Ultrathin metal-organic layer-mediated radiotherapy-radiodynamic therapy enhances immunotherapy of metastatic cancers

SUMMARY

Checkpoint blockade immunotherapy (CBI) is effective in promoting a systemic immune response against some metastatic tumors. The reliance on the pre-existing immune environment of the tumor, however, limits the efficacy of CBI on a broad spectrum of cancers. Herein, we report the design of a novel nanoscale metal-organic layer (nMOL), Hf-MOL, for effective treatment of local tumors by enabling radiotherapy-radiodynamic therapy (RT-RDT) with low-dose X-rays and, when in combination with an immune checkpoint inhibitor, regression of metastatic tumors by re-activating anti-tumor immunity and inhibiting myeloid-derived suppressor cells. Owing to the reduced dimensionality, nMOLs allow facile diffusion of reactive oxygen species and exhibit superior RT-RDT effects. The synergy of Hf-MOL-enabled RT-RDT immune activation and anti-programmed death ligand 1 (anti-PD-L1) CBI led to robust abscopal effects on a series of bilateral models of colon, head and neck, and breast cancers and significant anti-metastatic effects on an orthotopic model of breast cancer.

eTOC blurb

The authors report the design of novel nanoscale metal-organic layers to enhance radiotherapy-radiodynamic therapy using low-dose X-rays by facilitating the diffusion of reactive oxygen species. In combination with an immune checkpoint inhibitor, robust abscopal effects were observed on a series of bilateral models of colon, head and neck, and breast cancers. The combination treatment also exhibited strong anti-metastatic effects on an orthotopic model of breast cancer by re-activating anti-tumor immunity and inhibiting myeloid-derived suppressor cells.

Graphical Abstract

INTRODUCTION

Advanced tumors escape immune surveillance by dysregulating cell signaling pathways,^1,2 hijacking immunosuppressive cells/cytokines,^3,4 and deactivating effector cells/molecules.⁵ Targeting T cell inhibitory checkpoint signaling pathways, such as programmed cell death protein 1 (PD-1) and its ligand (PD-L1), with monoclonal antibodies has recently provided a promising strategy for tumor-specific immunotherapy.⁶⁻⁸ Several anti-PD-1 and anti-PD-L1 antibodies have been approved by the US Food and Drug Administration for the treatment of a subset of immunogenic tumors, including non-small cell lung cancers,^9,10 melanomas,^11,12 and genitourinary cancers.^13,14 Immune checkpoint inhibition, however, only elicits a durable response in a minority of cancer patients due to the reliance on high expression of PD-L1 on tumor cells and/or pre-existing tumor-infiltrating CD8⁺ T cells.¹⁵⁻¹⁷ Immunomodulatory adjuvant treatments are thus actively examined in combination with checkpoint inhibitors to overcome immune tolerance and potentiate anti-tumor immunity in the host system.¹⁸⁻²²

Radiotherapy (RT) is an effective local treatment clinically used on a broad spectrum of cancers.²³ Over a hundred clinical trials are currently evaluating hypofractionated, high-dose RT as an immunomodulatory adjuvant treatment to enhance checkpoint blockade immunotherapy (CBI) of a variety of cancers.^24,25 The reported RT-induced upregulation of PD-(L)1 in tumors supports the combination of anti-PD-(L)1 checkpoint blockade with hypofractionated RT.²⁶⁻²⁸ However, toxicity from high doses of X-ray radiation and nonsynchronous dosing regimens between RT and CBI present major hurdles for optimizing the synergy between these two treatment modalities.²⁹⁻³¹

We recently disclosed the enhancement of radiotherapeutic effects of X-rays using nanoscale metal-organic frameworks (nMOFs) via a unique radiotherapy–radiodynamic therapy (RT–RDT) mechanism of action.^32,33 Built from non-toxic heavy metal-oxo cluster secondary building units (SBUs) and photosensitizing bridging ligands, nMOFs increase hydroxyl radical (·OH) production (RT) via enhanced X-ray absorption and enable singlet oxygen (¹O₂) generation (RDT) via energy transfer from SBUs to photosensitizers. Such nMOF-enabled RT–RDT processes not only significantly reduce X-ray doses needed to eliminate irradiated tumors in mouse models but also potentiate CBI to consistently inhibit/regress distant, un-irradiated tumors by eliciting robust abscopal effects at X-ray doses used in conventional fractionated RT.³² We hypothesized that the RT-RDT effects of metal-organic nanomaterials could be further enhanced if the generated reactive oxygen species (ROS, including ·OH and ¹O₂) can readily diffuse into cell organelles and the nanomaterials could evenly distribute in tumors.^34,35 To that end, we herein report the design of nanoscale metal-organic layers (nMOLs), a two-dimensional (2D) version of nMOFs, for enhanced RT-RDT and cancer immunotherapy.³⁶

Hf-DBP (DBP = 5,15-di(p-benzoato)porphyrin) nMOL, Hf-MOL, was designed by combining electron-dense Hf₁₂O₈(OH)₁₄ SBUs as X-ray absorbers for ·OH generation and porphyrin-based photosensitizer bridging ligands for ¹O₂ production. Ultrathin Hf-MOL (1.6 nm thick) possess superior RT-RDT effects over previously reported nMOFs due to better tumor distribution and more facile ROS diffusion. Hf-MOL-enabled RT-RDT in conjunction with an anti-PD-L1 antibody not only eradicated local tumors but also rejected/regressed distant tumors through systemic anti-tumor immunity on several syngeneic bilateral tumor models, including CT26 colorectal cancer, squamous cell carcinoma (SCC) VII, and 4T1 triple-negative breast cancer. Furthermore, the synergistic combination of Hf-MOL-enabled RT-RDT and CBI eliminated lung metastases on a 4T1 orthotopic model by re-activating anti-tumor immunity and inhibiting myeloid-derived suppressor cells (MDSCs). We have thus developed a novel X-ray based local treatment strategy for the systemic rejection of primary and metastatic tumors in mouse models.

RESULTS

Synthesis and characterization of Hf-MOL

Hf-MOL was synthesized through a solvothermal reaction between HfCl4 and H₂DBP in N,N-dimethylformamide (DMF) at 80 °C with acetic acid (AcOH) and water as modulators. Hf-MOL is constructed from Hf₁₂(μ₃-O)₈(μ₃-OH)₈(μ₂-OH)₆ SBUs and DBP bridging ligands as a monolayer with an infinite 2D network of kagome dual (kgd) topology. Hf-MOL monolayers are vertically capped by acetate groups (via coordination to the Hf₁₂ SBUs) to afford a molecular formula of Hf₁₂(μ₃-O)₈(μ₃-OH)₈(μ₂-OH)₆(DBP)₆(AcO)₆ (Figure 1C). Transmission electron microscopy (TEM) imaging showed a flat-plate morphology of Hf-MOL with a diameter of ~150 nm (Figure 1A and Figure S1) and a thickness of ~1.6 nm by atomic force microscopy (AFM, Figures 1G–H, and Figure S2), consistent with the modeled height of Hf₁₂ SBUs capped with acetate groups (Figure S3). High resolution TEM (HRTEM) images of Hf-MOL, in which Hf₁₂ SBUs appear as black spots, and fast Fourier transform (FFT) patterns of the HRTEM image revealed six-fold symmetry that is consistent with its kgd topology (Figure 1B–C).

Figure 1. Characterization of Hf-MOL and Hf-MOF.

(A) TEM image, (B) topological structure and (C) HR-TEM image of Hf-MOL with its fast Fourier transform (FFT) pattern shown in inset of (C). (D) TEM image, (E) topological structure and (F) HR-TEM image of Hf-MOF with its FFT pattern shown in inset of (F). (G) AFM topography and (H) height profile of Hf-MOL. (I) Number-average diameter of Hf-MOL (black line) and Hf-MOF (red line) in water (n = 3). (J) AFM topography and (K) height profile of Hf-MOF. (L) ¹O₂ generation was detected using SOSG in water with Hf-MOL (black line) or Hf-MOF (red line). Scale bar = 200 nm (A, D and J), 5 nm (C), and 20 nm (F and G).

As a control, the 3D Hf-DBP nMOF (Hf-MOF) was also synthesized through a solvothermal reaction between HfCl₄ and H₂DBP as reported previously.³⁷ Hf-MOF displayed a 3D crystalline nanoplate morphology with a diameter of ~100 nm and a thickness of 15–50 nm (Figures 1D–F and Figures S4–5 and S7). Dynamic light scattering (DLS) measurements gave number-average sizes of 59.2 ± 0.6 nm and 80.6 ± 4.2 nm for Hf-MOL and Hf-MOF, respectively (Figure 1I). Singlet oxygen sensor green (SOSG) indicated ~1.5 times higher ¹O₂ presence in solutions of 2D Hf-MOL upon light irradiation over 3D Hf-MOF under identical conditions, likely due to enhanced diffusion of generated ¹O₂ which facilitates reaction with SOSG (Figure 1L).

To further evaluate the RDT effects of Hf-MOL, the previously reported nMOF, Hf-DBA [DBA = 2,5-di(p-benzoato)aniline], was synthesized and used as a control.³⁸ Hf-DBA is constructed from non-photosensitizing DBA bridging ligand and the same Hf₁₂(μ₃-O)₈(μ₃-OH)₈(μ₂-OH)₆ SBU and enhances RT only.³⁸ TEM and HRTEM imaging showed that crystalline Hf-DBA exhibited a crystalline thin plate-like morphology with a diameter of approximately 70 nm (Figures S6–7).

In vitro anti-cancer effect of Hf-MOL-enabled RT-RDT

We first demonstrated the stability of Hf-MOL in physiological environments. The powder X-ray diffraction (PXRD) patterns of Hf-MOL incubated in either 6 mM phosphate buffered saline (PBS) solution or serum for up to 10 days were identical to that of the pristine sample (Figure 2A), indicating the stability of Hf-MOL in physiological environments. This was further confirmed by the TEM image of Hf-MOL incubated in 6 mM PBS (Figure S8). In cell culture, both Hf-MOL and Hf-DBA were efficiently taken up by 4T1 cells, reaching similar intracellular Hf levels after 4 h by inductively coupled plasma-mass spectrometry (ICP-MS, Figure S9).

Figure 2. Stability of Hf-MOL in physiological environments and in vitro RT-RDT effects.

(A) PXRD patterns of Hf-MOL samples that were freshly prepared or incubated in 6 mM PBS for 2 or 10 days or serum for 5 days. Clonogenic assays to evaluate radioenhancement of Hf-DBA and Hf-MOL on 4T1 cells upon (B) orthovoltage X-ray and (C) ⁶⁰Co γ-ray irradiation (n = 6). (D) Cytotoxicity of Hf-DBA, H₂DBP, or Hf-MOL upon X-ray irradiation at a dose of 2 Gy on 4T1 cells (n = 6). (E) Annexin V/PI cell apoptosis/death analysis of 4T1 cells. Cells were incubated with PBS, Hf-DBA, H₂DBP, or Hf-MOL upon X-ray irradiation at a dose of 2 Gy. The quadrants from lower left to upper left (counter clockwise) represent healthy, early apoptotic, late apoptotic, and necrotic cells, respectively. The percentage of cells in each quadrant is shown on the graphs. (F) γ-H2AX assays showing DSBs in 4T1 cells treated with Hf-DBA, H₂DBP or Hf-MOL or PBS upon X-ray irradiation. Scale bar = 50 μm. (G) ¹O₂ generation was detected using SOSG in live cells treated with Hf-DBA, H₂DBP or Hf-MOL DBP or PBS upon X-ray irradiation by CLSM. Scale bar = 20 μm. (H) Representative images of crystal violet-stained invading 4T1 cells after treatment with PBS, Hf-DBA, H₂DBP or Hf-MOL and irradiated upon X-ray by transwell invasion assay. Scale bar = 50 μm.

Clonogenic assays were performed on cells first treated with Hf-MOL or Hf-DBA at a Hf concentration of 20 μM for 4 h followed by irradiation with either X-ray or ⁶⁰Co isotope γ-ray source at 0–16 Gy. The irradiated cells were cultured for an additional 10 days to form visible colonies, which were counted to determine the survival fraction. The radiation enhancement factor at 10% survival dose (REF₁₀) was calculated as the ratio of equivalent irradiation doses needed to give 10% survival rate for the PBS control group over that for the experimental group. At the same Hf concentration, Hf-MOL outperformed Hf-DBA with an REF10 value of 2.29 compared to 1.45 for Hf-DBA upon X-ray radiation (Figure 2B). Upon irradiation with γ-rays from a ⁶⁰Co source, Hf-MOL exhibited an REF₁₀ value of 1.45 compared to 1.25 for Hf-DBA on 4T1 cells (Figure 2C). This result suggests that Hf-MOL can be activated with γ-rays from linear accelerators commonly used in the clinic for RT treatment. For convenience, we denote PBS, Hf-MOL, H₂DBP, or Hf-DBA plus X-ray radiation as PBS(+), Hf-MOL(+), H₂DBP(+), or Hf-DBA(+), respectively, and PBS, Hf-MOL, or Hf-DBA without X-ray radiation as PBS(−), Hf-MOL(−), H₂DBP(−), or Hf-DBA(−), respectively.

MTS assays further showed that Hf-MOL(+) exhibited much higher acute cytotoxicity than Hf-DBA(+) and H₂DBP(+). At 2 Gy, the IC₅₀ value for Hf-MOL against 4T1 cells was calculated to be 8.18 ± 3.15 μM while IC₅₀ values for H₂DBP and Hf-DBA both exceeded 100 M (Figure 2D). No cytotoxicity was observed without irradiation for any group (Figure S10). The colony and MTS results indicate higher radioenhancing effects of Hf-MOL over Hf-DBA, likely due to the RT-RDT effect and the enhanced ROS diffusion in Hf-MOL. Cell death pathways were investigated with an Annexin V/Cell death kit. Significant amounts of cells underwent apoptosis/necrosis when treated with Hf-MOL(+) with only 38.2% healthy cells, compared to 90.2% and 86.5% healthy cells for H₂DBP(+) and Hf-DBA(+), respectively (Figure 2E). More than 90% cells remained healthy in dark controls and PBS(+) group, indicating that Hf-DBA, H₂DBP, and Hf-MOL are not intrinsically cytotoxic and low dose X-ray elicits negligible cytotoxicity with PBS treatment (Figure S11). These results were further confirmed by confocal laser scanning microscopy (CLSM) which showed the majority of cells after Hf-MOL(+) treatment were apoptotic with red and green fluorescence (Figure S12).

We have previously shown that nMOFs assembled from heavy metal clusters and photosensitizing bridging ligands can absorb X-ray energy to enhance RT by generating hydroxyl radicals (·OH) and elicit RDT by transferring energy from heavy metal SBUs to ligands to sensitize the generation of ¹O₂.³⁸ We thus performed DNA double-strand break (DSB) quantification to determine the RT effects from generated ·OH. Phosphorylated γ-H2AX was immunostained to quantify the DSBs in cells treated with PBS, Hf-DBA, H₂DBP, or Hf-MOL at an X-ray dose of 0 or 2 Gy to evaluate the RT enhancing effect. 6 h after irradiation, significantly higher red γ-H2AX fluorescence was observed in the group treated with Hf-MOL(+) than that treated with Hf-DBA(+), while no fluorescence was observed in groups either without X-ray irradiation or without Hf-based nanoparticle (Figure 2F and Figure S13). Flow cytometric analyses further showed that cells treated with Hf-MOL(+) exhibited stronger red γ-H2AX fluorescence than Hf-DBA(+) treatment, confirming the stronger RT enhancement of Hf-MOL (Figure S14). ¹O₂ generation was determined to probe the Hf-MOL enabled RDT process. No green fluorescence was detected in PBS(+), Hf-DBA(+), or H₂DBP(+) treated 4T1 cells or in the Hf-MOL(−) treatment group after co-cultured with SOSG. In contrast, 4T1 cells treated with Hf-MOL(+) presented strong green fluorescence in CLSM images, indicating the generation of ¹O₂ in Hf-MOL-enabled RDT process (Figure 2G and Figure S15).

Transwell invasion assay was then performed to evaluate the anti-migration effect of RTRDT treatment. Upon X-ray irradiation, Hf-MOL treatment represses invasion of 4T1 cells compared with other groups (Figure 2H and Figure S16). Taken together, Hf-MOL(+) elicits strong anti-cancer effects via a distinct RT-RDT mechanism of action through a combination of electron-dense Hf₁₂-based SBUs and porphyrin-based photosensitizing bridging ligands. The ultrathin 2D structure of Hf-MOL facilitates ROS diffusion to further enhance both acute and chronic cell death. Hf-MOL(+) treatment also exhibits anti-migration effect on tumor cells, prompting us to examine its ability to prevent metastasis of 4T1 tumor cells to distant sites.

Immunogenicity

Immunogenicity Calreticulin (CRT) is a chaperone protein abundant in the endoplasmic reticulum (ER). CRT is transposed to the cell surface in response to ER stress, providing an indicator for immunogenic cell death (ICD). CRT exposure on the cell membrane serves as an “eat-me” signal for macrophages and immature dendritic cells (DCs) to engulf dying tumor cells and their apoptotic debris. High mobility group box-1 (HMGB-1), a nucleosome-associated chromatin binding protein released from dead cells to interact with toll-like receptor-4 on DCs for DC maturation and tumor-associated antigen presentation, serves as another marker of ICD. We thus investigated ICD induced by Hf-MOL(+) treatment via detecting cell-surface exposure of CRT by flow cytometry and CLSM as well as HMGB-1 release and adenosine triphosphate (ATP) excretion by enzyme-linked immunosorbent assay (ELISA).³⁹ As shown in Figure 3A, quantitative flow cytometry analysis demonstrated that both Hf-DBA(+) and Hf-MOL(+) treated groups showed high cell surface CRT expression. The Hf-MOL(+) treated group exhibited higher CRT fluorescence than the Hf-DBA(+) treated group, suggesting that Hf-MOL(+) enabled RT-RDT induced more ICD than Hf-DBA(+)-mediated RT. CRT exposure on cell surface was further confirmed by CLSM imaging. Stronger green fluorescence was observed in the group treated with Hf-MOL(+) than in the groups treated with PBS(+), H₂DBP(+), and Hf-DBA(+), which well merged with CellMask, a red fluorophore labeling cell membranes. The CLSM results support higher immunogenicity induced by Hf-MOL(+) treatment (Figure 3B and Figure S17).

Figure 3. In vitro immunogenic cell death and in vivo cancer vaccination studies.

In vitro CRT exposure on the cell surface of 4T1 was assessed after incubation with PBS, Hf-DBA, H₂DBP, or Hf-MOL upon X-ray irradiation at a dose of 2 Gy by (A) flow cytometry and (B) immunofluorescence microscopy. (+) and (−) refer to with and without irradiation, respectively. (C) HMGB-1 release from the cells incubated with PBS, Hf-DBA, H₂DBP, or Hf-MOL with or without X-ray irradiation at a dose of 2 Gy (n = 3). (D) ATP extracellular secretion from the cells incubated with PBS, Hf-DBA, H₂DBP, or Hf-MOL with or without X-ray irradiation at a dose of 2 Gy (n = 3). (E) Volumes of challenge tumors (n = 6). 4T1 cells treated with Hf-MOL or PBS upon X-ray irradiation in vitro were inoculated subcutaneously in BALB/c mice. After 7 days, mice were challenged with live 4T1 cells.

To further validate ICD, HMGB-1 and ATP excretion were examined by ELISA. Compared to groups treated with either Hf-DBA(+) or H₂DBP(+), cells treated with Hf-MOL(+) showed higher excretion of HMGB-1 and ATP, providing further support to more ICD by Hf-MOL(+) treatment (Figures 3C–D). We then performed an anti-tumor vaccination experiment to confirm the ICD induced by Hf-MOL(+)-treated cells in vivo. 4T1 cells incubated with Hf-MOL followed by irradiation with X-rays were inoculated into BALB/c mice as a tumor vaccine. Seven days later, the mice were challenged with live 4T1 cells by subcutaneous transplantation to the contralateral flanks. As shown in Figure 3E, mice receiving Hf-MOL(+)-treated 4T1 cells were protected against challenge with live 4T1 cells. This result indicates that Hf-MOL(+) treatment induced strong ICD in 4T1 cells and Hf-MOL(+)-treated cells acted as an effective vaccine against live tumor cells in immunocompetent mice.

Abscopal effects of Hf-MOL-enabled RT-RDT plus immune checkpoint blockade

After demonstrating the anti-cancer effects of Hf-MOL(+) and its ability to induce ICD in vitro, we combined Hf-MOL(+) with CBI to extend the local RT-RDT treatment to systemic cancer management. We immunostained the tumor slices of 4T1-bearing mice intratumorally injected with Hf-MOL or PBS followed by X-ray irradiation to examine PD-L1 expression after nMOL-mediated RT-RDT treatment. The higher signal of the PD-L1 marker on the cell surface after Hf-MOL(+) treatment supports the strategy to combine local RT-RDT and systemic anti-PD-L1 CBI (Figure S18). A bilateral model of 4T1 tumor was then established to assess the systemic anticancer efficacy of Hf-MOL(+) in combination with anti-PD-L1 CBI. Hf-MOL was intratumorally injected to the primary tumors at a dose of 0.11 mg/mouse on days 10 and 14 post tumor inoculation, with daily X-ray irradiation at a dose of 1 Gy/fraction (225 kVp, 13 mA, 0.3 mm Cu filter) beginning on day 10 for a total of 8 fractions. 75 μg of anti-PD-L1 antibody (α-PD-L1, Clone: 10F.9G2, Catalog No. BE0101, BioXCell) was administered every three days by intraperitoneal injection for a total of 3 doses. As shown in Figures 4A–C, PBS(+) and PBS(−) groups did not show any difference in tumor growth, indicating that low dose X-rays alone had no radiotherapeutic effects. Anti-PD-L1 plus fractionated X-ray irradiation [α-PD-L1(+)] moderately delayed 4T1 tumor progression on both primary and distant tumors. Hf-MOL(+) alone regressed local tumors, but only moderately delayed the growth of distant tumors. The combination of Hf-MOL(−) and anti-PD-L1 [Hf-MOL(−)/α-PD-L1] showed similar modest inhibition of both primary and distant tumors as anti-PD-L1 alone. In contrast, the combination treatment of Hf-MOL(+) and anti-PD-L1 [Hf-MOL(+)/α-PD-L1] significantly regressed both primary and distant 4T1 tumors, indicating a strong synergy between Hf-MOL(+) and α-PD-L1. Histological analysis showed that Hf-MOL(+) caused apoptosis/necrosis in local tumors, while only Hf-MOL(+)/α-PD-L1 treatment afforded the highest levels of apoptosis/necrosis in the unirradiated distant tumor (Figure S19). In addition, no weight loss or obvious histopathological changes in the main organs were observed in the treated groups, indicating the absence of general systemic toxicity (Figures S20–21). After treatments described in Figures 4A–C, four out of six mice had both of their primary and distant tumors completely eradicated, affording a cure rate of 66.7%. Tumors in the other two mice shrank to minute sizes, but eventually regrew.

Figure 4. Abscopal effect of Hf-MOL-enabled RT-RDT on 4T1 bilateral model.

Tumor growth curves of (A) primary tumors and (B) distant tumors of 4T1 bilateral tumor-bearing mice treated with PBS (with or without X-ray irradiation), Hf-MOL upon X-ray irradiation (with or without anti-PD-L1 antibody), anti-PD-L1 antibody with X-ray irradiation or Hf-MOL plus anti-PD-L1 antibody without X-ray irradiation (n = 6). (C) Tumor weights with representative optical images of tumors sectioned from groups in (A) & (B) shown in inset. Top row: primary tumors; bottom row: distant tumors. From left to right: PBS(−), PBS(+), α-PD-L1(+), Hf-MOL(+), Hf-MOL(−)/α-PD-L1 and Hf-MOL(+)/α-PD-L1. Tumor growth curves of (D) primary tumors and (E) distant tumors of 4T1 bilateral tumor-bearing mice with T cell or B cell depletion and treatment with Hf-MOL plus anti-PD-L1 antibody upon X-ray irradiation (n = 6). (F) Growth curves of challenged tumor on tumor-free mice treated with Hf-MOL(+)/α-PD-L1 (n = 4). Treatment began on day 10 after tumor inoculation when the tumor reached a volume of 100–150 mm³. X-ray irradiation was carried out on mice 12 h after the i.t. injection of PBS or Hf-MOL on eight consecutive days at a dose of 1 Gy/fraction (120 kVp, 20 mA, 2-mm Cu filter). Antibody was given intraperitoneally every three days at a dose of 75 μg/mouse. Black, red, and green arrows refer to the times of PBS or Hf-MOL injections, X-ray irradiation, and antibody administration, respectively. (G) ELISPOT assay was performed to detect tumor-specific IFN-γ producing T cells. 10 days after the first treatment, the splenocytes were harvested and co-cultured with X-ray irradiated 4T1 cells as stimulation for 42 h. (I-M) The primary and distant tumors were collected from tumor-bearing mice with as-mentioned treatments for flow cytometry analysis and the percentages of tumor-infiltrating CD45⁺ cells (H), CD8⁺ T cells (I), CD4⁺ T cells (J), B cells (K) and NK cells (L) with respect to the total tumor cells. (+) and (−) refer to with and without irradiation, respectively. Data are expressed as means ± s.d. (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 by t-test. Central lines, bounds of box and whiskers represent mean values, 25% to 75% of the range of data and 1.5 fold of interquartile range away from outliers, respectively.

Tumor challenge and T/B cell depletion studies 30 days post tumor eradication, treated mice and naïve control mice were challenged with 2 × 10⁶ 4T1 cells subcutaneously. None of the treated mice developed tumors on the left flank within 27 days post tumor inoculation when the distant tumors of the control mice reached 2cm³ (Figure 4F). This result indicates that tumor specific immune memory was generated in mice after tumor eradication by the combination of Hf-MOL(+) and α-PD-L1.

We further confirmed immune involvement in the therapeutic effects of Hf-MOL(+)/α-PD-L1 by depleting CD4⁺ T cells, CD8⁺ T cells, or B cells. Mice receiving intraperitoneal injections of anti-CD4 (GK1.5, BioXCell), anti-CD8 (53–6.7, BioXCell), mouse IgG antibodies (MOPC-21, BioXCell) or B cell inhibitor ibrutinib (PCI-32765, Selleckchem) were treated simultaneously with Hf-MOL(+)/α-PD-L1. As shown in Figure 4D, mouse IgG did not have any effect on tumor growth, but T/B cell depletion groups all showed rapid tumor growth after cessation of Hf-MOL(+)/α-PD-L1 treatment. Significantly, all groups inhibiting the immune system showed no abscopal effect on distant tumors (Figure 4E). These results indicate that T and B cells play essential roles in the anticancer efficacy of both local RT-RDT treatment and distant abscopal effects. The depletion studies thus support the immunotherapic effect of the combination treatment and the rationale of using Hf-MOL(+) to potentiate CBI.

Abscopal effects of Hf-MOL-enabled RT-RDT plus immune checkpoint blockade

The inhibition of distant tumors and the rejection of challenged tumors suggest the effective induction of a systemic antitumor immune response. We first determined the presence of tumor-antigen specific cytotoxic T cells with an IFN-γ Enzyme-Linked ImmunoSpot (ELISPOT) assay. On day 10 after the first treatment, splenocytes were harvested from 4T1-bearing mice and stimulated with X-ray irradiated 4T1 cells for 42 hours and the IFN-γ spot forming cells were counted with an Immunospot Reader. The number of antigen-specific IFN-γ producing T cells per 10⁶ splenocytes significantly increased in tumor-bearing mice treated with Hf-MOL(+) and Hf-MOL(+)/α-PD-L1 (64.2 ± 33.7 and 108.3 ± 27.5 compared to 8.6 ±11.6 for PBS(−), Figure 4G), suggesting that Hf-MOL(+) effectively generates a tumor-specific T cell response. We further profiled infiltrating leukocytes in both primary and distant tumors. There was no significant difference between PBS(+) and PBS(−) treatment groups, demonstrating that low-dose X-ray irradiation did not influence the immunological environment of 4T1 tumors. Hf-MOL(+)/α-PD-L1 treatment group showed significant increase of tumor-infiltrating CD45⁺ leukocyte cells, CD4⁺ T cells and CD8⁺ T cells in both primary and distant tumors (Figures 4H–J). Specifically, after treatment with Hf-MOL(+)/α-PD-L1, the percentages of CD8⁺ T cells in the total primary and distant tumor cells significantly increased to 1.3 ± 0.6 % and 1.3 ± 1.0 % from 0.3 ± 0.2 % and 0.4 ± 0.6 % in PBS(−) group, respectively (Figure 4I). Hf-MOL(+)/α-PD-L1 group also showed significant increase of tumor-infiltrating B cells and NK cells (Figure 4K–L) in both tumors. These results suggest that the combination of Hf-MOL(+) and α-PD-L1 not only induces innate immune response but also augments tumor-specific adaptive response in both local, irradiated and distant, un-irradiated tumors.

Efficacy of Hf-MOL-enabled RT-RDT in other synergistic tumor models

The triple negative breast cancer 4T1 model has moderate PD-L1 expression and resistance to RT. To demonstrate the efficacy of Hf-MOL(+) andα-PD-L1 on a broad spectrum of cancers, we further evaluated the anti-tumor activity on a bilateral CT26 colorectal tumor model in Balb/c mice with high PD-L1 expression and low resistance to RT and a bilateral SCC VII squamous cell carcinoma tumor model in C3H mice with low PD-L1 expression and high resistance to RT. Similar tumor regression was observed in both CT26 and SCC VII tumor models after combination treatment with Hf-MOL(+) and α-PD-L1 (Figures S21–22). Tumor growth inhibition indices, defined as [1-(mean volume of treated tumors/mean volume of control tumors)]×100%, of primary and distant tumors are 99.5% and 98.0% for CT26 and 94.7% and 92.2% for SCC VII tumor models, respectively, which are significantly higher than control groups (Table 1). In addition, several other checkpoint inhibitors were tested in combination with Hf-MOL(+) to explore broader applications of RT-RDT in potentiating different CBIs. Hf-MOL(+) significantly enhanced the therapeutic efficacy of anti-PD-1 antibody (α-PD-1, Clone: RMP1–14, BioXCell) and anti-CTLA-4 antibody (α-CTLA-4, Clone: 9D9, BioXCell) on CT26 and SCC VII tumor models, leading to effective regression of both primary and distant tumors (Figure S22–23 and Table 1). These results demonstrate that Hf-MOL(+) synergizes with multiple checkpoint inhibitors to afford robust abscopal effects on several different mouse tumor models with varied immunogenicity, suggesting the potential of using Hf-MOL(+) to significantly boost the therapeutic efficacy of checkpoint blockade immunotherapies on a broad spectrum of cancers.

Table 1.

Tumor growth inhibition indices (TGIs) of CT26 and SCC VII tumor models with different treatments.

TGI (%)	CT26		SCC VII
	Primary	Distant	Primary	Distant
PBS(+)	6.1	4.8	0.3	0.0
α-CTLA-4(+)	49.6	25.3	-	-
α-PD-l(+)	28.0	16.6	42.0	38.0
α-PD-Ll(+)	30.6	44.4	-	-
Hf-MOL(+)	95.9	36.3	92.7	26.3
Hf-MOL(+)/α-CTLA-4	96.1	95.6	92.3	91.6
Hf-MOL(+)/α-PD-1	99.4	91.2	88.2	84.8
Hf-MOL(+)/α-PD-Ll	99.5	98.0	94.7	92.2

Anti-metastatic effect

We then investigated the antitumor activity and anti-metastatic effect of Hf-MOL(+) in combination with α-PD-L1 on an orthotopic 4T1 tumor model. 4T1 cells were implanted into the mammary fat pads of immunocompetent BALB/c mice and allowed to form primary breast tumors of sizes of 100–150 mm³ in volume. Hf-MOL at a dose of 0.11 mg/mouse was intratumorally injected followed by daily X-ray irradiation at a dose of 1 Gy/fraction (225 kVp, 13 mA, 0.2 mm-Cu filter) for a total of 8 fractions. Anti-PD-L1 antibody was administered every three days at a dose of 75 μg/mouse for a total of 3 doses. Body weights were monitored daily and no systemic toxicity was observed (Figure S24). As shown in Figure 5A–C, α-PD-L1 (+) moderately delayed 4T1 tumor progression whereas Hf-MOL(−) showed no effect on tumor growth. In comparison, Hf-MOL(+) significantly inhibited tumor growth initially, but tumors regrew 5 days after cessation of treatment. Notably, Hf-MOL(+)/α-PD-L1 treatment nearly eradicated primary 4T1 tumors with a TGI of 98.9% on day 17 post treatment. Combination treatment thus markedly improved the therapeutic efficacy over Hf-MOL(+) or α-PD-L1 alone. Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay showed that the Hf-MOL(+)/α-PD-L1 group induced the most DNA fragmentation and apoptosis (Figure 5D), supporting its superior anticancer efficacy over monotherapy controls.

Figure 5. Anti-metastasis effect of Hf-MOL-enabled RT-RDT on 4T1 orthotopic model.

Tumor growth curves of 4T1 orthotopic tumor-bearing mice treated with PBS (with or without X-ray irradiation), Hf-MOL upon X-ray irradiation (with or without anti-PD-L1 antibody), anti-PD-L1 antibody with X-ray irradiation or Hf-MOL plus anti-PD-L1 antibody without X-ray irradiation. (B) Tumor weights and (C) optical images of tumors sectioned from groups in (A) at the endpoint. Treatment began on day 7 after tumor inoculation when the tumor reached a volume of 100–150 mm³. X-ray irradiation was carried out on mice 12 h after the i.t. injection of PBS or Hf-MOL on ten consecutive days at a dose of 0.5 Gy/fraction (225 kVp, 13 mA, 0.3-mm Cu filter). Antibody was given intraperitoneally every three days at a dose of 75 μg/mouse. Black, red, and green arrows refer to the times of PBS or nanoparticles injections, X-ray irradiation, and antibody administration, respectively. n = 6. (D) TUNEL immunofluorescence staining of excised tumor slices for PBS (−), PBS (+), anti-PD-L1 (+), Hf-MOL (+), Hf-MOL plus anti-PD-L1 (−) or Hf-MOL plus anti-PD-L1 (+). (+) and (−) refer to with and without irradiation, respectively. Scale bar = 100 μm. (E) Representative pictures showing the gross appearance of tumor nodules in the lungs. Scale bar = 5000 μm. (F) Representative lung sections stained with H&E. Scale bar = 5000 μm. (G) Representative pictures showing the colonies formed after culturing in the presence of 6-thiogunine for 10 days. (H) The numbers of tumor nodules present in the lungs. (I) Percentage of metastasis area in lung. (J) Normalized absorbance of crystal violet in different treatment groups. *P < 0.05, ** P < 0.01, *** P < 0.001.

The anti-metastatic effect was evaluated by examining lung tissues for tumor nodules at the end of the study. As reported previously,^40,41 4T1 cells can rapidly metastasize to the lungs from the mammary fat pad. Compared to the PBS control group, Hf-MOL(+) or α-PD-L1 alone showed little effect preventing lung metastasis as shown in Figure 5E. In contrast, Hf-MOL(+)/α-PD-L1 significantly reduced tumor nodules in the lungs, resulting in a 93.3% decrease in the number of macroscopically visible pulmonary metastases. Examination of the lungs revealed that mice treated with PBS had an average of 39.7 ± 8.34 visible lesions while mice treated with Hf-MOL(+)/α-PD-L1 exhibited an average of 2.7 ± 0.6 macroscopic lung metastases. The proportion of the metastasis area relative to the whole lung was further quantified by H&E staining. As shown in Figures 5F–G, Hf-MOL(+) or α-PD-L1 alone only slightly suppressed spontaneous metastasis with metastatic nodules covering 34.5 ± 5.3 % and 21.8 ± 4.8 % of the lung, respectively, compared to nodules covering 42.7 ± 5.8% of the lung in the PBS group. Hf-MOL(+)/α-PD-L1 treatment on the other hand significantly decreased the presence of lung metastasis to 2.2 ± 1.6%. Combination treatment with Hf-MOL(+)/α-PD-L1 is thus much more effective in preventing lung metastasis than Hf-MOL(+) or α-PD-L1 alone.

We then performed colony assays to quantitatively assess lung metastases after various treatments. At the end of the treatment, lungs were digested and the cells were harvested and cultured in the presence of 60 μM 6-thioguanine for 10 days. After being fixed with methanol, colonies formed by clonogenic metastatic cancer cells were stained with 0.1% crystal violet. Because 4T1 tumor cells are resistant to 6-thioguanine, only metastasized tumor cells can proliferate to form colonies. As shown in Figure 5H, only the Hf-MOL(+)/α-PD-L1 treatment significantly reduced the number of colonies, decreasing the absorbance of crystal violet to only 10.0% of the PBS control group (Figure 5I). Hf-MOL(+) or α-PD-L1 treatment alone reduced the absorbance of crystal violet to 84.5% or 74.3% of the PBS control group, respectively. Clonogenic assay results indicate that mice treated with Hf-MOL(+)/α-PD-L1 have far fewer clonogenic metastatic 4T1 cells in the lungs than other treatment groups. We also examined metastases in other major organs as shown in Figure S25. The Hf-MOL(+)/α-PD-L1 group significantly reduced the number of macroscopically visible cardiac and hepatic metastases compared to the control groups.

Anti-metastatic mechanism

In order to understand the anti-metastatic mechanism, we examined the anti-tumor immunity of orthotopic 4T1 tumor-bearing mice treated with Hf-MOL(+)/α-PD-L1 by ELISPOT and immunostaining flow cytometry. We first determined the presence of tumor-antigen specific cytotoxic T cells with an IFN-γ ELISPOT assay. On day 12 after the first treatment, splenocytes were harvested from treated mice and stimulated with 4T1 cells, which exposed to ⁶⁰Co γ-ray irradiation at a dose of 50 Gy to release tumor antigens, for 42 hours and the IFN-γ spot forming cells were counted. The number of antigen-specific IFN-γ producing T cells significantly increased in tumor-bearing mice treated with Hf-MOL(+)/α-PD-L1 (Figure 6A), suggesting that Hf-MOL(+)/α-PD-L1 treatment effectively generates systemic tumor-specific T cell response on the orthotopic 4T1 model. We further profiled infiltrating leukocytes in both breast tumors and lungs. The Hf-MOL(+)/α-PD-L1 group showed a significant increase of CD45⁺ leukocyte cells, in particular CD4⁺ T cells and CD8⁺ T cells, in both primary tumors and distant lungs (Figures 6B–D). The increase of CD8⁺ T cells in the lungs of the Hf-MOL(+)/α-PD-L1 group was confirmed with immunostaining under CLSM (Figure S26). Interestingly, the Hf-MOL(+)/α-PD-L1 group showed significant increases of NK cells and B cells (Figures 6E–F) in the breast tumors but not in the lungs.

Figure 6. Mechanism of Anti-metastasis effect of Hf-MOL-enabled RT-RDT.

(A) ELISPOT assay was performed to detect tumor-specific IFN-γ producing T cells. Ten days after the first treatment, the splenocytes were harvested and co-cultured with X-ray irradiated 4T1 cells as stimulation for 42 h. (b-g) The tumors, lungs and bone marrows were collected from tumor-bearing mice with as-mentioned treatments for flow cytometry analysis and the percentages of tumor-infiltrating CD45⁺ cells (B), CD8⁺ T cells (C), CD4⁺ T cells (D), NK cells (E), B cells (F), mMDSCs (G) and gMDSCs (H) with respect to the total tumor cells. (+) and (−) refer to with and without irradiation, respectively. Spleen on 4T1 orthotopic tumor-bearing mice treated with PBS or Hf-MOL with or without anti-PD-L1 was collected, weighted and ground through the cell strainers to get single cell suspensions. Splenocytes were then counted by hemocytometer. The images (I) and weights (J) of spleens from as-treated groups. (K) The numbers of splenocytes. (L) gMDSCs with respect to the total splenocytes. Data are expressed as means ± s.d. (n = 6). *P < 0.05, **P < 0.01 and ***P < 0.001 by t-test. Central lines, bounds of box and whiskers represent mean values, 25% to 75% of the range of data and 1.5 fold of interquartile range away from outliers, respectively.

We also profiled both monocytic MDSCs (mMDSCs, CD11b⁺Ly6C^hiLy6G⁻ phenotype) and granulocytic MDSCs (gMDSCs, CD11b⁺Ly6C^lowLy6G⁺ phenotype) in primary breast tumors and distant lungs. As shown in Figure 6G, significant reduction of mMDSCs was observed in the primary tumors and lungs after Hf-MOL(+)/α-PD-L1 treatment. Significant reduction of gMDSCs was also observed in the lungs. It is well-established that both mMDSCs and gMDSCs suppress T cell-mediated anti-tumor immunity and directly inhibit T cell proliferation. Hf-MOL(+)/α-PD-L1 treatment thus relieves inhibitory effects of MDSCs and enhances T cell activation and proliferation in both primary tumors and distant sites to suppress metastasis. Furthermore, it was reported that gMDSCs play an important role in mesenchymal-epithelial transition (MET) of circulating tumor cells and gMDSCs in the hematopoietic system support metastasis formation.⁴² Immune cell profiling studies indicated a reduction of gMDSCs in bone marrow and spleen for the Hf-MOL(+)/α-PD-L1 group, suggesting that Hf-MOL(+)/α-PD-L1 inhibits metastasis through suppressing the pro-tumor effect of gMDSCs and weakening MET. The significant decrease of gMDSCs in both distant lungs and bone marrow further suggests the suppression of distant metastasis via worsening the environments for metastatic seeds.

Interestingly, we observed a correlation between the level of metastasis and spleen enlargement in this study. As a highly lung-metastatic breast tumor model, 4T1-bearing mice show severe spleen enlargement compared with other non-lung-metastatic breast tumor models such as TUBO and MCF-7. The spleens of the mice receiving different treatments were harvested, imaged and grounded. The splenocytes were harvested and counted. The spleens from Hf-MOL(+)/α-PD-L1-treated mice displayed a normal morphology as those from naïve mice, while the spleens from other treatment groups were enlarged (Figure 6I). The spleens from the mice treated with Hf-MOL(+)/α-PD-L1 had similar average weight as those from naïve mice but were only ~1/4 of those from other treatment groups (Figure 6J). The number of splenocytes from the mice treated with Hf-MOL(+)/α-PD-L1 were similar to those of naïve mice but only ~ 1/3 of those from other treatment groups (Figure 6K). We profiled the splenocytes and detected significate upregulation of gMDSCs in the groups with enlarged spleens. Only the Hf-MOL(+)/α-PD-L1 treatment afforded a significant decrease of gMDSC, indicating that MOL(+)/α-PD-L1 systemically depletes gMDSCs and prevents abnormal spleen enlargement which is a typical syndrome in mice with highly metastatic tumors.

DISCUSSION

Emerging as a new type of crystalline nanomaterials, nMOFs have shown great potential in biomedical and even clinical applications due to the flexibility in their synthesis, the ability to endow multifunctionality, and their intrinsic biocompatibility.⁴³⁻⁴⁹ The ordered structures and inherent porosity of nMOFs avoid self-quenching and facilitate the diffusion of ROSs to improve the efficacy of RT-RDT, RT and PDT.^{26,37,50−53} To further suppress diffusion barrier and self-quenching, we have synthesized nMOLs, a class of novel 2D metal-organic nanomaterials, for cancer treatment and other applications.⁵⁴⁻⁵⁷ In this work, we successfully synthesized Hf-MOL that is built from porphyrin-based ligands and Hf-oxo SBUs and exhibits excellent stability under physiological conditions.

To maximize the therapeutic effect of RT at tumor site while minimizing deleterious effects on the surrounding healthy tissues, high-Z element based radioenhancers, including metal or metal oxide nanoparticles, have been extensively explored.⁵⁸⁻⁶⁰ While selectively accumulated in the tumor, these radioenhancers can effectively enhance X-ray absorption in the tumor over surrounding healthy tissues. We recently discovered that porous nMOFs based on Hf-oxo cluster SBUs outperformed HfO₂ nanoparticles in radioenhancement by more than 3.2 times.³² The highly porous structure of nMOFs allows the interior Hf-oxo SBUs to absorb X-ray to produce ROS and the generated ROS to diffuse out of the porous matrix, while only the superficial layers of solid nanoparticle participate X-ray enhancement.⁶¹ More recently, we discovered an unprecedented RT-RDT process enabled by nMOFs built from heavy metal SBUs and photosensitizing ligands to generate both ·OH and ¹O₂.²⁶ By carefully controlling the synthetic conditions, we synthesized the Hf-DBP nMOL based on Hf-oxo SBUs and photosensitizing porphyrin-based ligands for RT-RDT. Reduction of the dimensionality of 3D nMOFs to 2D nMOLs further relieves the diffusion barrier for ROS, leading to superior RT-RDT effects as demonstrated by extensive in vitro assays and in vivo therapeutic efficacy studies.

Tumor metastasis is one of the major reasons for the failure of cancer management in the clinic.^62,63 Significant current efforts are focused on developing combination therapies that synergize local cancer treatment with systemic anti-metastasis efficacy.^40,64−68 We used the highly metastatic murine triple-negative breast cancer model 4T1 to evaluate anti-metastasis efficacy of Hf-MOL-enabled RT-RDT. MDSCs are known to play a significant role on tumor progression and metastasis. It is thus logical to develop anti-metastasis therapeutics by targeting MDSCs.^69,70 We observed ubiquitous spleen enlargement in the groups with poor therapeutic efficacy but no spleen enlargement in the Hf-MOL(+)/α-PD-L1 group. Similar spleen enlargement was reported for late stages of 4T1 model in several studies,^71,72 and a recent study established the correlation between spleen enlargement and tumor metastasis.⁴² Significant increase of gMDSCs in both bone marrow and spleen leads to pulmonary infiltration of gMDSCs to then enhance MET process and facilitate the formation of metastatic niches. In the meanwhile, the increase of infiltrated mMDSCs in primary tumor site induces epithelial–mesenchymal transition (EMT) process to facilitate tumor cell dissemination. We found the link between the lack of spleen enlargement and the decrease of gMDSC populations in the spleen, bone marrow, and lung, which is correlated with significantly reduced lung metastasis and better prognosis.

In summary, we have designed a novel Hf-MOL based on electron-dense SBUs and photosensitizing ligands for effective RT-RDT with low dose X-rays by taking advantage of enhanced ROS diffusion. The synergistic combination of Hf-MOL-enabled RT-RDT and immune checkpoint inhibitors led to superb anti-tumor efficacy on bilateral models of colon, head and neck, and breast cancers and significant anti-metastatic effects on an orthotopic model of lung-metastatic triple-negative breast cancer. This combination extends the local therapeutic effects of RT-RDT to distant tumors via systemic antitumor immunity and inhibits metastasis by re-activating T cells and inhibiting immunosuppressive MDSCs in both orthotopic tumors and metastatic lung lesions. Rational tuning of nMOL compositions and structures promises to lead to even more potent RT-RDT to potentiate CBI for the treatment of metastatic tumors. The scalable and tunable nature of MOL synthesis thus paves the way for optimizing clinical candidates for effective radioenhancement and combination with immune checkpoint inhibitors.

EXPERIMENTAL PROCEDURES

Cell lines and animals.

Murine triple-negative breast cancer cell line 4T1 was kindly provided by Dr. Stephen J. Kron at University of Chicago Murine colon adenocarcinoma cell CT26 and murine squamous cell carcinoma SCC VII were purchased from the American Type Culture Collection (Rockville, MD, USA). CT26 cells was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (GE Healthcare, USA). 4T1 and SCC VII cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium (GE Healthcare, USA). All media were supplemented with 10% FBS, 100 U/mL penicillin G sodium and 100 μg/mL streptomycin sulfate. Cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C. Mycoplasma was tested before use by MycoAlert detection kit (Lonza Nottingham, Ltd.) BALB/c mice (6 – 8 weeks) and C3H mice (6 – 8 weeks) were obtained from Harlan-Envigo Laboratories, Inc (USA). The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago.

Cytotoxicity.

The cytotoxicity of Hf-DBA, H₂DBP, or Hf-MOL was evaluated with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay (Promega, USA) with or without X-ray irradiation. 4T1 cells were seeded on 96-well plates at 1×10⁴/well and further cultured for 12 h. Hf-DBA, H₂DBP or Hf-MOL were added to the cells at an equivalent ligand dose of 0, 1, 2, 5, 10, 20, 50 and 100 μM and incubated for 4 h. The cells were then irradiated with X-rays at a dose of 2 Gy (Philips RT250 X-ray generator, Philips, USA, 250 KVp, 15 mA, 1 mm Cu filter). The cells were further incubated for 72 h before determining the cell viability by MTS assay.

Clonogenic assay.

The clonogenic assay was performed according to a modified protocol. 4T1 cells were cultured in a 6-well plate overnight and incubated with particles at a Hf concentration of 20 μM for 4 h followed by irradiation with 0, 1, 2, 4, 8 and 16 Gy X-ray (Philips RT250 X-ray generator, Philips, USA, 250 KVp, 15 mA, 1 mm Cu filter) or γ- ray (⁶⁰Co source, Atomic Energy Canada Limited, Canada). Cells were trypsinized and counted immediately. 200–2000 cells were seeded in a 6-well plate and cultured with 2 mL medium for 15 days. Once colony formation was observed, the culture medium was discarded. The plates were rinsed twice with PBS, then stained with 500 μL of 0.5% w/v crystal violet in 50% methanol/H₂O. The wells were rinsed with water for three times and the colonies were counted manually.

Apoptosis/necrosis.

4T1 cells were cultured in a 6-well plate overnight and incubated with particles at a Hf concentration of 20 μM for 4 h followed by irradiation with 0 or 2 Gy X-ray (250 kVp, 15 mA, 1 mm Cu filter). 24 h later, the cells were stained according to the AlexaFluor 488 Annexin V/dead cell apoptosis kit (Life technology, USA) and quantified by flow cytometry.

DNA damage.

4T1 cells were cultured in a 6-well plate overnight and incubated with PBS, Hf-DBA, H₂DBP, or Hf-MOL at an equivalent concentration of 20 μM for 4 h followed by irradiation at 0 and 2 Gy X-ray (250 kVp, 15 mA, 1 mm Cu filter). Cells were stained 6 h after irradiation with the HCS DNA damage kit (Life Technology, USA) for confocal laser scanning microscopy (CLSM, FV1000, Olympus, Japan) and flow cytometry.

Transwell invasion.

4T1 cells were cultured in a 6-well plate overnight and incubated with PBS, Hf-DBA, H₂DBP, or Hf-MOL at an equivalent concentration of 20 μM for 4 h followed by irradiation with 0 or 2 Gy X-ray (250 kVp, 15 mA, 1 mm Cu filter). The cells were collected, washed three times with PBS, and then adjusted to a concentration of 2 × 10⁵ cells/mL in serum-free medium. 200mL of the cell suspension was seeded onto the upper chamber of a Millicell Cell Culture Insert with 8.0 mm pores (Millipore, USA). The lower chamber contained 1 mL medium with 10% FBS. After 24 h, the non-invading cells on the upper surface were removed with a cotton swab and the invading cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The invading cells were observed and photographed under an Axioskop inverted microscope (Zeiss, Germany) at 10× magnification.

Immunogenic cell death.

4T1 cells were cultured in a 6-well plate overnight and incubated with PBS, Hf-DBA, H₂DBP, or Hf-MOL at an equivalent concentration of 20 μM for 4 h followed by irradiation with 0 or 2 Gy X-ray (250 kVp, 15 mA, 1 mm Cu filter). After incubation for 4 h, supernatant was harvested for extracellular HMGB-1 and ATP secretion assayed by HMGB-1 ELISA kit (Invitrogen, USA) and Chemiluminescence ATP Determination kit (Thermo Fisher, USA), respectively. The cells were washed three times with PBS, fixed with 4% paraformaldehyde, incubated with AlexaFluor 488-CRT (Enzo Life Sciences, USA) with 1: 100 dilution for 2 h, stained with DAPI, and observed by CLSM, or collected, incubated with AlexaFluor 488-CRT antibody for 2 h, and then stained with PI for analysis by flow cytometry (LSRFortessa, BD, USA).

Vaccination.

4T1 cells were cultured in a 3.5-cm dish overnight and incubated with Hf-MOL at an Hf concentration of 20 μM for 4 h followed by irradiation 4 Gy X-ray (250 kVp, 15 mA, 1 mm Cu filter). After incubation for 48 h, cells were harvested and injected subcutaneously as vaccine. Mice injected with same amount of PBS serves as control. 7 days after vaccination, mice were challenged with live 4T1 cells.

In vivo abscopal effect on bilateral models.

Three synergistic bilateral tumor models, 4T1, CT26 and SCC VII were established to evaluate the in vivo anti-cancer efficacy of combination of Hf-MOL-enabled RT-RDT and checkpoint blockade immunotherapy. For 4T1, 1×10⁶ and 5×10⁵ 4T1 cells were subcutaneously inoculated onto the right and left flanks of BALB/c mice for respective primary and secondary tumors. For CT26, 2×10⁶ and 1×10⁶ CT26 cells were subcutaneously inoculated onto the right and left flanks of BALB/c mice for respective primary and secondary tumors. For SCCVII, 1×10⁶ and 2×10⁵ SCC VII cells were by subcutaneously inoculated onto the right and left flanks of C3H mice for respective primary and secondary tumors. When the primary tumors reached 100–150 mm³ in volume, mice were injected intratumorally with Hf-MOL at a dose of 0.2 μmol Hf or PBS. 12 h after injection, mice were anaesthetized with 2% (v/v) isoflurane and the primary tumors were irradiated with 0.5 or 1 Gy X-ray/fraction (225 kVp, 13 mA, 0.3 mm-Cu filter) for a total of 8 or 10 daily fractions. Antibodies were given every three days by intraperitoneal injection at a dose of 75 μg/mouse. The tumor sizes were measured daily with a caliper where tumor volume equals (width² × length)/2. Mice with 4T1, CT26 or SCCVII were sacrificed on Day 27, 19 or 22, respectively.

T cell and B cell depletion.

The bilateral subcutaneous model was established as for the in vivo anti-cancer efficacy. When the primary tumors reached 100–150 mm³ in volume, mice were injected intratumorally with nMOFs at a dose of 0.11 mg/mouse or PBS. Anti-CD4 (GK1.5, BioXCell, USA), anti-CD8 (OKT-8, BioXCell, USA), mouse IgG (C1.18.4, BioXCell, USA) antibodies Or B cell inhibitor ibrutinib (PCI-32765, Selleckchem) were intraperitoneally injected into the mice (200 μg/mouse) on Day 0 and 5 after the first treatment. Twelve hours post-injection, mice were anesthetized with 2% (v/v) isoflurane, and tumors were irradiated with X-ray at 225 kVp and 13 mA with a 0.3-mm Cu filter. To evaluate the therapeutic efficacy, the tumor growth and body weight were monitored daily.

Tumor challenge studies.

When the tumors reached 100–150 mm³ in volume, mice were injected intratumorally with Hf-MOL at a dose of 0.11 mg/mouse or PBS. 12 h after injection, mice were anaesthetized with 2% (v/v) isoflurane and the primary tumors were irradiated with 1 Gy X-ray/fraction (120 kVp, 20 mA, 2 mm-Cu filter) for a total of 10 daily fractions. On day 50 post inoculation, mice were challenged with 2×10⁶ cells on the contralateral flank. Healthy mice were simultaneously inoculated as control. The mice were sacrificed when the tumors of the control mice reached 2 cm³. Statistical analysis was performed using the log-rank Kaplan-Meier estimation.

ELISPOT assay.

Tumor-specific immune responses to IFN-γ were measured in vitro by ELISPOT assay (Mouse IFN-γ ELISPOT Ready-SET-Go!; Cat. No. 88-7384-88; eBioscience). 4T1 cells were irradiated with X-ray irradiator at a dose of 50 Gy to expose tumor-specific antigen. A Millipore Multiscreen HTS-IP plate was coated overnight at 4 °C with anti-mouse IFN-γ capture antibody. Single-cell suspensions of splenocytes were obtained from 4T1 tumor-carrying mice and seeded onto the antibody-coated plate at a concentration of 2×10⁵ cells per well. Cells were incubated with or without antigen-exposed 4T1 cell stimulation (1×10⁴ cells per well) for 42 h at 37 °C and then discarded. The plate was then incubated with biotin-conjugated anti-IFN-γ detection antibody at r.t. for 2 h, followed by incubation with Avidin-HRP at r.t. for 2 h. 3-amino-9-ethylcarbazole substrate solution (Sigma, Cat. AEC101) was added for cytokine spot detection. Spots were imaged and quantified with a CTL ImmunoSpot Analyzer (Cellular Technology Ltd, USA).

Lymphocytes profiling.

Tumors were harvested, treated with 1 mg/ml collagenase I (Gibco, USA) for 1 h at 37 °C. Cells were filtered through nylon mesh filters with size of 40 μm and washed with PBS. Tumor-draining lymph nodes were collected and directly ground through the cell strainers. The single-cell suspension was incubated with anti-CD16/32 (clone 93) to reduce nonspecific binding to FcRs. Cells were further stained with the following fluorochrome-conjugated antibodies: CD45 (30-F11), TCRff (H57–597), CD4 (GK1.5), CD8 (53–6.7), Foxp3 (FJK-16s), CD25 (PC61.5), Nkp46 (29A1.4), F4/80 (BM8), B220 (RA3–6B2), CD11b (M1/70), Ly6C (HK1.4), Ly6G (RB6–8C5) and yellow-fluorescent reactive dye (all from eBioscience). Antibodies were used with the dilution of 1: 200. Representative gating strategies for different immune cells are shown in Figure S27. LSR Fortessa (BD Biosciences) was used for cell acquisition and data analysis was carried out with FlowJo software (Tree Star, Ashland, OR).

In vivo anti-metastasis effect.

Orthotopic 4T1 model was established by inoculating 4T1 cells into the mammary fat pads of BALB/c mice. When the primary tumors reached 100–150 mm³ in volume, mice were injected intratumorally with Hf-MOL at a dose of 0.2 μmol Hf or PBS. 12 h after injection, mice were anaesthetized with 2% (v/v) isoflurane and the orthotopic tumors were irradiated with 0.5 Gy X-ray/fraction (225 kVp, 13 mA, 0.3 mm-Cu filter) for a total of 10 daily fractions. Antibodies were given every three days by intraperitoneal injection at a dose of 75 μg/mouse. Body weights and tumor volumes were monitored and recorded over a period of 24 days. At the end of experiment, mice were sacrificed, and tumors were excised, weighed and photographed. Lungs were also harvested, observed for the gross examination of tumor nodules, or sectioned and stained with H&E for quantification of metastasis area, or digested with collagenase type IV/elastase cocktail and cultured with 60 μM 6-thiogunine for 10 days. The colonies formed by clonogenic metastatic cancer cells were then fixed with methanol and stained with 0.1% crystal violet. For quantification, the crystal violet stained colonies were dissolved with 10% acetic acid and their absorbance at 590 nm was measured and normalized to the PBS control group.

Statistical analysis.

Group sizes (n ≥ 5) were chosen to ensure proper statistical ANOVA analysis for efficacy studies. Student’s t-tests were used to determine if the variance between groups is similar. Statistical analysis was performed using OriginPro (OriginLab Corp.). Statistical significant was calculated using two-tailed Student’s t-tests and defined as * P < 0.05, ** P < 0.01, *** P < 0.001. Animal experiments were not performed in a blinded fashion and are represented as mean ± SD. The immune analysis was performed in a blinded fashion and are represented as median ± SD.

Scheme 1. Hf-MOL-enabled local RT-RDT treatment synergizes with CBI to elicit systemic anti-tumor and anti-metastatic effects.

Local injection of Hf-MOL in 4T1 orthotopic tumors enables RT-RDT with fractionated, low-dose X-rays. The RT-RDT effects potentiate CBI to systemically eliminate tumors and prevent metastasis through three mechanisms: regressing local tumors by direct killing effects, stimulating anti-tumor immunity via immunogenic cell death and tumor antigen release, and decreasing pro-metastatic MDSCs in bone marrow, spleen, and lungs. The combination of Hf-MOL-enabled RT-RDT and anti-PD-L1 antibody thus overcomes suppressive tumor microenvironments and activate cytotoxic T cells to eliminate lung metastasis.

Progress and Potential statement.

Immune checkpoint inhibitors targeting the anti-programmed death 1 (PD-1) and its ligand (PD-L1) have enjoyed clinical success by reactivating the suppressed immune surveillance, but they do not elicit durable responses in many cancers due to the reliance on inflamed tumor phenotypes. We recently disclosed radiotherapy–radiodynamic therapy (RT–RDT) using nanoscale metal-organic frameworks (nMOFs) to significantly enhance radiotherapeutic effects of X-ray radiotherapy. Here we report an ultrathin version of nMOFs, termed nanoscale metal-organic layers (nMOLs), with reduced dimensionality to facilitate the diffusion of reactive oxygen species generated by RT-RDT to kill cancer cells. MOL-enabled RT-RDT in conjunction with an anti-PD-L1 antibody eradicated local tumors and rejected/regressed distant tumors on several syngeneic bilateral tumor models as well as eliminated lung metastases by re-activating anti-tumor immunity and inhibiting myeloid-derived suppressor cells (MDSCs).

Highlights.

1. nMOLs outperform nMOFs in RT-RDT due to facile diffusion of ROS.

2. nMOL-mediated RT-RDT synergizes with CBI to reject both local and distant tumors.

3. The combination treatment has strong anti-metastatic effects via inhibition of MDSCs.