Immunogenic Treatment for Metastatic Breast Cancer Using Targeted Carbon Nanotube Mediated Photothermal Therapy in Combination with Anti-PD-1

doi:10.21203/rs.3.rs-2883356/v1

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Immunogenic Treatment for Metastatic Breast Cancer Using Targeted Carbon Nanotube Mediated Photothermal Therapy in Combination with Anti-PD-1

https://doi.org/10.21203/rs.3.rs-2883356/v1

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published 20 May, 2024

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The high prevalence of breast cancer is a global health concern, but there are no safe or effective treatments for it at its advanced stages. These facts urge the development of novel treatment strategies. Annexin A5 (ANXA5) is a natural human protein that binds with high specificity to phosphatidylserine, a phospholipid tightly maintained in the inner leaflet of the cell membrane on most healthy cells but externalized in tumor cells and the tumor vasculature. Here, we have developed a targeted photosensitizer for photothermal therapy (PTT) of solid tumors through the functionalization of single walled carbon nanotubes (SWCNTs) to ANXA5—the SWCNT-ANXA5 conjugate. The ablation of tumors through the SWCNT-ANXA5-mediated PTT synergizes with checkpoint inhibition, creating a systemic anti-cancer immune response. In vitro ablation of cells incubated with the conjugate promoted cell death in a dose-dependent and targeted manner. This treatment strategy was tested in vivo with the orthotopic EMT6 breast tumor model in female balb/cJ mice. Enhanced therapeutic effects were achieved by using intratumoral injection of the conjugate and treating tumors at a lower PTT temperature (45^oC). When combined with checkpoint inhibition of anti-PD-1, SWCNT-ANXA5-mediated PTT increased survival and 80% of the mice survived for 100 days. Evidence of immune system activation by flow cytometry of splenic cells strengthens the hypothesis of an abscopal effect as a mechanism of prolonged survival.

The incidence of cancer has been growing worldwide and so has its global mortality. Accordingly, cancer is predicted to be the primary cause of mortality and thus the single most critical barrier to improving life expectancy in every country on the planet in the 21st century, according to the World Health Organization (WHO) [1]. More specifically, one in every 20 women is affected by breast cancer globally, while this statistic rises to one in every eight women by age 85 who live in developed countries [2]. The absence of targeting receptors expression characterizes triple-negative breast cancer (TNBC). It affects approximately 10–20% of the women diagnosed with breast cancer [3, 4]. Early-stage TNBC has a 37% death rate in the first 5 years after surgery, and metastatic TNBC has short progression-free survival of 3 to 4 months [5–8]. Poor prognosis is strongly associated with the lack of targeted therapies. The limited treatment options also increase the incidence of cancer recurrence in TNBC patients [9].

Although the development of checkpoint inhibition, such as anti-PD-1, has established a breakthrough in cancer treatment, the lack of responsivity to immunotherapy has been correlated to the absence of tumor-infiltrating lymphocytes, such as associated CD8 + T cells, which characterize non-immunogenic tumors [10]. Breast cancer is one of the types of tumors that has been considered immunologically cold. The first immunotherapy approved for breast cancer was in combination with the chemotherapy of paclitaxel for TNBC. Other combinations of chemotherapy and checkpoint inhibition have been studied in clinical trials [11]. Even though immunotherapy is effective in some breast cancer patients, more studies are necessary to improve safety and efficacy.

While one of the hallmarks of cancers is the ability to avoid immune destruction, immunogenic cell death (ICD) can establish an anticancer adaptive immune response that might counterbalance the equilibrium between cancer cells and immune cells towards the induction of antitumoral immunity [12]. Indeed, during the oncogenic process, cancer develops mechanisms to avoid ICD to guarantee immune evasion [13]. There is evidence that the ablation of solid tumors generates danger signals and tumor antigens that convert it into an immunogenic environment. Thus, the combination of an ablation method and immunotherapy is promising for the generation of a synergistic effect that is capable of not only treating the primary tumor, but also generating a systemic immune response that suppresses metastasis. The stress during photothermal therapy (PTT)—an irradiation-based thermoablation, using photons from the near-infrared range (750–1350 nm) to illuminate a pertinent area—promotes an immune response associated with ICD. When stress is applied to cells, programmed cell death might generate inflammatory responses that can lead to the activation of cytotoxic T cells and subsequent establishment of immunological memory [13].

Here we combine the ablation of solid tumors through a photosensitizer-mediated photothermal therapy (PTT) with checkpoint inhibition for a systemic anti-cancer response. The PTT-mediated hyperthermia induces tissue damage in temperatures greater than 41ºC [14]. Due to limitations of laser penetration and heat transfer, temperatures of 45–60ºC are necessary to ablate most of the tumor volume efficiently [15, 16]. Due to the very localized destruction of tumor cells induced by PTT and the consequent induction of ICD, it is believed that combining immunotherapy with photothermal therapy creates a synergistic effect efficient for the treatment of cold tumors [17]. The benefits of exploiting PTT-induced ICD to enhance immunotherapy are remarkable. ICD generated by PTT allows for the successful removal of the treated tumor with the assistance of surrounding immune cells. At the same time, the tumor-specific antigens can function as an in situ anti-cancer vaccination [18]. While the native tumor antigens are weak at activating the immune response, their immunogenicity can be enhanced by the release of damage-associated molecular patterns (DAMPs) during PTT [19]. Tumor ablation is also accompanied by the release of pro-inflammatory cytokines that promote the attraction and activation of immune system cells [20]. Additionally, PTT mediated by photosensitizers is a highly targeted treatment option due to the dual selectivity strategies. First, the control of the region of irradiation by aiming the laser in the desirable area (tumor) prevents damage to unwanted parts of the body. Second, the localization of photosensitizers in the affected regions provides another level of selectivity. Their linkage to targeting agents can further increase the accumulation of photosensitizers in tumors. Invasive techniques, such as laparotomies and thoracotomies, can be avoided when treating deep-seated tumors using endoscopy and interventional procedures with optical fibers [21].

In this work, we have developed a targeted photosensitizer for photothermal therapy (PTT) for the efficient ablation of solid tumors based on the functionalization of single-walled carbon nanotubes (SWCNTs) to annexin A5 (ANXA5): SWCNT-ANXA5 conjugate. ANXA5 is a protein that has specificity for cancer due to the strong binding to externalized phosphatidylserine (PS) on the surface of tumoral cells and tumor vasculature [22–24]. PS is a biomarker of various types of cancers, including TNBC, but is absent on the surface of healthy non-apoptotic cells. SWCNTs efficiently absorb near-infrared (NIR) light, causing them to be heated, and SWCNTs that are bound to the tumor and treated with NIR light cause the tumor to be selectively heated. We assessed the combination of the SWCNT-ANXA5-mediated PTT with checkpoint inhibition of anti-PD-1, after determining the optimal temperature of irradiation, and evaluated the mechanisms of anticancer immunity in the combination therapy.

Materials

For ANXA5 protein production and purification, the plasmid encoding ANXA5, pET-30 Ek/LIC/ANX, was previously constructed in this laboratory [25]. Sodium hydroxide (NaOH), sodium chloride (NaCl), tryptone, kanamycin, yeast extract, isopropyl-beta-D-thiogalactopyranoside (IPTG), N- p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), phenylmethylsulfonyl fluoride (PMSF), β-mercaptoethanol, and sodium phosphate dibasic (Na₂HPO₄) were from Sigma Aldrich (St. Louis, MO). All cell lines and cell media were from American Type Culture Collection (ATCC, Manassas, VA, USA). Waymouth's MB 752/1 medium), was from Gibco (Thermo Fisher, Waltham, MA). Antibiotic-antimitotic (10,000 IU Penicillin, 10,000 µg/ml Streptomycin, and 25 µg/ml Amphotericin B) was from Corning (Kennebunk, MA, USA). Vascular cell basal medium and endothelial cell growth kit BBE (0.2% bovine brain extract, 5 ng/ml EGF, 10 mM L-glutamine, 0.75 Units/ml heparin sulfate, 1 µg/ml hydrocortisone, 50 µg/ml ascorbic acid, 2% FBS) was from ATCC. Trypsin/EDTA was from Thermo Fisher Scientific.Antibiotic-antimitotic (10,000 IU Penicillin, 10,000 µg/ml Streptomycin, and 25 µg/ml Amphotericin B) was from Corning (Kennebunk, MA, USA). Endothelial cell growth kit BBE (0.2% bovine brain extract, 5 ng/ml EGF, 10 mM L-glutamine, 0.75 Units/ml heparin sulfate, 1 µg/ml hydrocortisone, 50 µg/ml ascorbic acid, 2% FBS) was from ATCC. Trypsin/EDTA was from Thermo Fisher Scientific. HRV 3C protease was from Acro Biosystems (Newark, DE). The HisTrap chromatography column (5 ml) was from Cytiva (Marlborough, MA). The 12–14 kDa regenerated cellulose dialysis membrane was from Fisher Scientific (Pittsburgh, PA). Laemmli sample buffer, ladder (marker), 4–20% mini-protean TGX stain-free pre-casted polyacrylate gels, Bio-Rad 10x Tris-glycine-SDS buffer, and Bradford dye reagent were from Bio-Rad Laboratories (Hercules, CA). Imperial™ protein stain was from Thermo Scientific (Waltham, MA). For conjugation of ANXA5 to SWCNTs, purified and freeze dried (6,5) CoMoCAT SG65i SWCNTs were provided by Chasm Advanced Material Company (Norman, Oklahoma), with a tubular carbon purity of ≥ 97% as determined by TGA, Raman Q factor ≥ 0.97, an average length of 1 µm, an outer diameter of 0.78 ± 2 nm, and a (6,5) chirality composition of ≥ 40%, as determined by near-infrared fluorescence spectroscopy (NIRF). DSPE-PEG-mal (3.4 kDa) was from Creative PEGWorks (Winston Salem, NC). Spectra-Por® dialysis membranes (2 and 100 kDa) from Spectrum Laboratories, Inc. (Rancho Dominguez, CA). Bradford protein reagent was from Bio-Rad (Hercules, CA). L-cysteine amino acid was from Sigma Aldrich. For cell culture and in vitro assays, all cell lines and cell media were from American Type Culture Collection (ATCC, Manassas, VA, USA). For in vivo studies, BALB/cJ mice were from Jackson Laboratory (Bar Harbor, ME). ELISA kits, and flow cytometry staining antibodies were from Biolegend (San Diego, CA). Anti-PD-1 was from Bio X Cell (Lebanon, NH).

Annexin A5 production and purification

Recombinant ANXA5 was produced as previously described [26]. In brief, BL21(DE3) Escherichia coli harboring the plasmid containing pET-30 Ek/LIC/ANXA5 were initially inoculated and grown in TB medium with kanamycin (35 µg/ml) and then induced to produce ANXA5. The bacteria were collected and sonicated. The debris-free supernatant from cell lysate was loaded into a nickel HisTrap column. After washing, an endotoxin removal step was added using a 1% Triton X-100 wash [27], After another wash, ANXA5 protein (with an N-terminal six histidine tail) was eluted. After dialysis against sodium phosphate buffer, the (His)₆-tagged protein was cleaved with the HRV 3C protease. Final column purification was performed by loading cleaved protein into the affinity column and collecting the first flow-through with ANXA5 without the (His)₆ tag. The protein solution was dialyzed against a 20 mM sodium phosphate buffer containing 100 mM NaCl (pH 7.4) a final time before being aliquoted in cryogenic vials and flash-frozen in liquid nitrogen. Aliquots were placed in a -80°C freezer for long-term storage. The purified protein was quantified using the Bradford assay and analyzed with SDS-PAGE electrophoresis for purity.

Conjugation of SWCNTs to annexin A5

The conjugation was performed using a DSPE-PEG-maleimide linker (3.4 kD) as previously described [25]. Briefly, SWCNTs (6 mg) were added to 5 ml of a 1% sodium dodecyl sulfate (SDS) solution and sonicated for 1 h at 19.8 W of power (E = 35,640 J) (VirSonic 100 ultrasonic cell disruptor, VirTis). Sonicated SWCNTs were centrifuged in an ultracentrifuge (16,000 g) for 1 h. The DSPE-PEG-MAL linker (1.5 mg) dissolved in 1% SDS solution (1 mL) was added to 5 ml of the SWCNT suspension and then mixed at room temperature for 30 min with gentle shaking. The suspension was dialyzed in a 2-kDa dialysis membrane for 8 h against 3 liters of deionized water. The dialysate was changed after the first 4 h. SWCNT-linker suspension (2 ml) was mixed with the concentrated ANXA5 (1 ml of 5 mg/ml totaling 5 mg of ANXA5) at room temperature for 2 h with gentle shaking. Any unreacted linker sites were blocked with L-cysteine, which was added to the suspension and allowed to react for 1 h at room temperature with gentle shaking. The suspension was then dialyzed using a 100 kDa dialysis membrane to remove any unbound ANXA5 or L-cysteine for 8 h against 3 L of 20 mM sodium phosphate buffer (pH 7.4) with a dialysate change after 4 h. The final suspension was centrifuged at 16,000 g for 1 h to remove any aggregates. The conjugate was stored in a glass vial at 4^oC until use. Conjugate was used within 1 week of the end of conjugation.

Cell culture

EMT6 murine breast carcinoma cells were cultivated with Waymouth's MB 752/1 medium supplemented with 2 mM glutamine, 15% FBS, and 1% antibiotic-antimitotic (10,000 IU penicillin, 10,000 µg/ml streptomycin, and 25 µg/ml amphotericin B). Human umbilical vein endothelial cells (HUVEC) were cultured in vascular cell basal medium supplemented with endothelial cell growth kit BBE (0.2% bovine brain extract, 5 ng/ml EGF, 10 mM L-glutamine, 0.75 Units/ml heparin sulfate, 1 µg/ml hydrocortisone, 50 µg/ml ascorbic acid, and 2% FBS) and 1% antibiotic-antimycotic. Cells were passaged using 0.25% (w/v) trypsin in 0.53 mM EDTA before reaching 85% confluency. All cell lines were cultured under a 5% CO₂-supplemented atmosphere at 37°C. The medium was refreshed every 48 h. During assays, EMT6 cancer cell cultures were grown to less than 75% confluence, and HUVEC were grown to confluency for inhibition of PS expression upon confluency.

In vitro studies

The cells that were previously grown in T-75 flasks were seeded at 2⋅10⁵ into sterile surface-treated 24-well plates. At 24 h after seeding, cell media was aspirated, and fresh media enriched with 6 mg/L SWCNT-ANXA5 suspension and 2 mM of Ca²⁺ (CaCl₂) was added to the wells. Incubation was performed for 2 h at 37 ºC. The wells were washed three times with sterile phosphate buffer saline (PBS) to remove unbound SWCNT-ANXA5. Each group was evaluated at least in triplicate. Different controls were studied to guarantee the specificity of the assay.

For PTT treatment, each well subjected to the laser treatment was irradiated for different time periods at 980 nm at a power density of 1 W/cm² using a Diodevet-50 NIR laser (B&W Tek Inc., Newark, DE). The irradiation was always performed by having the plate sitting on top of a platform and aiming the optical fiber at the bottom of the plate, positioned a few centimeters away from the bottom to obtain the desired size beam. Each well was carefully adjusted to be centered with the laser beam. The size of the beam was adjusted using a laser sensitive paper (Zap-it® paper) which burns when exposed to a laser beam. The attenuation of power from the fiber and plates was determined by a power meter (Ophir Optronics Ltd., Israel). The power meter probe was placed on top of a microtiter plate (without the lid on), and the power was determined to reach a power level of 1 W/m². The irradiation was performed either at room temperature or inside an incubator at 37°C to mimic physiological temperatures.

The cell viability after PTT was determined by using the Alamar Blue assay. The cells were incubated with the reagent for 2–4 h at 37 ºC, until coloration of the media in untreated control wells turned pink. Changes in fluorescence were then quantified with a microplate reader (excitation wavelength at 530 nm and emission at 590 nm). The viability was determined by normalizing the results to the average fluorescence of the untreated control wells, which is set to 100%, giving each well the relative viability. The average background fluorescence (no cell wells) was also subtracted from sample fluorescence. Temperature measurement of cell media bulk temperature was performed with a type J thermocouple inserted to the bottom of the well.

Animal handling procedures

All animal studies were performed following the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Oklahoma and performed by staff with proper training. Animals were housed in a pathogen-free facility at the University of Oklahoma and monitored daily.

In vivo studies

For tumor induction, six-week-old female BALB/cJ mice were injected with 1x10⁶ EMT6 cells suspended in 100 µl of PBS using a 30-gauge needle. The injection was performed subcutaneously in the fourth mammary fat pad, close to the nipple, which characterizes an orthotopic tumor. Mice weight and tumor volumes were measured every 3–5 days during each study. Tumor volumes were measured using a caliper, and volume (V) was determined through the modified ellipsoid formula V = (1/2) ⋅ (L⋅W²), where length (L) is the longest diameter of the tumor and the perpendicular diameter is the width (W). For all the studies, mouse health was assessed every 3–5 days for signs of distress. Mice were euthanized when the tumor size was greater than 15 mm in any of the directions of measurement or when the mice were sick, characterized by dehydration, weight loss greater than 20%, recumbent posture, breathing difficulty, or loss of leg function due to tumor proximity. Mice were fed a standard chow diet. During photothermal irradiation of tumors with a NIR laser light, mice were anesthetized with 3% isoflurane and 97% oxygen using a nose cone. Treatments were performed when tumors reached a size of ~ 5 mm (approximately 60 mm³). SWCNT-ANXA5 injections were performed at a 1.2 mg/kg dose of SWCNT.

To evaluate the cytokine release related to tumor surface temperature EMT6-tumor-bearing mice were injected intratumorally (i.t.) with SWCNT-ANXA5 when the tumors were around 5 mm in diameter (~ 60 mm³). The tumors were treated by a 980-nm NIR laser at 1 W/cm². The tumor surface temperature was monitored during the irradiation by a thermal camera (FLIR E5-XT). Irradiation ceased immediately after the tumor surface reached the temperature assigned to the groups of 45, 50, 55, or 60°C. Mice were euthanized either 1 or 7 days after PTT for blood collection (respectively 24 h and 168 h). The concentration of four pro-inflammatory cytokines was measured in the serum by ELISA. The control group was i.t. injected with SWCNT-ANXA5 but not treated with PTT.

To assess the effects of SWCNT-ANXA5-mediated PTT at 45°C in combination with anti-PD-1, a long-term survival test was performed. Mice were inoculated with EMT6 tumors on day 0. On day 7, when tumors were around 3 mm in diameter, treatment started for the assigned groups with an intraperitoneal injection of anti-PD-1 checkpoint inhibitor at 10 mg/kg. The same injection was performed two more times on days 10 and 15. On day 11, mice were injected i.t. with SWCNT-ANXA5 when the tumors were around 5 mm in diameter (~ 60 mm³) and treated with PTT. The tumors were treated by 980-nm NIR laser at 1 W/cm². The tumor surface temperature was monitored during the irradiation by a thermal camera (FLIR e-5). Irradiation was ceased immediately after the tumor surface temperature reached 45°C. Mice health was monitored for 100 days after tumor induction, when the study was terminated. For the mechanistic analysis of the combination therapy, mice were treated following the same protocol for the long-term survival test, but the mice were euthanized by CO₂ asphyxiation 14 days after PTT, when the spleens were harvested for quantification of antitumoral immune effector cells.

To improve the efficiency of the PTT at 45°C, a tumor control study was performed with prolonged irradiation times. Tumor-bearing mice were injected i.t. with SWCNT-ANXA5 when the tumors were around ~ 100 mm³. The tumors were treated by a 980-nm NIR laser at 1 W/cm². Tumors were irradiated until the surface temperature reached 45°C and then held the temperature at 45°C ± 3°C by repeatedly turning the laser off and on. The cycles of turning off and on were repeated for the total time of the irradiation session of 0, 1, 2 and 5 min. After irradiation sessions, tumor volumes and body weights were monitored daily. The experiment was finalized 15 days after PTT when the remaining mice were euthanized.

To analyze the combination therapy of anti-PD-1 and PTT at 45°C for 5 min, tumor-bearing mice were treated with three injections of anti-PD-1 (intraperitoneal injection on days 7,10, and 15 at 10 mg/kg), and the tumors were treated by 980 nm NIR laser at 1 W/cm² after i.t. injection of SWCNT-ANXA5 (1.2 mg/kg) on day 11. The cycles of turning off and on were repeated for the total time of the irradiation session of 5 min. The group that received irradiation for 5 min was compared to an untreated control group, and another group treated with anti-PD-1 and PTT, but the irradiation stopped immediately when the tumor surface temperature reached 45°C.

Ex vivo SWCNT detection for biodistribution analysis

BALBc/J mice were inoculated with EMT6 tumors and were injected i.t. with SWCNT-ANXA5 (1.2 mg/kg of SWCNTs) when the tumors were around 5 mm in diameter (~ 60 mm³). The tumors were treated by a 980-nm NIR laser at 1 W/cm². Tumors were irradiated until the surface temperature reached 45°C. Mice were euthanized by CO₂ asphyxiation 1 week after PTT. Major organs were harvested and weighed. Organs were frozen and stored in a -80°C freezer. Tissue lysates for analysis of SWCNT concentration were prepared according to the protocol from Liu et al.[28]. Briefly, after thawing samples, organs were individually ground in a tissue grinder added to lysing buffer composed of 1% SDS, 1% Triton X-100, 10 nM DTT (dithiothreitol), and 40 mM tris-acetate-EDTA buffer to complete volume of 10 ml. Tissue was digested overnight in an incubator at 70°C. Each sample was sonicated for 1 h to resuspend SWCNTs from digested tissue. Samples were analyzed using intrinsic SWCNT fluorescence by single excitation fluorescence measurement using an NS MiniTracer (Applied Nanofluorescence, Houston, TX).

Statistical analysis

The statistical significance of in vitro results was assessed using an unpaired t-test with Welch’s correction for assumed unequal variances for experiments with only two groups. One-way ANOVA with Dunnett’s multiple comparisons was used for tests with more than two groups, comparing treated groups to the control group. The statistical significance of cytokine levels in serum, tumor volumes, and splenic cell counts in the in vivo studies was assessed by one-way ANOVA with Dunnett’s multiple comparisons comparing treated groups to the control group. In survival experiments with only three groups, one-way ANOVA with Tukey’s multiple comparisons comparing every pair of groups was used to assess statistical analysis between tumor volumes. The Mantel-Haenszel log-rank test determined the statistical significance of survival curves by comparing treated groups to the untreated control. All analyses were carried out in GraphPad Prism 8 software.

In vitro studies

In vitro studies with SWCNT-ANXA5-mediated PTT show the cytotoxic effect and selectivity of the treatment towards tumor cells. The NIR-laser treatment at room temperature induced a significant decrease in cell viability after 3 min of irradiation when the EMT6 cells were incubated for a short period of time and the unbound conjugate was washed off (Fig. 1a). The same decline in viability was not observed in the control group consisting of EMT6 cells irradiated with the laser and absent of the conjugate, even in higher irradiation times. In this experiment, the decrease in cell viability can be attributed to the bound SWCNT-ANXA5 to the cancer cells that express PS. Previous studies have shown evidence of the binding of the SWCNT-ANXA5 to PS-expressing cells [25, 29]. Therefore, the presence of the conjugate is crucial to the effectiveness of the cancer cell laser ablation, and its absence hinders the cytotoxic effects of the irradiation at the energy density studied in this experiment (up to 450 J/cm²). In a parallel experiment performed at the same conditions, the bulk temperature of the cell media after laser irradiation was measured (Fig. 1b). The cell media of EMT6 treated with SWCNT-ANXA5-mediated PTT showed an increased temperature elevation compared to the control treated with laser irradiation alone. While those results confirm that the presence of SWCNT-ANXA5 bound to the monolayer of EMT6 cells significantly increases the macroscale temperature compared to the laser alone, it cannot be used to correlate to the temperature at which cells suffer thermal stress. By comparing the results from Fig. 1a and b and assuming a conservative room temperature of 21°C, the higher macroscale temperature reached in those experiments was around 32°C, which induced cell death in 83% of cells. In vivo, tissue damage is believed to occur at temperatures greater than 41ºC [14]. This discrepancy is likely to be related to the ability of SWCNTs to induce a local nanoscale temperature rise, which cannot be assessed by the temperature measurement method used (type J thermocouple). It is hypothesized that the microscale temperature around the cells is much higher than the macroscale temperature assessed by the type J thermocouple in this experiment.

The previous in vitro experiments were performed at room temperature. EMT6 cells may not react the same at room temperature as at 37°C, especially considering the ablation temperatures in natural physiological conditions. Figure 1c shows that EMT6 cells treated with SWCNT-ANXA5-mediated PTT in an incubator at 37^oC had a significant cell viability decrease after 1 min of irradiation at 1 W/cm². Comparatively, cells treated with laser alone or PEGylated SWCNT-mediated PTT did not result in a significant cell viability drop even after 7 min of laser irradiation. These results suggest that ANXA5 is an effective targeting agent, since the untargeted SWCNTs were not effective at inducing a decrease in cell viability, results that are similar to the absence of SWCNT incubation (control in Fig. 3c). It is hypothesized that the untargeted PEGylated SWCNTs was washed away from the cell surface after four washing steps, therefore hindering the cytotoxic effect, while the targeted conjugate bound to cells after washing steps induces an effective photothermal effect resulting in cytotoxicity. The same targeting capabilities of SWCNT-ANXA5 are also displayed in Fig. 1d where confluent HUVEC were treated with the same conditions as EMT6 cells in Fig. 1c. Confluent HUVEC cells mimic healthy endothelial cells, therefore lacking PS expression on the outer leaflet of the phospholipid bilayer. Even after 7 min of treatment, corresponding to a power density of 420 J/cm², neither SWCNT-ANXA5 nor the control group had a significant cell viability decrease. These results suggest that for the 2 h incubation times, the SWCNT-ANXA5 did not bind confluent healthy endothelial cells and, therefore, was washed away during washing steps, resulting in an ineffective PTT. Comparing the results of healthy cells to the ones gathered from PS-expressing EMT6 tumor cells demonstrates the targeting capabilities of ANXA5 to PS-expressing cells.

Effect of tumor surface temperature in vivo on immune system activation

In vitro studies provide essential data on individual steps in mechanistic processes in cancer development and therapeutics. Nevertheless, in vivo studies are necessary to excel in modeling immunity interactions. In our previous study of PTT using SWCNTs targeted by conjugation to ANXA5, the conjugate was injected intravenously (i.v.), which resulted in the accumulation of SWCNTs in several organs, including the liver and kidneys, with slow clearance from these organs [30]. Therefore, in the current study, the SWCNT-ANXA5 conjugate was injected intratumorally (i.t.) at the same dosage used for i.v. injection in the previous study. First, we analyzed the immune activation of PTT by reaching various endpoint tumor surface temperatures of 45, 50, 55, and 60^oC (Fig. 2a, b, c). EMT6-tumor-bearing mice treated with SWCNT-ANXA5-mediated PTT that stopped at 45°C had an elevation of TNF-α (24 and 168 h after PTT) and IL-12 p70 (24 h after PTT), while higher temperatures did not significantly increase the level of those cytokines compared to control.

Various studies have shown efficient tumor ablation when the carbon nanotubes are used as photosensitizers for PTT [31–41]. Huang et al. [34] studied the photothermal therapy efficiency on orthotopic squamous cell carcinoma in mice. Tumor-bearing mice were injected i.t. with different doses of covalently PEGylated SWCNTs and subjected to 785 nm laser treatment at different energy density dosages. For each combination of SWCNTs and energy dosages, the superficial temperature of the tumors was measured. Additionally, tumor volumes were recorded 11 days after treatment, and survival curves were reported for 45 days. Results showed that the highest dosage of MWCNTs and power density (1 mg/ml and 0.2 W/cm² for 10 min) caused an increase of 56°C on the surface of the tumor, total tumor regression for 11 days and 62.5% survival for 45 days (n = 8). Zhou et al. [36] studied the therapeutic effects of PEGylated SWCNTs combined with laser irradiation in vivo. Mice that bore non-orthotopic breast cancer models were injected i.t. with 1 mg/kg and irradiated with a 980 nm laser (1 W/cm², 10 min). The tumor surface temperature reached 76°C after 5 min of irradiation. Tumor volume assessment showed complete tumor regression for the SWCNT + laser treatment group for 30 days and 75% survival for 100 days.

It can be noted that the temperatures obtained in those experiments were extreme, with reports of temperatures of 76°C. Those extreme temperatures are very efficient at ablating solid tumors due to the induction of instantaneous cell death [42]. Nonetheless, concerns about the safety of procedures that involve such high temperatures need to be addressed. Conversely, the fact that 45°C PTT increased the level of two pro-inflammatory cytokines at different time-points in our experiment is evidence that the lower temperature is enough to induce an immune response in an EMT6 tumor model. We hypothesize that higher temperatures induce very rapid cell death and possibly necrotic cell death, impeding the release of danger signals and that cause the recruitment of immune cells to the tumor site. Therefore, 45°C appears to be sufficient for the induction of immunogenic cell death. The decrease in the final temperature of the PTT is beneficial for the procedure's safety, reducing the chance of damage to surrounding tissue.

Combination of SWCNT-ANXA5-mediated PTT at 45°C and anti-PD-1 checkpoint inhibition

Following the results about tumor surface temperature at the end of irradiation, a long-term survival study was performed with the combination therapy of PTT and checkpoint inhibition with anti-PD-1 (Fig. 3). The combination therapy followed the schedule shown in Fig. 3a. EMT6 tumor-bearing mice were injected i.t. with SWCNT-ANXA5 when tumor sizes were ~ 5 mm. The NIR irradiation was immediately stopped when the tumor surface temperature reached 45°C. Three doses of anti-PD-1 were injected intraperitoneally in the assigned groups. The survival curve (Fig. 3b) shows that only the combination therapy was able to increase the survival compared to the untreated control group (**p < 0.01). In previous studies in our lab, the ablation of EMT6 tumors to temperatures up to 54°C was efficient at generating a complete recession within a week of PTT without the combination with checkpoint inhibition; however, it did not increase survival[30]. Here, the ablation of EMT6 tumors at a milder temperature of 45°C was not effective at generating a complete tumor recession by itself. Therefore, we hypothesize that even though the PTT at 45°C is more effective at generating an immune response, correlated to the higher levels of pro-inflammatory cytokines (Fig, 2a, b, c), this temperature is not sufficient to ablate the whole volume of the primary tumor.

We hypothesize that the combination therapy was efficient in generating a systemic immune response that effectively killed the remaining tumor that the PTT did not efficiently ablate at 45°C. By day 21 (12 days after PTT), four out of eight mice in the anti-PD-1 + PTT group had complete tumor recession. Those mice did not undergo tumor regrowth and survived until the end of the study (100 days after tumor inoculation), yielding a 50% survival at 100 days (Fig. 3b). The lack of complete tumor recession in the PTT group concurrent with complete tumor recession seen in some of the mice in the PTT + anti-PD-1 group is evidence of a synergistic effect of the combination therapy, which might be due to an anti-tumoral systemic immune response.

To study that hypothesis, an immunophenotypical analysis of splenic cells was performed. EMT6 tumor-bearing mice were treated using the same treatment schedule of the long-term survival experiment (Fig. 3a). Fourteen days after PTT, mice were euthanized, their spleens were collected, and splenic cells were stained for immunophenotyping through flow cytometry. Figure 3d, e shows that only the combination therapy was effective at increasing the total number of helper T cells (CD4 positive) and cytotoxic T cells (CD8 positive) in the spleen, which introduces an understanding of the mechanism of antitumor immunity. While helper CD4 + T cells assist other immune cells by cytokine stimulation and direct cell-cell contacts, mediating a humoral immune response, cytotoxic CD8 + T lymphocytes are directly responsible for killing tumor cells. Therefore, increased CD4 + and CD8 + cell numbers suggest systemic anti-cancer immune responses.

Another critical obstacle in cancer treatment is metastasis, which is the cause of 90% of the deaths related to cancer [43]. The results from the combination therapy of anti-PD-1 and PTT at 45°C were encouraging due to the cure of 50% of mice from an EMT6 tumor and evidence of an abscopal effect. Because EMT6 tumor models are known to establish metastatic tumors very early in tumor development, long-term survival is evidence that the combination therapy of PTT and anti-PD-1 elicits a systemic antitumoral response that efficiently suppresses metastatic tumors.

Increased efficiency of EMT6 tumor ablation at 45°C

Despite the encouraging results from the long-term survival test in Fig. 3, we hypothesized that if PTT efficiently ablates the tumor's whole volume, the combination therapy response would be even more effective. For that, longer irradiation times would allow heat transfer from the surface of the tumor, where most of the irradiation reaches, to deeper layers of cancer, thus promoting heat stress in the totality of the tumor volume. To test that hypothesis, we tested tumor control of a 45°C PTT keeping tumors at that temperature for extended periods (1, 2, and 5 min) by cycling the laser on/off to keep the tumor surface temperature at 45°C ± 3°C. Figure 4 shows the tumor control after irradiation of EMT6 tumors at 45°C for more extended time (mice were not treated with checkpoint inhibition). After PTT, the groups 1 min, 2 min, and 5 min were able to induce a decrease in tumor volume. The same decrease did not happen in the control group that was irradiated with PTT at 45°C and immediately stopped, which is an indication of how mild PTT is at 45°C. Additionally, this result strengthens the hypothesis of a synergistic effect of combination therapy with anti-PD-1 (Fig. 3), since the PTT at 45°C (0 min) did not induce tumor recession by itself. PTT at 45°C for 1 min or 2 min was not efficient in maintaining a statistically significant reduction of tumor volumes compared to control, whereas the 5 min irradiation of the tumor at 45°C reduced the tumor volumes significantly and maintained that difference until the end of the study 15 days after irradiation. This is evidence that better tumor control can be achieved with a milder PTT temperature of 45°C in EMT6 tumor, ultimately leading to a safer procedure.

Increased survival from prolonged SWCNT-ANXA5-mediated PTT at 45°C and anti-PD-1 checkpoint inhibition

The improved ablation of the primary tumor obtained by the 5 min irradiation at 45°C led to another long-term survival test (Fig. 5), which was performed to analyze the hypothesis that a more efficient ablation of the primary tumor at 45°C increases the efficiency of the combination therapy. The treatment schedule was the same as shown in Fig. 3a. For one treated group [anti-PD-1 + PTT (0 min)], NIR irradiation was immediately stopped when the tumor surface temperature reached 45°C. For the other treated group [anti-PD-1 + PTT (5 min)], NIR irradiation continued for 5 min after reaching 45°C, cycling the laser on/off to keep the tumor surface temperature at 45°C ± 3°C. The treated groups received three doses of anti-PD-1 injected intraperitoneally. Figure 5 shows that combining checkpoint inhibition of anti-PD-1 to the PTT keeping the tumor at 45°C for 5 min is a more effective treatment for EMT6 tumor than stopping the irradiation immediately after the tumor reaches 45°C. Eighty percent of the mice survived 100 days after PTT, which is evidence of complete cure.

One of the most relevant challenges in tumor treatment is hypoxic regions in the primary tumor because of the lack or minimal blood flow that prevents the delivery of therapeutic agents. Hypoxia is characterized by oxygen levels below physiological requirements correlated to malignant proliferation and dysfunctional vascularization. Hypoxia induces activation of complex cell signaling pathways that lead to metabolism changes related to cell quiescence, ultimately leading to treatment resistance and selection of aggressive cancer cells [44]. Tumor ablation is a very effective strategy for the treatment of hypoxic and drug-resistant cancers because the heat transfer in tissue, although ameliorated by vascularization, does not require blood flow to reach hypoxic areas of the tumor. Therefore, PTT can be an effective strategy to induce cell death in regions of tumors that were previously unreachable by common therapeutic approaches and prevent recurrence and metastasis related to untreated hypoxic areas of tumors [45]. This effect is further advanced by photosensitizers because of the more efficient heating patterns seen in the tumor with the addition of those agents during photothermal therapy.

Retention of SWCNT-ANXA5 in injection site

An experiment was performed to analyze the biodistribution of SWCNTs after i.t. injection. EMT6-tumor-bearing mice were injected i.t. with 1.2 mg/kg of SWCNT-ANXA5, treated with PTT at 45°C, and euthanized seven days after. Organs were harvested, weighed, and digested in lysing buffer. The concentration of SWCNTs in the tumor, liver, and kidney was determined by relative NIR fluorescence spectroscopy (Table 1). Tumors had a detectable amount of SWCNTs in the tumor, while SWCNTs could not be detected in the liver and kidneys. In previous studies with i.v. injection of the conjugate, SWCNTs tended to accumulate in the liver and kidney clearance organs as well as in the tumor and had a slow rate of clearance from the liver and kidneys over a period of 4 months [30]. Therefore, this is evidence that the i.t. injection of SWCNT-ANXA5 prevents the systemic distribution of the nanotubes, which is beneficial for avoiding accumulation in clearance organs. These results confirm the hypothesis that the SWCNTs are too long to diffuse out of the injection site and/or is prevented from leaving the tumor because of being bound to PS on the surface of tumor cells. Accordingly, one of the mice in this biodistribution study had complete tumor recession due to PTT, and the image of the skin injected with SWCNT-ANXA5 resembled a tattoo. Similar to the ink particles in a tattoo, SWCNTs are confined in the tissue.

Table 1

Concentration of SWCNTs in the tumor and clearance organs 1 week after photothermal therapy (1.2 mg/kg injection i.t. of SWNT and irradiation until tumor temperature of 45^oC). Data are shown as mean ± SE (n = 3).
Organ	SWCNT concentration (µg SWCNT/g tumor)^a
Tumor	13.8 ± 4.0
Liver	ND
Kidneys	ND
^aND: not detected

The i.t. injection of SWCNTs used in this study could be accomplished for non-superficial tumors using image-guided delivery techniques that are used clinically [46]. Using i.t. injection of SWCNTs prevents the systemic delivery of these nanoparticles, ultimately avoiding accumulation in major organs and lingering clearance.

Here, we demonstrate a novel combinatorial treatment modality where we obtain a high survival rate in mice with aggressive metastatic breast cancer, the EMT6 tumor model, using SWCNT-ANXA5-mediated photothermal therapy at a relatively low temperature combined with anti-PD-1-based checkpoint inhibition. The study of the temperature of photothermal therapy is crucial for the safety of the treatment and the potential translation to clinical trials. A lower PTT temperature in a relatively immunogenic tumor would be enough to generate a systemic immune response when combined with checkpoint inhibition. Additionally, the transition from i.v. to i.t. injection prevented the accumulation of the SWCNTs in major organs. Even though only the original tumor was given radiation treatment, it is interesting to note that mice with metastatic cancer that received combinatorial therapy lived longer. The combination of both treatment modalities enhanced the amount of CD4 + helper and CD8 + cytotoxic T cells in the EMT6 tumor model, according to a mechanistic investigation that quantified the splenic antitumor effector cells. According to our hypothesis, this rise in T cells results from an abscopal response, in which antitumoral effector cells prevent tumor spread.

Funding This study was supported by the Universidad Nacional de San Agustin, Arequipa, Peru and by the Jean Wheeler Sparks and Baxter Abbott Sparks Breast Cancer Research Fund at the University of Oklahoma Foundation.

Conflicts of interest/Competing interests The authors declare that there are no conflicts of interests or competing interests.

Data availability All data are fully available without restriction.

Code availability Not applicable

Author’s contributions Roger Harrison conceived the idea and directed the experimental work. Gabriela Faria and Roger Harrison wrote the manuscript. Gabriela Faria, Clement Karch, Alexis Woodward, and Adam Aissanou conducted the experiments. Gabriela Faria, Clement Karch, Alexis Woodward, Adam Aissanou, Sathish Lageshetty, Ricardo Prada Silvy,Daniel Resasco, Jorge Andres Ballon, and Roger Harrison read and approved the manuscript.

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Immunogenic Treatment for Metastatic Breast Cancer Using Targeted Carbon Nanotube Mediated Photothermal Therapy in Combination with Anti-PD-1

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