folds higher MFI in Sox2+, Oct4+, and Nanog+ cells, respectively. These results show that NCPs efficiently deliver therapeutics to CSCs in vivo.

CSC eradication prevents post-surgery recurrence and systemic metastasis

CSCs are considered as the root cause of tumor relapse and metastasis. As combination treatments of AA and NCPs effectively reduced cancer cell stemness, we determined whether the combination treatments could inhibit post-surgery recurrence and prevent metastasis of cancer. As shown in Fig. 5A, we established the colorectal cancer resection model by subcutaneous inoculation of CT26 cells followed by surgical resection of established tumors(26). After surgery, the mice were treated by i.p. injection of high-dose AA (4g/kg) twice daily, i.v. injection of OX/SN38 (3.5 mg/kg OX equivalent) once every three days, or a combination of AA (4g/kg) and OX/SN38 (1.75 mg/kg OX equivalent). Tumor recurrence was monitored and measured by an electronic caliper. All mice in the PBS group showed tumor regrowth by Day 18 post surgery (Fig. 5B and Fig. S13). AA treatment did not inhibit tumor recurrence with 6 out of 6 mice showing tumor regrowth. OX/SN38 treatment slightly inhibited tumor regrowth with 4 out of 6 mice showing tumor recurrence. In contrast, combination treatment with AA and OX/SN38 completely inhibited tumor recurrence with no sign of tumor regrowth until the end of the study (Day 87 post tumor resection). As expected, inhibition of tumor recurrence led to increase of mouse survival. While AA treatment only prolonged the median survival from 54.5 days to 70 days, OX/SN38 increased the median survival from 54.4 days to 80.5 days in the resection model (Fig. 5C). Combination treatment with AA and OX/SN38 increased the median survival to >100 days as no mouse showed any sign of tumor regrowth on Day 100. These results indicate that AA effectively cleared CT26-CSCs to enhance the therapeutic effect of OX/SN38 and prevent tumor recurrence. Combination treatment of AA and OX/SN38 is highly clinically relevant as many colorectal cancer patients experience tumor recurrence after successful post-surgery adjuvant chemotherapy.

Figure 5. AA and NCP combination treatments prevent post-surgery relapse and inhibit systemic metastasis.

Figure 5.

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(A) Establishment of CT26 tumor resection model. (B, C) AA and OX/SN38 combination treatment prevents post-surgery recurrence (B) and prolongs mouse survival (C). (D-G) AA and Carb/DTX combination treatment inhibits the growth of orthotopic 4T1 tumors (D) and reduced lung metastasis nodules (D-G). Scale bar, 300 μm. (I) AA and Carb/DTX combination treatment reduced pluripotency factors of 4T1 tumors in vivo.

We also treated tumors before surgical resection to evaluate the clearance of tumor CSCs by neoadjuvant combination treatment with AA and NCPs. As shown in Fig. S14, CT26 tumor-bearing mice were treated with AA, OX/SN38, or OX/SN38+AA for 3 doses before surgical removal of tumors at Day 13. Tumor recurrence was monitored, and the recurred tumors were measured with an electronic caliper. All mice in the PBS and OX/SN38 group showed tumor regrowth by Day 15 post surgery. OX/SN38 treatment showed no difference from PBS treatment. AA-treated mice also had tumor recurrence, but the growth of tumors was retarded with a TGI of 43.3%. In contrast, combination treatment with AA and OX/SN38 completely inhibited tumor recurrence with no sign of tumor regrowth. The results strongly indicate that AA and NCP combination treatment in both neoadjuvant and adjuvant settings can efficiently inhibit tumor recurrence after surgery.

We next examined if combination treatment of AA and NCPs could inhibit cancer metastasis using an orthotopic 4T1 model of TNBC which has been shown to readily metastasize to major organs including lungs and livers. Treatment of AA and Carb/DTX inhibited the growth of 4T1 primary tumors with TGI values of 47.5±11.0% and 86.1±4.0%, respectively (Fig. 5D, Fig. S15). Combination treatment of AA and Carb/DTX significantly inhibited the growth of 4T1 primary tumors with a TGI of 95.5±1.0%. We then evaluated the metastasis to lungs (Fig. 5E–H). AA treatment had little effect on 4T1 cancer metastasis into the lungs, reducing the average number of metastatic nodules from 25.3±11.0 to 23.5±11.0 at the PBS endpoint (Day 18 post treatment). Carb/DTX treatment moderately inhibited 4T1 cancer metastasis with an average number of metastatic nodules of 9.8±5.0. In contrast, combination treatment of AA and Carb/DTX greatly suppressed 4T1 cancer metastasis with an average number of metastatic nodules of 1.5±2.0. We further quantified the metastatic area in the lungs using H & E staining (Fig. 5E–F). AA treatment slightly reduced the metastatic area by 14.5±6.0% compared to PBS control. Carb/DTX treatment decreased the metastatic area by 81.8±9.0% while combination treatment of AA and Carb/DTX decreased the metastatic area by 96.7±4.3%. Taken together, AA potentiates the antitumor efficacy of Carb/DTX by clearing 4T1-CSCs to prevent cancer metastasis. Consistent with the results on the CT26 model, treatment of Carb/DTX increased the expressions of Sox2, Oct4, and Nanog stemness factors by 10.4±2.9%, 28.0±7.2%, and 23.4±6.2%, respectively, over the PBS control. Combination treatment of AA and Carb/DTX significantly reduced Sox2, Oct4, and Nanog expressions by 75.5±13.2%, 79.0±18.6%, and 78.1±26.1%, respectively, from the Carb/DTX group (Fig. 5I), confirming the ability of AA to clear CSCs in the orthotopic 4T1 model. The clearance of CSCs significantly reduced systemic metastasis of 4T1 cancer.

DISCUSSION

Although the failure of chemotherapy to eradicate tumors is attributed to many factors, a major contributor is the heterogeneity of oncogenic phenotypes and cellular hierarchies derived from CSCs. Targeting CSC populations has been proved to be challenging because of their resistance to conventional therapies. Stem cell biology has motivated the evaluation of many new treatment strategies. Metabolism plasticity has been noted as an essential feature of CSC differentiation in the evolving models of cancer hallmarks(27). Although all cancer cells display dysregulated metabolism, the differential growth pattern of CSCs indicates that this subpopulation adopts distinguishable metabolic profiles from bulk tumor cells(28, 29). Metabolic intervention of CSCs not only abolishes their ability of sustained proliferation, but also blocks the change of oncological phenotype(14).

Nanomedicines have been widely explored to preferentially deliver drugs to tumors by elongating blood circulation, avoiding clearance, and extravasation into tumor tissues(30–39). As a result, nanomedicines can more potently induce tumor plasticity than conventional chemotherapeutics. Here we show that high-dose AA targets CSC metabolic plasticity to improve two clinically relevant combination chemotherapeutic nanoparticles, Carb/DTX and OX/SN38. Particularly, high-dose AA metabolically differentiates CSCs to reduce their chemoresistance, resulting in strong inhibition of primary tumor progression, post-surgery relapse, and systemic metastasis. However, the underlying pathway(s) that account for epigenetic reprogramming remain to be elucidated. Although high-dose AA has been used to enhance chemotherapy in several clinical investigations(40, 41), our preclinical results suggest that pharmacological AA not only improves therapeutic efficacy of NCPs against primary tumors, but also overcomes the major limitations of conventional chemotherapy, such as drug resistance and tumor recurrence.

Beyond the improvements to chemotherapy, our study also provides potential explanations for many clinical observations. First, immunomodulation function of high-dose AA can be mediated through CSC vulnerability. Recently, Magri et al. showed that AA modulated antitumor immune responses by enhancing the activity of CD8+ T cells and synergized with immune checkpoint blockade in several cancer types(42). It is known that CSCs express low levels of antigens, which inhibits T cell response as a consequence of inefficient antigen presentation(43). Our data suggest that AA can metabolically change CSCs phenotype. However, whether AA can interfere CSC interactions with T cells and/or relieve inhibition on T cell activity remains to be studied. Second, AA-mediated mitochondria fission inhibition may play a role in reducing chemotherapy-associated toxicity. Ma et al. previously reported AA significantly decreased grade 1/2 toxicity caused by chemotherapy in a phase 1/2a clinical trial(23). Recent studies showed that chemotherapy toxicity could be ameliorated, at least partly, via inhibiting mitochondria fission in major organs(25, 44). Our results suggest that AA’s influence on mitochondria dynamics may be in part responsible for protecting patients from chemotoxicity. This effect on major organs needs further investigations.

MATERIALS AND METHODS

Synthesis of Carb/DTX and OX/SN38

Carb-bare and OX-bare were synthesized according to our previous reports(20). Briefly, an aqueous solution of Carb-biphosphate or OX-biphosphate prodrug (30 mg, 150 mg/mL) was added to a 5 mL of 0.3 M Triton X-100/1.5M 1-hexanol in cyclohexane and stirred vigorously for 15 min in the presence of 1,2-dioleolyl-sn-glycero-3-phosphate (sodium salt). An aqueous solution of Zn(NO3)2 (60mg, 600 mg/mL) was added to a 5 mL of 0.3M Triton X/1.5M 1-hexanol in cyclohexane and stirred vigorously for 5 min. The Zn2+ containing microemulsion was added dropwise to the Pt-containing microemulsion and stirred vigorously for 30 min at room temperature. After addition of 10 mL of ethanol, the bare particles were collected by centrifugation at 12000 rpm for 20 min. The resulting pellet was washed twice with THF/ethanol and finally redispersed in THF. Carb and OX loadings in particles were determined by ICP-MS (Agilent 7700×, Agilent Technologies, United States) after digestion in nitric acid.

Carb/DTX and OX/SN38 were prepared by adding a THF solution of 1,2-dioleolyl-sn-glycero-3-phosphocoline, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethylene glycol)-2000], Chol-drug conjugates (3:3:1.5:1), and bare particles to 500 μL of 30% (v/v) ethanol/water at room temperature. The THF and ethanol were evaporated in a 40°C water bath while stirring. The hydrodynamic size was measured by DLS using a Zetasizer (Nano ZS, Malvern, United Kingdom). Particle morphology was visualized by TEM (Tecnai Spirit, FEI, United States). The chol-DTX/SN38 loading was determined with LC-MS (Agilent 6540, Agilent Technologies, United States) after de-emulsification with Triton X and extraction with ethyl acetate.

In vitro cytotoxicity

Murine carcinoma cells CT26, MC38, and 4T1 were purchased from American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) or RPMI-1640 (Gibco, Grand Island, NY, United States), supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium and 100 g/mL streptomycin sulfate.

CT26-CSC and 4T1-CSC were separated from inoculated tumor on mice as previously described (45). Briefly, the tumor was harvested, digested into single-cell suspension, and filtered with 40 μm strainer. The cells were cultured in CSC tumorsphere culturing medium consisting of DMEM/F-12 supplemented with B27 supplements (1×), epidermal growth factor (20 ng/mL, PeproTech), basic fibroblast growth factor (20 ng/mL, PeproTech), and penicillin-streptomycin (1×), in ultralow attachment culture dish (Corning, United States). The cells were cultured to form tumor spheres with spheroid morphology under serum-free medium, and the obtained tumorsphere cells were used for further experiments.

CT26(-CSC) cells or 4T1(-CSC) cells were seeded in 96-well plates at a density of 2,000 cells/well and allowed to adhere for 24 hours. Cells were then pretreated with 5mM of AA for 2 h and replaced treatment to different concentrations of OX/SN38 or Carb/DTX. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay (Promega, Madison, MI) according to manufacturer’s instructions.

ROS generation

CT26 or CT26-CSC cells were treated with 5 mM AA for 2 h, then incubated with 10 μM H2DCFDA (Thermal Fisher, United States) for one additional hour. The cells were collected, washed twice with ice-cold PBS, and analyzed by flow cytometry.

Depolarization of mitochondria membrane potential

CT26-CSC cells were seed in six-well plate at a density of 5 × 104 cells/well, and then treated with 5 mM AA for 2 h. The cells were stained with 10 μM JC-1 (Abcam, United Kingdom) in their cultured state for 30 additional minutes. The cells were collected, washed twice with ice-cold PBS, and analyzed by flow cytometry.

Fluorescence lifetime imaging microscopy

CT26 and CT26-CSCs were seeded on a 25 mm2 cover glass at a density of 5 × 104 cells/well. The cells fully adhered and spread in 2 h when plated in complete medium, which is necessary for measuring their metabolic states and keeping their original stem or differentiated status. We conducted fluorescence lifetime imaging (FLIM) of the endogenous fluorophore FAD, a metabolic cofactor with short or long lifetimes reflecting its mitochondria protein bound (OXPHOS) or unbound (glycolysis), respectively. We performed all studies with Leica Stellaris 8 Laser Scanning Confocal Microscopy. To image the samples, an excitation wavelength 440 nm emitted from a white light laser was used with a repetition rate of 16 MHz. Each plane was scanned for 32 times using a 63 /1.4 UV oil immersion objective. A Notch filter centered at 440 nm was applied to minimize laser scattering to the detector.

Fitting of FLIM images was performed with the FLIMfit, an open-source software tool developed by Imperial College London. The instrument response function (IRF) was measured by imaging a blank sample with the same settings as data acquisition and then using the ‘Estimate IRF’ function within the FLIMfit software. Fitting of the fluorescence images was then performed pixelwise with a biexponential model, corresponding to two states of FAD, on all pixels above an intensity threshold of 10 photons. The intensity-weighted mean lifetime was calculated for each pixel.

Apoptosis assay

4T1-CSCs cells seeded in 6-well plate (5 × 104 cells/well) were pretreated with 5 mM AA and then replaced with 1 μM of Carb/DTX for 24 h. The cells were harvested, washed twice with ice-cold PBS, and stained with Alexa Fluor 488-Annexin V and propidium iodide for 15 min at room temperature. The cells were finally analyzed by flow cytometry (LSR II, BD, United States).

GAPDH activity assay

Tumorsphere cultured CT26-CSC cells were seeded at a density of 200,000 cells per well. 24 h later, the media was replaced with media containing 5 mM AA. After 2 h incubation, cell lysates were prepared, and GAPDH activity was determined by assaying the rate of NADH oxidation, which is proportional to the increase in absorbance at 450 nm over time. The detailed protocol can be found in the manufacturer’s instructions (Sigma Aldrich, Cat. No: MAK277).

Tumorsphere assay

For tumorsphere culture of CT26-CSC cell lines, 2000 cells were seeded into each well of 96-well ultralow attachment plate in 200 μL of serum-free medium which contains DMEM/F12 (1:1), epidermal growth factor (EGF, 20 ng/ml), basic fibroblast growth factor (bFGF; 20 ng/ml) (both from PeproTech, Rocky Hill, NJ, USA), 1 × B27 supplement (Thermal Fisher, United States) and 1 % P/S. Tumorspheres were counted under an optical microscope.

Mitochondrial morphology assessment

4T1 or 4T1-CSCs cells were seeded in a glass-bottom dish (Thermal Fisher, United States) at a density of 5 × 104 cells/mL. The cells were allowed to adhere for 24 h and then treated with 5mM AA for 2 h. After treatment, fresh medium was replaced and 100nM MitoTracker Orange was added to the culture medium, and incubated for 30 min. Nuclei were then stained with Hoechst 33342. Confocal images were obtained with live cells using Leica SP8 confocal laser microscope.

Animals and tumor models

BALB/c mice (female, 6 weeks, 18-22 g) and C57BL/6 (female, 6 weeks, 18-22 g) were provided by Harlan Laboratory, Inc (United States). All the animals were treated according to the Guide for Care and Use of Laboratory Animals, approved by the Institutional Animal Care and Use Committee at the University of Chicago.

2 × 106 CT26/MC38/4T1 cells were subcutaneously inoculated into the right flank regions of 6-week-old BALB/c or C57BL/6 mice. The tumors were allowed to grow for 7 days, when the tumors reached ~100 mm3. Carb/DTX was i.v. administered 5mg/kg once per week and OX/SN38 was i.v. administered once every 3 days. AA was i.p. administered 4g/kg twice per day. For combination treatment, Carb/DTX was lowered to 3mg/kg and OX/SN38 was lowered to 1.5mg/kg. Tumor size was measured and calculated according to the following formula: width2 × length × 0.5.

To establish orthotopic 4T1 model, 2 × 106 4T1 cells suspended in ice-cold PBS were inoculated into the mammary fat pads of 6-week-old female BALB/c mice. When tumor reached ~100 mm3, treatment began with the same dosing schedule as above.

To establish the tumor resection model, 2 × 106 CT26 cells suspended in ice-cold PBS were inoculated into the right flank of BALB/c mice (female, 6 weeks, 18-22 g). When tumor reached ~500 mm3, mice were randomly divided into five groups (n = 6) and tumors were resected, leaving ~1% residual tumor to mimic residual micro tumors after surgery. Briefly, mice were anaesthetized in an induction chamber using isoflurane (up to 5% for induction; 1–3% for maintenance), and anesthesia was maintained via a nose cone. Sterile instruments were used to remove ~99% of the tumor. Immediately after surgery, treatment began with the same dosing schedule as above.

Flow cytometry analysis of pluripotency factors

For in vitro quantification, the single-cell suspension is obtained from trypsinization. To quantify the expression of stemness-associated transcription factors, the cells were permeated with Triton-X (0.05%) and stained with Sox2, Oct4, and Nanog antibodies according to the manufacturer’s protocol (Invitrogen, Thermal Fisher Scientific). The stained cells were analyzed with flow cytometry (LSR II, BD). For in vivo studies, the tumors were collected from the mice, cut into small pieces, digested with collagenase, and filter with a membrane filter (pore size, 40 μm) to obtain single-cell suspensions.

NCPs delivery to CSCs

Two million CT26 cells suspended in ice-cold PBS were inoculated into the right flanks of BALB/c mice (female, 6 weeks,18-22 g). When the tumors reached ~100 mm3, the mice were randomly divided into 2 groups (n = 3). The tumors were excised at 24 h after intravenous injection of 100 μg of Pyro or Pyro NCP. Single-cell suspensions were obtained by cutting tumors into small pieces, digesting with collagenase, followed by membrane filter filtration. The cells were permeated with Triton-X (0.05%) and stained with Sox2, Oct4, and Nanog antibodies according to the manufacturer’s protocol (Invitrogen, Thermal Fisher Scientific). For flow cytometry, the Pyro fluorescence intensity of stained cells was gated on Sox2+, Oct4+, and Nanog+ cells.

H & E staining

The tumors and organs were collected from the mice and fixed with paraformaldehyde (4%). The tissues were embedded in paraffin and sectioned into 5-μm-thick slices. The tissue slices were stained with hematoxylin and eosin, then visualized with a Panoramic MIDI II Digital Slide Scanner in the Integrated Light Microscopy Core at the University of Chicago.

Immunofluorescence staining

Tumors were collected from the mice and snap-frozen in optimal cutting temperature medium. Tumors were then sectioned into 5-μm-thick slices, mounted on slides and stained with different primary antibodies: Sox2, Oct4, and Nanog before analyzing with a confocal microscope. All antibodies were diluted 40 times in the experiments.

Statistical analysis

All results are shown as mean ± s.d. The GraphPad Prism software 8.0 was used for all statistical analysis. Two-tailed Student’s t-test was used for statistical comparison between two groups. One-way analysis of variance (ANOVA) with a Tukey post hoc test was used for statistical comparison among multiple (more than two) groups. The threshold for statistical significance is defined as * p<0.05, **p<0.01, ***p<0.001.

Supplementary Material

1

Acknowledgements

We acknowledge the National Cancer Institute (1R01CA216436 and 1R01CA223184) for funding support.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Credit Author Statement

X.J., J.L., and W.L. conceived the project. X.J, J.L., J.M., W.H., Y.F., T.L., J.X., and M.L. performed the experiments and analyzed the results. X.J, J.L., and W.L. wrote the manuscript.

Competing Interests

W.L. is founder of Coordination Pharmaceuticals, which licensed the NCP technology from the University of Chicago. Other authors declare no competing interests.

Data availability

The authors declare that all the data supporting the findings of this study are available within the article and its Supplementary Information files or from the corresponding author upon reasonable request.

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Associated Data

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Supplementary Materials

1
NIHMS1875293-supplement-1.pdf (1.9MB, pdf)

Data Availability Statement

The authors declare that all the data supporting the findings of this study are available within the article and its Supplementary Information files or from the corresponding author upon reasonable request.

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