Harnessing immunotherapy to enhance the systemic anti-tumor effects of thermosensitive liposomes

Chemotherapy plays an important role in debulking tumors in advance of surgery and/or radiotherapy, tackling residual disease, and treating metastatic disease. In recent years many promising advanced drug delivery strategies have emerged that offer more targeted delivery approaches to chemotherapy treatment. For example, thermosensitive liposome-mediated drug delivery in combination with localized mild hyperthermia can increase local drug concentrations resulting in a reduction in systemic toxicity and an improvement in local disease control. However, the majority of solid tumor-associated deaths are due to metastatic spread. A therapeutic approach focused on a localized target area harbors the risk of overlooking and undertreating potential metastatic spread. Previous studies reported systemic, albeit limited, anti-tumor effects following treatment with thermosensitive liposomal chemotherapy and localized mild hyperthermia. This work explores the systemic treatment capabilities of a thermosensitive liposome formulation of the vinca alkaloid vinorelbine in combination with mild hyperthermia in an immunocompetent murine model of rhabdomyosarcoma. This treatment approach was found to be highly effective at heated, primary tumor sites. However, it demonstrated limited anti-tumor effects in secondary, distant tumors. As a result, the addition of immune checkpoint inhibition therapy was pursued to further enhance the systemic anti-tumor effect of this treatment approach. Once combined with immune checkpoint inhibition therapy, a significant improvement in systemic treatment capability was achieved. We believe this is one of the first studies to demonstrate that a triple combination of thermosensitive liposomes, localized mild hyperthermia, and immune checkpoint inhibition therapy can enhance the systemic treatment capabilities of thermosensitive liposomes.


Introduction
Surgery and radiotherapy remain the cornerstones of local cancer therapy [1]. While chemotherapy is commonly used as a systemic treatment approach to debulk tumors in preparation for surgery and/or radiotherapy, treat residual disease, and reach metastatic disease sites. Incomplete local disease control can lead to recurrence at the primary site [2]. Such instances of local recurrence are known to occur in a number of different cancers, including non-small cell lung cancer, colorectal cancer, malignant pleural mesothelioma, and rhabdomyosarcoma (RMS) [2]. In the case of RMS, up to one-third of patients experience recurrence and 70-80% of these cases occur at the primary disease site [3]. Reducing the likelihood of disease recurrence requires more aggressive therapeutic strategies. However, these are often accompanied by more severe adverse effects. Consideration of potential long-term adverse effects can play an important role in treatment selection, in particular in cancers such as RMS, where the majority of cases are present in patients under 10 years of age [4,5].
One option to improve local disease control is to employ drug delivery strategies, such as nanoparticles, that provide triggered drug release and result in an increase in tumor drug concentrations. These systems are administered systemically and designed to release their content in the presence of a specific trigger. To this end, delivery platforms exploiting both internal (e.g., pH or hypoxia) and/or external (e.g., temperature or ultrasound) triggers to stimulate drug release have been developed [6]. This includes thermosensitive liposomes, initially developed by Yatvin et al. over four decades ago with the goal of improving upon passively targeted liposomes [7]. Since then, various chemotherapy drugs have been formulated as thermosensitive liposomes with doxorubicin being the most frequently explored compound. In many cases, these triggered delivery systems have been evaluated in combination with localized heating and have afforded promising improvements in treatment efficacy in animal models of local disease [8][9][10]. ThermoDox ® , a thermosensitive liposome formulation encapsulating doxorubicin, is the only formulation that has been evaluated in clinical trials [11] However, as discussed elsewhere, ThermoDox has faced significant setbacks in its clinical development, in particular when evaluated in combination with radiofrequency ablation (temperatures > 50 °C) [11]. Nonetheless, several pre-clinical and clinical studies have demonstrated the potential of this treatment approach in combination with mild hyperthermia (temperatures of 39-45 °C; HT), with a number of studies achieving significant improvements in the drug's therapeutic index [12][13][14].
Currently, the majority of cancer-associated deaths are due to metastatic spread to secondary sites [15]. An important consideration regarding the treatment of metastatic disease is that achieving local control (e.g., via surgery or radiotherapy) is recognized as a pivotal step in reducing the probability of metastatic spread in the first place [16]. However, due to challenges in detecting metastatic sites, many patients are estimated to harbor undetected metastases at the time of initial surgery [16]. Thus, despite the need for local control in cancer therapy, there is clearly a need for a treatment approach that offers both local as well as systemic anti-tumor treatment capabilities. This raises the question: can systemically administered thermosensitive liposomes, that are designed to improve local disease control, also treat metastatic disease?
The concept of a local treatment approach inducing a systemic anti-tumor effect is commonly referred to as the "abscopal effect". The first mention of this effect dates back to a 1953 publication on whole-body irradiation, where Robin Mole asked the question "has irradiation of a mammal an effect at a distance from the volume irradiated?" [17]. However, to this day, the potential systemic effects of ionizing radiation remain highly questionable with few documented occurrences over the past several decades [18]. In fact, only 46 clinical cases of radiotherapy-induced abscopal effects were reported between 1969 and 2014 [19]. Yet, the emergence of immune checkpoint inhibition (ICI) therapy has led to a significant increase in pre-clinical and clinical reports of the abscopal effect [20]. While the exact mechanism remains to be fully understood, it has been shown that the immune system plays a key role [21,22].
More recently, the abscopal effect has been referenced in connection with other localized treatment strategies that induce a systemic effect (e.g., intratumoral chemotherapy, cryotherapy, ablation, and HT) [23]. If the therapeutic intervention is confined to a specific treatment volume, the resulting systemic effects can generally be described as abscopal. To this end, localized HT treatment (39-43 °C) has been shown to stimulate the immune system and enhance anti-tumor immune effects [24,25]. As far back as 2009, Skitzki et al. highlighted the potential of HT as a non-invasive, non-toxic, and readily available treatment modality that could be used as an adjuvant with existing immunotherapy approaches [25]. In the clinic, localized HT is commonly applied in combination with radiotherapy focusing on HT as an approach to boost radiationinduced abscopal effects [26,27]. The immune stimulating effects of HT treatment are currently under investigation in combination with immunotherapy (NCT03757858, NCT03393858).
The study presented herein is one of the first to combine ICI (i.e., PD-1 blockade alone) with HT-triggered thermosensitive liposome-mediated chemotherapy. First, we explored the treatment efficacy of our previously developed thermosensitive liposome formulation encapsulating vinorelbine (ThermoVRL) in an immunocompetent model of RMS. We then assessed potential systemic anti-tumor effects of this treatment approach in a bilateral tumor model. Lastly, we determined if the addition of ICI in the form of PD-1 blockade therapy enhances these effects. Overall, the results demonstrate the significant local treatment effect of ThermoVRL, as well as a limited effect on distant tumors, when combined with localized HT. Interestingly, the addition of PD-1 blockade therapy was found to significantly boost systemic anti-tumor effects. Moreover, we were able to demonstrate abscopal effects of localized HT alone with the addition of PD-1 blockade therapy. In summary, this study shows that the combination of ICI and HT-triggered thermosensitive liposome mediated chemotherapy has tremendous potential as a multimodal treatment approach for the management of local and metastatic disease.

Cell culture and cytotoxicity evaluation
The murine RMS cell line M3-9-M (obtained from the Mackall Laboratory at Stanford University) was grown in RPMI 1640 supplemented with 10% FBS, 1% P/S, 1% L-Glutamine, 1% NEAA, 1% sodium pyruvate, and 50 µM 2-mercaptoethanol. Cells were kept at 37 °C with 5% CO 2 , unless otherwise indicated. Cells with passage numbers between 5-15 were seeded in 96-well plates at 100 cells per well followed by treatment with VRL after 24 h of incubation. Cell viability was then assessed after 72 h, using an acid phosphatase assay. Specifically, 2 mg/mL phosphatase substrate p-nitrophenylphophate was added to cells for 1 h followed by the addition of 0.1 N NaOH and UV absorbance measurement at 405 nm. Half maximal inhibitory concentration (IC 50 ) values were obtained using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA) by fitting the data with a 4-parameter sigmoidal dose-response curve. To evaluate the effect of HT on VRL cytotoxicity, cells were incubated at 42 °C for 1 h followed by 71 h at 37 °C. The in vitro cytotoxicity data can be found in Fig. S1.

Liposome preparation
ThermoVRL was prepared as previously described [35]. In brief, a thin lipid film was prepared by dissolving DPPC, MSPC, and PEG 2k -DSPE at a molar ratio of 86/10/4 in chloroform. This solution was then dried using a rotary evaporator. The film was then hydrated with a TEA 8 SOS solution (0.22 M sulfate group concentration) to a total lipid concentration of 125 mM. The liposomes were then extruded (Lipex Extruder, Northern Lipids, Vancouver, BC, Canada) three times through double-stacked track-etch polycarbonate membranes with a 200 nm pore size (Whatman Inc., Clifton, NJ, USA). Following this, the resulting liposomes were extruded a further 10 times through 100 nm poresize membranes. Extruded liposomes were then chilled on ice for 10 min prior to overnight dialysis in HBS (20 mM HEPES, 150 mM sodium chloride, pH 6.5). VRL was loaded at 30 g VRL/mol lipid by incubating the liposomes with the drug at 35 °C for 60 min. ThermoVRL liposomes were subsequently chilled on ice for 10 min, purified by overnight dialysis in HBS, and concentrated via tangential flow filtration using a polysulfone MicroKros ® filter (Spectrum, Rancho Dominguez, CA, USA).
ThermoVRL liposomes were filtered through a 0.22 µm PES filter into a sterile rubber-sealed glass vial (ALK-Abelló Inc., Round Rock, TX, USA) prior to characterization or use in in vivo experiments. ThermoVRL was stored at 4 °C and used within 72 h.

Liposome characterization
The reader is referred to Regenold et al. [35] for a detailed description of the characterization of the liposomes. In brief, dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Malvern, WOR, UK) was used to determine the size of ThermoVRL liposomes after a 1:100 dilution in phosphate-buffered saline. The same instrument was used to determine the zeta potential following a 1:100 dilution in Milli Q water. Differential scanning calorimetry (TA Q100, TA Instruments, New Castle, DE, USA) was used to determine the melting phase transition temperature by heating ThermoVRL liposomes three times from 25 to 60 °C at a rate of 1 °C/min. VRL drug levels were determined via HPLC-MS analysis.

Tumor model and localized mild hyperthermia treatment
All animal studies were conducted in accordance with the guidelines of the Animal Care Committee of the University Health Network (UHN, Toronto, ON, Canada).

Single tumor study
An aliquot of 2 × 10 6 M3-9-M cells was injected subcutaneously into the right flank (Scheme 1: single tumor study (A) of 6-8-week-old male C57BL/6 mice to develop ectopic syngeneic tumors. Tumor volumes were calculated as V = 6 (w 2 )(l) from measurements of tumor width (w) and length (l) . Treatments were administered once tumors reached a volume > 90 mm 3 . It took approximately 10-days post-cell injection for tumors to reach this volume. Control and treatment groups included (i) saline, (ii) free VRL (10 mg VRL/kg body weight), and (iii) ThermoVRL (10 mg VRL/kg body weight). All treatments were administered without and with concurrent HT treatment localized to the tumor. A dose of 10 mg VRL/kg body weight was chosen since Aston et al. previously identified this as the maximum tolerated dose in C57BL/6 mice when administered as free VRL [36]. For treatment with ThermoVRL, a drug dose of 10 mg VRL/kg body weight did correspond to an approximate lipid dose of 460 µmol phospholipid/kg body weight. A tumor dimension of > 15 mm in any dimension or a body weight loss > 20% were selected as ethical study endpoints.

Double tumor study
Similar to the approach described in "Single tumor study" section, aliquots of 2 × 10 6 M3-9-M cells were injected subcutaneously into the right and left flanks (Scheme 1: double tumor study (B)) of 6-8-week-old male C57BL/6 mice to develop two ectopic syngeneic tumors. Tumor volume measurements were conducted as described previously ("Single tumor study" section) and similarly as above, treatments were commenced approximately 10 days post cell injection once one tumor had reached a volume > 90 mm 3 . In the bilateral tumor model, the larger of the two tumors was considered the primary tumor and thus determined to be the site of HT. Control and treatment groups included the following: (i) no treatment (untreated); (ii) intraperitoneal (i.p.) PD-1 blockade therapy (anti-PD-1); (iii) intravenous (i.v.) saline and HT (saline + HT); (iv) i.p. anti-PD-1 and HT (anti-PD-1 + HT); (v) ThermoVRL and HT (ThermoVRL + HT); and (vi) ThermoVRL, anti-PD-1, and HT (ThermoVRL + anti-PD-1 + HT). Anti-PD-1 treatment was administered i.p. as 200 µg anti-PD-1 antibody (RMP1-14, Bio X Cell). This treatment was started one day prior to ThermoVRL and/ or mild HT administration and was administered twice per week for 35 days. Similar to the "Single tumor study", Ther-moVRL was administered at 10 mg VRL/kg body weight which corresponds to approximately 460 µmol phospholipid/ kg body weight. A tumor dimension > 15 mm in any dimension, or a body weight loss > 20%, were selected as ethical endpoints. The bilateral tumor study (Scheme 1) was concluded as soon as no palpable tumors were detectable since the single tumor study (Fig. 1) demonstrated a very durable treatment response with no tumor re-growth past a specific study timepoint.

Mild hyperthermia treatment
A laser-based heating system developed by Dou et al., in combination with a single-point temperature probe, was used to administer HT localized to the tumor [37]. In brief, a centrally placed point-based optical fiber temperature probe (Luxtron Model 790, LumaSense Technologies Inc., Santa Clara, CA, USA) located within the tumor was used to monitor the temperature. Then, a custom-built illuminator (Spectralon, Labsphere Inc., North Sutton, NH, USA) was placed on top of the tumor receiving HT. A 763 nm diode laser (Model CD 403 laser, Ceralas, Jena, Germany) connected to the illuminator (Spectralon, Labsphere Inc., North Sutton, NH, USA) via a 400 µm fiber was then used to deliver homogenous light (± 15%) to the tumor. To maintain a target temperature of 42.5 °C, the power of the laser was manually adjusted between 0.2 to 0.65 W/cm 2 . Tumors were pre-heated to 42.5 °C for 5 min. Following this 5 min period, i.v. tail vein injection of ThermoVRL or saline was administered under constant heating, and heating was continued for another 20 min post treatment. The contralateral tumor remained unheated.

Statistical analysis
Statistical analysis was performed using SSPS Statistics 28.0 (IBM, Armonk, NY, USA). Animal weights on specific treatment days were compared by one-way ANOVA with Bonferroni post hoc testing. Survival differences were calculated and compared with a log-rank test and a Bonferroni correction for multiple comparisons. Tumor volumes of different treatment groups were compared by unpaired t-test.

Results
ThermoVRL was prepared as previously described [35]. Prior to in vivo administration, the drug-loaded liposomes were characterized in terms of size (98 ± 18 nm), zeta potential (− 32 ± 5 mV), melting phase transition temperature Scheme 1 Overview of the design of both the single tumor and double tumor studies. A To determine the treatment efficacy of VRL encapsulated in thermosensitive liposomes (ThermoVRL), single M3-9-M tumor-bearing male C57BL/6 mice were treated with free VRL or ThermoVRL in combination with mild hyperthermia (42.5 °C). Tumors were pre-heated for 5 min prior to intravenous treatment administration followed by another 20 min of heating. B Distant effects of this treatment approach were determined using a bilateral tumor model where one tumor was subjected to the same heating protocol as described in (A), while the second tumor remained unheated. As part of study (B), the addition of immune checkpoint inhibition therapy (anti-PD-1) was pursued to enhance distant anti-tumor effects. Specifically, animals treated with ThermoVRL and localized mild hyperthermia received anti-PD-1 treatment one day prior to chemotherapy administration. Bi-weekly anti-PD-1 treatment was then continued for a total of 5 weeks (40.51 ± 0.12 °C), as well as the concentration of VRL (2.8 ± 0.7 mg VRL/mL) and the data were in agreement with previously published results. The heat-triggered in vitro release of the ThermoVRL formulation was previously characterized over a broad range of temperatures in protein-containing release media [35]. In brief, these studies found < 5% VRL release over a duration of 10 min at 37 °C and 100% release after 30 s at 42 °C.
The pharmacokinetics and biodistribution of VRL administered as ThermoVRL in combination with HT localized to the tumor were previously investigated in a subcutaneous model of human RMS in immunocompromised mice [38]. In brief, administration of VRL (10 mg VRL/kg body weight) as ThermoVRL + HT resulted in a sevenfold increase in AUC ∞,blood and an eightfold reduction in clearance relative to administration as free VRL + HT [38]. Similarly, VRL encapsulation in ThermoVRL reduced the volume of distribution almost tenfold compared to administration as free VRL + HT. Moreover, ThermoVRL + HT and free VRL + HT afforded similar VRL concentrations in normal tissue (i.e., liver, kidney, spleen). However, ThermoVRL + HT resulted in VRL concentrations nearly fourfold higher in tumor tissue relative to free VRL + HT. These findings describe the general pharmacokinetics and biodistribution behavior of VRL following administration as ThermoVRL and in combination with localized mild HT in an immunocompromised animal model. Other groups have previously assessed the pharmacokinetics of doxorubicin administered via thermosensitive liposomes in both immunocompromised and immunocompetent mice [39][40][41]. In both cases, the circulation half-life for the drug is relatively short, particularly in comparison to administration in non-thermosensitive liposomes. However, it is important to recognize the difficulties in making comparisons between different animal studies. For example, in all of the studies referenced here, the liposome formulations were prepared independently by different research labs, and this introduces inherent variability. Moreover, other factors such as animal Fig. 1 In vivo efficacy study comparing the different treatment groups with and without the addition of mild hyperthermia (HT) localized to the tumor site. Male C57BL/6 mice bearing a single, subcutaneous M3-9-M tumor were treated once on day 0 with 10 mg VRL/ kg body weight. A Tumor volume change, B body weight change, and C Kaplan-Meier survival analysis are shown. The addition of localized HT to treatment with thermosensitive liposomes loaded with VRL (ThermoVRL) improved median survival times nearly ninefold (p < .03). n ≥ 5 mice per group strain, sex, and age, as well as tumor burden are known to also influence a drug (or formulation's) pharmacokinetics [42][43][44][45]. That being said, we do observe that a shorter halflife tends to be reported for drugs when administered in thermosensitive liposomes (relative to non-thermosensitive liposomes). This highlights the need to heat the tumor site to the target temperature prior to thermosensitive liposome administration in order to maximize the amount of drug that can be released at the tumor.
In this study, we evaluated the efficacy of ThermoVRL in an immunocompetent murine model of RMS [46]. Specifically, M3-9-M cells were used to grow subcutaneous tumors in male C57BL/6 mice. As shown in Scheme 1, tumors were heated to HT temperatures (HT; 42.4 ± 0.5 °C) for 5 min prior to i.v. administration of free VRL or ThermoVRL [47]. Pre-heating was performed to ensure maximum intravascular drug release prior to liposome clearance from the systemic circulation. Localized heating was then continued for another 20 min post administration.
To evaluate the efficacy of ThermoVRL (+ / − HT) in this immunocompetent animal model, controls of saline (+ / − HT) and free VRL (+ / − HT) were used. Hyperthermia is known to have direct cytotoxic effects particularly on cancer cells [48]. However, this cytotoxicity depends on both the temperature and the duration of heating [49]. As shown in Fig. 1C, localized HT treatment (compared to saline alone) did not increase median survival (11 ± 1 days versus 10 ± 1 days, respectively). The heating protocol employed was designed to effectively trigger intravascular drug release from thermosensitive liposomes. Higher temperatures or extended heating times would likely be required to result in hyperthermia-mediated cytotoxicity. Treatment with free VRL at 10 mg VRL/kg body weight increased the median survival time (20 ± 10 days) in comparison to the administration of saline (10 ± 1 days), albeit this increase was not statistically significant (Fig. 1C). However, the addition of HT further improved survival times of animals treated with free VRL (26 ± 5 days), leading to a significant difference in comparison to saline-treated animals (p = .04). In fact, 2 out of the 5 mice treated with free VRL + HT went into complete remission with no regrowth of tumors prior to the end of the study (120 days). Interestingly, in our previous study which explored the therapeutic effect of VRL and liposomal formulations of VRL in an immunocompromised model of RMS, we did not observe a benefit associated with the addition of HT to free drug treatment [38]. This does point toward a potential treatment benefit enabled by components of the immune system.
As expected, adding HT to ThermoVRL treatment significantly improved median survival times relative to treatment with ThermoVRL alone (14 ± 1 days versus > 120 days; p < .03; Fig. 1C). In fact, all animals treated with Ther-moVRL in combination with HT went into complete remission and no palpable tumors were detected for the remainder of the study.
On the other hand, no statistically significant difference in median survival between animals treated with free VRL or ThermoVRL alone (p = 1) was detected. Equally, no statistically significant difference in tumor growth on different days post treatment was detected between these two treatment groups (p > .2) (Fig. 1A). This data does demonstrate that the addition of HT is required for ThermoVRL to be effective.
The body weight of mice was recorded as a measure of treatment toxicity. A decrease in body weight was observed in animals administered VRL (free or ThermoVRL) immediately following treatment, but animals appeared to recover quickly with weights reaching baseline levels within 10 days post treatment. Previously, Drummond et al. evaluated the toxicity of unloaded traditional liposomes only containing TEA 8 SOS solution in Swiss-Webster mice [50]. These studies did not find any severe toxicity up to a dose of 583.2 µmol phospholipid/kg. ThermoVRL liposomes utilized in our studies contain TEA 8 SOS solution at the same concentration and are administered at approximately 460 µmol phospholipid/kg. Thus, the encapsulated TEA 8 SOS solution is not expected to contribute to the body weight loss observed in Fig. 1B. Additionally, Dunne et al. previously evaluated the toxicity of free doxorubicin treatment versus thermosensitive liposomal doxorubicin (i.e., same lipid composition as ThermoVRL) in terms of body weight changes in breast cancer-bearing SCID mice and did not report any increased toxicity of the liposome formulation relative to the free drug [51]. Additionally, all formulation components are commonly used in liposomal formulations and in the case of ThermoDox have been used in clinical trials previously [11]. Taken together, these studies indicate that toxicity due to the unloaded ThermoVRL formulation is highly unlikely.
Treatment of localized tumors with ThermoVRL + HT in this immunocompetent model of RMS proved to be highly effective. In order to investigate the systemic treatment capabilities of this approach, a bilateral tumor model was employed (Scheme 1). M3-9-M cells were injected subcutaneously into both the left and right hind limbs of each mouse. Following tumor development, the tumor with the larger volume was considered the "primary" tumor, while contralateral tumors were used as a proxy for metastatic disease. HT was applied locally to the primary tumor only, and the contralateral tumor remained unheated. The average volume of primary (i.e., heated) tumors was 162 ± 72 mm 3 compared to 85 ± 48 mm 3 for the contralateral (i.e., unheated) tumors (p < .001). Control groups for this study included animals bearing bilateral tumors that received (i) no treatment (untreated); (ii) i.p. PD-1 blockade therapy (anti-PD-1); and (iii) i.v. saline and HT (saline + HT). Free VRL was not included as a treatment group in the bilateral tumor study. The goal of this study was to evaluate the systemic anti-tumor effects of ThermoVRL + HT and to identify if combination with immunotherapy enhances these effects.
Interestingly, as shown in Fig. 2, the saline + HT group exhibited significantly smaller primary (i.e., heated) tumor volumes at day 11 compared to tumors of animals in the untreated group (p = .03). However, no difference in tumor volumes was observed between the contralateral (i.e., unheated) tumors of animals in the saline + HT group and tumors of animals in the untreated group. This resulted in no difference in median survival times between these treatment groups (Fig. 4). This indicates that under these conditions HT treatment does result in a reduced tumor growth; however, this effect was limited to the primary tumor and did not yield an abscopal effect. As expected, treatment with ThermoVRL + HT led to complete primary tumor remission in 9 out of 10 animals (Fig. 2). Volumes of contralateral tumors were significantly reduced compared to corresponding tumors of the saline + HT control group (on day 11, p < .001). Figure S2 provides a detailed overview of the tumor growth of contralateral (i.e., unheated) tumors from all control and treatment groups. The limited growth inhibition of contralateral (i.e., unheated) tumors in animals treated with ThermoVRL + HT led to animals reaching ethical endpoints within 37 days. Nonetheless, median survival times of animals treated with ThermoVRL + HT were significantly prolonged compared to animals treated with saline + HT or untreated animals (p < .01). In fact, the median survival time was found to be similar to that of mice receiving free drug treatment in the single tumor study (Scheme 1) (25 ± 1 days and 20 ± 10 days, respectively). Intravascular drug release triggered at the target site is known to increase the amount of drug delivered to the tumor [37,52]; however, a significant amount of released drug can access the systemic circulation [10]. It appears that drug molecules that enter the systemic circulation (following ThermoVRL + HT treatment) do significantly influence the growth of contralateral tumors (Fig. 2). Indeed, despite the heating being applied locally, this delivery approach does not preclude systemic drug exposure. Thus, off-target anti-tumor effects should not Fig. 2 Volume change of bilateral M3-9-M tumors in male C57BL/6 mice on day 11 post treatment. Thermosensitive liposomal vinorelbine (ThermoVRL) was administered at 10 mg/ kg body weight. Saline and ThermoVRL were administered once intravenously on day 0, while immune checkpoint inhibition (ICI) therapy via anti-PD-1 antibodies was administered intraperitoneally on day 1 and continued twice per week for a total of 5 weeks. The primary tumor was subjected to mild hyperthermia (HT; 42.5 °C, 25 min) treatment (i.e., heated), while the contralateral tumor remained unheated (i.e., unheated). Addition of ICI therapy to mild HT treatment alone resulted in a significant abscopal effect. More importantly, the addition of ICI to ThermoVRL + HT provided significant anenestic tumor effects resulting in complete contralateral tumor remission in 8 out of 10 mice on day 11 post treatment. n ≥ 8 mice per group be referred to as abscopal effects but rather as anenestic tumor effects [34]. Where, enestic describes a tumor lesion exposed to intratumoral treatment (here externally triggered intravascular release of chemotherapeutics) and anenestic refers to "non-injected" lesions. Here, the anenestic tumor effects of ThermoVRL + HT stand in stark contrast to the effect observed on primary, heated tumors (i.e., where complete remission was observed). This does highlight the tremendous potential of ThermoVRL + HT to provide local anti-tumor effects, but it also demonstrates the limited effects of this treatment approach on secondary, unheated tumors.
In order to extend beyond the localized treatment constraints of ThermoVRL + HT, a multimodal strategy including immunotherapy was evaluated. Specifically, animals received 200 µg of anti-PD-1 antibody administered i.p. approximately 24 h prior to treatment with Ther-moVRL + HT. Anti-PD-1 injections were subsequently continued twice per week for a total of 5 weeks or until animals reached the endpoint. No significant difference in tumor volume between primary (i.e., heated) and contralateral (i.e., unheated) tumors was observed within the anti-PD-1 + HT group on day 11 (Fig. 2). The tumor volumes for both the primary and contralateral tumors of the anti-PD-1 + HT animals were significantly reduced compared to the anti-PD-1 control (on day 11; p < .01). Thus, indicating that these abscopal effects are stimulated by anti-PD-1 + HT and not anti-PD-1 treatment alone. This combination (i.e., anti-PD-1 + HT) did induce complete remission of both the primary and contralateral tumors in 3 out of 10 animals (compared to 0 out of 8 mice treated with anti-PD-1 only) (Fig. 3). Moreover, 7 out of 10 of the primary (i.e., heated) tumors in mice receiving anti-PD-1 + HT were undetectable at the time of sacrifice or study termination. Overall, this led to a twofold increase in survival time for animals receiving anti-PD-1 + HT compared to anti-PD-1 alone (p < .01).
As shown in Fig. 2, combining ThermoVRL + HT with PD-1 blockade therapy significantly reduced the growth of contralateral (i.e., unheated) tumors compared to treatment with ThermoVRL + HT alone. This led to a more than 2.5-fold increase in median survival time (> 68 days versus 25 ± 1 days; p < .001) (Fig. 4). In fact, for 7 out of the 10 mice, both the primary (i.e., heated) and contralateral (i.e., unheated) tumors went into complete remission with no palpable tumor regrowth for the duration of the study. The increase in median survival time was significantly greater than that resulting from treatment with anti-PD-1 alone (19 ± 2 days; p < .001). While the triple combination, ThermoVRL + HT + anti-PD-1, prolonged survival compared to anti-PD-1 + HT almost 1.6-fold, this difference was not found to be statistically significant (> 68 days versus 40 ± 5 days; p = .633).

Treatment with thermosensitive liposomes and localized mild hyperthermia
Systemic administration of thermosensitive liposomes in combination with localized HT has been proven to be highly effective in the treatment of local disease [53][54][55]. These improvements are largely attributed to increased accumulation and distribution of drug molecules to the tumor site [56][57][58]. However, this delivery approach requires heating of the target region as a trigger for drug release. Advances in heating techniques, thermometry, and treatment planning have significantly expanded the types, volumes, and locations of tumors that are amenable to mild hyperthermic heating [59][60][61]. Thus, expanding the clinical scenarios in which thermosensitive liposomes can be explored. However, any therapeutic approach focused on a localized target risks undertreating metastatic disease. Indeed, metastatic spread continues to be the primary cause of death from cancer [62,63]. This raises the question of how to increase the systemic anti-tumor effects of this treatment approach. Previously, Viglianti et al. demonstrated that a formulation equivalent to ThermoDox was able to reduce tumor growth in secondary, unheated tumors in a bilateral immunodeficient mouse model [10]. The authors concluded that this effect "is most likely due to recirculation of intravascularly released drug" [10]. However, there are several factors that need to be considered prior to generalizing these findings. First, the drug's pharmacokinetic properties will govern its circulation behavior and thus impact systemic treatment effects. Second, it is important to note that this study employed water bath heating to achieve mild hyperthermic tumor temperatures. While this approach does lead to uniform heating of the animal's hind limb, heating is not constrained to the tumor [64]. Consequently, drug release occurs throughout the entire heated vasculature. Differences in heated tissue volume have been shown to affect the kinetics as well as total amount of intravascularly released drug, thus leading to varied amounts of free drug present in the systemic circulation [64]. The laser-based heating setup employed in this study provides more precise localized heating compared to water bath heating [47]. Nonetheless, combining thermosensitive liposomal VRL with localized HT treatment via this heating setup resulted in reduced growth of the contralateral (i.e., unheated) tumor. This reduction in tumor growth could be attributed to systemically available ThermoVRL and free VRL. However, since these studies were performed in an immunocompetent animal model, it could also be due to immune mediated anti-tumor effects stimulated by HT and/ or VRL chemotherapy. While it is interesting to observe this anti-tumor effect at the contralateral site, it is important to note that this is a bilateral tumor model used to assess systemic anti-tumor capabilities. In general, metastatic tumor cell spread follows a highly complex process that is not reflected by bilateral tumor implantation [65]. Nonetheless, following primarily localized drug release of VRL at the heated tumor we do observe a systemic anti-tumor effect. Thus, this study confirms the findings reported by Viglianti et al. albeit in a different animal model, and for a different thermosensitive liposome formulation.

Immunocompetent animal models of rhabdomyosarcoma
We previously evaluated the combination of ThermoVRL and HT in an immunocompromised murine model of RMS [38]. However, immunocompetent cancer models are necessary to evaluate the full potential of heat-triggered drug delivery approaches. While heating is used as the external trigger for drug release, it also elicits a series of effects that stimulate and enhance anti-tumor immune responses [25]. Most murine RMS models are based on human-derived xenografts and only a few syngeneic RMS animal models have been reported [66]. The M3-9-M RMS model employed was previously developed and characterized by Meadors et al. [46]. The authors demonstrated its immunogenicity and responsiveness to T-cell-based immunotherapy [46]. Additionally, Highfill et al. demonstrated the role of PD-1 signaling in the immune escape of the M3-9-M tumors. Interestingly, in this study, PD-1 blockade therapy showed limited tumor growth inhibition effects when initiated later in the tumor development process [67]. Generally, orthotopic tumor models better Fig. 3 Male C57BL/6 mice bearing bilateral, subcutaneous, M3-9-M tumors received either no treatment (untreated), intraperitoneal PD-1 blockade therapy (anti-PD-1), intravenous saline (saline), or intravenous thermosensitive liposomal vinorelbine (ThermoVRL) at 10 mg VRL/kg body weight. As indicated (+ HT), several of these treatments were administered simultaneously with mild hyperthermia (42.5 °C, 25 min) localized to the primary tumor. Tumor growth of contralateral (i.e., unheated) tumors is indicated in blue, while growth of primary (i.e., heated) tumors is shown in red. Comparing the tumor growth of animals that received saline + HT and animals treated with anti-PD-1 + HT reveals abscopal effects in the form of slower tumor growth in the contralateral tumors. Only one primary tumor in the Ther-moVRL + HT + anti-PD-1 group did not respond to therapy. This triple combination also afforded significantly slower tumor growth of contralateral tumors. n ≥ 8 mice per group recapitulate clinical disease; however, in the present study, tumors were grown subcutaneously due to limitations associated with the laser-based heating setup [47,[68][69][70]. Kheirolomoom et al. have previously investigated similar strategies to increase the systemic treatment capabilities of thermosensitive liposomes. For example, the combination of a thermosensitive liposome formulation of doxorubicin with ultrasound-mediated hyperthermia and intratumoral administration of CpG has previously been studied [71].

Previous studies combining thermosensitive liposomes and immune checkpoint inhibition
Here, CpG was employed as a local immune adjuvant to stimulate a more potent innate immune response. However, this therapy proved to be inefficient in treating distant (i.e., unheated) tumors. In a subsequent study, the authors showed that the addition of PD-1 blockade priming therapy to this treatment approach led to a potent anti-tumor T-cell response and successfully treated local as well as distant tumors [72]. The immunotherapy combination (i.e., i.t. CpG plus i.p. anti-PD-1) alone provided a potent systemic anti-tumor effect, without the addition of heat-triggered chemotherapy. Thus, based on this study, it is difficult to discern the contribution of thermosensitive liposomal doxorubicin to immunotherapy treatment. In our study, we also observed improvements in the survival of animals receiving immunotherapy. Furthermore, we saw a significant survival benefit in animals treated with immunotherapy (i.e., anti-PD-1) in combination with ThermoVRL and localized HT, compared to animals treated with immunotherapy alone. Albeit, in a different disease model and using a different treatment approach compared to the aforementioned study by Kheirolomoom et al. Ultimately, a comparison of the present study and the study conducted by Kheirolomoom et al. highlights that the integration of immunotherapy into multimodal treatment regimens and evaluation of their efficacy in murine models of cancer is non-trivial.

Mild hyperthermia and abscopal effects
As mentioned previously, the exact mechanism underlying the abscopal effect associated with localized therapies remains unclear [20]. However, it is generally recognized that a systemic cytotoxic T-cell-mediated anti-tumor response plays a key role. Interestingly, HT has been shown Efficacy study evaluating systemic treatment effects of thermosensitive liposome-mediated vinorelbine chemotherapy in combination with localized mild hyperthermia and immune checkpoint inhibition (ICI) therapy. Male C57BL/6 mice bearing two bilateral, subcutaneous, M3-9-M tumors received either saline (saline) or thermosensitive liposomes loaded with vinorelbine (ThermoVRL) at 10 mg/kg body weight via intravenous teil vein injection. Primary tumors were heated to mild hyperthermic temperatures (HT) 5 min prior to intravenous treatment administration followed by heating for another 20 min. Contralateral tumors remained unheated. Intraperitoneal administration of 200 µg anti-PD-1 antibody was commenced 24 h prior to treatment with saline, ThermoVRL, or mild HT alone. PD-1 blockade therapy was subsequently administered twice per week for a total of 5 weeks. The addition of ICI therapy to mild HT (i.e., saline + HT compared to anti PD-1 + HT) increased survival times 3.6-fold (p < .001). The addition of ICI to ThermoVRL (i.e., ThermoVRL + HT compared to Ther-moVRL + HT + anti-PD-1) therapy increased median survival times 2.7-fold (p < .001). n ≥ 8 mice per group to directly and indirectly influence components of the innate as well as an adaptive immune response [73]. However, it is important to note that in most of these studies heat is applied for extended periods of time (≥ 1 h), and/or wholebody hyperthermia is employed instead of localized heating. In fact, very few in vivo studies have evaluated the potential abscopal effects that may be induced by treatment with localized HT alone. HT is most commonly applied in combination with chemotherapy or radiotherapy treatment, thereby making it difficult to determine any effect of HT alone [74,75]. It was only recently that HT was combined with ICI to enhance abscopal effects [27,76]. Oei et al. evaluated hyperthermia alone and in combination with ICI in a highly metastatic mouse model of breast cancer (i.e., luciferasetransfected 4T1 cells). The authors observed an increase in lung metastases following treatment with a combination of localized HT and ICI (i.e., anti-PD-1 and anti-CTLA-4). In contrast, Ibuki et al. employed the non-transfected 4T1 parent cell line in a bilateral subcutaneous tumor model. Here, the combination of HT and ICI (i.e., anti-CTLA-4) led to strong tumor growth inhibition of both heated and unheated tumors [76]. The combination treatment also reduced metastatic spread to lungs (compared to ICI alone), resulting in an increase in overall survival. The contradictory results reported by Oei et al. and Ibuki et al. further highlight the need for more research into the combined effect of immunotherapy and HT [27,76]. While there are many differences in the study design and treatment strategies that might explain these results, there are four key differences that highlight the complexity of evaluating such a multimodal treatment approach. First, the immunogenic potential of the luciferasetransfected cell line needs to be considered when evaluating an anti-tumor immune response [77]. Second, the anti-tumor immune response is heavily influenced and directed by the tumor microenvironment (i.e., orthotopic versus ectopic) [78]. Third, the immune effects mediated by HT treatment depend on the temperature and duration of heating as well as the specific heating technique [79,80]. And lastly, the timing of treatment relative to disease burden is critical when evaluating the efficacy of treatment-induced anti-tumor immune effects. For example, the overall tumor burden has been shown to have an immunosuppressive effect [81]. In the present study, when PD-1 blockade monotherapy was initiated early (i.e., at a significantly smaller tumor volume) this led to complete tumor remission (albeit n = 2 mice), while delayed treatment resulted in limited tumor growth inhibition (Fig. S3). This is in agreement with the previous work done by Highfill et al. [67]. These studies demonstrate the complexity of this multimodal approach and highlight the need for detailed studies to better understand the parameters that influence treatment outcomes.

Contributions of chemotherapy to systemic anti-tumor immune response
In the last ten years, chemotherapy has increasingly been recognized for its potential impact on the immune system [28][29][30][31]. There is a growing body of evidence that suggests chemotherapy can exert a physiological response beyond a direct cytostatic or cytotoxic effect. In fact, some have proposed that the reason behind the success of certain chemotherapies lies in their ability to induce an anti-tumor immune response [82,83]. This is usually attributed to a specific form of regulated cell death, known as immunogenic cell death [84]. Indeed, several commonly used chemotherapeutic drugs have recently been identified as immunogenic cell death inducers. Among these are anthracyclines (e.g., doxorubicin) as well as microtubule inhibitors (e.g., taxanes and vinca alkaloids), including VRL [85][86][87]. The classification of chemotherapeutic drugs as immunogenic cell death inducers is an active area of research. However, there is evidence to suggest that both localized HT as well as VRL chemotherapy can create immunogenic tumors [30,[88][89][90][91][92][93]. This would certainly be advantageous, and potentially synergistic, when administered in combination with ICI therapy. Indeed, chemotherapy combined with ICI has been suggested as a promising approach to produce systemic anti-cancer immunity [29,32,33]. Thermosensitive liposome-mediated chemotherapy could be of particular interest since it not only targets the drug specifically to the tumor but also comes with the immune-stimulating effects of localized HT treatment. In the present study, such synergy is observed for the triple combination of ThermoVRL, HT, and anti-PD-1. Specifically, this is demonstrated by enhanced efficacy at the distant tumor sites. Furthermore, this idea of exploiting the potentially immunostimulatory effects of VRL is currently being investigated in clinical trials as a combination with immunotherapy (NCT03801304, NCT03518606, NCT04848454).
To conclude, ThermoVRL plus localized HT in a bilateral tumor model of RMS was shown to have a significant anti-tumor effect at the heated primary site, resulting in tumor remission. Moreover, an effect at distant tumor sites was also observed for this treatment approach, albeit limited to reduced tumor growth rates. Interestingly, combining ICI therapy with ThermoVRL and HT significantly improved the treatment of distant tumor sites. Moreover, the addition of ICI therapy to localized HT alone produced a measurable abscopal effect. The underlying mechanism of this synergistic treatment effect will require further investigation. Yet, to the authors' knowledge, this is the first study demonstrating that ICI therapy can enhance the systemic treatment capabilities of thermosensitive liposomes in combination with localized HT.